呼吸道融合病毒(Respiratory syncytial virus；RSV)為感染肺及呼吸通道之呼吸道病毒。RSV為全世界嬰兒中嚴重病毒性下呼吸道病痛之主要原因及老年人中呼吸道病痛之重要原因。在2023年批准兩種RSV蛋白次單位疫苗：ABRYSVO (Pfizer)及AREXVY (GSK)。然而，尚無RNA疫苗經批准用於預防RSV感染。Respiratory syncytial virus (RSV) is a respiratory virus that infects the lungs and respiratory tract. RSV is the leading cause of severe viral lower respiratory tract illness in infants worldwide and an important cause of respiratory tract illness in the elderly. Two RSV protein subunit vaccines were approved in 2023: ABRYSVO (Pfizer) and AREXVY (GSK). However, no RNA vaccines have been approved for the prevention of RSV infection.
RSV為肺病毒科之一員。其基因體由編碼11種蛋白質(包括九種結構蛋白(三種醣蛋白及六種內部蛋白)及兩種非結構蛋白)之單股、反義RNA分子組成。結構蛋白包括三種跨膜表面醣蛋白：附著蛋白G、融合蛋白F及疏水性小SH蛋白。存在兩種RSV子類型：A及B。其主要在G醣蛋白方面不同，而兩種子類型之間的F醣蛋白之序列更保守。RSV is a member of the Pneumoviridae family. Its genome consists of a single-stranded, negative-sense RNA molecule encoding 11 proteins, including nine structural proteins (three glycoproteins and six internal proteins) and two nonstructural proteins. The structural proteins include three transmembrane surface glycoproteins: attachment protein G, fusion protein F, and a small hydrophobic SH protein. There are two RSV subtypes: A and B. They differ primarily in the G glycoprotein, while the sequence of the F glycoprotein is more conserved between the two subtypes.
成熟F醣蛋白具有三個一般域：胞外域(ED)、跨膜域(TM)及細胞質尾(CT)。CT含有單個棕櫚醯化半胱胺酸殘基。The mature F glycoprotein has three general domains: the extracellular domain (ED), the transmembrane domain (TM), and the cytoplasmic tail (CT). The CT contains a single palmitylated cysteine residue.
人類RSV之F醣蛋白最初自mRNA轉譯為單個574胺基酸多肽前驅物(稱為「F0」或「F0前驅物」)，其在N端含有訊號肽序列(胺基酸1-25)。在轉譯後，訊號肽在內質網中被訊號肽酶移除。F0前驅物之剩餘部分(亦即殘基26-574)可在兩個多鹼基位點(a.a. 109/110及136/137)處被細胞蛋白酶(特定而言弗林蛋白酶(furin))裂解，從而移除稱為pep27 (胺基酸110-136)之27胺基酸插入序列且產生稱為F1 (C端部分；胺基酸137-574)及F2 (N端部分；胺基酸26-109)的兩個連接片段。F1在其N端處含有疏水性融合肽，及兩個七肽(heptad)重複區域(HRA及HRB)。HRA在融合肽附近，且HRB在TM域附近。F1及F2片段經由兩個雙硫鍵連接在一起。無訊號肽序列之未裂解的F0蛋白質或F1-F2異二聚體二者中之一者可形成RSV F原聚體。三種此類原聚體組裝以形成最終RSV F蛋白複合物，其為三種原聚體之同源三聚體。The F glycoprotein of human RSV is initially translated from mRNA as a single 574 amino acid polypeptide precursor (called "F0" or "F0 precursor"), which contains a signal peptide sequence (amino acids 1-25) at the N-terminus. After translation, the signal peptide is removed by signal peptidase in the endoplasmic reticulum. The remainder of the F0 promotor (i.e., residues 26-574) can be cleaved by cellular proteases (specifically furin) at two polybasic sites (a.a. 109/110 and 136/137), thereby removing a 27-amino acid insertion sequence called pep27 (amino acids 110-136) and generating two linked fragments called F1 (C-terminal portion; amino acids 137-574) and F2 (N-terminal portion; amino acids 26-109). F1 contains a hydrophobic fusion peptide at its N-terminus, and two heptad repeat regions (HRA and HRB). HRA is near the fusion peptide, and HRB is near the TM domain. The F1 and F2 fragments are linked together via two disulfide bonds. Either the uncleaved F0 protein or the F1-F2 heterodimer without the signal peptide sequence can form the RSV F protomer. Three such protomers assemble to form the final RSV F protein complex, which is a homotrimer of three protomers.
子類型A及B之F蛋白在胺基酸序列方面約90%一致。A子類型之F0前驅物多肽之實例序列提供於SEQ ID NO: 1 (A2株系；GenBank GI：138251；Swiss Prot P03420)中，且B子類型之F0前驅物多肽之實例序列提供於SEQ ID NO: 2 (18537株系；GenBank GI：138250；Swiss Prot P13843)中。SEQ ID NO: 1及SEQ ID NO: 2均為574胺基酸序列。SEQ ID NO: 1及SEQ ID NO: 2之訊號肽序列亦已報導為胺基酸1-25 (GenBank及UniProt)。在兩個序列中，TM域大約為胺基酸530至550，但可替代地報導為525-548。細胞質尾始於胺基酸548或550且結束於胺基酸574，其中棕櫚醯化半胱胺酸殘基位於胺基酸550處。The F proteins of subtypes A and B are approximately 90% identical in amino acid sequence. An example sequence of a F0 pro-promoter polypeptide of subtype A is provided in SEQ ID NO: 1 (A2 strain; GenBank GI: 138251; Swiss Prot P03420), and an example sequence of a F0 pro-promoter polypeptide of subtype B is provided in SEQ ID NO: 2 (18537 strain; GenBank GI: 138250; Swiss Prot P13843). SEQ ID NO: 1 and SEQ ID NO: 2 are both 574 amino acid sequences. The signal peptide sequences of SEQ ID NO: 1 and SEQ ID NO: 2 have also been reported as amino acids 1-25 (GenBank and UniProt). In both sequences, the TM domain is approximately amino acids 530 to 550, but may alternatively be reported as 525-548. The cytoplasmic tail begins at amino acids 548 or 550 and ends at amino acid 574, with a palmitylated cysteine residue at amino acid 550.
RSV F蛋白為探究RSV疫苗之初級抗原。RSV F蛋白三聚體介導病毒顆粒膜與宿主細胞膜之間的融合，且亦促進合胞體之形成。在與宿主細胞之膜融合之前的病毒顆粒中，最大的F分子群體形成棒棒糖形結構，其中TM域錨定於病毒套膜中[Dormitzer, P.R., Grandi, G., Rappuoli, R., Nature Reviews Microbiol, 10, 807, 2012.]。此構形稱為融合前構形。融合前RSV F被單株抗體(mAb) D25、AM22及MPE8識別，寡聚狀態之間無區別。融合前F三聚體被mAb AM14特異性識別[Gilman MS, Moin SM, Mas V等人, PLoS Pathogens,11(7), 2015]。在RSV進入細胞期間，F蛋白經由中間延伸結構自融合前狀態(其在本文中可稱為「pre-F」)重排為融合後狀態(「post-F」)。在此重排期間，融合前分子之C端捲曲螺旋解離成其三個組成股，接著其圍繞球狀頭部捲繞且接合三個額外螺旋以形成融合後六螺旋束。若融合前RSV F三聚體經受愈來愈苛刻的化學或物理條件(諸如高溫)，則其進行結構變化。起初，喪失三聚結構(至少在分子內局部地)且接著重排為融合後形式，且接著域變性。RSV F protein is the primary antigen for exploring RSV vaccines. RSV F protein trimers mediate the fusion between the viral particle membrane and the host cell membrane, and also promote the formation of syncytia. In the viral particle before fusion with the host cell membrane, the largest F molecule group forms a lollipop-shaped structure, in which the TM domain is anchored in the viral envelope [Dormitzer, P.R., Grandi, G., Rappuoli, R., Nature Reviews Microbiol, 10, 807, 2012.]. This configuration is called the pre-fusion configuration. Before fusion, RSV F is recognized by monoclonal antibodies (mAb) D25, AM22 and MPE8, and there is no difference between oligomeric states. The prefusion F trimer is specifically recognized by mAb AM14 [Gilman MS, Moin SM, Mas V et al., PLoS Pathogens, 11(7), 2015]. During RSV entry into cells, the F protein rearranges from the prefusion state (which may be referred to herein as "pre-F") to the postfusion state ("post-F") via an intermediate extension structure. During this rearrangement, the C-terminal coiled coil of the prefusion molecule dissociates into its three component strands, which then coil around the globular head and join three additional helices to form the postfusion six-helix bundle. If the prefusion RSV F trimer is subjected to increasingly harsh chemical or physical conditions (such as high temperature), it undergoes structural changes. Initially, the trimeric structure is lost (at least locally within the molecule) and then rearranged to a post-fusion form and then the domains are denatured.
為了防止病毒進入，F特異性中和抗體可能必須在病毒套膜與細胞膜融合之前結合病毒顆粒上之F的融合前構形或可能的延伸的中間物。因此，F蛋白之融合前形式被視為作為所需疫苗抗原的較佳構形[Ngwuta, J.O., Chen, M., Modjarrad, K., Joyce, M.G., Kanekiyo, M., Kumar, A., Yassine, H.M., Moin, S.M., Killikelly, A.M., Chuang, G.Y., Druz, A., Georgiev, I.S., Rundlet, E.J., Sastry, M., Stewart-Jones, G.B., Yang. Y., Zhang, B., Nason, M.C., Capella, C., Peeples, M., Ledgerwood, J. E., Mclellan, J.S., Kwong, P.D., Graham, B.S., Science Translat. Med., 14, 7, 309 (2015)]。在用界面活性劑(諸如曲拉通(Triton) X-100、曲拉通X-114、NP-40、Brij-35、Brij-58、吐溫(Tween) 20、吐溫80、辛基葡糖苷、辛基硫葡糖苷、SDS、CHAPS、CHAPSO)自膜萃取、或表現為胞外域、物理或化學脅迫、或儲存後，F醣蛋白容易地轉化成融合後形式[McLellan JS, Chen M, Leung S等人Structure of RSV fusion glycoprotein trimer bound to a pre-fusion-specific neutralizing antibody. Science 340, 1113-1117 (2013)；Chaiwatpongsakorn, S., Epand, R.F., Collins, P.L., Epand R.M., Peeples, M.E., J Virol. 85(8):3968-77 (2011)；Yunus, A.S., Jackson T.P., Crisafi, K., Burimski, I., Kilgore, N.R., Zoumplis, D., Allaway, G.P., Wild, C.T., Salzwedel, K. Virology. 2010年1月20日;396(2):226-37]。因此，製備融合前F作為疫苗抗原仍為一種挑戰。由於中和及藉由干擾病毒進入進行的保護性抗體功能，所以假定不引起融合前特異性抗體之F抗原預期不與引起融合前特異性抗體之F抗原一樣有效。因此，認為更需要利用含有呈融合前形式之F蛋白免疫原的F蛋白疫苗。已提供RSV F蛋白之突變體以增加融合前穩定性(參見例如PCT申請案第WO2017/109629號)且為有前景的疫苗候選者。To prevent viral entry, F-specific neutralizing antibodies may have to bind to the prefusion conformation of F or possible extended intermediates on the viral particle before the fusion of the viral envelope with the cell membrane. Therefore, the prefusion form of the F protein is considered to be the preferred conformation for the desired vaccine antigen [Ngwuta, J.O., Chen, M., Modjarrad, K., Joyce, M.G., Kanekiyo, M., Kumar, A., Yassine, H.M., Moin, S.M., Killikelly, A.M., Chuang, G.Y., Druz, A., Georgiev, I.S., Rundlet, E.J., Sastry, M., Stewart-Jones, G.B., Yang. Y., Zhang, B., Nason, M.C., Capella, C., Peeples, M., Ledgerwood, J. E., Mclellan, J.S., Kwong, P.D., Graham, B.S., Science Translat. Med., 14, 7, 309 (2015)]. After extraction from membranes with surfactants (e.g., Triton X-100, Triton X-114, NP-40, Brij-35, Brij-58, Tween 20, Tween 80, octyl glucoside, octylthioglucoside, SDS, CHAPS, CHAPSO), or expression as an extracellular domain, physical or chemical stress, or storage, F glycoprotein is readily converted to a post-fusion form [McLellan JS, Chen M, Leung S et al. Structure of RSV fusion glycoprotein trimer bound to a pre-fusion-specific neutralizing antibody. Science 340, 1113-1117 (2013); Chaiwatpongsakorn, S., Epand, R.F., Collins, P.L., Epand R.M., Peeples, M.E., J Virol. 85(8):3968-77 (2011); Yunus, A.S., Jackson T.P., Crisafi, K., Burimski, I., Kilgore, N.R., Zoumplis, D., Allaway, G.P., Wild, C.T., Salzwedel, K. Virology. 2010 Jan 20;396(2):226-37]. Therefore, the preparation of prefusion F as a vaccine antigen remains a challenge. F antigens that do not elicit prefusion-specific antibodies are not expected to be as effective as F antigens that elicit prefusion-specific antibodies due to neutralization and protective antibody function by interfering with viral entry. Therefore, it is believed that there is a greater need to utilize F protein vaccines that contain F protein immunogens in prefusion form. Mutants of the RSV F protein have been provided to increase prefusion stability (see, e.g., PCT Application No. WO2017/109629) and are promising vaccine candidates.
併入F蛋白抗原之RSV疫苗已在研發中。臨床研究已顯示，一些基於F蛋白次單元的疫苗候選者為安全的且具有免疫原性，但保護性功效及保護持久性方面需要提高。RSV vaccines incorporating F protein antigens are under development. Clinical studies have shown that some vaccine candidates based on F protein subunits are safe and immunogenic, but the protective efficacy and durability of protection need to be improved.
因此，需要改良的免疫原性組合物以保護免受RSV感染。Therefore, there is a need for improved immunogenic compositions to protect against RSV infection.
本發明提供尤其如本文所提供之對針對RSV感染之改良的免疫原性組合物之未滿足的需求。在一個態樣中，本發明提供用於預防、治療或改善個體之感染、疾病或病狀的免疫原性組合物及方法，其包含投與RNA分子，例如編碼胺基酸序列(例如免疫原性抗原，包含呼吸道融合病毒(RSV)蛋白、其免疫原性變體、或RSV蛋白之免疫原性片段或其免疫原性變體，例如抗原性肽或蛋白質)之免疫原性RNA聚核苷酸。因此，免疫原性抗原包含用於誘導個體中針對RSV之免疫反應之RSV蛋白的抗原決定基。投與編碼免疫原性抗原之RNA聚核苷酸以(在藉由適當目標細胞表現聚核苷酸之後)提供用於誘導(例如刺激、啟動及/或擴增)免疫反應(例如抗體及/或免疫效應細胞)之抗原。在一個態樣中，根據本發明誘導之免疫反應為B細胞介導之免疫反應(例如抗體介導之免疫反應)以及T細胞介導之免疫反應兩者。在一個態樣中，免疫反應為抗RSV免疫反應。The present invention provides unmet needs for improved immunogenic compositions for RSV infection, particularly as provided herein. In one aspect, the present invention provides immunogenic compositions and methods for preventing, treating or ameliorating infection, disease or condition in an individual, comprising administering an RNA molecule, such as an immunogenic RNA polynucleotide encoding an amino acid sequence (e.g., an immunogenic antigen, comprising a respiratory syncytial virus (RSV) protein, an immunogenic variant thereof, or an immunogenic fragment of a RSV protein or an immunogenic variant thereof, such as an antigenic peptide or protein). Thus, the immunogenic antigen comprises an antigenic determinant of an RSV protein for inducing an immune response in an individual against RSV. Administration of RNA polynucleotides encoding immunogenic antigens provides antigens for inducing (e.g., stimulating, activating and/or amplifying) immune responses (e.g., antibodies and/or immune effector cells) (after expression of the polynucleotides by appropriate target cells). In one aspect, the immune responses induced according to the present invention are both B cell-mediated immune responses (e.g., antibody-mediated immune responses) and T cell-mediated immune responses. In one aspect, the immune response is an anti-RSV immune response.
本文所描述之免疫原性組合物包含RNA分子，其包含可在接收者之細胞中轉譯成一或多種蛋白質之RNA (作為活性成分)。除編碼抗原序列之野生型、密碼子最佳化或突變型序列以外，RNA分子可含有針對就穩定性及轉譯效率而言RNA之最大功效最佳化的一或多種結構元件(5'帽、5' UTR、次基因體啟動子、3' UTR、多-A尾)。在一個態樣中，RNA分子含有所有此等元件。本文所描述之RNA分子可與脂質及/或蛋白質複合以產生RNA-顆粒(例如脂質奈米顆粒(LNP))用於投與。在一個態樣中，本文所描述之RNA分子與脂質複合以產生RNA-脂質奈米顆粒(例如RNA-LNP)用於投與。在一個態樣中，本文所描述之RNA分子與蛋白質複合用於投與。在一個態樣中，本文所描述之RNA分子與脂質及蛋白質複合用於投與。若使用不同RNA分子之組合，則RNA分子可一同與脂質及/或蛋白質一起複合或分開與脂質及/或蛋白質一起複合以產生RNA-顆粒用於投與。The immunogenic compositions described herein include RNA molecules that include RNA (as active ingredients) that can be translated into one or more proteins in the cells of the recipient. In addition to the wild-type, codon-optimized or mutant sequences encoding the antigen sequence, the RNA molecule may contain one or more structural elements (5' cap, 5' UTR, subgenomic promoter, 3' UTR, poly-A tail) optimized for maximum efficacy of RNA in terms of stability and translation efficiency. In one aspect, the RNA molecule contains all of these elements. The RNA molecules described herein can be compounded with lipids and/or proteins to produce RNA-particles (e.g., lipid nanoparticles (LNPs)) for administration. In one aspect, the RNA molecules described herein are compounded with lipids to produce RNA-lipid nanoparticles (e.g., RNA-LNPs) for administration. In one aspect, the RNA molecules described herein are complexed with proteins for administration. In one aspect, the RNA molecules described herein are complexed with lipids and proteins for administration. If a combination of different RNA molecules is used, the RNA molecules can be complexed with lipids and/or proteins together or separately to produce RNA-particles for administration.
本發明提供RNA分子及RNA-LNP，其包括至少一個編碼RSV抗原之開讀框(open reading frame；ORF)。在一些態樣中，RSV抗原為RSV多肽。在一些態樣中，RSV多肽為RSV F蛋白。在一些態樣中，RSV F蛋白為全長蛋白、截短蛋白、其片段或變體。在一些態樣中，RSV F蛋白包含至少一個突變。The present invention provides RNA molecules and RNA-LNPs, which include at least one open reading frame (ORF) encoding an RSV antigen. In some aspects, the RSV antigen is an RSV polypeptide. In some aspects, the RSV polypeptide is an RSV F protein. In some aspects, the RSV F protein is a full-length protein, a truncated protein, a fragment thereof, or a variant. In some aspects, the RSV F protein comprises at least one mutation.
本發明提供RNA分子及RNA-LNP，其包括至少一個編碼表1之RSV多肽之ORF。在一些態樣中，RSV多肽包含選自SEQ ID NO: 1至6或71至74之胺基酸序列。在一些態樣中，RSV多肽與表1之胺基酸序列中之任一者(例如SEQ ID NO: 1至6或71至74中之任一者)具有、具有至少或具有至多90%、91%、92%、93%、94%、95、96%、97%、98%或99%或更高一致性。在一些態樣中，RSV多肽由表1之胺基酸序列中之任一者(例如SEQ ID NO: 1至6或71至74中之任一者)組成。The invention provides RNA molecules and RNA-LNP, which include the ORF of the RSV polypeptide of at least one coding table 1. In some aspects, the RSV polypeptide comprises the amino acid sequence selected from SEQ ID NO: 1 to 6 or 71 to 74. In some aspects, the RSV polypeptide and the amino acid sequence of Table 1 have, have at least or have at most 90%, 91%, 92%, 93%, 94%, 95, 96%, 97%, 98% or 99% or higher consistency. In some aspects, the RSV polypeptide is composed of any one of the amino acid sequence of Table 1 (such as SEQ ID NO: 1 to 6 or 71 to 74).
本發明提供RNA分子及RNA-LNP，其包含至少一個轉錄自表2之至少一個DNA核酸之ORF。在一些態樣中，RNA分子轉錄自選自SEQ ID NO: 7至10或59至62之核酸序列。在一些態樣中，RNA分子包含轉錄自與表2之核酸序列中之任一者(例如SEQ ID NO: 7至10或59至62中之任一者)具有、具有至少或具有至多90%、91%、92%、93%、94%、95、96%、97%、98%或99%或更高一致性之核酸序列的ORF。在一些態樣中，RNA分子包含轉錄自由表2之核酸序列中之任一者(例如SEQ ID NO: 7至10或59至62中之任一者)組成之核酸序列的ORF。The present invention provides RNA molecules and RNA-LNPs comprising at least one ORF transcribed from at least one DNA nucleic acid of Table 2. In some aspects, the RNA molecule is transcribed from a nucleic acid sequence selected from SEQ ID NOs: 7 to 10 or 59 to 62. In some aspects, the RNA molecule comprises an ORF transcribed from a nucleic acid sequence having, having at least or having at most 90%, 91%, 92%, 93%, 94%, 95, 96%, 97%, 98% or 99% or higher identity to any one of the nucleic acid sequences of Table 2 (e.g., any one of SEQ ID NOs: 7 to 10 or 59 to 62). In some aspects, the RNA molecule comprises an ORF transcribed from a nucleic acid sequence consisting of any one of the nucleic acid sequences of Table 2 (e.g., any one of SEQ ID NOs: 7 to 10 or 59 to 62).
本發明進一步提供RNA分子及RNA-LNP，其包含至少一個包含表3之RNA核酸序列之ORF。在一些態樣中，RNA分子包含選自SEQ ID NO: 11至16或63至70之核酸序列。在一些態樣中，RNA分子包含與表3之核酸序列中之任一者(例如SEQ ID NO: 11至16或63至70中之任一者)具有、具有至少或具有至多90%、91%、92%、93%、94%、95、96%、97%、98%或99%一致性的核酸序列。在一些態樣中，RNA分子包含由表3之核酸序列中之任一者(例如SEQ ID NO: 11至16或63至70中之任一者)組成的核酸序列。在一些態樣中，SEQ ID NO: 11至16中之任一者之各尿苷經N1-甲基假尿苷(Ψ)置換(例如經修飾之RNA；modRNA)。The present invention further provides RNA molecules and RNA-LNPs comprising at least one ORF comprising an RNA nucleic acid sequence of Table 3. In some aspects, the RNA molecule comprises a nucleic acid sequence selected from SEQ ID NOs: 11 to 16 or 63 to 70. In some aspects, the RNA molecule comprises a nucleic acid sequence having, having at least or having at most 90%, 91%, 92%, 93%, 94%, 95, 96%, 97%, 98% or 99% identity to any one of the nucleic acid sequences of Table 3 (e.g., any one of SEQ ID NOs: 11 to 16 or 63 to 70). In some aspects, the RNA molecule comprises a nucleic acid sequence consisting of any one of the nucleic acid sequences of Table 3 (e.g., any one of SEQ ID NOs: 11 to 16 or 63 to 70). In some aspects, each uridine of any one of SEQ ID NOs: 11 to 16 is replaced by N1-methyl pseudouridine (Ψ) (e.g., modified RNA; modRNA).
本發明進一步提供RNA分子及RNA-LNP，其包括5'非轉譯區(5'-UTR)及/或3'非轉譯區(3'-UTR)。在一些態樣中，RNA分子包括5'非轉譯區(5'-UTR)。在一些態樣中，5' UTR包含選自SEQ ID NO: 17至19中之任一者的序列。在一些態樣中，5' UTR包含與SEQ ID NO: 17至19中之任一者具有至少90%、91%、92%、93%、94%、95、96%、97%、98%或99%或更高一致性的序列。在一些態樣中，5' UTR包含選自SEQ ID NO: 17至19中之任一者的序列。在一些態樣中，5' UTR包含由SEQ ID NO: 17至19中之任一者組成的序列。The present invention further provides RNA molecules and RNA-LNPs, which include a 5' non-translational region (5'-UTR) and/or a 3' non-translational region (3'-UTR). In some aspects, the RNA molecule includes a 5' non-translational region (5'-UTR). In some aspects, the 5'UTR includes a sequence selected from any one of SEQ ID NOs: 17 to 19. In some aspects, the 5'UTR includes a sequence having at least 90%, 91%, 92%, 93%, 94%, 95, 96%, 97%, 98% or 99% or higher identity with any one of SEQ ID NOs: 17 to 19. In some aspects, the 5'UTR includes a sequence selected from any one of SEQ ID NOs: 17 to 19. In some aspects, the 5'UTR includes a sequence consisting of any one of SEQ ID NOs: 17 to 19.
在一些態樣中，RNA分子及RNA-LNP包括3'非轉譯區(3'-UTR)。在一些態樣中，3' UTR包含選自SEQ ID NO: 20至25中之任一者的序列。在一些態樣中，3' UTR包含與SEQ ID NO: 20至25中之任一者具有至少90%、91%、92%、93%、94%、95、96%、97%、98%或99%或更高一致性的序列。在一些態樣中，3' UTR包含選自SEQ ID NO: 20至25中之任一者的序列。在一些態樣中，3' UTR包含由SEQ ID NO: 20至25中之任一者組成的序列。In some aspects, RNA molecules and RNA-LNPs include a 3' non-translated region (3'-UTR). In some aspects, the 3'UTR comprises a sequence selected from any one of SEQ ID NOs: 20 to 25. In some aspects, the 3'UTR comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95, 96%, 97%, 98% or 99% or higher identity to any one of SEQ ID NOs: 20 to 25. In some aspects, the 3'UTR comprises a sequence selected from any one of SEQ ID NOs: 20 to 25. In some aspects, the 3'UTR comprises a sequence consisting of any one of SEQ ID NOs: 20 to 25.
本發明進一步提供RNA分子及RNA-LNP，其包括5'帽部分。在一些態樣中，5'帽部分為(3'OMe) - m27,3'-OGppp (m12'-O)ApG。本發明進一步提供RNA分子及RNA-LNP，其包括3'多-A尾。在一些態樣中，多-A尾包含具有SEQ ID NO: 26之序列。The present invention further provides RNA molecules and RNA-LNPs, which include a 5' cap portion. In some aspects, the 5' cap portion is (3'OMe)-m27,3'-O Gppp (m12'-O )ApG. The present invention further provides RNA molecules and RNA-LNPs, which include a 3' poly-A tail. In some aspects, the poly-A tail comprises a sequence having SEQ ID NO: 26.
在一些態樣中，RNA分子包括5' UTR及3' UTR。在一些態樣中，RNA分子包括5'帽、5' UTR及3' UTR。在一些態樣中，RNA分子包括5'帽、5' UTR、3' UTR及多-A尾。在一些態樣中，RNA分子包括5' UTR、3' UTR及多-A尾。在一些態樣中，前述要素中之1、2、3者或更多者可排除在RNA分子外。在一些態樣中，5' UTR、3' UTR及多-A尾中之任一者之各尿苷經N1-甲基假尿苷(Ψ)置換(例如經修飾之RNA；modRNA)。In some aspects, the RNA molecule includes a 5'UTR and a 3'UTR. In some aspects, the RNA molecule includes a 5'cap, a 5'UTR, and a 3'UTR. In some aspects, the RNA molecule includes a 5'cap, a 5'UTR, a 3'UTR, and a poly-A tail. In some aspects, the RNA molecule includes a 5'UTR, a 3'UTR, and a poly-A tail. In some aspects, 1, 2, 3, or more of the aforementioned elements may be excluded from the RNA molecule. In some aspects, each uridine in any one of the 5'UTR, the 3'UTR, and the poly-A tail is replaced by N1-methyl pseudouridine (Ψ) (e.g., modified RNA; modRNA).
在一些態樣中，多-A尾長度可含有+1/-1 A。在一些態樣中，尿苷為N1-甲基假尿苷(Ψ)。In some aspects, the poly-A tail length may contain +1/-1 A. In some aspects, the uridine is N1-methylpseudouridine (Ψ).
本發明提供表5中所描述之RNA分子。在一些態樣中，RNA分子包含SEQ ID NO: 18之5' UTR、SEQ ID NO: 11之RSV ORF、SEQ ID NO: 21之3' UTR及/或SEQ ID NO: 26之多-A尾。在另一態樣中，RNA分子包含SEQ ID NO: 18之5' UTR、SEQ ID NO: 12之RSV ORF、SEQ ID NO: 21之3' UTR及/或SEQ ID NO: 26之多-A尾。在另一態樣中，RNA分子包含SEQ ID NO: 18之5' UTR、SEQ ID NO: 63之RSV ORF、SEQ ID NO: 21之3' UTR及/或SEQ ID NO: 26之多-A尾。在另一態樣中，RNA分子包含SEQ ID NO: 18之5' UTR、SEQ ID NO: 65之RSV ORF、SEQ ID NO: 21之3' UTR及/或SEQ ID NO: 26之多-A尾。在另一態樣中，RNA分子包含SEQ ID NO: 18之5' UTR、SEQ ID NO: 67之RSV ORF、SEQ ID NO: 21之3' UTR及/或SEQ ID NO: 26之多-A尾。在另一態樣中，RNA分子包含SEQ ID NO: 18之5' UTR、SEQ ID NO: 69之RSV ORF、SEQ ID NO: 21之3' UTR及/或SEQ ID NO: 26之多-A尾。在一些態樣中，RSV ORF進一步包含本文所描述之終止密碼子。在一些態樣中，多-A尾長度可含有+1/-1 A或+2/-2 A。在一些態樣中，RNA分子之各尿苷經N1-甲基假尿苷(Ψ)置換(例如經修飾之RNA；modRNA)。The present invention provides RNA molecules described in Table 5. In some aspects, the RNA molecule comprises the 5'UTR of SEQ ID NO: 18, the RSV ORF of SEQ ID NO: 11, the 3'UTR of SEQ ID NO: 21, and/or the poly-A tail of SEQ ID NO: 26. In another aspect, the RNA molecule comprises the 5'UTR of SEQ ID NO: 18, the RSV ORF of SEQ ID NO: 12, the 3'UTR of SEQ ID NO: 21, and/or the poly-A tail of SEQ ID NO: 26. In another aspect, the RNA molecule comprises the 5'UTR of SEQ ID NO: 18, the RSV ORF of SEQ ID NO: 63, the 3'UTR of SEQ ID NO: 21, and/or the poly-A tail of SEQ ID NO: 26. In another aspect, the RNA molecule comprises the 5'UTR of SEQ ID NO: 18, the RSV ORF of SEQ ID NO: 65, the 3'UTR of SEQ ID NO: 21, and/or the poly-A tail of SEQ ID NO: 26. In another aspect, the RNA molecule comprises the 5'UTR of SEQ ID NO: 18, the RSV ORF of SEQ ID NO: 67, the 3'UTR of SEQ ID NO: 21, and/or the poly-A tail of SEQ ID NO: 26. In another aspect, the RNA molecule comprises the 5'UTR of SEQ ID NO: 18, the RSV ORF of SEQ ID NO: 69, the 3'UTR of SEQ ID NO: 21, and/or the poly-A tail of SEQ ID NO: 26. In some aspects, the RSV ORF further comprises a stop codon described herein. In some aspects, the poly-A tail length may contain +1/-1 A or +2/-2 A. In some aspects, each uridine of the RNA molecule is replaced with N1-methylpseudouridine (Ψ) (eg, modified RNA; modRNA).
本發明進一步提供RNA分子，其包括至少一個由密碼子最佳化DNA產生之開讀框。在一些態樣中，開讀框包含至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的G/C含量：50%、51%、52%、53%、54%、55%、56%、57%、58%、59%、60%、61%、62%、63%、64%、65%、66%、67%、68%、69%、70%、71%、72%、73%、74%或75%，例如至少55%、至少60%、至少65%、至少70%、至少75%，為或為約50%至75%，或者為或為約55%至70%。在一些態樣中，G/C含量為或為約58%，為或為約66%，或者為或為約62%。The present invention further provides RNA molecules, which include at least one open reading frame produced by codon optimized DNA. In some aspects, the open reading frame comprises at least, at most, just below, or between any two of the following (inclusive or exclusive) G/C content: 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74% or 75%, such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, for or about 50% to 75%, or for or about 55% to 70%. In some aspects, the G/C content is for or about 58%, for or about 66%, or for or about 62%.
本發明進一步提供RNA分子，其包含經穩定化之RNA。本發明進一步提供RNA分子，其包括具有至少一個經修飾之核苷酸的RNA(例如經修飾之RNA；modRNA)。在一些態樣中，經修飾之核苷酸為假尿苷、N1-甲基假尿苷、N1-乙基假尿苷、2-硫尿苷、4'-硫尿苷、5-甲基胞嘧啶、5-甲基尿苷、2-硫-1-甲基-1-脫氮-假尿苷、2-硫-1-甲基-假尿苷、2-硫-5-氮雜-尿苷、2-硫-二氫假尿苷、2-硫-二氫尿苷、2-硫-假尿苷、4-甲氧基-2-硫-假尿苷、4-甲氧基-假尿苷、4-硫-1-甲基-假尿苷、4-硫-假尿苷、5-氮雜-尿苷、二氫假尿苷、5-甲氧基尿苷或2'-O-甲基尿苷。在一些態樣中，經修飾之核苷酸為N1-甲基假尿苷(Ψ)。在一些態樣中，前述經修飾之核苷酸中之1、2、3、4、5者或更多者可排除在RNA分子外。The present invention further provides RNA molecules comprising stabilized RNA. The present invention further provides RNA molecules comprising RNA having at least one modified nucleotide (eg, modified RNA; modRNA). In some aspects, the modified nucleotide is pseudouridine, N1-methyl pseudouridine, N1-ethyl pseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine or 2'-O-methyluridine. In some aspects, the modified nucleotide is N1-methylpseudouridine (Ψ). In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned modified nucleotides can be excluded from the RNA molecule.
本發明進一步提供RNA分子，其為信使RNA (mRNA)或自複製RNA。在一些態樣中，RNA為mRNA。The present invention further provides RNA molecules, which are messenger RNA (mRNA) or self-replicating RNA. In some aspects, the RNA is mRNA.
本發明進一步提供免疫原性組合物，其包括本文所描述之RNA分子。RNA分子可調配於此類免疫原性組合物中、囊封於其中、與其中之脂質奈米顆粒(LNP)複合、與LNP結合或吸附於LNP上(例如RSV RNA-LNP)。在一些態樣中，脂質奈米顆粒包括以下至少一者：陽離子脂質、聚合物結合脂質(例如聚乙二醇化脂質)及至少一種結構脂質(例如中性脂質及類固醇或類固醇類似物)。在一些態樣中，前述脂質中之1、2、3者或更多者可排除在脂質奈米顆粒外。The present invention further provides immunogenic compositions comprising RNA molecules described herein. RNA molecules can be formulated in such immunogenic compositions, encapsulated therein, complexed with lipid nanoparticles (LNPs) therein, bound to LNPs, or adsorbed on LNPs (e.g., RSV RNA-LNPs). In some aspects, the lipid nanoparticles include at least one of the following: cationic lipids, polymer-bound lipids (e.g., PEGylated lipids), and at least one structural lipid (e.g., neutral lipids and steroids or steroid analogs). In some aspects, 1, 2, 3, or more of the aforementioned lipids can be excluded from the lipid nanoparticles.
在一些態樣中，脂質奈米顆粒包括陽離子脂質。在一些態樣中，陽離子脂質為(4-羥丁基)氮二基)雙(己烷-6,1-二基)雙(2-己基癸酸酯) (ALC-0315)。In some embodiments, the lipid nanoparticles include a cationic lipid. In some embodiments, the cationic lipid is (4-hydroxybutyl) azodiyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315).
在一些態樣中，脂質奈米顆粒包括聚合物結合脂質。在一些態樣中，脂質奈米顆粒包括聚乙二醇化脂質，亦稱為PEG-脂質。在一些態樣中，聚乙二醇化脂質為PEG修飾之磷脂醯乙醇胺；PEG修飾之磷脂酸；PEG修飾之神經醯胺(例如PEG-CerC14或PEG-CerC20)；PEG修飾之二烷基胺、PEG修飾之二醯基甘油、PEG修飾之二烷基甘油；2-[(聚乙二醇)-2000]-N,N-二(十四烷基)乙醯胺；二醇-脂質，包括PEG-c-DOMG、PEG-c-DMA、PEG-s-DMG、N-[(甲氧基聚乙二醇)2000)胺甲醯基]-1,2-二肉豆蔻醯氧基丙基-3-胺(PEG-c-DMA)及PEG-2000-DMG；聚乙二醇化二醯基甘油(PEG-DAG)，諸如1-(單甲氧基-聚乙二醇)-2,3-二肉豆蔻醯基甘油(PEG-DMG)；聚乙二醇化磷脂醯乙醇胺(PEG-PE)；PEG丁二酸二醯基甘油(PEG-S-DAG)，諸如4-O-(2',3'-二(十四碳醯氧基)丙基-1-O-((ω-甲氧基(聚乙氧基)乙基)丁二酸酯(PEG-S-DMG)；聚乙二醇化神經醯胺(PEG-cer)；或PEG二烷氧基丙基胺基甲酸酯，諸如共聚甲氧基(聚乙氧基)乙基-N-(2,3-二(十四烷氧基)丙基)胺基甲酸酯或2,3-二(十四烷氧基)丙基-N-(ω-甲氧基(聚乙氧基)乙基)胺基甲酸酯。在一些態樣中，前述聚乙二醇化脂質中之1、2、3、4、5者或更多者可排除在RNA分子外。在一些態樣中，聚乙二醇化脂質為2-[(聚乙二醇)-2000]-N,N-二(十四烷基)乙醯胺(ALC-0159)。In some aspects, the lipid nanoparticles include polymer-bound lipids. In some aspects, the lipid nanoparticles include PEGylated lipids, also known as PEG-lipids. In some aspects, the PEGylated lipids are PEG-modified phosphatidylethanolamine; PEG-modified phosphatidic acid; PEG-modified ceramide (e.g., PEG-CerC14 or PEG-CerC20); PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol; 2-[(polyethylene glycol)-2000]-N,N-di(tetradecyl)acetamide; glycol-lipids, including PEG-c- DOMG, PEG-c-DMA, PEG-s-DMG, N-[(methoxypolyethylene glycol) 2000)aminomethyl]-1,2-dimyristoyloxypropyl-3-amine (PEG-c-DMA) and PEG-2000-DMG; polyethylene glycolated diacylglycerols (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG); polyethylene glycolated phospholipids PEG-PE; PEG-succinate diacylglycerol (PEG-S-DAG), such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-((ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG); PEGylated ceramide (PEG-cer); or PEG dialkoxypropylcarbamates, such as co-methoxy(polyethoxy)ethyl-N-(2,3 In some embodiments, the PEGylated lipid is 2-[(polyethylene glycol)-2000]-N,N-di(tetradecyloxy)propyl)carbamate or 2,3-di(tetradecyloxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate. In some embodiments, 1, 2, 3, 4, 5 or more of the aforementioned PEGylated lipids can be excluded from the RNA molecule. In some embodiments, the PEGylated lipid is 2-[(polyethylene glycol)-2000]-N,N-di(tetradecyl)acetamide (ALC-0159).
在一些態樣中，脂質奈米顆粒包括至少一種結構脂質，諸如中性脂質。在一些態樣中，中性脂質為1,2-二硬脂醯基-sn-甘油基-3-磷酸膽鹼(DSPC)、二硬脂醯基磷脂醯膽鹼(DSPC)、二油醯基磷脂醯膽鹼(DOPC)、二棕櫚醯基磷脂醯膽鹼(DPPC)、二油醯基磷脂醯甘油(DOPG)、二棕櫚醯基磷脂醯甘油(DPPG)、二油醯基-磷脂醯乙醇胺(DOPE)、棕櫚醯油醯基磷脂醯膽鹼(POPC)、棕櫚醯基-油醯基-磷脂醯乙醇胺(POPE)、二油醯基-磷脂醯乙醇胺4-(N-順丁烯二醯亞胺基甲基)-環己烷-1-甲酸酯(DOPE-mal)、二棕櫚醯基磷脂醯乙醇胺(DPPE)、二肉豆蔻醯基磷酸乙醇胺(DMPE)、二硬脂醯基磷脂醯乙醇胺(DSPE)、16-O-單甲基PE、16-O-二甲基PE、18-1-反式PE、1-硬脂醯基-2-油醯基磷脂醯乙醇胺(SOPE)及/或1,2-二反油醯基-sn-甘油基-3-磷酸乙醇胺(transDOPE)。在一些態樣中，前述結構脂質中之1、2、3、4、5者或更多者可排除在RNA分子外。在一些態樣中，中性脂質為1,2-二硬脂醯基-sn-甘油基-3-磷酸膽鹼(DSPC)。In some aspects, lipid nanoparticles include at least one structured lipid, such as a neutral lipid. In some aspects, the neutral lipid is 1,2-distearyl-sn-glycero-3-phosphocholine (DSPC), distearylphosphatidylcholine (DSPC), dioleylphosphatidylcholine (DOPC), dimalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylglycerol (DOPG), dimalmitoylphosphatidylglycerol (DPPG), dioleyl-phosphatidylethanolamine (DOPE), palmitoyloleylphosphatidylcholine (POPC), palmitoyl-oleyl-phosphatidylethanolamine (POPE), dioleyl-phosphatidyl 4-(N-cis-butylenediimidylmethyl)-cyclohexane-1-carboxylate (DOPE-mal), dimalcylidenephosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearyl-2-oleylphosphatidylethanolamine (SOPE) and/or 1,2-ditransoleyl-sn-glycero-3-phosphoethanolamine (transDOPE). In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned structural lipids can be excluded from the RNA molecule. In some aspects, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).
在一些態樣中，脂質奈米顆粒包括第二結構脂質，諸如類固醇或類固醇類似物。在一些態樣中，類固醇或類固醇類似物為膽固醇。In some aspects, the lipid nanoparticles include a second structural lipid, such as a steroid or a steroid analog. In some aspects, the steroid or steroid analog is cholesterol.
在一些態樣中，脂質奈米顆粒之平均直徑為約1至約500 nm，例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：1 nm、10 nm、20 nm、30 nm、40 nm、50 nm、60 nm、70 nm、80 nm、90 nm、100 nm、110 nm、120 nm、130 nm、140 nm、150 nm、160 nm、170 nm、180 nm、190 nm、200 nm、210 nm、220 nm、230 nm、240 nm、250 nm、260 nm、270 nm、280 nm、290 nm、300 nm、310 nm、320 nm、330 nm、340 nm、350 nm、360 nm、370 nm、380 nm、390 nm、400 nm、410 nm、420 nm、430 nm、440 nm、450 nm、460 nm、470 nm、480 nm、490 nm或500 nm。In some aspects, the lipid nanoparticles have an average diameter of about 1 to about 500 nm, for example at least, at most, just below, or between any two of the following (inclusive or exclusive): 1 nm, 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm, 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, 300 nm, 310 nm, 320 nm, 330 nm, 340 nm, 350 nm, 360 nm, 370 nm, 380 nm, 390 nm, nm, 400 nm, 410 nm, 420 nm, 430 nm, 440 nm, 450 nm, 460 nm, 470 nm, 480 nm, 490 nm or 500 nm.
在一些態樣中，RNA-LNP免疫原性組合物為液體RNA-LNP組合物，其包含濃度為至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的編碼本文所揭示之RSV多肽的RNA分子/聚核苷酸：0.01、0.15、0.30、0.45、0.60、0.75或0.90 mg/mL，較佳地為或為約0.01至0.09 mg/mL，其囊封於包含以下之脂質組成的LNP中：濃度為或為約0.8至0.95 mg/mL (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：0.80、0.81、0.82、0.83、0.84、0.85、0.86、0.87、0.88、0.89、0.90、0.91、0.92、0.93、0.94或0.95 mg/mL)的陽離子脂質、濃度為或為約0.05至0.15 mg/mL (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：0.05、0.06、0.07、0.08、0.09、0.10、0.11、0.12、0.13、0.14或0.15 mg/mL)的聚乙二醇化脂質、濃度為或為約0.1至0.25 mg/mL (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：0.10、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.20、0.21、0.22、0.23、0.24或0.25 mg/mL)的第一結構脂質、及濃度為或為約0.3至0.45 mg/mL (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：0.30、0.31、0.32、0.33、0.34、0.35、0.36、0.37、0.38、0.39、0.40、0.41、0.42、0.43、0.44或0.45 mg/mL)的第二結構脂質。在一些態樣中，液體組合物進一步包含緩衝組合物，其包含濃度為或為約0.1至0.3 mg/mL (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：0.10、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.20、0.21、0.22、0.23、0.24、0.25、0.26、0.27、0.28、0.29或0.30 mg/mL)的第一緩衝物、濃度為或為約1.25至1.4 mg/mL (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：1.25、1.26、1.27、1.28、1.29、1.30、1.31、1.32、1.33、1.34、1.35、1.36、1.37、1.38、1.39或1.40 mg/mL)的第二緩衝物、及濃度為或為約95至110 mg/mL (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：95、96、97、98、99、100、101、102、103、104、105、106、107、108、109或110 mg/mL)的穩定劑。在一些態樣中，前述要素中之1、2、3、4、5者或更多者可排除在液體RNA-LNP組合物外。在一些態樣中，前述要素濃度中之1、2、3、4、5者或更多者可排除在液體RNA-LNP組合物外。In some aspects, the RNA-LNP immunogenic composition is a liquid RNA-LNP composition comprising an RNA molecule/polynucleotide encoding an RSV polypeptide disclosed herein at a concentration of at least, at most, just below, or between any two of the following (inclusive or exclusive): 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably at or about 0.01 to 0.09 mg/mL, encapsulated in a LNP comprising a lipid composition of at or about 0.8 to 0.95 mg/mL. The invention relates to a cationic lipid in an amount of at least 0.05 to 0.15 mg/mL (e.g., at least, at most, just below, or between any two of the following, inclusive or exclusive): 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, or 0.95 mg/mL) and a cationic lipid in an amount of at least 0.05 to 0.15 mg/mL (e.g., at least, at most, just below, or between any two of the following, inclusive or exclusive): 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, or 0.15 mg/mL), a pegylated lipid at a concentration of or about 0.1 to 0.25 mg/mL (e.g., at least, at most, just below, or between any two of the following (inclusive or exclusive): 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25 mg/mL), and a first structure lipid at a concentration of or about 0.3 to 0.45 mg/mL (e.g., at least, at most, just below, or between any two of the following (inclusive or exclusive): 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, or 0.45 mg/mL) of a second structural lipid. In some aspects, the liquid composition further comprises a buffer composition comprising a first buffer at a concentration of or about 0.1 to 0.3 mg/mL (e.g., at least, at most, just below, or between any two of the following (inclusive or exclusive): 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, or 0.30 mg/mL), a first buffer at a concentration of or about 1.25 to 1.4 mg/mL The invention also provides a second buffer having a concentration of at least 95 to 110 mg/mL (e.g., at least, at most, just below, or between any two of the following, inclusive or exclusive: 1.25, 1.26, 1.27, 1.28, 1.29, 1.30, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, or 1.40 mg/mL), and a stabilizer having a concentration of at or about 95 to 110 mg/mL (e.g., at least, at most, just below, or between any two of the following, inclusive or exclusive: 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 mg/mL). In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned elements can be excluded from the liquid RNA-LNP composition. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned element concentrations can be excluded from the liquid RNA-LNP composition.
在特定態樣中，液體RNA-LNP免疫原性組合物包含濃度為至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的編碼本文所揭示之RSV多肽的RNA分子/聚核苷酸：0.01、0.15、0.30、0.45、0.60、0.75或0.90 mg/mL，較佳地為或為約0.01至0.09 mg/mL，其囊封於包含以下之脂質組成的LNP中：濃度為或為約0.8至0.95 mg/mL (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：0.80、0.81、0.82、0.83、0.84、0.85、0.86、0.87、0.88、0.89、0.90、0.91、0.92、0.93、0.94或0.95 mg/mL)的((4-羥丁基)氮二基)雙(己烷-6,1-二基)雙(2-己基癸酸酯) (ALC-0315)、濃度為或為約0.05至0.15 mg/mL (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：0.05、0.06、0.07、0.08、0.09、0.10、0.11、0.12、0.13、0.14或0.15 mg/mL)的2-[(聚乙二醇)-2000]-N,N-二(十四烷基)乙醯胺(ALC-0159)、濃度為或為約0.1至0.25 mg/mL (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：0.10、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.20、0.21、0.22、0.23、0.24或0.25 mg/mL)的1,2-二硬脂醯基-sn-甘油基-3-磷酸膽鹼(DSPC)、及濃度為或為約0.3至0.45 mg/mL (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：0.30、0.31、0.32、0.33、0.34、0.35、0.36、0.37、0.38、0.39、0.40、0.41、0.42、0.43、0.44或0.45 mg/mL)的膽固醇。在一些態樣中，液體組合物進一步包含Tris緩衝組合物，該Tris緩衝組合物包含濃度為或為約0.1至0.3 mg/mL (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：0.10、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.20、0.21、0.22、0.23、0.24、0.25、0.26、0.27、0.28、0.29或0.30 mg/mL)的緩血酸胺，及濃度為或為約1.25至1.4 mg/mL (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：1.25、1.26、1.27、1.28、1.29、1.30、1.31、1.32、1.33、1.34、1.35、1.36、1.37、1.38、1.39或1.40 mg/mL)的Tris鹽酸(HCl)、及濃度為或為約95至110 mg/mL (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：95、96、97、98、99、100、101、102、103、104、105、106、107、108、109或110 mg/mL)的蔗糖。在一些態樣中，前述要素中之1、2、3、4、5者或更多者可排除在液體RNA-LNP組合物外。在一些態樣中，前述要素濃度中之1、2、3、4、5者或更多者可排除在液體RNA-LNP組合物外。In certain aspects, the liquid RNA-LNP immunogenic composition comprises an RNA molecule/polynucleotide encoding an RSV polypeptide disclosed herein at a concentration of at least, at most, just below, or between any two of the following (inclusive or exclusive): 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably at or about 0.01 to 0.09 mg/mL, encapsulated in an LNP comprising a lipid composition of at or about 0.8 to 0.95 mg/mL (e.g., at least, at most, just below, or between any two of the following, inclusively or exclusively: 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, or 0.95 mg/mL) of ((4-hydroxybutyl)azinediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), at a concentration of or about 0.05 to 0.15 mg/mL (e.g., at least, at most, just less than, or between any two of the following (inclusive or exclusive): 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, or 0.15 mg/mL) of 2-[(polyethylene glycol)-2000]-N,N-di(tetradecyl)acetamide (ALC-0159) at a concentration of or about 0.1 to 0.25 mg/mL 1,2-distearyl-sn-glycero-3-phosphocholine (DSPC) (e.g., at least, at most, just less than, or between any two of the following (inclusive or exclusive): 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25 mg/mL), and a concentration of or about 0.3 to 0.45 mg/mL (e.g., at least, at most, just below, or between any two of the following, inclusively or exclusively: 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, or 0.45 mg/mL) of cholesterol. In some aspects, the liquid composition further comprises a Tris buffer composition comprising sulphuric acid at a concentration of or about 0.1 to 0.3 mg/mL (e.g., at least, at most, just below, or between any two of the following (inclusive or exclusive): 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, or 0.30 mg/mL) and sulphuric acid at a concentration of or about 1.25 to 1.4 mg/mL. Tris hydrochloric acid (HCl) at a concentration of at or about 95 to 110 mg/mL (e.g., at least, at most, just below, or between any two of the following, inclusive or exclusive: 1.25, 1.26, 1.27, 1.28, 1.29, 1.30, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, or 1.40 mg/mL), and sucrose at a concentration of at or about 95 to 110 mg/mL (e.g., at least, at most, just below, or between any two of the following, inclusive or exclusive: 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 mg/mL). In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned elements can be excluded from the liquid RNA-LNP composition. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned element concentrations can be excluded from the liquid RNA-LNP composition.
在一些態樣中，液體RNA-LNP免疫原性組合物包含濃度為至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)之編碼本文所揭示之RSV多肽的RNA分子/聚核苷酸：0.01、0.15、0.30、0.45、0.60、0.75或0.90 mg/mL，較佳地為或為約0.01至0.09 mg/mL，其囊封於LNP中，且其進一步包含為或為約5至15 mM的Tris緩衝物(例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：5、6、7、8、9、10、11、12、13、14或15 mM)及為或為約200至400 mM的蔗糖(例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：200、210、220、230、240、250、260、270、280、290、300、310、320、330、340、350、360、370、380、390或400 mM)，pH呈或呈約7.0至8.0 (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性) 7.0、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9或8.0)。在一些態樣中，前述要素中之1、2、3者或更多者可排除在液體RNA-LNP組合物外。在一些態樣中，前述要素濃度中之1、2、3、4、5者或更多者可排除在液體RNA-LNP組合物外。In some aspects, the liquid RNA-LNP immunogenic composition comprises an RNA molecule/polynucleotide encoding an RSV polypeptide disclosed herein at a concentration of at least, at most, just below, or between any two of the following (inclusive or exclusive): 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably at or about 0.01 to 0.09 mg/mL, encapsulated in LNP, and further comprising at or about 5 to 15 mM Tris buffer (e.g., at least, at most, just below, or between any two of the following (inclusive or exclusive): 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mM) and at or about 200 to 400 mM. mM sucrose (e.g., at least, at most, just below, or between any two of the following, inclusive or exclusive: 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, or 400 mM) at a pH of or about 7.0 to 8.0 (e.g., at least, at most, just below, or between any two of the following, inclusive or exclusive: 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0). In some aspects, one, two, three, or more of the aforementioned elements can be excluded from the liquid RNA-LNP composition. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned element concentrations may be excluded from the liquid RNA-LNP composition.
在一些態樣中，RNA-LNP免疫原性組合物為凍乾(復原) RNA-LNP組合物，其包含濃度為至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)之編碼本文所揭示之RSV多肽的RNA分子/聚核苷酸：0.01、0.15、0.30、0.45、0.60、0.75或0.90 mg/mL，較佳地為或為約0.01至0.09 mg/mL，其囊封於包含以下之脂質組成的LNP中：濃度為或為約0.8至0.95 mg/mL (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：0.80、0.81、0.82、0.83、0.84、0.85、0.86、0.87、0.88、0.89、0.90、0.91、0.92、0.93、0.94或0.95 mg/mL)的陽離子脂質、濃度為或為約0.05至0.15 mg/mL (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：0.05、0.06、0.07、0.08、0.09、0.10、0.11、0.12、0.13、0.14或0.15 mg/mL)的聚乙二醇化脂質、濃度為或為約0.1至0.25 mg/mL (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：0.10、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.20、0.21、0.22、0.23、0.24或0.25 mg/mL)的第一結構脂質、及濃度為或為約0.3至0.45 mg/mL (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：0.30、0.31、0.32、0.33、0.34、0.35、0.36、0.37、0.38、0.39、0.40、0.41、0.42、0.43、0.44或0.45 mg/mL)的第二結構脂質。在一些態樣中，凍乾組合物進一步包含以下各者：濃度為或為約0.01及0.15 mg/mL (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.10、0.11、0.12、0.13、0.14或0.15 mg/mL)的第一緩衝物、濃度為或為約0.5及0.65 mg/mL (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：0.50、0.51、0.52、0.53、0.54、0.55、0.56、0.57、0.58、0.59、0.60、0.61、0.62、0.63、0.64或0.65 mg/mL)的第二緩衝物、濃度為或為約35至50 mg/mL (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50 mg/mL)的穩定劑、及用於復原的濃度為或為約5至15 mg/mL (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：5、6、7、8、9、10、11、12、13、14或15 mg/mL)的鹽稀釋劑。在特定態樣中，凍乾組合物復原於或於約0.6至0.75 mL之鹽稀釋劑(例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：0.60、0.61、0.62、0.63、0.64、0.65、0.66、0.67、0.68、0.69、0.70、0.71、0.72、0.73、0.74或0.75 mL)中。凍乾RNA-LNP組合物中之濃度係在復原後測定。在一些態樣中，前述要素中之1、2、3、4、5者或更多者可排除在凍乾RNA-LNP組合物外。在一些態樣中，前述要素濃度中之1、2、3、4、5者或更多者可排除在凍乾RNA-LNP組合物外。In some aspects, the RNA-LNP immunogenic composition is a lyophilized (reconstituted) RNA-LNP composition comprising an RNA molecule/polynucleotide encoding an RSV polypeptide disclosed herein at a concentration of at least, at most, just below, or between any two of the following (inclusive or exclusive): 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably at or about 0.01 to 0.09 mg/mL, encapsulated in an LNP comprising a lipid composition of at or about 0.8 to 0.95 mg/mL The invention relates to a cationic lipid in an amount of at least 0.05 to 0.15 mg/mL (e.g., at least, at most, just below, or between any two of the following, inclusive or exclusive): 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, or 0.95 mg/mL) and a cationic lipid in an amount of at least 0.05 to 0.15 mg/mL (e.g., at least, at most, just below, or between any two of the following, inclusive or exclusive): 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, or 0.15 mg/mL), a pegylated lipid at a concentration of or about 0.1 to 0.25 mg/mL (e.g., at least, at most, just below, or between any two of the following (inclusive or exclusive): 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25 mg/mL), and a first structure lipid at a concentration of or about 0.3 to 0.45 mg/mL (e.g., at least, at most, just below, or between any two of the following (inclusive or exclusive): 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, or 0.45 mg/mL) of a second structural lipid. In some aspects, the lyophilized composition further comprises: a first buffer at a concentration of or about between 0.01 and 0.15 mg/mL (e.g., at least, at most, just below, or between any two of the following (inclusive or exclusive): 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, or 0.15 mg/mL), a first buffer at a concentration of or about between 0.5 and 0.65 mg/mL a second buffer (e.g., at least, at most, just below, or between any two of the following, inclusive or exclusive: 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, or 0.65 mg/mL), a stabilizer at a concentration of or about 35 to 50 mg/mL (e.g., at least, at most, just below, or between any two of the following, inclusive or exclusive: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mg/mL), and a concentration of or about 5 to 15 mg/mL for reconstitution In certain aspects, the lyophilized composition is reconstituted in or about 0.6 to 0.75 mL of a salt diluent (e.g., at least, at most, just below, or between any two of the following, inclusive or exclusive: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg/mL). In certain aspects, the lyophilized composition is reconstituted in or about 0.6 to 0.75 mL of a salt diluent (e.g., at least, at most, just below, or between any two of the following, inclusive or exclusive: 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, or 0.75 mL). The concentration in the lyophilized RNA-LNP composition is determined after reconstitution. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned elements can be excluded from the freeze-dried RNA-LNP composition. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned element concentrations can be excluded from the freeze-dried RNA-LNP composition.
在特定態樣中，凍乾(復原) RNA-LNP組合物包含濃度為至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)之編碼本文所揭示之RSV多肽的RNA聚核苷酸：0.01、0.15、0.30、0.45、0.60、0.75或0.90 mg/mL，較佳地為或為約0.01至0.09 mg/mL，其囊封於包含以下之脂質組成的LNP中：濃度為或為約0.8至0.95 mg/mL (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：0.80、0.81、0.82、0.83、0.84、0.85、0.86、0.87、0.88、0.89、0.90、0.91、0.92、0.93、0.94或0.95 mg/mL)的ALC-0315、濃度為或為約0.05至0.15 mg/mL (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：0.05、0.06、0.07、0.08、0.09、0.10、0.11、0.12、0.13、0.14或0.15 mg/mL)的ALC-0159、濃度為或為約0.1至0.25 mg/mL (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：0.10、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.20、0.21、0.22、0.23、0.24或0.25 mg/mL)的DSPC、及濃度為或為約0.3至0.45 mg/mL (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：0.30、0.31、0.32、0.33、0.34、0.35、0.36、0.37、0.38、0.39、0.40、0.41、0.42、0.43、0.44或0.45 mg/mL)的膽固醇，且進一步包含Tris緩衝組合物，該Tris緩衝組合物包含：濃度為或為約0.01至0.15 mg/mL (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.10、0.11、0.12、0.13、0.14或0.15 mg/mL)的緩血酸胺、及濃度為或為約0.5至0.65 mg/mL (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：0.50、0.51、0.52、0.53、0.54、0.55、0.56、0.57、0.58、0.59、0.60、0.61、0.62、0.63、0.64或0.65 mg/mL)的Tris HCl、濃度為或為約35至50 mg/mL (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50 mg/mL)的蔗糖、及用於復原的濃度為或為約5至15 mg/mL (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性) 5、6、7、8、9、10、11、12、13、14或15 mg/mL)的氯化鈉(NaCl)稀釋劑。在特定態樣中，凍乾組合物被復原於或於約0.6至0.75 mL之氯化鈉(例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：0.60、0.61、0.62、0.63、0.64、0.65、0.66、0.67、0.68、0.69、0.70、0.71、0.72、0.73、0.74或0.75 mL)中。凍乾RNA-LNP組合物中之濃度係在復原後測定。在一些態樣中，前述要素中之1、2、3、4、5者或更多者可排除在凍乾RNA-LNP組合物外。在一些態樣中，前述要素濃度中之1、2、3、4、5者或更多者可排除在凍乾RNA-LNP組合物外。In certain aspects, the lyophilized (reconstituted) RNA-LNP composition comprises an RNA polynucleotide encoding an RSV polypeptide disclosed herein at a concentration of at least, at most, just below, or between any two of the following (inclusive or exclusive): 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably at or about 0.01 to 0.09 mg/mL, encapsulated in an LNP comprising a lipid composition of at or about 0.8 to 0.95 mg/mL 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, or 0.15 mg/mL) of ALC-0315 (e.g., at least, at most, just below, or between any two of the following, inclusive or exclusive: 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, or 0.95 mg/mL) and a concentration of at or about 0.05 to 0.15 mg/mL (e.g., at least, at most, just below, or between any two of the following, inclusive or exclusive: 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, or 0.15 mg/mL), ALC-0159 at a concentration of or about 0.1 to 0.25 mg/mL (e.g., at least, at most, just below, or between any two of the following (inclusive or exclusive): 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25 mg/mL), and DSPC at a concentration of or about 0.3 to 0.45 mg/mL (e.g., at least, at most, just below, or between any two of the following, inclusive or exclusive: 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, or 0.45 mg/mL) of cholesterol, and further comprising a Tris buffer composition comprising: a concentration of or about 0.01 to 0.15 mg/mL sulphonamide (e.g., at least, at most, just below, or between any two of the following, inclusive or exclusive: 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, or 0.15 mg/mL) and Tris at a concentration of or about 0.5 to 0.65 mg/mL (e.g., at least, at most, just below, or between any two of the following, inclusive or exclusive: 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, or 0.65 mg/mL) HCl, sucrose at a concentration of at or about 35 to 50 mg/mL (e.g., at least, at most, just below, or between any two of the following, inclusively or exclusively: 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mg/mL), and a sodium chloride (NaCl) diluent for reconstitution at a concentration of at or about 5 to 15 mg/mL (e.g., at least, at most, just below, or between any two of the following, inclusively or exclusively: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg/mL). In certain aspects, the lyophilized composition is reconstituted in or about 0.6 to 0.75 mL of sodium chloride (e.g., at least, at most, just below, or between any two of the following (inclusive or exclusive): 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, or 0.75 mL). The concentration in the lyophilized RNA-LNP composition is determined after reconstitution. In some aspects, 1, 2, 3, 4, 5, or more of the aforementioned elements can be excluded from the lyophilized RNA-LNP composition. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned concentrations of elements may be excluded from the freeze-dried RNA-LNP composition.
本發明提供RNA分子、RNA-LNP及免疫原性組合物，其可以每次投與至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的劑量向個體投與：1 µg、15 µg、30 µg、45 µg、60 µg、75 µg、90 µg、100 µg或更高之囊封於LNP中的RSV RNA。在一些態樣中，可排除囊封於LNP中之RSV RNA之前述濃度中之1、2、3、4、5者或更多者。The present invention provides RNA molecules, RNA-LNPs, and immunogenic compositions that can be administered to a subject at a dose of at least, at most, just below, or between any two of the following (inclusive or exclusive) per administration: 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, 90 μg, 100 μg or more of RSV RNA encapsulated in LNPs. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned concentrations of RSV RNA encapsulated in LNPs can be excluded.
本發明提供RNA分子、RNA-LNP及免疫原性組合物，其可以單次劑量投與。本發明進一步提供RNA分子、RNA-LNP及免疫原性組合物，其可投與兩次(例如第0天及在或約第7天、第0天及在或約第14天、第0天及在或約第21天、第0天及在或約第28天、第0天及在或約第60天、第0天及在或約第90天、第0天及在或約第120天、第0天及在或約第150天、第0天及在或約第180天、第0天及在或約1個月後、第0天及在或約2個月後、第0天及在或約3個月後、第0天及在或約6個月後、第0天及在或約9個月後、第0天及在或約12個月後、第0天及在或約18個月後、第0天及在或約2年後、第0天及在或約5年後、或第0天及在或約10年後)。本發明進一步提供RNA分子、RNA-LNP及免疫原性組合物，其可在第0天及在或約2個月後投與兩次。本發明進一步提供RNA分子、RNA-LNP及免疫原性組合物，其可在第0天及在或約6個月後投與兩次。本發明進一步提供RNA分子、RNA-LNP及免疫原性組合物，其可投與三次、四次、五次、六次、七次、八次、九次、十次、十一次、十二次、十三次、十四次或更多次。在一些態樣中，可能需要以1-5年之間隔週期性加強來維持抗體之保護性水平。本發明進一步提供至少一次加強劑量之投與。在一些態樣中，可排除前述給藥方案中之1、2、3、4、5者或更多者。The present invention provides RNA molecules, RNA-LNPs, and immunogenic compositions that can be administered in a single dose. The present invention further provides RNA molecules, RNA-LNPs, and immunogenic compositions that can be administered twice (e.g., day 0 and at or about day 7, day 0 and at or about day 14, day 0 and at or about day 21, day 0 and at or about day 28, day 0 and at or about day 60, day 0 and at or about day 90, day 0 and at or about day 120, day 0 and at or about day 150). , day 0 and at or about day 180, day 0 and at or about 1 month later, day 0 and at or about 2 months later, day 0 and at or about 3 months later, day 0 and at or about 6 months later, day 0 and at or about 9 months later, day 0 and at or about 12 months later, day 0 and at or about 18 months later, day 0 and at or about 2 years later, day 0 and at or about 5 years later, or day 0 and at or about 10 years later). The present invention further provides RNA molecules, RNA-LNPs, and immunogenic compositions that can be administered twice, at day 0 and at or about 2 months later. The present invention further provides RNA molecules, RNA-LNPs, and immunogenic compositions that can be administered twice, at day 0 and at or about 6 months later. The present invention further provides RNA molecules, RNA-LNPs and immunogenic compositions that can be administered three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or more times. In some aspects, periodic boosts at intervals of 1-5 years may be required to maintain the protective level of the antibody. The present invention further provides for administration of at least one booster dose. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned dosing regimens may be excluded.
本發明提供一種誘導個體中針對RSV之免疫反應的方法，其包括向個體投與有效量之本文所描述之RNA分子、RNA-LNP及/或免疫原性組合物。本發明進一步提供一種本文所描述之RNA分子、RNA-LNP及/或免疫原性組合物之用途，其用於製造用於誘導個體中針對RSV之免疫反應的藥劑。The present invention provides a method for inducing an immune response against RSV in an individual, comprising administering to the individual an effective amount of the RNA molecules, RNA-LNPs and/or immunogenic compositions described herein. The present invention further provides a use of the RNA molecules, RNA-LNPs and/or immunogenic compositions described herein for the manufacture of a medicament for inducing an immune response against RSV in an individual.
本發明提供一種誘導個體中針對RSV之免疫反應之方法，其包括向個體投與有效量之本文所描述之包括至少一個編碼RSV多肽之開讀框的RNA分子及/或RNA-LNP、或免疫原性組合物。本發明進一步提供一種本文所描述之包括至少一個編碼RSV多肽之開讀框之RNA分子及/或RNA-LNP、或免疫原性組合物的用途，其用於製造用於誘導個體中針對RSV之免疫反應的藥劑。The present invention provides a method for inducing an immune response against RSV in an individual, comprising administering to the individual an effective amount of an RNA molecule and/or RNA-LNP, or an immunogenic composition described herein comprising at least one open reading frame encoding an RSV polypeptide. The present invention further provides a use of an RNA molecule and/or RNA-LNP, or an immunogenic composition described herein comprising at least one open reading frame encoding an RSV polypeptide for the manufacture of a medicament for inducing an immune response against RSV in an individual.
本發明提供一種誘導個體中針對RSV之免疫反應之方法，其包括向個體投與有效量之本文所描述之包括至少一個編碼所關注基因之多肽之開讀框的RNA分子及/或RNA-LNP、或組合物。本發明進一步提供一種本文所描述之包括至少一個編碼所關注基因之多肽之開讀框的RNA分子及/或RNA-LNP、或組合物的用途，其用於製造用於誘導個體中針對RSV之免疫反應的藥劑。The present invention provides a method for inducing an immune response against RSV in an individual, comprising administering to the individual an effective amount of an RNA molecule and/or RNA-LNP, or a composition as described herein, comprising at least one open reading frame encoding a polypeptide of a gene of interest. The present invention further provides a use of an RNA molecule and/or RNA-LNP, or a composition as described herein, comprising at least one open reading frame encoding a polypeptide of a gene of interest, for the manufacture of a medicament for inducing an immune response against RSV in an individual.
本發明提供一種預防、治療及/或改善個體之感染、疾病或病狀的方法，其包括向個體投與有效量之本文所描述之RNA分子、RNA-LNP及/或免疫原性組合物。本發明進一步提供一種本文所描述之RNA分子、RNA-LNP及/或免疫原性組合物之用途，其用於製造用於預防、治療及/或改善個體之感染、疾病或病狀的藥劑。在一些態樣中，感染、疾病或病狀與RSV相關。在一些態樣中，感染、疾病或病狀為急性下呼吸道感染(acute lower respiratory infection；ALRI)，包括肺炎及支氣管炎。在一些態樣中，感染、疾病或病狀為急性下呼吸道感染(ALRI)，包括肺炎及支氣管炎。The present invention provides a method for preventing, treating and/or improving an infection, disease or condition of an individual, comprising administering to an individual an effective amount of an RNA molecule, RNA-LNP and/or immunogenic composition described herein. The present invention further provides a use of an RNA molecule, RNA-LNP and/or immunogenic composition described herein for the manufacture of a medicament for preventing, treating and/or improving an infection, disease or condition of an individual. In some aspects, the infection, disease or condition is associated with RSV. In some aspects, the infection, disease or condition is an acute lower respiratory tract infection (ALRI), including pneumonia and bronchitis. In some aspects, the infection, disease or condition is an acute lower respiratory tract infection (ALRI), including pneumonia and bronchitis.
本發明提供一種預防、治療及/或改善個體之感染、疾病或病狀之方法，其包括向個體投與有效量之本文所描述之包括至少一個編碼RSV多肽之開讀框的RNA分子及/或RNA-LNP、或免疫原性組合物。本發明進一步提供一種本文所描述之包括至少一個編碼RSV多肽之開讀框的RNA分子及/或RNA-LNP、或免疫原性組合物之用途，其用於製造用於預防、治療及/或改善個體之感染、疾病或病狀的藥劑。在一些態樣中，感染、疾病或病狀與RSV相關。在一些態樣中，感染、疾病或病狀為急性下呼吸道感染(ALRI)，包括肺炎及支氣管炎。在一些態樣中，感染、疾病或病狀為急性下呼吸道感染(ALRI)，包括肺炎及支氣管炎。The present invention provides a method for preventing, treating and/or improving an infection, disease or condition of an individual, comprising administering to an individual an effective amount of an RNA molecule and/or RNA-LNP or an immunogenic composition comprising at least one open reading frame encoding an RSV polypeptide as described herein. The present invention further provides a method for preventing, treating and/or improving an infection, disease or condition of an individual as described herein, comprising administering to an individual an effective amount of an RNA molecule and/or RNA-LNP or an immunogenic composition comprising at least one open reading frame encoding an RSV polypeptide as described herein, for the manufacture of a medicament for preventing, treating and/or improving an infection, disease or condition of an individual. In some aspects, the infection, disease or condition is associated with RSV. In some aspects, the infection, disease or condition is an acute lower respiratory tract infection (ALRI), including pneumonia and bronchitis. In some aspects, the infection, disease or condition is an acute lower respiratory tract infection (ALRI), including pneumonia and bronchitis.
本發明進一步提供一種預防、治療及/或改善個體之感染、疾病或病狀之方法，其包括向個體投與有效量之本文所描述之包括至少一個編碼所關注基因之多肽之開讀框的RNA分子及/或RNA-LNP、或免疫原性組合物。本發明進一步提供一種本文所描述之包括至少一個編碼所關注基因之多肽之開讀框的RNA分子及/或RNA-LNP、或免疫原性組合物的用途，其用於製造用於預防、治療及/或改善個體之感染、疾病或病狀的藥劑。在一些態樣中，感染、疾病或病狀與所關注基因相關。The present invention further provides a method for preventing, treating and/or ameliorating an infection, disease or condition in an individual, comprising administering to the individual an effective amount of an RNA molecule and/or RNA-LNP or an immunogenic composition as described herein comprising at least one open reading frame encoding a polypeptide of a gene of interest. The present invention further provides a use of an RNA molecule and/or RNA-LNP or an immunogenic composition as described herein comprising at least one open reading frame encoding a polypeptide of a gene of interest for the manufacture of a medicament for preventing, treating and/or ameliorating an infection, disease or condition in an individual. In some aspects, the infection, disease or condition is associated with a gene of interest.
在一些態樣中，個體之年齡為至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：1、2、3、4、5、6、7、8、9、10、11或12個月，或年齡為1、5、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80歲或更大。在一些態樣中，個體之年齡為、至少為、至多為或為約小於1歲、1歲或更大、5歲或更大、10歲或更大、20歲或更大、30歲或更大、40歲或更大、50歲或更大、60歲或更大、70歲或更大，或更大。在一些態樣中，個體之年齡為或為約50歲或更大。在一些態樣中，前述年齡群組中之1、2、3、4、5者或更多者不投與RNA分子及/或RNA-LNP。In some aspects, the age of the individual is at least, at most, just under, or between any two of the following (inclusive or exclusive): 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or is 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 years or older. In some aspects, the age of the individual is, at least, at most, or about less than 1 year old, 1 year old or older, 5 years old or older, 10 years old or older, 20 years old or older, 30 years old or older, 40 years old or older, 50 years old or older, 60 years old or older, 70 years old or older, or older. In some aspects, the age of the individual is or is about 50 years old or older. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned age groups are not administered RNA molecules and/or RNA-LNPs.
在一些態樣中，個體具有免疫能力。在一些態樣中，個體免疫功能不全。In some aspects, the individual is immunocompetent. In some aspects, the individual is immunocompromised.
本發明提供一種本文所描述之方法或用途，其中RNA分子、RNA-LNP及/或免疫原性組合物係作為疫苗投與。本發明提供一種本文所描述之方法或用途，其中RNA分子、RNA-LNP及/或免疫原性組合物係藉由皮內、肌肉內或鼻內注射投與。The present invention provides a method or use as described herein, wherein the RNA molecule, RNA-LNP and/or immunogenic composition is administered as a vaccine. The present invention provides a method or use as described herein, wherein the RNA molecule, RNA-LNP and/or immunogenic composition is administered by intradermal, intramuscular or intranasal injection.
經考慮，本說明書中所論述之任何態樣可關於本發明之任何方法或組合物實施，且反之亦然。此外，本發明之組合物可用於實現本發明之方法。It is contemplated that any aspect discussed in this specification can be implemented with respect to any method or composition of the present invention, and vice versa. In addition, the composition of the present invention can be used to implement the method of the present invention.
在治療性、診斷性或生理學目的或作用之情形下的任何方法亦可描述於「用途」申請專利範圍語言中，諸如本文中所論述之任何化合物、組合物或藥劑用於達成或實施所描述之治療性、診斷性或生理學目的或作用「之用途」。可基於本文所描述之任何方法而採用一或多種組合物之用途。Any method in the context of a therapeutic, diagnostic, or physiological purpose or effect may also be described in "use" claim language, such as any compound, composition, or medicament discussed herein is used to achieve or perform the described therapeutic, diagnostic, or physiological purpose or effect "for use." One or more compositions may be used in conjunction with any method described herein.
本發明之其他目標、特徵及優勢將自以下實施方式而變得顯而易見。然而，應理解，實施方式及特定實例儘管指示本發明之特定態樣，但僅以說明方式給出，因為在本發明之精神及範疇內的各種變化及修改將由此實施方式而變得對熟習此項技術者顯而易見。Other objects, features and advantages of the present invention will become apparent from the following embodiments. However, it should be understood that the embodiments and specific examples, although indicating specific aspects of the present invention, are only given by way of illustration, because various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from the embodiments.
相關申請案之交互參考本申請案主張2023年9月26日申請之美國臨時申請案第63/585,254號及2022年10月27日申請之美國臨時申請案第63/381,238號的權益。前述申請案中之各者的全部內容以引用之方式併入本文中。CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63/585,254, filed on September 26, 2023, and U.S. Provisional Application No. 63/381,238, filed on October 27, 2022. Each of the foregoing applications is incorporated herein by reference in its entirety.
序列表之參考本申請案經由EFS網以電子方式申請且包括以.xml格式以電子方式提交的序列表。.xml檔案含有名稱為「PC072895A Sequence Listing.xml」之序列表，其在2023年9月25日創建且大小為145 KB。此.xml檔案中所含之序列表為說明書之一部分且以全文引用之方式併入本文中。Reference to Sequence Listing This application was filed electronically via the EFS website and includes a sequence listing submitted electronically in .xml format. The .xml file contains a sequence listing named "PC072895A Sequence Listing.xml", which was created on September 25, 2023 and is 145 KB in size. The sequence listing contained in this .xml file is part of the specification and is incorporated herein by reference in its entirety.
本發明提供一種RNA分子(例如RNA聚核苷酸)，其包含至少一個編碼呼吸道融合病毒(RSV)抗原之開讀框(ORF)。在一些態樣中，RSV抗原為RSV多肽。在一些態樣中，RSV多肽為RSV F多肽。在一些態樣中，RSV多肽包含表1中所闡述之胺基酸序列。在一些態樣中，RNA分子包含轉錄自表2之至少一個DNA核酸序列的ORF。在一些態樣中，RNA分子包含含有表3之RNA核酸序列的ORF。在一些態樣中，RNA分子包含5'帽、5' UTR、3' UTR及多-A尾中之至少一者。在其他態樣中，RNA分子包含5'帽、3' UTR及多-A尾中之至少一者。本發明提供一種RNA分子，其包含經修飾之核苷酸(例如經修飾之RNA；modRNA)。The present invention provides a kind of RNA molecule (such as RNA polynucleotide), which includes at least one open reading frame (ORF) encoding respiratory syncytial virus (RSV) antigen. In some aspects, the RSV antigen is an RSV polypeptide. In some aspects, the RSV polypeptide is an RSV F polypeptide. In some aspects, the RSV polypeptide comprises the amino acid sequence described inTable1. In some aspects, the RNA molecule comprises an ORF transcribed from at least one DNA nucleic acid sequence ofTable2. In some aspects, the RNA molecule comprises an ORF containing the RNA nucleic acid sequence ofTable3. In some aspects, the RNA molecule comprises at least one of a 5' cap, a 5' UTR, a 3' UTR and a poly-A tail. In other aspects, the RNA molecule comprises at least one of a 5' cap, a 3' UTR and a poly-A tail. The present invention provides a kind of RNA molecule, which comprises a modified nucleotide (such as a modified RNA; modRNA).
本發明提供一種免疫原性組合物，其包含編碼本文所描述之RSV多肽之RNA分子中之任一者，該等RNA分子與一或多種脂質複合、囊封於其中或用其調配且形成脂質奈米顆粒(RNA-LNP)。本發明進一步提供一種免疫原性組合物，其包含含有本文所描述之至少一種RNA核酸的RNA分子中之任一者，該等RNA分子與一或多種脂質複合、囊封於一或多種脂質中或與一或多種脂質一起調配且形成RNA-LNP。本發明進一步提供一種經由向個體投與有效量之本文所描述之RNA分子、RNA-LNP或免疫原性組合物來預防、治療或改善個體之感染、疾病或病狀(例如RSV感染相關呼吸道病痛，包括肺炎及支氣管炎)的方法。本發明進一步提供一種本文所描述之RNA分子、RNA-LNP及/或免疫原性組合物作為疫苗之用途。The present invention provides an immunogenic composition comprising any of the RNA molecules encoding the RSV polypeptides described herein, which are complexed with, encapsulated in, or formulated with one or more lipids and form lipid nanoparticles (RNA-LNPs). The present invention further provides an immunogenic composition comprising any of the RNA molecules containing at least one RNA nucleic acid described herein, which are complexed with, encapsulated in, or formulated with one or more lipids and form RNA-LNPs. The present invention further provides a method of preventing, treating, or ameliorating an infection, disease, or condition (e.g., RSV infection-related respiratory tract ailments, including pneumonia and bronchitis) of an individual by administering to the individual an effective amount of the RNA molecules, RNA-LNPs, or immunogenic compositions described herein. The present invention further provides a use of the RNA molecules, RNA-LNPs and/or immunogenic compositions described herein as vaccines.
藉由參考以下對本發明之實施例及其中所包括之實例之詳細描述可更容易理解本發明。應理解，本發明不限於特定的製造方法，其當然可以有所變化。亦應理解，本文所用之術語僅出於描述特定實施例之目的且不意欲為限制性的。The present invention may be more readily understood by reference to the following detailed description of the embodiments of the present invention and the examples included therein. It should be understood that the present invention is not limited to a particular method of manufacture, which may of course vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting.
本文所用之章節標題僅出於組織目的且不應解釋為限制所描述之主題。The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
本文所引用之所有參考文獻，包括專利申請案、專利公開案、UniProtKB寄存編號，均以引用之方式併入本文中，如同具體地且單獨地指出各個別參考文獻以全文引用之方式併入本文中一般。All references cited herein, including patent applications, patent publications, and UniProtKB deposit numbers, are incorporated herein by reference as if each individual reference was specifically and individually indicated to be incorporated herein by reference in its entirety.
I.定義之實例除非本文中另外定義，否則結合本發明中使用之科學及技術術語應具有一般熟習此項技術者通常所理解之含義。I.Examples of Definitions Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by one of ordinary skill in the art.
在整個本申請案中，術語「約」及「大約」及「實質上」係根據其在細胞及分子生物學領域中之普通且一般含義使用，以指示與其連接之一或多個值的±10%之偏差。因此，在任何所揭示態樣中，該等術語可替換為「在」指定內容「之[百分比]內」。在一個非限制性態樣中，該百分比包括0.1%、0.5%、1%、5%及10%。Throughout this application, the terms "about," "approximately," and "substantially" are used according to their ordinary and customary meanings in the art of cell and molecular biology to indicate a deviation of ±10% from one or more values to which they are attached. Thus, in any disclosed aspect, these terms may be replaced with "within [percentage] of" the specified content. In a non-limiting aspect, the percentages include 0.1%, 0.5%, 1%, 5%, and 10%.
本文中值的範圍之敍述僅僅意欲充當個別地提及處於該範圍內之各個別值的簡寫方法。除非本文中另外指示，否則各個別值均併入本說明書中，如同其在本文中個別地敍述一般。Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each individual value within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.
當與術語「包含」結合使用時，字組「一(a)」或「一(an)」之使用可意謂「一個」，但其亦與「一或多個」、「至少一個」及「一個或超過一個」之含義相符。When used in conjunction with the term "comprising", the use of the word "a" or "an" may mean "one", but it is also consistent with the meaning of "one or more", "at least one" and "one or more than one".
片語「及/或」意謂「及」或「或」。舉例而言，A、B及/或C包括：單獨的A、單獨的B、單獨的C、A與B之組合、A與C之組合、B與C之組合或A、B及C之組合。換言之，「及/或」作為包括性或操作。The phrase "and/or" means "and" or "or". For example, A, B and/or C includes: A alone, B alone, C alone, the combination of A and B, the combination of A and C, the combination of B and C, or the combination of A, B and C. In other words, "and/or" acts as an inclusive or operation.
片語「基本上全部」定義為「至少95%」；若群組之基本上全部成員具有某一特性，則該群組之至少95%成員具有該特性。在一些態樣中，基本上全部意謂等於以下中之任一者、以下中之至少任一者或在以下中之任何兩者之間的群組成員具有該特性：95%、96%、97%、98%、99%或100%。The phrase "substantially all" is defined as "at least 95%"; if substantially all members of a group have a certain characteristic, then at least 95% of the members of the group have that characteristic. In some aspects, substantially all means that equal to any one of the following, at least any one of the following, or between any two of the following, the members of the group have that characteristic: 95%, 96%, 97%, 98%, 99%, or 100%.
組合物及其使用方法可「包含」、「基本上由…組成」或「由…組成」：在整個本說明書中所揭示之成分或步驟中之任一者。在整個本說明書中，除非上下文另外要求，否則詞語「包含(comprising)」(及包含之任何形式，諸如「包含(comprise)」及「包含(comprises)」)、「具有(having)」(及具有之任何形式，諸如「具有(have)」及「具有(has)」)、「包括(including)」(及包括之任何形式，諸如「包括(includes)」及「包括(include)」)或「含有(containing)」(及含有之任何形式，諸如「含有(contains)」及「含有(contain)」)為包括性或開放性的，且應理解為暗示包括所陳述步驟或要素或者步驟或要素之群組，但不排除包括任何其他步驟或要素或者步驟或要素之群組。經考慮，本文在術語「包含」之上下文中描述的態樣亦可在術語「由…組成」或「基本上由…組成」的上下文中實施。「基本上由」所揭示之成分或步驟中之任一者「組成」的組合物及方法將申請專利範圍之範疇限制於不實質影響所主張發明之基本及新穎特徵的指定材料或步驟。詞語「由…組成(consisting of)」(及由…組成之任何形式，諸如「由…組成(consist of)」及「由…組成(consists of)」)意謂包括且限於接在片語「由…組成」後面的任何內容。因此，片語「由…組成」指示所列要素為所需或必選的，且不可存在其他要素。The compositions and methods of use thereof may "comprise," "consist essentially of," or "consist of": any of the ingredients or steps disclosed throughout this specification. Throughout this specification, unless the context requires otherwise, the words “comprising” (and any form of inclusion, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of inclusion, such as “includes” and “include”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended, and will be understood to imply the inclusion of stated steps or elements, or groups of steps or elements, but not the exclusion of the inclusion of any other steps or elements, or groups of steps or elements. It is contemplated that aspects described herein in the context of the term "comprising" may also be implemented in the context of the term "consisting of" or "consisting essentially of." Compositions and methods that "consist essentially of" any of the disclosed components or steps limit the scope of the claimed invention to the specified materials or steps that do not materially affect the basic and novel characteristics of the claimed invention. The phrase "consisting of" (and any form of consisting of, such as "consist of" and "consists of") is meant to include and be limited to whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or optional, and that no other elements may be present.
在整個本說明書中對「一個態樣」、「一態樣」、「特定態樣」、「相關態樣」、「某一態樣」、「額外態樣」或「另一態樣」或其組合之提及意謂結合該態樣描述之特定特徵、結構或特性包括在本發明之至少一個態樣中。因此，前述片語在整個本說明書中各處之出現未必皆指代同一態樣。此外，特定特徵、結構或特性可在一或多個態樣中以任何適合方式組合。References to "an aspect", "an aspect", "a specific aspect", "a related aspect", "an aspect", "an additional aspect", or "another aspect" or a combination thereof throughout this specification mean that the specific features, structures, or characteristics described in conjunction with the aspect are included in at least one aspect of the present invention. Therefore, the appearance of the aforementioned phrases in various places throughout this specification does not necessarily refer to the same aspect. In addition, specific features, structures, or characteristics may be combined in any suitable manner in one or more aspects.
術語「抑制(inhibiting)」、「降低(decreasing)」或「減小(reducing)」或此等術語之任何變化形式包括任何可量測的降低(例如5%、10%、15%、20%、25%、30%、35%、40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、95%或99%降低)或完全抑制以達成所需結果。術語「改良/提高(improve)」、「促進(promote)」或「增加(increase)」或此等術語之任何變化形式包括任何可量測增加(例如5%、10%、15%、20%、25%、30%、35%、40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、95%或99%增加)以達成蛋白質或分子之所需結果或產量。The terms "inhibiting," "decreasing," or "reducing," or any variation of these terms, include any measurable reduction (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% reduction) or complete inhibition to achieve the desired result. The terms "improve," "promote," or "increase," or any variations of these terms, include any measurable increase (e.g., a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% increase) to achieve a desired outcome or yield of a protein or molecule.
如本文所用，術語「參考」、「標準」或「對照」描述相對於其進行比較的值。舉例而言，將藥劑、個體、群體、樣品或所關注值與參考、標準或對照藥劑、個體、群體、樣品或所關注值進行比較。參考、標準或對照可實質上同時進行測試及/或測定及/或與藥劑、個體、群體、樣品或所關注值之所關注測試或測定一起，及/或可在與評定下之藥劑、個體、群體、樣品或所關注值相當之條件或情況下測定或表徵。As used herein, the term "reference," "standard," or "control" describes a value to which a comparison is made. For example, an agent, individual, population, sample, or value of interest is compared to a reference, standard, or control agent, individual, population, sample, or value of interest. A reference, standard, or control may be tested and/or determined substantially simultaneously and/or together with the test or determination of interest for the agent, individual, population, sample, or value of interest, and/or may be determined or characterized under conditions or circumstances equivalent to the agent, individual, population, sample, or value of interest under assessment.
術語「經分離」可指實質上不含其起源來源之細胞材料、細菌材料、病毒材料或培養基(當藉由重組DNA技術產生時)、或化學前驅物或其他化學物質(當以化學方式合成時)的核酸或多肽。此外，經分離化合物係指可以經分離化合物形式向個體投與之化合物；換言之，若化合物黏附至管柱或嵌入於瓊脂糖凝膠中，則其不可簡單地視為「經分離」。此外，「經分離核酸片段」或「經分離肽」為天然不以片段形式存在及/或通常不呈功能狀態及/或經由人工干預自天然狀態改變或移除的核酸或蛋白質片段。舉例而言，天然存在於活動物中之DNA並非「經分離」的，但合成DNA或自其天然狀態之共存材料部分或完全分離之DNA為「經分離」的。經分離核酸可以實質上經純化之形式存在，或可存在於非天然環境(諸如已遞送核酸至其中之細胞)中。The term "isolated" may refer to a nucleic acid or polypeptide that is substantially free of the cellular material, bacterial material, viral material, or culture medium (when produced by recombinant DNA techniques), or chemical precursors or other chemicals (when chemically synthesized) from which it originated. In addition, an isolated compound refers to a compound that can be administered to an individual in the form of an isolated compound; in other words, if the compound is attached to a column or embedded in an agarose gel, it cannot be simply considered "isolated." In addition, an "isolated nucleic acid fragment" or "isolated peptide" is a nucleic acid or protein fragment that does not exist in fragment form in nature and/or is not normally in a functional state and/or has been altered or removed from the natural state by artificial intervention. For example, DNA naturally present in living animals is not "isolated," but synthetic DNA or DNA that has been partially or completely separated from coexisting materials with which it is naturally present is "isolated." Isolated nucleic acids may exist in a substantially purified form or may exist in a non-natural environment, such as a cell into which the nucleic acid has been delivered.
如本文所用，「核酸」為包含核酸組分之分子，且係指DNA或RNA分子。其可與術語「聚核苷酸」互換使用。核酸分子為包含核苷酸單體或由核苷酸單體組成之聚合物，該等核苷酸單體藉由糖/磷酸酯主鏈之磷酸二酯鍵彼此共價連接。核酸亦可涵蓋經修飾之核酸分子，諸如鹼基修飾、糖修飾或主鏈修飾等DNA或RNA分子。核酸可以各種形式存在，諸如：併入序列之經分離之區段及重組載體或編碼多肽(諸如抗原或抗體之一或兩條鏈、或其片段、衍生物、突變蛋白或變體)之重組聚核苷酸；足夠用作雜交探針之聚核苷酸；PCR引子或用於鑑別、分析、突變或擴增編碼多肽之聚核苷酸的定序引子；用於抑制本文前文所描述之聚核苷酸、mRNA、saRNA、modRNA及互補序列之表現的反義核酸。核酸可編碼抗體可結合之抗原決定基。As used herein, "nucleic acid" is a molecule comprising a nucleic acid component and refers to a DNA or RNA molecule. It is used interchangeably with the term "polynucleotide". A nucleic acid molecule is a polymer comprising or composed of nucleotide monomers covalently linked to each other by phosphodiester bonds of a sugar/phosphate backbone. Nucleic acid may also encompass modified nucleic acid molecules, such as base-modified, sugar-modified, or backbone-modified DNA or RNA molecules. Nucleic acids can exist in various forms, such as: isolated segments and recombinant vectors incorporating sequences or recombinant polynucleotides encoding polypeptides (such as one or both chains of an antigen or antibody, or fragments, derivatives, mutants or variants thereof); polynucleotides sufficient for use as hybridization probes; PCR primers or sequencing primers for identifying, analyzing, mutating or amplifying polynucleotides encoding polypeptides; antisense nucleic acids for inhibiting the expression of polynucleotides, mRNA, saRNA, modRNA and complementary sequences described herein above. Nucleic acids can encode antigenic determinants to which antibodies can bind.
術語「抗原決定基」係指被免疫球蛋白(例如抗體或受體)結合組分特異性識別之部分。在一些態樣中，抗原決定基包含抗原上之複數個化學原子或基團。在一些態樣中，當抗原採用相關三維構形時，此類化學原子或基團為表面暴露的。在一些態樣中，當抗原採用此類構形時，此類化學原子或基團在空間中以物理方式彼此接近。在一些態樣中，當抗原採用替代構形(例如經線性化)時，至少一些此類化學原子或基團以物理方式彼此分開。The term "antigenic determinant" refers to a portion that is specifically recognized by an immunoglobulin (e.g., an antibody or a receptor) binding component. In some aspects, an antigenic determinant comprises a plurality of chemical atoms or groups on an antigen. In some aspects, when an antigen adopts a related three-dimensional configuration, such chemical atoms or groups are surface exposed. In some aspects, when an antigen adopts such a configuration, such chemical atoms or groups are physically close to each other in space. In some aspects, when an antigen adopts an alternative configuration (e.g., linearization), at least some of such chemical atoms or groups are physically separated from each other.
核酸可為單股或雙股，且可包含RNA及/或DNA核苷酸以及其人工變體(例如肽核酸)。在一些情況下，核酸序列可編碼具有其他異源編碼序列之多肽序列，例如以實現多肽之純化、運輸、分泌、轉譯後修飾，或實現治療益處，諸如靶向或功效。可向經修飾之多肽編碼序列中添加標籤或其他異源多肽，其中「異源」係指與經修飾多肽不同的多肽。Nucleic acids can be single-stranded or double-stranded, and can include RNA and/or DNA nucleotides, as well as artificial variants thereof (e.g., peptide nucleic acids). In some cases, a nucleic acid sequence can encode a polypeptide sequence with other heterologous coding sequences, for example, to achieve purification, transport, secretion, post-translational modification of the polypeptide, or to achieve therapeutic benefits, such as targeting or efficacy. Tags or other heterologous polypeptides can be added to the modified polypeptide coding sequence, where "heterologous" refers to a polypeptide that is different from the modified polypeptide.
術語「聚核苷酸」係指一種核酸分子，其可為重組的或已自總基因體核酸分離。術語「聚核苷酸」內包括寡核苷酸(100個殘基或更少殘基長度的核酸)、重組載體(包括例如質體、黏質體、噬菌體、病毒)及其類似者。在某些態樣中，聚核苷酸包括與其天然存在之基因或蛋白質編碼序列實質上分離的調控序列。聚核苷酸可為單股(編碼或反義)或雙股的，且可為RNA、DNA (基因體、cDNA或合成的)、其類似物或其組合。額外編碼或非編碼序列可(但未必)存在於聚核苷酸內。The term "polynucleotide" refers to a nucleic acid molecule that may be recombinant or that has been isolated from total genomic nucleic acid. Included within the term "polynucleotide" are oligonucleotides (nucleic acids of 100 residues or less in length), recombinant vectors (including, for example, plasmids, cosmids, phages, viruses), and the like. In certain aspects, a polynucleotide includes regulatory sequences that are substantially separated from the gene or protein coding sequence in which it is naturally found. A polynucleotide may be single-stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA, or synthetic), analogs thereof, or combinations thereof. Additional coding or non-coding sequences may (but need not) be present within a polynucleotide.
在某些態樣中，存在與本文所揭示之序列具有實質上一致性之聚核苷酸變體；使用本文所描述之方法(例如使用標準參數之BLAST分析)，與本文提供之聚核苷酸序列相比包含等於以下中之任一者、以下中之至少任一者、以下中之至多任一者或以下中之任何兩者之間的序列一致性的彼等者：70%、75%、80%、85%、90%、95%、96%、97%、98%或99%或更高。在某些態樣中，經分離聚核苷酸將包含編碼在整個序列長度上與本文所描述之胺基酸序列具有至少90%一致性之多肽的核苷酸序列；或與該經分離聚核苷酸互補之核苷酸序列。在一些態樣中，經分離聚核苷酸將包含編碼在整個序列長度上與本文所描述之胺基酸序列具有至少95%一致性之多肽的核苷酸序列；或與該經分離聚核苷酸互補之核苷酸序列。In certain aspects, there are polynucleotide variants that have substantial identity to the sequences disclosed herein; those that comprise a sequence identity equal to any of the following, at least any of the following, at most any of the following, or between any two of the following, as compared to the polynucleotide sequences provided herein using the methods described herein (e.g., BLAST analysis using standard parameters): 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more. In certain aspects, an isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide having at least 90% identity to an amino acid sequence described herein over the entire sequence length; or a nucleotide sequence that is complementary to the isolated polynucleotide. In some aspects, the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide having at least 95% identity over the entire length of the sequence to an amino acid sequence described herein; or a nucleotide sequence that is complementary to the isolated polynucleotide.
與編碼序列本身之長度無關，核酸區段可與其他核酸序列(諸如啟動子、多腺苷酸化訊號、其他限制酶位點、多個選殖位點、其他編碼區段及其類似者)組合，使得其整體長度可顯著變化。核酸可具有任何長度。該等核酸之長度可例如等於以下中之任一者、以下中之至少任一者、以下中之至多任一者或以下中之任何兩者之間：5、10、15、20、25、30、35、40、45、50、75、100、125、175、200、250、300、350、400、450、500、750、1000、1500、3000、5000、6000、7000、8000、9000、10000、11000、12000、13000、14000、15000或更多個核苷酸；及/或可包含一或多個額外序列(例如調節序列)；及/或可為較大核酸(例如載體)之一部分。因此考慮可使用幾乎任何長度之核酸片段，其中總長度受製備簡易性及預期重組核酸方案中之用途限制。Independent of the length of the coding sequence itself, a nucleic acid segment may be combined with other nucleic acid sequences (e.g., promoters, polyadenylation signals, other restriction enzyme sites, multiple cloning sites, other coding segments, and the like) so that its overall length may vary significantly. A nucleic acid may be of any length. The length of the nucleic acids can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000 or more nucleotides; and/or can comprise one or more additional sequences (e.g., regulatory sequences); and/or can be part of a larger nucleic acid (e.g., a vector). It is therefore contemplated that nucleic acid fragments of nearly any length may be used, with the overall length being limited by ease of preparation and intended use in recombinant nucleic acid protocols.
就此而言，術語「基因」用於指編碼蛋白質、多肽或肽之核酸(包括恰當轉錄、轉譯後修飾或定位所需之任何序列)。如熟習此項技術者將理解，此術語涵蓋基因體序列、表現卡匣、cDNA序列及較小的經工程改造之核酸區段，其表現或可經調適以表現蛋白質、多肽、域、肽、融合蛋白及突變體。編碼全部或一部分多肽之核酸可含有編碼全部或一部分此類多肽之連續核酸序列。亦考慮特定多肽可由含有變化之核酸編碼，該等核酸具有略微不同的核酸序列，但仍編碼相同或實質上類似的多肽。In this regard, the term "gene" is used to refer to a nucleic acid encoding a protein, polypeptide, or peptide (including any sequences required for proper transcription, post-translational modification, or localization). As will be understood by those skilled in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments whose expression may be adapted to express proteins, polypeptides, domains, peptides, fusion proteins, and mutants. A nucleic acid encoding all or a portion of a polypeptide may contain a contiguous nucleic acid sequence encoding all or a portion of such a polypeptide. It is also contemplated that a particular polypeptide may be encoded by nucleic acids containing variations that have slightly different nucleic acid sequences but still encode the same or substantially similar polypeptides.
如本文所用，術語核酸序列之「表現」係指核酸序列產生任何基因產物。在一些態樣中，基因產物可為轉錄本。在一些態樣中，基因產物可為多肽。在一些態樣中，核酸序列之表現涉及以下中之一或多者：(1)自DNA序列產生RNA模板(例如藉由轉錄)；(2)加工RNA轉錄本(例如藉由剪接、編輯等)；(3)將RNA轉譯成多肽或蛋白質；及/或(4)轉譯後修飾多肽或蛋白質。As used herein, the term "expression" of a nucleic acid sequence refers to the production of any gene product by the nucleic acid sequence. In some aspects, the gene product may be a transcript. In some aspects, the gene product may be a polypeptide. In some aspects, expression of a nucleic acid sequence involves one or more of the following: (1) generating an RNA template from a DNA sequence (e.g., by transcription); (2) processing the RNA transcript (e.g., by splicing, editing, etc.); (3) translating the RNA into a polypeptide or protein; and/or (4) post-translational modification of the polypeptide or protein.
一般而言，術語「經工程改造」係指已人工操控之態樣。舉例而言，當未以自然界中之次序連接在一起之兩個或更多個序列藉由人工操控以在經工程改造之聚核苷酸中彼此直接連接時，及/或當聚核苷酸中之特定殘基為非天然存在的及/或係經由人工的作用引起與在自然界中其未連接之實體或部分連接時，聚核苷酸被視為「經工程改造」。Generally speaking, the term "engineered" refers to a state that has been manipulated by human hands. For example, a polynucleotide is considered "engineered" when two or more sequences that are not linked together in the order in nature are directly linked to each other in an engineered polynucleotide by human manipulation, and/or when a specific residue in a polynucleotide is not naturally present and/or is caused by human action to be linked to an entity or part to which it is not linked in nature.
如本文所用，術語「DNA」意謂包含諸如去氧腺苷單磷酸、去氧胸苷單磷酸、去氧鳥苷單磷酸及去氧胞苷單磷酸單體之核苷酸的核酸分子，該等核苷酸由糖部分(去氧核糖)、鹼基部分及磷酸部分構成，且藉由特徵性主鏈結構聚合。主鏈結構通常藉由第一相鄰單體之核苷酸之糖部分(例如去氧核糖)與第二相鄰單體之磷酸部分之間的磷酸二酯鍵形成。單體之特定次序(例如連接至糖/磷酸主鏈之鹼基之次序)被稱為DNA序列。DNA可為單股或雙股。在雙股形式中，第一股之核苷酸通常例如藉由A/T鹼基配對及G/C鹼基配對與第二股之核苷酸雜交。DNA可包含全部或大部分去氧核糖核苷酸殘基。如本文所用，術語「去氧核糖核苷酸」意謂在β-D-呋喃核糖基之2'位置處缺乏羥基的核苷酸。在無任何限制之情況下，DNA可涵蓋雙股DNA、反義DNA、單股DNA、經分離DNA、合成DNA、以重組方式產生之DNA及經修飾之DNA。As used herein, the term "DNA" means a nucleic acid molecule comprising nucleotides such as deoxyadenosine monophosphate, deoxythymidine monophosphate, deoxyguanosine monophosphate, and deoxycytidine monophosphate monomers, which are composed of a sugar portion (deoxyribose), a base portion, and a phosphate portion, and are polymerized by a characteristic backbone structure. The backbone structure is usually formed by a phosphodiester bond between the sugar portion (e.g., deoxyribose) of the nucleotide of the first adjacent monomer and the phosphate portion of the second adjacent monomer. The specific order of the monomers (e.g., the order of the bases attached to the sugar/phosphate backbone) is called the DNA sequence. DNA can be single-stranded or double-stranded. In the double-stranded form, the nucleotides of the first strand are usually hybridized with the nucleotides of the second strand, such as by A/T base pairing and G/C base pairing. DNA may contain all or most deoxyribonucleotide residues. As used herein, the term "deoxyribonucleotide" means a nucleotide lacking a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. Without any limitation, DNA may encompass double-stranded DNA, antisense DNA, single-stranded DNA, isolated DNA, synthetic DNA, recombinantly produced DNA, and modified DNA.
如本文所用，術語「RNA」意謂包含諸如腺苷單磷酸、尿苷單磷酸、鳥苷單磷酸及胞苷單磷酸單體之核苷酸的核酸分子，該等核苷酸沿著所謂的主鏈彼此連接。主鏈藉由第一單體之糖(例如核糖)與第二相鄰單體之磷酸部分之間的磷酸二酯鍵形成。RNA可例如在細胞內藉由DNA序列之轉錄獲得。在真核細胞中，轉錄通常在細胞核或粒線體內進行。在活體內，DNA之轉錄可產生未成熟RNA，其被加工成信使RNA (mRNA)。例如在真核生物體中對未成熟RNA之加工包含各種轉錄後修飾，諸如剪接、5'加帽、多腺苷酸化、自細胞核或粒線體輸出。加工成熟信使RNA且提供可轉譯成肽或蛋白質之胺基酸序列的核苷酸序列。成熟mRNA可包含5'帽、5' UTR、開讀框、3' UTR及多腺苷酸尾序列。RNA可包含全部或大部分核糖核苷酸殘基。如本文所用，術語「核糖核苷酸」意謂在β-D-呋喃核糖基之2'位置處具有羥基的核苷酸。在一個態樣中，RNA可為與編碼肽或蛋白質之RNA轉錄本相關的信使RNA (mRNA)。如熟習此項技術者已知，mRNA一般含有5'非轉譯區(5' UTR)、多肽編碼區及3'非轉譯區(3' UTR)。在無任何限制之情況下，RNA可涵蓋雙股RNA、反義RNA、單股RNA、經分離RNA、合成RNA、以重組方式產生之RNA及經修飾之RNA (modRNA)。As used herein, the term "RNA" means a nucleic acid molecule comprising nucleotides such as adenosine monophosphate, uridine monophosphate, guanosine monophosphate and cytidine monophosphate monomers, which are linked to each other along a so-called backbone. The backbone is formed by a phosphodiester bond between a sugar (e.g., ribose) of a first monomer and a phosphate portion of a second adjacent monomer. RNA can be obtained, for example, by transcription of a DNA sequence in a cell. In eukaryotic cells, transcription is usually carried out in the nucleus or mitochondria. In vivo, transcription of DNA can produce immature RNA, which is processed into messenger RNA (mRNA). For example, processing of immature RNA in eukaryotic organisms includes various post-transcriptional modifications, such as splicing, 5' capping, polyadenylation, and export from the nucleus or mitochondria. Processing mature messenger RNA and providing a nucleotide sequence that can be translated into an amino acid sequence of a peptide or protein. Mature mRNA may include a 5' cap, a 5' UTR, an open reading frame, a 3' UTR, and a polyadenylic acid tail sequence. RNA may include all or most ribonucleotide residues. As used herein, the term "ribonucleotide" means a nucleotide having a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. In one aspect, RNA may be a messenger RNA (mRNA) associated with an RNA transcript encoding a peptide or protein. As known to those skilled in the art, mRNA generally contains a 5' non-translational region (5' UTR), a polypeptide coding region, and a 3' non-translational region (3' UTR). Without limitation, RNA may include double-stranded RNA, antisense RNA, single-stranded RNA, isolated RNA, synthetic RNA, recombinantly produced RNA, and modified RNA (modRNA).
「經分離RNA」定義為一種RNA分子，其可為重組的或已自總基因體核酸分離。經分離RNA分子或蛋白質可以實質上經純化之形式存在，或可存在於非天然環境(諸如宿主細胞)中。"Isolated RNA" is defined as an RNA molecule that may be recombinant or that has been separated from total genomic nucleic acid. An isolated RNA molecule or protein may exist in a substantially purified form, or may exist in a non-native environment such as a host cell.
「經修飾RNA」或「modRNA」係指與天然存在之RNA相比具有一或多個核苷酸之至少一個添加、缺失、取代及/或改變的RNA分子。此類改變可指將非核苷酸材料添加至內部RNA核苷酸，或添加至RNA之一或多個5'及/或3'端。在一個態樣中，此類modRNA含有至少一個經修飾核苷酸，諸如核苷酸之鹼基之改變。舉例而言，經修飾核苷酸可置換一或多個尿苷及/或胞苷核苷酸。舉例而言，此等置換可針對RNA序列中之尿苷及/或胞苷的每個例項進行，或可僅針對所選擇尿苷及/或胞苷核苷酸進行。RNA中標準核苷酸之此類改變可包括非標準核苷酸，諸如化學合成之核苷酸或去氧核苷酸。舉例而言，RNA序列中之至少一個尿苷核苷酸可經N1-甲基假尿苷置換。其他此類經改變之核苷酸為熟習此項技術者已知的。此類經改變之RNA分子被視為天然存在之RNA之類似物。在一些態樣中，RNA藉由使用DNA模板進行活體外轉錄而產生，其中DNA係指含有去氧核糖核苷酸之核酸。在一些態樣中，RNA可為複製子RNA (複製子)，尤其自複製RNA或自擴增RNA (saRNA)。"Modified RNA" or "modRNA" refers to an RNA molecule that has at least one addition, deletion, substitution, and/or alteration of one or more nucleotides compared to naturally occurring RNA. Such alterations may refer to the addition of non-nucleotide material to internal RNA nucleotides, or to one or more 5' and/or 3' ends of the RNA. In one aspect, such modRNA contains at least one modified nucleotide, such as a change in the base of the nucleotide. For example, the modified nucleotide may replace one or more uridine and/or cytidine nucleotides. For example, such replacements may be made for every instance of uridine and/or cytidine in the RNA sequence, or may be made only for selected uridine and/or cytidine nucleotides. Such alterations of standard nucleotides in RNA may include non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For example, at least one uridine nucleotide in the RNA sequence may be replaced by N1-methyl pseudouridine. Other such altered nucleotides are known to those skilled in the art. Such altered RNA molecules are considered analogs of naturally occurring RNA. In some aspects, RNA is produced by in vitro transcription using a DNA template, wherein DNA refers to a nucleic acid containing deoxyribonucleotides. In some aspects, RNA may be a replicon RNA (replicon), particularly a self-replicating RNA or a self-amplifying RNA (saRNA).
如本文中所涵蓋，不受任何限制，RNA可用作治療及/或預防哺乳動物(包括人類)之多種病狀的治療模態。本文所描述之方法包含向哺乳動物(諸如人類)投與本文所描述之RNA。舉例而言，在一個態樣中，使用RNA之此類方法包括編碼抗原之RNA疫苗以誘導穩定中和抗體及隨附/伴隨的T細胞反應，從而達成保護性免疫接種。在一些態樣中，投與最小疫苗劑量以誘導穩固中和抗體及伴隨/相伴T細胞反應以達成保護性免疫接種。在一個態樣中，所投與之RNA為活體外轉錄之RNA。舉例而言，此類RNA可用於編碼至少一種意欲在該哺乳動物中產生免疫反應之抗原。病原性抗原為來源於與傳染病相關之病原體的肽或蛋白質抗原。在特定態樣中，病原性物質為來源於RSV之肽或蛋白質抗原。可用本文所揭示之RNA治療之病狀及/或疾病包括但不限於由病毒感染引起及/或受其影響之彼等者。此類病毒包括但不限於RSV。As encompassed herein, without limitation, RNA can be used as a therapeutic modality for treating and/or preventing a variety of conditions in mammals, including humans. The methods described herein include administering RNA described herein to mammals, such as humans. For example, in one aspect, such methods using RNA include RNA vaccines encoding antigens to induce stable neutralizing antibodies and accompanying/concomitant T cell responses to achieve protective immunization. In some aspects, a minimal vaccine dose is administered to induce stable neutralizing antibodies and accompanying/concomitant T cell responses to achieve protective immunization. In one aspect, the administered RNA is an in vitro transcribed RNA. For example, such RNA can be used to encode at least one antigen intended to generate an immune response in the mammal. Pathogenic antigens are peptide or protein antigens derived from pathogens associated with infectious diseases. In a specific aspect, the pathogenic substance is a peptide or protein antigen derived from RSV. Conditions and/or diseases that can be treated with the RNA disclosed herein include, but are not limited to, those caused by and/or affected by viral infection. Such viruses include, but are not limited to RSV.
如本文所用，「預防(Prevent)」或「預防(prevention)」當結合疾病、病症及/或病狀之出現使用時，係指減少罹患疾病、病症及/或病狀之風險及/或延遲該疾病、病症或病狀之一或多個特徵或症狀之發作。當疾病、病症或病狀之發作已延遲預定時段時，可認為預防完成。As used herein, "Prevent" or "prevention" when used in conjunction with the occurrence of a disease, disorder and/or condition, refers to reducing the risk of developing a disease, disorder and/or condition and/or delaying the onset of one or more features or symptoms of the disease, disorder or condition. Prevention is considered accomplished when the onset of the disease, disorder or condition has been delayed for a predetermined period of time.
如將自上下文理解，疾病、病症及/或病狀之「風險」係指特定個體將罹患該疾病、病症及/或病狀之可能性。在一些態樣中，風險表示為百分比。在一些態樣中，風險為、至少為或至多為0、1%、2%、3%、4%、5%、6%、7%、8%、9%、10%、20%、30%、40%、50%、60%、70%、80%、90%直至100%。在一些態樣中，風險表示為相對於與參考樣品或參考樣品群組相關之風險的風險。在一些態樣中，參考樣品或參考樣品群組具有疾病、病症、病狀及/或事件之已知風險。在一些態樣中，參考樣品或參考樣品群組來自與特定個體類似之個體。在一些態樣中，風險可反映一或多種遺傳屬性，例如其可使個體傾向於罹患(或不罹患)特定疾病、病症及/或病狀。在一些態樣中，風險可反映一或多種表觀遺傳事件或屬性及/或一或多種生活方式或環境事件或屬性。易患上(Susceptible to)：「易患上」疾病、病症及/或病狀之個體為罹患該疾病、病症及/或病狀之風險高於一般公眾成員之個體。在一些態樣中，易患上疾病、病症及/或病狀之個體可能尚未診斷出該疾病、病症及/或病狀。在一些態樣中，易患上疾病、病症及/或病狀之個體可展現該疾病、病症及/或病狀之症狀。在一些態樣中，易患上疾病、病症及/或病狀之個體可能不展現該疾病、病症及/或病狀之症狀。在一些態樣中，易患上疾病、病症及/或病狀之個體將罹患該疾病、病症及/或病狀。在一些態樣中，易患上疾病、病症及/或病狀之個體將不罹患該疾病、病症及/或病狀。As will be understood from the context, the "risk" of a disease, illness and/or condition refers to the probability that a particular individual will suffer from the disease, illness and/or condition. In some aspects, the risk is expressed as a percentage. In some aspects, the risk is, at least or at most 0, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% to 100%. In some aspects, the risk is expressed as a risk relative to the risk associated with a reference sample or a reference sample group. In some aspects, a reference sample or a reference sample group has a known risk of a disease, illness, condition and/or event. In some aspects, a reference sample or a reference sample group is from an individual similar to a particular individual. In some aspects, risk may reflect one or more genetic attributes, e.g., that may predispose an individual to develop (or not develop) a particular disease, disorder, and/or condition. In some aspects, risk may reflect one or more epigenetic events or attributes and/or one or more lifestyle or environmental events or attributes. Susceptible to: An individual who is "susceptible to" a disease, disorder, and/or condition is an individual who is at a higher risk of developing the disease, disorder, and/or condition than the general public. In some aspects, an individual who is susceptible to a disease, disorder, and/or condition may not have been diagnosed with the disease, disorder, and/or condition. In some aspects, an individual who is susceptible to a disease, disorder, and/or condition may exhibit symptoms of the disease, disorder, and/or condition. In some aspects, an individual susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition. In some aspects, an individual susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some aspects, an individual susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.
術語「蛋白質」、「多肽」或「肽」在本文中用作同義詞且係指胺基酸單體之聚合物，例如包含至少兩個胺基酸殘基之分子。多肽可包括基因產物、天然存在之多肽、合成多肽、同源物、異種同源物、同種同源物、片段及前述者之其他等效物、變體及類似物。多肽可為單分子或可為多分子複合物，諸如二聚體、三聚體或四聚體。蛋白質包含一或多種肽或多肽，且可摺疊成蛋白質發揮其生物功能可能需要的3維形式。The terms "protein", "polypeptide" or "peptide" are used synonymously herein and refer to a polymer of amino acid monomers, such as a molecule comprising at least two amino acid residues. Polypeptides may include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, heterologs, homologs, fragments and other equivalents, variants and analogs of the foregoing. Polypeptides may be single molecules or may be multimolecular complexes, such as dimers, trimers or tetramers. Proteins contain one or more peptides or polypeptides and may fold into a 3-dimensional form that may be required for the protein to exert its biological function.
如本文所用，術語「野生型」或「WT」或「天然」係指在生物體中天然存在的分子之內源性形式。在一些態樣中，採用蛋白質或多肽之野生型形式，然而在本發明之其他態樣中，使用經修飾之蛋白質或多肽來產生免疫反應。上文所描述之術語可互換地使用。As used herein, the term "wild type" or "WT" or "native" refers to the endogenous form of a molecule that occurs naturally in an organism. In some aspects, the wild type form of a protein or polypeptide is used, whereas in other aspects of the invention, a modified protein or polypeptide is used to generate an immune response. The terms described above may be used interchangeably.
「經修飾之蛋白質」或「經修飾之多肽」或「變體」係指其化學結構，尤其其胺基酸序列相對於野生型蛋白質或多肽改變的蛋白質或多肽。在一些態樣中，經修飾之蛋白質/變異蛋白或多肽具有至少一種經修飾之活性或功能(認識到蛋白質或多肽可具有多種活性或功能)。尤其考慮，經修飾/變異蛋白或多肽可相對於一種活性或功能改變，但在其他態樣中保留野生型活性或功能，諸如免疫原性。當在本文中特定提及蛋白質時，其一般係指天然(野生型)或重組(經修飾)蛋白質。蛋白質可自天然的生物體直接分離，藉由重組DNA/外源性表現方法產生或藉由固相肽合成(solid-phase peptide synthesis；SPPS)或其他活體外方法產生。在特定態樣中，存在經分離核酸區段及重組載體，其併有編碼多肽(例如抗原或其片段)之核酸序列。術語「重組」可與多肽或特定多肽之名稱結合使用，且此一般係指由已經活體外操控之核酸分子產生的多肽或作為此類分子之複製產物的多肽。A "modified protein" or "modified polypeptide" or "variant" refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is altered relative to a wild-type protein or polypeptide. In some aspects, a modified protein/variant protein or polypeptide has at least one modified activity or function (recognizing that a protein or polypeptide may have multiple activities or functions). It is particularly contemplated that a modified/variant protein or polypeptide may be altered relative to one activity or function, but retain a wild-type activity or function in other aspects, such as immunogenicity. When a protein is specifically referred to herein, it is generally referred to as a natural (wild-type) or recombinant (modified) protein. Proteins can be isolated directly from natural organisms, produced by recombinant DNA/exogenous expression methods, or produced by solid-phase peptide synthesis (SPPS) or other in vitro methods. In certain aspects, there are isolated nucleic acid segments and recombinant vectors that combine a nucleic acid sequence encoding a polypeptide (e.g., an antigen or fragment thereof). The term "recombinant" may be used in conjunction with the name of a polypeptide or a specific polypeptide, and generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or a polypeptide that is a replication product of such a molecule.
參考胺基酸序列(肽或蛋白質)之術語「片段」係指胺基酸序列之一部分，亦即表示在N端及/或C端處縮短之胺基酸序列的序列。在C端處縮短之片段(N端片段)可例如藉由轉譯缺乏開讀框之3'端的截短開讀框而獲得。在N端處縮短之片段(C端片段)可例如藉由轉譯缺乏開讀框之5'端的截短開讀框而獲得，只要截短開讀框包含用以起始轉譯之起始密碼子即可。胺基酸序列之片段包含例如來自胺基酸序列之至少50%、至少60 %、至少70 %、至少80%、至少90%或至少99%之胺基酸殘基。在本發明中，多肽、DNA核酸或RNA核酸序列之片段係指與其所來源之多肽、DNA核酸或RNA核酸序列具有至少、至多、恰好以下或在以下中之任何兩者之間的序列一致性的序列：5%、10%、20%、30%、40%、50%、60%、70%、80%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%。The term "fragment" with reference to an amino acid sequence (peptide or protein) refers to a portion of the amino acid sequence, i.e., a sequence of an amino acid sequence shortened at the N-terminus and/or the C-terminus. A fragment shortened at the C-terminus (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame at the 3' end lacking an open reading frame. A fragment shortened at the N-terminus (C-terminal fragment) can be obtained, for example, by translating a truncated open reading frame at the 5' end lacking an open reading frame, as long as the truncated open reading frame includes a start codon for initiating translation. A fragment of an amino acid sequence includes, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% of the amino acid residues from the amino acid sequence. In the present invention, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having at least, at most, exactly less than, or between any two of the following sequence identities with the polypeptide, DNA nucleic acid or RNA nucleic acid sequence from which it is derived: 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
在一個態樣中，多肽、DNA核酸或RNA核酸序列之片段係指與其所來源之多肽、DNA核酸或RNA核酸序列具有至少70%之序列一致性的序列。在一個態樣中，多肽、DNA核酸或RNA核酸序列之片段係指與其所來源之多肽、DNA核酸或RNA核酸序列具有至少80%之序列一致性的序列。在一個態樣中，多肽、DNA核酸或RNA核酸序列之片段係指與其所來源之多肽、DNA核酸或RNA核酸序列具有至少85%之序列一致性的序列。在一個態樣中，多肽、DNA核酸或RNA核酸序列之片段係指與其所來源之多肽、DNA核酸或RNA核酸序列具有至少90%之序列一致性的序列。在一個態樣中，多肽、DNA核酸或RNA核酸序列之片段係指與其所來源之多肽、DNA核酸或RNA核酸序列具有至少95%之序列一致性的序列。在一個態樣中，多肽、DNA核酸或RNA核酸序列之片段係指與其所來源之多肽、DNA核酸或RNA核酸序列具有至少97%之序列一致性的序列。在一個態樣中，多肽、DNA核酸或RNA核酸序列之片段係指與其所來源之多肽、DNA核酸或RNA核酸序列具有至少99%之序列一致性的序列。In one aspect, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence that has at least 70% sequence identity with the polypeptide, DNA nucleic acid or RNA nucleic acid sequence from which it is derived. In one aspect, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence that has at least 80% sequence identity with the polypeptide, DNA nucleic acid or RNA nucleic acid sequence from which it is derived. In one aspect, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence that has at least 85% sequence identity with the polypeptide, DNA nucleic acid or RNA nucleic acid sequence from which it is derived. In one aspect, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence that has at least 90% sequence identity with the polypeptide, DNA nucleic acid or RNA nucleic acid sequence from which it is derived. In one aspect, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence that has at least 95% sequence identity with the polypeptide, DNA nucleic acid or RNA nucleic acid sequence from which it is derived. In one aspect, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having at least 97% sequence identity with the polypeptide, DNA nucleic acid or RNA nucleic acid sequence from which it is derived. In one aspect, a fragment of a polypeptide, DNA nucleic acid or RNA nucleic acid sequence refers to a sequence having at least 99% sequence identity with the polypeptide, DNA nucleic acid or RNA nucleic acid sequence from which it is derived.
如本文在分子(例如核酸、蛋白質或小分子)之上下文中所使用，術語「變體/變異(variant)」係指顯示與參考分子之顯著結構一致性但在結構上與該參考分子不同的分子，例如與參考實體相比在一或多個化學部分之存在或不存在方面或在一或多個化學部分之含量方面不同。在一些態樣中，變體亦在功能上與其參考分子不同。一般而言，特定分子是否恰當地視為參考分子之「變體」係基於其與參考分子之結構一致性的程度。如熟習此項技術者將瞭解，任何生物或化學參考分子均具有某些特徵結構元件。按照定義，變體為與參考分子共有一或多個此類特徵結構元件但在至少一個態樣上與參考分子不同的不同分子。在一些態樣中，由於胺基酸或核苷酸序列中之一或多個差異及/或作為多肽或核酸之共價組分(例如與多肽或核酸主鏈連接)之化學部分(例如碳水化合物、脂質、磷酸酯基團)中之一或多個差異，變異多肽或核酸可能與參考多肽或核酸不同。在一些態樣中，變異多肽或核酸與參考多肽或核酸顯示至少、至多、恰好以下或在以下中之任何兩者之間的總體序列一致性：85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%或99%。在一些態樣中，變異多肽或核酸不與參考多肽或核酸共有至少一個特徵序列元件。在一些態樣中，參考多肽或核酸具有一或多種生物活性。在一些態樣中，變異多肽或核酸共有參考多肽或核酸之生物活性中之一或多者。在一些態樣中，變異多肽或核酸缺乏參考多肽或核酸之生物活性中之一或多者。在一些態樣中，變異多肽或核酸與參考多肽或核酸相比顯示一或多種生物活性水平之降低。在一些態樣中，若所關注多肽或核酸之胺基酸或核苷酸序列與參考多肽或核酸之胺基酸或核苷酸序列一致但具有特定位置處之少數序列改變，則將其視為參考多肽或核酸之「變體」。較佳地，變異多肽或核酸序列與參考多肽或核酸序列相比具有至少一個修飾，例如1至約20個修飾。在一個態樣中，變異多肽或核酸序列與參考多肽或核酸序列相比具有1至約10個修飾。在一個態樣中，變異多肽或核酸序列與參考多肽或核酸序列相比具有1至約5個修飾。在一個態樣中，變異多肽或核酸序列與參考多肽或核酸序列相比具有1至約4個修飾。通常，與參考相比，變體中少於約20%、約15%、約10%、約9%、約8%、約7%、約6%、約5%、約4%、約3%或約2%之殘基經取代、插入或缺失。通常，相對於參考，變異多肽或核酸包含極少數目個(例如少於約5、約4、約3、約2或約1個)經取代、插入或缺失之功能性殘基(例如參與特定生物活性之殘基)。在一些態樣中，與參考相比，變異多肽或核酸包含約10、約9、約8、約7、約6、約5、約4、約3、約2或約1個經取代之殘基。在一些態樣中，與參考相比，變異多肽或核酸包含少於約25、約20、約19、約18、約17、約16、約15、約14、約13、約10、約9、約8、約7、約6個且通常少於約5、約4、約3或約2個添加或缺失。在一些態樣中，與參考相比，變異多肽或核酸包含不超過約5、約4、約3、約2或約1個添加或缺失，且在一些態樣中，不包含添加或缺失。As used herein in the context of molecules (e.g., nucleic acids, proteins, or small molecules), the term "variant" refers to a molecule that exhibits significant structural identity with a reference molecule but is structurally different from the reference molecule, for example, in the presence or absence of one or more chemical moieties or in the amount of one or more chemical moieties compared to the reference entity. In some aspects, a variant is also functionally different from its reference molecule. In general, whether a particular molecule is properly considered a "variant" of a reference molecule is based on the degree of its structural identity with the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. By definition, a variant is a different molecule that shares one or more of these characteristic structural elements with a reference molecule but differs from the reference molecule in at least one aspect. In some aspects, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid by one or more differences in the amino acid or nucleotide sequence and/or one or more differences in the chemical moiety (e.g., carbohydrate, lipid, phosphate group) that is a covalent component of the polypeptide or nucleic acid (e.g., linked to the polypeptide or nucleic acid backbone). In some aspects, a variant polypeptide or nucleic acid exhibits an overall sequence identity of at least, at most, just below, or between any two of the following: 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% with a reference polypeptide or nucleic acid. In some aspects, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some aspects, a reference polypeptide or nucleic acid has one or more biological activities. In some aspects, the variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid. In some aspects, the variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some aspects, the variant polypeptide or nucleic acid shows a reduction in the level of one or more biological activities compared to the reference polypeptide or nucleic acid. In some aspects, if the amino acid or nucleotide sequence of the polypeptide or nucleic acid of interest is consistent with the amino acid or nucleotide sequence of the reference polypeptide or nucleic acid but has a few sequence changes at a specific position, it is considered to be a "variant" of the reference polypeptide or nucleic acid. Preferably, the variant polypeptide or nucleic acid sequence has at least one modification, such as 1 to about 20 modifications, compared to the reference polypeptide or nucleic acid sequence. In one aspect, the variant polypeptide or nucleic acid sequence has 1 to about 10 modifications compared to the reference polypeptide or nucleic acid sequence. In one aspect, the variant polypeptide or nucleic acid sequence has 1 to about 5 modifications compared to a reference polypeptide or nucleic acid sequence. In one aspect, the variant polypeptide or nucleic acid sequence has 1 to about 4 modifications compared to a reference polypeptide or nucleic acid sequence. Typically, less than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in the variant are substituted, inserted, or deleted compared to the reference. Typically, the variant polypeptide or nucleic acid comprises a very small number (e.g., less than about 5, about 4, about 3, about 2, or about 1) of substituted, inserted, or deleted functional residues (e.g., residues involved in a specific biological activity) relative to the reference. In some aspects, the variant polypeptide or nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residue compared to the reference. In some aspects, the variant polypeptide or nucleic acid comprises less than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and usually less than about 5, about 4, about 3, or about 2 additions or deletions compared to the reference. In some aspects, the variant polypeptide or nucleic acid comprises no more than about 5, about 4, about 3, about 2, or about 1 addition or deletion compared to the reference, and in some aspects, comprises no additions or deletions.
在一些態樣中，參考多肽或核酸為自然界中存在之「野生型」或「WT」或「天然」序列，包括對偶基因變異。野生型多肽或核酸序列具有未經有意修飾之序列。出於本發明之目的，胺基酸序列(肽、蛋白質或多肽)之「變體」包含胺基酸插入變體、胺基酸添加變體、胺基酸缺失變體及/或胺基酸取代變體。核苷酸序列之「變體」包含核苷酸插入變體、核苷酸添加變體、核苷酸缺失變體及/或核苷酸取代變體。術語「變體」包括所有突變體、剪接變體、轉譯後修飾變體、構形、同功異型物、對偶基因變體、物種變體及物種同源物，特定言之，天然存在之上述物質。術語「變體」尤其包括胺基酸或核酸序列之片段。In some aspects, the reference polypeptide or nucleic acid is a "wild-type" or "WT" or "native" sequence found in nature, including allelic variants. A wild-type polypeptide or nucleic acid sequence has a sequence that has not been intentionally modified. For the purposes of the present invention, a "variant" of an amino acid sequence (peptide, protein or polypeptide) includes an amino acid insertion variant, an amino acid addition variant, an amino acid deletion variant and/or an amino acid substitution variant. A "variant" of a nucleotide sequence includes a nucleotide insertion variant, a nucleotide addition variant, a nucleotide deletion variant and/or a nucleotide substitution variant. The term "variant" includes all mutants, splice variants, post-translational modification variants, conformations, isoforms, allelic variants, species variants and species homologs, specifically, the above substances that occur in nature. The term "variant" particularly includes fragments of an amino acid or nucleic acid sequence.
可藉由突變向核酸中引入改變，從而引起該核酸所編碼之多肽(例如抗原或抗體或抗體衍生物)之胺基酸序列的改變。突變可使用此項技術中已知的任何技術引入。在一個態樣中，使用例如定點突變誘發方案來改變一或多個特定胺基酸殘基。在另一態樣中，使用例如隨機突變誘發方案來改變一或多個隨機選擇之殘基。在一些態樣中，無論以何種方式進行，可針對所需特性來表現及篩選突變多肽。Changes can be introduced into nucleic acids by mutations, thereby causing changes in the amino acid sequence of a polypeptide (e.g., an antigen or an antibody or an antibody derivative) encoded by the nucleic acid. Mutations can be introduced using any technique known in the art. In one aspect, one or more specific amino acid residues are changed using, for example, a site-directed mutagenesis induction scheme. In another aspect, one or more randomly selected residues are changed using, for example, a random mutagenesis induction scheme. In some aspects, no matter how it is performed, mutant polypeptides can be expressed and screened for desired properties.
可在不顯著改變核酸所編碼之多肽的生物活性的情況下將突變引入核酸中。舉例而言，吾人可進行在非必需胺基酸殘基處引起胺基酸取代的核苷酸取代。或者，可將選擇性改變核酸所編碼之多肽的生物活性的一或多個突變引入核酸中。舉例而言，突變可定量或定性地改變生物活性。定量改變之實例包括增加、降低或消除活性。定性改變之實例包括改變抗體之抗原特異性。Mutations can be introduced into a nucleic acid without significantly altering the biological activity of the polypeptide encoded by the nucleic acid. For example, one can perform nucleotide substitutions that result in amino acid substitutions at non-essential amino acid residues. Alternatively, one or more mutations that selectively alter the biological activity of a polypeptide encoded by a nucleic acid can be introduced into a nucleic acid. For example, mutations can quantitatively or qualitatively alter biological activity. Examples of quantitative alterations include increasing, decreasing, or eliminating activity. Examples of qualitative alterations include altering the antigenic specificity of an antibody.
「序列相似性」指示一致的或表示保守胺基酸取代之胺基酸之百分比。兩個胺基酸序列之間的「序列一致性」指示序列之間一致的胺基酸之百分比。兩個核酸序列之間的「序列一致性」指示序列之間一致的核苷酸之百分比。"Sequence similarity" indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences indicates the percentage of nucleotides that are identical between the sequences.
術語「一致% (% identical)」、「一致性% (% identity)」或類似術語意欲尤其指待比較之序列之間最佳比對中一致之核苷酸或胺基酸的百分比。該百分比純粹為統計的，且兩個序列之間的差異可能但未必隨機分佈於所比較之序列的整個長度上。兩個序列之比較通常藉由在最佳比對之後相對於比較片段或「窗口」比較序列來進行，以便鑑別相應序列之局部區域。用於比較之最佳比對可手動地或藉助於Smith及Waterman, 1981, Ads App. Math. 2, 482之局部同源性演算法、藉助於Neddleman及Wunsch, 1970, J. Mol. Biol. 48, 443之局部同源性演算法、藉助於Pearson及Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444之相似性搜尋演算法、或藉助於使用該等演算法之電腦程式(在Wisconsin遺傳學套裝軟體中之GAP、BESTFIT、FASTA、BLAST P、BLAST N及TFASTA，Genetics Computer Group)進行。在一些態樣中，使用可在United States National Center for Biotechnology Information (NCBI)網站獲得的BLASTN或BLASTP演算法測定兩個序列之一致性百分比。The terms "% identical", "% identity" or similar terms are intended to refer in particular to the percentage of identical nucleotides or amino acids in an optimal alignment between the sequences being compared. This percentage is purely statistical, and the differences between the two sequences are likely but not necessarily randomly distributed over the entire length of the sequences being compared. Comparison of two sequences is usually performed by comparing the sequences over comparison segments or "windows" after optimal alignment in order to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually or by means of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by means of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by means of the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or by means of computer programs that use such algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Suite, Genetics Computer Group). In some aspects, the percent identity between two sequences is determined using the BLASTN or BLASTP algorithm available at the United States National Center for Biotechnology Information (NCBI) website.
一致性百分比係藉由測定所比較之序列中之對應的相同位置之數目，將此數值除以所比較之位置數(例如，參考序列中之位置數)且將此結果乘以100來獲得。The percent identity is calculated by determining the number of corresponding identical positions in the compared sequences, dividing this number by the number of compared positions (eg, the number of positions in the reference sequence), and multiplying the result by 100.
在一些態樣中，針對參考序列之整個長度之至少、至多、恰好以下或在以下中之任何兩者之間的區域給出相似性或一致性程度：約50%、約60%、約70%、約80%、約90%或約100%。舉例而言，若參考核酸序列由200個核苷酸組成，則針對至少、至多、恰好以下或在以下中之任何兩者之間的核苷酸(在一些態樣中，連續核苷酸)給出一致性程度：約100、約120、約140、約160、約180或約200個。在一些態樣中，針對參考序列之整個長度給出相似性或一致性程度。In some aspects, similarity or degree of identity is given for at least, at most, just below or in any two of the whole length of the reference sequence: about 50%, about 60%, about 70%, about 80%, about 90% or about 100%. For example, if the reference nucleic acid sequence is composed of 200 nucleotides, then the degree of identity is given for at least, at most, just below or in any two of the nucleotides (in some aspects, continuous nucleotides) between: about 100, about 120, about 140, about 160, about 180 or about 200. In some aspects, similarity or degree of identity is given for the whole length of the reference sequence.
同源胺基酸序列可展現至少、至多、恰好以下或在以下中之任何兩者之間的胺基酸殘基之一致性：40%、50%、60%、70%、80%、90%、95%、98%或99%。在一個態樣中，同源胺基酸序列展現胺基酸殘基之至少95%一致性。在一個態樣中，同源胺基酸序列展現胺基酸殘基之至少98%一致性。在一個態樣中，同源胺基酸序列展現胺基酸殘基之至少99%一致性。Homologous amino acid sequences can show at least, at most, just below or the identity of any two of the following amino acid residues: 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%. In one aspect, homologous amino acid sequences show at least 95% identity of amino acid residues. In one aspect, homologous amino acid sequences show at least 98% identity of amino acid residues. In one aspect, homologous amino acid sequences show at least 99% identity of amino acid residues.
胺基酸序列(肽或蛋白質)之片段或變體可為「功能片段」或「功能變體」。術語胺基酸序列之「功能片段」或「功能變體」係指展現出與其所來源之胺基酸序列相同或類似之一或多種功能特性的任何片段或變體，例如其為功能上等效的。關於抗原或抗原序列，一種特定功能為由片段或變體所來源之胺基酸序列呈現的一或多種免疫原性活性。如本文所用，術語「功能片段」或「功能變體」尤其係指包含胺基酸序列之變體分子或序列，該胺基酸序列與親體分子或序列相比具有一或多個胺基酸之變化且仍能夠實現親體分子或序列之一或多種功能，例如誘導免疫反應。在一個態樣中，親體分子或序列之胺基酸序列中之修飾未顯著影響或改變該分子或序列之特徵。野生型RSV F蛋白之術語「突變體」、RSV F蛋白之「突變體」、「RSV F蛋白突變體」或「經修飾之RSV F蛋白」係指相對於野生型F蛋白呈現所引入突變且針對野生型F蛋白具有免疫原性的多肽。A fragment or variant of an amino acid sequence (peptide or protein) may be a "functional fragment" or "functional variant". The term "functional fragment" or "functional variant" of an amino acid sequence refers to any fragment or variant that exhibits one or more functional properties that are the same or similar to the amino acid sequence from which it is derived, for example, it is functionally equivalent. With respect to antigens or antigenic sequences, a specific function is one or more immunogenic activities exhibited by the amino acid sequence from which the fragment or variant is derived. As used herein, the term "functional fragment" or "functional variant" refers in particular to a variant molecule or sequence comprising an amino acid sequence that has one or more amino acid changes compared to the parent molecule or sequence and is still able to achieve one or more functions of the parent molecule or sequence, such as inducing an immune response. In one aspect, the modification in the amino acid sequence of the parent molecule or sequence does not significantly affect or change the characteristics of the molecule or sequence. The terms "mutant" of wild-type RSV F protein, "mutant" of RSV F protein, "RSV F protein mutant," or "modified RSV F protein" refer to a polypeptide that exhibits the introduced mutations relative to the wild-type F protein and is immunogenic against the wild-type F protein.
「來源於」指定胺基酸序列(肽、蛋白質或多肽)之胺基酸序列(肽、蛋白質或多肽)係指第一胺基酸序列之來源。較佳地，來源於特定胺基酸序列之胺基酸序列具有與該特定序列或其片段一致、基本上一致或同源的胺基酸序列。來源於特定胺基酸序列之胺基酸序列可為該特定序列或其片段之變體。舉例而言，一般熟習此項技術者應理解，可改變適用於本文中之抗原，使得其在序列上與其所來源之天然存在之序列或天然序列不同，同時保留天然序列之所需活性。An amino acid sequence (peptide, protein or polypeptide) "derived from" a specified amino acid sequence (peptide, protein or polypeptide) refers to the source of the first amino acid sequence. Preferably, the amino acid sequence derived from a specific amino acid sequence has an amino acid sequence that is consistent, substantially consistent or homologous to the specific sequence or a fragment thereof. The amino acid sequence derived from a specific amino acid sequence may be a variant of the specific sequence or a fragment thereof. For example, it will be understood by those skilled in the art that an antigen applicable to the present invention may be altered so that it differs in sequence from the naturally occurring sequence or native sequence from which it is derived while retaining the desired activity of the native sequence.
在本發明中，載體係指核酸分子，諸如人工核酸分子。載體可用於併入核酸序列，諸如包含開讀框之核酸序列。載體包括但不限於儲存載體、表現載體、選殖載體、轉移載體。載體可為RNA載體或DNA載體。在一些態樣中，載體為DNA分子。在一些態樣中，載體為質體載體。在一些態樣中，載體為病毒載體。通常，表現載體將含有所需編碼序列及在特定宿主生物體(例如細菌、酵母、植物、昆蟲或哺乳動物)中或在活體外表現系統中表現可操作地連接之編碼序列所必需的適當其他序列。選殖載體通常用以工程改造及擴增某一所需片段(通常DNA片段)，且可缺乏表現一或多種所需片段所需要的功能性序列。In the present invention, a vector refers to a nucleic acid molecule, such as an artificial nucleic acid molecule. A vector can be used to incorporate a nucleic acid sequence, such as a nucleic acid sequence containing an open reading frame. Vectors include, but are not limited to, storage vectors, expression vectors, cloning vectors, and transfer vectors. A vector can be an RNA vector or a DNA vector. In some aspects, the vector is a DNA molecule. In some aspects, the vector is a plasmid vector. In some aspects, the vector is a viral vector. Typically, an expression vector will contain the desired coding sequence and appropriate other sequences necessary for expressing an operably linked coding sequence in a specific host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are typically used to engineer and amplify a desired fragment (usually a DNA fragment) and may lack functional sequences required to express one or more desired fragments.
如本文所用，術語「醫藥組合物」係指與一或多種醫藥學上可接受之載劑一起調配之活性劑。醫藥組合物可為免疫原性組合物。在一些態樣中，活性劑以適合投與之單位劑量的量存在於治療方案中，其在向相關群體投與時顯示出統計學上顯著之達成預定治療作用的機率。在一些態樣中，醫藥組合物可專門調配用於非經腸投與，以例如無菌溶液或懸浮液、或持續釋放調配物形式，例如藉由皮下、肌肉內、靜脈內或硬膜外注射。As used herein, the term "pharmaceutical composition" refers to an active agent formulated with one or more pharmaceutically acceptable carriers. The pharmaceutical composition may be an immunogenic composition. In some aspects, the active agent is present in a treatment regimen in an amount of a unit dose suitable for administration, which shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some aspects, the pharmaceutical composition may be specifically formulated for parenteral administration, such as in the form of a sterile solution or suspension, or a sustained release formulation, such as by subcutaneous, intramuscular, intravenous or epidural injection.
如本文所用，術語「疫苗接種」係指投與意欲產生免疫反應之免疫原性組合物，例如疾病相關(例如致病)藥劑(例如病毒)。在一些態樣中，疫苗接種可在暴露於疾病相關藥劑之前、期間及/或之後投與，且在某些態樣中，在暴露於該藥劑之前、期間及/或之後不久投與。在一些態樣中，疫苗接種包括在時間上適當間隔開地多次投與疫苗組合物。在一些態樣中，疫苗接種產生針對傳染原之免疫反應。在一些態樣中，疫苗接種產生針對腫瘤之免疫反應；在一些此類態樣中，疫苗接種為「個人化」的，因為其部分或完全針對經測定在特定個體之腫瘤中存在的一或多種抗原決定基(例如其可為或包括一或多種新抗原決定基)。As used herein, the term "vaccination" refers to the administration of an immunogenic composition intended to produce an immune response, such as a disease-related (e.g., pathogenic) agent (e.g., a virus). In some aspects, vaccination can be administered before, during, and/or after exposure to a disease-related agent, and in some aspects, before, during, and/or shortly after exposure to the agent. In some aspects, vaccination includes multiple administrations of a vaccine composition appropriately spaced apart in time. In some aspects, vaccination produces an immune response against an infectious agent. In some aspects, vaccination produces an immune response against a tumor; in some such aspects, vaccination is "personalized" in that it is directed in part or in whole to one or more antigenic determinants determined to be present in a tumor of a particular individual (e.g., it may be or include one or more new antigenic determinants).
免疫反應係指生物體中之體液反應、細胞反應或體液反應及細胞反應兩者。免疫反應可藉由包括但不限於以下之分析量測：量測特異性識別蛋白質或細胞表面蛋白之抗體之存在或量的分析、量測T細胞活化或增殖的分析、及/或量測關於一或多種細胞介素之活性或表現之調節的分析。An immune response refers to a humoral response, a cellular response, or both a humoral response and a cellular response in an organism. An immune response can be measured by assays including, but not limited to, assays that measure the presence or amount of antibodies that specifically recognize a protein or cell surface protein, assays that measure T cell activation or proliferation, and/or assays that measure the modulation of the activity or expression of one or more cytokines.
如本文所用，術語「組合療法」係指個體同時暴露於兩種或更多種治療方案(例如兩種或更多種治療劑)之彼等情況。在一些態樣中，兩種或更多種方案可同時投與；在一些態樣中，此類方案可依序投與(例如第一方案之全部「劑量」在投與第二方案之任何劑量之前投與)；在一些態樣中，此類藥劑以重疊給藥方案投與。在一些態樣中，組合療法之「投與」可涉及向接受呈組合形式之一或多種其他藥劑或一或多種模態(modality)之個體投與一或多種藥劑或一或多種模態。出於清楚起見，組合療法不需要個別藥劑以單一組合物形式一起投與(或甚至必須在同時)，但在一些態樣中，兩種或更多種藥劑或其活性部分可以組合組合物形式或甚至以組合化合物形式(例如作為單一化學複合物或共價實體之一部分)一起投與。As used herein, the term "combination therapy" refers to those situations in which an individual is exposed to two or more treatment regimens (e.g., two or more therapeutic agents) simultaneously. In some aspects, the two or more regimens may be administered simultaneously; in some aspects, such regimens may be administered sequentially (e.g., all "doses" of the first regimen are administered before any doses of the second regimen are administered); in some aspects, such agents are administered in overlapping dosing regimens. In some aspects, "administration" of a combination therapy may involve administering one or more agents or one or more modalities to an individual who receives one or more other agents or one or more modalities in combination. For clarity, combination therapy does not require that the individual agents be administered together in a single composition (or even necessarily at the same time), but in some aspects, two or more agents or their active moieties may be administered together in a combined composition or even in a combined compound (e.g., as part of a single chemical complex or covalent entity).
熟習此項技術者將瞭解，術語「給藥方案」可用以係指個別地向個體投與之一組單位劑量(通常超過一個)，其通常間隔一段時間。在一些態樣中，給定治療劑具有推薦的給藥方案，其可涉及一或多次劑量。在一些態樣中，給藥方案包含複數個劑量，該等劑量中之各者在時間上與其他劑量分隔開。在一些態樣中，個別劑量彼此間隔長度相同之時段；在一些態樣中，給藥方案包含複數個劑量及分隔開個別劑量之至少兩個不同時段。在一些態樣中，給藥方案內之所有劑量均具有相同單位劑量的量。在一些態樣中，給藥方案內之不同劑量具有不同的量。在一些態樣中，給藥方案包含第一劑量的量之第一劑量，接著為不同於第一劑量的量之第二劑量的量的一或多次額外劑量。在一些態樣中，給藥方案包含第一劑量的量之第一劑量，接著為與第一劑量的量相同之第二劑量的量的一或多次額外劑量。在一些態樣中，給藥方案在跨越相關群體投與時與所需或有益結果相關(例如為治療性給藥方案)。Those skilled in the art will appreciate that the term "dosing regimen" may be used to refer to a set of unit doses (usually more than one) that are administered individually to an individual, usually at intervals of time. In some aspects, a given therapeutic agent has a recommended dosing regimen that may involve one or more doses. In some aspects, the dosing regimen comprises a plurality of doses, each of which is separated in time from the other doses. In some aspects, the individual doses are separated from each other by time periods of the same length; in some aspects, the dosing regimen comprises a plurality of doses and at least two different time periods separating the individual doses. In some aspects, all doses within a dosing regimen have the same unit dose amount. In some aspects, different doses within a dosing regimen have different amounts. In some aspects, the dosing regimen comprises a first dose of the amount of the first dose, followed by one or more additional doses of a second dose of the amount different from the amount of the first dose. In some aspects, the dosing regimen comprises a first dose of the amount of the first dose, followed by one or more additional doses of the second dose of the amount the same as the amount of the first dose. In some aspects, the dosing regimen is associated with a desired or beneficial outcome when administered across a relevant population (e.g., a therapeutic dosing regimen).
II.呼吸道融合病毒(RSV)本發明提供RNA分子(例如RNA聚核苷酸)，其包含至少一個編碼呼吸道融合病毒(RSV)多肽之開讀框。本發明進一步提供一種免疫原性組合物，其包含至少一種編碼RSV多肽之RNA分子，該RNA分子與一或多種脂質複合、囊封於其中或用其調配且形成脂質奈米顆粒(LNP)。本文所揭示之免疫原性組合物中所包括之RSV多肽可為呈融合前構形之任何RSV F蛋白。II.Respiratory syncytial virus(RSV) The present invention provides RNA molecules (e.g., RNA polynucleotides) comprising at least one open reading frame encoding a respiratory syncytial virus (RSV) polypeptide. The present invention further provides an immunogenic composition comprising at least one RNA molecule encoding a RSV polypeptide, the RNA molecule being complexed with, encapsulated in, or formulated with one or more lipids and forming lipid nanoparticles (LNPs). The RSV polypeptide included in the immunogenic compositions disclosed herein can be any RSV F protein in a pre-fusion conformation.
術語「融合前構形」係指(i)當RSV F蛋白或突變體呈單體或三聚體形式時可由抗體D25或AM22特異性結合，或(ii)當RSV F蛋白突變體呈三聚體形式時由抗體AM14特異性結合之RSV F蛋白或其突變體所採用的結構性構形。融合前三聚體構形為融合前構形之子集。如本文所用，呈融合前構形之RSV F蛋白或多肽或其突變體可表示為「RSV preF」。The term "prefusion conformation" refers to the structural conformation adopted by the RSV F protein or its mutant that can be specifically bound by antibody D25 or AM22 (i) when the RSV F protein or mutant is in monomeric or trimer form, or (ii) when the RSV F protein mutant is in trimer form by antibody AM14. The prefusion trimer conformation is a subset of the prefusion conformation. As used herein, the RSV F protein or polypeptide or its mutant in the prefusion conformation can be expressed as "RSV preF".
術語「融合後構形」係指不由D25、AM22或AM14特異性結合之RSV F蛋白所採用的結構性構形。天然F蛋白在病毒套膜與宿主細胞膜融合之後採用融合後構形。當自膜萃取時，當表現為胞外域時，或在儲存後，RSV F蛋白亦可在融合事件之情形之外呈現融合後構形，例如在脅迫條件(諸如加熱及低滲透壓)下。術語「AM14」係指WO 2008/147196 A2 (其以全文引用之方式併入本文中)中所描述之抗體。術語「AM22」係指WO 2011/043643 A1 (其以全文引用之方式併入本文中)中所描述之抗體。術語「D25」係指WO 2008/147196 A2 (其在此以全文引用之方式併入本文中)中所描述之抗體。The term "post-fusion conformation" refers to the structural conformation adopted by the RSV F protein that is not specifically bound by D25, AM22 or AM14. The native F protein adopts the post-fusion conformation after the fusion of the viral envelope and the host cell membrane. The RSV F protein can also present the post-fusion conformation outside the context of a fusion event when extracted from the membrane, when expressed as an extracellular domain, or after storage, for example under stress conditions such as heat and hypoosmotic pressure. The term "AM14" refers to the antibodies described in WO 2008/147196 A2 (which is incorporated herein by reference in its entirety). The term "AM22" refers to the antibodies described in WO 2011/043643 A1 (which is incorporated herein by reference in its entirety). The term "D25" refers to the antibody described in WO 2008/147196 A2, which is hereby incorporated by reference in its entirety.
在一些實施例中，RSV F蛋白為子類型A之RSV F蛋白。在一些實施例中，RSV F蛋白為子類型B之RSV F蛋白。如本文所用，術語「子類型」及「子群組」可互換地使用。如本文所用，術語「株系(strain)」係指各子類型或子群組內之特定分離株。在一些實施例中，RSV F蛋白為野生型RSV F蛋白之突變體。在一些實施例中，RSV F蛋白為子類型A之野生型RSV F蛋白之突變體。在一些實施例中，RSV F蛋白為子類型B之野生型RSV F蛋白之突變體。在一些實施例中，突變體相對於對應野生型RSV F蛋白之胺基酸序列在胺基酸序列中呈現所引入之突變，且針對呈融合前構形之野生型RSV F蛋白或針對包含野生型F蛋白之病毒具有免疫原性。相對於野生型RSV F蛋白，突變體中之胺基酸突變包括胺基酸取代、缺失或添加。In some embodiments, the RSV F protein is a RSV F protein of subtype A. In some embodiments, the RSV F protein is a RSV F protein of subtype B. As used herein, the terms "subtype" and "subgroup" are used interchangeably. As used herein, the term "strain" refers to a specific isolate within each subtype or subgroup. In some embodiments, the RSV F protein is a mutant of a wild-type RSV F protein. In some embodiments, the RSV F protein is a mutant of a wild-type RSV F protein of subtype A. In some embodiments, the RSV F protein is a mutant of a wild-type RSV F protein of subtype B. In some embodiments, the mutant exhibits an introduced mutation in the amino acid sequence relative to the amino acid sequence corresponding to the wild-type RSV F protein and is immunogenic against the wild-type RSV F protein in a prefusion conformation or against a virus comprising the wild-type F protein. The amino acid mutation in the mutant comprises an amino acid substitution, deletion, or addition relative to the wild-type RSV F protein.
在一些實施例中，RSV F蛋白為WO2017/109629 (其以全文引用之方式併入本文中)中所描述之RSV蛋白突變體。In some embodiments, the RSV F protein is a RSV protein mutant described in WO2017/109629, which is incorporated herein by reference in its entirety.
在一些實施例中，RSV F蛋白為野生型RSV F蛋白之突變體，其中所引入胺基酸突變為野生型RSV F蛋白中之一對胺基酸殘基突變為一對半胱胺酸(「經工程改造之雙硫鍵突變」)。所引入半胱胺酸殘基對允許在半胱胺酸殘基之間形成雙硫鍵，其使蛋白質之構形或寡聚狀態(諸如融合前構形)穩定。特定的此類突變對之實例包括：55C及188C；155C及290C；103C及148C；及142C及371C，諸如S55C及L188C；S155C及S290C；A103C及I148C；及L142C及N371C。In some embodiments, the RSV F protein is a mutant of a wild-type RSV F protein, wherein the introduced amino acid mutation is a mutation of a pair of amino acid residues in the wild-type RSV F protein to a pair of cysteines ("engineered disulfide mutations"). The introduced cysteine residue pair allows for the formation of disulfide bonds between the cysteine residues, which stabilizes the conformation or oligomeric state (such as the pre-fusion conformation) of the protein. Examples of specific such mutation pairs include: 55C and 188C; 155C and 290C; 103C and 148C; and 142C and 371C, such as S55C and L188C; S155C and S290C; A103C and I148C; and L142C and N371C.
在另其他實施例中，RSV F蛋白突變體包含作為一或多種空腔填充突變之胺基酸突變。可經空腔填充之目標置換之胺基酸之實例包括小脂族(例如Gly、Ala及Val)或小極性胺基酸(例如Ser及Thr)，以及融合前構形中埋入但融合後構形中暴露於溶劑的胺基酸。置換胺基酸之實例包括較大脂族胺基酸(Ile、Leu及Met)或較大芳族胺基酸(His、Phe、Tyr及Trp)。在一些特定實施例中，RSV F蛋白突變體包含選自由以下組成之群的空腔填充突變： (1)在位置55、62、155、190或290之S經I、Y、L、H或M取代； (2)在位置54、58、189、219或397之T經I、Y、L、H或M取代； (3)在位置151之G經A或H取代； (4)在位置147或298之A經I、L、H或M取代； (5)在位置164、187、192、207、220、296、300或495之V經I、Y、H取代；及 (6)在位置106之R經W取代。In yet other embodiments, RSV F protein mutants include amino acid mutations as one or more cavity-filling mutations. Examples of amino acids that can be targeted for cavity filling include small aliphatic (e.g., Gly, Ala, and Val) or small polar amino acids (e.g., Ser and Thr), and amino acids that are buried in the pre-fusion conformation but exposed to solvent in the post-fusion conformation. Examples of replaced amino acids include larger aliphatic amino acids (Ile, Leu, and Met) or larger aromatic amino acids (His, Phe, Tyr, and Trp). In some specific embodiments, the RSV F protein mutant comprises a cavity-filling mutation selected from the group consisting of:(1) S at position 55, 62, 155, 190 or 290 is substituted with I, Y, L, H or M;(2) T at position 54, 58, 189, 219 or 397 is substituted with I, Y, L, H or M;(3) G at position 151 is substituted with A or H;(4) A at position 147 or 298 is substituted with I, L, H or M;(5) V at position 164, 187, 192, 207, 220, 296, 300 or 495 is substituted with I, Y, H; and(6) R at position 106 is substituted with W.
在一些特定實施例中，RSV F蛋白突變體包含至少一個選自由以下組成之群的空腔填充突變：T54H、S190I及V296I。In some specific embodiments, the RSV F protein mutant comprises at least one cavity-filling mutation selected from the group consisting of: T54H, S190I, and V296I.
在另其他實施例中，RSV F蛋白突變體包含靜電突變，其降低摺疊結構中彼此接近之蛋白質中之殘基之間的離子排斥或增加其間的離子吸引。在若干實施例中，RSV F蛋白突變體包括降低與來自RSV F三聚體之另一原聚體之Glu487及Asp489之酸性殘基的排斥性離子相互作用或增加與其之吸引性離子相互作用的靜電取代。在一些特定實施例中，RSV F蛋白突變體包含選自由以下組成之群之靜電突變： (1)在位置82、92或487之E經D、F、Q、T、S、L或H取代； (2)在位置315、394或399之K經F、M、R、S、L、I、Q或T取代； (3)在位置392、486或489之D經H、S、N、T或P取代；及 (4)在位置106或339之R經F、Q、N或W取代。In yet other embodiments, the RSV F protein mutants comprise electrostatic mutations that reduce ionic repulsion or increase ionic attraction between residues in the protein that are close to each other in the folded structure. In several embodiments, the RSV F protein mutants include electrostatic substitutions that reduce repulsive ionic interactions with or increase attractive ionic interactions with acidic residues of Glu487 and Asp489 from another protomer of the RSV F trimer. In some specific embodiments, the RSV F protein mutant comprises an electrostatic mutation selected from the group consisting of:(1) E at position 82, 92 or 487 is substituted with D, F, Q, T, S, L or H;(2) K at position 315, 394 or 399 is substituted with F, M, R, S, L, I, Q or T;(3) D at position 392, 486 or 489 is substituted with H, S, N, T or P; and(4) R at position 106 or 339 is substituted with F, Q, N or W.
在另其他實施例中，RSV F蛋白突變體包含選自以下之兩種或更多種不同類型之突變的組合：經工程改造之雙硫鍵突變、空腔填充突變及靜電突變。在一些特定實施例中，RSV F蛋白突變體包含相對於對應野生型RSV F蛋白之突變之組合，其中突變之組合選自由以下組成之群：： (1) A103C、I148C、S190I及D486S之組合； (2) T54H、S55C、L188C、D486S之組合； (3) T54H、A103C、I148C、S190I、V296I及D486S之組合； (4) T54H、S55C、L142C、L188C、V296I及N371C之組合； (5) S55C、L188C及D486S之組合； (6) T54H、S55C、L188C及S190I之組合； (7) S55C、L188C、S190I及D486S之組合； (8) T54H、S55C、L188C、S190I及D486S之組合； (9) S155C、S190I、S290C及D486S之組合； (10) T54H、S55C、L142C、L188C、V296I、N371C、D486S、E487Q及D489S之組合； (11) T54H、S155C、S190I、S290C及V296I之組合；及 (12) S155C、S190F、S290C及V207L之組合。In yet other embodiments, the RSV F protein mutant comprises a combination of two or more different types of mutations selected from: engineered disulfide bond mutations, cavity-filling mutations, and electrostatic mutations. In some specific embodiments, the RSV F protein mutant comprises a combination of mutations relative to a corresponding wild-type RSV F protein, wherein the combination of mutations is selected from the group consisting of:(1) A103C, I148C, S190I and D486S;(2) T54H, S55C, L188C, D486S;(3) T54H, A103C, I148C, S190I, V296I and D486S;(4) T54H, S55C, L142C, L188C, V296I and N371C;(5) S55C, L188C and D486S;(6) T54H, S55C, L188C and S190I;(7) The combination of S55C, L188C, S190I and D486S;(8) The combination of T54H, S55C, L188C, S190I and D486S;(9) The combination of S155C, S190I, S290C and D486S;(10) The combination of T54H, S55C, L142C, L188C, V296I, N371C, D486S, E487Q and D489S;(11) The combination of T54H, S155C, S190I, S290C and V296I; and(12) The combination of S155C, S190F, S290C and V207L.
在一些實施例中，RSV F蛋白具有子類型A，且包含突變S155C、S190F、S290C及V207L。In some embodiments, the RSV F protein is of subtype A and comprises mutations S155C, S190F, S290C, and V207L.
在一些實施例中，RSV F蛋白具有子類型B，且包含突變S155C、S190F、S290C及V207L。In some embodiments, the RSV F protein is of subtype B and comprises mutations S155C, S190F, S290C, and V207L.
在一些實施例中，RSV F蛋白具有子類型A，且包含突變S155C、S190F及S290C。In some embodiments, the RSV F protein is of subtype A and comprises mutations S155C, S190F, and S290C.
在一些實施例中，RSV F蛋白具有子類型B，且包含突變S155C、S190F及S290C。In some embodiments, the RSV F protein is of subtype B and comprises mutations S155C, S190F, and S290C.
在一些實施例中，RSV F蛋白具有子類型A，且包含突變A103C、I148C、S190I及D486S。In some embodiments, the RSV F protein is of subtype A and comprises mutations A103C, I148C, S190I, and D486S.
在一些實施例中，RSV F蛋白具有子類型B，且包含突變A103C、I148C、S190I及D486S。In some embodiments, the RSV F protein is of subtype B and comprises mutations A103C, I148C, S190I, and D486S.
在一些實施例中，RSV F蛋白具有子類型A，且包含突變T54H、A103C、I148C、S190I及D486S。In some embodiments, the RSV F protein is of subtype A and comprises mutations T54H, A103C, I148C, S190I, and D486S.
在一些實施例中，RSV F蛋白具有子類型B，且包含突變T54H、A103C、I148C、S190I及D486S。In some embodiments, the RSV F protein is of subtype B and comprises mutations T54H, A103C, I148C, S190I, and D486S.
在一些實施例中，RSV F蛋白具有子類型A，且包含突變T54H、S55C、L188C及D486S。In some embodiments, the RSV F protein is of subtype A and comprises mutations T54H, S55C, L188C, and D486S.
在一些實施例中，RSV F蛋白具有子類型B，且包含突變T54H、S55C、L188C及D486S。In some embodiments, the RSV F protein is of subtype B and comprises mutations T54H, S55C, L188C, and D486S.
鑒於RSV F序列之實質上保守，一般熟習此項技術者可容易地比較不同天然RSV F序列之間的胺基酸位置，以鑑別不同RSV株系及子類型之間的對應RSV F胺基酸位置。舉例而言，在整個幾乎全部所鑑別天然RSV F0前驅物蛋白質中，弗林蛋白酶裂解位點落入相同胺基酸位置中。因此，整個株系及子類型中之天然RSV F蛋白序列之保守允許使用參考RSV F序列以用於比較RSV F蛋白中特定位置處之胺基酸。出於本發明之目的(除非上下文另外指示)，參考RSV A2株系之全長天然F前驅物多肽之胺基酸序列給出RSV F蛋白胺基酸位置；對應於GenInfo標識符GI 138251及Swiss Prot標識符P03420 (SEQ ID NO: 1)。In view of the substantial conservation of RSV F sequences, those skilled in the art can easily compare the amino acid positions between different natural RSV F sequences to identify the corresponding RSV F amino acid positions between different RSV strains and subtypes. For example, in almost all identified natural RSV F0 pro-driver proteins, the furin protease cleavage site falls into the same amino acid position. Therefore, the conservation of natural RSV F protein sequences in the entire strain and subtype allows the use of reference RSV F sequences for comparing the amino acids at specific positions in RSV F proteins. For the purposes of the present invention (unless the context indicates otherwise), RSV F protein amino acid positions are given with reference to the amino acid sequence of the full-length native F pro-propolypeptide of the RSV A2 strain; corresponding to GenInfo identifier GI 138251 and Swiss Prot identifier P03420 (SEQ ID NO: 1).
在一些實施例中，RSV F蛋白呈RSV F蛋白之成熟形式，其包含兩條個別多肽鏈，即F1多肽及F2多肽。在一些其他實施例中，F2多肽藉由一或兩個雙硫鍵與F1多肽連接以形成F2/F1異二聚體。在另其他實施例中，RSV F突變體呈單鏈蛋白質形式，其中F2多肽藉由肽鍵或肽連接子與F1多肽連接。可使用用於接合兩條多肽鏈在一起之任何適合的肽連接子。此類連接子之實例包括G、GG、GGG、GS及SAIG連接子序列。連接子亦可為全長pep27序列或其片段，該全長pep27序列對應於SEQ ID NO: 1之位置110-136處的胺基酸。In some embodiments, the RSV F protein is a mature form of the RSV F protein, which comprises two individual polypeptide chains, namely the F1 polypeptide and the F2 polypeptide. In some other embodiments, the F2 polypeptide is linked to the F1 polypeptide by one or two disulfide bonds to form an F2/F1 heterodimer. In yet other embodiments, the RSV F mutant is in the form of a single-chain protein, wherein the F2 polypeptide is linked to the F1 polypeptide by a peptide bond or a peptide linker. Any suitable peptide linker for joining two polypeptide chains together can be used. Examples of such linkers include G, GG, GGG, GS and SAIG linker sequences. The linker can also be a full-length pep27 sequence or a fragment thereof, and the full-length pep27 sequence corresponds to the amino acids at positions 110-136 of SEQ ID NO: 1.
突變體之F1多肽鏈與對應野生型RSV F蛋白之全長F1多肽可具有相同長度；然而，其亦可具有缺失，諸如缺失來自全長F1多肽之C端的1直至60個胺基酸殘基。RSV F突變體之全長F1多肽對應於天然RSV F0前驅物(SEQ ID NO: 1)之胺基酸位置137-574，且包括(自N端至C端)細胞外區(殘基137-524)、跨膜域(「TM」) (殘基525-550)及細胞質域(「CT」) (殘基551-574)。應注意，自天然F1多肽序列中之胺基酸殘基514起為本文提供之免疫原性組合物中所包括之RSV F蛋白之F1多肽中的視情況存在之序列，且因此可能不存在於突變體之F1多肽中。The F1 polypeptide chain of the mutant may have the same length as the full-length F1 polypeptide corresponding to the wild-type RSV F protein; however, it may also have a deletion, such as a deletion of 1 to 60 amino acid residues from the C-terminus of the full-length F1 polypeptide. The full-length F1 polypeptide of the RSV F mutant corresponds to amino acid positions 137-574 of the native RSV F0 promotor (SEQ ID NO: 1), and includes (from N-terminus to C-terminus) an extracellular region (residues 137-524), a transmembrane domain ("TM") (residues 525-550), and a cytoplasmic domain ("CT") (residues 551-574). It should be noted that amino acid residue 514 onwards in the native F1 polypeptide sequence is a sequence that optionally occurs in the F1 polypeptide of the RSV F protein included in the immunogenic compositions provided herein, and thus may not be present in the F1 polypeptide of the mutant.
在一些實施例中，RSV F突變體之F1多肽缺乏整個細胞質域。在其他實施例中，F1多肽缺乏細胞質域以及跨膜域之一部分或所有全部。在一些特定實施例中，突變體包含F1多肽，其中來自位置510、511、512、513、514、515、520、525或530直至574之胺基酸殘基不存在。通常，對於與三聚域(諸如摺疊子)連接之突變體而言，胺基酸514直至574可不存在。因此，在一些特定實施例中，胺基酸殘基514直至574不存在於突變體之F1多肽中。在另其他特定實施例中，RSV F突變體之F1多肽包含以下或由以下組成：天然F0多肽序列(SEQ ID NO: 1)之胺基酸殘基137-513，諸如RSV 847A-摺疊子多肽(SEQ ID NO: 74)；或替代性F0前驅物序列中之任一者，諸如WO2017109629 (其以全文引用之方式併入本文中)之SEQ ID NO: 1、2、4、6及81-270中所揭示之彼等者。In some embodiments, the F1 polypeptide of the RSV F mutant lacks the entire cytoplasmic domain. In other embodiments, the F1 polypeptide lacks a portion or all of the cytoplasmic domain and the transmembrane domain. In some specific embodiments, the mutant comprises an F1 polypeptide in which amino acid residues from positions 510, 511, 512, 513, 514, 515, 520, 525, or 530 to 574 are absent. Typically, for mutants connected to a trimerization domain (such as a fold), amino acids 514 to 574 may not be present. Therefore, in some specific embodiments, amino acid residues 514 to 574 are not present in the F1 polypeptide of the mutant. In yet other specific embodiments, the F1 polypeptide of the RSV F mutant comprises or consists of amino acid residues 137-513 of a native F0 polypeptide sequence (SEQ ID NO: 1), such as the RSV 847A-folder polypeptide (SEQ ID NO: 74); or any of the alternative F0 promotor sequences, such as those disclosed in SEQ ID NOs: 1, 2, 4, 6, and 81-270 of WO2017109629, which is incorporated herein by reference in its entirety.
其中引入一或多個突變之RSV F蛋白突變體之F1多肽及F2多肽可來自此項技術中已知或未來發現之任何野生型RSV F蛋白，包括但不限於RSV子類型A及子類型B株系(包括A2 Ontario及Buenos Aires)或任何其他子類型之F蛋白胺基酸序列。在一些實施例中，RSV F突變體包含其中引入一或多個突變之來自RSV A病毒之F1及/或F2多肽，例如來自WO2017109629之SEQ ID NO: 1、2、4、6及81-270 (該等序列以全文引用之方式併入本文中)中之任一者中所闡述之來自RSV F0前驅物蛋白質的F1及/或F2多肽。在一些其他實施例中，RSV F突變體包含其中引入一或多個突變之來自RSV B病毒之F1及/或F2多肽，例如WO2017/109629之SEQ ID NO: 2及211-263 (該等序列以全文引用之方式併入本文中)中之任一者中所闡述之來自RSV F0前驅物蛋白質的F1及/或F2多肽。在另其他實施例中，RSV F突變體包含其中引入一或多個突變之來自RSV牛病毒之F1及/或F2多肽，例如WO2017109629之SEQ ID NO: 264-270 (該等序列以全文引用之方式併入本文中)中之任一者中所闡述之來自RSV F0前驅物蛋白質的F1及/或F2多肽。The F1 polypeptide and F2 polypeptide of the RSV F protein mutant into which one or more mutations are introduced can be from any wild-type RSV F protein known in the art or discovered in the future, including but not limited to RSV subtype A and subtype B strains (including A2 Ontario and Buenos Aires) or any other subtype F protein amino acid sequences. In some embodiments, the RSV F mutant comprises an F1 and/or F2 polypeptide from an RSV A virus into which one or more mutations are introduced, such as an F1 and/or F2 polypeptide from an RSV F0 pro-driver protein described in any one of SEQ ID NOs: 1, 2, 4, 6, and 81-270 of WO2017109629 (these sequences are incorporated herein by reference in their entirety). In some other embodiments, RSV F mutants include F1 and/or F2 polypeptides from RSV B virus into which one or more mutations are introduced, such as WO2017/109629 SEQ ID NO: 2 and 211-263 (these sequences are incorporated herein by reference in their entirety) F1 and/or F2 polypeptides from RSV F0 pro-driver protein as described in any one of them. In yet other embodiments, RSV F mutants include F1 and/or F2 polypeptides from RSV bovine virus into which one or more mutations are introduced, such as WO2017109629 SEQ ID NO: 264-270 (these sequences are incorporated herein by reference in their entirety) F1 and/or F2 polypeptides from RSV F0 pro-driver protein as described in any one of them.
術語「F0多肽」(F0)係指RSV F蛋白之前驅物多肽，其由訊號多肽序列、F1多肽序列、pep27多肽序列及F2多肽序列構成。在罕見例外情況下，已知RSV株系之F0多肽由574個胺基酸組成。The term "F0 polypeptide" (F0) refers to the RSV F protein precursor polypeptide, which is composed of the signal polypeptide sequence, the F1 polypeptide sequence, the pep27 polypeptide sequence, and the F2 polypeptide sequence. With rare exceptions, the F0 polypeptide of known RSV strains consists of 574 amino acids.
術語「F1多肽」(F1)係指成熟RSV F蛋白之多肽鏈。天然F1包括RSV F0前驅物之大致殘基137-574且由以下構成(自N端至C端)：細胞外區(大致殘基137-524)、跨膜域(「TM」) (大致殘基525-550)及細胞質尾區(「CT」) (大致殘基551-574)。如本文所用，該術語涵蓋天然F1多肽及包括來自天然序列之修飾(例如胺基酸取代、插入或缺失)之F1多肽兩者，該等修飾例如設計成使RSV F蛋白突變體穩定或增強RSV F蛋白突變體之免疫原性的修飾。The term "F1 polypeptide" (F1) refers to the polypeptide chain of the mature RSV F protein. Native F1 includes approximately residues 137-574 of the RSV F0 promotor and is composed of (from N-terminus to C-terminus): an extracellular region (approximately residues 137-524), a transmembrane domain ("TM") (approximately residues 525-550), and a cytoplasmic tail ("CT") (approximately residues 551-574). As used herein, the term encompasses both native F1 polypeptides and F1 polypeptides that include modifications (e.g., amino acid substitutions, insertions, or deletions) from the native sequence, such as modifications designed to stabilize RSV F protein mutants or enhance the immunogenicity of RSV F protein mutants.
術語「F2多肽」(F2)係指成熟RSV F蛋白之多肽鏈。天然F2包括RSV F0前驅物之大致殘基26-109。如本文所用，該術語涵蓋天然F2多肽及包括來自天然序列之修飾(例如胺基酸取代、插入或缺失)之F2多肽兩者，該等修飾例如設計成使呈融合前構形之RSV F蛋白突變體穩定或增強RSV F蛋白突變體之免疫原性的修飾。在天然RSV F蛋白中，F2多肽藉由兩個雙硫鍵與F1多肽連接以形成F2-F1異二聚體。術語「摺疊子(foldon)」或「摺疊子域」係指能夠形成三聚體之胺基酸序列。此類摺疊子域之一個實例為來源於噬菌體T4纖維蛋白之肽序列，其具有GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 45)之序列。The term "F2 polypeptide" (F2) refers to the polypeptide chain of the mature RSV F protein. Native F2 includes approximately residues 26-109 of the RSV F0 promotor. As used herein, the term encompasses both native F2 polypeptides and F2 polypeptides that include modifications (e.g., amino acid substitutions, insertions, or deletions) from the native sequence, such as modifications designed to stabilize RSV F protein mutants in a prefusion conformation or enhance the immunogenicity of RSV F protein mutants. In the native RSV F protein, the F2 polypeptide is linked to the F1 polypeptide by two disulfide bonds to form an F2-F1 heterodimer. The term "foldon" or "foldon domain" refers to an amino acid sequence that is capable of forming a trimer. An example of such a fold subdomain is a peptide sequence derived from bacteriophage T4 fiber protein having the sequence GYIPEAPRDGQAYVRKDGEWVLLSTFL (SEQ ID NO: 45).
在一些態樣中，RNA分子編碼以下中所揭示之RSV F蛋白突變體：WO2009/079796、WO2010/149745、WO2011/008974、WO2014/160463、WO2014/174018、WO2014/202570、WO2015/013551、WO2015/177312、WO2017/005848、WO2017/174564、WO2017/005844及WO2018/109220。此等參考文獻中所揭示之RSV F蛋白以全文引用之方式併入本文中。In some aspects, the RNA molecule encodes RSV F protein mutants disclosed in WO2009/079796, WO2010/149745, WO2011/008974, WO2014/160463, WO2014/174018, WO2014/202570, WO2015/013551, WO2015/177312, WO2017/005848, WO2017/174564, WO2017/005844 and WO2018/109220. The RSV F proteins disclosed in these references are incorporated herein by reference in their entirety.
針對RSV F蛋白之抗體在天然感染之後及在疫苗接種之後為普遍的，且已顯示在活體外中和病毒活性。如本文所用，術語「呼吸道融合病毒」或「RSV」不限於任何特定株系或變體。Antibodies to the RSV F protein are prevalent following natural infection and following vaccination, and have been shown to neutralize viral activity in vitro. As used herein, the term "respiratory syncytial virus" or "RSV" is not limited to any particular strain or variant.
在一些態樣中，RNA分子包含編碼RSV抗原之開讀框。在一些態樣中，RSV抗原為RSV多肽。在一些態樣中，RSV多肽為RSV醣蛋白或其片段或變體。在一些態樣中，RNA分子編碼RSV F蛋白。In some aspects, the RNA molecule comprises an open reading frame encoding an RSV antigen. In some aspects, the RSV antigen is an RSV polypeptide. In some aspects, the RSV polypeptide is an RSV glycoprotein or a fragment or variant thereof. In some aspects, the RNA molecule encodes an RSV F protein.
在一些態樣中，RSV多肽為全長RSV多肽。在一些態樣中，RSV多肽為截短RSV多肽。在一些態樣中，RSV多肽為RSV多肽之變體。在一些態樣中，RSV多肽為RSV多肽之片段。In some aspects, the RSV polypeptide is a full-length RSV polypeptide. In some aspects, the RSV polypeptide is a truncated RSV polypeptide. In some aspects, the RSV polypeptide is a variant of the RSV polypeptide. In some aspects, the RSV polypeptide is a fragment of the RSV polypeptide.
在一些態樣中，RSV多肽為全長RSV F蛋白。在一些態樣中，RSV多肽為截短RSV F蛋白。在一些態樣中，RSV多肽為RSV F蛋白之變體。在一些態樣中，RSV多肽為RSV F蛋白之片段。In some aspects, the RSV polypeptide is a full-length RSV F protein. In some aspects, the RSV polypeptide is a truncated RSV F protein. In some aspects, the RSV polypeptide is a variant of the RSV F protein. In some aspects, the RSV polypeptide is a fragment of the RSV F protein.
在一些態樣中，RSV F蛋白包含至少一個突變。在一些態樣中，RSV F蛋白包含至少兩個突變。在一些態樣中，RSV F蛋白包含至少三個突變。在一些態樣中，RSV F蛋白包含至少四個突變。在一些態樣中，RSV F蛋白包含4個突變。在一些態樣中，RSV F蛋白包含至少五個突變。In some aspects, the RSV F protein comprises at least one mutation. In some aspects, the RSV F protein comprises at least two mutations. In some aspects, the RSV F protein comprises at least three mutations. In some aspects, the RSV F protein comprises at least four mutations. In some aspects, the RSV F protein comprises 4 mutations. In some aspects, the RSV F protein comprises at least five mutations.
在一些態樣中，RNA分子編碼表1中所闡述之RSV F蛋白(參見實例6)。在一些態樣中，RNA分子編碼包含SEQ ID NO: 1至6及71至74中之任一者之胺基酸序列的RSV F蛋白，或其片段或變體。在一些態樣中，RSV F多肽與表1之胺基酸序列中之任一者(例如SEQ ID NO: 1至6及71至74中之任一者)可具有至少、至多、恰好以下或在以下中之任何兩者之間的一致性：70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%。在一些態樣中，RSV F蛋白由表1之胺基酸序列中之任一者(例如SEQ ID NO: 1至6及71至74中之任一者)組成。In some aspects, the RNA molecule encodes the RSV F protein described inTable1 (see Example 6). In some aspects, the RNA molecule encodes an RSV F protein comprising an amino acid sequence of any one of SEQ ID NOs: 1 to 6 and 71 to 74, or a fragment or variant thereof. In some aspects, the RSV F polypeptide and any one of the amino acid sequences ofTable1 (e.g., any one of SEQ ID NOs: 1 to 6 and 71 to 74) may have an identity of at least, at most, just below, or between any two of the following: 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some aspects, the RSV F protein consists of any one of the amino acid sequences ofTable1 (eg, any one of SEQ ID NOs: 1-6 and 71-74).
在一些態樣中，RNA分子序列轉錄自表2之DNA核酸序列(DNA聚核苷酸) (參見實例6)。在一些態樣中，RNA分子包含轉錄自SEQ ID NO: 7至10及59至62中之任一者之核酸序列或其片段或變體的ORF。在一些態樣中，RNA分子包含轉錄自與表2之核酸序列中之任一者(例如SEQ ID NO: 7至10及59至62中之任一者)可具有至少、至多、恰好以下或在以下中之任何兩者之間的一致性之核酸序列的ORF：70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%。在一些態樣中，RNA分子包含轉錄自由表2之核酸序列中之任一者(例如SEQ ID NO: 7至10及59至62中之任一者)組成之核酸序列的ORF。In some aspects, the RNA molecule sequence is transcribed from a DNA nucleic acid sequence (DNA polynucleotide) ofTable2 (see Example 6). In some aspects, the RNA molecule comprises an ORF transcribed from a nucleic acid sequence of any one of SEQ ID NOs: 7 to 10 and 59 to 62, or a fragment or variant thereof. In some aspects, the RNA molecule comprises an ORF transcribed from a nucleic acid sequence that may have at least, at most, just below, or between any two of the following identities to any one of the nucleic acid sequences ofTable2 (e.g., any one of SEQ ID NOs: 7 to 10 and 59 to 62): 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some aspects, the RNA molecule comprises an ORF that transcribes a nucleic acid sequence consisting of any one of the nucleic acid sequences inTable2 (eg, any one of SEQ ID NOs: 7 to 10 and 59 to 62).
在一些態樣中，RNA分子包含含有表3之RNA核酸序列(RNA聚核苷酸) (參見實例6)的ORF。在一些態樣中，RNA分子包含含有SEQ ID NO: 11至16及63至70中之任一者之核酸序列或其片段或變體的ORF。在一些態樣中，RNA分子包含含有與表3之RNA核酸序列中之任一者(例如SEQ ID NO: 11至16及63至70中之任一者)可具有至少、至多、恰好以下或在以下中之任何兩者之間的一致性之核酸序列的ORF：70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%。在一些態樣中，RNA分子包含含有由表3之RNA核酸序列中之任一者(例如SEQ ID NO: 11至16及63至70中之任一者)組成之核酸序列的ORF。In some aspects, the RNA molecule comprises an ORF comprising an RNA nucleic acid sequence (RNA polynucleotide) ofTable3 (see Example 6). In some aspects, the RNA molecule comprises an ORF comprising a nucleic acid sequence of any one of SEQ ID NOs: 11 to 16 and 63 to 70, or a fragment or variant thereof. In some aspects, the RNA molecule comprises an ORF comprising a nucleic acid sequence that may have at least, at most, just less than, or between any two of the following identities to any one of the RNA nucleic acid sequences ofTable3 (e.g., any one of SEQ ID NOs: 11 to 16 and 63 to 70): 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some aspects, the RNA molecule comprises an ORF comprising a nucleic acid sequence consisting of any one of the RNA nucleic acid sequences inTable3 (eg, any one of SEQ ID NOs: 11-16 and 63-70).
在一些態樣中，RNA分子包含穩定化RNA。在一些態樣中，RNA分子包含至少一個尿苷經N1-甲基假尿苷置換之核酸序列。在一些態樣中，RNA分子包含所有尿苷經N1-甲基假尿苷置換之序列(表示為「Ψ」)。在一些態樣中，RNA分子包含含有SEQ ID NO: 11至16及63至70中之任一者之核酸序列的ORF，其中所有尿苷已經N1-甲基假尿苷(表示為「Ψ」)置換。In some aspects, the RNA molecule comprises stabilized RNA. In some aspects, the RNA molecule comprises a nucleic acid sequence in which at least one uridine is replaced by N1-methyl pseudouridine. In some aspects, the RNA molecule comprises a sequence in which all uridines are replaced by N1-methyl pseudouridine (denoted as "Ψ"). In some aspects, the RNA molecule comprises an ORF containing a nucleic acid sequence of any one of SEQ ID NOs: 11 to 16 and 63 to 70, wherein all uridines have been replaced by N1-methyl pseudouridine (denoted as "Ψ").
在一些態樣中，RNA分子包含編碼與SEQ ID NO: 1至6及71 至74 (表1)之RSV F蛋白序列中之任一者或本文所描述之其他RSV融合前F蛋白可至少、至多、恰好以下或在以下中之任何兩者之間一致之RSV F蛋白胺基酸序列的開讀框：70%、80%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%。在一些態樣中，RNA分子包含編碼由SEQ ID NO: 1至6及71 至74 (表1)之RSV F蛋白序列中之任一者或本文所描述之其他RSV融合前F蛋白組成之RSV F蛋白胺基酸序列的開讀框。In some aspects, the RNA molecule comprises an open reading frame encoding an RSV F protein amino acid sequence that is at least, at most, just below, or between any two ofthe following: 70%, 80%, 85%,86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the RSV F protein sequences of SEQ ID NOs: 1 to 6 and 71 to 74 (Table1) , or other RSV prefusion F proteins described herein. In some aspects, the RNA molecule comprises an open reading frame encoding an RSV F protein amino acid sequence consisting of any one of the RSV F protein sequences of SEQ ID NOs: 1 to 6 and 71 to 74 (Table 1), or other RSV prefusion F proteins described herein.
在一些態樣中，RNA分子包含轉錄自與SEQ ID NO: 7至10及59至62 (表2)之核酸序列中之任一者或本文所描述之其他核酸可至少、至多、恰好以下或在以下中之任何兩者之間一致之DNA核酸序列的開讀框：70%、80%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%。在一些態樣中，RNA分子包含轉錄自由SEQ ID NO: 7至10及59至62 (表2)之核酸序列中之任一者或本文所描述之其他核酸組成之DNA核酸序列的開讀框。In some aspects, the RNA molecule comprises an open reading frame transcribed from a DNA nucleic acid sequence that is at least, at most, just below, or between any two ofthe following: 70%, 80%, 85%,86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the nucleic acid sequences of SEQ ID NOs: 7 to 10 and 59 to 62 (Table2), or other nucleic acids described herein. In some aspects, the RNA molecule comprises an open reading frame transcribed from a DNA nucleic acid sequence consisting of any one of the nucleic acid sequences of SEQ ID NOs: 7 to 10 and 59 to 62 (Table 2 ), or other nucleic acids described herein.
在一些態樣中，RNA分子包含含有與SEQ ID NO: 11至16及63至70 (表3)之核酸序列中之任一者或本文所描述之其他核酸可至少、至多、恰好以下或在以下中之任何兩者之間一致之RNA核酸序列的開讀框：70%、80%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%。在一些態樣中，RNA分子包含含有由SEQ ID NO: 11至16及63至70 (表3)之核酸序列中之任一者或本文所描述之其他核酸組成之RNA核酸序列的開讀框。在一些態樣中，RNA分子包含含有SEQ ID NO: 11至16及63至70 (表3)中之任一者之核酸序列的ORF，其中所有尿苷已經N1-甲基假尿苷(表示為「Ψ」)置換。In some aspects, the RNA molecule comprises an open reading frame comprising an RNA nucleic acid sequence that is at least, at most, just below, or between any two ofthe following: 70%, 80%, 85%, 86%,87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the nucleic acid sequences of SEQ ID NOs: 11 to 16 and 63 to 70 (Table3), or other nucleic acids described herein. In some aspects, the RNA molecule comprises an open reading frame comprising an RNA nucleic acid sequence consisting of any one of the nucleic acid sequences of SEQ ID NOs: 11 to 16 and 63 to 70 (Table 3 ), or other nucleic acids described herein. In some aspects, the RNA molecule comprises an ORF comprising a nucleic acid sequence of any one of SEQ ID NOs: 11 to 16 and 63 to 70 (Table3 ), wherein all uridines have been replaced with N1-methylpseudouridine (denoted as "Ψ").
III. RNA分子在一些態樣中，本文所描述之RNA分子為編碼RNA分子。編碼RNA包括可轉譯成肽或多肽之功能性RNA分子。在一些態樣中，編碼RNA分子包括至少一個編碼至少一種肽或多肽之開讀框(ORF)。開讀框包含可轉譯成肽或蛋白質之密碼子序列。編碼RNA分子可包括一個(單順反子)、兩個(雙順反子)或更多個(多順反子) ORF，其可為可轉譯成所關注多肽或蛋白質之密碼子序列。III. RNAmolecules In some aspects, the RNA molecules described herein are coding RNA molecules. Coding RNA includes functional RNA molecules that can be translated into peptides or polypeptides. In some aspects, the coding RNA molecule includes at least one open reading frame (ORF) that encodes at least one peptide or polypeptide. The open reading frame contains a codon sequence that can be translated into a peptide or protein. The coding RNA molecule can include one (monostronic), two (bi-stronic) or more (poly-stronic) ORFs, which can be codon sequences that can be translated into a polypeptide or protein of interest.
編碼RNA分子可為信使RNA (mRNA)分子、病毒RNA分子或自擴增RNA分子(saRNA，亦稱為複製子)。在一些態樣中，RNA分子為mRNA。較佳地，本發明之RNA分子為mRNA。在一些態樣中，RNA分子為modRNA。在一些態樣中，RNA分子為saRNA。在一些態樣中，saRNA分子可為編碼RNA分子。The coding RNA molecule can be a messenger RNA (mRNA) molecule, a viral RNA molecule, or a self-amplifying RNA molecule (saRNA, also known as a replicon). In some aspects, the RNA molecule is mRNA. Preferably, the RNA molecule of the present invention is mRNA. In some aspects, the RNA molecule is modRNA. In some aspects, the RNA molecule is saRNA. In some aspects, the saRNA molecule can be a coding RNA molecule.
RNA分子可編碼一種所關注多肽或更多，諸如一種抗原或超過一種抗原，例如兩種、三種、四種、五種、六種、七種、八種、九種、十種或更多種多肽。或者或另外，一個RNA分子亦可編碼超過一種所關注多肽，諸如抗原，例如編碼不同或相同抗原的雙順反子或三順反子RNA分子。The RNA molecule may encode one polypeptide of interest or more, such as one antigen or more than one antigen, such as two, three, four, five, six, seven, eight, nine, ten or more polypeptides. Alternatively or additionally, one RNA molecule may encode more than one polypeptide of interest, such as an antigen, such as a bicistronic or tricistronic RNA molecule encoding different or the same antigen.
RNA分子之序列可經密碼子最佳化或去最佳化以供在所需宿主(諸如人類細胞)中表現。在一些態樣中，與野生型編碼序列相比，本文所描述之所關注基因(例如抗原)由密碼子最佳化及/或鳥苷/胞苷(G/C)含量增加的編碼序列編碼。在一些態樣中，與野生型編碼序列之對應序列區域相比，編碼序列之一或多個序列區域經密碼子最佳化及/或其G/C含量增加。在一些態樣中，密碼子最佳化及/或增加G/C含量不會改變所編碼胺基酸序列之序列。The sequence of the RNA molecule can be codon optimized or deoptimized for expression in a desired host, such as a human cell. In some aspects, the gene of interest described herein, such as an antigen, is encoded by a coding sequence that is codon optimized and/or has an increased guanosine/cytidine (G/C) content compared to the wild-type coding sequence. In some aspects, one or more sequence regions of the coding sequence are codon optimized and/or have an increased G/C content compared to the corresponding sequence region of the wild-type coding sequence. In some aspects, codon optimization and/or increased G/C content does not change the sequence of the encoded amino acid sequence.
熟習此項技術者應瞭解，術語「密碼子最佳化」係指改變核酸分子之編碼區中之密碼子以反映宿主生物體之典型密碼子使用而不改變由核酸分子編碼之胺基酸序列。在本發明之上下文內，在一些態樣中，編碼區經密碼子最佳化以供在待使用本文所描述之RNA聚核苷酸治療之個體中最佳表現。密碼子最佳化係基於以下發現：轉譯效率亦由細胞中tRNA分子之不同出現頻率決定。因此，RNA之序列可經修飾，使得可獲得頻繁出現之tRNA分子的密碼子插入在「罕見密碼子」之位置。Those skilled in the art will appreciate that the term "codon optimization" refers to changing the codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of the host organism without changing the amino acid sequence encoded by the nucleic acid molecule. In the context of the present invention, in some aspects, the coding region is codon optimized for optimal performance in an individual to be treated with the RNA polynucleotides described herein. Codon optimization is based on the discovery that translation efficiency is also determined by the different frequencies of occurrence of tRNA molecules in the cell. Therefore, the sequence of the RNA can be modified so that codons that can obtain frequently occurring tRNA molecules are inserted at the position of "rare codons".
在一些態樣中，與編碼所關注基因之野生型RNA之對應編碼序列的G/C含量相比，RNA之(例如所關注基因序列；開讀框(ORF)之)編碼區之G/C含量增加，其中在一些態樣中，與由野生型RNA編碼之胺基酸序列相比，由該RNA編碼之胺基酸序列未經修飾。RNA序列之此修飾係基於以下事實：待轉譯的任何RNA區之序列對於該mRNA之高效轉譯而言為重要的。G (鳥苷)/C (胞苷)含量增加之序列與A (腺苷)/U (尿苷)含量增加之序列相比更穩定。相對於若干密碼子編碼同一個胺基酸之事實(所謂的遺傳密碼簡併)，可確定對於穩定性最有利之密碼子(所謂的替代密碼子使用)。視由RNA編碼之胺基酸而定，與其野生型序列相比，RNA序列之修飾存在各種可能性。特定言之，含有A及/或U核苷之密碼子可藉由用其他密碼子取代此等密碼子而經修飾，該等其他密碼子編碼相同胺基酸但不含A及/或U或含有更低含量之A及/或U核苷。因此，在一些態樣中，與野生型RNA之編碼區之G/C含量相比，本文所描述之RNA之編碼區的G/C含量增加至少、至多、恰好以下或在以下中之任何兩者之間：10%、20%、30%、40%、50%、55%或甚至更多。在一些態樣中，本文所描述之RSV RNA之編碼區包含以下之G/C含量：至少50%、至少55%、至少60%、至少65%、至少70%、至少75%或約80%。在一些態樣中，本文所描述之RSV RNA之編碼區包含以下之G/C含量：約50%至75%、約55%至70%、約50%至60%、約60%至70%、約70%至80%、約50%至55%、約55%至60%、約60%至65%、約65%至70%、約70%至75%、或約75%至80%。在一些態樣中，本文所描述之RSV RNA之編碼區包含以下之G/C含量：約50%、約51%、約52%、約53%、約54%、約55%、約56%、約57%、約58%、約59%、約60%、約61%、約62%、約63%、約64%、約65%、約66%、約67%、約68%、約69%、約70%、約71%、約72%、約73%、約74%或約75%。在一些態樣中，本文所描述之RSV RNA之編碼區包含約58%、約66%或約62%之G/C含量。In some aspects, the G/C content of the coding region of the RNA (e.g., the sequence of the gene of interest; the open reading frame (ORF)) is increased compared to the G/C content of the corresponding coding sequence of the wild-type RNA encoding the gene of interest, wherein in some aspects, the amino acid sequence encoded by the RNA is not modified compared to the amino acid sequence encoded by the wild-type RNA. This modification of the RNA sequence is based on the fact that the sequence of any RNA region to be translated is important for the efficient translation of the mRNA. Sequences with increased G (guanosine)/C (cytidine) content are more stable than sequences with increased A (adenosine)/U (uridine) content. Relative to the fact that several codons encode the same amino acid (so-called genetic codon simplification), the codons that are most favorable for stability (so-called alternative codon usage) can be determined. Depending on the amino acid encoded by RNA, there are various possibilities for the modification of RNA sequence compared with its wild-type sequence. Specifically, the codon containing A and/or U nucleosides can be modified by replacing these codons with other codons, and these other codons encode the same amino acid but do not contain A and/or U or contain A and/or U nucleosides of lower content. Therefore, in some aspects, compared with the G/C content of the coding region of wild-type RNA, the G/C content of the coding region of the RNA described herein increases at least, at most, just below or between any two of the following: 10%, 20%, 30%, 40%, 50%, 55% or even more. In some aspects, the coding region of the RSV RNA described herein includes the following G/C content: at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or about 80%. In some aspects, the coding region of the RSV RNA described herein comprises the following G/C content: about 50% to 75%, about 55% to 70%, about 50% to 60%, about 60% to 70%, about 70% to 80%, about 50% to 55%, about 55% to 60%, about 60% to 65%, about 65% to 70%, about 70% to 75%, or about 75% to 80%. In some aspects, the coding region of the RSV RNA described herein comprises the following G/C content: about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74% or about 75%. In some aspects, the coding region of the RSV RNA described herein comprises a G/C content of about 58%, about 66%, or about 62%.
在一些態樣中，RNA分子包括約20至約100,000個核苷酸(例如30至50、30至100、30至250、30至500、30至1,000、30至1,500、30至3,000、30至5,000、30至7,000、30至10,000、30至25,000、30至50,000、30至70,000、100至250、100至500、100至1,000、100至1,500、100至3,000、100至5,000、100至7,000、100至10,000、100至25,000、100至50,000、100至70,000、100至100,000、500至1,000、500至1,500、500至2,000、500至3,000、500至5,000、500至7,000、500至10,000、500至25,000、500至50,000、500至70,000、500至100,000、1,000至1,500、1,000至2,000、1,000至3,000、1,000至5,000、1,000至7,000、1,000至10,000、1,000至25,000、1,000至50,000、1,000至70,000、1,000至100,000、1,500至3,000、1,500至5,000、1,500至7,000、1,500至10,000、1,500至25,000、1,500至50,000、1,500至70,000、1,500至100,000、2,000至3,000、2,000至5,000、2,000至7,000、2,000至10,000、2,000至25,000、2,000至50,000、2,000至70,000、及2,000至100,000個核苷酸)。In some aspects, the RNA molecule comprises from about 20 to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500 0, 100 to 3,000, 100 to 5,000, 100 to 7,000, 100 to 10,000, 100 to 25,000, 100 to 50,000, 100 to 70,000, 100 to 100,000, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 3,000, 500 to 5,000, 500 to 7,000, 500 to 10,000, 500 to 25,000, 500 to 50,000 , 500 to 70,000, 500 to 100,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 3,000, 1,000 to 5,000, 1,000 to 7,000, 1,000 to 10,000, 1,000 to 25,000, 1,000 to 50,000, 1,000 to 70,000, 1,000 to 100,000, 1,500 to 3,000, 1,500 to 5,000, 1,500 to 00, 1,500 to 10,000, 1,500 to 25,000, 1,500 to 50,000, 1,500 to 70,000, 1,500 to 100,000, 2,000 to 3,000, 2,000 to 5,000, 2,000 to 7,000, 2,000 to 10,000, 2,000 to 25,000, 2,000 to 50,000, 2,000 to 70,000, and 2,000 to 100,000 nucleotides).
在一些態樣中，RNA分子具有至少、至多、恰好以下或在以下中之任何兩者之間的核苷酸：約20、40、60、80、100、120、140、160、180、200、220、240、260、280、300、320、340、360、380、400、420、440、460、480、500、520、540、560、580、600、620、640、660、680、700、720、740、760、780、800、820、840、860、880、900、920、940、960、980、1000、1200、1400、1600、1800、2000、2200、2400、2600、2800、3000、3200、3400、3600、3800、4000、4200、4400、4600、4800、5000、5200、5400、5600、5800、6000、6200、6400、6600、6800、7000、7200、7400、7600、7800、8000、8200、8400、8600、8800、9000、9200、9400、9600、9800、10000、12000、14000、16000、18000、20000、22000、24000、26000、28000、30000、32000、34000、36000、38000、40000、42000、44000、46000、48000、50000、52000、54000、56000、58000、60000、62000、64000、66000、68000、70000、72000、74000、76000、78000、80000、82000、84000、86000、88000、90000、92000、94000、96000、98000或100000個。In some aspects, the RNA molecule has at least, at most, just below, or between about 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600, 620, 640, 660, 680, 700, 720, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950 0, 760, 780, 800, 820, 840, 860, 880, 900, 920, 940, 960, 980, 1000, 1200, 1400, 1600, 1800, 2000, 2200, 2400, 2600, 2800, 3000, 3200, 3400, 3600, 3800, 4000, 4200, 4400, 4600, 4800, 5000, 5200, 5400, 5600, 5800, 6000, 6200 , 6400, 6600, 6800, 7000, 7200, 7400, 7600, 7800, 8000, 8200, 8400, 8600, 8800, 9000, 9200, 9400, 9600, 9800, 10000, 12000, 14000, 16000, 18000, 20000, 22000, 24000, 26000, 28000, 30000, 32000, 34000, 36000, 38000, 400 00, 42000, 44000, 46000, 48000, 50000, 52000, 54000, 56000, 58000, 60000, 62000, 64000, 66000, 68000, 70000, 72000, 74000, 76000, 78000, 80000, 82000, 84000, 86000, 88000, 90000, 92000, 94000, 96000, 98000, or 100000.
在一些態樣中，RNA分子包括至少100個核苷酸。舉例而言，在一些態樣中，RNA之長度在100與15,000個核苷酸之間；在7,000與16,000個核苷酸之間；在8,000與15,000個核苷酸之間；在9,000與12,500個核苷酸之間；在11,000與15,000個核苷酸之間；在13,000與16,000個核苷酸之間；在7,000與25,000個核苷酸之間。在一些態樣中，RNA分子具有至少、至多、恰好以下或在以下中之任何兩者之間的核苷酸：約100、150、200、250、300、350、400、450、500、550、600、650、700、750、800、850、900、950、1000、1050、1100、1150、1200、1250、1300、1350、1400、1450、1500、1550、1600、1650、1700、1750、1800、1850、1900、1950、2000、2050、2100、2150、2200、2250、2300、2350、2400、2450、2500、2550、2600、2650、2700、2750、2800、2850、2900、2950、3000、3050、3100、3150、3200、3250、3300、3350、3400、3450、3500、3550、3600、3650、3700、3750、3800、3850、3900、3950、4000、4050、4100、4150、4200、4250、4300、4350、4400、4450、4500、4550、4600、4650、4700、4750、4800、4850、4900、4950、5000、5050、5100、5150、5200、5250、5300、5350、5400、5450、5500、5550、5600、5650、5700、5750、5800、5850、5900、5950、6000、6050、6100、6150、6200、6250、6300、6350、6400、6450、6500、6550、6600、6650、6700、6750、6800、6850、6900、6950、7000、7050、7100、7150、7200、7250、7300、7350、7400、7450、7500、7550、7600、7650、7700、7750、7800、7850、7900、7950、8000、8050、8100、8150、8200、8250、8300、8350、8400、8450、8500、8550、8600、8650、8700、8750、8800、8850、8900、8950、9000、9050、9100、9150、9200、9250、9300、9350、9400、9450、9500、9550、9600、9650、9700、9750、9800、9850、9900、9950、10000、10050、10100、10150、10200、10250、10300、10350、10400、10450、10500、10550、10600、10650、10700、10750、10800、10850、10900、10950、11000、11050、11100、11150、11200、11250、11300、11350、11400、11450、11500、11550、11600、11650、11700、11750、11800、11850、11900、11950、12000、12050、12100、12150、12200、12250、12300、12350、12400、12450、12500、12550、12600、12650、12700、12750、12800、12850、12900、12950、13000、13050、13100、13150、13200、13250、13300、13350、13400、13450、13500、13550、13600、13650、13700、13750、13800、13850、13900、13950、14000、14050、14100、14150、14200、14250、14300、14350、14400、14450、14500、14550、14600、14650、14700、14750、14800、14850、14900、14950或15000個。In some aspects, the RNA molecule comprises at least 100 nucleotides. For example, in some aspects, the length of the RNA is between 100 and 15,000 nucleotides; between 7,000 and 16,000 nucleotides; between 8,000 and 15,000 nucleotides; between 9,000 and 12,500 nucleotides; between 11,000 and 15,000 nucleotides; between 13,000 and 16,000 nucleotides; between 7,000 and 25,000 nucleotides. In some aspects, the RNA molecule has at least, at most, just below, or between about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 0, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, 3000, 3050, 3100, 3150, 3200, 3250, 3300, 3350, 3400, 3450, 3500, 3550, 3600, 3650, 3700, 3750, 3800, 3850, 3 900、3950、4000、4050、4100、4150、4200、4250、4300、4350、4400、4450、4500、4550、4600、4650、4700、4750、4800、4850、4900、4950、5000、5050、5100、5150、5200、5250、5300、5350、5400、5450、5500、5550、5600、5650、5700、5750、5800、5850、590 0, 5950, 6000, 6050, 6100, 6150, 6200, 6250, 6300, 6350, 6400, 6450, 6500, 6550, 6600, 6650, 6700, 6750, 6800, 6850, 6900, 6950, 7000, 7050, 7100, 7150, 7200, 7250, 7300, 7350, 7400, 7450, 7500, 7550, 7600, 7650, 7700, 7750, 7800, 7850, 7900, 7 950、8000、8050、8100、8150、8200、8250、8300、8350、8400、8450、8500、8550、8600、8650、8700、8750、8800、8850、8900、8950、9000、9050、9100、9150、9200、9250、9300、9350、9400、9450、9500、9550、9600、9650、9700、9750、9800、9850、9900、995 0, 10000, 10050, 10100, 10150, 10200, 10250, 10300, 10350, 10400, 10450, 10500, 10550, 10600, 10650, 10700, 10750, 10800, 10850, 10900, 10950, 11000, 11050, 11100, 11150, 11200, 11250, 11300, 11350, 11400, 11450, 11500, 11550, 11600, 11650 50、11700、11750、11800、11850、11900、11950、12000、12050、12100、12150、12200、12250、12300、12350、12400、12450、12500、12550、12600、12650、12700、12750、12800、12850、12900、12950、13000、13050、13100、13150、13200、13250、13300、13 350, 13400, 13450, 13500, 13550, 13600, 13650, 13700, 13750, 13800, 13850, 13900, 13950, 14000, 14050, 14100, 14150, 14200, 14250, 14300, 14350, 14400, 14450, 14500, 14550, 14600, 14650, 14700, 14750, 14800, 14850, 14900, 14950, or 15000.
本發明之RNA分子可藉由此項技術中已知之任何方法製備，包括化學合成及活體外方法，諸如RNA活體外轉錄。在有一些態樣中，本發明之RNA係使用活體外轉錄製備。The RNA molecules of the present invention can be prepared by any method known in the art, including chemical synthesis and in vitro methods, such as RNA in vitro transcription. In some aspects, the RNA of the present invention is prepared using in vitro transcription.
在一些態樣中，本發明之RNA分子例如藉由過濾來純化，該過濾可經由例如超濾、透濾或例如切向流超濾/透濾進行。In some aspects, RNA molecules of the invention are purified, for example, by filtration, which can be performed, for example, by ultrafiltration, diafiltration, or, for example, tangential flow ultrafiltration/diafiltration.
在一些態樣中，本發明之RNA分子凍乾成溫度穩定的。In some aspects, the RNA molecules of the invention are freeze-dried to be temperature stable.
在本發明之一些態樣中，RNA為或包含信使RNA (mRNA)，其與編碼多肽之RNA轉錄本有關。在一些態樣中，本文所揭示之RNA包含：5'帽，其包含本文所揭示之5'帽；5'非轉譯區(5' UTR)，其包含帽近端序列；編碼蛋白質(例如多肽) (例如RSV融合前F蛋白)之序列；3'非轉譯區(3' UTR)；及/或多腺苷酸化(多-A)序列。In some aspects of the invention, the RNA is or comprises a messenger RNA (mRNA), which is associated with an RNA transcript encoding a polypeptide. In some aspects, the RNA disclosed herein comprises: a 5' cap comprising a 5' cap disclosed herein; a 5' non-translational region (5' UTR) comprising a cap proximal sequence; a sequence encoding a protein (e.g., a polypeptide) (e.g., RSV pre-fusion F protein); a 3' non-translational region (3' UTR); and/or a polyadenylation (poly-A) sequence.
在一些態樣中，本文所揭示之RNA在5'至3'取向上包含以下組分：5'帽，其包含本文所揭示之5'帽；5'非轉譯區(5' UTR)，其包含帽近端序列；編碼蛋白質(例如多肽) (例如RSV融合前F蛋白)之序列；3'非轉譯區(3' UTR)；及多-A序列。In some aspects, the RNA disclosed herein comprises the following components in the 5' to 3' orientation: a 5' cap comprising the 5' cap disclosed herein; a 5' non-translated region (5' UTR) comprising a cap-proximal sequence; a sequence encoding a protein (e.g., a polypeptide) (e.g., RSV prefusion F protein); a 3' non-translated region (3' UTR); and a poly-A sequence.
在一些態樣中，本文所揭示之RNA進一步包含訊號肽。訊號肽以及編碼此類肽之胺基酸及核酸序列之非限制性實例可見於例如WO2017/109629 (其揭示內容以全文引用之方式併入本文中)中。In some aspects, the RNA disclosed herein further comprises a signal peptide. Non-limiting examples of signal peptides and amino acid and nucleic acid sequences encoding such peptides can be found in, for example, WO2017/109629 (the disclosure of which is incorporated herein by reference in its entirety).
在一些態樣中，本文所揭示之RNA編碼抗原性融合蛋白。因此，所編碼之一或多種抗原可包括兩種或更多種接合在一起之蛋白質(例如蛋白質及/或蛋白質片段)。或者，與蛋白質抗原融合之蛋白質不促進針對自身之強免疫反應，而是促進針對抗原之強免疫反應。在一些態樣中，抗原性融合蛋白保留各初始蛋白質之功能特性。在一些態樣中，本文所揭示之RNA編碼包含與骨架部分連接之抗原的融合蛋白。在一些態樣中，RNA進一步編碼位於融合蛋白之至少一個或各域之間的連接子。此類骨架部分及連接子之非限制性實例可見於例如WO 2022/067010 (其揭示內容以全文引用之方式併入本文中)中。In some aspects, the RNA disclosed herein encodes an antigenic fusion protein. Therefore, one or more antigens encoded may include two or more proteins (e.g., proteins and/or protein fragments) joined together. Alternatively, the protein fused to the protein antigen does not promote a strong immune response against itself, but promotes a strong immune response against the antigen. In some aspects, the antigenic fusion protein retains the functional properties of each initial protein. In some aspects, the RNA disclosed herein encodes a fusion protein comprising an antigen connected to a backbone portion. In some aspects, the RNA further encodes a linker located between at least one or each domain of the fusion protein. Non-limiting examples of such backbone portions and linkers can be found in, for example, WO 2022/067010 (the disclosure of which is incorporated herein by reference in its entirety).
A.經修飾之核鹼基在本發明之一些態樣中，RNA分子未經化學修飾且包含由腺苷、鳥苷、胞嘧啶及尿苷組成之標準核糖核苷酸。在一些態樣中，本發明之核苷酸及核苷包含標準核苷殘基，諸如所轉錄RNA中存在之彼等者(例如A、G、C及/或U)。在一些態樣中，本發明之核苷酸及核苷包含標準去氧核糖核苷，諸如DNA中存在之彼等者(例如dA、dG、dC及/或dT)。A.Modified Nucleobases In some aspects of the invention, the RNA molecule is not chemically modified and comprises standard ribonucleotides composed of adenosine, guanosine, cytosine, and uridine. In some aspects, the nucleotides and nucleosides of the invention comprise standard nucleoside residues such as those present in transcribed RNA (e.g., A, G, C, and/or U). In some aspects, the nucleotides and nucleosides of the invention comprise standard deoxyribonucleosides such as those present in DNA (e.g., dA, dG, dC, and/or dT).
在本發明之其他態樣中，RNA分子可包含經修飾之核鹼基，該等經修飾之核鹼基可併入經修飾之核苷及核苷酸中。在一些態樣中，RNA分子可包括一或多種經修飾之核苷酸。在一些態樣中，RNA分子可包括一或多種經修飾之核苷酸。天然存在之核苷酸修飾為此項技術中已知的。在一些態樣中，RNA分子可包括經修飾之核苷酸。可包括在RNA分子中的經修飾之核苷酸之非限制性實例包括假尿苷、N1-甲基假尿苷、5-甲基尿苷、3-甲基-尿苷、5-甲氧基-尿苷、5-氮雜-尿苷、6-氮雜-尿苷、2-硫-5-氮雜-尿苷、2-硫-尿苷、4-硫-尿苷、4-硫-假尿苷、2-硫-假尿苷、5-羥基-尿苷、5-胺基烯丙基-尿苷、5-鹵基-尿苷(例如5-碘-尿苷或5-溴-尿苷)、尿苷5-氧乙酸、尿苷5-氧乙酸甲酯、5-羧甲基-尿苷、1-羧甲基-假尿苷、5-羧基羥甲基-尿苷、5-羧基羥甲基-尿苷甲酯、5-甲氧基羰基甲基-尿苷、5-甲氧基羰基甲基-2-硫-尿苷、5-胺甲基-2-硫-尿苷、5-甲胺基甲基-尿苷、1-乙基-假尿苷、5-甲胺基甲基-2-硫-尿苷、5-甲胺基甲基-2-硒基-尿苷、5-胺甲醯基甲基-尿苷、5-羧甲基胺甲基-尿苷、5-羧甲基胺甲基-2-硫-尿苷、5-丙炔基-尿苷、1-丙炔基-假尿苷、5-牛磺酸甲基-尿苷、1-牛磺酸甲基-假尿苷、5-牛磺酸甲基-2-硫-尿苷、1-牛磺酸甲基-4-硫-假尿苷、5-甲基-2-硫-尿苷、1-甲基-4-硫-假尿苷、4-硫-1-甲基-假尿苷、3-甲基-1-假尿苷、2-硫-1-甲基-假尿苷、1-甲基-1-脫氮-假尿苷、2-硫-1-甲基-1-脫氮-假尿苷、二氫尿苷、二氫假尿苷、5,6-二氫尿苷、5-甲基-二氫尿苷、2-硫-二氫尿苷、2-硫-二氫假尿苷、2-甲氧基-尿苷、2-甲氧基-4-硫-尿苷、4-甲氧基-假尿苷、4-甲氧基-2-硫-假尿苷、N1-甲基-假尿苷、3-(3-胺基-3-羧丙基)尿苷、1-甲基-3-(3-胺基-3-羧丙基)假尿苷、5-(異戊烯基胺甲基)尿苷、5-(異戊烯基胺甲基)-2-硫-尿苷、a-硫-尿苷、2'-O-甲基-尿苷、5,2'-O-二甲基-尿苷、2'-O-甲基-假尿苷、2-硫-2'-O-甲基-尿苷、5-甲氧基羰基甲基-2'-O-甲基-尿苷、5-胺甲醯基甲基-2'-O-甲基-尿苷、5-羧甲基胺甲基-2'-O-甲基-尿苷、3,2'-O-二甲基-尿苷、5-(異戊烯基胺甲基)-2'-O-甲基-尿苷、1-硫-尿苷、去氧胸苷、2'-F-阿糖尿苷、2'-F-尿苷、2'-OH-阿糖尿苷、5-(2-甲氧羰基乙烯基)尿苷、5-[3-(1-E-丙烯基胺基)尿苷、此項技術中已知的任何其他經修飾之尿苷，或其組合。在一些態樣中，前述經修飾之核苷酸中之1、2、3、4、5者或更多者可排除在本文所揭示之RNA分子外。In other aspects of the invention, RNA molecules may include modified nucleobases that may be incorporated into modified nucleosides and nucleotides. In some aspects, RNA molecules may include one or more modified nucleotides. In some aspects, RNA molecules may include one or more modified nucleotides. Naturally occurring nucleotide modifications are known in the art. In some aspects, RNA molecules may include modified nucleotides. Non-limiting examples of modified nucleotides that can be included in the RNA molecule include pseudouridine, N1-methylpseudouridine, 5-methyluridine, 3-methyl-uridine, 5-methoxy-uridine, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine, 4-thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine, 5-aminoallyl-uridine, 5-halogen-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid, uridine 5-oxyacetic acid methyl ester, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine, 5-carboxyhydroxymethyl-uridine methyl ester, 5-methoxycarbonylmethyl-uridine, 5-methoxycarbonylmethyl-2-thio-uridine, 5-aminomethyl-2-thio-uridine, 5-methylaminomethyl-uridine, 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine, 5-methylaminomethyl-2-seleno-uridine, 5-aminoformylmethyl-uridine, 5-carboxymethylaminomethyl-uridine, 5-carboxymethylaminomethyl-2-thio-uridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinemethyl-uridine, 1-taurinemethyl-pseudouridine, 5-taurinemethyl-2-thio-uridine, 1-taurinemethyl-4-thio-pseudouridine, 5-methyl-2-thio-uridine, 1-methyl-4-thio-pseudouridine, 4-thio-1-methyl-pseudouridine, 3-methyl-1-pseudouridine uridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4- Methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, 5-(isopentenylaminomethyl)uridine, 5-(isopentenylaminomethyl)-2-thio-uridine, a-thio-uridine, 2'-O-methyl-uridine uridine, 5,2'-O-dimethyl-uridine, 2'-O-methyl-pseudouridine, 2-thio-2'-O-methyl-uridine, 5-methoxycarbonylmethyl-2'-O-methyl-uridine, 5-aminoformylmethyl-2'-O-methyl-uridine, 5-carboxymethylaminomethyl-2'-O-methyl-uridine, 3,2'-O-dimethyl-uridine, 5-(isopentenylaminomethyl)-2'-O-methyl-uridine, 1-thio-uridine, deoxythymidine, 2'-F-arabinouridine, 2'-F-uridine, 2'-OH-arabinouridine, 5-(2-methoxycarbonylvinyl)uridine, 5-[3-(1-E-propenylamino)uridine, any other modified uridine known in the art, or a combination thereof. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned modified nucleotides may be excluded from the RNA molecules disclosed herein.
RNA分子中可存在之修飾進一步包括但不限於例如以下：ms2io6A (2-甲基硫-(N6-(順式-羥基異戊烯基)腺苷)；ms2m6A (2-甲基硫-N6-甲基腺苷)；ms2t6A 2-甲基硫-N6-蘇胺醯基胺甲醯基腺苷；g6A (N6-甘胺醯基胺甲醯基腺苷)；i6A (N6-異戊烯基腺苷)；m6A (N6-甲基腺苷)；t6A (N6-蘇胺醯基胺甲醯基腺苷)；m'Am (1,2'-O-二甲基腺苷)；m1A (1-甲基腺苷)；2'-O-甲基腺苷；Ar(p) (2'-O-核糖基腺苷(磷酸酯))；2-甲基腺苷；2-甲基硫-N6-異戊烯基腺苷；ms2hn6A (2-甲基硫-N6-羥基正纈胺醯基胺甲醯基腺苷)；2-O-甲基腺苷；Am (2-1-O-甲基腺苷)；2'-O-核糖基腺苷(磷酸酯)；異戊烯基腺苷；io6A N6-(順式-羥基異戊烯基)腺苷；m6Am (N6,2'-O-二甲基腺苷)；m62Am (N6,N6,2'-O-三甲基腺苷)；m62A (N6,N6-二甲基腺苷)；ac6A (N6-乙醯基腺苷)；hn6A (N6-羥基正纈胺醯基胺甲醯基腺苷)；m6t6A (N6-甲基-N6-蘇胺醯基胺甲醯基腺苷)；m2A (2-甲基腺苷)；ms2i6A (2-甲基硫-N6-異戊烯基腺苷)；7-脫氮-腺苷；N1-甲基-腺苷；N6,N6-(二甲基)腺嘌呤；N6-順式-羥基-異戊烯基-腺苷；a-硫-腺苷；2-(胺基)腺嘌呤；2-(胺基丙基)腺嘌呤；2-(甲基硫)-N6-(異戊烯基)腺嘌呤；2-(烷基)腺嘌呤；2-(胺基烷基)腺嘌呤；2-(胺基丙基)腺嘌呤；2-(鹵基)腺嘌呤；2-(鹵基)腺嘌呤；2-(丙基)腺嘌呤；2'-胺基-2'-去氧-ATP；2'-疊氮基-2'-去氧-ATP；2'-去氧-2'-a-胺基腺苷TP；2'-去氧-2'-a-疊氮基腺苷TP；6-(烷基)腺嘌呤；6-(甲基)腺嘌呤；6-(烷基)腺嘌呤；6-(甲基)腺嘌呤；7-(脫氮)腺嘌呤；8-(烯基)腺嘌呤；8-(炔基)腺嘌呤；8-(胺基)腺嘌呤；8-(硫烷基)腺嘌呤；8-(烯基)腺嘌呤；8-(烷基)腺嘌呤；8-(炔基)腺嘌呤；8-(胺基)腺嘌呤；8-(鹵基)腺嘌呤；8-(羥基)腺嘌呤；8-(硫烷基)腺嘌呤；8-(硫醇基)腺嘌呤；8-疊氮基-腺苷；8-側氧基-腺嘌呤；氮雜腺嘌呤；脫氮腺嘌呤；N6-(甲基)腺嘌呤；N6-(異戊基)腺嘌呤；7-脫氮-8-氮雜-腺苷；7-甲基腺嘌呤；1-脫氮腺苷TP；2'氟-N6-Bz-去氧腺苷TP；2'-OMe-2-胺基-ATP；2'O-甲基-N6-Bz-去氧腺苷TP；2'-a-乙炔基腺苷TP；2-胺基腺嘌呤；2-胺基腺苷TP；2-胺基-ATP；2'-a-三氟甲基腺苷TP；2-疊氮基腺苷TP；2'-b-乙炔基腺苷TP；2-溴腺苷TP；2'-b-三氟甲基腺苷TP；2-氯腺苷TP；2'-去氧-2',2'-二氟腺苷TP；2'-去氧-2'-a-巰基腺苷TP；2'-去氧-2'-a-硫甲氧基腺苷TP；2'-去氧-2'-b-胺基腺苷TP；2'-去氧-2'-b-疊氮基腺苷TP；2'-去氧-2'-b-溴腺苷TP；2'-去氧-2'-b-氯腺苷TP；2'-去氧-2'-b-氟腺苷TP；2'-去氧-2'-b-碘腺苷TP；2'-去氧-2'-b-巰基腺苷TP；2'-去氧-2'-b-硫甲氧基腺苷TP；2-氟腺苷TP；2-碘腺苷TP；2-巰基腺苷TP；2-甲氧基-腺嘌呤；2-甲基硫-腺嘌呤；2-三氟甲基腺苷TP；3-脫氮-3-溴腺苷TP；3-脫氮-3-氯腺苷TP；3-脫氮-3-氟腺苷TP；3-脫氮-3-碘腺苷TP；3-脫氮腺苷TP；4'-疊氮基腺苷TP；4'-碳環腺苷TP；4'-乙炔基腺苷TP；5'-高(Homo)-腺苷TP；8-氮雜-ATP；8-溴-腺苷TP；8-三氟甲基腺苷TP；9-脫氮腺苷TP；2-胺基嘌呤；經取代之7-脫氮嘌呤；7-脫氮-7取代之嘌呤；7-脫氮-8取代之嘌呤；7-脫氮-2,6-二胺基嘌呤；7-脫氮-8-氮雜-2,6-二胺基嘌呤；7-脫氮-8-氮雜-2-胺基嘌呤；2,4-二胺基嘌呤；2,6-二胺基嘌呤；7-脫氮-8-氮雜-腺嘌呤；7-脫氮-2-胺基嘌呤；8-氮雜嘌呤；s2C (2-硫胞苷)；m3C (3-甲基胞苷)；f5C (5-甲醯基胞苷)；hm5C (5-羥基甲基胞苷)；m5C (5-甲基胞苷)；ac4C (N4-乙醯基胞苷)；Cm (2'-O-甲基胞苷)；m5Cm (5,2'-O-二甲基胞苷)；f5Cm (5-甲醯基-2'-O-甲基胞苷)；k2C (立西啶(Lysidine))；m4Cm (N4,2'-O-二甲基胞苷)；ac4Cm (N4-乙醯基-2'-O-甲基胞苷)；m4C (N4-甲基胞苷)；N4,N4-二甲基-2'-OMe-胞苷TP；4-甲基胞苷；5-氮雜-胞苷；假-異-胞苷；吡咯并-胞苷；a-硫-胞苷；2-(硫)胞嘧啶；2'-胺基-2'-去氧-CTP；2'-疊氮基-2'-去氧-CTP；2'-去氧-2'-a-胺基胞苷TP；2'-去氧-2'-a-疊氮基胞苷TP；3-(脫氮) 5-(氮雜)胞嘧啶；3-(甲基)胞嘧啶；3-(烷基)胞嘧啶；3-(脫氮) 5-(氮雜)胞嘧啶；3-(甲基)胞苷；4,2'-O-二甲基胞苷；5-(鹵基)胞嘧啶；5-(甲基)胞嘧啶；5-(丙炔基)胞嘧啶；5-(三氟甲基)胞嘧啶；5-氯胞嘧啶；5-氟胞嘧啶；5-溴胞嘧啶；5-羥基胞嘧啶；5-甲基胞嘧啶；5-(烷基)胞嘧啶；5-(烯基)胞嘧啶；5-(炔基)胞嘧啶；5-(鹵基)胞嘧啶；5-(丙炔基)胞嘧啶；5-(三氟甲基)胞嘧啶；5-溴-胞苷；5-碘-胞苷；5-丙炔基胞嘧啶；6-(偶氮)胞嘧啶；6-氮雜-胞苷；氮雜胞嘧啶；脫氮胞嘧啶；N4-(乙醯基)胞嘧啶；1-甲基-1-脫氮-假異胞苷；1-甲基-假異胞苷；2-甲氧基-5-甲基-胞苷；2-甲氧基-胞苷；2-硫-5-甲基-胞苷；4-甲氧基-1-甲基-假異胞苷；4-甲氧基-假異胞苷；4-硫-1-甲基-1-脫氮-假異胞苷；4-硫-1-甲基-假異胞苷；4-硫-假異胞苷；5-氮雜-澤布拉林(zebularine)；5-甲基-澤布拉林；吡咯并-假異胞苷；澤布拉林；(E)-5-(2-溴-乙烯基)胞苷TP；2,2'-脫水-胞苷TP鹽酸鹽；2'氟-N4-Bz-胞苷TP；2'氟-N4-乙醯基-胞苷TP；2'-O-甲基-N4-乙醯基-胞苷TP；2'O-甲基-N4-Bz-胞苷TP；2'-a-乙炔基胞苷TP；2'-a-三氟甲基胞苷TP；2'-b-乙炔基胞苷TP；2'-b-三氟甲基胞苷TP；2'-去氧-2',2'-二氟胞苷TP；2'-去氧-2'-a-巰基胞苷TP；2'-去氧-2'-a-硫甲氧基胞苷TP；2'-去氧-2'-b-胺基胞苷TP；2'-去氧-2'-b-疊氮基胞苷TP；2'-去氧-2'-b-溴胞苷TP；2'-去氧-2'-b-氯胞苷TP；2'-去氧-2'-b-氟胞苷TP；2'-去氧-2'-b-碘胞苷TP；2'-去氧-2'-b-巰基胞苷TP；2'-去氧-2'-b-硫甲氧基胞苷TP；2'-O-甲基-5-(1-丙炔基)胞苷TP；3'-乙炔基胞苷TP；4'-疊氮基胞苷TP；4'-碳環胞苷TP；4'-乙炔基胞苷TP；5-(1-丙炔基)阿糖-胞苷TP；5-(2-氯-苯基)-2-硫胞苷TP；5-(4-胺基-苯基)-2-硫胞苷TP；5-胺基烯丙基-CTP；5-氰基胞苷TP；5-乙炔基阿糖-胞苷TP；5-乙炔基胞苷TP；5'-高-胞苷TP；5-甲氧基胞苷TP；5-三氟甲基-胞苷TP；N4-胺基-胞苷TP；N4-苯甲醯基-胞苷TP；假異胞苷；mimG (甲基鳥苷)；m7G (7-甲基鳥苷)；m2Gm (N2,2'-O-二甲基鳥苷)；m2G (N2-甲基鳥苷)；imG (懷俄苷(Wyosine))；m1Gm (1,2'-O-二甲基鳥苷)；m1G (1-甲基鳥苷)；2'-O-甲基鳥苷；2'-O-核糖基鳥苷(磷酸酯)；Gm (2'-O-甲基鳥苷)；Gr(p) (2'-O-核糖基鳥苷(磷酸酯))；preQi (7-胺基甲基-7-脫氮鳥苷)；preQo (7-氰基-7-脫氮鳥苷)；G* (古嘌苷(Archaeosine))；甲基懷俄苷；m2'7G (N2,7-二甲基鳥苷)；m22Gm (N2,N2,2'-O-三甲基鳥苷)；m2'2'7G (N2,N2,7-三甲基鳥苷)；m22G (N2,N2-二甲基鳥苷)；N2,7,2'-O-三甲基鳥苷；6-硫-鳥苷；7-脫氮-鳥苷；8-側氧基-鳥苷；N1-甲基-鳥苷；a-硫-鳥苷；2-(丙基)鳥嘌呤；2-(烷基)鳥嘌呤；2'-胺基-2'-去氧-GTP；2'-疊氮基-2'-去氧-GTP；2'-去氧-2'-a-胺基鳥苷TP；2'-去氧-2'-a-疊氮基鳥苷TP；N2-二甲基鳥嘌呤；6-(甲基)鳥嘌呤；6-(烷基)鳥嘌呤；6-(甲基)鳥嘌呤；6-甲基-鳥苷；6-硫鳥嘌呤；7-(烷基)鳥嘌呤；7-脫氮-7取代之鳥嘌呤；7-脫氮-7-(C2-C6)炔基鳥嘌呤；7-脫氮-8取代之鳥嘌呤；7-(甲基)鳥嘌呤；7-(烷基)鳥嘌呤；7-(脫氮)鳥嘌呤；7-(甲基)鳥嘌呤；8-氮雜鳥嘌呤；8-羥基鳥嘌呤；8-側氧基鳥嘌呤；8-(烷基)鳥嘌呤；8-(炔基)鳥嘌呤；8-(鹵基)鳥嘌呤；8-(硫烷基)鳥嘌呤；8-(烯基)鳥嘌呤；8-(烷基)鳥嘌呤；8-(炔基)鳥嘌呤；8-(胺基)鳥嘌呤；8-(鹵基)鳥嘌呤；8-(羥基)鳥嘌呤；8-(硫烷基)鳥嘌呤；8-(硫醇基)鳥嘌呤；氮雜鳥嘌呤；脫氮鳥嘌呤；N (甲基)鳥嘌呤；N-(甲基)鳥嘌呤；1-甲基-6-硫-鳥苷；6-甲氧基-鳥苷；6-硫-7-脫氮-8-氮雜-鳥苷；6-硫-7-脫氮-鳥苷；6-硫-7-甲基-鳥苷；7-脫氮-8-氮雜-鳥苷；7-甲基-8-側氧基-鳥苷；N2,N2-二甲基-6-硫-鳥苷；N2-甲基-6-硫-鳥苷；1-me-GTP；2'氟-N2-異丁基-鳥苷TP；2'-O-甲基-N2-異丁基-鳥苷TP；2'-a-乙炔基鳥苷TP；2'-a-三氟甲基鳥苷TP；2'-b-乙炔基鳥苷TP；2'-b-三氟甲基鳥苷TP；2'-去氧-2',2'-二氟鳥苷TP；2'-去氧-2'-a-巰基鳥苷TP；2'-去氧-2'-a-硫甲氧基鳥苷TP；2'-去氧-2'-b-胺基鳥苷TP；2'-去氧-2'-b-疊氮基鳥苷TP；2'-去氧-2'-b-溴鳥苷TP；2'-去氧-2'-b-氯鳥苷TP；2'-去氧-2'-b-氟鳥苷TP；2'-去氧-2'-b-碘鳥苷TP；2'-去氧-2'-b-巰基鳥苷TP；2'-去氧-2'-b-硫甲氧基鳥苷TP；4'-疊氮基鳥苷TP；4'-碳環鳥苷TP；4'-乙炔基鳥苷TP；5'-高-鳥苷TP；8-溴-鳥苷TP；9-脫氮鳥苷TP；N2-異丁基-鳥苷TP；miI (1-甲基肌苷)；I (肌苷)；m'lm (1,2'-O-二甲基肌苷)；2'-O-甲基肌苷；7-甲基肌苷；Tm (2'-O-甲基肌苷)；oQ (環氧基Q核苷(queuosine))；galQ (半乳糖苷-Q核苷)；manQ (甘露糖基Q核苷)；Q (Q核苷)；烯丙基胺基-胸苷；氮雜胸苷；脫氮胸苷；去氧-胸苷；Um (2'-O-甲基尿苷)；s2U (2-硫尿苷)；m3U (3-甲基尿苷)；cm5U (5-羧基甲基尿苷)；ho5U (5-羥基尿苷)；m5U (5-甲基尿苷)；tm5s2U (5-牛磺酸甲基-2-硫尿苷)；5-牛磺酸甲基尿苷；D (二氫尿苷)；假尿苷；acp3U (3-(3-胺基-3-羧基丙基)尿苷)；1-甲基-3-(3-胺基-5-羧基丙基)假尿苷；1-甲基假尿苷；1-乙基-假尿苷；2'-O-甲基尿苷；2'-O-甲基假尿苷；2'-O-甲基尿苷；s2Um (2-硫-2'-O-甲基尿苷)；3-(3-胺基-3-羧基丙基)尿苷；m3Um (3,2'-O-二甲基尿苷)；3-甲基-假-尿苷TP；s4U (4-硫尿苷)；chm5U (5-(羧基羥基甲基)尿苷)；mchm5U (5-(羧基羥基甲基)尿苷甲酯)；m5Um (5,2'-O-二甲基尿苷)；5,6-二氫-尿苷；nm5s2U (5-胺基甲基-2-硫尿苷)；ncm5Um (5-胺甲醯基甲基-2'-O-甲基尿苷)；ncm5U (5-胺甲醯基甲基尿苷)；5-羧基羥基甲基尿苷；5-羧基羥基甲基尿苷甲酯；cnmm5Um (5-羧基甲基胺基甲基-2'-O-甲基尿苷)；cmnm5s2U (5-羧基甲基胺基甲基-2-硫尿苷)；5-羧基甲基胺基甲基尿苷；cmnm5U (5-羧基甲基胺基甲基尿苷)；5-胺甲醯基甲基尿苷TP；mcm5Um (5-甲氧基羰基甲基-2'-O-甲基尿苷)；mcm5s2U (5-甲氧基羰基甲基-2-硫尿苷)；mcm5U (5-甲氧基羰基甲基尿苷)；mo5U (5-甲氧基尿苷)；m5s2U (5-甲基-2-硫尿苷)；mnm5se2U (5-甲基胺基甲基-2-硒基尿苷)；mnm5s2U (5-甲基胺基甲基-2-硫尿苷)；mnm5U (5-甲基胺基甲基尿苷)；m5D (5-甲基二氫尿苷)；5-氧基乙酸-尿苷TP；5-氧基乙酸-甲酯-尿苷TP；二氫尿嘧啶；假尿嘧啶；N1-甲基-假-尿嘧啶；N1-乙基-假-尿嘧啶；cmo5U (尿苷5-氧基乙酸)；mcmo5U (尿苷5-氧基乙酸甲酯)；3-(3-胺基-3-羧基丙基)-尿苷TP；5-(異-戊烯基胺基甲基)-2-硫尿苷TP；5-(異-戊烯基胺基甲基)-2'-O-甲基尿苷TP；5-(異-戊烯基胺基甲基)尿苷TP；5-丙炔基尿嘧啶；a-硫-尿苷；1-(胺基烷基胺基-羰基乙烯基)-2(硫)-假尿嘧啶；1-(胺基烷基胺基-羰基乙烯基)-2,4-(二硫)假尿嘧啶；1-(胺基烷基胺基-羰基乙烯基)-4-(硫)假尿嘧啶；1-(胺基烷基胺基-羰基乙烯基)-假尿嘧啶；1-(胺基羰基乙烯基)-2(硫)-假尿嘧啶；1-(胺基羰基乙烯基)-2,4-(二硫)假尿嘧啶；1-(胺基羰基乙烯基)-4-(硫)假尿嘧啶；1-(胺基羰基乙烯基)-假尿嘧啶；1-取代之2(硫)-假尿嘧啶；1-取代之2,4-(二硫)假尿嘧啶；1-取代之4-(硫)假尿嘧啶；1-取代之假尿嘧啶；1-(胺基烷基胺基-羰基乙烯基)-2-(硫)-假尿嘧啶；1-甲基-3-(3-胺基-3-羧基丙基) 假尿苷TP；1-甲基-3-(3-胺基-3-羧基丙基)假-UTP；1-甲基-假-UTP；1-乙基-假-UTP；2-(硫)假尿嘧啶；2' 去氧尿苷；2' 氟尿苷；2-(硫)尿嘧啶；2,4-(二硫)假尿嘧啶；2'甲基、2'胺基、2'疊氮基、2'氟-鳥苷；2'-胺基-2'-去氧-UTP；2'-疊氮基-2'-去氧-UTP；2'-疊氮基-去氧尿苷TP；2'-O-甲基假尿苷；2' 去氧尿苷；2' 氟尿苷；2'-去氧-2'-a-胺基尿苷TP；2'-去氧-2'-a-疊氮基尿苷TP；2-甲基假尿苷；3-(3-胺基-3-羧基丙基)尿嘧啶；4-(硫)假尿嘧啶；4-(硫)假尿嘧啶；4-(硫)尿嘧啶；4-硫尿嘧啶；5-胺基尿嘧啶；5-(1,3-二唑-1-烷基)尿嘧啶；5-(2-胺基丙基)尿嘧啶；5-(胺基烷基)尿嘧啶；5-(二甲基胺基烷基)尿嘧啶；5-(胍基烷基)尿嘧啶；5-(甲氧基羰基甲基)-2-(硫)尿嘧啶；5-(甲氧基羰基-甲基)尿嘧啶；5-(甲基) 2-(硫)尿嘧啶；5-(甲基) 2,4-(二硫)尿嘧啶；5-(甲基)-4-(硫)尿嘧啶；5-(甲基胺基甲基)-2-(硫)尿嘧啶；5-(甲基胺基甲基)-2,4-(二硫)尿嘧啶；5-(甲基胺基甲基)-4-(硫)尿嘧啶；5-(丙炔基)尿嘧啶；5-(三氟甲基)尿嘧啶；5-(2-胺基丙基)尿嘧啶；5-(烷基)-2-(硫)假尿嘧啶；5-(烷基)-2,4-(二硫)假尿嘧啶；5-(烷基)-4-(硫)假尿嘧啶；5-(烷基)假尿嘧啶；5-(烷基)尿嘧啶；5-(烯基)尿嘧啶；5-(炔基)尿嘧啶；5-(烯丙基胺基)尿嘧啶；5-(氰基烷基)尿嘧啶；5-(二烷基胺基烷基)尿嘧啶；5-(二甲基胺基烷基)尿嘧啶；5-(胍基烷基)尿嘧啶；5-(鹵基)尿嘧啶；5-(1,3-二唑-1-烷基)尿嘧啶；5-(甲氧基)尿嘧啶；5-(甲氧基羰基甲基)-2-(硫)尿嘧啶；5-(甲氧基羰基-甲基)尿嘧啶；5-(甲基) 2(硫)尿嘧啶；5-(甲基)-2,4-(二硫)尿嘧啶；5-(甲基)-4-(硫)尿嘧啶；5-(甲基)-2-(硫)假尿嘧啶；5-(甲基)-2,4-(二硫)假尿嘧啶；5-(甲基)-4-(硫)假尿嘧啶；5-(甲基)假尿嘧啶；5-(甲基胺基甲基)-2-(硫)尿嘧啶；5-(甲基胺基甲基)-2,4(二硫)尿嘧啶；5-(甲基胺基甲基)-4-(硫)尿嘧啶；5-(丙炔基)尿嘧啶；5-(三氟甲基)尿嘧啶；5-胺基烯丙基-尿苷；5-溴-尿苷；5-碘-尿苷；5-尿嘧啶；6-(偶氮)尿嘧啶；6-(偶氮)尿嘧啶；6-氮雜-尿苷；烯丙基胺基-尿嘧啶；氮雜尿嘧啶；脫氮尿嘧啶；5-甲基尿嘧啶；5-(羥基甲基)尿嘧啶；5-氯尿嘧啶；5-氟尿嘧啶；5-溴尿嘧啶；N3-(甲基)尿嘧啶；假-UTP-1-2-乙酸；假尿嘧啶；4-硫-假-UTP；1-羧基甲基-假尿苷；1-甲基-1-脫氮-假尿苷；1-丙炔基-尿苷；1-牛磺酸甲基-1-甲基-尿苷；1-牛磺酸甲基-4-硫-尿苷；1-牛磺酸甲基-假尿苷；2-甲氧基-4-硫-假尿苷；2-硫-1-甲基-1-脫氮-假尿苷；2-硫-1-甲基-假尿苷；2-硫-5-氮雜-尿苷；2-硫-二氫假尿苷；2-硫-二氫尿苷；2-硫-假尿苷；4-甲氧基-2-硫-假尿苷；4-甲氧基-假尿苷；4-硫-1-甲基-假尿苷；4-硫-假尿苷；5-氮雜-尿苷；二氫假尿苷；(±)1-(2-羥基丙基)假尿苷TP；(2R)-1-(2-羥基丙基)假尿苷TP；(2S)-1-(2-羥基丙基)假尿苷TP；(E)-5-(2-溴-乙烯基)阿糖-尿苷TP；(E)-5-(2-溴-乙烯基)尿苷TP；(Z)-5-(2-溴-乙烯基)阿糖-尿苷TP；(Z)-5-(2-溴-乙烯基)尿苷TP；1-(2,2,2-三氟乙基)-假-UTP；1-(2,2,3,3,3-五氟丙基)假尿苷TP；1-(2,2-二乙氧基乙基)假尿苷TP；1-(2,4,6-三甲基苯甲基)假尿苷TP；1-(2,4,6-三甲基-苯甲基)假-UTP；1-(2,4,6-三甲基-苯基)假-UTP；1-(2-胺基-2-羧基乙基)假-UTP；1-(2-胺基-乙基)假-UTP；1-(2-羥基乙基)假尿苷TP；1-(2-甲氧基乙基)假尿苷TP；1-(3,4-雙-三氟甲氧基苯甲基)假尿苷TP；1-(3,4-二甲氧基苯甲基)假尿苷TP；1-(3-胺基-3-羧基丙基)假-UTP；1-(3-胺基-丙基)假-UTP；1-(3-環丙基-丙-2-炔基)假尿苷TP；1-(4-胺基-4-羧基丁基)假-UTP；1-(4-胺基-苯甲基)假-UTP；1-(4-胺基-丁基)假-UTP；11(4-胺基-苯基)假-UTP；1-(4-疊氮基苯甲基)假尿苷TP；1-(4-溴苯甲基)假尿苷TP；1-(4-氯苯甲基)假尿苷TP；1-(4-氟苯甲基)假尿苷TP；1-(4-碘苯甲基)假尿苷TP；1-(4-甲磺醯基苯甲基)假尿苷TP；1-(4-甲氧基苯甲基)假尿苷TP；1-(4-甲氧基-苯甲基)假-UTP；1-(4-甲氧基-苯基)假-UTP；1-(4-甲基苯甲基)假尿苷TP；1-(4-甲基-苯甲基)假-UTP；1-(4-硝基苯甲基)假尿苷TP；1-(4-硝基-苯甲基)假-UTP；1(4-硝基-苯基)假-UTP；1-(4-硫甲氧基苯甲基)假尿苷TP；1-(4-三氟甲氧基苯甲基)假尿苷TP；1-(4-三氟甲基苯甲基)假尿苷TP；1-(5-胺基-戊基)假-UTP；1-(6-胺基-己基)假-UTP；1,6-二甲基-假-UTP；1-[3-(2-{2-[2-(2-胺基乙氧基)-乙氧基]-乙氧基}-乙氧基)-丙醯基]假尿苷TP；1-{3-[2-(2-胺基乙氧基)-乙氧基]-丙醯基} 假尿苷TP；1-乙醯基假尿苷TP；1-烷基-6-(1-丙炔基)-假-UTP；1-烷基-6-(2-丙炔基)-假-UTP；1-烷基-6-烯丙基-假-UTP；1-烷基-6-乙炔基-假-UTP；1-烷基-6-高烯丙基-假-UTP；1-烷基-6-乙烯基-假-UTP；1-烯丙基假尿苷TP；1-胺基甲基-假-UTP；1-苯甲醯基假尿苷TP；1-苯甲基氧基甲基假尿苷TP；1-苯甲基-假-UTP；1-生物素基-PEG2-假尿苷TP；1-生物素基假尿苷TP；1-丁基-假-UTP；1-氰基甲基假尿苷TP；1-環丁基甲基-假-UTP；1-環丁基-假-UTP；1-環庚基甲基-假-UTP；1-環庚基-假-UTP；1-環己基甲基-假-UTP；1-環己基-假-UTP；1-環辛基甲基-假-UTP；1-環辛基-假-UTP；1-環戊基甲基-假-UTP；1-環戊基-假-UTP；1-環丙基甲基-假-UTP；1-環丙基-假-UTP；1-乙基-假-UTP；1-己基-假-UTP；1-高烯丙基假尿苷TP；1-羥基甲基假尿苷TP；1-異-丙基-假-UTP；1-me-2-硫-假-UTP；1-me-4-硫-假-UTP；1-me-α-硫-假-UTP；1-甲磺醯基甲基假尿苷TP；1-甲氧基甲基假尿苷TP；1-甲基-6-(2,2,2-三氟乙基)假-UTP；1-甲基-6-(4-(N-𠰌啉基))-假-UTP；1-甲基-6-(4-硫(N-𠰌啉基))-假-UTP；1-甲基-6-(經取代之苯基)假-UTP；1-甲基-6-胺基-假-UTP；1-甲基-6-疊氮基-假-UTP；1-甲基-6-溴-假-UTP；1-甲基-6-丁基-假-UTP；1-甲基-6-氯-假-UTP；1-甲基-6-氰基-假-UTP；1-甲基-6-二甲基胺基-假-UTP；1-甲基-6-乙氧基-假-UTP；1-甲基-6-羧酸乙酯-假-UTP；1-甲基-6-乙基-假-UTP；1-甲基-6-氟-假-UTP；1-甲基-6-甲醯基-假-UTP；1-甲基-6-羥基胺基-假-UTP；1-甲基-6-羥基-假-UTP；1-甲基-6-碘-假-UTP；1-甲基-6-異-丙基-假-UTP；1-甲基-6-甲氧基-假-UTP；1-甲基-6-甲基胺基-假-UTP；1-甲基-6-苯基-假-UTP；1-甲基-6-丙基-假-UTP；1-甲基-6-三級丁基-假-UTP；1-甲基-6-三氟甲氧基-假-UTP；1-甲基-6-三氟甲基-假-UTP；1-(N-𠰌啉基)甲基假尿苷TP；1-戊基-假-UTP；1-苯基-假-UTP；1-三甲基乙醯基假尿苷TP；1-炔丙基假尿苷TP；1-丙基-假-UTP；1-丙炔基-假尿苷；1-對甲苯基-假-UTP；1-三級丁基-假-UTP；1-硫甲氧基甲基假尿苷TP；1-硫(N-𠰌啉基)甲基假尿苷TP；1-三氟乙醯基假尿苷TP；1-三氟甲基-假-UTP；1-乙烯基假尿苷TP；2,2'-脫水-尿苷TP；2'-溴-去氧尿苷TP；2'-F-5-甲基-2'-去氧-UTP；2'-OMe-5-me-UTP；2'-OMe-假-UTP；2'-a-乙炔基尿苷TP；2'-a-三氟甲基尿苷TP；2'-b-乙炔基尿苷TP；2'-b-三氟甲基尿苷TP；2'-去氧-2',2'-二氟尿苷TP；2'-去氧-2'-a-巰基尿苷TP；2'-去氧-2'-a-硫甲氧基尿苷TP；2'-去氧-2'-b-胺基尿苷TP；2'-去氧-2'-b-疊氮基尿苷TP；2'-去氧-2'-b-溴尿苷TP；2'-去氧-2'-b-氯尿苷TP；2'-去氧-2'-b-氟尿苷TP；2'-去氧-2'-b-碘尿苷TP；2'-去氧-2'-b-巰基尿苷TP；2'-去氧-2'-b-硫甲氧基尿苷TP；2-甲氧基-4-硫-尿苷；2-甲氧基尿苷；2'-O-甲基-5-(1-丙炔基)尿苷TP；3-烷基-假-UTP；4'-疊氮基尿苷TP；4'-碳環尿苷TP；4'-乙炔基尿苷TP；5-(1-丙炔基)阿糖-尿苷TP；5-(2-呋喃基)尿苷TP；5-氰基尿苷TP；5-二甲基胺基尿苷TP；5'-高-尿苷TP；5-碘-2'-氟-去氧尿苷TP；5-苯基乙炔基尿苷TP；5-三氘代甲基-6-氘代尿苷TP；5-三氟甲基-尿苷TP；5-乙烯基阿糖尿苷TP；6-(2,2,2-三氟乙基)-假-UTP；6-(4-(N-𠰌啉基))-假-UTP；6-(4-硫(N-𠰌啉基))-假-UTP；6-(經取代之苯基)-假-UTP；6-胺基-假-UTP；6-疊氮基-假-UTP；6-溴-假-UTP；6-丁基-假-UTP；6-氯-假-UTP；6-氰基-假-UTP；6-二甲基胺基-假-UTP；6-乙氧基-假-UTP；6-羧酸乙酯-假-UTP；6-乙基-假-UTP；6-氟-假-UTP；6-甲醯基-假-UTP；6-羥基胺基-假-UTP；6-羥基-假-UTP；6-碘-假-UTP；6-異-丙基-假-UTP；6-甲氧基-假-UTP；6-甲基胺基-假-UTP；6-甲基-假-UTP；6-苯基-假-UTP；6-苯基-假-UTP；6-丙基-假-UTP；6-三級丁基-假-UTP；6-三氟甲氧基-假-UTP；6-三氟甲基-假-UTP；α-硫-假-UTP；假尿苷1-(4-甲基苯磺酸) TP；假尿苷1-(4-甲基苯甲酸) TP；假尿苷TP 1-[3-(2-乙氧基)]丙酸；假尿苷TP 1-[3-{2-(2-[2-(2-乙氧基)-乙氧基]-乙氧基)-乙氧基}]丙酸；假尿苷TP 1-[3-{2-(2-[2-{2(2-乙氧基)-乙氧基}-乙氧基]-乙氧基)-乙氧基}]丙酸；假尿苷TP 1-[3-{2-(2-[2-乙氧基]-乙氧基)-乙氧基}]丙酸；假尿苷TP 1-[3-{2-(2-乙氧基)-乙氧基}] 丙酸；假尿苷TP 1-甲基膦酸；假尿苷TP 1-甲基膦酸二乙酯；假-UTP-N1-3-丙酸；假-UTP-N1-4-丁酸；假-UTP-N1-5-戊酸；假-UTP-N1-6-己酸；假-UTP-N1-7-庚酸；假-UTP-N1-甲基-對苯甲酸；假-UTP-N1-對苯甲酸；yW (懷俄丁苷(Wybutosine))；OHyW (羥基懷俄丁苷)；imG2 (異懷俄苷)；o2yW (過氧基懷俄丁苷)；OHyW* (欠修飾之羥基懷俄丁苷)；imG-14 (4-去甲基懷俄苷)；2,6-(二胺基)嘌呤；1-(氮雜)-2-(硫)-3-(氮雜)-啡㗁𠯤-1-基；1,3-(二氮雜)-2-(側氧基)-啡噻𠯤-1-基；1,3-(二氮雜)-2-(側氧基)-啡㗁𠯤-1-基；1,3,5-(三氮雜)-2,6-(二氧雜)-萘；2-(胺基)嘌呤；2,4,5-(三甲基)苯基；2' 甲基、2'胺基、2'疊氮基、2'氟-胞苷；2' 甲基、2'胺基、2'疊氮基、2'氟-腺嘌呤；2'甲基、2'胺基、2'疊氮基、2'氟-尿苷；2'-胺基-2'-去氧核糖；2-胺基-6-氯-嘌呤；2-氮雜-肌苷基；2'-疊氮基-2'-去氧核糖；2'氟-2'-去氧核糖；2'-氟-經修飾之鹼基；2'-O-甲基-核糖；2-側氧基-7-胺基吡啶并嘧啶-3-基；2-側氧基-吡啶并嘧啶-3-基；2-吡啶酮；3-硝基吡咯；3-(甲基)-7-(丙炔基)異喹諾酮基(carbostyrilyl)；3-(甲基)異喹諾酮基；4-(氟)-6-(甲基)苯并咪唑；4-(甲基)苯并咪唑；4-(甲基)吲哚基；4,6-(二甲基)吲哚基；5-硝基吲哚；5-取代之嘧啶；5-(甲基)異喹諾酮基；5-硝基吲哚；6-(氮雜)嘧啶；6-(偶氮)胸腺嘧啶；6-(甲基)-7-(氮雜)吲哚基；6-氯-嘌呤；6-苯基-吡咯并-嘧啶-2-酮-3-基；7-(胺基烷基羥基)-1-(氮雜)-2-(硫)-3-(氮雜)-啡噻𠯤-1-基；7-(胺基烷基羥基)-1-(氮雜)-2-(硫)-3-(氮雜)-啡㗁𠯤-1-基；7-(胺基烷基羥基)-1,3-(二氮雜)-2-(側氧基)-啡㗁𠯤-1-基；7-(胺基烷基羥基)-1,3-(二氮雜)-2-(側氧基)-啡噻𠯤-1-基；7-(胺基烷基羥基)-1,3-(二氮雜)-2-(側氧基)-啡㗁𠯤-1-基；7-(氮雜)吲哚基；7-(胍基烷基羥基)-1-(氮雜)-2-(硫)-3-(氮雜)-啡㗁𠯤l-基；7-(胍基烷基羥基)-1-(氮雜)-2-(硫)-3-(氮雜)-啡噻𠯤-1-基；7-(胍基烷基羥基)-1-(氮雜)-2-(硫)-3-(氮雜)-啡㗁𠯤-1-基；7-(胍基烷基羥基)-1,3-(二氮雜)-2-(側氧基)-啡㗁𠯤-1-基；7-(胍基烷基-羥基)-1,3-(二氮雜)-2-(側氧基)-啡噻𠯤-1-基；7-(胍基烷基羥基)-1,3-(二氮雜)-2-(側氧基)-啡㗁𠯤-1-基；7-(丙炔基)異喹諾酮基；7-(丙炔基)異喹諾酮基；丙炔基-7-(氮雜)吲哚基；7-脫氮-肌苷基；7取代之1-(氮雜)-2-(硫)-3-(氮雜)-啡㗁𠯤-1-基；7取代之1,3-(二氮雜)-2-(側氧基)-啡㗁𠯤-1-基；9-(甲基)-咪唑并吡啶基；胺基吲哚基；蒽基；雙-鄰位-(胺基烷基羥基)-6-苯基-吡咯并-嘧啶-2-酮-3-基；雙-鄰位取代之6-苯基-吡咯并-嘧啶-2-酮-3-基；二氟甲苯基；次黃嘌呤；咪唑并吡啶基；肌苷基；異喹諾酮基；異鳥苷(isoguanisine)；N2-取代之嘌呤；N6-甲基-2-胺基-嘌呤；N6取代之嘌呤；N-烷基化衍生物；萘基；硝基苯并咪唑基；硝基咪唑基；硝基吲唑基；硝基吡唑基；魯布拉潤(nubularine)；O6-取代之嘌呤；O-烷基化衍生物；鄰位-(胺基烷基羥基)-6-苯基-吡咯并-嘧啶-2-酮-3-基；鄰位取代之-6-苯基-吡咯并-嘧啶-2-酮-3-基；氧間型黴素(oxoformycin) TP；對(胺基烷基羥基)-6-苯基-吡咯并-嘧啶-2-酮-3-基；對位取代之6-苯基-吡咯并-嘧啶-2-酮-3-基；稠五苯基；丙烯合蒽基；苯基；丙炔基-7-(氮雜)吲哚基；芘基；吡啶并嘧啶-3-基；吡啶并嘧啶-3-基；2-側氧基-7-胺基-吡啶并嘧啶-3-基；吡咯并-嘧啶-2-酮-3-基；吡咯并嘧啶基；吡咯并吡𠯤基；均二苯乙烯基(stilbenzyl)；經取代之1,2,4-三唑；并四苯基；殺結核菌素(tubercidine)；黃嘌呤；黃苷-5'-TP；2-硫-澤布拉林；5-氮雜-2-硫-澤布拉林；7-脫氮-2-胺基-嘌呤；吡啶-4-酮核糖核苷；2-胺基核糖苷-TP；間型黴素(formycin) A TP；間型黴素B TP；吡咯離胺酸(pyrrolosine) TP；2'-OH-阿糖-腺苷TP；2'-OH-阿糖-胞苷TP；2'-OH-阿糖-尿苷TP；2'-OH-阿糖-鳥苷TP；5-(2-甲氧羰基乙烯基)尿苷TP；N6-(19-胺基-五氧雜十九烷基)腺苷TP；氫(無鹼基殘基)；及2'-O-甲基-U。在一些態樣中，RNA分子包括前述經修飾之核鹼基中之至少兩者(例如2、3、4者或更多者)之組合。在一些態樣中，前述修飾中之1、2、3、4、5者或更多者可排除在本文所揭示之RNA分子外。Modifications that may be present in the RNA molecule further include, but are not limited to, for example, the following: ms2io6A (2-methylthio-(N6-(cis-hydroxyisopentenyl)adenosine); ms2m6A (2-methylthio-N6-methyladenosine); ms2t6A 2-methylthio-N6-threonamidoylaminomethyladenosine; g6A (N6-glycinamidoylaminomethyladenosine); i6A (N6-isopentenyladenosine); m6A (N6-methyladenosine); t6A (N6-threonamidoylaminomethyladenosine); m'Am (1,2'-O-dimethyladenosine); m1A (1-methyladenosine); 2'-O-methyladenosine; Ar(p) (2'-O-ribosyladenosine (phosphate)); 2-methyladenosine; 2-methylthio-N6-isopentenyladenosine; ms2hn6A (2-methylthio-N6-hydroxyn-valeramidoylaminoformyladenosine); 2-O-methyladenosine; Am (2-1-O-methyladenosine); 2'-O-ribosyladenosine (phosphate); isopentenyladenosine; io6A N6-(cis-hydroxyisopentenyl)adenosine; m6Am (N6,2'-O-dimethyladenosine); m62Am (N6,N6,2'-O-trimethyladenosine); m62A (N6,N6-dimethyladenosine); ac6A (N6-acetyladenosine); hn6A (N6-hydroxyn-valeramidomethyladenosine); m6t6A (N6-methyl-N6-threamidomethyladenosine); m2A (2-methyladenosine); ms2i6A (2-methylthio-N6-isopentenyladenosine); 7-deazinated adenosine; N1-methyl-adenosine; N6,N6-(dimethyl)adenine; N6-cis-hydroxy-isopentenyl-adenosine; α-thio-adenosine; 2-(amino)adenine; 2-(aminopropyl)adenine; 2-(methylthio)-N6-(isopentenyl)adenine; 2-(alkyl)adenine; 2-(aminoalkyl)adenine; 2-(aminopropyl)adenine; 2 -(halogen)adenine; 2-(halogen)adenine; 2-(propyl)adenine; 2'-amino-2'-deoxy-ATP; 2'-azido-2'-deoxy-ATP; 2'-deoxy-2'-a-aminoadenosine TP; 2'-deoxy-2'-a-azidoadenosine TP; 6-(alkyl)adenine; 6-(methyl)adenine; 6-(alkyl)adenine; 6-(methyl)adenine; 7-(deazinated)adenine; 8-(ene)adenine; 8-(alkenyl)adenine; 8-(alkyl)adenine; 8-(alkynyl)adenine; 8-(amino)adenine; 8-(sulfanyl)adenine; 8-(alkenyl)adenine; 8-(alkyl)adenine; 8-(alkynyl)adenine; 8-(amino)adenine; 8-(halogen)adenine; 8-(hydroxy)adenine; 8-(sulfanyl)adenine; 8-(thiol)adenine; 8-azido-adenosine; 8-oxo-adenine; aza-adenine; deazinated adenine; N6 -(methyl)adenine; N6-(isopentyl)adenine; 7-deaza-8-aza-adenosine; 7-methyladenine; 1-deazaadenosine TP; 2'-fluoro-N6-Bz-deoxyadenosine TP; 2'-OMe-2-amino-ATP; 2'O-methyl-N6-Bz-deoxyadenosine TP; 2'-a-ethynyladenosine TP; 2-aminoadenine; 2-aminoadenosine TP; 2-amino-ATP; 2'-a-trifluoromethyladenosine TP; 2-azidoadenosine TP; 2'-b-ethynyladenosine TP; 2-bromoadenosine TP; 2'-b-trifluoromethyladenosine TP; 2-chloroadenosine TP; 2'-deoxy-2',2'-difluoroadenosine TP; 2'-deoxy-2'-a-hydroxyadenosine TP; 2'-deoxy-2'-a-thiomethoxyadenosine TP; 2'-deoxy-2'-b-aminoadenosine TP; 2'-deoxy-2'-b-azidoadenosine TP; '-deoxy-2'-b-bromoadenosine TP; 2'-deoxy-2'-b-chloroadenosine TP; 2'-deoxy-2'-b-fluoroadenosine TP; 2'-deoxy-2'-b-iodoadenosine TP; 2'-deoxy-2'-b-hydroxyadenosine TP; 2'-deoxy-2'-b-thiomethoxyadenosine TP; 2-fluoroadenosine TP; 2-iodoadenosine TP; 2-hydroxyadenosine TP; 2-methoxy-adenine; 2-methylthio-adenine; 2-triazine Fluoromethyladenosine TP; 3-deaza-3-bromoadenosine TP; 3-deaza-3-chloroadenosine TP; 3-deaza-3-fluoroadenosine TP; 3-deaza-3-iodoadenosine TP; 3-deazaadenosine TP; 4'-azidoadenosine TP; 4'-carbocyclic adenosine TP; 4'-ethynyladenosine TP; 5'-homo-adenosine TP; 8-aza-ATP; 8-bromoadenosine TP; 8-trifluoromethyladenosine TP; 9-deazaadenosine TP; 2-aminopurine; substituted 7-deazapurine; 7-deaza-7-substituted purine; 7-deaza-8-substituted purine; 7-deaza-2,6-diaminopurine; 7-deaza-8-aza-2,6-diaminopurine; 7-deaza-8-aza-2-aminopurine; 2,4-diaminopurine; 2,6-diaminopurine; 7-deaza-8-aza-adenine; 7-deaza-2-aminopurine; 8-azapurine; s2C (2-thiocytidine); m3C (3-methylcytidine); f5C (5-methylcytidine); hm5C (5-hydroxymethylcytidine); m5C (5-methylcytidine); ac4C (N4-acetylcytidine); Cm (2'-O-methylcytidine); m5Cm (5,2'-O-dimethylcytidine); f5Cm (5-methyl-2'-O-methylcytidine); k2C (lysidine); m4Cm (N4,2'-O-dimethylcytidine); ac4Cm (N4-acetyl-2'-O-methylcytidine); m4C (N4-methylcytidine); N4,N4-dimethyl-2'-OMe-cytidine TP; 4-methylcytidine; 5-aza-cytidine; pseudo-iso-cytidine; pyrrolo-cytidine; a-thio-cytidine; 2-(thio)cytidine; 2'-amino-2'-deoxy-CTP; 2'-azido-2'-deoxy-CTP; 2'-deoxy-2'-a-aminocytidine TP; 2'-deoxy-2'-a-azidocytidine TP; 3-(deaza) 5-(aza)cytidine; 3-(methyl)cytosine; 3-(alkyl)cytosine; 3-(deaza) 5-(aza)cytosine; 3-(methyl)cytidine; 4,2'-O-dimethylcytidine; 5-(halogen)cytosine; 5-(methyl)cytosine; 5-(propynyl)cytosine; 5-(trifluoromethyl)cytosine; 5-chlorocytosine; 5-fluorocytosine; 5-bromocytosine; 5-hydroxycytosine; 5-methylcytosine; 5-(alkyl)cytosine; 5-( 5-(alkenyl)cytosine; 5-(alkynyl)cytosine; 5-(halogen)cytosine; 5-(propynyl)cytosine; 5-(trifluoromethyl)cytosine; 5-bromo-cytidine; 5-iodo-cytidine; 5-propynylcytosine; 6-(azo)cytosine; 6-aza-cytidine; azacytosine; deazocytidine; N4-(acetyl)cytosine; 1-methyl-1-deazocytidine Isocytidine; 1-methyl-pseudoisocytidine; 2-methoxy-5-methyl-cytidine; 2-methoxy-cytidine; 2-thio-5-methyl-cytidine; 4-methoxy-1-methyl-pseudoisocytidine; 4-methoxy-pseudoisocytidine; 4-thio-1-methyl-1-deaza-pseudoisocytidine; 4-thio-1-methyl-pseudoisocytidine; 4-thio-pseudoisocytidine; 5-aza-zeb Zebularine; 5-methyl-zebularine; pyrrolo-pseudoisocytidine; zebularine; (E)-5-(2-bromo-vinyl)cytidine TP; 2,2'-dehydro-cytidine TP hydrochloride; 2'-fluoro-N4-Bz-cytidine TP; 2'-fluoro-N4-acetyl-cytidine TP; 2'-O-methyl-N4-acetyl-cytidine TP; 2'O-methyl-N4-Bz-cytidine TP; 2'-a-ethynylcytidine TP; 2'-a-trifluoromethylcytidine TP; 2'-b-ethynylcytidine TP; 2'-b-trifluoromethylcytidine TP; 2'-deoxy-2',2'-difluorocytidine TP; 2'-deoxy-2'-a-hydroxycytidine TP; 2'-deoxy-2'-a-thiomethoxycytidine TP; 2'-deoxy-2'-b-aminocytidine TP; 2'-deoxy-2'-b-azidocytidine TP; 2'-deoxy-2'-b-bromocytidine TP; 2'-deoxy-2'-b-chlorocytidine TP; 2'-deoxy-2'-b-fluorocytidine TP; 2'-deoxy-2'-b-iodocytidine TP; 2'-deoxy-2'-b-hydroxycytidine TP ; 2'-deoxy-2'-b-thiomethoxycytidine TP; 2'-O-methyl-5-(1-propynyl)cytidine TP; 3'-ethynylcytidine TP; 4'-azidocytidine TP; 4'-carbocyclic cytidine TP; 4'-ethynylcytidine TP; 5-(1-propynyl)arabino-cytidine TP; 5-(2-chloro-phenyl)-2-thiocytidine TP; 5-(4-amino-phenyl)-2-thiocytidine TP; 5-aminoallyl-CTP; 5-cyanocytidine TP; 5-ethynylarabino-cytidine TP; 5-ethynylcytidine TP; 5'-homo-cytidine TP; 5-methoxycytidine TP; 5-trifluoromethyl-cytidine TP; N4-amino-cytidine TP; N4-benzoyl-cytidine TP; pseudoisocytidine; mimG (methylguanosine); m7G (7-methylguanosine); m2Gm (N2,2'-O-dimethylguanosine); m2G (N2-methylguanosine); imG (wyosine); m1Gm (1,2'-O-dimethylguanosine); m1G (1-methylguanosine); 2'-O-methylguanosine; 2'-O-ribosylguanosine (phosphate); Gm (2'-O-methylguanosine); Gr(p) (2'-O-ribosylguanosine (phosphate)); preQi (7-aminomethyl-7-deazaguanosine); preQo (7-cyano-7-deazaguanosine); G* (archaeosine); methyl wyosine; m2'7G (N2,7-dimethylguanosine); m22Gm (N2,N2,2'-O-trimethylguanosine); m2'2'7G (N2,N2,7-trimethylguanosine); m22G (N2,N2-dimethylguanosine); N2,7,2'-O-trimethylguanosine; 6-thio-guanosine; 7-deaza-guanosine; 8-oxo-guanosine; N1-methyl-guanosine; a-thio-guanosine; 2-(propyl)guanine; 2-(alkyl)guanine; 2'-amino-2'-deoxy-GTP; 2'-azido-2'-deoxy -GTP; 2'-deoxy-2'-a-aminoguanosine TP; 2'-deoxy-2'-a-hydrazineguanosine TP; N2-dimethylguanine; 6-(methyl)guanine; 6-(alkyl)guanine; 6-(methyl)guanine; 6-methyl-guanosine; 6-thioguanine; 7-(alkyl)guanine; 7-deaza-7-substituted guanine guanine; 7-deaza-7-(C2-C6)alkynylguanine; 7-deaza-8-substituted guanine; 7-(methyl)guanine; 7-(alkyl)guanine; 7-(deaza)guanine; 7-(methyl)guanine; 8-azaguanine; 8-hydroxyguanine; 8-oxoguanine; 8-(alkyl)guanine; 8-(alkynyl)guanine guanine; 8-(halogen)guanine; 8-(sulfanyl)guanine; 8-(alkenyl)guanine; 8-(alkyl)guanine; 8-(alkynyl)guanine; 8-(amino)guanine; 8-(halogen)guanine; 8-(hydroxy)guanine; 8-(sulfanyl)guanine; 8-(thiol)guanine; azaguanine; deazaguanine; N (Methyl)guanine; N-(methyl)guanine; 1-methyl-6-thio-guanosine; 6-methoxy-guanosine; 6-thio-7-deaza-8-aza-guanosine; 6-thio-7-deaza-guanosine; 6-thio-7-methyl-guanosine; 7-deaza-8-aza-guanosine; 7-methyl-8-oxo-guanosine; N2,N2-dimethyl-6-thio-guanosine; N2- Methyl-6-thio-guanosine; 1-me-GTP; 2'-fluoro-N2-isobutyl-guanosine TP; 2'-O-methyl-N2-isobutyl-guanosine TP; 2'-a-ethynylguanosine TP; 2'-a-trifluoromethylguanosine TP; 2'-b-ethynylguanosine TP; 2'-b-trifluoromethylguanosine TP; 2'-deoxy-2',2'-difluoroguanosine TP; 2'-deoxy-2'-a-isoguanosine TP; 2'-deoxy-2'-a-thiomethoxyguanosine TP; 2'-deoxy-2'-b-aminoguanosine TP; 2'-deoxy-2'-b-hydrazineguanosine TP; 2'-deoxy-2'-b-bromoguanosine TP; 2'-deoxy-2'-b-chloroguanosine TP; 2'-deoxy-2'-b-fluoroguanosine TP; 2' -deoxy-2'-b-iodoguanosine TP; 2'-deoxy-2'-b-hydroxyguanosine TP; 2'-deoxy-2'-b-thiomethoxyguanosine TP; 4'-azoguanosine TP; 4'-carbocyclicguanosine TP; 4'-ethynylguanosine TP; 5'-homoguanosine TP; 8-bromoguanosine TP; 9-deazaguanosine TP; N2-isobutylguanosine TP; miI (1-methylinosine); I (inosine); m'lm (1,2'-O-dimethylinosine); 2'-O-methylinosine; 7-methylinosine; Tm (2'-O-methylinosine); oQ (queuosine); galQ (galactoside-Q nucleoside); manQ (mannosyl Q nucleoside); Q (Q nucleoside); allylamino-thymidine; azathymidine; deoxythymidine; deoxy-thymidine; Um (2'-O-methyluridine); s2U (2-thiouridine); m3U (3-methyluridine); cm5U (5-carboxymethyluridine); ho5U (5-hydroxyuridine); m5U (5-methyluridine); tm5s2U (5-tauromethyl-2-thiouridine); 5-tauromethyluridine; D (dihydrouridine); pseudouridine; acp3U (3-(3-amino-3-carboxypropyl) uridine); 1-methyl-3-(3-amino-5-carboxypropyl) pseudouridine; 1-methyl pseudouridine; 1-ethyl-pseudouridine; 2'-O-methyl uridine; 2'-O-methyl pseudouridine; 2'-O-methyl uridine; s2Um (2-thio-2'-O-methyl uridine); 3-(3-amino-3-carboxypropyl) uridine; m3Um (3,2'-O-dimethyl uridine); 3-methyl-pseudo-uridine TP; s4U (4-thiouridine); chm5U (5-(carboxyhydroxymethyl) uridine); mchm5U (5-(carboxyhydroxymethyl) uridine methyl ester); m5Um (5,2'-O-dimethyluridine); 5,6-dihydro-uridine; nm5s2U (5-aminomethyl-2-thiouridine); ncm5Um (5-aminomethylmethyl-2'-O-methyluridine); ncm5U (5-aminomethylmethyluridine); 5-carboxyhydroxymethyluridine; 5-carboxyhydroxymethyluridine methyl ester; cnmm5Um (5-carboxymethylaminomethyl-2'-O-methyluridine); cmnm5s2U (5-carboxymethylaminomethyl-2-thiouridine); 5-carboxymethylaminomethyluridine; cmnm5U (5-carboxymethylaminomethyluridine); 5-aminomethylmethyluridine TP; mcm5Um (5-methoxycarbonylmethyl-2'-O-methyluridine); mcm5s2U (5-methoxycarbonylmethyl-2-thiouridine); mcm5U (5-methoxycarbonylmethyluridine); mo5U (5-methoxyuridine); m5s2U (5-methyl-2-thiouridine); mnm5se2U (5-methylaminomethyl-2-selenouridine); mnm5s2U (5-methylaminomethyl-2-thiouridine); mnm5U (5-methylaminomethyluridine); m5D (5-methyldihydrouridine); 5-oxyacetate-uridine TP; 5-oxyacetate-methyl-uridine TP; dihydrouracil; pseudouracil; N1-methyl-pseudo-uracil; N1-ethyl-pseudo-uracil; cmo5U (uridine 5-oxyacetic acid); mcmo5U (Uridine 5-oxyacetic acid methyl ester); 3-(3-amino-3-carboxypropyl)-uridine TP; 5-(iso-pentenylaminomethyl)-2-thiouridine TP; 5-(iso-pentenylaminomethyl)-2'-O-methyluridine TP; 5-(iso-pentenylaminomethyl)uridine TP; 5-propynyluracil; a-thio-uridine; 1-(aminoalkylamino-carbonylvinyl)-2(thio)-pseudouracil; 1-(aminoalkylamino-carbonylvinyl)-2,4-(dithio)pseudouracil; 1-(aminoalkylamino-carbonylvinyl)-4-(thio)pseudouracil; 1-(aminoalkylamino-carbonylvinyl)- Amino-carbonylvinyl)-pseudouracil; 1-(aminocarbonylvinyl)-2(thio)-pseudouracil; 1-(aminocarbonylvinyl)-2,4-(dithio)pseudouracil; 1-(aminocarbonylvinyl)-4-(thio)pseudouracil; 1-(aminocarbonylvinyl)-pseudouracil; 1-substituted 2(thio)-pseudouracil; 1-substituted 2,4-(dithio)pseudouracil; 1-substituted 4-(thio)pseudouracil; 1-substituted pseudouracil; 1-(aminoalkylamino-carbonylvinyl)-2-(thio)-pseudouracil; 1-methyl-3-(3-amino-3-carboxypropyl) Pseudouridine TP; 1-methyl-3-(3-amino-3-carboxypropyl) pseudo-UTP; 1-methyl-pseudo-UTP; 1-ethyl-pseudo-UTP; 2-(thio)pseudouracil; 2'deoxyuridine; 2'fluorouridine; 2-(thio)uracil; 2,4-(dithio)pseudouracil; 2'methyl, 2'amino, 2'azido, 2'fluoro-guanosine; 2'-amino-2'-deoxy-UTP; 2'-azido-2'-deoxy-UTP; 2'-azido-deoxyuridine TP; 2'-O-methylpseudouridine; 2'deoxyuridine; 2' Fluorouridine; 2'-deoxy-2'-a-aminouridine TP; 2'-deoxy-2'-a-azidouridine TP; 2-methylpseudouridine; 3-(3-amino-3-carboxypropyl)uracil; 4-(thio)pseudouridine; 4-(thio)pseudouridine; 4-(thio)uracil; 4-thiouracil; 5-aminouracil; 5-(1,3-oxadiazole-1-alkyl)uracil; 5-(2-aminopropyl)uracil; 5-(aminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil; 5-(guanidinoalkyl)uracil; 5-(methoxycarbonylmethyl)-2-(thio)uracil; 5-(methoxycarbonyl-methyl)uracil; 5-(methyl) 2-(thio)uracil; 5-(methyl) 2,4-(dithio)uracil; 5-(methyl)-4-(thio)uracil; 5-(methylaminomethyl)-2-(thio)uracil; 5-(methylaminomethyl)-2,4-(dithio)uracil; 5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil; 5-(trifluoromethyl)uracil; 5-(2-aminopropyl)uracil; 5-(alkyl)-2-(thio)pseudouracil; 5-(alkyl)-2,4-(dithio)pseudouracil; 5-(alkyl)-4-(thio)pseudouracil; 5-(alkyl)- 5-(alkyl) uracil; 5-(alkenyl) uracil; 5-(alkynyl) uracil; 5-(allylamino) uracil; 5-(cyanoalkyl) uracil; 5-(dialkylaminoalkyl) uracil; 5-(dimethylaminoalkyl) uracil; 5-(guanidinoalkyl) uracil; 5-(halogen) uracil; 5-(1,3-oxadiazol-1-alkyl) uracil; 5-(methoxy) uracil; 5-(methoxycarbonylmethyl)-2-(thio) uracil; 5-(methoxycarbonyl-methyl) uracil; 5-(methyl) 2(thio)uracil; 5-(methyl)-2,4-(dithio)uracil; 5-(methyl)-4-(thio)uracil; 5-(methyl)-2-(thio)pseudouracil; 5-(methyl)-2,4-(dithio)pseudouracil; 5-(methyl)-4-(thio)pseudouracil; 5-(methyl)pseudouracil; 5-(methyl)pseudouracil; 5-(methylaminomethyl)-2-(thio)uracil; 5-(methylaminomethyl)-2,4(dithio)uracil; 5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil; 5-(trifluoromethyl)uracil pyrimidine; 5-aminoallyl-uridine; 5-bromo-uridine; 5-iodo-uridine; 5-uracil; 6-(azo)uracil; 6-(azo)uracil; 6-aza-uridine; allylamino-uracil; aza-uracil; deaza-uracil; 5-methyluracil; 5-(hydroxymethyl)uracil; 5-chlorouracil; 5-fluorouracil; 5-bromouracil; N3-(methyl)uracil; pseudo-UTP-1-2-acetic acid; pseudouracil; 4-thio-pseudo-UTP; 1-carboxymethyl-pseudouridine; 1-methyl-1-deaza-pseudouridine; 1- Propynyl-uridine; 1-taurine methyl-1-methyl-uridine; 1-taurine methyl-4-thio-uridine; 1-taurine methyl-pseudouridine; 2-methoxy-4-thio-pseudouridine; 2-thio-1-methyl-1-deaza-pseudouridine; 2-thio-1-methyl-pseudouridine; 2-thio-5-aza-uridine; 2-thio-dihydropseudouridine; 2-thio-dihydrouridine; 2-thio-pseudouridine; 4-methoxy-2-thio-pseudouridine; 4-methoxy-pseudouridine; 4-thio-1-methyl-pseudouridine; 4-thio-pseudouridine; 5-aza-uridine; dihydropseudouridine uridine; (±)1-(2-hydroxypropyl)pseudouridine TP; (2R)-1-(2-hydroxypropyl)pseudouridine TP; (2S)-1-(2-hydroxypropyl)pseudouridine TP; (E)-5-(2-bromo-vinyl)arabino-uridine TP; (E)-5-(2-bromo-vinyl)uridine TP; (Z)-5-(2-bromo-vinyl)arabino-uridine TP; (Z)-5-(2-bromo-vinyl)uridine TP; 1-(2,2,2-trifluoroethyl)-pseudouridine; 1-(2,2,3,3,3-pentafluoropropyl)pseudouridine TP; 1-(2,2-diethoxyethyl)pseudouridine TP; 1-(2,4,6-trimethylbenzyl)pseudouridine TP; 1-(2,4,6-trimethyl-benzyl)pseudouridine TP; 1-(2,4,6-trimethyl-phenyl)pseudouridine TP; 1-(2-amino-2-carboxyethyl)pseudouridine TP; 1-(2-amino-ethyl)pseudouridine TP; 1-(2-hydroxyethyl)pseudouridine TP; 1-(2-methoxyethyl)pseudouridine TP; 1-(3,4-bis-trifluoromethoxybenzyl)pseudouridine TP; 1-(3, 1-(4-dimethoxybenzyl)pseudouridine TP; 1-(3-amino-3-carboxypropyl)pseudouridine TP; 1-(3-amino-propyl)pseudouridine TP; 1-(3-cyclopropyl-prop-2-ynyl)pseudouridine TP; 1-(4-amino-4-carboxybutyl)pseudouridine TP; 1-(4-amino-benzyl)pseudouridine TP; 1-(4-amino-butyl)pseudouridine TP; 11(4-amino-phenyl)pseudouridine TP; 1-(4-azidobenzyl)pseudouridine TP; 1-(4-bromobenzyl)pseudouridine TP; 1-(4-chlorobenzyl) 1-(4-fluorobenzyl)pseudouridine TP; 1-(4-iodobenzyl)pseudouridine TP; 1-(4-methylsulfonylbenzyl)pseudouridine TP; 1-(4-methoxybenzyl)pseudouridine TP; 1-(4-methoxy-benzyl)pseudouridine TP; 1-(4-methoxy-benzyl)pseudouridine TP; 1-(4-methoxy-phenyl)pseudouridine TP; 1-(4-methylbenzyl)pseudouridine TP; 1-(4-methyl-benzyl)pseudouridine TP; 1-(4-nitrobenzyl)pseudouridine TP; 1-(4-nitro-benzyl)pseudouridine TP; 1(4-nitro-benzyl)pseudouridine phenyl) pseudo-UTP; 1-(4-thiomethoxybenzyl) pseudo-UTP; 1-(4-trifluoromethoxybenzyl) pseudo-UTP; 1-(4-trifluoromethylbenzyl) pseudo-UTP; 1-(5-amino-pentyl) pseudo-UTP; 1-(6-amino-hexyl) pseudo-UTP; 1,6-dimethyl-pseudo-UTP; 1-[3-(2-{2-[2-(2-aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl] pseudo-UTP; 1-{3-[2-(2-aminoethoxy)-ethoxy]-propionyl} Pseudouridine TP; 1-acetyl pseudouridine TP; 1-alkyl-6-(1-propynyl)-pseudouridine TP; 1-alkyl-6-(2-propynyl)-pseudouridine TP; 1-alkyl-6-allyl-pseudouridine TP; 1-alkyl-6-ethynyl-pseudouridine TP; 1-alkyl-6-homoallyl-pseudouridine TP; 1-alkyl-6-vinyl-pseudouridine TP; 1-allyl pseudouridine TP; 1-aminomethyl-pseudouridine TP; 1-benzoyl pseudouridine TP; 1-benzyloxymethyl pseudouridine TP; 1-benzyl-pseudouridine TP; 1-biotinyl-PEG2-pseudouridine TP; 1-biotinyl pseudouridine TP; 1-butyl-pseudouridine TP; 1-cyanomethyl pseudouridine TP; 1-cyclobutylmethyl-pseudouridine TP; 1-cyclobutyl-pseudouridine UTP; 1-cycloheptylmethyl-pseudo-UTP; 1-cycloheptyl-pseudo-UTP; 1-cyclohexylmethyl-pseudo-UTP; 1-cyclohexyl-pseudo-UTP; 1-cyclooctylmethyl-pseudo-UTP; 1-cyclooctyl-pseudo-UTP; 1-cyclopentylmethyl-pseudo-UTP; 1-cyclopentyl-pseudo-UTP; 1-cyclopropylmethyl-pseudo-UTP; 1-cyclopropyl-pseudo-UTP ; 1-ethyl-pseudo-UTP; 1-hexyl-pseudo-UTP; 1-homoallyl-pseudouridine TP; 1-hydroxymethyl-pseudouridine TP; 1-iso-propyl-pseudouridine TP; 1-me-2-thio-pseudouridine TP; 1-me-4-thio-pseudouridine TP; 1-me-α-thio-pseudouridine TP; 1-methylsulfonylmethyl-pseudouridine TP; 1-methoxymethyl-pseudouridine TP; 1- Methyl-6-(2,2,2-trifluoroethyl) pseudo-UTP; 1-methyl-6-(4-(N-thiophenyl))-pseudo-UTP; 1-methyl-6-(4-thio(N-thiophenyl))-pseudo-UTP; 1-methyl-6-(substituted phenyl) pseudo-UTP; 1-methyl-6-amino-pseudo-UTP; 1-methyl-6-azido-pseudo-UTP; 1-methyl-6-bromo-pseudo-UTP; 1-methyl-6-butyl-pseudo-UTP; 1-methyl-6-chloro-pseudo-UTP; 1-methyl-6-cyano-pseudo-UTP; 1-methyl-6-dimethylamino-pseudo-UTP; 1-methyl-6-ethoxy-pseudo-UTP; 1-methyl-6-carboxylic acid ethyl ester-pseudo-UTP; 1-methyl-6-ethyl-pseudo-UTP; 1-methyl-6- Fluoro-pseudo-UTP; 1-methyl-6-methyl-pseudo-UTP; 1-methyl-6-hydroxylamino-pseudo-UTP; 1-methyl-6-hydroxyl-pseudo-UTP; 1-methyl-6-iodo-pseudo-UTP; 1-methyl-6-iso-propyl-pseudo-UTP; 1-methyl-6-methoxy-pseudo-UTP; 1-methyl-6-methylamino-pseudo-UTP; 1-methyl-6-phenyl-pseudo-UTP; 1-methyl-6-propyl-pseudo-UTP; 1-methyl-6-tert-butyl-pseudo-UTP; 1-methyl-6-trifluoromethoxy-pseudo-UTP; 1-methyl-6-trifluoromethyl-pseudo-UTP; 1-(N-oxo-1-indole)methylpseudouridine TP; 1-pentyl-pseudouridine TP; 1-phenyl-pseudouridine TP; 1-trimethylacetyl-pseudouridine TP; 1-propargyl pseudouridine TP; 1-propyl-pseudo-UTP; 1-propynyl-pseudo-UTP; 1-p-tolyl-pseudo-UTP; 1-tert-butyl-pseudo-UTP; 1-thiomethoxymethyl pseudouridine TP; 1-thio(N-thiophenyl)methyl pseudouridine TP; 1-trifluoroacetyl pseudouridine TP; 1-trifluoromethyl-pseudo-UTP; 1-vinyl pseudouridine TP; 2,2'-dehydro-uridine TP; 2'-bromo-deoxyuridine TP; 2'-F-5-methyl-2'-deoxy-UTP; 2'-OMe-5-me-UTP; 2'-OMe-pseudo-UTP; 2'-a-ethynyl uridine TP; 2'-a-trifluoromethyl uridine TP; 2'-b-ethynyl uridine TP; 2'-b-trifluoromethyl uridine TP; 2'-deoxy-2',2'- Difluorouridine TP; 2'-deoxy-2'-a-hydroxyuridine TP; 2'-deoxy-2'-a-thiomethoxyuridine TP; 2'-deoxy-2'-b-aminouridine TP; 2'-deoxy-2'-b-azidouridine TP; 2'-deoxy-2'-b-bromouridine TP; 2'-deoxy-2'-b-chlorouridine TP; 2'-deoxy-2'-b-fluorouridine TP; 2'-deoxy-2'-b-iodouridine TP; 2'-deoxy-2'-b-butyluridine TP; 2'-deoxy-2'-b-thiomethoxyuridine TP; 2-methoxy-4-thio-uridine; 2-methoxyuridine; 2'-O-methyl-5-(1-propynyl)uridine TP; 3-alkyl-pseudo-UTP; 4'-azidouridine TP; 4'-carbocyclic uridine TP; 4'-ethyl Alkynyl uridine TP; 5-(1-propynyl) arabino-uridine TP; 5-(2-furanyl) uridine TP; 5-cyanouridine TP; 5-dimethylamino uridine TP; 5'-homo-uridine TP; 5-iodo-2'-fluoro-deoxyuridine TP; 5-phenylethynyl uridine TP; 5-trideuteromethyl-6-deuterouridine TP; 5-trifluoromethyl-uridine TP; 5-vinyl arabinose Uridine TP; 6-(2,2,2-trifluoroethyl)-pseudo-UTP; 6-(4-(N-thiophenyl))-pseudo-UTP; 6-(4-thio(N-thiophenyl))-pseudo-UTP; 6-(substituted phenyl)-pseudo-UTP; 6-amino-pseudo-UTP; 6-azido-pseudo-UTP; 6-bromo-pseudo-UTP; 6-butyl-pseudo-UTP; 6-chloro-pseudo-UTP UTP; 6-cyano-pseudo-UTP; 6-dimethylamino-pseudo-UTP; 6-ethoxy-pseudo-UTP; 6-carboxylic acid ethyl-pseudo-UTP; 6-ethyl-pseudo-UTP; 6-fluoro-pseudo-UTP; 6-methyl-pseudo-UTP; 6-hydroxyamino-pseudo-UTP; 6-hydroxy-pseudo-UTP; 6-iodo-pseudo-UTP; 6-iso-propyl-pseudo-UTP; 6-methoxy-pseudo-UTP; 6-methylamino-pseudo-UTP; 6-methyl-pseudo-UTP; 6-phenyl-pseudo-UTP; 6-phenyl-pseudo-UTP; 6-propyl-pseudo-UTP; 6-tert-butyl-pseudo-UTP; 6-trifluoromethoxy-pseudo-UTP; 6-trifluoromethyl-pseudo-UTP; α-thio-pseudo-UTP; pseudouridine 1-(4-methylbenzenesulfonic acid) TP; Pseudouridine 1-(4-methylbenzoic acid) TP; Pseudouridine TP 1-[3-(2-ethoxy)] propionic acid; Pseudouridine TP 1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}] propionic acid; Pseudouridine TP 1-[3-{2-(2-[2-{2(2-ethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}] propionic acid; Pseudouridine TP 1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}] propionic acid; Pseudouridine TP 1-[3-{2-(2-ethoxy)-ethoxy}] propionic acid; Pseudouridine TP 1-[3-{2-(2-ethoxy)-ethoxy}] propionic acid; Pseudouridine TP 1-methylphosphonic acid; Pseudouridine TP 1-methylphosphonic acid diethyl ester; pseudo-UTP-N1-3-propionic acid; pseudo-UTP-N1-4-butyric acid; pseudo-UTP-N1-5-pentanoic acid; pseudo-UTP-N1-6-hexanoic acid; pseudo-UTP-N1-7-heptanoic acid; pseudo-UTP-N1-methyl-p-benzoic acid; pseudo-UTP-N1-p-benzoic acid; yW (wybutosine); OHyW (hydroxywybutosine); imG2 (isowybutosine); o2yW (peroxywybutosine); OHyW* (unmodified hydroxywybutosine); imG-14 (4-demethyl ruthenium); 2,6-(diamino)purine; 1-(aza)-2-(thio)-3-(aza)-phenanthia-1-yl; 1,3-(diaza)-2-(oxo)-phenanthia-1-yl; 1,3-(diaza)-2-(oxo)-phenanthia-1-yl; 1,3,5-(triaza)-2,6-(dioxo)-naphthalene; 2-(amino)purine; 2,4,5-(trimethyl)phenyl; 2'methyl, 2'amino, 2'azido, 2'fluoro-cytidine; 2' methyl, 2'amino, 2'azido, 2'fluoro-adenine; 2'methyl, 2'amino, 2'azido, 2'fluoro-uridine; 2'-amino-2'-deoxyribose; 2-amino-6-chloro-purine; 2-aza-inosyl; 2'-azido-2'-deoxyribose; 2'fluoro-2'-deoxyribose; 2'-fluoro-modified base; 2'-O-methyl-ribose; 2-oxo-7-aminopyridopyrimidin-3-yl; 2- Oxy-pyridopyrimidin-3-yl; 2-pyridone; 3-nitropyrrole; 3-(methyl)-7-(propynyl)isoquinolone (carbostyrilyl); 3-(methyl)isoquinolone; 4-(fluoro)-6-(methyl)benzimidazole; 4-(methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl; 5-nitroindole; 5-substituted pyrimidine; 5-(methyl)isoquinolone; 5 -nitroindole; 6-(aza)pyrimidine; 6-(azo)thymine; 6-(methyl)-7-(aza)indolyl; 6-chloro-purine; 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; 7-(aminoalkylhydroxyl)-1-(aza)-2-(thio)-3-(aza)-phenanthridine-1-yl; 7-(aminoalkylhydroxyl)-1-(aza)-2-(thio)-3-(aza)-phenanthridine-1-yl; 7-(aminoalkylhydroxyl)-1-(aza)-2-(thio)-3-(aza)-phenanthridine-1-yl; alkylhydroxyl)-1,3-(diaza)-2-(oxo)-phenanthridine-1-yl; 7-(aminoalkylhydroxyl)-1,3-(diaza)-2-(oxo)-phenanthridine-1-yl; 7-(aminoalkylhydroxyl)-1,3-(diaza)-2-(oxo)-phenanthridine-1-yl; 7-(aza)indolyl; 7-(guanidinoalkylhydroxyl)-1-(aza)-2-(thio)-3-(aza)-phenanthridine-1-yl ; 7-(Guanidinylalkylhydroxyl)-1-(aza)-2-(thio)-3-(aza)-phenanthridine-1-yl; 7-(Guanidinylalkylhydroxyl)-1-(aza)-2-(thio)-3-(aza)-phenanthridine-1-yl; 7-(Guanidinylalkylhydroxyl)-1,3-(diaza)-2-(oxo)-phenanthridine-1-yl; 7-(Guanidinylalkyl-hydroxyl)-1,3-(diaza)-2-(oxo)-phenanthridine-1-yl ; 7-(Guanidinylalkylhydroxyl)-1,3-(diaza)-2-(oxo)-phenanthroline-1-yl; 7-(propynyl)isoquinolone; 7-(propynyl)isoquinolone; propynyl-7-(aza)indolyl; 7-deaza-inosinyl; 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenanthroline-1-yl; 7-substituted 1,3-(diaza)-2-(oxo)-phenanthroline-1-yl; 9-(methyl)- 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; bis-ortho-substituted 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; difluorotolyl; hypoxanthine; imidazopyridinyl; inosinyl; isoquinolone; isoguanisine; N2-substituted purine; N6-methyl-2-amino-purine; N6 substituted Purine; N-alkylated derivatives; naphthyl; nitrobenzimidazolyl; nitroimidazolyl; nitroindazolyl; nitropyrazolyl; nubularine; O6-substituted purine; O-alkylated derivatives; o-(aminoalkylhydroxyl)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; o-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; oxoformycin TP; p-(aminoalkylhydroxyl)-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; p-substituted 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl; fused pentaphenyl; allyl anthracenyl; phenyl; propynyl-7-(aza)indolyl; pyrenyl; pyridopyrimidin-3-yl; pyridopyrimidin-3-yl; 2-oxo-7-amino-pyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-one-3-yl; pyrrolopyrimidine pyrrolopyridine; stilbenzyl; substituted 1,2,4-triazole; tetraphenyl; tubercidine; xanthine; xanthine-5'-TP; 2-thio-zebularine; 5-aza-2-thio-zebularine; 7-deaza-2-amino-purine; pyridine-4-one ribonucleoside; 2-aminoriboside-TP; formycin A TP; metamycin B TP; pyrrolosine TP; 2'-OH-arabino-adenosine TP; 2'-OH-arabino-cytidine TP; 2'-OH-arabino-uridine TP; 2'-OH-arabino-guanosine TP; 5-(2-methoxycarbonylvinyl)uridine TP; N6-(19-amino-pentaoxadecyl)adenosine TP; hydrogen (without a base residue); and 2'-O-methyl-U. In some aspects, the RNA molecule includes a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned modifications may be excluded from the RNA molecules disclosed herein.
在一些態樣中，RNA分子中之經修飾之核鹼基包含假尿苷(ψ)、2-硫尿苷(s2U)、4'-硫尿苷、5-甲基胞嘧啶、2-硫-1-甲基-1-脫氮-假尿苷、2-硫-1-甲基-假尿苷、2-硫-5-氮雜-尿苷、2-硫-二氫假尿苷、2-硫-二氫尿苷、2-硫-假尿苷、4-甲氧基-2-硫-假尿苷、4-甲氧基-假尿苷、4-硫-1-甲基-假尿苷、4-硫-假尿苷、5-氮雜-尿苷、二氫假尿苷、5-甲基尿苷、5-甲氧基尿苷、2'-O-甲基尿苷、1-甲基-假尿苷(m1ψ)、1-乙基-假尿苷(e1ψ)、5-甲氧基-尿苷(mo5U)、5-甲基-胞苷(m5C)、a-硫-鳥苷、a-硫-腺苷、5-氰基尿苷、4'-硫尿苷7-脫氮-腺嘌呤、1-甲基-腺苷(m1A)、2-甲基-腺嘌呤(m2A)、N6-甲基-腺苷(m6A)、2,6-二胺基嘌呤、肌苷(I)、1-甲基-肌苷(m1I)、懷俄苷(imG)、甲基懷俄苷(mimG)、7-脫氮-鳥苷、7-氰基-7-脫氮-鳥苷(preQO)、7-胺基甲基-7-脫氮-鳥苷(preQl)、7-甲基-鳥苷(m7G)、1-甲基-鳥苷(m1G)、8-側氧基-鳥苷、7-甲基-8-側氧基-鳥苷、2,8-二甲基腺苷、2-香葉基硫尿苷、2-立西啶、2-硒基尿苷、3-(3-胺基-3-羧基丙基)-5,6-二氫尿苷、3-(3-胺基-3-羧基丙基)假尿苷、3-甲基假尿苷、5-(羧基羥基甲基)-2'-O-甲基尿苷甲酯、5-胺基甲基-2-香葉基硫尿苷、5-胺基甲基-2-硒基尿苷、5-胺基甲基尿苷、5-胺甲醯基羥基甲基尿苷、5-胺甲醯基甲基-2-硫尿苷、5-羧基甲基-2-硫尿苷、5-羧基甲基胺基甲基-2-香葉基硫尿苷、5-羧基甲基胺基甲基-2-硒基尿苷、5-氰基甲基尿苷、5-羥基胞苷、5-甲基胺基甲基-2-香葉基硫尿苷、7-胺基羧基丙基-去甲基懷俄苷、7-胺基羧基丙基懷俄苷、7-胺基羧基丙基懷俄苷甲酯、8-甲基腺苷、N4,N4-二甲基胞苷、N6-甲醯基腺苷、N6-羥基甲基腺苷、阿格嗎特啶(agmatidine)、環狀N6-蘇胺醯基胺甲醯基腺苷、麩胺醯基-Q核苷、甲基化的欠修飾之羥基懷俄丁苷、N4,N4,2'-O-三甲基胞苷、香葉基化的5-甲基胺基甲基-2-硫尿苷、香葉基化的5-羧基甲基胺基甲基-2-硫尿苷、Q鹼基、preQO鹼基、preQI鹼基，及其兩者或更多者之組合。在一些態樣中，RNA分子包括前述經修飾之核鹼基中之至少兩者(例如2、3、4者或更多者)之組合，包括但不限於化學修飾。在一些態樣中，前述經修飾之核鹼基中之1、2、3、4、5者或更多者為排除在本文所揭示之RNA分子外。In some aspects, the modified nucleobase in the RNA molecule comprises pseudouridine (ψ), 2-thiouridine (s2U), 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, 2'-O-methyluridine, 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ), 5-methoxy-uridine guanosine (mo5U), 5-methyl-cytidine (m5C), α-thio-guanosine, α-thio-adenosine, 5-cyanouridine, 4'-thiouridine 7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), 2,6-diaminopurine, inosine (I), 1-methyl-inosine (m1I), imG, methyl imG, 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQO), 7-aminomethyl-7-deaza-guanosine (preQl), 7-methyl-guanosine (m7G), 1-methyl-guanosine (m1G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine glycosides, 2,8-dimethyladenosine, 2-geranylthiouridine, 2-licidin, 2-selenouridine, 3-(3-amino-3-carboxypropyl)-5,6-dihydrouridine, 3-(3-amino-3-carboxypropyl)pseudouridine, 3-methylpseudouridine, 5-(carboxyhydroxymethyl)-2'-O-methyluridine methyl ester, 5-aminomethyl-2-geranylthiouridine, 5-aminomethyl-2-selenouridine, 5-aminomethyluridine, 5-aminoformylhydroxymethyluridine, 5-aminoformylmethyl-2-thiouridine, 5-carboxymethyl-2-thiouridine, 5-carboxymethylaminomethyl-2-geranylthiouridine, 5-carboxymethylaminomethyl-2-selenouridine, 5-cyanomethyluridine, 5-hydroxycytidine, 5- Methylaminomethyl-2-geranylthiouridine, 7-aminocarboxypropyl-demethyl uaustoside, 7-aminocarboxypropyl uaustoside, 7-aminocarboxypropyl uaustoside methyl ester, 8-methyladenosine, N4,N4-dimethylcytidine, N6-methyladenosine, N6-hydroxymethyladenosine, agmatidine, cyclic N6-threonine In some embodiments, the RNA molecule comprises a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases, including but not limited to chemical modifications. In some embodiments, 1, 2, 3, 4, 5 or more of the aforementioned modified nucleobases are excluded from the RNA molecules disclosed herein.
具有經修飾之胞嘧啶的例示性核鹼基及核苷包括5-氮雜-胞苷、6-氮雜-胞苷、假異胞苷、3-甲基-胞苷(m3C)、N4-乙醯基-胞苷(ac4C)、5-甲醯基-胞苷(f5C)、N4-甲基-胞苷(m4C)、5-甲基-胞苷(m5C)、5-鹵基-胞苷(例如5-碘-胞苷)、5-羥基甲基-胞苷(hm5C)、1-甲基-假異胞苷、吡咯并-胞苷、吡咯并-假異胞苷、2-硫-胞苷(s2C)、2-硫-5-甲基-胞苷、4-硫-假異胞苷、4-硫-1-甲基-假異胞苷、4-硫-1-甲基-1-脫氮-假異胞苷、1-甲基-1-脫氮-假異胞苷、澤布拉林、5-氮雜-澤布拉林、5-甲基-澤布拉林、5-氮雜-2-硫-澤布拉林、2-硫-澤布拉林、2-甲氧基-胞苷、2-甲氧基-5-甲基-胞苷、4-甲氧基-假異胞苷、4-甲氧基-1-甲基-假異胞苷、立西啶(k2C)、a-硫-胞苷、2'-O-甲基-胞苷(Cm)、5,2'-O-二甲基-胞苷(m5Cm)、N4-乙醯基-2'-O-甲基-胞苷(ac4Cm)、N4,2'-O-二甲基-胞苷(m4Cm)、5-甲醯基-2'-O-甲基-胞苷(f5Cm)、N4,N4,2'-O-三甲基-胞苷(m42Cm)、1-硫-胞苷、2'-F-阿糖-胞苷、2'-F-胞苷及2'-OH-阿糖-胞苷。在一些態樣中，前述經修飾之胞嘧啶中之1、2、3、4、5者或更多者可排除在本文所揭示之RNA分子外。Exemplary nucleobases and nucleosides having modified cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl-cytidine (ac4C), 5-methyl-cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halogen-cytidine (e.g., 5-iodo-cytidine), 5-hydroxy 2-Hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine , 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, ricinidine (k2C), a-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl-cytidine (m5Cm ), N4-acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (f5Cm), N4,N4,2'-O-trimethyl-cytidine (m42Cm), 1-thio-cytidine, 2'-F-arabino-cytidine, 2'-F-cytidine and 2'-OH-arabino-cytidine. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned modified cytosines may be excluded from the RNA molecules disclosed herein.
在一些態樣中，經修飾之核鹼基為經修飾之尿苷。具有經修飾之尿嘧啶的例示性核鹼基及核苷包括假尿苷(ψ)、吡啶-4-酮核糖核苷、5-氮雜-尿苷、6-氮雜-尿苷、2-硫-5-氮雜-尿苷、2-硫-尿苷(s2U)、4-硫-尿苷(s4U)、4-硫-假尿苷、2-硫-假尿苷、5-羥基-尿苷(ho5U)、5-胺基烯丙基-尿苷、5-鹵基-尿苷(例如5-碘-尿苷或5-溴-尿苷)、5-氰基尿苷、3-甲基-尿苷(m3U)、5-甲氧基-尿苷(mo5U)、尿苷5-氧基乙酸(cmo5U)、尿苷5-氧基乙酸甲酯(mcmo5U)、5-羧基甲基-尿苷(cm5U)、1-羧基甲基-假尿苷、5-羧基羥基甲基-尿苷(chm5U)、5-羧基羥基甲基-尿苷甲酯(mchm5U)、5-甲氧基羰基甲基-尿苷(mcm5U)、5-甲氧基羰基甲基-2-硫-尿苷(mcm5s2U)、5-胺基甲基-2-硫-尿苷(nm5s2U)、5-甲基胺基甲基-尿苷(mnm5U)、5-甲基胺基甲基-2-硫-尿苷(mnm5s2U)、5-甲基胺基甲基-2-硒基-尿苷(mnm5se2U)、5-胺甲醯基甲基-尿苷(ncm5U)、5-羧基甲基胺基甲基-尿苷(cmnm5U)、5-羧基甲基胺基甲基-2-硫-尿苷(cmnmVU)、5-丙炔基-尿苷、1-丙炔基-假尿苷、5-牛磺酸甲基-尿苷(xm5U)、1-牛磺酸甲基-假尿苷、5-牛磺酸甲基-2-硫-尿苷(xmVu)、1-牛磺酸甲基-4-硫-假尿苷、5-甲基-尿苷(m5U，例如具有核鹼基去氧胸腺嘧啶)、1-甲基-假尿苷(m1Ψ)、1-乙基-假尿苷(e1ψ)、5-甲基-2-硫-尿苷(m5s2U)、1-甲基-4-硫-假尿苷(m1s4Ψ)、4-硫-1-甲基-假尿苷、3-甲基-假尿苷(m3Ψ)、2-硫-1-甲基-假尿苷、1-甲基-1-脫氮-假尿苷、2-硫-1-甲基-1-脫氮-假尿苷、二氫尿苷(D)、二氫假尿苷、5,6-二氫尿苷、5-甲基-二氫尿苷(m5D)、2-硫-二氫尿苷、2-硫-二氫假尿苷、2-甲氧基-尿苷、2-甲氧基-4-硫-尿苷、4-甲氧基-假尿苷、4-甲氧基-2-硫-假尿苷、N1-甲基-假尿苷、3-(3-胺基-3-羧基丙基)尿苷(acp3U)、1-甲基-3-(3-胺基-3-羧基丙基)假尿苷(acp3 ψ)、5-(異戊烯基胺基甲基)尿苷(inm5U)、5-(異戊烯基胺基甲基)-2-硫-尿苷(inm5s2U)、a-硫-尿苷、2'-O-甲基-尿苷(Um)、5,2'-O-二甲基-尿苷(m5Um)、2'-O-甲基-假尿苷(Ψm)、2-硫-2'-O-甲基-尿苷(s2Um)、5-甲氧基羰基甲基-2'-O-甲基-尿苷(mcm5Um)、5-胺甲醯基甲基-2'-O-甲基-尿苷(ncm5Um)、5-羧基甲基胺基甲基-2'-O-甲基-尿苷(cmnm5Um)、3,2'-O-二甲基-尿苷(m3Um)、及5-(異戊烯基胺基甲基)-2'-O-甲基-尿苷(inm5Um)、1-硫-尿苷、去氧胸苷、2'-F-阿糖-尿苷、2'-F-尿苷、2'-OH-阿糖-尿苷、5-(2-羰基甲氧基乙烯基) 尿苷及5-[3-(l-E-丙烯基胺基)]尿苷。在一些態樣中，前述經修飾之尿苷中之1、2、3、4、5者或更多者可排除在本文所揭示之RNA分子外。In some aspects, the modified nucleobase is a modified uridine. Exemplary nucleobases and nucleosides having a modified uracil include pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halogen-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 5-cyanouridine, 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5- Carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-aminomethylmethyl-uridine (ncm5U), 5-carboxymethyl aminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnmVU), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinemethyl-uridine (xm5U), 1-taurinemethyl-pseudouridine, 5-taurinemethyl-2-thio-uridine (xmVu), 1-taurinemethyl-4-thio-pseudouridine, 5-methyl-uridine (m5U, for example, with a nucleobase deoxythymidine), 1-methyl-pseudouridine (m1Ψ), 1-ethyl-pseudouridine (e1ψ), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4Ψ), 4-thio-1-methyl -pseudouridine, 3-methyl-pseudouridine (m3Ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3 ψ), 5-(isoprenylaminylaminomethyl)uridine (inm5U), 5-(isoprenylaminylaminomethyl)-2-thio-uridine (inm5s2U), a-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (Ψm), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-amino Formylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-arabino-uridine, 2'-F-uridine, 2'-OH-arabino-uridine, 5-(2-carbonylmethoxyvinyl) uridine and 5-[3-(1-E-propenylamino)] uridine. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned modified uridines may be excluded from the RNA molecules disclosed herein.
在本發明之一些態樣中，經修飾之核苷酸包括N1-甲基假尿苷及/或假尿苷中之任一者。In some aspects of the invention, the modified nucleotide includes any one of N1-methylpseudouridine and/or pseudouridine.
在一些態樣中，RNA分子包含經N1-甲基假尿苷修飾之核苷酸。在一些態樣中，RNA分子包含經假尿苷修飾之核苷酸。In some aspects, the RNA molecule comprises nucleotides modified with N1-methyl pseudouridine. In some aspects, the RNA molecule comprises nucleotides modified with pseudouridine.
在一些態樣中，RNA包含經修飾之核苷來代替至少一個尿苷。在一些態樣中，RNA包含代替各尿苷的經修飾之核苷。在一些態樣中，RNA分子包含至少一個尿苷經N1-甲基假尿苷置換之序列。在一些態樣中，RNA分子包含所有尿苷經N1-甲基假尿苷置換之序列。N1-甲基假尿苷在序列中表示為「Ψ」。如本文所用，術語「尿嘧啶」描述可出現在RNA之核酸中的一種核鹼基。如本文所用，術語「尿苷」描述可出現在RNA中的一種核苷。「假尿苷」為經修飾之核苷之一個實例，其為尿苷之異構物，其中尿嘧啶經由碳-碳鍵而非氮-碳醣苷鍵連接至戊醣環。In some aspects, the RNA comprises a modified nucleoside in place of at least one uridine. In some aspects, the RNA comprises a modified nucleoside in place of each uridine. In some aspects, the RNA molecule comprises a sequence in which at least one uridine is replaced by N1-methylpseudouridine. In some aspects, the RNA molecule comprises a sequence in which all uridines are replaced by N1-methylpseudouridine. N1-methylpseudouridine is represented as "Ψ" in the sequence. As used herein, the term "uracil" describes a nucleoside that can appear in the nucleic acid of RNA. As used herein, the term "uridine" describes a nucleoside that can appear in RNA. "Pseudouridine" is an example of a modified nucleoside that is an isomer of uridine in which uracil is linked to the pentose ring via a carbon-carbon bond rather than a nitrogen-carbon glycosidic bond.
在一些態樣中，RNA分子包含至少一個尿苷經N1-甲基假尿苷及/或假尿苷置換之核酸序列。在一些態樣中，RNA分子包含至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的尿苷經N1-甲基假尿苷及/或假尿苷置換的核酸序列：1%、2%、3%、4%、5%、6%、7%、8%、9%、10%、11%、12%、13%、14%、15%、16%、17%、18%、19%、20%、21%、22%、23%、24%、25%、26%、27%、28%、29%、30%、31%、32%、33%、34%、35%、36%、37%、38%、39%、40%、41%、42%、43%、44%、45%、46%、47%、48%、49%、50%、51%、52%、53%、54%、55%、56%、57%、58%、59%、60%、61%、62%、63%、64%、65%、66%、67%、68%、69%、70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%。在一些態樣中，RNA分子包含所有尿苷經N1-甲基假尿苷及/或假尿苷置換之核酸序列。In some aspects, the RNA molecule comprises at least one uridine replaced by N1-methylpseudouridine and/or pseudouridine. In some aspects, the RNA molecule comprises at least, at most, exactly or between any two of the following (inclusive or exclusive) uridine replaced by N1-methylpseudouridine and/or pseudouridine: 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42% , 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. In some aspects, the RNA molecule comprises a nucleic acid sequence in which all uridines are replaced by N1-methylpseudouridine and/or pseudouridine.
在一些態樣中，經修飾之核鹼基為經修飾之腺嘌呤。具有經修飾之腺嘌呤的例示性核鹼基及核苷包括2-胺基-嘌呤、2、6-二胺基嘌呤、2-胺基-6-鹵基-嘌呤(例如2-胺基-6-氯-嘌呤)、6-鹵基-嘌呤(例如6-氯-嘌呤)、2-胺基-6-甲基-嘌呤、8-疊氮基-腺苷、7-脫氮-腺嘌呤、7-脫氮-8-氮雜-腺嘌呤、7-脫氮-2-胺基-嘌呤、7-脫氮-8-氮雜-2-胺基-嘌呤、7-脫氮-2,6-二胺基嘌呤、7-脫氮-8-氮雜-2,6-二胺基嘌呤、1-甲基-腺苷(m1A)、2-甲基-腺嘌呤(m2A)、N6-甲基-腺苷(m6A)、2-甲基硫-N6-甲基-腺苷(ms2m6A)、N6-異戊烯基-腺苷(i6A)、2-甲基硫-N6-異戊烯基-腺苷(ms2i6A)、N6-(順式-羥基異戊烯基)腺苷(io6A)、2-甲基硫-N6-(順式-羥基異戊烯基)腺苷(ms2io6A)、N6-甘胺醯基胺甲醯基-腺苷(g6A)、N6-蘇胺醯基胺甲醯基-腺苷(t6A)、N6-甲基-N6-蘇胺醯基胺甲醯基-腺苷(m6t6A)、2-甲基硫-N6-蘇胺醯基胺甲醯基-腺苷(ms2g6A)、N6,N6-二甲基-腺苷(m62A)、N6-羥基正纈胺醯基胺甲醯基-腺苷(hn6A)、2-甲基硫-N6-羥基正纈胺醯基胺甲醯基-腺苷(ms2hn6A)、N6-乙醯基-腺苷(ac6A)、7-甲基-腺嘌呤、2-甲基硫-腺嘌呤、2-甲氧基-腺嘌呤、a-硫-腺苷、2'-O-甲基-腺苷(Am)、N6,2'-O-二甲基-腺苷(m6Am)、N6,N6,2'-O-三甲基-腺苷(m62Am)、1,2'-O-二甲基-腺苷(m1Am)、2'-O-核糖基腺苷(磷酸酯) (Ar(p))、2-胺基-N6-甲基-嘌呤、1-硫-腺苷、8-疊氮基-腺苷、2'-F-阿糖-腺苷、2'-F-腺苷、2'-OH-阿糖-腺苷、及N6-(19-胺基-五氧雜十九烷基)-腺苷。在一些態樣中，前述經修飾之腺嘌呤中之1、2、3、4、5者或更多者可排除在本文所揭示之RNA分子外。In some aspects, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halogen-purine (e.g., 2-amino-6-chloro-purine), 6-halogen-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6 -diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxy isopentenyl)adenosine (ms2io6A), N6-glycinamidomethyladenosine (g6A), N6-threonamidomethyladenosine (t6A), N6-methyl-N6-threonamidomethyladenosine (m6t6A), 2-methylthio-N6-threonamidomethyladenosine (ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxyn-valeramidomethyladenosine (hn6A), 2-methylthio-N6-hydroxy N-aminomethyladenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, a-thio-adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O-trimethyl-adenosine (m62Am), 1,2'-O-dimethyl-adenosine (m1Am), 2'-O-ribosyl-adenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-arabino-adenosine, 2'-F-adenosine, 2'-OH-arabino-adenosine, and N6-(19-amino-pentaoxadecyl)-adenosine. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned modified adenines may be excluded from the RNA molecules disclosed herein.
在一些態樣中，經修飾之核鹼基為經修飾之鳥嘌呤。具有經修飾之鳥嘌呤的例示性核鹼基及核苷包括肌苷(I)、1-甲基-肌苷(m1I)、懷俄苷(imG)、甲基懷俄苷(mimG)、4-去甲基-懷俄苷(imG-14)、異懷俄苷(imG2)、懷俄丁苷(yW)、過氧基懷俄丁苷(o2yW)、羥基懷俄丁苷(OhyW)、欠修飾之羥基懷俄丁苷(OhyW*)、7-脫氮-鳥苷、Q核苷(Q)、環氧基Q核苷(oQ)、半乳糖苷-Q核苷(galQ)、甘露糖基-Q核苷(manQ)、7-氰基-7-脫氮-鳥苷(preQo)、7-胺基甲基-7-脫氮-鳥苷(preQ1)、古嘌苷(G+)、7-脫氮-8-氮雜-鳥苷、6-硫-鳥苷、6-硫-7-脫氮-鳥苷、6-硫-7-脫氮-8-氮雜-鳥苷、7-甲基-鳥苷(m7G)、6-硫-7-甲基-鳥苷、7-甲基-肌苷、6-甲氧基-鳥苷、1-甲基-鳥苷(m1G)、N2-甲基-鳥苷(m2G)、N2,N2-二甲基-鳥苷(m22G)、N2,7-二甲基-鳥苷(m2'7G)、N2、N2,7-二甲基-鳥苷(m2'2'7G)、8-側氧基-鳥苷、7-甲基-8-側氧基-鳥苷、1-甲基-6-硫-鳥苷、N2-甲基-6-硫-鳥苷、N2,N2-二甲基-6-硫-鳥苷、a-硫-鳥苷、2'-O-甲基-鳥苷(Gm)、N2-甲基-2'-O-甲基-鳥苷(m2Gm)、N2,N2-二甲基-2'-O-甲基-鳥苷(m22Gm)、1-甲基-2'-O-甲基-鳥苷、N2,7-二甲基-2'-O-甲基-鳥苷(m2'7Gm)、2'-O-甲基-肌苷(Im)、1,2'-O-二甲基-肌苷(m1Im)、2'-O-核糖基鳥苷(磷酸酯) (Gr(p))、1-硫-鳥苷、O6-甲基-鳥苷、2'-F-阿糖-鳥苷及2'-F-鳥苷。在一些態樣中，前述經修飾之鳥嘌呤中之1、2、3、4、5者或更多者可排除在本文所揭示之RNA分子外。In some aspects, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1-methyl-inosine (m1I), yohistoside (imG), methylyohistoside (mimG), 4-demethyl-yohistoside (imG-14), isoyohistoside (imG2), yohistoside (yW), peroxyyohistoside (o2yW), hydroxyyohistoside (OhyW), undermodified hydroxyyohistoside (OhyW*), 7-deaza-guanosine, Q nucleoside (Q), epoxy Q nucleoside (oQ), galactosyl-Q nucleoside (galQ), mannosyl-Q nucleoside (manQ), 7-cyano-7-deaza-guanosine (preQo), 7-aminomethyl-7-deaza-guanosine (preQ1), archaeosine (G+), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine , 6-methoxy-guanosine, 1-methyl-guanosine (m1G), N2-methyl-guanosine (m2G), N2,N2-dimethyl-guanosine (m22G), N2,7-dimethyl-guanosine (m2'7G), N2,N2,7-dimethyl-guanosine (m2'2'7G), 8-oxoguanosine, 7-methyl-8-oxoguanosine, 1-methyl-6-thioguanosine, N2-methyl-6-thioguanosine, N2,N2-dimethyl-6-thioguanosine, a -thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m22Gm), 1-methyl-2'-O-methyl-guanosine, N2,7-dimethyl-2'-O-methyl-guanosine (m2'7Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m1Im), 2'-O-ribosylguanosine (phosphate) (Gr(p)), 1-thio-guanosine, O6-methyl-guanosine, 2'-F-arabino-guanosine and 2'-F-guanosine. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned modified guanines can be excluded from the RNA molecules disclosed herein.
在一些態樣中，對於特定修飾而言，RNA分子均勻地經修飾(例如完全修飾，在整個序列中經修飾)。在一些態樣中，RNA分子可沿分子之整個長度部分地或完全(例如均勻地)經修飾。舉例而言，本發明之聚核苷酸中或其給定預定序列區中，一或多種或所有或給定類型之核苷酸(例如嘌呤及/或嘧啶，或A、G、U、C中之任何一或多種或所有)可均勻地經修飾。在一些態樣中，本發明之聚核苷酸中(或其給定序列區中)之所有核苷酸X為經修飾之核苷酸，其中X可為核苷酸A、G、U、C中之任一者，及/或組合A+G、A+U、A+C、G+U、G+C、U+C、A+G+U、A+G+C、G+U+C及/或A+G+C中之任一者。舉例而言，聚核苷酸可均勻地經假尿苷修飾，意謂RNA序列中之所有尿苷殘基均經假尿苷置換。類似地，聚核苷酸可藉由用經修飾之殘基(諸如上文所闡述之彼等者)進行置換而對序列中存在之任何類型的核苷殘基進行均勻地修飾。經修飾之核苷酸可經具有單一獨特結構之一種化合物置換，或可經複數種具有不同結構(例如2、3、4種或更多種獨特結構)之化合物置換。In some aspects, for a particular modification, the RNA molecule is uniformly modified (e.g., fully modified, modified throughout the entire sequence). In some aspects, the RNA molecule can be partially or completely (e.g., uniformly) modified along the entire length of the molecule. For example, one or more or all or a given type of nucleotide (e.g., purine and/or pyrimidine, or any one or more or all of A, G, U, C) can be uniformly modified in a polynucleotide of the present invention or in a given predetermined sequence region thereof. In some aspects, all nucleotides X in a polynucleotide of the invention (or in a given sequence region thereof) are modified nucleotides, wherein X can be any of the nucleotides A, G, U, C, and/or any of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C, and/or A+G+C. For example, a polynucleotide can be uniformly modified with pseudouridine, meaning that all uridine residues in an RNA sequence are replaced with pseudouridine. Similarly, a polynucleotide can be uniformly modified with any type of nucleoside residue present in the sequence by replacing them with modified residues, such as those described above. The modified nucleotide may be replaced with one compound having a single unique structure, or may be replaced with a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures).
RNA分子可含有為或為約1%至100%之經修飾之核苷酸(與總體核苷酸含量有關，或與一或多種類型之核苷酸(例如A、G、U及/或C中之任何一或多者)有關) (例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99或100%)或任何中間百分比(例如1%至20%、1%至25%、1%至50%、1%至60%、1%至70%、1%至80%、1%至90%、1%至95%、10%至20%、10%至25%、10%至50%、10%至60%、10%至70%、10%至80%、10%至90%、10%至95%、10%至100%、20%至25%、20%至50%、20%至60%、20%至70%、20%至80%、20%至90%、20%至95%、20%至100%、50%至60%、50%至70%、50%至80%、50%至90%、50%至95%、50%至100%、70%至80%、70%至90%、70%至95%、70%至100%、80%至90%、80%至95%、80%至100%、90%至95%、90%至100%、及95%至100%)。應理解，任何剩餘百分比由未經修飾A、G、U及/或C之存在補足。The RNA molecule can contain between or about 1% to 100% modified nucleotides (with respect to the overall nucleotide content, or with respect to one or more types of nucleotides (e.g., any one or more of A, G, U and/or C) (e.g., at least, at most, exactly less than, or between any two of the following (inclusive or exclusive): 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%) or any percentage in between % to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10% to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 70%, 20% to 80%, 20% to 90%, 10% to 95%, 10% to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 7 ... 0% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100%). It is understood that any remaining percentages are made up by the presence of unmodified A, G, U and/or C.
在一些態樣中，RNA分子可包括胺基磷酸酯、硫代磷酸酯及/或甲基磷酸酯鍵聯。In some aspects, RNA molecules can include phosphoramidate, phosphorothioate, and/or methylphosphonate linkages.
在一些態樣中，RNA分子可包括一或多種賦予聚核苷酸適用特性之結構及/或化學修飾及/或改變，在一些態樣中，包括細胞或生物體中降低的降解及/或缺乏對其中引入RNA分子之細胞之先天性免疫反應的實質上誘導。如本文所用，「結構」特徵或修飾為兩個或更多個連接核苷酸在RNA分子中插入、缺失、複製、倒置及/或隨機化而不對核苷酸本身進行顯著化學修飾的特徵或修飾。因為化學鍵將必然斷裂且重新形成以實現結構修飾，因此結構修飾具有化學性質且因此為化學修飾。然而，結構修飾將產生不同核苷酸序列。舉例而言，聚核苷酸「ATCG」可化學修飾為「AT-5meC-G」。相同聚核苷酸可自「ATCG」結構修飾為「ATCCCG」。此處，已插入二核苷酸「CC」，從而引起聚核苷酸之結構修飾。In some aspects, RNA molecules can include one or more structural and/or chemical modifications and/or alterations that impart useful properties to polynucleotides, including, in some aspects, reduced degradation in cells or organisms and/or lack of substantial induction of an innate immune response to cells into which the RNA molecule is introduced. As used herein, a "structural" feature or modification is one in which two or more linked nucleotides are inserted, deleted, duplicated, inverted, and/or randomized in an RNA molecule without significant chemical modification of the nucleotides themselves. Because chemical bonds will necessarily break and reform to achieve the structural modification, the structural modification is chemical in nature and is therefore a chemical modification. However, the structural modification will result in a different nucleotide sequence. For example, the polynucleotide "ATCG" can be chemically modified to "AT-5meC-G". The same polynucleotide can be structurally modified from "ATCG" to "ATCCCG". Here, the dinucleotide "CC" has been inserted, thereby causing the structural modification of the polynucleotide.
在一些態樣中，相對於包含標準核苷酸及核苷之未經修飾之核酸，引入細胞或生物體中之經修飾之RNA分子分別展現細胞或生物體中降低的降解。在一些態樣中，相對於包含標準核苷酸及核苷之未經修飾之核酸，引入細胞或生物體中之經修飾之RNA分子可分別展現細胞或生物體中降低的免疫原性(例如降低的先天性反應)。In some aspects, the modified RNA molecules introduced into a cell or organism exhibit reduced degradation in the cell or organism, respectively, relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides. In some aspects, the modified RNA molecules introduced into a cell or organism can exhibit reduced immunogenicity (e.g., reduced innate response) in the cell or organism, respectively, relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.
在一些態樣中，RNA分子可包括一或多個除任何5'帽結構以外的經修飾之核苷酸。在一些態樣中，RNA分子不包括經修飾之核苷酸，例如不包括經修飾之核鹼基，且RNA分子中之所有核苷酸均為習知標準核糖核苷酸A、U、G及C，除可包括例如7-甲基鳥苷之視情況存在之5'帽以外，其在下文進一步描述。在一些態樣中，RNA可包括包含7'-甲基鳥苷之5'帽，且前1、2或3個5'核糖核苷酸可在核糖之2'位置處甲基化。In some aspects, the RNA molecule may include one or more modified nucleotides in addition to any 5' cap structure. In some aspects, the RNA molecule does not include modified nucleotides, e.g., does not include modified nucleobases, and all nucleotides in the RNA molecule are known standard ribonucleotides A, U, G, and C, except for an optional 5' cap that may include, e.g., 7-methylguanosine, which is further described below. In some aspects, the RNA may include a 5' cap comprising 7'-methylguanosine, and the first 1, 2, or 3 5' ribonucleotides may be methylated at the 2' position of the ribose.
B.5'帽在一些態樣中，本文所描述之RNA分子包括5'帽，其通常對RNA之5'端「加帽」且使RNA分子穩定。B.5'Cap In some aspects, the RNA molecules described herein include a 5' cap, which generally "caps" the 5' end of the RNA and stabilizes the RNA molecule.
在一些態樣中，5'帽部分為天然5'帽。「天然5'帽」定義為包括經由5'-5'三磷酸酯鍵聯連接至mRNA分子之5'端之7-甲基鳥苷的帽。在一些態樣中，包括在5'帽中之鳥苷核苷可例如藉由鹼基(鳥嘌呤)上之一或多個位置處(例如7位置處)之甲基化及/或藉由核糖之一或多個位置處之甲基化而經修飾。在一些態樣中，包括在5'帽中之鳥苷核苷包含核糖處之3'O甲基化(3'OMeG)。在一些態樣中，包括在5'帽中之鳥苷核苷包含鳥嘌呤之7位置處之甲基化(m7G)。在一些態樣中，包括在5'帽中之鳥苷核苷包含鳥嘌呤之7位置處之甲基化及核糖處之3'O甲基化(m7(3'OMeG))。5'帽可在RNA合成期間併入(例如，共轉錄加帽)，或可在RNA轉錄之後酶促工程改造(例如，轉錄後加帽)。在一些態樣中，用本文所揭示之帽進行共轉錄加帽與用適當參考比較物進行共轉錄加帽相比，RNA之加帽效率增加。在一些態樣中，提高加帽效率可增加RNA之轉譯效率及/或轉譯速率，及/或增加所編碼多肽之表現。在一些態樣中，加帽在RNA分子之純化(例如切向流過濾)之後進行。In some aspects, the 5' cap portion is a natural 5' cap. A "natural 5' cap" is defined as a cap that includes a 7-methylguanosine linked to the 5' end of the mRNA molecule via a 5'-5' triphosphate linkage. In some aspects, the guanosine nucleoside included in the 5' cap can be modified, for example, by methylation at one or more positions on the base (guanine) (e.g., at the 7 position) and/or by methylation at one or more positions of the ribose. In some aspects, the guanosine nucleoside included in the 5' cap includes 3'O methylation at the ribose (3'OMeG). In some aspects, the guanosine nucleoside included in the 5' cap includes methylation at the 7 position of guanine (m7G). In some aspects, the guanosine nucleoside included in the 5' cap comprises methylation at the 7 position of guanine and 3'O methylation at the ribose (m7(3'OMeG)). The 5' cap can be incorporated during RNA synthesis (e.g., co-transcriptional capping), or can be enzymatically engineered after RNA transcription (e.g., post-transcriptional capping). In some aspects, the capping efficiency of RNA is increased by co-transcriptional capping with the cap disclosed herein compared to co-transcriptional capping with an appropriate reference comparator. In some aspects, improving the capping efficiency can increase the translation efficiency and/or translation rate of the RNA, and/or increase the expression of the encoded polypeptide. In some aspects, capping is performed after purification of the RNA molecule (e.g., tangential flow filtration).
在一些態樣中，本文所描述之RNA包含5'帽或5'帽類似物，例如帽0、帽1或帽2。在一些態樣中，所提供之RNA不具有未加帽的5'-三磷酸酯。在一些態樣中，RNA之5'端以經修飾之核糖核苷酸加帽。在一些態樣中，5'帽部分為5'帽類似物。在一些態樣中，RNA可用5'帽類似物加帽。帽結構包括但不限於7mG(5')ppp(5')N1pN2p (帽0)、7mG(5')ppp(5')N1mpNp (帽1)、及7mG(5')ppp(5')N1mpN2mp (帽2)。在一些態樣中，前述帽結構中之1、2、3、4、5者或更多者可排除在本文所揭示之RNA分子外。In some aspects, the RNA described herein comprises a 5' cap or a 5' cap analog, such as cap 0, cap 1, or cap 2. In some aspects, the RNA provided does not have an uncapped 5'-triphosphate. In some aspects, the 5' end of the RNA is capped with a modified ribonucleotide. In some aspects, the 5' cap portion is a 5' cap analog. In some aspects, the RNA can be capped with a 5' cap analog. Cap structures include, but are not limited to, 7mG(5')ppp(5')N1pN2p(cap 0),7mG (5')ppp(5 ')N1mpNp (cap 1), and7mG (5')ppp(5')N1mpN2mp (cap 2). In some aspects, 1, 2,3, 4 , 5 or more of the aforementioned cap structures may be excluded from the RNA molecules disclosed herein.
在一些態樣中，本文所描述之RNA包含帽0。在一些態樣中，帽0為N7-甲基鳥苷，且帽0結構包含在鳥嘌呤之7位置處甲基化的鳥苷核苷(m7G)。在一些態樣中，帽0結構經由5'-5'三磷酸酯鍵聯連接至RNA，且在本文中亦稱為m7G、m7Gppp及/或m7G(5')ppp(5')。5'帽可用結構7mG(5')ppp(5')N1pN2p (帽0)或其衍生物甲基化，其中N為攜帶5'帽之核酸的末端5'核苷酸，通常mRNA之5'端。用於加帽之例示性酶促反應可包括使用包括mRNA三磷酸酶、鳥苷醯基轉移酶及鳥嘌呤-7-甲基轉移酶之牛痘病毒加帽酶(VCE)，其催化N7-單甲基化帽0結構之構築。帽0結構在維持RNA分子之穩定性及轉譯功效方面起重要作用。在細胞中，帽0結構對於攜載帽之mRNA的高效轉譯為至關重要的。In some aspects, the RNA described herein comprises a cap 0. In some aspects, the cap 0 is an N7-methylguanosine, and the cap 0 structure comprises a guanosine nucleoside methylated at the 7 position of the guanine (m7G). In some aspects, the cap 0 structure is linked to the RNA via a 5'-5' triphosphate linkage, and is also referred to herein as m7G, m7Gppp, and/or m7G(5')ppp(5'). The 5' cap canbe methylated with the structure7 mG(5')ppp(5')N1pN2p (cap 0) or a derivative thereof, where N is the terminal 5' nucleotide of the nucleic acid carrying the 5' cap, typically the 5' end of the mRNA. An exemplary enzymatic reaction for capping may include the use of vaccinia virus capping enzyme (VCE) including mRNA triphosphatase, guanosyl transferase and guanine-7-methyltransferase, which catalyzes the construction of N7-monomethylated cap 0 structure. The cap 0 structure plays an important role in maintaining the stability and translation efficiency of RNA molecules. In cells, the cap 0 structure is crucial for the efficient translation of capped mRNA.
在一些態樣中，本文所描述之RNA包含例如本文所描述之帽1。RNA分子之5'帽可進一步由2'-O-甲基轉移酶對2'O位置進行修飾，該2'-O-甲基轉移酶引起帽1結構(m7Gppp [m2'-Ο] N)之產生，其可進一步增加轉譯效率。在一些態樣中，帽1結構包含在鳥嘌呤之7位置處甲基化之鳥苷核苷(m7G)及RNA中經2'O甲基化之第一個核苷酸(2'OmeN1)。在一些態樣中，帽1結構經由5'-5'三磷酸酯鍵聯連接至RNA且在本文中亦稱為m7GpppNm，其中Nm表示具有2'O甲基化、7mG(5')ppp(5')N1mpNp、m7Gppp(2'OMeN1)及/或m7G(5')ppp(5')(2'OMeN1)之任何核苷酸。在一些態樣中，N1選自A、C、G或U。在一些態樣中，N1為A。在一些態樣中，N1為C。在一些態樣中，N1為G。在一些態樣中，N1為U。在一些態樣中，m7G(5')ppp(5')(2'OmeN1)帽1結構包含第二核苷酸N2，其為位置2處之帽近端核苷酸且係選自A、G、C或U (m7G(5')ppp(5')(2'OmeN1)N2)。在一些態樣中，N2為A。在一些態樣中，N2為C。在一些態樣中，N2為G。在一些態樣中，N2為U。In some aspects, the RNA described herein comprises, for example, Cap 1 as described herein. The 5' cap of the RNA molecule can be further modified at the 2'O position by a 2'-O-methyltransferase, which results in the generation of a Cap 1 structure (m7Gppp [m2'-O] N), which can further increase translation efficiency. In some aspects, the Cap 1 structure comprises a guanosine nucleoside methylated at the 7 position of guanine (m7G) and the first nucleotide in the RNA that is methylated at 2'O (2'OmeN1 ). In some embodiments, the cap 1 structure is linked to the RNA via a 5'-5' triphosphate linkage and is also referred to herein asm7GpppNm , whereinNm represents any nucleotide with 2'O methylation,m7G (5')ppp (5')N1mpNp , m7Gppp(2'OMeN1 ) and/or m7G(5 ')ppp(5')(2'OMeN1). In some embodiments,N1 is selected from A, C, G or U. In some embodiments,N1 is A. In some embodiments,N1 is C. In some embodiments,N1 is G. In some embodiments,N1 is U. In some aspects, the m7G(5')ppp(5')(2'OmeN1 )cap 1 structure comprises a second nucleotide N2 which is the cap-proximal nucleotide at position 2 and is selected from A, G, C, or U (m7G(5')ppp( 5')(2'OmeN1 )N2 ). In some aspects, N2 is A. In some aspects, N2 is C. In some aspects, N 2 is G. In some aspects, N2 is U.
在一些態樣中，帽1結構包含在鳥嘌呤之7位置處甲基化之鳥苷核苷(m7G)及一或多個額外修飾(例如核糖上之甲基化)以及RNA中經2'O甲基化之第一核苷酸。在一些態樣中，帽1結構包含在鳥嘌呤之7位置處甲基化之鳥苷核苷、核糖處之3'O甲基化(m7(3'OMeG))及RNA中經2'O甲基化之第一核苷酸(2'OMeN1)。在一些態樣中，帽1結構經由5'-5'三磷酸酯鍵聯連接至RNA，且在本文中亦稱為m7(3'OMeG)ppp(2'OMeN1)及/或m7(3'OMeG)(5')ppp(5')(2'OMeN1)。在一些態樣中，N1選自A、C、G或U。在一些態樣中，N1為A。在一些態樣中，N1為C。在一些態樣中，N1為G。在一些態樣中，N1為U。在一些態樣中，m7(3'OMeG)(5')ppp(5')(2'OMeN1)帽1結構包含第二核苷酸N2，其為位置2處之帽近端核苷酸且係選自A、G、C或U (m7(3'OMeG)(5')ppp(5')(2'OmeN1)N2)。在一些態樣中，N2為A。在一些態樣中，N2為C。在一些態樣中，N2為G。在一些態樣中，N2為U。在一些態樣中，前述帽1結構中之1、2、3、4、5者或更多者可排除在本文所揭示之RNA分子外。In some aspects, the cap 1 structure comprises a guanosine nucleoside methylated at the 7-position of guanine (m7G) and one or more additional modifications (e.g., methylation on the ribose) and the first nucleotide methylated by 2'O in the RNA. In some aspects, the cap 1 structure comprises a guanosine nucleoside methylated at the 7-position of guanine, 3'O methylation at the ribose (m7(3'OMeG)) and the first nucleotide methylated by 2'O in the RNA (2'OMeN1 ). In some aspects, the cap 1 structure is linked to the RNA via a 5'-5' triphosphate bond and is also referred to herein as m7(3'OMeG)ppp(2'OMeN1 ) and/or m7(3'OMeG)(5')ppp(5')(2'OMeN1 ). In some aspects, N1 is selected from A, C, G or U. In some aspects,N1 is A. In some aspects,N1 is C. In some aspects,N1 is G. In some aspects,N1 is U. In some aspects, the m7(3'OMeG)(5')ppp(5')(2'OMeN1 )cap1 structure comprises a second nucleotideN2 , which is the cap-proximal nucleotide at position 2 and is selected from A, G, C or U (m7(3'OMeG)(5')ppp(5')(2'OmeN1 )N2 ). In some aspects,N2 is A. In some aspects,N2 is C. In some aspects,N2 is G. In some aspects,N2 is U. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned cap1 structures can be excluded from the RNA molecules disclosed herein.
在一些態樣中，帽1結構中之第二核苷酸可包含一或多種修飾，例如甲基化。在一些態樣中，本文所描述之RNA包含帽2。在一些態樣中，包含含有2'O甲基化之第二核苷酸的帽1結構為帽2結構。In some aspects, the second nucleotide in the cap 1 structure can comprise one or more modifications, such as methylation. In some aspects, the RNA described herein comprises a cap 2. In some aspects, the cap 1 structure comprising a second nucleotide comprising a 2'O methylation is a cap 2 structure.
在一些態樣中，RNA分子可使用牛痘鳥苷醯基轉移酶、鳥苷三磷酸及S-腺苷基-L-甲硫胺酸在5'端處酶促加帽，得到帽0結構。倒置的7-甲基鳥苷帽經由5'-5'三磷酸酯橋添加。或者，使用2'O-甲基轉移酶與牛痘鳥苷醯基轉移酶得到帽1結構，其中除帽0結構以外，倒數第二個核苷酸上之2'OH基團經甲基化。S-腺苷-L-甲硫胺酸(SAM)為用作甲基轉移試劑之輔因子。5'帽結構之非限制性實例為與此項技術中已知的合成5'帽結構(或野生型、天然或生理5'帽結構)相比，尤其帽結合多肽之結合增強、半衰期增加、對5'核酸內切酶之敏感性降低及/或5'去帽減少的彼等結構。In some aspects, RNA molecules can be enzymatically capped at the 5' end using vaccinia guanosyltransferase, guanosine triphosphate, and S-adenosyl-L-methionine to give a Cap 0 structure. An inverted 7-methylguanosine cap is added via a 5'-5' triphosphate bridge. Alternatively, a 2'O-methyltransferase and vaccinia guanosyltransferase are used to give a Cap 1 structure, in which, in addition to the Cap 0 structure, the 2'OH group on the penultimate nucleotide is methylated. S-adenosyl-L-methionine (SAM) is a cofactor used as a methyl transfer reagent. Non-limiting examples of 5' cap structures are those that provide enhanced binding, increased half-life, reduced susceptibility to 5' endonucleases, and/or reduced 5' decapping, particularly of the cap-bound polypeptide, compared to synthetic 5' cap structures known in the art (or wild-type, natural or physiological 5' cap structures).
舉例而言，重組牛痘病毒加帽酶及重組2' O-甲基轉移酶可在mRNA之5'末端核苷酸與鳥嘌呤帽核苷酸之間產生典型5'-5'三磷酸酯鍵聯，其中帽鳥嘌呤包括N7甲基化且mRNA之5'末端核苷酸包括2'-O-甲基。此類結構被稱為帽1結構。與例如此項技術中已知的其他5'帽類似物結構相比，此帽使得轉譯能力及細胞穩定性更高以及細胞促炎性細胞介素之活化減少。For example, recombinant vaccinia virus capping enzymes and recombinant 2'O-methyltransferases can generate a typical 5'-5' triphosphate linkage between the 5' terminal nucleotide of the mRNA and the guanine cap nucleotide, wherein the cap guanine includes N7 methylation and the 5' terminal nucleotide of the mRNA includes a 2'-O-methyl group. Such structures are referred to as cap 1 structures. Compared to other 5' cap analog structures known in the art, for example, this cap results in higher translational capacity and cell stability and reduced activation of cellular proinflammatory cytokines.
帽物種可包括一或多種經修飾之核苷及/或連接子部分。舉例而言，帽可包括在其5'位置處藉由三磷酸酯鍵聯接合的鳥嘌呤核苷酸及在7位置處甲基化之鳥嘌呤(G)核苷酸，例如m7G(5')ppp(5')G，通常書寫為m7GpppG。帽物種亦可為抗反向帽類似物。可能的帽物種之非限制性清單包括m7GpppG、m7Gpppm7G、m73'dGpppG、m27,O3'GpppG、m27,O3'GppppG、m27,O2'GppppG、m7Gpppm7G、m73'dGpppG、m27,O3'GpppG、m27,O3'GppppG及m27,O2'GppppG。在一些態樣中，前述帽物種中之1、2、3、4、5者或更多者可排除在本文所揭示之RNA分子外。Cap species may include one or more modified nucleosides and/or linker moieties. For example, a cap may include a guanine nucleotide joined at its 5' position by a triphosphate linkage and a guanine (G) nucleotide methylated at the 7 position, such as m7G(5')ppp(5')G, typically written as m7GpppG. Cap species may also be anti-reverse cap analogs. A non-limiting list of possible cap species includes m7GpppG, m7Gpppm7G, m73'dGpppG, m27,03'GpppG, m27,03'GppppG, m27,02'GppppG, m7Gpppm7G, m73'dGpppG, m27,03'GpppG, m27,03'GppppG, and m27,02'GppppG. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned cap species can be excluded from the RNA molecules disclosed herein.
在一些態樣中，5'端帽包括帽類似物，例如5'端帽可包括鳥嘌呤類似物。例示性鳥嘌呤類似物包括但不限於肌苷、N1-甲基-鳥苷、2'-氟-鳥苷、7-脫氮-鳥苷、8-側氧基-鳥苷、2-胺基-鳥苷、LNA-鳥苷及2-疊氮基-鳥苷。在一些態樣中，前述鳥嘌呤類似物中之1、2、3、4、5者或更多者可排除在本文所揭示之帽結構外。In some embodiments, the 5' end cap includes a cap analog, for example, the 5' end cap may include a guanine analog. Exemplary guanine analogs include but are not limited to inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. In some embodiments, 1, 2, 3, 4, 5 or more of the aforementioned guanine analogs may be excluded from the cap structure disclosed herein.
在一些態樣中，加帽區可包括單一帽或形成帽之一系列核苷酸。在此態樣中，加帽區之長度可為1至10，例如2-9、3-8、4-7、1-5、5-10個，或至少2個或10個或更少核苷酸。在此態樣中，加帽區之長度為至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：1、2、3、4、5、6、7、8、9或10個核苷酸。在一些態樣中，不存在帽。在一些態樣中，第一及第二可操作區之長度可在3至40個核苷酸，例如5-30、10-20、15、或至少4個或30個或更少之範圍內，且除起始及/或終止密碼子以外可包含一或多個訊號及/或限制性酶切序列。在一些態樣中，第一及第二操作區之長度為至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39或40個核苷酸，且除起始及/或終止密碼子以外可包含一或多個訊號及/或限制性酶切序列。In some aspects, the capping region may include a single cap or a series of nucleotides that form a cap. In this aspect, the length of the capping region may be 1 to 10, such as 2-9, 3-8, 4-7, 1-5, 5-10, or at least 2 or 10 or fewer nucleotides. In this aspect, the length of the capping region is at least, at most, just below, or between any two of the following (inclusive or exclusive): 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In some aspects, there is no cap. In some aspects, the length of the first and second operable regions may be in the range of 3 to 40 nucleotides, such as 5-30, 10-20, 15, or at least 4 or 30 or fewer, and may include one or more signal and/or restriction enzyme sequences in addition to the start and/or stop codons. In some aspects, the length of the first and second operating regions is at least, at most, just below, or between any two of the following (inclusive or exclusive): 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides and may comprise one or more signal and/or restriction enzyme sequences in addition to the start and/or stop codons.
5'帽結構之其他實例包括但不限於甘油基、倒置的去氧無鹼基殘基(部分)、4',5'-亞甲基核苷酸、1-(β-D-赤呋喃醣基)核苷酸、4'-硫核苷酸、碳環核苷酸、1,5-無水己糖醇核苷酸、L-核苷酸、α-核苷酸、經修飾之鹼基核苷酸、蘇-呋喃戊醣基核苷酸、非環狀3',4'-開環核苷酸、非環狀3,4-二羥丁基核苷酸、非環狀3,5-二羥基戊基核苷酸、3'-3'-倒置的核苷酸部分、3'-3'-倒置的無鹼基部分、3'-2'-倒置的核苷酸部分、3'-2'-倒置的無鹼基部分、1,4-丁二醇磷酸酯、3'-胺基磷酸酯、己基磷酸酯、胺基己基磷酸酯、3'-磷酸酯、3'-硫代磷酸酯、二硫代磷酸酯、及/或橋聯或非橋聯甲基膦酸酯部分。在一些態樣中，前述5'帽結構中之1、2、3、4、5者或更多者可排除在本文所揭示之RNA分子外。Other examples of 5' cap structures include, but are not limited to, glyceryl, inverted deoxy abasic residue (part), 4',5'-methylene nucleotide, 1-(β-D-erythrofuranosyl) nucleotide, 4'-thionucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotide, α-nucleotide, modified alkali nucleotide, threo-furanopentosyl nucleotide, non-cyclic 3',4'-open ring nucleotide, non-cyclic 3,4-dihydroxybutyl nucleoside. In some embodiments, the 5'-cap structure may be a 5'-capped RNA molecule. In some embodiments, the 5'-capped RNA molecule may be a 5'-capped RNA molecule. In some embodiments, the 5'-capped RNA molecule may be a 5'-capped RNA molecule. In some embodiments, the 5'-capped RNA molecule may be a 5'-capped RNA molecule. In some embodiments, the 5'-capped RNA molecule may be a 5'-capped RNA molecule. In some embodiments, the 5'-capped RNA molecule may be a 5'-capped RNA molecule. In some embodiments, the 5'-capped RNA molecule may be a 5'-capped RNA molecule. In some embodiments, the 5'-capped RNA molecule may be a 5'-capped RNA molecule.
在一些態樣中，本發明之RNA分子包含至少一個5'帽結構。在一些態樣中，本發明之RNA分子不包含5'帽結構。In some aspects, the RNA molecules of the present invention comprise at least one 5' cap structure. In some aspects, the RNA molecules of the present invention do not comprise a 5' cap structure.
多種合成5'帽類似物已研發出且為此項技術中已知的，以增強mRNA穩定性及可譯性(參見例如Grudzien-Nogalska, E., Kowalska, J., Su, W., Kuhn, A.N., Slepenkov, S.V., Darynkiewicz, E., Sahin, U., Jemielity, J.及Rhoads, R.E., Synthetic mRNAs with superior translation and stability properties in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology 69 (Rabinovich, P.H.編), 2013)。在一個態樣中，5'加帽結構包含經修飾之5'帽1結構(m7G+m3'-5'-ppp-5'-Am)。在一個態樣中，5'加帽結構包含(3'OMe)-m27,3'-OGppp(m12'-O)ApG (TriLink BioTechnologies)。此分子與天然RNA帽結構一致，因為其以在N7處甲基化之鳥苷開始，且藉由5'-5'三磷酸酯鍵聯連接至所轉錄RNA之第一所編碼核苷酸(在此情況下為腺苷)。此鳥苷亦在核糖之3'羥基處甲基化以緩解帽分子之可能反向併入。腺苷上之核糖之2'羥基經甲基化，從而賦予帽1結構。A variety of synthetic 5' cap analogs have been developed and are known in the art to enhance mRNA stability and translatability (see, e.g., Grudzien-Nogalska, E., Kowalska, J., Su, W., Kuhn, AN, Slepenkov, SV, Darynkiewicz, E., Sahin, U., Jemielity, J., and Rhoads, RE, Synthetic mRNAs with superior translation and stability properties in Synthetic Messenger RNA and Cell Metabolism Modulation in Methods in Molecular Biology 69 (Rabinovich, PH ed.), 2013). In one aspect, the 5' capping structure comprises a modified 5' cap 1 structure (m7G+ m3'-5'-ppp-5'-Am). In one aspect, the 5' capping structure comprises (3'OMe)-m27, 3'-O Gppp(m12'-O )ApG (TriLink BioTechnologies). This molecule is consistent with the natural RNA cap structure in that it begins with a guanosine methylated at N7 and is linked to the first encoded nucleotide of the transcribed RNA (in this case, adenosine) via a 5'-5' triphosphate linkage. This guanosine is also methylated at the 3' hydroxyl of the ribose to mitigate possible reverse incorporation of the cap molecule. The 2' hydroxyl of the ribose on the adenosine is methylated, thereby conferring the cap 1 structure.
C.非轉譯區(UTR)5' UTR為位於蛋白質開讀框之5'端的調節區域，其轉錄成mRNA但不轉譯成胺基酸序列，及/或RNA聚核苷酸(諸如mRNA分子)中之對應區。非轉譯區(UTR)可存在於開讀框之5'端(上游) (5' UTR)及/或開讀框之3'端(下游) (3' UTR)。C.Non-translated region(UTR) The 5' UTR is a regulatory region located at the 5' end of the protein open reading frame that is transcribed into mRNA but not translated into amino acid sequence, and/or the corresponding region in RNA polynucleotides (such as mRNA molecules). The non-translated region (UTR) can be present at the 5' end (upstream) of the open reading frame (5' UTR) and/or the 3' end (downstream) of the open reading frame (3' UTR).
在一些態樣中，UTR來源於在mRNA表現所靶向之特定組織(例如淋巴組織)中天然豐富的mRNA。在一些態樣中，UTR增加蛋白質合成。不受機制或理論所束縛，UTR可藉由增加mRNA在轉譯聚核糖體中之保持時間(訊息穩定性)及/或核糖體基於訊息起始轉譯之速率(訊息轉譯效率)來增加蛋白質合成。因此，UTR序列可以組織特異性方式延長蛋白質合成。In some aspects, UTRs are derived from mRNAs that are naturally abundant in a specific tissue to which mRNA expression is targeted (e.g., lymphoid tissue). In some aspects, UTRs increase protein synthesis. Without being bound by mechanism or theory, UTRs can increase protein synthesis by increasing the retention time of mRNA in translating polyribosomes (message stability) and/or the rate at which ribosomes initiate translation based on the message (message translation efficiency). Thus, UTR sequences can prolong protein synthesis in a tissue-specific manner.
在一些態樣中，UTR之調節特徵可併入本發明之RNA中以尤其增強分子之穩定性。亦可併入特異性特徵，以確保轉錄本在被誤導至不希望的器官部位之情況下的受控下調。各種5' UTR及3' UTR序列為此項技術中已知且可用的。In some aspects, regulatory features of UTRs can be incorporated into the RNA of the present invention to, among other things, enhance the stability of the molecule. Specific features can also be incorporated to ensure controlled downregulation in the event that the transcript is misdirected to an undesirable organ site. Various 5'UTR and 3'UTR sequences are known and available in the art.
應理解，來自任何基因之任何UTR可併入本發明之RNA之區域中。此外，可利用任何已知基因之多種野生型UTR。提供不為野生型區域之變體之人工UTR亦在本發明之範疇內。此等UTR或其部分之置放取向可與其所選自之轉錄本中相同，或其取向及/或位置可變化。因此，5'及/或3' UTR可倒置、縮短、延長，及/或一或多種其他5' UTR或3' UTR亦如此。如本文所用，術語「改變」在與UTR序列有關時，意謂UTR已相對於參考序列以某種方式進行變化。舉例而言，5' UTR及/或3' UTR可藉由上文所教示改變其取向及/或位置而相對於野生型或天然UTR進行改變，及/或可藉由包括額外核苷酸、缺失核苷酸、交換及/或轉位核苷酸而進行改變。此等變化中之任一者產生「改變」的UTR (無論5'及/或3')，包括變異UTR。It should be understood that any UTR from any gene can be incorporated into the region of the RNA of the present invention. In addition, a variety of wild-type UTRs of any known gene can be used. Providing artificial UTRs that are not variants of wild-type regions is also within the scope of the present invention. The placement orientation of these UTRs or parts thereof can be the same as in the transcript from which they are selected, or their orientation and/or position can be varied. Therefore, 5' and/or 3' UTRs can be inverted, shortened, extended, and/or one or more other 5' UTRs or 3' UTRs are also the same. As used herein, the term "alteration" when related to a UTR sequence means that the UTR has been changed in some way relative to a reference sequence. For example, the 5'UTR and/or 3'UTR can be altered relative to the wild-type or native UTR by changing its orientation and/or position as taught above, and/or can be altered by including additional nucleotides, deleting nucleotides, exchanging and/or transposing nucleotides. Any of these changes results in an "altered" UTR (whether 5' and/or 3'), including a variant UTR.
在一些實施例中，可使用雙重、三重或四重UTR，諸如5'及/或3' UTR。如本文所用，「雙」UTR為同一UTR之兩個複本串聯或實質上串聯編碼的UTR。舉例而言，可使用雙重β-球蛋白3' UTR。具有模式化UTR亦在本發明之範疇內。如本文所用，「模式化UTR」為反映重複或交替模式之彼等UTR，諸如AB AB AB或AABBAABBAABB或ABCABCABC或其變體重複一次、兩次或超過3次。在此等模式中，各字母A、B或C表示在核苷酸層級的不同UTR。In some embodiments, a double, triple or quadruple UTR may be used, such as the 5' and/or 3' UTR. As used herein, a "double" UTR is a UTR in which two copies of the same UTR are coded in tandem or substantially in tandem. For example, a double β-globin 3' UTR may be used. It is also within the scope of the present invention to have a patterned UTR. As used herein, a "patterned UTR" is one that reflects a repeating or alternating pattern, such as AB AB AB or AABBAABBABB or ABCABCABC or variants thereof repeated once, twice or more than 3 times. In these patterns, each letter A, B or C represents a different UTR at the nucleotide level.
RNA可編碼屬於在特定細胞、組織中及/或在發育期間之一些時間表現的蛋白質家族之所關注多肽。在一些態樣中，來自此等基因中之任一者之UTR可交換相同或不同的蛋白質家族之任何其他UTR以產生新的RNA分子。如本文所用，「蛋白質家族」以最廣泛的意義使用，係指共用至少一種功能、結構、特徵、定位、起源及/或表現模式的一組兩種或更多種所關注多肽。The RNA may encode a polypeptide of interest belonging to a family of proteins that are expressed in a particular cell, tissue, and/or at some time during development. In some aspects, a UTR from any of these genes may be exchanged for any other UTR of the same or different protein family to generate a new RNA molecule. As used herein, "protein family" is used in the broadest sense to refer to a group of two or more polypeptides of interest that share at least one function, structure, characteristic, localization, origin, and/or expression pattern.
在一些態樣中，5' UTR及3' UTR序列以計算方式得出。在一些態樣中，5' UTR及3' UTR來源於組織中天然豐富的mRNA。該組織可為例如肝臟、幹細胞及/或淋巴組織。淋巴組織可包括例如淋巴球(例如B淋巴球、輔助T淋巴球、細胞毒性T淋巴細胞、調節T淋巴球及/或自然殺手細胞)、巨噬細胞、單核球、樹突狀細胞、嗜中性球、嗜酸性球及網狀紅血球中之任一者。在一些態樣中，5' UTR及3' UTR來源於α病毒(alphavirus)。在一些態樣中，5' UTR及3' UTR來自野生型α病毒。In some aspects, 5'UTR and 3'UTR sequences are derived computationally. In some aspects, 5'UTR and 3'UTR are derived from mRNAs that are naturally abundant in tissues. The tissue may be, for example, liver, stem cells, and/or lymphoid tissue. Lymphoid tissue may include, for example, lymphocytes (e.g., B lymphocytes, helper T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, and/or natural killer cells), macrophages, monocytes, dendritic cells, neutrophils, eosinophils, and any one of reticulocytes. In some aspects, 5'UTR and 3'UTR are derived from alphaviruses. In some aspects, 5'UTR and 3'UTR are derived from wild-type alphaviruses.
在一些態樣中，非轉譯區亦可包括轉譯強化子元件(TEE)。作為一非限制性實例，TEE可包括美國申請案第20090226470號(其以全文引用之方式併入本文中)中所描述之彼等者以及此項技術中已知之彼等者。In some aspects, the non-translation region may also include a translation enhancement element (TEE). As a non-limiting example, the TEE may include those described in U.S. Application No. 20090226470 (which is incorporated herein by reference in its entirety) and those known in the art.
i.5'UTR在一些態樣中，本文所揭示之RNA包含5' UTR。5' UTR (若存在)位於5'端處且以蛋白質編碼區之起始密碼子上游之轉錄起始位點開始。5' UTR在5'帽(若存在)下游，例如直接鄰近於5'帽。5' UTR可含有各種調節元件，例如5'帽結構、莖環結構及內部核糖體進入位點(IRES)，其可在轉譯起始之控制中起作用。5' UTR可含有標誌，如Kozak序列，其亦涉及核糖體藉以起始許多基因轉譯之過程。5' UTR亦可形成涉及延伸因子結合之二級結構。i.5'UTR In some aspects, the RNA disclosedherein comprises a 5'UTR. The 5'UTR (if present) is located at the 5' end and begins with the transcription initiation site upstream of the start codon of the protein coding region. The 5'UTR is downstream of the 5'cap (if present), for example, directly adjacent to the 5'cap. The 5'UTR may contain various regulatory elements, such as a 5'cap structure, a stem-loop structure, and an internal ribosome entry site (IRES), which may play a role in the control of translation initiation. The 5'UTR may contain markers, such as a Kozak sequence, which is also involved in the process by which the ribosome initiates the translation of many genes. The 5'UTR may also form a secondary structure involved in the binding of elongation factors.
在一些態樣中，本文所揭示之5' UTR包含例如本文所揭示之帽近端序列。在一些態樣中，帽近端序列包含鄰近於5'帽之序列。在一些態樣中，帽近端序列包含RNA聚核苷酸之位置+1、+2、+3、+4及/或+5之核苷酸。In some aspects, the 5'UTR disclosed herein comprises a cap-proximal sequence, e.g., as disclosed herein. In some aspects, the cap-proximal sequence comprises a sequence adjacent to the 5' cap. In some aspects, the cap-proximal sequence comprises nucleotides at positions +1, +2, +3, +4, and/or +5 of the RNA polynucleotide.
在一些態樣中，帽結構包含帽近端序列之一或多個聚核苷酸。在一些態樣中，帽結構包含m7鳥苷帽及RNA聚核苷酸之核苷酸+1 (N1)。在一些態樣中，帽結構包含m7鳥苷帽及RNA聚核苷酸之核苷酸+2 (N2)。在一些態樣中，帽結構包含m7鳥苷帽及RNA聚核苷酸之核苷酸+1及+2 (N1及N2)。In some aspects, the cap structure comprises one or more polynucleotides of the cap-proximal sequence. In some aspects, the cap structure comprises an m7 guanosine cap and nucleotide +1 (N1 ) of the RNA polynucleotide. In some aspects, the cap structure comprises an m7 guanosine cap and nucleotide +2 (N2 ) of the RNA polynucleotide. In some aspects, the cap structure comprises an m7 guanosine cap and nucleotides +1 and +2 (N1 andN2 ) of the RNA polynucleotide.
閱讀本發明之熟習此項技術者應瞭解，在一些態樣中，帽近端序列(例如殘基+1、+2、+3、+4及/或+5中之一或多者)之一或多個殘基可藉助於包括於帽實體(例如帽1結構等)中而包括於RNA中；或者，在一些態樣中，帽近端序列中之至少一些殘基可以酶促方式添加(例如藉由聚合酶，諸如T7聚合酶)。舉例而言，在其中利用(m27,3'-O)Gppp(m2'-O)ApG帽之某些例示性態樣中，+1及+2殘基為帽之(m27,3'-O) A及G殘基，且+3、+4及+5殘基藉由聚合酶(例如T7聚合酶)添加。Those skilled in the art who read the present invention will appreciate that in some aspects, one or more residues in the cap-proximal sequence (e.g., one or more of residues +1, +2, +3, +4, and/or +5) can be included in the RNA by inclusion in a cap entity (e.g., a cap 1 structure, etc.); or, in some aspects, at least some of the residues in the cap-proximal sequence can be added enzymatically (e.g., by a polymerase, such as T7 polymerase). For example, in certain exemplary aspects in which a (m27,3'-0 )Gppp(m2'-0 )ApG cap is utilized, the +1 and +2 residues are the (m27,3'-0 )A and G residues of the cap, and the +3, +4, and +5 residues are added by a polymerase (e.g., T7 polymerase).
在一些態樣中，帽近端序列包含帽結構之N1及/或N2，其中N1及N2為任何核苷酸，例如A、C、G或U。在一些態樣中，N1為A。在一些態樣中，N1為C。在一些態樣中，N1為G。在一些態樣中，N1為U。在一些態樣中，N2為A。在一些態樣中，N2為C。在一些態樣中，N2為G。在一些態樣中，N2為U。在一些態樣中，帽近端序列包含帽結構之N1及N2以及N3、N4及N5，其中N1至N5對應於RNA聚核苷酸之位置+1、+2、+3、+4及/或+5。在一些態樣中，N1、N2、N3、N4或N5為任何核苷酸，例如A、C、G或U。在一些態樣中，N1N2包含以下中之任一者：AA、AC、AG、AU、CA、CC、CG、CU、GA、GC、GG、GU、UA、UC、UG或UU。在一些態樣中，N1N2包含AG，且N3N4N5包含以下中之任一者：AAA、ACA、AGA、AUA、AAG、AGG、ACG、AUG、AAC、ACC、AGC、AUC、AAU、ACU、AGU、AUU、CAA、CCA、CGA、CUA、CAG、CGG、CCG、CUG、CAC、CCC、CGC、CUC、CAU、CCU、CGU、CUU、GAA、GCA、GGA、GUA、GAG、GGG、GCG、GUG、GAC、GCC、GGC、GUC、GAU、GCU、GGU、GUU、UAA、UCA、UGA、UUA、UAG、UGG、UCG、UUG、UAC、UCC、UGC、UUC、UAU、UCU、UGU或UUU。In some aspects, the cap-proximal sequence comprisesN1 and/orN2 of the cap structure, whereinN1 andN2 are any nucleotides, such as A, C, G, or U. In some aspects,N1 is A. In some aspects,N1 is C. In some aspects,N1 is G. In some aspects,N1 is U. In some aspects,N2 is A. In some aspects,N2 is C. In some aspects,N2 is G. In some aspects,N2 is U. In some aspects, the cap-proximal sequence comprisesN1 andN2 of the cap structure andN3 ,N4 , andN5 , whereinN1 toN5 correspond to positions +1, +2, +3, +4, and/or +5 of the RNA polynucleotide. In some aspects,N1 ,N2 ,N3 ,N4 orN5 is any nucleotide, such as A, C, G or U. In some aspects,N1N2 comprises any ofthe following: AA, AC, AG, AU, CA, CC, CG, CU, GA, GC, GG, GU, UA, UC, UG or UU. In some aspects,N1N2 comprises AG andN3N4N5 comprises any of AAA,ACA , AGA, AUA, AAG, AGG, ACG, AUG, AAC,ACC , AGC, AUC, AAU, ACU, AGU, AUU, CAA, CCA, CGA, CUA, CAG, CGG, CCG, CUG, CAC, CCC, CGC, CUC, CAU, CCU, CGU, CUU, GAA, GCA, GGA, GUA, GAG, GGG, GCG, GUG, GAC, GCC, GGC, GUC, GAU, GCU, GGU, GUU, UAA, UCA, UGA, UUA, UAG, UGG, UCG, UUG, UAC, UCC, UGC, UUC, UAU, UCU, UGU, or UUU.
在一些態樣中，帽近端序列包含帽結構之N1及N2，以及包含A3A4X5(SEQ ID NO: 46；其中X5為A、G、C或U)之序列，其中N1及N2各自獨立地選自：A、C、G或U。在一些態樣中，N1為A且N2為G。在一些態樣中，X5選自A、C、G或U。在一些態樣中，X5為A。在一些態樣中，X5為C。在一些態樣中，X5為G。在一些態樣中，X5為U。In some aspects, the cap-proximal sequence comprisesN1 andN2 of the cap structure, and a sequence comprisingA3A4X5 (SEQ ID NO: 46; whereinX5 is A, G, C, or U) , whereinN1 andN2 are each independently selected from: A, C,G , or U. In some aspects,N1 is A andN2 is G. In some aspects,X5 is selected from A, C, G, or U. In some aspects,X5 is A. In some aspects,X5 is C. In some aspects,X5 is G. In some aspects,X5 is U.
在一些態樣中，帽近端序列包含帽結構之N1及N2，以及包含C3A4X5(SEQ ID NO: 47；其中X5為A、G、C或U)之序列，其中N1及N2各自獨立地選自：A、C、G或U。在一些態樣中，N1為A且N2為G。在一些態樣中，X5選自A、C、G或U。在一些態樣中，X5為A。在一些態樣中，X5為C。在一些態樣中，X5為G。在一些態樣中，X5為U。In some aspects, the cap-proximal sequence comprisesN1 andN2 of the cap structure, and a sequence comprisingC3A4X5 (SEQ ID NO:47 ; whereinX5 is A, G, C, or U) , whereinN1 andN2 are each independently selected from: A, C, G, or U. In some aspects,N1 is A andN2 is G. In some aspects,X5 is selected from A, C, G, or U. In some aspects,X5 is A. In some aspects,X5 is C. In some aspects,X5 is G. In some aspects,X5 is U.
在一些態樣中，帽近端序列包含帽結構之N1及N2，以及包含X3Y4X5(SEQ ID NO: 48；其中X3或X5各自獨立地選自A、G、C或U；且Y4不為C)之序列。在一些態樣中，N1且N2各自獨立地選自：A、C、G或U。在一些態樣中，N1為A且N2為G。在一些態樣中，X3及X5各自獨立地選自：A、C、G或U。在一些態樣中，X3及/或X5為A。在一些態樣中，X3及/或X5為C。在一些態樣中，X3及/或X5為G。在一些態樣中，X3及/或X5為U。在一些態樣中，Y4為C。在其他態樣中，Y4不為C。在一些態樣中，Y4為A。在一些態樣中，Y4為G。在其他態樣中，Y4不為G。在一些態樣中，Y4為U。In some aspects, the cap-proximal sequence comprisesN1 andN2 of the cap structure, anda sequence comprisingX3Y4X5 (SEQ ID NO:48 ; whereinX3 orX5 is each independently selected from A, G, C or U; andY4 is not C). In some aspects,N1 andN2 are each independently selected from: A, C, G or U. In some aspects,N1 is A andN2 is G. In some aspects,X3 andX5 are each independently selected from: A, C, G or U. In some aspects,X3 and/orX5 are A. In some aspects,X3 and/orX5 are C. In some aspects,X3 and/or X5 are G. In some aspects, X3and/ orX5 are U. In some aspects,Y4 is C. In other aspects, Y4 is not C. In some aspects, Y4 is A. In some aspects, Y4 is G. In other aspects, Y4 is not G. In some aspects, Y4 is U.
在一些態樣中，帽近端序列包含帽結構之N1及N2以及包含A3C4A5(SEQ ID NO: 49)之序列。在一些態樣中，N1且N2各自獨立地選自：A、C、G或U。在一些態樣中，N1為A且N2為G。In some aspects, the cap-proximal sequence comprisesN1 andN2 of the cap structure and a sequence comprisingA3C4A5 (SEQ ID NO: 49). In some aspects,N1 andN2 are each independently selected from: A,C , G or U. In some aspects,N1 is A andN2 is G.
在一些態樣中，帽近端序列包含帽結構之N1及N2以及包含A3U4G5(SEQ ID NO: 50)之序列。在一些態樣中，N1且N2各自獨立地選自：A、C、G或U。在一些態樣中，N1為A且N2為G。In some aspects, the cap-proximal sequence comprisesN1 andN2 of the cap structure and a sequence comprisingA3U4G5 (SEQ ID NO: 50). In some aspects,N1 andN2 are each independently selected from: A,C , G orU. In some aspects,N1 is A andN2 is G.
在一些態樣中，前述帽近端序列中之1、2、3、4、5者或更多者可排除在本文所揭示之RNA分子之5' UTR外。In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned cap-proximal sequences can be excluded from the 5'UTR of the RNA molecules disclosed herein.
在本發明之一些態樣中，5' UTR為異源UTR，例如為在自然界中發現與不同ORF結合的UTR。在另一態樣中，5' UTR為例如在自然界中不存在的合成UTR。合成UTR包括已突變或合成以改良其特性(例如以增加基因表現)之UTR。在一些態樣中，5' UTR功能性地連接至ORF，例如與ORF結合，以使得其可發揮功能，例如與包含參考5' UTR之參考RNA分子或缺乏5' UTR之RNA分子相比，增加、增強、穩定及/或延長自RNA分子之蛋白質產生，及/或增加自RNA分子之蛋白質表現及/或總蛋白質產生。在一些態樣中，可排除前述5' UTR功能中之1、2、3、4、5者或更多者。In some aspects of the present invention, the 5'UTR is a heterologous UTR, for example, a UTR found in nature to be bound to a different ORF. In another aspect, the 5'UTR is a synthetic UTR, for example, not found in nature. Synthetic UTRs include UTRs that have been mutated or synthesized to improve their properties (e.g., to increase gene expression). In some aspects, the 5'UTR is functionally linked to the ORF, for example, bound to the ORF, so that it can function, for example, compared to a reference RNA molecule comprising a reference 5'UTR or an RNA molecule lacking a 5'UTR, increase, enhance, stabilize and/or extend protein production from the RNA molecule, and/or increase protein expression and/or total protein production from the RNA molecule. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned 5'UTR functions may be excluded.
例示性5' UTR包括來源於非洲爪蟾屬(Xenopus)或人類α球蛋白或β球蛋白、人類細胞色素b-245 a、羥基類固醇(17b)脫氫酶、菸草蝕刻病毒、CMV即刻早期1 (IE1)基因、TEV、HSP705'、c-Jun或前述中之任一者之同源物、片段或變體的5' UTR。在一些態樣中，5' UTR為：缺乏5' TOP模體(寡嘧啶段(tract))之TOP基因之5' UTR的片段、同源物或變體；來源於核糖體蛋白大32 (L32)基因的5' UTR；來源於羥基類固醇(17p)脫氫酶4基因(HSD17B4)之5' UTR的5' UTR；或來源於ATP5A1之5' UTR的5' UTR。在一些態樣中，5' UTR來源於專利申請案WO2013/143700 (其揭示內容以全文引用之方式併入本文中)之SEQ ID NO: 1-1363、SEQ ID NO: 1395、SEQ ID NO: 1421及SEQ ID NO: 1422，或與前述序列中之任一者具有至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的一致性之序列：99%、98%、97%、96%、95%、90%、85%或80%。亦可使用序列GGGAUCCUACC。在一些態樣中，前述5' UTR序列中之1、2、3、4、5者或更多者可排除在本文所揭示之RNA分子外。Exemplary 5'UTRs include those derived fromXenopus or human alpha or beta globulin, human cytochrome b-245a, hydroxysteroid (17b) dehydrogenase, tobacco etch virus, CMV immediate early 1 (IE1) gene, TEV, HSP705', c-Jun, or a homolog, fragment or variant of any of the foregoing. In some aspects, the 5'UTR is: a fragment, homolog or variant of the 5'UTR of a TOP gene lacking a 5'TOP motif (oligopyrimidine tract); a 5'UTR derived from the ribosomal protein large 32 (L32) gene; a 5'UTR derived from the 5'UTR of the hydroxysteroid (17p) dehydrogenase 4 gene (HSD17B4); or a 5'UTR derived from the 5'UTR of ATP5A1. In some aspects, the 5'UTR is derived from SEQ ID NO: 1-1363, SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 of patent application WO2013/143700 (the disclosure of which is incorporated herein by reference in its entirety), or a sequence having at least, at most, just below, or between any two of the following (inclusive or exclusive) identity to any of the foregoing sequences: 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80%. The sequence GGGAUCCUACC may also be used. In some aspects, 1, 2, 3, 4, 5 or more of the foregoing 5'UTR sequences may be excluded from the RNA molecules disclosed herein.
在一些態樣中，5' UTR包含來自編碼以下各者之基因之5' UTR區域或來自其同源物、片段或變體的序列：RPSA、RPS2、RPS3、RPS3A、RPS4、RPS5、RPS6、RPS7、RPS8、RPS9、RPS10、RPS1 1、RPS12、RPS13、RPS14、RPS15、RPS15A、RPS16、RPS17、RPS18、RPS19、RPS20、RPS21、RPS23、RPS24、RPS25、RPS26、RPS27、RPS27A、RPS28、RPS29、RPS30、RPL3、RPL4、RPL5、RPL6、RPL7、RPL7A、RPL8、RPL9、RPL10、RPL10A、RPL1 1、RPL12、RPL13、RPL13A、RPL14、RPL15、RPL17、RPL18、RPL18A、RPL19、RPL21、RPL22、RPL23、RPL23A、RPL24、RPL26、RPL27、RPL27A、RPL28、RPL29、RPL30、RPL31、RPL32、RPL34、RPL35、RPL35A、RPL36、RPL36A、RPL37、RPL37A、RPL38、RPL39、RPL40、RPL41、RPLPO、RPLP1、RPLP2、RPLP3、RPLPO、RPLP1、RPLP2、EEF1A1、EEF1 B2、EEF1 D、EEF1 G、EEF2、EIF3E、EIF3F、EIF3H、EIF2S3、EIF3C、EIF3K、EIF3EIP、EIF4A2、PABPC1、HNRNPA1、TPT1、TUBB1、UBA52、NPM1、ATP5G2、GNB2L1、NME2、UQCRB；或與前述基因序列中之任一者具有至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的一致性之基因序列：99%、98%、97%、96%、95%、90%、85%或80%。在一些態樣中，前述5' UTR序列中之1、2、3、4、5者或更多者可排除在本文所揭示之RNA分子外。In some aspects, the 5'UTR comprises a sequence from the 5'UTR region of a gene encoding RPSA, RPS2, RPS3, RPS3A, RPS4, RPS5, RPS6, RPS7, RPS8, RPS9, RPS10, RPS11, RPS12, RPS13, RPS14, RPS15, RPS15A, RPS16, RPS17, RPS18, RPS19, RPS20, RPS21, RPS23, RPS24, RPS25, RPS26, RPS27, RPS27A, RPS28, RPS29, RPS30, RPL3, RPL4, RPL5, RPL6, RPL7, RPL7A, RPL8, RPL9, RPL10, RPL10A, RPL11, RPS12, RPS13, RPS14, RPS15 1. RPL12, RPL13, RPL13A, RPL14, RPL15, RPL17, RPL18, RPL18A, RPL19, RPL21, RPL22, RPL23, RPL23A, RPL24, RPL26, RPL27, RPL27A, RPL28, RPL29, RPL30, RPL3 1. RPL32, RPL34, RPL35, RPL35A, RPL36, RPL36A, RPL37, RPL37A, RPL38, RPL39, RPL40, RPL41, RPLPO, RPLP1, RPLP2, RPLP3, RPLPO, RPLP1, RPLP2, EEF1A1, EEF1 B2, EEF1 D, EEF1 G, EEF2, EIF3E, EIF3F, EIF3H, EIF2S3, EIF3C, EIF3K, EIF3EIP, EIF4A2, PABPC1, HNRNPA1, TPT1, TUBB1, UBA52, NPM1, ATP5G2, GNB2L1, NME2, UQCRB; or a gene sequence having at least, at most, just below, or between any two of the following (inclusive or exclusive) identity with any of the aforementioned gene sequences: 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80%. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned 5'UTR sequences can be excluded from the RNA molecules disclosed herein.
在一個態樣中，編碼本文所揭示之5' UTR之DNA包含與SEQ ID NO: 17具有至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的一致性之序列：99%、98%、97%、96%、95%、90%、85%或80%。在一個態樣中，編碼5' UTR之DNA包含SEQ ID NO: 17之序列。在一個態樣中，本文所揭示之RNA包含含有與SEQ ID NO: 18或19 (其中所轉錄5'帽結構加底線)中之任一者中所提供之5' UTR具有至少、至多、恰好以下或在以下中之任何兩者之間的一致性之序列的5' UTR：99%、98%、97%、96%、95%、90%、85%或80%。在一個態樣中，5' UTR包含SEQ ID NO: 18或19 (其中所轉錄5'帽結構加底線)中之任一者之序列。 SEQ ID NO: 17 (DNA)AGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCC SEQ ID NO:18 (RNA)AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCC SEQ ID NO: 19 (RNA)AGAAΨAAACΨAGΨAΨΨCΨΨCΨGGΨCCCCACAGACΨCAGAGAGAACCCIn one aspect, the DNA encoding the 5'UTR disclosed herein comprises a sequence having at least, at most, just below, or between any two of the following (inclusive or exclusive) identity to SEQ ID NO: 17: 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%. In one aspect, the DNA encoding the 5'UTR comprises the sequence of SEQ ID NO: 17. In one aspect, the RNA disclosed herein comprises a 5'UTR comprising a sequence having at least, at most, just below, or between any two of the following identity to the 5'UTR provided in any one of SEQ ID NO: 18 or 19 (wherein the transcribed 5' cap structure is underlined): 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%. In one aspect, the 5'UTR comprises the sequence of either SEQ ID NO: 18 or 19 (wherein the transcribed 5' cap structure is underlined). SEQ ID NO: 17 (DNA)AG AATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCC SEQ ID NO: 18 (RNA) AGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCC SEQ ID NO: 19 (RNA)AG AAΨAAACΨAGΨAΨΨCΨΨCΨGGΨCCCCACAGACΨCAGAGAGAACCC
在一個態樣中，編碼本文所揭示之5' UTR之DNA包含與SEQ ID NO: 51具有至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的一致性之序列：99%、98%、97%、96%、95%、90%、85%或80%。在一個態樣中，編碼5' UTR之DNA包含SEQ ID NO: 51之序列。在一個態樣中，本文所揭示之RNA包含含有與SEQ ID NO: 52或53中之任一者中所提供之5' UTR具有至少、至多、恰好以下或在以下中之任何兩者之間的一致性之序列的5' UTR：99%、98%、97%、96%、95%、90%、85%或80%。在一個態樣中，5' UTR包含SEQ ID NO: 52或53 (其中所轉錄5'帽結構加底線)中之任一者之序列。 SEQ ID NO: 51 (DNA)GATAGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAA SEQ ID NO: 52 (RNA)GAUAGGCGGCGCAUGAGAGAAGCCCAGACCAAUUACCUACCCAAA SEQ ID NO: 53 (RNA)GAΨAGGCGGCGCAΨGAGAGAAGCCCAGACCAAΨΨACCΨACCCAAAIn one aspect, the DNA encoding the 5'UTR disclosed herein comprises a sequence having at least, at most, just below, or between any two of the following (inclusive or exclusive) identity to SEQ ID NO: 51: 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%. In one aspect, the DNA encoding the 5'UTR comprises the sequence of SEQ ID NO: 51. In one aspect, the RNA disclosed herein comprises a 5'UTR comprising a sequence having at least, at most, just below, or between any two of the following identity to the 5'UTR provided in any one of SEQ ID NO: 52 or 53: 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%. In one aspect, the 5'UTR comprises the sequence of any one of SEQ ID NO: 52 or 53 (wherein the transcribed 5' cap structure is underlined). SEQ ID NO: 51 (DNA)G ATAGGCGGCGCATGAGAGAAGCCCAGACCAATTACCTACCCAAA SEQ ID NO: 52 (RNA)G AUAGGCGGCGCAUGAGAGAAGCCCAGACCAAUUACCUACCCAAA SEQ ID NO: 53 (RNA)G AΨAGGCGGCGCAΨGAGAGAAGCCCAGACCAAΨΨACCΨACCCAAA
在一些態樣中，前述5' UTR序列中之1、2、3者或更多者可排除在本文所揭示之RNA分子外。In some aspects, 1, 2, 3 or more of the aforementioned 5'UTR sequences can be excluded from the RNA molecules disclosed herein.
ii. 3' UTR在一些態樣中，本文所揭示之RNA包含3' UTR。3' UTR (若存在)位於蛋白編碼序列開讀框下游，例如蛋白質編碼區之終止密碼子下游。3' UTR通常為位於mRNA之蛋白質編碼序列與多-A尾之間的mRNA部分。因此，在一些態樣中，3' UTR在多-A序列(若存在)上游，例如直接鄰近於多-A序列。3' UTR可涉及調節過程，包括轉錄本裂解、穩定性及多腺苷酸化、轉譯、及mRNA定位。ii. 3'UTR In some aspects, the RNA disclosed herein comprises a 3'UTR. The 3'UTR, if present, is located downstream of the protein coding sequence opening frame, e.g., downstream of the termination codon of the protein coding region. The 3'UTR is typically the portion of the mRNA between the protein coding sequence and the poly-A tail of the mRNA. Thus, in some aspects, the 3'UTR is upstream of the poly-A sequence, if present, e.g., directly adjacent to the poly-A sequence. The 3'UTR may be involved in regulatory processes, including transcript cleavage, stability and polyadenylation, translation, and mRNA localization.
天然或野生型3' UTR包含腺苷及尿苷之延伸部分。此等富AU標誌在周轉率較高之基因中尤其普遍。基於其序列特徵及功能特性，富AU元件(ARE)可分成三類：I類ARE在富U區域內含有數個分散的AUUUA模體複本。II類ARE具有兩個或更多個重疊UUAUUUA(U/A)(U/A)九聚體。III類ARE不含AUUUA模體。已知大部分與ARE結合之蛋白質使分子不穩定。因此，3' UTR ARE之引入、移除及/或修飾可用於調節本發明之核酸(例如RNA)之穩定性。當工程改造特定核酸時，在一些態樣中，可引入一或多個ARE複本以使得RNA較不穩定且因此減少轉譯且降低所得蛋白質之產生。同樣，在一些態樣中，可鑑別及移除及/或突變ARE以增加細胞內穩定性且因此增加轉譯及所得蛋白質之產生。可在相關細胞株中使用本發明之核酸進行轉染實驗，且可在轉染後之不同時間點分析蛋白質產生。舉例而言，可用不同的ARE工程改造分子且藉由使用針對相關蛋白質之ELISA套組來轉染細胞且在轉染後6小時、12小時、24小時、48小時及7天分析所產生之蛋白質。在一些態樣中，3' UTR可使一或多個富AU序列被移除。或者，富AU序列可保持在3' UTR中。The natural or wild-type 3'UTR comprises an extension of adenosine and uridine. These AU-rich markers are particularly common in genes with a higher turnover rate. Based on their sequence characteristics and functional properties, AU-rich elements (AREs) can be divided into three categories: Class I AREs contain several dispersed copies of the AUUUA motif in the U-rich region. Class II AREs have two or more overlapping UUAUUUA (U/A) (U/A) 9-mers. Class III AREs do not contain the AUUUA motif. It is known that most proteins bound to AREs make the molecules unstable. Therefore, the introduction, removal and/or modification of 3'UTR AREs can be used to regulate the stability of nucleic acids (e.g., RNA) of the present invention. When engineering a specific nucleic acid, in some aspects, one or more ARE copies can be introduced to make the RNA less stable and thus reduce translation and reduce the production of the resulting protein. Similarly, in some aspects, ARE can be identified and removed and/or mutated to increase intracellular stability and thus increase the production of translation and resulting protein. Transfection experiments can be performed using the nucleic acids of the present invention in relevant cell lines, and protein production can be analyzed at different time points after transfection. For example, different ARE engineered molecules can be used and cells can be transfected using ELISA kits for relevant proteins and the produced proteins can be analyzed 6 hours, 12 hours, 24 hours, 48 hours and 7 days after transfection. In some aspects, 3'UTR can remove one or more AU-rich sequences. Alternatively, the AU-rich sequence can remain in the 3'UTR.
3' UTR亦可包含不在轉錄RNA之模板中編碼但在轉錄之後在成熟期間添加的元件，例如多-A尾。mRNA之3' UTR不轉譯成胺基酸序列。在一些態樣中，本文所揭示之RNA包含含有F元件及/或I元件之3' UTR。在一些態樣中，3' UTR或其近端序列包含限制位點。在一些態樣中，限制位點為BamHI位點。在一些態樣中，限制位點為Xhol位點。3'UTR may also include elements that are not encoded in the template of the transcribed RNA but are added during maturation after transcription, such as a poly-A tail. The 3'UTR of mRNA is not translated into an amino acid sequence. In some aspects, the RNA disclosed herein includes a 3'UTR containing an F element and/or an I element. In some aspects, the 3'UTR or its proximal sequence includes a restriction site. In some aspects, the restriction site is aBamHI site. In some aspects, the restriction site isan Xhol site.
在本發明之一些態樣中，3' UTR為異源UTR，例如為在自然界中發現與不同ORF結合的UTR。在另一態樣中，3' UTR為例如在自然界中不存在的合成UTR。在一些態樣中，3' UTR功能性地連接至ORF，例如與ORF結合，以使得其可發揮功能，例如與包含參考3' UTR之參考RNA分子或缺乏3' UTR之RNA分子相比，增加、增強、穩定及/或延長自RNA分子之蛋白質產生，及/或增加自RNA分子之蛋白質表現及/或總蛋白質產生。在一些態樣中，可排除前述3' UTR功能中之1、2、3、4、5者或更多者。In some aspects of the present invention, the 3'UTR is a heterologous UTR, such as a UTR found in nature in combination with a different ORF. In another aspect, the 3'UTR is a synthetic UTR, such as one that does not exist in nature. In some aspects, the 3'UTR is functionally linked to the ORF, such as in combination with the ORF, so that it can function, such as increasing, enhancing, stabilizing and/or extending protein production from the RNA molecule, and/or increasing protein expression and/or total protein production from the RNA molecule compared to a reference RNA molecule comprising a reference 3'UTR or an RNA molecule lacking a 3'UTR. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned 3'UTR functions can be excluded.
例示性3' UTR包括：來源於以下之3' UTR：白蛋白基因、a-球蛋白基因、β-球蛋白基因、核糖體蛋白基因、酪胺酸羥化酶基因、脂肪加氧酶基因及膠原蛋白α基因(諸如膠原蛋白α1 (1)基因；或來源於包含以下之基因之3' UTR的同源物、片段或變體：白蛋白基因、a-球蛋白基因、β-球蛋白基因、核糖體蛋白基因、酪胺酸羥化酶基因、脂肪加氧酶基因及/或膠原蛋白α基因(諸如膠原蛋白α1 (1)基因)，根據專利申請案WO2013/143700 (其揭示內容以全文引用之方式併入本文中)之SEQ ID NO: 1369-1390，或與前述序列中之任一者具有至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的一致性的序列：99%、98%、97%、96%、95%、90%、85%或80%。在一些態樣中，使用序列UUUGAAUU。在一些態樣中，前述3' UTR序列中之1、2、3、4、5者或更多者可排除在本文所揭示之RNA分子外。Exemplary 3'UTRs include: 3'UTRs derived from albumin gene, α-globulin gene, β-globulin gene, ribosomal protein gene, tyrosine hydroxylase gene, lipoxygenase gene and collagen α gene (such as collagen α1 (1) gene); or homologs, fragments or variants derived from 3'UTRs comprising albumin gene, α-globulin gene, β-globulin gene, ribosomal protein gene, tyrosine hydroxylase gene, lipoxygenase gene and/or collagen α gene (such as collagen α1 (1) gene), according to SEQ ID NO: 1369-1390, or a sequence having at least, at most, just less than, or between any two of the following (inclusive or exclusive) identity to any of the aforementioned sequences: 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%. In some aspects, the sequence UUUGAAUU is used. In some aspects, 1, 2, 3, 4, 5, or more of the aforementioned 3'UTR sequences can be excluded from the RNA molecules disclosed herein.
在一些態樣中，3' UTR包含：轉錄本之序列，其包括NM_000661.4、NM_001024921.2、NM_000967.3、NM_001033853.1、NMJD00968.3、NM_000969.3、NM_001024662.1、NM_000970.3、NM_000971.3、NMJD00972.2、NM_000975.3、NM_001 199802.1、NM_000976.3、NM__000977.3、NM_033251.2、NMJ 01243130.1、NM_001243131、NM_000978.3、NM_000979.3、NM_001270490.1、NMJD00980.3、NM_000981.3、NM_000982.3、NM_000983.3、NM_000984.5、NM_000985.4、NM_001035006.2、NM_001 199340.1、NM_001 199341.1、NMJD01 199342.1、NM_001 199343.1、NM_001 199344.1、NM_001 199345.1、NM_000986.3、NM_000987.3、NM_000988.3、NM_000989.3、NM_000990.4、NM_001 136134.1、NMJD00991.4、NM_001 136135.1、NM_001 136136.1、NM_001 136137.1、NM_000992.2、NM_000993.4、NM_001098577.2、NM_001099693.1、NM_000994.3、NM_001007073.1、NM_001007074.1、NM_000996.2、M_000997.4、NM_000998.4、NM_000999.3、NM_001035258.1、NM_001000.3、NM_001002.3、NM_053275.3、NM_001003.2、NM_213725.1、NM_001004.3、NM_001005.4、NM_001256802.1、NM_001260506.1、NM_001260507.1、NM_001006.4、NM_001267699.1、NM_001007.4、NM_001008.3、N _001009.3、NM_001010.2、NM_00101 1.3、NM_001012.1、NM_001013.3、NM_001203245.2、NM_001014.4、NM_001204091.1、NM_001015.4、NM_001016.3、NM_001017.2、NM_001018.3、NM_001030009.1、NM_001019.4、NM_001020.4、NM_001022.3、NM_001 146227.1、NM_001023.3、NM_001024.3、NM_001025.4、NM_001028.2、NM_001029.3、NM_001030.4、NM_002954、NM_001 135592.2、NM_001 177413.1、NM_001031.4、NM_001032.4、NM_001030001.2、NM_002948.3、NM_001253379.1、NM_001253380.1、NM_001253382.1、NM_001253383.1、NM_001253384.1、NM_002952.3、NM_001034996.2、NM_001025071.1、NM_001025070.1、NM_005617.3、NM_006013.3、NM_001256577.1、NM_001256580.1、NM_007104.4、NM_007209.3、NM_012423.3、NM_001270491.1、NM_033643.2、NM_015414.3、NM_021029.5、NM_001 199972.1、NM_021 104.1、NM_022551.2、NM_033022.3、NM_001 142284.1、NM_001026.4、NM_001 142285.1、NM_001 142283.1、NM_001 142282.1、NM_000973.3、NM_033301.1、NM_000995.3、NM_033625.2、NM_001021.3、NM_002295.4、NM_001012321.1、NM_001033930.1、NM_003333.3、NM_001997.4、NM_001099645.1、NM_001021.3、NM_052969.1、NM_080746.2、NM_001001.4、NM_005061.2、NM_015920.3、NM_016093.2、NM_198486.2、NG_01 1 172.1、NG_01 1253.1、NG_000952.4、NR_002309.1、NG_010827.2、NG_009952.2或NG_009517.1；或與前述轉錄本中之任一者具有至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的一致性之轉錄本的序列：99%、98%、97%、96%、95%、90%、85%或80%。在一些態樣中，前述3' UTR序列中之1、2、3、4、5者或更多者可排除在本文所揭示之RNA分子外。In some aspects, the 3'UTR comprises a transcript sequence including NM_000661.4, NM_001024921.2, NM_000967.3, NM_001033853.1, NMJD00968.3, NM_000969.3, NM_001024662.1, NM_000970.3, NM_000971.3, NMJD00972.2, NM_000975.3, NM_001 199802.1, NM_000976.3, NM_000977.3, NM_033251.2, NMJD00968.3, NM_000969.3, NM_001024662.1, NM_000970.3, NM_000971.3, NMJD00972.2, NM_000975.3, NM_001 199802.1, NM_000976.3, NM_000977.3, NM_033251.2, NMJD00968.3, NM_000969.3, 01243130.1, NM_001243131, NM_000978.3, NM_000979.3, NM_001270490.1, NMJD00980.3, NM_ 000981.3, NM_000982.3, NM_000983.3, NM_000984.5, NM_000985.4, NM_001035006.2, NM_001 199340.1, NM_001 199341.1, NMJD01 199342.1, NM_001 199343.1, NM_001 199344.1, NM_001 199345.1, NM_000986.3, NM_000987.3, NM_000988.3, NM_000989.3, NM_000990.4, NM_001 136134.1, NMJD00991.4, NM_001 136135.1, NM_001 136136.1, NM_001 136137.1, NM_000992.2, NM_000993.4, NM_001098577.2, NM_001099693.1, NM_000994.3, NM_001007073 .1, NM_001007074.1, NM_000996.2, M_000997.4, NM_000998.4, NM_000999.3, NM_001035258.1, NM_0010 00.3, NM_001002.3, NM_053275.3, NM_001003.2, NM_213725.1, NM_001004.3, NM_001005.4, NM_0012568 02.1, NM_001260506.1, NM_001260507.1, NM_001006.4, NM_001267699.1, NM_001007.4, NM_001008.3, N _001009.3, NM_001010.2, NM_00101 1.3, NM_001012.1, NM_001013.3, NM_001203245.2, NM_001014.4, NM_001204091.1, NM_001015.4, NM_00 1016.3, NM_001017.2, NM_001018.3, NM_001030009.1, NM_001019.4, NM_001020.4, NM_001022.3, NM_001 146227.1, NM_001023.3, NM_001024.3, NM_001025.4, NM_001028.2, NM_001029.3, NM_001030.4, NM_002954, NM_001 135592.2, NM_001 177413.1, NM_001031.4, NM_001032.4, NM_001030001.2, NM_002948.3, NM_001253379.1, NM_001253 380.1, NM_001253382.1, NM_001253383.1, NM_001253384.1, NM_002952.3, NM_001034996.2, NM_0010 25071.1, NM_001025070.1, NM_005617.3, NM_006013.3, NM_001256577.1, NM_001256580.1, NM_00710 4.4, NM_007209.3, NM_012423.3, NM_001270491.1, NM_033643.2, NM_015414.3, NM_021029.5, NM_001 199972.1, NM_021 104.1, NM_022551.2, NM_033022.3, NM_001 142284.1, NM_001026.4, NM_001 142285.1, NM_001 142283.1, NM_001 142282.1, NM_000973.3, NM_033301.1, NM_000995.3, NM_033625.2, NM_001021.3, NM_002295.4, NM_001012321.1, NM_001033930.1, NM_003333.3, NM _001997.4, NM_001099645.1, NM_001021.3, NM_052969.1, NM_080746.2, NM_001001.4, NM_005061.2, NM_015920.3, NM_016093.2, NM_198486.2, NG_01 1 172.1, NG_01 1253.1, NG_000952.4, NR_002309.1, NG_010827.2, NG_009952.2, or NG_009517.1; or a transcript sequence having at least, at most, just less than, or between any two of the following (inclusive or exclusive) identity to any of the foregoing transcripts: 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%. In some aspects, 1, 2, 3, 4, 5, or more of the foregoing 3'UTR sequences can be excluded from the RNA molecules disclosed herein.
在一些態樣中，3' UTR包含：來自編碼核糖體蛋白之基因之3' UTR區域的序列，該核糖體蛋白例如核糖體蛋白L9 (RPL9)、核糖體蛋白L3 (RPL3)、核糖體蛋白L4 (RPL4)、核糖體蛋白L5 (RPL5)、核糖體蛋白L6 (RPL6)、核糖體蛋白L7 (RPL7)、核糖體蛋白L7a (RPL7A)、核糖體蛋白L11 (RPL11 )、核糖體蛋白L12 (RPL12)、核糖體蛋白L13 (RPL13)、核糖體蛋白L23 (RPL23)、核糖體蛋白L18 (RPL18)、核糖體蛋白L18a (RPL18A)、核糖體蛋白L19 (RPL19)、核糖體蛋白L21 (RPL21 )、核糖體蛋白L22 (RPL22)、核糖體蛋白L23a (RPL23A)、核糖體蛋白L17 (RPL17)、核糖體蛋白L24 (RPL24)、核糖體蛋白L26 (RPL26)、核糖體蛋白L27 (RPL27)、核糖體蛋白L30 (RPL30)、核糖體蛋白L27a (RPL27A)、核糖體蛋白L28 (RPL28)、核糖體蛋白L29 (RPL29)、核糖體蛋白L31 (RPL31 )、核糖體蛋白L32 (RPL32)、核糖體蛋白L35a (RPL35A)、核糖體蛋白L37 (RPL37)、核糖體蛋白L37a (RPL37A)、核糖體蛋白L38 (RPL38)、核糖體蛋白L39 (RPL39)、核糖體蛋白大P0 (RPLP0)、核糖體蛋白大P1 (RPLP1 )、核糖體蛋白大P2 (RPLP2)、核糖體蛋白S3 (RPS3)、核糖體蛋白S3A (RPS3A)、X連鎖的核糖體蛋白S4 (RPS4X)、Y連鎖的核糖體蛋白S4 1 (RPS4Y1 )、核糖體蛋白S5 (RPS5)、核糖體蛋白S6 (RPS6)、核糖體蛋白S7 (RPS7)、核糖體蛋白S8 (RPS8)、核糖體蛋白S9 (RPS9)、核糖體蛋白S10 (RPS10)、核糖體蛋白S11 (RPS11 )、核糖體蛋白S12 (RPS12)、核糖體蛋白S13 (RPS13)、核糖體蛋白S15 (RPS15)、核糖體蛋白S15a (RPS15A)、核糖體蛋白S16 (RPS16)、核糖體蛋白S19 (RPS19)、核糖體蛋白S20 (RPS20)、核糖體蛋白S21 (RPS21 )、核糖體蛋白S23 (RPS23)、核糖體蛋白S25 (RPS25)、核糖體蛋白S26 (RPS26)、核糖體蛋白S27 (RPS27)、核糖體蛋白S27a (RPS27a)、核糖體蛋白S28 (RPS28)、核糖體蛋白S29 (RPS29)、核糖體蛋白L15 (RPL15)、核糖體蛋白S2 (RPS2)、核糖體蛋白L14 (RPL14)、核糖體蛋白S14 (RPS14)、核糖體蛋白L10 (RPL10)、核糖體蛋白L10a (RPL10A)、核糖體蛋白L35 (RPL35)、核糖體蛋白L13a (RPL13A)、核糖體蛋白L36 (RPL36)、核糖體蛋白L36a (RPL36A)、核糖體蛋白L41 (RPL41 )、核糖體蛋白S18 (RPS18)、核糖體蛋白S24 (RPS24)、核糖體蛋白L8 (RPL8)、核糖體蛋白L34 (RPL34)、核糖體蛋白S17 (RPS17)、核糖體蛋白SA (RPSA)或核糖體蛋白S17 (RPS17)；或編碼與前述核糖體基因蛋白質序列中之任一者具有至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的一致性之核糖體蛋白之基因的序列：99%、98%、97%、96%、95%、90%、85%或80%。在一些態樣中，前述3' UTR序列中之1、2、3、4、5者或更多者可排除在本文所揭示之RNA分子外。In some aspects, the 3'UTR comprises a sequence from the 3'UTR region of a gene encoding a ribosomal protein, such as ribosomal protein L9 (RPL9), ribosomal protein L3 (RPL3), ribosomal protein L4 (RPL4), ribosomal protein L5 (RPL5), ribosomal protein L6 (RPL6), ribosomal protein L7 (RPL7), ribosomal protein L7a (RPL7A), ribosomal protein L11 (RPL11), ribosomal protein L12 (RPL12), ribosomal protein L13 (RPL13), ribosomal protein L23 (RPL23), ribosomal protein L18 (RPL18), ribosomal protein L18a (RPL18A), ribosomal protein L19 (RPL19), ribosomal protein L21 (RPL21), ribosomal protein L22 (RPL22), ribosomal protein L23a (RPL23), (RPL23A), ribosomal protein L17 (RPL17), ribosomal protein L24 (RPL24), ribosomal protein L26 (RPL26), ribosomal protein L27 (RPL27), ribosomal protein L30 (RPL30), ribosomal protein L27a (RPL27A), ribosomal protein L28 (RPL28), ribosomal protein L29 (RPL29), ribosomal protein L31 (RPL31), ribosomal protein L32 (RPL32), ribosomal protein L35a (RPL35A), ribosomal protein L37 (RPL37), ribosomal protein L37a (RPL37A), ribosomal protein L38 (RPL38), ribosomal protein L39 (RPL39), ribosomal protein large P0 (RPLP0), ribosomal protein large P1 (RPLP1), ribosomal protein large P2 (RPLP2), ribosomal protein S3 (RPS3), ribosomal protein S3A (RPS3A), X-linked ribosomal protein S4 (RPS4X), Y-linked ribosomal protein S4 1 (RPS4Y1), ribosomal protein S5 (RPS5), ribosomal protein S6 (RPS6), ribosomal protein S7 (RPS7), ribosomal protein S8 (RPS8), ribosomal protein S9 (RPS9), ribosomal protein S10 (RPS10), ribosomal protein S11 (RPS11), ribosomal protein S12 (RPS12), ribosomal protein S13 (RPS13), ribosomal protein S15 (RPS15), ribosomal protein S15a (RPS15A), ribosomal protein S16 (RPS16), ribosomal protein S19 (RPS19), ribosomal protein S20 (RPS20), ribosomal protein S21 (RPS21), ribosomal protein S23 (RPS23), ribosomal protein S25 (RPS25), ribosomal protein S26 (RPS26), ribosomal protein S27 (RPS27), ribosomal protein S27a (RPS27a), ribosomal protein S28 (RPS28), ribosomal protein S29 (RPS29), ribosomal protein L15 (RPL15), ribosomal protein S2 (RPS2), ribosomal protein L14 (RPL14), ribosomal protein S14 (RPS14), ribosomal protein L10 (RPL10), ribosomal protein L10a (RPL10A), ribosomal protein L35 (RPL35), ribosomal protein L13a (RPL13A), ribosomal protein L36 (RPL36), ribosomal protein L36a (RPL36A), ribosomal protein L41 (RPL41), ribosomal protein S18 (RPS18), ribosomal protein S24 (RPS24), ribosomal protein L8 (RPL8), ribosomal protein L34 (RPL34), ribosomal protein S17 (RPS17), ribosomal protein SA (RPSA), or ribosomal protein S17 (RPS17); or a sequence of a gene encoding a ribosomal protein having at least, at most, exactly less than, or between any two of the following (inclusive or exclusive) identity to any of the aforementioned ribosomal gene protein sequences: 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%. In some aspects, 1, 2, 3, 4, 5, or more of the aforementioned 3'UTR sequences can be excluded from the RNA molecules disclosed herein.
在一些態樣中，3' UTR包含：來自編碼核糖體蛋白之基因之3' UTR區域或來自包含以下之基因的序列：泛蛋白A-52殘基核糖體蛋白融合產物1 (UBA52)、廣泛表現(FAU)的芬克爾-比斯基斯-賴利(Finkel-Biskis-Reilly)鼠類肉瘤病毒(FBR-MuSV)、核糖體蛋白L22樣1 (RPL22L1)、核糖體蛋白L39樣(RPL39L)、核糖體蛋白L10樣(RPL10L)、核糖體蛋白L36a樣(RPL36AL)、核糖體蛋白L3樣(RPL3L)、核糖體蛋白S27樣(RPS27L)、核糖體蛋白L26樣1 (RPL26L1)、核糖體蛋白L7樣1 (RPL7L1)、核糖體蛋白L13a假基因(RPL13AP)、核糖體蛋白L37a假基因8 (RPL37AP8)、核糖體蛋白S10假基因5 (RPS10P5)、核糖體蛋白S26假基因1 1 (RPS26P1 1)、核糖體蛋白L39假基因5 (RPL39P5)、核糖體蛋白大PO假基因6 (RPLP0P6)及核糖體蛋白L36假基因14 (RPL36P14)；及/或編碼與前述基因蛋白質序列中之任一者具有至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的一致性之蛋白質之基因的序列：99%、98%、97%、96%、95%、90%、85%或80%。在一些態樣中，前述3' UTR序列中之1、2、3、4、5者或更多者可排除在本文所揭示之RNA分子外。In some aspects, the 3'UTR comprises: a 3'UTR region from a gene encoding a ribosomal protein or a sequence from a gene comprising: ubiquitin A-52 residue ribosomal protein fusion product 1 (UBA52), ubiquitously expressed (FAU) Finkel-Biskis-Reilly murine sarcoma virus (FBR-MuSV), ribosomal protein L22-like 1 (RPL22L1), ribosomal protein L39-like (RPL39L), ribosomal protein L10-like (RPL10L), ribosomal protein L36a-like (RPL36AL), ribosomal protein L3-like (RPL3L), ribosomal protein S27-like (RPS27L), ribosomal protein L26-like 1 (RPL26L1), ribosomal protein L7-like 1 (RPL26L1), (RPL7L1), ribosomal protein L13a pseudogene (RPL13AP), ribosomal protein L37a pseudogene 8 (RPL37AP8), ribosomal protein S10 pseudogene 5 (RPS10P5), ribosomal protein S26 pseudogene 1 1 (RPS26P1 1), ribosomal protein L39 pseudogene 5 (RPL39P5), ribosomal protein large PO pseudogene 6 (RPLP0P6) and ribosomal protein L36 pseudogene 14 (RPL36P14); and/or a sequence of a gene encoding a protein that has at least, at most, exactly less than, or between any two of the following (inclusive or exclusive) identity to any of the protein sequences of the aforementioned genes: 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80%. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned 3'UTR sequences can be excluded from the RNA molecules disclosed herein.
一般熟習此項技術者應理解，可使用異源及/或合成的5' UTR與任何所需3' UTR序列，且反之亦然。舉例而言，可使用異源5' UTR與合成及/或異源3' UTR。One of ordinary skill in the art will appreciate that a heterologous and/or synthetic 5'UTR can be used with any desired 3'UTR sequence, and vice versa. For example, a heterologous 5'UTR can be used with a synthetic and/or heterologous 3'UTR.
在一個態樣中，本文所揭示之編碼3' UTR的DNA包含與SEQ ID NO: 20具有至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的一致性之序列：99%、98%、97%、96%、95%、90%、85%或80%。在一個態樣中，編碼3' UTR之DNA包含SEQ ID NO: 20之序列。在一些態樣中，本文所揭示之RNA包含含有與SEQ ID NO: 21或22中之任一者中所提供之3' UTR具有至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的一致性之序列的3' UTR：99%、98%、97%、96%、95%、90%、85%或80%。在一個態樣中，3' UTR包含SEQ ID NO: 21或22中之任一者之序列。 SEQ ID NO: 20 (DNA) CTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGC SEQ ID NO: 21 (RNA) CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUAGC SEQ ID NO: 22 (RNA) CΨCGAGCΨGGΨACΨGCAΨGCACGCAAΨGCΨAGCΨGCCCCΨΨΨCCCGΨCCΨGGGΨACCCCGAGΨCΨCCCCCGACCΨCGGGΨCCCAGGΨAΨGCΨCCCACCΨCCACCΨGCCCCACΨCACCACCΨCΨGCΨAGΨΨCCAGACACCΨCCCAAGCACGCAGCAAΨGCAGCΨCAAAACGCΨΨAGCCΨAGCCACACCCCCACGGGAAACAGCAGΨGAΨΨAACCΨΨΨAGCAAΨAAACGAAAGΨΨΨAACΨAAGCΨAΨACΨAACCCCAGGGΨΨGGΨCAAΨΨΨCGΨGCCAGCCACACCCΨGGAGCΨAGCIn one aspect, the DNA encoding the 3'UTR disclosed herein comprises a sequence having at least, at most, just below, or between any two of the following (inclusive or exclusive) identity to SEQ ID NO: 20: 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%. In one aspect, the DNA encoding the 3'UTR comprises the sequence of SEQ ID NO: 20. In some aspects, the RNA disclosed herein comprises a 3'UTR containing a sequence having at least, at most, just below, or between any two of the following (inclusive or exclusive) identity to the 3'UTR provided in any one of SEQ ID NO: 21 or 22: 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%. In one aspect, the 3'UTR comprises the sequence of any one of SEQ ID NO: 21 or 22.SEQ ID NO: 20 (DNA) CTCGAGCTGGTACTGCATGCACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCGACCTCGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTCCAGACACCTCCCAAGC ACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACACCCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAGCTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCCTGGAGCTAGC SEQ ID NO: 21 (RNA) CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGAGUCUCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGC ACGCAGCAAUGCAGCUCAAAACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACACCCUGGAGCUAGC SEQ ID NO: 22 (RNA) CΨCGAGCΨGGΨACΨGCAΨGCACGCAAΨGCΨAGCΨGCCCCΨΨΨCCCGΨCCΨGGGΨACCCCGAGΨCΨCCCCCGACC ΨCGGGΨCCCAGGΨAΨGCΨCCCACCΨCCACCΨGCCCCACΨCACCACCΨCΨGCΨAGΨΨCCAGACACCΨCCCAAGCA CGCAGCAAΨGCAGCΨCAAACGCΨΨAGCCΨAGCCACACCCCCACGGGAAACAGCAGΨGAΨΨAACCΨΨΨAGCAAΨAAACGAAAGΨΨΨAACΨAAGCΨAΨACΨAACCCCAGGGΨΨGGΨCAAΨΨΨCGΨGCCAGCCACACCCΨGGAGCΨAGC
在一個態樣中，本文所揭示之編碼3' UTR的DNA包含與SEQ ID NO: 23具有至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的一致性之序列：99%、98%、97%、96%、95%、90%、85%或80%。在一個態樣中，編碼3' UTR之DNA包含SEQ ID NO: 23之序列。在一個態樣中，本文所揭示之RNA包含含有與SEQ ID NO: 24或25中之任一者中所提供之3' UTR具有至少、至多、恰好以下或在以下中之任何兩者之間的一致性之序列的3' UTR：99%、98%、97%、96%、95%、90%、85%或80%。在一個態樣中，3' UTR包含SEQ ID NO: 24或25中之任一者之序列。 SEQ ID NO: 23 (DNA) ATACAGCAGCAATTGGCAAGCTGCTTACATAGAACTCGCGGCGATTGGCATGCCGCCTTAAAATTTTTATTTTATTTTTCTTTTCTTTTCCGAATCGGATTTTGTTTTTAATATTTC SEQ ID NO: 24 (RNA) AUACAGCAGCAAUUGGCAAGCUGCUUACAUAGAACUCGCGGCGAUUGGCAUGCCGCCUUAAAAUUUUUAUUUUAUUUUUCUUUUCUUUUCCGAAUCGGAUUUUGUUUUUAAUAUUUC SEQ ID NO: 25 (RNA) AΨACAGCAGCAAΨΨGGCAAGCΨGCΨΨACAΨAGAACΨCGCGGCGAΨΨGGCAΨGCCGCCΨΨAAAAΨΨΨΨΨAΨΨΨΨAΨΨΨΨΨCΨΨΨΨCΨΨΨΨCCGAAΨCGGAΨΨΨΨGΨΨΨΨΨAAΨAΨΨΨCIn one aspect, the DNA encoding the 3'UTR disclosed herein comprises a sequence having at least, at most, just below, or between any two of the following (inclusive or exclusive) identity to SEQ ID NO: 23: 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%. In one aspect, the DNA encoding the 3'UTR comprises the sequence of SEQ ID NO: 23. In one aspect, the RNA disclosed herein comprises a 3'UTR comprising a sequence having at least, at most, just below, or between any two of the following identity to the 3'UTR provided in any one of SEQ ID NO: 24 or 25: 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%. In one aspect, the 3'UTR comprises the sequence of any one of SEQ ID NO: 24 or 25.SEQ ID NO: 23 (DNA) ATACAGCAGCAATTGGCAAGCTGCTTACATAGAACTCGCGGCGATTGGCATGCCGCCTTAAAATTTTTATTTTATTTTTCTTTTCTTTTCCGAATCGGATTTTGTTTTTAATATTTC SEQ ID NO: 24 (RNA) AUACAGCAGCAAUUGGCAAGCUGCUUACAUAGAACUCGCGGCGAUUGGCAUGCCGCCUUAAAAUUUUAUUUUUUUUCUUUUCUUUUCCGAAUCGGAUUUUGUUUUUAAUAUUUC SEQ ID NO: 25 (RNA) AΨACAGCAGCAAΨΨGGCAAGCΨGCΨΨACAΨAGAACΨCGCGGCGAΨΨGGCAΨGCCGCCΨ ΨAAAAΨΨΨΨΨAΨΨΨΨAΨΨΨΨΨΨCΨΨΨΨCΨΨΨΨCCGAAΨCGGAΨΨΨΨGΨΨΨΨΨΨΨAAΨAΨΨΨC
在一些態樣中，前述3' UTR序列中之1、2、3、4、5者或更多者可排除在本文所揭示之RNA分子外。In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned 3'UTR sequences can be excluded from the RNA molecules disclosed herein.
D.開讀框(ORF)5'及3' UTR可操作地連接至開讀框(ORF)，其可為能夠轉譯成所關注多肽之密碼子序列。開讀框可為若干DNA或RNA核苷酸三聯體之序列，其可轉譯成肽或蛋白質。ORF可以其5'端及下一區處之起始密碼子開始，該起始密碼子例如通常編碼胺基酸甲硫胺酸之三個連續核苷酸之組合(ATG或AUG)，其長度通常為3個核苷酸之倍數。開讀框可以至少一個終止密碼子結束，該終止密碼子包括但不限於TAA、TAG、TGA或UAA、UAG或UGA，或其任何組合。在一些態樣中，開讀框可以一個、兩個、三個、四個或更多個終止密碼子結束，該等終止密碼子包括但不限於TAATAA (SEQ ID NO: 27)、TAATAG (SEQ ID NO: 28)、TAATGA (SEQ ID NO: 29)、TAGTGA (SEQ ID NO: 30)、TAGTAA (SEQ ID NO: 31)、TAGTAG (SEQ ID NO: 32)、TGATGA (SEQ ID NO: 33)、TGATAG (SEQ ID NO: 34)、TGATAA (SEQ ID NO: 35)或UAAUAA (SEQ ID NO: 36)、UAAUAG (SEQ ID NO: 37)、UAAUGA (SEQ ID NO: 38)、UAGUGA (SEQ ID NO: 39)、UAGUAA (SEQ ID NO:40)、UAGUAG (SEQ ID NO: 41)、UGAUGA (SEQ ID NO: 42)、UGAUAG (SEQ ID NO: 43)、UGAUAA (SEQ ID NO: 44)或其任何組合。開讀框可為分離的，或其可併入較長核酸序列中，例如併入載體或mRNA中。開讀框亦可被稱為「(蛋白質)編碼區」或「編碼序列」。D.Open reading frame(ORF) The 5' and 3' UTRs are operably linked to an open reading frame (ORF), which can be a codon sequence that can be translated into a polypeptide of interest. The open reading frame can be a sequence of several DNA or RNA nucleotide triplets that can be translated into a peptide or protein. The ORF can begin with a start codon at its 5' end and the next region, such as a combination of three consecutive nucleotides (ATG or AUG) that usually encodes the amino acid methionine, and its length is usually a multiple of 3 nucleotides. The open reading frame can end with at least one stop codon, which includes but is not limited to TAA, TAG, TGA or UAA, UAG or UGA, or any combination thereof. In some aspects, the open reading frame can end with one, two, three, four or more stop codons, including but not limited to TAATAA (SEQ ID NO: 27), TAATAG (SEQ ID NO: 28), TAATGA (SEQ ID NO: 29), TAGTGA (SEQ ID NO: 30), TAGTAA (SEQ ID NO: 31), TAGTAG (SEQ ID NO: 32), TGATGA (SEQ ID NO: 33), TGATAG (SEQ ID NO: 34), TGATAA (SEQ ID NO: 35), or UAAUAA (SEQ ID NO: 36), UAAUAG (SEQ ID NO: 37), UAAUGA (SEQ ID NO: 38), UAGUGA (SEQ ID NO: 39), UAGUAA (SEQ ID NO: 40), UAGUAG (SEQ ID NO: 41), UGAUGA (SEQ ID NO: 42), UGAUAG (SEQ ID NO: 43), UGAUAA (SEQ ID NO: 44) or any combination thereof. The open reading frame may be isolated, or it may be incorporated into a longer nucleic acid sequence, for example, into a vector or mRNA. The open reading frame may also be referred to as a "(protein) coding region" or a "coding sequence".
如本文所陳述，RNA分子可包括一個(單順反子)、兩個(雙順反子)或更多個(多順反子)開讀框。As described herein, an RNA molecule can include one (monostronic), two (bistronic), or more (polystronic) open reading frames.
在一些態樣中，ORF編碼非結構病毒基因。在一些態樣中，ORF進一步包括一或多個次基因體啟動子。在一些態樣中，RNA分子包括可操作地連接至ORF之次基因體啟動子。在一些態樣中，第一RNA分子不包括編碼任何所關注多肽之ORF，而第二RNA分子包括編碼所關注多肽之ORF。在一些態樣中，第一RNA分子不包括次基因體啟動子。In some aspects, the ORF encodes a nonstructural viral gene. In some aspects, the ORF further comprises one or more subgenomic promoters. In some aspects, the RNA molecule comprises a subgenomic promoter operably linked to the ORF. In some aspects, the first RNA molecule does not include an ORF encoding any polypeptide of interest, and the second RNA molecule includes an ORF encoding a polypeptide of interest. In some aspects, the first RNA molecule does not include a subgenomic promoter.
本發明提供一種RNA分子，其包含至少一個編碼呼吸道融合病毒(RSV)多肽之開讀框。在一些態樣中，RNA分子包含至少一個編碼RSV F蛋白之開讀框。在一較佳態樣中，RNA分子包含至少一個編碼呼吸道融合病毒(RSV)融合前F蛋白(preF)多肽之開讀框。The present invention provides an RNA molecule comprising at least one open reading frame encoding a respiratory syncytial virus (RSV) polypeptide. In some aspects, the RNA molecule comprises at least one open reading frame encoding a RSV F protein. In a preferred aspect, the RNA molecule comprises at least one open reading frame encoding a respiratory syncytial virus (RSV) pre-fusion F protein (preF) polypeptide.
E.所關注基因本文所描述之RNA分子可包括所關注基因。所關注基因編碼所關注多肽。所關注多肽之非限制性實例包括例如生物製劑、抗體、疫苗、治療性多肽或肽、細胞穿透肽、分泌性多肽、質膜多肽、細胞質或細胞骨架多肽、細胞內膜結合多肽、核多肽、與人類疾病相關之多肽、靶向部分、由已確定無治療適應症但其在研究及探索領域中仍具有效用之人類基因體編碼的彼等多肽，或其組合。在一些態樣中，可排除前述所關注多肽中之1、2、3、4、5者或更多者。特定所關注基因之序列容易由熟習此項技術者使用公共及私有資料庫(例如GENBANK®)鑑別。E.Genes of interest The RNA molecules described herein may include genes of interest. Genes of interest encode polypeptides of interest. Non-limiting examples of polypeptides of interest include, for example, biological agents, antibodies, vaccines, therapeutic polypeptides or peptides, cell penetrating peptides, secreted polypeptides, plasma membrane polypeptides, cytoplasmic or cytoskeletal polypeptides, intracellular membrane-bound polypeptides, nuclear polypeptides, polypeptides associated with human diseases, targeting moieties, those polypeptides encoded by human genomes that have been determined to have no therapeutic indications but still have utility in the field of research and exploration, or combinations thereof. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned polypeptides of interest may be excluded. The sequence of a particular gene of interest is easily identified by those skilled in the art using public and private databases (e.g., GENBANK®).
在一些態樣中，RNA分子包括所關注基因之編碼區。在一些態樣中，所關注基因為或包含抗原性多肽或其免疫原性變體或免疫原性片段。在一些態樣中，抗原性多肽包含來自抗原之一個抗原決定基。在一些態樣中，抗原性多肽包含複數個來自抗原之不同抗原決定基。在一些態樣中，包含複數個來自抗原之不同抗原決定基的抗原性多肽為多抗原決定基的。在一些態樣中，抗原性多肽包含：來自過敏原之抗原性多肽、病毒抗原性多肽、細菌抗原性多肽、真菌抗原性多肽、寄生蟲抗原性多肽、來自傳染原之抗原性多肽、來自病原體之抗原性多肽、腫瘤抗原性多肽或自身抗原性多肽。在一些態樣中，可排除前述抗原性多肽中之1、2、3、4、5者或更多者。In some aspects, the RNA molecule includes the coding region of the gene of interest. In some aspects, the gene of interest is or comprises an antigenic polypeptide or an immunogenic variant or immunogenic fragment thereof. In some aspects, the antigenic polypeptide comprises one antigenic determinant from an antigen. In some aspects, the antigenic polypeptide comprises a plurality of different antigenic determinants from an antigen. In some aspects, the antigenic polypeptide comprising a plurality of different antigenic determinants from an antigen is polyantigenic. In some aspects, the antigenic polypeptide comprises: an antigenic polypeptide from an allergen, a viral antigenic polypeptide, a bacterial antigenic polypeptide, a fungal antigenic polypeptide, a parasite antigenic polypeptide, an antigenic polypeptide from an infectious agent, an antigenic polypeptide from a pathogen, a tumor antigenic polypeptide, or an autoantigenic polypeptide. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned antigenic polypeptides may be excluded.
術語「抗原」可係指能夠由免疫系統(例如由後天性免疫系統)識別且能夠例如藉由形成抗體及/或抗原特異性T細胞來誘發抗原特異性免疫反應作為後天性免疫反應之一部分的物質。抗原可為或可包含可藉由MHC呈現至T細胞之肽或蛋白質。抗原可為所提供核酸分子(例如本文所描述之包含至少一個編碼序列的RNA分子)之轉譯產物。另外，抗原(諸如肽或蛋白質)之包含至少一個抗原決定基之片段、變體及衍生物理解為抗原。The term "antigen" may refer to a substance that can be recognized by the immune system (e.g., by the acquired immune system) and can induce an antigen-specific immune response as part of an acquired immune response, such as by forming antibodies and/or antigen-specific T cells. An antigen may be or may comprise a peptide or protein that can be presented to T cells via MHC. An antigen may be a translation product of a provided nucleic acid molecule (e.g., an RNA molecule comprising at least one coding sequence as described herein). In addition, fragments, variants, and derivatives of an antigen (e.g., a peptide or protein) comprising at least one antigenic determinant are understood to be antigens.
在一些態樣中，編碼所關注基因(例如抗原)之RNA在所治療個體之細胞中表現以提供所關注基因(例如抗原)。在一些態樣中，RNA在個體之細胞中短暫表現。在一些態樣中，所關注基因(例如抗原)之表現係在細胞表面處。在一些態樣中，所關注基因(例如抗原)在MHC之環境下表現及呈現。在一些態樣中，所關注基因(例如抗原)表現至細胞外空間中，例如抗原被分泌出。In some aspects, RNA encoding a gene of interest (e.g., an antigen) is expressed in cells of the treated individual to provide the gene of interest (e.g., an antigen). In some aspects, RNA is expressed transiently in cells of the individual. In some aspects, the expression of the gene of interest (e.g., an antigen) is at the cell surface. In some aspects, the gene of interest (e.g., an antigen) is expressed and presented in the context of MHC. In some aspects, the gene of interest (e.g., an antigen) is expressed in the extracellular space, for example, the antigen is secreted.
在一些態樣中，RNA分子包括所關注基因(例如抗原)之編碼區。在一些態樣中，RNA分子包括來源於與傳染病相關之病原體之所關注基因(例如抗原)的編碼區。在一些態樣中，RNA分子包括來源於RSV之所關注基因(例如抗原)的編碼區。In some aspects, the RNA molecule includes the coding region of a gene of interest (e.g., an antigen). In some aspects, the RNA molecule includes the coding region of a gene of interest (e.g., an antigen) derived from a pathogen associated with an infectious disease. In some aspects, the RNA molecule includes the coding region of a gene of interest (e.g., an antigen) derived from RSV.
在一些態樣中，RNA分子編碼RSV preF蛋白或其片段或變體。In some aspects, the RNA molecule encodes RSV preF protein or a fragment or variant thereof.
在一些態樣中，本文所描述之RNA聚核苷酸或包含其之組合物或醫藥製劑包含本文所揭示之核苷酸序列。在一些態樣中，RNA聚核苷酸包含與本文所揭示之核苷酸序列具有至少80%一致性之序列。在一些態樣中，RNA聚核苷酸包含編碼與本文所揭示之多肽序列具有至少80%一致性之多肽的序列。在一些態樣中，本文所描述之RNA聚核苷酸或包含其之組合物或醫藥製劑由DNA模板轉錄。在一些態樣中，用於轉錄本文所描述之RNA聚核苷酸的DNA模板包含與RNA聚核苷酸互補之序列。在一些態樣中，本文所描述之所關注基因由包含本文所揭示之核苷酸序列的本文所描述之RNA聚核苷酸編碼。在一些態樣中，RNA聚核苷酸編碼與本文所揭示之多肽序列具有至少80%一致性之多肽。在一些態樣中，本文所描述之多肽係由RNA聚核苷酸編碼，該RNA聚核苷酸係由包含與RNA聚核苷酸互補之序列的DNA模板轉錄。In some aspects, the RNA polynucleotide described herein or a composition or pharmaceutical preparation comprising it comprises a nucleotide sequence disclosed herein. In some aspects, the RNA polynucleotide comprises a sequence having at least 80% identity with a nucleotide sequence disclosed herein. In some aspects, the RNA polynucleotide comprises a sequence encoding a polypeptide having at least 80% identity with a polypeptide sequence disclosed herein. In some aspects, the RNA polynucleotide described herein or a composition or pharmaceutical preparation comprising it is transcribed by a DNA template. In some aspects, the DNA template for transcribing the RNA polynucleotide described herein comprises a sequence complementary to the RNA polynucleotide. In some aspects, the gene of interest described herein is encoded by an RNA polynucleotide described herein comprising a nucleotide sequence disclosed herein. In some aspects, the RNA polynucleotide encodes a polypeptide having at least 80% identity with a polypeptide sequence disclosed herein. In some aspects, a polypeptide described herein is encoded by an RNA polynucleotide that is transcribed from a DNA template comprising a sequence that is complementary to the RNA polynucleotide.
在一些態樣中，RNA分子編碼RSV preF蛋白，該RSV preF蛋白包含SEQ ID NO: 1-6及71-74中之任一者的序列、或其片段或變體。In some aspects, the RNA molecule encodes a RSV preF protein comprising the sequence of any one of SEQ ID NOs: 1-6 and 71-74, or a fragment or variant thereof.
在一些態樣中，RNA分子編碼自核酸序列或其片段或變體合成之RSV preF蛋白，該核酸序列包含SEQ ID NO: 7至10及59至62中之任一者。In some aspects, the RNA molecule encodes a RSV preF protein synthesized from a nucleic acid sequence comprising any one of SEQ ID NOs: 7-10 and 59-62, or a fragment or variant thereof.
F.多-A尾在一些態樣中，本文所揭示之RNA分子包含例如本文所描述之多腺苷酸(多-A)序列。在一些態樣中，多-A序列位於3' UTR下游，例如鄰近於3' UTR。「多-A尾」或「多-A序列」係指可連接至RNA分子之3'端的連續腺嘌呤殘基延伸部分，其例如具有長達或長達約400個腺苷核苷酸，例如為或為約20至約400個，較佳地為或為約50至約400個，更佳地為或為約50至約300個，甚至更佳地為或為約50至約250個，最佳地為或為約60至約250個腺苷核苷酸。多-A序列為熟習此項技術者已知的，且可接在本文所描述之RNA分子中之3' UTR後面。多-A尾可增加RNA分子之穩定性、半衰期及/或轉譯效率。F.Poly-Atail In some aspects, the RNA molecules disclosed herein include a polyadenylic acid (poly-A) sequence, such as described herein. In some aspects, the poly-A sequence is located downstream of the 3' UTR, such as adjacent to the 3' UTR. "Poly-A tail" or "poly-A sequence" refers to a continuous stretch of adenine residues that can be attached to the 3' end of an RNA molecule, for example, having a length of up to or about 400 adenosine nucleotides, such as from or about 20 to about 400, preferably from or about 50 to about 400, more preferably from or about 50 to about 300, even more preferably from or about 50 to about 250, and most preferably from or about 60 to about 250 adenosine nucleotides. Poly-A sequences are known to those skilled in the art and can be attached to the 3' UTR in the RNA molecules described herein. The poly-A tail can increase the stability, half-life and/or translation efficiency of RNA molecules.
在裂解之後，大部分pre-mRNA獲得多腺苷酸化尾，其中包括以組蛋白莖-環替代多-A序列結束之複製依賴性組蛋白轉錄本的pre-mRNA例外。在此上下文中，3'端加工為一種促進將mRNA自細胞核運輸至細胞質且影響mRNA之穩定性及轉譯的核共轉錄過程。此3'端之形成發生在藉由裂解/多腺苷酸化機制引導之兩步反應中，且視mRNA前驅物(pre-mRNA)中之兩個序列元件：六核苷酸多腺苷酸化訊號及下游富G/U序列之存在而定。在第一步驟中，此等兩個元件之間的pre-mRNA裂解成游離3'羥基。在第二步驟中，新形成的3'端藉由多腺苷酸化或添加多-A序列而延長。After cleavage, most pre-mRNAs acquire a polyadenylated tail, including the exception of replication-dependent histone transcripts that end with a histone stem-loop instead of a poly-A sequence. In this context, 3'-end processing is a nuclear co-transcriptional process that facilitates the transport of mRNA from the cell nucleus to the cytoplasm and affects the stability and translation of the mRNA. The formation of this 3' end occurs in a two-step reaction guided by a cleavage/polyadenylation mechanism and depends on the presence of two sequence elements in the pre-mRNA: a hexanucleotide polyadenylation signal and a downstream G/U-rich sequence. In the first step, the pre-mRNA between these two elements is cleaved into a free 3' hydroxyl group. In the second step, the newly formed 3' end is extended by polyadenylation or the addition of a poly-A sequence.
多腺苷酸化係指將多-A序列添加至RNA分子，例如添加至未成熟mRNA。多腺苷酸化可由所謂的多腺苷酸化訊號誘導。此訊號可位於接近於待多腺苷酸化之RNA分子之3'端或在其3'端處的核苷酸延伸部分內。人工核酸分子之3' UTR亦可包含多腺苷酸化訊號。多腺苷酸化訊號通常包含由腺嘌呤及尿嘧啶/胸腺嘧啶核苷酸組成之六聚體，較佳地六聚體序列AAUAAA，但亦可以想到其他序列(較佳地六聚體序列)。多腺苷酸化通常發生在pre-mRNA (亦稱為未成熟mRNA)之加工期間。通常，RNA成熟(自pre-mRNA至成熟mRNA)包含多腺苷酸化之步驟。活體外轉錄mRNA之多-A加尾可使用多種途徑達成，該等途徑包括但不限於將聚T段選殖至DNA模板中，或藉由使用多-A聚合酶進行轉錄後添加。該術語可指作為細胞過程之RNA的多腺苷酸化，或指藉由活體外酶促反應用適合酶(諸如大腸桿菌(E. col) 多-A聚合酶)或藉由化學合成進行多腺苷酸化。Polyadenylation refers to the addition of a poly-A sequence to an RNA molecule, for example to an immature mRNA. Polyadenylation can be induced by a so-called polyadenylation signal. This signal can be located close to the 3' end of the RNA molecule to be polyadenylated or within a nucleotide extension at its 3' end. The 3' UTR of the artificial nucleic acid molecule may also comprise a polyadenylation signal. The polyadenylation signal typically comprises a hexamer composed of adenine and uracil/thymine nucleotides, preferably the hexamer sequence AAUAAA, but other sequences (preferably hexamer sequences) are also conceivable. Polyadenylation typically occurs during the processing of pre-mRNA (also called immature mRNA). Typically, RNA maturation (from pre-mRNA to mature mRNA) comprises the step of polyadenylation. Poly-A tailing of in vitro transcribed mRNA can be achieved using a variety of approaches, including but not limited to cloning poly-T stretches into the DNA template, or by post-transcriptional addition using a poly-A polymerase. The term can refer to polyadenylation of RNA as a cellular process, or to polyadenylation by an in vitro enzymatic reaction with a suitable enzyme (such as E. coli (E. col ) poly-A polymerase) or by chemical synthesis.
本文所揭示之RNA分子可具有在轉錄之後藉由不依賴於模板之RNA聚合酶連接至RNA之游離3'端的多-A序列，或由DNA編碼且由依賴於模板之RNA聚合酶轉錄的多-A序列。在一些態樣中，多-A序列係在RNA轉錄期間，例如在活體外轉錄RNA之製備期間連接，其係基於與編碼股互補之股中包含重複dT核苷酸(去氧胸苷酸)的DNA模板。The RNA molecules disclosed herein can have a poly-A sequence that is attached to the free 3' end of the RNA by a template-independent RNA polymerase after transcription, or a poly-A sequence that is encoded by a DNA and transcribed by a template-dependent RNA polymerase. In some aspects, the poly-A sequence is attached during RNA transcription, for example, during preparation of an in vitro transcribed RNA based on a DNA template that includes repeated dT nucleotides (deoxythymidylic acid) in the strand complementary to the coding strand.
編碼多-A序列之DNA序列(編碼股)被稱為多-A卡匣。在一些態樣中，DNA之編碼股中存在之多-A卡匣基本上由dA核苷酸組成，但間雜有四種核苷酸(dA、dC、dG及dT)之隨機序列。此類隨機序列之長度可為至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的核苷酸：5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50個。此類卡匣揭示於例如WO 2016/005324 A1 (其以引用之方式併入本文中)中。WO 2016/005324 A1中所揭示之任何多-A卡匣均可用於本發明中。基本上由dA核苷酸組成但間雜有具有四種核苷酸(dA、dC、dG、dT)之相等分佈且長度為例如5至50個核苷酸之隨機序列的多-A卡匣在DNA層級上顯示質體DNA在大腸桿菌中之不斷繁殖，且在RNA層級上仍與同支持RNA穩定性及轉譯效率有關之有益特性相關。在一些態樣中，本文所描述之RNA聚核苷酸中所含有之多-A序列基本上由腺苷核苷酸組成，但間雜有四種核苷酸(A、C、G、U)之隨機序列。此類隨機序列之長度可為至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的核苷酸：5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50個。A DNA sequence encoding a poly-A sequence (coding strand) is called a poly-A cassette. In some aspects, the poly-A cassette present in the coding strand of the DNA consists essentially of dA nucleotides but is interspersed with random sequences of the four nucleotides (dA, dC, dG, and dT). The length of such random sequences can be at least, at most, just below, or between any two of the following (inclusive or exclusive) nucleotides: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. Such cassettes are disclosed, for example, in WO 2016/005324 A1, which is incorporated herein by reference. Any poly-A cassette disclosed in WO 2016/005324 A1 can be used in the present invention. Poly-A cassettes consisting essentially of dA nucleotides but interspersed with random sequences having an equal distribution of four nucleotides (dA, dC, dG, dT) and having a length of, for example, 5 to 50 nucleotides, show continuous propagation of plasmid DNA in E. coli at the DNA level and are still associated with beneficial properties related to supporting RNA stability and translation efficiency at the RNA level. In some aspects, the poly-A sequence contained in the RNA polynucleotides described herein consists essentially of adenosine nucleotides but is interspersed with random sequences of four nucleotides (A, C, G, U). The length of such random sequences may be at least, at most, exactly less than, or between any two of the following (inclusive or exclusive): 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides.
多-A序列可位於3' UTR內之任何位置處。在一些態樣中，沒有除腺苷核苷酸以外的核苷酸在多-A序列之3'端側接該多-A序列，例如多-A序列之3'端不被除腺苷以外的核苷酸掩蓋或多-A序列之3'端後面不跟隨除腺苷以外的核苷酸。在一些態樣中，多-A序列可位於3' UTR之3'末端處，例如3' UTR不含有超過12、11、10、9、8、7、6、5、4、3、2或1個核苷酸位於多-A序列之3'；更佳地，3' UTR不含有其他元件位於多-A序列之3'。在一些態樣中，多-A序列位於RNA分子之3'末端處，例如人工核酸分子不含有超過12、11、10、9、8、7、6、5、4、3、2或1個核苷酸位於多-A序列之3'。或者，多-A序列可位於3' UTR之5'末端處，例如位於緊鄰人工核酸分子之ORF之3'處，或位於3' UTR內，例如側接在其他3' UTR元件之5'側及3'側。在一些態樣中，多-A序列側接在聚C序列及/或組蛋白莖-環序列之3'側。或者或另外，多-A序列可側接在來源於例如人類白蛋白或球蛋白基因之3' UTR元件的5'側。The poly-A sequence may be located at any position within the 3'UTR. In some aspects, no nucleotides other than adenosine nucleotides flank the poly-A sequence at the 3' end of the poly-A sequence, e.g., the 3' end of the poly-A sequence is not masked by nucleotides other than adenosine or the 3' end of the poly-A sequence is not followed by nucleotides other than adenosine. In some aspects, the poly-A sequence may be located at the 3' end of the 3'UTR, e.g., the 3'UTR does not contain more than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotides located 3' to the poly-A sequence; more preferably, the 3'UTR does not contain other elements located 3' to the poly-A sequence. In some aspects, the poly-A sequence is located at the 3' end of the RNA molecule, e.g., the artificial nucleic acid molecule does not contain more than 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotides located 3' to the poly-A sequence. Alternatively, the poly-A sequence may be located at the 5' end of the 3'UTR, e.g., located 3' to the ORF of the adjacent artificial nucleic acid molecule, or located within the 3'UTR, e.g., flanked by other 3'UTR elements on the 5' and 3' sides. In some aspects, the poly-A sequence is flanked by the 3' side of a poly C sequence and/or a histone stem-loop sequence. Alternatively or additionally, the poly-A sequence may be flanked by the 5' side of a 3'UTR element derived from, e.g., a human albumin or globulin gene.
在一些態樣中，RNA分子可進一步包括緊接在多-A尾序列下游的核酸內切酶識別位點序列。RNA分子可進一步包括多-A聚合酶識別序列(例如多腺苷酸化訊號) (例如AAUAAA)在其3'端附近。在一些態樣中，多腺苷酸化訊號位於3' UTR中所包含之多-A序列的3'。在一些態樣中，多-A序列藉由包含至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的核苷酸或由其組成之核苷酸序列與多腺苷酸化訊號分隔開：1、2、3、4、5、6、7、8、9、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、95、100、105、110、115、120、125、130、135、140、145或150個，其中核苷酸序列較佳地不包含超過10、9、8、7、6、5、4、3或2個連續腺嘌呤核苷酸。在一些態樣中，將多-A序列與多腺苷酸化訊號分隔開之核苷酸序列包含或包含約1至約200個核苷酸，例如10至90、20至85、30至80、40至80、50至75、或55至85個核苷酸，更佳地55至80個核苷酸，且核苷酸序列不包含超過10、9、8、7、6、5、4、3或2個連續腺嘌呤核苷酸。In some aspects, the RNA molecule may further include an endonuclease recognition site sequence immediately downstream of the poly-A tail sequence. The RNA molecule may further include a poly-A polymerase recognition sequence (e.g., a polyadenylation signal) (e.g., AAUAAA) near its 3' end. In some aspects, the polyadenylation signal is located 3' of the poly-A sequence contained in the 3' UTR. In some aspects, the poly-A sequence is separated from the polyadenylation signal by a nucleotide sequence comprising or consisting of at least, at most, exactly below, or between any two of the following (inclusive or exclusive), 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150, wherein the nucleotide sequence preferably does not comprise more than 10, 9, 8, 7, 6, 5, 4, 3, or 2 consecutive adenine nucleotides. In some aspects, the nucleotide sequence separating the poly-A sequence from the polyadenylation signal comprises or comprises about 1 to about 200 nucleotides, such as 10 to 90, 20 to 85, 30 to 80, 40 to 80, 50 to 75, or 55 to 85 nucleotides, more preferably 55 to 80 nucleotides, and the nucleotide sequence does not contain more than 10, 9, 8, 7, 6, 5, 4, 3 or 2 consecutive adenine nucleotides.
在一些態樣中，多腺苷酸化訊號包含共有序列NN(U/T)ANA，其中N = A或U，較佳地為AA(U/T)AAA或A(U/T)(U/T)AAA。此類共有序列可被大部分動物及細菌細胞系統識別，例如被多腺苷酸化因子(諸如與CstF、PAP、PAB2、CFI及/或CFII協作之裂解/多腺苷酸化特異性因子(CPSF))識別。在一些態樣中，多腺苷酸化訊號(例如共有序列NNUANA)位於本文所定義之3' UTR元件之3'端下游的小於或小於約50個核苷酸處，例如位於至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的核苷酸處：1、5、10、15、20、25、30、35、40、45或50個，使得RNA分子之轉錄將產生含有多腺苷酸化訊號在其3' UTR下游的未成熟RNA且隨後將多-A序列連接至未成熟RNA。因此，所得RNA可包含含有至少一個多-A序列之3' UTR，且其中3' UTR之後為額外多-A序列。In some aspects, the polyadenylation signal comprises a consensus sequence NN(U/T)ANA, wherein N = A or U, preferably AA(U/T)AAA or A(U/T)(U/T)AAA. Such consensus sequences are recognized by most animal and bacterial cell systems, for example, by polyadenylation factors such as cleavage/polyadenylation specific factor (CPSF) that cooperates with CstF, PAP, PAB2, CFI and/or CFII. In some aspects, the polyadenylation signal (e.g., consensus sequence NNUANA) is located less than or less than about 50 nucleotides downstream of the 3' end of the 3'UTR element defined herein, such as at least, at most, just below, or between any two of the following (inclusive or exclusive) nucleotides: 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50, such that transcription of the RNA molecule will produce an immature RNA containing the polyadenylation signal downstream of its 3'UTR and subsequently ligating the poly-A sequence to the immature RNA. Thus, the resulting RNA may comprise a 3'UTR comprising at least one poly-A sequence, and wherein the 3'UTR is followed by an additional poly-A sequence.
多-A序列可具有任何長度。在一些態樣中，多-A尾之長度可為5至300個核苷酸。在一些態樣中，RNA分子包括多-A尾，該多-A尾包含以下、基本上由以下組成或由以下組成：為或為約25至約400個腺苷核苷酸之序列、為或為約50至約400個腺苷核苷酸之序列、為或為約50至約300腺苷核苷酸之序列、為或為約50至約250個腺苷核苷酸之序列、為或為約60至約250個腺苷核苷酸之序列、或者為或為約40至約100個腺苷核苷酸之序列。在一些態樣中，多-A尾包含至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的腺苷核苷酸、基本上由其組成或由其組成：5、10、15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90、91、92、93、94、95、96、97、98、99、100、105、110、115、120、125、130、135、140、145、150、155、160、165、170、175、180、185、190、195、200、205、210、215、220、225、230、235、240、245、250、255、260、265、270、275、280、285、290、295、300、305、310、315、320、325、330、335、340、345、350、355、360、365、370、375、380、385、390、395、400、405、410、415、420、425、430、435、440、445、450、455、460、465、470、475、480、485、490、495、500、505、510、515、520、525、530、535、540、545、550、555、560、565、570、575、580、585、590、595、600、605、610、615、620、625、630、635、640、645、650、655、660、665、670、675、680、685、690、695、700、705、710、715、720、725、730、735、740、745、750、755、760、765、770、775、780、785、790、795、800、805、810、815、820、825、830、835、840、845、850、855、860、865、870、875、880、885、890、895、900、905、910、915、920、925、930、935、940、945、950、955、960、965、970、975、980、985、990、995或1000個。在此上下文中，「基本上由…組成」意指多-A序列中之大部分核苷酸，多-A序列中通常至少75%、至少80%、至少85%、至少90%、至少95%、至少96%、至少97%、至少98%或至少99%數目之核苷酸為腺苷核苷酸，但允許其餘核苷酸為除腺苷核苷酸以外的核苷酸，諸如尿苷、鳥苷及/或胞嘧啶。在此上下文中，「由…組成」意指多-A序列中之所有核苷酸(亦即多-A序列中100%數目的核苷酸)為腺苷核苷酸。Poly-A sequences can have any length. In some aspects, the length of poly-A tails can be 5 to 300 nucleotides. In some aspects, RNA molecules include poly-A tails, which are comprised of, substantially comprised of, or comprised of: a sequence of about 25 to about 400 adenosine nucleotides, a sequence of about 50 to about 400 adenosine nucleotides, a sequence of about 50 to about 300 adenosine nucleotides, a sequence of about 50 to about 250 adenosine nucleotides, a sequence of about 60 to about 250 adenosine nucleotides, or a sequence of about 40 to about 100 adenosine nucleotides. In some aspects, the poly-A tail comprises, consists essentially of, or consists of at least, at most, exactly less than, or between any two of the following (inclusive or exclusive): 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 200, 210, 211, 212, 213, 214, 215 90, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460 ,465,470,475,480,485,490,495,500,505,510,515,520,525,530,535,540,545,550,555,560,565,570,575,580,585,590,595,600,605,610,615,620,625,630,635,640,645,650,655,660,665,670,675,680,685,690,695,700,705,710,715,720,725,730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995 or 1000. In this context, "consisting essentially of" means that most of the nucleotides in the poly-A sequence, usually at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% of the nucleotides in the poly-A sequence are adenosine nucleotides, but the remaining nucleotides are allowed to be nucleotides other than adenosine nucleotides, such as uridine, guanosine and/or cytosine. In this context, "consisting of" means that all nucleotides in the poly-A sequence (i.e., 100% of the nucleotides in the poly-A sequence) are adenosine nucleotides.
在一些態樣中，RNA分子包括含有大於30個腺苷核苷酸之序列的多-A尾。在一些態樣中，RNA分子包括含有或含有約40個腺苷核苷酸之多-A尾。在一些態樣中，RNA分子包括含有或含有約80個腺苷核苷酸之多-A尾。在一些態樣中，3'多-A尾具有至少10個連續腺苷殘基且至多300個連續腺苷殘基的延伸部分。在一些特定態樣中，RNA分子包括或包括約40個連續腺苷殘基。在一些態樣中，RNA分子包括或包括約80個連續腺苷殘基。多-A尾可在增強轉譯效率及調節mRNA品質控制及降解之效率中起關鍵調節作用。短序列或超多腺苷酸化可傳訊RNA降解。In some embodiments, the RNA molecule includes a poly-A tail containing a sequence greater than 30 adenosine nucleotides. In some embodiments, the RNA molecule includes a poly-A tail containing or containing about 40 adenosine nucleotides. In some embodiments, the RNA molecule includes a poly-A tail containing or containing about 80 adenosine nucleotides. In some embodiments, the 3' poly-A tail has an extension of at least 10 consecutive adenosine residues and up to 300 consecutive adenosine residues. In some specific embodiments, the RNA molecule includes or includes about 40 consecutive adenosine residues. In some embodiments, the RNA molecule includes or includes about 80 consecutive adenosine residues. The poly-A tail can play a key regulatory role in enhancing translation efficiency and regulating the efficiency of mRNA quality control and degradation. Short sequences or super polyadenylation can signal RNA degradation.
在一些態樣中，多-A尾可位於RNA分子或其他核酸分子內，諸如位於載體中，例如位於充當例如藉由轉錄載體來產生RNA (例如mRNA)之模板的載體中。在一些態樣中，RNA分子可不包括多-A尾。In some aspects, the poly-A tail can be located within an RNA molecule or other nucleic acid molecule, such as in a vector, for example, in a vector that serves as a template for producing RNA (e.g., mRNA), for example, by transcribing the vector. In some aspects, the RNA molecule may not include a poly-A tail.
在一些態樣中，多-A尾可位於RNA分子或其他核酸分子內，諸如位於載體中，例如位於充當例如藉由轉錄載體來產生RNA (例如mRNA)之模板的載體中。在一些態樣中，RNA分子可不包括多-A尾。In some aspects, the poly-A tail can be located within an RNA molecule or other nucleic acid molecule, such as in a vector, for example, in a vector that serves as a template for producing RNA (e.g., mRNA), for example, by transcribing the vector. In some aspects, the RNA molecule may not include a poly-A tail.
在一個態樣中，本文所揭示之編碼多-A尾的DNA包含與SEQ ID NO: 26具有至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的一致性之序列：99%、98%、97%、96%、95%、90%、85%或80%。在一個態樣中，編碼多-A尾之DNA包含SEQ ID NO: 26之序列。在一個態樣中，本文所揭示之RNA包含含有與SEQ ID NO: 26具有至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的一致性之序列的多-A尾：99%、98%、97%、96%、95%、90%、85%或80%。在一個態樣中，多-A尾包含SEQ ID NO: 26之序列。在一個態樣中，多-A尾包含SEQ ID NO: 26 +/- 2個腺苷(A)核苷酸之序列。在一個態樣中，多-A尾包含SEQ ID NO: 26 +/- 1個腺苷(A)核苷酸之序列。在一個態樣中，多-A尾包含SEQ ID NO: 26之序列。在一個態樣中，多-A尾包含SEQ ID NO: 26 +/- 2個腺苷(A)核苷酸之序列。在一個態樣中，多-A尾包含SEQ ID NO: 26 +/- 1個腺苷(A)核苷酸之序列。在一些態樣中，多-A尾包含SEQ ID NO: 26之序列。In one aspect, the DNA encoding the poly-A tail disclosed herein comprises a sequence having at least, at most, just below, or between any two of the following (inclusive or exclusive) identity to SEQ ID NO: 26: 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80%. In one aspect, the DNA encoding the poly-A tail comprises the sequence of SEQ ID NO: 26. In one aspect, the RNA disclosed herein comprises a poly-A tail comprising a sequence having at least, at most, just below, or between any two of the following (inclusive or exclusive) identity to SEQ ID NO: 26: 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80%. In one aspect, the poly-A tail comprises the sequence of SEQ ID NO: 26. In one aspect, the poly-A tail comprises the sequence of SEQ ID NO: 26 +/- 2 adenosine (A) nucleotides. In one aspect, the poly-A tail comprises a sequence of SEQ ID NO: 26 +/- 1 adenosine (A) nucleotides. In one aspect, the poly-A tail comprises a sequence of SEQ ID NO: 26. In one aspect, the poly-A tail comprises a sequence of SEQ ID NO: 26 +/- 2 adenosine (A) nucleotides. In one aspect, the poly-A tail comprises a sequence of SEQ ID NO: 26 +/- 1 adenosine (A) nucleotides. In some aspects, the poly-A tail comprises a sequence of SEQ ID NO: 26.
在一些態樣中，前述多-A序列中之1、2、3、4、5者或更多者可排除在本文所揭示之RNA分子外。 SEQ ID NO: 26 (DNA, RNA) AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAIn some embodiments, 1, 2, 3, 4, 5 or more of the aforementioned poly-A sequences may be excluded from the RNA molecules disclosed herein.SEQ ID NO: 26 (DNA, RNA)AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
G.其他元件在本發明之一些態樣中，RNA分子另外包括鏈終止核苷。舉例而言，鏈終止核苷可包括在其糖基之2'及/或3'位置處去氧的彼等核苷。此類物種可包括3'-去氧腺苷(蛹蟲草菌素(cordycepin))、3'去氧尿苷、3'去氧胞嘧啶、3'去氧鳥苷、3'去氧胸腺嘧啶及2',3'-雙去氧核苷(諸如2',3'-雙去氧腺苷、2',3'-雙去氧尿苷、2',3'-雙去氧胞嘧啶、2',3'-雙去氧鳥苷及2',3'-雙去氧胸腺嘧啶)。在一些態樣中，前述鏈終止核苷中之1、2、3、4、5者或更多者可排除在本文所揭示之RNA分子外。在一些態樣中，將鏈終止核苷酸併入mRNA中(例如在3'末端)，可引起mRNA之穩定化，例如國際專利公開案第WO 2013/103659號中所描述。G.Other Elements In some aspects of the invention, the RNA molecule additionally includes chain-terminating nucleosides. For example, chain-terminating nucleosides may include those nucleosides that are deoxygenated at the 2' and/or 3' position of their sugar moiety. Such species may include 3'-deoxyadenosine (cordycepin), 3'deoxyuridine, 3'deoxycytosine, 3'deoxyguanosine, 3'deoxythymidine, and 2',3'-dideoxynucleosides (such as 2',3'-dideoxyadenosine, 2',3'-dideoxyuridine, 2',3'-dideoxycytosine, 2',3'-dideoxyguanosine, and 2',3'-dideoxythymidine). In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned chain-terminating nucleosides may be excluded from the RNA molecules disclosed herein. In some aspects, incorporation of chain-terminating nucleotides into mRNA (e.g., at the 3' end) may result in stabilization of mRNA, such as described in International Patent Publication No. WO 2013/103659.
在本發明之一些態樣中，RNA分子另外包括莖環，諸如組蛋白莖環。莖環可包括2、3、4、5、6、7、8或更多個核苷酸鹼基對。舉例而言，莖環可包括4、5、6、7或8個核苷酸鹼基對。莖環可位於mRNA之任何區域中。舉例而言，莖環可位於非轉譯區(5' UTR或3' UTR)、編碼區或多-A序列或尾中、之前或之後。在一些態樣中，莖環可影響mRNA之一或多種功能，諸如轉譯之起始、轉譯效率及/或轉錄終止。此類組蛋白莖-環序列可為WO 2012/019780 (其揭示內容以全文引用之方式併入本文中)中所揭示之組蛋白莖-環序列。組蛋白莖環結構及編碼此類結構之核酸序列的其他非限制性實例可見於例如WO 2016/091391 (其揭示內容以全文引用之方式併入本文中)中。In some aspects of the present invention, the RNA molecule further includes a stem loop, such as a histone stem loop. The stem loop may include 2, 3, 4, 5, 6, 7, 8 or more nucleotide base pairs. For example, the stem loop may include 4, 5, 6, 7 or 8 nucleotide base pairs. The stem loop may be located in any region of the mRNA. For example, the stem loop may be located in, before or after a non-translated region (5'UTR or 3'UTR), a coding region or a poly-A sequence or tail. In some aspects, the stem loop may affect one or more functions of the mRNA, such as the initiation of translation, translation efficiency and/or transcription termination. Such histone stem-loop sequences may be histone stem-loop sequences disclosed in WO 2012/019780 (the disclosure of which is incorporated herein by reference in its entirety). Other non-limiting examples of histone stem-loop structures and nucleic acid sequences encoding such structures can be found, for example, in WO 2016/091391 (the disclosure of which is incorporated herein by reference in its entirety).
在一些態樣中，多-A序列或多腺苷酸化訊號與至少一個組蛋白莖-環(即使兩者在自然界中表示替代性機制)之組合協同起作用以增加蛋白質表現超出個別元件中之任一者的情況下所觀測到的含量。在一些態樣中，多-A與至少一個組蛋白莖-環之組合的協同作用不視元件之次序及/或多-A序列之長度而定。In some aspects, the combination of a poly-A sequence or a polyadenylation signal and at least one histone stem-loop (even if the two represent alternative mechanisms in nature) acts synergistically to increase protein expression over that observed with either of the individual elements. In some aspects, the synergistic effect of the combination of poly-A and at least one histone stem-loop is independent of the order of the elements and/or the length of the poly-A sequence.
在一些態樣中，RNA不包含組蛋白下游元件(histone downstream element；HDE)。HDE包括天然存在之莖-環3'之大約15至20個核苷酸之富嘌呤聚核苷酸延伸部分，其表示U7 snRNA之結合位點，其涉及將組蛋白pre-mRNA加工成成熟組蛋白mRNA。In some aspects, the RNA does not comprise a histone downstream element (HDE). The HDE includes a naturally occurring purine-rich polynucleotide stretch of approximately 15 to 20 nucleotides 3' to the stem-loop that represents the binding site for U7 snRNA, which is involved in processing histone pre-mRNA into mature histone mRNA.
在一些態樣中，組蛋白莖-環一般來源於組蛋白基因，且包括兩個相鄰的部分或完全反向互補序列的分子內鹼基配對，該等互補序列藉由間隔子分隔開，由短序列組成，形成結構之環。未配對環區通常不能與莖環元件中之任一者進行鹼基配對。莖-環結構之穩定性一般視配對區域之長度、錯配或隆起之數目及/或鹼基組成而定。在一些態樣中，可產生擺動鹼基配對(非沃森-克里克(Watson-Crick)鹼基配對)。在一些態樣中，至少一個組蛋白莖-環序列包含15至45個核苷酸之長度。In some aspects, the histone stem-loop is generally derived from a histone gene and includes intramolecular base pairing of two adjacent partially or completely reverse complementary sequences separated by a spacer and composed of short sequences to form a loop of the structure. Unpaired loop regions are generally unable to base pair with any of the stem-loop elements. The stability of the stem-loop structure generally depends on the length of the paired region, the number of mismatches or ridges, and/or the base composition. In some aspects, wobbly base pairing (non-Watson-Crick base pairing) can occur. In some aspects, at least one histone stem-loop sequence comprises a length of 15 to 45 nucleotides.
在一些態樣中，RNA分子包括(例如在3' UTR內)聚(C)序列。在一些態樣中，聚C序列具有至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的胞苷：10、20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190或200個。在一些態樣中，聚C序列具有或具有約30個胞苷。In some aspects, the RNA molecule includes (e.g., within the 3'UTR) a poly(C) sequence. In some aspects, the poly C sequence has at least, at most, just below, or between any two of the following (inclusive or exclusive) cytidines: 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200. In some aspects, the poly C sequence has or has about 30 cytidines.
在一些態樣中，RNA分子包括內部核糖體進入位點(internal ribosome entry site；IRES)序列或IRES模體。在一些態樣中，例如若RNA編碼兩種或更多種肽或蛋白質，則IRES序列將ORF分隔開。若RNA分子為雙順反子或多順反子核酸分子，則IRES序列因此可為適用的。In some aspects, the RNA molecule includes an internal ribosome entry site (IRES) sequence or IRES motif. In some aspects, for example, if the RNA encodes two or more peptides or proteins, the IRES sequence separates the ORFs. If the RNA molecule is a bicistronic or polycistronic nucleic acid molecule, the IRES sequence may therefore be applicable.
在一些態樣中，RNA不包含內含子。在一些態樣中，RNA可替代地或另外包括微小RNA結合位點。In some aspects, RNA does not comprise introns. In some aspects, RNA may alternatively or additionally comprise a microRNA binding site.
包括本文所揭示之元件之組合之代表性RNA分子在5'-至-3'方向上可包括但不限於以下： ORF - 多-A序列； ORF - IRES - ORF - 多-A序列； ORF - 3' UTR - 多-A序列； ORF - 多-A序列 - 3' UTR； ORF - 3' UTR - 多-A序列 - 聚(C)序列 - 組蛋白莖-環； ORF - 3' UTR - 多-A序列 - 聚(C)序列 - 多-A序列； ORF - 3' UTR - 多-A序列 - 組蛋白莖-環 - 多-A序列； 5' UTR - ORF - 3' UTR； 5' UTR - ORF - 多-A序列； 5' UTR - ORF - 多-A序列 - 聚(C)序列 - 組蛋白莖-環； 5' UTR - ORF - 多-A序列 - 聚(C)序列 - 多-A序列； 5' UTR - ORF - 多-A序列 - 組蛋白莖-環 - 多-A序列； 5' UTR - ORF - 3' UTR - 多-A序列； 5' UTR - ORF - 3' UTR - 多-A序列 - 聚(C)序列 5' UTR - ORF - 3' UTR - 多-A序列 - 聚(C)序列 - 組蛋白莖-環； 5'-帽 - 5' UTR - ORF - 3' UTR； 5'-帽 - 5' UTR - ORF - 多-A序列； 5'-帽 - 5' UTR - ORF - 3' UTR - 多-A序列； 5'-帽 - 5' UTR - ORF - 3' UTR - 多-A序列 - 聚(C)序列；或 5'-帽 - 5' UTR - ORF - 3' UTR - 多-A序列 - 聚(C)序列 - 組蛋白莖-環。Representative RNA molecules comprising a combination of elements disclosed herein may include, but are not limited to, the following in the 5'- to -3' direction:ORF - poly-A sequence;ORF - IRES - ORF - poly-A sequence;ORF - 3' UTR - poly-A sequence;ORF - poly-A sequence - 3' UTR;ORF - 3' UTR - poly-A sequence - poly(C) sequence - histone stem-loop;ORF - 3' UTR - poly-A sequence - poly(C) sequence - poly-A sequence;ORF - 3' UTR - poly-A sequence - histone stem-loop - poly-A sequence;5' UTR - ORF - 3' UTR;5' UTR - ORF - poly-A sequence;5' UTR - ORF - poly-A sequence - poly(C) sequence - Histone stem-loop;5' UTR - ORF - poly-A sequence - poly(C) sequence - poly-A sequence;5' UTR - ORF - poly-A sequence - histone stem-loop - poly-A sequence;5' UTR - ORF - 3' UTR - poly-A sequence - poly(C) sequence5' UTR - ORF - 3' UTR - poly-A sequence - poly(C) sequence - histone stem-loop;5'-cap - 5' UTR - ORF - 3' UTR;5'-cap - 5' UTR - ORF - poly-A sequence;5'-cap - 5' UTR - ORF - 3' UTR - poly-A sequence - poly(C) sequence - histone stem-loop;5'-cap - 5' UTR - ORF - 3' UTR;5'-cap - 5' UTR - ORF - poly-A sequence;5'-cap - 5' UTR - ORF - 3' UTR - poly-A sequence;5'-cap - 5' UTR - ORF - 3' UTR - poly-A sequence - poly(C) sequence; or5'-cap - 5' UTR - ORF - 3' UTR - poly-A sequence - poly(C) sequence - histone stem-loop.
在一些態樣中，前述元件中之1、2、3、4、5者或更多者可排除在本文所揭示之RNA分子外。In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned elements can be excluded from the RNA molecules disclosed herein.
H.自擴增RNA (saRNA)在一些態樣中，RNA分子可為saRNA。「自擴增RNA」、「saRNA」及「複製子」係指能夠自我複製之RNA。自擴增RNA分子可藉由使用來源於例如α病毒之複製元件且用編碼所關注多肽之核苷酸序列取代結構病毒多肽來產生。自擴增RNA分子通常為可在遞送至細胞之後直接轉譯的正股分子，且此轉譯提供RNA依賴性RNA聚合酶，其接著自所遞送RNA產生反義及有義轉錄本兩者。所遞送RNA引起多個子RNA分子之產生。此等子RNA分子以及共線次基因體轉錄本可自身經轉譯以提供所編碼之所關注基因(例如病毒抗原)之原位表現，及/或可經轉錄以提供與經轉譯以提供抗原之原位表現的所遞送RNA同義的其他轉錄本。此系列轉錄之總體結果為所引入saRNA分子之數目擴增，且因此所編碼之所關注基因(例如病毒抗原)變成細胞之主要多肽產物。H.Self-amplifyingRNA (saRNA) In some aspects, the RNA molecule may be saRNA. "Self-amplifying RNA", "saRNA" and "replicon" refer to RNA that is capable of self-replication. Self-amplifying RNA molecules can be produced by using replication elements derived from, for example, alphaviruses and replacing structural viral polypeptides with nucleotide sequences encoding polypeptides of interest. Self-amplifying RNA molecules are typically positive strand molecules that can be directly translated after delivery to cells, and this translation provides RNA-dependent RNA polymerases, which then produce both antisense and sense transcripts from the delivered RNA. The delivered RNA results in the production of multiple daughter RNA molecules. These daughter RNA molecules, as well as colinear subgenomic transcripts, can themselves be transcribed to provide in situ expression of the encoded gene of interest (e.g., viral antigen), and/or can be transcribed to provide additional transcripts that are synonymous with the delivered RNA that are translated to provide in situ expression of the antigen. The overall result of this series of transcriptions is an expansion in the number of introduced saRNA molecules, and thus the encoded gene of interest (e.g., viral antigen) becomes the major polypeptide product of the cell.
在一些態樣中，自擴增RNA包括至少一種或多種基因，包括病毒複製酶、病毒蛋白酶、病毒解螺旋酶及其他非結構病毒蛋白中之任一者，或其組合。在一些態樣中，前述基因中之1、2、3者或更多者可排除在本文所揭示之自擴增RNA分子外。在一些態樣中，自擴增RNA亦可包括5'端及3'端牽引複製序列，及視情況存在之編碼所需胺基酸序列(例如所關注抗原)之異源序列。引導異源序列之表現的次基因體啟動子可包括在自擴增RNA中。視情況，異源序列(例如所關注抗原)可框內融合至自擴增RNA中之其他編碼區及/或可處於內部核糖體進入位點(IRES)之控制下。In some aspects, the self-amplifying RNA includes at least one or more genes, including any one of viral replicase, viral protease, viral helicase and other non-structural viral proteins, or a combination thereof. In some aspects, 1, 2, 3 or more of the aforementioned genes may be excluded from the self-amplifying RNA molecules disclosed herein. In some aspects, the self-amplifying RNA may also include 5' and 3' end induction replication sequences, and optionally heterologous sequences encoding the required amino acid sequence (e.g., an antigen of interest). A subgenomic promoter that directs the expression of the heterologous sequence may be included in the self-amplifying RNA. Optionally, the heterologous sequence (e.g., an antigen of interest) may be fused in-frame to other coding regions in the self-amplifying RNA and/or may be under the control of an internal ribosome entry site (IRES).
在一些態樣中，本文所描述之自擴增RNA分子編碼：(i)可由自擴增RNA分子轉錄RNA之RNA依賴性RNA聚合酶；及(ii)所關注多肽，例如病毒抗原。在一些態樣中，聚合酶可為α病毒複製酶，例如包括α病毒蛋白質nsP1、nsP2、nsP3、nsP4中之任一者，或其任何組合。在一些態樣中，前述α病毒蛋白質中之1、2、3者或更多者可排除在本文所揭示之RNA分子外。In some aspects, the self-amplifying RNA molecules described herein encode: (i) an RNA-dependent RNA polymerase that can transcribe RNA from the self-amplifying RNA molecule; and (ii) a polypeptide of interest, such as a viral antigen. In some aspects, the polymerase can be an alphavirus replicase, for example, including any of the alphavirus proteins nsP1, nsP2, nsP3, nsP4, or any combination thereof. In some aspects, one, two, three or more of the aforementioned alphavirus proteins can be excluded from the RNA molecules disclosed herein.
在一些態樣中，自擴增RNA分子可具有兩個開讀框。第一(5')開讀框可編碼複製酶；第二(3')開讀框可編碼包含所關注抗原之多肽。在一些態樣中，RNA可具有額外(例如下游)開讀框，例如以編碼其他抗原或編碼輔助多肽。In some aspects, the self-amplifying RNA molecule may have two open reading frames. The first (5') open reading frame may encode a replicase; the second (3') open reading frame may encode a polypeptide comprising an antigen of interest. In some aspects, the RNA may have an additional (e.g., downstream) open reading frame, for example, to encode other antigens or to encode auxiliary polypeptides.
在一些態樣中，saRNA分子進一步包括：(1) α病毒5'複製識別序列；及(2) α病毒3'複製識別序列。在一些態樣中，自擴增RNA分子之5'序列經選擇以確保與所編碼之複製酶相容。In some aspects, the saRNA molecule further comprises: (1) an alphavirus 5' replication recognition sequence; and (2) an alphavirus 3' replication recognition sequence. In some aspects, the 5' sequence of the self-amplifying RNA molecule is selected to ensure compatibility with the encoded replication enzyme.
在一些態樣中，自擴增RNA分子可編碼單一多肽抗原，或視情況編碼兩種或更多種多肽抗原，該等多肽抗原以序列中之各者在以胺基酸序列形式表現時保持其一致性的方式連接在一起(例如串聯連接)。由自擴增RNA產生之多肽接著可以融合多肽之形式產生，或以使得產生分離多肽或肽序列之方式經工程改造。In some aspects, the self-amplifying RNA molecule can encode a single polypeptide antigen, or optionally encode two or more polypeptide antigens, which are linked together (e.g., tandemly linked) in a manner that maintains the identity of each of the sequences when expressed in the form of amino acid sequences. The polypeptide produced by the self-amplifying RNA can then be produced in the form of a fusion polypeptide, or engineered in a manner that produces separate polypeptide or peptide sequences.
在一些態樣中，本文所描述之自擴增RNA可編碼包括一系列抗原決定基之一或多種多肽抗原。在一些態樣中，本文所描述之自擴增RNA可編碼能夠引起輔助T細胞反應或細胞毒性T細胞反應或兩者的抗原決定基。In some aspects, the self-amplifying RNA described herein can encode one or more polypeptide antigens including a series of antigenic determinants. In some aspects, the self-amplifying RNA described herein can encode antigenic determinants capable of eliciting a helper T cell response or a cytotoxic T cell response or both.
在一個態樣中，本文所揭示之自擴增RNA包含含有與SEQ ID NO: 54具有至少、至多、恰好以下或在以下中之任何兩者之間的一致性之序列的次基因體啟動子：99%、98%、97%、96%、95%、90%、85%或80%。在一個態樣中，次基因體啟動子包含SEQ ID NO: 54之序列。 SEQ ID NO: 54 (RNA) CCUGAAUGGACUACGACAUAGUCUAGUCCGCCAAGIn one aspect, the self-amplifying RNA disclosed herein comprises a subgenomic promoter comprising a sequence having at least, at most, just below, or between any two of the following identities to SEQ ID NO: 54: 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%. In one aspect, the subgenomic promoter comprises the sequence of SEQ ID NO: 54.SEQ ID NO: 54 (RNA)CCUGAAUGGACUACGACAUAGUCUAGUCCGCCAAG
在一些態樣中，本文所描述之自擴增RNA分子編碼：(i)可由自擴增RNA分子轉錄RNA之RNA依賴性RNA聚合酶；及(ii)所關注多肽，例如病毒抗原。在一些態樣中，聚合酶可為α病毒複製酶，例如包括α病毒蛋白質nsP1、nsP2、nsP3、nsP4中之任一者及其任何組合。In some aspects, the self-amplifying RNA molecules described herein encode: (i) an RNA-dependent RNA polymerase that can transcribe RNA from the self-amplifying RNA molecule; and (ii) a polypeptide of interest, such as a viral antigen. In some aspects, the polymerase can be an alphavirus replicase, such as any one of the alphavirus proteins nsP1, nsP2, nsP3, nsP4, and any combination thereof.
在一個態樣中，本文所揭示之自擴增RNA包含α病毒複製酶，例如包括α病毒蛋白質nsP1、nsP2、nsP3、nsP4中之任一者及其任何組合，其包含分別與SEQ ID NO: 55-58具有至少、至多、恰好以下或在以下中之任何兩者之間的一致性之序列：99%、98%、97%、96%、95%、90%、85%或80%。在一個態樣中，α病毒蛋白質nsP1、nsP2、nsP3及nsP4各自分別包含SEQ ID NO: 55-58之序列。 SEQ ID NO: 55 (nsP1 RNA) AUGGAGAAAGUUCACGUUGACAUCGAGGAAGACAGCCCAUUCCUCAGAGCUUUGCAGCGGAGCUUCCCGCAGUUUGAGGUAGAAGCCAAGCAGGUCACUGAUAAUGACCAUGCUAAUGCCAGAGCGUUUUCGCAUCUGGCUUCAAAACUGAUCGAAACGGAGGUGGACCCAUCCGACACGAUCCUUGACAUUGGAAGUGCGCCCGCCCGCAGAAUGUAUUCUAAGCACAAGUAUCAUUGUAUCUGUCCGAUGAGAUGUGCGGAAGAUCCGGACAGAUUGUAUAAGUAUGCAACUAAGCUGAAGAAAAACUGUAAGGAAAUAACUGAUAAGGAAUUGGACAAGAAAAUGAAGGAGCUCGCCGCCGUCAUGAGCGACCCUGACCUGGAAACUGAGACUAUGUGCCUCCACGACGACGAGUCGUGUCGCUACGAAGGGCAAGUCGCUGUUUACCAGGAUGUAUACGCGGUUGACGGACCGACAAGUCUCUAUCACCAAGCCAAUAAGGGAGUUAGAGUCGCCUACUGGAUAGGCUUUGACACCACCCCUUUUAUGUUUAAGAACUUGGCUGGAGCAUAUCCAUCAUACUCUACCAACUGGGCCGACGAAACCGUGUUAACGGCUCGUAACAUAGGCCUAUGCAGCUCUGACGUUAUGGAGCGGUCACGUAGAGGGAUGUCCAUUCUUAGAAAGAAGUAUUUGAAACCAUCCAACAAUGUUCUAUUCUCUGUUGGCUCGACCAUCUACCACGAGAAGAGGGACUUACUGAGGAGCUGGCACCUGCCGUCUGUAUUUCACUUACGUGGCAAGCAAAAUUACACAUGUCGGUGUGAGACUAUAGUUAGUUGCGACGGGUACGUCGUUAAAAGAAUAGCUAUCAGUCCAGGCCUGUAUGGGAAGCCUUCAGGCUAUGCUGCUACGAUGCACCGCGAGGGAUUCUUGUGCUGCAAAGUGACAGACACAUUGAACGGGGAGAGGGUCUCUUUUCCCGUGUGCACGUAUGUGCCAGCUACAUUGUGUGACCAAAUGACUGGCAUACUGGCAACAGAUGUCAGUGCGGACGACGCGCAAAAACUGCUGGUUGGGCUCAACCAGCGUAUAGUCGUCAACGGUCGCACCCAGAGAAACACCAAUACCAUGAAAAAUUACCUUUUGCCCGUAGUGGCCCAGGCAUUUGCUAGGUGGGCAAAGGAAUAUAAGGAAGAUCAAGAAGAUGAAAGGCCACUAGGACUACGAGAUAGACAGUUAGUCAUGGGGUGUUGUUGGGCUUUUAGAAGGCACAAGAUAACAUCUAUUUAUAAGCGCCCGGAUACCCAAACCAUCAUCAAAGUGAACAGCGAUUUCCACUCAUUCGUGCUGCCCAGGAUAGGCAGUAACACAUUGGAGAUCGGGCUGAGAACAAGAAUCAGGAAAAUGUUAGAGGAGCACAAGGAGCCGUCACCUCUCAUUACCGCCGAGGACGUACAAGAAGCUAAGUGCGCAGCCGAUGAGGCUAAGGAGGUGCGUGAAGCCGAGGAGUUGCGCGCAGCUCUACCACCUUUGGCAGCUGAUGUUGAGGAGCCCACUCUGGAAGCCGAUGUCGACUUGAUGUUACAAGAGGCUGGGGCC SEQ ID NO: 56 (NSP2 RNA) GGCUCAGUGGAGACACCUCGUGGCUUGAUAAAGGUUACCAGCUACGAUGGCGAGGACAAGAUCGGCUCUUACGCUGUGCUUUCUCCGCAGGCUGUACUCAAGAGUGAAAAAUUAUCUUGCAUCCACCCUCUCGCUGAACAAGUCAUAGUGAUAACACACUCUGGCCGAAAAGGGCGUUAUGCCGUGGAACCAUACCAUGGUAAAGUAGUGGUGCCAGAGGGACAUGCAAUACCCGUCCAGGACUUUCAAGCUCUGAGUGAAAGUGCCACCAUUGUGUACAACGAACGUGAGUUCGUAAACAGGUACCUGCACCAUAUUGCCACACAUGGAGGAGCGCUGAACACUGAUGAAGAAUAUUACAAAACUGUCAAGCCCAGCGAGCACGACGGCGAAUACCUGUACGACAUCGACAGGAAACAGUGCGUCAAGAAAGAACUAGUCACUGGGCUAGGGCUCACAGGCGAGCUGGUGGAUCCUCCCUUCCAUGAAUUCGCCUACGAGAGUCUGAGAACACGACCAGCCGCUCCUUACCAAGUACCAACCAUAGGGGUGUAUGGCGUGCCAGGAUCAGGCAAGUCUGGCAUCAUUAAAAGCGCAGUCACCAAAAAAGAUCUAGUGGUGAGCGCCAAGAAAGAAAACUGUGCAGAAAUUAUAAGGGACGUCAAGAAAAUGAAAGGGCUGGACGUCAAUGCCAGAACUGUGGACUCAGUGCUCUUGAAUGGAUGCAAACACCCCGUAGAGACCCUGUAUAUUGACGAAGCUUUUGCUUGUCAUGCAGGUACUCUCAGAGCGCUCAUAGCCAUUAUAAGACCUAAAAAGGCAGUGCUCUGCGGGGAUCCCAAACAGUGCGGUUUUUUUAACAUGAUGUGCCUGAAAGUGCAUUUUAACCACGAGAUUUGCACACAAGUCUUCCACAAAAGCAUCUCUCGCCGUUGCACUAAAUCUGUGACUUCGGUCGUCUCAACCUUGUUUUACGACAAAAAAAUGAGAACGACGAAUCCGAAAGAGACUAAGAUUGUGAUUGACACUACCGGCAGUACCAAACCUAAGCAGGACGAUCUCAUUCUCACUUGUUUCAGAGGGUGGGUGAAGCAGUUGCAAAUAGAUUACAAAGGCAACGAAAUAAUGACGGCAGCUGCCUCUCAAGGGCUGACCCGUAAAGGUGUGUAUGCCGUUCGGUACAAGGUGAAUGAAAAUCCUCUGUACGCACCCACCUCAGAACAUGUGAACGUCCUACUGACCCGCACGGAGGACCGCAUCGUGUGGAAAACACUAGCCGGCGACCCAUGGAUAAAAACACUGACUGCCAAGUACCCUGGGAAUUUCACUGCCACGAUAGAGGAGUGGCAAGCAGAGCAUGAUGCCAUCAUGAGGCACAUCUUGGAGAGACCGGACCCUACCGACGUCUUCCAGAAUAAGGCAAACGUGUGUUGGGCCAAGGCUUUAGUGCCGGUGCUGAAGACCGCUGGCAUAGACAUGACCACUGAACAAUGGAACACUGUGGAUUAUUUUGAAACGGACAAAGCUCACUCAGCAGAGAUAGUAUUGAACCAACUAUGCGUGAGGUUCUUUGGACUCGAUCUGGACUCCGGUCUAUUUUCUGCACCCACUGUUCCGUUAUCCAUUAGGAAUAAUCACUGGGAUAACUCCCCGUCGCCUAACAUGUACGGGCUGAAUAAAGAAGUGGUCCGUCAGCUCUCUCGCAGGUACCCACAACUGCCUCGGGCAGUUGCCACUGGAAGAGUCUAUGACAUGAACACUGGUACACUGCGCAAUUAUGAUCCGCGCAUAAACCUAGUACCUGUAAACAGAAGACUGCCUCAUGCUUUAGUCCUCCACCAUAAUGAACACCCACAGAGUGACUUUUCUUCAUUCGUCAGCAAAUUGAAGGGCAGAACUGUCCUGGUGGUCGGGGAAAAGUUGUCCGUCCCAGGCAAAAUGGUUGACUGGUUGUCAGACCGGCCUGAGGCUACCUUCAGAGCUCGGCUGGAUUUAGGCAUCCCAGGUGAUGUGCCCAAAUAUGACAUAAUAUUUGUUAAUGUGAGGACCCCAUAUAAAUACCAUCACUAUCAGCAGUGUGAAGACCAUGCCAUUAAGCUUAGCAUGUUGACCAAGAAAGCUUGUCUGCAUCUGAAUCCCGGCGGAACCUGUGUCAGCAUAGGUUAUGGUUACGCUGACAGGGCCAGCGAAAGCAUCAUUGGUGCUAUAGCGCGGCAGUUCAAGUUUUCCCGGGUAUGCAAACCGAAAUCCUCACUUGAAGAGACGGAAGUUCUGUUUGUAUUCAUUGGGUACGAUCGCAAGGCCCGUACGCACAAUCCUUACAAGCUUUCAUCAACCUUGACCAACAUUUAUACAGGUUCCAGACUCCACGAAGCCGGAUGU SEQ ID NO: 57 (NSP3 RNA) GCACCCUCAUAUCAUGUGGUGCGAGGGGAUAUUGCCACGGCCACCGAAGGAGUGAUUAUAAAUGCUGCUAACAGCAAAGGACAACCUGGCGGAGGGGUGUGCGGAGCGCUGUAUAAGAAAUUCCCGGAAAGCUUCGAUUUACAGCCGAUCGAAGUAGGAAAAGCGCGACUGGUCAAAGGUGCAGCUAAACAUAUCAUUCAUGCCGUAGGACCAAACUUCAACAAAGUUUCGGAGGUUGAAGGUGACAAACAGUUGGCAGAGGCUUAUGAGUCCAUCGCUAAGAUUGUCAACGAUAACAAUUACAAGUCAGUAGCGAUUCCACUGUUGUCCACCGGCAUCUUUUCCGGGAACAAAGAUCGACUAACCCAAUCAUUGAACCAUUUGCUGACAGCUUUAGACACCACUGAUGCAGAUGUAGCCAUAUACUGCAGGGACAAGAAAUGGGAAAUGACUCUCAAGGAAGCAGUGGCUAGGAGAGAAGCAGUGGAGGAGAUAUGCAUAUCCGACGACUCUUCAGUGACAGAACCUGAUGCAGAGCUGGUGAGGGUGCAUCCGAAGAGUUCUUUGGCUGGAAGGAAGGGCUACAGCACAAGCGAUGGCAAAACUUUCUCAUAUUUGGAAGGGACCAAGUUUCACCAGGCGGCCAAGGAUAUAGCAGAAAUUAAUGCCAUGUGGCCCGUUGCAACGGAGGCCAAUGAGCAGGUAUGCAUGUAUAUCCUCGGAGAAAGCAUGAGCAGUAUUAGGUCGAAAUGCCCCGUCGAAGAGUCGGAAGCCUCCACACCACCUAGCACGCUGCCUUGCUUGUGCAUCCAUGCCAUGACUCCAGAAAGAGUACAGCGCCUAAAAGCCUCACGUCCAGAACAAAUUACUGUGUGCUCAUCCUUUCCAUUGCCGAAGUAUAGAAUCACUGGUGUGCAGAAGAUCCAAUGCUCCCAGCCUAUAUUGUUCUCACCGAAAGUGCCUGCGUAUAUUCAUCCAAGGAAGUAUCUCGUGGAAACACCACCGGUAGACGAGACUCCGGAGCCAUCGGCAGAGAACCAAUCCACAGAGGGGACACCUGAACAACCACCACUUAUAACCGAGGAUGAGACCAGGACUAGAACGCCUGAGCCGAUCAUCAUCGAAGAGGAAGAAGAGGAUAGCAUAAGUUUGCUGUCAGAUGGCCCGACCCACCAGGUGCUGCAAGUCGAGGCAGACAUUCACGGGCCGCCCUCUGUAUCUAGCUCAUCCUGGUCCAUUCCUCAUGCAUCCGACUUUGAUGUGGACAGUUUAUCCAUACUUGACACCCUGGAGGGAGCUAGCGUGACCAGCGGGGCAACGUCAGCCGAGACUAACUCUUACUUCGCAAAGAGUAUGGAGUUUCUGGCGCGACCGGUGCCUGCGCCUCGAACAGUAUUCAGGAACCCUCCACAUCCCGCUCCGCGCACAAGAACACCGUCACUUGCACCCAGCAGGGCCUGCUCGAGAACCAGCCUAGUUUCCACCCCGCCAGGCGUGAAUAGGGUGAUCACUAGAGAGGAGCUCGAGGCGCUUACCCCGUCACGCACUCCUAGCAGGUCGGUCUCGAGAACCAGCCUGGUCUCCAACCCGCCAGGCGUAAAUAGGGUGAUUACAAGAGAGGAGUUUGAGGCGUUCGUAGCACAACAACAAUGACGGUUUGAUGCGGGUGCA SEQ ID NO: 58 (NSP4 RNA) UACAUCUUUUCCUCCGACACCGGUCAAGGGCAUUUACAACAAAAAUCAGUAAGGCAAACGGUGCUAUCCGAAGUGGUGUUGGAGAGGACCGAAUUGGAGAUUUCGUAUGCCCCGCGCCUCGACCAAGAAAAAGAAGAAUUACUACGCAAGAAAUUACAGUUAAAUCCCACACCUGCUAACAGAAGCAGAUACCAGUCCAGGAAGGUGGAGAACAUGAAAGCCAUAACAGCUAGACGUAUUCUGCAAGGCCUAGGGCAUUAUUUGAAGGCAGAAGGAAAAGUGGAGUGCUACCGAACCCUGCAUCCUGUUCCUUUGUAUUCAUCUAGUGUGAACCGUGCCUUUUCAAGCCCCAAGGUCGCAGUGGAAGCCUGUAACGCCAUGUUGAAAGAGAACUUUCCGACUGUGGCUUCUUACUGUAUUAUUCCAGAGUACGAUGCCUAUUUGGACAUGGUUGACGGAGCUUCAUGCUGCUUAGACACUGCCAGUUUUUGCCCUGCAAAGCUGCGCAGCUUUCCAAAGAAACACUCCUAUUUGGAACCCACAAUACGAUCGGCAGUGCCUUCAGCGAUCCAGAACACGCUCCAGAACGUCCUGGCAGCUGCCACAAAAAGAAAUUGCAAUGUCACGCAAAUGAGAGAAUUGCCCGUAUUGGAUUCGGCGGCCUUUAAUGUGGAAUGCUUCAAGAAAUAUGCGUGUAAUAAUGAAUAUUGGGAAACGUUUAAAGAAAACCCCAUCAGGCUUACUGAAGAAAACGUGGUAAAUUACAUUACCAAAUUAAAAGGACCAAAAGCUGCUGCUCUUUUUGCGAAGACACAUAAUUUGAAUAUGUUGCAGGACAUACCAAUGGACAGGUUUGUAAUGGACUUAAAGAGAGACGUGAAAGUGACUCCAGGAACAAAACAUACUGAAGAACGGCCCAAGGUACAGGUGAUCCAGGCUGCCGAUCCGCUAGCAACAGCGUAUCUGUGCGGAAUCCACCGAGAGCUGGUUAGGAGAUUAAAUGCGGUCCUGCUUCCGAACAUUCAUACACUGUUUGAUAUGUCGGCUGAAGACUUUGACGCUAUUAUAGCCGAGCACUUCCAGCCUGGGGAUUGUGUUCUGGAAACUGACAUCGCGUCGUUUGAUAAAAGUGAGGACGACGCCAUGGCUCUGACCGCGUUAAUGAUUCUGGAAGACUUAGGUGUGGACGCAGAGCUGUUGACGCUGAUUGAGGCGGCUUUCGGCGAAAUUUCAUCAAUACAUUUGCCCACUAAAACUAAAUUUAAAUUCGGAGCCAUGAUGAAAUCUGGAAUGUUCCUCACACUGUUUGUGAACACAGUCAUUAACAUUGUAAUCGCAAGCAGAGUGUUGAGAGAACGGCUAACCGGAUCACCAUGUGCAGCAUUCAUUGGAGAUGACAAUAUCGUGAAAGGAGUCAAAUCGGACAAAUUAAUGGCAGACAGGUGCGCCACCUGGUUGAAUAUGGAAGUCAAGAUUAUAGAUGCUGUGGUGGGCGAGAAAGCGCCUUAUUUCUGUGGAGGGUUUAUUUUGUGUGACUCCGUGACCGGCACAGCGUGCCGUGUGGCAGACCCCCUAAAAAGGCUGUUUAAGCUUGGCAAACCUCUGGCAGCAGACGAUGAACAUGAUGAUGACAGGAGAAGGGCAUUGCAUGAAGAGUCAACACGCUGGAACCGAGUGGGUAUUCUUUCAGAGCUGUGCAAGGCAGUAGAAUCAAGGUAUGAAACCGUAGGAACUUCCAUCAUAGUUAUGGCCAUGACUACUCUAGCUAGCAGUGUUAAAUCAUUCAGCUACCUGAGAGGGGCCCCUAUAACUCUCUACGGCUAA。In one aspect, the self-amplifying RNA disclosed herein comprises an alphavirus replicase, such as any one of the alphavirus proteins nsP1, nsP2, nsP3, nsP4, and any combination thereof, comprising a sequence having at least, at most, just below, or between any two of the following identities to SEQ ID NOs: 55-58, respectively: 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80%. In one aspect, the alphavirus proteins nsP1, nsP2, nsP3, and nsP4 each comprise the sequence of SEQ ID NOs: 55-58, respectively.SEQ ID NO: 55 (nsP1 RNA) AUGGAGAAAGUUCACGUUGACAUCGAGGAAGACAGCCCAUUCCUCAGAGCUUUGCAGCGGAGCUUCCCGCAGUUUGAGGUAGAAGCCAAGCAGGUCACUGAUAAUGACCAUGCUAAUGCCAGAGCGUUUUCGCAUCUGGCUUCAAAACUGAUCGAAACGGAGGUGGACCCAUCCGACACGAUCCUUGACAUUGGAAGUGC GCCCGCCCGCAGAAUGUAUUCUAAGCACAAGUAUCAUUGUAUCUCCGAUGAGAUGUGCGGAAGAUCCGGACAGAUUGUAUAAGUAUGCAACUAAGCUGAAGAAAAACUGUAAGGAAAUAACUGAUAAGGAAUUGGACAAGAAAAUGAAGGAGCUCGCCGCCGUCAUGAGCGACCCUGACCUGGAAACUGAGACUAUGUG CCUCCACGACGACGAGUCGUGUCGCUACGAAGGGCAAGUCGCUGUUUACCAGGAUGUAUACGCGGUUGACGGACCGACAAGUCUCUAUCACCAAGCCAAUAAGGGAGUUAGAGUCGCCUACUGGAUAGGCUUUGACACCACCCCUUUAUGUUUAAGAACUUGGCUGGAGCAUAUCCAUCAUACUCUACCAACUGGGCCGA CGAAACCGUGUUAACGGCUCGUAACAUAGGCCUAUGCAGCUCUGACGUUAUGGAGCGGUCACGUAGAGGGAUGUCCAUUCUUAGAAAGAAGUAUUUGAAACCACAAUGUUCUAUUCUCUGUUGGCUCGACCAUCUACCACGAGAAGAGGGACUUACUGAGGAGCUGGCACCUGCCGUCUGUAUUUCACUUACGUGG CAAGCAAAAUUACACAUGUCGGUGAGACUAUAGUUAGUUGCGACGGGUACGUCGUUAAAAGAAUAGCUAUCAGUCCAGGCCUGUAUGGGAAGCCUUCAGGCUAUGCUGCUACGAUGCACCGCGAGGGAUUCUUGUGCUGCAAAGUGACAGACACAUUGAACGGGGAGAGGGUCUCUUUUCCCGUGUGCACGUAUGUGC CAGCUACAUUGUGUGACCAAAUGACUGGCAUACUGGCAACAGAUGUCAGUGCGGACGACGCGCAAAAACUGCUGGUUGGGCUCAACCAGCGUAUAGUCGUCAACGGUCGCACCCAGAGAAACACCAAUACCAUGAAAAAUUACCUUUUGCCCGUAGUGGCCCAGGCAUUUGCUAGGUGGGCAAAGGAAUAUAAGGAAGAUC AAGAAGAUGAAAGGCCACUAGGACUACGAGAUAGACAGUUAGUCAUGGGGUUGUUGGGCUUUUAGAAGGCACAAGAUAACAUCUAUUUAUAAGCGCCCGGAUACCCAAACCAUCAUCAAAGUGAACAGCGAUUUCCACUCAUUCGUGCUGCCCAGGAUAGGCAGUAACACAUUGGAGAUCGGGCUGAGAACAAGAAUC AGGAAAAUGUUAGAGGAGCACAAGGAGCCGUCACCUCUCAUUACCGCCGAGGACGUACAAGAAGCUAAGUGCGCAGCCGAUGAGGCUAAGGAGGUGCGUGAAGCCGAGGAGUUGCGCGCAGCUCUACCACCUUUGGCAGCUGAUGUUGAGGAGCCCACUCUGGAAGCCGAUGUCGACUUGAUGUUACAAGAGGCUGGGGCC SEQ ID NO: 56 (NSP2 RNA) GGCUCAGUGGAGACACCUCGUGGCUUGAUAAAGGUUACCAGCUACGAUGGCGAGGACAAGAUCGGCUCUUACGCUGUGCUUUCUCCGCAGGCUGUACUCAAGAGUGAAAAAUUAUCUUGCAUCCACCCUCUCGCUGAACAAGUCAUAG UGAUAACACACUCUGGCCGAAAAGGGCGUUAUGCCUGGAACCAUACCAUGGUAAAGUAGUGGUGCCAGAGGGACAUGCAAUACCCGUCCAGGACUUUCAAGCUCUGAGUGAAAGUGCCACCAUUGUGUACAACGAACGUGAGUUCGUA AACAGGUACCUGCACCAUAUUGCCACACAUGGAGGAGCGCUGAACACUGAUGAAGAAUAUUACAAAACUGUCAAGCCCAGCGAGCACGACGGCGAAUACCUGUACGACAUCGACAGGAAACAGUGCGUCAAGAAAGAACUAGUCACUGG GCUAGGGCUCACAGGCGAGCUGGUGGAUCCUCCCUUCCAUGAAUUCGCCUACGAGAGUCUGAGAACACGACCAGCCGCUCCUUACCAAGUACCAACCAUAGGGGUGUAUGGCGUGCCAGGAUCAGGCAAGUCUGGCAUCAUUAAAAGCG CAGUCACCAAAAAAGAUCUAGUGGUGAGCGCCAAGAAAGAAAACUGUGCAGAAAUUAUAAGGGACGUCAAGAAAAUGAAAGGGCUGGACGUCAAUGCCAGAACUGUGGACUCAGUGCUCUUGAAUGGAUGCAAACAACCCCGUAGAGACC CUGUAUAUUGACGAAGCUUUUGCUUGUCAUGCAGGUACUCUCAGAGCGCUCAUAGCCAUUAUAAGACCUAAAAAGGCAGUGCUCUGCGGGGAUCCCAAACAGUGCGGUUUUUUAACAUGAUGGCCCUGAAAGUGCAUUUUAACCACGA GAUUUGCACACAAGUCUUCCACAAAAGCAUCUCUCGCCGUUGCACUAAAUCUGUGACUUCGGUCGUCUCAACCUUGUUUUACGACAAAAAAAUGAGAACGACGAAUCCGAAAGAGACUAAGAUUGUGAUUGACACUACCGGCAGUACCA AACCUAAGCAGGACGAUCUCAUUCUCACUUGUUUCAGAGGGUGGGUGAAGCAGUUGCAAAUAGAUUACAAAGGCAACGAAAUAAUGACGGCAGCUGCCUCUCAAGGGCUGACCCGUAAAGGUGUGUAUGCCGUUCGGUACAAGGUGAAU GAAAAUCCUCUGUACGCACCCACCUCAGAACAUGUGAACGUCCUACUGACCCGCACGGAGGACCGCAUCGUGUGGAAAACACUAGCCGGCGACCCAUGGAUAAAAACACUGACUGCCAAGUACCCUGGGAAUUUCACUGCCACGAUAGA GGAGUGGCAAGCAGAGCAUGAUGCCAUCAUGAGGCACAUCUUGGAGAGACCGGACCCUACCGACGUCUUCCAGAAUAAGGCAAACGUGUGUUGGGCCAAGGCUUUAAGUGCCGGUGCUGAAGACCGCUGGCAUAGACAUGACCACUGAAC AAUGGAACACUGUGGAUUAUUUUGAAACGGACAAAGCUCACUCAGCAGAGAUAGUAUGAACCAACUAUGCGUGAGGUUCUUUGGACUCGAUCUGGACUCCGGUCUAUUUUCUGCACCCACUGUUCCGUUAUCCAUUAGGAAUAAUCAC UGGGAUAACUCCCCGUCGCCUAACAUGUACGGGCUGAAUAAAGAAGUGGUCCGUCAGCUCUCGCAGGUACCCACAACUGCCUCGGGCAGUUGCCACUGGAAGAGUCUAUGACAUGAACACUGGUACACUGCGCAAUUAUGAUCCGCG CAUAAACCUAGUACCUGUAAACAGAAGACUGCCUCAUGCUUUAGUCCUCCACCAUAAUGAACACCCACAGAGUGACUUUUCUUCAUUCGUCAGCAAAUUGAAGGGCAGAACUGUCCUGGUGGUCGGGGAAAAGUUGUCCGUCCCAGGCA AAAUGGUUGACUGGUUGUCAGACCGGCCUGAGGCUACCUUCAGAGCUCGGCUGGAUUUAGGCAUCCCAGGUGAUGUGCCCAAAUAUGACAUAAUAUUUGUUAAUGUGAGGACCCCAUAUAAAUACCAUCACUAUCAGCAGUGUGAAGAC CAUGCCAUUAAGCUUAGCAUGUUGACCAAGAAAGCUUGUCUGCAUCUGAAUCCCGGCGGAACCUGUGUCAGCAUAGGUUAUGGUUACGCUGACAGGGCCAGCGAAAGCAUCAUUGGUGCUAUAGCGCGGCAGUUCAAGUUUUCCCGGG UAUGCAAACCGAAAUCCUCACUUGAAGAGACGGAAGUUCUGUUUGUAUUCAUUGGGUACGAUCGCAAGGCCCGUACGCACAAUCCUUACAAGCUUUCAUCAACCUUGACCAACAUUUAUACAGGUUCCAGACUCCACGAAGCCGGAUGU SEQ ID NO: 57 (NSP3 RNA) GCACCCUCAUAUCAUGUGGUGCGAGGGGAUAUUGCCACGGCCACCGAAGGAGUGAUUAUAAAUGCUGCUAACAGCAAAGGACAACCUGGCGGAGGGGUGCGGAGCGCUGUAUAAGAAAUUCCCGGAAAGCUUCGAUUUACAGCCGAUCGAAGUAGGAAAAGCGCGACUGGUCAAAGGUGCAGCUAAACAUAUCAUUCAUGCCGUAGG ACCAAACUUCAACAAAGUUUCGGAGGUUGAAGGUGACAAACAGUUGGCAGAGGCUUAUGAGUCCAUCGCUAAGAUUGUCAACGAUAACAAUUAGUCAGUAGCGAUUCCACUGUUGUCCACCGGCAUCUUUUCCGGGAACAAAGAUCGACUAACCCAAUCAUUGAACCAUUUGCUGACAGCUUUAGACACCACUGAUGCAGAUGUAG CCAUAUACUGCAGGGACAAGAAAUGGGAAAUGACUCUCAAGGAAGCAGUGGCUAGGAGAGAAGCAGUGGAGGAGAUAUGCAUAUCCGACGACUCUUCAGUGACAGAACCUGAUGCAGAGCUGGUGAGGGUGCAUCCGAAGAGUUCUUUGGCUGGAAGGAAGGGCUACAGCACAAGCGAUGGCAAAACUUUCUCAUAUUUGGAAGGGACC AAGUUUCACCAGGCGGCCAAGGAUAUAGCAGAAAUUAAUGCCAUGUGGCCCGUUGCAACGGAGGCCAAUGAGCAGGUAUGCAUGUAUAUCCUCGGAGAAAGCAUGAGCAGUAUUAGGUCGAAAUGCCCGUCGAAGAGUCGGAAGCCUCCACACCACCUAGCACGCUGCCUUGCUUGUGCAUCCAUGCCAUGACUCCAGAAAGAGUACA GCGCCUAAAAGCCUCACGUCCAGAACAAAUUCUGUGGCUCAUCCUUUCCAUUGCCGAAGUAUAGAAUCACUGGUGUGCAGAAGAUCCAAUGCUCCCAGCCUAUAUUGUUCUCACCGAAAGUGCCUGCGUAUAUUCAUCCAAGGAAGUAUCUCGUGGAAACACCACCGGUAGACGAGACUCCGGAGCCAUCGGCAGAGAACCAAUCCA CAGAGGGGACACCUGAACAACCACCACUUAUAACCGAGGAUGAGACCAGGACUAGAACGCCUGAGCCGAUCAUCAUCGAAGAGGAAGAAGAGGAUAGCAUAAGUUUGCUGUCAGAUGGCCCGACCCACCAGGUGCUGCAAGUCGAGGCAGACAUUCACGGGCCGCCCUCUGUAUCUAGCUCAUCCUGGUCCAUUCCUCAUGCAUCCGAC UUUGAUGUGGACAGUUUAUCCAUACUUGACACCCUGGAGGGAGCUAGCGUGACCAGCGGGGCAACGUCAGCCGAGACUAACUCUUACUUCGCAAAGAGUAUGGAGUUUCUGGCCGACCGGUGCCUGCGCCUCGAACAGUAUUCAGGAACCCUCCACAUCCCGCUCCGCGCACAAGAACACCGUCACUUGCACCCAGCAGGGCCUGCU CGAGAACCAGCCUAGUUUCCACCCCGCCAGGCGUGAAUAGGGUGAUCACUAGAGAGGAGCUCGAGGCGCUUACCCCGUCACGCACCUAGCAGGUCGGUCGAGAACCAGCCUGGUCUCCAACCCGCCAGGCGUAAAUAGGGUGAUUACAAGAGGAGUUUGAGGCGUUCGUAGCACAACAACAAUGACGGUUUGAUGCGGGUGCA SEQ ID NO: 58 (NSP4 RNA) UACAUCUUUUCCUCCGACACCGGUCAAGGGCAUUUACAACAAAAAUCAGUAAGGCAAACGGUGCUAUCCGAAGUGGUGUUGGAGAGGACCGAAUUGGAGAUUUCGUAUGCCCCGCGCCUCGACCAAGAAAAAGAAGAAUUACUACGCAAGAAAUUACAGUUAAAUCCCACACCUGCUAACAGAAGCAGAUACCAGUCCAGGAAGGUGGAGAACAUGAAAGCCAUAACA GCUAGACGUAUUCUGCAAGGCCUAGGGCAUUAUUUGAAGGCAGAAGGAAAAGUGGAGUGCUACCGAACCCUGCAUCCUGUUCCUUUGUAUUCAUCUAGUGUGAACCGUGCCUUU UCAAGCCCCAAGGUCGCAGUGGAAGCCUGUAACGCCAUGUUGAAAGAGAACUUUCCGACUGUGGCUUCUUACUGUAUUAUUCCAGAGUACGAUGCCUAUUUGGACAUGGUUGAC GGAGCUUCAUGCUGCUUAGACACUGCCAGUUUUUGCCCUGCAAAGCUGCGCAGCUUUCCAAAGAAACACUCCUAUUUGGAACCCACAAUACGAUCGGCAGUGCCUUCAGCGAUCCAGAACACGCUCCAGAACGUCCUGGCAGCUGCCACAAAAAGAAAUUGCAAUGUCACGCAAAUGAGAGAAUUGCCCGUAUUGGAUUCGGCGGCCUUUAAUGUGGAAUGCUUCAAG AAAUAUGCGUGUAAUAAUGAAUAUUGGGAAACGUUUAAAGAAAACCCCAUCAGGCUUACUGAAGAAAACGUGGUAAAUUACAUUACCAAAUUAAAAGGACCAAAAGCUGCUGCUCUUUUUGCGAAGACACAUAAUUUGAAUAUGUUGCAGGACAUACCAAUGGACAGGUUUGUAAUGGACUUAAAGAGAGACGUGAAAGUGACUCCAGGAACAAAACAUACUGAAGAA CGGCCCAAGGUACAGGUGAUCCAGGCUGCCGAUCCGCUAGCAACAGCGUAUCUGUGCGGAAUCCACCGAGAGCUGGUUAGGAGAUUAAAUGCGGUCCUGCUUCCGAACAUUCAUACACUGUUUGAUAUGUCGGCUGAAGACUUUGACGCUAUUAUAGCCGAGCACUUCCAGCCUGGGGAUUGUGUUCUGGAAACUGACAUCGCGUCGUUUGAUAAAAGUGAGGACGAC GCCAUGGCUCUGACCGCGUUAAUGAUUCUGGAAGACUUAGGUGUGGACGCAGAGCUGUUGACGCUGAUUGAGGCGGCUUUCGGCGAAAUUUCAUCAAUACAUUUGCCCACUAAAACUAAAUUUAAAUUCGGAGCCAUGAUGAAAUCUGGAAUGUUCCUCACACUGUUUGUGAACACAGUCAUUAACAUUGUAAUCGCAAGCAGAGUGUUGAGAGAACGGCUAACCGGA UCACCAUGUGCAGCAUUCAUUGGAGAUGACAAUAUCGUGAAAGGAGUCAAAUCGGACAAAUUAAUGGCAGACAGGUGCGCCACCUGGUUGAAUAUGGAAGUCAAGAUUAUAGAUGCUGUGGUGGGCGAGAAAGCGCCUUAUUUCUGUGGAGGGUUUAUUUUGUGUGACUCCGUGACCGGCACAGCGUGCCGUGUGGCAGACCCCCUAAAAAGGCUGUUUAAGCUUGGC AAACCUCUGGCAGCAGACGAUGAACAUGAUGAUGACAGGAGAAGGGCAUUGCAUGAAGAGUCAACACGGCUGGAACCGAGUGGGUAUUCUUCAGAGCUGUGCAAGGCAGUAGAAUCAAGGUAUGAAAACCGUAGGAACUUCCAUCAUAGUUAUGGCCAUGACUACUCUAGCUAGCAGUGUUAAAUCAUUCAGCUACCUGAGAGGGGCCCCUAUAACUCUCUACGGCUAA.
IV.RNA轉錄在一些態樣中，本文所揭示之RNA係藉由活體外轉錄或化學合成產生。在本發明之上下文中，術語「轉錄」係關於其中DNA序列中之遺傳密碼轉錄成RNA的過程。隨後，RNA可轉譯成肽或蛋白質。IV.RNAtranscription In some aspects, the RNA disclosed herein is produced by in vitro transcription or chemical synthesis. In the context of the present invention, the term "transcription" refers to the process in which the genetic code in a DNA sequence is transcribed into RNA. Subsequently, the RNA can be translated into a peptide or protein.
根據本發明，「轉錄」包含「活體外轉錄」或「IVT」，其係指其中轉錄在非細胞系統中活體外發生以產生用於各種應用之合成RNA產物，包括例如產生蛋白質或多肽的過程。用於活體外轉錄mRNA之方法為此項技術中熟知的。(參見例如Losick, R. 1972.In vitrotranscription,Ann Rev Biochem, 41 409-46；Kamakaka, R. T.及Kraus, W. L. 2001.In vitroTranscription,Current Protocols in Cell Biology, 2:11.6:11.6.1-11.6.17；Beckert, B.及Masquida, B. 2010. Synthesis of RNA byIn vitroTranscription in RNA,Methods in Molecular Biology, 703 (Neilson, H.編), New York, N.Y. Humana Press, 2010；Brunelle, J.L.及Green, R., 2013,第五章-In vitrotranscription from plasmid or PCR-amplified DNA,Methods in Enzymology530:101-114；其全部均以引用之方式併入本文中)。According to the present invention, "transcription" includes "in vitro transcription" or "IVT", which refers to a process in which transcription occurs in vitro in a non-cellular system to produce a synthetic RNA product for various applications, including, for example, the production of proteins or polypeptides. Methods for in vitro transcription of mRNA are well known in the art. (See, e.g., Losick, R. 1972.In vitro transcription,Ann Rev Biochem , 41 409-46; Kamakaka, RT and Kraus, WL 2001.In vitro Transcription,Current Protocols in Cell Biology , 2:11.6:11.6.1-11.6.17; Beckert, B. and Masquida, B. 2010. Synthesis of RNA byIn vitro Transcription in RNA,Methods in Molecular Biology , 703 (Neilson, H. ed.), New York, NY Humana Press, 2010; Brunelle, JL and Green, R., 2013, Chapter 5 -In vitro transcription from plasmid or PCR-amplified DNA,Methods in Enzymology 530:101-114; all of which are incorporated herein by reference).
可應用選殖載體來產生轉錄本。此等選殖載體一般被稱為轉錄載體，且根據本發明由術語「載體」涵蓋。根據特定態樣，所使用RNA為活體外轉錄之RNA (IVT-RNA)，且可藉由適當DNA模板之活體外轉錄獲得。可用此項技術中熟知之適當技術製備模板DNA用於自多種來源進行活體外轉錄，該等技術包括但不限於質體DNA及聚合酶連鎖反應擴增(參見Linpinsel, J.L及Conn, G.L., General protocols for preparation of plasmid DNA template，及Bowman, J.C., Azizi, B., Lenz, T.K., Ray, P.及Williams, L.D., RNAin vitrotranscription and RNA purification by denaturing PAGE in Recombinant andin vitroRNA syntheses,Methods941 Conn G.L. (編), New York, N.Y. Humana Press, 2012，其各自以引用之方式併入本文中)。用於控制轉錄之啟動子可為任何RNA聚合酶之任何啟動子。RNA聚合酶之特定實例為T7、T3及SP6 RNA聚合酶。較佳地，根據本發明之活體外轉錄受T7或SP6啟動子控制。用於活體外轉錄之DNA模板可藉由選殖核酸(尤其cDNA)且將其引入用於活體外轉錄之適當載體中而獲得。cDNA可藉由RNA之反轉錄獲得。Cloning vectors can be used to produce transcripts. Such cloning vectors are generally referred to as transcription vectors and are covered by the term "vector" according to the present invention. According to a specific embodiment, the RNA used is an in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of an appropriate DNA template. Template DNA can be prepared for in vitro transcription from a variety of sources using appropriate techniques well known in the art, including but not limited to plasmid DNA and polymerase chain reaction amplification (see Linpinsel, JL and Conn, GL, General protocols for preparation of plasmid DNA template, and Bowman, JC, Azizi, B., Lenz, TK, Ray, P. and Williams, LD, RNAin vitro transcription and RNA purification by denaturing PAGE in Recombinant andin vitro RNA syntheses,Methods 941 Conn GL (ed.), New York, NY Humana Press, 2012, each of which is incorporated herein by reference). The promoter used to control transcription can be any promoter of any RNA polymerase. Specific examples of RNA polymerases are T7, T3 and SP6 RNA polymerases. Preferably, the in vitro transcription according to the present invention is controlled by the T7 or SP6 promoter. The DNA template for in vitro transcription can be obtained by cloning nucleic acid (especially cDNA) and introducing it into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.
合成IVT RNA產物可活體外轉譯或直接引入細胞中，該等產物可在該等細胞中轉譯。就RNA而言，術語「表現」或「轉譯」係關於在細胞之核糖體中mRNA股引導胺基酸序列之組裝以產生肽或蛋白質的過程。此類合成RNA產物包括但不限於例如mRNA分子、saRNA分子、反義RNA分子、shRNA分子、長非編碼RNA分子、核糖核酸酶、適體、引導RNA分子(例如對於CRISPR)、核糖體RNA分子、小核RNA分子、小核仁RNA分子及其類似者。在一些態樣中，可排除前述合成RNA產物中之1、2、3、4、5者或更多者。IVT反應通常利用如本文所描述及/或利用之DNA模板(例如線性DNA模板)、核糖核苷酸(例如未經修飾之核糖核苷酸三磷酸或經修飾之核糖核苷酸三磷酸)及適當RNA聚合酶。Synthetic IVT RNA products can be translated in vitro or introduced directly into cells where they can be translated. With respect to RNA, the term "expression" or "translation" refers to the process by which mRNA strands direct the assembly of amino acid sequences in the ribosomes of a cell to produce a peptide or protein. Such synthetic RNA products include, but are not limited to, for example, mRNA molecules, saRNA molecules, antisense RNA molecules, shRNA molecules, long noncoding RNA molecules, ribonucleases, aptamers, guide RNA molecules (e.g., for CRISPR), ribosomal RNA molecules, small nuclear RNA molecules, small nucleolar RNA molecules, and the like. In some aspects, 1, 2, 3, 4, 5, or more of the foregoing synthetic RNA products can be excluded. An IVT reaction typically utilizes a DNA template (e.g., a linear DNA template), ribonucleotides (e.g., unmodified ribonucleotide triphosphates or modified ribonucleotide triphosphates), and an appropriate RNA polymerase as described and/or utilized herein.
在一些態樣中，mRNA係藉由活體外轉錄使用DNA模板製造，其中DNA係指含有去氧核糖核苷酸之核酸。在一些態樣中，本文所揭示之RNA為活體外轉錄之RNA (IVT-RNA)，且可藉由適當DNA模板之活體外轉錄獲得。用於控制轉錄之啟動子可為任何RNA聚合酶之任何啟動子。用於活體外轉錄之DNA模板可藉由選殖核酸(尤其cDNA)且將其引入用於活體外轉錄之適當載體中而獲得。cDNA可藉由RNA之反轉錄獲得。In some aspects, mRNA is produced by in vitro transcription using a DNA template, wherein DNA refers to a nucleic acid containing deoxyribonucleotides. In some aspects, the RNA disclosed herein is an in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of an appropriate DNA template. The promoter used to control transcription can be any promoter of any RNA polymerase. The DNA template used for in vitro transcription can be obtained by cloning nucleic acid (especially cDNA) and introducing it into an appropriate vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.
在一些態樣中，IVT之起始材料可包括線性化DNA模板、核苷酸、RNA酶抑制劑、焦磷酸酶及/或聚合酶(例如T7 RNA聚合酶)。核苷酸可內部製造，可獲自供應商，或可合成。核苷酸可為但不限於本文所描述之彼等者，包括天然及非天然(經修飾)核苷酸。可使用任何數目之RNA聚合酶或變體，包括但不限於：噬菌體RNA聚合酶，例如T7 RNA聚合酶、T3 RNA聚合酶、SP6 RNA聚合酶；及/或突變聚合酶，諸如但不限於能夠併入經修飾之核酸及/或經修飾之核苷酸(包括化學修飾之核酸及/或核苷酸)的聚合酶。在一些態樣中，前述RNA聚合酶中之1、2、3、4、5者或更多者可排除在外。一些實施例排除使用DNA酶。In some aspects, the starting materials for IVT may include a linearized DNA template, nucleotides, an RNase inhibitor, a pyrophosphatase, and/or a polymerase (e.g., T7 RNA polymerase). The nucleotides may be made in-house, may be obtained from a supplier, or may be synthesized. The nucleotides may be, but are not limited to, those described herein, including natural and non-natural (modified) nucleotides. Any number of RNA polymerases or variants may be used, including, but not limited to: bacteriophage RNA polymerases, such as T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase; and/or mutant polymerases, such as, but not limited to, polymerases capable of incorporating modified nucleic acids and/or modified nucleotides (including chemically modified nucleic acids and/or nucleotides). In some aspects, 1, 2, 3, 4, 5, or more of the aforementioned RNA polymerases may be excluded. Some embodiments exclude the use of DNA enzymes.
在一些態樣中，IVT過程在生物反應器中進行。生物反應器可包含混合器。在一些態樣中，核苷酸可在整個IVT過程期間添加至生物反應器中。In some aspects, the IVT process is performed in a bioreactor. The bioreactor may include a mixer. In some aspects, nucleotides may be added to the bioreactor throughout the IVT process.
在一些態樣中，在生物反應器中，在IVT過程後，向包含RNA之IVT混合物中添加一或多種IVT後藥劑。例示性IVT後藥劑可包括經組態以消化線性化DNA模板之DNA酶I及/或經組態以消化DNA酶I及T7 RNA聚合酶之蛋白酶K。在一些態樣中，在IVT之後，將IVT後藥劑與生物反應器中之混合物一起培育。在一些態樣中，生物反應器可含有至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的IVT混合物：60、70、80、90、100、110、120、130、140、150 ,160、170、180、190、200、210、220、230、240、250、260、270、280、290、300、310、320、330、340、350、360、370、380、390、400、410、420、430、440、450、460、470、480、490及500公升或更多。IVT混合物可具有為或不為至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的RNA濃度：3.3、3.4、3.5、3.6、3.7、3.8、3.9、4.0、4.1、4.2、4.3、4.4、4.5、4.6、4.7、4.8、4.9、5.0、5.1、5.2、5.3、5.4、5.5、5.6、5.7、5.8、5.9、6.0、7.0、8.0、9.0、10、11、12、13、14、15、16、17、18、19、20、30、40、50、60、70、80、90及100 mg/mL RNA或更多。In some aspects, one or more post-IVT reagents are added to the IVT mixture containing RNA after the IVT process in the bioreactor. Exemplary post-IVT reagents can include DNase I configured to digest linearized DNA templates and/or proteinase K configured to digest DNase I and T7 RNA polymerase. In some aspects, after IVT, the post-IVT reagents are incubated with the mixture in the bioreactor. In some aspects, the bioreactor may contain at least, at most, just less than, or between any two of the following (inclusive or exclusive): 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, and 500 liters or more of the IVT mixture. The IVT mixture may have an RNA concentration that is or is not at least, at most, just below, or between any two of the following, inclusively or exclusively: 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 7.0, 8.0, 9.0, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, and 100 mg/mL RNA or more.
在一些態樣中，IVT混合物可包括殘餘亞精胺、殘餘DNA、殘餘蛋白質、肽、HEPES、EDTA、硫酸銨、陽離子(例如Mg2+、Na+、Ca2+)、RNA片段、殘餘核苷酸、游離磷酸酯或其任何組合。在一些態樣中，前述中之1、2、3、4、5者或更多者可排除在IVT混合物外。In some aspects, the IVT mixture may include residual spermidine, residual DNA, residual protein, peptide, HEPES, EDTA, ammonium sulfate, cations (e.g., Mg2+ , Na+ , Ca2+ ), RNA fragments, residual nucleotides, free phosphates, or any combination thereof. In some aspects, 1, 2, 3, 4, 5 or more of the foregoing may be excluded from the IVT mixture.
本文所描述之核酸之分離及/或純化可包括但不限於苯酚/氯仿萃取及/或在一價陽離子或氯化鋰存在下用任一醇(乙醇、異丙醇)沉澱，以用於核酸清除、品質保證及品質對照。純化程序之額外非限制性實例包括AGENCOURT®珠粒(Beckman Coulter Genomics, Danvers, MA)、聚T珠粒、LNATM寡聚T捕獲探針(EXIQON® Inc, Vedbaek, Denmark)、基於HPLC之純化方法(諸如但不限於強陰離子交換HPLC、弱陰離子交換HPLC、逆相HPLC (RP-HPLC)及疏水相互作用HPLC (HIC-HPLC))、尺寸排阻層析及基於二氧化矽之親和層析與聚丙烯醯胺凝膠電泳。可使用各種市售套組進行純化，該等套組包括但不限於SV總分離系統(Promega)及活體外轉錄清除及濃縮套組(Norgen Biotek)。在一些態樣中，可排除前述純化中之1、2、3、4、5者或更多者。Isolation and/or purification of nucleic acids described herein may include, but are not limited to, phenol/chloroform extraction and/or precipitation with any alcohol (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride for nucleic acid cleanup, quality assurance, and quality control. Additional non-limiting examples of purification procedures include AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads, LNATM oligo-T capture probes (EXIQON® Inc, Vedbaek, Denmark), HPLC-based purification methods (such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC)), size exclusion chromatography, and silica-based affinity chromatography and polyacrylamide gel electrophoresis. Purification can be performed using a variety of commercially available kits, including but not limited to the SV Total Isolation System (Promega) and the In Vitro Transcript Cleanup and Concentration Kit (Norgen Biotek). In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned purifications may be excluded.
當關於核酸使用時，術語「經純化」，諸如「經純化核酸」係指與至少一種污染物分離之一者。「污染物(contaminant)」為使得另一物質不合適、不純或較差的任何物質。因此，經純化核酸(例如DNA及RNA)以不同於其在自然界中發現的形式或設定存在，或以與在對其進行處理及/或純化方法之前存在的形式或設定不同的形式或設定存在。The term "purified" when used with respect to nucleic acids, such as "purified nucleic acid" refers to one that is separated from at least one contaminant. A "contaminant" is any substance that makes another substance unsuitable, impure, or inferior. Thus, purified nucleic acids (e.g., DNA and RNA) exist in a form or setting different from that in which they are found in nature, or in a form or setting different from that which existed prior to subjecting them to a treatment and/or purification method.
在一些態樣中，IVT混合物之至少一部分經過濾。IVT混合物可經由超濾及/或透濾來過濾，以自IVT混合物移除至少一些雜質及/或改變至少一部分IVT混合物之緩衝溶液以產生濃縮RNA溶液作為滲餘物。In some embodiments, at least a portion of the IVT mixture is filtered. The IVT mixture can be filtered by ultrafiltration and/or diafiltration to remove at least some impurities from the IVT mixture and/or to modify at least a portion of the buffer solution of the IVT mixture to produce a concentrated RNA solution as a raffinate.
在一些態樣中，「超濾」及「透濾」均係指膜過濾製程。超濾通常使用孔徑為至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的膜：0.001、0.002、0.003、0.004、0.005、0.006、0.007、0.008、0.009、0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09及0.1 µm。在一些態樣中，超濾膜通常藉由截留分子量(MWCO)而非孔徑來進行分類。舉例而言，MWCO可為至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：30 kDa、40 kDa、50 kDa、60 kDa、70 kDa、80 kDa、90 kDa、100 kDa、110 kDa、120 kDa、130 kDa、140 kDa、150 kDa、160 kDa、170 kDa、180 kDa、190 kDa、200 kDa、210 kDa、220 kDa、230 kDa、240 kDa、250 kDa、260 kDa、270 kDa、280 kDa、290 kDa、300 kDa、310 kDa、320 kDa、330 kDa、340 kDa、350 kDa、360 kDa、370 kDa、380 kDa、390 kDa、400 kDa、500 kDa、600 kDa、700 kDa、800 kDa、900 kDa、1000 kDa、2000 kDa、3000 kDa、4000 kDa、5000 kDa、6000 kDa、7000 kDa、8000 kDa、9000 kDa及10000 kDa。熟習此項技術者應理解，過濾膜可包含不同的適合材料，包括例如聚合物、纖維素、陶瓷等，視應用而定。在一些態樣中，膜過濾可更合乎大體積純化製程需要。In some aspects, "ultrafiltration" and "filtration" refer to membrane filtration processes. Ultrafiltration typically uses membranes with pore sizes of at least, at most, just below, or between any two of the following (inclusive or exclusive): 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, and 0.1 μm. In some aspects, ultrafiltration membranes are typically classified by molecular weight cutoff (MWCO) rather than pore size. For example, the MWCO can be at least, at most, exactly below, or between any two of the following (inclusive or exclusive): 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 110 kDa, 120 kDa, 130 kDa, 140 kDa, 150 kDa, 160 kDa, 170 kDa, 180 kDa, 190 kDa, 200 kDa, 210 kDa, 220 kDa, 230 kDa, 240 kDa, 250 kDa, 260 kDa, 270 kDa, 280 kDa, 290 kDa, 300 kDa, 310 kDa, 320 kDa, 330 kDa, 340 kDa, 350 kDa, 360 kDa, 370 kDa, 380 kDa, kDa, 390 kDa, 400 kDa, 500 kDa, 600 kDa, 700 kDa, 800 kDa, 900 kDa, 1000 kDa, 2000 kDa, 3000 kDa, 4000 kDa, 5000 kDa, 6000 kDa, 7000 kDa, 8000 kDa, 9000 kDa and 10000 kDa. Those skilled in the art will appreciate that the filter membrane may comprise different suitable materials, including, for example, polymers, cellulose, ceramics, etc., depending on the application. In some aspects, membrane filtration may be more suitable for bulk purification processes.
在一些態樣中，用於純化RNA的IVT混合物之超濾及透濾可包括：(1)直流過濾(DFF)，亦稱為「死端」過濾，其應用垂直於膜表面之進料流且試圖使100%的流體通過膜；及/或(2)切向流過濾(TFF)，亦稱為掃流過濾，其中進料流平行於膜表面傳遞，其中一個部分通過膜(滲透物)，而剩餘物(滲餘物)保留及/或再循環回至進料槽。In some embodiments, ultrafiltration and diafiltration of the IVT mixture used to purify RNA can include: (1) direct flow filtration (DFF), also known as "dead-end" filtration, which applies feed flow perpendicular to the membrane surface and attempts to pass 100% of the fluid through the membrane; and/or (2) tangential flow filtration (TFF), also known as swept flow filtration, in which the feed flow is passed parallel to the membrane surface, a portion of which passes through the membrane (permeate), and the remainder (retentate) is retained and/or recycled back to the feed tank.
在一些態樣中，IVT混合物之過濾係經由TFF進行，該TFF包含超濾步驟、第一透濾步驟及第二透濾步驟。在一些態樣中，第一透濾步驟在硫酸銨存在下進行。第一透濾步驟可經組態以自IVT混合物移除大部分雜質。在一些態樣中，第二透濾步驟在不存在硫酸銨之情況下進行。第二透濾步驟可經組態以將RNA轉移至DS緩衝液調配物中。In some embodiments, filtration of the IVT mixture is performed by TFF, which comprises an ultrafiltration step, a first filtration step, and a second filtration step. In some embodiments, the first filtration step is performed in the presence of ammonium sulfate. The first filtration step can be configured to remove a majority of impurities from the IVT mixture. In some embodiments, the second filtration step is performed in the absence of ammonium sulfate. The second filtration step can be configured to transfer the RNA into a DS buffer formulation.
可選擇具有適當MWCO之過濾膜用於TFF製程中之超濾。TFF膜之MWCO決定哪些溶質可通過膜進入濾液中及哪些溶質保留在滲餘物中。TFF膜之MWCO可經選擇，使得實質上所有所關注溶質(例如所需合成RNA物種)保留在滲餘物中，而非所需組分(例如過量的核糖核苷酸、小核酸片段(諸如經消化或水解之DNA模板)、肽片段(諸如經消化之蛋白質)及/或其他雜質)傳遞至濾液中。在一些態樣中，包含所需合成RNA物種之滲餘物可再循環至進料儲槽中以在額外循環中再過濾。在一些態樣中，TFF膜之MWCO可為至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：30 kDa、40 kDa、50 kDa、60 kDa、70 kDa、80 kDa、90 kDa或更多。在一些態樣中，TFF膜之MWCO可為至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：100 kDa、150 kDa、200 kDa、250 kDa、300 kDa、350 kDa、400 kDa或更多。在一些態樣中，TFF膜之MWCO可為或為約250-350 kDa。在一些態樣中，TFF膜(例如基於纖維素之膜)之MWCO可為或為約30-300 kDa；50-300 kDa、100-300 kDa或200-300 kDa。A filter membrane with an appropriate MWCO can be selected for ultrafiltration in a TFF process. The MWCO of a TFF membrane determines which solutes can pass through the membrane into the filtrate and which solutes are retained in the retentate. The MWCO of a TFF membrane can be selected so that substantially all solutes of interest (e.g., desired synthetic RNA species) are retained in the retentate, while undesired components (e.g., excess ribonucleotides, small nucleic acid fragments (e.g., digested or hydrolyzed DNA templates), peptide fragments (e.g., digested proteins), and/or other impurities) are passed into the filtrate. In some embodiments, the retentate containing the desired synthetic RNA species can be recycled to the feed tank for refiltration in an additional cycle. In some aspects, the MWCO of the TFF membrane can be at least, at most, just below, or between any two of the following (inclusive or exclusive): 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, or more. In some aspects, the MWCO of the TFF membrane can be at least, at most, just below, or between any two of the following (inclusive or exclusive): 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, or more. In some aspects, the MWCO of the TFF membrane can be or is about 250-350 kDa. In some aspects, the MWCO of a TFF membrane (e.g., a cellulose-based membrane) can be or is about 30-300 kDa; 50-300 kDa, 100-300 kDa, or 200-300 kDa.
透濾可不連續地或者連續地進行。舉例而言，在連續透濾中，透濾溶液可以與產生濾液相同的速率添加至樣品進料儲槽中。以此方式，樣品儲槽之容積保持恆定，但移除可自由滲透通過膜之小分子(例如鹽、溶劑等)。使用溶劑移除作為實例，各額外透濾體積(DV)進一步降低溶劑濃度。在不連續透濾中，溶液首先經稀釋且接著濃縮回至起始體積。接著重複此製程直至達到儲槽中剩餘之小分子(例如鹽、溶劑等)之所需濃度。各額外透濾體積(DV)進一步降低小分子(例如溶劑)濃度。連續透濾通常需要用於待過濾分子之給定降低的最小體積。另一方面，不連續透濾准許滲餘物狀態(諸如pH、鹽含量及其類似者)之快速變化。在一些態樣中，以至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的量進行第一透濾步驟：2、3、4、5、6、7、8、9、10個或更多個透濾體積。在一些態樣中，以至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的量進行第二透濾步驟：5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20個或更多個透濾體積。在一些態樣中，以5個透濾體積進行第一透濾步驟，且以10個透濾體積進行第二透濾步驟。Filtration can be performed discontinuously or continuously. For example, in continuous filtration, the filtration solution can be added to the sample feed reservoir at the same rate as the filtration solution is produced. In this way, the volume of the sample reservoir is kept constant, but small molecules (such as salts, solvents, etc.) that can freely permeate through the membrane are removed. Using solvent removal as an example, each additional filtration volume (DV) further reduces the solvent concentration. In discontinuous filtration, the solution is first diluted and then concentrated back to the starting volume. This process is then repeated until the desired concentration of small molecules (such as salts, solvents, etc.) remaining in the reservoir is reached. Each additional filtration volume (DV) further reduces the concentration of small molecules (e.g., solvent). Continuous filtration generally requires a minimum volume for a given reduction of the molecules to be filtered. On the other hand, discontinuous filtration allows for rapid changes in retentate conditions (e.g., pH, salt content, and the like). In some aspects, the first filtration step is performed in an amount of at least, at most, just below, or between any two of the following (inclusive or exclusive): 2, 3, 4, 5, 6, 7, 8, 9, 10 or more filtration volumes. In some aspects, the second filtration step is performed at least, at most, just below, or between any two of the following (inclusive or exclusive): 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more filtration volumes. In some aspects, the first filtration step is performed at 5 filtration volumes and the second filtration step is performed at 10 filtration volumes.
在一些態樣中，對於超濾及/或透濾，IVT混合物以至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的速率過濾：100、110、120、130、140、150、160、170、180、190、200、210、220、230、240、250、260、270、280、290、300、310、320、330、340、350、360、370、380、390、400、410、420、430、440、450、500、600、700、800、900或1000 L/m2過濾器面積/小時或更大。濃縮RNA溶液可包含至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的單股RNA：2.0、2.1、2.2、2.3、2.4或2.5 mg/mL。In some aspects, for ultrafiltration and/or filtration, the IVT mixture is filtered at a rate of at least, at most, just below, or between any two of the following, inclusively or exclusively: 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 500, 600, 700, 800, 900, or 1000 L/m2 filter area/hour or more. The concentrated RNA solution can contain single-stranded RNA of at least, at most, just below, or between any two of the following (inclusive or exclusive): 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 mg/mL.
在一些態樣中，亦可減少經由過濾獲得RNA產物溶液的濃縮RNA溶液之生物負荷。可使用一或多個過濾器進行過濾以用於減少生物負荷。該一或多個過濾器可包括孔徑為或不為至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的過濾器：0.2 µm、0.45 µm、0.65 µm、0.8 µm，或經組態以移除生物負荷之任何其他孔徑。In some aspects, the bioburden of the concentrated RNA solution obtained by filtering the RNA product solution can also be reduced. Filtering can be performed using one or more filters for reducing bioburden. The one or more filters may include a filter having a pore size of or not at least, at most, just below, or between any two of the following (inclusive or exclusive): 0.2 μm, 0.45 μm, 0.65 μm, 0.8 μm, or any other pore size configured to remove bioburden.
作為一個實例，減少生物負荷可包括對含有獲自超濾及/或透濾之滲餘物的滲餘物貯槽進行排液以獲得滲餘物。減少生物負荷可包括使用洗滌緩衝溶液來沖洗用於超濾及/或透濾之過濾系統，以獲得包含過濾系統中剩餘之殘餘RNA的洗滌池溶液。可過濾滲餘物以獲得經過濾之滲餘物。可使用第一0.2 µm過濾器過濾洗滌池溶液以獲得經過濾之洗滌池溶液。可使用第一0.2 µm過濾器或另一0.2 µm過濾器過濾滲餘物。As an example, reducing the bioburden may include draining a raffinate tank containing raffinate obtained from ultrafiltration and/or filtration to obtain a raffinate. Reducing the bioburden may include flushing a filtration system used for ultrafiltration and/or filtration with a wash buffer solution to obtain a wash tank solution comprising residual RNA remaining in the filtration system. The raffinate may be filtered to obtain a filtered raffinate. The wash tank solution may be filtered using a first 0.2 µm filter to obtain a filtered wash tank solution. The remnant can be filtered using the first 0.2 µm filter or a second 0.2 µm filter.
可合併經過濾之洗滌池溶液及經過濾之滲余物以形成經合併之池溶液。可使用第二0.2 µm過濾器來過濾經合併之池溶液以獲得經過濾之合併的池溶液，該經過濾之合併的池溶液使用第三0.2 µm過濾器進一步過濾以產生RNA產物溶液。The filtered wash pool solution and the filtered retentate can be combined to form a combined pool solution. The combined pool solution can be filtered using a second 0.2 μm filter to obtain a filtered combined pool solution, which is further filtered using a third 0.2 μm filter to produce an RNA product solution.
品質保證及/或品質控制檢查可使用諸如但不限於凝膠電泳、UV吸光度及/或分析型HPLC之方法進行。Quality assurance and/or quality control checks may be performed using methods such as, but not limited to, gel electrophoresis, UV absorbance, and/or analytical HPLC.
在一些態樣中，可藉由包括但不限於逆轉錄酶-PCR之方法對核酸進行定序。In some aspects, nucleic acids can be sequenced by methods including, but not limited to, reverse transcriptase-PCR.
在一些態樣中，可使用諸如但不限於紫外線可見光譜分析(UV/Vis)之方法對核酸進行定量。UV/Vis光譜儀之非限制性實例為NANODROP®光譜儀(ThermoFisher, Waltham, MA)。可分析定量的核酸以便判定核酸是否可具有適當大小及/或評定降解。核酸之降解可藉由諸如但不限於以下之方法評定：瓊脂糖凝膠電泳；基於HPLC之純化方法，諸如但不限於強陰離子交換HPLC、弱陰離子交換HPLC、逆相HPLC (RP-HPLC)及疏水相互作用HPLC (HIC-HPLC)；液相層析質譜法(LCMS)；毛細管電泳(CE)；及毛細管凝膠電泳(CGE)。在一些態樣中，可排除前述評定方法中之1、2、3、4、5者或更多者。In some aspects, nucleic acids can be quantified using methods such as, but not limited to, ultraviolet visible spectroscopy (UV/Vis). A non-limiting example of a UV/Vis spectrometer is a NANODROP® spectrometer (ThermoFisher, Waltham, MA). The quantified nucleic acids can be analyzed to determine whether the nucleic acids are of appropriate size and/or to assess degradation. Degradation of nucleic acids can be assessed by methods such as, but not limited to, agarose gel electrophoresis; HPLC-based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC); liquid chromatography mass spectrometry (LCMS); capillary electrophoresis (CE); and capillary gel electrophoresis (CGE). In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned assessment methods may be excluded.
V. RNA囊封RNA產物溶液中之RNA可經囊封，且RNA溶液可進一步包含至少一種囊封劑。在一個態樣中，囊封劑包含脂質、脂質奈米顆粒(LNP)、脂質複合物、聚合物顆粒、聚合複合物、整體型遞送系統或其組合。在一些態樣中，可排除前述元件中之1、2、3、4、5者或更多者作為囊封劑。V. RNAEncapsulation The RNA in the RNA product solution may be encapsulated, and the RNA solution may further comprise at least one encapsulating agent. In one aspect, the encapsulating agent comprises a lipid, a lipid nanoparticle (LNP), a lipoplex, a polymer particle, a polymer complex, an integral delivery system, or a combination thereof. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned elements may be excluded as an encapsulating agent.
在一個態樣中，囊封劑為脂質，且產生脂質奈米顆粒(LNP)囊封之RNA。不意欲受任何理論所束縛，咸信，陽離子或陽離子可離子化脂質或脂質樣材料及/或陽離子聚合物與核酸組合在一起以形成聚集物，且此聚集產生膠態穩定的顆粒。In one embodiment, the encapsulating agent is a lipid, and lipid nanoparticles (LNPs) encapsulated RNA are produced. Without intending to be bound by any theory, it is believed that cations or cations can ionize lipids or lipid-like materials and/or cationic polymers and nucleic acids to form aggregates, and this aggregation produces colloidally stable particles.
脂質可為天然存在之脂質或合成脂質。然而，脂質通常為生物物質。生物脂質為此項技術中所熟知，且包括例如中性脂肪、磷脂、磷酸甘油酯、類固醇、萜類、溶血脂質、鞘醣脂、糖脂質、硫脂、具有醚及酯連接之脂肪酸之脂質及可聚合脂質，及其組合。脂質為不溶於水且可用有機溶劑萃取之物質。除本文專門描述之彼等化合物以外的化合物由熟習此項技術者理解為脂質，且由本發明之組合物及方法涵蓋。脂質組分及非脂質可彼此共價或非共價連接。Lipids can be naturally occurring lipids or synthetic lipids. However, lipids are generally biological substances. Biological lipids are well known in the art and include, for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenoids, lysolipids, sphingolipids, glycolipids, sulfolipids, lipids with ether- and ester-linked fatty acids, and polymerizable lipids, and combinations thereof. Lipids are substances that are insoluble in water and can be extracted with organic solvents. Compounds other than those specifically described herein are understood by those skilled in the art to be lipids and are encompassed by the compositions and methods of the present invention. Lipid components and non-lipids can be covalently or non-covalently linked to each other.
在一些態樣中，LNP可設計成保護RNA分子(例如saRNA、mRNA)免於細胞外RNA酶，及/或可經工程改造用於將RNA全身性遞送至目標細胞。在一些態樣中，此類LNP可尤其適用於當向有需要之個體靜脈內投與RNA分子時遞送RNA分子(例如saRNA、mRNA)。在一些態樣中，此類LNP可尤其適用於當向有需要之個體肌肉內投與RNA分子時遞送RNA分子(例如saRNA、mRNA)。在一些態樣中，此類LNP可尤其適用於當向有需要之個體皮內投與RNA分子時遞送RNA分子(例如saRNA、mRNA)。在一些態樣中，此類LNP可尤其適用於當向有需要之個體鼻內投與RNA分子時遞送RNA分子(例如saRNA、mRNA)。In some aspects, LNPs can be designed to protect RNA molecules (e.g., saRNA, mRNA) from extracellular RNases and/or can be engineered for systemic delivery of RNA to target cells. In some aspects, such LNPs may be particularly suitable for delivering RNA molecules (e.g., saRNA, mRNA) when the RNA molecules are administered intravenously to an individual in need. In some aspects, such LNPs may be particularly suitable for delivering RNA molecules (e.g., saRNA, mRNA) when the RNA molecules are administered intramuscularly to an individual in need. In some aspects, such LNPs may be particularly suitable for delivering RNA molecules (e.g., saRNA, mRNA) when the RNA molecules are administered intradermally to an individual in need. In some aspects, such LNPs may be particularly suitable for delivering RNA molecules (e.g., saRNA, mRNA) when the RNA molecules are administered intranasally to an individual in need.
在一個態樣中，RNA產物溶液中之RNA之濃度＜ 1 mg/mL。在另一態樣中，RNA之濃度為至少或至少約0.05 mg/mL。在另一態樣中，RNA之濃度為至少或至少約0.5 mg/mL。在另一態樣中，RNA之濃度為至少或至少約1 mg/ml。在另一態樣中，RNA濃度為或為約0.05 mg/mL至約0.5 mg/mL。在另一態樣中，RNA之濃度為至少10 mg/mL。在另一態樣中，RNA之濃度為至少50 mg/mL。在一些態樣中，RNA之濃度為或不為至少、至多、恰好以下、在以下中之任何兩者之間(包括性或排他性)或約以下：0.05 mg/mL、0.5 mg/mL、1 mg/mL、10 mg/mL、50 mg/mL、75 mg/mL、100 mg/mL、150 mg/mL、200 mg/mL、250 mg/mL、300 mg/mL、400 mg/mL或更多。In one aspect, the concentration of RNA in the RNA product solution is < 1 mg/mL. In another aspect, the concentration of RNA is at least or at least about 0.05 mg/mL. In another aspect, the concentration of RNA is at least or at least about 0.5 mg/mL. In another aspect, the concentration of RNA is at least or at least about 1 mg/ml. In another aspect, the concentration of RNA is or is about 0.05 mg/mL to about 0.5 mg/mL. In another aspect, the concentration of RNA is at least 10 mg/mL. In another aspect, the concentration of RNA is at least 50 mg/mL. In some aspects, the concentration of the RNA is or is not at least, at most, just below, between any two of (inclusive or exclusive) or about below 0.05 mg/mL, 0.5 mg/mL, 1 mg/mL, 10 mg/mL, 50 mg/mL, 75 mg/mL, 100 mg/mL, 150 mg/mL, 200 mg/mL, 250 mg/mL, 300 mg/mL, 400 mg/mL, or more.
本發明提供一種RNA產物溶液及一種其脂質製劑混合物或組合物，其包含至少一種編碼例如抗原(例如RSV融合前F蛋白)之RNA，該至少一種RNA與一或多種脂質複合、囊封於其中及/或用其調配，且形成脂質奈米顆粒(LNP)、脂質體、脂質複合物及/或奈米脂質體。在一些態樣中，組合物包含脂質奈米顆粒。The present invention provides a kind of RNA product solution and a kind of lipid preparation mixture or composition thereof, it comprises at least one RNA encoding, for example, an antigen (for example, RSV prefusion F protein), the at least one RNA is complexed with one or more lipids, encapsulated therein and/or formulated therewith, and forms lipid nanoparticles (LNPs), liposomes, lipid complexes and/or nanoliposomes. In some aspects, the composition comprises lipid nanoparticles.
脂質奈米顆粒或LNP係指當陽離子脂質及視情況一或多種其他脂質例如在水性環境中及/或在RNA存在下合併時所產生之任何形態的顆粒。在一些態樣中，脂質奈米顆粒包括在調配物中，該調配物可用於將活性劑或治療劑(諸如核酸(例如mRNA))遞送至所關注之目標部位(例如細胞、組織、器官、腫瘤及其類似者)。在一些態樣中，本發明之脂質奈米顆粒包含核酸(例如mRNA)。此類脂質奈米顆粒通常包含陽離子脂質及一或多種賦形劑，例如一或多種中性脂質、帶電脂質、類固醇、聚合物結合脂質或其組合。在一些態樣中，LNP包含至少一種陽離子(例如可離子化)脂質、至少一種中性(例如非陽離子)脂質、至少一種結構脂質(例如類固醇)及/或至少一種聚合物結合脂質(例如聚乙二醇(PEG)修飾之脂質)。在一些態樣中，前述賦形劑中之1、2、3者或更多者可排除在LNP外。Lipid nanoparticles or LNPs refer to particles of any form produced when a cationic lipid and, optionally, one or more other lipids are combined, for example, in an aqueous environment and/or in the presence of RNA. In some aspects, lipid nanoparticles are included in formulations that can be used to deliver active agents or therapeutic agents, such as nucleic acids (e.g., mRNA), to target sites of interest (e.g., cells, tissues, organs, tumors, and the like). In some aspects, the lipid nanoparticles of the present invention comprise nucleic acids (e.g., mRNA). Such lipid nanoparticles typically comprise cationic lipids and one or more shaping agents, such as one or more neutral lipids, charged lipids, steroids, polymer-bound lipids, or combinations thereof. In some embodiments, LNPs include at least one cationic (e.g., ionizable) lipid, at least one neutral (e.g., non-cationic) lipid, at least one structural lipid (e.g., steroid) and/or at least one polymer-bound lipid (e.g., polyethylene glycol (PEG)-modified lipid). In some embodiments, 1, 2, 3 or more of the aforementioned excipients may be excluded from LNPs.
在一些態樣中，LNP包含20-60 mol%之一或多種陽離子(例如可離子化)脂質。舉例而言，LNP可包含20-50 mol%、20-40 mol%、20-30 mol%、30-60 mol%、30-50 mol%、30-40 mol%、40-60 mol%、40-50 mol%或50-60 mol%之一或多種陽離子(例如可離子化)脂質。在一些態樣中，LNP包含或不包含至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的一或多種陽離子(例如可離子化)脂質：20 mol%、30 mol%、40 mol%、50 mol%或60 mol%。在一些態樣中，LNP包含45至55莫耳%(mol%)之一或多種陽離子(例如可離子化)脂質。舉例而言，LNP可包含或不包含至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的一或多種陽離子(例如可離子化)脂質：45、46、47、48、49、50、51、52、53、54或55 mol% 。In some aspects, LNPs include 20-60 mol% of one or more cationic (e.g., ionizable) lipids. For example, LNPs may include 20-50 mol%, 20-40 mol%, 20-30 mol%, 30-60 mol%, 30-50 mol%, 30-40 mol%, 40-60 mol%, 40-50 mol%, or 50-60 mol% of one or more cationic (e.g., ionizable) lipids. In some aspects, LNPs include or do not include at least, at most, just below, or between any two of the following (inclusive or exclusive) one or more cationic (e.g., ionizable) lipids: 20 mol%, 30 mol%, 40 mol%, 50 mol%, or 60 mol%. In some aspects, the LNP comprises 45 to 55 mol% of one or more cationic (e.g., ionizable) lipids. For example, the LNP may or may not comprise at least, at most, just below, or between any two of the following (inclusive or exclusive) one or more cationic (e.g., ionizable) lipids: 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mol%.
在一些態樣中，LNP包含5-25 mol%一或多種中性(例如非陽離子)脂質。舉例而言，LNP可包含5-20 mol%、5-15 mol%、5-10 mol%、10-25 mol%、10-20 mol%、10-25 mol%、15-25 mol%、15-20 mol%或20-25 mol%之一或多種中性(例如非陽離子)脂質。在一些態樣中，LNP為或不為至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的一或多種中性(例如非陽離子)脂質：5 mol%、10 mol%、15 mol%、20 mol%或25 mol%。在一些態樣中，LNP包含5至15 mol%之一或多種中性(例如非陽離子)脂質。舉例而言，LNP可包含至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的一或多種中性(例如非陽離子)脂質：5、6、7、8、9、10、11、12、13、14或15 mol%。In some aspects, the LNP comprises 5-25 mol% of one or more neutral (e.g., non-cationic) lipids. For example, the LNP may comprise 5-20 mol%, 5-15 mol%, 5-10 mol%, 10-25 mol%, 10-20 mol%, 10-25 mol%, 15-25 mol%, 15-20 mol%, or 20-25 mol% of one or more neutral (e.g., non-cationic) lipids. In some aspects, the LNP is or is not at least, at most, just below, or between any two of the following (inclusive or exclusive) one or more neutral (e.g., non-cationic) lipids: 5 mol%, 10 mol%, 15 mol%, 20 mol%, or 25 mol%. In some aspects, the LNP comprises 5 to 15 mol% of one or more neutral (e.g., non-cationic) lipids. For example, the LNP may comprise at least, at most, just below, or between any two of the following (inclusive or exclusive) one or more neutral (e.g., non-cationic) lipids: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mol%.
在一些態樣中，LNP包含25-55 mol%之一或多種結構脂質(例如類固醇)。舉例而言，LNP可包含25-50 mol%、25-45 mol%、25-40 mol%、25-35 mol%、25-30 mol%、30-55 mol%、30-50 mol%、30-45 mol%、30-40 mol%、30-35 mol%、35-55 mol%、35-50 mol%、35-45 mol%、35-40 mol%、40-55 mol%、40-50 mol%、40-45 mol%、45-55 mol%、45-50 mol%或50-55 mol%之一或多種結構脂質(例如類固醇)。在一些態樣中，LNP為或不為至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的一或多種結構脂質(例如類固醇)：25 mol%、30 mol%、35 mol%、40 mol%、45 mol%、50 mol%或55 mol%。在一些態樣中，LNP包含35至40 mol%之一或多種結構脂質(例如類固醇)。舉例而言，LNP可包含至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的一或多種結構脂質(例如類固醇)：35、36、37、38、39或40 mol%。In some aspects, LNPs comprise 25-55 mol% of one or more structural lipids (e.g., steroids). For example, LNPs may comprise 25-50 mol%, 25-45 mol%, 25-40 mol%, 25-35 mol%, 25-30 mol%, 30-55 mol%, 30-50 mol%, 30-45 mol%, 30-40 mol%, 30-35 mol%, 35-55 mol%, 35-50 mol%, 35-45 mol%, 35-40 mol%, 40-55 mol%, 40-50 mol%, 40-45 mol%, 45-55 mol%, 45-50 mol%, or 50-55 mol% of one or more structural lipids (e.g., steroids). In some aspects, LNP is or is not at least, at most, just below, or between any two of the following (inclusive or exclusive) one or more structural lipids (e.g., steroids): 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, or 55 mol%. In some aspects, LNP comprises 35 to 40 mol% of one or more structural lipids (e.g., steroids). For example, LNP may comprise at least, at most, just below, or between any two of the following (inclusive or exclusive) one or more structural lipids (e.g., steroids): 35, 36, 37, 38, 39, or 40 mol%.
在一些態樣中，LNP包含0.5-15 mol%之一或多種聚合物結合脂質(例如聚乙二醇(PEG)修飾之脂質)。舉例而言，脂質奈米顆粒可包含0.5-10 mol%、0.5-5 mol%、1-15 mol%、1-10 mol%、1-5 mol%、2-15 mol%、2-10 mol%、2-5 mol%、5-15 mol%、5-10 mol%或10-15 mol%之一或多種聚合物結合脂質(例如聚乙二醇(PEG)修飾之脂質)。在一些態樣中，脂質LNP為或不為至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的一或多種聚合物結合脂質(例如聚乙二醇(PEG)修飾之脂質)：0.5 mol%、1 mol%、2 mol%、3 mol%、4 mol%、5 mol%、6 mol%、7 mol%、8 mol%、9 mol%、10 mol%、11 mol%、12 mol%、13 mol%、14 mol%或15 mol%。在一些態樣中，LNP包含1至2 mol%之一或多種聚合物結合脂質(例如聚乙二醇(PEG)修飾之脂質)。舉例而言，LNP可包含至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的一或多種聚合物結合脂質(例如聚乙二醇(PEG)修飾之脂質)：1、1.5或2 mol%。In some aspects, the LNP comprises 0.5-15 mol% of one or more polymer-bound lipids (e.g., lipids modified with polyethylene glycol (PEG)). For example, the lipid nanoparticles may comprise 0.5-10 mol%, 0.5-5 mol%, 1-15 mol%, 1-10 mol%, 1-5 mol%, 2-15 mol%, 2-10 mol%, 2-5 mol%, 5-15 mol%, 5-10 mol% or 10-15 mol% of one or more polymer-bound lipids (e.g., lipids modified with polyethylene glycol (PEG)). In some aspects, lipid LNPs are or are not at least, at most, just below, or between any two of the following (inclusive or exclusive) one or more polymer-bound lipids (e.g., lipids modified with polyethylene glycol (PEG)): 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or 15 mol%. In some aspects, LNPs comprise 1 to 2 mol% of one or more polymer-bound lipids (e.g., lipids modified with polyethylene glycol (PEG)). For example, LNPs may comprise at least, at most, just below, or between any two of the following (inclusive or exclusive) one or more polymer-bound lipids (e.g., lipids modified with polyethylene glycol (PEG)): 1, 1.5, or 2 mol%.
在一些態樣中，LNP包含：20-75 mol%之一或多種陽離子(例如可離子化)脂質(例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：20%、25%、30%、35%、40%、45%、50%、55%、60%、65%、70%及75%)、0.5-25 mol%之一或多種中性(例如非陽離子)脂質(例如至少、至多、恰好以下或在以下之間(包括性或排他性)：0.5%、2.25%、4%、5.75%、7.5%、9.25%、11%、12.75%、14.5%、16.25%、18%、19.75%、21.5%、23.25%及25%)、5-55 mol%之一或多種結構脂質(例如類固醇) (例如至少、至多、恰好以下或在以下之間(包括性或排他性)：5%、10%、15%、20%、25%、30%、35%、40%、45%、50%及55%)及0.5-20 mol%之一或多種聚合物結合脂質(例如聚乙二醇(PEG)修飾之脂質) (例如至少、至多、恰好以下或在以下之間(包括性或排他性)：0.5%、2%、3.5%、5%、6.5%、8%、9.5%、11%、12.5%、14%、15.5%、17%、18.5%及20%)。在一些態樣中，該等脂質中之1、2、3者或更多者可排除在LNP外。In some aspects, the LNP comprises: 20-75 mol% of one or more cationic (e.g., ionizable) lipids (e.g., at least, at most, just below, or between any two of the following (inclusive or exclusive): 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% and 75%), 0.5-25 mol% of one or more neutral (e.g., non-cationic) lipids (e.g., at least, at most, just below, or between the following (inclusive or exclusive): 0.5%, 2.25%, 4%, 5.75%, 7.5%, 9.25%, 11%, 12.75%, 14.5%, 16.25%, 18%, 19.75%, 21.5%, 23.25% and 25%), 5-55 mol% of one or more structural lipids (e.g., steroids) (e.g., at least, at most, just below, or between (inclusive or exclusive): 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, and 55%) and 0.5-20 mol% of one or more polymer-bound lipids (e.g., lipids modified with polyethylene glycol (PEG)) (e.g., at least, at most, just below, or between (inclusive or exclusive): 0.5%, 2%, 3.5%, 5%, 6.5%, 8%, 9.5%, 11%, 12.5%, 14%, 15.5%, 17%, 18.5%, and 20%). In some aspects, 1, 2, 3, or more of the lipids may be excluded from the LNP.
在一些非限制性態樣中，莫耳脂質比率為50/10/38.5/1.5 (mol%陽離子脂質/中性脂質/結構脂質/聚合物結合脂質)、60/7.5/31/1.5 (mol%陽離子脂質/中性脂質/結構脂質/聚合物結合脂質)、57.5/7.5/31.5/3.5 (mol%陽離子脂質/中性脂質/結構脂質/聚合物結合脂質)、57.2/7.1/34.3/1.4 (mol%陽離子脂質/中性脂質/結構脂質/聚合物結合脂質)、40/15/40/5 (mol%陽離子脂質/中性脂質/結構脂質/聚合物結合脂質)、50/10/35/4.5/0.5 (mol%陽離子脂質/中性脂質/結構脂質/聚合物結合脂質)、50/10/35/5 (mol%陽離子脂質/中性脂質/結構脂質/聚合物結合脂質)、40/10/40/10 (mol%陽離子脂質/中性脂質/結構脂質/聚合物結合脂質)、35/15/40/10 (mol%陽離子脂質/中性脂質/結構脂質/聚合物結合脂質)、或52/13/30/5 (mol%陽離子脂質/中性脂質/結構脂質/聚合物結合脂質)。In some non-limiting aspects, the molar lipid ratio is 50/10/38.5/1.5 (mol% cationic lipid/neutral lipid/structural lipid/polymer-bound lipid), 60/7.5/31/1.5 (mol% cationic lipid/neutral lipid/structural lipid/polymer-bound lipid), 57.5/7.5/31.5/3.5 (mol% cationic lipid/neutral lipid/structural lipid/polymer-bound lipid), 57.2/7.1/34.3/1.4 (mol% cationic lipid/neutral lipid/structural lipid/polymer-bound lipid), 40/15/40/5 (mol% cationic lipids/neutral lipids/structural lipids/polymer-bound lipids), 50/10/35/4.5/0.5 (mol% cationic lipids/neutral lipids/structural lipids/polymer-bound lipids), 50/10/35/5 (mol% cationic lipids/neutral lipids/structural lipids/polymer-bound lipids), 40/10/40/10 (mol% cationic lipids/neutral lipids/structural lipids/polymer-bound lipids), 35/15/40/10 (mol% cationic lipids/neutral lipids/structural lipids/polymer-bound lipids), or 52/13/30/5 (mol% cationic lipids/neutral lipids/structural lipids/polymer-bound lipids).
在一些態樣中，活性劑或治療劑(諸如核酸(例如mRNA))可囊封於脂質奈米顆粒之脂質部分及/或被脂質奈米顆粒之脂質部分中之一些或所有包裹的水性空間中，從而保護其免於酵素降解或由宿主生物體或細胞之機制誘導的其他非所需作用，例如不良免疫反應。核酸(例如mRNA)或其部分亦可與脂質奈米顆粒結合及複合。脂質奈米顆粒可包含能夠形成連接核酸及/或囊封一或多種核酸之顆粒的任何脂質。In some aspects, active agents or therapeutic agents such as nucleic acids (e.g., mRNA) can be encapsulated in the lipid portion of the lipid nanoparticle and/or in the aqueous space enclosed by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the host organism or cell mechanism, such as adverse immune responses. Nucleic acids (e.g., mRNA) or portions thereof can also be bound and complexed with lipid nanoparticles. Lipid nanoparticles can include any lipid capable of forming particles that are linked to nucleic acids and/or encapsulate one or more nucleic acids.
在一些態樣中，所提供RNA分子(例如saRNA、mRNA)可用LNP調配。在一些態樣中，脂質奈米顆粒可具有或可不具有為或不為約1至500 nm之平均直徑(例如至少、至多、恰好以下或在以下之間(包括性或排他性)：1、10、20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190、200、210、220、230、240、250、260、270、280、290、300、310、320、330、340、350、360、370、380、390、400、410、420、430、440、450、460、470、480、490或500 nm)。在一些態樣中，脂質奈米顆粒之平均直徑為或為約30 nm至約150 nm、約40 nm至約150 nm、約50 nm至約150 nm、約60 nm至約130 nm、約70 nm至約110 nm、約70 nm至約100 nm、約80 nm至約100 nm、約90 nm至約100 nm、約70至約90 nm、約80 nm至約90 nm、約70 nm至約80 nm，或為至少、至多、恰好以下或在以下之間(包括性或排他性)：30 nm、35 nm、40 nm、45 nm、50 nm、55 nm、60 nm、65 nm、70 nm、75 nm、80 nm、85 nm、90 nm、95 nm、100 nm、105 nm、110 nm、115 nm、120 nm、125 nm、130 nm、135 nm、140 nm、145 nm或150 nm，且為實質上無毒的。術語「平均直徑」係指使用所謂的累積量演算法藉由動態雷射光散射(DLS)與資料分析所量測的顆粒之平均流體動力學直徑，其結果提供長度維度之所謂的Z平均值及無維度的多分散性指數(PI) (Koppel, D.,J. Chem. Phys. 57, 1972,第4814-4820頁, ISO 13321)。此處，顆粒之「平均直徑」、「直徑」或「尺寸」與Z平均值同義使用。In some aspects, the provided RNA molecules (e.g., saRNA, mRNA) can be formulated with LNPs. In some aspects, the lipid nanoparticles may or may not have a density of about 1 to 500 30, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 nm). In some aspects, the average diameter of the lipid nanoparticles is or is between about 30 nm and about 150 nm, about 40 nm and about 150 nm, about 50 nm and about 150 nm, about 60 nm and about 130 nm, about 70 nm and about 110 nm, about 70 nm and about 100 nm, about 80 nm and about 100 nm, about 90 nm and about 100 nm, about 70 to about 90 nm, about 80 nm and about 90 nm, about 70 nm and about 80 nm, or is at least, at most, just below, or between, inclusively or exclusively, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, The term "mean diameter" refers to the mean hydrodynamic diameter of the particles measured by dynamic laser light scattering (DLS) and data analysis using the so-called cumulative volume method, the result of which provides the so-called Z-average value in the length dimension and the polydispersity index (PI) without dimension (Koppel, D.,J. Chem. Phys . 57, 1972, pp. 4814-4820, ISO 13321). Herein, the "mean diameter", "diameter" or "size" of the particles are used synonymously with the Z-average value.
本文所描述之LNP可展現小於或小於約0.5、0.4、0.3或0.2或更小之多分散性指數。藉助於實例，LNP可展現或可不展現至少、至多、恰好以下或在以下之間(包括性或排他性)的多分散性指數：0.1、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.2、0.21、0.22、0.23、0.24、0.25、0.26、0.27、0.28、0.29、0.3、0.31、0.32、0.33、0.34、0.35、0.36、0.37、0.38、0.39、0.4、0.41、0.42、0.43、0.44、0.45、0.46、0.47、0.48、0.49或0.5。在一些態樣中，多分散性指數係基於動態光散射量測藉由「平均直徑」之定義中所提及之所謂的累積量分析來計算。在某些先決條件下，其可視為奈米顆粒總體之尺寸分佈之量測值。The LNPs described herein can exhibit a polydispersity index of less than or less than about 0.5, 0.4, 0.3, or 0.2, or less. By way of example, the LNPs may or may not exhibit a polydispersity index of at least, at most, just below, or between, inclusively or exclusively, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5. In some aspects, the polydispersity index is calculated based on dynamic light scattering measurements by so-called cumulative amount analysis as mentioned in the definition of "mean diameter". Under certain prerequisites, it can be regarded as a measure of the size distribution of the nanoparticle population.
在一些態樣中，本發明之LNP包含或不包含為或為約2:1至約30:1之N:P比，例如至少、至多、恰好以下或在以下之間(包括性或排他性)的N:P比：2:1、3:1、4:1、5:1、6:1、7:1、8:1、9:1、10:1、11:1、12:1、13:1、14:1、15:1、16:1、17:1、18:1、19:1、20:1、21:1、22:1、23:1、24:1、25:1、26:1、27:1、28:1、29:1或30:1。在一些態樣中，本發明之LNP包含為或為約6:1之N:P比。在一些態樣中，本發明之LNP包含為或為約3:1之N:P比。In some aspects, the LNPs of the invention comprise or do not comprise an N:P ratio of or about 2: 1 to about 30: 1, such as an N:P ratio of at least, at most, just below, or between (inclusive or exclusive) 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, 11: 1, 12: 1, 13: 1, 14: 1, 15: 1, 16: 1, 17: 1, 18: 1, 19: 1, 20: 1, 21: 1, 22: 1, 23: 1, 24: 1, 25: 1, 26: 1, 27: 1, 28: 1, 29: 1, or 30: 1. In some aspects, the LNPs of the invention comprise or about 6: 1. In some aspects, the LNPs of the invention comprise an N:P ratio of at or about 3:1.
在一些態樣中，本發明之LNP包含或不包含為或為約5:1至約100:1之陽離子脂質組分與RNA之wt/wt比，例如至少、至多、恰好以下或在以下之間(包括性或排他性)的陽離子脂質組分與RNA之wt/wt比：5:1、6:1、7:1、8:1、9:1、10:1、11:1、12:1、13:1、14:1、15:1、16:1、17:1、18:1、19:1、20:1、21:1、22:1、23:1、24:1、25:1、26:1、27:1、28:1、29:1、30:1、31:1、32:1、33:1、34:1、35:1、36:1、37:1、38:1、39:1、40:1、41:1、42:1、43:1、44:1、45:1、46:1、47:1、48:1、49:1、50:1、51:1、52:1、53:1、54:1、55:1、56:1、57:1、58:1、59:1、60:1、61:1、62:1、63:1、64:1、65:1、66:1、67:1、68:1、69:1、70:1、71:1、72:1、73:1、74:1、75:1、76:1、77:1、78:1、79:1、80:1、81:1、82:1、83:1、84:1、85:1、86:1、87:1、88:1、89:1、90:1、91:1、92:1、93:1、94:1、95:1、96:1、97:1、98:1、99:1或100:1。在一些態樣中，本發明之LNP包含為或為約20:1之可離子化陽離子脂質組分與RNA之wt/wt比。在一些態樣中，本發明之LNP包含為或為約10:1之可離子化陽離子脂質組分與RNA之wt/wt比。In some aspects, the LNPs of the present invention include or do not include a wt/wt ratio of a cationic lipid component to RNA of or about 5:1 to about 100:1, such as a wt/wt ratio of a cationic lipid component to RNA of at least, at most, just below, or between (inclusive or exclusive): 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1 1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1, 29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1, 41:1, 42:1, 43 :1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1, 53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1, 60:1, 61:1, 62:1, 63:1, 64:1, 65:1, 66:1, 67:1, 68:1, 69:1, 70:1, 71:1, 72:1 2:1, 73:1, 74:1, 75:1, 76:1, 77:1, 78:1, 79:1, 80:1, 81:1, 82:1, 83:1, 84:1, 85:1, 86:1, 87:1, 88:1, 89:1, 90:1, 91:1, 92:1, 93:1, 94:1, 95:1, 96:1, 97:1, 98:1, 99:1 or 100:1. In some aspects, the LNPs of the present invention comprise a wt/wt ratio of an ionizable cationic lipid component to RNA of or about 20:1. In some aspects, the LNPs of the present invention comprise a wt/wt ratio of an ionizable cationic lipid component to RNA of or about 10:1.
在某些態樣中，核酸(例如RNA分子) (當所提供LNP中存在時)在水溶液中對於用核酸酶降解具有抗性。在一些態樣中，LNP為靶向肝臟之脂質奈米顆粒。在一些態樣中，LNP為包含一或多種陽離子脂質(例如本文所描述之彼等者)的陽離子脂質奈米顆粒。在一些態樣中，陽離子LNP可包含至少一種陽離子脂質、至少一種聚合物結合脂質及至少一種輔助脂質(例如至少一種中性脂質)。In some aspects, nucleic acids (e.g., RNA molecules) (when present in provided LNPs) are resistant to degradation by nucleases in aqueous solutions. In some aspects, LNPs are lipid nanoparticles targeted to the liver. In some aspects, LNPs are cationic lipid nanoparticles comprising one or more cationic lipids (e.g., those described herein). In some aspects, cationic LNPs may comprise at least one cationic lipid, at least one polymer-bound lipid, and at least one auxiliary lipid (e.g., at least one neutral lipid).
在某些態樣中，RNA溶液及其脂質製劑混合物或組合物可具有至少、至多、恰好以下、在以下之間(包括性或排他性)或約以下的特定脂質、脂質類型或非脂質組分(諸如脂質樣材料及/或陽離子聚合物及/或佐劑)、抗原、肽、多肽、糖、核酸或本文所揭示或熟習此項技術者將已知之其他材料：1%、2%、3%、4% 5%、6%、7%、8%、9%、10%、11%、12%、13%、14%、15%、16%、17%、18%、19%、20%、21%、22%、23%、24%、25%、26%、27%、28%、29%、30%、31%、32%、33%、34%、35%、36%、37%、38%、39%、40%、41%、42%、43%、44%、45%、46%、47%、48%、49%、50%、51%、52%、53%、54%、55%、56%、57%、58%、59%、60%、61%、62%、63%、64%、65%、66%、67%、68%、69%、70%、71%、72%、73%、74%、75%、76%、77%、78%、79%、80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%。In certain aspects, RNA solutions and lipid preparation mixtures or compositions thereof may have at least, at most, just below, between (inclusive or exclusive) or about below a particular lipid, lipid type or non-lipid component (such as lipid-like material and/or cationic polymer and/or adjuvant), antigen, peptide, polypeptide, sugar, nucleic acid or other material disclosed herein or known to those skilled in the art: 1%, 2%, 3%, 4% 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.
本文所描述之LNP可使用此項技術中一般已知之組分、組合物及方法來產生，參見例如PCT/US2016/052352、PCT/US2016/068300、PCT/US2017/037551、PCT/US2015/027400、PCT/US2016/047406、PCT/US2016000129、PCT/US2016/014280、PCT/US2016/014280、PCT/US2017/038426、PCT/US2014/027077、PCT/US2014/055394、PCT/US2016/52117、PCT/US2012/069610、PCT/US2017/027492、PCT/US2016/059575及PCT/US2016/069491，其全部均以全文引用之方式併入本文中。用於製備LNP之方法之其他非限制性實例可見於例如WO 2022/032154中，其揭露內容以全文引用之方式併入本文中。The LNPs described herein can be produced using components, compositions, and methods generally known in the art, see, e.g., PCT/US2016/052352, PCT/US2016/068300, PCT/US2017/037551, PCT/US2015/027400, PCT/US2016/047406, PCT/US2016000129, PCT/US2016/014280, PCT/US2 016/014280, PCT/US2017/038426, PCT/US2014/027077, PCT/US2014/055394, PCT/US2016/52117, PCT/US2012/069610, PCT/US2017/027492, PCT/US2016/059575 and PCT/US2016/069491, all of which are incorporated herein by reference in their entirety. Other non-limiting examples of methods for preparing LNPs can be found, for example, in WO 2022/032154, the disclosure of which is incorporated herein by reference in its entirety.
舉例而言，製備LNP之方法可涉及自至少一種陽離子或陽離子可離子化脂質或脂質樣材料及/或至少一種陽離子聚合物獲得膠體，及將膠體與核酸混合，以獲得核酸顆粒。如本文所用，術語「膠體」係關於分散顆粒不會沉澱出來的一種均勻混合物。混合物中之不可溶顆粒為微小的，其粒徑在1與1000奈米之間。混合物可被稱為膠體或膠態懸浮液。有時，術語「膠體」僅係指混合物中之顆粒而非整個懸浮液。For example, a method for preparing LNPs may involve obtaining a colloid from at least one cationic or cationically ionizable lipid or lipid-like material and/or at least one cationic polymer, and mixing the colloid with a nucleic acid to obtain nucleic acid particles. As used herein, the term "colloid" refers to a uniform mixture of dispersed particles that do not precipitate out. The insoluble particles in the mixture are tiny, with a particle size between 1 and 1000 nanometers. The mixture may be referred to as a colloid or a colloidal suspension. Sometimes, the term "colloid" refers only to the particles in the mixture rather than the entire suspension.
為了製備包含至少一種陽離子或陽離子可離子化脂質或脂質樣材料及/或至少一種陽離子聚合物的膠體，在本文中可應用習知用於製備脂質體囊泡且經適當調適之方法。用於製備脂質體囊泡之最常用方法共用以下基本階段：(i)將脂質溶解於有機溶劑中，(ii)對所得溶液進行乾燥，及(iii)對乾燥脂質進行水合(使用多種水性介質)。In order to prepare colloids comprising at least one cationic or cationically ionizable lipid or lipid-like material and/or at least one cationic polymer, methods known for preparing liposomal vesicles and appropriately adapted can be applied herein. The most commonly used methods for preparing liposomal vesicles share the following basic stages: (i) dissolving the lipid in an organic solvent, (ii) drying the resulting solution, and (iii) hydrating the dried lipid (using a variety of aqueous media).
在膜水合方法中，首先將脂質溶解於適合的有機溶劑中且乾燥，在燒瓶底部得到薄膜。使用適當水性介質對所獲得脂質膜進行水合，產生脂質體分散液。此外，可包括額外縮小尺寸步驟。In the membrane hydration method, the lipid is first dissolved in a suitable organic solvent and dried to obtain a thin film at the bottom of a flask. The resulting lipid film is hydrated using a suitable aqueous medium to produce a liposome dispersion. In addition, an additional size reduction step may be included.
逆相蒸發為一種膜水合以用於製備脂質體囊泡之替代方法，該方法涉及在水相與含有脂質之有機相之間形成油包水乳劑。此混合物之短暫音波處理為系統均勻化所需。在減壓下移除有機相，得到乳白色凝膠，其隨後轉變成脂質體懸浮液。Reverse phase evaporation is an alternative method to membrane hydration for the preparation of liposome vesicles, which involves the formation of a water-in-oil emulsion between an aqueous phase and an organic phase containing lipids. Brief sonication of this mixture is required to homogenize the system. Removal of the organic phase under reduced pressure yields a milky gel, which is then converted into a liposome suspension.
術語「乙醇注射技術」係指其中包含脂質之乙醇溶液經由針頭快速注射至水溶液中的製程。此操作使脂質分散在整個溶液中且促進脂質結構形成，例如脂質囊泡形成，諸如脂質體形成。一般而言，本文所描述之RNA脂質複合物顆粒可藉由將RNA添加至膠態脂質體分散液中而獲得。使用乙醇注射技術，在一些態樣中，此類膠體脂質體分散液如下形成：在攪拌下將包含脂質(諸如陽離子脂質及額外脂質)之乙醇溶液注射至水溶液中。在一些態樣中，本文所描述之RNA脂質複合物顆粒可在無擠出步驟的情況下獲得。術語「擠出(extruding)」或「擠出(extrusion)」係指產生具有固定橫截面輪廓之顆粒。特定言之，其係指縮小顆粒之尺寸，其中迫使顆粒通過具有所限定孔之過濾器。The term "ethanol injection technique" refers to a process in which an ethanol solution containing lipids is rapidly injected into an aqueous solution through a needle. This operation disperses the lipids throughout the solution and promotes lipid structure formation, such as lipid vesicle formation, such as liposome formation. In general, the RNA lipid complex particles described herein can be obtained by adding RNA to a colloidal liposome dispersion. Using the ethanol injection technique, in some embodiments, such colloidal liposome dispersions are formed as follows: an ethanol solution containing lipids (such as cationic lipids and additional lipids) is injected into an aqueous solution under stirring. In some embodiments, the RNA lipid complex particles described herein can be obtained without an extrusion step. The term "extruding" or "extrusion" refers to the production of particles with a constant cross-sectional profile. Specifically, it refers to the reduction of the size of particles, wherein the particles are forced through a filter with defined pores.
根據本發明，亦可使用用於製備具有無有機溶劑特徵之膠體的其他方法。Other methods for preparing colloids having organic solvent-free characteristics may also be used according to the present invention.
在一些態樣中，LNP囊封RNA可藉由以下來製造：在使得觸發一或多種脂質組分之溶解性之急劇變化(其驅動脂質以LNP形式自組裝)的條件下，將本文所描述之RNA溶液(例如RNA產物溶液)與本文所描述之脂質製劑(包含例如至少一種陽離子脂質及視情況一或多種其他脂質組分於有機溶劑中)快速混合。在一些態樣中，適合的緩衝劑包含tris、組胺酸、檸檬酸鹽、乙酸鹽、磷酸鹽及/或丁二酸鹽。在一些態樣中，排除前述緩衝劑中之1、2、3者或更多者。液體調配物之pH與囊封劑(例如陽離子脂質)之pKa有關。酸化緩衝液之pH可比囊封劑(例如陽離子脂質)之pKa小至少一半pH標度，且最終緩衝液之pH可比囊封劑(例如陽離子脂質)之pKa大至少一半pH標度。在一些態樣中，選擇陽離子脂質之特性以使得顆粒之初始形成係藉由與核酸(例如RNA)之帶相反電荷之主鏈結合而發生。以此方式，圍繞核酸形成顆粒，在一些態樣中，其例如可產生比在核酸與脂質組分中之至少一者之間不存在相互作用的情況下達成的囊封效率高得多的囊封效率。在某些態樣中，核酸當存在於脂質奈米顆粒中時，其在水溶液中耐核酸酶降解。In some aspects, LNP encapsulated RNA can be made by rapidly mixing an RNA solution described herein (e.g., an RNA product solution) with a lipid preparation described herein (comprising, for example, at least one cationic lipid and, optionally, one or more other lipid components in an organic solvent) under conditions that trigger a dramatic change in the solubility of one or more lipid components, which drives the lipids to self-assemble in the form of LNPs. In some aspects, suitable buffers include tris, histidine, citrate, acetate, phosphate, and/or succinate. In some aspects, 1, 2, 3, or more of the aforementioned buffers are excluded. The pH of the liquid formulation is related to the pKa of the encapsulating agent (e.g., cationic lipid). The pH of the acidifying buffer may be at least half the pH scale less than the pKa of the encapsulating agent (e.g., cationic lipid), and the pH of the final buffer may be at least half the pH scale greater than the pKa of the encapsulating agent (e.g., cationic lipid). In some aspects, the properties of the cationic lipid are selected so that the initial formation of the particle occurs by binding to the oppositely charged backbone of the nucleic acid (e.g., RNA). In this way, particles are formed around the nucleic acid, which in some aspects, for example, can produce an encapsulation efficiency that is much higher than the encapsulation efficiency achieved in the absence of an interaction between the nucleic acid and at least one of the lipid components. In certain aspects, the nucleic acid, when present in the lipid nanoparticle, is resistant to nuclease degradation in aqueous solution.
包含核酸之脂質奈米顆粒及其製備方法揭示於例如美國專利公開案第2004/0142025號、第2007/0042031號及PCT公開案第WO 2013/016058號及第WO 2013/086373號中，該等文獻之全部揭示內容出於所有目的以全文引用之方式併入本文中。Lipid nanoparticles containing nucleic acids and methods for preparing the same are disclosed in, for example, U.S. Patent Publication Nos. 2004/0142025, 2007/0042031, and PCT Publication Nos. WO 2013/016058 and WO 2013/086373, the entire disclosures of which are incorporated herein by reference in their entirety for all purposes.
本文所描述之一些態樣係關於涉及超過一種，例如2、3、4、5、6或甚至更多種核酸物種(諸如RNA物種)的組合物、方法及用途。在LNP調配物中，有可能將各核酸物種分開調配為個別LNP調配物。在該情況下，各個別LNP調配物將包含一種核酸物種。個別LNP調配物可以分開的實體之形式存在，例如存在於分開的容器中。此類調配物可藉由分開提供各核酸物種(通常各自呈含核酸溶液之形式)以及允許形成LNP之適合陽離子或陽離子可離子化脂質或脂質樣材料及陽離子聚合物來獲得。各別顆粒將僅含有當形成顆粒時提供的特定核酸物種(個別顆粒調配物)。Some aspects described herein are related to compositions, methods and uses involving more than one, for example, 2, 3, 4, 5, 6 or even more nucleic acid species (such as RNA species). In LNP formulations, it is possible to separately formulate each nucleic acid species into individual LNP formulations. In this case, each individual LNP formulation will contain a nucleic acid species. Individual LNP formulations can exist in the form of separate entities, such as in separate containers. Such formulations can be obtained by separately providing each nucleic acid species (usually each in the form of a nucleic acid-containing solution) and suitable cations or cationically ionizable lipids or lipid-like materials and cationic polymers that allow the formation of LNPs. Individual particles will only contain the specific nucleic acid species (individual particle formulations) provided when forming particles.
在一些態樣中，諸如醫藥組合物之組合物包含超過一種個別LNP調配物。各別醫藥組合物被稱為混合LNP調配物。根據本發明之混合的LNP調配物可藉由分開地形成如上文所描述之個別LNP調配物，隨後將個別LNP調配物混合之步驟而獲得。藉由混合之步驟，可獲得包含含有核酸之LNP的混合群體的調配物。個別LNP群體可一起在一個容器中，其包含個別LNP調配物之混合群體。In some embodiments, a composition such as a pharmaceutical composition comprises more than one individual LNP formulation. Individual pharmaceutical compositions are referred to as mixed LNP formulations. Mixed LNP formulations according to the present invention can be obtained by separately forming individual LNP formulations as described above, followed by a step of mixing the individual LNP formulations. By the step of mixing, a formulation comprising a mixed population of LNPs containing nucleic acids can be obtained. Individual LNP populations can be together in one container, which comprises a mixed population of individual LNP formulations.
或者，有可能的是，不同核酸物種調配在一起作為組合的LNP調配物。此類調配物可藉由提供不同RNA物種之組合調配物(通常為組合溶液)以及允許形成LNP之適合陽離子或陽離子可離子化脂質或脂質樣材料及陽離子聚合物來獲得。與混合LNP調配物相反，組合LNP調配物將通常包含含有超過一種RNA物種之LNP。在組合LNP組合物中，不同RNA物種通常一起存在於單一顆粒中。Alternatively, it is possible that different nucleic acid species are formulated together as a combined LNP formulation. Such formulations can be obtained by providing a combined formulation (usually a combined solution) of different RNA species and suitable cations or cationically ionizable lipids or lipid-like materials and cationic polymers that allow the formation of LNPs. In contrast to mixed LNP formulations, combined LNP formulations will typically include LNPs containing more than one RNA species. In combined LNP compositions, different RNA species are typically present together in a single particle.
A.陽離子聚合材料鑒於聚合材料之高度化學可撓性，通常將聚合材料用於基於奈米顆粒之遞送。通常，使用陽離子材料使帶負電的核酸靜電凝聚成奈米顆粒。此等帶正電基團通常由質子化狀態在5.5與7.5之間的pH範圍內改變的胺組成，認為該改變會引起導致胞內體破裂之離子不平衡。聚合物(諸如聚-L-離胺酸、聚醯胺基胺、魚精蛋白及聚乙烯亞胺)以及天然存在之聚合物(諸如幾丁糖)全部均已應用於核酸遞送且適用作適用於本文中之一些態樣中的陽離子材料。另外，一些研究者已合成特定用於核酸遞送之聚合材料。特定言之，聚(P-胺基酯)由於其易於合成及可生物降解性而廣泛用於核酸遞送中。在一些態樣中，此類合成材料可適用作本文中之陽離子材料。A.Cationic Polymeric Materials Given their high chemical flexibility, polymeric materials are often used for nanoparticle-based delivery. Typically, cationic materials are used to electrostatically condense negatively charged nucleic acids into nanoparticles. These positively charged groups are typically composed of amines whose protonation states change in the pH range between 5.5 and 7.5, which is believed to cause an ionic imbalance that leads to endosomal disruption. Polymers such as poly-L-lysine, polyamidoamine, protamine, and polyethyleneimine, as well as naturally occurring polymers such as chitosan, have all been used for nucleic acid delivery and are suitable as cationic materials for use in some aspects herein. In addition, some researchers have synthesized polymeric materials specifically for nucleic acid delivery. In particular, poly(p-amino esters) are widely used in nucleic acid delivery due to their ease of synthesis and biodegradability. In some aspects, such synthetic materials can be suitable for use as cationic materials herein.
如本文所用，「聚合材料」具有其普通含義，例如包含藉由共價鍵連接的一或多個重複單元(單體)的分子結構。在一些態樣中，此類重複單元可全部均一致；或者，在一些情況下，聚合材料內可存在超過一種類型之重複單元。在一些情況下，聚合材料為以生物方式衍生的，例如生物聚合物，諸如蛋白質。在一些情況下，聚合材料中亦可存在額外部分，例如靶向部分，諸如本文所描述之彼等靶向部分。As used herein, "polymeric material" has its ordinary meaning, e.g., a molecular structure comprising one or more repeating units (monomers) connected by covalent bonds. In some aspects, such repeating units may all be identical; or, in some cases, more than one type of repeating unit may be present in a polymeric material. In some cases, the polymeric material is biologically derived, e.g., a biopolymer, such as a protein. In some cases, additional moieties, such as targeting moieties, such as those described herein, may also be present in the polymeric material.
熟習此項技術者應瞭解，當聚合物(或聚合部分)內存在超過一種類型之重複單元時，則聚合物(或聚合部分)稱為「共聚物」。在一些態樣中，根據本發明使用之聚合物(或聚合部分)可為共聚物。形成共聚物之重複單元可以任何方式排列。舉例而言，在一些態樣中，重複單元可以隨機次序排列；或者或另外，在一些態樣中，重複單元可以交替次序排列，或排列為「嵌段」共聚物，其例如包含一或多個各自含有第一重複單元(例如第一嵌段)之區域及一或多個各自含有第二重複單元(例如第二嵌段)之區域等。嵌段共聚物可具有兩種(二嵌段共聚物)、三種(三嵌段共聚物)或更多數目種不同嵌段。Those skilled in the art will appreciate that when more than one type of repeating unit is present in a polymer (or polymeric portion), then the polymer (or polymeric portion) is referred to as a "copolymer." In some aspects, the polymer (or polymeric portion) used in accordance with the present invention may be a copolymer. The repeating units forming the copolymer may be arranged in any manner. For example, in some aspects, the repeating units may be arranged in a random order; alternatively or additionally, in some aspects, the repeating units may be arranged in an alternating order, or arranged as a "block" copolymer, which, for example, comprises one or more regions each containing a first repeating unit (e.g., a first block) and one or more regions each containing a second repeating unit (e.g., a second block), etc. Block copolymers may have two (diblock copolymers), three (triblock copolymers), or more different blocks.
在某些態樣中，根據本發明使用之聚合材料為生物相容性的。生物相容性材料為在適中濃度下通常不導致顯著細胞死亡的材料。在某些態樣中，生物相容性材料為可生物降解的，例如能夠在生理環境內，諸如在身體內以化學及/或生物方式降解。在某些態樣中，聚合材料可為或包含魚精蛋白或聚伸烷基亞胺，特定言之魚精蛋白。In some aspects, the polymeric material used according to the present invention is biocompatible. A biocompatible material is a material that does not generally cause significant cell death at moderate concentrations. In some aspects, a biocompatible material is biodegradable, for example, capable of chemically and/or biologically degrading in a physiological environment, such as in the body. In some aspects, the polymeric material can be or include protamine or a polyalkylene imine, particularly protamine.
熟習此項技術者應瞭解，術語「魚精蛋白」常用以指具有相對較低分子量、富含精胺酸且發現在各種動物(如魚)之精子細胞中尤其與DNA而非體細胞組蛋白結合的各種強鹼性蛋白質中之任一者。特定言之，術語「魚精蛋白」常用以指存在於魚精子中的呈強鹼性、可溶於水、加熱不凝固且在水解時主要產生精胺酸的蛋白質。在純化形式中，其用於胰島素之長效調配物中且用以中和肝素之抗凝血劑作用。Those skilled in the art will appreciate that the term "protamine" is often used to refer to any of a variety of strongly alkaline proteins of relatively low molecular weight, rich in arginine, and found in sperm cells of various animals (such as fish) that are particularly associated with DNA rather than somatic histones. Specifically, the term "protamine" is often used to refer to a protein present in fish sperm that is strongly alkaline, soluble in water, does not coagulate when heated, and produces primarily arginine when hydrolyzed. In purified form, it is used in long-acting formulations of insulin and to neutralize the anticoagulant effect of heparin.
在一些態樣中，如本文所用，術語「魚精蛋白」係指獲自或來源於天然或生物學來源之魚精蛋白胺基酸序列，包括其片段及/或該胺基酸序列或其片段之多聚形式，以及為人工的且專門設計用於特定目的並且不能與天然或生物學來源分離之(合成)多肽。In some aspects, as used herein, the term "protamine" refers to a protamine amino acid sequence obtained or derived from a natural or biological source, including fragments thereof and/or polymeric forms of the amino acid sequence or its fragments, as well as (synthetic) polypeptides that are artificial and specifically designed for a specific purpose and cannot be separated from a natural or biological source.
在一些態樣中，聚伸烷基亞胺包含聚伸乙亞胺及/或聚伸丙亞胺。在一些態樣中，聚伸烷基亞胺為聚乙亞胺(PEI)。在一些態樣中，聚伸烷基亞胺為直鏈聚伸烷基亞胺，例如直鏈聚乙亞胺(PEI)。In some embodiments, the polyalkyleneimine comprises polyethyleneimine and/or polypropyleneimine. In some embodiments, the polyalkyleneimine is polyethyleneimine (PEI). In some embodiments, the polyalkyleneimine is a linear polyalkyleneimine, such as a linear polyethyleneimine (PEI).
預期用於本文中之陽離子材料(例如聚合材料，包括多陽離子聚合物)包括能夠靜電結合核酸的彼等陽離子材料。在一些態樣中，預期用於本文中之陽離子聚合材料包括例如藉由與核酸形成複合物或形成圍封或囊封核酸之囊泡而可與核酸結合之任何陽離子聚合材料。Cationic materials (e.g., polymeric materials, including polycationic polymers) contemplated for use herein include those cationic materials capable of electrostatically binding nucleic acids. In some aspects, cationic polymeric materials contemplated for use herein include any cationic polymeric material that can bind to nucleic acids, for example, by forming a complex with the nucleic acid or forming a vesicle that encloses or encapsulates the nucleic acid.
在一些態樣中，本文所描述之顆粒可包含除陽離子聚合物以外之聚合物，例如非陽離子聚合材料及/或陰離子聚合材料。陰離子及中性聚合材料在本文中統稱為非陽離子聚合材料。In some aspects, the particles described herein may include polymers other than cationic polymers, such as non-cationic polymeric materials and/or anionic polymeric materials. Anionic and neutral polymeric materials are collectively referred to herein as non-cationic polymeric materials.
B.脂質及脂質樣材料術語「脂質」及「脂質樣材料」在本文中用於指包含一或多種疏水性部分或基團以及視情況一或多種親水性部分或基團之分子。根據本發明，脂質及脂質樣材料可為陽離子、陰離子或中性的。在所選pH下，中性脂質或脂質樣材料以不帶電或中性兩性離子形式存在。B.Lipids and lipid-like materials The terms "lipid" and "lipid-like material" are used herein to refer to molecules comprising one or more hydrophobic parts or groups and, optionally, one or more hydrophilic parts or groups. According to the present invention, lipids and lipid-like materials can be cationic, anionic or neutral. At a selected pH, neutral lipids or lipid-like materials exist in an uncharged or neutral zwitterionic form.
術語「脂質」係指特徵為不可溶於水中但可溶於許多有機溶劑中的一群有機化合物。通常，脂質可分為八類：脂肪酸及其衍生物(包括三酸甘油酯、二酸甘油酯、單酸甘油酯及磷脂)、甘油脂、甘油磷脂、鞘脂、醣脂、聚酮化合物、固醇脂質以及含固醇代謝物(諸如膽固醇)及異戊烯醇脂質。脂肪酸之實例包括但不限於脂肪酯及脂肪醯胺。甘油脂之實例包括但不限於糖基甘油(glycosylglycerol)及甘油磷脂(例如磷脂醯膽鹼、磷脂醯乙醇胺、磷脂醯絲胺酸)。鞘脂之實例包括但不限於神經醯胺鞘磷脂(phosphosphingolipid) (例如鞘磷脂(sphingomyelin)、磷酸膽鹼)及醣神經鞘脂質(例如腦苷脂、神經節苷脂)。固醇脂質之實例包括但不限於膽固醇及其衍生物以及生育酚及其衍生物。在一些態樣中，該等脂質中之1、2、3、4、5者或更多者可排除在本發明之LNP外。The term "lipid" refers to a group of organic compounds that are characterized by being insoluble in water but soluble in many organic solvents. Generally, lipids can be divided into eight categories: fatty acids and their derivatives (including triglycerides, diglycerides, monoglycerides and phospholipids), glycerolipids, glycerophospholipids, sphingolipids, carbohydrates, polyketides, sterol lipids, and lipids containing sterol metabolites (such as cholesterol) and prenols. Examples of fatty acids include, but are not limited to, fatty esters and fatty amides. Examples of glycerolipids include, but are not limited to, glycosylglycerol and glycerophospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine). Examples of sphingolipids include, but are not limited to, phosphosphingolipids (e.g., sphingomyelin, phosphocholine) and glycosphingolipids (e.g., cerebrosides, gangliosides). Examples of sterol lipids include, but are not limited to, cholesterol and its derivatives and tocopherol and its derivatives. In some aspects, 1, 2, 3, 4, 5 or more of these lipids may be excluded from the LNP of the present invention.
術語「脂質樣材料」、「脂質樣化合物」或「脂質樣分子」係指結構上及/或功能上與脂質有關但在嚴格意義上可能不視為脂質之物質。舉例而言，該術語包括在其存在於水性環境中之囊泡、多層/單層脂質體或膜中時能夠形成兩親性層的化合物，且包括界面活性劑或具有親水性及疏水性部分兩者之合成化合物。一般言之，該術語係指包含具有不同結構組織之親水性及疏水性部分的分子，其可能與脂質類似或可能不類似。The term "lipid-like material", "lipid-like compound" or "lipid-like molecule" refers to substances that are structurally and/or functionally related to lipids but may not be considered lipids in the strict sense. For example, the term includes compounds that are capable of forming an amphiphilic layer when they are present in a vesicle, multilamellar/unilamellar liposome or membrane in an aqueous environment, and includes surfactants or synthetic compounds that have both hydrophilic and hydrophobic parts. In general, the term refers to molecules that contain hydrophilic and hydrophobic parts with different structural organizations, which may or may not be similar to lipids.
在一些態樣中，RNA溶液及其脂質製劑混合物或組合物可包含陽離子脂質、中性脂質、膽固醇及/或聚合物(例如聚乙二醇)結合脂質，其形成包圍RNA分子之脂質奈米顆粒。因此，在一些態樣中，LNP可包含陽離子脂質及一或多種賦形劑，例如一或多種中性脂質、帶電脂質、類固醇或類固醇類似物(例如膽固醇)、聚合物結合脂質(例如PEG-脂質)或其組合。在一些態樣中，前述賦形劑中之1、2、3者或更多者可排除在本發明之LNP外。在一些態樣中，脂質以有效形成脂質奈米顆粒且遞送治療劑(例如RNA分子)用於治療所關注之特定疾病或病狀(例如與RSV相關之彼等者)之量存在於組合物中。在一些態樣中，LNP包涵或囊封核酸分子。In some aspects, the RNA solution and its lipid preparation mixture or composition may include cationic lipids, neutral lipids, cholesterol and/or polymer (e.g., polyethylene glycol) bound lipids, which form lipid nanoparticles surrounding RNA molecules. Therefore, in some aspects, LNPs may include cationic lipids and one or more excipients, such as one or more neutral lipids, charged lipids, steroids or steroid analogs (e.g., cholesterol), polymer bound lipids (e.g., PEG-lipids), or combinations thereof. In some aspects, 1, 2, 3 or more of the aforementioned excipients may be excluded from the LNPs of the present invention. In some aspects, the lipid is present in the composition in an amount effective to form lipid nanoparticles and deliver a therapeutic agent (e.g., an RNA molecule) for treating a specific disease or condition of interest (e.g., those associated with RSV). In some aspects, the LNP contains or encapsulates a nucleic acid molecule.
i.陽離子脂質陽離子或陽離子可離子化脂質或脂質樣材料係指能夠帶正電且能夠靜電結合核酸之脂質或脂質樣材料。如本文所用，「陽離子脂質」或「陽離子脂質樣材料」係指具有淨正電荷的脂質或脂質樣材料。陽離子脂質或脂質樣材料藉由靜電相互作用結合帶負電之核酸。一般而言，陽離子脂質具有親脂性部分，諸如固醇、醯基鏈、二醯基或更多醯基鏈，且脂質之頭部基團通常攜帶正電荷。例示性陽離子脂質包括攜帶正電荷的一或多個胺基。陽離子脂質可囊封帶負電之RNA。i.Cationic lipids Cationic or cationically ionizable lipids or lipid-like materials refer to lipids or lipid-like materials that are able to carry a positive charge and are able to electrostatically bind to nucleic acids. As used herein, "cationic lipids" or "cationic lipid-like materials" refer to lipids or lipid-like materials with a net positive charge. Cationic lipids or lipid-like materials bind to negatively charged nucleic acids through electrostatic interactions. Generally speaking, cationic lipids have a lipophilic portion, such as a sterol, an acyl chain, a diacyl group or more acyl chains, and the head group of the lipid usually carries a positive charge. Exemplary cationic lipids include one or more amine groups carrying a positive charge. Cationic lipids can encapsulate negatively charged RNA.
在一些態樣中，陽離子脂質為可離子化的，使得視pH而定，其可以帶正電或中性形式存在。陽離子脂質之離子化影響脂質奈米顆粒在不同pH條件下之表面電荷。不希望受理論所束縛，認為此可離子化行為經由幫助胞內體逃逸以及與在生理pH下保持陽離子性之顆粒相比降低毒性來促進功效。出於本發明之目的，除非與情形相矛盾，否則術語「陽離子脂質」或「陽離子脂質樣材料」包含此類「陽離子可離子化」脂質或脂質樣材料。In some aspects, the cationic lipid is ionizable, so that it can exist in a positively charged or neutral form depending on the pH. The ionization of the cationic lipid affects the surface charge of the lipid nanoparticles under different pH conditions. Without wishing to be bound by theory, it is believed that this ionizable behavior promotes efficacy by helping intracellular escaping and reducing toxicity compared to particles that maintain cationicity at physiological pH. For the purposes of the present invention, unless contradictory to the situation, the term "cationic lipid" or "cationic lipid-like material" includes such "cationic ionizable" lipids or lipid-like materials.
在一些態樣中，陽離子脂質可包含或包含約10 mol%至約100 mol%、約20 mol%至約100 mol%、約30 mol%至約100 mol%、約40 mol%至約100 mol%、或約50 mol%至約100 mol%之顆粒中存在的總脂質。在一些態樣中，陽離子脂質可為或可不為至少、至多、恰好以下或在以下之間(包括性或排他性)的顆粒中存在的總脂質：10 mol%、20 mol%、30 mol%、40 mol%、50 mol%、60 mol%、70 mol%、80 mol%、90 mol%或100 mol%，或其中可導出的任何範圍或值。In some aspects, the cationic lipid may comprise or comprise about 10 mol% to about 100 mol%, about 20 mol% to about 100 mol%, about 30 mol% to about 100 mol%, about 40 mol% to about 100 mol%, or about 50 mol% to about 100 mol% of the total lipid present in the particle. In some aspects, the cationic lipid may or may not be at least, at most, just below, or between (inclusive or exclusive) 10 mol%, 20 mol%, 30 mol%, 40 mol%, 50 mol%, 60 mol%, 70 mol%, 80 mol%, 90 mol% or 100 mol%, or any range or value derivable therein.
陽離子脂質之實例包括但不限於：((4-羥丁基)氮二基)雙(己烷-6,1-二基)雙(2-己基癸酸酯)、1,2-二油醯基-3-三甲基銨丙烷(DOTAP)、N,N-二甲基-2,3-二油醯氧基丙胺(DODMA)、1,2-二-O-十八碳烯基-3-三甲基銨丙烷(DOTMA)、3-(N-(N',N'-二甲基胺基乙烷)-胺甲醯基)膽固醇(DC-Chol)、二甲基二(十八烷基)銨(DDAB)、1,2-二油醯基-3-二甲基銨-丙烷(DODAP)、1,2-二醯氧基-3-二甲基銨丙烷、1,2-二烷氧基-3-二甲基銨丙烷；氯化二(十八基)二甲基銨(DODAC)、1,2-二硬脂基氧基-N,N-二甲基-3-胺基丙烷(DSDMA)、2,3-二(十四烷氧基)丙基-(2-羥乙基)-二甲基銨氮(DMRIE)、1,2-二肉豆蔻醯基-sn-甘油基-3-乙基磷酸膽鹼(DMEPC)、1,2-二肉豆蔻醯基-3-三甲基銨丙烷(DMTAP)、溴化1,2-二油醯基氧基丙基-3-二甲基-羥乙基銨(DORIE)、2,3-二油醯氧基-N-[2(精胺甲醯胺)乙基]-N,N-二甲基-1-丙醯胺鎓(propanamium)三氟乙酸鹽(DOSPA)、1,2-二亞油氧基-N,N-二甲基胺基丙烷(DLinDMA)、1,2-二次亞麻氧基-N,N-二甲基胺基丙烷(DLenDMA)、雙十八烷基醯胺基甘胺醯基精胺(DOGS)、3-二甲胺基-2-(膽固醇-5-en-3-β-氧基丁-4-氧基)-1-(順式,順式-9,12-十八碳二烯氧基)丙烷(CLinDMA)、2-[5'-(膽固醇-5-烯-3-β-氧基)-3'-氧雜戊烯氧基)-3-二甲基-1-(順式,順式-9',12'-十八碳二烯氧基)丙烷(CpLinDMA)、N,N-二甲基-3,4-二油烯基氧基苯甲胺(DMOBA)、1,2-N,N'-二油烯基胺甲醯基-3-二甲基胺基丙烷(DOcarbDAP)、2,3-二亞油醯基氧基-N,N-二甲基丙胺(DLinDAP)、1,2-N,N'-二亞油基胺甲醯基-3-二甲基胺基丙烷(DLincarbDAP)、1,2-二亞油醯基胺甲醯基-3-二甲基胺基丙烷(DLinCDAP)、2,2-二亞油基-4-二甲胺基甲基-[1,3]-二氧雜環戊烷(DLin-K-DMA)、2,2-二亞油基-4-二甲基胺基乙基-[1,3]-二氧雜環戊烷(DLin-K-XTC2-DMA)、2,2-二亞油基-4-(2-二甲基胺基乙基)-[1,3]-二氧雜環戊烷(DLin-KC2-DMA)、三十七碳-6,9,28,31-四烯-19-基-4-(二甲胺基)丁酸酯(DLin-MC3-DMA)、溴化N-(2-羥乙基)-N,N-二甲基-2,3-雙(十四烷氧基)-1-丙胺鎓(DMRIE)、(±)-溴化N-(3-胺基丙基)-N,N-二甲基-2,3-雙(順式-9-十四碳烯基氧基)-1-丙胺鎓(GAP-DMORIE)、(±)-溴化N-(3-胺基丙基)-N,N-二甲基-2,3-雙(十二烷氧基)-1-丙胺鎓(GAP-DLRIE)、(±)-溴化N-(3-胺基丙基)-N,N-二甲基-2,3-雙(十四烷氧基)-1-丙胺鎓(GAP-DMRIE)、溴化N-(2-胺基乙基)-N,N-二甲基-2,3-雙(十四烷氧基)-1-丙胺鎓(bAE-DMRIE)、N-(4-羧基苯甲基)-N,N-二甲基-2,3-雙(油醯基氧基)丙-1-銨(DOBAQ)、2-({8-[(3b)-膽固醇-5-烯-3-基氧基]辛基}氧基)-N,N-二甲基-3-[(9Z,12Z)-十八碳-9,12-二烯-1-基氧基]丙-1-胺(Octyl-CLinDMA)、1,2-二肉豆蔻醯基-3-二甲基銨-丙烷(DMDAP)、1,2-二棕櫚醯基-3-二甲基銨-丙烷(DPDAP)、N1-[2-((1S)-1-[(3-胺基丙基)胺基]-4-[二(3-胺基-丙基)胺基]丁基甲醯胺基)乙基]-3,4-二[油氧基]-苯甲醯胺(MVL5)、1,2-二油醯基-sn-甘油基-3-乙基磷酸膽鹼(DOEPC)、溴化2,3-雙(十二烷氧基)-N-(2-羥乙基)-N,N-二甲基丙-1-銨(DLRIE)、溴化N-(2-胺基乙基)-N,N-二甲基-2,3-雙(十四烷氧基)丙-1-銨(DMORIE)、8,8'-((((2(二甲胺基)乙基)硫)羰基)氮二基)二辛酸二((Z)-壬-2-烯-1-基酯)(ATX)、N,N-二甲基-2,3-雙(十二烷氧基)丙-1-胺(DLDMA)、N,N-二甲基-2,3-雙(十四烷氧基)丙-1-胺(DMDMA)、二((Z)-壬-2-烯-1-基)-9-((4-(二甲基胺基丁醯基)氧基)十七烷二酸酯(L319)、N-十二基-3-((2-十二烷基胺甲醯基-乙基)-{2-[(2-十二烷基胺甲醯基-乙基)-2-{(2-十二烷基胺甲醯基-乙基)-[2-(2-十二烷基胺甲醯基-乙胺基)-乙基]-胺基}-乙胺基)丙醯胺(類脂質98N12-5)、1-[2-[雙(2-羥基十二烷基)胺基]乙基-[2-[4-[2-[雙(2羥基十二烷基)胺基]乙基]哌𠯤-1-基]乙基]胺基]十二烷-2-醇(類脂質02-200)；C12-200；或8-((2-羥乙基)(6-側氧基-6-(十一烷氧基)己基)胺基)辛酸十七烷-9-基酯(SM-102)。在一些態樣中，前述陽離子脂質中之1、2、3、4、5者或更多者可排除在本發明之LNP外。Examples of cationic lipids include, but are not limited to: ((4-hydroxybutyl) azodiyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate), 1,2-dioleyl-3-trimethylammonium propane (DOTAP), N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3-(N-( N',N'-dimethylaminoethane)-aminoformyl) cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB), 1,2-dioleyl-3-dimethylammonium-propane (DODAP), 1,2-diacyloxy-3-dimethylammonium propane, 1,2-dialkoxy-3-dimethylammonium propane; dioctadecyldimethylammonium chloride (DODAC), 1,2-diol DSDMA, 2,3-di(tetradecyloxy)propyl-(2-hydroxyethyl)-dimethylammonium nitrogen (DMRIE), 1,2-dimyristyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristyl-3-trimethylammonium propane (DMTAP), 1,2-dioleyloxypropyl bromide -3-Dimethyl-hydroxyethylammonium (DORIE), 2,3-dioleyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propanamium trifluoroacetate (DOSPA), 1,2-dilinoleyl-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyl-N,N-dimethylaminopropane (D LenDMA), dioctadecylaminoglycinylspermine (DOGS), 3-dimethylamino-2-(cholesterol-5-en-3-β-oxybut-4-oxy)-1-(cis,cis-9,12-octadecadienyloxy)propane (CLinDMA), 2-[5'-(cholesterol-5-en-3-β-oxy)-3'-oxopentenyloxy)-3-dimethyl-1- (cis,cis-9',12'-octadecadienyloxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylaminomethyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N , N'-dilinoleylaminomethyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoleylaminomethyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane 2-(2-Dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 6,9,28,31-triacon ... Methyl-2,3-bis(tetradecyloxy)-1-propylamine (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propylamine bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propylamine bromide (GA P-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propylamine bromide (GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propylamine bromide (bAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3- Bis(oleyloxy)propan-1-ammonium (DOBAQ), 2-({8-[(3b)-cholesterol-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadec-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA), 1,2-dimyristoyl-3-dimethylammonium-propane (DMDAP ）、1,2-dipalmitoyl-3-dimethylammonium-propane (DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyl]-benzamide (MVL5), 1,2-dioleyl-sn-glycero-3-ethylphosphocholine (DOEPC), bromide 2,3-Bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropane-1-ammonium (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propane-1-ammonium bromide (DMORIE), 8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)nitrogendiyl)dioctanoate ((Z)-non-2-en-1-yl) (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutyryl)oxy)heptadecanedioate (L319), N-dodecyl-3-((2-dodecylaminomethyl)propan 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]-[2-[(2-dodecylaminomethyl-ethyl)-2-{(2-dodecylaminomethyl-ethyl)-[2-(2-dodecylaminomethyl-ethyl)-ethyl]-amino}-ethylamino)propionamide (lipid 98N12-5), 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]- []ethyl]piperidin-1-yl]ethyl]amino]dodecan-2-ol (lipid 02-200); C12-200; or 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoic acid heptadecan-9-yl ester (SM-102). In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned cationic lipids can be excluded from the LNP of the present invention.
在一些態樣中，本發明之可離子化陽離子脂質包含式(I)化合物：， 或其醫藥學上可接受之鹽、互變異構物、前藥或立體異構物，其中： R1為C5-30烷基、C5-20烯基、-R*YR"、-YR"或-R"M'R'； R2及R3獨立地為H、C1-14烷基、C2-14烯基、-R*YR"、-YR"或-R*OR"，及/或R2及R3與其所連接之原子一起形成雜環或碳環； R4為C3-6碳環、-(CH2)nQ、-(CH2)nCHQR、-CHQR、-CQ(R)2或未經取代之C1-6烷基，其中Q為碳環、雜環、-OR、-O(CH2)nN(R)2、-C(O)OR、-OC(O)R、-CX3、-CX2H、-CXH2、-CN、-N(R)2、-C(O)N(R)2、-N(R)C(O)R、-N(R)S(O)2R、-N(R)C(O)N(R)2、-N(R)C(S)N(R)2、-N(R)R8、-O(CH2)nOR、-N(R)C(=NR9)N(R)2、-N(R)C(=CHR9)N(R)2、-OC(O)N(R)2、-N(R)C(O)OR、-N(OR)C(O)R、-N(OR)S(O)2R、-N(OR)C(O)OR、-N(OR)C(O)N(R)2、-N(OR)C(S)N(R)2、-N(OR)C(=NR9)N(R)2、-N(OR)C(=CHR9)N(R)2、-C(=NR9)N(R)2、-C(=NR9)R、-C(O)N(R)OR或-C(R)N(R)2C(O)OR，及/或各n獨立地為1、2、3、4或5； 各R5獨立地為C1-3烷基、C2-3烯基或H； 各R6獨立地為C1-3烷基、C2-3烯基或H； M及M'獨立地為-C(O)O-、-OC(O)-、-C(O)N(R')-、-N(R')C(O)-、-C(O)-、-C(S)-、-C(S)S-、-SC(S)-、-CH(OH)-、-P(O)(OR')O-、-S(O)2-、-S-S-、芳基或雜芳基； R7為C1-3烷基、C2-3烯基或H； R8為C3-6碳環或雜環； R9為H、CN、NO2、C1-6烷基、-OR、-S(O)2R、-S(O)2N(R)2、C2-6烯基、C3-6碳環或雜環； 各R為C1-3烷基、C2-3烯基或H； 各R'為C1-18烷基、C2-18烯基、-R*YR"、-YR"或H； 各R"為C3-14烷基或C3-14烯基； 各R*獨立地為C1-12烷基或C2-12烯基； 各Y獨立地為C3-6碳環； 各X獨立地為F、Cl、Br或I；及 m為5、6、7、8、9、10、11、12或13。In some embodiments, the ionizable cationic lipid of the present invention comprises a compound of formula (I): , or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: R1 is C5-30 alkyl, C5-20 alkenyl, -R*YR", -YR" or -R"M'R'; R2 and R3 are independently H, C1-14 alkyl, C2-14 alkenyl, -R*YR", -YR" or -R*OR", and/or R2 and R3 together with the atoms to which they are attached form a heterocyclic or carbocyclic ring; R4 is C3-6 carbocyclic ring, -(CH2 )n Q, -(CH2 )n CHQR, -CHQR, -CQ(R)2 or unsubstituted C1-6 alkyl, wherein Q is a carbocyclic ring, a heterocyclic ring, -OR, -O(CH2 )n N(R)2 , -C(O)OR, -OC(O)R, -CX3 , -CX2 H , -CXH2 , -CN, -N(R)2 , -C(O)N(R)2 , -N(R)C(O)R, -N(R)S(O)2 R, -N(R)C(O)N(R)2 , -N(R)C(S)N(R)2 , -N(R)R8 , -O(CH2 )n OR, -N(R)C(=NR9 )N(R)2 , -N(R)C(=CHR9 )N(R)2 , -OC(O)N(R)2 , -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2 R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2 , -N(OR)C(S)N(R)2 , -N(OR)C(=NR9 )N(R)2 , -N(OR)C(=CHR9 )N(R)2 , -C(=NR9 )N(R)2 , -C(=NR9 )R, -C(O)N(R)OR or -C(R)N(R)2 C(O)OR, and/or each n is independently 1, 2, 3, 4 or 5; each R5 is independently C1-3 alkyl, C2-3 alkenyl or H; each R6 is independently C1-3 alkyl, C2-3 alkenyl or H; M and M' are independently -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2 -, -SS-, aryl or heteroaryl; R7 is C1-3 alkyl, C2-3 alkenyl or H; R8 is C3-6 carbocyclic or heterocyclic; R9 is H, CN, NO2 , C1-6 alkyl, -OR, -S(O)2 R, -S(O)2 N(R)2 , C2-6 alkenyl, C3-6 carbocyclic or heterocyclic; each R is C1-3 alkyl, C 2-3 alkenyl, C 3-6 carbocyclic or heterocyclic. each R' isC1-18 alkyl, C2-18 alkenyl, -R*YR", -YR" or H; each R" is C3-14 alkyl or C3-14 alkenyl; each R* is independently C1-12 alkyl or C2-12 alkenyl; each Y is independently C3-6 carbocycle; each X is independently F, Cl, Br or I; and m is 5, 6, 7, 8, 9, 10, 11, 12 or 13.
在一些態樣中，式(I)化合物之子集包括如下之彼等者：其中當R4為-(CH2)nQ、-(CH2)nCHQR、-CHQR或-CQ(R)2時，則(i)當n為1、2、3、4、或5時，Q不為-N(R)2，或(ii)當n為1或2時，Q不為5、6或7員雜環烷基。In some aspects, a subset of compounds of formula (I) include those wherein when R4 is -(CH2 )n Q, -(CH2 )n CHQR, -CHQR or -CQ(R)2 , then (i) when n is 1, 2, 3, 4, or 5, Q is not -N(R)2 , or (ii) when n is 1 or 2, Q is not a 5-, 6- or 7-membered heterocycloalkyl.
在一些態樣中，式(I)化合物之另一子集包括如下之彼等者：其中R1為C5-30烷基、C5-20烯基、-R*YR"、-YR"或-R"M'R'； R2及R3獨立地為H、C1-14烷基、C2-14烯基、-R*YR"、-YR"或-R*OR"，及/或R2及R3與其所連接之原子一起形成雜環或碳環； R4為C3-6碳環、-(CH2)nQ、-(CH2)nCHQR、-CHQR、-CQ(R)2或未經取代之C1-6烷基，其中Q為C3-6碳環、具有一或多個包含N、O或S之雜原子的5至14員雜芳基、-OR、-O(CH2)nN(R)2、-C(O)OR、-OC(O)R、-CX3、-CX2H、-CXH2、-CN、-C(O)N(R)2、-N(R)C(O)R、-N(R)S(O)2R、-N(R)C(O)N(R)2、-N(R)C(S)N(R)2、-CRN(R)2C(O)OR、-N(R)R8、-O(CH2)nOR、-N(R)C(=NR9)N(R)2、-N(R)C(=CHR9)N(R)2、-OC(O)N(R)2、-N(R)C(O)OR、-N(OR)C(O)R、-N(OR)S(O)2R、-N(OR)C(O)OR、-N(OR)C(O)N(R)2、-N(OR)C(S)N(R)2、-N(OR)C(=NR9)N(R)2、-N(OR)C(=CHR9)N(R)2、-C(=NR9)N(R)2、-C(=NR9)R、-C(O)N(R)OR或具有一或多個包含N、O及S之雜原子的5至14員雜環烷基(其經一或多個包含側氧基(=O)、OH、胺基、單或二烷基胺基、或C1-3烷基之取代基取代)，及/或各n獨立地為1、2、3、4或5； 各R5獨立地為C1-3烷基、C2-3烯基或H； 各R6獨立地為C1-3烷基、C2-3烯基或H； M及M'獨立地為-C(O)O-、-OC(O)-、-C(O)N(R')-、-N(R')C(O)-、-C(O)-、-C(S)-、-C(S)S-、-SC(S)-、-CH(OH)-、-P(O)(OR')O-、-S(O)2-、-S-S-、芳基或雜芳基； R7為C1-3烷基、C2-3烯基或H； R8為C3-6碳環或雜環； R9為H、CN、NO2、C1-6烷基、-OR、-S(O)2R、-S(O)2N(R)2、C2-6烯基、C3-6碳環或雜環； 各R獨立地為C1-3烷基、C2-3烯基或H； 各R'獨立地為C1-18烷基、C2-18烯基、-R* YR"、-YR"或H； 各R"獨立地為C3-14烷基或C3-14烯基； 各R*獨立地為C1-12烷基或C2-12烯基； 各Y獨立地為C3-6碳環； 各X獨立地為F、Cl、Br或I；及 m為5、6、7、8、9、10、11、12或13；及/或其醫藥學上可接受之鹽、互變異構物、前藥或立體異構物。In some embodiments, another subset of compounds of formula (I) includes those wherein R1 is C5-30 alkyl, C5-20 alkenyl, -R*YR", -YR" or -R"M'R'; R2 and R3 are independently H, C1-14 alkyl, C2-14 alkenyl, -R*YR", -YR" or -R*OR", and/or R2 and R3 together with the atoms to which they are attached form a heterocyclic or carbocyclic ring; R4 is C3-6 carbocyclic ring, -(CH2 )n Q, -(CH2 )n CHQR, -CHQR, -CQ(R)2 or unsubstituted C1-6 alkyl, wherein Q is C3-6 carbocyclic ring, 5-14 membered heteroaryl having one or more heteroatoms including N, O or S, -OR, -O(CH2 )n N(R)2 , -C(O)OR, -OC(O)R, -CX3 , -CX2 H, -CXH2 , -CN, -C(O)N(R)2 , -N(R)C(O)R, -N(R)S(O)2 R, -N(R)C(O)N(R)2 , -N(R)C(S)N(R)2 , -CRN(R)2 C(O)OR, -N(R)R8 , -O(CH2 )n OR, -N(R)C(=NR9 )N(R)2 , -N(R)C(=CHR9 )N(R)2 -OC(O)N(R)2 , -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R , -N(OR)C(O)OR, -N(OR)C(O)N(R)2 , -N(OR)C(S)N(R)2 , -N(OR)C(=NR9 )N(R)2 , -N(OR)C(=CHR9 )N(R)2 , -C(=NR9 )N(R)2 , -C(=NR9 )R, -C(O)N(R)OR, or a 5- to 14-membered heterocycloalkyl group having one or more heteroatoms comprising N, O and S (which is terminated by one or more pendant oxy groups (=O), OH, amino groups, mono- or dialkylamino groups, or C each R5 is independently C1-3 alkyl, C 2-3 alkenyl or H; each R6 is independently C1-3 alkyl, C2-3 alkenyl or H; M and M' are independently -C(O) O-,-OC (O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2 -, -SS-, aryl or heteroaryl; R7 is C1-3 alkyl, C2-3 alkenyl or H; R8 is C3-6 carbocyclic or heterocyclic;9 is H, CN, NO2 , C1-6 alkyl, -OR, -S(O)2 R, -S(O)2 N(R)2 , C2-6 alkenyl, C3-6 carbocyclic ring or heterocyclic ring; each R is independently a C1-3 alkyl, C2-3 alkenyl or H; each R' is independently a C1-18 alkyl, C2-18 alkenyl, -R*YR", -YR" or H; each R" is independently a C3-14 alkyl or C3-14 alkenyl; each R* is independently a C1-12 alkyl or C2-12 alkenyl; each Y is independently a C3-6 carbocyclic ring; Each X is independently F, Cl, Br or I; and m is 5, 6, 7, 8, 9, 10, 11, 12 or 13; and/or pharmaceutically acceptable salts, tautomers, prodrugs or stereoisomers thereof.
在一些態樣中，式(I)化合物之另一子集包括如下之彼等者，其中： R1為C5-30烷基、C5-20烯基、-R*YR"、-YR"或-R"M'R'； R2及R3獨立地為H、C1-14烷基、C2-14烯基、-R*YR"、-YR"或-R*OR"，及/或R2及R3與其所連接之原子一起形成雜環或碳環； R4為C3-6碳環、-(CH2)nQ、-(CH2)nCHQR、-CHQR、-CQ(R)2或未經取代之C1-6烷基，其中Q為C3-6碳環、具有一或多個包含N、O或S之雜原子的5至14員雜環、-OR、-O(CH2)nN(R)2、-C(O)OR、-OC(O)R、-CX3、-CX2H、-CXH2、-CN、-C(O)N(R)2、-N(R)C(O)R、-N(R)S(O)2R、-N(R)C(O)N(R)2、-N(R)C(S)N(R)2、-CRN(R)2C(O)OR、-N(R)R8、-O(CH2)nOR、-N(R)C(=NR9)N(R)2、-N(R)C(=CHR9)N(R)2、-OC(O)N(R)2、-N(R)C(O)OR、-N(OR)C(O)R、-N(OR)S(O)2R、-N(OR)C(O)OR、-N(OR)C(O)N(R)2、-N(OR)C(S)N(R)2、-N(OR)C(=NR9)N(R)2、-N(OR)C(=CHR9)N(R)2、-C(=NR9)R、-C(O)N(R)OR或-C(=NR9)N(R)2，及/或各n獨立地為1、2、3、4或5；及/或當Q為5至14員雜環時且(i) R4為-(CH2)nQ，其中n為1或2，或(ii) R4為-(CH2)nCHQR，其中n為1，或(iii) R4為-CHQR及-CQ(R)2，則Q為5至14員雜芳基或8至14員雜環烷基； 各R5獨立地為C1-3烷基、C2-3烯基或H； 各R6獨立地為C1-3烷基、C2-3烯基或H； M及M'獨立地為-C(O)O-、-OC(O)-、-C(O)N(R')-、-N(R')C(O)-、-C(O)-、-C(S)-、-C(S)S-、-SC(S)-、-CH(OH)-、-P(O)(OR')O-、-S(O)2-、-S-S-、芳基或雜芳基； R7為C1-3烷基、C2-3烯基或H； R8為C3-6碳環或雜環； R9為H、CN、NO2、C1-6烷基、-OR、-S(O)2R、-S(O)2N(R)2、C2-6烯基、C3-6碳環或雜環； 各R獨立地為C1-3烷基、C2-3烯基或H； 各R'獨立地為C1-18烷基、C2-18烯基、-R*YR"、-YR"或H； 各R"獨立地為C3-14烷基或C3-14烯基； 各R*獨立地為C1-12烷基或C2-12烯基； 各Y獨立地為C3-6碳環； 各X獨立地為F、Cl、Br或I；及 m為5、6、7、8、9、10、11、12或13；及/或其醫藥學上可接受之鹽、互變異構物、前藥或立體異構物。In some embodiments, another subset of compounds of formula (I) includes those wherein: R1 is C5-30 alkyl, C5-20 alkenyl, -R*YR", -YR" or -R"M'R'; R2 and R3 are independently H, C1-14 alkyl, C2-14 alkenyl, -R*YR", -YR" or -R*OR", and/or R2 and R3 together with the atoms to which they are attached form a heterocyclic or carbocyclic ring; R4 is C3-6 carbocyclic ring, -(CH2 )n Q, -(CH2 )n CHQR, -CHQR, -CQ(R)2 or unsubstituted C1-6 alkyl, wherein Q is C3-6 carbon ring, 5-14 membered heterocyclic ring having one or more heteroatoms including N, O or S, -OR, -O(CH2 )n N(R)2 , -C(O)OR, -OC(O)R, -CX3 , -CX2 H, -CXH2 , -CN, -C(O)N(R)2 , -N(R)C(O)R, -N(R)S(O)2 R, -N(R)C(O)N(R)2 , -N(R)C(S)N(R)2 , -CRN(R)2 C(O)OR, -N(R)R8 , -O(CH2 )n OR, -N(R)C(=NR9 )N(R)2 , -N(R)C(=CHR9 )N(R)2 wherein: -OC(O)N(R)2 , -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R , -N(OR)C(O)OR, -N(OR)C(O)N(R)2 , -N(OR)C(S)N(R)2 , -N(OR)C(=NR9 )N(R)2 , -N(OR)C(=CHR9 )N(R)2 , -C(=NR9 )R, -C(O)N(R)OR or -C(=NR9 )N(R)2 , and/or each n is independently 1, 2, 3, 4 or 5; and/or when Q is a 5-14 membered heterocyclic ring and (i)R4 is -(CH2 )nQ , wherein n is 1 or 2, or (ii) RR4 is -(CH2 )nCHQR , wherein n is 1, or (iii)R4 is -CHQR and -CQ(R)2 , then Q is a 5- to 14-membered heteroaryl group or an 8- to 14-membered heterocycloalkyl group; eachR5 is independently aC1-3 alkyl group, aC2-3 alkenyl group or H; eachR6 is independently aC1-3 alkyl group, aC2-3 alkenyl group or H; M and M' are independently -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2- , -SS-, aryl or heteroaryl;R7 is C R8 is a C3-6carbocyclic ring or a heterocyclic ring; R9 is H, CN, NO2 ,a C1-6 alkyl, -OR, -S(O)2 R, -S(O)2 N(R)2 , a C2-6 alkenyl, a C3-6 carbocyclic ring or a heterocyclic ring; each R is independently a C1-3 alkyl, a C2-3 alkenyl or H; each R' is independently a C1-18 alkyl, a C2-18 alkenyl, -R*YR", -YR" or H; each R" is independently a C3-14 alkyl or a C3-14 alkenyl; each R* is independently a C1-12 alkyl or a C2-12 alkenyl; each Y is independently a C3-6 carbocyclic ring; Each X is independently F, Cl, Br or I; and m is 5, 6, 7, 8, 9, 10, 11, 12 or 13; and/or pharmaceutically acceptable salts, tautomers, prodrugs or stereoisomers thereof.
在一些態樣中，式(I)化合物之另一子集包括如下之彼等者，其中： R1為C5-30烷基、C5-20烯基、-R*YR"、-YR"或-R"M'R'； R2及R3獨立地為H、C1-14烷基、C2-14烯基、-R*YR"、-YR"或-R*OR"，及/或R2及R3與其所連接之原子一起形成雜環或碳環； R4為C3-6碳環、-(CH2)nQ、-(CH2)nCHQR、-CHQR、-CQ(R)2或未經取代之C1-6烷基，其中Q為C3-6碳環、具有一或多個包含N、O或S之雜原子的5至14員雜芳基、-OR、-O(CH2)nN(R)2、-C(O)OR、-OC(O)R、-CX3、-CX2H、-CXH2、-CN、-C(O)N(R)2、-N(R)C(O)R、-N(R)S(O)2R、-N(R)C(O)N(R)2、-N(R)C(S)N(R)2、-CRN(R)2C(O)OR、-N(R)R8、-O(CH2)nOR、-N(R)C(=NR9)N(R)2、-N(R)C(=CHR9)N(R)2、-OC(O)N(R)2、-N(R)C(O)OR、-N(OR)C(O)R、-N(OR)S(O)2R、-N(OR)C(O)OR、-N(OR)C(O)N(R)2、-N(OR)C(S)N(R)2、-N(OR)C(=NR9)N(R)2、-N(OR)C(=CHR9)N(R)2、-C(=NR9)R、-C(O)N(R)OR或-C(=NR9)N(R)2，及/或各n獨立地為1、2、3、4、或5； 各R5獨立地為C1-3烷基、C2-3烯基或H； 各R6獨立地為C1-3烷基、C2-3烯基或H； M及M'獨立地為-C(O)O-、-OC(O)-、-C(O)N(R')-、-N(R')C(O)-、-C(O)-、-C(S)-、-C(S)S-、-SC(S)-、-CH(OH)-、-P(O)(OR')O-、-S(O)2-、-S-S-、芳基或雜芳基； R7為C1-3烷基、C2-3烯基或H； R8為C3-6碳環或雜環； R9為H、CN、NO2、C1-6烷基、-OR、-S(O)2R、-S(O)2N(R)2、C2-6烯基、C3-6碳環或雜環； 各R獨立地為C1-3烷基、C2-3烯基或H； 各R'獨立地為C1-18烷基、C2-18烯基、-R*YR"、-YR"或H； 各R"獨立地為C3-14烷基或C3-14烯基； 各R*獨立地為C1-12烷基或C2-12烯基； 各Y獨立地為C3-6碳環； 各X獨立地為F、Cl、Br或I；及 m為5、6、7、8、9、10、11、12或13；及/或其醫藥學上可接受之鹽、互變異構物、前藥或立體異構物。In some embodiments, another subset of compounds of formula (I) includes those wherein: R1 is C5-30 alkyl, C5-20 alkenyl, -R*YR", -YR" or -R"M'R'; R2 and R3 are independently H, C1-14 alkyl, C2-14 alkenyl, -R*YR", -YR" or -R*OR", and/or R2 and R3 together with the atoms to which they are attached form a heterocyclic or carbocyclic ring; R4 is C3-6 carbocyclic ring, -(CH2 )n Q, -(CH2 )n CHQR, -CHQR, -CQ(R)2 or unsubstituted C1-6 alkyl, wherein Q is C3-6 carbocyclic ring, 5-14 membered heteroaryl having one or more heteroatoms including N, O or S, -OR, -O(CH2 )n N(R)2 , -C(O)OR, -OC(O)R, -CX3 , -CX2 H, -CXH2 , -CN, -C(O)N(R)2 , -N(R)C(O)R, -N(R)S(O)2 R, -N(R)C(O)N(R)2 , -N(R)C(S)N(R)2 , -CRN(R)2 C(O)OR, -N(R)R8 , -O(CH2 )n OR, -N(R)C(=NR9 )N(R)2 , -N(R)C(=CHR9 )N(R)2 wherein: -OC(O)N(R)2 , -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R , -N(OR)C(O)OR, -N(OR)C(O)N(R)2 , -N(OR)C(S)N(R)2 , -N(OR)C(=NR9 )N(R)2 , -N(OR)C(=CHR9 )N(R)2 , -C(=NR9 )R, -C(O)N(R)OR, or -C(=NR9 )N(R)2 , and/or each n is independently 1, 2, 3, 4, or 5; eachR5 is independentlyC1-3 alkyl,C2-3 alkenyl, or H; eachR6 is independentlyC1-3 alkyl, C R7 isC1-3 alkyl, C2-3 alkenyl or H; R8 is C3-6 carbocyclic or heterocyclic; R9 is H, CN, NO2,C1-6 alkyl, -OR, -S(O)2R, -S(O) 2N(R)2,C2-6alkenyl,C3-6carbocyclic orheterocyclic; each R is independently C1-3 alkyl, C2-3 alkenyl or H; each R' is independently C1-18 alkyl, C2-18 alkenyl, -R*YR", -YR" or H; each R" is independently C3-14 alkyl or C3-14 alkenyl; each R* is independently C1-12 alkyl or C2-12 alkenyl; each Y is independently C3-6 carbocycle; each X is independently F, Cl, Br or I; and m is 5, 6, 7, 8, 9, 10, 11, 12 or 13; and/or pharmaceutically acceptable salts, tautomers, prodrugs or stereoisomers thereof.
在一些態樣中，式(I)化合物之另一子集包括如下之彼等者，其中： R1為C5-30烷基、C5-20烯基、-R*YR"、-YR"或-R"M'R'； R2及R3獨立地為H、C2-14烷基、C2-14烯基、-R*YR"、-YR"或-R*OR"，及/或R2及R3與其所連接之原子一起形成雜環或碳環； R4為-(CH2)nQ或-(CH2)nCHQR，其中Q為-N(R)2，及/或n為3、4或5； 各R5獨立地為C1-3烷基、C2-3烯基或H； 各R6獨立地為C1-3烷基、C2-3烯基或H； M及M'獨立地為-C(O)O-、-OC(O)-、-C(O)N(R')-、-N(R')C(O)-、-C(O)-、-C(S)-、-C(S)S-、-SC(S)-、-CH(OH)-、-P(O)(OR')O-、-S(O)2-、-S-S-、芳基或雜芳基； R7為C1-3烷基、C2-3烯基或H； 各R獨立地為C1-3烷基、C2-3烯基或H； 各R'獨立地為C1-18烷基、C2-18烯基、-R*YR"、-YR"或H； 各R"獨立地為C3-14烷基或C3-14烯基； 各R*獨立地為C1-12烷基或C1-12烯基； 各Y獨立地為C3-6碳環； 各X獨立地為F、Cl、Br或I；及 m為5、6、7、8、9、10、11、12或13；及/或其醫藥學上可接受之鹽、互變異構物、前藥或立體異構物。In some embodiments, another subset of compounds of formula (I) includes those wherein: R1 is C5-30 alkyl, C5-20 alkenyl, -R*YR", -YR" or -R"M'R'; R2 and R3 are independently H, C2-14 alkyl, C2-14 alkenyl, -R*YR", -YR" or -R*OR", and/or R2 and R3 together with the atoms to which they are attached form a heterocyclic or carbocyclic ring; R4 is -(CH2 )n Q or -(CH2 )n CHQR, wherein Q is -N(R)2 , and/or n is 3, 4 or 5; each R5 is independently C1-3 alkyl, C2-3 alkenyl or H; each R6 is independently C1-3 alkyl, C2-3 alkenyl or H; M and M' are independently -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2 -, -SS-, aryl or heteroaryl;R7 isC1-3 alkyl,C2-3 alkenyl or H; each R is independentlyC1-3 alkyl,C2-3 alkenyl or H; each R' is independentlyC1-18 alkyl,C2-18 alkenyl, -R*YR", -YR" or H; each R" is independentlyC3-14 alkyl orC3-14 alkenyl; each R* is independentlyC1-12 alkyl or C1-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently F, Cl, Br or I; and m is 5, 6, 7, 8, 9, 10, 11, 12 or 13; and/or pharmaceutically acceptable salts, tautomers, prodrugs or stereoisomers thereof.
在一些態樣中，式(I)化合物之另一子集包括如下之彼等者，其中： R1為C5-30烷基、C5-20烯基、-R*YR"、-YR"或-R"M'R'； R2及R3獨立地為C1-14烷基、C2-14烯基、-R*YR"、-YR"或-R*OR"，及/或R2及R3與其所連接之原子一起形成雜環或碳環； R4為-(CH2)nQ、-(CH2)nCHQR、-CHQR或-CQ(R)2，其中Q為-N(R)2，及/或n為1、2、3、4、或5； 各R5獨立地為C1-3烷基、C2-3烯基或H； 各R6獨立地為C1-3烷基、C2-3烯基或H； M及M'獨立地為-C(O)O-、-OC(O)-、-C(O)N(R')-、-N(R')C(O)-、-C(O)-、-C(S)-、-C(S)S-、-SC(S)-、-CH(OH)-、-P(O)(OR')O-、-S(O)2-、-S-S-、芳基或雜芳基； R7為C1-3烷基、C2-3烯基或H； 各R獨立地為C1-3烷基、C2-3烯基或H； 各R'獨立地為C1-18烷基、C2-18烯基、-R*YR"、-YR"或H； 各R"獨立地為C3-14烷基或C3-14烯基； 各R*獨立地為C1-12烷基或C1-12烯基； 各Y獨立地為C3-6碳環； 各X獨立地為F、Cl、Br或I；及 m為5、6、7、8、9、10、11、12或13；及/或其醫藥學上可接受之鹽、互變異構物、前藥或立體異構物。In some embodiments, another subset of compounds of formula (I) includes those wherein: R1 is C5-30 alkyl, C5-20 alkenyl, -R*YR", -YR" or -R"M'R'; R2 and R3 are independently C1-14 alkyl, C2-14 alkenyl, -R*YR", -YR" or -R*OR", and/or R2 and R3 together with the atoms to which they are attached form a heterocyclic or carbocyclic ring; R4 is -(CH2 )n Q, -(CH2 )n CHQR, -CHQR or -CQ(R)2 , wherein Q is -N(R)2 , and/or n is 1, 2, 3, 4, or 5; each R5 is independently C1-3 alkyl, C2-3 alkenyl or H; each RR 6 is independently C1-3 alkyl, C2-3 alkenyl or H; M and M' are independently -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2 -, -SS-, aryl or heteroaryl; R7 is C1-3 alkyl, C2-3 alkenyl or H; each R is independently C1-3 alkyl, C2-3 alkenyl or H; each R' is independently C1-18 alkyl, C2-18 alkenyl, -R*YR", -YR" or H; each R" is independently C3-14 alkyl or C each R* is independently a C1-12 alkyl ora C1-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently F, Cl, Br or I; and m is 5, 6, 7, 8, 9, 10, 11, 12 or 13; and/or pharmaceutically acceptable salts, tautomers, prodrugs or stereoisomers thereof.
在一些態樣中，式(I)化合物之子集包括式(IA)之彼等者：， 或其醫藥學上可接受之鹽、互變異構物、前藥或立體異構物，其中I為1、2、3、4或5；m為5、6、7、8或9；M1為鍵或M'；R4為未經取代之C1-3烷基或-(CH2)nQ，其中Q為OH、-NHC(S)N(R)2、-NHC(O)N(R)2、-N(R)C(O)R、-N(R)S(O)2R、-N(R)R8、-NHC(=NR9)N(R)2、-NHC(=CHR9)N(R)2、-OC(O)N(R)2、-N(R)C(O)OR、雜芳基或雜環烷基；M及M'獨立地為-C(O)O-、-OC(O)-、-C(O)N(R')-、-P(O)(OR')O-、-S-S-、芳基或雜芳基；且R2及R3獨立地為H、C1-14烷基或C2-14烯基。In some aspects, a subset of compounds of formula (I) include those of formula (IA): , or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein I is 1, 2, 3, 4 or 5; m is 5, 6, 7, 8 or 9; M1 is a bond or M'; R4 is an unsubstituted C1-3 alkyl group or -(CH2 )n Q, wherein Q is OH, -NHC(S)N(R)2 , -NHC(O)N(R)2 , -N(R)C(O)R, -N(R)S(O)2 R, -N(R)R8 , -NHC(=NR9 )N(R)2 , -NHC(=CHR9 )N(R)2 , -OC(O)N(R)2 , -N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M' are independently -C(O)O-, -OC(O)-, -C(O)N(R')-, -P(O)(OR')O-, -SS-, aryl or heteroaryl; andR2 andR3 are independently H,C1-14 alkyl orC2-14 alkenyl.
在一些態樣中，式(I)化合物之子集包括式(II)之彼等者：或其醫藥學上可接受之鹽、互變異構物、前藥或立體異構物，其中I為1、2、3、4或5；M1為鍵或M'；R4為未經取代之C1-3烷基或-(CH2)nQ，其中n為2、3或4，且Q為OH、-NHC(S)N(R)2、-NHC(O)N(R)2、-N(R)C(O)R、-N(R)S(O)2R、-N(R)R8、-NHC(=NR9)N(R)2、-NHC(=CHR9)N(R)2、-OC(O)N(R)2、-N(R)C(O)OR、雜芳基或雜環烷基；M及M'獨立地為-C(O)O-、-OC(O)-、-C(O)N(R')-、-P(O)(OR')O-、-S-S-、芳基或雜芳基；且R2及R3獨立地為H、C1-14烷基或C2-14烯基。在一些態樣中，式(I)化合物之子集包括式(Ila)、(lIb)、(lIc)或(lIe)之彼等者：， 或其醫藥學上可接受之鹽、互變異構物、前藥或立體異構物，其中R4如本文所描述。In some aspects, a subset of compounds of formula (I) include those of formula (II): or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein I is 1, 2, 3, 4 or 5; M1 is a bond or M'; R4 is an unsubstituted C1-3 alkyl group or -(CH2 )n Q, wherein n is 2, 3 or 4, and Q is OH, -NHC(S)N(R)2 , -NHC(O)N(R)2 , -N(R)C(O)R, -N(R)S(O)2 R, -N(R)R8 , -NHC(=NR9 )N(R)2 , -NHC(=CHR9 )N(R)2 , -OC(O)N(R)2 , -N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M' are independently -C(O)O-, -OC(O)-, -C(O)N(R')-, -P(O)(OR')O-, -SS-, aryl or heteroaryl; and R2 and R3 are independently H, C1-14 alkyl or C2-14 alkenyl. In some aspects, a subset of compounds of formula (I) includes those of formula (Ila), (Ib), (Ic) or (Ie): , or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein R4 is as described herein.
在一些態樣中，式(I)化合物之子集包括式(IId)之彼等者：， 或其醫藥學上可接受之鹽、互變異構物、前藥或立體異構物，其中n為2、3或4；且m、R'、R "及R2至R6如本文所描述。舉例而言，R2及R3中之各者可獨立地為C5-14烷基或C5-14烯基。In some aspects, a subset of compounds of formula (I) include those of formula (IId): , or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein n is 2, 3 or 4; and m, R', R", andR2 toR6 are as described herein. For example, each ofR2 andR3 can independently be aC5-14 alkyl or aC5-14 alkenyl.
在一些態樣中，本發明之可離子化陽離子脂質包含具有以下結構之化合物：(化合物I)。In some aspects, the ionizable cationic lipids of the present invention include compounds having the following structure: (Compound I).
在一些態樣中，本發明之可離子化陽離子脂質包含具有以下結構之化合物：(化合物II)。In some aspects, the ionizable cationic lipids of the present invention include compounds having the following structure: (Compound II).
在一些態樣中，本發明之可離子化陽離子脂質包含具有以下結構之化合物：或其醫藥學上可接受之鹽、互變異構物、前藥或立體異構物，其中： L1或L2中之一者為-O(C=O)-、-(C=O)O-、-C(=O)-、-O-、-S(O)x-、-S-S-、-C(=O)S-、SC(=O)-、-NRaC(=O)-、-C(=O) =NRa-、NRaC(=O)NRa-、-OC(=O)NRa-或-NRaC(=O)O-，且L1或L2中之另一者為-O(C=O)-、-(C=O)O-、-C(=O)-、-O-、-S(O)x-、-S-S-、-C(=O)S-、SC(=O)-、-NRaC(=O)-、-C(=O)NRa-、NRaC(=O)NRa-、-OC(=O)NRa-或-NRaC(=O)O-，或為直接鍵； G1及G2各自獨立地為未經取代之C1-C12伸烷基或C1-C12伸烯基；G為C1-C24伸烷基、C1-C24伸烯基、C3-C8伸環烷基、C3-C8伸環烯基； Ra為H或C1-C12烷基； R1及R2各自獨立地為C6-C24烷基或C6-C24烯基； R3為H、OR5、CN、-C(=O)OR4、-OC(=O)R4或-NR5C(=O)R4； R4為C1-C12烷基； R5為H或C1-C6烷基；及 x為0、1或2。In some aspects, the ionizable cationic lipids of the present invention include compounds having the following structure: or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: one ofL1 orL2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -SS-, -C(=O)S- , SC(=O)-, -NRaC(=O)-,-C (=O)=NRa-, NRaC(=O)NRa- ,-OC (=O)NRa- or -NRaC(=O)O-, and the other ofL1 orL2 is-O (C =O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -SS-, -C(=O)S-,SC (=O)-, -NRaC(=O)-, -C(=O)=NRa-,NRaC (=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O-; -, NRa C(=O)NRa -, -OC(=O)NRa - or -NRa C(=O)O-, or is a direct bond; G1 and G2 are each independently unsubstituted C1 -C12 alkylene or C1 -C12 alkenylene; G is C1 -C24 alkylene, C1 -C24 alkenylene, C3 -C8 cycloalkylene, C3 -C8 cycloalkenylene;Ra is H or C1 -C12 alkyl; R1 and R2 are each independently C6 -C24 alkyl or C6 -C24 alkenyl; R3 is H, OR5 , CN, -C(=O)OR4 , -OC(=O)R4 or -NR5 C(=O)R4 ; R4 is C1 -C12 alkyl; R5 is H or C1 -C6 alkyl; and x is 0, 1 or 2.
在有一些前述態樣中，可離子化陽離子脂質包含具有以下結構中之一者的化合物：或其醫藥學上可接受之鹽、互變異構物、前藥或立體異構物，其中： A為3至8員環烷基或伸環烷基環； R6在各次出現時獨立地為H、OH或C1-C24烷基；及 n為在1至15範圍內之整數。In some of the foregoing aspects, the ionizable cationic lipid comprises a compound having one of the following structures: or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: A is a 3- to 8-membered cycloalkyl or cycloalkylene ring;R6 at each occurrence is independently H, OH orC1 -C24 alkyl; and n is an integer in the range of 1 to 15.
在有一些前述態樣中，可離子化陽離子脂質包含具有以下結構中之一者的化合物：或其醫藥學上可接受之鹽、互變異構物、前藥或立體異構物，其中y及z各自獨立地為在1至12範圍內之整數。In some of the foregoing aspects, the ionizable cationic lipid comprises a compound having one of the following structures: or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein y and z are each independently an integer in the range of 1 to 12.
在前述態樣中之任一者中，L1或L2中之一者為-OCCO)-。舉例而言，在一些態樣中，L1及L2中之各者為-O(C=O)-。在前述任一者之一些態樣中，L1及L2各自獨立地為-(C=O)O-或-O(C=O)-。舉例而言，在一些態樣中，L1及L2中之各者為-(C=O)O-。In any of the foregoing aspects, one ofL1 orL2 is -OCCO)-. For example, in some aspects, each ofL1 andL2 is -O(C=O)-. In some aspects of any of the foregoing, each ofL1 andL2 is independently -(C=O)O- or -O(C=O)-. For example, in some aspects, each ofL1 andL2 is -(C=O)O-.
在有一些前述態樣中，可離子化陽離子脂質包含具有以下結構中之一者的化合物：或其醫藥學上可接受之鹽、互變異構物、前藥或立體異構物。In some of the foregoing aspects, the ionizable cationic lipid comprises a compound having one of the following structures: or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.
在有一些前述態樣中，可離子化陽離子脂質包含具有以下結構中之一者的化合物：或其醫藥學上可接受之鹽、互變異構物、前藥或立體異構物。In some of the foregoing aspects, the ionizable cationic lipid comprises a compound having one of the following structures: or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof.
在有一些前述態樣中，n為在2至12，例如2至8或2至4範圍內之整數。舉例而言，在一些態樣中，n為3、4、5或6。在一些態樣中，n為3。在一些態樣中，n為4。在一些態樣中，n為5。在一些態樣中，n為6。In some of the foregoing aspects, n is an integer in the range of 2 to 12, such as 2 to 8 or 2 to 4. For example, in some aspects, n is 3, 4, 5, or 6. In some aspects, n is 3. In some aspects, n is 4. In some aspects, n is 5. In some aspects, n is 6.
在有一些前述態樣中，y及z各自獨立地為在2至10範圍內之整數。舉例而言，在一些態樣中，y及z各自獨立地為在4至9或4至6範圍內之整數。In some of the foregoing aspects, y and z are each independently an integer in the range of 2 to 10. For example, in some aspects, y and z are each independently an integer in the range of 4 to 9 or 4 to 6.
在有一些前述態樣中，R6為H。在前述實施例中之其他者中，R6為C1-C24烷基。在其他態樣中，R6為OH。在一些實施例中，G未經取代。在其他態樣中，G3經取代。在各種不同態樣中，G3為直鏈C1-C24伸烷基或直鏈C1-C24伸烯基。In some of the foregoing aspects,R6 is H. In others of the foregoing embodiments,R6 isC1 -C24 alkyl. In other aspects,R6 is OH. In some embodiments, G is unsubstituted. In other aspects,G3 is substituted. In various aspects,G3 is a straight chainC1 -C24 alkylene or a straight chainC1 -C24 alkenylene.
在一些其他前述實施例中，R1或R2或兩者為C6-C24烯基。舉例而言，在一些實施例中，R1及R2各自獨立地具有以下結構：其中： R7a及R7b在各次出現時獨立地為H或C1-C12烷基；且a為2至12之整數， 其中R7a、R7b及a各自經選擇以使得R1及R2各自獨立地包含6至20個碳原子。舉例而言，在一些實施例中，a為在5至9或8至12範圍內之整數。In some other aforementioned embodiments, R1 or R2 or both are C6 -C24 alkenyl. For example, in some embodiments, R1 and R2 each independently have the following structure: wherein: R7a and R7b are independently H or C1 -C12 alkyl at each occurrence; and a is an integer from 2 to 12, wherein R7a , R7b and a are each selected such that R1 and R2 each independently contain 6 to 20 carbon atoms. For example, in some embodiments, a is an integer ranging from 5 to 9 or 8 to 12.
在有一些前述態樣中，R7a之至少一次出現為H。舉例而言，在一些態樣中，R7a在各次出現時為H。在前述之其他不同態樣中，R7b之至少一次出現為C1-C8烷基。舉例而言，在一些實施例中，C1-C8烷基為甲基、乙基、正丙基、異丙基、正丁基、異丁基、三級丁基、正己基或正辛基。In some of the foregoing aspects, at least one occurrence of R7a is H. For example, in some aspects, R7a is H at each occurrence. In other different aspects of the foregoing, at least one occurrence of R7b is C1 -C8 alkyl. For example, in some embodiments, C1 -C8 alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.
在不同態樣中，R1或R2或兩者具有以下中之一者：In various embodiments,R1 orR2 or both are one of the following:
在有一些前述態樣中，R為OH、CN、-C(=O)OR4-OC(=O)R4或-NHC(=O)R4。在一些態樣中，R4為甲基或乙基。In some of the foregoing aspects, R is OH, CN, -C(=O)OR4 -OC(=O)R4 or -NHC(=O)R4 . In some aspects, R4 is methyl or ethyl.
應理解，上文所闡述之化合物之任何態樣以及上文所闡述之化合物中之任何特定取代基及/或變數可獨立地與其他態樣及/或化合物之取代基及/或變數組合，以形成上文未特定闡述之本發明的態樣。另外，在一特定實施例及/或技術方案中針對任何特定取代基及/或變數列舉取代基及/或變數之清單的情況下，應理解，可自特定態樣及/或技術方案缺失各個別取代基及/或變數，以及取代基及/或變數之殘餘清單將視為在本發明之範疇內。應理解，在本說明書中，所描繪化學式之取代基及/或變數之組合僅當此類作用產生穩定化合物時才容許。It is understood that any aspect of the compounds described above and any specific substituents and/or variables in the compounds described above can be independently combined with substituents and/or variables of other aspects and/or compounds to form aspects of the invention not specifically described above. In addition, where a list of substituents and/or variables is listed for any specific substituent and/or variable in a particular embodiment and/or technical scheme, it is understood that each individual substituent and/or variable may be missing from the particular aspect and/or technical scheme, and the remaining list of substituents and/or variables will be considered within the scope of the present invention. It is understood that in this specification, combinations of substituents and/or variables of the depicted chemical formulas are permitted only when such effects produce stable compounds.
在一些實施例中，陽離子脂質為In some embodiments, the cationic lipid is
在一些實施例中，陽離子脂質為In some embodiments, the cationic lipid is
在一些態樣中，脂質奈米顆粒包含一或多種陽離子脂質。在一個態樣中，脂質奈米顆粒包含(4-羥丁基)氮二基)雙(己烷-6,1-二基)雙(2-己基癸酸酯) (ALC-0315)，其具有下式：In some aspects, the lipid nanoparticles comprise one or more cationic lipids. In one aspect, the lipid nanoparticles comprise (4-hydroxybutyl) azodiyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), which has the following formula:
例示性陽離子脂質揭示於例如U.S. 10,166,298中，其全部揭示內容出於所有目的以全文引用之方式併入本文中。代表性陽離子脂質包括：Exemplary cationic lipids are disclosed, for example, in US 10,166,298, the entire disclosure of which is incorporated herein by reference in its entirety for all purposes. Representative cationic lipids include:
在一些態樣中，前述陽離子脂質中之1、2、3、4、5者或更多者可排除在本發明之LNP外。In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned cationic lipids may be excluded from the LNP of the present invention.
在一些態樣中，RNA-LNP包含陽離子脂質、本文所描述之RNA分子及以下中之一或多者：中性脂質、類固醇、聚乙二醇化脂質或其組合。若將超過一種陽離子脂質併入LNP內，則此等百分比應用於經合併之陽離子脂質。在一個態樣中，陽離子脂質係以諸如至少、至多、恰好以下或在以下之間(包括性或排他性)或約以下的量存在於LNP中：40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59或60莫耳% (mol%)。在一些態樣中，將兩種或更多種陽離子脂質併入LNP內。若將超過一種陽離子脂質併入LNP內，則前述百分比應用於經合併之陽離子脂質。In some aspects, the RNA-LNP comprises a cationic lipid, an RNA molecule described herein, and one or more of the following: a neutral lipid, a steroid, a pegylated lipid, or a combination thereof. If more than one cationic lipid is incorporated into the LNP, these percentages apply to the incorporated cationic lipids. In one aspect, the cationic lipid is present in the LNP in an amount such as at least, at most, just below, or between (inclusive or exclusive) or about below: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol %. In some embodiments, two or more cationic lipids are incorporated into LNPs. If more than one cationic lipid is incorporated into LNPs, the aforementioned percentages apply to the incorporated cationic lipids.
在本發明之一些態樣中，LNP包含上文所描述之任何脂質之組合或混合物。In some aspects of the invention, the LNP comprises a combination or mixture of any of the lipids described above.
ii.聚合物結合脂質在一些態樣中，LNP包含聚合物結合脂質。術語「聚合物結合脂質」係指包含脂質部分及聚合物部分兩者之分子。聚合物結合脂質之實例為聚乙二醇化脂質(例如聚乙二醇-脂質、PEG-脂質)。在某些態樣中，LNP包含額外穩定化脂質，其為聚乙二醇化脂質。術語「聚乙二醇化脂質」係指包含脂質部分及聚乙二醇部分兩者之分子。ii.Polymer-bound lipids In some aspects, the LNP comprises a polymer-bound lipid. The term "polymer-bound lipid" refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer-bound lipid is a pegylated lipid (e.g., polyethylene glycol-lipid, PEG-lipid). In certain aspects, the LNP comprises an additional stabilizing lipid, which is a pegylated lipid. The term "pegylated lipid" refers to a molecule comprising both a lipid portion and a polyethylene glycol portion.
聚乙二醇化脂質為此項技術中已知的且包括但不限於PEG修飾之磷脂醯乙醇胺、PEG修飾之磷脂酸、PEG修飾之神經醯胺(例如PEG-CerC14或PEG-CerC20)、PEG修飾之二烷基胺、PEG修飾之二醯基甘油、PEG修飾之二烷基甘油、2-[(聚乙二醇)-2000]-N,N-二(十四烷基)乙醯胺及其混合物。代表性聚乙二醇-脂質包括PEG-c-DOMG、PEG-c-DMA、PEG-DSG、PEG-DPG及PEG-s-DMG (1-(單甲氧基-聚乙二醇)-2,3-二肉豆蔻醯基甘油)。在一個態樣中，聚乙二醇-脂質為N-[(甲氧基聚乙二醇)2000)胺甲醯基]-1,2-二肉豆蔻醯氧基丙基-3-胺(PEG-c-DMA)。在一個態樣中，聚乙二醇-脂質為PEG-2000-DMG。在一個態樣中，聚乙二醇-脂質為PEG-c-DOMG。在其他態樣中，LNP包含聚乙二醇化二醯基甘油(PEG-DAG) (諸如1-(單甲氧基-聚乙二醇)-2,3-二肉豆蔻醯基甘油(PEG-DMG))、聚乙二醇化磷脂醯乙醇胺(PEG-PE)、PEG丁二酸二醯基甘油(PEG-S-DAG) (諸如4-O-(2',3'-二(十四碳醯氧基)丙基-1-O-((ω-甲氧基(聚乙氧基)乙基)丁二酸酯(PEG-S-DMG))、聚乙二醇化神經醯胺(PEG-cer)、或PEG二烷氧基丙基胺基甲酸酯(諸如共聚甲氧基(聚乙氧基)乙基-N-(2,3-二(十四烷氧基)丙基)胺基甲酸酯或2,3-二(十四烷氧基)丙基-N-(ω-甲氧基(聚乙氧基)乙基)胺基甲酸酯)。PEG-脂質揭示於例如U.S. 9,737,619中，其全部揭示內容出於所有目的以全文引用之方式併入本文中。在一些態樣中，前述聚乙二醇化脂質中之1、2、3、4、5者或更多者可排除在本發明之LNP外。PEGylated lipids are known in the art and include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, 2-[(polyethylene glycol)-2000]-N,N-di(tetradecyl)acetamide, and mixtures thereof. Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, PEG-DSG, PEG-DPG, and PEG-s-DMG (1-(monomethoxy-polyethylene glycol)-2,3-dimyristylglycerol). In one embodiment, the polyethylene glycol-lipid is N-[(methoxy polyethylene glycol) 2000) aminomethyl]-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA). In one embodiment, the polyethylene glycol-lipid is PEG-2000-DMG. In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG. In other embodiments, the LNP comprises polyethylene glycol diacylglycerol (PEG-DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristylglycerol (PEG-DMG)), polyethylene glycol phospholipid ethanolamine (PEG-PE), PEG succinate diacylglycerol (PEG-S-DAG) (such as 4-O-(2',3'-di-tetradecanoyloxy)propyl-1-O-((ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG)), PEGylated ceramide (PEG-cer), or PEG dialkoxypropyl carbamate (such as co-methoxy(polyethoxy)ethyl-N-(2,3-di-tetradecanoyloxy)propyl)carbamate or 2,3-di-tetradecanoyloxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate). PEG-lipids are disclosed in, for example, U.S. 9,737,619, the entire disclosure of which is incorporated herein by reference in its entirety for all purposes. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned PEGylated lipids may be excluded from the LNPs of the present invention.
在一些態樣中，組合物包含具有以下結構之聚乙二醇化脂質：或其醫藥學上可接受之鹽、互變異構物或立體異構物，其中： R8及R9各自獨立地為含有10至30個碳原子之直鏈或分支鏈、飽和或不飽和烷基鏈，其中該烷基鏈視情況間雜有一或多個酯鍵；且w具有在30至60範圍內之平均值。在一些態樣中，R8及R9各自獨立地為含有12至16個碳原子之直鏈飽和烷基鏈。在一些態樣中，w具有在43至53範圍內之平均值。在其他態樣中，平均w為或為約45。在其他不同實施例中，平均w為或為約49。In some aspects, the composition comprises a PEGylated lipid having the following structure: or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: R8 and R9 are each independently a linear or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, wherein the alkyl chain is optionally doped with one or more ester bonds; and w has an average value in the range of 30 to 60. In some embodiments, R8 and R9 are each independently a linear saturated alkyl chain containing 12 to 16 carbon atoms. In some embodiments, w has an average value in the range of 43 to 53. In other embodiments, the average w is or is about 45. In other different embodiments, the average w is or is about 49.
在一些態樣中，脂質奈米顆粒包含聚合物結合脂質。在一個態樣中，脂質奈米顆粒包含2-[(聚乙二醇)-2000]-N,N-二(十四烷基)乙醯胺(ALC-0159)，其具有下式：In some aspects, the lipid nanoparticles comprise polymer-bound lipids. In one aspect, the lipid nanoparticles comprise 2-[(polyethylene glycol)-2000]-N,N-di(tetradecyl)acetamide (ALC-0159), which has the following formula:
在各種態樣中，陽離子脂質與聚乙二醇化脂質之莫耳比在或約100:1至約20:1之範圍內，例如20:1、25:1、30:1、35:1、40:1、45:1、50:1、55:1、60:1、65:1、70:1、75:1、80:1、85:1、90:1、95:1或100:1，或其中可導出之任何範圍或值。In various aspects, the molar ratio of the cationic lipid to the pegylated lipid is in the range of or about 100:1 to about 20:1, e.g., 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, or 100:1, or any range or value derivable therein.
在某些態樣中，PEG-脂質以或不以相對於奈米顆粒之總脂質含量為或為約1至約10莫耳% (mol%) (例如至少、至多、恰好以下或在以下之間(包括性或排他性)：1、2、3、4、5、6、7、8、9或10 mol%)之量存在於LNP中。In some aspects, the PEG-lipid is or is not present in the LNP in an amount of at or about 1 to about 10 mole % (mol %) (e.g., at least, at most, just below, or between (inclusive or exclusive): 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mol %) relative to the total lipid content of the nanoparticle.
在一些態樣中，脂質奈米顆粒調配物中PEG之比率可增加或降低，及/或PEG脂質之碳鏈長度可修改以改變脂質奈米顆粒調配物之藥物動力學及/或生物分佈。In some aspects, the ratio of PEG in the lipid nanoparticle formulation can be increased or decreased, and/or the carbon chain length of the PEG lipid can be modified to alter the pharmacokinetic and/or biodistribution of the lipid nanoparticle formulation.
iii.額外脂質在某些態樣中，LNP包含一或多種額外脂質或脂質樣材料，其使顆粒在其形成期間穩定。適合的穩定化或結構脂質包括非陽離子脂質，例如中性脂質及陰離子脂質。不受任何理論所束縛，藉由添加其他疏水性部分，諸如膽固醇及除可離子化/陽離子脂質或脂質樣材料以外的脂質使LNP之調配最佳化可增強顆粒穩定性及核酸遞送功效。iii.Additional lipids In certain aspects, LNPs contain one or more additional lipids or lipid-like materials that stabilize the particles during their formation. Suitable stabilizing or structuring lipids include non-cationic lipids, such as neutral lipids and anionic lipids. Without being bound by any theory, optimizing the formulation of LNPs by adding other hydrophobic moieties, such as cholesterol and lipids other than ionizable/cationic lipids or lipid-like materials can enhance particle stability and nucleic acid delivery efficacy.
如本文所用，「陰離子脂質」係指在所選pH下帶負電的任何脂質。術語「中性脂質」係指在生理pH下以不帶電或中性兩性離子形式存在的多種脂質物種中之任一者。在一些態樣中，額外脂質包含以下中性脂質組分中之一者：(1)磷脂；(2)膽固醇或其衍生物；或(3)磷脂及膽固醇或其衍生物之混合物。As used herein, "anionic lipid" refers to any lipid that is negatively charged at a selected pH. The term "neutral lipid" refers to any of a variety of lipid species that exist in an uncharged or neutral zwitterionic form at physiological pH. In some aspects, the additional lipid comprises one of the following neutral lipid components: (1) a phospholipid; (2) cholesterol or a derivative thereof; or (3) a mixture of a phospholipid and cholesterol or a derivative thereof.
代表性中性脂質包括磷脂醯膽鹼、磷脂醯乙醇胺、磷脂醯甘油、磷脂酸、磷脂醯絲胺酸、神經醯胺、鞘磷脂、二氫-鞘磷脂、腦磷脂及腦苷脂。例示性磷脂包括例如：磷脂醯膽鹼，例如二醯基磷脂醯膽鹼，諸如二硬脂醯基磷脂醯膽鹼(DSPC)、二油醯基磷脂醯膽鹼(DOPC)、二肉豆蔻醯基磷脂醯膽鹼(DMPC)、二十五碳醯磷脂醯膽鹼、二月桂醯基膽鹼磷脂、二棕櫚醯基磷脂醯膽鹼(DPPC)、二油醯基磷脂醯甘油(DOPG)、二棕櫚醯基磷脂醯甘油(DPPG)、二花生醯膽鹼磷脂(DAPC)、二山崳醯基磷脂醯膽鹼(DBPC)、二(二十三烷醯基)磷脂醯膽鹼(DTPC)、二(二十四烷醯基)磷脂醯膽鹼(DLPC)、棕櫚醯油醯基-磷脂醯膽鹼(POPC)、1,2-二-O-十八碳烯基-sn-甘油基-3-磷酸膽鹼(18:0 Diether PC)、1-油醯基-2-膽固醇半丁二醯基-sn-甘油基-3-磷酸膽鹼(OChemsPC)及1-十六烷基-sn-甘油基-3-磷酸膽鹼(C16 Lyso PC)；及磷脂醯乙醇胺，例如二醯基磷脂醯乙醇胺，諸如二油醯基-磷脂醯乙醇胺(DOPE)、1,2-雙十一碳醯基-sn-甘油基-磷酸膽鹼(DUPC)、棕櫚醯油醯基磷脂醯膽鹼(POPC)、棕櫚醯油醯基-磷脂醯乙醇胺(POPE)及二油醯基-磷脂醯乙醇胺4-(N-順丁烯二醯亞胺基甲基)-環己烷-1-甲酸酯(DOPE-mal)、二棕櫚醯基磷脂醯乙醇胺(DPPE)、二肉豆蔻醯基磷酸乙醇胺(DMPE)、二月桂醯基-磷脂醯乙醇胺(DLPE)、二硬脂醯基-磷脂醯乙醇胺(DSPE)、1-植烷醯基-磷脂醯乙醇胺(DpyPE)、16-O-單甲基PE、16-O-二甲基PE、18-1-反式PE、1-硬脂醯基-2-油醯基磷脂醯乙醇胺(SOPE)、1,2-二反油醯基-sn-甘油基-3-磷酸乙醇胺(transDOPE)、1,2-二亞麻醯基-sn-甘油基-3-磷酸膽鹼、1,2-二花生四烯醯基-sn-甘油基-3-磷酸膽鹼、1,2-雙二十二碳六烯醯基-sn-甘油基-3-磷酸膽鹼、1,2-二植烷醯基-sn-甘油基-3-磷酸乙醇胺(ME 16.0 PE)、1,2-二硬脂醯基-sn-甘油基-3-磷酸乙醇胺、1,2-二亞麻醯基-sn-甘油基-3-磷酸乙醇胺、1,2-二亞麻醯基-sn-甘油基-3-磷酸乙醇胺、1,2-二花生四烯醯基-sn-甘油基-3-磷酸乙醇胺、1,2-雙二十二碳六烯醯基-sn-甘油基-3-磷酸乙醇胺、1,2-二油醯基-sn-甘油基-3-磷酸-外消旋-(1-甘油)鈉鹽(DOPG)、鞘磷脂及其混合物。在一些態樣中，前述中性脂質中之1、2、3、4、5者或更多者可排除在本發明之LNP外。Representative neutral lipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, ceramide, sphingomyelin, dihydro-sphingomyelin, cephalin and cerebroside. Exemplary phospholipids include, for example, phospholipid acyl choline, for example, diacyl phospholipid acyl choline, such as distearyl phospholipid acyl choline (DSPC), dioleyl phospholipid acyl choline (DOPC), dimyristoyl phospholipid acyl choline (DMPC), pentacosyl phospholipid acyl choline, dilauryl choline phospholipid, dipalmitoyl phospholipid acyl choline (DPPC), dioleoyl phospholipid acyl glycerol (DOPG), dipalmitoyl , diarachidyl phosphatidyl glycerol (DPPG), diarachidyl choline (DAPC), dibehenyl phosphatidyl choline (DBPC), di(tricosyl) phosphatidyl choline (DTPC), di(tetracosyl) phosphatidyl choline (DLPC), palmitoyloleyl-phosphatidyl choline (POPC), 1,2-di-O-octadecenyl-sn-glyceryl-3-phosphocholine (18:0 Diether PC), 1-oleyl-2-cholesterol hemisuccinyl-sn-glyceryl-3-phosphocholine (OChemsPC) and 1-hexadecyl-sn-glyceryl-3-phosphocholine (C16 Lyso PC); and phospholipid ethanolamines, for example diacyl phosphatidylethanolamines, such as dioleoyl-phosphatidylethanolamine (DOPE), 1,2-diundecyl-sn-glycero-phosphocholine (DUPC), palmitoyloleylphosphatidylcholine (POPC), palmitoyloleyl-phosphatidylethanolamine (POPE) and dioleoyl DOPE-mal, 4-(N-cis-butylenediimidomethyl)-cyclohexane-1-carboxylate, dimalcylidene phosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), dilauryl-phosphatidylethanolamine (DLPE), distearyl-phosphatidylethanolamine (DSPE), 1-phytanyl-phosphatidylethanolamine (DpyPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearyl-2-oleylphosphatidylethanolamine (SOPE), 1,2-ditransoleyl-sn-glycero-3-phosphoethanolamine (transDOPE), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diacetoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-diarachidonyl-sn-glyceryl-3-phosphoethanolamine, 1,2-bis-docosahexaenoyl-sn-glyceryl-3-phosphoethanolamine, 1,2-dioleyl-sn-glyceryl-3-phospho-racem-(1-glycerol) sodium salt (DOPG), sphingomyelin and mixtures thereof. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned neutral lipids may be excluded from the LNP of the present invention.
在一個態樣中，中性脂質為1,2-二硬脂醯基-sn-甘油基-3-磷酸膽鹼(DSPC)，其具有下式：In one aspect, the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), which has the following formula:
在一些態樣中，LNP包含中性脂質，且中性脂質包含以下中之一或多者：DSPC、DPPC、DMPC、DOPC、POPC、DOPE及/或SM。在一些態樣中，前述中性脂質中之1、2、3、4、5者或更多者可排除在本發明之LNP外。In some aspects, LNP comprises neutral lipid, and neutral lipid comprises one or more of the following: DSPC, DPPC, DMPC, DOPC, POPC, DOPE and/or SM. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned neutral lipid can be excluded from LNP of the present invention.
在各種態樣中，LNP進一步包含類固醇或類固醇類似物。「類固醇」為包含以下碳骨架之化合物：。In various aspects, the LNP further comprises a steroid or a steroid analog. "Steroid" is a compound comprising the following carbon skeleton: .
在某些態樣中，類固醇或類固醇類似物為膽固醇、糞固醇、植物固醇、麥角固醇、菜油固醇、豆固醇、菜子固醇、番茄次鹼、熊果酸、α-生育酚及其混合物。在一些態樣中，前述類固醇或類固醇類似物中之1、2、3、4、5者或更多者可排除在本發明之LNP外。在某些態樣中，類固醇或類固醇類似物為膽固醇。膽固醇衍生物之實例包括但不限於膽甾烷醇、膽甾烷酮、膽甾烯酮、糞甾醇、膽固醇基-2'-羥乙基醚、膽固醇基-4'-羥丁基醚、生育酚及其衍生物，以及其混合物。在一些態樣中，前述膽固醇衍生物中之1、2、3、4、5者或更多者可排除在本發明之LNP外。在一個態樣中，膽固醇具有下式：In some aspects, the steroid or steroid analog is cholesterol, natriol, phytosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatosterol, ursolic acid, α-tocopherol and mixtures thereof. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned steroids or steroid analogs may be excluded from the LNP of the present invention. In some aspects, the steroid or steroid analog is cholesterol. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, natriol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof. In some embodiments, 1, 2, 3, 4, 5 or more of the aforementioned cholesterol derivatives may be excluded from the LNP of the present invention. In one embodiment, cholesterol has the following formula:
不受任何理論所束縛，與至少一種額外脂質之量相比，至少一種陽離子脂質之量可影響重要核酸顆粒特徵，諸如核酸之電荷、粒徑、穩定性、組織選擇性及生物活性。因此，在一些態樣中，陽離子脂質與中性脂質之莫耳比在或約2:1至約8:1之範圍內，或在或約10:0至約1:9、約4:1至約1:2或約3:1至約1:1之範圍內。Without being bound by any theory, the amount of at least one cationic lipid compared to the amount of at least one additional lipid can affect important nucleic acid particle characteristics, such as nucleic acid charge, particle size, stability, tissue selectivity and biological activity. Thus, in some aspects, the molar ratio of cationic lipid to neutral lipid is in the range of or about 2:1 to about 8:1, or in the range of or about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1.
在一些態樣中，非陽離子脂質(例如中性脂質(例如一或多種磷脂及/或膽固醇))可包含在或在約0 mol%至約90 mol%、在或在約0 mol%至約80 mol%、在或在約0 mol%至約70 mol%、在或在約0 mol%至約60 mol%、或者在或在約0 mol%至約50 mol%之顆粒中存在的總脂質。在一些態樣中，非陽離子脂質(例如中性脂質(例如一或多種磷脂及/或膽固醇))可為或可不為至少、至多、恰好以下或在以下之間(包括性或排他性)的顆粒中存在之總脂質：0 mol%、10 mol%、20 mol%、30 mol%、40 mol%、50 mol%、60 mol%、70 mol%、80 mol%或90 mol%。In some aspects, non-cationic lipids (e.g., neutral lipids (e.g., one or more phospholipids and/or cholesterol)) may be included in or at about 0 mol% to about 90 mol%, at or at about 0 mol% to about 80 mol%, at or at about 0 mol% to about 70 mol%, at or at about 0 mol% to about 60 mol%, or at or at about 0 mol% to about 50 mol% of the total lipid present in the particle. In some aspects, the non-cationic lipid (e.g., neutral lipid (e.g., one or more phospholipids and/or cholesterol)) may or may not be at least, at most, just below, or between (inclusive or exclusive) 0 mol%, 10 mol%, 20 mol%, 30 mol%, 40 mol%, 50 mol%, 60 mol%, 70 mol%, 80 mol%, or 90 mol% of the total lipid present in the particle.
VI. RNA分子之表徵及分析本文所描述之RNA分子可使用各種方法分析及表徵。分析可在加帽之前及/或之後進行。或者，分析可在基於多-A捕獲之親和純化之前及/或之後進行。在另一態樣中，分析可在額外純化步驟，例如陰離子交換層析及其類似者之前及/或之後進行。舉例而言，RNA模板品質可使用基於生物分析儀晶片之電泳系統來測定。在其他態樣中，RNA模板純度係使用分析型逆相HPLC來分析。加帽效率可使用例如總核酸酶消化，隨後二核苷酸帽物種相對於未加帽GTP物種之MS/MS定量來分析。活體外功效可藉由例如將RNA分子轉染至人類細胞株中來分析。所關注多肽之蛋白質表現可使用諸如ELISA及/或流式細胞分析技術之方法來定量。免疫原性可藉由例如將RNA分子轉染至指示先天性免疫刺激之細胞株(例如PBMC)中來分析。細胞介素誘導可使用例如諸如ELISA之方法來分析，以定量細胞介素，例如干擾素-α。生物分佈可藉由例如生物發光量測來分析。在一些態樣中，可排除前述分析方法中之1、2、3、4、5者或更多者。VI.Characterization and Analysis ofRNAMolecules The RNA molecules described herein can be analyzed and characterized using a variety of methods. Analysis can be performed before and/or after capping. Alternatively, analysis can be performed before and/or after affinity purification based on poly-A capture. In another aspect, analysis can be performed before and/or after additional purification steps, such as anion exchange chromatography and the like. For example, RNA template quality can be determined using an electrophoresis system based on a bioanalyzer chip. In other aspects, RNA template purity is analyzed using analytical reverse phase HPLC. Capping efficiency can be analyzed using, for example, total nuclease digestion, followed by MS/MS quantification of dinucleotide cap species relative to uncapped GTP species. In vitro efficacy can be analyzed by, for example, transfecting RNA molecules into human cell lines. Protein expression of the polypeptide of interest can be quantified using methods such as ELISA and/or flow cytometry techniques. Immunogenicity can be analyzed, for example, by transfecting RNA molecules into cell lines (e.g., PBMCs) that indicate innate immune stimulation. Cytokine induction can be analyzed using methods such as ELISA to quantify cytokines, such as interferon-α. Biodistribution can be analyzed by, for example, bioluminescence measurement. In some aspects, 1, 2, 3, 4, 5, or more of the aforementioned analytical methods can be excluded.
在一些態樣中，本文所揭示之RNA聚核苷酸之特徵在於，當在投與包含RNA聚核苷酸之組合物或醫藥製劑的生物體中評定時，相對於適當參考觀測到：所關注基因(例如抗原)之表現升高；所關注基因(例如抗原)之表現持續時間增加(例如延長表現)；所關注基因(例如抗原)之表現升高且表現持續時間增加(例如延長表現)；與RNA聚核苷酸之IFIT1的相互作用減少；及/或RNA聚核苷酸之轉譯增加。在一些態樣中，前述特徵中之1、2、3、4、5者或更多者可能不係在投與包含本發明之RNA分子的組合物或醫藥製劑之後觀測到。In some aspects, the RNA polynucleotides disclosed herein are characterized in that, when assessed in an organism administered a composition or pharmaceutical formulation comprising the RNA polynucleotide, relative to an appropriate reference, the following is observed: increased expression of a gene of interest (e.g., an antigen); increased duration of expression of a gene of interest (e.g., an antigen); increased and increased duration of expression of a gene of interest (e.g., an antigen); decreased interaction with IFIT1 of the RNA polynucleotide; and/or increased translation of the RNA polynucleotide. In some aspects, 1, 2, 3, 4, 5, or more of the aforementioned characteristics may not be observed after administration of a composition or pharmaceutical formulation comprising an RNA molecule of the invention.
在一些態樣中，參考包含投與在其他方面類似但不具有m7(3'OMeG)(5')ppp(5')(2'OMeAi)pG2帽的RNA聚核苷酸。在一些態樣中，參考包含投與在其他方面類似但不具有本文所揭示之帽近端序列之RNA聚核苷酸的生物體。在一些態樣中，參考包含投與在其他方面類似但具有自雜交序列之RNA聚核苷酸的生物體。In some aspects, the reference comprises administration of an otherwise similar RNA polynucleotide but without the m7(3'OMeG)(5')ppp(5')(2'OMeAi)pG2 cap. In some aspects, the reference comprises administration of an organism with an otherwise similar RNA polynucleotide but without the cap-proximal sequence disclosed herein. In some aspects, the reference comprises administration of an organism with an otherwise similar RNA polynucleotide but with a self-hybridizing sequence.
在一些態樣中，在投與包含RNA聚核苷酸之組合物或醫藥製劑之後至少24小時、至少48小時、至少72小時、至少96小時或至少120小時測定升高之表現。在一些態樣中，在投與包含RNA聚核苷酸之組合物或醫藥製劑之後至少24小時測定升高之表現。在一些態樣中，在投與包含RNA聚核苷酸之組合物或醫藥製劑之後至少48小時測定升高之表現。在一些態樣中，在投與包含RNA聚核苷酸之組合物或醫藥製劑之後至少72小時測定升高之表現。在一些態樣中，在投與包含RNA聚核苷酸之組合物或醫藥製劑之後至少96小時測定升高之表現。在一些態樣中，在投與包含RNA聚核苷酸之組合物或醫藥製劑之後至少120小時測定升高之表現。In some aspects, the elevated expression is measured at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after administration of a composition or pharmaceutical formulation comprising an RNA polynucleotide. In some aspects, the elevated expression is measured at least 24 hours after administration of a composition or pharmaceutical formulation comprising an RNA polynucleotide. In some aspects, the elevated expression is measured at least 48 hours after administration of a composition or pharmaceutical formulation comprising an RNA polynucleotide. In some aspects, the elevated expression is measured at least 72 hours after administration of a composition or pharmaceutical formulation comprising an RNA polynucleotide. In some aspects, the elevated expression is measured at least 96 hours after administration of a composition or pharmaceutical formulation comprising an RNA polynucleotide. In some aspects, the increased expression is measured at least 120 hours after administration of the composition or pharmaceutical formulation comprising the RNA polynucleotide.
在一些態樣中，在投與包含RNA聚核苷酸之組合物或醫藥製劑之後在或在約24-120小時測定升高之表現。在一些態樣中，在投與包含RNA聚核苷酸之組合物或醫藥製劑之後在或在約以下時測定升高之表現：24-110小時、24-100小時、24-90小時、24-80小時、24-70小時、24-60小時、24-50小時、24-40小時、24-30小時、30-120小時、40-120小時、50-120小時、60-120小時、70-120小時、80-120小時、90-120小時、100-120小時或110-120小時。In some aspects, the increased expression is measured at or about 24-120 hours after administration of a composition or pharmaceutical formulation comprising an RNA polynucleotide. In some aspects, the increased expression is measured at or about 24-110 hours, 24-100 hours, 24-90 hours, 24-80 hours, 24-70 hours, 24-60 hours, 24-50 hours, 24-40 hours, 24-30 hours, 30-120 hours, 40-120 hours, 50-120 hours, 60-120 hours, 70-120 hours, 80-120 hours, 90-120 hours, 100-120 hours, or 110-120 hours after administration of a composition or pharmaceutical formulation comprising an RNA polynucleotide.
在一些態樣中，所關注基因(例如抗原)之表現升高或不升高至少2倍至至少10倍。在一些態樣中，所關注基因(例如抗原)之表現升高至少2倍。在一些態樣中，所關注基因(例如抗原)之表現升高至少3倍。在一些態樣中，所關注基因(例如抗原)之表現升高至少4倍。在一些態樣中，所關注基因(例如抗原)之表現升高至少6倍。在一些態樣中，所關注基因(例如抗原)之表現升高至少8倍。在一些態樣中，所關注基因(例如抗原)之表現升高至少10倍。In some aspects, the expression of the gene of interest (e.g., antigen) is increased or not increased by at least 2 times to at least 10 times. In some aspects, the expression of the gene of interest (e.g., antigen) is increased by at least 2 times. In some aspects, the expression of the gene of interest (e.g., antigen) is increased by at least 3 times. In some aspects, the expression of the gene of interest (e.g., antigen) is increased by at least 4 times. In some aspects, the expression of the gene of interest (e.g., antigen) is increased by at least 6 times. In some aspects, the expression of the gene of interest (e.g., antigen) is increased by at least 8 times. In some aspects, the expression of the gene of interest (e.g., antigen) is increased by at least 10 times.
在一些態樣中，所關注基因(例如抗原)之表現升高或升高約2倍至約50倍。在一些態樣中，所關注基因(例如抗原)之表現升高或升高約2倍至約45倍、約2倍至約40倍、約2倍至約30倍、約2倍至約25倍、約2倍至約20倍、約2倍至約15倍、約2倍至約10倍、約2倍至約8倍、約2倍至約5倍、約5倍至約50倍、約10倍至約50倍、約15倍至約50倍、約20倍至約50倍、約25倍至約50倍、約30倍至約50倍、約40倍至約50倍、或約45倍至約50倍。在一些態樣中，所關注基因(例如抗原)之表現升高或不升高至少、至多、恰好以下或在以下之間(包括性或排他性)：2倍、3倍、4倍、5倍、6倍、7倍、8倍、9倍、10倍、11倍、12倍、13倍、14倍、15倍、16倍、17倍、18倍、19倍、20倍、21倍、22倍、23倍、24倍、25倍、26倍、27倍、28倍、29倍、30倍、31倍、32倍、33倍、34倍、35倍、36倍、37倍、38倍、39倍、40倍、41倍、42倍、43倍、44倍、45倍、46倍、47倍、48倍、49倍或50倍，或其中可導出之任何範圍或值。In some aspects, the expression of a gene of interest (e.g., an antigen) is elevated or increased by about 2-fold to about 50-fold. In some aspects, the expression of a gene of interest (e.g., an antigen) is elevated or increased by about 2-fold to about 45-fold, about 2-fold to about 40-fold, about 2-fold to about 30-fold, about 2-fold to about 25-fold, about 2-fold to about 20-fold, about 2-fold to about 15-fold, about 2-fold to about 10-fold, about 2-fold to about 8-fold, about 2-fold to about 5-fold, about 5-fold to about 50-fold, about 10-fold to about 50-fold, about 15-fold to about 50-fold, about 20-fold to about 50-fold, about 25-fold to about 50-fold, about 30-fold to about 50-fold, about 40-fold to about 50-fold, or about 45-fold to about 50-fold. In some aspects, the expression of a gene of interest (e.g., an antigen) is or is not increased by at least, at most, just below, or between (inclusive or exclusive) 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, 30-fold, 31-fold, 32-fold, 33-fold, 34-fold, 35-fold, 36-fold, 37-fold, 38-fold, 39-fold, 40-fold, 41-fold, 42-fold, 43-fold, 44-fold, 45-fold, 46-fold, 47-fold, 48-fold, 49-fold, or 50-fold, or any range or value derivable therein.
在一些態樣中，所關注基因(例如抗原)之表現升高(例如表現持續時間增加)在投與包含RNA聚核苷酸之組合物或醫藥製劑之後持續至少、至多、恰好以下或在以下之間(包括性或排他性)：24小時、48小時、72小時、96小時或120小時。在一些態樣中，所關注基因(例如抗原)之表現升高在投與之後持續至少24小時。在一些態樣中，所關注基因(例如抗原)之表現升高在投與之後持續至少48小時。在一些態樣中，所關注基因(例如抗原)之表現升高在投與之後持續至少72小時。在一些態樣中，所關注基因(例如抗原)之表現升高在投與之後持續至少96小時。在一些態樣中，所關注基因(例如抗原)之表現升高在投與包含RNA聚核苷酸之組合物或醫藥製劑之後持續至少120小時。In some aspects, the increased expression (e.g., increased duration of expression) of the gene of interest (e.g., antigen) persists at least, at most, just below, or between (inclusive or exclusive) 24 hours, 48 hours, 72 hours, 96 hours, or 120 hours after administration of a composition or pharmaceutical formulation comprising an RNA polynucleotide. In some aspects, the increased expression of the gene of interest (e.g., antigen) persists for at least 24 hours after administration. In some aspects, the increased expression of the gene of interest (e.g., antigen) persists for at least 48 hours after administration. In some aspects, the increased expression of the gene of interest (e.g., antigen) persists for at least 72 hours after administration. In some aspects, the increased expression of the gene of interest (e.g., antigen) persists for at least 96 hours after administration. In some aspects, the increased expression of the gene of interest (eg, antigen) persists for at least 120 hours after administration of the composition or pharmaceutical formulation comprising the RNA polynucleotide.
在一些態樣中，所關注基因(例如抗原)之表現升高在投與包含RNA聚核苷酸之組合物或醫藥製劑之後持續或持續約24-120小時。在一些態樣中，表現升高在投與包含RNA聚核苷酸之組合物或醫藥製劑之後持續或持續約24-110小時、24-100小時、24-90小時、24-80小時、24-70小時、24-60小時、24-50小時、24-40小時、24-30小時、30-120小時、40-120小時、50-120小時、60-120小時、70-120小時、80-120小時、90-120小時、100-120小時或110-120小時。在一些態樣中，所關注基因(例如抗原)之表現升高持續或不持續至少、至多、恰好以下或在以下之間(包括性或排他性)：24小時、36小時、48小時、60小時、72小時、84小時、96小時、108小時或120小時，或其中可導出之任何範圍或值。In some aspects, the increased expression of the gene of interest (eg, antigen) persists or lasts for about 24-120 hours after administration of the composition or pharmaceutical formulation comprising the RNA polynucleotide. In some aspects, the increased expression persists or lasts for about 24-110 hours, 24-100 hours, 24-90 hours, 24-80 hours, 24-70 hours, 24-60 hours, 24-50 hours, 24-40 hours, 24-30 hours, 30-120 hours, 40-120 hours, 50-120 hours, 60-120 hours, 70-120 hours, 80-120 hours, 90-120 hours, 100-120 hours, or 110-120 hours after administration of the composition or pharmaceutical formulation comprising the RNA polynucleotide. In some aspects, the elevated expression of a gene of interest (e.g., an antigen) persists or does not persist for at least, at most, just below, or between (inclusive or exclusive) 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, or 120 hours, or any range or value derivable therein.
VII.免疫反應及分析如本文所論述，本發明係關於誘發或誘導個體中針對RSV蛋白(例如野生型或變異RSV F蛋白)之免疫反應。在一個態樣中，免疫反應可保護個體免於罹患感染或相關疾病(特定言之與RSV相關之彼等者)，或治療患有、疑似患有該感染或相關疾病或處於罹患該感染或相關疾病之風險下的個體。本發明之免疫原性組合物的一種用途為藉由對個體進行接種或疫苗接種來預防RSV感染。在一些態樣中，免疫原性組合物使個體免疫RSV長達1年(例如針對單個RSV季節)。在一些態樣中，免疫原性組合物使個體免疫RSV長達2年。在一些態樣中，免疫原性組合物使個體免疫RSV超過2年。在一些態樣中，免疫原性組合物使個體免疫RSV超過3年。在一些態樣中，免疫原性組合物使個體免疫RSV超過4年。在一些態樣中，免疫原性組合物使個體免疫RSV 5-10年。VII.Immune Response and Analysis As discussed herein, the present invention relates to inducing or inducing an immune response in an individual to an RSV protein (e.g., wild-type or variant RSV F protein). In one aspect, the immune response can protect an individual from infection or related diseases (specifically those associated with RSV), or treat an individual who has, is suspected of having, or is at risk of having the infection or related disease. One use of the immunogenic composition of the present invention is to prevent RSV infection by inoculating or vaccinating an individual. In some aspects, the immunogenic composition immunizes an individual to RSV for up to 1 year (e.g., for a single RSV season). In some aspects, the immunogenic composition immunizes an individual to RSV for up to 2 years. In some aspects, the immunogenic composition immunizes an individual to RSV for more than 2 years. In some aspects, the immunogenic composition immunizes an individual against RSV for more than 3 years. In some aspects, the immunogenic composition immunizes an individual against RSV for more than 4 years. In some aspects, the immunogenic composition immunizes an individual against RSV for 5-10 years.
A.免疫分析本發明包括實施血清學分析以評估免疫反應是否係由本發明之組合物誘導或誘發以及誘導或誘發至何種程度。存在可實施之許多類型的免疫分析。本發明涵蓋之免疫分析包括但不限於美國專利4,367,110 (雙重單株抗體夾心分析)及美國專利4,452,901 (西方墨點(western blot))中所描述之彼等者。其他分析包括活體外及活體內的經標記配體之免疫沉澱及免疫細胞化學。A.Immunoassays The present invention includes performing serological assays to assess whether and to what extent an immune response is induced or elicited by the compositions of the present invention. There are many types of immunoassays that can be performed. Immunoassays encompassed by the present invention include, but are not limited to, those described in U.S. Pat. No. 4,367,110 (double monoclonal antibody sandwich assay) and U.S. Pat. No. 4,452,901 (western blot). Other assays include immunoprecipitation of labeled ligands in vitro and in vivo and immunocytochemistry.
免疫分析一般為結合分析。在一些態樣中，免疫分析為此項技術中已知之各種類型之酶聯免疫吸附分析(ELISA)及放射免疫分析(RIA)。使用組織切片進行之免疫組織化學偵測亦為尤其適用的。在一個實例中，抗體或抗原固定在所選擇表面上，諸如聚苯乙烯微量滴定盤中之孔、試紙或管柱載體上。接著，將懷疑含有所需抗原或抗體之測試組合物(諸如臨床樣品)添加至該等孔。在結合及洗滌以移除非特異性結合之免疫複合物之後，可偵測結合之抗原或抗體。偵測一般藉由添加連接至可偵測標記之對所需抗原或抗體具有特異性的另一種抗體來達成。此類型ELISA被稱為「夾心ELISA」。偵測亦可藉由添加對所需抗原具有特異性之第二抗體，隨後添加對於第二抗體具有結合親和力之第三抗體來達成，其中第三抗體連接至可偵測標記。Immunoassays are generally binding assays. In some embodiments, immunoassays are various types of enzyme-linked immunosorbent assays (ELISA) and radioimmunoassays (RIA) known in the art. Immunohistochemical detection using tissue sections is also particularly suitable. In one example, antibodies or antigens are fixed on a selected surface, such as a well in a polystyrene microtiter plate, a test paper, or a column carrier. Then, a test composition (such as a clinical sample) suspected of containing the desired antigen or antibody is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen or antibody can be detected. Detection is generally achieved by adding another antibody that is specific to the desired antigen or antibody and is linked to a detectable marker. This type of ELISA is called a "sandwich ELISA." Detection can also be achieved by adding a second antibody specific for the desired antigen, followed by the addition of a third antibody that has binding affinity for the second antibody, where the third antibody is linked to a detectable label.
競爭ELISA亦為其中測試樣品與已知量之經標記抗原或抗體競爭結合的可能實施方式。未知樣品中反應性物種之量係藉由在與經塗佈之孔一起培育之前或期間將樣品與已知標記物種混合來測定。樣品中反應性物種之存在用以減少可用於結合至孔之經標記物種的量，且因此減少最終訊號。無論採用何種形式，ELISA均具有某些共同特徵，諸如塗佈、培育或結合、洗滌以移除非特異性結合之物種，及偵測經結合之免疫複合物。Competition ELISA is also a possible implementation in which the test sample competes for binding to a known amount of labeled antigen or antibody. The amount of reactive species in the unknown sample is determined by mixing the sample with the known labeled species before or during incubation with the coated wells. The presence of reactive species in the sample serves to reduce the amount of labeled species available for binding to the wells and thus reduces the final signal. Regardless of the format, ELISAs have certain common features, such as coating, incubation or binding, washing to remove nonspecifically bound species, and detection of bound immune complexes.
抗原或抗體亦可連接至固體載體，諸如呈盤、珠粒、試紙、膜或管柱基質形式之固體載體，且待分析之樣品施加至經固定之抗原或抗體。在用抗原或抗體塗佈培養盤時，一般將該培養盤之孔與抗原或抗體溶液一起培育過夜或指定時間。接著洗滌該培養盤之孔以移除不完全吸附之物質。接著，該等孔之任何殘留可用表面利用對測試抗血清呈抗原中性之非特異性蛋白質「塗佈」。該等蛋白質包括牛血清白蛋白(BSA)、酪蛋白及奶粉溶液。該塗佈允許阻斷固定表面上之非特異性吸附位點且因此減少由該表面上抗血清之非特異性結合引起的背景。Antigens or antibodies may also be attached to a solid support, such as a solid support in the form of a disk, bead, test paper, membrane or column matrix, and the sample to be analyzed is applied to the immobilized antigen or antibody. When coating a culture plate with antigen or antibody, the wells of the culture plate are generally incubated with the antigen or antibody solution overnight or for a specified time. The wells of the culture plate are then washed to remove incompletely adsorbed material. Any residues in the wells can then be "coated" with a nonspecific protein that is antigenically neutral to the test antiserum. Such proteins include bovine serum albumin (BSA), casein, and milk powder solutions. The coating allows blocking of nonspecific adsorption sites on the immobilization surface and thus reducing the background caused by nonspecific binding of antisera on the surface.
B. RSV感染之診斷本發明涵蓋以多種方式使用RSV多肽、蛋白質及/或肽，包括偵測RSV之存在以診斷感染。根據本發明，偵測感染存在之方法涉及獲得疑似經一或多種RSV株系感染之樣品的步驟，該樣品諸如獲自個體之樣品，例如獲自血液、唾液、組織、骨骼、肌肉、軟骨或皮膚之樣品。在分離樣品之後，可進行利用本發明之多肽、蛋白質及/或肽之診斷分析以偵測RSV之存在，且用於測定樣品中之此類存在的此類分析技術為熟習此項技術者所熟知的，且包括諸如放射免疫分析、西方墨點分析及ELISA分析之方法。B.Diagnosis ofRSV Infection The present invention encompasses the use of RSV polypeptides, proteins and/or peptides in a variety of ways, including detecting the presence of RSV to diagnose infection. According to the present invention, methods of detecting the presence of infection involve the step of obtaining a sample suspected of being infected with one or more strains of RSV, such as a sample obtained from an individual, for example, a sample obtained from blood, saliva, tissue, bone, muscle, cartilage or skin. After isolating the sample, a diagnostic assay utilizing the polypeptides, proteins and/or peptides of the present invention may be performed to detect the presence of RSV, and such analytical techniques for determining such presence in a sample are well known to those skilled in the art and include methods such as radioimmunoassays, Western blots, and ELISA assays.
一般而言，根據本發明，涵蓋一種診斷感染之方法，其中已將根據本發明之多肽、蛋白質或肽添加至懷疑感染RSV之樣品中，且RSV係藉由與該等多肽、蛋白質及/或肽之抗體結合或與樣品中之抗體的多肽、蛋白質及/或肽結合來指示。Generally speaking, according to the present invention, a method of diagnosing infection is encompassed, wherein a polypeptide, protein or peptide according to the present invention has been added to a sample suspected of being infected with RSV, and RSV is indicated by binding of antibodies to the polypeptides, proteins and/or peptides or binding of the polypeptides, proteins and/or peptides to antibodies in the sample.
亦涵蓋一種測試樣品之方法，已向該樣品中添加根據本發明之多肽、蛋白質或肽，疑似感染RSV、先前已感染RSV或感染RSV，且RSV係藉由與該等多肽、蛋白質及/或肽之抗體結合或與樣品中之抗體的多肽、蛋白質及/或肽結合來指示。Also encompassed is a method of testing a sample to which a polypeptide, protein or peptide according to the invention has been added, suspected of being infected with RSV, previously infected with RSV or infected with RSV, and RSV is indicated by binding of antibodies to the polypeptides, proteins and/or peptides or binding of the polypeptides, proteins and/or peptides to antibodies in the sample.
因此，編碼根據本發明之RSV多肽、蛋白質及/或肽的RNA分子可用於治療、預防由RSV感染(例如主動或被動免疫接種)引起之感染所致的疾病或降低其嚴重程度或適用作研究工具。Therefore, RNA molecules encoding RSV polypeptides, proteins and/or peptides according to the present invention can be used to treat, prevent or reduce the severity of diseases caused by RSV infection (e.g., active or passive vaccination) or are suitable as research tools.
上述多肽、蛋白質及/或肽中之任一者可直接用可偵測標籤標記以便鑑別及定量RSV。適用於免疫分析之標籤一般為熟習此項技術者已知的，且包括酶、放射性同位素、及螢光、發光及發色物質，包括著色顆粒，諸如膠體金或乳膠珠粒。適合的免疫分析包括酶聯免疫吸附分析(ELISA)。Any of the above polypeptides, proteins and/or peptides can be directly labeled with a detectable label to facilitate identification and quantification of RSV. Labels suitable for use in immunoassays are generally known to those skilled in the art and include enzymes, radioisotopes, and fluorescent, luminescent and chromogenic substances, including colored particles such as colloidal gold or latex beads. Suitable immunoassays include enzyme-linked immunosorbent assays (ELISA).
C.保護性免疫在本發明之一些態樣中，編碼RSV preF多肽之RNA分子、RNA-LNP及其組合物向個體賦予保護性免疫。保護性免疫係指建立特異性免疫反應之身體能力，其保護個體免於患上特定疾病或病狀，其涉及針對其存在免疫反應之藥劑。免疫原性有效量能夠賦予個體保護性免疫。在一些態樣中，本發明之編碼RSV多肽的RNA分子、RNA-LNP及其組合物可用於誘導針對RSV之平衡免疫反應(包含細胞及體液免疫)，而無與減毒病毒疫苗接種相關之許多風險。「體液」免疫反應係指由抗體分子(包括例如分泌性(IgA)或IgG分子)介導之免疫反應，而「細胞」免疫反應為由T淋巴球(例如CD4+輔助T細胞及/或CD8+ T細胞(例如CTL)及/或其他白血球介導之免疫反應。C.Protective Immunity In some aspects of the invention, RNA molecules encoding RSV preF polypeptides, RNA-LNPs, and compositions thereof confer protective immunity to an individual. Protective immunity refers to the body's ability to mount a specific immune response that protects the individual from a specific disease or condition, to which an agent against which an immune response exists. An immunogenic effective amount is capable of conferring protective immunity to an individual. In some aspects, RNA molecules encoding RSV polypeptides, RNA-LNPs, and compositions thereof of the invention can be used to induce a balanced immune response (including cellular and humoral immunity) against RSV without the many risks associated with vaccination with attenuated virus vaccines. A "humoral" immune response refers to an immune response mediated by antibody molecules (including, for example, secretory (IgA) or IgG molecules), while a "cellular" immune response is an immune response mediated by T lymphocytes (e.g., CD4+ helper T cells and/or CD8+ T cells (e.g., CTLs) and/or other white blood cells.
如本文所用，片語「免疫反應(immune response)」或其等效片語「免疫反應(immunological response)」係指產生針對抗原之體液反應(抗體介導)、細胞反應(由抗原特異性T細胞或其分泌產物介導)或體液及細胞反應兩者。此類反應可為主動反應或被動反應。細胞免疫反應係藉由多肽抗原決定基與I類或II類MHC分子結合呈現以活化抗原特異性CD4 (+) T輔助細胞及/或CD8 (+)細胞毒性T細胞來引發。反應亦可涉及單核球、巨噬細胞、NK細胞、嗜鹼性球、樹突狀細胞、星形膠質細胞、微神經膠質細胞、嗜酸性球或先天性免疫之其他組分的活化。如本文所用，「主動免疫」係指藉由回應於抗原(例如由本發明之RNA分子編碼的RSV F蛋白)之存在產生抗體而賦予個體之任何免疫。As used herein, the phrase "immune response" or its equivalent phrase "immunological response" refers to the production of a humoral response (antibody-mediated), a cellular response (mediated by antigen-specific T cells or their secreted products), or both humoral and cellular responses to an antigen. Such responses may be active or passive. Cellular immune responses are induced by the presentation of polypeptide antigenic determinants in conjunction with class I or class II MHC molecules to activate antigen-specific CD4 (+) T helper cells and/or CD8 (+) cytotoxic T cells. The response may also involve the activation of monocytes, macrophages, NK cells, basophils, dendritic cells, astrocytes, microneurocytes, eosinophils, or other components of innate immunity. As used herein, "active immunity" refers to any immunity conferred to an individual by the production of antibodies in response to the presence of an antigen (e.g., RSV F protein encoded by an RNA molecule of the invention).
如本文所用，「被動免疫」包括但不限於投與活化之免疫效應子，包括免疫反應之細胞介體或蛋白質介體(例如單株及/或多株抗體)。單株或多株抗體組合物可用於被動免疫接種以治療、預防由攜帶抗體所識別之抗原的生物體感染引起之疾病或降低其嚴重程度。抗體組合物可包括結合於多種抗原之抗體，該等抗原可繼而與各種生物體結合。抗體組分可為多株抗血清。在某些態樣中，一或多種抗體係自已用一或多種抗原攻擊之動物或第二個體親和純化得到。或者，可使用抗體混合物，其為針對相同、相關或不同微生物或生物體(諸如病毒，包括但不限於RSV)中存在之抗原的單株及/或多株抗體之混合物。As used herein, "passive immunization" includes, but is not limited to, the administration of activated immune effectors, including cellular mediators or protein mediators of immune responses (e.g., monoclonal and/or polyclonal antibodies). Monoclonal or polyclonal antibody compositions can be used for passive immunization to treat, prevent, or reduce the severity of diseases caused by infection with organisms carrying antigens recognized by the antibodies. Antibody compositions may include antibodies that bind to a variety of antigens, which in turn can bind to a variety of organisms. The antibody component may be a polyclonal antiserum. In certain aspects, one or more antibodies are affinity purified from an animal or a second individual that has been challenged with one or more antigens. Alternatively, an antibody mixture may be used, which is a mixture of monoclonal and/or polyclonal antibodies directed against antigens present in the same, related, or different microorganisms or organisms (such as viruses, including but not limited to RSV).
可藉由向患者投與獲自具有已知免疫反應性之供體或其他非患者來源的免疫球蛋白(Ig)及/或其他免疫因子而向患者或個體賦予被動免疫。在其他態樣中，可向個體投與本發明之免疫原性組合物，該個體隨後充當含有針對RSV或其他生物體之抗體的球蛋白之來源或供體，該球蛋白係回應於免疫原性組合物之攻擊而產生(「超免疫球蛋白」)。由此治療之個體將供給血漿，接著經由習知血漿分離方法自其中獲得超免疫球蛋白，且投與給另一個體以賦予針對RSV感染之抗性或治療RSV感染。Passive immunity can be conferred to a patient or individual by administering to the patient immunoglobulins (Ig) and/or other immune factors obtained from a donor of known immunoreactivity or other non-patient source. In other aspects, an immunogenic composition of the invention can be administered to an individual who then serves as a source or donor of globulins containing antibodies to RSV or other organisms produced in response to challenge with the immunogenic composition ("hyperimmune globulins"). The individual thus treated would be given plasma from which hyperimmune globulins would then be obtained by known plasma separation methods and administered to another individual to confer resistance to or treat RSV infection.
出於本說明書及隨附申請專利範圍之目的，術語「抗原決定基」及「抗原決定子」可互換地用於指B及/或T細胞回應或識別之抗原上之位點。B細胞抗原決定基可由藉由蛋白質之三級摺疊而並置的連續胺基酸或非連續胺基酸形成。由連續胺基酸形成的抗原決定基通常在暴露於變性溶劑後保留，而藉由三級摺疊形成的抗原決定基通常在用變性溶劑處理後消失。抗原決定基通常包括呈獨特空間構形之至少3個且更通常至少5個或8至10個胺基酸。測定抗原決定基之空間構形之方法包括例如x射線結晶學及2維核磁共振。參見例如Epitope Mapping Protocols (1996)。識別相同抗原決定基之抗體可在顯示一種抗體阻斷另一抗體與目標抗原之結合的能力的簡單免疫分析中鑑別。T細胞針對CD8細胞識別約九個胺基酸之連續抗原決定基或針對CD4細胞識別約13至15個胺基酸之連續抗原決定基。識別抗原決定基之T細胞可藉由活體外分析、藉由抗原依賴性殺死(細胞毒性T淋巴細胞分析，Tigges等人, 1996)或藉由細胞介素分泌來鑑別，該等活體外分析量測抗原依賴性增殖，其係藉由回應於抗原決定基之致敏T細胞引起之3H-胸苷併入所測定(Burke等人, 1994)。For the purposes of this specification and the accompanying patent applications, the terms "antigenic determinant" and "antigenic determinant" are used interchangeably to refer to sites on antigens that B and/or T cells respond to or recognize. B cell antigenic determinants can be formed by consecutive amino acids or non-consecutive amino acids juxtaposed by tertiary folding of proteins. Antigenic determinants formed by consecutive amino acids are generally retained after exposure to denaturing solvents, while antigenic determinants formed by tertiary folding generally disappear after treatment with denaturing solvents. Antigenic determinants generally include at least 3 and more generally at least 5 or 8 to 10 amino acids in a unique spatial configuration. Methods for determining the spatial configuration of antigenic determinants include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. See, e.g., Epitope Mapping Protocols (1996). Antibodies that recognize the same antigenic determinant can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to the target antigen. T cells recognize a continuous antigenic determinant of about nine amino acids for CD8 cells or about 13 to 15 amino acids for CD4 cells. T cells that recognize the antigenic determinant can be identified by in vitro assays that measure antigen-dependent proliferation as measured by3H -thymidine incorporation by primed T cells in response to the antigenic determinant (Burke et al., 1994), by antigen-dependent killing (cytotoxic T lymphocyte assay, Tigges et al., 1996), or by interleukin secretion.
細胞介導之免疫反應的存在可藉由增殖分析(CD4 (+) T細胞)或CTL (細胞毒性T淋巴細胞)分析法來測定。體液及細胞反應對免疫原性組合物之保護或治療作用之相對貢獻可藉由分別自經免疫接種之同基因型動物分離IgG及T細胞且量測第二個體中之保護或治療作用來區分。The presence of a cell-mediated immune response can be determined by proliferation assays (CD4 (+) T cells) or CTL (cytotoxic T lymphocyte) assays. The relative contribution of humoral and cellular responses to the protective or therapeutic effects of an immunogenic composition can be distinguished by isolating IgG and T cells, respectively, from immunized animals of the same genotype and measuring protection or therapeutic effects in a second individual.
如本文所用，術語「抗體」或「免疫球蛋白」可互換使用，且係指作為動物或接受者之免疫反應之一部分起作用的若干類別之結構相關蛋白質中之任一者，該等蛋白質包括IgG、IgD、IgE、IgA、IgM及相關蛋白質。在正常生理條件下，抗體存在於血漿及其他體液中以及某些細胞之膜中，且由類型指示為B細胞之淋巴細胞或其功能等效物產生。As used herein, the terms "antibody" or "immunoglobulin" are used interchangeably and refer to any of several classes of structurally related proteins that function as part of an immune response in an animal or recipient, including IgG, IgD, IgE, IgA, IgM, and related proteins. Under normal physiological conditions, antibodies are present in plasma and other body fluids and in the membranes of certain cells, and are produced by lymphocytes of a type indicated as B cells or their functional equivalents.
如本文所用，術語「免疫原性劑」或「免疫原」或「抗原」可互換使用以描述在單獨、與佐劑結合或呈現於呈遞媒劑上向接受者投與時能夠誘導針對其自身之免疫反應的分子。As used herein, the terms "immunogenic agent" or "immunogen" or "antigen" are used interchangeably to describe a molecule that is capable of inducing an immune response against itself when administered to a recipient, alone, in combination with an adjuvant, or presented in a presentation vehicle.
VIII.組合物在一些態樣中，本文所揭示之RNA分子及/或RNA-LNP可以醫藥組合物或藥劑形式投與且可以任何適合醫藥組合物之形式投與。在一些態樣中，醫藥組合物係用於治療性及/或防治性治療。在一個態樣中，本發明係關於一種用於向宿主投與之組合物。在一些態樣中，該宿主為人類。在其他態樣中，該宿主為非人類。VIII.Compositions In some aspects, the RNA molecules and/or RNA-LNPs disclosed herein can be administered in the form of a pharmaceutical composition or medicament and can be administered in the form of any suitable pharmaceutical composition. In some aspects, the pharmaceutical composition is used for therapeutic and/or prophylactic treatment. In one aspect, the present invention relates to a composition for administration to a host. In some aspects, the host is a human. In other aspects, the host is non-human.
本文所描述之疫苗組合物之調配物可藉由藥理學技術中已知或此後研發之任何方法來製備。一般而言，此類製備方法包括以下之步驟：使活性成分(例如RNA分子及/或RNA-LNP)與賦形劑及/或一或多種其他輔助成分結合，及接著必要及/或需要時，將產物分割、塑形及/或封裝成所需單劑量或多劑量單位。根據本發明之醫藥組合物或調配物可以散裝形式、以單一單位劑量形式及/或以複數個單一單位劑量形式製備、封裝及/或出售。根據本發明之醫藥組合物中活性成分、醫藥學上可接受之賦形劑及/或任何額外成分之相對量將視治療之個體之身分、體型及/或病狀且進一步視組合物投與之途徑而變化。藉助於實例，組合物可包含0.1%至100%之間，例如0.5至50%之間、1至30%之間、5至80%之間、至少80%(w/w)，或至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的活性成分：0.1%、0.15%、0.2%、0.25%、0.3%、0.35%、0.4%、0.45%、0.5%、0.55%、0.6%、0.65%、0.7%、0.75%、0.8%、0.85%、0.9%、0.95%、1%、5%、10%、15%、20%、25%、30%、35%、40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、95%或100% (w/w)。諸如本文所描述之組合物之醫藥劑的調配及/或製造中之一般考慮因素可見於例如Remington: The Science and Practice of Pharmacy第21版, Lippincott Williams及Wilkins, 2005年(以全文引用之方式併入本文中)中。The formulations of the vaccine compositions described herein can be prepared by any method known in the art of pharmacology or developed thereafter. In general, such preparation methods include the steps of combining the active ingredient (e.g., RNA molecule and/or RNA-LNP) with a formulator and/or one or more other auxiliary ingredients, and then dividing, shaping, and/or packaging the product into the desired single or multiple dosage units when necessary and/or desired. The pharmaceutical compositions or formulations according to the present invention can be prepared, packaged, and/or sold in bulk form, in a single unit dosage form, and/or in a plurality of single unit dosage forms. The relative amounts of the active ingredient, the pharmaceutically acceptable excipient and/or any additional ingredients in the pharmaceutical composition according to the present invention will vary depending on the identity, size and/or condition of the subject to be treated and further on the route of administration of the composition. By way of example, the composition may comprise between 0.1% and 100% active ingredient, for example, between 0.5 and 50%, between 1 and 30%, between 5 and 80%, at least 80% (w/w), or at least, at most, just below, or between any two of the following (inclusive or exclusive), inclusively or exclusively: 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% (w/w). General considerations in the formulation and/or manufacture of pharmaceuticals such as the compositions described herein can be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).
在一些態樣中，本文所揭示之RNA分子及/或RNA-LNP可以醫藥組合物形式投與，該醫藥組合物可調配成固體、半固體、液體、凍乾、冷凍及/或氣體形式之製劑。在一些態樣中，本文所揭示之RNA分子及/或RNA-LNP可以醫藥組合物形式投與，該醫藥組合物可包含醫藥學上可接受之載劑且可視情況包含一或多種佐劑、穩定劑、鹽、緩衝劑、防腐劑及視情況存在之其他治療劑。在一些態樣中，本文所揭示之醫藥組合物包含一或多種醫藥學上可接受之載劑、稀釋劑及/或賦形劑。在一些態樣中，醫藥組合物不包括佐劑(例如，其不含佐劑)。In some aspects, the RNA molecules and/or RNA-LNP disclosed herein can be administered in the form of a pharmaceutical composition, which can be formulated into a solid, semi-solid, liquid, lyophilized, frozen and/or gaseous preparation. In some aspects, the RNA molecules and/or RNA-LNP disclosed herein can be administered in the form of a pharmaceutical composition, which can contain a pharmaceutically acceptable carrier and can optionally contain one or more adjuvants, stabilizers, salts, buffers, preservatives and other therapeutic agents as appropriate. In some aspects, the pharmaceutical composition disclosed herein contains one or more pharmaceutically acceptable carriers, diluents and/or excipients. In some aspects, the pharmaceutical composition does not include an adjuvant (e.g., it is adjuvant-free).
如本文所用，術語「賦形劑」係指本發明之醫藥組合物中可存在但不為活性成分之物質。賦形劑之實例包括但不限於載劑、稀釋劑(例如溶劑、分散介質及/或其他液體媒劑、分散液或懸浮助劑)、造粒劑及/或分散劑、界面活性劑、等張劑、增稠劑及/或乳化劑、防腐劑、黏合劑、潤滑劑及/或油、著色劑、甜味劑及/或調味劑、穩定劑、抗氧化劑、抗微生物劑及/或抗真菌劑、重量莫耳滲透濃度調節劑、pH調節劑、緩衝劑、螯合劑、低溫保護劑及/或增積劑。在一些態樣中，前述賦形劑中之1、2、3、4、5者或更多者可排除在本文所揭示之醫藥組合物外。As used herein, the term "excipient" refers to a substance that may be present in the pharmaceutical composition of the present invention but is not an active ingredient. Examples of excipients include, but are not limited to, carriers, diluents (e.g., solvents, dispersion media and/or other liquid vehicles, dispersion or suspension aids), granulating and/or dispersing agents, surfactants, isotonic agents, thickeners and/or emulsifiers, preservatives, binders, lubricants and/or oils, colorants, sweeteners and/or flavoring agents, stabilizers, antioxidants, antimicrobial and/or antifungal agents, weight molar osmotic concentration adjusters, pH adjusters, buffers, chelating agents, low temperature protective agents and/or bulking agents. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned excipients may be excluded from the pharmaceutical compositions disclosed herein.
術語「載劑」係指可為天然、合成、有機或無機之組分，活性組分合併於該組分中以便有助於、增強及/或實現醫藥組合物之投與。如本文所用，載劑可為一或多種適合於向個體投與的相容固體或液體填充劑、稀釋劑或囊封物質。適合載劑包括但不限於無菌水、林格氏溶液(Ringer's solution)、林格氏乳酸鹽溶液、無菌氯化鈉溶液、等張鹽水、聚伸烷二醇、氫化萘，以及尤其生物相容性乳酸交酯聚合物、乳酸交酯/乙交酯共聚物或聚氧乙烯/聚氧丙烯共聚物。在一些態樣中，本發明之醫藥組合物包括氯化鈉。在一些態樣中，前述載劑中之1、2、3、4、5者或更多者可排除在本文所揭示之醫藥組合物外。The term "carrier" refers to a component that may be natural, synthetic, organic or inorganic, into which the active ingredient is incorporated in order to facilitate, enhance and/or enable administration of the pharmaceutical composition. As used herein, a carrier may be one or more compatible solid or liquid fillers, diluents or encapsulating materials suitable for administration to an individual. Suitable carriers include, but are not limited to, sterile water, Ringer's solution, Ringer's lactate solution, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes, and especially biocompatible lactide polymers, lactide/glycolide copolymers or polyoxyethylene/polyoxypropylene copolymers. In some aspects, the pharmaceutical composition of the present invention includes sodium chloride. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned carriers may be excluded from the pharmaceutical compositions disclosed herein.
術語「稀釋劑」係關於稀釋及/或稀化劑。此外，術語「稀釋劑」包括流體、液體或固體懸浮液及/或混合介質中之任何一或多者。適合用於本發明之醫藥組合物中之稀釋劑之實例包括但不限於乙醇、甘油、鹽水、水、碳酸鈣或碳酸鈉、磷酸鈣、磷酸氫鈣、磷酸鈉、乳糖、蔗糖、纖維素、微晶纖維素、高嶺土、甘露糖醇、山梨糖醇等，及/或其組合。在一些態樣中，前述稀釋劑中之1、2、3、4、5者或更多者可排除在本文所揭示之醫藥組合物外。The term "diluent" refers to a diluent and/or a thinning agent. In addition, the term "diluent" includes any one or more of a fluid, a liquid or solid suspension and/or a mixed medium. Examples of diluents suitable for use in the pharmaceutical composition of the present invention include, but are not limited to, ethanol, glycerol, saline, water, calcium carbonate or sodium carbonate, calcium phosphate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, etc., and/or combinations thereof. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned diluents may be excluded from the pharmaceutical composition disclosed herein.
適合造粒劑及/或分散劑之實例包括但不限於澱粉、預糊化澱粉、或微晶澱粉、褐藻酸、瓜爾豆膠、瓊脂、聚(乙烯基-吡咯啶酮) (聚維酮(providone))、交聯化聚(乙烯基-吡咯啶酮) (交聯聚維酮)、纖維素、甲基纖維素、羧甲基纖維素、交聯化羧甲基纖維素鈉(交聯羧甲纖維素)、矽酸鎂鋁(VEEGUM®)、月桂基硫酸鈉等，及/或其組合。在一些態樣中，前述造粒劑及/或分散劑中之1、2、3、4、5者或更多者可排除在本文所揭示之醫藥組合物外。Examples of suitable granulating agents and/or dispersing agents include, but are not limited to, starch, pregelatinized starch, or microcrystalline starch, alginic acid, guar gum, agar, poly(vinyl-pyrrolidone) (providone), cross-linked poly(vinyl-pyrrolidone) (cross-linked providone), cellulose, methylcellulose, carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (cross-linked carboxymethylcellulose), magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, etc., and/or combinations thereof. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned granulating agents and/or dispersing agents may be excluded from the pharmaceutical compositions disclosed herein.
適合用於本發明之醫藥組合物中之界面活性劑之實例包括但不限於天然乳化劑(例如阿拉伯膠、瓊脂、褐藻酸、褐藻酸鈉、黃蓍膠、鹿角菜、膽固醇、黃原膠、果膠、明膠、蛋黃、酪蛋白、羊毛脂、膽固醇、蠟及卵磷脂)、脫水山梨糖醇脂肪酸酯(例如聚氧乙烯脫水山梨糖醇單油酸酯[TWEEN®80]、脫水山梨糖醇單棕櫚酸酯[SPAN®40]、單油酸甘油酯、聚氧乙烯酯、聚乙二醇脂肪酸酯(例如CREMOPHOR®)、聚氧乙烯醚(例如聚氧乙烯月桂基醚[BRIJ®30])、PLUORINC®F 68、POLOXAMER®188等，及/或其組合。在一些態樣中，前述界面活性劑中之1、2、3、4、5者或更多者可排除在本文所揭示之醫藥組合物外。Examples of surfactants suitable for use in the pharmaceutical compositions of the present invention include, but are not limited to, natural emulsifiers (e.g., gum arabic, agar, alginic acid, sodium alginate, tragacanth gum, carrageenan, cholesterol, xanthan gum, pectin, gelatin, egg yolk, casein, lanolin, cholesterol, wax, and lecithin), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monooleate [TWEEN® 80], sorbitan monopalmitate [SPAN® 40], glyceryl monooleate, polyoxyethylene esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR®), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [BRIJ® 30]), PLUORINC® F 68, POLOXAMER® 188, etc., and/or combinations thereof. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned surfactants may be excluded from the pharmaceutical composition disclosed herein.
適合用於本發明之醫藥組合物中之防腐劑之實例包括但不限於氯化烷基二甲基苄基銨、氯丁醇、對羥基苯甲酸酯、硫柳汞、維生素A、維生素C、維生素E、β-胡蘿蔔素、檸檬酸、抗壞血酸、丁基化羥基甲氧苯、乙二胺、月桂基硫酸鈉(SLS)、月桂基醚硫酸鈉(SLES)等，及其組合。在一些態樣中，前述防腐劑中之1、2、3、4、5者或更多者可排除在本文所揭示之醫藥組合物外。Examples of preservatives suitable for use in the pharmaceutical composition of the present invention include, but are not limited to, alkyl dimethyl benzyl ammonium chloride, chlorobutanol, p-hydroxybenzoate, thimerosal, vitamin A, vitamin C, vitamin E, β-carotene, citric acid, ascorbic acid, butylated hydroxymethoxyphenyl, ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), etc., and combinations thereof. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned preservatives may be excluded from the pharmaceutical composition disclosed herein.
適合用於本發明之醫藥組合物中之抗微生物劑及/或抗真菌劑之實例包括但不限於苯紮氯銨、苄索氯銨、對羥基苯甲酸甲酯、對羥基苯甲酸乙酯、對羥基苯甲酸丙酯、對羥基苯甲酸丁酯、苯甲酸、羥基苯甲酸、苯甲酸鉀或苯甲酸鈉、山梨酸鉀或山梨酸鈉、丙酸鈉、山梨酸等，及其組合。在一些態樣中，前述抗微生物劑及/或抗真菌劑中之1、2、3、4、5者或更多者可排除在本文所揭示之醫藥組合物外。Examples of antimicrobial and/or antifungal agents suitable for use in the pharmaceutical composition of the present invention include, but are not limited to, benzothiazolidine, benzethonium chloride, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, propyl p-hydroxybenzoate, butyl p-hydroxybenzoate, benzoic acid, hydroxybenzoic acid, potassium or sodium benzoate, potassium or sodium sorbate, sodium propionate, sorbic acid, and the like, and combinations thereof. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned antimicrobial and/or antifungal agents may be excluded from the pharmaceutical composition disclosed herein.
適合用於本發明之醫藥組合物中之黏合劑之實例包括但不限於澱粉、明膠、糖(例如蔗糖、葡萄糖、右旋糖、糊精、糖蜜、乳糖、乳糖醇、甘露糖醇)、胺基酸(例如甘胺酸)、天然及合成膠(例如阿拉伯膠、褐藻酸鈉)、乙基纖維素、羥乙基纖維素、羥丙基甲基纖維素等，及其組合。在一些態樣中，前述黏合劑中之1、2、3、4、5者或更多者可排除在本文所揭示之醫藥組合物外。Examples of binders suitable for use in the pharmaceutical compositions of the present invention include, but are not limited to, starch, gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol), amino acids (e.g., glycine), natural and synthetic gums (e.g., gum arabic, sodium alginate), ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, etc., and combinations thereof. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned binders may be excluded from the pharmaceutical compositions disclosed herein.
適合用於本發明之醫藥組合物中之潤滑劑及/或油之實例包括但不限於硬脂酸鎂、硬脂酸鈣、硬脂酸、二氧化矽、滑石、麥芽、氫化植物油、聚乙二醇、苯甲酸鈉、月桂基硫酸鈉或月桂基硫酸鎂等，及其組合。在一些態樣中，前述潤滑劑及/或油中之1、2、3、4、5者或更多者可排除在本文所揭示之醫藥組合物外。Examples of lubricants and/or oils suitable for use in the pharmaceutical compositions of the present invention include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silicon dioxide, talc, malt, hydrogenated vegetable oil, polyethylene glycol, sodium benzoate, sodium lauryl sulfate or magnesium lauryl sulfate, and combinations thereof. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned lubricants and/or oils may be excluded from the pharmaceutical compositions disclosed herein.
適合用於本發明之醫藥組合物中之抗氧化劑之實例包括但不限於α生育酚、抗壞血酸、抗壞血酸棕櫚酸酯、苯甲醇、丁基化羥基甲氧苯、間甲酚、甲硫胺酸、丁基化羥基甲苯、單硫甘油、偏亞硫酸氫鈉或偏亞硫酸氫鉀、丙酸、沒食子酸丙酯、抗壞血酸鈉等，及其組合。在一些態樣中，前述抗氧化劑中之1、2、3、4、5者或更多者可排除在本文所揭示之醫藥組合物外。Examples of antioxidants suitable for use in the pharmaceutical composition of the present invention include, but are not limited to, alpha-tocopherol, ascorbic acid, ascorbic acid palmitate, benzyl alcohol, butylated hydroxymethoxyphenyl, meta-cresol, methionine, butylated hydroxytoluene, monothioglycerol, sodium metabisulfite or potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, and the like, and combinations thereof. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned antioxidants may be excluded from the pharmaceutical composition disclosed herein.
適合用於本發明之醫藥組合物中之重量莫耳滲透濃度調節劑、pH調節劑及緩衝劑之實例包括但不限於磷酸鈉、檸檬酸鈉、丁二酸鈉、組胺酸(或組胺酸-HCl)、蘋果酸鈉、碳酸鈉等，及/或其組合。在一些態樣中，前述重量莫耳滲透濃度調節劑中之1、2、3、4、5者或更多者可排除在本文所揭示之醫藥組合物外。Examples of weight molar osmotic concentration regulators, pH regulators and buffers suitable for use in the pharmaceutical compositions of the present invention include, but are not limited to, sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium appletate, sodium carbonate, etc., and/or combinations thereof. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned weight molar osmotic concentration regulators may be excluded from the pharmaceutical compositions disclosed herein.
適合用於本發明之醫藥組合物中之螯合劑之實例包括但不限於乙二胺四乙酸(EDTA)、單水合檸檬酸、乙二胺四乙酸二鈉、反丁烯二酸、蘋果酸、磷酸、乙二胺四乙酸鈉、酒石酸、乙二胺四乙酸三鈉等，及其組合。在一些態樣中，前述螯合劑中之1、2、3、4、5者或更多者可排除在本文所揭示之醫藥組合物外。Examples of chelating agents suitable for use in the pharmaceutical compositions of the present invention include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium ethylenediaminetetraacetate, fumaric acid, apple acid, phosphoric acid, sodium ethylenediaminetetraacetate, tartaric acid, trisodium ethylenediaminetetraacetate, and combinations thereof. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned chelating agents may be excluded from the pharmaceutical compositions disclosed herein.
適合用於本發明之醫藥組合物中之低溫保護劑之實例包括但不限於甘露糖醇、蔗糖、海藻糖、乳糖、甘油、右旋糖等，及其組合。在一些態樣中，前述低溫保護劑中之1、2、3、4、5者或更多者可排除在本文所揭示之醫藥組合物外。Examples of cryopreservatives suitable for use in the pharmaceutical compositions of the present invention include, but are not limited to, mannitol, sucrose, trehalose, lactose, glycerol, dextrose, and combinations thereof. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned cryopreservatives may be excluded from the pharmaceutical compositions disclosed herein.
適合增積劑之實例包括但不限於蔗糖、海藻糖、甘露糖醇、甘胺酸、乳糖、棉子糖及其組合。在一些態樣中，前述增積劑中之1、2、3、4、5者或更多者可排除在本文所揭示之醫藥組合物外。Examples of suitable bulking agents include, but are not limited to, sucrose, trehalose, mannitol, glycine, lactose, raffinose, and combinations thereof. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned bulking agents may be excluded from the pharmaceutical composition disclosed herein.
組合物可使用一或多種賦形劑(例如一或多種載體及/或稀釋劑)調配以例如：(1)增加穩定性；(2)增加細胞轉染；(3)允許持續及/或延遲釋放(例如自儲槽式調配物)；(4)改變生物分佈(例如靶向特定組織及/或細胞類型)；(5)增加活體內所編碼蛋白質之轉譯；及/或(6)改變活體內所編碼蛋白質(抗原)之釋放概況。在一些態樣中，可排除前述賦形劑目的中之1、2、3、4、5者或更多者。用於治療用途之醫藥學上可接受之賦形劑(例如載劑及/或稀釋劑)為醫藥技術中熟知的，且描述於例如Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro編輯. 1985)中。The composition can be formulated using one or more excipients (e.g., one or more carriers and/or diluents) to, for example: (1) increase stability; (2) increase cell transfection; (3) allow for sustained and/or delayed release (e.g., self-reservoir formulations); (4) alter biodistribution (e.g., targeting to specific tissues and/or cell types); (5) increase translation of the encoded protein in vivo; and/or (6) alter the release profile of the encoded protein (antigen) in vivo. In some aspects, 1, 2, 3, 4, 5, or more of the foregoing excipients may be excluded. Pharmaceutically acceptable excipients (e.g., carriers and/or diluents) for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro, ed. 1985).
可關於預期投與途徑及標準醫藥實踐來選擇醫藥賦形劑(例如載劑及/或稀釋劑)。The choice of pharmaceutical formulation (e.g., carrier and/or diluent) may be determined with regard to the intended route of administration and standard pharmaceutical practice.
在一些態樣中，組合物包含含有編碼免疫原性多肽之開讀框的RNA分子。在一些態樣中，免疫原性多肽包含RSV抗原。在一些態樣中，RSV抗原為RSV F蛋白或其片段或變體。In some aspects, the composition comprises an RNA molecule containing an open reading frame encoding an immunogenic polypeptide. In some aspects, the immunogenic polypeptide comprises an RSV antigen. In some aspects, the RSV antigen is an RSV F protein or a fragment or variant thereof.
在一些態樣中，組合物包含有包含編碼全長RSV F蛋白之開讀框的RNA分子。在一些態樣中，所編碼之免疫原性多肽為截短RSV F蛋白。在一些態樣中，所編碼之免疫原性多肽為RSV F蛋白之變異體。在一些態樣中，所編碼之免疫原性多肽為RSV F蛋白之片段。In some aspects, the composition comprises an RNA molecule comprising an open reading frame encoding a full-length RSV F protein. In some aspects, the encoded immunogenic polypeptide is a truncated RSV F protein. In some aspects, the encoded immunogenic polypeptide is a variant of the RSV F protein. In some aspects, the encoded immunogenic polypeptide is a fragment of the RSV F protein.
A.包括LNP之免疫原性組合物在一些態樣中，醫藥組合物包含與基於脂質之遞送系統一起調配的本文所揭示之RNA分子(例如聚核苷酸)。因此，在一些態樣中，組合物包括基於脂質之遞送系統(例如LNP) (例如基於脂質之疫苗)，其將核酸分子遞送至細胞內部，該核酸分子接著可在細胞內部複製、抑制所關注蛋白質表現及/或表現所編碼之所關注多肽。遞送系統可具有增強所編碼抗原之免疫原性的佐劑作用。在一些態樣中，組合物包含至少一個編碼RSV多肽之RNA分子，該至少一個RNA分子與一或多種脂質複合、囊封於一或多種脂質中及/或與一或多種脂質一起調配，且形成脂質奈米顆粒(LNP)、脂質體、脂質複合物及/或奈米脂質體。在一些態樣中，組合物包含脂質奈米顆粒。因此，在某些態樣中，本發明係關於包含一或多種與核酸或多肽/肽結合之脂質(例如RSV RNA-LNP)的組合物。A.Immunogenic compositionscomprisingLNPs In some aspects, pharmaceutical compositions comprise an RNA molecule (e.g., a polynucleotide) disclosed herein formulated with a lipid-based delivery system. Thus, in some aspects, a composition comprises a lipid-based delivery system (e.g., LNP) (e.g., a lipid-based vaccine) that delivers a nucleic acid molecule to the interior of a cell, where the nucleic acid molecule can then replicate, inhibit expression of a protein of interest, and/or express the encoded polypeptide of interest. The delivery system can have an adjuvant effect that enhances the immunogenicity of the encoded antigen. In some aspects, the composition comprises at least one RNA molecule encoding a RSV polypeptide, which is complexed with one or more lipids, encapsulated in one or more lipids, and/or formulated with one or more lipids to form lipid nanoparticles (LNPs), liposomes, lipid complexes, and/or nanoliposomes. In some aspects, the composition comprises lipid nanoparticles. Therefore, in some aspects, the present invention relates to a composition comprising one or more lipids (e.g., RSV RNA-LNPs) bound to a nucleic acid or polypeptide/peptide.
在一些情況下，包括基於脂質之遞送系統的免疫原性組合物可進一步包括一或多種鹽及/或一或多種醫藥學上可接受之界面活性劑、防腐劑、載劑、稀釋劑及/或賦形劑。在一些態樣中，包括基於脂質之遞送系統的免疫原性組合物進一步包括醫藥學上可接受之媒劑。在一些態樣中，緩衝劑、穩定劑及視情況存在之鹽中之每一者可包括在包括基於脂質之遞送系統的免疫原性組合物中。在其他態樣中，緩衝劑、穩定劑、鹽、界面活性劑、防腐劑及賦形劑中之任何一或多者可排除在包括基於脂質之遞送系統的免疫原性組合物外。In some cases, the immunogenic composition comprising a lipid-based delivery system may further include one or more salts and/or one or more pharmaceutically acceptable surfactants, preservatives, carriers, diluents and/or excipients. In some aspects, the immunogenic composition comprising a lipid-based delivery system further includes a pharmaceutically acceptable vehicle. In some aspects, each of a buffer, a stabilizer, and optionally a salt may be included in the immunogenic composition comprising a lipid-based delivery system. In other aspects, any one or more of buffers, stabilizers, salts, surfactants, preservatives, and excipients may be excluded from the immunogenic composition comprising the lipid-based delivery system.
在另一態樣中，包括基於脂質之遞送系統的免疫原性組合物進一步包含穩定劑。在一些態樣中，穩定劑包含蔗糖、甘露糖、山梨糖醇、棉子糖、海藻糖、甘露糖醇、肌醇、氯化鈉、精胺酸、乳糖、羥乙基澱粉、聚葡萄糖、聚乙烯吡咯啶酮、甘胺酸或其組合。在一些態樣中，前述穩定劑中之1、2、3、4、5者或更多者可排除在本文所揭示之免疫原性組合物外。在一些態樣中，穩定劑為雙醣或糖。在一個態樣中，穩定劑為蔗糖。在另一態樣中，穩定劑為海藻糖。在另一態樣中，穩定劑為蔗糖與海藻糖之組合。在一些態樣中，組合物中一或多種穩定劑之總濃度為或為約5%至約10% w/v。舉例而言，穩定劑之總濃度可為或可不為至少、至多、恰好等於以下或在以下之間(包括性或排他性)：1%、2%、3%、4%、5%、6%、7%、8%、9%、10%、11%、12%、13%、14%、15%、16%、17%、18%、19%或20% w/v，或其中可導出之任何範圍或值。在一些態樣中，穩定劑濃度包括但不限於為或為約10 mg/mL至約400 mg/mL、約100 mg/mL至約200 mg/mL、約100 mg/mL至約150 mg/mL、約100 mg/mL至約140 mg/mL、約100 mg/mL至約130 mg/mL、約100 mg/mL至約120 mg/mL、約100 mg/mL至約110 mg/mL、或約100 mg/mL至約105 mg/mL之濃度。在一些態樣中，穩定劑之濃度為或不為至少、至多、恰好等於以下或在以下之間(包括性或排他性)：10 mg/mL、20 mg/mL、50 mg/mL、100 mg/mL、101 mg/mL、102 mg/mL、103 mg/mL、104 mg/mL、105 mg/mL、106 mg/mL、107 mg/mL、108 mg/mL、109 mg/mL、110 mg/mL、150 mg/mL、200 mg/mL、300 mg/mL、400 mg/mL或更高。In another aspect, the immunogenic composition comprising a lipid-based delivery system further comprises a stabilizer. In some aspects, the stabilizer comprises sucrose, mannose, sorbitol, raffinose, trehalose, mannitol, inositol, sodium chloride, arginine, lactose, hydroxyethyl starch, polydextrose, polyvinylpyrrolidone, glycine or a combination thereof. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned stabilizers may be excluded from the immunogenic composition disclosed herein. In some aspects, the stabilizer is a disaccharide or sugar. In one aspect, the stabilizer is sucrose. In another aspect, the stabilizer is trehalose. In another aspect, the stabilizer is a combination of sucrose and trehalose. In some aspects, the total concentration of one or more stabilizers in the composition is or is about 5% to about 10% w/v. For example, the total concentration of stabilizers may or may not be at least, at most, exactly equal to, or between (inclusive or exclusive) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% w/v, or any range or value derivable therein. In some aspects, the stabilizer concentration includes, but is not limited to, a concentration of or about 10 mg/mL to about 400 mg/mL, about 100 mg/mL to about 200 mg/mL, about 100 mg/mL to about 150 mg/mL, about 100 mg/mL to about 140 mg/mL, about 100 mg/mL to about 130 mg/mL, about 100 mg/mL to about 120 mg/mL, about 100 mg/mL to about 110 mg/mL, or about 100 mg/mL to about 105 mg/mL. In some aspects, the concentration of the stabilizer is or is not at least, at most, exactly equal to, or between, inclusively or exclusively, 10 mg/mL, 20 mg/mL, 50 mg/mL, 100 mg/mL, 101 mg/mL, 102 mg/mL, 103 mg/mL, 104 mg/mL, 105 mg/mL, 106 mg/mL, 107 mg/mL, 108 mg/mL, 109 mg/mL, 110 mg/mL, 150 mg/mL, 200 mg/mL, 300 mg/mL, 400 mg/mL, or more.
在另一態樣中，穩定劑之質量的量與RNA之質量的量呈特定比率。在一個態樣中，穩定劑與RNA之質量的量之比率不超過5000。在另一態樣中，穩定劑與RNA之質量的量之比率不超過2000。在另一態樣中，穩定劑與RNA之質量的量之比率不超過1000。在另一態樣中，穩定劑與RNA之質量的量之比率不超過500。在另一態樣中，穩定劑與RNA之質量的量之比率不超過100。在另一態樣中，穩定劑與醫藥物質之質量的量之比率不超過50。在另一態樣中，穩定劑與RNA之質量的量之比率不超過10。在另一態樣中，穩定劑與RNA之質量的量之比率不超過1。在另一態樣中，穩定劑與RNA之質量的量之比率不超過0.5。在另一態樣中，穩定劑與RNA之質量的量之比率不超過0.1。在另一態樣中，穩定劑及RNA包含為或為約200至2000:1之質量比的穩定劑及RNA。In another aspect, the amount of the mass of the stabilizer is in a specific ratio to the amount of the mass of the RNA. In one aspect, the ratio of the amount of the mass of the stabilizer to the amount of the mass of the RNA is no more than 5000. In another aspect, the ratio of the amount of the mass of the stabilizer to the amount of the mass of the RNA is no more than 2000. In another aspect, the ratio of the amount of the mass of the stabilizer to the amount of the mass of the RNA is no more than 1000. In another aspect, the ratio of the amount of the mass of the stabilizer to the amount of the mass of the RNA is no more than 500. In another aspect, the ratio of the amount of the mass of the stabilizer to the amount of the mass of the RNA is no more than 100. In another aspect, the ratio of the amount of the mass of the stabilizer to the amount of the mass of the pharmaceutical substance is no more than 50. In another aspect, the ratio of the amount of the mass of the stabilizer to the amount of the mass of the RNA is no more than 10. In another embodiment, the ratio of the mass of the stabilizer to the RNA is no more than 1. In another embodiment, the ratio of the mass of the stabilizer to the RNA is no more than 0.5. In another embodiment, the ratio of the mass of the stabilizer to the RNA is no more than 0.1. In another embodiment, the stabilizer and the RNA comprise a stabilizer and the RNA at a mass ratio of or about 200 to 2000:1.
在一些態樣中，包括基於脂質之遞送系統的免疫原性組合物進一步包含緩衝劑。緩衝劑之實例包括但不限於檸檬酸鹽緩衝溶液、乙酸鹽緩衝溶液、磷酸鹽緩衝溶液、氯化銨、碳酸鈣、氯化鈣、檸檬酸鈣、葡乳醛酸鈣、葡庚糖酸鈣、葡糖酸鈣、d-葡萄糖酸、甘油磷酸鈣、乳酸鈣、乳糖酸鈣、丙酸、乙醯丙酸鈣、戊酸、磷酸氫鈣、磷酸、磷酸鈣、磷酸氫氧化鈣、乙酸鉀、氯化鉀、葡糖酸鉀、鉀混合物、磷酸氫二鉀、磷酸二氫鉀、磷酸鉀混合物、乙酸鈉、碳酸氫鈉、氯化鈉、檸檬酸鈉、乳酸鈉、磷酸氫二鈉、磷酸二氫鈉、磷酸鈉混合物、緩血酸胺、Tris鹽酸(HCl)、胺基磺酸鹽緩衝液(例如HEPES)、氫氧化鎂、氫氧化鋁、褐藻酸、無熱原水、等張鹽水、林格氏溶液、乙醇及/或其組合。在一些態樣中，前述緩衝劑中之1、2、3、4、5者或更多者可排除在本文所揭示之免疫原性組合物外。在一些態樣中，緩衝劑為HEPES緩衝液、Tris緩衝液及/或PBS緩衝液。在一個態樣中，緩衝劑為Tris緩衝液。在另一態樣中，緩衝劑為HEPES緩衝液。在另一態樣中，緩衝劑為PBS緩衝液。舉例而言，緩衝液濃度可為或可不為至少、至多、恰好等於以下或在以下之間(包括性或排他性)：1 mM、2 mM、3 mM、4 mM、5 mM、6 mM、7 mM、8 mM、9 mM、10 mM、11 mM、12 mM、13 mM、14 mM、15 mM、16 mM、17 mM、18 mM、19 mM、或20 mM，或其中可導出之任何範圍或值。緩衝液可呈中性pH、pH 6.5至8.5、pH 7.0至pH 8.0、或pH 7.2至pH 7.6。舉例而言，緩衝液可呈或可不呈至少、至多、恰好以下pH或以下之間(包括性或排他性)的pH：6.5、6.6、6.7、6.8、6.9、7、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9、8、8.1、8.2、8.3、8.4、或8.5，或其中可導出之任何範圍或值。在特定態樣中，緩衝液呈pH 7.4。In some aspects, the immunogenic composition comprising a lipid-based delivery system further comprises a buffer. Examples of buffers include, but are not limited to, citrate buffer, acetate buffer, phosphate buffer, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glucuronate, calcium glucoheptonate, calcium gluconate, d-gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propionic acid, calcium acetylpropionic acid, valeric acid, calcium hydrogen phosphate, phosphoric acid, calcium phosphate, calcium hydrogen phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium Mixture, potassium dihydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, sodium dihydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate mixture, buffer amine, Tris hydrochloric acid (HCl), sulfamate buffer (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethanol and/or combinations thereof. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned buffers may be excluded from the immunogenic composition disclosed herein. In some embodiments, the buffer is HEPES buffer, Tris buffer and/or PBS buffer. In one embodiment, the buffer is Tris buffer. In another embodiment, the buffer is HEPES buffer. In another embodiment, the buffer is PBS buffer. For example, the buffer concentration may or may not be at least, at most, exactly equal to, or between (inclusive or exclusive) 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM, or any range or value derivable therein. The buffer may be at a neutral pH, pH 6.5 to 8.5, pH 7.0 to pH 8.0, or pH 7.2 to pH 7.6. For example, the buffer may or may not be at least, at most, just below, or below (inclusive or exclusive) 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, or 8.5, or any range or value derivable therein. In a specific aspect, the buffer is at pH 7.4.
在一些態樣中，包括基於脂質之遞送系統的免疫原性組合物可進一步包含鹽。鹽之實例包括但不限於鈉鹽及/或鉀鹽。在一個態樣中，鹽為鈉鹽。在一特定態樣中，鈉鹽為氯化鈉。在一個態樣中，鹽為鉀鹽。在一些態樣中，鉀鹽包含氯化鉀。在一些態樣中，前述鹽中之任何一或多者可排除在本文所揭示之免疫原性組合物外。組合物中鹽之濃度可為或為約70 mM至約140 mM。舉例而言，鹽濃度可為或不可為至少、至多、恰好等於以下或在以下之間(包括性或排他性)：50 mM、60 mM、70 mM、80 mM、90 mM、100 mM、120 mM、130 mM、140 mM、150 mM、160 mM、170 mM、180 mM、190 mM或200 mM。In some aspects, the immunogenic composition comprising a lipid-based delivery system may further comprise a salt. Examples of salts include, but are not limited to, sodium salts and/or potassium salts. In one aspect, the salt is a sodium salt. In a particular aspect, the sodium salt is sodium chloride. In one aspect, the salt is a potassium salt. In some aspects, the potassium salt comprises potassium chloride. In some aspects, any one or more of the aforementioned salts may be excluded from the immunogenic composition disclosed herein. The concentration of the salt in the composition may be or is about 70 mM to about 140 mM. For example, the salt concentration may or may not be at least, at most, exactly equal to, or between, inclusively or exclusively, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM.
在一些態樣中，鹽濃度包括但不限於為或為約1 mg/mL至約100 mg/mL、約1 mg/mL至約50 mg/mL、約1 mg/mL至約40 mg/mL、約1 mg/mL至約30 mg/mL、約1 mg/mL至約20 mg/mL、約1 mg/mL至約10 mg/mL、或約1 mg/mL至約15 mg/mL之濃度。在一些態樣中，鹽之濃度為或不為至少、至多、恰好等於以下或在以下之間(包括性或排他性)：1 mg/mL、2 mg/mL、3 mg/mL、4 mg/mL、5 mg/mL、6 mg/mL、7 mg/mL、8 mg/mL、9 mg/mL、10 mg/mL、11 mg/mL、12 mg/mL、13 mg/mL、14 mg/mL、15 mg/mL、16 mg/mL、17 mg/mL、18 mg/mL、19 mg/mL、20 mg/mL或更高。鹽可呈中性pH、pH 6.5至8.5、pH 7.0至pH 8.0、或pH 7.2至pH 7.6。舉例而言，鹽可呈或可不呈至少、至多、恰好等於以下或在以下之間(包括性或排他性)的pH：6.5、6.6、6.7、6.8、6.9、7、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9、8、8.1、8.2、8.3、8.4或8.5。In some aspects, the salt concentration includes, but is not limited to, a concentration of or about 1 mg/mL to about 100 mg/mL, about 1 mg/mL to about 50 mg/mL, about 1 mg/mL to about 40 mg/mL, about 1 mg/mL to about 30 mg/mL, about 1 mg/mL to about 20 mg/mL, about 1 mg/mL to about 10 mg/mL, or about 1 mg/mL to about 15 mg/mL. In some aspects, the concentration of the salt is or is not at least, at most, exactly equal to, or between, inclusively or exclusively, 1 mg/mL, 2 mg/mL, 3 mg/mL, 4 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, 9 mg/mL, 10 mg/mL, 11 mg/mL, 12 mg/mL, 13 mg/mL, 14 mg/mL, 15 mg/mL, 16 mg/mL, 17 mg/mL, 18 mg/mL, 19 mg/mL, 20 mg/mL or more. The salt may be at a neutral pH, pH 6.5 to 8.5, pH 7.0 to pH 8.0, or pH 7.2 to pH 7.6. For example, the salt may or may not be at a pH of at least, at most, exactly, or between, inclusively or exclusively, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, or 8.5.
在一些態樣中，包括基於脂質之遞送系統的免疫原性組合物進一步包含界面活性劑、防腐劑、任何其他賦形劑或其組合。如本文所用，「任何其他賦形劑」包括但不限於抗氧化劑、麩胱甘肽、EDTA、甲硫胺酸、甲磺酸去鐵胺(desferal)、抗氧化劑、金屬清除劑及/或游離基清除劑。在一個態樣中，界面活性劑、防腐劑、賦形劑或其組合為無菌注射用水(sWFI)、抑菌注射用水(BWFI)、鹽水、右旋糖溶液、聚山梨醇酯、泊洛沙姆(poloxamer)、曲拉通、二價陽離子、乳酸林格氏液、胺基酸、糖、多元醇、聚合物及/或環糊精。在一些態樣中，前述賦形劑中之1、2、3、4、5者或更多者可排除在本文所揭示之免疫原性組合物外。In some aspects, the immunogenic composition comprising a lipid-based delivery system further comprises a surfactant, a preservative, any other excipient, or a combination thereof. As used herein, "any other excipient" includes, but is not limited to, antioxidants, glutathione, EDTA, methionine, desferal, antioxidants, metal scavengers, and/or free radical scavengers. In one aspect, the surfactant, preservative, excipient, or a combination thereof is sterile water for injection (sWFI), bacteriostatic water for injection (BWFI), saline, dextrose solution, polysorbate, poloxamer, triton, divalent cations, lactated Ringer's solution, amino acids, sugars, polyols, polymers, and/or cyclodextrins. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned excipients may be excluded from the immunogenic compositions disclosed herein.
賦形劑係指免疫原性組合物中不為活性成分之成分，其其他實例包括但不限於載劑、黏合劑、稀釋劑、潤滑劑、增稠劑、界面活性劑、防腐劑、穩定劑、乳化劑、緩衝劑、調味劑、崩解劑、包衣、塑化劑、壓縮劑、濕式造粒劑及/或著色劑。如本文所用，「醫藥學上可接受之載劑」包括任何及所有水性溶劑(例如水、醇/水溶液、鹽水溶液、非經腸媒劑，諸如氯化鈉、林格氏右旋糖(Ringer's dextrose)等)、非水性溶劑(例如丙二醇、聚乙二醇、植物油及諸如油酸乙酯之可注射有機酯)、分散介質、包衣、界面活性劑、抗氧化劑、防腐劑(例如抗細菌或抗真菌劑、抗氧化劑、螯合劑及惰性氣體)、等張劑、吸收延遲劑、鹽、藥物、藥物穩定劑、凝膠、黏合劑、賦形劑、崩解劑、潤滑劑、甜味劑、調味劑、染料、流體及營養補充劑，諸如一般熟習此項技術者將已知之物質及其組合。稀釋劑或者稀釋或稀化劑包括但不限於乙醇、甘油、水、糖(諸如乳糖、蔗糖、甘露糖醇及山梨糖醇)及來源於小麥、玉米水稻及馬鈴薯之澱粉；及纖維素，諸如微晶纖維素。組合物中稀釋劑之量按總組合物之重量計可在或在約10%至約90%之範圍內，例如在或在約25%至約75%、約30%至約60%、或約12%至約60%之範圍內。用於本文所揭示之組合物中的防腐劑包括但不限於苯紮氯銨、氯丁醇、對羥基苯甲酸酯及硫柳汞。在一些態樣中，前述賦形劑中之1、2、3、4、5者或更多者可排除在本文所揭示之免疫原性組合物外。Excipients refer to ingredients in the immunogenic composition that are not active ingredients. Other examples include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, disintegrants, coatings, plasticizers, compressing agents, wet granulating agents, and/or coloring agents. As used herein, "pharmaceutically acceptable carriers" include any and all aqueous solvents (e.g., water, alcohol/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, dextrose), etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oils and injectable organic esters such as ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, antioxidants, chelating agents and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavorings, dyes, fluids and nutritional supplements, as well as substances and combinations thereof known to those skilled in the art. Diluents or diluents or thinning agents include, but are not limited to, ethanol, glycerol, water, sugars (such as lactose, sucrose, mannitol and sorbitol) and starches from wheat, corn, rice and potato; and cellulose, such as microcrystalline cellulose. The amount of diluent in the composition may be in the range of or about 10% to about 90% by weight of the total composition, for example, in the range of or about 25% to about 75%, about 30% to about 60%, or about 12% to about 60%. Preservatives used in the compositions disclosed herein include, but are not limited to, benzoic acid ammonium chloride, chlorobutanol, para-hydroxybenzoate and thimerosal. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned excipients may be excluded from the immunogenic compositions disclosed herein.
包括基於脂質之遞送系統的免疫原性組合物中各種組分之pH及確切濃度係根據熟知參數來進行調節。此類介質及藥劑用於醫藥活性物質之用途為此項技術中熟知的。除非任何習知介質或藥劑與活性成分不相容，否則涵蓋其在免疫原性、預防性及/或治療性組合物中之用途。The pH and exact concentration of the various components of immunogenic compositions including lipid-based delivery systems are adjusted according to well-known parameters. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any known media or agents are incompatible with the active ingredient, their use in immunogenic, prophylactic and/or therapeutic compositions is contemplated.
在一個態樣中，醫藥組合物包含編碼本文所揭示之RSV多肽的RSV RNA分子，該RSV RNA分子與一或多種脂質複合、囊封於一或多種脂質中及/或與一或多種脂質一起調配以形成RSV RNA-LNP。在一些態樣中，RSV RNA-LNP組合物為液體。在一些態樣中，RSV RNA-LNP組合物為冷凍的。在一些態樣中，RSV RNA-LNP組合物為凍乾的。在一些態樣中，RSV RNA-LNP組合物包含編碼本文所揭示之RSV多肽的RSV RNA聚核苷酸分子，其囊封於具有陽離子脂質、聚乙二醇化脂質(亦即PEG-脂質)及一或多種結構性脂質(例如中性脂質)之脂質組成的LNP中。在一些態樣中，前述脂質中之任何一或多者可排除在本文所揭示之醫藥組合物之LNP外。In one aspect, the pharmaceutical composition comprises an RSV RNA molecule encoding the RSV polypeptide disclosed herein, which is compounded with one or more lipids, encapsulated in one or more lipids and/or formulated with one or more lipids to form RSV RNA-LNP. In some aspects, the RSV RNA-LNP composition is a liquid. In some aspects, the RSV RNA-LNP composition is frozen. In some aspects, the RSV RNA-LNP composition is freeze-dried. In some aspects, the RSV RNA-LNP composition comprises an RSV RNA polynucleotide molecule encoding the RSV polypeptide disclosed herein, which is encapsulated in a LNP composed of a lipid having a cationic lipid, a PEGylated lipid (i.e., a PEG-lipid) and one or more structural lipids (e.g., a neutral lipid). In some aspects, any one or more of the aforementioned lipids may be excluded from the LNPs of the pharmaceutical compositions disclosed herein.
在一些態樣中，RSV RNA-LNP組合物包含陽離子脂質。陽離子脂質可包含本文所揭示之任何一或多種陽離子脂質。In some aspects, the RSV RNA-LNP composition comprises a cationic lipid. The cationic lipid can comprise any one or more cationic lipids disclosed herein.
陽離子脂質可包含本文所揭示之任何一或多種陽離子脂質。在特定態樣中，陽離子脂質包含((4-羥丁基)氮雜二基)雙(己烷-6,1-二基)雙(2-己基癸酸酯) (ALC-0315)。在一些態樣中，陽離子脂質(例如ALC-0315)以或不以至少以下、至多以下、以下之間(包括性或排他性)或恰好以下之濃度包括在組合物中：0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.2、0.21、0.22、0.23、0.24、0.25、0.26、0.27、0.28、0.29、0.3、0.31、0.32、0.33、0.34、0.35、0.36、0.37、0.38、0.39、0.4、0.41、0.42、0.43、0.44、0.45、0.46、0.47、0.48、0.49、0.5、0.51、0.52、0.53、0.54、0.55、0.56、0.57、0.58、0.59、0.6、0.61、0.62、0.63、0.64、0.65、0.66、0.67、0.68、0.69、0.7、0.71、0.72、0.73、0.74、0.75、0.76、0.77、0.78、0.79、0.8、0.81、0.82、0.83、0.84、0.85、0.86、0.87、0.88、0.89、0.9、0.91、0.92、0.93、0.94、0.95、0.96、0.97、0.98、0.99、1、1.01、1.02、1.03、1.04、1.05、1.06、1.07、1.08、1.09、1.1、1.11、1.12、1.13、1.14、1.15、1.16、1.17、1.18、1.19、1.2、1.21、1.22、1.23、1.24、1.25、1.26、1.27、1.28、1.29、1.3、1.31、1.32、1.33、1.34、1.35、1.36、1.37、1.38、1.39、1.4、1.41、1.42、1.43、1.44、1.45、1.46、1.47、1.48、1.49、1.5、1.51、1.52、1.53、1.54、1.55、1.56、1.57、1.58、1.59、1.6、1.61、1.62、1.63、1.64、1.65、1.66、1.67、1.68、1.69、1.7、1.71、1.72、1.73、1.74、1.75、1.76、1.77、1.78、1.79、1.8、1.81、1.82、1.83、1.84、1.85、1.86、1.87、1.88、1.89、1.9、1.91、1.92、1.93、1.94、1.95、1.96、1.97、1.98、1.99或2 ng/µg/mg/mL。在一些態樣中，陽離子脂質(例如ALC-0315)以或不以至少以下、至多以下、以下之間(包括性或排他性)或恰好以下之濃度包括在組合物中：0.4、0.41、0.42、0.43、0.44、0.45、0.46、0.47、0.48、0.49、0.5、0.51、0.52、0.53、0.54、0.55、0.56、0.57、0.58、0.59、0.6、0.61、0.62、0.63、0.64、0.65、0.66、0.67、0.68、0.69、0.7、0.71、0.72、0.73、0.74、0.75、0.76、0.77、0.78、0.79、0.8、0.81、0.82、0.83、0.84、0.85、0.86、0.87、0.88、0.89、0.9、0.91、0.92、0.93、0.94、0.95、0.96、0.97、0.98、0.99或1 mg/mL。在一些態樣中，陽離子脂質(例如ALC-0315)以至少0.4 mg/mL、至少0.45 mg/mL、至少0.5 mg/mL、至少0.55 mg/mL、至少0.6 mg/mL、至少0.65 mg/mL、至少0.7 mg/mL、至少0.75 mg/mL、至少0.8 mg/mL、至少0.85 mg/mL、至少0.9 mg/mL、至少0.95 mg/mL、或至少1 mg/mL之濃度包括在組合物中。在一些態樣中，陽離子脂質(例如ALC-0315)以0.4與0.5 mg/mL之間、0.5與0.6 mg/mL之間、0.6與0.7 mg/mL之間、0.7與0.8 mg/mL之間、0.8與0.9 mg/mL之間、或0.9與1 mg/mL之間的濃度包括在組合物中。在一些態樣中，陽離子脂質(例如ALC-0315)以0.4與0.45 mg/mL之間、0.45與0.5 mg/mL之間、0.5與0.55 mg/mL之間、0.55與0.6 mg/mL之間、0.6與0.65 mg/mL之間、0.65與0.7 mg/mL之間、0.7與0.75 mg/mL之間、0.75與0.8 mg/mL之間、0.8與0.85 mg/mL之間、0.85與0.9 mg/mL之間、0.9與0.95 mg/mL之間、或0.95與1 mg/mL之間的濃度包括在組合物中。The cationic lipid may comprise any one or more cationic lipids disclosed herein. In a particular aspect, the cationic lipid comprises ((4-hydroxybutyl) azadiyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315). In some aspects, the cationic lipid (e.g., ALC-0315) is or is not included in the composition at a concentration of at least 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13 , 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0 .4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66 , 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.9 3. 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1. 2, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.5, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7, 1.71, 1.72, 1.7 3. 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86 , 1.87, 1.88, 1.89, 1.9, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99 or 2 ng/µg/mg/mL. In some aspects, the cationic lipid (e.g., ALC-0315) is or is not included in the composition at a concentration of at least, at most, between (inclusive or exclusive) or just below 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95 63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, or 1 mg/mL. In some aspects, the cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of at least 0.4 mg/mL, at least 0.45 mg/mL, at least 0.5 mg/mL, at least 0.55 mg/mL, at least 0.6 mg/mL, at least 0.65 mg/mL, at least 0.7 mg/mL, at least 0.75 mg/mL, at least 0.8 mg/mL, at least 0.85 mg/mL, at least 0.9 mg/mL, at least 0.95 mg/mL, or at least 1 mg/mL. In some aspects, the cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of between 0.4 and 0.5 mg/mL, between 0.5 and 0.6 mg/mL, between 0.6 and 0.7 mg/mL, between 0.7 and 0.8 mg/mL, between 0.8 and 0.9 mg/mL, or between 0.9 and 1 mg/mL. In some aspects, the cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of between 0.4 and 0.45 mg/mL, between 0.45 and 0.5 mg/mL, between 0.5 and 0.55 mg/mL, between 0.55 and 0.6 mg/mL, between 0.6 and 0.65 mg/mL, between 0.65 and 0.7 mg/mL, between 0.7 and 0.75 mg/mL, between 0.75 and 0.8 mg/mL, between 0.8 and 0.85 mg/mL, between 0.85 and 0.9 mg/mL, between 0.9 and 0.95 mg/mL, or between 0.95 and 1 mg/mL.
在特定態樣中，陽離子脂質(例如ALC-0315)以0.8至0.95 mg/mL之濃度包括在組合物中。在特定態樣中，陽離子脂質(例如ALC-0315)以或以約0.8至0.9 mg/mL之濃度包括在組合物中。在特定態樣中，陽離子脂質(例如ALC-0315)以或以約0.85至0.9 mg/mL之濃度包括在組合物中。在特定態樣中，陽離子脂質(例如ALC-0315)以或不以以下之濃度或至少、至多、恰好以下或在以下任何兩者之間(包括性或排他性)或約以下之濃度包括在組合物中：0.8、0.81、0.82、0.83、0.84、0.85、0.86、0.87、0.88、0.89、0.9、0.91、0.92、0.93、0.94或0.95 mg/mL。在特定態樣中，陽離子脂質(例如ALC-0315)以或以約0.86 mg/mL之濃度包括在組合物中。凍乾組合物之濃度係在復原後測定。In certain aspects, the cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of 0.8 to 0.95 mg/mL. In certain aspects, the cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of or about 0.8 to 0.9 mg/mL. In certain aspects, the cationic lipid (e.g., ALC-0315) is included in the composition at a concentration of or about 0.85 to 0.9 mg/mL. In certain aspects, the cationic lipid (e.g., ALC-0315) is included in the composition at or below a concentration of at least, at most, just below, or between any two of the following (inclusive or exclusive) or about 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, or 0.95 mg/mL. In certain aspects, the cationic lipid (e.g., ALC-0315) is included in the composition at or about 0.86 mg/mL. The concentration of the lyophilized composition is determined after reconstitution.
在一些態樣中，RSV RNA-LNP組合物進一步包含聚乙二醇化脂質(亦即PEG-脂質)。In some aspects, the RSV RNA-LNP composition further comprises a polyethylene glycolated lipid (ie, PEG-lipid).
聚乙二醇化脂質可包含本文所揭示之任何一或多種聚乙二醇化脂質。在特定態樣中，聚乙二醇化脂質包含2-[(聚乙二醇)-2000]-N,N-二(十四烷基)乙醯胺(ALC-0159)。在一些態樣中，聚乙二醇化脂質(例如ALC-0159)以或不以至少以下、至多以下、以下之間(包括性或排他性)或恰好以下之濃度包括在組合物中：0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.2、0.21、0.22、0.23、0.24、0.25、0.26、0.27、0.28、0.29、0.3、0.31、0.32、0.33、0.34、0.35、0.36、0.37、0.38、0.39、0.4、0.41、0.42、0.43、0.44、0.45、0.46、0.47、0.48、0.49、0.5、0.51、0.52、0.53、0.54、0.55、0.56、0.57、0.58、0.59、0.6、0.61、0.62、0.63、0.64、0.65、0.66、0.67、0.68、0.69、0.7、0.71、0.72、0.73、0.74、0.75、0.76、0.77、0.78、0.79、0.8、0.81、0.82、0.83、0.84、0.85、0.86、0.87、0.88、0.89、0.9、0.91、0.92、0.93、0.94、0.95、0.96、0.97、0.98、0.99、1、1.01、1.02、1.03、1.04、1.05、1.06、1.07、1.08、1.09、1.1、1.11、1.12、1.13、1.14、1.15、1.16、1.17、1.18、1.19、1.2、1.21、1.22、1.23、1.24、1.25、1.26、1.27、1.28、1.29、1.3、1.31、1.32、1.33、1.34、1.35、1.36、1.37、1.38、1.39、1.4、1.41、1.42、1.43、1.44、1.45、1.46、1.47、1.48、1.49、1.5、1.51、1.52、1.53、1.54、1.55、1.56、1.57、1.58、1.59、1.6、1.61、1.62、1.63、1.64、1.65、1.66、1.67、1.68、1.69、1.7、1.71、1.72、1.73、1.74、1.75、1.76、1.77、1.78、1.79、1.8、1.81、1.82、1.83、1.84、1.85、1.86、1.87、1.88、1.89、1.9、1.91、1.92、1.93、1.94、1.95、1.96、1.97、1.98、1.99或2 ng/µg/mg/mL。在一些態樣中，聚乙二醇化脂質(例如ALC-0159)以或不以至少以下、至多以下、以下之間(包括性或排他性)或恰好以下之濃度包括在組合物中：0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.2、0.21、0.22、0.23、0.24、0.25、0.26、0.27、0.28、0.29、0.3、0.31、0.32、0.33、0.34、0.35、0.36、0.37、0.38、0.39、0.4、0.41、0.42、0.43、0.44、0.45、0.46、0.47、0.48、0.49或0.5 mg/mL。在一些態樣中，聚乙二醇化脂質(例如ALC-0159)以至少0.01 mg/mL、至少0.05 mg/mL、至少0.1 mg/mL、至少0.15 mg/mL、至少0.2 mg/mL、至少0.25 mg/mL、至少0.3 mg/mL、至少0.35 mg/mL、至少0.4 mg/mL、至少0.45 mg/mL、或至少0.5 mg/mL之濃度包括在組合物中。在一些態樣中，聚乙二醇化脂質(例如ALC-0159)以0.01與0.05 mg/mL之間、0.05與0.1 mg/mL之間、0.1與0.15 mg/mL之間、0.15與0.2 mg/mL之間、或0.2與0.25 mg/mL之間的濃度包括在組合物中。The PEGylated lipid may comprise any one or more of the PEGylated lipids disclosed herein. In a particular aspect, the PEGylated lipid comprises 2-[(polyethylene glycol)-2000]-N ,N -di(tetradecyl)acetamide (ALC-0159). In some aspects, the PEGylated lipid (e.g., ALC-0159) is or is not included in the composition at a concentration of at least, at most, between (inclusive or exclusive) or just below 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.6 6, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0. 93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1 .2, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, 1.5, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7, 1.71, 1.72, 1.7 3. 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86 , 1.87, 1.88, 1.89, 1.9, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99 or 2 ng/µg/mg/mL. In some aspects, the pegylated lipid (e.g., ALC-0159) is or is not included in the composition at a concentration of at least 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 10. 8, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5 mg/mL. In some aspects, the PEGylated lipid (e.g., ALC-0159) is included in the composition at a concentration of at least 0.01 mg/mL, at least 0.05 mg/mL, at least 0.1 mg/mL, at least 0.15 mg/mL, at least 0.2 mg/mL, at least 0.25 mg/mL, at least 0.3 mg/mL, at least 0.35 mg/mL, at least 0.4 mg/mL, at least 0.45 mg/mL, or at least 0.5 mg/mL. In some aspects, the PEGylated lipid (e.g., ALC-0159) is included in the composition at a concentration of between 0.01 and 0.05 mg/mL, between 0.05 and 0.1 mg/mL, between 0.1 and 0.15 mg/mL, between 0.15 and 0.2 mg/mL, or between 0.2 and 0.25 mg/mL.
在特定態樣中，聚乙二醇化脂質(例如ALC-0159)以或以約0.05至0.15 mg/mL之濃度包括在組合物中。在特定態樣中，聚乙二醇化脂質(例如ALC-0159)以或以約0.10至0.15 mg/mL之濃度包括在組合物中。在特定態樣中，聚乙二醇化脂質(例如ALC-0159)以或不以以下之濃度或至少、至多、恰好以下或在以下任何兩者之間(包括性或排他性)或約以下之濃度包括在組合物中：0.05、0.06、0.07、0.08、0.09、0.1、0.11、0.12、0.13、0.14或0.15 mg/mL。在特定態樣中，聚乙二醇化脂質(例如ALC-0159)以或以約0.11 mg/mL之濃度包括在組合物中。凍乾組合物之濃度係在復原後測定。In certain aspects, the pegylated lipid (e.g., ALC-0159) is included in the composition at a concentration of or about 0.05 to 0.15 mg/mL. In certain aspects, the pegylated lipid (e.g., ALC-0159) is included in the composition at a concentration of or about 0.10 to 0.15 mg/mL. In certain aspects, the pegylated lipid (e.g., ALC-0159) is included in the composition at a concentration of or about 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, or 0.15 mg/mL. In certain aspects, a pegylated lipid (e.g., ALC-0159) is included in the composition at a concentration of or about 0.11 mg/mL. The concentration of the lyophilized composition is determined after reconstitution.
在一些態樣中，RSV RNA-LNP組合物進一步包含一或多種結構性脂質。In some aspects, the RSV RNA-LNP composition further comprises one or more structural lipids.
一或多種結構性脂質可包含本文所揭示之任何一或多種結構性脂質。在特定態樣中，一或多種結構性脂質包含中性脂質及類固醇或類固醇類似物。在特定態樣中，一或多種結構性脂質包含1,2-二硬脂醯基-sn-甘油基-3-磷酸膽鹼(DSPC)及膽固醇。在一些態樣中，一或多種結構性脂質(例如DSPC及膽固醇)以或不以至少以下、至多以下、以下之間(包括性或排他性)或恰好以下之濃度包括在組合物中：0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.2、0.21、0.22、0.23、0.24、0.25、0.26、0.27、0.28、0.29、0.3、0.31、0.32、0.33、0.34、0.35、0.36、0.37、0.38、0.39、0.4、0.41、0.42、0.43、0.44、0.45、0.46、0.47、0.48、0.49、0.5、0.51、0.52、0.53、0.54、0.55、0.56、0.57、0.58、0.59、0.6、0.61、0.62、0.63、0.64、0.65、0.66、0.67、0.68、0.69、0.7、0.71、0.72、0.73、0.74、0.75、0.76、0.77、0.78、0.79、0.8、0.81、0.82、0.83、0.84、0.85、0.86、0.87、0.88、0.89、0.9、0.91、0.92、0.93、0.94、0.95、0.96、0.97、0.98、0.99、1、1.01、1.02、1.03、1.04、1.05、1.06、1.07、1.08、1.09、1.1、1.11、1.12、1.13、1.14、1.15、1.16、1.17、1.18、1.19、1.2、1.21、1.22、1.23、1.24、1.25、1.26、1.27、1.28、1.29、1.3、1.31、1.32、1.33、1.34、1.35、1.36、1.37、1.38、1.39、1.4、1.41、1.42、1.43、1.44、1.45、1.46、1.47、1.48、1.49、1.5、1.51、1.52、1.53、1.54、1.55、1.56、1.57、1.58、1.59、1.6、1.61、1.62、1.63、1.64、1.65、1.66、1.67、1.68、1.69、1.7、1.71、1.72、1.73、1.74、1.75、1.76、1.77、1.78、1.79、1.8、1.81、1.82、1.83、1.84、1.85、1.86、1.87、1.88、1.89、1.9、1.91、1.92、1.93、1.94、1.95、1.96、1.97、1.98、1.99或2 ng/µg/mg/mL。在一些態樣中，一或多種結構性脂質(例如DSPC及膽固醇)以或不以至少以下、至多以下、以下之間(包括性或排他性)或恰好以下之濃度包括在組合物中：0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.2、0.21、0.22、0.23、0.24、0.25、0.26、0.27、0.28、0.29、0.3、0.31、0.32、0.33、0.34、0.35、0.36、0.37、0.38、0.39、0.4、0.41、0.42、0.43、0.44、0.45、0.46、0.47、0.48、0.49或0.5 mg/mL。在一些態樣中，一或多種結構性脂質(例如DSPC及膽固醇)以至少0.05 mg/mL、至少0.1 mg/mL、至少0.15 mg/mL、至少0.2 mg/mL、至少0.25 mg/mL、至少0.3 mg/mL、至少0.35 mg/mL、至少0.4 mg/mL、至少0.45 mg/mL、至少0.5 mg/mL、至少0.55 mg/mL、至少0.6 mg/mL、至少0.65 mg/mL、至少0.7 mg/mL、至少0.75 mg/mL、至少0.8 mg/mL、至少0.85 mg/mL、至少0.9 mg/mL、至少0.95 mg/mL、或至少1 mg/mL之濃度包括在組合物中。在一些態樣中，一或多種結構性脂質(例如DSPC及膽固醇)以0.05與0.1 mg/mL之間、0.1與0.15 mg/mL之間、0.15與0.2 mg/mL之間、0.2與0.25 mg/mL之間、0.25與0.3 mg/mL之間、0.3與0.35 mg/mL之間、0.35與0.4 mg/mL之間、0.4與0.45 mg/mL之間、0.45與0.5 mg/mL之間、0.5與0.55 mg/mL之間、0.55與0.6 mg/mL之間、0.6與0.65 mg/mL之間、0.65與0.7 mg/mL之間、0.7與0.75 mg/mL之間、0.75與0.8 mg/mL之間、0.8與0.85 mg/mL之間、0.85與0.9 mg/mL之間、0.9與0.95 mg/mL之間、或0.95與1 mg/mL之間的濃度包括在組合物中。One or more structured lipids may include any one or more structured lipids disclosed herein. In a specific aspect, one or more structured lipids include neutral lipids and steroids or steroid analogs. In a specific aspect, one or more structured lipids include 1,2-distearoyl-sn -glycero-3-phosphocholine (DSPC) and cholesterol. In some aspects, one or more structured lipids (e.g., DSPC and cholesterol) are included in the composition with or without a concentration of at least 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0. 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.3 9, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0. 66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0 .93、0.94、0.95、0.96、0.97、0.98、0.99、1、1.01、1.02、1.03、1.04、1.05、1.06、1.07、1.08、1.09、1.1、1.11、1.12、1.13、1.14、1.15、1.16、1.17、1.18、1.19、 1.2, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46 , 1.47, 1.48, 1.49, 1.5, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.6, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.7, 1.71, 1.72, 1.7 3. 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86 , 1.87, 1.88, 1.89, 1.9, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99 or 2 ng/µg/mg/mL. In some aspects, one or more structured lipids (e.g., DSPC and cholesterol) are or are not included in the composition at a concentration of at least, at most, between (inclusive or exclusive) or just below 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5 mg/mL. In some aspects, one or more structured lipids (e.g., DSPC and cholesterol) are included in the composition at a concentration of at least 0.05 mg/mL, at least 0.1 mg/mL, at least 0.15 mg/mL, at least 0.2 mg/mL, at least 0.25 mg/mL, at least 0.3 mg/mL, at least 0.35 mg/mL, at least 0.4 mg/mL, at least 0.45 mg/mL, at least 0.5 mg/mL, at least 0.55 mg/mL, at least 0.6 mg/mL, at least 0.65 mg/mL, at least 0.7 mg/mL, at least 0.75 mg/mL, at least 0.8 mg/mL, at least 0.85 mg/mL, at least 0.9 mg/mL, at least 0.95 mg/mL, or at least 1 mg/mL. In some aspects, one or more structured lipids (e.g., DSPC and cholesterol) are present at between 0.05 and 0.1 mg/mL, between 0.1 and 0.15 mg/mL, between 0.15 and 0.2 mg/mL, between 0.2 and 0.25 mg/mL, between 0.25 and 0.3 mg/mL, between 0.3 and 0.35 mg/mL, between 0.35 and 0.4 mg/mL, between 0.4 and 0.45 mg/mL, between 0.45 and 0.5 mg/mL, between 0.5 and 0.55 mg/mL, between 0.55 and 0.6 mg/mL, between 0.6 and 0.65 mg/mL, between 0.65 and 0.7 mg/mL, between 0.7 and 0.75 mg/mL, between 0.75 and 0.8 mg/mL, between 0.8 and 0.85 Concentrations between 100 and 200 mg/mL, between 0.85 and 0.9 mg/mL, between 0.9 and 0.95 mg/mL, or between 0.95 and 1 mg/mL are included in the composition.
在特定態樣中，一或多種結構性脂質包括DSPC，且DSPC以或以約0.1至0.25 mg/mL之濃度包括在組合物中。在特定態樣中，一或多種結構性脂質包括DSPC，且DSPC以或以約0.15至0.25 mg/mL之濃度包括在組合物中。在特定態樣中，一或多種結構性脂質包括DSPC，且DSPC以或不以以下之濃度或至少、至多、恰好以下或在以下任何兩者之間(包括性或排他性)或約以下之濃度包括在組合物中：0.1、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.2、0.21、0.22、0.23、0.24或0.25 mg/mL。在特定態樣中，DSPC以或以約0.19 mg/mL之濃度包括在組合物中。In a specific aspect, one or more structural lipids include DSPC, and DSPC is included in the composition at a concentration of or about 0.1 to 0.25 mg/mL. In a specific aspect, one or more structural lipids include DSPC, and DSPC is included in the composition at a concentration of or about 0.15 to 0.25 mg/mL. In a specific aspect, one or more structural lipids include DSPC, and DSPC is included in the composition at or about 0.15 to 0.25 mg/mL. In a specific aspect, one or more structural lipids include DSPC, and DSPC is included in the composition at or not at the following concentration or at least, at most, just below, or between any two of the following (inclusive or exclusive) or about the following concentration: 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, or 0.25 mg/mL. In certain aspects, DSPC is included in the composition at a concentration of or about 0.19 mg/mL.
在特定態樣中，一或多種結構性脂質包括膽固醇，且膽固醇以或以約0.3至0.45 mg/mL之濃度包括在組合物中。在特定態樣中，一或多種結構性脂質包括膽固醇，且膽固醇以或以約0.3至0.4 mg/mL之濃度包括在組合物中。在特定態樣中，一或多種結構性脂質包括膽固醇，且膽固醇以或以約0.35至0.45 mg/mL之濃度包括在組合物中。在特定態樣中，一或多種結構性脂質包括膽固醇，且膽固醇以或不以以下之濃度或至少、至多、恰好以下或在以下任何兩者之間(包括性或排他性)或約以下之濃度包括在組合物中：0.30、0.31、0.32、0.33、0.34、0.35、0.36、0.37、0.38、0.39、0.40、0.41、0.42、0.43、0.44或0.45 mg/mL。在特定態樣中，膽固醇以及/或以約0.37 mg/mL之濃度包括在組合物中。凍乾組合物之濃度係在復原後測定。In certain aspects, the one or more structured lipids include cholesterol, and cholesterol is included in the composition at a concentration of or about 0.3 to 0.45 mg/mL. In certain aspects, the one or more structured lipids include cholesterol, and cholesterol is included in the composition at a concentration of or about 0.3 to 0.4 mg/mL. In certain aspects, the one or more structured lipids include cholesterol, and cholesterol is included in the composition at a concentration of or about 0.35 to 0.45 mg/mL. In a specific aspect, the one or more structured lipids include cholesterol, and cholesterol is included in the composition at or below a concentration of at least, at most, just below, or between any two of the following (inclusive or exclusive) or about 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, or 0.45 mg/mL. In a specific aspect, cholesterol is included in the composition at a concentration of about 0.37 mg/mL. The concentration of the lyophilized composition is determined after reconstitution.
在一些態樣中，RSV RNA-LNP組合物進一步包含一或多種緩衝劑及穩定劑以及視情況存在之鹽稀釋劑。因此，在一些態樣中，RSV RNA-LNP組合物包含陽離子脂質、聚乙二醇化脂質、一或多種結構性脂質、一或多種緩衝劑、穩定劑及視情況存在之鹽稀釋劑。在一些態樣中，前述要素中之1、2、3者或更多者排除在RSV RNA-LNP組合物外。In some aspects, the RSV RNA-LNP composition further comprises one or more buffers and stabilizers and, optionally, a salt diluent. Thus, in some aspects, the RSV RNA-LNP composition comprises a cationic lipid, a pegylated lipid, one or more structured lipids, one or more buffers, stabilizers, and, optionally, a salt diluent. In some aspects, 1, 2, 3 or more of the aforementioned elements are excluded from the RSV RNA-LNP composition.
在一些態樣中，RSV RNA-LNP組合物包含一或多種緩衝劑。In some aspects, the RSV RNA-LNP composition comprises one or more buffers.
一或多種緩衝劑可包含本文所揭示之任何一或多種緩衝劑。在特定態樣中，組合物包含Tris緩衝液，其包含至少第一緩衝劑及第二緩衝劑。在一些態樣中，第一緩衝劑為緩血酸胺。在一些態樣中，第二緩衝劑為Tris鹽酸(HCl)。在一些態樣中，Tris緩衝液之第一緩衝劑及第二緩衝劑(例如緩血酸胺及Tris HCl)以或不以至少以下、至多以下、以下之間(包括性或排他性)或恰好以下之濃度包括在組合物中：0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.2、0.21、0.22、0.23、0.24、0.25、0.26、0.27、0.28、0.29、0.3、0.31、0.32、0.33、0.34、0.35、0.36、0.37、0.38、0.39、0.4、0.41、0.42、0.43、0.44、0.45、0.46、0.47、0.48、0.49、0.5、0.51、0.52、0.53、0.54、0.55、0.56、0.57、0.58、0.59、0.6、0.61、0.62、0.63、0.64、0.65、0.66、0.67、0.68、0.69、0.7、0.71、0.72、0.73、0.74、0.75、0.76、0.77、0.78、0.79、0.8、0.81、0.82、0.83、0.84、0.85、0.86、0.87、0.88、0.89、0.9、0.91、0.92、0.93、0.94、0.95、0.96、0.97、0.98、0.99、1、1.01、1.02、1.03、1.04、1.05、1.06、1.07、1.08、1.09、1.1、1.11、1.12、1.13、1.14、1.15、1.16、1.17、1.18、1.19、1.2、1.21、1.22、1.23、1.24、1.25、1.26、1.27、1.28、1.29、1.3、1.31、1.32、1.33、1.34、1.35、1.36、1.37、1.38、1.39、1.4、1.41、1.42、1.43、1.44、1.45、1.46、1.47、1.48、1.49、1.5、1.51、1.52、1.53、1.54、1.55、1.56、1.57、1.58、1.59、1.6、1.61、1.62、1.63、1.64、1.65、1.66、1.67、1.68、1.69、1.7、1.71、1.72、1.73、1.74、1.75、1.76、1.77、1.78、1.79、1.8、1.81、1.82、1.83、1.84、1.85、1.86、1.87、1.88、1.89、1.9、1.91、1.92、1.93、1.94、1.95、1.96、1.97、1.98、1.99或2 ng/µg/mg/mL。凍乾組合物之濃度係在復原後測定。The one or more buffers may include any one or more buffers disclosed herein. In a particular aspect, the composition includes a Tris buffer solution, which includes at least a first buffer and a second buffer. In some aspects, the first buffer is sulfamethoxazole. In some aspects, the second buffer is Tris hydrochloric acid (HCl). In some aspects, the first buffer and the second buffer of the Tris buffer solution (e.g., sulfamethoxazole and Tris HCl) is or is not included in the composition in a concentration of at least 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55 .17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0. 43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0. 69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.9 5, 0.96, 0.97, 0.98, 0.99, 1, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.2, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.3, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, 1.4, 1.41, 1.42, 1.43, 1.44, 1.45, 1.46, 1.47, 1.48、1.49、1.5、1.51、1.52、1.53、1.54、1.55、1.56、1.57、1.58、1.59、1.6、1.61、1.62、1.63、1.64、1.65、1.66、1.67、1.68、1.69、1.7、1.71、1.72、1.73、1 .74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.8, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89, 1.9, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99, or 2 ng/µg/mg/mL. The concentration of the lyophilized composition was determined after reconstitution.
在一些態樣中，RSV RNA-LNP組合物為包含Tris緩衝液之液體組合物。In some aspects, the RSV RNA-LNP composition is a liquid composition comprising Tris buffer.
在一些態樣中，Tris緩衝液包含第一緩衝劑。在一些態樣中，第一緩衝劑為緩血酸胺。在一些態樣中，第一緩衝劑(例如緩血酸胺)以或不以至少以下、至多以下、以下之間(包括性或排他性)或恰好以下之濃度包括在液體組合物中：0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.2、0.21、0.22、0.23、0.24、0.25、0.26、0.27、0.28、0.29、0.3、0.31、0.32、0.33、0.34、0.35、0.36、0.37、0.38、0.39、0.4、0.41、0.42、0.43、0.44、0.45、0.46、0.47、0.48、0.49或0.5 mg/mL。在一些態樣中，第一緩衝劑(例如緩血酸胺)以至少0.1 mg/mL、至少0.05 mg/mL、至少0.1 mg/mL、至少0.15 mg/mL、至少0.2 mg/mL、至少0.25 mg/mL、至少0.3 mg/mL、至少0.35 mg/mL、至少0.4 mg/mL、至少0.45 mg/mL、至少0.5 mg/mL、至少0.55 mg/mL、至少0.6 mg/mL、至少0.65 mg/mL、至少0.7 mg/mL、至少0.75 mg/mL、至少0.8 mg/mL、至少0.85 mg/mL、至少0.9 mg/mL、至少0.95 mg/mL、或至少1 mg/mL之濃度包括在液體組合物中。在一些態樣中，第一緩衝劑(例如緩血酸胺)以0.05與0.15 mg/mL之間、0.15與0.25 mg/mL之間、0.25與0.35 mg/mL之間、0.35與0.45 mg/mL之間、0.45與0.55 mg/mL之間、0.55與0.65 mg/mL之間、0.65與0.75 mg/mL之間、0.75與0.85 mg/mL之間、或0.85與0.95 mg/mL之間的濃度包括在液體組合物中。在一些態樣中，第一緩衝劑(例如緩血酸胺)以0.05與0.1 mg/mL之間、0.1與0.15 mg/mL之間、0.15與0.2 mg/mL之間、0.2與0.25 mg/mL之間、0.25與0.3 mg/mL之間、0.3與0.35 mg/mL之間、0.35與0.4 mg/mL之間、0.4與0.45 mg/mL之間、0.45與0.5 mg/mL之間、0.5與0.55 mg/mL之間、0.55與0.6 mg/mL之間、0.6與0.65 mg/mL之間、0.65與0.7 mg/mL之間、0.7與0.75 mg/mL之間、0.75與0.8 mg/mL之間、0.8與0.85 mg/mL之間、0.85與0.9 mg/mL之間、0.9與0.95 mg/mL之間、或0.95與1 mg/mL之間的濃度包括在液體組合物中。In some embodiments, the Tris buffer comprises a first buffer. In some embodiments, the first buffer is a sulphuric acid amine. In some embodiments, the first buffer (e.g., a sulphuric acid amine) is or is not included in the liquid composition at a concentration of at least, at most, between (inclusive or exclusive) or just below: 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5 mg/mL. In some aspects, the first buffer (e.g., sulphonamide) is included in the liquid composition at a concentration of at least 0.1 mg/mL, at least 0.05 mg/mL, at least 0.1 mg/mL, at least 0.15 mg/mL, at least 0.2 mg/mL, at least 0.25 mg/mL, at least 0.3 mg/mL, at least 0.35 mg/mL, at least 0.4 mg/mL, at least 0.45 mg/mL, at least 0.5 mg/mL, at least 0.55 mg/mL, at least 0.6 mg/mL, at least 0.65 mg/mL, at least 0.7 mg/mL, at least 0.75 mg/mL, at least 0.8 mg/mL, at least 0.85 mg/mL, at least 0.9 mg/mL, at least 0.95 mg/mL, or at least 1 mg/mL. In some aspects, the first buffer (e.g., sulphonamide) is included in the liquid composition at a concentration of between 0.05 and 0.15 mg/mL, between 0.15 and 0.25 mg/mL, between 0.25 and 0.35 mg/mL, between 0.35 and 0.45 mg/mL, between 0.45 and 0.55 mg/mL, between 0.55 and 0.65 mg/mL, between 0.65 and 0.75 mg/mL, between 0.75 and 0.85 mg/mL, or between 0.85 and 0.95 mg/mL. In some aspects, the first buffer (e.g., sulfamethoxazole) is present at between 0.05 and 0.1 mg/mL, between 0.1 and 0.15 mg/mL, between 0.15 and 0.2 mg/mL, between 0.2 and 0.25 mg/mL, between 0.25 and 0.3 mg/mL, between 0.3 and 0.35 mg/mL, between 0.35 and 0.4 mg/mL, between 0.4 and 0.45 mg/mL, between 0.45 and 0.5 mg/mL, between 0.5 and 0.55 mg/mL, between 0.55 and 0.6 mg/mL, between 0.6 and 0.65 mg/mL, between 0.65 and 0.7 mg/mL, between 0.7 and 0.75 mg/mL, between 0.75 and 0.8 mg/mL, between 0.8 and 0.85 Concentrations between 100 and 200 mg/mL, between 0.85 and 0.9 mg/mL, between 0.9 and 0.95 mg/mL, or between 0.95 and 1 mg/mL are included in the liquid composition.
在特定態樣中，第一緩衝劑(例如緩血酸胺)以或以約0.1至0.3 mg/mL之濃度包括在液體組合物中。在特定態樣中，第一緩衝劑(例如緩血酸胺)以或以約0.15至0.25 mg/mL之濃度包括在液體組合物中。在特定態樣中，第一緩衝劑(例如緩血酸胺)以或不以以下之濃度或至少、至多、恰好以下或在以下任何兩者之間(包括性或排他性)或約以下之濃度包括在液體組合物中：0.1、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.20、0.21、0.22、0.23、0.24、0.25、0.26、0.27、0.28、0.29或0.3 mg/mL。在特定態樣中，第一緩衝劑(例如緩血酸胺)以或以約0.20 mg/mL之濃度包括在液體組合物中。In certain aspects, the first buffer (e.g., sulfamethoxazole) is included in the liquid composition at a concentration of about 0.1 to 0.3 mg/mL. In certain aspects, the first buffer (e.g., sulfamethoxazole) is included in the liquid composition at a concentration of about 0.15 to 0.25 mg/mL. In certain aspects, the first buffer (e.g., sulfamethoxazole) is included in the liquid composition at or below a concentration of at least, at most, just below, or between any two of the following (inclusive or exclusive) or about 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, or 0.3 mg/mL. In certain aspects, the first buffer (e.g., sulfamethoxazole) is included in the liquid composition at or about 0.20 mg/mL.
在一些態樣中，RSV RNA-LNP組合物為包含有包含第二緩衝劑之Tris緩衝液的液體組合物。In some aspects, the RSV RNA-LNP composition is a liquid composition comprising a Tris buffer comprising a second buffer.
在一些態樣中，第二緩衝劑包含Tris HCl。在一些態樣中，第二緩衝劑(例如Tris HCl)以或不以至少以下、至多以下、以下之間(包括性或排他性)或恰好以下之濃度包括在液體組合物中：0.5、0.55、1、1.01、1.02、1.03、1.04、1.05、1.06、1.07、1.08、1.09、1.1、1.11、1.12、1.13、1.14、1.15、1.16、1.17、1.18、1.19、1.2、1.21、1.22、1.23、1.24、1.25、1.26、1.27、1.28、1.29、1.3、1.31、1.32、1.33、1.34、1.35、1.36、1.37、1.38、1.39、1.4、1.41、1.42、1.43、1.44、1.45、1.46、1.47、1.48、1.49或1.5 mg/mL。在一些態樣中，第二緩衝劑(例如Tris HCl)以至少0.5 mg/mL、至少0.55 mg/mL、至少0.6 mg/mL、至少0.65 mg/mL、至少0.7 mg/mL、至少0.75 mg/mL、至少0.8 mg/mL、至少0.85 mg/mL、至少0.9 mg/mL、至少0.95 mg/mL、至少1 mg/mL、至少1.05 mg/mL、至少1.10 mg/mL、至少1.15 mg/mL、至少1.20 mg/mL、至少1.25 mg/mL、至少1.30 mg/mL、至少1.35 mg/mL、至少1.40 mg/mL、至少1.45 mg/mL、或至少1.50 mg/mL之濃度包括在液體組合物中。在一些態樣中，第二緩衝劑(例如Tris HCl)以0.5與0.6 mg/mL之間、0.6與0.7 mg/mL之間、0.7與0.8 mg/mL之間、0.8與0.9 mg/mL之間、0.9與1 mg/mL之間、1與1.10 mg/mL之間、1.10與1.20 mg/mL之間、1.20與1.30 mg/mL之間、1.30與1.40 mg/mL之間、或1.40與1.50 mg/mL之間的濃度包括在液體組合物中。In some aspects, the second buffer comprises Tris HCl. In some aspects, the second buffer (e.g., Tris HCl) is or is not included in the liquid composition at a concentration of at least, at most, between (inclusive or exclusive), or just below: 0.5, 0.55, 1, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.1, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, 1.44, 1.45, 1.46, 1.47, 1.48, 1.49, or 1.5 mg/mL. In some aspects, the second buffer (e.g., Tris HCl) is included in the liquid composition at a concentration of at least 0.5 mg/mL, at least 0.55 mg/mL, at least 0.6 mg/mL, at least 0.65 mg/mL, at least 0.7 mg/mL, at least 0.75 mg/mL, at least 0.8 mg/mL, at least 0.85 mg/mL, at least 0.9 mg/mL, at least 0.95 mg/mL, at least 1 mg/mL, at least 1.05 mg/mL, at least 1.10 mg/mL, at least 1.15 mg/mL, at least 1.20 mg/mL, at least 1.25 mg/mL, at least 1.30 mg/mL, at least 1.35 mg/mL, at least 1.40 mg/mL, at least 1.45 mg/mL, or at least 1.50 mg/mL. In some aspects, the second buffer (e.g., Tris HCl) is included in the liquid composition at a concentration between 0.5 and 0.6 mg/mL, between 0.6 and 0.7 mg/mL, between 0.7 and 0.8 mg/mL, between 0.8 and 0.9 mg/mL, between 0.9 and 1 mg/mL, between 1 and 1.10 mg/mL, between 1.10 and 1.20 mg/mL, between 1.20 and 1.30 mg/mL, between 1.30 and 1.40 mg/mL, or between 1.40 and 1.50 mg/mL.
在特定態樣中，第二緩衝劑(例如Tris HCl)以或以約1.25至1.40 mg/mL之濃度包括在液體組合物中。在特定態樣中，第二緩衝劑(例如Tris HCl)以或以約1.30至1.40 mg/mL之濃度包括在液體組合物中。在特定態樣中，第二緩衝劑(例如Tris HCl)以或不以以下之濃度或至少、至多、恰好以下或在以下任何兩者之間(包括性或排他性)或約以下之濃度包括在液體組合物中：1.25、1.26、1.27、1.28、1.29、1.30、1.31、1.32、1.33、1.34、或1.35、1.36、1.37、1.38、1.39或1.40 mg/mL。在特定態樣中，第二緩衝劑(例如Tris HCl)以或以約1.32 mg/mL之濃度包括在液體組合物中。In certain aspects, the second buffer (e.g., Tris HCl) is included in the liquid composition at a concentration of or about 1.25 to 1.40 mg/mL. In certain aspects, the second buffer (e.g., Tris HCl) is included in the liquid composition at a concentration of or about 1.30 to 1.40 mg/mL. In certain aspects, the second buffer (e.g., Tris HCl) is included in the liquid composition at or not at a concentration of 1.25, 1.26, 1.27, 1.28, 1.29, 1.30, 1.31, 1.32, 1.33, 1.34, or 1.35, 1.36, 1.37, 1.38, 1.39, or 1.40 mg/mL. In certain aspects, the second buffer (e.g., Tris HCl) is included in the liquid composition at or at a concentration of about 1.32 mg/mL.
在一些態樣中，RSV RNA-LNP組合物為包含Tris緩衝液之凍乾組合物。在一些態樣中，Tris緩衝液包含第一緩衝劑。在一些態樣中，第一緩衝劑為緩血酸胺。在一些態樣中，第一緩衝劑(例如緩血酸胺)以或不以至少以下、至多以下、以下之間(包括性或排他性)或恰好以下之復原後濃度包括在凍乾組合物中：0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.2、0.21、0.22、0.23、0.24、0.25、0.26、0.27、0.28、0.29、0.3、0.31、0.32、0.33、0.34、0.35、0.36、0.37、0.38、0.39、0.4、0.41、0.42、0.43、0.44、0.45、0.46、0.47、0.48、0.49或0.5 mg/mL。在一些態樣中，第一緩衝劑(例如緩血酸胺)以至少0.01、至少0.05、至少0.1、至少0.15、至少0.2、至少0.25、至少0.3、至少0.35、至少0.4、至少0.45或至少0.5 mg/mL之復原後濃度包括在凍乾組合物中。在一些態樣中，第一緩衝劑(例如緩血酸胺(Tris鹼))以0.01與0.05 mg/mL之間、0.05與0.1 mg/mL之間、0.1與0.15 mg/mL之間、0.15與0.2 mg/mL之間、0.2與0.25 mg/mL之間、0.25與0.3 mg/mL之間、0.3與0.35 mg/mL之間、0.35與0.4 mg/mL之間、0.4與0.45 mg/mL之間、或0.45與0.5 mg/mL之間的復原後濃度包括在凍乾組合物中。In some embodiments, the RSV RNA-LNP composition is a freeze-dried composition comprising a Tris buffer. In some embodiments, the Tris buffer comprises a first buffer. In some embodiments, the first buffer is a sulphuric acid amine. In some embodiments, the first buffer (e.g., a sulphuric acid amine) is or is not included in the freeze-dried composition at a reconstituted concentration of at least, at most, between (inclusive or exclusive) or just below: 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18 , 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5 mg/mL. In some aspects, the first buffer (e.g., sulfadiazine) is included in the lyophilized composition at a post-reconstitution concentration of at least 0.01, at least 0.05, at least 0.1, at least 0.15, at least 0.2, at least 0.25, at least 0.3, at least 0.35, at least 0.4, at least 0.45, or at least 0.5 mg/mL. In some aspects, the first buffer (e.g., Tris base) is included in the lyophilized composition at a post-reconstitution concentration of between 0.01 and 0.05 mg/mL, between 0.05 and 0.1 mg/mL, between 0.1 and 0.15 mg/mL, between 0.15 and 0.2 mg/mL, between 0.2 and 0.25 mg/mL, between 0.25 and 0.3 mg/mL, between 0.3 and 0.35 mg/mL, between 0.35 and 0.4 mg/mL, between 0.4 and 0.45 mg/mL, or between 0.45 and 0.5 mg/mL.
在特定態樣中，第一緩衝劑(例如緩血酸胺)以或以約0.01與0.15 mg/mL之復原後濃度包括在凍乾組合物中。在特定態樣中，第一緩衝劑(例如緩血酸胺)以或以約0.01與0.10 mg/mL之復原後濃度包括在凍乾組合物中。在特定態樣中，第一緩衝劑(例如緩血酸胺)以或以約0.05與0.15 mg/mL之復原後濃度包括在凍乾組合物中。在特定態樣中，第一緩衝劑(例如緩血酸胺)以或不以以下之復原後濃度或至少、至多、恰好以下或在以下任何兩者之間(包括性或排他性)或約以下之復原後濃度包括在凍乾組合物中：0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.11、0.12、0.13、0.14或0.15 mg/mL。在特定態樣中，第一緩衝劑(例如緩血酸胺)以或以約0.09 mg/mL之復原後濃度包括在凍乾組合物中。In certain aspects, the first buffer (e.g., sulfamethoxazole) is included in the lyophilized composition at or about 0.01 and 0.15 mg/mL after reconstitution. In certain aspects, the first buffer (e.g., sulfamethoxazole) is included in the lyophilized composition at or about 0.01 and 0.10 mg/mL after reconstitution. In certain aspects, the first buffer (e.g., sulfamethoxazole) is included in the lyophilized composition at or about 0.05 and 0.15 mg/mL after reconstitution. In certain aspects, the first buffer (e.g., sulfamethoxazole) is included in the lyophilized composition at or without a concentration after reconstitution of at least, at most, just below, or between any two of the following (inclusive or exclusive) or about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, or 0.15 mg/mL. In certain aspects, the first buffer (e.g., sulfamethoxazole) is included in the lyophilized composition at or about 0.09 mg/mL after reconstitution.
在一些態樣中，RSV RNA-LNP組合物為包含有包含第二緩衝劑之Tris緩衝液的凍乾組合物。在一些態樣中，第二緩衝劑包含Tris HCl。在一些態樣中，第二緩衝劑(例如Tris HCl)以或不以至少以下、至多以下、以下之間(包括性或排他性)或恰好以下之復原後濃度包括在凍乾組合物中：0.1、0.15、0.2、0.25、0.3、0.35、0.4、0.45、0.5、0.51、0.52、0.53、0.54、0.55、0.56、0.57、0.58、0.59、0.6、0.61、0.62、0.63、0.64、0.65、0.66、0.67、0.68、0.69、0.7、0.71、0.72、0.73、0.74、0.75、0.76、0.77、0.78、0.79、0.8、0.81、0.82、0.83、0.84、0.85、0.86、0.87、0.88、0.89、0.9、0.91、0.92、0.93、0.94、0.95、0.96、0.97、0.98、0.99或1 mg/mL。在一些態樣中，第二緩衝劑(例如Tris HCl)以至少0.1 mg/mL、至少0.2 mg/mL、至少0.3 mg/mL、至少0.4 mg/mL、至少0.5 mg/mL、至少0.6 mg/mL、至少0.7 mg/mL、至少0.8 mg/mL、至少0.9 mg/mL、或至少1 mg/mL之復原後濃度包括在凍乾組合物中。在一些態樣中，第二緩衝劑(例如Tris HCl)以0.1與0.2 mg/mL之間、0.2與0.3 mg/mL之間、0.3與0.4 mg/mL之間、0.4與0.5 mg/mL之間、0.5與0.6 mg/mL之間、0.6與0.7 mg/mL之間、0.7與0.8 mg/mL之間、0.8與0.9 mg/mL之間、或0.9與1 mg/mL之間的復原後濃度包括在凍乾組合物中。In some embodiments, the RSV RNA-LNP composition is a freeze-dried composition comprising a Tris buffer comprising a second buffer. In some embodiments, the second buffer comprises Tris HCl. In some embodiments, the second buffer (e.g., Tris HCl) is or is not included in the freeze-dried composition at a post-reconstitution concentration of at least 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96 .66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, or 1 mg/mL. In some aspects, the second buffer (e.g., Tris HCl) is included in the lyophilized composition at a post-reconstitution concentration of at least 0.1 mg/mL, at least 0.2 mg/mL, at least 0.3 mg/mL, at least 0.4 mg/mL, at least 0.5 mg/mL, at least 0.6 mg/mL, at least 0.7 mg/mL, at least 0.8 mg/mL, at least 0.9 mg/mL, or at least 1 mg/mL. In some aspects, a second buffer (e.g., Tris HCl) is included in the lyophilized composition at a post-reconstitution concentration of between 0.1 and 0.2 mg/mL, between 0.2 and 0.3 mg/mL, between 0.3 and 0.4 mg/mL, between 0.4 and 0.5 mg/mL, between 0.5 and 0.6 mg/mL, between 0.6 and 0.7 mg/mL, between 0.7 and 0.8 mg/mL, between 0.8 and 0.9 mg/mL, or between 0.9 and 1 mg/mL.
在特定態樣中，第二緩衝劑(例如Tris HCl)以或以約0.5與0.65 mg/mL之復原後濃度包括在凍乾組合物中。在特定態樣中，第二緩衝劑(例如Tris HCl)以或以約0.5與0.6 mg/mL之復原後濃度包括在凍乾組合物中。在特定態樣中，第二緩衝劑(例如Tris HCl)以約0.55與0.65 mg/mL之復原後濃度包括在凍乾組合物中。在特定態樣中，第二緩衝劑(例如Tris HCl)以或不以以下之復原後濃度或至少、至多、恰好以下或在以下任何兩者之間(包括性或排他性)或約以下之復原後濃度包括在凍乾組合物中：0.5、0.51、0.52、0.53、0.54、0.55、0.56、0.57、0.58、0.59、0.6、0.61、0.62、0.63、0.64或0.65 mg/mL。在特定態樣中，第二緩衝劑(例如Tris HCl)以或以約0.57 mg/mL之復原後濃度包括在凍乾組合物中。In certain aspects, the second buffer (e.g., Tris HCl) is included in the lyophilized composition at or about 0.5 and 0.65 mg/mL post-reconstitution concentration. In certain aspects, the second buffer (e.g., Tris HCl) is included in the lyophilized composition at or about 0.5 and 0.6 mg/mL post-reconstitution concentration. In certain aspects, the second buffer (e.g., Tris HCl) is included in the lyophilized composition at or about 0.5 and 0.6 mg/mL post-reconstitution concentration. In a specific aspect, the second buffer (e.g., Tris HCl) is included in the lyophilized composition at or without a post-reconstitution concentration of at least, at most, just below, or between any two of the following (inclusive or exclusive) or about 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, or 0.65 mg/mL. In a specific aspect, the second buffer (e.g., Tris HCl) is included in the lyophilized composition at or about 0.57 mg/mL post-reconstitution concentration.
在一些態樣中，RSV RNA-LNP組合物包含穩定劑。穩定劑可包含本文所揭示之任何一或多種穩定劑。在一些態樣中，穩定劑亦充當低溫保護劑。在特定態樣中，穩定劑包含蔗糖。在一些態樣中，穩定劑(例如蔗糖)以或不以至少以下、至多以下、以下之間(包括性或排他性)或恰好以下之濃度包括在組合物中：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199或200 ng/µg/mg/mL。In some aspects, the RSV RNA-LNP composition comprises a stabilizer. The stabilizer may comprise any one or more stabilizers disclosed herein. In some aspects, the stabilizer also acts as a low temperature protectant. In a specific aspect, the stabilizer comprises sucrose. In some aspects, a stabilizer (e.g., sucrose) is or is not included in the composition at a concentration of at least, at most, between (inclusive or exclusive) or just below: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 ,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153 ,154,155,156,157,158,159,160,161,162,163,164,165,166,167,168,169,170,171,172,173,174,175,176,1 77, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199 or 200 ng/µg/mg/mL.
在一些態樣中，RSV RNA-LNP組合物為液體組合物，且穩定劑(例如蔗糖)以或不以至少以下、至多以下、以下之間(包括性或排他性)或恰好以下之濃度包括在液體組合物中：70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129或130 mg/mL。在一些態樣中，穩定劑(例如蔗糖)以至少70、至少75、至少80、至少85、至少90、至少95、至少100、至少105、至少110、至少115、至少120、至少125或至少130 mg/mL之濃度包括在液體組合物中。在一些態樣中，穩定劑(例如蔗糖)以70與80 mg/mL之間、80與90 mg/mL之間、90與100 mg/mL之間、100與110 mg/mL之間、110與120 mg/mL之間、或120與130 mg/mL之間的濃度包括在液體組合物中。In some aspects, the RSV RNA-LNP composition is a liquid composition, and the stabilizer (e.g., sucrose) is or is not included in the liquid composition at a concentration of at least, at most, between (inclusive or exclusive) or just below: 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125 In some aspects, the stabilizer (e.g., sucrose) is included in the liquid composition at a concentration of at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 105, at least 110, at least 115, at least 120, at least 125, or at least 130 mg/mL. In some aspects, a stabilizer (e.g., sucrose) is included in the liquid composition at a concentration between 70 and 80 mg/mL, between 80 and 90 mg/mL, between 90 and 100 mg/mL, between 100 and 110 mg/mL, between 110 and 120 mg/mL, or between 120 and 130 mg/mL.
在特定態樣中，穩定劑(例如蔗糖)以或以約95至110 mg/mL之濃度包括在液體組合物中。在特定態樣中，穩定劑(例如蔗糖)以或以約95至105 mg/mL之濃度包括在液體組合物中。在特定態樣中，穩定劑(例如蔗糖)以或以約100至110 mg/mL之濃度包括在液體組合物中。在特定態樣中，穩定劑(例如蔗糖)以或不以以下之濃度或至少、至多、恰好以下、在以下中之任何兩者之間(包括性或排他性)或約以下之濃度包括在液體組合物中：95、96、97、98、99、100、101、102、103、104、105、106、107、108、109或110 mg/mL。在特定態樣中，穩定劑(例如蔗糖)以或以約103 mg/mL之濃度包括在液體組合物中。In certain aspects, the stabilizer (e.g., sucrose) is included in the liquid composition at a concentration of or about 95 to 110 mg/mL. In certain aspects, the stabilizer (e.g., sucrose) is included in the liquid composition at a concentration of or about 95 to 105 mg/mL. In certain aspects, the stabilizer (e.g., sucrose) is included in the liquid composition at a concentration of or about 100 to 110 mg/mL. In certain aspects, the stabilizer (e.g., sucrose) is or is not included in the liquid composition at a concentration of at least, at most, just below, between any two of the following (inclusive or exclusive), or about 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or 110 mg/mL. In certain aspects, the stabilizer (e.g., sucrose) is included in the liquid composition at a concentration of about 103 mg/mL.
在一些態樣中，RSV RNA-LNP組合物為凍乾組合物，且穩定劑(例如蔗糖)以或不以至少以下、至多以下、以下之間(包括性或排他性)或恰好以下之復原後濃度包括在凍乾組合物中：20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79或80 mg/mL。在一些態樣中，穩定劑(例如蔗糖)以至少20、至少25、至少30、至少35、至少40、至少45、至少50、至少55、至少60、至少65、至少70、至少75或至少80 mg/mL之復原後濃度包括在凍乾組合物中。在一些態樣中，穩定劑(例如蔗糖)以20至30 mg/mL之間、30至40 mg/mL之間、40至50 mg/mL之間、50至60 mg/mL之間、60至70 mg/mL之間、或70至80 mg/mL之間的復原後濃度包括在凍乾組合物中。In some aspects, the RSV RNA-LNP composition is a freeze-dried composition, and the stabilizer (e.g., sucrose) is or is not included in the freeze-dried composition at a post-reconstitution concentration of at least the following, at most the following, between the following (inclusive or exclusive), or just below: 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 In some aspects, the stabilizer (e.g., sucrose) is included in the lyophilized composition at a post-reconstitution concentration of at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, or at least 80 mg/mL. In some aspects, a stabilizer (e.g., sucrose) is included in the lyophilized composition at a post-reconstitution concentration of between 20 and 30 mg/mL, between 30 and 40 mg/mL, between 40 and 50 mg/mL, between 50 and 60 mg/mL, between 60 and 70 mg/mL, or between 70 and 80 mg/mL.
在特定態樣中，穩定劑(例如蔗糖)以或以約35至50 mg/mL之復原後濃度包括在凍乾組合物中。在特定態樣中，穩定劑(例如蔗糖)以或以約35至45 mg/mL之復原後濃度包括在凍乾組合物中。在特定態樣中，穩定劑(例如蔗糖)以或以約40至50 mg/mL之復原後濃度包括在凍乾組合物中。在特定態樣中，穩定劑(例如蔗糖)以或不以以下之復原後濃度或至少、至多、恰好以下、在以下中之任何兩者之間(包括性或排他性)或約以下之復原後濃度包括在凍乾組合物中：35、36、37、38、39、40、41、42、43、44、45、46、47、48、49或50 mg/mL。在特定態樣中，穩定劑(例如蔗糖)以或以約44 mg/mL之復原後濃度包括在凍乾組合物中。In certain aspects, the stabilizer (e.g., sucrose) is included in the lyophilized composition at or about 35 to 50 mg/mL post-reconstitution concentration. In certain aspects, the stabilizer (e.g., sucrose) is included in the lyophilized composition at or about 35 to 45 mg/mL post-reconstitution concentration. In certain aspects, the stabilizer (e.g., sucrose) is included in the lyophilized composition at or about 40 to 50 mg/mL post-reconstitution concentration. In certain aspects, the stabilizer (e.g., sucrose) is or is not included in the lyophilized composition at a post-reconstitution concentration of at least, at most, just below, between any two of the following (inclusive or exclusive), or about 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 mg/mL. In certain aspects, the stabilizer (e.g., sucrose) is included in the lyophilized composition at a post-reconstitution concentration of at or about 44 mg/mL.
在一些態樣中，凍乾組合物在適合載劑及/或稀釋劑中復原。載劑及/或稀釋劑可包含本文所揭示之任何一或多種載劑及/或稀釋劑。在特定態樣中，載劑及/或稀釋劑包含鹽稀釋劑，諸如氯化鈉(NaCl) (例如鹽水，例如生理鹽水(physiological/normal saline))。氯化鈉可包含用於注射之0.9%氯化鈉。在一些態樣中，凍乾組合物在或不在至少以下、至多以下、以下之間(包括性或排他性)或恰好以下之鹽水中復原：0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、0.10、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.20、0.21、0.22、0.23、0.24、0.25、0.26、0.27、0.28、0.29、0.30、0.31、0.32、0.33、0.34、0.35、0.36、0.37、0.38、0.39、0.40、0.41、0.42、0.43、0.44、0.45、0.46、0.47、0.48、0.49、0.50、0.51、0.52、0.53、0.54、0.55、0.56、0.57、0.58、0.59、0.60、0.61、0.62、0.63、0.64、0.65、0.66、0.67、0.68、0.69、0.70、0.71、0.72、0.73、0.74、0.75、0.76、0.77、0.78、0.79、0.80、0.81、0.82、0.83、0.84、0.85、0.86、0.87、0.88、0.89、0.90、0.91、0.92、0.93、0.94、0.95、0.96、0.97、0.98、0.99或1 mL。在一些態樣中，凍乾組合物在至少0.1、至少0.2、至少0.3、至少0.4、至少0.5、至少0.6、至少0.7、至少0.8、至少0.9或至少1 mL之氯化鈉中復原。In some aspects, the lyophilized composition is reconstituted in a suitable carrier and/or diluent. The carrier and/or diluent may comprise any one or more of the carriers and/or diluents disclosed herein. In a particular aspect, the carrier and/or diluent comprises a salt diluent, such as sodium chloride (NaCl) (e.g., saline, such as physiological/normal saline). The sodium chloride may comprise 0.9% sodium chloride for injection. In some aspects, the freeze-dried composition is or is not reconstituted in saline water at least below, at most below, between (inclusive or exclusive) or just below: 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0. 19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.40, 0.41, 0.42, 0.43, 0.44, 0.45, 0. 46, 0.47, 0.48, 0.49, 0.50, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0 In some aspects, the freeze-dried composition is reconstituted in at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, or at least 1 mL of sodium chloride.
在特定態樣中，凍乾組合物在或在約0.6至0.75 mL之氯化鈉/鹽水中復原。在特定態樣中，凍乾組合物在或在約0.65至0.75 mL之氯化鈉/鹽水中復原。在特定態樣中，凍乾組合物在或不在至少、至多、恰好以下、在以下中之任何兩者之間(包括性或排他性)或約以下之氯化鈉/鹽水中復原：0.6、0.61、0.62、0.63、0.64、0.65、0.66、0.67、0.68、0.69、0.7、0.71、0.72、0.73、0,74或0.75 mL。In certain aspects, the lyophilized composition is reconstituted in or about 0.6 to 0.75 mL of sodium chloride/brine. In certain aspects, the lyophilized composition is reconstituted in or about 0.65 to 0.75 mL of sodium chloride/brine. In certain aspects, the lyophilized composition is reconstituted in or about 0.65 to 0.75 mL of sodium chloride/brine. In certain aspects, the lyophilized composition is reconstituted in or about at least, at most, just below, between any two of the following (inclusive or exclusive), or about 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0,74, or 0.75 mL of sodium chloride/brine.
在一些態樣中，鹽稀釋劑(例如NaCl)以或不以至少以下、至多以下、以下之間(包括性或排他性)或恰好以下之復原後濃度包括在凍乾組合物中：每毫升0.5、1、1.5、2、2.5、3、3.5、4、4.5、5、5.5、6、6.5、7、7.5、8、8.5、9、9.5、10、10.5、11、11.5、12、12.5、13、13.5、14、14.5、15、15.5、16、16.5、17、17.5、18、18.5、19、19.5、20、20.5、21、21.5、22、22.5、23、23.5、24、24.5、25、25.5、26、26.5、27、27.5、28、28.5、29、29.5、30、30.5、31、31.5、32、32.5、33、33.5、34、34.5、35、35.5、36、36.5、37、37.5、38、38.5、39、39.5、40、40.5、41、41.5、42、42.5、43、43.5、44、44.5、45、45.5、46、46.5、47、47.5、48、48.5、49、49.5或50 ng/µg/mg。在一些態樣中，鹽稀釋劑(例如NaCl)以或不以至少以下、至多以下、以下之間(包括性或排他性)或恰好以下之復原後濃度包括在凍乾組合物中：1、1.5、2、2.5、3、3.5、4、4.5、5、5.5、6、6.5、7、7.5、8、8.5、9、9.5、10、10.5、11、11.5、12、12.5、13、13.5、14、14.5、15、15.5、16、16.5、17、17.5、18、18.5、19、19.5或20 mg/mL。在一些態樣中，鹽稀釋劑(例如NaCl)以至少1、至少2、至少3、至少4、至少5、至少6、至少7、至少8、至少9、至少10、至少11、至少12、至少13、至少14、至少15或至少20 mg/mL之復原後濃度包括在凍乾組合物中。In some aspects, a salt diluent (e.g., NaCl) is or is not included in the lyophilized composition at a post-reconstitution concentration of at least, at most, between (inclusive or exclusive) or just below 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21, 21.5, 2 2, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31, 31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 3 6, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41, 41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5 or 50 ng/µg/mg. In some aspects, a salt diluent (e.g., NaCl) is or is not included in the lyophilized composition at a post-reconstitution concentration of at least, at most, between (inclusive or exclusive) or just below 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 mg/mL. In some aspects, a salt diluent (e.g., NaCl) is included in the lyophilized composition at a post-reconstitution concentration of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 20 mg/mL.
在特定態樣中，鹽稀釋劑(例如NaCl)以在5與15 mg/mL之間或在約其間之復原後濃度包括在凍乾組合物中。在一些態樣中，鹽稀釋劑(例如NaCl)以在5與10 mg/mL之間或在約其間之復原後濃度包括在凍乾組合物中。在特定態樣中，鹽稀釋劑(例如NaCl)以或不以以下之復原後濃度或至少、至多、恰好以下、在以下中之任何兩者之間(包括性或排他性)或約以下之復原後濃度包括在凍乾組合物中：5、6、7、8、9、10、11、12、13、14或15 mg/mL。在特定態樣中，鹽稀釋劑(例如NaCl)以或以約9 mg/mL之復原後濃度包括在凍乾組合物中。In certain aspects, the salt diluent (e.g., NaCl) is included in the lyophilized composition at a post-reconstitution concentration between 5 and 15 mg/mL or about therebetween. In some aspects, the salt diluent (e.g., NaCl) is included in the lyophilized composition at a post-reconstitution concentration between 5 and 10 mg/mL or about therebetween. In certain aspects, the salt diluent (e.g., NaCl) is included in the lyophilized composition at a post-reconstitution concentration of or at least, at most, just below, between any two of the following (inclusive or exclusive), or about below: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mg/mL. In certain aspects, a salt diluent (e.g., NaCl) is included in the lyophilized composition at or at a post-reconstitution concentration of about 9 mg/mL.
RSV RNA-LNP組合物之pH可呈或可不呈至少、至多、恰好以下或在以下之間(包括性或排他性)的pH：6.5、6.6、6.7、6.8、6.9、7.0、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9、8.0、8.1、8.2、8.3、8.4或8.5，或其中可導出的任何範圍或值。在一些態樣中，RSV RNA-LNP組合物呈至少6.5、至少7.0、至少7.5、至少8.0或至少8.5之pH。在特定態樣中，RSV RNA-LNP組合物呈6.0與7.5之間、6.5與7.5之間、7.0與8.0之間、7.5與8.5之間的pH。在特定態樣中，RSV RNA-LNP組合物在7.0與8.0之間的pH。在特定態樣中，RSV RNA-LNP組合物呈或不呈至少、至多、恰好以下、在以下中之任何兩者之間(包括性或排他性)或約以下的pH：7.0、7.1、7.2、7.3、7.4、7.5、7.6、7.7、7.8、7.9或8.0。在特定態樣中，RSV RNA-LNP組合物呈或呈約pH 7.4。在一些態樣中，氫氧化鈉緩衝液可用於緩衝液pH調節。The pH of the RSV RNA-LNP composition may or may not be at least, at most, just below, or between (inclusive or exclusive) 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5, or any range or value derivable therein. In some aspects, the RSV RNA-LNP composition is at least 6.5, at least 7.0, at least 7.5, at least 8.0, or at least 8.5. In specific aspects, the RSV RNA-LNP composition is at a pH between 6.0 and 7.5, between 6.5 and 7.5, between 7.0 and 8.0, between 7.5 and 8.5. In certain aspects, the RSV RNA-LNP composition is at a pH between 7.0 and 8.0. In certain aspects, the RSV RNA-LNP composition is or is not at a pH of at least, at most, just below, between any two of the following (inclusive or exclusive), or about below: 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In certain aspects, the RSV RNA-LNP composition is at or about pH 7.4. In some aspects, a sodium hydroxide buffer can be used for buffer pH adjustment.
在特定態樣中，RSV RNA-LNP組合物包含本文所揭示之編碼RSV多肽的RSV RNA聚核苷酸，該聚核苷酸囊封於具有以下之脂質組成的LNP中：濃度為或為約0.8至0.95 mg/mL的陽離子脂質、濃度為或為約0.05至0.15 mg/mL的聚乙二醇化脂質、濃度為或為約0.1至0.25 mg/mL的第一結構脂質、及濃度為或為約0.3至0.45 mg/mL的第二結構脂質。在一些態樣中，前述要素中之一或多者可排除在RSV RNA-LNP組合物外。In a specific aspect, the RSV RNA-LNP composition comprises an RSV RNA polynucleotide encoding an RSV polypeptide disclosed herein, encapsulated in an LNP having the following lipid composition: a cationic lipid at a concentration of or about 0.8 to 0.95 mg/mL, a pegylated lipid at a concentration of or about 0.05 to 0.15 mg/mL, a first structural lipid at a concentration of or about 0.1 to 0.25 mg/mL, and a second structural lipid at a concentration of or about 0.3 to 0.45 mg/mL. In some aspects, one or more of the aforementioned elements may be excluded from the RSV RNA-LNP composition.
在特定態樣中，RSV RNA-LNP組合物包含本文所揭示之編碼RSV多肽的RSV RNA聚核苷酸，該聚核苷酸囊封於具有以下之脂質組成的LNP中：濃度為或為約0.8至0.95 mg/mL的ALC-0315、濃度為或為約0.05至0.15 mg/mL的ALC-0159、濃度為或為約0.1至0.25 mg/mL的DSPC、及濃度為或為約0.3至0.45 mg/mL的膽固醇。在一些態樣中，前述要素中之一或多者可排除在RSV RNA-LNP組合物外。In a specific aspect, the RSV RNA-LNP composition comprises an RSV RNA polynucleotide encoding an RSV polypeptide disclosed herein, encapsulated in an LNP having the following lipid composition: ALC-0315 at a concentration of or about 0.8 to 0.95 mg/mL, ALC-0159 at a concentration of or about 0.05 to 0.15 mg/mL, DSPC at a concentration of or about 0.1 to 0.25 mg/mL, and cholesterol at a concentration of or about 0.3 to 0.45 mg/mL. In some aspects, one or more of the aforementioned elements may be excluded from the RSV RNA-LNP composition.
在特定態樣中，RSV RNA-LNP組合物為液體RSV RNA-LNP組合物，且液體RSV RNA-LNP組合物進一步包含含有以下之緩衝組合物：濃度為或為約0.15至0.3 mg/mL的第一緩衝劑、濃度為或為約1.25至1.4 mg/mL的第二緩衝劑、及濃度為或為約95至110 mg/mL的穩定劑。在特定態樣中，RSV RNA-LNP組合物為液體RSV RNA-LNP組合物，且液體RSV RNA-LNP組合物進一步包含含有以下之Tris緩衝組合物：濃度為或為約0.1至0.3 mg/mL的緩血酸胺、濃度為或為約1.25至1.4 mg/mL的Tris HCl、及濃度為或為約95至110 mg/mL的蔗糖。在一些態樣中，前述要素中之一或多者可排除在RSV RNA-LNP組合物外。In a specific embodiment, the RSV RNA-LNP composition is a liquid RSV RNA-LNP composition, and the liquid RSV RNA-LNP composition further comprises a buffer composition containing: a first buffer at a concentration of or about 0.15 to 0.3 mg/mL, a second buffer at a concentration of or about 1.25 to 1.4 mg/mL, and a stabilizer at a concentration of or about 95 to 110 mg/mL. In a specific embodiment, the RSV RNA-LNP composition is a liquid RSV RNA-LNP composition, and the liquid RSV RNA-LNP composition further comprises a Tris buffer composition containing: sulphuric acid amine at a concentration of or about 0.1 to 0.3 mg/mL, Tris HCl at a concentration of or about 1.25 to 1.4 mg/mL, and sucrose at a concentration of or about 95 to 110 mg/mL. In some embodiments, one or more of the aforementioned elements may be excluded from the RSV RNA-LNP composition.
因此，在特定態樣中，液體RSV RNA-LNP組合物包含：陽離子脂質，其濃度為或為約0.8至0.95 mg/mL；聚乙二醇化脂質，其濃度為或為約0.05至0.15 mg/mL；第一結構脂質，其濃度為或為約0.1至0.25 mg/mL；第二結構脂質，其濃度為或為約0.3至0.45 mg/mL；且進一步包含：第一緩衝劑，其濃度為或為約0.1至0.3 mg/mL；第二緩衝劑，其濃度為或為約1.25至1.4 mg/mL；及穩定劑，其濃度為或為約95至110 mg/mL。在一些態樣中，前述要素中之一或多者可排除在RSV RNA-LNP組合物外。Thus, in a particular embodiment, the liquid RSV RNA-LNP composition comprises: a cationic lipid at a concentration of or about 0.8 to 0.95 mg/mL; a pegylated lipid at a concentration of or about 0.05 to 0.15 mg/mL; a first structural lipid at a concentration of or about 0.1 to 0.25 mg/mL; a second structural lipid at a concentration of or about 0.3 to 0.45 mg/mL; and further comprises: a first buffer at a concentration of or about 0.1 to 0.3 mg/mL; a second buffer at a concentration of or about 1.25 to 1.4 mg/mL; and a stabilizer at a concentration of or about 95 to 110 mg/mL. In some aspects, one or more of the aforementioned elements may be excluded from the RSV RNA-LNP composition.
因此，在特定態樣中，液體RSV RNA-LNP組合物包含：ALC-0315，其濃度為或為約0.8至0.95 mg/mL；ALC-0159，其濃度為或為約0.05至0.15 mg/mL；DSPC，其濃度為或為約0.1至0.25 mg/mL；膽固醇，其濃度為或為約0.3至0.45 mg/mL；且進一步包含Tris緩衝組合物，該Tris緩衝組合物包含：緩血酸胺，其濃度為或為約0.1至0.3 mg/mL；Tris HCl，其濃度為或為約1.25至1.4 mg/mL；及蔗糖，其濃度為或為約95至110 mg/mL。在一些態樣中，前述要素中之一或多者可排除在RSV RNA-LNP組合物外。Thus, in a particular embodiment, the liquid RSV RNA-LNP composition comprises: ALC-0315 at a concentration of or about 0.8 to 0.95 mg/mL; ALC-0159 at a concentration of or about 0.05 to 0.15 mg/mL; DSPC at a concentration of or about 0.1 to 0.25 mg/mL; cholesterol at a concentration of or about 0.3 to 0.45 mg/mL; and further comprises a Tris buffer composition comprising: thiazide at a concentration of or about 0.1 to 0.3 mg/mL; Tris HCl at a concentration of or about 1.25 to 1.4 mg/mL; and sucrose at a concentration of or about 95 to 110 mg/mL. In some aspects, one or more of the aforementioned elements may be excluded from the RSV RNA-LNP composition.
在特定態樣中，RSV RNA-LNP組合物為凍乾RSV RNA-LNP組合物，且凍乾RSV RNA-LNP組合物進一步包含(在復原之後)：第一緩衝劑，其濃度為或為約0.01至0.15 mg/mL；第二緩衝劑，其濃度為或為約0.5至0.65 mg/mL；穩定劑，其濃度為或為約35至50 mg/mL；及鹽稀釋劑，其濃度為或為約5至15 mg/mL。在一些態樣中，前述要素中之一或多者可排除在RSV RNA-LNP組合物外。In a specific embodiment, the RSV RNA-LNP composition is a freeze-dried RSV RNA-LNP composition, and the freeze-dried RSV RNA-LNP composition further comprises (after reconstitution): a first buffer at a concentration of or about 0.01 to 0.15 mg/mL; a second buffer at a concentration of or about 0.5 to 0.65 mg/mL; a stabilizer at a concentration of or about 35 to 50 mg/mL; and a salt diluent at a concentration of or about 5 to 15 mg/mL. In some embodiments, one or more of the aforementioned elements may be excluded from the RSV RNA-LNP composition.
在特定態樣中，RSV RNA-LNP組合物為凍乾RSV RNA-LNP組合物，且凍乾RSV RNA-LNP組合物進一步包含(在復原之後)Tris緩衝組合物，該Tris緩衝組合物包含：緩血酸胺，其濃度為或為約0.01至0.15 mg/mL；Tris HCl，其濃度為或為約0.5至0.65 mg/mL；蔗糖，其濃度為或為約35至50 mg/mL；及氯化鈉(NaCl)，其濃度為或為約5至15 mg/mL。在一些態樣中，前述要素中之一或多者可排除在RSV RNA-LNP組合物外。In a specific embodiment, the RSV RNA-LNP composition is a freeze-dried RSV RNA-LNP composition, and the freeze-dried RSV RNA-LNP composition further comprises (after reconstitution) a Tris buffer composition comprising: sulfhydryl, its concentration is or is about 0.01 to 0.15 mg/mL; Tris HCl, its concentration is or is about 0.5 to 0.65 mg/mL; sucrose, its concentration is or is about 35 to 50 mg/mL; and sodium chloride (NaCl), its concentration is or is about 5 to 15 mg/mL. In some embodiments, one or more of the aforementioned elements can be excluded from the RSV RNA-LNP composition.
因此，在特定態樣中，凍乾RSV RNA-LNP組合物包含(在復原之後)：陽離子脂質，其濃度為或為約0.8至0.95 mg/mL；聚乙二醇化脂質，其濃度為或為約0.05至0.15 mg/mL；第一結構脂質，其濃度為或為約0.1至0.25 mg/mL；第二結構脂質，其濃度為或為約0.3至0.45 mg/mL；且進一步包含：第一緩衝劑，其濃度為或為約0.01至0.15 mg/mL；第二緩衝劑，其濃度為或為約0.5至0.65 mg/mL；穩定劑，其濃度為或為約35至50 mg/mL；及鹽稀釋劑，其濃度為或為約5至15 mg/mL。在特定態樣中，凍乾組合物在0.6至0.75 mL之鹽稀釋劑中復原。在一些態樣中，前述要素中之一或多者可排除在RSV RNA-LNP組合物外。Thus, in a particular embodiment, the freeze-dried RSV RNA-LNP composition comprises (after reconstitution): a cationic lipid at a concentration of or about 0.8 to 0.95 mg/mL; a pegylated lipid at a concentration of or about 0.05 to 0.15 mg/mL; a first structure lipid at a concentration of or about 0.1 to 0.25 mg/mL; a second structure lipid at a concentration of or about 0.3 to 0.45 mg/mL; and further comprises: a first buffer at a concentration of or about 0.01 to 0.15 mg/mL; a second buffer at a concentration of or about 0.5 to 0.65 mg/mL; a stabilizer at a concentration of or about 35 to 50 mg/mL; and a saline diluent at a concentration of or about 5 to 15 mg/mL. In a specific embodiment, the lyophilized composition is reconstituted in 0.6 to 0.75 mL of the saline diluent. In some embodiments, one or more of the aforementioned elements may be excluded from the RSV RNA-LNP composition.
因此，在一些態樣中，凍乾RSV RNA-LNP組合物包含(在復原之後)：ALC-0315，其濃度為或為約0.8至0.95 mg/mL；ALC-0159，其濃度為或為約0.05至0.15 mg/mL；DSPC，其濃度為或為約0.1至0.25 mg/mL；膽固醇，其濃度為或為約0.3至0.45 mg/mL；且進一步包含：緩血酸胺，其濃度為或為約0.01至0.15 mg/mL；Tris HCl，其濃度為或為約0.5至0.65 mg/mL；蔗糖，其濃度為或為約35至50 mg/mL；及NaCl，其濃度為或為約5至15 mg/mL。在特定態樣中，凍乾組合物在0.6至0.75 mL之NaCl (鹽水)中復原。在一些態樣中，前述要素中之一或多者可排除在RSV RNA-LNP組合物外。Thus, in some aspects, the freeze-dried RSV RNA-LNP composition comprises (after reconstitution): ALC-0315 at a concentration of or about 0.8 to 0.95 mg/mL; ALC-0159 at a concentration of or about 0.05 to 0.15 mg/mL; DSPC at a concentration of or about 0.1 to 0.25 mg/mL; cholesterol at a concentration of or about 0.3 to 0.45 mg/mL; and further comprises: thiazide at a concentration of or about 0.01 to 0.15 mg/mL; Tris HCl at a concentration of or about 0.5 to 0.65 mg/mL; sucrose at a concentration of or about 35 to 50 mg/mL; and NaCl at a concentration of or about 5 to 15 mg/mL. In certain aspects, the freeze-dried composition is reconstituted in 0.6 to 0.75 mL of NaCl (saline). In some aspects, one or more of the aforementioned elements may be excluded from the RSV RNA-LNP composition.
以上凍乾RSV RNA-LNP組合物中之濃度係在復原後測定。The concentrations in the above freeze-dried RSV RNA-LNP compositions were measured after reconstitution.
在一些態樣中，RSV RNA-LNP組合物(凍乾前)包含：陽離子脂質，其濃度為或為約1.0至3.0 mg/mL；聚乙二醇化脂質，其濃度為或為約0.10至0.35 mg/mL；第一結構脂質，其濃度為或為約0.4至0.55 mg/mL；第二結構脂質，其濃度為或為約0.85至1.0 mg/mL；且進一步包含：第一緩衝劑，其濃度為或為約0.1至0.3 mg/mL；第二緩衝劑，其濃度為或為約1.25至1.40 mg/mL；穩定劑，其濃度為或為約95至110 mg/mL。在一些態樣中，前述要素中之一或多者可排除在RSV RNA-LNP組合物外。In some embodiments, the RSV RNA-LNP composition (before lyophilization) comprises: a cationic lipid at a concentration of or about 1.0 to 3.0 mg/mL; a pegylated lipid at a concentration of or about 0.10 to 0.35 mg/mL; a first structural lipid at a concentration of or about 0.4 to 0.55 mg/mL; a second structural lipid at a concentration of or about 0.85 to 1.0 mg/mL; and further comprises: a first buffer at a concentration of or about 0.1 to 0.3 mg/mL; a second buffer at a concentration of or about 1.25 to 1.40 mg/mL; and a stabilizer at a concentration of or about 95 to 110 mg/mL. In some aspects, one or more of the aforementioned elements may be excluded from the RSV RNA-LNP composition.
因此，在一些態樣中，RSV RNA-LNP組合物(凍乾前)包含：ALC-0315，其濃度為或為約1.0至3.0 mg/mL；ALC-0159，其濃度為或為約0.10至0.35 mg/mL；DSPC，其濃度為或為約0.4至0.55 mg/mL；膽固醇，其濃度為或為約0.85至1.0 mg/mL；且進一步包含：緩血酸胺，其濃度為或為約0.1至0.3 mg/mL；Tris HCl，其濃度為或為約1.25至1.40 mg/mL；蔗糖，其濃度為或為約95至110 mg/m。在一些態樣中，前述要素中之一或多者可排除在RSV RNA-LNP組合物外。Thus, in some aspects, the RSV RNA-LNP composition (before freeze drying) comprises: ALC-0315 at a concentration of or about 1.0 to 3.0 mg/mL; ALC-0159 at a concentration of or about 0.10 to 0.35 mg/mL; DSPC at a concentration of or about 0.4 to 0.55 mg/mL; cholesterol at a concentration of or about 0.85 to 1.0 mg/mL; and further comprises: sulphuric acid amine at a concentration of or about 0.1 to 0.3 mg/mL; Tris HCl at a concentration of or about 1.25 to 1.40 mg/mL; sucrose at a concentration of or about 95 to 110 mg/mL. In some aspects, one or more of the aforementioned elements may be excluded from the RSV RNA-LNP composition.
RSV RNA-LNP組合物進一步包含囊封於LNP中之本文所描述之RSV RNA。The RSV RNA-LNP composition further comprises the RSV RNA described herein encapsulated in the LNP.
在特定態樣中，RSV RNA-LNP組合物為液體RSV RNA-LNP組合物，其包含濃度為至少、至多、恰好以下或在以下之間(包括性或排他性)的本文所揭示之編碼RSV多肽的RSV RNA聚核苷酸：0.01、0.15、0.30、0.45、0.60、0.75或0.90 mg/mL，較佳地為或為約0.01至0.09 mg/mL，該等聚核苷酸囊封於具有以下之脂質組成的LNP中：陽離子脂質，其濃度為或為約0.8至0.95 mg/mL；聚乙二醇化脂質，其濃度為或為約0.05至0.15 mg/mL；第一結構脂質，其濃度為或為約0.1至0.25 mg/mL；及第二結構脂質，其濃度為或為約0.3至0.45 mg/mL；且其進一步包含緩衝組合物，該緩衝組合物包含：第一緩衝劑，其濃度為或為約0.15至0.3 mg/mL；第二緩衝劑，其濃度為或為約1.25至1.4 mg/mL；及穩定劑，其濃度為或為約95至110 mg/mL。在一些態樣中，前述要素中之一或多者可排除在RSV RNA-LNP組合物外。In a specific aspect, the RSV RNA-LNP composition is a liquid RSV RNA-LNP composition comprising a RSV RNA polynucleotide encoding an RSV polypeptide disclosed herein at a concentration of at least, at most, just below, or between the following (inclusive or exclusive): 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably at or about 0.01 to 0.09 mg/mL, encapsulated in an LNP having a lipid composition of: a cationic lipid at a concentration of at or about 0.8 to 0.95 mg/mL; a pegylated lipid at a concentration of at or about 0.05 to 0.15 mg/mL; a first structure lipid at a concentration of at or about 0.1 to 0.25 mg/mL; and a second structural lipid at a concentration of or about 0.3 to 0.45 mg/mL; and further comprising a buffer composition comprising: a first buffer at a concentration of or about 0.15 to 0.3 mg/mL; a second buffer at a concentration of or about 1.25 to 1.4 mg/mL; and a stabilizer at a concentration of or about 95 to 110 mg/mL. In some aspects, one or more of the aforementioned elements may be excluded from the RSV RNA-LNP composition.
在特定態樣中，液體RSV RNA-LNP組合物包含濃度為至少、至多、恰好以下或在以下之間(包括性或排他性)的本文所揭示之編碼RSV多肽的RSV RNA聚核苷酸：0.01、0.15、0.30、0.45、0.60、0.75或0.90 mg/mL，較佳地為或為約0.01至0.09 mg/mL，且更佳地為或為約0.06 mg/mL，該聚核苷酸囊封於具有以下之脂質組成的LNP中：ALC-0315，其濃度為或為約0.8至0.95 mg/mL；ALC-0159，其濃度為或為約0.05至0.15 mg/mL；DSPC，其濃度為或為約0.1至0.25 mg/mL；及膽固醇，其濃度為或為約0.3至0.45 mg/mL；且進一步包括Tris緩衝組合物，該Tris緩衝組合物包含：緩血酸胺，其濃度為或為約0.1至0.3 mg/mL；Tris HCl，其濃度為或為約1.25至1.4 mg/mL；及蔗糖，其濃度為或為約95至110 mg/mL。在一些態樣中，前述要素中之一或多者可排除在RSV RNA-LNP組合物外。In certain aspects, the liquid RSV RNA-LNP composition comprises a RSV RNA polynucleotide encoding an RSV polypeptide disclosed herein at a concentration of at least, at most, just below, or between, inclusively or exclusively, 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably at or about 0.01 to 0.09 mg/mL, and more preferably at or about 0.06 mg/mL, encapsulated in an LNP having a lipid composition of ALC-0315 at or about 0.8 to 0.95 mg/mL; ALC-0159 at or about 0.05 to 0.15 mg/mL; DSPC at or about 0.1 to 0.25 mg/mL; and cholesterol at a concentration of or about 0.3 to 0.45 mg/mL; and further comprising a Tris buffer composition comprising: sulfadiazine at a concentration of or about 0.1 to 0.3 mg/mL; Tris HCl at a concentration of or about 1.25 to 1.4 mg/mL; and sucrose at a concentration of or about 95 to 110 mg/mL. In some aspects, one or more of the aforementioned elements may be excluded from the RSV RNA-LNP composition.
在特定態樣中，RSV RNA-LNP組合物為凍乾RSV RNA-LNP組合物，其包含濃度為至少、至多、恰好以下或在以下之間(包括性或排他性)的本文所揭示之編碼RSV多肽的RSV RNA聚核苷酸：0.01、0.15、0.30、0.45、0.60、0.75或0.90 mg/mL，較佳地為或為約0.01至0.09 mg/mL，該聚核苷酸囊封於具有以下之脂質組成的LNP中：陽離子脂質，其濃度為或為約0.8至0.95 mg/mL；聚乙二醇化脂質，其濃度為或為約0.05至0.15 mg/mL；第一結構脂質，其濃度為或為約0.1至0.25 mg/mL；及第二結構脂質，其濃度為或為約0.3至0.45 mg/mL；且進一步包含：第一緩衝劑，其濃度為或為約0.01至0.15 mg/mL；第二緩衝劑，其濃度為或為約0.5至0.65 mg/mL；穩定劑，其濃度為或為約35至50 mg/mL；及鹽稀釋劑，其濃度為或為約5至15 mg/mL。在一些態樣中，前述要素中之一或多者可排除在RSV RNA-LNP組合物外。在特定態樣中，凍乾組合物在0.6至0.75 mL之鹽稀釋劑中復原。凍乾RSV RNA-LNP組合物中之濃度係在復原後測定。In certain aspects, the RSV RNA-LNP composition is a freeze-dried RSV RNA-LNP composition comprising a RSV RNA polynucleotide encoding an RSV polypeptide disclosed herein at a concentration of at least, at most, just below, or between the following (inclusive or exclusive): 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably at or about 0.01 to 0.09 mg/mL, encapsulated in an LNP having a lipid composition of: a cationic lipid at a concentration of at or about 0.8 to 0.95 mg/mL; a pegylated lipid at a concentration of at or about 0.05 to 0.15 mg/mL; a first structure lipid at a concentration of at or about 0.1 to 0.25 mg/mL; and a second structural lipid at a concentration of or about 0.3 to 0.45 mg/mL; and further comprising: a first buffer at a concentration of or about 0.01 to 0.15 mg/mL; a second buffer at a concentration of or about 0.5 to 0.65 mg/mL; a stabilizer at a concentration of or about 35 to 50 mg/mL; and a salt diluent at a concentration of or about 5 to 15 mg/mL. In some aspects, one or more of the aforementioned elements may be excluded from the RSV RNA-LNP composition. In a specific aspect, the freeze-dried composition is reconstituted in 0.6 to 0.75 mL of the salt diluent. The concentration in the freeze-dried RSV RNA-LNP composition is determined after reconstitution.
在特定態樣中，凍乾RSV RNA-LNP組合物包含濃度為至少、至多、恰好以下或在以下之間(包括性或排他性)的本文所揭示之編碼RSV多肽的RSV RNA聚核苷酸：0.01、0.15、0.30、0.45、0.60、0.75或0.90 mg/mL，較佳地為或為約0.01至0.09 mg/mL，且更佳地為或為約0.06 mg/mL，該聚核苷酸囊封於具有以下之脂質組成的LNP中：ALC-0315，其濃度為或為約0.8至0.95 mg/mL；ALC-0159，其濃度為或為約0.05至0.15 mg/mL；DSPC，其濃度為或為約0.1至0.25 mg/mL；及膽固醇，其濃度為或為約0.3至0.45 mg/mL；且進一步包含：緩血酸胺，其濃度為或為約0.01至0.15 mg/mL；Tris HCl，其濃度為或為約0.5至0.65 mg/mL；蔗糖，其濃度為或為約35至50 mg/mL；及NaCl，其濃度為或為約5至15 mg/mL。在一些態樣中，前述要素中之一或多者可排除在RSV RNA-LNP組合物外。在特定態樣中，凍乾組合物在0.6至0.75 mL之NaCl稀釋劑(鹽水)中復原。凍乾RSV RNA-LNP組合物中之濃度係在復原後測定。In certain aspects, the freeze-dried RSV RNA-LNP composition comprises a RSV RNA polynucleotide encoding an RSV polypeptide disclosed herein at a concentration of at least, at most, just below, or between, inclusively or exclusively, 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably at or about 0.01 to 0.09 mg/mL, and more preferably at or about 0.06 mg/mL, encapsulated in an LNP having a lipid composition of ALC-0315 at or about 0.8 to 0.95 mg/mL; ALC-0159 at or about 0.05 to 0.15 mg/mL; DSPC at or about 0.1 to 0.25 mg/mL; mg/mL; and cholesterol at a concentration of or about 0.3 to 0.45 mg/mL; and further comprising: sulphuric acid at a concentration of or about 0.01 to 0.15 mg/mL; Tris HCl at a concentration of or about 0.5 to 0.65 mg/mL; sucrose at a concentration of or about 35 to 50 mg/mL; and NaCl at a concentration of or about 5 to 15 mg/mL. In some aspects, one or more of the aforementioned elements may be excluded from the RSV RNA-LNP composition. In a specific aspect, the freeze-dried composition is reconstituted in 0.6 to 0.75 mL of NaCl diluent (saline). The concentration in the freeze-dried RSV RNA-LNP composition is determined after reconstitution.
在一些態樣中，RSV RNA-LNP組合物(凍乾前)包含濃度為至少、至多、恰好以下或在以下之間(包括性或排他性)的本文所揭示之編碼RSV多肽的RSV RNA聚核苷酸：0.01、0.15、0.30、0.45、0.60、0.75或0.90 mg/mL，較佳地為或為約0.01至0.09 mg/mL，該聚核苷酸囊封於具有以下之脂質組成的LNP中：陽離子脂質，其濃度為或為約1.0至3.0 mg/mL；聚乙二醇化脂質，其濃度為或為約0.10至0.35 mg/mL；第一結構脂質，其濃度為或為約0.4至0.55 mg/mL；第二結構脂質，其濃度為或為約0.85至1.0 mg/mL；且進一步包含：第一緩衝劑，其濃度為或為約0.1至0.3 mg/mL；第二緩衝劑，其濃度為或為約1.25至1.40 mg/mL；穩定劑，其濃度為或為約95至110 mg/mL。在一些態樣中，前述要素中之一或多者可排除在RSV RNA-LNP組合物外。In some aspects, the RSV RNA-LNP composition (before lyophilization) comprises a RSV RNA polynucleotide encoding an RSV polypeptide disclosed herein at a concentration of at least, at most, just below, or between (inclusive or exclusive) 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably at or about 0.01 to 0.09 mg/mL, encapsulated in an LNP having a lipid composition of: a cationic lipid at or about 1.0 to 3.0 mg/mL; a pegylated lipid at or about 0.10 to 0.35 mg/mL; a first structure lipid at or about 0.4 to 0.55 mg/mL; a second structural lipid at a concentration of or about 0.85 to 1.0 mg/mL; and further comprising: a first buffer at a concentration of or about 0.1 to 0.3 mg/mL; a second buffer at a concentration of or about 1.25 to 1.40 mg/mL; and a stabilizer at a concentration of or about 95 to 110 mg/mL. In some aspects, one or more of the aforementioned elements may be excluded from the RSV RNA-LNP composition.
因此，在一些態樣中，RSV RNA-LNP組合物(凍乾前)包含濃度為至少、至多、恰好以下或在以下之間(包括性或排他性)的本文所揭示之編碼RSV多肽的RSV RNA聚核苷酸：0.01、0.15、0.30、0.45、0.60、0.75或0.90 mg/mL，較佳地為或為約0.01至0.09 mg/mL，且更佳地0.15 mg/mL，該聚核苷酸囊封於具有包含以下之脂質組成的LNP中：ALC-0315，其濃度為或為約1.0至3.0 mg/mL；ALC-0159，其濃度為或為約0.10至0.35 mg/mL；DSPC，其濃度為或為約0.4至0.55 mg/mL；膽固醇，其濃度為或為約0.85至1.0 mg/mL；且進一步包含：緩血酸胺，其濃度為或為約0.1至0.3 mg/mL；Tris HCl，其濃度為或為約1.25至1.40 mg/mL；蔗糖，其濃度為或為約95至110 mg/mL。在一些態樣中，前述要素中之一或多者可排除在RSV RNA-LNP組合物外。Thus, in some aspects, the RSV RNA-LNP composition (before lyophilization) comprises a RSV RNA polynucleotide encoding an RSV polypeptide disclosed herein at a concentration of at least, at most, just below, or between (inclusive or exclusive) 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably at or about 0.01 to 0.09 mg/mL, and more preferably 0.15 mg/mL, encapsulated in an LNP having a lipid composition comprising: ALC-0315 at or about 1.0 to 3.0 mg/mL; ALC-0159 at or about 0.10 to 0.35 mg/mL; DSPC at or about 0.4 to 0.55 mg/mL; mg/mL; cholesterol at a concentration of or about 0.85 to 1.0 mg/mL; and further comprising: sulphuric acid amine at a concentration of or about 0.1 to 0.3 mg/mL; Tris HCl at a concentration of or about 1.25 to 1.40 mg/mL; sucrose at a concentration of or about 95 to 110 mg/mL. In some aspects, one or more of the aforementioned elements may be excluded from the RSV RNA-LNP composition.
在一些態樣中，液體RNA-LNP免疫原性組合物包含濃度為至少、至多、恰好以下或在以下之間(包括性或排他性)的本文所揭示之編碼RSV多肽的RNA分子/聚核苷酸：0.01、0.15、0.30、0.45、0.60、0.75或0.90 mg/mL，較佳地為或為約0.01至0.09 mg/mL，該聚核苷酸囊封於LNP中，且進一步包含或包含約5至15 mM Tris緩衝液及pH呈或呈約7.0至8.0的約200至400 mM蔗糖。在一些態樣中，前述要素中之一或多者可排除在液體RNA-LNP免疫原性組合物外。In some aspects, the liquid RNA-LNP immunogenic composition comprises an RNA molecule/polynucleotide encoding a RSV polypeptide disclosed herein at a concentration of at least, at most, just below, or between, inclusively or exclusively: 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably about 0.01 to 0.09 mg/mL, encapsulated in LNP, and further comprises or comprises about 5 to 15 mM Tris buffer and about 200 to 400 mM sucrose at or about 7.0 to 8.0. In some aspects, one or more of the aforementioned elements may be excluded from the liquid RNA-LNP immunogenic composition.
在一些態樣中，液體RNA-LNP免疫原性組合物包含濃度為至少、至多、恰好以下或在以下之間(包括性或排他性)的本文所揭示之編碼RSV多肽的RNA分子/聚核苷酸：0.01、0.15、0.30、0.45、0.60、0.75或0.90 mg/mL，較佳地為或為約0.01至0.09 mg/mL，且更佳地為或為約0.06 mg/mL，該RNA分子/聚核苷酸囊封於LNP中，且進一步包含或包含約10 mM Tris緩衝液及pH呈或呈約7.4的300 mM蔗糖。在一些態樣中，前述要素中之一或多者可排除在液體RNA-LNP免疫原性組合物外。In some aspects, the liquid RNA-LNP immunogenic composition comprises an RNA molecule/polynucleotide encoding a RSV polypeptide disclosed herein at a concentration of at least, at most, just below, or between, inclusively or exclusively: 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably at or about 0.01 to 0.09 mg/mL, and more preferably at or about 0.06 mg/mL, encapsulated in LNP, and further comprising or comprising about 10 mM Tris buffer and 300 mM sucrose at or about pH 7.4. In some aspects, one or more of the aforementioned elements may be excluded from the liquid RNA-LNP immunogenic composition.
在一些態樣中，RNA-LNP免疫原性組合物(凍乾前)包含濃度為至少、至多、恰好以下或在以下之間(包括性或排他性)的本文所揭示之編碼RSV多肽的RNA分子/聚核苷酸：0.01、0.15、0.30、0.45、0.60、0.75或0.90 mg/mL，較佳地為或為約0.01至0.09 mg/mL，該RNA分子/聚核苷酸囊封於LNP中，且進一步包含或包含約5至15 mM Tris緩衝液及pH呈或呈約7.0至8.0的200至400 mM蔗糖，且用0.9%氯化鈉稀釋劑復原。在一些態樣中，前述要素中之一或多者可排除在RNA-LNP免疫原性組合物外。In some aspects, the RNA-LNP immunogenic composition (before lyophilization) comprises a concentration of at least, at most, just below, or between (inclusive or exclusive) 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably about 0.01 to 0.09 mg/mL, of an RNA molecule/polynucleotide encoding a RSV polypeptide disclosed herein, encapsulated in LNP, and further comprising or comprising about 5 to 15 mM Tris buffer and 200 to 400 mM sucrose at or about 7.0 to 8.0, and reconstituted with 0.9% sodium chloride diluent. In some aspects, one or more of the aforementioned elements may be excluded from the RNA-LNP immunogenic composition.
在一些態樣中，RNA-LNP免疫原性組合物(凍乾前)包含濃度為至少、至多、恰好以下或在以下之間(包括性或排他性)的本文所揭示之編碼RSV多肽的RNA分子/聚核苷酸：0.01、0.15、0.30、0.45、0.60、0.75或0.90 mg/mL，較佳地為或為約0.01至0.09 mg/mL，且更佳地0.15 mg/mL，該RNA分子/聚核苷酸囊封於LNP中，且進一步包含或包含約10 mM Tris緩衝液及pH呈或呈約7.4的300 mM蔗糖，且用0.9%氯化鈉稀釋劑復原。在一些態樣中，前述要素中之一或多者可排除在RNA-LNP免疫原性組合物外。In some aspects, the RNA-LNP immunogenic composition (before lyophilization) comprises an RNA molecule/polynucleotide encoding a RSV polypeptide disclosed herein at a concentration of at least, at most, just below, or between (inclusive or exclusive): 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL, preferably at or about 0.01 to 0.09 mg/mL, and more preferably 0.15 mg/mL, encapsulated in LNP, and further comprising or comprising about 10 mM Tris buffer and 300 mM sucrose at or about pH 7.4, and reconstituted with 0.9% sodium chloride diluent. In some aspects, one or more of the aforementioned elements may be excluded from the RNA-LNP immunogenic composition.
B.疫苗在一些態樣中，本文所描述之醫藥組合物為用於誘導免疫反應之免疫原性組合物。舉例而言，在一些態樣中，免疫原性組合物為疫苗。在一些態樣中，本文所描述之組合物包括至少一種本文所描述之經分離核酸或多肽分子。在特定態樣中，免疫原性組合物包含核酸，且免疫原性組合物為核酸疫苗。在一些態樣中，免疫原性組合物包含RNA (例如mRNA、saRNA)，且疫苗為RNA疫苗。在其他態樣中，免疫原性組合物包含DNA，且疫苗為DNA疫苗。在又其他態樣中，免疫原性組合物包含多肽，且疫苗為多肽疫苗。可用核酸及/或肽或多肽組合物治療之病狀及/或疾病包括但不限於由感染引起及/或影響之彼等病狀及/或疾病、癌症、罕見病以及由蛋白質或核酸之過度產生、不足產生及/或不當產生引起之其他疾病或病狀。B.Vaccines In some aspects, the pharmaceutical compositions described herein are immunogenic compositions for inducing an immune response. For example, in some aspects, the immunogenic compositions are vaccines. In some aspects, the compositions described herein include at least one isolated nucleic acid or polypeptide molecule described herein. In specific aspects, the immunogenic composition comprises a nucleic acid, and the immunogenic composition is a nucleic acid vaccine. In some aspects, the immunogenic composition comprises RNA (e.g., mRNA, saRNA), and the vaccine is an RNA vaccine. In other aspects, the immunogenic composition comprises DNA, and the vaccine is a DNA vaccine. In yet other aspects, the immunogenic composition comprises a polypeptide, and the vaccine is a polypeptide vaccine. Conditions and/or diseases that may be treated with nucleic acid and/or peptide or polypeptide compositions include, but are not limited to, those caused and/or affected by infection, cancer, rare diseases, and other diseases or conditions caused by overproduction, underproduction, and/or inappropriate production of proteins or nucleic acids.
在一些態樣中，組合物實質上不含一或多種雜質或污染物，且例如包括以下純度之核酸或多肽分子：至少、至多、恰好等於90%、91%、92%、93%、94%、95%、96%、97%、98%或99%純度或其中之間(包括性或排他性)的純度；至少98%純度；或至少99%純度。In some aspects, the composition is substantially free of one or more impurities or contaminants, and, for example, includes nucleic acid or polypeptide molecules that are at least, at most, exactly equal to, or equal to 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure, or a purity therebetween (inclusive or exclusive); at least 98% pure; or at least 99% pure.
本發明包括用於預防、治療及/或改善個體之感染、疾病或病狀的方法，其包括向個體投與有效量的包括至少一個編碼本文所描述之多肽或組合物之開讀框的RNA分子。因此，本發明涵蓋用於主動及被動免疫接種態樣之疫苗。提議適用作疫苗之免疫原性組合物可由編碼一或多種多肽(諸如RSV preF多肽)之RNA分子製備。在某些態樣中，免疫原性組合物經凍乾以便更容易調配至所需媒劑中。The present invention includes methods for preventing, treating and/or ameliorating an infection, disease or condition in a subject comprising administering to the subject an effective amount of an RNA molecule comprising at least one open reading frame encoding a polypeptide or composition described herein. Thus, the present invention encompasses vaccines for active and passive vaccination. It is proposed that immunogenic compositions suitable for use as vaccines can be prepared from RNA molecules encoding one or more polypeptides (such as RSV preF polypeptides). In certain aspects, the immunogenic composition is lyophilized for easier formulation into a desired medium.
含有核酸及/或肽或多肽作為活性成分之疫苗的製備一般為此項技術中充分瞭解的，如由美國專利4,608,251；4,601,903；4,599,231；4,599,230；4,596,792；及4,578,770所例示，以上所有者均以引用之方式併入本文中。通常，此類疫苗係以呈液體溶液或懸浮液之可注射劑形式製備；亦可製備適合於在注射之前在液體中形成溶液或在液體中形成懸浮液的固體形式。製劑亦可乳化。活性免疫原性成分通常與醫藥學上可接受的且與活性成分相容的賦形劑混合。適合之賦形劑例如為水、鹽水、右旋糖、甘油、乙醇或其類似者及其組合。另外，必要時，疫苗可含有大量輔助物質，諸如濕潤劑或乳化劑、pH緩衝劑或增強疫苗之有效性的佐劑。在特定態樣中，疫苗用物質組合調配，如美國專利6,793,923及6,733,754中所描述，其以引用之方式併入本文中。在一些態樣中，前述要素中之一或多者可排除在疫苗外。The preparation of vaccines containing nucleic acids and/or peptides or polypeptides as active ingredients is generally well understood in the art, as exemplified by U.S. Patents 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, all of which are incorporated herein by reference. Typically, such vaccines are prepared in the form of injectables that are liquid solutions or suspensions; solid forms suitable for forming solutions in liquids or suspensions in liquids prior to injection may also be prepared. The formulation may also be emulsified. The active immunogenic ingredient is typically mixed with a pharmaceutically acceptable excipient that is compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if necessary, the vaccine may contain a large amount of auxiliary substances, such as wetting agents or emulsifiers, pH buffers or adjuvants that enhance the effectiveness of the vaccine. In a specific aspect, the vaccine is formulated with a combination of substances, such as described in U.S. Patents 6,793,923 and 6,733,754, which are incorporated herein by reference. In some aspects, one or more of the aforementioned elements may be excluded from the vaccine.
疫苗可習知地藉由例如皮下或肌肉內注射以非經腸方式投與。適合於其他投與模式之額外調配物包括栓劑及在一些情況下口服調配物。對於栓劑而言，傳統黏合劑及載劑可包括例如聚伸烷二醇或三酸甘油酯；此類栓劑可由含有在或約在0.5%至約10%之範圍內之活性成分的混合物形成。在一些態樣中，栓劑可由含有在或約在1%至約2%之範圍內之活性成分的混合物形成。口服調配物包括常用賦形劑，諸如醫藥級甘露糖醇、乳糖、澱粉、硬脂酸鎂、糖精鈉、纖維素、碳酸鎂及其類似物。在一些態樣中，前述賦形劑中之1、2、3、4、5者或更多者可排除在口服調配物外。此等組合物採用溶液、懸浮液、錠劑、丸劑、膠囊、持續釋放調配物或散劑之形式，且含有或含有約10%至約95%之活性成分。Vaccines are known to be administered parenterally, for example, by subcutaneous or intramuscular injection. Additional formulations suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from a mixture containing at or about 0.5% to about 10% of the active ingredient. In some aspects, suppositories may be formed from a mixture containing at or about 1% to about 2% of the active ingredient. Oral formulations include commonly used excipients such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned excipients may be excluded from oral formulations. Such compositions are in the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain or contain about 10% to about 95% active ingredient.
可將編碼多肽之核酸構築體以及多肽調配成呈中性或鹽形式之疫苗。「醫藥學上可接受之鹽」包括酸加成鹽及鹼加成鹽兩者。「醫藥學上可接受之酸加成鹽」係指保持游離鹼之生物有效性及特性的彼等鹽，其在生物學或其他方面無不良反應，且由以下形成：無機酸，諸如但不限於鹽酸、氫溴酸、硫酸、硝酸、磷酸及其類似物；及有機酸，諸如但不限於乙酸、2,2-二氯乙酸、己二酸、海藻酸、抗壞血酸、天冬胺酸、苯磺酸、苯甲酸、4-乙醯胺基苯甲酸、樟腦酸、樟腦-10-磺酸、癸酸、己酸、辛酸、碳酸、肉桂酸、檸檬酸、環己胺磺酸、十二烷基硫酸、乙烷-1,2-二磺酸、乙烷磺酸、2-羥基乙烷磺酸、甲酸、反丁烯二酸、半乳糖二酸、龍膽酸、葡糖庚酸、葡萄糖酸、葡糖醛酸、麩胺酸、戊二酸、2-側氧基-戊二酸、甘油磷酸、乙醇酸、馬尿酸、異丁酸、乳酸、乳糖酸、月桂酸、順丁烯二酸、蘋果酸、丙二酸、杏仁酸、甲烷磺酸、黏液酸、萘-1,5-二磺酸、萘-2-磺酸、1-羥基-2-萘甲酸、菸鹼酸、油酸、乳清酸、草酸、棕櫚酸、雙羥萘酸、丙酸、焦麩胺酸、丙酮酸、柳酸、4-胺基柳酸、癸二酸、硬脂酸、丁二酸、酒石酸、硫氰酸、對甲苯磺酸、三氟乙酸、十一碳烯酸及其類似者。在一些態樣中，可排除前述無機酸中之1、2、3、4、5者或更多者。「醫藥學上可接受之鹼加成鹽」係指保留游離酸之生物有效性及特性，合乎生物學或其他方面需要之鹽。此等鹽由無機鹼或有機鹼與游離酸加成製備。衍生自無機鹼之鹽包括但不限於鈉鹽、鉀鹽、鋰鹽、銨鹽、鈣鹽、鎂鹽、鐵鹽、鋅鹽、銅鹽、錳鹽、鋁鹽及其類似者。在一些態樣中，可排除前述無機鹼中之1、2、3、4、5者或更多者。較佳無機鹽為銨鹽、鈉鹽、鉀鹽、鈣鹽及鎂鹽。衍生自有機鹼之鹽包括但不限於以下各者之鹽：一級胺、二級胺及三級胺、經取代之胺(包括天然存在之經取代胺)、環胺及鹼性離子交換樹脂，諸如氨、異丙胺、三甲胺、二乙胺、三乙胺、三丙胺、二乙醇胺、乙醇胺、丹醇(deanol)、2-二甲胺基乙醇、2-二乙胺基乙醇、二環己胺、離胺酸、精胺酸、組胺酸、咖啡鹼、普魯卡因(procaine)、海卓胺(hydrabamine)、膽鹼、甜菜鹼、苄苯乙胺、苄星青黴素(benzathine)、乙二胺、葡糖胺、甲基還原葡糖胺、可可豆鹼、三乙醇胺、緩血酸胺、嘌呤、哌𠯤、哌啶、N-乙基哌啶、多元胺樹脂及其類似者。尤其較佳有機鹼為異丙胺、二乙胺、乙醇胺、三甲胺、二環己胺、膽鹼及咖啡鹼。在一些態樣中，可排除前述有機鹼中之1、2、3、4、5者或更多者。Nucleic acid constructs encoding polypeptides and polypeptides can be formulated into vaccines in neutral or salt form. "Pharmaceutically acceptable salts" include both acid addition salts and base addition salts. "Pharmaceutically acceptable acid addition salts" means those salts which retain the biological effectiveness and properties of the free bases, have no adverse biological or other effects, and are formed from inorganic acids such as, but not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclohexylaminesulfonic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, methanesulfonic acid, Acid, fumaric acid, galactaric acid, gentianic acid, glucoheptanoic acid, gluconic acid, glucuronic acid, glutamine, glutaric acid, 2-hydroxy-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, apple acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, niacin, oleic acid, orotic acid, oxalic acid, palmitic acid, bishydroxynaphthoic acid, propionic acid, pyroglutamine, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid and the like. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned inorganic acids may be excluded. "Pharmaceutically acceptable base addition salts" refer to salts that retain the biological effectiveness and properties of the free acid and meet biological or other requirements. These salts are prepared by addition of inorganic bases or organic bases to free acids. Salts derived from inorganic bases include but are not limited to sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts and the like. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned inorganic bases may be excluded. Preferred inorganic salts are ammonium, sodium, potassium, calcium and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, di- and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, diamine The organic bases include arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benzylphenethylamine, benzathine, ethylenediamine, glucosamine, methylglucosamine, theobrene, triethanolamine, styrene, purine, piperidine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned organic bases may be excluded.
編碼多肽之核酸構築體以及多肽或其醫藥學上可接受之鹽可含有一或多個不對稱中心，且因此可產生鏡像異構物、非鏡像異構物以及就絕對立體化學而言可對於胺基酸界定為(R)-或(S)-或界定為(D)-或(L)-的其他立體異構形式。本發明意謂包括所有該等可能的異構物，以及其外消旋及光學純形式。具光學活性之(+)及(-)、(R)-及(S)-、或(D)-及(L)-異構物可使用對掌性合成子或對掌性試劑來製備，或使用習知技術(例如層析及分步結晶)來解析。用於製備/分離個別鏡像異構物之習知技術包括自適合的光學純前驅物進行對掌性合成或使用例如對掌性高壓液相層析(HPLC)對外消旋體(或鹽或衍生物之外消旋體)進行解析。當本文所描述之化合物含有烯系雙鍵或其他幾何不對稱中心時且除非另外說明，否則意欲化合物包括E與Z型幾何異構物。同樣，亦意欲包括所有互變異構形式。「立體異構物」係指由相同鍵所鍵結之相同原子構成但具有不可互換的不同三維結構之化合物。本發明涵蓋各種立體異構物及其混合物且包括「鏡像異構物」，鏡像異構物係指分子彼此間為不可重疊之鏡像的兩種立體異構物。「互變異構物」係指質子自分子之一個原子轉移至同一分子之另一原子。本發明揭示任何該等化合物之互變異構物。Nucleic acid constructs encoding polypeptides and polypeptides or pharmaceutically acceptable salts thereof may contain one or more asymmetric centers and may therefore give rise to mirror image isomers, non-mirror image isomers, and other stereoisomeric forms that may be defined as (R)- or (S)- or as (D)- or (L)- for amino acids in terms of absolute stereochemistry. The present invention is intended to include all such possible isomers, as well as racemic and optically pure forms thereof. Optically active (+) and (-), (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using known techniques such as analysis and fractional crystallization. Known techniques for preparing/isolating individual mirror image isomers include chiral synthesis from suitable optically pure precursors or resolution of racemates (or racemates of salts or derivatives) using, for example, chiral high pressure liquid chromatography (HPLC). When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry and unless otherwise specified, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are intended to be included. "Stereoisomers" refer to compounds composed of the same atoms bonded by the same bonds but having different three-dimensional structures that are not interchangeable. The present invention encompasses various stereoisomers and mixtures thereof and includes "mirror isomers", which are two stereoisomers of a molecule that are non-superimposable mirror images of each other. "Tautomers" are tautomers in which a proton is transferred from one atom of a molecule to another atom of the same molecule. The present invention discloses tautomers of any of these compounds.
以游離鹼或酸形式存在之本文所描述之化合物可藉由用適當無機或有機鹼或酸，藉由熟習此項技術者已知之方法來轉化成其醫藥學上可接受之鹽。化合物之鹽可藉由標準技術轉化成其游離鹼或酸形式。The compounds described herein that exist in free base or acid form can be converted into their pharmaceutically acceptable salts by using an appropriate inorganic or organic base or acid by methods known to those skilled in the art. Salts of compounds can be converted into their free base or acid form by standard techniques.
熟習此項技術者應瞭解，在本文所描述之方法中，中間化合物之官能基可能需要由適合的保護基保護。此類官能基包括羥基、胺基、巰基及羧酸。羥基之適合的保護基包括三烷基矽烷基或二芳基烷基矽烷基(例如三級丁基二甲基矽烷基、三級丁基二苯基矽烷基或三甲基矽烷基)、四氫哌喃基、苯甲基及類似者。胺基、甲脒基及胍基之適合的保護基包括三級丁氧羰基、苯甲氧羰基及類似者。巰基之適合保護基包括-C(O)-R" (其中R"為烷基、芳基或芳烷基)、對甲氧基苯甲基、三苯甲基及類似者。適用於羧酸之保護基包括烷基、芳基或芳基烷基酯。如熟習此項技術者將瞭解，保護基亦可為聚合物樹脂，如王樹脂(Wang resin)、林克樹脂(Rink resin)或2-氯三苯甲基氯化物樹脂。在一些態樣中，可排除前述保護基中之1、2、3、4、5者或更多者。可根據熟習此項技術者已知(參見例如Green, T.W.及P.G.M. Wutz, Protective Groups in Organic Synthesis (1999), 第3版, Wiley)及本文所描述之標準技術來添加或移除保護基。Those skilled in the art will appreciate that in the methods described herein, the functional groups of the intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxyl, amine, alkyl and carboxylic acid. Suitable protecting groups for hydroxyl groups include trialkylsilyl or diarylalkylsilyl (e.g., tertiary butyldimethylsilyl, tertiary butyldiphenylsilyl or trimethylsilyl), tetrahydropyranyl, benzyl and the like. Suitable protecting groups for amine, amidino and guanidino groups include tertiary butyloxycarbonyl, benzyloxycarbonyl and the like. Suitable protecting groups for alkyl groups include -C(O)-R" (wherein R" is alkyl, aryl or aralkyl), p-methoxybenzyl, trityl and the like. Suitable protecting groups for carboxylic acids include alkyl, aryl or arylalkyl esters. As will be appreciated by those skilled in the art, the protecting group may also be a polymer resin, such as Wang resin, Rink resin, or 2-chlorotrityl chloride resin. In some aspects, 1, 2, 3, 4, 5, or more of the aforementioned protecting groups may be excluded. Protecting groups may be added or removed according to standard techniques known to those skilled in the art (see, e.g., Green, T.W. and P.G.M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd edition, Wiley) and described herein.
熟習此項技術者亦應瞭解，雖然本發明化合物之此類受保護衍生物可能不具有同樣的藥理學活性，但其可投與哺乳動物且此後在體內代謝，從而形成具有藥理學活性之本發明化合物。此類衍生物可因此描述為「前藥」。本發明化合物之所有前藥包括在本發明之範疇內。Those skilled in the art will also appreciate that, although such protected derivatives of the compounds of the present invention may not have the same pharmacological activity, they can be administered to mammals and thereafter metabolized in vivo to form pharmacologically active compounds of the present invention. Such derivatives can therefore be described as "prodrugs". All prodrugs of the compounds of the present invention are included within the scope of the present invention.
通常，疫苗以與劑型相容之方式，且以諸如將為治療有效及免疫原性的量投與。待投與之數量取決於待治療之個體，包括個體免疫系統合成抗體之能力及所需防護等級。需要投與之活性成分的精確量視從業者之判斷而定。然而，適合的劑量範圍為每次疫苗接種大約數百微克活性成分。適合於初次投與及加強注射的方案亦為可變的，但典型的是進行初次投與，接著進行後續接種及/或其他投與。Generally, vaccines are administered in a manner compatible with the dosage form, and in such amounts as will be therapeutically effective and immunogenic. The amount to be administered depends on the individual to be treated, including the capacity of the individual's immune system to synthesize antibodies and the degree of protection desired. The exact amount of active ingredient required to be administered depends on the judgment of the practitioner. However, suitable dosages range from about several hundred micrograms of active ingredient per vaccination. Suitable regimens for initial administration and booster injections are also variable, but typically an initial administration is performed, followed by subsequent vaccinations and/or other administrations.
施用方式可廣泛變化。可應用用於投與疫苗之習知方法中之任一種。咸信此等方法包括在生理學上可接受之固體基質內口服施用或在生理學上可接受之分散液中藉由注射或其類似方式而非經腸施用。疫苗劑量將視投與途徑而定且將根據個體之體型及健康狀況而變化。The mode of administration may vary widely. Any of the known methods for administering vaccines may be used. It is believed that these methods include oral administration in a physiologically acceptable solid matrix or by injection or the like in a physiologically acceptable dispersion rather than enteral administration. The vaccine dose will depend on the route of administration and will vary according to the size and health of the individual.
在某些態樣中，將需要投與一次疫苗。在一些態樣中，將需要多次投與疫苗，例如投與2、3、4、5、6或更多次。疫苗接種可間隔1、2、3、4、5、6、7、8至5、6、7、8、9、10、11或12週，包括其間所有範圍。在一些態樣中，疫苗接種可間隔1、2、3、4、5、6、7、8、9、10、11或12個月，包括其間所有範圍。可能需要以1至5年之間隔週期性加強來維持抗體之保護性水平。In some aspects, it will be necessary to administer the vaccine once. In some aspects, it will be necessary to administer the vaccine multiple times, for example, 2, 3, 4, 5, 6 or more times. Vaccinations may be spaced 1, 2, 3, 4, 5, 6, 7, 8 to 5, 6, 7, 8, 9, 10, 11 or 12 weeks apart, including all ranges therebetween. In some aspects, vaccinations may be spaced 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months apart, including all ranges therebetween. Periodic boosts at intervals of 1 to 5 years may be required to maintain protective levels of antibodies.
i.載劑醫藥學上可接受之載劑可包括組合物之液體或非液體基體。若組合物以液體形式提供，則載劑可為水，諸如無熱原水；等張鹽水或緩衝(水)溶液，例如磷酸鹽、檸檬酸鹽緩衝溶液。可使用水或含有鈉鹽、鈣鹽及/或鉀鹽之緩衝液，諸如水性緩衝液。鈉鹽、鈣鹽及/或鉀鹽可以其鹵化物(例如氯化物、碘化物或溴化物)之形式存在，以其氫氧化物、碳酸鹽、碳酸氫鹽或硫酸鹽等形式存在。鈉鹽之實例包括但不限於NaCl、NaI、NaBr、Na2CO3、NaHCO3、Na2SO4、Na2HPO4、Na2HPO4·2H2O，鉀鹽之實例包括但不限於KCl、KI、KBr、K2CO3、KHCO3、K2SO4、KH2PO4，且鈣鹽之實例包括但不限於CaCl2、CaI2、CaBr2、CaCO3、CaSO4、Ca(OH)2。其他載劑之實例可包括糖，諸如乳糖、葡萄糖、海藻糖及蔗糖；澱粉，諸如玉米澱粉或馬鈴薯澱粉；右旋糖；纖維素及其衍生物，諸如羧甲基纖維素鈉、乙基纖維素、乙酸纖維素；粉末黃蓍膠；麥芽；明膠；動物脂；固體滑動劑，諸如硬脂酸、硬脂酸鎂；硫酸鈣；植物油，諸如花生油、棉籽油、芝麻油、橄欖油、玉米油及來自可可的油；多元醇，諸如聚丙二醇、甘油、山梨糖醇、甘露糖醇及聚乙二醇；褐藻酸。其他載劑之實例可包括膠體氧化矽、硬脂酸鎂、纖維素及月桂基硫酸鈉。額外適合之醫藥載劑及稀釋劑以及其使用之醫藥需求描述於Remington's Pharmaceutical Sciences中。在一些態樣中，可排除前述載劑中之1、2、3、4、5者或更多者。i.Carriers Pharmaceutically acceptable carriers may include liquid or non-liquid bases of the composition. If the composition is provided in liquid form, the carrier may be water, such as pyrogen-free water; isotonic saline or buffered (aqueous) solutions, such as phosphate, citrate buffered solutions. Water or buffers containing sodium, calcium and/or potassium salts, such as aqueous buffers, may be used. Sodium, calcium and/or potassium salts may be present in the form of their halides (e.g., chlorides, iodides or bromides), in the form of their hydroxides, carbonates, bicarbonates or sulfates. Examples of sodium salts include, but are not limited to, NaCl, NaI, NaBr, Na2 CO3 , NaHCO3 , Na2 SO4 , Na2 HPO4 , Na2 HPO4 ·2 H2 O, examples of potassium salts include, but are not limited to, KCl, KI, KBr, K2 CO3 , KHCO3 , K2 SO4 , KH2 PO4 , and examples of calcium salts include, but are not limited to, CaCl2 , CaI2 , CaBr2 , CaCO3 , CaSO4 , Ca(OH)2 . Examples of other carriers may include sugars such as lactose, glucose, trehalose and sucrose; starches such as corn starch or potato starch; dextrose; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin; animal fat; solid lubricants such as stearic acid, magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and oil from cocoa; polyols such as polypropylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid. Examples of other carriers may include colloidal silica, magnesium stearate, cellulose, and sodium lauryl sulfate. Additional suitable pharmaceutical carriers and diluents and the pharmaceutical requirements for their use are described in Remington's Pharmaceutical Sciences. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned carriers may be excluded.
ii.佐劑適合佐劑包括所有可接受之免疫刺激化合物，諸如細胞介素、毒素或合成組合物。多種佐劑可用於增強抗體反應。佐劑包括但不限於水包油乳液、油包水乳液、礦物鹽、聚核苷酸及天然物質。可使用之特定佐劑包括弗氏佐劑(Freund's adjuvant)、油(諸如MONTANIDE® ISA51)、IL1、IL2、IL3、IL4、IL5、IL6、IL7、IL8、IL9、IL10、IL12、α-干擾素、PTNGg、GM-CSF、GMCSP、BCG、LT-a、鋁鹽(諸如氫氧化鋁或其他鋁化合物)、MDP化合物(諸如thur-MDP及nor-MDP)、CGP (MTP-PE)、脂質A、單磷醯基脂質A (MPL)、脂肽(例如Pam3Cys)。RIBI，其含有於2%角鯊烯/吐溫80乳液中的自細菌提取之三種組分，亦即MPL、海藻糖二黴菌酸酯(TDM)及細胞壁骨架(CWS)。甚至可使用MHC抗原。ii.Adjuvants Suitable adjuvants include all acceptable immunostimulatory compounds, such as interleukins, toxins or synthetic compositions. A variety of adjuvants can be used to enhance antibody responses. Adjuvants include but are not limited to oil-in-water emulsions, water-in-oil emulsions, mineral salts, polynucleotides and natural substances. Specific adjuvants that may be used include Freund's adjuvant, oils (such as MONTANIDE® ISA51), IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, α-interferon, PTNGg, GM-CSF, GMCSP, BCG, LT-a, aluminum salts (such as aluminum hydroxide or other aluminum compounds), MDP compounds (such as thur-MDP and nor-MDP), CGP (MTP-PE), lipid A, monophosphoryl lipid A (MPL), lipopeptides (such as Pam3Cys). RIBI, which contains three components extracted from bacteria, namely MPL, trehalose dimelatin (TDM) and cell wall skeleton (CWS) in a 2% squalene/Tween 80 emulsion. Even MHC antigens can be used.
達成針對疫苗之佐劑效應的各種方法分別包括：使用諸如氫氧化鋁或磷酸鋁(明礬)之藥劑，其常以於磷酸鹽緩衝鹽水中之約0.05%至約0.1%溶液之形式使用；與以約0.25%溶液形式使用之合成糖聚合物(CARBOPOL®)混合；藉由在約70℃至約101℃之間的溫度下熱處理30秒至2分鐘時間而使疫苗中之蛋白質聚集。亦可採用以下步驟來產生佐劑效應：藉由用針對白蛋白的經胃蛋白酶處理之(Fab)抗體再活化來發生聚集；與細菌細胞(例如微小隱孢子蟲(C. parvum))、內毒素或革蘭氏陰性細菌之脂多醣組分混合；在生理學上可接受之油媒劑(諸如二縮甘露糖醇單油酸酯(Aracel A))中乳化；或使用用作阻斷替代物之全氟化碳(FLUOSOL-DA®)之20%溶液乳化。Various methods of achieving an adjuvant effect for vaccines include: using agents such as aluminum hydroxide or aluminum phosphate (aluminum), which are usually used in the form of about 0.05% to about 0.1% solutions in phosphate-buffered saline; mixing with a synthetic sugar polymer (CARBOPOL®) used in the form of about 0.25% solution; aggregating the proteins in the vaccine by heat treatment at temperatures between about 70°C and about 101°C for 30 seconds to 2 minutes. Adjuvant effects can also be produced by aggregation by reactivation with pepsin-treated (Fab) antibodies directed against albumin; mixing with bacterial cells (e.g., Cryptosporidiumparvum ), endotoxins, or lipopolysaccharide components of Gram-negative bacteria; emulsification in a physiologically acceptable oil vehicle such as Aracel A; or emulsification using a 20% solution of a perfluorocarbon (FLUOSOL-DA®) used as a blocking surrogate.
在一些態樣中，可排除前述佐劑中之1、2、3、4、5者或更多者。In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned adjuvants may be excluded.
除佐劑之外，可能需要共同投與生物反應調節劑(BRM)以增強免疫反應。BRM已顯示上調T細胞免疫或下調抑制細胞活性。此類BRM包括但不限於西咪替丁(Cimetidine) (CIM；1200 mg/d) (Smith/Kline, PA)；或低劑量環磷醯胺(CYP；300 mg/m2) (Johnson/Mead, NJ)及細胞介素，諸如γ-干擾素、IL-2、或IL-12、或編碼免疫輔助功能中涉及之蛋白質的基因，諸如B-7。In addition to adjuvants, co-administration of biological response modifiers (BRMs) may be necessary to enhance the immune response. BRMs have been shown to upregulate T cell immunity or downregulate suppressor cell activity. Such BRMs include, but are not limited to, cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA); or low-dose cyclophosphamide (CYP; 300 mg/m2 ) (Johnson/Mead, NJ) and interleukins such as gamma-interferon, IL-2, or IL-12, or genes encoding proteins involved in immune helper functions, such as B-7.
C.組合療法本發明之組合物及相關方法，特定言之編碼RSV preF多肽之RNA分子的投與，亦可與一或多種其他治療劑之投與組合使用。此等包括但不限於傳統療法之投與，該等傳統療法例如抗病毒療法，諸如阿昔洛韋(acyclovir)、發昔洛韋(valacyclovir)及泛昔洛韋(famciclovir)，或抗病毒劑之各種組合。亦包括投與一或多種用以治療RSV感染之一或多個症狀的療法，包括但不限於類固醇(包括皮質類固醇)、抗炎劑(包括乙醯胺苯酚或布洛芬(ibuprofen))、止痛劑、緩解瘙癢之乳膏或洗劑、冷敷法或其各種組合。在一些態樣中，可排除前述治療劑中之1、2、3、4、5者或更多者。C.Combination Therapies The compositions and related methods of the present invention, in particular the administration of RNA molecules encoding RSV preF polypeptides, can also be used in combination with the administration of one or more other therapeutic agents. These include, but are not limited to, the administration of conventional therapies, such as antiviral therapies, such as acyclovir, valacyclovir, and famciclovir, or various combinations of antiviral agents. Also included is the administration of one or more therapies to treat one or more symptoms of RSV infection, including but not limited to steroids (including corticosteroids), anti-inflammatory agents (including acetaminophen or ibuprofen), analgesics, creams or lotions to relieve itching, cold compresses, or various combinations thereof. In some aspects, 1, 2, 3, 4, 5 or more of the aforementioned therapies may be excluded.
此類組合療法包括本發明之組合物及一或多種額外活性劑之單一醫藥劑型的投與、以及以其自身分開的醫藥劑型形式之本發明之組合物及各活性劑的投與。舉例而言，本發明之組合物及其他活性劑可以單一劑量組合物(諸如注射或錠劑或膠囊)形式一起向患者投與，或各藥劑以分開的口服劑量調配物形式投與。當使用分開的劑量調配物時，本發明之化合物及一或多種額外活性劑可在基本上相同的時間(例如並行)或在分開的錯開時間(例如依序)投與；組合療法應理解為包括所有此等方案。Such combination therapy includes administration of a composition of the invention and one or more additional active agents in a single pharmaceutical dosage form, as well as administration of the composition of the invention and each active agent in its own separate pharmaceutical dosage form. For example, the composition of the invention and the other active agent may be administered to the patient together in a single dosage composition (such as an injection or tablet or capsule), or each agent may be administered in a separate oral dosage formulation. When a separate dosage formulation is used, the compound of the invention and one or more additional active agents may be administered at substantially the same time (e.g., concurrently) or at separate staggered times (e.g., sequentially); combination therapy should be understood to include all such regimens.
在一個態樣中，經考慮，疫苗及/或療法與抗病毒治療結合使用。或者，疫苗及/或療法可以在數分鐘至數週之範圍內的間隔在用另一藥劑處理之前或之後。在分開地投與其他藥劑及/或疫苗之態樣中，通常將確保在每次遞送之間有意義的時段沒有到期，以使得藥劑及免疫原性組合物將仍能夠對個體發揮有利的組合作用。在此類態樣中，經考慮，吾人可彼此間隔或間隔約12-24 h或彼此間隔或間隔約6-12 h (例如間隔至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23或24小時)投與兩種模態。在一些情況中，可能需要顯著地延長投與時段，其中在各別投與之間流逝若干天(2、3、4、5、6、7或更長)至若干週(1、2、3、4、5、6、7、8週或更長)。In one aspect, it is contemplated that the vaccine and/or therapy is used in conjunction with an antiviral therapy. Alternatively, the vaccine and/or therapy may precede or follow treatment with the other agent at intervals ranging from minutes to weeks. In aspects where the other agent and/or vaccine are administered separately, it will generally be ensured that a meaningful period of time has not expired between each delivery so that the agents and immunogenic composition will still be able to exert a beneficial combined effect on the individual. In such aspects, it is contemplated that one may administer the two modalities at or about 12-24 h apart from each other, or at or about 6-12 h apart from each other (e.g., at least, at most, just below, or between any two of the following (inclusive or exclusive): 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours apart). In some cases, it may be desirable to significantly extend the administration period, with several days (2, 3, 4, 5, 6, 7 or more) to several weeks (1, 2, 3, 4, 5, 6, 7, 8 weeks or more) passing between respective administrations.
可採用各種組合，例如抗病毒療法「A」及作為免疫療法方案「B」之一部分給與的免疫原性多肽： A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/AVarious combinations may be used, such as an antiviral therapy "A" and an immunogenic peptide given as part of an immunotherapy regimen "B":A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/BB/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/AB/A/B/A B/A/A/A/B A/A/A/B B/A/A/A A/B/A/A
本發明之免疫原性組合物向患者/個體之投與將遵循用於投與此類化合物之一般方案，考慮RSV RNA疫苗組合物或本文所描述之其他組合物之毒性(若存在)。預期治療週期將視需要重複進行。亦考慮，不同標準療法(諸如保濕)可與所描述之療法組合應用。Administration of the immunogenic compositions of the present invention to patients/individuals will follow the general regimen for administering such compounds, taking into account the toxicity (if any) of the RSV RNA vaccine composition or other compositions described herein. It is expected that treatment cycles will be repeated as needed. It is also contemplated that different standard therapies (such as moisturizing) may be used in combination with the described therapies.
D.投與本文所描述之組合物之投與可經由用於起類似功用之藥劑的任一可接受投與模式進行。在一些態樣中，本文所描述之醫藥組合物可經靜脈內、鼻內、皮下、皮內或肌肉內投與。在特定態樣中，RSV RNA分子及/或RNA-LNP組合物經肌肉內投與。在某些態樣中，醫藥組合物經調配用於局部投與或全身投與。全身投與可包括經腸投與(其涉及經由胃腸道吸收)或非經腸投與。如本文所用，「非經腸投與」係指以除經由胃腸道以外之任何方式投與，諸如藉由靜脈內注射。在一個態樣中，醫藥組合物經調配用於肌肉內投與。在另一態樣中，醫藥組合物經調配用於全身投與，例如用於靜脈內投與。在一些態樣中，可排除前述投與途徑中之1、2、3者或更多者。D.Administration Administration of the compositions described herein can be performed via any acceptable mode of administration for similarly functioning agents. In some aspects, the pharmaceutical compositions described herein can be administered intravenously, intranasally, subcutaneously, intradermally, or intramuscularly. In a particular aspect, RSV RNA molecules and/or RNA-LNP compositions are administered intramuscularly. In certain aspects, the pharmaceutical compositions are formulated for topical or systemic administration. Systemic administration may include enteral administration (which involves absorption through the gastrointestinal tract) or parenteral administration. As used herein, "parenteral administration" refers to administration in any manner other than through the gastrointestinal tract, such as by intravenous injection. In one aspect, the pharmaceutical composition is formulated for intramuscular administration. In another aspect, the pharmaceutical composition is formulated for systemic administration, such as intravenous administration. In some aspects, 1, 2, 3 or more of the aforementioned routes of administration may be excluded.
醫藥組合物可調配成呈固體、半固體、液體、凍乾、冷凍及/或氣體形式之製劑，諸如錠劑、膠囊、散劑、顆粒、軟膏、溶液、懸浮液、栓劑、注射劑、吸入劑、凝膠、微球體及氣霧劑。在一些態樣中，可排除前述製劑中之1、2、3者或更多者。投與此類醫藥組合物之典型途徑包括但不限於口服、局部、經皮、吸入、非經腸、舌下、經頰、經直腸、經陰道及鼻內。如本文所使用之術語非經腸包括皮下注射、靜脈內、肌肉內、皮內、鼻內、胸骨內注射或輸注技術。本文所描述之醫藥組合物經調配以便允許在向患者投與組合物時其中所含之活性成分為生物可用的。將向個體或患者投與之組合物呈一或多個劑量單位之形式，其中例如錠劑可為單一劑量單位，且呈氣霧劑形式之化合物之容器可容納複數個劑量單位。待投與之組合物將在任何情況下含有治療及/或預防有效量的在本發明之範疇內的化合物或其醫藥學上可接受之鹽，以根據本文所描述之教示治療所關注之疾病或病狀。The pharmaceutical composition can be formulated into a solid, semisolid, liquid, lyophilized, frozen and/or gaseous preparation, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols. In some embodiments, 1, 2, 3 or more of the aforementioned preparations may be excluded. Typical routes of administration of such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal and intranasal. The term parenteral as used herein includes subcutaneous injection, intravenous, intramuscular, intradermal, intranasal, intrasternal injection or infusion techniques. The pharmaceutical compositions described herein are formulated so as to allow the active ingredients contained therein to be bioavailable when the composition is administered to a patient. The composition to be administered to an individual or patient is in the form of one or more dosage units, where, for example, a tablet can be a single dosage unit, and a container of a compound in aerosol form can accommodate multiple dosage units. The composition to be administered will in any case contain a therapeutically and/or prophylactically effective amount of a compound within the scope of the present invention or a pharmaceutically acceptable salt thereof to treat the disease or condition of interest according to the teachings described herein.
在本發明之範疇內的醫藥組合物可呈固體或液體之形式，且可經冷凍或凍乾。在一個態樣中，一或多種載劑為微粒，以使得組合物例如呈錠劑或散劑形式。一或多種載劑可為液體，使得組合物為例如口服糖漿、可注射液體或適用於例如吸入投與之氣霧劑。在一些態樣中，當意欲用於口服投與時，醫藥組合物呈固體或液體形式，其中半固體、半液體、懸浮液及凝膠形式包括在本文視為固體或液體之形式內。Pharmaceutical compositions within the scope of the present invention may be in solid or liquid form and may be frozen or freeze-dried. In one aspect, one or more carriers are microparticles, so that the composition is, for example, in the form of a tablet or powder. One or more carriers may be liquids, so that the composition is, for example, an oral syrup, an injectable liquid, or an aerosol suitable for administration by, for example, inhalation. In some aspects, when intended for oral administration, the pharmaceutical composition is in solid or liquid form, wherein semi-solid, semi-liquid, suspension, and gel forms are included in the forms considered as solid or liquid herein.
作為用於口服投與之固體組合物，醫藥組合物可調配成散劑、顆粒、壓縮錠劑、丸劑、膠囊、口嚼錠、粉片或其類似形式。此類固體組合物將通常含有一或多種惰性稀釋劑或可食載劑。另外，可存在或排除以下中之一或多者：黏合劑，諸如羧甲基纖維素、乙基纖維素、微晶纖維素、黃蓍膠或明膠；賦形劑，諸如澱粉、乳糖或糊精；崩解劑，諸如褐藻酸、褐藻酸鈉、PRIMOJEL®、玉米澱粉及其類似物；潤滑劑，諸如硬脂酸鎂或STEROTEX®；滑動劑，諸如膠體二氧化矽；甜味劑，諸如蔗糖或糖精；調味劑，諸如胡椒薄荷、水楊酸甲酯或橙味調味劑；及著色劑。當醫藥組合物呈膠囊(例如明膠膠囊)形式時，除以上類型之物質之外，其可含有諸如聚乙二醇或油之液體載劑。在一些態樣中，前述要素中之1、2、3者或更多者可排除在固體組合物外。As solid compositions for oral administration, the pharmaceutical composition can be formulated into powders, granules, compressed tablets, pills, capsules, chewable tablets, powder tablets or the like. Such solid compositions will generally contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present or excluded: binders such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth or gelatin; formulators such as starch, lactose or dextrin; disintegrants such as alginic acid, sodium alginate, PRIMOJEL®, corn starch and the like; lubricants such as magnesium stearate or STEROTEX®; slip agents such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; flavorings such as peppermint, methyl salicylate or orange flavoring; and coloring agents. When the pharmaceutical composition is in the form of a capsule (e.g., a gelatin capsule), it may contain, in addition to the above-mentioned types of substances, a liquid carrier such as polyethylene glycol or oil. In some aspects, 1, 2, 3 or more of the aforementioned elements may be excluded from the solid composition.
醫藥組合物可呈液體形式，例如酏劑、糖漿、溶液、乳液或懸浮液。舉兩個例子，液體可用於口服投與或用於藉由注射遞送。在一些態樣中，當意欲用於口服投與時，除本發明化合物以外，組合物亦含有甜味劑、防腐劑、染料/著色劑及增香劑中之一或多者。在意欲藉由注射投與之組合物中，可包括或排除界面活性劑、防腐劑、濕潤劑、分散劑、懸浮劑、緩衝劑、穩定劑及等張劑中之一或多者。The pharmaceutical composition may be in the form of a liquid, such as an elixir, syrup, solution, emulsion or suspension. To cite two examples, the liquid may be used for oral administration or for delivery by injection. In some aspects, when intended for oral administration, in addition to the compounds of the invention, the composition also contains one or more of a sweetener, a preservative, a dye/colorant and a flavoring agent. In a composition intended for administration by injection, one or more of a surfactant, a preservative, a wetting agent, a dispersant, a suspending agent, a buffer, a stabilizer and an isotonic agent may be included or excluded.
液體醫藥組合物，無論其為溶液、懸浮液或其他類似形式，均可包括或排除以下佐劑中之一或多者：無菌稀釋劑，諸如注射用水、鹽水溶液(例如生理鹽水)、林格氏溶液、等張氯化鈉、不揮發性油(諸如可充當溶劑或懸浮介質之合成單酸甘油酯或二酸甘油酯)、聚乙二醇、甘油、丙二醇或其他溶劑；抗細菌劑，諸如苯甲醇或對羥基苯甲酸甲酯；抗氧化劑，諸如抗壞血酸或亞硫酸氫鈉；螯合劑，諸如乙二胺四乙酸；緩衝劑，諸如乙酸鹽、檸檬酸鹽或磷酸鹽；及張力調節劑，諸如氯化鈉或右旋糖；充當低溫保護劑之藥劑，諸如蔗糖或海藻糖。非經腸製劑可封裝於由玻璃或塑膠製成之安瓿、拋棄式注射器或多劑量小瓶中。在一個態樣中，生理鹽水為佐劑。在一個態樣中，可注射醫藥組合物為無菌的。意欲用於非經腸或口服投與之液體醫藥組合物應含有一定數量之化合物，使得將獲得適合劑量。Liquid pharmaceutical compositions, whether in the form of solutions, suspensions or other similar forms, may include or exclude one or more of the following adjuvants: sterile diluents, such as water for injection, saline solutions (e.g., physiological saline), Ringer's solution, isotonic sodium chloride, involatile oils (such as synthetic mono- or di-glycerides that can serve as solvents or suspending media), polyethylene glycols, , glycerol, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and tonicity regulators such as sodium chloride or dextrose; agents acting as low-temperature protectants such as sucrose or trehalose. Parenteral preparations can be packaged in ampoules, disposable syringes or multi-dose vials made of glass or plastic. In one aspect, physiological saline is an adjuvant. In one aspect, the injectable pharmaceutical composition is sterile. Liquid pharmaceutical compositions intended for parenteral or oral administration should contain an amount of the compound such that a suitable dosage will be obtained.
醫藥組合物可包括各種材料，該等材料調節固體或液體劑量單位之物理形式。舉例而言，組合物可包括圍繞活性成分形成包覆殼層之材料。形成包覆殼層之材料通常為惰性的，且可為例如糖、蟲膠或其他腸溶包衣劑。Pharmaceutical compositions may include various materials that adjust the physical form of the solid or liquid dosage unit. For example, the composition may include materials that form a coating around the active ingredient. The materials that form the coating are generally inert and may be, for example, sugar, wormwood or other enteric coating agents.
醫藥組合物可包括可以氣霧劑形式投與之劑量單位。術語氣霧劑表示介於膠態性質之彼等系統至由加壓封裝組成之系統之範圍內的多種系統。遞送可藉由液化或壓縮氣體或藉由分配活性成分的適合泵系統。化合物之氣霧劑可以單相、雙相或三相系統遞送以便遞送一或多種活性成分。氣霧劑之遞送包括必需容器、活化劑、閥門、次容器及其類似者，其在一起可形成套組。熟習此項技術者無需過度實驗即可確定較佳氣霧劑。Pharmaceutical compositions may include dosage units that can be administered in the form of an aerosol. The term aerosol refers to a variety of systems ranging from those of a colloidal nature to those consisting of pressurized packaging. Delivery can be by liquefied or compressed gases or by a suitable pump system that distributes the active ingredient. Aerosols of compounds can be delivered in a single-phase, two-phase or three-phase system to deliver one or more active ingredients. The delivery of an aerosol includes the necessary containers, activators, valves, subcontainers and the like, which together can form a kit. Those skilled in the art can determine the preferred aerosol without undue experimentation.
醫藥組合物可藉由醫藥技術中熟知之方法製備。舉例而言，可藉由將核酸或多肽與無菌蒸餾水或其他載劑組合以便形成溶液來製備意欲藉由注射投與之醫藥組合物。可添加界面活性劑以促進形成均勻溶液或懸浮液。界面活性劑為與同本文中之教示一致的化合物非共價相互作用以便促進該化合物溶解或均勻懸浮於水性遞送系統中的化合物。Pharmaceutical compositions can be prepared by methods well known in the pharmaceutical art. For example, pharmaceutical compositions intended for administration by injection can be prepared by combining a nucleic acid or polypeptide with sterile distilled water or other carrier to form a solution. Surfactants may be added to facilitate the formation of a uniform solution or suspension. Surfactants are compounds that non-covalently interact with a compound consistent with the teachings herein to facilitate dissolution or uniform suspension of the compound in an aqueous delivery system.
根據本發明之醫藥組合物或其醫藥學上可接受之鹽一般以「治療有效量」或「預防有效量」且以「醫藥學上可接受之製劑」施用。術語「醫藥學上可接受」係指不與醫藥組合物之活性組分之作用相互作用的物質之非毒性。術語「治療有效量」及「預防有效量」係指單獨或與其他劑量一起達成所需反應或所需作用的量。在治療特定疾病的情況下，在一個態樣中，所需反應係關於抑制該疾病之病程。此包含減緩疾病進展，且尤其中斷及/或逆轉疾病進展。治療疾病之所需反應亦可為延遲該疾病或該病狀之發作及/或預防該疾病或該病狀之發作。The pharmaceutical composition according to the present invention or a pharmaceutically acceptable salt thereof is generally administered in a "therapeutically effective amount" or a "prophylactically effective amount" and in a "pharmaceutically acceptable formulation". The term "pharmaceutically acceptable" refers to the non-toxicity of a substance that does not interact with the action of the active component of the pharmaceutical composition. The terms "therapeutically effective amount" and "prophylactically effective amount" refer to an amount that achieves the desired response or desired effect alone or together with other doses. In the case of treating a specific disease, in one embodiment, the desired response is related to inhibiting the course of the disease. This includes slowing the progression of the disease, and in particular interrupting and/or reversing the progression of the disease. The desired response for treating a disease may also be to delay the onset of the disease or the condition and/or prevent the onset of the disease or the condition.
本發明範疇內之組合物以治療及/或預防有效量投與，該治療及/或預防有效量將視各種因素變化，包括：所採用之特定治療劑及/或預防劑之活性；治療劑及/或預防劑之代謝穩定性及作用時長；患者之個體參數，包括患者之年齡、體重、一般健康狀況、性別及飲食；投與之模式、時間及/或持續時間；分泌速率；藥物組合；特定病症或病狀之嚴重程度；及個體正進行之療法。在一些態樣中，該等因素中之1、2、3、4、5者或更多者可排除在確定治療及/或預防有效量外。因此，本文所描述之組合物之投與劑量可視各種此類參數而定。在患者中之反應在初始劑量下不充分的情況中，可使用較高劑量(或藉由另一更局部的投與途徑來達成之有效較高劑量)。在一些態樣中，組合物(例如RSV RNA-LNP組合物)可以足以遞送以下量之劑量水平投與：每天每公斤個體體重0.0001 ng/µg/mg至100 ng/µg/mg、0.001 ng/µg/mg至0.05 ng/µg/mg、0.005 ng/µg/mg至0.05 ng/µg/mg、0.001 ng/µg/mg至0.005 ng/µg/mg、0.05 ng/µg/mg至0.5 ng/µg/mg、0.01 ng/µg/mg至50 ng/µg/mg、0.1 ng/µg/mg至40 ng/µg/mg、0.5 ng/µg/mg至30 ng/µg/mg、0.01 ng/µg/mg至10 ng/µg/mg、0.1 ng/µg/mg至10 ng/µg/mg、或1 ng/µg/mg至25 ng/µg/mg，一天一或多次、每週、每月等投與，以獲得所需治療性、診斷性、預防性及/或成像作用(參見例如國際公開案第WO2013/078199號中所描述之單位劑量範圍，該文獻以全文引用之方式併入本文中)。在一些態樣中，組合物(例如RSV RNA-LNP組合物)可以或可不以足以遞送至少、至多、恰好以下或在以下任何兩者之間(包括性或排他性)的量之劑量水平投與：每天每公斤個體體重0.0001、0.0002、0.0003、0.0004、0.0005、0.0006、0.0007、0.0008、0.0009、0.001、0.002、0.003、0.004、0.005、0.006、0.007、0.008、0.009、0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99或100 ng/µg/mg，一天一或多次、每週、每月等投與，以獲得所需治療性、診斷性、預防性及/或成像作用。The compositions within the scope of the present invention are administered in a therapeutically and/or prophylactically effective amount, which will vary depending on various factors, including: the activity of the specific therapeutic and/or prophylactic agent employed; the metabolic stability and duration of action of the therapeutic and/or prophylactic agent; the individual parameters of the patient, including the patient's age, weight, general health, sex and diet; the mode, time and/or duration of administration; the rate of secretion; the drug combination; the severity of the specific disease or condition; and the individual's ongoing therapy. In some aspects, 1, 2, 3, 4, 5 or more of these factors may be excluded from determining the therapeutically and/or prophylactically effective amount. Thus, the dosage of the compositions described herein administered may depend on a variety of such parameters. In cases where the response in a patient is inadequate at an initial dose, a higher dose (or an effectively higher dose achieved by another more localized route of administration) may be used. In some aspects, the composition (e.g., RSV RNA-LNP composition) can be administered at a dosage level sufficient to deliver 0.0001 ng/µg/mg to 100 ng/µg/mg, 0.001 ng/µg/mg to 0.05 ng/µg/mg, 0.005 ng/µg/mg to 0.05 ng/µg/mg, 0.001 ng/µg/mg to 0.005 ng/µg/mg, 0.05 ng/µg/mg to 0.5 ng/µg/mg, 0.01 ng/µg/mg to 50 ng/µg/mg, 0.1 ng/µg/mg to 40 ng/µg/mg, 0.5 ng/µg/mg to 30 ng/µg/mg, 0.01 ng/µg/mg to 10 ng/µg/mg, 0.1 ng/µg/mg to 10 ng/µg/mg, or 1 ng/µg/mg to 25 ng/µg/mg, administered one or more times a day, weekly, monthly, etc., to achieve the desired therapeutic, diagnostic, preventive and/or imaging effects (see, e.g., the unit dose ranges described in International Publication No. WO2013/078199, which is incorporated herein by reference in its entirety). In some aspects, the composition (e.g., RSV The RNA-LNP composition may or may not be administered at a dosage level sufficient to deliver at least, at most, just below, or any two of the following (inclusive or exclusive): 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0. ... .002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 4, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 ng/µg/mg, administered one or more times a day, weekly, monthly, etc., to achieve the desired therapeutic, diagnostic, preventive and/or imaging effect.
在一些態樣中，組合物(例如RSV RNA-LNP組合物)可以或可不以至少、至多、恰好以下總劑量或在以下任何兩者之間(包括性或排他性)的總劑量投與或以足以遞送至少、至多、恰好以下總劑量或在以下任何兩者之間的總劑量之劑量水平投與：每天0.0001、0.0002、0.0003、0.0004、0.0005、0.0006、0.0007、0.0008、0.0009、0.001、0.002、0.003、0.004、0.005、0.006、0.007、0.008、0.009、0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99或100 ng/µg/mg，一天一或多次、每週、每月等投與，以獲得所需治療性、診斷性、預防性及/或成像作用。In some aspects, a composition (e.g., a RSV RNA-LNP composition) may or may not be administered at a total dose of at least, at most, just below, or between any two of the following (inclusive or exclusive) or at a dose level sufficient to deliver at least, at most, just below, or between any two of the following: 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.0010, 0.0011, 0.0012, 0.0013, 0.0014, 0.0015, 0.0016, 0.0017, 0.0018, 0.0019, 0.0111, 0.0112, 0.0113, 0.0114, 0.0115, 0.0116, 0.0117, 0.0118, 0.0119, 0.0121, 0.0122, 0.0123, 0.0124, 0.0125, 0.0126, 0.0127, 0.0128, 0.0129, , 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0 .9,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 ng/µg/mg, administered one or more times a day, weekly, monthly, etc., to obtain the desired therapeutic, diagnostic, preventive and/or imaging effect.
在特定態樣中，組合物(例如RSV RNA-LNP組合物)可以或可不以至少、至多、恰好以下總劑量或在以下其中任何兩者之間(包括性或排他性)的總劑量投與或以足以遞送至少、至多、恰好以下總劑量或在以下其中任何兩者之間的總劑量之劑量水平投與：0.0001、0.0002、0.0003、0.0004、0.0005、0.0006、0.0007、0.0008、0.0009、0.001、0.002、0.003、0.004、0.005、0.006、0.007、0.008、0.009、0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99或100 mg/mL之囊封於LNP中之RSV RNA。In certain aspects, the composition (e.g., RSV RNA-LNP composition) may or may not be administered at a total dose of at least, at most, just below, or between any two of the following (inclusive or exclusive) or at a dose level sufficient to deliver at least, at most, just below, or between any two of the following: 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.010, 0.011, 0.012, 0.013, 0.014, 0.015, 0.016, 0.017, 0.018, 0.019, 0.021, 0.022, 0.023, 0.024, 0.025, 0.026, 0.027, 0.028, 0.029, 0.030, 0.031, 0.032, 0.033, 0.034, 0.035, 0.036, 0.037, 0.038, 0.039, 0.040, 0.041, 0.042, 0.043, 0.044, 0.045, 0.046, 0.047, 0.048, 0.049, 0.050, 0.051, 0.052, 0.053, 0.054, 0.055, 0.056, 0.057, 0.058, 0.059, 0.061, 7, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 94, 95, 96, 97, 98, 99, or 100 mg/mL of RSV RNA encapsulated in LNPs.
在例示性態樣中，組合物(例如RSV RNA-LNP組合物)可以或可不以至少、至多、恰好以下劑量水平或在以下其中任何兩者之間(包括性或排他性)的劑量水平投與：0.01、0.15、0.30、0.45、0.60、0.75或0.90 mg/mL之囊封於LNP中之RSV RNA。在例示性態樣中，組合物(例如RSV RNA-LNP組合物)可以或可不以至少、至多、恰好以下劑量水平或在以下其中任何兩者之間(包括性或排他性)的劑量水平投與：0.01、0.15、0.30、0.45、0.60、0.75或0.90 mg之囊封於LNP中之RSV RNA。In exemplary aspects, the composition (e.g., RSV RNA-LNP composition) may or may not be administered at a dosage level of at least, at most, just below, or between any two of the following (inclusive or exclusive): 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg/mL of RSV RNA encapsulated in LNP. In exemplary aspects, the composition (e.g., RSV RNA-LNP composition) may or may not be administered at a dosage level of at least, at most, just below, or between any two of the following (inclusive or exclusive): 0.01, 0.15, 0.30, 0.45, 0.60, 0.75, or 0.90 mg of RSV RNA encapsulated in LNP.
在特定態樣中，組合物(例如RSV RNA-LNP組合物)可以或可不以至少、至多、恰好以下總劑量或在以下其中任何兩者之間(包括性或排他性)的總劑量投與或以足以遞送至少、至多、恰好以下總劑量或在以下其中任何兩者之間(包括性或排他性)的總劑量之劑量水平投與：0.0001、0.0002、0.0003、0.0004、0.0005、0.0006、0.0007、0.0008、0.0009、0.001、0.002、0.003、0.004、0.005、0.006、0.007、0.008、0.009、0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99或100 µg/mL之囊封於LNP中之RSV RNA。In certain aspects, the composition (e.g., RSV RNA-LNP composition) may or may not be administered at a total dose of at least, at most, just below, or between any two of the following (inclusive or exclusive) or at a dose level sufficient to deliver at least, at most, just below, or between any two of the following (inclusive or exclusive): 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.0010, 0.0011, 0.0012, 0.0013, 0.0014, 0.0015, 0.0016, 0.0017, 0.0018, 0.0019, 0.0110, 0.0121, 0.0132, 0.0133, 0.0134, 0.0135, 0.0136, 0.0137, 0.0138, 0.0139, 0.021 6. 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 99, 100 µg/mL of RSV RNA encapsulated in LNPs.
在例示性態樣中，組合物(例如RSV RNA-LNP組合物)可以或可不以至少、至多、恰好以下劑量水平或在以下其中任何兩者之間(包括性或排他性)的劑量水平投與：1、15、30、45、60、75、90、100或更高微克/毫升之囊封於LNP中之RSV RNA。在例示性態樣中，組合物(例如RSV RNA-LNP組合物)可以或可不以至少、至多、恰好以下劑量水平或在以下其中任何兩者之間(包括性或排他性)的劑量水平投與：1、15、30、45、60、75、90、100或更多微克之囊封於LNP中之RSV RNA。In exemplary aspects, the composition (e.g., RSV RNA-LNP composition) may or may not be administered at a dosage level of at least, at most, just below, or between any two of the following (inclusive or exclusive): 1, 15, 30, 45, 60, 75, 90, 100 or more micrograms/ml of RSV RNA encapsulated in LNP. In exemplary aspects, the composition (e.g., RSV RNA-LNP composition) may or may not be administered at a dosage level of at least, at most, just below, or between any two of the following (inclusive or exclusive): 1, 15, 30, 45, 60, 75, 90, 100 or more micrograms of RSV RNA encapsulated in LNP.
所需劑量可一天多次(例如一天1、2、3、4、5或更多次)、每隔一天、每三天、每週、每兩週、每三週、每四週、每2個月、每三個月、每6個月或每年等遞送。在某些態樣中，所需劑量可使用單次劑量投與進行遞送。在某些態樣中，所需劑量可使用多次投與(例如兩次、三次、四次、五次、六次、七次、八次、九次、十次、十一次、十二次、十三次、十四次或更多次投與)進行遞送。當採用多次投與時，可使用分次給藥方案。組合物之初次投與與組合物之後續投與之間的投與時間可為但不限於1分鐘、2分鐘、3分鐘、4分鐘、5分鐘、6分鐘、7分鐘、8分鐘、9分鐘、10分鐘、15分鐘、20分鐘、35分鐘、40分鐘、45分鐘、50分鐘、55分鐘、1小時、2小時、3小時、4小時、5小時、6小時、7小時、8小時、9小時、10小時、11小時、12小時、13小時、14小時、15小時、16小時、17小時、18小時、19小時、20小時、21小時、22小時、23小時、1天、36小時、2天、3天、4天、5天、6天、1週、10天、2週、3週、1個月、2個月、3個月、4個月、5個月、6個月、7個月、8個月、9個月、10個月、11個月、1年、18個月、2年、3年、4年、5年、6年、7年、8年、9年、10年、11年、12年、13年、14年、15年、16年、17年、18年、19年、20年、25年、30年、35年、40年、45年、50年、55年、60年、65年、70年、75年、80年、85年、90年、95年或超過99年。The desired dose can be delivered multiple times a day (e.g., 1, 2, 3, 4, 5 or more times a day), every other day, every three days, every week, every two weeks, every three weeks, every four weeks, every two months, every three months, every six months, or every year. In certain aspects, the desired dose can be delivered using a single dose administration. In certain aspects, the desired dose can be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or more administrations). When multiple administrations are used, a split dosing regimen can be used. The administration time between the initial administration of the composition and the subsequent administration of the composition can be, but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours. 10 days, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years or more than 99 years.
在一些態樣中，組合物(例如RSV RNA-LNP組合物)可以單次劑量投與。在一些態樣中，組合物(例如RSV RNA-LNP組合物)可投與兩次(例如第0天及在或在約第7天、第0天及在或在約第14天、第0天及在或在約第21天、第0天及在或在約第28天、第0天及在或在約第60天、第0天及在或在約第90天、第0天及在或在約第120天、第0天及在或在約第150天、第0天及在或在約第180天、第0天及在或在約1個月後、第0天及在或在約2個月後、第0天及在或在約3個月後、第0天及在或在約6個月後、第0天及在或在約9個月後、第0天及在或在約12個月後、第0天及在或在約18個月後、第0天及在或在約2年後、第0天及在或在約5年後、或第0天及在或在約10年後)，其中每次投與至少、至多、恰好以下總劑量或在以下其中任何兩者之間(包括性或排他性)的總劑量或足以遞送至少、至多、恰好以下總劑量或在以下其中任何兩者之間(包括性或排他性)的總劑量之劑量水平：0.0001、0.0002、0.0003、0.0004、0.0005、0.0006、0.0007、0.0008、0.0009、0.001、0.002、0.003、0.004、0.005、0.006、0.007、0.008、0.009、0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99或100 ng/µg/mg之囊封於LNP中之RSV RNA。本發明涵蓋較高及較低之投與劑量及頻率。舉例而言，組合物(例如RSV RNA-LNP組合物)可投與三次或四次。In some aspects, a composition (e.g., an RSV RNA-LNP composition) can be administered in a single dose. In some aspects, a composition (e.g., an RSV RNA-LNP composition) can be administered twice (e.g., day 0 and at or about day 7, day 0 and at or about day 14, day 0 and at or about day 21, day 0 and at or about day 28, day 0 and at or about day 60, day 0 and at or about day 90, day 0 and at or about day 120, day 0 and at or about day 150, day 0 and at or about day 180, day 0 and at or about 1 month later, day 0 and at or about 2 months later, day 0 and at or about 3 months later, day 0 and at or about 6 months later, day 0 and at or about 9 months later, day 0 and at or about 12 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.0010, 0.0011, 0.0012, 0.0013, 0.0014, 0.0015, 0.0016, 0.0017, 0.0018, 0.0019, 0.0020, 0.0021, 0.0022, 0.0023, 0.0024, 0.0025, 0.0026, 0.0027, 0.0028, 0.0029, 0.0030, 0.0031, 0.0032, 0.0033, 0.0034, 0.0035, 0.0036, 0.0037, 0.0038, 0.0039, 0.014, 0.015 0.001、0.002、0.003、0.004、0.005、0.006、0.007、0.008、0.009、0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 ng/µg/mg of RSV RNA encapsulated in LNPs. The present invention encompasses higher and lower dosages and frequencies of administration. For example, a composition (eg, RSV RNA-LNP composition) can be administered three or four times.
可能需要以1至5年之間隔週期性加強來維持抗體之保護性水平。如本文所用，術語「加強(booster)」係指組合物(例如RSV RNA-LNP組合物)之額外投與。加強可在組合物之早期投與之後給與。Periodic boosts at intervals of 1 to 5 years may be required to maintain protective levels of antibodies. As used herein, the term "booster" refers to additional administration of a composition (e.g., RSV RNA-LNP composition). The boost can be given after an earlier administration of the composition.
在一些態樣中，組合物(例如RSV RNA-LNP組合物)以或不以至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的單次劑量向個體投與：0.0001、0.0002、0.0003、0.0004、0.0005、0.0006、0.0007、0.0008、0.0009、0.001、0.002、0.003、0.004、0.005、0.006、0.007、0.008、0.009、0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99或100 ng/µg/mg之囊封於LNP中之RSV RNA。在一些態樣中，組合物(例如RSV RNA-LNP組合物)以或不以至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的單次劑量向個體投與：1 µg、15 µg、30 µg、45 µg、60 µg、75 µg、90 µg、100 µg或更高劑量之囊封於LNP中之RSV RNA。In some aspects, a composition (e.g., a RSV RNA-LNP composition) is administered to a subject with or without a single dose of at least, at most, just below, or between any two of the following (inclusive or exclusive): 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.00 3. 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 5, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 ng/µg/mg of RSV RNA encapsulated in LNPs. In some aspects, a composition (e.g., a RSV RNA-LNP composition) is administered to a subject with or without a single dose of at least, at most, just below, or between any two of the following (inclusive or exclusive): 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, 90 μg, 100 μg or more of RSV RNA encapsulated in LNPs.
在一些態樣中，組合物(例如RSV RNA-LNP組合物)以或不以至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的兩次劑量向個體投與：0.0001、0.0002、0.0003、0.0004、0.0005、0.0006、0.0007、0.0008、0.0009、0.001、0.002、0.003、0.004、0.005、0.006、0.007、0.008、0.009、0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99或100 ng/µg/mg之囊封於LNP中之RSV RNA。在一些態樣中，組合物(例如RSV RNA-LNP組合物)以或不以至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的兩次劑量向個體投與：1 µg、15 µg、30 µg、45 µg、60 µg、75 µg、90 µg、100 µg或更高劑量之囊封於LNP中之RSV RNA。In some aspects, a composition (e.g., a RSV RNA-LNP composition) is administered to a subject in two doses or not in at least, at most, just below, or between any two of the following (inclusive or exclusive): 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.00 3. 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55 5, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 ng/µg/mg of RSV RNA encapsulated in LNPs. In some aspects, a composition (e.g., a RSV RNA-LNP composition) is administered to a subject with or without two doses of at least, at most, just below, or between any two of the following (inclusive or exclusive): 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, 90 μg, 100 μg or more of RSV RNA encapsulated in LNPs.
在特定態樣中，組合物(例如RSV RNA-LNP組合物)可投與可不投與兩次(例如第0天及第28天、第0天及第60天、第0天及第180天、第0天及2個月後、第0天及6個月後、第0天及一年後等)，其中每次投與至少、至多、恰好以下總劑量或在以下其中任何兩者之間(包括性或排他性)的總劑量或足以遞送至少、至多、恰好以下總劑量或在以下其中任何兩者之間(包括性或排他性)的總劑量之劑量水平：1 µg、15 µg、30 µg、45 µg、60 µg、75 µg、90 µg、100 µg或更高劑量之囊封於LNP中之RSV RNA。In certain aspects, a composition (e.g., a RSV RNA-LNP composition) may or may not be administered twice (e.g., day 0 and day 28, day 0 and day 60, day 0 and day 180, day 0 and 2 months later, day 0 and 6 months later, day 0 and one year later, etc.), wherein each administration is at least, at most, exactly the following total dose, or a total dose between any two of the following (inclusive or exclusive), or a dose level sufficient to deliver at least, at most, exactly the following total dose, or a total dose between any two of the following (inclusive or exclusive): 1 μg, 15 μg, 30 μg, 45 μg, 60 μg, 75 μg, 90 μg, 100 μg or more of RSV RNA encapsulated in LNPs.
IX.使用方法本文提供用於預防及/或治療人類及其他哺乳動物之RSV的組合物(例如包含RSV RNA分子及/或RSV RNA-LNP之醫藥組合物)、方法、套組及試劑。IX.Methods of Use Provided herein are compositions (eg, pharmaceutical compositions comprising RSV RNA molecules and/or RSV RNA-LNPs), methods, kits, and reagents for preventing and/or treating RSV in humans and other mammals.
RSV RNA組合物(例如RSV RNA-LNP組合物)可用作治療劑及/或預防劑。其可用於藥物中以預防及/或治療傳染病。在例示性態樣中，RSV RNA組合物用於提供對任何基因型、株系或分離株之急性下呼吸道感染(ALRI)的預防保護。設想可存在其中人處於感染超過一種RSV株系之風險下的情況。RSV RNA組合物(例如RSV RNA-LNP組合物)尤其適合於組合疫苗接種途徑，此係由於包括但不限於以下之多種因素：製造速度、快速調整疫苗以適應感知到的地理威脅之能力及其類似者。此外，因為RSV RNA組合物(例如RSV RNA-LNP組合物)使用人體來產生抗原性蛋白質，所以RSV RNA組合物(例如RSV RNA-LNP組合物)適合於產生較大、較複雜的抗原性蛋白質，從而允許在人類個體中適當摺疊、表面表現、抗原呈現等。為了保護免於超過一種RSV株系，可投與組合RSV RNA組合物，其包括編碼第一RSV之至少一種抗原性多肽蛋白質(或其抗原性部分)的RNA且進一步包括編碼第二RSV之至少一種抗原性多肽蛋白質(或其抗原性部分)的RNA。本發明之RSV疫苗可用於預防RSV(感染相關病痛，包括肺炎及支氣管炎)，且尤其可用於預防及/或治療免疫功能不全且老年患者以預防RSV感染或降低其嚴重程度及/或持續時間。RSV RNA compositions (e.g., RSV RNA-LNP compositions) can be used as therapeutic and/or prophylactic agents. They can be used in medicines to prevent and/or treat infectious diseases. In an exemplary aspect, RSV RNA compositions are used to provide prophylactic protection against acute lower respiratory tract infections (ALRI) of any genotype, strain, or isolate. It is envisioned that there may be situations in which a person is at risk of infection with more than one RSV strain. RSV RNA compositions (e.g., RSV RNA-LNP compositions) are particularly suitable for combination vaccine inoculation routes due to a variety of factors including, but not limited to, speed of manufacture, the ability to quickly adjust vaccines to adapt to perceived geographic threats, and the like. Furthermore, because RSV RNA compositions (e.g., RSV RNA-LNP compositions) use the human body to produce antigenic proteins, RSV RNA compositions (e.g., RSV RNA-LNP compositions) are suitable for producing larger, more complex antigenic proteins, thereby allowing for proper folding, surface expression, antigenic presentation, etc. in human subjects. To protect against more than one RSV strain, a combination RSV RNA composition can be administered that includes RNA encoding at least one antigenic polypeptide protein (or an antigenic portion thereof) of a first RSV and further includes RNA encoding at least one antigenic polypeptide protein (or an antigenic portion thereof) of a second RSV. The RSV vaccine of the present invention can be used to prevent RSV (infection-related illnesses, including pneumonia and bronchitis), and can be particularly used to prevent and/or treat immunocompromised and elderly patients to prevent RSV infection or reduce its severity and/or duration.
在一些態樣中，向個體(例如哺乳動物個體，諸如人類個體)投與本發明之RSV RNA組合物(例如RSV RNA-LNP組合物)，且RNA聚核苷酸在活體內轉譯以產生抗原性多肽。可誘導RSV RNA組合物(例如RSV RNA-LNP組合物)以在細胞、組織或生物體中轉譯多肽(例如抗原或免疫原)。在例示性實施例中，此類轉譯發生在活體內，但可設想存在其中此類轉譯離體、在培養物中或在活體外發生的實施例。在例示性實施例中，將細胞、組織或生物體與有效量之RSV RNA組合物(例如RSV RNA-LNP組合物)接觸，該RSV RNA組合物包括具有至少一個編碼抗原性多肽(例如RSV抗原)之可轉譯區域的RNA分子。In some aspects, an RSV RNA composition (e.g., RSV RNA-LNP composition) of the invention is administered to an individual (e.g., a mammalian individual, such as a human individual), and the RNA polynucleotide is translated in vivo to produce an antigenic polypeptide. RSV RNA compositions (e.g., RSV RNA-LNP compositions) can be induced to translate polypeptides (e.g., antigens or immunogens) in cells, tissues, or organisms. In exemplary embodiments, such translation occurs in vivo, but embodiments are envisioned in which such translation occurs in vitro, in culture, or in vitro. In exemplary embodiments, a cell, tissue, or organism is contacted with an effective amount of an RSV RNA composition (e.g., an RSV RNA-LNP composition) that includes an RNA molecule having at least one translateable region encoding an antigenic polypeptide (e.g., an RSV antigen).
在一些態樣中，本發明之RSV RNA組合物可用於致敏免疫效應細胞，例如離體活化周邊血液單核細胞(PBMC)，接著將其輸注(再輸注)至個體中。In some aspects, the RSV RNA compositions of the invention can be used to prime immune effector cells, such as peripheral blood mononuclear cells (PBMCs) activated in vitro, which are then infused (re-infused) into an individual.
在一些態樣中，在投與本文所描述之RSV RNA分子(例如調配為RNA-LNP)之後，將至少一部分之RNA遞送至目標細胞。In some aspects, following administration of a RSV RNA molecule described herein (e.g., formulated as RNA-LNP), at least a portion of the RNA is delivered to a target cell.
在一些態樣中，RNA之至少一部分遞送至目標細胞之胞溶質。在一些態樣中，RNA由目標細胞轉譯以產生其編碼之多肽或蛋白質。在一些態樣中，目標細胞為脾臟細胞。在一些態樣中，目標細胞為脾臟中之抗原呈現細胞，諸如專職抗原呈現細胞。在一些態樣中，目標細胞為樹突狀細胞及/或巨噬細胞。RNA分子(諸如本文所描述之RNA-LNP)可用於將RNA遞送至此類目標細胞。因此，本發明亦係關於一種用於將RNA遞送至個體中之目標細胞的方法，其包含向該個體投與本文所描述之RNA顆粒。In some aspects, at least a portion of the RNA is delivered to the cytosol of a target cell. In some aspects, the RNA is translated by the target cell to produce a polypeptide or protein encoded by it. In some aspects, the target cell is a spleen cell. In some aspects, the target cell is an antigen presenting cell in the spleen, such as a professional antigen presenting cell. In some aspects, the target cell is a dendritic cell and/or a macrophage. RNA molecules (such as RNA-LNPs described herein) can be used to deliver RNA to such target cells. Therefore, the present invention also relates to a method for delivering RNA to a target cell in an individual, comprising administering to the individual an RNA particle described herein.
在一些態樣中，RNA被遞送至目標細胞之胞溶質。在一些態樣中，RNA由目標細胞轉譯以產生RNA編碼之多肽或蛋白質。「編碼」係指諸如基因、cDNA或mRNA之聚核苷酸中之核苷酸之特異性序列在生物過程中充當合成其他聚合物及大分子之模板的固有特性，該等聚合物及大分子具有已確定之核苷酸序列(例如rRNA、tRNA及mRNA)或已確定之胺基酸序列及由其獲得之生物特性。因此，若與基因相對應之mRNA之轉錄及轉譯在細胞或其他生物系統中產生蛋白質，則基因編碼該蛋白質。核苷酸序列與mRNA序列一致且通常提供於序列表中的編碼股與用作基因或cDNA轉錄之模板的非編碼股均可被稱為編碼該基因或cDNA之蛋白質或其他產物。In some aspects, the RNA is delivered to the cytosol of a target cell. In some aspects, the RNA is translated by the target cell to produce a polypeptide or protein encoded by the RNA. "Coding" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide such as a gene, cDNA or mRNA to serve as a template for the synthesis of other polymers and macromolecules in biological processes, which have a defined nucleotide sequence (e.g., rRNA, tRNA and mRNA) or a defined amino acid sequence and the biological properties derived therefrom. Therefore, if the transcription and translation of the mRNA corresponding to the gene produces a protein in a cell or other biological system, then the gene encodes the protein. The coding strands whose nucleotide sequence is consistent with the mRNA sequence and is usually provided in the sequence listing and the non-coding strands used as templates for transcription of a gene or cDNA can all be referred to as encoding the protein or other product of the gene or cDNA.
在一些態樣中，本文所描述之核酸組合物(例如包含RSV RNA-LNP之組合物)之特徵在於(例如當向個體投與時)隨細胞中之抗原產生變化的誘導及/或加強的免疫反應。抗原產生增加可藉由例如以下來證實：細胞轉染(經RNA疫苗轉染之細胞的百分比)增加、自聚核苷酸之蛋白質轉譯增加、核酸降解減少(例如藉由自經修飾之聚核苷酸之蛋白質轉譯的持續時間增加所證實)及/或宿主細胞之抗原特異性免疫反應改變。In some aspects, the nucleic acid compositions described herein (e.g., compositions comprising RSV RNA-LNPs) are characterized by induction and/or enhanced immune responses that vary with antigen production in cells (e.g., when administered to an individual). Increased antigen production can be demonstrated, for example, by increased cell transfection (percentage of cells transfected with the RNA vaccine), increased protein translation from polynucleotides, decreased nucleic acid degradation (e.g., as demonstrated by increased duration of protein translation from modified polynucleotides), and/or altered antigen-specific immune responses of host cells.
在一些態樣中，本發明係關於一種誘導個體中針對RSV之免疫反應的方法。該方法包括向個體投與有效量之本文所描述之RNA分子、RNA-LNP及/或組合物以產生針對RSV之免疫反應。In some aspects, the present invention relates to a method of inducing an immune response against RSV in an individual. The method comprises administering to the individual an effective amount of the RNA molecules, RNA-LNPs and/or compositions described herein to produce an immune response against RSV.
在另一態樣中，本發明係關於一種對個體進行疫苗接種之方法。該方法包括向有需要之個體投與有效量的本文所描述之RNA分子、RNA-LNP及/或組合物。In another aspect, the present invention relates to a method for vaccinating an individual. The method comprises administering an effective amount of the RNA molecules, RNA-LNPs and/or compositions described herein to an individual in need thereof.
在另一態樣中，本發明係關於一種治療及/或預防傳染病的方法。該方法包括向個體投與有效量的本文所描述之RNA分子、RNA-LNP及/或組合物。In another aspect, the present invention relates to a method for treating and/or preventing infectious diseases, comprising administering to an individual an effective amount of the RNA molecules, RNA-LNPs and/or compositions described herein.
在另一態樣中，本發明係關於一種治療及/或預防RSV感染及/或由RSV引起之病痛及/或降低其嚴重程度的方法。該方法包括向個體投與有效量之本文所描述之RNA分子、RNA-LNP及/或組合物。In another aspect, the present invention relates to a method for treating and/or preventing RSV infection and/or illness caused by RSV and/or reducing its severity. The method comprises administering to an individual an effective amount of the RNA molecules, RNA-LNPs and/or compositions described herein.
在另一態樣中，本發明係關於一種藉由例如誘導個體中針對傳染病之免疫反應來治療及/或預防個體患傳染病及/或降低其嚴重程度的方法。在一些態樣中，該方法包括投與包括有效量之本文所描述之RNA分子、RNA-LNP及/或組合物的初打組合物，及投與包括有效量之RNA分子、RNA-LNP及/或組合物之加強組合物。在一些態樣中，組合物引發免疫反應，包括抗體反應。在一些態樣中，組合物引發免疫反應，包括T細胞反應及/或B細胞反應。在一些態樣中，免疫反應包含T細胞反應及B細胞反應。在一些態樣中，組合物引發中和免疫反應。中和免疫反應為作為中和抗體反應及/或有效中和T細胞反應之免疫反應。在一些實施例中，中和抗體反應產生滿足或超過血清保護臨限值之抗體水平。在一些態樣中，組合物引發有效T細胞反應。有效T細胞反應為製造基線水平之傳染病活化的及/或傳染病特異性T細胞(包括CD8+及CD4+ T輔助1型細胞)的反應。在一些態樣中，相對於基線水平(在未處理個體中)，有效T細胞包含高比例之CD8+ T細胞及/或CD4+ T細胞。在一些實施例中，此等T細胞朝向共表現CD27及CD28之早期分化的記憶表型分化。In another aspect, the present invention relates to a method for treating and/or preventing an individual from contracting an infectious disease and/or reducing its severity by, for example, inducing an immune response to an infectious disease in an individual. In some aspects, the method comprises administering a priming composition comprising an effective amount of RNA molecules, RNA-LNPs and/or compositions described herein, and administering a booster composition comprising an effective amount of RNA molecules, RNA-LNPs and/or compositions. In some aspects, the composition induces an immune response, including an antibody response. In some aspects, the composition induces an immune response, including a T cell response and/or a B cell response. In some aspects, the immune response comprises a T cell response and a B cell response. In some aspects, the composition induces a neutralizing immune response. Neutralizing immune response is an immune response as a neutralizing antibody response and/or an effective neutralizing T cell response. In some embodiments, the neutralizing antibody response produces an antibody level that meets or exceeds the serum protection threshold. In some aspects, the composition triggers an effective T cell response. An effective T cell response is a response to infectious disease activation and/or infectious disease-specific T cells (including CD8+ and CD4+ T helper type 1 cells) at a baseline level. In some aspects, relative to the baseline level (in untreated individuals), effective T cells include a high proportion of CD8+ T cells and/or CD4+ T cells. In some embodiments, these T cells are differentiated toward a memory phenotype of early differentiation that co-expresses CD27 and CD28.
在另一態樣中，本發明係關於一種藉由例如誘導個體中針對RSV之免疫反應來治療及/或預防個體感染RSV及/或由RSV引起之病痛及/或降低其嚴重程度的方法。在一些態樣中，該方法包括投與包括有效量的本文所描述之RNA分子、RNA-LNP及/或組合物的初打組合物，及投與包括有效量的本文所描述之RNA分子、RNA-LNP及/或組合物的加強組合物。在一些態樣中，組合物引發免疫反應，包括抗體反應。在一些態樣中，組合物引發免疫反應，包括T細胞反應及/或B細胞反應。在一些態樣中，免疫反應包含T細胞反應及B細胞反應。在一些態樣中，組合物引發中和免疫反應。中和免疫反應為作為中和抗體反應及/或有效中和T細胞反應之免疫反應。在一些實施例中，中和抗體反應產生滿足或超過血清保護臨限值之抗體水平。在一些態樣中，組合物引發有效T細胞反應。有效T細胞反應為製造基線水平之傳染病活化的及/或傳染病特異性T細胞(包括CD8+及CD4+ T輔助1型細胞)的反應。在一些態樣中，相對於基線水平(在未處理個體中)，有效T細胞包含高比例之CD8+ T細胞及/或CD4+ T細胞。在一些實施例中，此等T細胞朝向共表現CD27及CD28之早期分化的記憶表型分化。In another aspect, the present invention relates to a method for treating and/or preventing an individual from being infected with RSV and/or suffering caused by RSV and/or reducing the severity thereof by, for example, inducing an immune response to RSV in an individual. In some aspects, the method comprises administering a primary composition comprising an effective amount of RNA molecules, RNA-LNPs and/or compositions described herein, and administering a booster composition comprising an effective amount of RNA molecules, RNA-LNPs and/or compositions described herein. In some aspects, the composition induces an immune response, including an antibody response. In some aspects, the composition induces an immune response, including a T cell response and/or a B cell response. In some aspects, the immune response comprises a T cell response and a B cell response. In some aspects, the composition induces a neutralizing immune response. Neutralizing immune response is an immune response as a neutralizing antibody response and/or an effective neutralizing T cell response. In some embodiments, the neutralizing antibody response produces an antibody level that meets or exceeds the serum protection threshold. In some aspects, the composition triggers an effective T cell response. An effective T cell response is a response to infectious disease activation and/or infectious disease-specific T cells (including CD8+ and CD4+ T helper type 1 cells) at a baseline level. In some aspects, relative to the baseline level (in untreated individuals), effective T cells include a high proportion of CD8+ T cells and/or CD4+ T cells. In some embodiments, these T cells are differentiated toward a memory phenotype of early differentiation that co-expresses CD27 and CD28.
本文所揭示之方法可涉及向個體投與包含至少一種具有編碼至少一種RSV抗原性多肽之開讀框的RSV RNA分子之RSV RNA-LNP組合物，從而在個體中誘導對RSV抗原性多肽具有特異性之免疫反應，其中相對於疫苗接種預防有效劑量(例如在臨床上可接受含量下預防感染病毒之治療有效劑量)之針對RSV之傳統疫苗的個體中之抗抗原性多肽抗體力價，個體中之抗抗原性多肽抗體力價在疫苗接種之後增加。「抗抗原性多肽抗體」為特異性結合於抗原性多肽之血清抗體。在一些態樣中，相對於投與預防有效劑量之針對RSV之傳統組合物(例如重組或經純化RSV蛋白疫苗、減毒或失活RSV疫苗或RSV VLP疫苗之標準照護劑量)之個體中之抗抗原性多肽抗體力價，個體中之抗抗原性多肽抗體力價在投與RSV RNA-LNP組合物後增加或不增加至少以下、至多以下、以下中之任何兩者之間(包括性或排他性)或恰好以下的量：1、2、3、4、5、6、7、8、9或10 log。在一些態樣中，相對於投與預防有效劑量之針對RSV之傳統組合物(例如重組或經純化RSV蛋白疫苗、減毒或失活RSV疫苗或RSV VLP疫苗之標準照護劑量)之個體中之抗抗原性多肽抗體力價，個體中之抗抗原性多肽抗體力價在投與RSV RNA-LNP組合物後增加或不增加至少以下、至多以下、以下中之任何兩者之間(包括性或排他性)或恰好以下的量：1、2、3、4、5、6、7、8、9、10、100或1000倍。The methods disclosed herein may involve administering to an individual an RSV RNA-LNP composition comprising at least one RSV RNA molecule having an open reading frame encoding at least one RSV antigenic polypeptide, thereby inducing an immune response specific for the RSV antigenic polypeptide in the individual, wherein the anti-antigenic polypeptide antibody titer in the individual is increased after vaccination relative to the anti-antigenic polypeptide antibody titer in the individual after vaccination with a prophylactic effective dose (e.g., a therapeutically effective dose to prevent infection with the virus at a clinically acceptable level) of a traditional vaccine for RSV. "Anti-antigenic polypeptide antibodies" are serum antibodies that specifically bind to antigenic polypeptides. In some aspects, the titer of anti-antigenic polypeptide antibodies in an individual is increased or not increased after administration of the RSV RNA-LNP composition by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 logs, relative to the titer of anti-antigenic polypeptide antibodies in an individual administered a prophylactically effective dose of a traditional composition against RSV (e.g., a standard of care dose of a recombinant or purified RSV protein vaccine, an attenuated or inactivated RSV vaccine, or an RSV VLP vaccine). In some aspects, the titer of anti-antigenic polypeptide antibodies in an individual is increased or not increased after administration of the RSV RNA-LNP composition by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, or 1000-fold, at most, between any two of the following (inclusive or exclusive), or exactly the following, relative to the titer of anti-antigenic polypeptide antibodies in an individual administered a prophylactically effective dose of a traditional composition against RSV (e.g., a standard of care dose of a recombinant or purified RSV protein vaccine, an attenuated or inactivated RSV vaccine, or an RSV VLP vaccine).
在一些態樣中，相對於針對RSV之傳統組合物(例如重組或經純化RSV蛋白疫苗、減毒或失活RSV疫苗或RSV VLP疫苗之標準照護劑量)，有效量之包含至少一種具有編碼至少一種RSV抗原性多肽之開讀框的RSV RNA分子之RSV RNA-LNP組合物引起針對RSV之血清中和抗體增加2倍至200倍(例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：2、3、4、5、6、7、8、9、10、20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、180、190或200倍)。In some aspects, an effective amount of an RSV RNA-LNP composition comprising at least one RSV RNA molecule having an open reading frame encoding at least one RSV antigenic polypeptide induces a 2-fold to 200-fold increase in serum neutralizing antibodies against RSV (e.g., at least, at most, just below, or between any two of the following (inclusive or exclusive): 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200-fold) relative to a traditional composition against RSV (e.g., a standard of care dose of a recombinant or purified RSV protein vaccine, an attenuated or inactivated RSV vaccine, or an RSV VLP vaccine).
在一些態樣中，包含至少一種具有編碼至少一種RSV抗原性多肽之開讀框的RSV RNA分子之RSV RNA-LNP組合物的有效量為等效於針對RSV之傳統組合物之標準照護劑量的至少2倍降低的劑量。舉例而言，RSV RNA-LNP組合物之有效量可為或可不為等效於針對RSV之傳統組合物之標準照護劑量的至少以下降低的劑量：2、3、4、5、6、7、8、9、10、20、50、100、250、500或1000倍。在一些實施例中，投與有效量之RSV RNA-LNP組合物之個體中產生的抗RSV抗原性多肽抗體力價等效於投與針對RSV之傳統組合物之標準照護劑量之對照個體中產生的抗RSV抗原性多肽抗體力價。在一些實施例中，RSV RNA-LNP組合物之有效量為或不為等效於針對RSV之傳統組合物之標準照護劑量的2倍至1000倍降低(例如至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：2、3、4、5、6、7、8、9、10、20、30、40、50、60、70、80、90、100、110、120、130、140、150、160、170、1280、190、200、210、220、230、240、250、260、270、280、290、300、310、320、330、340、350、360、370、380、390、400、410、420、430、440、450、4360、470、480、490、500、510、520、530、540、550、560、5760、580、590、600、610、620、630、640、650、660、670、680、690、700、710、720、730、740、750、760、770、780、790、800、810、820、830、840、850、860、870、880、890、900、910、920、930、940、950、960、970、980、990或1000倍降低)的劑量，其中個體中產生的抗RSV抗原性多肽抗體力價等效於投與針對RSV之傳統組合物之標準照護劑量之對照個體中產生的抗RSV抗原性多肽抗體力價。在一些態樣中，個體中產生的抗RSV抗原性多肽抗體力價等效於投與針對RSV之傳統組合物之標準照護劑量之對照個體中產生的抗RSV抗原性多肽抗體力價。In some aspects, the effective amount of the RSV RNA-LNP composition comprising at least one RSV RNA molecule having an open reading frame encoding at least one RSV antigenic polypeptide is an at least 2-fold reduced dose equivalent to the standard of care dose of a traditional composition for RSV. For example, the effective amount of the RSV RNA-LNP composition may or may not be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 250, 500, or 1000-fold reduced dose equivalent to the standard of care dose of a traditional composition for RSV. In some embodiments, the anti-RSV antigenic polypeptide antibody titer produced in an individual administered an effective amount of the RSV RNA-LNP composition is equivalent to the anti-RSV antigenic polypeptide antibody titer produced in a control individual administered a standard of care dose of a traditional composition for RSV. In some embodiments, the effective amount of the RSV RNA-LNP composition is or is not 2-fold to 1000-fold lower (e.g., at least, at most, just below, or between any two of the following (inclusive or exclusive): 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600 0, 140, 150, 160, 170, 1280, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 4360, 470, 480 ,490,500,510,520,530,540,550,560,5760,580,590,600,610,620,630,640,650,660,670,680,690,700,710,720,730,740,750,760,770,780,790,800,810,820,830, In some embodiments, the present invention relates to an antibody titer of anti-RSV antigenic polypeptide produced in an individual that is equivalent to the titer of anti-RSV antigenic polypeptide antibody produced in a control individual administered a standard of care dose of a traditional composition for RSV. In some embodiments, the titer of anti-RSV antigenic polypeptide antibody produced in an individual is equivalent to the titer of anti-RSV antigenic polypeptide antibody produced in a control individual administered a standard of care dose of a traditional composition for RSV.
如本文所用，針對RSV之傳統組合物係指除本文所描述之RNA分子、RNA-LNP及/或組合物以外的組合物。舉例而言，傳統組合物包括但不限於活微生物疫苗、經殺滅的微生物疫苗、減毒疫苗、次單位疫苗、含有異源表現系統中產生或自大量病原性生物體純化之重組蛋白的蛋白質抗原疫苗、DNA疫苗、含有病毒殼體蛋白質(例如融合前及/或融合後F蛋白)但缺乏病毒基因體之病毒樣顆粒(VLP)疫苗等。在例示性實施例中，傳統疫苗為已實現監管批准及/或由國家藥物監管機構(例如美國食品與藥物管理局(Food and Drug Administration；FDA)或歐洲藥物管理局(European Medicines Agency；EMA))登記的疫苗。如本文所提供，「標準照護」係指醫學或精神治療指南且可為通用或特異性的。「標準照護」基於科學證據及涉及給定病狀之治療之醫學專業人士之間的協作規定適當治療。其為醫師/臨床醫師應遵循某一類型之患者、病痛或臨床情形的診斷及治療方法。如本文所提供，「標準照護劑量」係指醫師/臨床醫師或其他醫療專業人士將向個體投與以治療及/或預防RSV或RSV相關病狀，同時遵循用於治療及/或預防RSV或RSV相關病狀之標準照護指南的針對RSV之傳統組合物的劑量。As used herein, a traditional composition for RSV refers to a composition other than the RNA molecules, RNA-LNPs and/or compositions described herein. For example, traditional compositions include, but are not limited to, live microbial vaccines, killed microbial vaccines, attenuated vaccines, subunit vaccines, protein antigen vaccines containing recombinant proteins produced in heterologous expression systems or purified from large quantities of pathogenic organisms, DNA vaccines, virus-like particles (VLP) vaccines containing viral capsid proteins (e.g., pre-fusion and/or post-fusion F proteins) but lacking viral genomes, etc. In an exemplary embodiment, a traditional vaccine is a vaccine that has achieved regulatory approval and/or is registered by a national drug regulatory agency (e.g., the U.S. Food and Drug Administration (FDA) or the European Medicines Agency (EMA)). As provided herein, "standard of care" refers to medical or psychiatric treatment guidelines and can be general or specific. "Standard of care" stipulates appropriate treatment based on scientific evidence and collaboration between medical professionals involved in the treatment of a given condition. It is the diagnostic and treatment approach that a physician/clinician should follow for a certain type of patient, ailment, or clinical situation. As provided herein, "standard of care dosage" refers to the dosage of a traditional composition for RSV that a physician/clinician or other medical professional would administer to an individual to treat and/or prevent RSV or an RSV-related condition while following standard care guidelines for the treatment and/or prevention of RSV or an RSV-related condition.
在一些態樣中，本文所描述之RNA分子、RNA-LNP及/或組合物(例如包含至少一種具有編碼至少一種RSV抗原性多肽之開讀框的RSV RNA分子之RSV RNA-LNP組合物)在個體之血液或血清中產生預防及/或治療有效水平、濃度及/或力價的抗原特異性抗體。如本文所定義，術語抗體力價係指在個體(例如人類個體)中產生之抗原特異性抗體的量。在例示性實施例中，抗體力價表示為仍產生陽性結果之最大稀釋度(在連續稀釋中)之倒數。在例示性態樣中，藉由酶聯免疫吸附分析(ELISA)測定或量測抗體力價。在例示性實施例中，藉由中和分析，例如藉由微中和分析測定或量測抗體力價。在某些態樣中，抗體力價量測表示為比率，諸如1:40、1:100等。In some aspects, the RNA molecules, RNA-LNPs and/or compositions described herein (e.g., RSV RNA-LNP compositions comprising at least one RSV RNA molecule having an open reading frame encoding at least one RSV antigenic polypeptide) produce antigen-specific antibodies at a preventive and/or therapeutically effective level, concentration and/or titer in the blood or serum of an individual. As defined herein, the term antibody titer refers to the amount of antigen-specific antibodies produced in an individual (e.g., a human individual). In exemplary embodiments, the antibody titer is expressed as the reciprocal of the maximum dilution (in a series of dilutions) that still produces a positive result. In exemplary aspects, the antibody titer is determined or measured by an enzyme-linked immunosorbent assay (ELISA). In exemplary embodiments, the antibody titer is determined or measured by a neutralization assay, such as a microneutralization assay. In some aspects, the resistance value measurement is expressed as a ratio, such as 1:40, 1:100, etc.
在例示性態樣中，有效的本文所描述之RNA分子、RNA-LNP及/或組合物(例如包含至少一種具有編碼至少一種RSV抗原性多肽之開讀框的RSV RNA分子之RSV RNA-LNP組合物)產生大於1:10、大於1:100、大於1:400、大於1:1000、大於1:2000、大於1:3000、大於1:4000、大於1:5000、大於1:6000、大於1:7500或大於1:10000之抗體力價。在例示性態樣中，在疫苗接種之後10天、疫苗接種之後20天、疫苗接種之後30天、疫苗接種之後40天或疫苗接種之後50天或更多天產生或達到該抗體力價。在例示性態樣中，在向個體投與之單次劑量之疫苗之後產生或達到該力價。在其他態樣中，在多次劑量後，例如在第一次及第二次劑量(例如加強劑量)後產生或達到該力價。In exemplary aspects, the effective RNA molecules, RNA-LNPs and/or compositions described herein (e.g., RSV RNA-LNP compositions comprising at least one RSV RNA molecule having an open reading frame encoding at least one RSV antigenic polypeptide) produce an antibody titer of greater than 1:10, greater than 1:100, greater than 1:400, greater than 1:1000, greater than 1:2000, greater than 1:3000, greater than 1:4000, greater than 1:5000, greater than 1:6000, greater than 1:7500, or greater than 1:10000. In exemplary aspects, the antibody titer is produced or achieved 10 days after vaccination, 20 days after vaccination, 30 days after vaccination, 40 days after vaccination, or 50 days after vaccination or more. In an exemplary embodiment, the potency is produced or achieved after a single dose of the vaccine administered to an individual. In other embodiments, the potency is produced or achieved after multiple doses, such as after the first and second doses (e.g., booster doses).
本文所揭示之方法可涉及向個體投與包含至少一種具有編碼至少一種RSV抗原性多肽之開讀框的RSV RNA分子之RSV RNA-LNP組合物，從而在個體中誘導對RSV抗原性多肽具有特異性之免疫反應，其中個體中之免疫反應等效於投與針對RSV之傳統組合物之個體之免疫反應，相對於RNA組合物，其為或不為至少以下、至多以下、以下中之任何兩者之間(包括性或排他性)或恰好以下量的劑量水平：2、4、6、8、10、12、14、16、18、20、22、24、26、28、30、32、34、36、38、40、42、44、46、48、50、52、54、56、58、60、62、64、66、68、70、72、74、76、78、80、82、84、86、88、90、92、94、96、98或100倍。The methods disclosed herein may involve administering to a subject an RSV RNA-LNP composition comprising at least one RSV RNA molecule having an open reading frame encoding at least one RSV antigenic polypeptide, thereby inducing an immune response in the subject that is specific for the RSV antigenic polypeptide, wherein the immune response in the subject is equivalent to the immune response of the subject administered a traditional composition directed against RSV, which is or is not a dosage level of at least the following, at most the following, between any two of the following (inclusive or exclusive), or exactly the following amounts, relative to the RNA composition: 2, 4, 6, 8, 1 0, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98 or 100 times.
在一些態樣中，RNA分子、RNA-LNP及/或組合物用作疫苗。在一些態樣中，RNA分子、RNA-LNP及/或組合物可在各種治療或預防方法中使用以用於預防、治療或改善急性下呼吸道感染(ALRI)或與呼吸道病痛相關之病症，包括肺炎及支氣管炎。In some aspects, RNA molecules, RNA-LNPs and/or compositions are used as vaccines. In some aspects, RNA molecules, RNA-LNPs and/or compositions can be used in various treatment or prevention methods to prevent, treat or ameliorate acute lower respiratory tract infection (ALRI) or symptoms associated with respiratory tract diseases, including pneumonia and bronchitis.
在一些態樣中，RNA分子、RNA-LNP及/或組合物可在各種治療或預防方法中使用以用於預防、治療或改善急性下呼吸道感染(ALRI)，包括肺炎及支氣管炎。In some aspects, RNA molecules, RNA-LNPs and/or compositions can be used in various therapeutic or preventive methods for preventing, treating or ameliorating acute lower respiratory tract infections (ALRI), including pneumonia and bronchitis.
在一些態樣中，本發明之方法係關於對疑似患有傳染病(例如RSV)、患有傳染病(例如RSV)、處於患傳染病(例如RSV)及/或患傳染病(例如RSV)之症狀之風險下的個體的預後、診斷、測試、監測及/或治療。該個體可能患有傳染病(例如RSV)之一或多種症狀。可藉由一或多種診斷測試(例如用於偵測皮膚病變中之RSV的PCR測試；Tzanck塗片；IgM血清測試；用於IgG偵測之ELISA、基於醣蛋白之ELISA、乳膠凝集及/或間接螢光抗體；直接螢光抗體分析、病毒培養等)，來測試個體之一或多種來自傳染病(例如RSV)的抗原性多肽或蛋白質(或其抗原性部分)。在一些態樣中，藉由一或多種診斷測試(例如用於偵測RSV之PCR測試；Tzanck塗片；IgM血清測試；用於IgG偵測之ELISA、基於醣蛋白之ELISA、乳膠凝集及/或間接螢光抗體；直接螢光抗體分析、病毒培養物等)，基於對來自個體之樣品(例如血液、唾液、組織、骨骼、肌肉、軟骨及/或皮膚)中之一或多種來自傳染病(例如RSV)之抗原性多肽或蛋白質(或其抗原性部分)進行的量測、分析或偵測，對已患有傳染病(例如RSV)、患有傳染病(例如RSV)、處於患傳染病(例如RSV)及/或患有傳染病(例如RSV)之症狀之風險下的個體進行預後、診斷、監測及/或治療傳染病(例如RSV)。In some aspects, the methods of the invention relate to the prognosis, diagnosis, testing, monitoring and/or treatment of an individual suspected of having an infectious disease (e.g., RSV), having an infectious disease (e.g., RSV), at risk of having an infectious disease (e.g., RSV) and/or having symptoms of an infectious disease (e.g., RSV). The individual may have one or more symptoms of an infectious disease (e.g., RSV). An individual can be tested for one or more antigenic polypeptides or proteins (or antigenic portions thereof) from an infectious disease (e.g., RSV) by one or more diagnostic tests (e.g., PCR test for detection of RSV in skin lesions; Tzanck smear; IgM serum test; ELISA for IgG detection, glycoprotein-based ELISA, latex agglutination and/or indirect fluorescent antibodies; direct fluorescent antibody analysis, viral culture, etc.). In some embodiments, the virus is detected by one or more diagnostic tests (e.g., PCR test for RSV detection; Tzanck smear; IgM serum test; ELISA for IgG detection, glycoprotein-based ELISA, latex agglutination and/or indirect fluorescent antibodies; direct fluorescent antibody analysis, viral culture, etc.) based on the analysis of samples (e.g., blood, saliva, tissue, bone, muscle, cartilage, and/or skin) from an individual. The invention relates to the measurement, analysis or detection of one or more antigenic polypeptides or proteins (or antigenic portions thereof) from an infectious disease (e.g., RSV) in a subject who has an infectious disease (e.g., RSV), has an infectious disease (e.g., RSV), is at risk of having an infectious disease (e.g., RSV) and/or has symptoms of an infectious disease (e.g., RSV).
可向健康個體或在感染早期在潛伏期期間或在症狀發作之後的活動性感染期間預防性地投與RSV RNA組合物。在一些態樣中，個體具有免疫能力。在一些態樣中，個體免疫功能不全。RSV RNA compositions can be administered prophylactically to healthy individuals or in the early stages of infection during the latent period or during active infection after the onset of symptoms. In some aspects, the individual is immunocompetent. In some aspects, the individual is immunocompromised.
在一些態樣中，RNA分子、RNA-LNP及/或組合物以單次劑量投與。在一些態樣中，可給與第二、第三或第四劑量。在一些態樣中，RNA分子、RNA-LNP及/或組合物以多次劑量投與。In some aspects, the RNA molecules, RNA-LNPs and/or compositions are administered in a single dose. In some aspects, a second, third or fourth dose may be given. In some aspects, the RNA molecules, RNA-LNPs and/or compositions are administered in multiple doses.
在一些態樣中，RNA分子、RNA-LNP及/或組合物經肌肉內(IM)或皮內(ID)投與。In some aspects, the RNA molecules, RNA-LNPs and/or compositions are administered intramuscularly (IM) or intradermally (ID).
本發明進一步提供一種套組，其包含RNA分子、RNA-LNP及/或組合物。The present invention further provides a kit comprising RNA molecules, RNA-LNPs and/or compositions.
在一些態樣中，向小於約1歲、或約1歲至約10歲、或約10歲至約20歲、或約20歲至約50歲、或約60歲至約70歲或更大之個體投與本文所描述之RNA分子、RNA-LNP及/或組合物。In some aspects, the RNA molecules, RNA-LNPs, and/or compositions described herein are administered to an individual less than about 1 year old, or between about 1 year old and about 10 years old, or between about 10 years old and about 20 years old, or between about 20 years old and about 50 years old, or between about 60 years old and about 70 years old, or older.
在一些態樣中，個體為至少、至多、恰好以下歲數或其中任何兩者之間的歲數：小於1歲、大於1歲、大於5歲、大於10歲、大於20歲、大於30歲、大於40歲、大於50歲、大於60歲、大於70歲或更大。在一些態樣中，個體大於50歲。In some aspects, the subject is at least, at most, exactly, or any two of the following ages: less than 1 year old, greater than 1 year old, greater than 5 years old, greater than 10 years old, greater than 20 years old, greater than 30 years old, greater than 40 years old, greater than 50 years old, greater than 60 years old, greater than 70 years old, or older. In some aspects, the subject is greater than 50 years old.
在一些態樣中，個體為至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的年齡：約1歲或更大、約5歲或更大、約10歲或更大、約20歲或更大、約30歲或更大、約40歲或更大、約50歲或更大、約60歲或更大、約70歲或更大，或更大。在一些態樣中，個體之年齡可為約50歲或更大。In some aspects, the subject is at least, at most, just below, or between any two of the following (inclusive or exclusive): about 1 year old or older, about 5 years old or older, about 10 years old or older, about 20 years old or older, about 30 years old or older, about 40 years old or older, about 50 years old or older, about 60 years old or older, about 70 years old or older, or older. In some aspects, the age of the subject may be about 50 years old or older.
在一些態樣中，個體為至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)的年齡：1歲或更大、5歲或更大、10歲或更大、20歲或更大、30歲或更大、40歲或更大、50歲或更大、60歲或更大、70歲或更大，或更大。在一些態樣中，個體可為50歲或更大。In some aspects, the subject is at least, at most, just below, or between any two of the following (inclusive or exclusive): 1 year or older, 5 years or older, 10 years or older, 20 years or older, 30 years or older, 40 years or older, 50 years or older, 60 years or older, 70 years or older, or older. In some aspects, the subject may be 50 years or older.
X.臨床研究本發明之RSV RNA-LNP疫苗包含編碼來自RSV之融合前F (preF)多肽之核苷修飾之mRNA (經修飾之RNA；modRNA)。RSV RNA-LNP疫苗可包含RNA，該RNA包含在進入細胞之後轉譯的單股、5'加帽且多腺苷酸化的經修飾之RNA。RNA包含編碼RSV preF多肽之變體的開讀框(ORF)。此外，如本文所描述，RNA可包含針對RNA之高功效最佳化的結構元件，諸如非轉譯區(UTR)。RSV RNA-LNP可包含本文所揭示之實例1之表5中所提供之RNA。RSV RNA-LNP可包含本文所揭示之實例6之表1至3中所提供之RNA。RNA亦可包含1-甲基-假尿苷對尿苷之取代以減少先天免疫感測器(諸如鐸樣受體(TLR) 7及8)對疫苗RNA之識別，從而引起先天免疫活化之降低及蛋白質轉譯之增加。X.Clinical Studies The RSV RNA-LNP vaccines of the present invention comprise nucleoside-modified mRNA (modified RNA; modRNA) encoding a prefusion F (preF) polypeptide from RSV. The RSV RNA-LNP vaccine may comprise RNA comprising a single-stranded, 5'-capped, polyadenylated modified RNA that is translated after entering the cell. The RNA comprises an open reading frame (ORF) encoding a variant of the RSV preF polypeptide. In addition, as described herein, the RNA may comprise structural elements optimized for high efficacy of the RNA, such as a non-translated region (UTR). The RSV RNA-LNP may comprise the RNA provided in Table 5 of Example 1 disclosed herein. The RSV RNA-LNP may comprise the RNA provided in Tables 1 to 3 of Example 6 disclosed herein. The RNA may also contain 1-methyl-pseudouridine substitutions for uridine to reduce recognition of the vaccine RNA by innate immune sensors such as toll-like receptors (TLRs) 7 and 8, thereby resulting in decreased innate immune activation and increased protein translation.
本文所描述之RNA分子經調配/囊封於脂質奈米顆粒(LNP)中，以使得能夠在例如肌肉內(IM)、皮內(ID)或鼻內(IN)注射之後將RNA遞送至宿主細胞中。LNP調配物可包含兩種功能性脂質(ALC-0315及ALC-0159)及兩種結構脂質(DSPC (1,2-二硬脂醯基-sn-甘油基-3-磷酸膽鹼)及膽固醇)。在一些態樣中，前述脂質中之1、2、3者或更多者可排除在LNP調配物外。RNA疫苗之效能藉由LNP囊封最佳化，LNP囊封保護RNA免於細胞外RNase降解且有助於在細胞中之遞送。在IM注射RSV RNA-LNP疫苗之後，LNP由細胞吸收，且RNA釋放至胞溶質中。在胞溶質中，RNA被轉譯，且產生所編碼病毒抗原。The RNA molecules described herein are formulated/encapsulated in lipid nanoparticles (LNPs) to enable delivery of the RNA to host cells following, for example, intramuscular (IM), intradermal (ID), or intranasal (IN) injection. The LNP formulation may include two functional lipids (ALC-0315 and ALC-0159) and two structural lipids (DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) and cholesterol). In some aspects, one, two, three or more of the aforementioned lipids may be excluded from the LNP formulation. The efficacy of RNA vaccines is optimized by LNP encapsulation, which protects the RNA from degradation by extracellular RNases and facilitates delivery in cells. Following IM injection of the RSV RNA-LNP vaccine, the LNPs are taken up by cells and the RNA is released into the cytosol, where it is translated and the encoded viral antigen is produced.
本文中之實例證實本發明之RSV RNA-LNP疫苗在小鼠中具有免疫原性且在小鼠中誘導體液免疫反應及細胞介導之免疫反應兩者。The examples herein demonstrate that the RSV RNA-LNP vaccine of the present invention is immunogenic in mice and induces both humoral and cell-mediated immune responses in mice.
本發明之臨床研究評估針對RSV之RSV RNA-LNP疫苗的安全性、耐受性及免疫原性。舉例而言，可指示RSV RNA-LNP疫苗用於主動免疫接種以預防成年人(例如≥45、≥50、≥55、≥60、≥70歲等或50至69歲)之急性下呼吸道感染(ALRI)，包括由RSV引起之肺炎及支氣管炎。RSV RNA-LNP疫苗可以本文所描述之不同的一或多種劑量水平、劑量調配物、劑量次數及給藥時程進行投與，包括但不限於： -單次劑量時程或兩次劑量時程(例如第0天及在或在約2個月後，或第0天及在或在約6個月後) -以不同劑量水平(例如每次投與為或為約15 µg、30 µg、60 µg、90 µg、100 µg或更高) -以不同調配物(非凍乾及/或凍乾)The clinical studies of the present invention evaluate the safety, tolerability and immunogenicity of RSV RNA-LNP vaccines against RSV. For example, RSV RNA-LNP vaccines may be indicated for active immunization to prevent acute lower respiratory tract infections (ALRI) in adults (e.g., ≥45, ≥50, ≥55, ≥60, ≥70 years old, etc. or 50 to 69 years old), including pneumonia and bronchitis caused by RSV. RSV RNA-LNP vaccines may be administered at one or more of the various dose levels, dose formulations, dose times, and dosing schedules described herein, including but not limited to: - a single dose schedule or a two-dose schedule (e.g., day 0 and at or about 2 months later, or day 0 and at or about 6 months later) - at different dose levels (e.g., at or about 15 µg, 30 µg, 60 µg, 90 µg, 100 µg or more per administration) - in different formulations (non-lyophilized and/or lyophilized)
RSV RNA-LNP可以液體或凍乾調配物形式呈現。RSV RNA-LNP疫苗之投與可以或可不以在或在約以下之範圍內投藥：每劑量15 µg、30 µg、60 µg、90 µg、100 µg或更高，其中注射體積為或為約0.25至1 mL (例如為或為約0.25、0.5、1 mL)。可能需要用無菌0.9%氯化鈉(生理鹽水)進行稀釋。RSV RNA-LNP can be presented in liquid or lyophilized formulations. Administration of RSV RNA-LNP vaccines may or may not be in the range of or about 15 μg, 30 μg, 60 μg, 90 μg, 100 μg or more per dose, with an injection volume of or about 0.25 to 1 mL (e.g., or about 0.25, 0.5, 1 mL). Dilution with sterile 0.9% sodium chloride (normal saline) may be required.
RSV RNA-LNP臨床研究之目標可包括但不限於： -描述以所選劑量水平及時程投與之RSV RNA-LNP疫苗在參與者中之安全性及耐受性概況。 -描述藉由以所選擇劑量水平及時程投與之市售的針對RSV之疫苗以及RSV RNA-LNP疫苗在參與者中引起的免疫反應。Objectives of RSV RNA-LNP clinical studies may include but are not limited to:-Describe the safety and tolerability profile of RSV RNA-LNP vaccines in participants administered at selected dose levels and schedules.-Describe the immune responses elicited in participants by commercially available vaccines against RSV and RSV RNA-LNP vaccines administered at selected dose levels and schedules.
在一些態樣中，本文所揭示之編碼RSV多肽之RNA分子、RNA-LNP及其組合物針對RSV的功效(或有效性)大於或不大於50% (例如至少、至多、恰好以下或在以下中之任何兩者之間：50%、60%、70%、80%、90%、或更多)。可使用標準分析評定疫苗功效(參見例如Weinberg等人,J Infect Dis. 2010年6月1日;201(11): 1607-10)。舉例而言，可藉由雙盲、隨機分組的臨床受控試驗(諸如本文所描述之彼等者)來量測疫苗功效。疫苗功效可表示為未經疫苗接種(ARU)與經疫苗接種(ARV)研究小組之間的疾病侵襲率(AR)之成比例降低，且可使用以下公式由疫苗接種組中之疾病之相對風險(RR)來計算： 功效= (ARU ‒ ARV)/ARU × 100；及 功效= (1-RR) × 100。In some aspects, the RNA molecules encoding RSV polypeptides disclosed herein, RNA-LNPs, and compositions thereof have an efficacy (or effectiveness) against RSV of greater than or less than 50% (e.g., at least, at most, just below, or between any two of the following: 50%, 60%, 70%, 80%, 90%, or more). Vaccine efficacy can be assessed using standard assays (see, e.g., Weinberg et al.,J Infect Dis . 2010 Jun 1; 201(11): 1607-10). For example, vaccine efficacy can be measured by double-blind, randomized controlled clinical trials (such as those described herein). Vaccine efficacy can be expressed as the proportional reduction in disease attack rate (AR) between unvaccinated (ARU) and vaccinated (ARV) study groups and can be calculated from the relative risk (RR) of disease in the vaccinated group using the following formulas: Efficacy = (ARU ‒ ARV)/ARU × 100; and Efficacy = (1-RR) × 100.
同樣地，可使用標準分析來評定疫苗有效性(參見例如Weinberg等人,J Infect Dis. 2010年6月1日;201(11): 1607-10)。疫苗有效性為對疫苗(其可能已證實具有高疫苗功效)如何減少群體中之疾病的評定。此量測可評定疫苗接種程序而非僅疫苗本身在天然現場條件下而非在受控臨床試驗中之益處與不良作用的淨平衡。疫苗有效性與疫苗功效(效力)成比例，但亦受群體中之目標群免疫接種之程度以及影響住院治療、動態問診及/或費用之真實結果的其他非疫苗相關因素影響。舉例而言，可使用回溯性病例控制分析，其中比較一組感染病例及適當對照當中的疫苗接種比率。疫苗有效性可表示為率差，其中使用在疫苗接種後仍發生感染的幾率比(OR)： 有效性= (1 ‒ OR) × 100。Similarly, standard analyses can be used to assess vaccine effectiveness (see, e.g., Weinberg et al.,J Infect Dis . 2010 Jun 1; 201(11): 1607-10). Vaccine effectiveness is an assessment of how well a vaccine (which may have demonstrated high vaccine efficacy) reduces disease in a population. This measure assesses the net balance of benefits and adverse effects of a vaccination program, rather than just the vaccine itself, under natural field conditions rather than in controlled clinical trials. Vaccine effectiveness is proportional to vaccine efficacy (effectiveness), but is also affected by the extent to which target herd immunity in a population is vaccinated, as well as other non-vaccine-related factors that affect the true outcomes of hospitalizations, ambulatory visits, and/or costs. For example, a retrospective case-control analysis can be used, in which the vaccine vaccination rates in a group of infected cases and appropriate controls are compared. Vaccine effectiveness can be expressed as a rate difference, using the odds ratio (OR) of developing an infection after vaccination: Effectiveness = (1 ‒ OR) × 100.
在一些態樣中，相對於未進行疫苗接種的對照個體，RSV多肽、RNA-LNP及其組合物之功效為至少60%。舉例而言，相對於未進行疫苗接種之對照個體，功效可為至少、至多、恰好以下或在以下中之任何兩者之間(包括性或排他性)：65%、70%、75%、80%、85%、95%、98%或100%。In some aspects, the efficacy of RSV polypeptides, RNA-LNPs, and compositions thereof is at least 60% relative to a control subject that has not been vaccinated. For example, the efficacy relative to a control subject that has not been vaccinated may be at least, at most, just below, or between any two of the following (inclusive or exclusive): 65%, 70%, 75%, 80%, 85%, 95%, 98%, or 100%.
實例下文為用於實施本發明之特定態樣的實例。包括以下實例以說明本發明之態樣。實例僅出於說明性目的提供，且不意欲以任何方式限制本發明之範疇。熟習此項技術者應瞭解，以下實例中所揭示之技術代表本發明人發現的在本發明之實踐中發揮良好作用的技術。然而，熟習此項技術者應理解，根據本發明，在不背離本發明之精神及範疇的情況下可對所揭示之特定態樣作出許多改變且仍獲得相同或類似結果。已儘力確保關於所用數字(例如量、溫度等)之準確性，但當然應該允許一些實驗誤差及偏差。Examples The following are examples for implementing specific aspects of the present invention. The following examples are included to illustrate aspects of the present invention. Examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques that the inventors have discovered to work well in the practice of the present invention. However, those skilled in the art will appreciate that, according to the present invention, many changes may be made to the disclosed specific aspects without departing from the spirit and scope of the present invention and still obtaining the same or similar results. Every effort has been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), but of course some experimental errors and deviations should be allowed.
實例1.產生RSV preF modRNA構築體本文中產生之RNA構築體編碼RSV F蛋白野生型(WT)及RSV F蛋白變體/突變體(亦即RSV融合前F蛋白)。表4顯示WT F蛋白(WT F)及變異RSV preF蛋白。表4.RSV F蛋白及描述
製備編碼RSV F蛋白之DNA序列且將其用於活體外轉錄反應以產生RNA。RNA之活體外轉錄為此項技術中已知且在本文中描述。將DNA模板選殖至具有用於改良RNA穩定性及轉譯效率之主鏈序列元件(T7啟動子、5'及3' UTR、多-A尾)之質體載體中。DNA經純化，以分光光度法定量，且在三核苷酸帽1類似物((m27,3'-O)Gppp(m2'-O)ApG) (TriLink)存在下藉由T7 RNA聚合酶活體外轉錄，且其中N1-甲基假尿苷(Ψ)置換尿苷(經修飾之RNA (modRNA))。A DNA sequence encoding the RSV F protein was prepared and used in an in vitro transcription reaction to produce RNA. In vitro transcription of RNA is known in the art and described herein. The DNA template was cloned into a plasmid vector with backbone sequence elements (T7 promoter, 5' and 3' UTR, poly-A tail) for improved RNA stability and translation efficiency. The DNA was purified, quantified spectrophotometrically, and transcribed in vitro by T7 RNA polymerase in the presence of a trinucleotide cap 1 analog ((m27,3'-O )Gppp(m2'-O )ApG) (TriLink) and in which N1-methylpseudouridine (Ψ) replaces uridine (modified RNA (modRNA)).
RSV RNA由密碼子最佳化(CO) DNA產生以實現穩定性及優良蛋白質表現。表5顯示包含5' UTR、編碼呼吸道融合病毒(RSV)多肽之開讀框、3' UTR及多-A尾的本發明之RNA構築體及對應序列。表5.RSV F modRNA構築體
實例2.產生RSV preF saRNA構築體saRNA合成經由活體外轉錄(IVT)進行且藉由超濾/透濾(UFDF-1)純化。接著，saRNA被酶促加帽且藉由層析及最終UFDF-2純化，隨後最終過濾且施配。表6.按次序包含5'帽-5'UTR-nsP1-nsP2-nsP3-nsP4-次基因體啟動子-RSV [ORF]-3'UTR-多-A尾之RSV F 847A saRNA構築體(編碼具有SEQ ID NO: 4之RSV F蛋白)
實例3.製備調配於LNP中之RSV preF RNALNP調配物含有2種功能性脂質ALC-0315及ALC-0159，以及2種結構脂質DSPC (1,2-二硬脂醯基-sn-甘油基-3-磷酸膽鹼)及膽固醇。4種脂質之物理化學特性及結構顯示於下表8中。Example3.Preparation ofRSV preF RNA LNP formulationsinLNPs contained two functional lipids, ALC-0315 and ALC-0159, and two structural lipids, DSPC (1,2-distearyl-sn-glycero-3-phosphocholine) and cholesterol. The physicochemical properties and structures of the four lipids are shown inTable8 below.
根據美國專利9737619 (PCT公開案第WO2015/199952號)及美國專利10166298 (WO 2017/075531)及WO2020/146805 (其各自以全文引用之方式併入本文中)中所描述之通用程序來製備及測試脂質奈米顆粒。簡言之，陽離子脂質、DSPC、膽固醇及PEG-脂質以約47.5:10:40.7:1.8之莫耳比溶解於乙醇中。表8. LNP調配物中之脂質
實例4. RSV preF表現此實例用以捕獲針對本文所描述之呼吸道融合病毒(RSV) modRNA脂質奈米顆粒(LNP)藥物產品所產生之活體外表現(in vitro expression；IVE)結果。LNP中囊封之構築體分別編碼來自A株系及B株系病毒之全長RSV三聚融合體醣蛋白(F-蛋白)。表9.抗體之清單
以下方案之描述係針對全染色(表面+細胞內蛋白質)，對於僅表面染色方案而言，改變固定及洗滌緩衝液以移除滲透劑，所有其他步驟及試劑均相同。簡言之，以2.5×105個細胞/孔之密度對96孔培養盤接種HEK293F細胞，且將其置於振盪培育箱(350 RPM，37℃，潮濕，5% CO2)中，同時準備樣品滴定。將LNP藥物產品稀釋於DPBS中至80 ng/μL之濃度，且以4之稀釋因子連續稀釋8點。接著，自培育箱移出96孔培養盤，且將50 μL各步驟之所稀釋LNP添加至96孔培養盤之重複孔中，以產生在8,000 ng/孔-1.95 ng/孔之範圍內的滴定曲線。將96孔培養盤放回振盪培育箱中過夜。在培育之後，將250 μL細胞轉移至96孔u形底聚苯乙烯培養盤中，且使用擺動鬥式離心機(500 rcf，5 min，在室溫)集結(pelleted)。移除上清液，且將細胞再懸浮於100 μL Aqua405活/死染色溶液中。在室溫避光培育培養盤30 min。在培育之後，細胞用洗滌緩衝液洗滌且使用離心(500 rcf，5 min，在室溫)集結。移除上清液，且將細胞再懸浮於100 μL固定/滲透緩衝液中，且在2-8℃避光培育培養盤30分鐘。一旦培育完成，則使用擺動鬥式離心機(500 rcf，5 min，在室溫)使細胞集結。移除上清液，且細胞用250 μL洗滌緩衝液再懸浮，重複此總共2次洗滌。在最終洗滌步驟之後，使細胞集結，移除上清液，且再懸浮於50 μL一級抗體溶液中。將培養盤密封且在2-8℃避光培育45分鐘。一旦完成，則使用擺動鬥式離心機(500 rcf，5 min，在室溫)使細胞集結。移除上清液，且細胞用250 μL洗滌緩衝液再懸浮，重複此總共2次洗滌。在最終洗滌步驟之後，使細胞集結，移除上清液，且再懸浮於50 μL二級抗體溶液中。將培養盤密封且在2-8℃避光培育45分鐘。一旦完成，則使用擺動鬥式離心機(500 rcf，5 min，在室溫)使細胞集結。移除上清液，且細胞用250 μL洗滌緩衝液再懸浮，重複此總共2次洗滌。在最終洗滌步驟之後，使細胞集結，移除上清液，且再懸浮於200 μL洗滌緩衝液中，並且藉由流式細胞分析技術獲取資料。The following protocol is described for full staining (surface + intracellular proteins). For the surface staining protocol only, the fixation and wash buffers were changed to remove the permeabilizing agent, and all other steps and reagents were the same. Briefly, 96-well plates were seeded with HEK293F cells at a density of 2.5×105 cells/well and placed in a shaking incubator (350 RPM, 37°C, humidified, 5% CO2 ) while sample titrations were prepared. The LNP drug product was diluted in DPBS to a concentration of 80 ng/μL and serially diluted by a dilution factor of 4 for 8 points. Next, the 96-well plate was removed from the incubator and 50 μL of the diluted LNPs from each step were added to duplicate wells of the 96-well plate to generate a titration curve ranging from 8,000 ng/well to 1.95 ng/well. The 96-well plate was returned to the shaking incubator overnight. After incubation, 250 μL of cells were transferred to a 96-well u-bottom polystyrene plate and pelleted using a swinging bucket centrifuge (500 rcf, 5 min, at room temperature). The supernatant was removed and the cells were resuspended in 100 μL of Aqua405 live/dead staining solution. The plate was incubated at room temperature in the dark for 30 min. After incubation, cells were washed with wash buffer and pelleted using centrifugation (500 rcf, 5 min at room temperature). The supernatant was removed and the cells were resuspended in 100 μL of fixation/permeabilization buffer and the plates were incubated in the dark at 2-8°C for 30 minutes. Once the incubation was complete, the cells were pelleted using a swinging bucket centrifuge (500 rcf, 5 min at room temperature). The supernatant was removed and the cells were resuspended with 250 μL of wash buffer, and this was repeated for a total of 2 washes. After the final wash step, the cells were pelleted, the supernatant was removed, and resuspended in 50 μL of primary antibody solution. The plates were sealed and incubated at 2-8°C in the dark for 45 minutes. Once complete, the cells were pelleted using a swinging bucket centrifuge (500 rcf, 5 min at room temperature). The supernatant was removed and the cells were resuspended in 250 μL of wash buffer, and this was repeated for a total of 2 washes. After the final wash step, the cells were pelleted, the supernatant was removed, and resuspended in 50 μL of secondary antibody solution. The plates were sealed and incubated at 2-8°C in the dark for 45 minutes. Once complete, the cells were pelleted using a swinging bucket centrifuge (500 rcf, 5 min at room temperature). The supernatant was removed and the cells were resuspended in 250 μL of wash buffer, and this was repeated a total of 2 washes. After the final wash step, the cells were pelleted, the supernatant removed, and resuspended in 200 μL of wash buffer, and data were acquired by flow cytometry.
藉由轉染HEK293F細胞，用劑量滴定曲線，以及進行抗體染色評定modRNA LNP藥物產品之活體外表現(IVE)，該等抗體為對三聚RSV F-蛋白具有特異性的RSV mAb1及對總RSV F-蛋白具有特異性的L4-6。已顯示此等抗體識別A株系及B株系RSV F-蛋白，且用於用滲透或非滲透條件之分析中以評定RSV F-蛋白之總細胞含量與細胞表面含量。所量測之陽性細胞% (2,000 ng/孔輸入)及藥物產品批次之劑量反應曲線的EC50顯示於表11中。表11.藥物產品IVE結果
實例5.免疫反應(活體內實驗)在第0天及第21天，以不同劑量，對雌性BALB/c小鼠免疫接種WO2017/109629中所描述的呈二價蛋白質次單元形式(RSV 847A + 847B)之RSV融合前F (847)或本文所描述的呈一價(RSV 847A)或二價(RSV 847A + 847B)形式之modRNA-LNP調配物或呈二價調配物形式之saRNA-LNP。藉由量測RSV中和抗體反應及RSV F特異性T細胞反應來評估免疫原性。在第21天及第35天(劑量2後(PD2) 2週)收集血清用於RSV中和分析，且在第35天(PD2 2週)收集脾臟用於T細胞分析(ELISpot及細胞內細胞介素染色ICS分析)。Example5.Immune response(in vivo experiment) Female BALB/c mice were immunized with RSV prefusion F (847) described in WO2017/109629 in bivalent protein subunit form (RSV 847A + 847B) or modRNA-LNP formulations described herein in monovalent (RSV 847A) or bivalent (RSV 847A + 847B) form or saRNA-LNP in bivalent formulation form at different doses on days 0 and 21. Immunogenicity was assessed by measuring RSV neutralizing antibody responses and RSV F-specific T cell responses. Serum was collected on days 21 and 35 (2 weeks after dose 2 (PD2)) for RSV neutralization analysis, and spleen was collected on day 35 (2 weeks after PD2) for T cell analysis (ELISpot and intracellular interleukin staining ICS analysis).
中和分析RSV微中和分析為使用A549細胞(人類肺泡基底上皮細胞)進行之3天分析，其用於量測血清中中和RSV活性從而預防宿主細胞單層感染之功能性抗體。在第0天，以2.5×104個細胞/孔將A549細胞(人類肺泡基礎上皮細胞；ATCC，目錄號CCL-185)接種於96孔組織培養處理的培養盤中，且培育至少20小時，以形成匯合單層。在第1天，將稀釋的病毒(RSV A，M37；RSV B，B18537；500 FFU/孔)添加至所製備的熱滅活測試血清之3倍連續稀釋液中，重複兩次，且培育1小時，以使抗體與病毒結合。接著，將中和反應轉移至所製備的A549細胞單層上且培育2小時。在過夜培育(至少16小時)之前，將額外介質補充至培養盤上。在第2天，培養盤用甲醇固定且用小鼠抗RSV F (Pfizer, N50-9)一級抗體染色，隨後用Alexa 488螢光標記之二級抗體染色以偵測病毒灶。50%中和力價計算為與僅含有病毒之孔相比50%病毒被中和之最後血清稀釋度倒數。力價報導為各樣品之兩個重複力價之幾何平均力價(GMT)。分析力價範圍為20至43,740。將力價＞43,740之任何樣品預稀釋且重複以擴大力價極限。低於偵測下限(LLOD)之任何樣品均以LLOD為20報導。Neutralization Assay The RSV microneutralization assay is a 3-day assay using A549 cells (human alveolar basal epithelial cells) that measures functional antibodies in serum that neutralize RSV activity and thereby prevent infection of host cell monolayers. On day 0, A549 cells (human alveolar basal epithelial cells; ATCC, catalog number CCL-185) were seeded at 2.5×104 cells/well in 96-well tissue culture-treated plates and incubated for at least 20 hours to form confluent monolayers. On day 1, diluted virus (RSV A, M37; RSV B, B18537; 500 FFU/well) was added to 3-fold serial dilutions of prepared heat-killed test serum, repeated twice, and incubated for 1 hour to allow antibody to bind to virus. Next, the neutralization reaction was transferred to the prepared A549 cell monolayer and incubated for 2 hours. Additional medium was added to the culture plates before overnight incubation (at least 16 hours). On day 2, the culture plates were fixed with methanol and stained with mouse anti-RSV F (Pfizer, N50-9) primary antibody, followed by staining with Alexa 488 fluorescent-labeled secondary antibody to detect viral foci. The 50% neutralization titer was calculated as the reciprocal of the last serum dilution at which 50% of the virus was neutralized compared to wells containing virus alone. The titer was reported as the geometric mean titer (GMT) of two replicates for each sample. The assay titer range was 20 to 43,740. Any sample with a titer > 43,740 was pre-diluted and repeated to expand the titer limit. Any sample below the lower limit of detection (LLOD) was reported as LLOD 20.
T細胞反應量測藉由在RSV F (A+B)肽池存在下離體刺激脾細胞以活化抗原特異性T細胞中各種細胞介素(諸如IFN-γ)之產生來評定疫苗誘導之針對RSV F的T細胞反應。可藉由ELISpot (表示為斑點形成細胞SFC/百萬細胞)來量測分泌在細胞外部的細胞介素，或可阻斷藉由ICS (表示為表現細胞介素之CD4+ T細胞及CD8+ T細胞之百分比)量測之細胞內部的細胞介素分泌。Tcellresponse measurement Vaccine-induced T cell responses against RSV F were assessed by stimulating splenocytes ex vivo in the presence of RSV F (A+B) peptide pools to activate the production of various cytokines (such as IFN-γ) in antigen-specific T cells. Cytokines secreted externally of cells can be measured by ELISpot (expressed as spot forming cell SFC/million cells), or internal cell cytokine secretion measured by ICS (expressed as the percentage of CD4+ T cells and CD8+ T cells expressing cytokines) can be blocked.
在ELISpot之情況下，活化T細胞所分泌的細胞介素IFN-γ被塗覆至微量盤上孔底之聚偏二氟乙烯(PVDF)膜上的抗IFN-γ抗體捕獲。所捕獲IFN-γ藉由另一非競爭性生物素標記之抗IFN-γ二級抗體顯色為斑點，隨後使用鏈球菌親生物素蛋白-鹼性磷酸酶(ALP)結合物及受質溶液硝基藍四唑鎓及5-溴-4-氯-3'-吲哚磷酸(BCIP/NBT-plus)進行酶促顏色反應，其產生深紫色沉澱或斑點。使用Mabtech小鼠IFN-γ ELISpot PLUS套組(ALP)來量測T細胞IFN-γ反應，且其表示為斑點形成細胞(SFC)/百萬細胞。In the case of ELISpot, the interleukin IFN-γ secreted by activated T cells is captured by anti-IFN-γ antibodies coated on the polyvinylidene fluoride (PVDF) membrane at the bottom of the wells on the microtiter plate. The captured IFN-γ is visualized as spots by another non-competitive biotin-labeled anti-IFN-γ secondary antibody, followed by an enzymatic color reaction using streptococcal avidin-alkaline phosphatase (ALP) conjugate and the substrate solution nitro blue tetrazolium and 5-bromo-4-chloro-3'-indolyl phosphate (BCIP/NBT-plus), which produces a dark purple precipitate or spot. T cell IFN-γ responses were measured using the Mabtech Mouse IFN-γ ELISpot PLUS Panel (ALP) and expressed as spot forming cells (SFC)/million cells.
ICS染色可偵測在抗原肽刺激之後在CD4+及CD8+ T細胞中產生的多種細胞介素，包括IFN-γ。在具有培養基-DMSO (未刺激)或表示RSV F A+B之特定肽池(15aa，11aa重疊，2 µg/mL/肽)之cRPMI中，在37℃，在抗CD107a APC抗體及蛋白質運輸抑制劑GolgiPlug及GolgiStop存在下，離體培養脾細胞之單細胞懸浮液(2×106個細胞/孔)5小時。在刺激之後，將脾細胞與針對表面蛋白質CD19、CD3、CD4、CD8、CD44之螢光結合抗體一起培育(25 ± 5分鐘，在18-25℃)，隨後固定及滲透且針對IFN-γ、TNF-α、IL-2及CD40L/CD154進行染色(25 ± 5分鐘，在18-25℃)。在染色之後，洗滌細胞且再懸浮於FC緩衝液中。在LSR Fortessa上收集細胞且藉由FlowJo (10.7.1)分析資料。結果為減去背景(培養基-DMSO)的，且顯示為表現細胞介素之CD4+ T細胞及CD8+ T細胞之百分比。ICS staining can detect a variety of interleukins, including IFN-γ, produced in CD4+ and CD8+ T cells after antigenic peptide stimulation. Single cell suspensions of splenocytes (2×10 6 cells/well) were cultured in vitro for 5 hours at 37°C in cRPMI with medium-DMSO (unstimulated) or specific peptide pools expressing RSV FA+B (15aa,11aa stack, 2 µg/mL/peptide) in the presence of anti-CD107a APC antibody and protein transport inhibitors GolgiPlug and GolgiStop. After stimulation, spleen cells were incubated with fluorescent conjugated antibodies against surface proteins CD19, CD3, CD4, CD8, CD44 (25 ± 5 min at 18-25°C), followed by fixation and permeabilization and staining for IFN-γ, TNF-α, IL-2 and CD40L/CD154 (25 ± 5 min at 18-25°C). After staining, cells were washed and resuspended in FC buffer. Cells were collected on LSR Fortessa and data were analyzed by FlowJo (10.7.1). Results are background (medium-DMSO) subtracted and shown as the percentage of CD4+ T cells and CD8+ T cells expressing interleukins.
結果result::
活體外表現(IVE)RNA-LNP之IVE分析之結果在表11中詳述。在RSV F設計之modRNA-LNP調配物中，基於三聚構形特異性RSVmAb1，847A-摺疊子、DS-Cav1及WT顯示相對更低的表現，但總F表現(L4-6)顯示類似含量。此外，對於三聚F及總F表現，與modRNA-LNP相比，RSV 847A及847B之saRNA-LNP調配物顯示類似的更佳表現。此等結果表明，與來自RSV子群組A或B之全長847、851及852抗原相比，自mRNA編碼之部分穩定的全長DS-Cav1及天然全長WT F抗原呈現降低比例的呈融合前構形之抗原。在847之可溶性形式(亦即847-摺疊子)之情況下觀測到之有限融合前mAb結合係歸因於在短暫轉染之後很大程度上存在於細胞培養物上清液中之蛋白質，且IVE方法偵測定位於胞內或僅定位於細胞表面的蛋白質。In vitro expression(IVE) The results of IVE analysis of RNA-LNPs are detailed inTable11. In the modRNA-LNP formulations designed for RSV F, RSV mAb1, 847A-fold, DS-Cav1 and WT showed relatively lower expression based on the trimeric conformation specificity, but the total F expression (L4-6) showed similar levels. In addition, for trimeric F and total F expression, saRNA-LNP formulations of RSV 847A and 847B showed similar better expression compared to modRNA-LNPs. These results indicate that the partially stabilized full-length DS-Cav1 and native full-length WT F antigens encoded from mRNA present a reduced proportion of antigen in the prefusion conformation compared to the full-length 847, 851 and 852 antigens from RSV subgroups A or B. The limited prefusion mAb binding observed with the soluble form of 847 (i.e., 847-fold) is due to the protein being largely present in the cell culture supernatant after a short transfection, whereas the IVE method detects proteins that are either localized intracellularly or exclusively on the cell surface.
免疫原性在第35天(2週PD2)之中和分析之結果揭露，對小鼠免疫接種呈一價RSV 847A或二價形式RSV 847A及847B之RSV 847的modRNA-LNP調配物誘導針對RSV A及RSV B兩者之強力中和抗體反應，該中和抗體反應與二價蛋白質次單元形式相比更高(圖1A及圖1B、表10)。此外，由modRNA-LNP之二價調配物RSV847A+RSV847B誘導之RSV B中和反應比一價調配物RSV847A的反應更高(圖1B、表12)，支持對RSV preFmodRNA疫苗之二價形式用於人類中之最佳免疫反應的需求。Immunogenicity The results of the neutralization analysis at day 35 (2 weeks PD2) revealed that the modRNA-LNP formulations of RSV 847 immunized mice with either monovalent RSV 847A or bivalent forms of RSV 847A and 847B induced potent neutralizing antibody responses against both RSV A and RSV B, which were higher than the bivalent protein subunit forms (Figure1Aand Figure1B ,Table10 ). In addition, the RSV B neutralization response induced by the bivalent formulation of modRNA-LNP RSV847A+RSV847B was higher than that of the monovalent formulation RSV847A (Figure1B ,Table12 ), supporting the need for a bivalent form of RSV preF modRNA vaccine for optimal immune responses in humans.
吾人接下來評估在小鼠中由此等RSV preF之modRNA-LNP調配物誘導之T細胞反應。IFN-γ ELISpot結果顯示，RSV 847 modRNA疫苗誘導強力T細胞反應，趨勢與RSV中和反應類似(圖1C、表12)。此外，ICS分析結果揭露，RSV 847 modRNA疫苗以劑量依賴性方式誘導高頻率之F特異性的表現IFN-γ之CD4+ T細胞以及CD8+ T細胞(圖1D及圖1E、表12)。值得注意的是，由二價調配物(RSV 847A + 847B)誘導之RSV A+B F特異性T細胞反應比一價調配物(RSV 847A)更高，進一步支持RSV 847之二價形式誘導更高量值的T細胞反應，與中和反應類似。We next evaluated T cell responses induced by these RSV preF modRNA-LNP formulations in mice. IFN-γ ELISpot results showed that RSV 847 modRNA vaccine induced a potent T cell response with a trend similar to RSV neutralization response (Figure1C ,Table12 ). In addition, ICS analysis results revealed that RSV 847 modRNA vaccine induced a high frequency of F-specific IFN-γ-expressing CD4+ T cells and CD8+ T cells in a dose-dependent manner (Figure1Dand1E ,Table12 ). Notably, RSV A+BF-specific T cell responses induced by the bivalent formulation (RSV 847A + 847B) were higher than those induced by the monovalent formulation (RSV 847A), further supporting that the bivalent form of RSV 847 induces higher magnitude T cell responses, similar to neutralization responses.
總而言之，研究結果證實與未處理小鼠中之蛋白質次單元疫苗相比，RSV融合前F構築體之modRNA-LNP調配物的免疫原性更高。表12.小鼠中由蛋白質次單元及RSV融合前F之modRNA-LNP調配物誘導之免疫反應
接下來，評估本文所描述之編碼不同RSV融合前F (preF)抗原設計之modRNA-LNP的免疫原性。中和分析結果揭露，與DS-CAV1及WT以及設計為具有摺疊子之胞外域的847A-摺疊子相比，呈全長形式之突變型融合前設計847、851及852誘導更高力價(圖2)。此等結果進一步確認，當自mRNA編碼時，使突變以及RSV F抗原之全長(胞外域+TM+CT)形式穩定之融合前對於RSV preF抗原之最佳免疫原性至關重要。Next, the immunogenicity of modRNA-LNPs encoding different RSV prefusion F (preF) antigen designs described herein was evaluated. Neutralization analysis results revealed that mutant prefusion designs 847, 851, and 852 induced higher potency in full-length form compared to DS-CAV1 and WT, as well as 847A-fold designed as an ectodomain with a fold (Figure2 ). These results further confirm that when encoded from mRNA, stabilizing the prefusion of mutations and the full-length (ectodomain+TM+CT) form of the RSV F antigen is critical for optimal immunogenicity of the RSV preF antigen.
自擴增RNA (saRNA)為利用RNA複製酶來擴增編碼抗原之mRNA且預期比modRNA平台更強效的mRNA平台。使用RSVpreF之二價(A及B)調配物，將saRNA-LNP之免疫原性與modRNA-LNP及蛋白質次單元比較。在劑量1後3週(3W PD1)，在由針對RSV A及B病毒之modRNA及saRNA疫苗誘導之中和反應中觀測到劑量依賴性影響。此外，由在0.02 µg劑量下之saRNA誘導之中和反應實質上高於接受0.2 µg劑量之小鼠中之modRNA疫苗(圖3A及圖3B)，表明由saRNA誘導之劑量節省反應。在劑量2後2週(2W PD2)，由saRNA誘導出強力劑量依賴性中和反應，且對於RSV A及B，該反應與在0.2 µg劑量水平下之modRNA相當(圖3C及圖3D)。在2W PD2藉由ICS分析所分析的脾臟中之T細胞反應顯示，由modRNA及saRNA疫苗誘導出穩固且劑量依賴性的RSV A+B F特異性的表現IFN-γ之CD4+以及CD8+ T細胞反應(圖3E及圖3F)。由在0.02 µg劑量水平下之saRNA群組誘導的CD8+ T細胞反應實質上高於在0.2 µg劑量下之modRNA疫苗群組(圖3E及圖3F)，表明由saRNA誘導之劑量節省反應。Self-amplifying RNA (saRNA) is an mRNA platform that utilizes RNA replicase to amplify antigen-encoding mRNA and is expected to be more potent than modRNA platforms. Using bivalent (A and B) formulations of RSVpreF, the immunogenicity of saRNA-LNPs was compared to modRNA-LNPs and protein subunits. At 3 weeks after dose 1 (3W PD1), dose-dependent effects were observed in the neutralization responses induced by modRNA and saRNA vaccines against RSV A and B viruses. In addition, the neutralization responses induced by saRNA at a dose of 0.02 µg were substantially higher than those of modRNA vaccines in mice receiving a dose of 0.2 µg (Figures3Aand3B ), indicating a dose-saving response induced by saRNA. At 2 weeks after dose 2 (2W PD2), saRNA induced potent dose-dependent neutralization responses that were comparable to modRNA at the 0.2 µg dose level for RSV A and B (Figures3Cand3D ). T cell responses in spleen analyzed by ICS analysis at 2W PD2 showed that modRNA and saRNA vaccines induced robust and dose-dependent RSV A+BF-specific IFN-γ-expressing CD4+ and CD8+ T cell responses (Figures3Eand3F ). The CD8+ T cell responses induced by the saRNA group at the 0.02 µg dose level were substantially higher than those of the modRNA vaccine group at the 0.2 µg dose (Figure 3E and Figure 3F), indicating a dose-sparing response induced by saRNA.
總而言之，此等全面活體外表現及免疫原性結果證實，編碼RSV preF設計之modRNA-LNP及saRNA-LNP支持在小鼠中RSV preF抗原之穩固表現且產生高效RSV F特異性免疫反應。In summary, these comprehensive in vitro performance and immunogenicity results demonstrate that modRNA-LNPs and saRNA-LNPs encoding RSV preF design support robust expression of RSV preF antigen in mice and generate efficient RSV F-specific immune responses.
實例6. RSV抗原本發明之RSV抗原/多肽、RSV DNA及RSV RNA之序列提供於表1-3中。序列可包含任何終止密碼子，包括但不限於下表中所提供之終止密碼子。表1.RSV多肽
以下段落描述本發明之額外態樣： 1. 一種RNA分子，其包含至少一個編碼呼吸道融合病毒(RSV)多肽之開讀框。 2. 如段落1之RNA分子，其中該RSV多肽為RSV醣蛋白。 3. 如段落2之RNA分子，其中該RSV醣蛋白為RSV F蛋白。 4. 如段落1至3中任一項之RNA分子，其中該RSV多肽為全長多肽、截短多肽、其片段或變體。 5. 如段落1至4中任一項之RNA分子，其中該RSV多肽包含至少一個突變。 6. 如段落1至5中任一項之RNA分子，其中該RSV多肽包含表1之胺基酸，包括但不限於SEQ ID NO: 1至6或71至74中之任一者。 7. 如段落1至6中任一項之RNA分子，其中該RSV多肽與SEQ ID NO: 1至6或71至74中之任一者之胺基酸序列具有至少90%、95%、96%、97%、98%或99%一致性。 8. 如段落1至7中任一項之RNA分子，其中該RSV多肽包含SEQ ID NO: 1至6或71至74中之任一者之胺基酸序列。 9. 如段落1至8中任一項之RNA分子，其中該開讀框係轉錄自表2之核酸序列，包括但不限於SEQ ID NO: 7至10或59至62中之任一者。 10. 如段落1至9中任一項之RNA分子，其中該開讀框係轉錄自與SEQ ID NO: 7至10或59至62中之任一者之序列具有至少90%、95、96%、97%、98%或99%一致性的核酸序列。 11. 如段落1至10中任一項之RNA分子，其中該開讀框包含表3之核酸序列，包括但不限於SEQ ID NO: 11至16或63至70中之任一者。 12. 如段落1至11中任一項之RNA分子，其中該開讀框包含與SEQ ID NO: 11至16或63至70中之任一者之序列具有至少90%、95、96%、97%、98%或99%一致性的核酸序列。 13. 如段落1至12中任一項之RNA分子，其中該開讀框包含SEQ ID NO: 11至16或63至70之核酸序列。 14. 如段落1至13中任一項之RNA分子，其中各尿苷經N1-甲基假尿苷(Ψ)置換。 15. 如段落1至14中任一項之RNA分子，其進一步包含5'非轉譯區(5' UTR)。 16. 如段落15之RNA分子，其中該5' UTR包含選自SEQ ID NO: 17至19中之任一者的序列。 17. 如段落1至16中任一項之RNA分子，其進一步包含3'非轉譯區(3' UTR)。 18. 如段落17之組合物，其中該3' UTR包含選自SEQ ID NO: 20至25中之任一者的序列。 19. 如段落1至18中任一項之RNA分子，其中該RNA分子包含5'帽部分。 20. 如段落1至19中任一項之RNA分子，其進一步包含3'多-A尾。 21. 如段落20之RNA分子，其中該多-A尾包含具有SEQ ID NO: 26之序列，其視情況包含+/- 1或+/- 2腺苷(A)。 22. 如段落1至21中任一項之RNA分子，其中該RNA分子包含5' UTR及3' UTR。 23. 如段落1至22中任一項之RNA分子，其中該RNA分子包含5'帽、5' UTR及3' UTR。 24. 如段落1至23中任一項之RNA分子，其中該RNA分子包含5'帽、5' UTR、3' UTR及多-A尾。 25. 如段落1至24中任一項之RNA分子，其包含：5' UTR，其包含SEQ ID NO: 18或19的序列；開讀框，其包含SEQ ID NO: 11至16或63至70中之任一者的序列；及3' UTR，其包含SEQ ID NO: 20至25的序列。 26. 如段落1至25中任一項之RNA分子，其包含：5' UTR，其包含SEQ ID NO: 18或19中之任一者的序列；開讀框，其包含SEQ ID NO: 11至16或63至70中之任一者的序列；3' UTR，其包含SEQ ID NO: 20至25中之任一者的序列；及多-A尾，其包含SEQ ID NO: 26中之任一者的序列。 27. 如段落1至26中任一項之RNA分子，其中該開讀框係由密碼子最佳化DNA產生。 28. 如段落1至27中任一項之RNA分子，其中該開讀框包含至少55%、至少60%、至少65%、至少70%或至少75%、為或為約50%至75%或55%至70%、或者為或為約58%、66%或62%的G/C含量。 29. 如段落1至28中任一項之RNA分子，其中所編碼RSV多肽位於細胞膜中、位於高基氏體中及/或錨定在膜中且分泌。 30. 如段落1至29中任一項之RNA分子，其中該RNA分子包含經穩定化之RNA。 31. 如段落1至30中任一項之RNA分子，其中該RNA包含至少一個經修飾之核苷酸。 32. 如段落31之RNA分子，其中該經修飾之核苷酸為假尿苷、N1-甲基假尿苷、N1-乙基假尿苷、2-硫尿苷、4'-硫尿苷、5-甲基胞嘧啶、5-甲基尿苷、2-硫-1-甲基-1-脫氮-假尿苷、2-硫-1-甲基-假尿苷、2-硫-5-氮雜-尿苷、2-硫-二氫假尿苷、2-硫-二氫尿苷、2-硫-假尿苷、4-甲氧基-2-硫-假尿苷、4-甲氧基-假尿苷、4-硫-1-甲基-假尿苷、4-硫-假尿苷、5-氮雜-尿苷、二氫假尿苷、5-甲氧基尿苷或2'-O-甲基尿苷。 33. 如段落32之RNA分子，其中該經修飾之核苷酸為N1-甲基假尿苷(Ψ)。 34. 如段落1至33中任一項之RNA分子，其中該RNA為mRNA。 35. 如段落34之RNA分子，其中該RNA為modRNA或saRNA。 36. 一種組合物，其包含如段落1至35中任一項之RNA分子，其中該RNA分子調配於脂質奈米顆粒中(RNA-LNP)。 37. 如段落36之組合物，其中脂質奈米顆粒包含陽離子脂質、PEG-脂質、中性脂質及類固醇或類固醇類似物中之至少一者。 38. 如段落36或37之組合物，其中該脂質奈米顆粒包含陽離子脂質。 39. 如段落38之組合物，其中該陽離子脂質為(4-羥丁基)氮二基)雙(己烷-6,1-二基)雙(2-己基癸酸酯) (ALC-0315)。 40. 如段落36至39中任一項之組合物，其中該脂質奈米顆粒包含聚乙二醇化脂質。 41. 如段落40之組合物，其中該聚乙二醇化脂質為PEG修飾之磷脂醯乙醇胺、PEG修飾之磷脂酸、PEG修飾之神經醯胺(例如PEG-CerC14或PEG-CerC20)、PEG修飾之二烷基胺、PEG修飾之二醯基甘油、PEG修飾之二烷基甘油、2-[(聚乙二醇)-2000]-N,N-二(十四烷基)乙醯胺、二醇-脂質(包括PEG-c-DOMG、PEG-c-DMA、PEG-s-DMG、N-[(甲氧基聚乙二醇)2000)胺甲醯基]-1,2-二肉豆蔻醯氧基丙基-3-胺(PEG-c-DMA)及PEG-2000-DMG)、聚乙二醇化二醯基甘油(PEG-DAG) (諸如1-(單甲氧基-聚乙二醇)-2,3-二肉豆蔻醯基甘油(PEG-DMG))、聚乙二醇化磷脂醯乙醇胺(PEG-PE)、PEG丁二酸二醯基甘油(PEG-S-DAG) (諸如4-O-(2',3'-二(十四碳醯氧基)丙基-1-O-((ω-甲氧基(聚乙氧基)乙基)丁二酸酯(PEG-S-DMG))、聚乙二醇化神經醯胺(PEG-cer)、或PEG二烷氧基丙基胺基甲酸酯(諸如共聚-甲氧基(聚乙氧基)乙基-N-(2,3-(十四烷氧基)丙基)胺基甲酸酯或2,3-二(十四烷氧基)丙基-N-(ω-甲氧基(聚乙氧基)乙基)胺基甲酸酯)。 42. 如段落40或41之組合物，其中該PEG-脂質為2-[(聚乙二醇)-2000]-N,N-二(十四烷基)乙醯胺(ALC-0159)。 43. 如段落36至42中任一項之組合物，其中該脂質奈米顆粒包含中性脂質。 44. 如段落43之組合物，其中該中性脂質為二硬脂醯基磷脂醯膽鹼(DSPC)、二油醯基磷脂醯膽鹼(DOPC)、二棕櫚醯基磷脂醯膽鹼(DPPC)、二油醯基磷脂醯甘油(DOPG)、二棕櫚醯基磷脂醯甘油(DPPG)、二油醯基-磷脂醯乙醇胺(DOPE)、棕櫚醯油醯基磷脂醯膽鹼(POPC)、棕櫚醯油醯基-磷脂醯乙醇胺(POPE)及二油醯基-磷脂醯乙醇胺4-(N-順丁烯二醯亞胺基甲基)-環己烷-1-甲酸酯(DOPE-mal)、二棕櫚醯基磷脂醯乙醇胺(DPPE)、二肉豆蔻醯基磷酸乙醇胺(DMPE)、二硬脂醯基-磷脂醯乙醇胺(DSPE)、16-O-單甲基PE、16-O-二甲基PE、18-1-反式PE、1-硬脂醯基-2-油醯基磷脂醯乙醇胺(SOPE)或1,2-二反油醯基-sn-甘油基-3-磷酸乙醇胺(transDOPE)。 45. 如段落43或44之組合物，其中該中性脂質為1,2-二硬脂醯基-sn-甘油基-3-磷酸膽鹼(DSPC)。 46. 如段落36至45中任一項之組合物，其中該脂質奈米顆粒包含類固醇或類固醇類似物。 47. 如段落46之組合物，其中該類固醇或類固醇類似物為膽固醇。 48. 如段落36至47中任一項之組合物，其中脂質奈米顆粒之平均直徑為約1至約500 nm。 49. 如段落36至48中任一項之組合物，其中該RNA來自RSV子類型A。 50. 如段落36至48中任一項之組合物，其中該RNA來自RSV子類型B。 51. 如段落36至48中任一項之組合物，其中該RNA來自：(i) RSV子類型A及(ii) RSV子類型B。 52. 如段落36至51中任一項之組合物，其包含濃度為或為約0.01至0.09 mg/mL之調配於脂質奈米顆粒(LNP)中的RNA分子，該脂質奈米顆粒包含：陽離子脂質，其濃度為或為約0.8至0.95 mg/mL；聚乙二醇化脂質，其濃度為或為約0.05至0.15 mg/mL；中性脂質，其濃度為或為約0.1至0.25 mg/mL；及類固醇或類固醇類似物，其濃度為或為約0.3至0.45 mg/mL。 53. 如段落36至52中任一項之組合物，其包含濃度為或為約0.01至0.09 mg/mL之調配於脂質奈米顆粒(LNP)中的RNA分子，該脂質奈米顆粒包含：(4-羥丁基)氮二基)雙(己烷-6,1-二基)雙(2-己基癸酸酯) (ALC-0315)，其濃度為或為約0.8至0.95 mg/mL；2-[(聚乙二醇)-2000]-N,N-二(十四烷基)乙醯胺(ALC-0159)，其濃度為或為約0.05至0.15 mg/mL；1,2-二硬脂醯基-sn-甘油基-3-磷酸膽鹼(DSPC)，其濃度為或為約0.1至0.25 mg/mL；膽固醇，其濃度為或為約0.3至0.45 mg/mL。 54. 如段落36至53中任一項之組合物，其包含濃度為或為約0.06 mg/mL之調配於脂質奈米顆粒(LNP)中的RNA分子。 55. 如段落36至54中任一項之組合物，其進一步包含緩衝劑、穩定劑、鹽、界面活性劑、防腐劑、賦形劑及/或佐劑中之至少一者。 56. 如段落36至55中任一項之組合物，其進一步包含至少緩衝劑及穩定劑及視情況存在之鹽稀釋劑。 57. 如段落36或56之組合物，其中該緩衝劑為Tris緩衝液。 58. 如段落57之組合物，其中該Tris緩衝液包含緩血酸胺及Tris鹽酸(HCl)。 59. 如段落58之組合物，其中該緩血酸胺之濃度為或為約0.1至0.3 mg/mL、或者為或為約0.01至0.15 mg/mL。 60. 如段落57或58之組合物，其中該Tris HCl之濃度為或為約1.25至1.40 mg/mL、或者為或為約0.5至0.65 mg/mL。 61. 如段落56至60中任一項之組合物，其中該穩定劑為蔗糖。 62. 如段落61之組合物，其中該蔗糖之濃度為或為約95至110 mg/mL、或者為或為約35至50 mg/mL。 63. 如段落55至62中任一項之組合物，其中用於復原之該鹽稀釋劑為氯化鈉。 64. 如段落63之組合物，其中該氯化鈉之濃度為或為約5至15 mg/mL。 65. 如段落36至64中任一項之組合物，其中該組合物為液體或凍乾的。 66. 如段落65之組合物，其包含濃度為或為約0.01至0.09 mg/mL之調配於脂質奈米顆粒(LNP)中的RNA分子，該脂質奈米顆粒包含：陽離子脂質，其濃度為或為約0.8至0.95 mg/mL；聚乙二醇化脂質，其濃度為或為約0.05至0.15 mg/mL；中性脂質，其濃度為或為約0.1至0.25 mg/mL；及類固醇或類固醇類似物，其濃度為或為約0.3至0.45 mg/mL；且進一步包含Tris緩衝液，該Tris緩衝液包含：緩血酸胺，其濃度為或為約0.1至0.3 mg/mL；及Tris鹽酸(HCl)，其濃度為或為約1.25至1.40 mg/mL；及蔗糖，其濃度為或為約95至110 mg/mL，其中該組合物為液體組合物。 67. 如段落65之組合物，其包含濃度為或為約0.01至0.09 mg/mL之調配於脂質奈米顆粒(LNP)中的RNA分子，該脂質奈米顆粒包含：陽離子脂質，其濃度為或為約0.8至0.95 mg/mL；聚乙二醇化脂質，其濃度為或為約0.05至0.15 mg/mL；中性脂質，其濃度為或為約0.1至0.25 mg/mL；及類固醇或類固醇類似物，其濃度為或為約0.3至0.45 mg/mL；且進一步包含Tris緩衝液，該Tris緩衝液包含：緩血酸胺，其濃度為或為約0.01至0.15 mg/mL；及Tris鹽酸(HCl)，其濃度為或為約0.5至0.65 mg/mL；蔗糖，其濃度為或為約35至50 mg/mL。 68. 如段落65之組合物，其中該組合物用濃度為或為約5至15 mg/mL之氯化鈉復原。 69. 如段落65之組合物，其中該組合物用或用約0.6至0.75 mg/mL氯化鈉復原。 70. 如段落65之組合物，其進一步包含或包含約5至15 mM Tris緩衝液、pH呈或呈約7.0至8.0的200至400 mM蔗糖及視情況存在之用於復原之0.9%氯化鈉稀釋劑。 71. 一種誘導個體中之免疫反應之方法，其包含向該個體投與有效量之如段落1至70中任一項之RNA分子、RNA-LNP及/或組合物。 72. 一種預防、治療或改善個體之感染、疾病或病狀之方法，其包含向個體投與有效量之如段落1至70中任一項之RNA分子、RNA-LNP及/或組合物。 73. 如段落72之方法，其中該感染、疾病或病狀與RSV相關。 74. 如段落72或73之方法，其中該感染、疾病或病狀為RSV感染誘導之急性呼吸道病痛，包括肺炎及支氣管炎。 75. 一種如段落1至70中任一項之RNA分子、RNA-LNP及/或組合物之用途，其用於製造用於誘導個體中針對RSV之免疫反應的藥劑。 76. 一種如段落1至70中任一項之RNA分子、RNA-LNP及/或組合物之用途，其用於製造用於預防、治療或改善個體之感染、疾病或病狀的藥劑。 77. 如段落76之用途，其中該感染、疾病或病狀與RSV相關。 78. 如段落76或77之用途，其中該感染、疾病或病狀為RSV感染相關呼吸道病痛，包括肺炎及支氣管炎。 79. 如段落71至78中任一項之方法或用途，其中該個體之年齡為小於約1歲、約1歲或更大、約5歲或更大、約10歲或更大、約20歲或更大、約30歲或更大、約40歲或更大、約50歲或更大、約60歲或更大、約70歲或更大，或更大。 80. 如段落71至79中任一項之方法或用途，其中該個體之年齡為約50歲或更大。 81. 如段落71至79中任一項之方法或用途，其中該個體為孕婦。 82. 如段落71至81中任一項之方法或用途，其中該RNA分子或組合物係作為疫苗投與。 83. 如段落71至82中任一項之方法或用途，其中該RNA分子或組合物係藉由皮內或肌肉內注射投與。 84. 如段落83之RNA分子，其中該modRNA包含具有SEQ ID NO: 13之核苷酸。 85. 如段落83之RNA分子，其中該modRNA包含具有SEQ ID NO: 14之核苷酸。 86. 如段落83之RNA分子，其中該saRNA包含具有SEQ ID NO: 15之核苷酸。 87. 如段落83之RNA分子，其中該modRNA包含具有SEQ ID NO: 16之核苷酸。 88. 如段落71至87中任一項之方法或用途，其中向該個體投與單次劑量、兩次劑量、三次劑量或更多次劑量的該RNA分子及/或組合物。 89. 如段落71至88中任一項之方法或用途，其中向該個體投與單次劑量的該RNA分子及/或組合物。 90. 如段落71至89中任一項之方法或用途，其中向該個體投與兩次劑量的該RNA分子及/或組合物。 91. 如段落71至90中任一項之方法或用途，其中在第0天及在或在約2個月後向該個體投與兩次劑量的該RNA分子及/或組合物。 92. 如段落71至91中任一項之方法或用途，其中在第0天及在或在約6個月後向該個體投與兩次劑量的該RNA分子及/或組合物。 93. 如段落71至92中任一項之方法或用途，其中向該個體投與至少一次加強劑量的該RNA分子及/或組合物。 94. 如段落71至93中任一項之方法或用途，其中每次投與向該個體投與至少或至少約15 μg、30 μg、60 μg、90 μg、100 μg或更高劑量的RNA分子及/或組合物。 95. 如段落71至94中任一項之方法或用途，其中向該個體投與體積為或為約0.25至1 mL，包括但不限於為或為約0.25、0.5、1 mL的注射液。The following paragraphs describe additional aspects of the invention: 1. An RNA molecule comprising at least one open reading frame encoding a respiratory syncytial virus (RSV) polypeptide. 2. The RNA molecule of paragraph 1, wherein the RSV polypeptide is a RSV glycoprotein. 3. The RNA molecule of paragraph 2, wherein the RSV glycoprotein is a RSV F protein. 4. The RNA molecule of any of paragraphs 1 to 3, wherein the RSV polypeptide is a full-length polypeptide, a truncated polypeptide, a fragment or a variant thereof. 5. The RNA molecule of any of paragraphs 1 to 4, wherein the RSV polypeptide comprises at least one mutation. 6. The RNA molecule of any of paragraphs 1 to 5, wherein the RSV polypeptide comprises an amino acid of Table 1, including but not limited to any one of SEQ ID NOs: 1 to 6 or 71 to 74. 7. The RNA molecule of any of paragraphs 1 to 6, wherein the RSV polypeptide has at least 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of any of SEQ ID NOs: 1 to 6 or 71 to 74. 8. The RNA molecule of any of paragraphs 1 to 7, wherein the RSV polypeptide comprises the amino acid sequence of any of SEQ ID NOs: 1 to 6 or 71 to 74. 9. The RNA molecule of any of paragraphs 1 to 8, wherein the open reading frame is transcribed from a nucleic acid sequence of Table 2, including but not limited to any of SEQ ID NOs: 7 to 10 or 59 to 62. 10. The RNA molecule of any of paragraphs 1 to 9, wherein the open reading frame is transcribed from a nucleic acid sequence having at least 90%, 95, 96%, 97%, 98% or 99% identity to the sequence of any of SEQ ID NOs: 7 to 10 or 59 to 62. 11. The RNA molecule of any one of paragraphs 1 to 10, wherein the open reading frame comprises a nucleic acid sequence of Table 3, including but not limited to any one of SEQ ID NOs: 11 to 16 or 63 to 70. 12. The RNA molecule of any one of paragraphs 1 to 11, wherein the open reading frame comprises a nucleic acid sequence having at least 90%, 95, 96%, 97%, 98% or 99% identity to the sequence of any one of SEQ ID NOs: 11 to 16 or 63 to 70. 13. The RNA molecule of any one of paragraphs 1 to 12, wherein the open reading frame comprises a nucleic acid sequence of SEQ ID NOs: 11 to 16 or 63 to 70. 14. The RNA molecule of any one of paragraphs 1 to 13, wherein each uridine is replaced by N1-methylpseudouridine (Ψ). 15. The RNA molecule of any one of paragraphs 1 to 14, further comprising a 5' non-translated region (5'UTR). 16. The RNA molecule of paragraph 15, wherein the 5'UTR comprises a sequence selected from any one of SEQ ID NOs: 17 to 19. 17. The RNA molecule of paragraphs 1 to 16, further comprising a 3' non-translated region (3'UTR). 18. The composition of paragraph 17, wherein the 3'UTR comprises a sequence selected from any one of SEQ ID NOs: 20 to 25. 19. The RNA molecule of paragraphs 1 to 18, wherein the RNA molecule comprises a 5' cap portion. 20. The RNA molecule of paragraphs 1 to 19, further comprising a 3' poly-A tail. 21. The RNA molecule of paragraph 20, wherein the poly-A tail comprises a sequence having SEQ ID NO: 26, optionally comprising +/- 1 or +/- 2 adenosines (A). 22. The RNA molecule of any one of paragraphs 1 to 21, wherein the RNA molecule comprises a 5'UTR and a 3'UTR. 23. The RNA molecule of any one of paragraphs 1 to 22, wherein the RNA molecule comprises a 5' cap, a 5'UTR and a 3'UTR. 24. The RNA molecule of any one of paragraphs 1 to 23, wherein the RNA molecule comprises a 5' cap, a 5'UTR, a 3'UTR and a poly-A tail. 25. The RNA molecule of any one of paragraphs 1 to 24, comprising: a 5'UTR comprising the sequence of SEQ ID NO: 18 or 19; an open reading frame comprising the sequence of any one of SEQ ID NO: 11 to 16 or 63 to 70; and a 3'UTR comprising the sequence of SEQ ID NO: 20 to 25. 26. The RNA molecule of any of paragraphs 1 to 25, comprising: a 5'UTR comprising the sequence of any of SEQ ID NOs: 18 or 19; an open reading frame comprising the sequence of any of SEQ ID NOs: 11 to 16 or 63 to 70; a 3'UTR comprising the sequence of any of SEQ ID NOs: 20 to 25; and a poly-A tail comprising the sequence of any of SEQ ID NOs: 26. 27. The RNA molecule of any of paragraphs 1 to 26, wherein the open reading frame is generated from codon-optimized DNA. 28. The RNA molecule of any of paragraphs 1 to 27, wherein the open reading frame comprises a G/C content of at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, is or is about 50% to 75%, or is or is about 55% to 70%, or is or is about 58%, 66%, or 62%. 29. The RNA molecule of any of paragraphs 1 to 28, wherein the encoded RSV polypeptide is located in the cell membrane, in the Golgi apparatus and/or is anchored in the membrane and secreted. 30. The RNA molecule of any of paragraphs 1 to 29, wherein the RNA molecule comprises stabilized RNA. 31. The RNA molecule of any of paragraphs 1 to 30, wherein the RNA comprises at least one modified nucleotide. 32. The RNA molecule of paragraph 31, wherein the modified nucleotide is pseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 5-methyluridine, 2-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methyl-pseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methyl-pseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine or 2'-O-methyluridine. 33. The RNA molecule of paragraph 32, wherein the modified nucleotide is N1-methyl pseudouridine (Ψ). 34. The RNA molecule of any of paragraphs 1 to 33, wherein the RNA is mRNA. 35. The RNA molecule of paragraph 34, wherein the RNA is modRNA or saRNA. 36. A composition comprising the RNA molecule of any of paragraphs 1 to 35, wherein the RNA molecule is formulated in a lipid nanoparticle (RNA-LNP). 37. The composition of paragraph 36, wherein the lipid nanoparticle comprises at least one of a cationic lipid, a PEG-lipid, a neutral lipid, and a steroid or a steroid analog. 38. The composition of paragraphs 36 or 37, wherein the lipid nanoparticle comprises a cationic lipid. 39. The composition of paragraph 38, wherein the cationic lipid is (4-hydroxybutyl) azodiyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315). 40. The composition of any one of paragraphs 36 to 39, wherein the lipid nanoparticles comprise pegylated lipids. 41. The composition of paragraph 40, wherein the PEGylated lipid is PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, 2-[(polyethylene glycol)-2000]-N,N-di(tetradecyl)acetamide, glycol-lipids (including PEG-c-DOMG, PEG-c-DMA, PEG-s-DMG, N-[(methoxypolyethylene glycol) 2000) aminomethyl]-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA) and PEG-2000-DMG), PEGylated diacylglycerol (PEG-DAG) (such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristyl glycerol (PEG-DMG)), polyethylene glycol phospholipid ethanolamine (PEG-PE), PEG succinate diacylglycerol (PEG-S-DAG) (such as 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-((ω-methoxy(polyethoxy)ethyl)succinate (PEG-S-DMG)), polyethylene glycol ceramide (PEG-cer), or PEG dialkoxypropyl carbamate (such as co-methoxy(polyethoxy)ethyl-N-(2,3-(tetradecanoyloxy)propyl)carbamate or 2,3-di(tetradecanoyloxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate). 42. The composition of paragraph 40 or 41, wherein the PEG-lipid 43. The composition of any one of paragraphs 36 to 42, wherein the lipid nanoparticles comprise neutral lipids. 44. The composition of paragraph 43, wherein the neutral lipid is distearylphosphatidylcholine (DSPC), dioleylphosphatidylcholine (DOPC), dimalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylglycerol (DOPG), dimalmitoylphosphatidylglycerol (DPPG), dioleyl-phosphatidylethanolamine (DOPE), Palmitoylphosphatidylcholine (POPC), palmitoylphosphatidylethanolamine (POPE) and dioleylphosphatidylethanolamine 4-(N-cis-butylenediimidylmethyl)-cyclohexane-1-carboxylate (DOPE-mal), dimalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearyl-2-oleylphosphatidylethanolamine (SOPE) or 1,2 45. The composition of paragraphs 43 or 44, wherein the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). 46. The composition of any of paragraphs 36 to 45, wherein the lipid nanoparticles comprise a steroid or a steroid analog. 47. The composition of paragraph 46, wherein the steroid or steroid analog is cholesterol. 48. The composition of any of paragraphs 36 to 47, wherein the average diameter of the lipid nanoparticles is about 1 to about 500 nm. 49. The composition of any of paragraphs 36 to 48, wherein the RNA is from RSV subtype A. 50. The composition of any of paragraphs 36 to 48, wherein the RNA is from RSV subtype B. 51. The composition of any of paragraphs 36 to 48, wherein the RNA is from: (i) RSV subtype A and (ii) RSV subtype B. 52. The composition of any of paragraphs 36 to 51, comprising an RNA molecule formulated in a lipid nanoparticle (LNP) at a concentration of or about 0.01 to 0.09 mg/mL, the lipid nanoparticle comprising: a cationic lipid at a concentration of or about 0.8 to 0.95 mg/mL; a pegylated lipid at a concentration of or about 0.05 to 0.15 mg/mL; a neutral lipid at a concentration of about 0.1 to about 0.25 mg/mL; and a steroid or steroid analog at a concentration of about 0.3 to about 0.45 mg/mL. 53. The composition of any of paragraphs 36 to 52, comprising an RNA molecule formulated in a lipid nanoparticle (LNP) at a concentration of about 0.01 to about 0.09 mg/mL, the lipid nanoparticle comprising: (4-hydroxybutyl) azodiyl) bis(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315) at a concentration of about 0.8 to about 0.95 mg/mL; 2-[(polyethylene glycol)-2000]-N,N-di(tetradecyl)acetamide (ALC-0159) at a concentration of or about 0.05 to 0.15 mg/mL; 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) at a concentration of or about 0.1 to 0.25 mg/mL; cholesterol at a concentration of or about 0.3 to 0.45 mg/mL. 54. The composition of any of paragraphs 36 to 53, comprising a concentration of or about 0.06 55. The composition of any one of paragraphs 36 to 54, further comprising at least one of a buffer, a stabilizer, a salt, a surfactant, a preservative, a shaping agent and/or an adjuvant. 56. The composition of any one of paragraphs 36 to 55, further comprising at least a buffer and 57. The composition of paragraph 36 or 56, wherein the buffer is a Tris buffer. 58. The composition of paragraph 57, wherein the Tris buffer comprises sulfadiazine and Tris hydrochloric acid (HCl). 59. The composition of paragraph 58, wherein the concentration of sulfadiazine is or is about 0.1 to 0.3 60. The composition of paragraphs 57 or 58, wherein the concentration of Tris HCl is at or about 1.25 to 1.40 mg/mL, or at or about 0.5 to 0.65 mg/mL. 61. The composition of any of paragraphs 56 to 60, wherein the stabilizer is sucrose. 62. The composition of paragraph 61, wherein the concentration of sucrose is at or about 95 to 110 mg/mL, or at or about 35 to 50 mg/mL. 63. The composition of any of paragraphs 55 to 62, wherein the salt diluent used for reconstitution is sodium chloride. 64. The composition of paragraph 63, wherein the concentration of sodium chloride is at or about 5 to 15 mg/mL. 65. The composition of any of paragraphs 36 to 64, wherein the composition is liquid or freeze-dried. 66. The composition of paragraph 65, comprising an RNA molecule formulated in a lipid nanoparticle (LNP) at a concentration of or about 0.01 to 0.09 mg/mL, the lipid nanoparticle comprising: a cationic lipid at a concentration of or about 0.8 to 0.95 mg/mL; a pegylated lipid at a concentration of or about 0.05 to 0.15 mg/mL; a neutral lipid at a concentration of or about 0.1 to 0.25 mg/mL; and a steroid or steroid analog at a concentration of or about 0.3 to 0.45 mg/mL; and further comprising a Tris buffer, the Tris buffer comprising: ammonium sulfate at a concentration of or about 0.1 to 0.3 mg/mL; and Tris hydrochloric acid (HCl) at a concentration of or about 1.25 to 1.40 mg/mL; and sucrose at a concentration of or about 95 to 110 mg/mL, wherein the composition is a liquid composition. 67. The composition of paragraph 65, comprising an RNA molecule formulated in a lipid nanoparticle (LNP) at a concentration of or about 0.01 to 0.09 mg/mL, the lipid nanoparticle comprising: a cationic lipid at a concentration of or about 0.8 to 0.95 mg/mL; a pegylated lipid at a concentration of or about 0.05 to 0.15 mg/mL; a neutral lipid at a concentration of or about 0.1 to 0.25 mg/mL; and a steroid or steroid analog at a concentration of or about 0.3 to 0.45 mg/mL; and further comprising a Tris buffer comprising: sulfhydryl acetate at a concentration of or about 0.01 to 0.15 mg/mL; and Tris hydrochloric acid (HCl) at a concentration of or about 0.5 to 0.65 mg/mL; and sucrose at a concentration of or about 35 to 50 mg/mL. 68. The composition of paragraph 65, wherein the composition is reconstituted with sodium chloride at a concentration of or about 5 to 15 mg/mL. 69. The composition of paragraph 65, wherein the composition is reconstituted with or about 0.6 to 0.75 70. The composition of paragraph 65, further comprising or including about 5 to 15 mM Tris buffer, 200 to 400 mM sucrose at or about pH 7.0 to 8.0, and optionally a 0.9% sodium chloride diluent for reconstitution. 71. A method for inducing an immune response in an individual, comprising administering to the individual an effective amount of an RNA molecule, RNA-LNP and/or composition as described in any one of paragraphs 1 to 70. 72. A method for preventing, treating or ameliorating an infection, disease or condition in an individual, comprising administering to the individual an effective amount of an RNA molecule, RNA-LNP and/or composition as described in any one of paragraphs 1 to 70. 73. The method of paragraph 72, wherein the infection, disease or condition is related to RSV. 74. The method of paragraph 72 or 73, wherein the infection, disease or condition is acute respiratory distress induced by RSV infection, including pneumonia and bronchitis. 75. Use of an RNA molecule, RNA-LNP and/or composition as described in any one of paragraphs 1 to 70 for the manufacture of a medicament for inducing an immune response against RSV in a subject. 76. Use of an RNA molecule, RNA-LNP and/or composition as described in any one of paragraphs 1 to 70 for the manufacture of a medicament for preventing, treating or ameliorating an infection, disease or condition in a subject. 77. The use of paragraph 76, wherein the infection, disease or condition is associated with RSV. 78. The use of paragraphs 76 or 77, wherein the infection, disease or condition is respiratory tract ailments associated with RSV infection, including pneumonia and bronchitis. 79. The method or use of any one of paragraphs 71 to 78, wherein the age of the individual is less than about 1 year old, about 1 year old or older, about 5 years old or older, about 10 years old or older, about 20 years old or older, about 30 years old or older, about 40 years old or older, about 50 years old or older, about 60 years old or older, about 70 years old or older, or older. 80. The method or use of any of paragraphs 71 to 79, wherein the individual is about 50 years of age or older. 81. The method or use of any of paragraphs 71 to 79, wherein the individual is a pregnant woman. 82. The method or use of any of paragraphs 71 to 81, wherein the RNA molecule or composition is administered as a vaccine. 83. The method or use of any of paragraphs 71 to 82, wherein the RNA molecule or composition is administered by intradermal or intramuscular injection. 84. The RNA molecule of paragraph 83, wherein the modRNA comprises a nucleotide having SEQ ID NO: 13. 85. The RNA molecule of paragraph 83, wherein the modRNA comprises a nucleotide having SEQ ID NO: 14. 86. The RNA molecule of paragraph 83, wherein the saRNA comprises a nucleotide having SEQ ID NO: 15. 87. The RNA molecule of paragraph 83, wherein the modRNA comprises a nucleotide having SEQ ID NO: 16 nucleotides. 88. The method or use of any of paragraphs 71 to 87, wherein a single dose, two doses, three doses or more of the RNA molecule and/or composition is administered to the individual. 89. The method or use of any of paragraphs 71 to 88, wherein a single dose of the RNA molecule and/or composition is administered to the individual. 90. The method or use of any of paragraphs 71 to 89, wherein two doses of the RNA molecule and/or composition are administered to the individual. 91. The method or use of any of paragraphs 71 to 90, wherein Two doses of the RNA molecule and/or composition are administered to the individual on day 0 and at or about 2 months later. 92. The method or use of any of paragraphs 71 to 91, wherein two doses of the RNA molecule and/or composition are administered to the individual on day 0 and at or about 6 months later. 93. The method or use of any of paragraphs 71 to 92, wherein at least one booster dose of the RNA molecule and/or composition is administered to the individual. 94. The method or use of any of paragraphs 71 to 93, wherein at least or at least about 15 95. The method or use of any one of paragraphs 71 to 94, wherein the subject is administered an injection of or about 0.25 to 1 mL, including but not limited to or about 0.25, 0.5, 1 mL.
本文所揭示及主張之所有方法可在不依據本發明進行不當實驗之情況下進行及執行。雖然本發明之組合物及方法已根據某些態樣加以描述，但熟習此項技術者將顯而易見的係，變化形式可適用於本文所描述之方法以及方法之步驟或方法之步驟順序，而不脫離本發明之概念、精神及範疇。更確切而言，顯而易知化學上及生理學上均相關之某些藥劑可取代本文所描述之藥劑，同時達成相同或類似結果。熟習此項技術者顯而易知的所有此類類似取代及修改視為在由隨附申請專利範圍所界定的本發明之精神、範疇及概念內。All methods disclosed and claimed herein may be performed and executed without undue experimentation according to the present invention. Although the compositions and methods of the present invention have been described according to certain aspects, it will be apparent to those skilled in the art that variations may be applied to the methods and steps of the methods or the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the present invention. More specifically, it is apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while achieving the same or similar results. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the present invention as defined by the scope of the accompanying patent applications.
在本申請案中敍述的所有引用之參考文獻(包括如在整個本申請案中引用之文獻參考、發佈專利、公開專利申請案及GENBANK®寄存編號)之內容在其提供補充本文所闡述之彼等者之例示性程序或其他細節的程度上在此特定且明確地以引用之方式併入。當以引用之方式併入本文中的文獻中之術語之定義與本文所使用之定義衝突時，以本文所使用之定義為準。The contents of all cited references described in this application (including literature references, published patents, published patent applications, and GENBANK® deposit numbers as cited throughout this application) are specifically and expressly incorporated by reference herein to the extent they provide exemplary procedural or other details supplementary to those set forth herein. In the event of a conflict between a definition of a term in a document incorporated by reference and the definition used herein, the definition used herein controls.
圖1A至圖1E顯示小鼠中RSV 847之modRNA-LNP調配物的免疫原性。在第0及21天，以指示劑量，對雌性BALB/c小鼠(10隻/群組)肌肉內免疫接種呈二價蛋白質次單元(RSV 847A+B)形式或呈一價(RSV 847A)或二價(RSV 847A+B)之modRNA-LNP調配物形式的RSV 847構築體。在第35天(劑量2後(PD2) 2週)，收集血清用於RSV中和分析，且收集脾用於T細胞分析(ELISpot及細胞內細胞介素染色ICS分析)。圖1A及圖1B顯示RSV A及B之中和分析結果，其表示為50%中和力價(各符號表示來自個別動物之力價。條杠表示幾何平均力價(GMT))。圖1C顯示ELISpot分析結果，其量測分泌IFN-γ之RSV A+B F特異性細胞之數目，且表示為斑點形成細胞(SFC)/百萬細胞。圖1D及圖1E顯示ICS分析結果，其量測CD4+及CD8+ T細胞內之表現RSV A+B F特異性IFN-γ之細胞，表示為IFN-γ+細胞之百分比。條杠及誤差條杠描繪中位數與四分位數範圍。NA：未分析。圖2顯示小鼠中編碼不同RSV A融合前F (preF)設計之modRNA-LNP調配物之免疫原性。在第0及21天，以0.5 µg劑量，對雌性BALB/c小鼠(10隻/群組)肌肉內免疫接種本文所描繪之編碼RSV A融合前F (preF)設計的modRNA-LNP調配物。在第35天(2W PD2)，分析血清之RSV A中和反應，表示為50%中和力價。各符號表示來自個別動物之力價。條杠表示幾何平均力價(GMT)。圖3A至圖3F顯示小鼠中RSV融合前F (preF)之modRNA-LNP及saRNA-LNP調配物之免疫原性。在第0及21天，以指示劑量，對雌性BALB/c小鼠(10隻/群組)肌肉內免疫接種呈二價蛋白質次單元(RSV preF A+B)或二價modRNA-LNP調配物或二價saRNA-LNP調配物形式之RSV preF構築體。在第21 (3W PD1)及35天(2W PD2)，收集血清用於RSV中和分析，且在第35天收集脾臟用於T細胞分析(細胞內細胞介素染色ICS分析)。顯示RSV A及B之中和分析結果，表示為在3W PD1 (圖3A及圖3B)或2W PD2 (圖3C及圖3D)之50%中和力價。各符號表示來自個別動物之力價。條杠表示幾何平均力價(GMT)。圖3E及圖3F顯示ICS分析結果，其量測CD4+ T細胞及CD8+ T細胞-內之表現RSV preF A+B F特異性IFN-γ的細胞。條杠及誤差條杠描繪中位數與四分位數範圍。NT：未測試。圖4示意性地繪示野生型(WT) RSV F蛋白(RSV WT)及變異RSV F蛋白構築體，其中「SP」係指訊號肽序列(各構築體之胺基酸殘基1-25)，「TM」係指對應於跨越細胞膜之蛋白質部分的跨膜肽序列，「CT」係指對應於延伸至細胞細胞質中之蛋白質部分的細胞質尾肽序列，且「胞外域」係指對應於延伸至細胞外空間中之蛋白質部分的肽序列，其中胞外域包含胺基酸殘基1-513 (無TM及CT，由「ΔTM & CT」表示)。其中指示各部分(亦即SP、F2、pep27、F1)之胺基酸位置或各構築體之突變，例如各構築體之SP跨越各構築體之胺基酸殘基1-25。Figures1Ato1E show the immunogenicity of modRNA-LNP formulations of RSV 847 in mice. Female BALB/c mice (10/group) were immunized intramuscularly with RSV 847 constructs in the form of bivalent protein subunits (RSV 847A+B) or in the form of monovalent (RSV 847A) or bivalent (RSV 847A+B) modRNA-LNP formulations at the indicated doses on days 0 and 21. On day 35 (2 weeks after dose 2 (PD2)), serum was collected for RSV neutralization analysis and spleens were collected for T cell analysis (ELISpot and intracellular interleukin staining ICS analysis).Figures1A and1B show the results of neutralization assays for RSV A and B, expressed as 50% neutralization titers (each symbol represents the titer from an individual animal. The bar represents the geometric mean titer (GMT)).Figure1C shows the results of ELISpot assays, which measure the number of RSV A+BF-specific cells secreting IFN- γ and are expressed as spot forming cells (SFC)/million cells.Figures1D and1E show the results of ICS assays, which measure cells expressing RSVA +BF-specific IFN-γ in CD4+ and CD8+ T cells, expressed as the percentage of IFN-γ+ cells. Bars and error bars depict the median and interquartile range. NA: Not analyzed.Figure2 shows the immunogenicity of modRNA-LNP formulations encoding different RSV A pre-fusion F (preF) designs in mice. Female BALB/c mice (10/group) were intramuscularly immunized with modRNA-LNP formulations encoding RSV A pre-fusion F (preF) designs described herein at a dose of 0.5 µg on days 0 and 21. On day 35 (2W PD2), serum was analyzed for RSV A neutralization reactions, expressed as 50% neutralization titers. Each symbol represents the titer from an individual animal. The bar represents the geometric mean titer (GMT).Figures3Ato3F show the immunogenicity of modRNA-LNP and saRNA-LNP formulations of RSV pre-fusion F (preF) in mice. Female BALB/c mice (10/group) were immunized intramuscularly with RSV preF constructs in the form of bivalent protein subunits (RSV preF A+B) or bivalent modRNA-LNP formulations or bivalent saRNA-LNP formulations at the indicated doses on days 0 and 21. Serum was collected for RSV neutralization assays on days 21 (3W PD1) and 35 (2W PD2), and spleens were collected on day 35 for T cell analysis (intracellular interleukin staining ICS analysis). Neutralization assay results for RSV A and B are shown, expressed as 50% neutralization titers at 3W PD1 (Figures3A and3B) or 2W PD2 (Figures3C and3D) . Each symbol represents the titer from an individual animal. The bar represents the geometric mean titer (GMT).Figures3E and3F show the results of ICS analysis, which measures cells expressing RSV preF A+BF-specificIFN -γ in CD4+ T cells and CD8+ T cells. Bars and error bars depict medians and interquartile ranges. NT: not tested.FIG4 schematically depicts wild-type (WT) RSV F protein (RSV WT) and variant RSV F protein constructs, wherein "SP" refers to the signal peptide sequence (aminoacid residues 1-25 of each construct), "TM" refers to the transmembrane peptide sequence corresponding to the portion of the protein that spans the cell membrane, "CT" refers to the cytoplasmic tail peptide sequence corresponding to the portion of the protein that extends into the cell cytoplasm, and "ectodomain" refers to the peptide sequence corresponding to the portion of the protein that extends into the extracellular space, wherein the ectodomain includes amino acid residues 1-513 (without TM and CT, indicated by "ΔTM &CT"). The amino acid positions of each portion (i.e., SP, F2, pep27, F1) or mutations of each construct are indicated, for example, the SP of each construct spans amino acid residues 1-25 of each construct.
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| EP4608440A1 (en) | 2025-09-03 |
| PE20251592A1 (en) | 2025-06-16 |
| AU2023369585A1 (en) | 2025-04-10 |
| IL320459A (en) | 2025-06-01 |
| MX2025004821A (en) | 2025-06-02 |
| CN120153078A (en) | 2025-06-13 |
| WO2024089633A1 (en) | 2024-05-02 |
| CO2025005088A2 (en) | 2025-05-08 |
| TW202424187A (en) | 2024-06-16 |
| KR20250096808A (en) | 2025-06-27 |
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