相关申请的交叉引用CROSS-REFERENCE TO RELATED APPLICATIONS
本申请要求以下临时申请的权益：2021年5月10日提交的美国临时申请序列号63/186,664；2021年6月15日提交的美国临时申请序列号63/210,882；2022年3月21日提交的美国临时申请序列号63/321,921；2022年3月31日提交的美国临时申请序列号63/362,295；2021年9月1日提交的美国临时申请序列号63/239,671；2021年6月15日提交的美国临时申请序列号63/210,866；2022年1月11日提交的美国临时申请序列号63/298,587；以及2022年3月9日提交的美国临时申请序列号63/318,201，这些临时申请在不与本文提供的公开内容相冲突的情况下其各自全文以引用方式并入本文。This application claims the benefit of the following provisional applications: U.S. Provisional Application Serial No. 63/186,664, filed May 10, 2021; U.S. Provisional Application Serial No. 63/210,882, filed June 15, 2021; U.S. Provisional Application Serial No. 63/321,921, filed March 21, 2022; U.S. Provisional Application Serial No. 63/362,295, filed March 31, 2022; U.S. Provisional Application Serial No. 63/362,295, filed September 1, 2021 U.S. Provisional Application Serial No. 63/239,671; U.S. Provisional Application Serial No. 63/210,866, filed on June 15, 2021; U.S. Provisional Application Serial No. 63/298,587, filed on January 11, 2022; and U.S. Provisional Application Serial No. 63/318,201, filed on March 9, 2022, each of which is incorporated herein by reference in its entirety to the extent it does not conflict with the disclosure provided herein.
技术领域Technical Field
本文提供了用于调节mRNA剪接的组合物和方法。具体地，提供了用于通过诱导外显子跳跃来调节感兴趣的蛋白质的表达或活性的组合物和方法，例如以在RNA转录物中引入移码，其可导致RNA转录物的无义介导的衰变。Provided herein are compositions and methods for regulating mRNA splicing. Specifically, provided are compositions and methods for regulating the expression or activity of a protein of interest by inducing exon skipping, such as to introduce a frameshift in an RNA transcript that can result in nonsense-mediated decay of the RNA transcript.
背景技术Background Art
基因是编码功能性基因产物诸如蛋白质的脱氧核糖核酸(DNA)序列。将基因编码转化成功能性基因产物的过程包括从遗传DNA转录RNA(转录物)和将RNA翻译成蛋白质的步骤。RNA首先从DNA转录为未成熟的“前mRNA”，其经历加工而变成成熟的信使RNA(mRNA)，该mRNA可被翻译成蛋白质。在真核生物中，加工步骤包括将经单核苷酸修饰的鸟嘌呤(G)核苷酸帽添加到RNA的5'端；将聚腺苷序列添加到RNA的3'端(聚A尾)；以及RNA剪接。A gene is a deoxyribonucleic acid (DNA) sequence that encodes a functional gene product such as a protein. The process of converting a gene code into a functional gene product includes the steps of transcribing RNA (transcript) from the genetic DNA and translating RNA into protein. RNA is first transcribed from DNA as an immature "pre-mRNA", which undergoes processing to become a mature messenger RNA (mRNA), which can be translated into protein. In eukaryotes, the processing steps include adding a mononucleotide-modified guanine (G) nucleotide cap to the 5' end of the RNA; adding a polyadenosine sequence to the 3' end of the RNA (poly A tail); and RNA splicing.
剪接是指从前mRNA去除内含子(间插序列)并将外显子(编码序列)连接在一起形成成熟mRNA的过程。Splicing refers to the process of removing introns (intervening sequences) from pre-mRNA and joining exons (coding sequences) together to form mature mRNA.
许多哺乳动物基因被选择性剪接，其中前mRNA序列中的不同外显子被包含或排除在成熟mRNA转录物中，使得一种基因可以生成不同的mRNA信息，其被翻译成具有不同大小和/或功能的蛋白质(同种型)。Many mammalian genes are alternatively spliced, where different exons in the pre-mRNA sequence are included or excluded from the mature mRNA transcript, so that one gene can generate different mRNA messages that are translated into proteins (isoforms) of different sizes and/or functions.
选择性剪接可涉及转录物的外显子区和/或内含子区内的隐蔽剪接位点。隐蔽剪接位点是通常不使用的剪接位点，但是可以在常用剪接位点被阻断或不可用时或者在突变导致正常非活性位点变成活性剪接位点时使用。在隐蔽剪接中，剪接机制识别隐蔽剪接位点而不是典型剪接位点。通常，隐蔽剪接导致在mRNA中包含或排除部分或整个内含子或外显子序列。Alternative splicing can involve cryptic splice sites in the exon region and/or intron region of the transcript. Cryptic splice sites are splice sites that are not normally used, but can be used when conventional splice sites are blocked or unavailable or when mutations cause normal inactive sites to become active splice sites. In cryptic splicing, the splicing mechanism recognizes cryptic splice sites instead of typical splice sites. Typically, cryptic splicing results in the inclusion or exclusion of part or all of an intron or exon sequence in the mRNA.
前mRNA剪接的反义调节已被用于恢复隐蔽剪接，改变选择性剪接的基因的水平(同种型转换)，以及用于外显子跳跃，例如恢复破坏的阅读框或敲低不需要的基因的功能(Aartsma-Rus和Ommen，RNA(2007),13:1609-1624)。Antisense regulation of pre-mRNA splicing has been used to restore cryptic splicing, alter the levels of alternatively spliced genes (isoform switching), and for exon skipping, for example to restore disrupted reading frames or knock down the function of unwanted genes (Aartsma-Rus and Ommen, RNA (2007), 13:1609-1624).
在治疗剂中使用反义化合物的主要问题包括当全身施用时它们获得进入细胞内区室的能力有限，它们实现广泛或特异性靶向的组织分布的能力有限，以及获得靶向RNA的足够特异性以最小化脱靶效应的挑战。反义化合物的细胞内递送可通过使用载体系统(诸如聚合物、阳离子脂质体)或通过构建体的化学修饰(例如通过胆固醇分子的共价附接)来促进。然而，细胞内递送效率很低并且组织分布可能较窄。另外，现有技术仍然受到脱靶相互作用的阻碍。因此，仍然需要改进的递送系统以增加这些反义方法的有效性，并且仍然存在对有效组合物的未满足的需要，所述有效组合物将反义化合物广泛递送到细胞内区室的所有受影响的组织类型以特异性靶向给定基因产物，从而治疗由例如异常基因转录、剪接和/或翻译引起的疾病。The major problems of using antisense compounds in therapeutic agents include their limited ability to obtain access to intracellular compartments when systemically administered, their limited ability to achieve extensive or specifically targeted tissue distribution, and the challenge of obtaining sufficient specificity of targeted RNA to minimize off-target effects. The intracellular delivery of antisense compounds can be promoted by using carrier systems (such as polymers, cationic liposomes) or by chemical modification of constructs (e.g., covalent attachment by cholesterol molecules). However, intracellular delivery efficiency is very low and tissue distribution may be narrow. In addition, the prior art is still hindered by off-target interactions. Therefore, there is still a need for improved delivery systems to increase the effectiveness of these antisense methods, and there is still an unmet need for effective compositions, which deliver antisense compounds extensively to all affected tissue types of intracellular compartments to specifically target a given gene product, thereby treating diseases caused by, for example, abnormal gene transcription, splicing and/or translation.
发明内容Summary of the invention
本公开整体涉及用于调节基因诸如与疾病相关的基因的靶转录物(例如，前mRNA)的剪接的化合物、组合物和方法。在实施方案中，本公开涉及包含治疗性部分(TM)和细胞穿透肽(CPP)的化合物和组合物。TM可以是反义化合物(AC)，其结合靶转录物以调节靶转录物的剪接。在实施方案中，AC结合靶转录物的剪接元件(SE)或顺式作用剪接调控元件(SRE)的至少一部分，或者接近靶转录物的剪接元件或顺式作用剪接调控元件，以调节靶转录物的剪接。在实施方案中，AC与靶转录物的结合导致由靶转录物表达的蛋白质的表达或活性的下调。The present disclosure as a whole relates to compounds, compositions and methods for regulating the splicing of target transcripts (e.g., pre-mRNA) of genes such as genes associated with diseases. In embodiments, the present disclosure relates to compounds and compositions comprising a therapeutic moiety (TM) and a cell penetrating peptide (CPP). The TM can be an antisense compound (AC) that binds to a target transcript to regulate the splicing of the target transcript. In embodiments, AC binds to at least a portion of a splicing element (SE) or a cis-acting splicing regulatory element (SRE) of a target transcript, or a splicing element or a cis-acting splicing regulatory element close to a target transcript to regulate the splicing of a target transcript. In embodiments, the binding of AC to a target transcript results in downregulation of the expression or activity of a protein expressed by the target transcript.
在实施方案中，AC与靶转录物的结合导致外显子的跳跃。在实施方案中，外显子的跳跃导致移码。在实施方案中，移码得到提前终止密码子。在实施方案中，移码得到无义介导的衰变。在实施方案中，移码得到提前终止密码子和无义介导的衰变。In embodiments, binding of AC to the target transcript results in exon skipping. In embodiments, exon skipping results in a frameshift. In embodiments, the frameshift results in a premature stop codon. In embodiments, the frameshift results in nonsense-mediated decay. In embodiments, the frameshift results in a premature stop codon and nonsense-mediated decay.
本文描述了其中本文所述的化合物或组合物用于治疗疾病的方法。在实施方案中，疾病是遗传性疾病。在实施方案中，所述化合物或组合物用于通过调节与遗传性疾病相关的基因的剪接来治疗该疾病。在实施方案中，所述化合物或组合物通过调节与遗传性疾病相关的基因转录物的剪接来治疗该疾病。在实施方案中，所述方法包括将本文所述的化合物或组合物施用于对其有需要的受试者。在实施方案中，对其有需要的受试者是患有遗传性疾病或具有患遗传性疾病的风险的患者。在实施方案中，所述方法包括将治疗有效量的本文所述的化合物或组合物施用于对其有需要的受试者。在实施方案中，遗传性疾病是与IRF-5、DUX4或GYS1或其遗传变体的异常表达相关的疾病。Described herein is a method for treating a disease in which a compound or composition as described herein is used. In an embodiment, the disease is a hereditary disease. In an embodiment, the compound or composition is used to treat the disease by regulating the splicing of a gene associated with a hereditary disease. In an embodiment, the compound or composition treats the disease by regulating the splicing of a gene transcript associated with a hereditary disease. In an embodiment, the method includes administering a compound or composition as described herein to a subject in need thereof. In an embodiment, the subject in need thereof is a patient suffering from a hereditary disease or having a risk of suffering from a hereditary disease. In an embodiment, the method includes administering a therapeutically effective amount of a compound or composition as described herein to a subject in need thereof. In an embodiment, a hereditary disease is a disease associated with abnormal expression of IRF-5, DUX4 or GYS1 or its genetic variants.
CPP可以增强AC的细胞内递送以增强AC调节靶转录物的剪接的有效性。CPP可以是环状CPP(cCPP)。The CPP may enhance the intracellular delivery of the AC to enhance the effectiveness of the AC in regulating splicing of target transcripts. The CPP may be a cyclic CPP (cCPP).
本文所述的化合物可包含内体逃逸载体(EEV)，其被构造成允许在内体中内化到细胞中的化合物或其部分逃出内体并进入细胞溶质或细胞区室以允许AC作用于靶转录物并调节剪接。在实施方案中，EEV包含CPP，诸如cCPP。The compounds described herein may comprise an endosomal escape vector (EEV) that is constructed to allow a compound or portion thereof internalized into a cell in an endosome to escape the endosome and enter the cytosol or cellular compartment to allow the AC to act on the target transcript and regulate splicing. In an embodiment, the EEV comprises a CPP, such as a cCPP.
在实施方案中，cCPP具有下式(A)：In an embodiment, the cCPP has the following formula (A):
或其质子化形式，其中：or a protonated form thereof, wherein:
R1、R2和R3各自独立地是H或氨基酸的芳族或杂芳族侧链；R1 , R2 and R3 are each independently H or an aromatic or heteroaromatic side chain of an amino acid;
R1、R2和R3中的至少一者是氨基酸的芳族或杂芳族侧链；At least one of R1 , R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid;
R4、R5、R6、R7独立地是H或氨基酸侧链；R4 , R5 , R6 , and R7 are independently H or an amino acid side chain;
R4、R5、R6、R7中的至少一者是3-胍基-2-氨基丙酸、4-胍基-2-氨基丁酸、精氨酸、高精氨酸、N-甲基精氨酸、N,N-二甲基精氨酸、2,3-二氨基丙酸、2,4-二氨基丁酸、赖氨酸、N-甲基赖氨酸、N,N-二甲基赖氨酸、N-乙基赖氨酸、N,N,N-三甲基赖氨酸、4-胍基苯丙氨酸、瓜氨酸、N,N-二甲基赖氨酸、β-高精氨酸、3-(1-哌啶基)丙氨酸的侧链；At least one of R4 , R5 , R6 , and R7 is a side chain of 3-guanidino-2-aminopropionic acid, 4-guanidino-2-aminobutyric acid, arginine, homoarginine, N-methylarginine, N,N-dimethylarginine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, lysine, N-methyllysine, N,N-dimethyllysine, N-ethyllysine, N,N,N-trimethyllysine, 4-guanidinophenylalanine, citrulline, N,N-dimethyllysine, β-homoarginine, or 3-(1-piperidinyl)alanine;
AASC是氨基酸侧链；并且AASC is an amino acid side chain; and
q是1、2、3或4。q is 1, 2, 3, or 4.
在实施方案中，式(A)的cCPP具有下式(I)：In an embodiment, the cCPP of formula (A) has the following formula (I):
或其质子化形式或盐，or a protonated form or salt thereof,
其中每个m独立地是0-3的整数。wherein each m is independently an integer from 0 to 3.
在实施方案中，式(A)的cCPP具有下式(I-1)：In an embodiment, the cCPP of formula (A) has the following formula (I-1):
或其质子化形式或盐。在实施方案中，cCPP具有式(A)，具有下式(I-2)：或其质子化形式或盐。在实施方案中，式(A)的cCPP具有下式(I-3)： or a protonated form or salt thereof. In an embodiment, the cCPP has formula (A) having the following formula (I-2): or a protonated form or salt thereof. In an embodiment, the cCPP of formula (A) has the following formula (I-3):
或其质子化形式或盐。 or a protonated form or salt thereof.
在实施方案中，式(A)的cCPP具有下式(I-4)：In an embodiment, the cCPP of formula (A) has the following formula (I-4):
或其质子化形式或盐。 or a protonated form or salt thereof.
在实施方案中，式(A)的cCPP具有下式(I-5)：In an embodiment, the cCPP of formula (A) has the following formula (I-5):
或其质子化形式或盐。 or a protonated form or salt thereof.
在实施方案中，式(A)的cCPP具有下式(I-6)：In an embodiment, the cCPP of formula (A) has the following formula (I-6):
或其质子化形式或盐。在实施方案中，cCPP具有下式(II)： or a protonated form or salt thereof. In an embodiment, the cCPP has the following formula (II):
其中：in:
AASC是氨基酸侧链；AASC is the amino acid side chain;
R1a、R1b和R1c各自独立地是6至14元芳基或6至14元杂芳基；R1a , R1b and R1c are each independently 6- to 14-membered aryl or 6- to 14-membered heteroaryl;
R2a、R2b、R2c和R2d独立地是氨基酸侧链；R2a , R2b , R2c and R2d are independently amino acid side chains;
R2a、R2b、R2c和R2d中的至少一者是或其质子化形式或盐；At least one of R2a , R2b , R2c and R2d is or a protonated form or salt thereof;
R2a、R2b、R2c和R2d中的至少一者是胍或其质子化形式或盐；At least one of R2a , R2b , R2c and R2d is guanidine or a protonated form or salt thereof;
每个n”独立地是0至5的整数；Each n" is independently an integer from 0 to 5;
每个n'独立地是0至3的整数；并且Each n' is independently an integer from 0 to 3; and
如果n'是0，则R2a、R2b、R2b或R2d不存在。If n' is 0, then R2a , R2b , R2d or R2d is not present.
在实施方案中，式(II)的cCPP具有下式(II-1)：In an embodiment, the cCPP of formula (II) has the following formula (II-1):
在实施方案中，式(II)的cCPP具有下式(IIa)：In an embodiment, the cCPP of formula (II) has the following formula (IIa):
在实施方案中，式(II)的cCPP具有下式(IIb)：In an embodiment, the cCPP of formula (II) has the following formula (IIb):
在实施方案中，式(II)的cCPP具有下式(IIc)：In an embodiment, the cCPP of formula (II) has the following formula (IIc):
或其质子化形式或盐。在实施方案中，cCPP具有以下结构： or a protonated form or salt thereof. In an embodiment, the cCPP has the following structure:
或其质子化形式或盐，其中氨基酸侧链的至少一个原子被所述治疗性部分或接头替换或者至少一个孤对形成与所述治疗性部分或所述接头的键。 or a protonated form or salt thereof, wherein at least one atom of the amino acid side chain is replaced by the therapeutic moiety or the linker or at least one lone pair forms a bond to the therapeutic moiety or the linker.
在实施方案中，cCPP具有以下结构：In an embodiment, the cCPP has the following structure:
或其质子化形式或盐，其中氨基酸侧链的至少一个原子被所述治疗性部分或接头替换或者至少一个孤对形成与所述治疗性部分或所述接头的键。 or a protonated form or salt thereof, wherein at least one atom of the amino acid side chain is replaced by the therapeutic moiety or the linker or at least one lone pair forms a bond to the therapeutic moiety or the linker.
在实施方案中，化合物包含环外肽(EP)。在实施方案中，EP包含以下序列之一：KK、KR、RR、HH、HK、HR、RH、KKK、KGK、KBK、KBR、KRK、KRR、RKK、RRR、KKH、KHK、HKK、HRR、HRH、HHR、HBH、HHH、HHHH、KHKK、KKHK、KKKH、KHKH、HKHK、KKKK、KKRK、KRKK、KRRK、RKKR、RRRR、KGKK、KKGK、HBHBH、HBKBH、RRRRR、KKKKK、KKKRK、RKKKK、KRKKK、KKRKK、KKKKR、KBKBK、RKKKKG、KRKKKG、KKRKKG、KKKKRG、RKKKKB、KRKKKB、KKRKKB、KKKKRB、KKKRKV、RRRRRR、HHHHHH、RHRHRH、HRHRHR、KRKRKR、RKRKRK、RBRBRB、KBKBKB、PKKKRKV、PGKKRKV、PKGKRKV、PKKGRKV、PKKKGKV、PKKKRGV或PKKKRKG，其中B是β-丙氨酸。In an embodiment, the compound comprises an exocyclic peptide (EP). In an embodiment, the EP comprises one of the following sequences: KK, KR, RR, HH, HK, HR, RH, KKK, KGK, KBK, KBR, KRK, KRR, RKK, RRR, KKH, KHK, HKK, HRR, HRH, HHR, HBH, HHH, HHHH, KHKK, KKHK, KKKH, KHKH, HKHK, KKKK, KKRK, KRKK, KRRK, RKKR, RRRR, KGKK, KKGK, HBHBH, HBKBH, RRRRR, KKKKK, KKKRK, RKKKK, KRKKK, KKRKK, KKKKR, KBKBK, RKKKKG, KRKKKG, KKRKKG, KKKKRG, RKKKKB, KRKKKB, KKRKKB, KKKKRB, KKKRKV, RRRRRR, HHHHHH, RHRHRH, HRHRHR, KRKRKR, RKRKRK, RBRBRB, KBKBKB, PKKKRKV, PGKKRKV, PKGKRKV, PKKGRKV, PKKKGKV, PKKKRGV or PKKKRKG, where B is beta-alanine.
在实施方案中，化合物具有下式(C)：In an embodiment, the compound has the following formula (C):
或其质子化形式或盐，其中：or a protonated form or salt thereof, wherein:
R1、R2和R3各自独立地是H或包含芳基或杂芳基基团的侧链，其中R1、R2和R3中的至少一者是包含芳基或杂芳基基团的侧链；R1 , R2 and R3 are each independently H or a side chain comprising an aryl or heteroaryl group, wherein at least one of R1 , R2 and R3 is a side chain comprising an aryl or heteroaryl group;
R4和R7独立地是H或氨基酸侧链；R4 andR7 are independently H or an amino acid side chain;
EP是环外肽；EP is an exocyclic peptide;
每个m独立地是0-3的整数；Each m is independently an integer from 0 to 3;
n是0-2的整数；n is an integer from 0 to 2;
x'是1-23的整数；x' is an integer from 1 to 23;
y是1-5的整数；y is an integer from 1 to 5;
q是1-4的整数；q is an integer from 1 to 4;
z'是1-23的整数；并且z' is an integer from 1 to 23; and
货物是AC。The goods are AC.
在实施方案中，化合物包含式(C-1)、(C-2)、(C-3)或(C-4)的结构：In an embodiment, the compound comprises a structure of Formula (C-1), (C-2), (C-3), or (C-4):
或其质子化形式或盐，其中EP是环外肽，并且寡核苷酸是AC。or a protonated form or salt thereof, wherein EP is an exocyclic peptide and the oligonucleotide is AC.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1A至图1B是示出剪接调控元件的示意图，包括剪接位点(A)和一般剪接反应(两个酯交换反应)(B)。1A-1B are schematic diagrams showing splicing regulatory elements, including splicing sites (A) and general splicing reactions (two transesterification reactions) (B).
图2是示出反义化合物介导的外显子跳跃以产生最终导致靶转录物的无义介导的衰变的提前终止密码子的示意图。2 is a schematic diagram showing antisense compound-mediated exon skipping to generate a premature stop codon that ultimately leads to nonsense-mediated decay of the target transcript.
图3示出了在本文所述的反义寡核苷酸中使用的经修饰的核苷酸。结构1-3(1＝硫代磷酸酯；2＝(SC5-Rp)-α,β-CAN；3＝PMO)是磷酸酯主链修饰；4(2-硫代-dT)是碱基修饰；5-8(5＝2'-OMe-RNA；6＝2'O-MOE-RNA；7＝2'F-RNA；8＝2'F-ANA)是2'糖修饰；9-11是限制性核苷酸；12-14(9＝LNA；10＝(S)-cET；11＝tcDNA；12＝FHNA；13＝(S)5'-C-甲基；14＝UNA)是另外的糖修饰；并且15-18(15＝E-VP；16＝膦酸甲酯；17＝5'硫代磷酸酯；18＝(S)-5'-C-甲基与磷酸酯)是5'磷酸酯稳定修饰；19是吗啉代糖。由Khvorova,A.等人，Nat.Biotechnol.(2017)3月；35(3):238-248改良而得。FIG. 3 shows modified nucleotides used in the antisense oligonucleotides described herein. Structures 1-3 (1 = phosphorothioate; 2 = (SC5 -Rp )-α,β-CAN; 3 = PMO) are phosphate backbone modifications; 4 (2-thio-dT) is a base modification; 5-8 (5 = 2'-OMe-RNA; 6 = 2'O-MOE-RNA; 7 = 2'F-RNA; 8 = 2'F-ANA) are 2' sugar modifications; 9-11 are limiting nucleotides; 12-14 (9 = LNA; 10 = (S)-cET; 11 = tcDNA; 12 = FHNA; 13 = (S)5'-C-methyl; 14 = UNA) are additional sugar modifications; and 15-18 (15 = E-VP; 16 = methyl phosphonate; 17 = 5'phosphorothioate; 18 = (S)-5'-C-methyl with phosphate) are 5' phosphate stabilizing modifications; 19 is a morpholino sugar. Modified from Khvorova, A. et al., Nat. Biotechnol. (2017) Mar;35(3):238-248.
图4A至图4D提供了用于合成二氨基磷酸酯连接的吗啉代寡聚物(PMO)的腺嘌呤(A)、胞嘧啶(B)、鸟嘌呤(C)和胸腺嘧啶(D)吗啉代亚基单体的结构。4A-4D provide structures of adenine (A), cytosine (B), guanine (C), and thymine (D) morpholino subunit monomers used to synthesize phosphorodiamidate-linked morpholino oligomers (PMOs).
图5A至图5D示出了用于将反义化合物(AC)连接到肽诸如环状细胞穿透肽(cCPP)的缀合化学。图5A示出了用于具有N-羟基琥珀酰亚胺活化酯的肽(上图)或具有游离羧酸的肽(下图)与AC的5'端的伯胺之间的酰胺键形成反应的试剂。图5B示出了用于AC的3'端的伯胺或仲胺与具有四氟苯基(TFP)活化酯的肽的酰胺键形成反应的试剂。图5C示出了用于肽-叠氮化物经由无铜叠氮化物-炔环加成与经5'环辛炔修饰的AC的缀合的试剂。图5D示出了用于经3'修饰的环辛炔AC或经3'修饰的叠氮化物AC与含有接头-叠氮化物或接头-炔/环辛炔部分的肽诸如cCPP之间分别经由无铜叠氮化物-炔环加成或铜催化的叠氮化物-炔环加成(点击反应)的缀合的另一种示例性试剂。Figures 5A to 5D show conjugation chemistry for connecting antisense compounds (AC) to peptides such as cyclic cell penetrating peptides (cCPP). Figure 5A shows reagents for amide bond formation reaction between peptides with N-hydroxysuccinimide activated esters (upper figure) or peptides with free carboxylic acids (lower figure) and primary amines at the 5' end of AC. Figure 5B shows reagents for amide bond formation reaction between primary or secondary amines at the 3' end of AC and peptides with tetrafluorophenyl (TFP) activated esters. Figure 5C shows reagents for conjugation of peptide-azide via copper-free azide-alkyne cycloaddition with 5' cyclooctyne-modified AC. 5D shows another exemplary reagent for conjugation between 3'-modified cyclooctyne AC or 3'-modified azide AC and peptides such as cCPP containing linker-azide or linker-alkyne/cyclooctyne moieties via copper-free azide-alkyne cycloaddition or copper-catalyzed azide-alkyne cycloaddition (click reaction), respectively.
图6示出了使用图5所示的缀合化学，用含有聚乙二醇(PEG)部分的另外的接头形式连接AC和CPP的缀合化学。指出了纯化方法。Figure 6 shows the conjugation chemistry of connecting AC and CPP using the conjugation chemistry shown in Figure 5 with an additional linker format containing a polyethylene glycol (PEG) moiety. Purification methods are indicated.
图7A至图7D示出了在GAA敲除小鼠模型中未处理的小鼠、用PMO处理的小鼠和用各种浓度的EEV-PMO处理的小鼠的横膈膜(A和B)和心脏(C和D)中的GYS1蛋白(A和C)和GYS1mRNA(B和D)的水平。(P>0.05＝NS；P≤0.05＝*；P≤0.01＝**；P≤0.001＝***)Figures 7A to 7D show the levels of GYS1 protein (A and C) and GYS1 mRNA (B and D) in the diaphragm (A and B) and heart (C and D) of untreated mice, mice treated with PMO, and mice treated with various concentrations of EEV-PMO in the GAA knockout mouse model. (P>0.05=NS; P≤0.05=*; P≤0.01=**; P≤0.001=***)
图8A至图8D示出了未处理小鼠、用PMO处理的小鼠和用EEV-PMO处理的小鼠在处理后不同时间点的心脏(A)、横膈膜(B)、四头肌(C)和三头肌(D)中GYS1 mRNA水平的图。(P>0.05＝NS；P≤0.05＝*；P≤0.01＝**；P≤0.001＝***)Figures 8A to 8D show graphs of GYS1 mRNA levels in the heart (A), diaphragm (B), quadriceps (C), and triceps (D) of untreated mice, mice treated with PMO, and mice treated with EEV-PMO at different time points after treatment. (P>0.05=NS; P≤0.05=*; P≤0.01=**; P≤0.001=***)
图9A至图9D示出了未处理小鼠、用PMO处理的小鼠和用EEV-PMO处理的小鼠在处理后不同时间点的心脏(A)、横膈膜(B)、四头肌(C)和三头肌(D)中GYS1蛋白水平的图。(P>0.05＝NS；P≤0.05＝*；P≤0.01＝**；P≤0.001＝***)Figures 9A to 9D show graphs of GYS1 protein levels in the heart (A), diaphragm (B), quadriceps (C), and triceps (D) of untreated mice, mice treated with PMO, and mice treated with EEV-PMO at different time points after treatment. (P>0.05=NS; P≤0.05=*; P≤0.01=**; P≤0.001=***)
图10A至图10C是示出在用各种浓度的EEV-PMO处理的小鼠的肝脏(A)、小肠(B)和胫骨前肌(C)中IRF5 mRNA表达水平的图。(P>0.05＝NS；P≤0.05＝*；P≤0.01＝**；P≤0.001＝***)。MPK(mpk)＝mg/kg。Figures 10A to 10C are graphs showing IRF5 mRNA expression levels in the liver (A), small intestine (B), and tibialis anterior muscle (C) of mice treated with various concentrations of EEV-PMO. (P>0.05=NS; P≤0.05=*; P≤0.01=**; P≤0.001=***). MPK (mpk)=mg/kg.
图11A至图11B是示出用各种浓度的EEV#1-PMO、EEV#2-PMO、EEV#3-PMO和EEV#4-PMO处理小鼠巨噬细胞的体外实验中IRF5蛋白表达水平的图。(P>0.05＝NS；P≤0.05＝*；P≤0.01＝**；P≤0.001＝***)Figures 11A to 11B are graphs showing the expression levels of IRF5 protein in in vitro experiments in which mouse macrophages were treated with various concentrations of EEV#1-PMO, EEV#2-PMO, EEV#3-PMO, and EEV#4-PMO. (P>0.05=NS; P≤0.05=*; P≤0.01=**; P≤0.001=***)
图12是示出在用各种浓度的PMO 220或EEV-PMO 220-814处理后野生型小鼠成肌细胞系C2C12中GYS1 mRNA水平的敲低的图。通过student t检验，相对于0(未处理)，N＝3，*p<0.05，**p<0.01。Figure 12 is a graph showing knockdown of GYS1 mRNA levels in wild-type mouse myoblast cell line C2C12 after treatment with various concentrations of PMO 220 or EEV-PMO 220-814. *p<0.05, **p<0.01 by student t-test, relative to 0 (untreated), N=3.
图13A至图13B是示出在用各种浓度的PMO 220处理后小鼠成肌细胞(A)和小鼠成纤维细胞(B)中GYS1 mRNA水平的敲低的图。通过student t-检验，相对于NT(未处理)，N＝2，*p<0.05。13A-13B are graphs showing knockdown of GYS1 mRNA levels in mouse myoblasts (A) and mouse fibroblasts (B) after treatment with various concentrations of PMO 220. N=2, *p<0.05, relative to NT (untreated) by student t-test.
图14A至图14D是示出在用PMO 220或各种浓度的PMO-EEV 220-814处理GAA敲除小鼠后心脏(A)、横膈膜(B)、三头肌(C)和四头肌(D)中GYS1 mRNA水平的图。MPK(mpk)＝mg/kg。Figures 14A to 14D are graphs showing GYS1 mRNA levels in the heart (A), diaphragm (B), triceps (C), and quadriceps (D) of GAA knockout mice after treatment with PMO 220 or various concentrations of PMO-EEV 220-814. MPK (mpk) = mg/kg.
图15是示出在用PMO 220或各种浓度的PMO-EEV 220-814处理GAA敲除小鼠后肝脏中GYS2 mRNA水平的图。MPK(mpk)＝mg/kg。Figure 15 is a graph showing GYS2 mRNA levels in the liver after treatment of GAA knockout mice with PMO 220 or various concentrations of PMO-EEV 220-814. MPK (mpk) = mg/kg.
图16A至图16D是示出在用PMO 220或各种浓度的PMO-EEV 220-1055处理GAA敲除小鼠后心脏(A)、横膈膜(B)、三头肌(C)和四头肌(D)中GYS1 mRNA水平的图。16A-16D are graphs showing GYS1 mRNA levels in the heart (A), diaphragm (B), triceps (C), and quadriceps (D) muscles of GAA knockout mice after treatment with PMO 220 or various concentrations of PMO-EEV 220-1055.
图17A至图17D是示出在用20mpk的PMO-EEV 220-1055处理GAA敲除小鼠后的不同时间点的心脏(A)、横膈膜(B)、三头肌(C)和四头肌(D)中GYS1蛋白水平的图。MPK(mpk)＝mg/kg。Figures 17A to 17D are graphs showing GYS1 protein levels in the heart (A), diaphragm (B), triceps (C), and quadriceps (D) at different time points after treatment of GAA knockout mice with 20 mpk of PMO-EEV 220-1055. MPK (mpk) = mg/kg.
图18A至图18D是示出在用20mpk的PMO 220或20mpk的PMO-EEV 220-1055处理GAA敲除小鼠后的不同时间点的心脏(A)、横膈膜(B)、三头肌(C)和四头肌(D)中药物暴露水平的图。MPK(mpk)＝mg/kg。Figures 18A to 18D are graphs showing drug exposure levels in the heart (A), diaphragm (B), triceps (C), and quadriceps (D) at different time points after treatment of GAA knockout mice with 20 mpk of PMO 220 or 20 mpk of PMO-EEV 220-1055. MPK (mpk) = mg/kg.
图19A至图19D是示出野生型小鼠、GAA敲除小鼠和用各种浓度的EEV-PMO 220-1120处理的GAA敲除小鼠的心脏(A)、横膈膜(B)、三头肌(C)和四头肌(D)中GYS1 mRNA水平的图。MPK(mpk)＝mg/kg。Figures 19A to 19D are graphs showing GYS1 mRNA levels in the heart (A), diaphragm (B), triceps (C), and quadriceps (D) of wild-type mice, GAA knockout mice, and GAA knockout mice treated with various concentrations of EEV-PMO 220-1120. MPK (mpk) = mg/kg.
图20A至图20D是示出野生型小鼠、GAA敲除小鼠和用各种浓度的EEV-PMO 220-1120处理的GAA敲除小鼠的心脏(A)、横膈膜(B)、三头肌(C)和四头肌(D)中GYS1蛋白水平的图。MPK(mpk)＝mg/kg。Figures 20A to 20D are graphs showing GYS1 protein levels in the heart (A), diaphragm (B), triceps (C), and quadriceps (D) of wild-type mice, GAA knockout mice, and GAA knockout mice treated with various concentrations of EEV-PMO 220-1120. MPK (mpk) = mg/kg.
图21A至图21D是示出野生型小鼠、GAA敲除小鼠和用多个剂量的EEV-PMO 220-1055处理的GAA敲除小鼠的心脏(A)、横膈膜(B)和四头肌(C)中GYS1蛋白水平的图。21A-21D are graphs showing GYS1 protein levels in the heart (A), diaphragm (B), and quadriceps (C) of wild-type mice, GAA knockout mice, and GAA knockout mice treated with various doses of EEV-PMO 220-1055.
图22A至图22B是示出野生型小鼠、GAA敲除小鼠和用多个剂量的EEV-PMO 220-1055处理的GAA敲除小鼠的肝脏中GYS1(A)和GYS2(B)水平的图。22A-22B are graphs showing GYS1 (A) and GYS2 (B) levels in livers of wild-type mice, GAA knockout mice, and GAA knockout mice treated with various doses of EEV-PMO 220-1055.
图23A至图23C示出了在用两个剂量的PMO或EEV-PMO 278-1120处理小鼠后小鼠TiA组织(A)、肝脏组织(B)和小肠组织(C)中IRF-5的表达水平。MPK(mpk)＝mg/kg。Figures 23A to 23C show the expression levels of IRF-5 in mouse TiA tissue (A), liver tissue (B), and small intestinal tissue (C) after mice were treated with two doses of PMO or EEV-PMO 278-1120. MPK (mpk) = mg/kg.
图24A至图24C示出了在用一个剂量的PMO 278或PMO-EEV 278-1120处理小鼠后小鼠肝脏(A)、肾脏(B)和胫骨前肌(C)组织中的IRF-5表达水平。P>0.05＝NS；P≤0.05＝*；P≤0.01＝**；P≤0.001＝***。Figures 24A to 24C show the expression levels of IRF-5 in mouse liver (A), kidney (B) and tibialis anterior muscle (C) tissues after mice were treated with one dose of PMO 278 or PMO-EEV 278-1120. P>0.05=NS; P≤0.05=*; P≤0.01=**; P≤0.001=***.
图25A至图25B示出了在用各种浓度的EEV-PMO构建体220-814处理小鼠后，使用对GYS1非特异性的GYS抗体，在四头肌(A)和三头肌(B)中的GYS1蛋白水平。Figures 25A-25B show GYS1 protein levels in quadriceps (A) and triceps (B) muscles after treatment of mice with various concentrations of EEV-PMO construct 220-814 using a GYS antibody non-specific for GYS1.
图26A至图26C示出了在用各种浓度的EEV-PMO构建体220-814处理小鼠后，使用GYS1特异性抗体，在横膈膜(A)、心脏(B)和三头肌(C)中的GYS1蛋白水平。Figures 26A-26C show GYS1 protein levels in the diaphragm (A), heart (B), and triceps (C) using a GYS1-specific antibody after treatment of mice with various concentrations of EEV-PMO construct 220-814.
图27A示出了用各种浓度的PMO-EEV 277-1120和278-1120处理后RAW 264.7单核细胞/巨噬细胞的IRF-5表达水平。P>0.05＝NS；P≤0.05＝*；P≤0.01＝**；P≤0.001＝***。Figure 27A shows the IRF-5 expression levels of RAW 264.7 monocytes/macrophages after treatment with various concentrations of PMO-EEV 277-1120 and 278-1120. P>0.05=NS; P≤0.05=*; P≤0.01=**; P≤0.001=***.
图27B是在用EEV-PMO 278-1120处理RAW 264.7单核细胞/巨噬细胞后的不同时间点的外显子跳跃百分比的柱状图。NT＝未处理。Figure 27B is a bar graph of the percentage of exon skipping at various time points following treatment of RAW 264.7 monocytes/macrophages with EEV-PMO 278-1120. NT = untreated.
图28A至图28B是示出在用各种浓度的各种EEV-PMO处理接着进行R848刺激后RAW264.7单核细胞/巨噬细胞中IRF-5表达水平(A)和外显子4跳跃百分比(B)的柱状图。28A-28B are bar graphs showing IRF-5 expression levels (A) and percentage of exon 4 skipping (B) in RAW264.7 monocytes/macrophages after treatment with various concentrations of various EEV-PMOs followed by R848 stimulation.
图29A至图29B是示出在用各种浓度的各种EEV-PMO处理后人THP1细胞中的IRF-5外显子4和外显子5跳跃水平的图。29A-29B are graphs showing the levels of IRF-5 exon 4 and exon 5 skipping in human THP1 cells after treatment with various concentrations of various EEV-PMOs.
具体实施方式DETAILED DESCRIPTION
剪接Editing
前mRNA分子在细胞核中制备，并且在转运到细胞质进行翻译之前或期间被加工。前mRNA的加工包括向转录物的3'端添加5'甲基化的鸟嘌呤帽和约200-250个碱基的聚(A)尾。前mRNA加工还包括剪接，其发生在约90％至约95％的哺乳动物mRNA的成熟中。内含子(或间插序列)是初级转录物(或编码它的DNA)的不包含在成熟mRNA的编码序列中的区域。外显子是初级转录物的当其到达细胞质时保留在成熟mRNA中的区域。转录物可具有多个内含子和外显子。外显子被剪接在一起形成成熟mRNA序列。剪接连接也称为剪接位点，其中连接的5'侧通常称为“5'剪接位点”或“剪接供体位点”，而3'侧称为“3'剪接位点”或“剪接受体位点”。在剪接中，上游外显子的3'端与下游外显子的5'端连接。因此，转录物(例如，前mRNA)在内含子的5'端具有外显子/内含子连接，并且在内含子的3'端具有内含子/外显子连接。去除内含子后，外显子在成熟mRNA中有时被称为外显子/外显子连接或边界的位置是连续的。隐蔽剪接位点是较少使用但当常用剪接位点被阻断或不可用时可以使用的那些。选择性剪接(定义为将外显子的不同组合剪接在一起)通常得到来自单个基因的多个mRNA转录物。Pre-mRNA molecules are prepared in the nucleus and processed before or during transport to the cytoplasm for translation. The processing of pre-mRNA includes adding a 5'methylated guanine cap and a poly (A) tail of about 200-250 bases to the 3' end of the transcript. Pre-mRNA processing also includes splicing, which occurs in the maturation of about 90% to about 95% of mammalian mRNA. Introns (or intervening sequences) are regions of the primary transcript (or the DNA encoding it) that are not included in the coding sequence of the mature mRNA. Exons are regions of the primary transcript that are retained in the mature mRNA when it reaches the cytoplasm. Transcripts may have multiple introns and exons. Exons are spliced together to form mature mRNA sequences. Splice connections are also referred to as splice sites, where the 5' side of the connection is usually referred to as a "5' splice site" or "splicing donor site", and the 3' side is referred to as a "3' splice site" or "splicing acceptor site". In splicing, the 3' end of the upstream exon is connected to the 5' end of the downstream exon. Thus, a transcript (e.g., pre-mRNA) has an exon/intron junction at the 5' end of an intron and an intron/exon junction at the 3' end of an intron. After removal of the introns, the exons are contiguous at positions sometimes referred to as exon/exon junctions or boundaries in mature mRNA. Cryptic splice sites are those that are less used but can be used when the usual splice sites are blocked or unavailable. Alternative splicing (defined as splicing together different combinations of exons) typically results in multiple mRNA transcripts from a single gene.
前mRNA中的内含子的去除由剪接体(一种核糖核蛋白(RNP)复合体，其包含五种小核核糖核蛋白(snRNP))和许多其他蛋白质催化(Will和Lührmann，Cold SpringHarb.Perspect.Biol.(2011),3(7):a003707；Havens等人，Wiley Interdiscip.RNA(2014),4(3),247-266.doi:10.1002/wrna.1158)。剪接部分地由剪接元件(SE)控制。如本文所用，“剪接元件”是存在于前mRNA中的发生剪接诸如典型剪接所必需的序列元件(图1A)。SE包含5'剪接位点(5'ss)和3'剪接位点(3'ss)。5'ss也称为供体剪接位点，包括几乎不变的“GU”二核苷酸序列以及较不保守的下游残基。5'剪接位点还包含外显子/内含子连接。如本文所用，外显子/内含子连接是上游10个核苷酸和从5'ss的GU序列的G起的10个核苷酸(+10和-10)的核苷酸序列。3'ss或受体剪接位点包含三个保守元件：分支剪接点(BSP)(有时称为分支点)、聚嘧啶或Py束和末端“AG”。BSP通常是位于3'ss中约18至约40个核苷酸的腺苷。Py束通常包含约15至约20个嘧啶残基，具体地尿嘧啶(U)(在图1A中示出为Xn)。然而，存在非典型的分支点；它们更远离3'剪接位点并且/或者利用非腺苷碱基(Montes等人Trends Genet.(2019),35(1):68-87)。3'ss还包含内含子/外显子连接。如本文所用，内含子/外显子连接是上游10个核苷酸和从3'ss的AG序列的G起的10个核苷酸(+10和-10)的核苷酸序列。The removal of introns in pre-mRNA is catalyzed by the spliceosome, a ribonucleoprotein (RNP) complex that includes five small nuclear ribonucleoproteins (snRNPs) and many other proteins (Will and Lührmann, Cold Spring Harb. Perspect. Biol. (2011), 3(7): a003707; Havens et al., Wiley Interdiscip. RNA (2014), 4(3), 247-266. doi: 10.1002/wrna.1158). Splicing is controlled in part by splicing elements (SEs). As used herein, "splicing elements" are sequence elements present in pre-mRNA that are necessary for splicing such as canonical splicing to occur (Figure 1A). SEs include a 5' splice site (5'ss) and a 3' splice site (3'ss). The 5'ss is also called a donor splice site and includes a nearly invariant "GU" dinucleotide sequence and less conserved downstream residues. 5' splice site also includes exon/intron connection. As used herein, exon/intron connection is a nucleotide sequence of 10 nucleotides (+10 and -10) from the G of the GU sequence of 5'ss in the upstream 10 nucleotides. 3'ss or acceptor splice site includes three conservative elements: branch splice point (BSP) (sometimes referred to as branch point), polypyrimidine or Py bundle and terminal "AG". BSP is usually an adenosine located at about 18 to about 40 nucleotides in 3'ss. Py bundle usually includes about 15 to about 20 pyrimidine residues, specifically uracil (U) (shown asXn in Figure 1A). However, there are atypical branch points; they are further away from the 3' splice site and/or utilize non-adenosine bases (Montes et al. Trends Genet. (2019), 35 (1): 68-87). 3'ss also includes intron/exon connection. As used herein, an intron/exon junction is the nucleotide sequence of the upstream 10 nucleotides and the 10 nucleotides (+10 and -10) from the G of the AG sequence of the 3' ss.
外显子在大多数剪接反应中通过与五种小核糖核蛋白(snRNP)的小核RNA(snRNA)组分的特异性碱基配对相互作用而被识别；U1、U2、U4、U5和U6(Havens等人，(2014)WileyInterdiscip.RNA.2013,4(3),247-266.doi:10.1002/wrna.1158；Wahl M.C.等人，Cell(2009),136:701-718)。每个snRNP包含被构造成识别特定核苷酸序列的小核RNA和一种或多种蛋白质。外显子剪接包括两个连续的剪接体催化的酯交换反应(图1B)。一般来讲，剪接反应由U1结合5'ss引发，随后U2结合分支剪接点(BPS)，最后U4、U5和U6在5'和3'剪接位点附近结合。然后置换U1和U4，随后进行第一酯交换反应，其中内含子内的分支点核苷酸(如图1B所示的A)的2'-OH对5'剪接位点处的内含子的第一核苷酸(如图1B所示的G)进行亲核攻击，从而形成套索中间体。在第二反应中，释放的5'外显子的3'-OH对3'剪接位点处的内含子的最后一个核苷酸(如图1B所示的G)进行亲核攻击，从而连接外显子并释放内含子套索。U4、U5和U6也被释放。Exons are recognized in most splicing reactions by specific base pairing interactions with the small nuclear RNA (snRNA) components of five small nuclear ribonucleoproteins (snRNPs); U1, U2, U4, U5 and U6 (Havens et al., (2014) Wiley Interdiscip. RNA. 2013, 4(3), 247-266. doi: 10.1002/wrna.1158; Wahl M.C. et al., Cell (2009), 136: 701-718). Each snRNP contains a small nuclear RNA and one or more proteins that are constructed to recognize a specific nucleotide sequence. Exon splicing involves two consecutive spliceosome-catalyzed transesterification reactions (Figure 1B). Generally speaking, the splicing reaction is initiated by U1 binding to the 5'ss, followed by U2 binding to the branch splice site (BPS), and finally U4, U5 and U6 binding near the 5' and 3' splice sites. U1 and U4 are then replaced, followed by a first transesterification reaction in which the 2'-OH of the branch point nucleotide (A as shown in FIG. 1B ) within the intron nucleophilically attacks the first nucleotide of the intron at the 5' splice site (G as shown in FIG. 1B ), thereby forming a lariat intermediate. In the second reaction, the 3'-OH of the released 5' exon nucleophilically attacks the last nucleotide of the intron at the 3' splice site (G as shown in FIG. 1B ), thereby connecting the exons and releasing the intron lariat. U4, U5, and U6 are also released.
除SE之外，剪接部分地由剪接调控元件(SRE)调控。SRE包含顺式调控元件和反式作用剪接因子。顺式调控元件和反式作用剪接因子可促进典型剪接、选择性剪接或隐蔽剪接。In addition to SEs, splicing is regulated in part by splicing regulatory elements (SREs). SREs contain cis-regulatory elements and trans-acting splicing factors. Cis-regulatory elements and trans-acting splicing factors can promote canonical splicing, alternative splicing, or cryptic splicing.
顺式调控元件是转录物内抑制或增强剪接的核苷酸序列。反式作用剪接因子是不位于转录物内并且用于增强或抑制剪接的蛋白质和/或寡核苷酸。顺式调控元件通常用于募集反式作用剪接因子，该反式作用剪接因子激活或抑制剪接。反式作用剪接因子通过与顺式调控元件结合来调控剪接。反式作用剪接因子包括富含丝氨酸/精氨酸(富含SR)的蛋白和异质核核糖核蛋白(hnRNPs)。Cis-regulatory elements are nucleotide sequences within transcripts that inhibit or enhance splicing. Trans-acting splicing factors are proteins and/or oligonucleotides that are not located within transcripts and are used to enhance or inhibit splicing. Cis-regulatory elements are typically used to recruit trans-acting splicing factors that activate or inhibit splicing. Trans-acting splicing factors regulate splicing by binding to cis-regulatory elements. Trans-acting splicing factors include serine/arginine-rich (SR-rich) proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs).
剪接顺式调控元件包括外显子剪接增强子(ESE)序列、外显子剪接沉默子(ESS)序列、内含子剪接增强子(ISE)序列和内含子剪接沉默子(ISS)序列(图1A)。ESE序列促进它们所驻留的外显子包含在mRNA中。ESS序列抑制它们所驻留的外显子包含在mRNA中。ISE序列从其在内含子内的位置增强选择性剪接位点的使用。ISS序列从其在内含子内的位置抑制选择性剪接位点的使用。通常，ISS的长度介于8个和16个核苷酸之间，并且比外显子-内含子连接处的剪接位点的保守性低。Splicing cis-regulatory elements include exon splicing enhancer (ESE) sequences, exon splicing silencer (ESS) sequences, intron splicing enhancer (ISE) sequences, and intron splicing silencer (ISS) sequences (Figure 1A). ESE sequences promote the inclusion of the exons in which they reside in mRNA. ESS sequences inhibit the inclusion of the exons in which they reside in mRNA. ISE sequences enhance the use of alternative splicing sites from their positions in introns. ISS sequences inhibit the use of alternative splicing sites from their positions in introns. Typically, the length of ISS is between 8 and 16 nucleotides and is less conservative than the splicing sites at the exon-intron junction.
前mRNA剪接还可通过在转录物内形成可影响剪接体或其他调控蛋白的结合的二级结构诸如末端茎环(TSL)来调控。末端茎环序列可以是SRE并且通常为约12个至约24个核苷酸，并且由于在12个至24个核苷酸序列内的互补性以及因此的结合而形成二级环结构。Pre-mRNA splicing can also be regulated by forming secondary structures such as terminal stem loops (TSLs) within the transcript that can affect the binding of spliceosomes or other regulatory proteins. The terminal stem loop sequence can be a SRE and is generally about 12 to about 24 nucleotides, and forms a secondary loop structure due to complementarity within the 12 to 24 nucleotide sequence and thus binding.
每个SE和/或顺式作用SRE通过间插序列(IS)与相邻的顺式作用SRE和/或SE分开。Each SE and/or cis-acting SRE is separated from adjacent cis-acting SREs and/or SEs by an intervening sequence (IS).
外显子跳跃Exon skipping
大多数真核生物前mRNA可以不同方式剪接，通常通过跳过外显子，以在称为选择性剪接的过程中产生不同的成熟mRNA同种型。术语“选择性剪接”是指以不同组合连接外显子(例如，连接不同的5'和3'剪接位点)。选择性剪接可以插入或去除氨基酸，使阅读框移位和/或引入终止密码子，这有助于基因和由基因表达的蛋白质的复杂性、灵活性和丰度。选择性剪接还可通过去除或插入调控元件、控制翻译、mRNA稳定性和/或定位来影响基因表达。据估计，破坏剪接的突变占所有致病突变的多达三分之一(Havens等人(2014)WileyInterdiscip.RNA.2013,4(3),247-266.doi:10.1002/wrna.1158；Lim K.H.等人，Proc.Natl.Acad.Sci.USA(2011),108:11093-11098；Faustino和Cooper，Genes&Dev.(2003),17:419-437；以及Sterne-Weiler T.等人，Genome Res.(2011),21:1563-1571)。Most eukaryotic pre-mRNAs can be spliced in different ways, usually by skipping exons, to produce different mature mRNA isoforms in a process called alternative splicing. The term "alternative splicing" refers to the joining of exons in different combinations (e.g., joining different 5' and 3' splice sites). Alternative splicing can insert or remove amino acids, shift the reading frame and/or introduce stop codons, which contributes to the complexity, flexibility and abundance of genes and proteins expressed by genes. Alternative splicing can also affect gene expression by removing or inserting regulatory elements, controlling translation, mRNA stability and/or positioning. It is estimated that mutations that disrupt splicing account for up to one-third of all pathogenic mutations (Havens et al. (2014) Wiley Interdiscip. RNA. 2013, 4(3), 247-266. doi: 10.1002/wrna.1158; Lim K.H. et al., Proc. Natl. Acad. Sci. USA (2011), 108: 11093-11098; Faustino and Cooper, Genes & Dev. (2003), 17: 419-437; and Sterne-Weiler T. et al., Genome Res. (2011), 21: 1563-1571).
影响剪接过程的突变可以许多不同的方式发生(Havens等人，(2014)WileyInterdiscip.RNA.2013,4(3),247-266.doi:10.1002/wrna.1158)。例如，内含子突变可以破坏核心剪接位点(5'ss或3'ss、Py束或BPS内的序列)，导致突变的剪接位点(5'ss和/或3ss)上游或下游外显子的跳跃或内含子的保留。通常，当剪接位点突变时，在侧接外显子或内含子内激活假剪接位点，其在剪接后产生选择性转录物。内含子内的突变也可以破坏或产生从头剪接沉默子和/或增强子和/或产生从头隐蔽剪接位点。内含子剪接位点突变可占疾病突变的约10％-15％(Havens等人(2014)Wiley Interdiscip.RNA.2013,4(3),247-266.doi:10.1002/wrna.1158；Stenson P.D.等人，The Human Gene Mutation Database:2008update.Genome Med 2009,1:13)。发生在编码外显子内的突变(外显子突变)可导致从头隐蔽剪接位点的产生、具有调控功能的RNA二级结构的破坏和/或剪接沉默子或增强子的破坏，使得剪接位点不能被剪接所需的序列特异性RNA结合蛋白识别。对外显子突变的分析预测外显子内多达25％的突变可改变剪接(Ibid；Proc.Natl.Acad.Sci.USA(2011),108:11093-11098)。隐蔽剪接是由前mRNA中的序列引起的，所述序列通常不被用作剪接位点，但被突变激活，所述突变使典型剪接位点失活或产生以前不存在的剪接位点(Arechavala-Gomeza等人，The Application of Clinical Genetics(2014),4(7),245-252；Roca X.等人Genes Dev.(2013)；27(2):129-144)。另外，促成由前mRNA生成的不同蛋白质的选择性剪接可通过将表达从一种同种型转移至与疾病相关的不同同种型而引起疾病(Ibid)。Mutations that affect the splicing process can occur in many different ways (Havens et al., (2014) Wiley Interdiscip. RNA. 2013, 4 (3), 247-266. doi: 10.1002 / wrna.1158). For example, intron mutations can destroy core splice sites (5'ss or 3'ss, Py bundle or sequence within BPS), resulting in the skipping of exons upstream or downstream of the mutated splice site (5'ss and / or 3ss) or the retention of introns. Usually, when the splice site mutates, a pseudo splice site is activated in the flanking exon or intron, which produces a selective transcript after splicing. Mutations in introns can also destroy or generate de novo splicing silencers and / or enhancers and / or generate de novo hidden splice sites. Intronic splice site mutations can account for about 10%-15% of disease mutations (Havens et al. (2014) Wiley Interdiscip. RNA. 2013, 4 (3), 247-266. doi: 10.1002/wrna.1158; Stenson P.D. et al., The Human Gene Mutation Database: 2008 update. Genome Med 2009, 1: 13). Mutations occurring within coding exons (exonic mutations) can lead to the generation of de novo cryptic splice sites, the destruction of RNA secondary structures with regulatory functions, and/or the destruction of splicing silencers or enhancers, making the splice sites unable to be recognized by sequence-specific RNA binding proteins required for splicing. Analysis of exonic mutations predicts that up to 25% of mutations within exons can alter splicing (Ibid; Proc. Natl. Acad. Sci. USA (2011), 108: 11093-11098). Cryptic splicing is caused by sequences in the pre-mRNA that are not normally used as splice sites, but are activated by mutations that inactivate typical splice sites or create splice sites that did not previously exist (Arechavala-Gomeza et al., The Application of Clinical Genetics (2014), 4(7), 245-252; Roca X. et al. Genes Dev. (2013); 27(2): 129-144). In addition, alternative splicing that promotes the production of different proteins from pre-mRNA can cause disease by shifting expression from one isoform to a different isoform associated with the disease (Ibid).
靶向剪接反应或参与剪接的剪接元件(例如，SE和/或SRE)以诱导异常剪接可用于破坏参与疾病发病机制的蛋白质的基因表达。例如，可靶向剪接以引起外显子跳跃，从而引入导致非功能性或截短的蛋白质或降解RNA转录物的移码或终止密码子(Stenson P.D.等人，Genome Med.2008；1(13))。剪接诱导的阅读框校正、再成框和/或靶转录物的无义介导的衰变提供了治疗许多疾病和病症的机会。Targeting splicing reactions or splicing elements involved in splicing (e.g., SEs and/or SREs) to induce aberrant splicing can be used to disrupt gene expression of proteins involved in disease pathogenesis. For example, splicing can be targeted to cause exon skipping, thereby introducing frameshifts or stop codons that lead to non-functional or truncated proteins or degraded RNA transcripts (Stenson P.D. et al., Genome Med. 2008; 1(13)). Splicing-induced reading frame correction, reframing, and/or nonsense-mediated decay of target transcripts provides opportunities for treating many diseases and disorders.
化合物Compound
本文公开了调节感兴趣的基因的表达和/或活性的化合物。在实施方案中，该化合物调节靶基因的靶转录物的剪接。在实施方案中，该化合物包含至少一种细胞穿透肽(CPP)和至少一个结合靶核苷酸序列的治疗性部分(TM)。在实施方案中，TM是反义化合物(AC)。在实施方案中，靶核苷酸序列包括接近或包含顺式作用剪接调控元件(SRE)的至少一部分和/或接近或包含剪接元件(SE)的至少一部分的核苷酸序列。Disclosed herein are compounds that regulate the expression and/or activity of a gene of interest. In embodiments, the compound regulates the splicing of a target transcript of a target gene. In embodiments, the compound comprises at least one cell penetrating peptide (CPP) and at least one therapeutic moiety (TM) that binds a target nucleotide sequence. In embodiments, the TM is an antisense compound (AC). In embodiments, the target nucleotide sequence comprises a nucleotide sequence that is proximal to or comprises at least a portion of a cis-acting splicing regulatory element (SRE) and/or proximal to or comprises at least a portion of a splicing element (SE).
如本文所用，“剪接的调节”和“调节剪接”是指改变前mRNA转录物的加工，使得剪接的mRNA分子由于外显子跳跃或外显子包含、一个或多个外显子中的缺失或通常不存在于剪接的mRNA中的序列(例如，内含子序列)的缺失或添加而含有外显子的不同组合。调节剪接可包括中断或促进剪接过程的一个或多个步骤。如本文所用，术语“剪接过程”涵盖剪接反应的所有步骤，例如包括各种snRNP(例如，U1、U2、U3、U4和U5)与剪接元件和/或顺式作用剪接调控元件的结合、各种蛋白质和/或寡核苷酸与顺式调控元件的结合以及两个连续的酯交换反应，如例如图1B所示。As used herein, "regulation of splicing" and "regulating splicing" refer to altering the processing of pre-mRNA transcripts so that spliced mRNA molecules contain different combinations of exons due to exon skipping or exon inclusion, deletions in one or more exons, or deletions or additions of sequences that are not normally present in the spliced mRNA (e.g., intronic sequences). Regulating splicing can include interrupting or promoting one or more steps of the splicing process. As used herein, the term "splicing process" encompasses all steps of the splicing reaction, including, for example, the binding of various snRNPs (e.g., U1, U2, U3, U4, and U5) to splicing elements and/or cis-acting splicing regulatory elements, the binding of various proteins and/or oligonucleotides to cis-regulatory elements, and two consecutive transesterification reactions, as shown, for example, in FIG. 1B .
治疗性部分Therapeutic part
在实施方案中，本公开描述了包括一个或多个治疗性部分(TM)的化合物，所述治疗性部分能够调节感兴趣的转录物从感兴趣的基因的剪接。在实施方案中，感兴趣的基因可以是致病基因。In embodiments, the disclosure describes compounds comprising one or more therapeutic moieties (TM) that are capable of modulating the splicing of a transcript of interest from a gene of interest. In embodiments, the gene of interest may be a disease-causing gene.
TM与靶核苷酸序列结合(例如，杂交)。靶核苷酸序列通常包含在感兴趣的基因的靶转录物中。例如，靶向感兴趣的基因的TM可以结合靶转录物内的靶核苷酸序列(例如，剪接元件)。The TM binds to (e.g., hybridizes to) a target nucleotide sequence. The target nucleotide sequence is typically contained in a target transcript of a gene of interest. For example, a TM targeted to a gene of interest can bind to a target nucleotide sequence (e.g., a splicing element) within the target transcript.
TM可以是反义化合物(AC)、与成簇规律间隔短回文重复序列(CRISPR)基因编辑机制相关的一种或多种元件、多肽或它们的组合。The TM can be an antisense compound (AC), one or more elements associated with the clustered regularly interspaced short palindromic repeats (CRISPR) gene editing mechanism, a polypeptide, or a combination thereof.
反义化合物(AC)Antisense Compounds (AC)
在实施方案中，治疗性部分包括可调节靶基因的靶转录物的剪接的反义化合物(AC)。AC是包含DNA碱基、经修饰的DNA碱基、RNA碱基、经修饰的RNA碱基、经修饰的核苷间连接、传统的核苷间连接、传统的DNA糖、经修饰的DNA糖、传统的RNA糖、经修饰的RNA糖或它们的组合的寡核苷酸。在实施方案中，AC包含与存在于靶转录物内的靶核苷酸序列互补的核苷酸序列。在实施方案中，AC包含与靶核苷酸序列互补的核苷酸序列，所述靶核苷酸序列接近或包含靶转录物内的剪接元件和/或剪接调控元件的至少一部分。In an embodiment, the therapeutic moiety includes an antisense compound (AC) that can modulate the splicing of a target transcript of a target gene. AC is an oligonucleotide comprising a DNA base, a modified DNA base, an RNA base, a modified RNA base, a modified internucleoside connection, a traditional internucleoside connection, a traditional DNA sugar, a modified DNA sugar, a traditional RNA sugar, a modified RNA sugar, or a combination thereof. In an embodiment, AC comprises a nucleotide sequence complementary to a target nucleotide sequence present in a target transcript. In an embodiment, AC comprises a nucleotide sequence complementary to a target nucleotide sequence, and the target nucleotide sequence is close to or comprises at least a portion of a splicing element and/or a splicing regulatory element within a target transcript.
本文所述的AC可含有一个或多个不对称中心，并且因此产生对映体、非对映体和其他立体异构构型，这些构型可根据绝对立体化学定义为(R)或(S)；α或β；或(D)或(L)。本文提供的反义化合物包括所有这些可能的异构体，以及它们的外消旋和光学纯形式。The ACs described herein may contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric configurations, which may be defined in terms of absolute stereochemistry as (R) or (S); α or β; or (D) or (L). The antisense compounds provided herein include all such possible isomers, as well as their racemic and optically pure forms.
在实施方案中，AC诱导导致靶转录物中核苷酸的添加或缺失的选择性剪接。在一些实施方案中，AC诱导导致靶转录物的单个外显子内核苷酸的添加或缺失的选择性剪接。在实施方案中，AC诱导导致靶转录物的单个外显子内的核苷酸缺失的选择性剪接。在实施方案中，单个外显子内核苷酸的缺失导致截短蛋白质的翻译。在实施方案中，截短蛋白质对细胞的毒性低于未截短蛋白质。In embodiments, AC induces alternative splicing resulting in addition or deletion of nucleotides in the target transcript. In some embodiments, AC induces alternative splicing resulting in addition or deletion of nucleotides within a single exon of the target transcript. In embodiments, AC induces alternative splicing resulting in deletion of nucleotides within a single exon of the target transcript. In embodiments, deletion of nucleotides within a single exon results in translation of a truncated protein. In embodiments, the truncated protein is less toxic to cells than the untruncated protein.
在实施方案中，AC被设计成使得外显子被跳过(有时称为外显子跳跃)，导致靶蛋白和/或由靶基因调控的下游蛋白的表达或活性增加或降低。在实施方案中，提供了生成编码截短蛋白质和/或非功能性蛋白质的mRNA的AC。在实施方案中，提供了通过选择性剪接生成编码截短蛋白质和/或非功能性蛋白质的mRNA的AC。在实施方案中，提供了触发靶转录物降解的AC，例如通过无义介导的衰变。在实施方案中，提供了生成具有有益特性的替代mRNA同种型的反义化合物(AC)。In an embodiment, AC is designed so that exons are skipped (sometimes referred to as exon skipping), resulting in an increase or decrease in the expression or activity of a target protein and/or a downstream protein regulated by a target gene. In an embodiment, an AC is provided for generating mRNA encoding a truncated protein and/or a non-functional protein. In an embodiment, an AC is provided for generating mRNA encoding a truncated protein and/or a non-functional protein by alternative splicing. In an embodiment, an AC is provided for triggering degradation of a target transcript, for example, by nonsense-mediated decay. In an embodiment, an antisense compound (AC) is provided for generating an alternative mRNA isoform having beneficial properties.
反义化合物(AC)可用于以任何合适的方式调节剪接。在实施方案中，AC可被设计成在空间上阻断对剪接位点、或剪接元件(SE)和/或顺式作用剪接调控元件(SRE)的至少一部分的接近，从而将剪接重定向到隐蔽或从头剪接位点。在实施方案中，AC可靶向剪接增强子序列(例如，ESE和/或ISE)或剪接沉默子序列(例如，ESS和/或ISS)以防止反式作用调控剪接因子结合在靶位点处并且有效地阻断或促进剪接。在实施方案中，AC可被设计成在剪接调控茎环的碱基上进行碱基配对以增强茎环结构。Antisense compounds (AC) can be used to regulate splicing in any suitable manner. In an embodiment, AC can be designed to spatially block access to at least a portion of a splicing site, or a splicing element (SE) and/or a cis-acting splicing regulatory element (SRE), thereby redirecting splicing to a hidden or de novo splicing site. In an embodiment, AC can target a splicing enhancer sequence (e.g., ESE and/or ISE) or a splicing silencer sequence (e.g., ESS and/or ISS) to prevent trans-acting regulatory splicing factors from binding at the target site and effectively blocking or promoting splicing. In an embodiment, AC can be designed to perform base pairing on the base of the splicing regulatory stem loop to enhance the stem loop structure.
在实施方案中，AC诱导所得经加工的转录物诸如mRNA中一个或多个核苷酸的添加或缺失。如果从开放读段添加或去除的核苷酸的数量可被三整除以产生整数，则所得的转录物可被翻译成具有比从转录物表达的对应物蛋白质更多或更少氨基酸的功能性或非功能性蛋白质，但在其他方面具有与从未添加或去除核苷酸的转录物表达的蛋白质相同的氨基酸序列(除了添加或缺失的氨基酸)。如果从开放阅读框添加或去除的核苷酸数量不能被三整除以产生整数，则所得的经加工的转录物诸如mRNA的开放读框被移位。例如，为诱导这种“移码”改变而添加或缺失的核苷酸数量可以是1、2、4、5、7、8、10、11、13、14、16、17、19、20、22、23等。由于遗传密码的三联体性质，不可被三整除的核苷酸数量的添加或缺失将所得的经加工的转录物诸如mRNA的阅读框移位到移码的下游。移位的阅读框可导致无义介导的衰变，可导致移码的无义下游内的提前终止密码子，并且/或者可导致具有完全不同氨基酸序列的蛋白质在移码下游的表达。In an embodiment, AC induces the addition or deletion of one or more nucleotides in the processed transcript such as mRNA. If the number of nucleotides added or removed from the open reading segment is divisible by three to produce an integer, the resulting transcript can be translated into a functional or non-functional protein with more or less amino acids than the counterpart protein expressed from the transcript, but otherwise has the same amino acid sequence (except for the added or deleted amino acids) as the protein expressed from the transcript to which nucleotides have never been added or removed. If the number of nucleotides added or removed from the open reading frame cannot be divided by three to produce an integer, the resulting processed transcript such as the open reading frame of mRNA is shifted. For example, the number of nucleotides added or deleted to induce this "frameshift" change can be 1, 2, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, etc. Due to the triplet nature of the genetic code, the addition or deletion of the number of nucleotides that cannot be divided by three shifts the reading frame of the processed transcript such as mRNA to the downstream of the frameshift. A shifted reading frame can result in nonsense-mediated decay, can result in a premature stop codon within the nonsense downstream of the frameshift, and/or can result in the expression of a protein with a completely different amino acid sequence downstream of the frameshift.
在实施方案中，AC诱导将提前终止密码子(PTC)引入开放阅读框中。如本文所用，“提前终止密码子”是与翻译起始密码子同相并且位于与翻译起始密码子同相的生理终止密码子上游的终止密码子。具有PTC的靶转录物可通过包括无义介导的衰变在内的各种机制去稳定化和降解。In an embodiment, AC induces the introduction of a premature stop codon (PTC) into an open reading frame. As used herein, a "premature stop codon" is a stop codon that is in phase with a translation start codon and is located upstream of a physiological stop codon that is in phase with a translation start codon. Target transcripts with PTCs can be destabilized and degraded by various mechanisms including nonsense-mediated decay.
无义介导的衰变是一种监视机制，其识别起始核酸外切和核酸内切降解途径以去除具有PTC的mRNA转录物，从而防止可能对细胞具有有害作用的截短蛋白质的表达。已经设想并综述了若干种无义介导的衰变途径(Lejeune等人，Biomedicines(2020),10(1):141；Brogna等人，Nature Structural and Molecular Biology(2009),16,108-113；Karousis等人，Wiley Interdiscip.Rev.RNA(2016),7(5):661-682)。在其中靶基因在疾病中过表达的实施方案中，诱导无义介导的衰变可用于降低靶蛋白的浓度，并因此治疗疾病。Nonsense-mediated decay is a surveillance mechanism that identifies the initial exonucleolytic and endonucleolytic degradation pathways to remove mRNA transcripts with PTCs, thereby preventing the expression of truncated proteins that may have deleterious effects on cells. Several nonsense-mediated decay pathways have been envisioned and reviewed (Lejeune et al., Biomedicines (2020), 10 (1): 141; Brogna et al., Nature Structural and Molecular Biology (2009), 16, 108-113; Karousis et al., Wiley Interdiscip. Rev. RNA (2016), 7 (5): 661-682). In embodiments where the target gene is overexpressed in a disease, inducing nonsense-mediated decay can be used to reduce the concentration of the target protein, and thus treat the disease.
在实施方案中，AC诱导外显子跳跃以导致靶转录物的无义介导的衰变。这与常规外显子跳跃形成对比，常规外显子跳跃旨在跳过外显子以诱导特定蛋白同种型的表达，从而校正错误剪接、选择性剪接并且/或者避免特定外显子中的有害突变。In embodiments, AC induces exon skipping to result in nonsense-mediated decay of the target transcript. This is in contrast to conventional exon skipping, which aims to skip exons to induce expression of specific protein isoforms, thereby correcting mis-splicing, alternative splicing and/or avoiding deleterious mutations in specific exons.
在实施方案中，AC诱导靶转录物内外显子的外显子跳跃，其中外显子具有不能被三整除的核苷酸数量。在实施方案中，AC诱导具有不能被三整除的核苷酸数量的外显子的外显子跳跃，得到靶转录物内的PTC。在实施方案中，AC诱导具有不能被三整除的核苷酸数量的外显子的外显子跳跃，得到靶转录物内的PCT，其导致靶转录物的无义介导的衰变。在实施方案中，诱导靶转录物的无义介导的衰变导致靶转录物浓度降低。在实施方案中，诱导靶转录物的无义介导的衰变导致由靶转录物编码的靶蛋白的浓度降低。在实施方案中，诱导靶转录物的无义介导的衰变导致由靶基因调控的下游基因的蛋白水平增加和/或降低。In embodiments, AC induces exon skipping of exons within a target transcript, wherein the exons have a number of nucleotides that are not divisible by three. In embodiments, AC induces exon skipping of exons having a number of nucleotides that are not divisible by three, resulting in a PTC within the target transcript. In embodiments, AC induces exon skipping of exons having a number of nucleotides that are not divisible by three, resulting in a PCT within the target transcript, which results in nonsense-mediated decay of the target transcript. In embodiments, inducing nonsense-mediated decay of a target transcript results in a decrease in the concentration of the target transcript. In embodiments, inducing nonsense-mediated decay of a target transcript results in a decrease in the concentration of a target protein encoded by the target transcript. In embodiments, inducing nonsense-mediated decay of a target transcript results in an increase and/or decrease in the protein level of a downstream gene regulated by the target gene.
图2示出了AC诱导的外显子跳跃导致靶转录物的无义介导的衰变或蛋白质的翻译的过早终止的示例。AC结合前mRNA。在例示性实施方案中，AC结合在外显子三的内含子/外显子连接处。在其他实施方案中，AC可以在各种其他位置结合靶转录物以诱导外显子跳跃，从而导致靶转录物的无义介导的衰变(如别处论述)。外显子三中的核苷酸数量不能被三整除，例如52、106、232、365等。AC与内含子/外显子连接的结合通过多种可能的机制诱导外显子三的外显子跳跃。例如，AC与内含子/外显子连接的结合防止剪接机制接近剪接元件。除此之外或另选地，AC与内含子/外显子连接的结合防止完成剪接过程所需的一个或两个酯交换反应的完成。作为AC结合靶转录物的结果，外显子三被跳过，并且所得的转录物包含与外显子四连接的外显子二。作为AC结合靶转录物并跳过外显子三的结果，所得转录物的外显子四中的阅读框被移位。在例示的实施方案中，阅读框的移位将PTC引入所得的转录物中。作为AC结合靶转录物的结果，跳过外显子三和具有PTC的外显子四，所得的转录物被靶向并经历无义介导的衰变。FIG. 2 shows an example of AC-induced exon skipping leading to nonsense-mediated decay of a target transcript or premature termination of translation of a protein. AC binds to pre-mRNA. In an exemplary embodiment, AC binds to the intron/exon junction of exon three. In other embodiments, AC can bind to the target transcript at various other positions to induce exon skipping, thereby leading to nonsense-mediated decay of the target transcript (as discussed elsewhere). The number of nucleotides in exon three cannot be divided by three, such as 52, 106, 232, 365, etc. The binding of AC to the intron/exon junction induces exon skipping of exon three through a variety of possible mechanisms. For example, the binding of AC to the intron/exon junction prevents the splicing machinery from approaching the splicing element. In addition or alternatively, the binding of AC to the intron/exon junction prevents the completion of one or two transesterification reactions required to complete the splicing process. As a result of AC binding to the target transcript, exon three is skipped, and the resulting transcript contains exon two connected to exon four. As a result of AC binding to the target transcript and skipping exon three, the reading frame in exon four of the resulting transcript is shifted. In the illustrated embodiment, the shift of the reading frame introduces a PTC into the resulting transcript. As a result of AC binding to the target transcript, exon three and exon four with a PTC are skipped, and the resulting transcript is targeted and undergoes nonsense-mediated decay.
确定靶序列和设计反义化合物(AC)以诱导外显子跳跃可使用各种不同的方法来完成，包括例如由Aartsma-Rus,A.等人，Molecular Therapy(2008),17(3)548-553；以及Aartsma-Rus,A.等人，RNA(2007),13(10)1609-1624公开的那些。在实施方案中，AC与包含靶转录物的剪接元件(SE)的至少一部分的靶核苷酸序列杂交。在实施方案中，AC与包含靶转录物的整个SE的靶核苷酸序列杂交。在实施方案中，AC与包含靶转录物的多个SE的靶核苷酸序列杂交。在实施方案中，AC与包含靶转录物的多个SE以及SE之间的间插序列的靶核苷酸序列杂交。Determining the target sequence and designing antisense compounds (AC) to induce exon skipping can be accomplished using a variety of different methods, including, for example, those disclosed by Aartsma-Rus, A. et al., Molecular Therapy (2008), 17 (3) 548-553; and Aartsma-Rus, A. et al., RNA (2007), 13 (10) 1609-1624. In an embodiment, AC hybridizes to a target nucleotide sequence comprising at least a portion of a splicing element (SE) of a target transcript. In an embodiment, AC hybridizes to a target nucleotide sequence comprising the entire SE of a target transcript. In an embodiment, AC hybridizes to a target nucleotide sequence comprising multiple SEs of a target transcript. In an embodiment, AC hybridizes to a target nucleotide sequence comprising multiple SEs of a target transcript and intervening sequences between SEs.
在实施方案中，AC与包含靶转录物的SRE的至少一部分的靶核苷酸序列杂交。在实施方案中，AC与包含靶转录物的整个SRE的靶核苷酸序列杂交。在实施方案中，AC与包含靶转录物的多个SRE的靶核苷酸序列杂交。在实施方案中，AC与包含靶转录物的多个SRE以及SRE之间的间插序列的靶核苷酸序列杂交。In an embodiment, AC hybridizes to a target nucleotide sequence comprising at least a portion of a SRE of a target transcript. In an embodiment, AC hybridizes to a target nucleotide sequence comprising the entire SRE of a target transcript. In an embodiment, AC hybridizes to a target nucleotide sequence comprising multiple SREs of a target transcript. In an embodiment, AC hybridizes to a target nucleotide sequence comprising multiple SREs of a target transcript and intervening sequences between SREs.
在实施方案中，靶核苷酸序列包含整个SE和/或SRE以及位于靶转录物的SE和/或SRE的上游和/或下游的一个或多个旁侧序列。在实施方案中，靶核苷酸序列包含SE和/或SRE的一部分而非全部，以及位于靶转录物的SE和/或SRE的上游和/或下游的一个或多个旁侧序列。In an embodiment, the target nucleotide sequence comprises the entire SE and/or SRE and one or more flanking sequences located upstream and/or downstream of the SE and/or SRE of the target transcript. In an embodiment, the target nucleotide sequence comprises a portion but not all of the SE and/or SRE and one or more flanking sequences located upstream and/or downstream of the SE and/or SRE of the target transcript.
在实施方案中，旁侧序列在SE和/或SRE的一侧或两侧包含1个或多个、2个或更多个、3个或更多个、4个或更多个、5个或更多个、10个或更多个、15个或更多个、或20个或更多个碱基。在实施方案中，旁侧序列在SE和/或SRE的一侧或两侧包含25个或更少个、20个或更少个、15个或更少个、10个或更少个、5个或更少个、4个或更少个、3个或更少个、或2个或更少个碱基。在实施方案中，旁侧序列在SE和/或SRE的一侧或两侧包含1个至25个、1个至20个、1个至15个、1个至10个、1个至5个、1个至4个、1个至3个、或1至2个碱基。在实施方案中，旁侧序列在SE和/或SRE的一侧或两侧包含2个至25个、2个至20个、2个至15个、2个至10个、2个至5个、2个至4个、或2个至3个碱基。在实施方案中，旁侧序列在SE和/或SRE的一侧或两侧包含3个至25个、3个至20个、3个至15个、3个至10个、3个至5个、或3个至4个碱基。在实施方案中，旁侧序列在SE和/或SRE的一侧或两侧包含4个至25个、4个至20个、4个至15个、4个至10个、或4个至5个碱基。在实施方案中，旁侧序列在SE和/或SRE的一侧或两侧包含5个至25个、5个至20个、5个至15个、或5个至10个碱基。在实施方案中，旁侧序列在SE和/或SRE的一侧或两侧包含10个至25个、10个至20个、或10个至15个碱基。在实施方案中，旁侧序列在SE和/或SRE的一侧或两侧包含15个至25个或15个至20个碱基。在实施方案中，旁侧序列在SE和/或SRE的一侧或两侧包含20个至25个碱基。在实施方案中，旁侧序列包含间插序列或其一部分。In embodiments, the flanking sequence comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, or 20 or more bases on one or both sides of the SE and/or SRE. In embodiments, the flanking sequence comprises 25 or less, 20 or less, 15 or less, 10 or less, 5 or less, 4 or less, 3 or less, or 2 or less bases on one or both sides of the SE and/or SRE. In embodiments, the flanking sequence comprises 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 bases on one or both sides of the SE and/or SRE. In embodiments, the flanking sequence comprises 2 to 25, 2 to 20, 2 to 15, 2 to 10, 2 to 5, 2 to 4, or 2 to 3 bases on one or both sides of the SE and/or SRE. In embodiments, the flanking sequence comprises 3 to 25, 3 to 20, 3 to 15, 3 to 10, 3 to 5, or 3 to 4 bases on one or both sides of the SE and/or SRE. In embodiments, the flanking sequence comprises 4 to 25, 4 to 20, 4 to 15, 4 to 10, or 4 to 5 bases on one or both sides of the SE and/or SRE. In embodiments, the flanking sequence comprises 5 to 25, 5 to 20, 5 to 15, or 5 to 10 bases on one or both sides of the SE and/or SRE. In an embodiment, the flanking sequence comprises 10 to 25, 10 to 20, or 10 to 15 bases on one or both sides of the SE and/or SRE. In an embodiment, the flanking sequence comprises 15 to 25 or 15 to 20 bases on one or both sides of the SE and/or SRE. In an embodiment, the flanking sequence comprises 20 to 25 bases on one or both sides of the SE and/or SRE. In an embodiment, the flanking sequence comprises an intervening sequence or a portion thereof.
在实施方案中，AC与包含靶转录物的5'ss的至少一部分的靶核苷酸序列杂交。在实施方案中，AC与包含靶转录物的外显子/内含子连接的至少一部分的靶核苷酸序列杂交。在实施方案中，AC与包含靶转录物的3'ss的至少一部分的靶核苷酸序列杂交。在实施方案中，AC与包含靶转录物的Py束、BPS、末端“AG”和/或内含子/外显子连接的至少一部分的靶核苷酸序列杂交。In an embodiment, AC hybridizes to a target nucleotide sequence comprising at least a portion of the 5'ss of a target transcript. In an embodiment, AC hybridizes to a target nucleotide sequence comprising at least a portion of an exon/intron junction of a target transcript. In an embodiment, AC hybridizes to a target nucleotide sequence comprising at least a portion of the 3'ss of a target transcript. In an embodiment, AC hybridizes to a target nucleotide sequence comprising at least a portion of a Py tract, a BPS, a terminal "AG", and/or an intron/exon junction of a target transcript.
在实施方案中，AC与包含靶转录物的剪接调控元件(SRE)的至少一部分的靶核苷酸序列杂交。在实施方案中，AC与包含靶转录物的整个SRE的靶核苷酸序列杂交。在实施方案中，AC与包含靶转录物的多个SRE的靶核苷酸序列杂交。在实施方案中，AC与包含靶转录物的多个SRE以及靶转录物的SRE之间的间插序列的靶核苷酸序列杂交。在实施方案中，AC与包含靶转录物的ESE的至少一部分的靶核苷酸序列杂交。在实施方案中，AC与包含ISE的至少一部分的靶核苷酸序列杂交。在实施方案中，AC与包含靶转录物的ESS的至少一部分的靶核苷酸序列杂交。在实施方案中，AC与包含靶转录物的ISS的至少一部分的靶核苷酸序列杂交。In an embodiment, AC hybridizes to a target nucleotide sequence comprising at least a portion of a splicing regulatory element (SRE) of a target transcript. In an embodiment, AC hybridizes to a target nucleotide sequence comprising the entire SRE of a target transcript. In an embodiment, AC hybridizes to a target nucleotide sequence comprising multiple SREs of a target transcript. In an embodiment, AC hybridizes to a target nucleotide sequence comprising multiple SREs of a target transcript and an intervening sequence between the SREs of a target transcript. In an embodiment, AC hybridizes to a target nucleotide sequence comprising at least a portion of an ESE of a target transcript. In an embodiment, AC hybridizes to a target nucleotide sequence comprising at least a portion of an ISE. In an embodiment, AC hybridizes to a target nucleotide sequence comprising at least a portion of an ESS of a target transcript. In an embodiment, AC hybridizes to a target nucleotide sequence comprising at least a portion of an ISS of a target transcript.
在实施方案中，AC与包含靶转录物的末端茎环(TLS)的至少一部分的靶核苷酸序列杂交。In embodiments, the AC hybridizes to a target nucleotide sequence comprising at least a portion of a terminal stem-loop (TLS) of a target transcript.
在实施方案中，AC与靶转录物的异常SE和/或SRE的至少一部分杂交，其中异常SE和/或SRE由靶基因中的突变产生。In embodiments, the AC hybridizes to at least a portion of an aberrant SE and/or SRE of a target transcript, wherein the aberrant SE and/or SRE results from a mutation in the target gene.
在实施方案中，AC与包含靶转录物的SE和/或SRE、外显子/内含子连接或内含子/外显子连接的至少一部分的靶核苷酸序列杂交。在实施方案中，AC与由于靶转录物的重排或缺失而包含异常融合连接的靶核苷酸序列杂交。在实施方案中，AC与靶转录物的选择性剪接的mRNA中的特定外显子杂交。In an embodiment, the AC hybridizes to a target nucleotide sequence comprising at least a portion of an SE and/or SRE, an exon/intron junction, or an intron/exon junction of a target transcript. In an embodiment, the AC hybridizes to a target nucleotide sequence comprising an aberrant fusion junction due to a rearrangement or deletion of the target transcript. In an embodiment, the AC hybridizes to a specific exon in an alternatively spliced mRNA of a target transcript.
在实施方案中，AC与包含IRF-5、GYS1和/或DUX4靶转录物的剪接元件(SE)的至少一部分的靶核苷酸序列杂交。在实施方案中，AC与包含IRF-5、GYS1和/或DUX4靶转录物的整个SE的靶核苷酸序列杂交。在实施方案中，AC与包含IRF-5、GYS1和/或DUX4靶转录物的多个SE的靶核苷酸序列杂交。在实施方案中，AC与包含靶转录物的多个SE以及IRF-5、GYS1和/或DUX4靶转录物的SE之间的间插序列的靶核苷酸序列杂交。在实施方案中，AC与IRF-5、GYS1和/或DUX4靶转录物的SE的至少一部分和SE的一个或多个旁侧序列杂交。In an embodiment, AC hybridizes to a target nucleotide sequence comprising at least a portion of a splicing element (SE) of an IRF-5, GYS1, and/or DUX4 target transcript. In an embodiment, AC hybridizes to a target nucleotide sequence comprising the entire SE of an IRF-5, GYS1, and/or DUX4 target transcript. In an embodiment, AC hybridizes to a target nucleotide sequence comprising multiple SEs of an IRF-5, GYS1, and/or DUX4 target transcript. In an embodiment, AC hybridizes to a target nucleotide sequence comprising multiple SEs of a target transcript and an intervening sequence between SEs of an IRF-5, GYS1, and/or DUX4 target transcript. In an embodiment, AC hybridizes to at least a portion of an SE of an IRF-5, GYS1, and/or DUX4 target transcript and one or more flanking sequences of an SE.
在实施方案中，AC与包含IRF-5靶转录物的5'ss的至少一部分的靶核苷酸序列杂交。在实施方案中，AC与包含IRF-5靶转录物的外显子/内含子连接的至少一部分的靶核苷酸序列杂交。在实施方案中，AC与包含IRF-5靶转录物的3'ss的至少一部分的靶核苷酸序列杂交。在实施方案中，AC与包含IRF-5靶转录物的Py束、BPS、末端“AG”和/或内含子/外显子连接的至少一部分的靶核苷酸序列杂交。In an embodiment, AC hybridizes to a target nucleotide sequence comprising at least a portion of the 5'ss of an IRF-5 target transcript. In an embodiment, AC hybridizes to a target nucleotide sequence comprising at least a portion of an exon/intron junction of an IRF-5 target transcript. In an embodiment, AC hybridizes to a target nucleotide sequence comprising at least a portion of the 3'ss of an IRF-5 target transcript. In an embodiment, AC hybridizes to a target nucleotide sequence comprising at least a portion of a Py tract, a BPS, a terminal "AG", and/or an intron/exon junction of an IRF-5 target transcript.
在实施方案中，AC结合不包含靶转录物的SE的至少一部分或SRE的至少一部分的靶核苷酸序列。在实施方案中，AC结合与SE和/或SRE足够接近的靶核苷酸序列以调节靶转录物的剪接。在实施方案中，结合不包含靶转录物的SE的至少一部分或SRE的至少一部分的靶核苷酸序列并且调节靶转录物的剪接的AC可以结合靶转录物并且在空间上阻断翻译因子或反式作用调节因子与SE或SRE的结合。In an embodiment, the AC binds to a target nucleotide sequence that does not comprise at least a portion of the SE or at least a portion of the SRE of the target transcript. In an embodiment, the AC binds to a target nucleotide sequence that is sufficiently close to the SE and/or SRE to regulate the splicing of the target transcript. In an embodiment, an AC that binds to a target nucleotide sequence that does not comprise at least a portion of the SE or at least a portion of the SRE of the target transcript and regulates the splicing of the target transcript can bind to the target transcript and sterically block the binding of a translation factor or a trans-acting regulatory factor to the SE or SRE.
在实施方案中，AC结合靶核苷酸序列，该靶核苷酸序列的3'端和/或5'端与靶转录物的SE和/或SRE的5'端和/或3'端相距1个或多个、2个或更多个、3个或更多个、4个或更多个、5个或更多个、10个或更多个、15个或更多个、或20个或更多个核苷酸。在实施方案中，AC结合靶核苷酸序列，该靶核苷酸序列的3'端和/或5'端与靶转录物的SE和/或SRE的5'端和/或3'端相距25个或更少个、20个或更少个、15个或更少个、10个或更少个、5个或更少个、4个或更少个、3个或更少个、或2个或更少个核苷酸。在实施方案中，AC结合靶核苷酸序列，该靶核苷酸序列的3'端和/或5'端与靶转录物的SE和/或SRE的5'端和/或3'端相距1个至25个、1个至20个、1个至15个、1个至10个、1个至5个、1个至4个、1个至3个、或1个至2个核苷酸。在实施方案中，AC结合靶核苷酸序列，该靶核苷酸序列的3'端和/或5'端与靶转录物的SE和/或SRE的5'端和/或3'端相距2个至25个、2个至20个、2个至15个、2个至10个、2个至5个、2个至4个、或2个至3个核苷酸。在实施方案中，AC结合靶核苷酸序列，该靶核苷酸序列的3'端和/或5'端与靶转录物的SE和/或SRE的5'端和/或3'端相距3个至25个、3个至20个、3个至15个、3个至10个、3个至5个、或3个至4个核苷酸。在实施方案中，AC结合靶核苷酸序列，该靶核苷酸序列的3'端和/或5'端与靶转录物的SE和/或SRE的5'端和/或3'端相距4个至25个、4个至20个、4个至15个、4个至10个、或4个至5个核苷酸。在实施方案中，AC结合靶核苷酸序列，该靶核苷酸序列的3'端和/或5'端与靶转录物的SE和/或SRE的5'端和/或3'端相距5个至25个、5个至20个、5个至15个、或5个至10个核苷酸。在实施方案中，AC结合靶核苷酸序列，该靶核苷酸序列的3'端和/或5'端与靶转录物的SE和/或SRE的5'端和/或3'端相距10个至25个或10个至20个核苷酸。在实施方案中，AC结合靶核苷酸序列，该靶核苷酸序列的3'端和/或5'端与靶转录物的SE和/或SRE的5'端和/或3'端相距20个至25个核苷酸。In embodiments, AC binds to a target nucleotide sequence whose 3' and/or 5' ends are 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, or 20 or more nucleotides away from the 5' and/or 3' ends of the SE and/or SRE of the target transcript. In embodiments, AC binds to a target nucleotide sequence whose 3' and/or 5' ends are 25 or less, 20 or less, 15 or less, 10 or less, 5 or less, 4 or less, 3 or less, or 2 or less nucleotides away from the 5' and/or 3' ends of the SE and/or SRE of the target transcript. In embodiments, AC binds to a target nucleotide sequence whose 3' and/or 5' ends are 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 nucleotides away from the 5' and/or 3' ends of the SE and/or SRE of the target transcript. In embodiments, AC binds to a target nucleotide sequence whose 3' and/or 5' ends are 2 to 25, 2 to 20, 2 to 15, 2 to 10, 2 to 5, 2 to 4, or 2 to 3 nucleotides away from the 5' and/or 3' ends of the SE and/or SRE of the target transcript. In embodiments, AC binds to a target nucleotide sequence whose 3' and/or 5' ends are 3 to 25, 3 to 20, 3 to 15, 3 to 10, 3 to 5, or 3 to 4 nucleotides away from the 5' and/or 3' ends of the SE and/or SRE of the target transcript. In embodiments, AC binds to a target nucleotide sequence whose 3' and/or 5' ends are 4 to 25, 4 to 20, 4 to 15, 4 to 10, or 4 to 5 nucleotides away from the 5' and/or 3' ends of the SE and/or SRE of the target transcript. In embodiments, AC binds to a target nucleotide sequence whose 3' and/or 5' ends are 5 to 25, 5 to 20, 5 to 15, or 5 to 10 nucleotides away from the 5' and/or 3' ends of the SE and/or SRE of the target transcript. In an embodiment, AC binds to a target nucleotide sequence whose 3' and/or 5' ends are 10 to 25 or 10 to 20 nucleotides away from the 5' and/or 3' ends of the SE and/or SRE of the target transcript. In an embodiment, AC binds to a target nucleotide sequence whose 3' and/or 5' ends are 20 to 25 nucleotides away from the 5' and/or 3' ends of the SE and/or SRE of the target transcript.
在实施方案中，AC与长度为约5个至约50个核酸的靶核苷酸序列杂交。在实施方案中，AC与靶核苷酸序列的长度相同。在实施方案中，AC与靶核苷酸序列的长度不同。在实施方案中，AC比靶核酸序列长。In an embodiment, AC hybridizes to a target nucleotide sequence of about 5 to about 50 nucleic acids in length. In an embodiment, AC is the same length as the target nucleotide sequence. In an embodiment, AC is different in length from the target nucleotide sequence. In an embodiment, AC is longer than the target nucleic acid sequence.
在实施方案中，AC的长度为5个或更多个、10个或更多个、15个或更多个、20个或更多个、25个或更多个、30个或更多个、35个或更多个、40个或更多个、或45个或更多个核酸。在实施方案中，AC的长度为50个或更少个、45个或更少个、40个或更少个、35个或更少个、30个或更少个、25个或更少个、20个或更少个、15个或更少个、或10个或更少个核酸。在实施方案中，AC的长度为5个至50个、5个至45个、5个至40个、5个至35个、5个至30个、5个至25个、5个至20个、5个至15个、或5个至10个核酸。在实施方案中，AC的长度为10个至50个、10个至45个、10个至40个、10个至35个、10个至30个、10个至25个、10个至20个、或10个至15个核酸。在实施方案中，AC的长度为15个至50个、15个至45个、15个至40个、15个至35个、15个至30个、15个至25个、或15个至20个核酸。在实施方案中，AC的长度为20个至50个、20个至45个、20个至40个、20个至35个、20个至30个、或20个至25个核酸。在实施方案中，AC的长度为25个至50个、25个至45个、25个至40个、25个至35个、或25个至30个核酸。在实施方案中，AC的长度为30个至50个、30个至45个、30个至40个、或30个至35个核酸。在实施方案中，AC的长度为35个至50个、35个至45个、或35个至40个核酸。在实施方案中，AC的长度为40个至50个或40个至45个核酸。在实施方案中，AC的长度为45个至50个核酸。在实施方案中，AC的长度为5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、26个、27个、28个、29个、30个、31个、32个、33个、34个、35个、36个、37个、38个、39个、40个、41个、42个、43个、44个、45个、46个、47个、48个、49个或50个核酸。In embodiments, AC is 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, or 45 or more nucleic acids in length. In embodiments, AC is 50 or less, 45 or less, 40 or less, 35 or less, 30 or less, 25 or less, 20 or less, 15 or less, or 10 or less nucleic acids in length. In embodiments, AC is 5 to 50, 5 to 45, 5 to 40, 5 to 35, 5 to 30, 5 to 25, 5 to 20, 5 to 15, or 5 to 10 nucleic acids in length. In embodiments, AC is 10 to 50, 10 to 45, 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, or 10 to 15 nucleic acids in length. In embodiments, AC is 15 to 50, 15 to 45, 15 to 40, 15 to 35, 15 to 30, 15 to 25, or 15 to 20 nucleic acids in length. In embodiments, AC is 20 to 50, 20 to 45, 20 to 40, 20 to 35, 20 to 30, or 20 to 25 nucleic acids in length. In embodiments, AC is 25 to 50, 25 to 45, 25 to 40, 25 to 35, or 25 to 30 nucleic acids in length. In embodiments, AC is 30 to 50, 30 to 45, 30 to 40, or 30 to 35 nucleic acids in length. In embodiments, AC is 35 to 50, 35 to 45, or 35 to 40 nucleic acids in length. In embodiments, AC is 40 to 50 or 40 to 45 nucleic acids in length. In embodiments, AC is 45 to 50 nucleic acids in length. In embodiments, AC is 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 nucleic acids in length.
在实施方案中，AC与靶核苷酸序列具有100％互补性。在实施方案中，AC与靶核苷酸序列不具有100％互补性。如本文所用，术语“互补百分比”是指与寡聚化合物或核酸(例如，靶核苷酸序列)的对应核碱基具有核碱基互补性的AC的核碱基数量除以AC的总长度(核碱基数量)。本领域技术人员认识到，在不消除反义化合物活性的情况下，包含错配是可能的。In an embodiment, AC has 100% complementarity with a target nucleotide sequence. In an embodiment, AC does not have 100% complementarity with a target nucleotide sequence. As used herein, the term "complementarity percentage" refers to the number of nucleobases of AC that have nucleobase complementarity with the corresponding nucleobase of an oligomeric compound or nucleic acid (e.g., a target nucleotide sequence) divided by the total length (number of nucleobases) of AC. It is recognized by those skilled in the art that it is possible to include mismatches without eliminating the activity of antisense compounds.
在实施方案中，AC包含与靶核苷酸序列的20％或更少、15％或更少、10％或更少、5％或更少、或零的错配。在一些实施方案中，AC包含与靶核苷酸序列的5％或更多、10％或更多、或15％或更多的错配。在实施方案中，AC包含与靶核苷酸序列的零至5％、零至10％、零至15％、或零至20％的错配。在实施方案中，AC包含与靶核苷酸序列的5％至10％、5％至15％、或5％至20％的错配。在实施方案中，AC包含与靶核苷酸序列的10％至15％或10％至20％的错配。在实施方案中，AC包含与靶核苷酸序列的10％至20％的错配。In embodiments, AC comprises 20% or less, 15% or less, 10% or less, 5% or less, or zero mismatches with the target nucleotide sequence. In some embodiments, AC comprises 5% or more, 10% or more, or 15% or more mismatches with the target nucleotide sequence. In embodiments, AC comprises zero to 5%, zero to 10%, zero to 15%, or zero to 20% mismatches with the target nucleotide sequence. In embodiments, AC comprises 5% to 10%, 5% to 15%, or 5% to 20% mismatches with the target nucleotide sequence. In embodiments, AC comprises 10% to 15% or 10% to 20% mismatches with the target nucleotide sequence. In embodiments, AC comprises 10% to 20% mismatches with the target nucleotide sequence.
在实施方案中，AC与靶核苷酸序列具有80％或更大、85％或更大、90％或更大、95％或更大、96％或更大、97％或更大、98％或更大、或99％或更大的互补性。在实施方案中，AC与靶核苷酸序列具有100％或更小、99％或更小、98％或更小、97％或更小、96％或更小、95％或更小、90％或更小、85％或更小的互补性。在实施方案中，AC与靶核苷酸序列具有80％至100％、80％至99％、80％至98％、80％至97％、80％至96％、80％至95％、80％至90％、或80％至85％的互补性。在实施方案中，AC与靶核苷酸序列具有85％至100％、85％至99％、85％至98％、85％至97％、85％至96％、85％至95％、或85％至90％的互补性。在实施方案中，AC与靶核苷酸序列具有90％至100％、90％至99％、90％至98％、90％至97％、90％至96％、或90％至95％的互补性。在实施方案中，AC与靶核苷酸序列具有95％至100％、95％至99％、95％至98％、95％至97％、或95％至96％的互补性。在实施方案中，AC与靶核苷酸序列具有96％至100％、96％至99％、96％至98％、或96％至97％的互补性。在实施方案中，AC与靶核苷酸序列具有97％至100％、97％至99％、或97％至98％的互补性。在实施方案中，AC与靶核苷酸序列具有98％至100％或98％至99％的互补性。在实施方案中，AC与靶核苷酸序列具有99％至100％的互补性。通过将互补核碱基的数量除以寡核苷酸的核碱基总数来计算寡核苷酸的互补性百分比。In embodiments, AC has 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more complementarity to the target nucleotide sequence. In embodiments, AC has 100% or less, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 90% or less, 85% or less complementarity to the target nucleotide sequence. In embodiments, AC has 80% to 100%, 80% to 99%, 80% to 98%, 80% to 97%, 80% to 96%, 80% to 95%, 80% to 90%, or 80% to 85% complementarity to the target nucleotide sequence. In embodiments, AC has 85% to 100%, 85% to 99%, 85% to 98%, 85% to 97%, 85% to 96%, 85% to 95%, or 85% to 90% complementarity with the target nucleotide sequence. In embodiments, AC has 90% to 100%, 90% to 99%, 90% to 98%, 90% to 97%, 90% to 96%, or 90% to 95% complementarity with the target nucleotide sequence. In embodiments, AC has 95% to 100%, 95% to 99%, 95% to 98%, 95% to 97%, or 95% to 96% complementarity with the target nucleotide sequence. In embodiments, AC has 96% to 100%, 96% to 99%, 96% to 98%, or 96% to 97% complementarity with the target nucleotide sequence. In embodiments, AC has 97% to 100%, 97% to 99%, or 97% to 98% complementarity with the target nucleotide sequence. In embodiments, AC has 98% to 100% or 98% to 99% complementarity with the target nucleotide sequence. In embodiments, AC has 99% to 100% complementarity with the target nucleotide sequence. The percent complementarity of an oligonucleotide is calculated by dividing the number of complementary nucleobases by the total number of nucleobases of the oligonucleotide.
在实施方案中，相对于AC杂交的靶核酸序列，AC包含1个、2个、3个、4个或5个错配。在实施方案中，相对于AC杂交的靶核酸序列，AC包含1个或2个错配。在实施方案中，相对于AC杂交的靶核酸序列，AC不包含错配。In embodiments, AC comprises 1, 2, 3, 4 or 5 mismatches relative to the target nucleic acid sequence to which AC hybridizes. In embodiments, AC comprises 1 or 2 mismatches relative to the target nucleic acid sequence to which AC hybridizes. In embodiments, AC comprises no mismatches relative to the target nucleic acid sequence to which AC hybridizes.
在实施方案中，与未修饰的化合物相比，掺入核苷酸亲和力修饰允许更大数量的错配。类似地，某些寡核苷酸序列可比其他寡核苷酸序列更耐受错配。本领域普通技术人员能够确定AC与靶核苷酸序列之间的适当数量的错配，诸如通过确定热解链温度(Tm)。Tm或ΔTm可通过本领域普通技术人员熟悉的技术来计算。例如，Freier等人(Nucleic AcidsResearch(1997),25,22:4429-4443)描述的技术允许本领域普通技术人员评价核苷酸修饰提高RNA:DNA双链体解链温度的能力。In an embodiment, compared with unmodified compounds, incorporation of nucleotide affinity modifications allows a greater number of mismatches. Similarly, certain oligonucleotide sequences may be more tolerant to mismatches than other oligonucleotide sequences. One of ordinary skill in the art can determine the appropriate number of mismatches between AC and the target nucleotide sequence, such as by determining the thermal melting temperature (Tm). Tm or ΔTm can be calculated by techniques familiar to one of ordinary skill in the art. For example, the techniques described by Freier et al. (Nucleic Acids Research (1997), 25, 22: 4429-4443) allow one of ordinary skill in the art to evaluate the ability of nucleotide modifications to improve RNA: DNA duplex melting temperatures.
在实施方案中，AC包含在严格条件下与靶转录物杂交的序列，并且包含在严格条件下不与靶转录物杂交的序列。在实施方案中，AC包含在严格条件下不与靶序列杂交的第一序列、在严格条件下不与靶序列杂交的第二序列和在严格条件下与靶序列杂交的第三序列，其中第三序列位于第一序列与第二序列之间。In an embodiment, AC comprises a sequence that hybridizes to the target transcript under stringent conditions, and comprises a sequence that does not hybridize to the target transcript under stringent conditions. In an embodiment, AC comprises a first sequence that does not hybridize to the target sequence under stringent conditions, a second sequence that does not hybridize to the target sequence under stringent conditions, and a third sequence that hybridizes to the target sequence under stringent conditions, wherein the third sequence is located between the first sequence and the second sequence.
在实施方案中，AC与包含IRF-5、GYS1和/或DUX4靶转录物的剪接调控元件(SRE)的至少一部分的靶核苷酸序列杂交。在实施方案中，AC与包含IRF-5、GYS1和/或DUX4靶转录物的整个SRE的靶核苷酸序列杂交。在实施方案中，AC与包含IRF-5、GYS1和/或DUX4靶转录物的多个SRE的靶核苷酸序列杂交。在实施方案中，AC与包含IRF-5、GYS1和/或DUX4靶转录物的多个SRE以及SRE之间的间插序列的靶核苷酸序列杂交。在实施方案中，AC与IRF-5、GYS1和/或DUX4靶转录物的SE的至少一部分和SE的一个或多个旁侧序列杂交。In an embodiment, AC hybridizes to a target nucleotide sequence comprising at least a portion of a splicing regulatory element (SRE) of an IRF-5, GYS1, and/or DUX4 target transcript. In an embodiment, AC hybridizes to a target nucleotide sequence comprising the entire SRE of an IRF-5, GYS1, and/or DUX4 target transcript. In an embodiment, AC hybridizes to a target nucleotide sequence comprising multiple SREs of an IRF-5, GYS1, and/or DUX4 target transcript. In an embodiment, AC hybridizes to a target nucleotide sequence comprising multiple SREs of an IRF-5, GYS1, and/or DUX4 target transcript and an intervening sequence between SREs. In an embodiment, AC hybridizes to at least a portion of an SE of an IRF-5, GYS1, and/or DUX4 target transcript and one or more flanking sequences of an SE.
在实施方案中，AC与包含IRF-5、GYS1和/或DUX4靶转录物的ESE的至少一部分的靶核苷酸序列杂交。在实施方案中，AC与包含IRF-5、GYS1和/或DUX4靶转录物的ISE的至少一部分的靶核苷酸序列杂交。在实施方案中，AC与包含IRF-5、GYS1和/或DUX4靶转录物的ESS的至少一部分的靶核苷酸序列杂交。在实施方案中，AC与包含IRF-5、GYS1和/或DUX4靶转录物的ISS的至少一部分的靶核苷酸序列杂交。In an embodiment, AC hybridizes to a target nucleotide sequence comprising at least a portion of an ESE of an IRF-5, GYS1, and/or DUX4 target transcript. In an embodiment, AC hybridizes to a target nucleotide sequence comprising at least a portion of an ISE of an IRF-5, GYS1, and/or DUX4 target transcript. In an embodiment, AC hybridizes to a target nucleotide sequence comprising at least a portion of an ESS of an IRF-5, GYS1, and/or DUX4 target transcript. In an embodiment, AC hybridizes to a target nucleotide sequence comprising at least a portion of an ISS of an IRF-5, GYS1, and/or DUX4 target transcript.
在实施方案中，AC与包含IRF-5、GYS1和/或DUX4靶转录物的末端茎环(TLS)的至少一部分的靶核苷酸序列杂交。In embodiments, the AC hybridizes to a target nucleotide sequence comprising at least a portion of a terminal stem-loop (TLS) of an IRF-5, GYS1 and/or DUX4 target transcript.
在实施方案中，AC与IRF-5、GYS1和/或DUX4靶转录物的异常SE和/或SRE的至少一部分杂交，其中异常SE和/或SRE由IRF-5、GYS1和/或DUX4靶转录物中的突变产生。In embodiments, the AC hybridizes to at least a portion of an aberrant SE and/or SRE of an IRF-5, GYS1 and/or DUX4 target transcript, wherein the aberrant SE and/or SRE results from a mutation in the IRF-5, GYS1 and/or DUX4 target transcript.
在实施方案中，AC与包含IRF-5、GYS1和/或DUX4靶转录物的外显子-外显子连接、内含子-外显子连接和/或外显子-内含子连接的至少一部分的靶核苷酸序列杂交。在实施方案中，AC与由于IRF-5、GYS1和/或DUX4靶转录物的一部分的重排或缺失而包含异常融合连接的靶核苷酸序列杂交。在实施方案中，AC与IRF-5、GYS1和/或DUX4靶转录物中选择性剪接的mRNA中的特定外显子杂交。In an embodiment, AC hybridizes to a target nucleotide sequence comprising at least a portion of an exon-exon junction, an intron-exon junction, and/or an exon-intron junction of an IRF-5, GYS1, and/or DUX4 target transcript. In an embodiment, AC hybridizes to a target nucleotide sequence comprising an abnormal fusion junction due to a rearrangement or deletion of a portion of an IRF-5, GYS1, and/or DUX4 target transcript. In an embodiment, AC hybridizes to a specific exon in an alternatively spliced mRNA in an IRF-5, GYS1, and/or DUX4 target transcript.
在实施方案中，AC结合不包含IRF-5、GYS1和/或DUX4靶转录物的ISS的SE的至少一部分或SRE的至少一部分的靶核苷酸序列。在实施方案中，AC结合与SE和/或SRE足够接近的靶核苷酸序列以调节IRF-5、GYS1和/或DUX4靶转录物的ISS的剪接。In an embodiment, AC binds to a target nucleotide sequence that does not include at least a portion of a SE or at least a portion of a SRE of an ISS of an IRF-5, GYS1, and/or DUX4 target transcript. In an embodiment, AC binds to a target nucleotide sequence that is sufficiently close to a SE and/or SRE to modulate splicing of an ISS of an IRF-5, GYS1, and/or DUX4 target transcript.
在实施方案中，AC结合靶核苷酸序列，该靶核苷酸序列的3'端和/或5'端与IRF-5、GYS1和/或DUX4靶转录物的SE和/或SRE的5'端和/或3'端相距1个或多个、2个或更多个、3个或更多个、4个或更多个、5个或更多个、10个或更多个、15个或更多个、或20个或更多个核苷酸。在实施方案中，AC结合靶核苷酸序列，该靶核苷酸序列的3'端和/或5'端与IRF-5、GYS1和/或DUX4靶转录物的SE和/或SRE的5'端和/或3'端相距25个或更少个、20个或更少个、15个或更少个、10个或更少个、5个或更少个、4个或更少个、3个或更少个、或2个或更少个核苷酸。在实施方案中，AC结合靶核苷酸序列，该靶核苷酸序列的3'端和/或5'端与IRF-5、GYS1和/或DUX4靶转录物的SE和/或SRE的5'端和/或3'端相距1个至25个、1个至20个、1个至15个、1个至10个、1个至5个、1个至4个、1个至3个、或1个至2个核苷酸。在实施方案中，AC结合靶核苷酸序列，该靶核苷酸序列的3'端和/或5'端与IRF-5、GYS1和/或DUX4靶转录物的SE和/或SRE的5'端和/或3'端相距2个至25个、2个至20个、2个至15个、2个至10个、2个至5个、2个至4个、或2个至3个核苷酸。在实施方案中，AC结合靶核苷酸序列，该靶核苷酸序列的3'端和/或5'端与IRF-5、GYS1和/或DUX4靶转录物的SE和/或SRE的5'端和/或3'端相距3个至25个、3个至20个、3个至15个、3个至10个、3个至5个、或3个至4个核苷酸。在实施方案中，AC结合靶核苷酸序列，该靶核苷酸序列的3'端和/或5'端与IRF-5、GYS1和/或DUX4靶转录物的SE和/或SRE的5'端和/或3'端相距4个至25个、4个至20个、4个至15个、4个至10个、或4个至5个核苷酸。在实施方案中，AC结合靶核苷酸序列，该靶核苷酸序列的3'端和/或5'端与IRF-5、GYS1和/或DUX4靶转录物的SE和/或SRE的5'端和/或3'端相距5个至25个、5个至20个、5个至15个、或5个至10个核苷酸。在实施方案中，AC结合靶核苷酸序列，该靶核苷酸序列的3'端和/或5'端与IRF-5、GYS1和/或DUX4靶转录物的SE和/或SRE的5'端和/或3'端相距10个至25个或10个至20个核苷酸。在实施方案中，AC结合靶核苷酸序列，该靶核苷酸序列的3'端和/或5'端与IRF-5、GYS1和/或DUX4靶转录物的SE和/或SRE的5'端和/或3'端相距20个至25个核苷酸。In embodiments, AC binds to a target nucleotide sequence whose 3' and/or 5' ends are 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, or 20 or more nucleotides away from the 5' and/or 3' ends of the SE and/or SRE of the IRF-5, GYS1, and/or DUX4 target transcripts. In embodiments, AC binds to a target nucleotide sequence whose 3' and/or 5' ends are 25 or less, 20 or less, 15 or less, 10 or less, 5 or less, 4 or less, 3 or less, or 2 or less nucleotides away from the 5' and/or 3' ends of the SE and/or SRE of the IRF-5, GYS1, and/or DUX4 target transcripts. In embodiments, AC binds to a target nucleotide sequence whose 3' and/or 5' ends are 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 nucleotides away from the 5' and/or 3' ends of the SE and/or SRE of the IRF-5, GYS1, and/or DUX4 target transcripts. In embodiments, AC binds to a target nucleotide sequence whose 3' and/or 5' ends are 2 to 25, 2 to 20, 2 to 15, 2 to 10, 2 to 5, 2 to 4, or 2 to 3 nucleotides away from the 5' and/or 3' ends of the SE and/or SRE of the IRF-5, GYS1, and/or DUX4 target transcripts. In embodiments, AC binds to a target nucleotide sequence whose 3' and/or 5' ends are 3 to 25, 3 to 20, 3 to 15, 3 to 10, 3 to 5, or 3 to 4 nucleotides away from the 5' and/or 3' ends of the SE and/or SRE of the IRF-5, GYS1, and/or DUX4 target transcripts. In embodiments, AC binds to a target nucleotide sequence whose 3' and/or 5' ends are 4 to 25, 4 to 20, 4 to 15, 4 to 10, or 4 to 5 nucleotides away from the 5' and/or 3' ends of the SE and/or SRE of the IRF-5, GYS1, and/or DUX4 target transcripts. In embodiments, AC binds to a target nucleotide sequence whose 3' and/or 5' ends are 5 to 25, 5 to 20, 5 to 15, or 5 to 10 nucleotides away from the 5' and/or 3' ends of the SE and/or SRE of the IRF-5, GYS1, and/or DUX4 target transcripts. In embodiments, AC binds to a target nucleotide sequence whose 3' and/or 5' ends are 10 to 25 or 10 to 20 nucleotides away from the 5' and/or 3' ends of the SE and/or SRE of the IRF-5, GYS1, and/or DUX4 target transcripts. In embodiments, AC binds to a target nucleotide sequence whose 3' and/or 5' ends are 20 to 25 nucleotides away from the 5' and/or 3' ends of the SE and/or SRE of the IRF-5, GYS1, and/or DUX4 target transcripts.
在实施方案中，AC与IRF-5、GYS1和/或DUX4靶转录物的长度为约5个至约50个核酸的靶核苷酸序列杂交。在实施方案中，AC与靶核苷酸序列的长度相同。在实施方案中，AC与靶核苷酸序列的长度不同。在实施方案中，AC比靶核酸序列长。In an embodiment, AC hybridizes to a target nucleotide sequence of about 5 to about 50 nucleic acids in length of an IRF-5, GYS1 and/or DUX4 target transcript. In an embodiment, AC is the same length as the target nucleotide sequence. In an embodiment, AC is different in length from the target nucleotide sequence. In an embodiment, AC is longer than the target nucleic acid sequence.
在实施方案中，AC与IRF-5、GYS1和/或DUX4靶转录物的靶核苷酸序列具有100％互补性。在实施方案中，AC与靶核苷酸序列不具有100％互补性。如本文所用，术语“互补百分比”是指与寡聚化合物或核酸(例如，靶核苷酸序列)的对应核碱基具有核碱基互补性的AC的核碱基数量除以AC的总长度(核碱基数量)。本领域技术人员认识到，在不消除反义化合物活性的情况下，包含错配是可能的。In an embodiment, AC has 100% complementarity with the target nucleotide sequence of IRF-5, GYS1 and/or DUX4 target transcripts. In an embodiment, AC does not have 100% complementarity with the target nucleotide sequence. As used herein, the term "complementarity percentage" refers to the number of nucleobases of AC that have nucleobase complementarity with the corresponding nucleobase of an oligomeric compound or nucleic acid (e.g., a target nucleotide sequence) divided by the total length (number of nucleobases) of AC. It is recognized by those skilled in the art that it is possible to include mismatches without eliminating the activity of antisense compounds.
在实施方案中，AC包含与IRF-5、GYS1和/或DUX4靶核苷酸序列的20％或更少、15％或更少、10％或更少、5％或更少、或零的错配。在一些实施方案中，AC包含与IRF-5、GYS1和/或DUX4靶转录物的靶核苷酸序列的5％或更多、10％或更多、或15％或更多的错配。在实施方案中，AC包含与IRF-5、GYS1和/或DUX4靶转录物的靶核苷酸序列的零至5％、零至10％、零至15％、或零至20％的错配。在实施方案中，AC包含与IRF-5、GYS1和/或DUX4靶转录物的靶核苷酸序列的5％至10％、5％至15％、或5％至20％的错配。在实施方案中，AC包含与IRF-5、GYS1和/或DUX4靶转录物的靶核苷酸序列的10％至15％或10％至20％的错配。在实施方案中，AC包含与IRF-5、GYS1和/或DUX4靶转录物的靶核苷酸序列的10％至20％的错配。In embodiments, AC comprises 20% or less, 15% or less, 10% or less, 5% or less, or zero mismatches with IRF-5, GYS1, and/or DUX4 target nucleotide sequences. In some embodiments, AC comprises 5% or more, 10% or more, or 15% or more mismatches with the target nucleotide sequences of IRF-5, GYS1, and/or DUX4 target transcripts. In embodiments, AC comprises zero to 5%, zero to 10%, zero to 15%, or zero to 20% mismatches with the target nucleotide sequences of IRF-5, GYS1, and/or DUX4 target transcripts. In embodiments, AC comprises 5% to 10%, 5% to 15%, or 5% to 20% mismatches with the target nucleotide sequences of IRF-5, GYS1, and/or DUX4 target transcripts. In embodiments, AC comprises 10% to 15% or 10% to 20% mismatches with the target nucleotide sequence of the IRF-5, GYS1 and/or DUX4 target transcript. In embodiments, AC comprises 10% to 20% mismatches with the target nucleotide sequence of the IRF-5, GYS1 and/or DUX4 target transcript.
在实施方案中，AC与IRF-5、GYS1和/或DUX4靶转录物的靶核苷酸序列具有80％或更大、85％或更大、90％或更大、95％或更大、96％或更大、97％或更大、98％或更大、或99％或更大的互补性。在实施方案中，AC与IRF-5、GYS1和/或DUX4靶转录物的靶核苷酸序列具有100％或更小、99％或更小、98％或更小、97％或更小、96％或更小、95％或更小、90％或更小、85％或更小的互补性。在实施方案中，AC与IRF-5、GYS1和/或DUX4靶转录物的靶核苷酸序列具有80％至100％、80％至99％、80％至98％、80％至97％、80％至96％、80％至95％、80％至90％、或80％至85％的互补性。在实施方案中，AC与IRF-5、GYS1和/或DUX4靶转录物的靶核苷酸序列具有85％至100％、85％至99％、85％至98％、85％至97％、85％至96％、85％至95％、或85％至90％的互补性。在实施方案中，AC与IRF-5、GYS1和/或DUX4靶转录物的靶核苷酸序列具有90％至100％、90％至99％、90％至98％、90％至97％、90％至96％、或90％至95％的互补性。在实施方案中，AC与IRF-5、GYS1和/或DUX4靶转录物的靶核苷酸序列具有95％至100％、95％至99％、95％至98％、95％至97％、或95％至96％的互补性。在实施方案中，AC与IRF-5、GYS1和/或DUX4靶转录物的靶核苷酸序列具有96％至100％、96％至99％、96％至98％、或96％至97％的互补性。在实施方案中，AC与IRF-5、GYS1和/或DUX4靶转录物的靶核苷酸序列具有97％至100％、97％至99％、或97％至98％的互补性。在实施方案中，AC与靶核苷酸序列具有98％至100％或98％至99％的互补性。在实施方案中，AC与IRF-5、GYS1和/或DUX4靶转录物的靶核苷酸序列具有99％至100％的互补性。通过将互补核碱基的数量除以寡核苷酸的核碱基总数来计算寡核苷酸的互补性百分比。In embodiments, AC has 80% or greater, 85% or greater, 90% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater complementarity to a target nucleotide sequence of an IRF-5, GYS1, and/or DUX4 target transcript. In embodiments, AC has 100% or less, 99% or less, 98% or less, 97% or less, 96% or less, 95% or less, 90% or less, 85% or less complementarity to a target nucleotide sequence of an IRF-5, GYS1, and/or DUX4 target transcript. In embodiments, AC has 80% to 100%, 80% to 99%, 80% to 98%, 80% to 97%, 80% to 96%, 80% to 95%, 80% to 90%, or 80% to 85% complementarity to the target nucleotide sequence of the IRF-5, GYS1 and/or DUX4 target transcript. In embodiments, AC has 85% to 100%, 85% to 99%, 85% to 98%, 85% to 97%, 85% to 96%, 85% to 95%, or 85% to 90% complementarity to the target nucleotide sequence of the IRF-5, GYS1 and/or DUX4 target transcript. In embodiments, AC has 90% to 100%, 90% to 99%, 90% to 98%, 90% to 97%, 90% to 96%, or 90% to 95% complementarity with the target nucleotide sequence of the IRF-5, GYS1 and/or DUX4 target transcript. In embodiments, AC has 95% to 100%, 95% to 99%, 95% to 98%, 95% to 97%, or 95% to 96% complementarity with the target nucleotide sequence of the IRF-5, GYS1 and/or DUX4 target transcript. In embodiments, AC has 96% to 100%, 96% to 99%, 96% to 98%, or 96% to 97% complementarity with the target nucleotide sequence of the IRF-5, GYS1 and/or DUX4 target transcript. In embodiments, AC has 97% to 100%, 97% to 99%, or 97% to 98% complementarity with a target nucleotide sequence of an IRF-5, GYS1, and/or DUX4 target transcript. In embodiments, AC has 98% to 100% or 98% to 99% complementarity with a target nucleotide sequence. In embodiments, AC has 99% to 100% complementarity with a target nucleotide sequence of an IRF-5, GYS1, and/or DUX4 target transcript. The percent complementarity of an oligonucleotide is calculated by dividing the number of complementary nucleobases by the total number of nucleobases of the oligonucleotide.
反义机理Antisense mechanism
在实施方案中，AC调节蛋白质转录、翻译和表达的一个或多个方面。在实施方案中，AC与靶转录物的靶核苷酸序列的杂交调节前mRNA剪接的一个或多个方面。在实施方案中，AC与靶转录物的靶核苷酸序列的杂交恢复对突变的转录物序列的天然剪接。在实施方案中，AC与靶转录物的靶核苷酸序列的杂交导致靶转录物的选择性剪接。In embodiments, AC modulates one or more aspects of protein transcription, translation and expression. In embodiments, hybridization of AC to a target nucleotide sequence of a target transcript modulates one or more aspects of pre-mRNA splicing. In embodiments, hybridization of AC to a target nucleotide sequence of a target transcript restores native splicing to a mutated transcript sequence. In embodiments, hybridization of AC to a target nucleotide sequence of a target transcript results in alternative splicing of the target transcript.
在实施方案中，AC杂交导致一个或多个外显子的外显子包含或外显子跳跃。在实施方案中，外显子跳跃增加由所得mRNA表达的蛋白质的活性。在实施方案中，外显子跳跃降低由所得mRNA表达的蛋白质的活性。在实施方案中，跳过一个或多个外显子诱导mRNA转录物中的移码。在实施方案中，移码得到编码具有降低活性的蛋白质的mRNA。在实施方案中，移码得到截短的或非功能性的蛋白质。在实施方案中，跳过一个或多个外显子导致在mRNA中引入提前终止密码子。在实施方案中，跳过一个或多个外显子导致mRNA转录物通过无义介导的衰变而降解。在实施方案中，跳过的外显子序列包含核酸缺失、取代或插入。在实施方案中，跳过的外显子不包含序列突变。在实施方案中，反义寡核苷酸与靶前mRNA转录物内的靶核苷酸序列杂交导致不同蛋白同种型的表达。In embodiments, AC hybridization results in exon inclusion or exon skipping of one or more exons. In embodiments, exon skipping increases the activity of the protein expressed by the resulting mRNA. In embodiments, exon skipping reduces the activity of the protein expressed by the resulting mRNA. In embodiments, skipping one or more exons induces frameshifting in mRNA transcripts. In embodiments, frameshifting results in mRNA encoding a protein with reduced activity. In embodiments, frameshifting results in truncated or non-functional proteins. In embodiments, skipping one or more exons results in the introduction of premature termination codons in mRNA. In embodiments, skipping one or more exons results in the degradation of mRNA transcripts by nonsense-mediated decay. In embodiments, the skipped exon sequence comprises a nucleic acid deletion, substitution or insertion. In embodiments, the skipped exon does not comprise a sequence mutation. In embodiments, hybridization of antisense oligonucleotides with a target nucleotide sequence within a target pre-mRNA transcript results in the expression of different protein isoforms.
在实施方案中，AC与靶转录物的靶核苷酸序列的杂交防止在成熟mRNA分子中包含内含子序列。在实施方案中，AC与靶转录物的靶核苷酸序列的杂交导致蛋白质同种型的表达增加。在实施方案中，AC与靶转录物的靶核苷酸序列的杂交导致蛋白质同种型的表达减少。在实施方案中，AC与靶转录物的靶核苷酸序列的杂交导致包含蛋白质的无活性片段的重新剪接蛋白质的表达。In embodiments, hybridization of AC to a target nucleotide sequence of a target transcript prevents inclusion of intron sequences in mature mRNA molecules. In embodiments, hybridization of AC to a target nucleotide sequence of a target transcript results in increased expression of a protein isoform. In embodiments, hybridization of AC to a target nucleotide sequence of a target transcript results in decreased expression of a protein isoform. In embodiments, hybridization of AC to a target nucleotide sequence of a target transcript results in expression of a re-spliced protein comprising an inactive fragment of a protein.
在实施方案中，AC包含DNA，并且AC与靶转录物的杂交导致经由RNA酶H的转录物降解。在实施方案中，AC包含被设计成不支持RNA酶H活性的核苷酸修饰。不支持RNA酶H活性的反义化合物的核苷酸修饰是已知的，包括但不限于2'-O-甲氧基乙基/硫代磷酸酯(MOE)修饰。有利地，具有MOE修饰的AC提高了对靶RNA的亲和力并且提高了核酸酶稳定性。In an embodiment, the AC comprises DNA, and hybridization of the AC to the target transcript results in transcript degradation via RNase H. In an embodiment, the AC comprises nucleotide modifications designed to not support RNase H activity. Nucleotide modifications of antisense compounds that do not support RNase H activity are known, including but not limited to 2'-O-methoxyethyl/thiophosphate (MOE) modifications. Advantageously, ACs with MOE modifications have increased affinity for target RNA and increased nuclease stability.
在实施方案中，AC通过空间阻断来调控转录、翻译或蛋白质表达。以下综述文章描述了空间阻断的机理及其应用，并且其全文以引用方式并入本文：Roberts等人，NatureReviews Drug Discovery(2020)19:673-694。In an embodiment, AC regulates transcription, translation or protein expression by steric blocking. The following review article describes the mechanism and application of steric blocking, and its entirety is incorporated herein by reference: Roberts et al., Nature Reviews Drug Discovery (2020) 19: 673-694.
AC的功效可通过评价由其施用所影响的反义活性来评估。如本文所用，术语“反义活性”是指可归因于反义化合物与其靶核苷酸序列杂交的任何可检测和/或可测量的活性。此类检测和/或测量可以是直接或间接的。在实施方案中，反义活性通过检测和/或测量由感兴趣的转录物表达的蛋白质的量来评估。在实施方案中，反义活性通过检测和/或测量感兴趣的转录物的量来评估。在实施方案中，反义活性通过检测和/或测量选择性剪接的RNA的量和/或从靶转录物翻译的蛋白质同种型的量来评估。在实施方案中，反义活性通过检测和/或测量由感兴趣的基因调控的下游转录物和/或蛋白质的量来评估。The efficacy of AC can be assessed by evaluating the antisense activity affected by its administration. As used herein, the term "antisense activity" refers to any detectable and/or measurable activity attributable to the hybridization of an antisense compound with its target nucleotide sequence. Such detection and/or measurement can be direct or indirect. In an embodiment, antisense activity is assessed by detecting and/or measuring the amount of protein expressed by a transcript of interest. In an embodiment, antisense activity is assessed by detecting and/or measuring the amount of a transcript of interest. In an embodiment, antisense activity is assessed by detecting and/or measuring the amount of alternatively spliced RNA and/or the amount of protein isoforms translated from a target transcript. In an embodiment, antisense activity is assessed by detecting and/or measuring the amount of downstream transcripts and/or proteins regulated by a gene of interest.
反义化合物设计Antisense compound design
AC的设计将取决于靶基因。将AC靶向特定的靶核苷酸序列可以是多步骤过程。该方法通常从识别感兴趣的基因开始。分析感兴趣的基因的转录物，并且识别靶核苷酸序列。在实施方案中，靶核苷酸序列包含剪接元件和/或剪接调控元件的至少一部分。在实施方案中，靶基因是IRF-5。在实施方案中，靶基因是GYS1。在实施方案中，靶基因是DUX4。The design of the AC will depend on the target gene. Targeting the AC to a specific target nucleotide sequence can be a multi-step process. The method generally starts with identifying the gene of interest. The transcript of the gene of interest is analyzed and the target nucleotide sequence is identified. In an embodiment, the target nucleotide sequence comprises at least a portion of a splicing element and/or a splicing regulatory element. In an embodiment, the target gene is IRF-5. In an embodiment, the target gene is GYS1. In an embodiment, the target gene is DUX4.
本领域技术人员将能够设计、合成和筛选不同核碱基序列的AC，以识别产生反义活性的序列。例如，可以设计抑制靶基因表达的反义化合物。根据针对预选靶核酸和/或靶基因的反义活性设计、合成和筛选AC的方法可见于例如由Stanley T.Crooke编辑的“Antisense Drug Technology,Principles,Strategies,and Applications”,CRC Press,Boca Raton,Florida，其全文以引用方式并入用于所有目的。Those skilled in the art will be able to design, synthesize and screen ACs of different nucleobase sequences to identify sequences that produce antisense activity. For example, antisense compounds that inhibit the expression of a target gene can be designed. Methods for designing, synthesizing and screening ACs for antisense activity against preselected target nucleic acids and/or target genes can be found, for example, in "Antisense Drug Technology, Principles, Strategies, and Applications", edited by Stanley T. Crooke, CRC Press, Boca Raton, Florida, which is incorporated by reference in its entirety for all purposes.
AC结构AC Structure
AC包括寡核苷酸和/或寡核苷。寡核苷酸和/或寡核苷是通过核苷间连接进行连接的核苷。核苷包含戊糖(例如，核糖或脱氧核糖)和与糖共价附接的含氮碱基。存在于DNA和/或RNA中的天然存在的(传统)碱基是腺嘌呤(A)、鸟嘌呤(G)、胸腺嘧啶(T)、胞嘧啶(C)和尿嘧啶(U)。存在于DNA和/或RNA中的天然存在的(传统)糖是脱氧核糖(DNA)和核糖(RNA)。天然存在的(传统)核苷连接是磷酸二酯键。在实施方案中，本公开的AC可具有所有天然糖、碱基和核苷间连接。AC includes oligonucleotides and/or oligonucleosides. Oligonucleotides and/or oligonucleosides are nucleosides connected by internucleoside connections. Nucleosides include pentoses (e.g., ribose or deoxyribose) and nitrogenous bases covalently attached to sugars. The naturally occurring (traditional) bases present in DNA and/or RNA are adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). The naturally occurring (traditional) sugars present in DNA and/or RNA are deoxyribose (DNA) and ribose (RNA). The naturally occurring (traditional) nucleoside connections are phosphodiester bonds. In an embodiment, AC of the present disclosure may have all natural sugars, bases and internucleoside connections.
经化学修饰的核苷通常用于掺入反义化合物中以增强一种或多种特性，诸如核酸酶抗性、药代动力学或对靶RNA的亲和力。在实施方案中，本公开的AC可具有一种或多种经修饰的核苷。在实施方案中，本公开的AC可具有一种或多种经修饰的糖。在实施方案中，本发明的AC可具有一种或多种经修饰的碱基。在实施方案中，本公开的AC可具有一种或多种经修饰的核苷间连接。Chemically modified nucleosides are often used for incorporation into antisense compounds to enhance one or more properties, such as nuclease resistance, pharmacokinetics, or affinity for target RNA. In an embodiment, the AC of the present disclosure may have one or more modified nucleosides. In an embodiment, the AC of the present disclosure may have one or more modified sugars. In an embodiment, the AC of the present disclosure may have one or more modified bases. In an embodiment, the AC of the present disclosure may have one or more modified internucleoside connections.
通常，核碱基是含有能够与另一核酸的碱基氢键合的一个或多个原子或原子团的任何基团。除“未修饰的”或“天然的”核碱基(A、G、T、C和U)之外，本领域技术人员已知的许多经修饰的核碱基或核碱基模拟物适用于本文所述的化合物。通常，经修饰的核碱基是指在结构上与亲本核碱基相当类似的核碱基，诸如7-脱氮嘌呤、5-甲基胞嘧啶、2-硫代-dT(图3)或G型夹。通常，核碱基模拟物是包含比经修饰的核碱基更复杂的结构的核碱基，诸如三环吩嗪核碱基模拟物。用于制备上述经修饰的核碱基的方法是本领域技术人员熟知的。In general, a nucleobase is any group containing one or more atoms or groups of atoms that can hydrogen bond with a base of another nucleic acid. In addition to "unmodified" or "natural" nucleobases (A, G, T, C, and U), many modified nucleobases or nucleobase mimetics known to those skilled in the art are suitable for use in the compounds described herein. In general, a modified nucleobase refers to a nucleobase that is structurally quite similar to a parent nucleobase, such as 7-deazapurine, 5-methylcytosine, 2-thio-dT (FIG. 3), or a G-clamp. In general, a nucleobase mimetic is a nucleobase that contains a more complex structure than a modified nucleobase, such as tricyclic phenoxy groups. Methods for preparing the above-mentioned modified nucleobases are well known to those skilled in the art.
在实施方案中，AC可包含一种或多种具有经修饰的糖部分的核苷。在实施方案中，天然核苷的呋喃糖基糖可具有2'修饰、制备受限核苷的修饰等(参见图3)。例如，在实施方案中，天然核苷的呋喃糖基糖环可以多种方式修饰，包括但不限于添加取代基、桥接两个非偕环原子以形成双环核酸(BNA)或锁核酸；用C或N交换呋喃糖基环的氧；和/或取代这样的原子或基团(参见图3)。经修饰的糖是众所周知的，可用于增加或降低AC对其靶核苷酸序列的亲和力。经修饰的糖也可用于增加AC对核酸酶的抗性。糖也可用糖模拟基团等替换。在实施方案中，AC的核苷的一种或多种糖被如图3中的19所示的亚甲基吗啉环置换。In an embodiment, AC may comprise one or more nucleosides having a modified sugar moiety. In an embodiment, the furanosyl sugar of a natural nucleoside may have a 2' modification, a modification of a preparation-restricted nucleoside, etc. (see FIG. 3 ). For example, in an embodiment, the furanosyl sugar ring of a natural nucleoside may be modified in a variety of ways, including but not limited to adding a substituent, bridging two non-geminal ring atoms to form a bicyclic nucleic acid (BNA) or a locked nucleic acid; exchanging the oxygen of the furanosyl ring with C or N; and/or replacing such an atom or group (see FIG. 3 ). Modified sugars are well known and can be used to increase or decrease the affinity of AC for its target nucleotide sequence. Modified sugars can also be used to increase the resistance of AC to nucleases. Sugars can also be replaced with sugar-mimicking groups, etc. In an embodiment, one or more sugars of the nucleoside of AC are replaced by a methylene morpholine ring as shown in 19 in FIG. 3 .
在实施方案中，AC包含一种或多种核苷，所述核苷包含双环修饰糖(BNA；有时称为桥接核酸)。BNA的示例包括但不限于LNA(4'-(CH2)-O-2'桥)、2'-硫代-LNA(4'-(CH2)-S-2'桥)、2'-氨基-LNA(4'-(CH2)-NR-2'桥)、ENA(4'-(CH2)2-O-2'桥)、4'-(CH2)3-2'桥接BNA、4'-(CH2CH(CH3))-2'桥接BNA"cEt(4'-(CH(CH3)-O-2'桥)和cMOE BNA(4'-(CH(CH2OCH3)-O-2'桥)。一些示例示于图3中。在专利文献以及科学文献中已制备并公开了BNA(Srivastava等人，J.Am.Chem.Soc.(2007),ACS Advanced online publication,10.1021/ja071106y；Albaek等人，J.Org.Chem.(2006),71,7731-7740；Fluiter等人，Chembiochem(2005),6,1104-1109；Singh等人，Chem.Commun.(1998),4,455-456；Koshkin等人，Tetrahedron(1998),54,3607-3630；Wahlestedt等人，Proc.Natl.Acad.Sci.U.S.A.(2000),97,5633-5638；Kumar等人，Bioorg.Med.Chem.Lett.(1998),8,2219-2222；WO 94/14226；WO 2005/021570；Singh等人，J.Org.Chem.(1998),63,10035-10039；WO 2007/090071；美国专利号7053207；6,268,490；6,770,748；6,794,499；7,034,133；和6,525,191；以及美国授权前公开号2004-0171570；2004-0219565；2004-0014959；2003-0207841；2004-0143114；和20030082807)。In an embodiment, AC comprises one or more nucleosides comprising a bicyclic modified sugar (BNA; sometimes referred to as a bridged nucleic acid). Examples of BNAs include, but are not limited to, LNA (4'-(CH2 )-0-2' bridge), 2'-thio-LNA (4'-(CH2 )-S-2' bridge), 2'-amino-LNA (4'-(CH2 )-NR-2' bridge), ENA (4'-(CH2 )2 -0-2' bridge), 4'-(CH2 )3 -2' bridged BNA, 4'-(CH2 CH(CH3 ))-2' bridged BNA "cEt (4'-(CH(CH3 )-0-2' bridge), and cMOE BNA (4'-(CH(CH2 OCH3 )-0-2' bridge). Some examples are shown in FIG3 . BNAs have been prepared and disclosed in the patent literature as well as in the scientific literature (Srivastava et al., J. Am. Chem. Soc. (2007), ACS Advanced Online publication, 10.1021/ja071106y; Albaek et al., J. Org. Chem. (2006), 71, 7731-7740; Fluiter et al., Chembiochem (2005), 6, 1104-1109; Singh et al., Chem. Commun. (1998), 4, 455-456; Koshkin et al., Tetrahedron (1998), 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. USA (2000), 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett. (1998), 8, 2219-2222; WO 94/14226; WO 2005/021570; Singh et al., J. Org. Chem. (1998), 63, 10035-10039; WO 2007/090071; U.S. Patent Nos. 7053207; 6,268,490; 6,770,748; 6,794,499; 7,034,133; and 6,525,191; and U.S. Pre-Grant Publication Nos. 2004-0171570; 2004-0219565; 2004-0014959; 2003-0207841; 2004-0143114; and 20030082807).
在实施方案中，AC包含一种或多种核苷，所述核苷包含锁核酸(LNA)。在LNA中，核糖基糖环的2'-羟基基团与糖环的4'碳原子连接，从而形成2'-C,4'-C-氧亚甲基连接以形成双环糖部分(综述于Elayadi等人，Curr.Opinion Invens.Drugs(2001),2,558-561；Braasch等人，Chem.Biol.(2001),8,1-7；以及Orum等人，Curr.Opinion Mol.Ther.(2001),3,239-243；还参见美国专利：6,268,490和6,670,461)。该连接可以是桥接2'氧原子和4'碳原子的亚甲基(-CH2-)基团，为此术语LNA用于双环部分；在该位置上为亚乙基基团的情况下，使用术语ENATM(Singh等人，Chem.Commun.(1998),4,455-456；ENATM；Morita等人，Bioorganic Medicinal Chemistry(2003),11,2211-2226)。LNA和其他双环糖类似物显示出与互补DNA和RNA的非常高的双链体热稳定性(Tm＝+3℃至+10℃)、对3'-外切核酸降解的稳定性和良好的溶解特性。已经描述了含有LNA的有效且无毒的反义寡核苷酸(Wahlestedt等人，Proc.Natl.Acad.Sci.U.S.A.(2000),97,5633-5638)。In an embodiment, AC comprises one or more nucleosides comprising locked nucleic acid (LNA). In LNA, the 2'-hydroxyl group of the ribosyl sugar ring is connected to the 4' carbon atom of the sugar ring to form a 2'-C, 4'-C-oxymethylene connection to form a bicyclic sugar portion (reviewed in Elayadi et al., Curr. Opinion Invens. Drugs (2001), 2, 558-561; Braasch et al., Chem. Biol. (2001), 8, 1-7; and Orum et al., Curr. Opinion Mol. Ther. (2001), 3, 239-243; see also U.S. Patents: 6,268,490 and 6,670,461). The link may be a methylene (-CH2- ) group bridging the 2' oxygen atom and the 4' carbon atom, for which the term LNA is used for the bicyclic portion; in the case of an ethylene group at this position, the term ENA™ is used (Singh et al., Chem. Commun. (1998), 4, 455-456; ENA™ ; Morita et al., Bioorganic Medicinal Chemistry (2003), 11, 2211-2226). LNA and other bicyclic sugar analogs show very high duplex thermal stability with complementary DNA and RNA (Tm = +3°C to +10°C), stability to 3'-exonucleolytic degradation and good solubility properties. Effective and non-toxic antisense oligonucleotides containing LNA have been described (Wahlestedt et al., Proc. Natl. Acad. Sci. USA (2000), 97, 5633-5638).
也已研究的LNA异构体是α-L-LNA，其已显示对3'-外切核酸酶具有优异的稳定性。将α-L-LNA掺入显示出有效反义活性的反义gapmer和嵌合体中(Frieden等人，NucleicAcids Research(2003),21,6365-6372)。An LNA isomer that has also been studied is α-L-LNA, which has been shown to have excellent stability against 3'-exonucleases. α-L-LNA has been incorporated into antisense gapmers and chimeras that have shown potent antisense activity (Frieden et al., Nucleic Acids Research (2003), 21, 6365-6372).
LNA单体腺嘌呤、胞嘧啶、鸟嘌呤、5-甲基-胞嘧啶、胸腺嘧啶和尿嘧啶的合成和制备以及它们的寡聚化和核酸识别特性已有描述(Koshkin等人，Tetrahedron(1998),54,3607-3630)。LNA及其制备也描述于WO 98/39352和WO 99/14226中。The synthesis and preparation of the LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil as well as their oligomerization and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron (1998), 54, 3607-3630). LNA and its preparation are also described in WO 98/39352 and WO 99/14226.
还制备了LNA的类似物，诸如硫代磷酸-LNA和2'-硫代-LNA(Kumar等人，Bioorg.Med.Chem.Lett.,1998,8,2219-2222)。含有寡脱氧核糖核苷酸双链体作为核酸聚合酶底物的LNA类似物的制备也已有描述(WO 99/14226)。此外，一种构象受限的高亲和力寡核苷酸类似物2'-氨基-LNA的合成已有描述(Singh等人，J.Org.Chem.(1998),63,10035-10039)。另外，已制备2'-氨基-LNA和2'-甲基氨基-LNA，并且先前已报道它们与互补RNA和DNA链的双链体的热稳定性。Analogs of LNA have also been prepared, such as phosphorothioate-LNA and 2'-thio-LNA (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). The preparation of LNA analogs containing oligodeoxyribonucleotide duplexes as nucleic acid polymerase substrates has also been described (WO 99/14226). In addition, the synthesis of a conformationally restricted high affinity oligonucleotide analog 2'-amino-LNA has been described (Singh et al., J. Org. Chem. (1998), 63, 10035-10039). In addition, 2'-amino-LNA and 2'-methylamino-LNA have been prepared, and their thermal stability in duplexes with complementary RNA and DNA chains has been previously reported.
在实施方案中，反义化合物是“三环-DNA(tc-DNA)”，其是指一类受约束的DNA类似物，其中每个核苷酸通过引入环丙烷环而被修饰以限制主链的构象柔性并且增强扭转角γ的主链几何形状。含同碱基腺嘌呤和胸腺嘧啶的tc-DNA与互补的RNA形成非常稳定的A-T碱基对。In an embodiment, the antisense compound is "tricyclic-DNA (tc-DNA)", which refers to a class of constrained DNA analogs in which each nucleotide is modified by the introduction of a cyclopropane ring to restrict the conformational flexibility of the backbone and enhance the backbone geometry of the torsion angle γ. The tc-DNA containing the same bases adenine and thymine forms very stable A-T base pairs with complementary RNA.
用于制备经修饰的糖的方法是本领域技术人员熟知的。一些教导制备此类经修饰的糖的代表性专利和出版物包括但不限于美国专利：4,981,957；5,118,800；5,319,080；5,359,044；5,393,878；5,446,137；5,466,786；5,514,785；5,519,134；5,567,811；5,576,427；5,591,722；5,597,909；5,610,300；5,627,053；5,639,873；5,646,265；5,658,873；5,670,633；5,792,747；5,700,920；和6,600,032；以及WO 2005/121371。Methods for preparing modified sugars are well known to those skilled in the art. Some representative patents and publications that teach the preparation of such modified sugars include, but are not limited to, U.S. Patents: 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; 5,700,920; and 6,600,032; and WO 2005/121371.
核苷间连接Internucleoside linkage
本文描述了核苷间连接基团，其将核苷或另外经修饰的核苷单体单元连接在一起，从而形成寡核苷酸和/或含寡核苷酸的AC。AC可包括天然存在的核苷间连接、非天然的核苷间连接或两者。Described herein are internucleoside linking groups that link nucleosides or otherwise modified nucleoside monomer units together to form oligonucleotides and/or oligonucleotide-containing ACs. ACs can include naturally occurring internucleoside linkages, non-natural internucleoside linkages, or both.
在天然存在的DNA和RNA中，核苷间连接基团是将相邻核苷彼此共价连接以形成线性聚合化合物的磷酸二酯。在天然存在的DNA和RNA中，磷酸二酯与糖的2'、3'或5'羟基部分连接。在寡核苷酸内，磷酸基团通常被称为形成寡核苷酸的核苷间主链。在天然存在的DNA和RNA中，RNA和DNA的连接或主链是3'至5'磷酸二酯连接。在实施方案中，AC的核苷间连接基团是磷酸二酯。在实施方案中，AC的核苷间连接基团是3'至5'磷酸二酯键。In naturally occurring DNA and RNA, the internucleoside linking group is a phosphodiester that covalently links adjacent nucleosides to each other to form a linear polymeric compound. In naturally occurring DNA and RNA, the phosphodiester is linked to the 2', 3' or 5' hydroxyl moiety of the sugar. In an oligonucleotide, the phosphate group is generally referred to as forming the internucleoside backbone of the oligonucleotide. In naturally occurring DNA and RNA, the connection or backbone of the RNA and DNA is a 3' to 5' phosphodiester connection. In an embodiment, the internucleoside linking group of AC is a phosphodiester. In an embodiment, the internucleoside linking group of AC is a 3' to 5' phosphodiester bond.
非天然的核苷间连接基团的两个主要类别通过磷原子的存在或不存在来定义。代表性的含磷核苷间连接包括但不限于磷酸三酯、甲基膦酸酯、氨基磷酸酯和硫代磷酸酯。代表性的不含磷的核苷间连接基团包括但不限于亚甲基甲基亚氨基(-CH2-N(CH3)-O-CH2-)、硫代二酯(-O-C(O)-S-)、硫代氨基甲酸酯(-O-C(O)(NH)-S-)；硅氧烷(-O-Si(H2-O-)；以及N,N'-二甲肼(-CH2-N(CH3)-N(CH3)-)。具有磷核苷间连接基团的AC称为寡核苷酸。具有非磷核苷间连接基团的反义化合物称为寡核苷。与天然磷酸二酯连接相比，经修饰的核苷间连接可用于改变(通常增加)反义化合物的核酸酶抗性。具有手性原子的核苷间连接可被制备为外消旋的、手性的或制备为混合物。代表性的手性核苷间连接包括但不限于烷基膦酸酯和硫代磷酸酯。含磷和不含磷的连接的制备方法是本领域技术人员熟知的。The two main categories of non-natural internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linkages include, but are not limited to, phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates. Representative non-phosphorus-free internucleoside linking groups include, but are not limited to, methylenemethylimino (-CH2- N(CH3 )-O-CH2- ), thiodiester (-OC(O)-S-), thiocarbamate (-OC(O)(NH)-S-); siloxane (-O-Si(H2- O-); and N,N'-dimethylhydrazine (-CH2- N(CH3 )-N(CH3 )-). ACs with phosphorus internucleoside linking groups are referred to as oligonucleotides. Antisense compounds with non-phosphorus internucleoside linking groups are referred to as oligonucleosides. Modified internucleoside linkages can be used to alter (usually increase) the nuclease resistance of antisense compounds compared to natural phosphodiester linkages. Internucleoside linkages with chiral atoms can be prepared as racemic, chiral, or as a mixture. Representative chiral internucleoside linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods for preparing phosphorus-containing and non-phosphorus-containing linkages are well known to those skilled in the art.
在实施方案中，具有经修饰的糖和/或经修饰的核碱基的两个或更多个核苷可使用氨基磷酸酯连接。在实施方案中，具有亚甲基吗啉环的两个或更多个核苷可通过如图3中的20所示的氨基磷酸酯核苷间连接进行连接，其中B1和B2是经修饰的或天然的核碱基。包含具有通过氨基磷酸酯核苷间连接进行连接的亚甲基吗啉环的核碱基的反义化合物可被称为氨基磷酸酯吗啉代寡聚物(PMO)。In an embodiment, two or more nucleosides with modified sugar and/or modified nucleobase can be connected using phosphoramidate.In an embodiment, two or more nucleosides with methylene morpholine ring can be connected by phosphoramidate internucleoside connection as shown in 20 in Figure 3, wherein B1 and B2 are modified or natural nucleobase.Antisense compounds comprising the nucleobase with the methylene morpholine ring connected by phosphoramidate internucleoside connection can be referred to as phosphoramidate morpholino oligomer (PMO).
缀合基团Conjugated Group
在实施方案中，AC通过一个或多个缀合基团的共价附接来修饰。一般来讲，缀合基团修饰AC的一种或多种特性，包括但不限于药效学、药代动力学、结合、吸收、细胞分布、细胞摄取、电荷和清除。缀合基团在化学领域中是常规使用的，并且直接地或经由任选的连接部分或连接基团连接到亲本化合物诸如AC。缀合基团包括但不限于嵌入剂、报道分子、聚胺、聚酰胺、聚乙二醇、硫醚、聚醚、胆固醇、硫胆固醇、胆酸部分、叶酸、脂质、磷脂、生物素、吩嗪、菲啶、蒽醌、金刚烷、吖啶、荧光素、罗丹明、香豆素和染料。在实施方案中，缀合基团是聚乙二醇(PEG)，并且PEG与AC或CPP(本文别处论述的CPP)缀合。In an embodiment, AC is modified by the covalent attachment of one or more conjugated groups. Generally speaking, the conjugated group modifies one or more properties of AC, including but not limited to pharmacodynamics, pharmacokinetics, binding, absorption, cell distribution, cellular uptake, charge and removal. Conjugated groups are conventionally used in the chemical field, and are directly or via an optional linking part or linking group connected to a parent compound such as AC. Conjugated groups include but are not limited to intercalators, reporters, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, bile acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin and dyes. In an embodiment, the conjugated group is polyethylene glycol (PEG), and PEG is conjugated to AC or CPP (CPP discussed elsewhere herein).
在实施方案中，缀合基团包括脂质部分，诸如胆固醇部分(Letsinger等人，Proc.Natl.Acad.Sci.USA(1989),86,6553)；胆酸(Manoharan等人，Bioorg.Med.Chem.Lett.(1994),4,1053)；硫醚，例如己基-S-三苯甲基硫醇(Manoharan等人，Ann.N.Y.Acad.Sci.(1992),660,306；Manoharan等人，Bioorg.Med.Chem.Let.(1993),3,2765)；硫胆固醇(Oberhauser等人，Nucl.Acids Res.(1992),20,533)；脂族链，例如十二烷二醇或十一烷基残基(Saison-Behmoaras等人，EMBO J.(1991),10,111；Kabanov等人，FEBS Lett.(1990),259,327；Svinarchuk等人，Biochimie(1993),75,49)；磷脂，例如二十六烷基外消旋甘油或三乙基铵-1,2-二-O-十六烷基外消旋甘油-3-H-膦酸酯(Manoharan等人，Tetrahedron Lett.(1995),36,3651；Shea等人，Nucl.Acids Res.(1990),18,3777)；聚胺或聚乙二醇链(Manoharan等人，Nucleosides&Nucleotides(1995),14,969)；金刚烷乙酸(Manoharan等人，Tetrahedron Lett.(1995),36,3651)；棕榈基部分(Mishra等人，Biochim.Biophys.Acta.(1995),1264,229)；或十八胺或己基氨基-羰基-氧胆固醇部分(Crooke等人，J.Pharmacol.Exp.Ther.(1996),277,923)。In embodiments, the conjugate group includes a lipid moiety, such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA (1989), 86, 6553); cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett. (1994), 4, 1053); a thioether, for example, hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci. (1992), 660, 306; Manoharan et al., Bioorg. Med. Chem. Let. (1993), 3, 2765); thiocholesterol (Oberhauser et al., Nucl. Acids Res. (1992), 20, 533); an aliphatic chain, for example, dodecanediol or an undecyl residue (Saison-Behmoaras et al., EMBO J. (1991), 10, 111; Kabanov et al., FEBS Lett. (1990), 259, 327; Svinarchuk et al., Biochimie (1993), 75, 49); phospholipids, such as hexadecyl racemic glycerol or triethylammonium-1,2-di-O-hexadecyl racemic glycerol-3-H-phosphonate (Manoharan et al., Tetrahedron Lett. (1995), 36, 3651; Shea et al., Nucl. Acids Res. (1990), 18, 3777); polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides (1995), 14, 969); adamantane acetic acid (Manoharan et al., Tetrahedron Lett. (1995), 36, 3651); a palmityl moiety (Mishra et al., Biochim. Biophys. Acta. (1995), 1264, 229); or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther. (1996), 277, 923).
反义化合物的类型Types of Antisense Compounds
可使用各种类型的AC，例如包括反义寡核苷酸、siRNA、微小RNA、antagomir、适体、核酶、supermir、miRNA模拟物、miRNA抑制剂或它们的组合。Various types of ACs can be used, including, for example, antisense oligonucleotides, siRNAs, microRNAs, antagomirs, aptamers, ribozymes, supermirs, miRNA mimics, miRNA inhibitors, or combinations thereof.
反义寡核苷酸Antisense Oligonucleotides
在各种实施方案中，反义化合物(AC)是与靶核苷酸序列互补的反义寡核苷酸(ASO)。术语“反义寡核苷酸(ASO)”或简称“反义”意在包括与靶核苷酸序列互补的寡核苷酸。该术语还包括可能不与期望靶核苷酸序列完全互补的ASO。ASO包括与所选靶核苷酸序列或靶基因互补的单链DNA和/或RNA。ASO可包含一个或多个经修饰的DNA和/或RNA碱基、经修饰的糖和/或非天然的核苷间连接。在实施方案中，ASO可包含一个或多个氨基磷酸酯核苷间连接。在实施方案中，ASO是氨基磷酸酯吗啉代寡聚物(PMO)。ASO可具有任何特征、任何长度、结合任何剪接元件并且实现关于AC所述的任何机制。在实施方案中，ASO诱导外显子跳跃以引入提前终止密码子并最终导致靶转录物的无义介导的降解。在实施方案中，ASO是PMO并且诱导外显子跳跃以引入提前终止密码子并最终导致靶转录物的无义介导的降解。In various embodiments, antisense compounds (AC) are antisense oligonucleotides (ASOs) complementary to target nucleotide sequences. The term "antisense oligonucleotides (ASOs)" or "antisense" for short is intended to include oligonucleotides complementary to target nucleotide sequences. The term also includes ASOs that may not be completely complementary to the desired target nucleotide sequence. ASOs include single-stranded DNA and/or RNA complementary to a selected target nucleotide sequence or target gene. ASOs may include one or more modified DNA and/or RNA bases, modified sugars and/or non-natural internucleosides. In embodiments, ASOs may include one or more phosphoramidate internucleosides. In embodiments, ASOs are phosphoramidate morpholino oligomers (PMOs). ASOs may have any features, any length, be combined with any splicing element and realize any mechanism described about AC. In embodiments, ASOs induce exon skipping to introduce premature termination codons and ultimately result in nonsense-mediated degradation of target transcripts. In embodiments, ASOs are PMOs and induce exon skipping to introduce premature termination codons and ultimately result in nonsense-mediated degradation of target transcripts.
反义寡核苷酸已被证明能够有效作为蛋白质合成的靶向抑制剂，并且因此可用于通过靶向基因特异性抑制蛋白质合成。ASO抑制蛋白质合成的功效已被充分确定。迄今为止，这些化合物已在若干种体外和体内模型中显示出前景，包括炎性疾病、癌症和HIV的模型(Agrawal,Trends in Biotech.(1996),14:376-387)。反义也可通过与染色体DNA特异性杂交来影响细胞活性。Antisense oligonucleotides have been shown to be effective as targeted inhibitors of protein synthesis, and therefore can be used to specifically inhibit protein synthesis by targeting genes. The efficacy of ASO in inhibiting protein synthesis has been fully determined. So far, these compounds have shown promise in several in vitro and in vivo models, including models of inflammatory diseases, cancer and HIV (Agrawal, Trends in Biotech. (1996), 14: 376-387). Antisense can also affect cell activity by specific hybridization with chromosomal DNA.
产生ASO的方法是本领域已知的，并且可以容易地适用于产生结合本公开的靶核苷酸序列的ASO。对于给定靶序列具有特异性的ASO序列的选择基于对所选靶核苷酸序列的分析以及对二级结构、Tm、结合能和相对稳定性的确定。反义寡核苷酸可基于它们相对不能形成将减少或抑制与宿主细胞中的靶核苷酸序列的特异性结合的二聚体、发夹或其他二级结构来选择。这些二级结构分析和靶位点选择考虑可例如使用第4版的OLIGO引物分析软件(Molecular Biology Insights)和/或BLASTN 2.0.5算法软件来进行(Altschul等人，Nucleic Acids Res.1997,25(17):3389-402)。The method for producing ASO is known in the art, and can be easily applied to produce ASO in conjunction with the target nucleotide sequence of the present disclosure. The selection of ASO sequence with specificity for a given target sequence is based on the analysis of the selected target nucleotide sequence and the determination of secondary structure, Tm, binding energy and relative stability. Antisense oligonucleotides can be selected based on their relative inability to form dimers, hairpins or other secondary structures that will reduce or inhibit the specific binding with the target nucleotide sequence in the host cell. These secondary structure analyses and target site selection considerations can be carried out, for example, using the 4th edition of OLIGO primer analysis software (Molecular Biology Insights) and/or BLASTN 2.0.5 algorithm software (Altschul et al., Nucleic Acids Res. 1997, 25 (17): 3389-402).
RNA干扰RNA interference
在实施方案中，AC包含介导RNA干扰(RNAi)的分子。如本文所用，短语“介导RNAi”是指以序列特异性方式沉默靶转录物的能力。尽管不希望受理论束缚，但据信沉默使用RNAi机制或方法和引导RNA，例如约21个至约23个核苷酸的siRNA和/或miRNA化合物。在实施方案中，AC靶向靶转录物以进行降解。如此，在实施方案中，RNAi分子可用于破坏感兴趣的基因或多核苷酸的表达。在实施方案中，RNAi分子用于诱导靶转录物诸如前mRNA或成熟mRNA的降解。In an embodiment, AC comprises a molecule that mediates RNA interference (RNAi). As used herein, the phrase "mediating RNAi" refers to the ability to silence a target transcript in a sequence-specific manner. Although not wishing to be bound by theory, it is believed that silencing uses RNAi mechanisms or methods and guide RNAs, such as siRNA and/or miRNA compounds of about 21 to about 23 nucleotides. In an embodiment, AC targets a target transcript for degradation. Thus, in an embodiment, RNAi molecules can be used to disrupt the expression of a gene or polynucleotide of interest. In an embodiment, RNAi molecules are used to induce degradation of a target transcript such as pre-mRNA or mature mRNA.
在实施方案中，AC包含引发RNAi应答的小干扰RNA(siRNA)。在实施方案中，AC包含引发RNAi应答的微小RNA(miRNA)。In embodiments, the AC comprises a small interfering RNA (siRNA) that elicits an RNAi response. In embodiments, the AC comprises a micro RNA (miRNA) that elicits an RNAi response.
小干扰RNA(siRNA)是通常约16个至约30个核苷酸长的核酸双链体，其可与称为RNAi诱导的沉默复合物(RISC)的细胞质多蛋白复合物结合。负载有siRNA的RISC介导同源转录物的降解，因此siRNA可被设计成以高特异性敲低蛋白质表达。与其他反义技术不同，siRNA通过经非编码RNA进化以控制基因表达的天然机理发挥作用。包括靶向临床相关靶标的siRNA在内的多种RNAi试剂目前正处于药物开发中，如例如de Fougerolles,A.等人，Nature Reviews(2007)6:443-453中所述。Small interfering RNA (siRNA) is a nucleic acid duplex of about 16 to about 30 nucleotides in length, which can be combined with a cytoplasmic multiprotein complex called RNAi-induced silencing complex (RISC). RISC loaded with siRNA mediates the degradation of homologous transcripts, so siRNA can be designed to knock down protein expression with high specificity. Unlike other antisense technologies, siRNA plays a role by evolving through non-coding RNA to control the natural mechanism of gene expression. A variety of RNAi agents including siRNA targeting clinically relevant targets are currently in drug development, such as, for example, de Fougerolles, A. et al., Nature Reviews (2007) 6: 443-453.
RNAi的治疗应用非常广泛，因为siRNA和miRNA构建体可用针对靶蛋白的任何核苷酸序列来合成。迄今为止，siRNA构建体已显示出在体外和体内模型以及临床研究中特异性下调靶蛋白的能力。The therapeutic applications of RNAi are very broad, as siRNA and miRNA constructs can be synthesized with any nucleotide sequence against a target protein. To date, siRNA constructs have demonstrated the ability to specifically downregulate target proteins in in vitro and in vivo models as well as in clinical studies.
虽然最先描述的RNAi分子是同时包含RNA有义链和RNA反义链的RNA:RNA杂交体，但现已证明DNA有义链：RNA反义杂交体、RNA有义链：DNA反义杂交体和DNA:DNA杂交体能够介导RNAi(Lamberton,J.S.和Christian,A.T.,Molecular Biotechnology(2003),24:111-119)。在实施方案中，使用包括这些不同类型的双链分子中的任一种的RNAi分子。此外，应当理解，RNAi分子可以多种形式被使用和引入细胞。因此，如本文所用，RNAi分子包括能够在细胞中介导RNAi的任何和所有分子，包括但不限于包含两条分开的链即有义链和反义链的双链寡核苷酸，例如小干扰RNA(siRNA)；包括通过非核苷酸接头连接在一起的两条分开的链的双链寡核苷酸；包含形成双链区的互补序列的发夹环的寡核苷酸，例如shRNAi分子，以及表达一种或多种多核苷酸的表达载体，所述一种或多种多核苷酸能够单独或与另一种多核苷酸组合形成双链多核苷酸。Although the first described RNAi molecules were RNA: RNA hybrids containing both RNA sense strands and RNA antisense strands, it has now been demonstrated that DNA sense strand: RNA antisense hybrids, RNA sense strand: DNA antisense hybrids, and DNA: DNA hybrids can mediate RNAi (Lamberton, J.S. and Christian, A.T., Molecular Biotechnology (2003), 24: 111-119). In embodiments, RNAi molecules comprising any of these different types of double-stranded molecules are used. In addition, it should be understood that RNAi molecules can be used and introduced into cells in a variety of forms. Therefore, as used herein, RNAi molecules include any and all molecules capable of mediating RNAi in a cell, including but not limited to double-stranded oligonucleotides comprising two separate strands, namely a sense strand and an antisense strand, such as small interfering RNA (siRNA); double-stranded oligonucleotides comprising two separate strands linked together by a non-nucleotide linker; oligonucleotides comprising a hairpin loop of complementary sequences forming a double-stranded region, such as shRNAi molecules, and expression vectors expressing one or more polynucleotides that are capable of forming a double-stranded polynucleotide alone or in combination with another polynucleotide.
如本文所用，“单链siRNA化合物”是由单个分子组成的siRNA化合物。它可包括由链内配对形成的双链体区，例如，它可以是或包括发夹或盘柄结构。单链siRNA化合物对于靶分子可以是反义的。As used herein, a "single-stranded siRNA compound" is an siRNA compound consisting of a single molecule. It may include a duplex region formed by intrachain pairing, for example, it may be or include a hairpin or coiled handle structure. The single-stranded siRNA compound may be antisense to the target molecule.
单链siRNA化合物可以足够长以使其能够进入RISC并参与RISC介导的靶mRNA的切割。单链siRNA化合物的长度是至少约14、至少约15、至少约20、至少约25、至少约30、至少约35、至少约40或至多约50个核苷酸。在某些实施方案中，单链siRNA的长度小于约200、约100或约60个核苷酸。The single-stranded siRNA compound can be long enough to enable it to enter RISC and participate in the cutting of the target mRNA mediated by RISC. The length of the single-stranded siRNA compound is at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40 or at most about 50 nucleotides. In certain embodiments, the length of the single-stranded siRNA is less than about 200, about 100 or about 60 nucleotides.
发夹siRNA化合物可具有等于或至少约17、约18、约19、约20、约21、约22、约23、约24或约25个核苷酸对的双链体区。双链体区的长度可等于或小于约200、约100或约50个核苷酸对。在某些实施方案中，双链体区的长度范围是约15至约30、约17至约23、约19至约23和约19至约21个核苷酸对。发夹可具有单链突出端或末端未配对区。在某些实施方案中，突出端的长度是约2至约3个核苷酸。在实施方案中，突出端位于发夹的同一侧，并且在实施方案中位于发夹的反义侧。The hairpin siRNA compound can have a duplex region equal to or at least about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24 or about 25 nucleotide pairs. The length of the duplex region can be equal to or less than about 200, about 100 or about 50 nucleotide pairs. In certain embodiments, the length range of the duplex region is about 15 to about 30, about 17 to about 23, about 19 to about 23 and about 19 to about 21 nucleotide pairs. The hairpin can have a single-stranded overhang or an unpaired terminal region. In certain embodiments, the length of the overhang is about 2 to about 3 nucleotides. In embodiments, the overhang is located on the same side of the hairpin, and in embodiments, is located on the antisense side of the hairpin.
如本文所用，“双链siRNA化合物”是包括多于一条并且在一些情况下两条链的siRNA化合物，其中链间杂交可形成双链体结构的区域。As used herein, a "double-stranded siRNA compound" is a siRNA compound that includes more than one, and in some cases two, strands, wherein hybridization between the strands may form a region of a duplex structure.
双链siRNA化合物的反义链的长度可等于或至少约14、约15、约16、约17、约18、约19、约20、约25、约30、约40或约60个核苷酸。其长度可等于或小于约200、约100或约50个核苷酸。长度范围可以是约17至约25、约19至约23、和约19至约21个核苷酸。如本文所用，术语“反义链”意指siRNA化合物的与靶分子(例如靶转录物的靶核苷酸序列)充分互补的链。The length of the antisense strand of a double-stranded siRNA compound may be equal to or at least about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 40, or about 60 nucleotides. Its length may be equal to or less than about 200, about 100, or about 50 nucleotides. The length range may be about 17 to about 25, about 19 to about 23, and about 19 to about 21 nucleotides. As used herein, the term "antisense strand" means a strand of a siRNA compound that is fully complementary to a target molecule (e.g., a target nucleotide sequence of a target transcript).
双链siRNA化合物的有义链的长度可等于或至少约14、约15、约16、约17、约18、约19、约20、约25、约30、约40或约60个核苷酸。其长度可等于或小于约200、约100或约50个核苷酸。长度范围可以是约17至约25、约19至约23、和约19至约21个核苷酸。The sense strand of the double-stranded siRNA compound may be equal to or at least about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 40, or about 60 nucleotides in length. It may be equal to or less than about 200, about 100, or about 50 nucleotides in length. The length may range from about 17 to about 25, about 19 to about 23, and about 19 to about 21 nucleotides.
双链siRNA化合物的双链部分的长度可等于或至少约14、约15、约16、约17、约18、约19、约20、约21、约22、约23、约24、约25、约30、约40或约60个核苷酸对，其长度可等于或小于约200、约100或约50个核苷酸对。长度范围可以是约15至约30、约17至约23、约19至约23和约19至约21个核苷酸对。The length of the double-stranded portion of the double-stranded siRNA compound can be equal to or at least about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 30, about 40, or about 60 nucleotide pairs, and its length can be equal to or less than about 200, about 100, or about 50 nucleotide pairs. The length range can be about 15 to about 30, about 17 to about 23, about 19 to about 23, and about 19 to about 21 nucleotide pairs.
在实施方案中，siRNA化合物足够大以使其能够被内源分子(例如，被Dicer)切割，以产生较小的siRNA化合物，例如siRNA剂。In embodiments, the siRNA compound is large enough to enable it to be cleaved by an endogenous molecule (eg, by Dicer) to produce a smaller siRNA compound, eg, a siRNA agent.
可选择有义链和反义链，使得双链siRNA化合物在分子的一端或两端包含单链或未配对区。因此，双链siRNA化合物可含有配对的有义链和反义链以含有突出端，例如一个或两个5'或3'突出端，或1至3个核苷酸的3'突出端。突出端可以是一条链比另一条链长的结果，或者是相同长度的两条链交错的结果。一些实施方案将具有至少一个3'突出端。在实施方案中，siRNA分子的两端将具有3'突出端。在实施方案中，突出端是2个核苷酸。The sense strand and antisense strand can be selected so that the double-stranded siRNA compound comprises a single strand or unpaired region at one or both ends of the molecule. Therefore, the double-stranded siRNA compound can contain paired sense strands and antisense strands to contain overhangs, such as one or two 5' or 3' overhangs, or 3' overhangs of 1 to 3 nucleotides. The overhang can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. Some embodiments will have at least one 3' overhang. In an embodiment, the two ends of the siRNA molecule will have 3' overhangs. In an embodiment, the overhang is 2 nucleotides.
在实施方案中，双链体区的长度是约15至约30、或约18、约19、约20、约21、约22或约23个核苷酸，例如，在以上讨论的ssiRNA(具有粘性突出端的siRNA)化合物范围内。ssiRNA化合物可在长度和结构上类似于来自长dsiRNA的天然Dicer加工产物。还包括其中ssiRNA化合物的两条链连接(例如共价连接)的实施方案。在实施方案中，包括发夹或提供双链区的其他单链结构和3'突出端。In embodiments, the length of the duplex region is about 15 to about 30, or about 18, about 19, about 20, about 21, about 22, or about 23 nucleotides, for example, within the range of the ssiRNA (siRNA with sticky overhangs) compounds discussed above. The ssiRNA compound can be similar in length and structure to the natural Dicer processing product from a long dsiRNA. Also included are embodiments in which the two strands of the ssiRNA compound are linked (e.g., covalently linked). In embodiments, a hairpin or other single-stranded structure providing a double-stranded region and a 3' overhang is included.
本文所述的siRNA化合物(包括双链siRNA化合物和单链siRNA化合物)可介导靶RNA(例如mRNA，例如编码蛋白质的基因的转录物)的沉默。为方便起见，此类mRNA在本文中也称为待沉默的mRNA。此类基因也被称为靶基因。通常，待沉默的RNA是内源基因。The siRNA compounds described herein (including double-stranded siRNA compounds and single-stranded siRNA compounds) can mediate the silencing of target RNA (e.g., mRNA, such as transcripts of genes encoding proteins). For convenience, such mRNA is also referred to herein as mRNA to be silenced. Such genes are also referred to as target genes. Typically, the RNA to be silenced is an endogenous gene.
在实施方案中，siRNA化合物与靶转录物“充分互补”，使得siRNA化合物沉默由靶mRNA编码的蛋白质的产生。在实施方案中，siRNA化合物与靶转录物的至少一部分“充分互补”，使得siRNA化合物沉默由靶转录物编码的基因产物的产生。在另一个实施方案中，siRNA化合物与靶核苷酸序列(例如，靶转录物的一部分)“精确互补”，使得靶核苷酸序列和siRNA化合物退火，例如在精确互补区域中形成仅由沃森-克里克碱基对组成的杂交体。与靶核苷酸序列“充分互补”可包括与靶核苷酸序列精确互补的内部区域(例如，至少约10个核苷酸)。此外，在某些实施方案中，siRNA化合物特异性区分单核苷酸差异。在这种情况下，如果在单核苷酸差异的区域(例如，在7个核苷酸内)发现精确互补，则siRNA化合物仅介导RNAi。In an embodiment, the siRNA compound is "fully complementary" to the target transcript, so that the siRNA compound silences the production of a protein encoded by the target mRNA. In an embodiment, the siRNA compound is "fully complementary" to at least a portion of the target transcript, so that the siRNA compound silences the production of a gene product encoded by the target transcript. In another embodiment, the siRNA compound is "precisely complementary" to the target nucleotide sequence (e.g., a portion of the target transcript), so that the target nucleotide sequence and the siRNA compound anneal, such as forming a hybrid consisting of only Watson-Crick base pairs in the precise complementary region. "Fully complementary" to the target nucleotide sequence may include an internal region (e.g., at least about 10 nucleotides) that is precisely complementary to the target nucleotide sequence. In addition, in certain embodiments, the siRNA compound specifically distinguishes between single nucleotide differences. In this case, if precise complementarity is found in the region of single nucleotide differences (e.g., within 7 nucleotides), the siRNA compound only mediates RNAi.
RNAi的治疗应用非常广泛，因为siRNA和miRNA构建体可用针对靶蛋白的任何核苷酸序列来合成。迄今为止，siRNA构建体已显示出在体外和体内模型以及临床研究中特异性下调靶蛋白的能力。The therapeutic applications of RNAi are very broad, as siRNA and miRNA constructs can be synthesized with any nucleotide sequence against a target protein. To date, siRNA constructs have demonstrated the ability to specifically downregulate target proteins in in vitro and in vivo models as well as in clinical studies.
微小RNAMicroRNA
在实施方案中，AC包括微小RNA分子。微小RNA(miRNA)是一类高度保守的小RNA分子，其从植物和动物基因组中的DNA转录，但不翻译成蛋白质。经加工的miRNA是单链17-25个核苷酸的RNA分子，其被整合到RNA诱导的沉默复合物(RISC)中并且已被鉴定为发育、细胞增殖、凋亡和分化的关键调节因子。据信它们通过与特定mRNA的3'-非翻译区结合而在基因表达的调控中发挥作用。RISC通过翻译抑制、转录切割或两者介导基因表达的下调。RISC还与多种真核生物细胞核中的转录沉默有关。In an embodiment, AC includes microRNA molecules. MicroRNA (miRNA) is a class of highly conserved small RNA molecules that are transcribed from DNA in plant and animal genomes but are not translated into proteins. Processed miRNA is a single-stranded RNA molecule of 17-25 nucleotides that is integrated into the RNA-induced silencing complex (RISC) and has been identified as a key regulator of development, cell proliferation, apoptosis and differentiation. It is believed that they play a role in the regulation of gene expression by binding to the 3'-untranslated region of specific mRNA. RISC mediates the downregulation of gene expression by translational inhibition, transcriptional cleavage or both. RISC is also related to transcriptional silencing in the nucleus of a variety of eukaryotic organisms.
AntagomirAntagomir
在实施方案中，AC是antagomir。Antagomir是RNA样寡核苷酸，其具有针对RNA酶保护和药理学特性(诸如增强的组织和细胞摄取)的各种修饰。它们与正常RNA的不同之处在于例如糖、硫代磷酸酯主链和例如3'-端的胆固醇部分的完全2'-0-甲基化。Antagomir可用于通过形成包括antagomir和内源miRNA的双链体来有效地沉默内源miRNA，从而防止miRNA诱导的基因沉默。antagomir介导的miRNA沉默的示例是miR-122的沉默，描述于Krutzfeldt等人，Nature(2005),438:685-689中，其全文以引用方式明确并入本文。Antagomir RNA可使用标准固相寡核苷酸合成方案来合成(美国专利申请号11/502,158和11/657,341；其各自的公开内容以引用方式并入本文)。In the embodiment, AC is antagomir.Antagomir is an RNA-like oligonucleotide with various modifications for RNA enzyme protection and pharmacological properties (such as enhanced tissue and cell uptake). They differ from normal RNA in that, for example, sugar, phosphorothioate backbone and, for example, 3'-end cholesterol moiety are completely 2'-O-methylated.Antagomir can be used to effectively silence endogenous miRNA by forming a duplex including antagomir and endogenous miRNA, thereby preventing miRNA-induced gene silencing.An example of antagomir-mediated miRNA silencing is the silencing of miR-122, described in Krutzfeldt et al., Nature (2005), 438:685-689, which is expressly incorporated herein by reference in its entirety.Antagomir RNA can be synthesized using standard solid phase oligonucleotide synthesis protocols (U.S. Patent Application Nos. 11/502,158 and 11/657,341; their respective disclosures are incorporated herein by reference).
antagomir可包括配体缀合的单体亚基和用于寡核苷酸合成的单体。单体描述于美国申请号10/916,185中。antagomir可具有ZXY结构，诸如PCT申请号PCT/US2004/07070中所述。antagomir可与两亲性部分复合。与寡核苷酸剂一起使用的两亲性部分描述于PCT申请号PCT/US2004/07070中。Antagomirs can include ligand-conjugated monomer subunits and monomers for oligonucleotide synthesis. Monomers are described in U.S. Application No. 10/916,185. Antagomirs can have a ZXY structure, such as described in PCT Application No. PCT/US2004/07070. Antagomirs can be compounded with amphipathic moieties. Amphipathic moieties used with oligonucleotide agents are described in PCT Application No. PCT/US2004/07070.
适体Aptamer
在实施方案中，AC包括适体。适体是以高亲和力和特异性与感兴趣的特定分子结合的核酸或肽分子(Tuerk和Gold,Science 249:505(1990)；Ellington和Szostak,Nature346:818(1990))。已成功生产了结合从大蛋白质到小有机分子的许多不同实体的DNA或RNA适体(Eaton,Curr.Opin.Chem.Biol.(1997),1:10-16；Famulok,Curr.Opin.Struct.Biol.(1999),9:324-9；以及Hermann和Patel,Science(2000),287:820-5)。适体可以是基于RNA或DNA的，并且可包含核开关。核开关是可直接结合小靶分子的mRNA分子的一部分，并且其与靶的结合影响基因的活性。因此，含有核开关的mRNA直接参与调节其自身的活性，这取决于其靶分子的存在与否。通常，通过重复几轮的体外选择或等效的SELEX(通过指数富集的配体的系统进化)来工程化适体以结合多种分子靶标，诸如小分子、蛋白质、核酸，以及甚至细胞、组织和生物体。适体可通过任何已知方法制备，包括合成、重组和纯化方法，并且可单独使用或与对相同靶标特异性的其他适体组合使用。此外，术语“适体”还包括含有共有序列的“二级适体”，该共有序列源自将两种或更多种已知适体与给定靶标进行比较。在实施方案中，适体是“细胞内适体”或“intramer”，其特异性识别细胞内靶标(Famulok等人，ChemBiol.(2001),8(10):931-939；Yoon和Rossi,Adv.Drug Deliv.Rev.(2018),134:22-35；其各自以引用方式并入本文)。In an embodiment, AC comprises an aptamer. An aptamer is a nucleic acid or peptide molecule that binds to a specific molecule of interest with high affinity and specificity (Tuerk and Gold, Science 249:505 (1990); Ellington and Szostak, Nature 346:818 (1990)). DNA or RNA aptamers that bind to many different entities from large proteins to small organic molecules have been successfully produced (Eaton, Curr. Opin. Chem. Biol. (1997), 1:10-16; Famulok, Curr. Opin. Struct. Biol. (1999), 9:324-9; and Hermann and Patel, Science (2000), 287:820-5). Aptamers can be RNA or DNA based and can include nuclear switches. A nuclear switch is a part of an mRNA molecule that can directly bind to a small target molecule, and its binding to the target affects the activity of the gene. Therefore, the mRNA containing the nuclear switch is directly involved in regulating its own activity, which depends on the presence or absence of its target molecule. Typically, aptamers are engineered to bind to a variety of molecular targets, such as small molecules, proteins, nucleic acids, and even cells, tissues, and organisms, by repeated rounds of in vitro selection or equivalent SELEX (systematic evolution of ligands by exponential enrichment). Aptamers can be prepared by any known method, including synthesis, recombination, and purification methods, and can be used alone or in combination with other aptamers specific for the same target. In addition, the term "aptamer" also includes "secondary aptamers" containing a consensus sequence, which is derived from comparing two or more known aptamers to a given target. In an embodiment, the aptamer is an "intracellular aptamer" or "intramer", which specifically recognizes an intracellular target (Famulok et al., ChemBiol. (2001), 8(10):931-939; Yoon and Rossi, Adv. Drug Deliv. Rev. (2018), 134:22-35; each of which is incorporated herein by reference).
核酶Ribozyme
在实施方案中，AC是核酶。核酶是具有特定催化结构域的RNA分子复合物，其具有核酸内切酶活性(Kim和Cech,Proc.Natl.Acad.Sci.USA(1987),84(24):8788-92；Forster和Symons,Cell(1987)24,49(2):211-20)。例如，大量核酶以高度特异性加速磷酸酯转移反应，通常仅切割寡核苷酸底物中若干种磷酸酯中的一种(Cech等人，Cell(1981),27(3Pt2):487-96；Michel和Westhof,J.Mol.Biol.(1990),5,216(3):585-610；Reinhold-Hurek和Shub,Nature(1992),14,357(6374):173-6)。这种特异性归因于需要底物在化学反应前经由特异性碱基配对相互作用与核酶的内部引导序列(IGS)结合。In an embodiment, AC is a ribozyme. Ribozymes are RNA molecule complexes with specific catalytic domains that have endonuclease activity (Kim and Cech, Proc. Natl. Acad. Sci. USA (1987), 84 (24): 8788-92; Forster and Symons, Cell (1987) 24, 49 (2): 211-20). For example, a large number of ribozymes accelerate phosphate transfer reactions with high specificity, usually only cutting one of several phosphates in an oligonucleotide substrate (Cech et al., Cell (1981), 27 (3Pt2): 487-96; Michel and Westhof, J. Mol. Biol. (1990), 5, 216 (3): 585-610; Reinhold-Hurek and Shub, Nature (1992), 14, 357 (6374): 173-6). This specificity is attributed to the requirement for substrate binding to the internal guide sequence (IGS) of the ribozyme via specific base-pairing interactions prior to chemical reaction.
目前已知至少六种基本种类的天然存在的酶促RNA。在生理条件下，每一种都可反式催化RNA磷酸二酯键的水解(并因此可切割其他RNA分子)，通常，酶促核酸通过首先与靶RNA结合来起作用。此类结合通过酶促核酸的靶结合部分发生，该靶结合部分保持紧密接近于用于切割靶RNA的分子的酶促部分。因此，酶促核酸首先识别、然后通过互补碱基配对结合靶RNA，并且一旦结合到正确的位点，就起到酶促作用以切割靶RNA。此类靶RNA的策略性切割将破坏其指导编码蛋白合成的能力。在酶促核酸已结合并切割其RNA靶标后，其从该RNA中释放以寻找另一个靶标并且可重复地结合并切割新的靶标。At least six basic types of naturally occurring enzymatic RNA are currently known. Under physiological conditions, each can trans-catalyze the hydrolysis of RNA phosphodiester bonds (and therefore can cut other RNA molecules), and generally, enzymatic nucleic acids work by first binding to a target RNA. Such binding occurs through the target binding portion of the enzymatic nucleic acid, which remains in close proximity to the enzymatic portion of the molecule for cutting the target RNA. Therefore, the enzymatic nucleic acid first recognizes and then binds to the target RNA through complementary base pairing, and once bound to the correct site, it acts enzymatically to cut the target RNA. The strategic cutting of such target RNA will destroy its ability to guide the synthesis of encoded proteins. After the enzymatic nucleic acid has bound and cut its RNA target, it is released from the RNA to find another target and can repeatedly bind and cut a new target.
例如，酶促核酸分子可在锤头、发夹、δ型肝炎病毒、I组内含子或RNaseP RNA(与RNA引导序列结合)或脉孢菌属VS RNA基序中形成。锤头基序的具体示例由Rossi等人Nucleic Acids Res.(1992),20(17):4559-65描述。发夹基序的示例由以下文献描述：Hampel等人(欧洲专利申请公开号EP 0360257)；Hampel和Tritz,Biochemistry(1989),28(12):4929-33；Hampel等人，Nucleic Acids Res.(1990),18(2):299-304和美国专利5,631,359。肝炎病毒基序的示例由Perrotta和Been,Biochemistry(1992),31(47):11843-52描述；RNaseP基序的示例由Guerrier-Takada等人，Cell(1983),35(3Pt2):849-57描述；脉孢菌属VS RNA核酶基序由Collins(Saville和Collins,Cell(1990),61(4):685-96；Saville和Collins,Proc.Natl.Acad.Sci.USA(1991),88(19):8826-30；Collins和Olive,Biochemistry(1993),32(11):2795-9)描述；并且I族内含子的示例描述于美国专利4,987,071中。在实施方案中，酶促核苷酸分子具有与一个或多个靶基因DNA或RNA区域互补的特异性底物结合位点，并且它们在该底物结合位点内或周围具有赋予该分子RNA切割活性的核苷酸序列。因此，核酶构建体不需要限于本文提到的特定基序。For example, the enzymatic nucleic acid molecule can be formed in a hammerhead, hairpin, hepatitis delta virus, group I intron or RNaseP RNA (in combination with an RNA guide sequence) or Neurospora VS RNA motif. Specific examples of hammerhead motifs are described by Rossi et al. Nucleic Acids Res. (1992), 20(17):4559-65. Examples of hairpin motifs are described by Hampel et al. (European Patent Application Publication No. EP 0360257); Hampel and Tritz, Biochemistry (1989), 28(12):4929-33; Hampel et al., Nucleic Acids Res. (1990), 18(2):299-304 and U.S. Pat. No. 5,631,359. Examples of hepatitis virus motifs are described by Perrotta and Been, Biochemistry (1992), 31(47):11843-52; examples of RNaseP motifs are described by Guerrier-Takada et al., Cell (1983), 35(3Pt2):849-57; the Neurospora VS RNA ribozyme motif is described by Collins (Saville and Collins, Cell (1990), 61(4):685-96; Saville and Collins, Proc. Natl. Acad. Sci. USA (1991), 88(19):8826-30; Collins and Olive, Biochemistry (1993), 32(11):2795-9); and examples of Group I introns are described in U.S. Patent 4,987,071. In an embodiment, the enzymatic nucleotide molecules have a specific substrate binding site complementary to one or more target gene DNA or RNA regions, and they have a nucleotide sequence within or around the substrate binding site that imparts RNA cleavage activity to the molecule. Therefore, ribozyme constructs do not need to be limited to the specific motifs mentioned herein.
核酶可如国际专利申请公开号WO 93/23569和国际专利申请公开号WO 94/02595中所述进行设计，各自特别地以引入方式本文，并且如其中所述合成用于体外和体内测试。在实施方案中，核酶靶向靶转录物的靶核苷酸序列。Ribozymes can be designed as described in International Patent Application Publication No. WO 93/23569 and International Patent Application Publication No. WO 94/02595, each specifically incorporated herein, and synthesized as described therein for in vitro and in vivo testing. In embodiments, the ribozyme targets a target nucleotide sequence of a target transcript.
核酶活性可通过改变核酶结合臂的长度或化学合成具有以下修饰的核酶来增加：防止其被血清核糖核酸酶降解的修饰(参见例如国际专利申请公开号WO 92/07065；国际专利申请公开号WO 93/15187；国际专利申请公开号WO 91/03162；欧洲专利申请公开号92110298.4；美国专利5334711；和国际专利申请公开号WO 94/13688，其描述了可对酶促RNA分子的糖部分进行的各种化学修饰)，增强其在细胞中的功效以及去除茎∏碱基以缩短RNA合成时间并降低化学需求的修饰。Ribozyme activity can be increased by changing the length of the ribozyme binding arm or chemically synthesizing ribozymes with the following modifications: modifications that prevent them from being degraded by serum ribonucleases (see, e.g., International Patent Application Publication No. WO 92/07065; International Patent Application Publication No. WO 93/15187; International Patent Application Publication No. WO 91/03162; European Patent Application Publication No. 92110298.4; U.S. Pat. No. 5334711; and International Patent Application Publication No. WO 94/13688, which describe various chemical modifications that can be made to the sugar portion of the enzymatic RNA molecule), enhancing their efficacy in cells, and removing stem ∏ bases to shorten RNA synthesis time and reduce chemical requirements.
SupermirSupermir
在实施方案中，AC是supermir。supermir是指RNA或DNA聚合物或两者或其修饰物的单链、双链或部分双链寡聚体或多聚体，其具有与miRNA基本上相同的核苷酸序列并且相对于其靶标而言是反义的，该术语包括由天然存在的核碱基、糖和共价核苷间(主链)连接组成的寡核苷酸，并且其含有至少一个功能类似的非天然存在的部分。此类经修饰或经取代的寡核苷酸具有期望的特性，诸如例如增强的细胞摄取、增强的对核酸靶标的亲和力和在核酸酶存在下增加的稳定性。在实施方案中，supermir不包含有义链，并且在另一个实施方案中，supermir不以显著的程度自杂交。supermir可具有二级结构，但其在生理条件下基本上是单链的。基本上为单链的supermir是单链的，其程度为小于约50％(例如，小于约40％、约30％、约20％、约10％或约5％)的supermir与其自身双链体化。supermir可包含发夹片段，例如序列，例如在3'端可自杂交并形成双链体区，例如至少约1、约2、约3或约4或小于约8、约7、约6或约5个核苷酸或约5个核苷酸的双链体区。双链体区可通过接头连接，例如核苷酸接头，例如约3、约4、约5或约6个dT，例如经修饰的dT。在另一个实施方案中，supermir与例如长度为约5、约6、约7、约8、约9或约10个核苷酸的较短寡核苷酸(例如在supermir的3'和5'端之一或两者处或者在一端和非末端或中间)双链体化。In an embodiment, AC is a supermir. Supermir refers to a single-stranded, double-stranded or partially double-stranded oligomer or polymer of RNA or DNA polymer or both or their modifications, which has a nucleotide sequence substantially identical to miRNA and is antisense relative to its target, the term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (main chain) connections, and it contains at least one non-naturally occurring part with a similar function. Such modified or substituted oligonucleotides have desired properties, such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid targets and increased stability in the presence of nucleases. In an embodiment, supermir does not include a sense strand, and in another embodiment, supermir does not self-hybridize to a significant extent. Supermir may have a secondary structure, but it is substantially single-stranded under physiological conditions. Substantially single-stranded supermir is single-stranded, and its extent is less than about 50% (e.g., less than about 40%, about 30%, about 20%, about 10% or about 5%) of supermir and its own duplex. Supermir can comprise hairpin fragment, for example sequence, for example, can self-hybridize and form duplex region at 3' end, for example, at least about 1, about 2, about 3 or about 4 or less than about 8, about 7, about 6 or about 5 nucleotides or duplex region of about 5 nucleotides. The duplex region can be connected by a linker, for example, a nucleotide linker, for example, about 3, about 4, about 5 or about 6 dT, for example, modified dT. In another embodiment, supermir is duplexed with shorter oligonucleotides (for example, at one or both of 3' and 5' ends of supermir or at one end and non-terminal or middle) of, for example, about 5, about 6, about 7, about 8, about 9 or about 10 nucleotides in length.
miRNA模拟物miRNA mimics
在实施方案中，AC是miRNA模拟物。miRNA模拟物代表一类可用于模拟一种或多种miRNA的基因沉默能力的分子。因此，术语“微小RNA模拟物”是指能够进入RNAi途径并调控基因表达的合成非编码RNA(即，miRNA不是通过从内源miRNA的来源纯化获得的)。miRNA模拟物可被设计为成熟分子(例如单链)或模拟前体(例如初级或前miRNA)。miRNA模拟物可包括核酸(经修饰或经修饰的核酸)，包括寡核苷酸，其包括但不限于RNA、经修饰的RNA、DNA、经修饰的DNA、锁核酸、或2'-0,4'-C-乙烯桥联核酸(ENA)、或上述的任何组合(包括DNA-RNA杂交体)。此外，miRNA模拟物可包括能够影响递送、细胞内区室化、稳定性、特异性、功能性、链使用和/或效力的缀合物。在一个设计中，miRNA模拟物是双链分子(例如，具有长度在约16与约31个核苷酸之间的双链体区)并且含有与给定miRNA的成熟链具有同一性的一个或多个序列。修饰可包括在分子的一条或两条链上的2'修饰(包括2'-0甲基修饰和2'F修饰)和增强核酸稳定性和/或特异性的核苷间修饰(例如硫代磷酸酯修饰)。此外，miRNA模拟物可包含突出端。突出端可在任一条链的3'或5'端包含约1至约6个核苷酸，并且可被修饰以增强稳定性或功能性。在实施方案中，miRNA模拟物包含约16至约31个核苷酸的双链体区和以下化学修饰模式中的一者或多者：有义链含有核苷酸1和2的2'-0-甲基修饰(从有义寡核苷酸的5'端计数)，以及所有C和U；反义链修饰可包括所有C和U的2'F修饰、寡核苷酸5'端的磷酸化、以及与2个核苷酸3'突出端相关的稳定化核苷间连接。In embodiments, AC is miRNA mimics. MiRNA mimics represent a class of molecules that can be used to simulate the gene silencing ability of one or more miRNAs. Therefore, the term "microRNA mimics" refers to synthetic non-coding RNAs (i.e., miRNAs are not obtained by purification from the source of endogenous miRNAs) that can enter the RNAi pathway and regulate gene expression. MiRNA mimics can be designed as mature molecules (e.g., single strands) or simulated precursors (e.g., primary or pre-miRNAs). MiRNA mimics may include nucleic acids (modified or modified nucleic acids), including oligonucleotides, including but not limited to RNA, modified RNA, DNA, modified DNA, locked nucleic acids, or 2'-0,4'-C-ethylene bridged nucleic acids (ENA), or any combination thereof (including DNA-RNA hybrids). In addition, miRNA mimics may include conjugates that can affect delivery, intracellular compartmentalization, stability, specificity, functionality, chain usage, and/or efficacy. In one design, the miRNA mimics are double-stranded molecules (e.g., duplex regions having a length between about 16 and about 31 nucleotides) and contain one or more sequences identical to the mature strand of a given miRNA. Modifications may include 2' modifications (including 2'-O methyl modifications and 2'F modifications) on one or both strands of the molecule and internucleoside modifications (e.g., phosphorothioate modifications) that enhance nucleic acid stability and/or specificity. In addition, the miRNA mimics may include overhangs. The overhangs may include about 1 to about 6 nucleotides at the 3' or 5' end of either strand and may be modified to enhance stability or functionality. In an embodiment, the miRNA mimic comprises a duplex region of about 16 to about 31 nucleotides and one or more of the following chemical modification patterns: the sense strand contains 2'-0-methyl modifications of nucleotides 1 and 2 (counted from the 5' end of the sense oligonucleotide), and all C and U; the antisense strand modifications may include 2'F modifications of all C and U, phosphorylation of the 5' end of the oligonucleotide, and a stabilized internucleoside linkage associated with a 2 nucleotide 3' overhang.
miRNA抑制剂miRNA inhibitors
在实施方案中，AC是miRNA抑制剂。术语“antimir”、“微小RNA抑制剂”、“miR抑制剂”或“miRNA抑制剂”是同义的，并且是指干扰特定miRNA能力的寡核苷酸或经修饰的寡核苷酸。通常，抑制剂本质上是核酸或经修饰的核酸，包括寡核苷酸，其包括RNA、经修饰的RNA、DNA、经修饰的DNA、锁定核酸(LNA)或上述的任何组合。In an embodiment, AC is a miRNA inhibitor. The terms "antimir", "microRNA inhibitor", "miR inhibitor" or "miRNA inhibitor" are synonymous and refer to oligonucleotides or modified oligonucleotides that interfere with the ability of a specific miRNA. Typically, inhibitors are nucleic acids or modified nucleic acids in nature, including oligonucleotides, which include RNA, modified RNA, DNA, modified DNA, locked nucleic acids (LNA), or any combination of the above.
修饰包括可影响递送、稳定性、特异性、细胞内区室化或效力的2'修饰(包括2'-0烷基修饰和2'F修饰)和核苷间修饰(例如硫代磷酸酯修饰)。此外，miRNA抑制剂可包括能够影响递送、细胞内区室化、稳定性和/或效力的缀合物。抑制剂可采用多种构型，包括单链、双链(RNA/RNA或RNA/DNA双链体)和发夹设计，通常，微小RNA抑制剂包括与待靶向的miRNA的成熟链(或多条链)互补或部分互补的一个或多个序列或序列的部分，此外，miRNA抑制剂还可包括位于成熟miRNA的反向互补序列的5'和3'处的附加序列。附加序列可以是成熟miRNA所来源的初级miRNA中与成熟miRNA相邻的序列的反向互补序列，或者附加序列可以是任意序列(具有A、G、C或U的混合物)。在实施方案中，附加序列中的一者或两者是能够形成发夹的任意序列。因此，在实施方案中，作为miRNA的反向互补的序列在5'侧和3'侧侧接发夹结构。当是双链时，微小RNA抑制剂可包括相反链上的核苷酸之间的错配。此外，可将微小RNA抑制剂连接到缀合部分以促进抑制剂摄入细胞中。例如，微小RNA抑制剂可与5-(双(4-甲氧基苯基)(苯基)甲氧基)-3羟基戊基氨基甲酸胆固醇酯连接，这允许微小RNA抑制剂被动摄入细胞内。微小RNA抑制剂，包括发夹miRNA抑制剂，详细描述于Vermeulen等人，“Double-Stranded Regions Are Essential Design Components Of Potent Inhibitorsof RISC Function,”RNA 13:723-730(2007)以及WO2007/095387和WO 2008/036825中，其各自全文以引用方式并入本文。本领域普通技术人员可从数据库中选择期望miRNA的序列并设计可用于本文公开的方法的抑制剂。Modifications include 2' modifications (including 2'-0 alkyl modifications and 2'F modifications) and internucleoside modifications (such as thiophosphate modifications) that can affect delivery, stability, specificity, intracellular compartmentalization or efficacy. In addition, miRNA inhibitors may include conjugates that can affect delivery, intracellular compartmentalization, stability and/or efficacy. Inhibitors may adopt a variety of configurations, including single-stranded, double-stranded (RNA/RNA or RNA/DNA duplexes) and hairpin designs. Typically, microRNA inhibitors include one or more sequences or portions of sequences that are complementary or partially complementary to the mature strand (or strands) of the miRNA to be targeted. In addition, miRNA inhibitors may also include additional sequences at 5' and 3' of the reverse complementary sequence of the mature miRNA. The additional sequence may be the reverse complementary sequence of the sequence adjacent to the mature miRNA in the primary miRNA from which the mature miRNA is derived, or the additional sequence may be an arbitrary sequence (with a mixture of A, G, C or U). In an embodiment, one or both of the additional sequences are arbitrary sequences that can form a hairpin. Therefore, in an embodiment, the reverse complementary sequence of miRNA is flanked by a hairpin structure at the 5' side and the 3' side. When it is double-stranded, the microRNA inhibitor may include a mismatch between the nucleotides on the opposite strand. In addition, the microRNA inhibitor may be connected to a conjugated portion to facilitate the inhibitor to be taken into the cell. For example, the microRNA inhibitor may be connected to 5-(bis(4-methoxyphenyl)(phenyl)methoxy)-3-hydroxypentylcarbamic acid cholesterol ester, which allows the microRNA inhibitor to be passively taken into the cell. MicroRNA inhibitors, including hairpin miRNA inhibitors, are described in detail in Vermeulen et al., "Double-Stranded Regions Are Essential Design Components Of Potent Inhibitorsof RISC Function," RNA 13:723-730 (2007) and WO 2007/095387 and WO 2008/036825, each of which is incorporated herein by reference in its entirety. One of ordinary skill in the art may select the sequence of the desired miRNA from a database and design an inhibitor that can be used for the method disclosed herein.
CRISPR基因编辑机制CRISPR gene editing mechanism
在实施方案中，治疗性部分包含CRISPR基因编辑机制的一个或多个元件。如本文所用，“CRISPR基因编辑机制”是指可用于编辑基因组的蛋白质、核酸或它们的组合。基因编辑机制的非限制性示例包括引导RNA(gRNA)、核酸酶、核酸酶抑制剂以及它们的组合和复合物。以下专利文献描述了CRISPR基因编辑机制：美国专利号8,697,359、美国专利号8,771,945、美国专利号8,795,965、美国专利号8,865,406、美国专利号8,871,445、美国专利号8,889,356、美国专利号8,895,308、美国专利号8,906,616、美国专利号8,932,814、美国专利号8,945,839、美国专利号8,993,233、美国专利号8,999,641、美国专利申请号14/704,551和美国专利申请号13/842,859。上述专利文献各自全文以引用方式并入。In an embodiment, the therapeutic moiety comprises one or more elements of a CRISPR gene editing mechanism. As used herein, "CRISPR gene editing mechanism" refers to a protein, nucleic acid, or combination thereof that can be used to edit a genome. Non-limiting examples of gene editing mechanisms include guide RNA (gRNA), nucleases, nuclease inhibitors, and combinations and complexes thereof. The following patent documents describe CRISPR gene editing mechanisms: U.S. Patent No. 8,697,359, U.S. Patent No. 8,771,945, U.S. Patent No. 8,795,965, U.S. Patent No. 8,865,406, U.S. Patent No. 8,871,445, U.S. Patent No. 8,889,356, U.S. Patent No. 8,895,308, U.S. Patent No. 8,906,616, U.S. Patent No. 8,932,814, U.S. Patent No. 8,945,839, U.S. Patent No. 8,993,233, U.S. Patent No. 8,999,641, U.S. Patent Application No. 14/704,551, and U.S. Patent Application No. 13/842,859. Each of the above patent documents is incorporated by reference in its entirety.
gRNAgRNA
在实施方案中，TM包括gRNA。gRNA靶向原核或真核细胞中的基因组基因座。In an embodiment, the TM comprises a gRNA. The gRNA targets a genomic locus in a prokaryotic or eukaryotic cell.
在实施方案中，gRNA是单分子引导RNA(sgRNA)。sgRNA包括间隔序列和支架序列。间隔序列是用于将核酸酶(例如，Cas9核酸酶)靶向至感兴趣的特定核苷酸区域(例如，待切割的基因组DNA序列)的短核酸序列。在实施方案中，间隔区的长度可以是约17-24个碱基，诸如长度约20个碱基。在实施方案中，间隔区的长度可以是约15、约16、约17、约18、约19、约20、约21、约22、约23、约24、约25、约26、约27、约28、约29或约30个碱基。在实施方案中，间隔区的长度可以是至少15、至少16、至少17、至少18、至少19、至少20、至少21、至少22、至少23、至少24、至少25、至少26、至少27、至少28、至少29或至少30个碱基。在实施方案中，间隔区的长度可以是约15、约16、约17、约18、约19、约20、约21、约22、约23、约24、约25、约26、约27、约28、约29或约30个碱基。在实施方案中，间隔序列具有在约40％至约80％之间的GC含量。In embodiments, gRNA is a single molecule guide RNA (sgRNA). sgRNA includes a spacer sequence and a scaffold sequence. Spacer sequences are short nucleic acid sequences for targeting nucleases (e.g., Cas9 nucleases) to specific nucleotide regions of interest (e.g., genomic DNA sequences to be cut). In embodiments, the length of the spacer region can be about 17-24 bases, such as about 20 bases in length. In embodiments, the length of the spacer region can be about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29 or about 30 bases. In embodiments, the length of the spacer region can be at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29 or at least 30 bases. In embodiments, the length of the spacer region can be about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 bases. In embodiments, the spacer sequence has a GC content between about 40% to about 80%.
在实施方案中，间隔区结合紧邻5'原间隔区相邻基序(PAM)之前的靶核苷酸序列。可基于期望的核酸酶选择PAM序列。例如，PAM序列可以是下表13中所示的PAM序列中的任何一种，其中N是指任何核酸，R是指A或G，Y是指C或T，W是指A或T，并且V是指A或C或G。In an embodiment, the spacer binds to a target nucleotide sequence immediately preceding the 5' protospacer adjacent motif (PAM). The PAM sequence can be selected based on the desired nuclease. For example, the PAM sequence can be any of the PAM sequences shown in Table 13 below, where N refers to any nucleic acid, R refers to A or G, Y refers to C or T, W refers to A or T, and V refers to A or C or G.
表13.核酸酶和PAM序列Table 13. Nuclease and PAM sequences
在实施方案中，间隔区结合靶基因诸如人基因的哺乳动物靶转录物的靶核苷酸序列。在实施方案中，间隔区可结合靶转录物的靶核苷酸序列。在实施方案中，间隔区可结合靶核苷酸序列，该靶核苷酸序列包含靶转录物的剪接元件(SE)和/或剪接调控元件(SRE)的至少一部分，或者与靶转录物的SE和/或SRE足够接近以调节剪接。In an embodiment, the spacer binds to a target nucleotide sequence of a target gene, such as a mammalian target transcript of a human gene. In an embodiment, the spacer may bind to a target nucleotide sequence of a target transcript. In an embodiment, the spacer may bind to a target nucleotide sequence that comprises at least a portion of a splicing element (SE) and/or a splicing regulatory element (SRE) of the target transcript, or is sufficiently close to a SE and/or SRE of the target transcript to regulate splicing.
支架序列是sgRNA内负责核酸酶(例如，Cas9)结合的序列。支架序列不包括间隔/靶向序列。在实施方案中，支架的长度可以是约1至约10、约10至约20、约20至约30、约30至约40、约40至约50、约50至约60、约60至约70、约70至约80、约80至约90、约90至约100、约100至约110、约110至约120或约120至约130个核苷酸。在实施方案中，支架的长度可以是约1、约2、约3、约4、约5、约6、约7、约8、约9、约10、约11、约12、约13、约14、约15、约16、约17、约18、约19、约20、约21、约22、约23、约24、约25、约26、约27、约28、约29、约30、约31、约32、约33、约34、约35、约36、约37、约38、约39、约40、约41、约42、约43、约44、约45、约46、约47、约48、约49、约50、约51、约52、约53、约54、约55、约56、约57、约58、约59、约60,about 60、约61、约62、约63、约64、约65、约66、约67、约68、约69、约70、约71、约72、约73、约74、约75、约76、约77、约78、约79、约80、约81、约82、约83、约84、约85、约86、约87、约88、约89、约90、约91、约92、约93、约94、约95、约96、约97、约98、约99、约100、约101、约102、约103、约104、约105、约106、约107、约108、约109、约110、约111、约112、约113、约114、约115、约116、约117、约118、约119、约120、约121、约122、约123、约124、或约125个核苷酸。在实施方案中，支架的长度可以是至少10、至少20、至少30、至少40、至少50、至少60、至少70、至少80、至少90、至少100、至少110、至少120或至少125个核苷酸。The scaffold sequence is a sequence within the sgRNA that is responsible for nuclease (e.g., Cas9) binding. The scaffold sequence does not include a spacer/targeting sequence. In embodiments, the length of the scaffold can be about 1 to about 10, about 10 to about 20, about 20 to about 30, about 30 to about 40, about 40 to about 50, about 50 to about 60, about 60 to about 70, about 70 to about 80, about 80 to about 90, about 90 to about 100, about 100 to about 110, about 110 to about 120, or about 120 to about 130 nucleotides. In embodiments, the length of the stent can be about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, 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, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, In some embodiments, the scaffold may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 110, at least 120, or at least 125 nucleotides in length.
在实施方案中，gRNA是双分子引导RNA，例如crRNA和tracrRNA。在实施方案中，gRNA还可包含聚(A)尾。In an embodiment, the gRNA is a double-molecule guide RNA, such as a crRNA and a tracrRNA. In an embodiment, the gRNA may further comprise a poly (A) tail.
在实施方案中，多种gRNA可以在单一化合物中用作TM。在实施方案中，TM包含约1、约2、约3、约4、约5、约6、约7、约8、约9、约10、约11、约12、约13、约14、约15、约16、约17、约18、约19或约20个gRNA。在实施方案中，gRNA识别相同的靶标。在实施方案中，gRNA识别不同的靶标。在实施方案中，包含gRNA的核酸包含编码启动子的序列，其中启动子驱动gRNA的表达。In embodiments, multiple gRNAs can be used as TMs in a single compound. In embodiments, the TM comprises about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19 or about 20 gRNAs. In embodiments, the gRNAs recognize the same target. In embodiments, the gRNAs recognize different targets. In embodiments, the nucleic acid comprising the gRNA comprises a sequence encoding a promoter, wherein the promoter drives the expression of the gRNA.
核酸酶Nuclease
在实施方案中，TM包括核酸酶。在实施方案中，核酸酶是II型、V-A型、V-B型、VC型、V-U型、VI-B型核酸酶。在实施方案中，核酸酶是类转录激活因子效应物核酸酶(TALEN)、大范围核酸酶或锌指核酸酶。在实施方案中，核酸酶是Cas9、Cas12a(CF3)、Cas12b、Cas12c、Tnp-B样、Cas13a(C2c2)、Cas13b或Cas14核酸酶。例如，在实施方案中，核酸酶是Cas9核酸酶或Cpf1核酸酶。In embodiments, the TM comprises a nuclease. In embodiments, the nuclease is a type II, type V-A, type V-B, type VC, type V-U, type VI-B nuclease. In embodiments, the nuclease is a transcription activator effector nuclease (TALEN), a meganuclease, or a zinc finger nuclease. In embodiments, the nuclease is a Cas9, Cas12a (CF3), Cas12b, Cas12c, Tnp-B-like, Cas13a (C2c2), Cas13b, or Cas14 nuclease. For example, in embodiments, the nuclease is a Cas9 nuclease or a Cpf1 nuclease.
在实施方案中，核酸酶是Cas9、Cas12a(Cpf1)、Cas12b、Cas12c、Tnp-B样、Cas13a(C2c2)、Cas13b或Cas14核酸酶的修饰形式或变体。在实施方案中，核酸酶是TAL核酸酶、大范围核酸酶或锌指核酸酶的修饰形式或变体。“修饰的”或“变体”核酸酶是例如截短的、与另一种蛋白质(诸如另一种核酸酶)融合的、催化失活的等核酸酶。在实施方案中，核酸酶可与天然存在的Cas9、Cas12a(Cpf1)、Cas12b、Cas12c、Tnp-B样、Cas13a(C2c2)、Cas13b、Cas14核酸酶或TALEN、大范围核酸酶或锌指核酸酶具有至少约80％、至少约85％、至少约90％、至少约95％、至少约98％、至少约99％或约100％序列同一性。在实施方案中，核酸酶是源自化脓性链球菌(S.pyogenes)的Cas9核酸酶(SpCas9)。在实施方案中，核酸酶与源自化脓性链球菌的Cas9核酸酶(SpCas9)具有至少约90％、至少约95％、至少约96％、至少约97％、至少约98％或至少约99％的序列同一性。在实施方案中，核酸酶是源自金黄色葡萄球菌(S.aureus)的Cas9(SaCas9)。在实施方案中，核酸酶与源自金黄色葡萄球菌的Cas9(SaCas9)具有至少约90％、至少约95％、至少约96％、至少约97％、至少约98％或至少约99％的序列同一性。在实施方案中，Cpf1是来自氨基酸球菌属(Acidaminococcus)(物种BV3L6，UniProt登录号U2UMQ6)的Cpf1酶。在实施方案中，核酸酶与来自氨基酸球菌属(物种BV3L6，UniProt登录号U2UMQ6)的Cpf1酶具有至少约90％、至少约95％、至少约96％、至少约97％、至少约98％或至少约99％的序列同一性。In an embodiment, the nuclease is a modified form or variant of a Cas9, Cas12a (Cpf1), Cas12b, Cas12c, Tnp-B-like, Cas13a (C2c2), Cas13b, or Cas14 nuclease. In an embodiment, the nuclease is a modified form or variant of a TAL nuclease, a meganuclease, or a zinc finger nuclease. A "modified" or "variant" nuclease is, for example, a truncated nuclease, a fusion with another protein (such as another nuclease), a catalytically inactive nuclease, etc. In an embodiment, the nuclease may have at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100% sequence identity with a naturally occurring Cas9, Cas12a (Cpf1), Cas12b, Cas12c, Tnp-B-like, Cas13a (C2c2), Cas13b, Cas14 nuclease, or a TALEN, a meganuclease, or a zinc finger nuclease. In embodiments, the nuclease is a Cas9 nuclease (SpCas9) derived from Streptococcus pyogenes (S.pyogenes). In embodiments, the nuclease has at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the Cas9 nuclease (SpCas9) derived from Streptococcus pyogenes. In embodiments, the nuclease is Cas9 (SaCas9) derived from Staphylococcus aureus (S.aureus). In embodiments, the nuclease has at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% sequence identity with the Cas9 (SaCas9) derived from Staphylococcus aureus. In embodiments, Cpf1 is a Cpf1 enzyme from Acidaminococcus (species BV3L6, UniProt accession number U2UMQ6). In embodiments, the nuclease has at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the Cpf1 enzyme from Acidaminococcus (species BV3L6, UniProt Accession No. U2UMQ6).
在实施方案中，Cpf1是来自毛螺菌科(Lachnospiraceae)的Cpf1酶(物种ND2006，UniProt登录号A0A182DWE3)。在实施方案中，核酸酶与来自毛螺菌科的Cpf1酶具有至少约90％、至少约95％、至少约96％、至少约97％、至少约98％或至少约99％的序列同一性。在实施方案中，对编码核酸酶的序列进行密码子优化以在哺乳动物细胞中表达。在实施方案中，对编码核酸酶的序列进行密码子优化以在人细胞或小鼠细胞中表达。In embodiments, Cpf1 is a Cpf1 enzyme from Lachnospiraceae (species ND2006, UniProt accession number A0A182DWE3). In embodiments, the nuclease has at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the Cpf1 enzyme from Lachnospiraceae. In embodiments, the sequence encoding the nuclease is codon optimized for expression in mammalian cells. In embodiments, the sequence encoding the nuclease is codon optimized for expression in human cells or mouse cells.
在实施方案中，核酸酶是可溶性蛋白质。In embodiments, the nuclease is a soluble protein.
在实施方案中，TM是编码核酸酶的核苷酸序列。在实施方案中，编码核酸酶的核酸包含编码启动子的序列，其中启动子驱动核酸酶的表达。In an embodiment, the TM is a nucleotide sequence encoding a nuclease. In an embodiment, the nucleic acid encoding the nuclease comprises a sequence encoding a promoter, wherein the promoter drives expression of the nuclease.
gRNA和核酸酶组合gRNA and nuclease combination
在实施方案中，本公开的化合物包含gRNA和核酸酶或编码核酸酶的核苷酸序列作为TM。在实施方案中，编码核酸酶和gRNA的核酸包含编码启动子的序列，其中启动子驱动核酸酶和gRNA的表达。在实施方案中，编码核酸酶和gRNA的核酸包含两个启动子，其中第一启动子控制核酸酶的表达，并且第二启动子控制gRNA的表达。在实施方案中，编码gRNA和核酸酶的核酸编码约1至约20种gRNA，或约1、约2、约3、约4、约5、约6、约7、约8、约9、约10、约11、约12、约13、约14、约15、约16、约17、约18或约19以及至多约20种gRNA。在实施方案中，gRNA识别不同的靶标。在实施方案中，gRNA识别相同的靶标。In embodiments, the compounds of the present disclosure include gRNA and nuclease or nucleotide sequences encoding nuclease as TM. In embodiments, nucleic acids encoding nuclease and gRNA include sequences encoding promoters, wherein promoter drives expression of nuclease and gRNA. In embodiments, nucleic acids encoding nuclease and gRNA include two promoters, wherein the first promoter controls expression of nuclease, and the second promoter controls expression of gRNA. In embodiments, nucleic acids encoding gRNA and nuclease encode about 1 to about 20 kinds of gRNA, or about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18 or about 19 and up to about 20 kinds of gRNA. In embodiments, gRNA recognizes different targets. In embodiments, gRNA recognizes the same target.
在实施方案中，本公开的化合物包含核糖核蛋白(RNP)，该核糖核蛋白包含gRNA和核酸酶作为TM。In an embodiment, a compound of the present disclosure comprises a ribonucleoprotein (RNP) comprising a gRNA and a nuclease as a TM.
在实施方案中，组合物被递送到细胞，该组合物包含：(a)第一化合物，该第一化合物包含gRNA TM，以及(b)第二化合物，该第二化合物是或包含核酸酶。在实施方案中，组合物被递送到细胞，该组合物包含：(a)第一化合物，该第一化合物包含作为TM的核酸酶、CPP，以及(b)第二分子，该第二分子是或包含gRNA。在实施方案中，组合物被递送到细胞，该组合物包含：(a)第一化合物，该第一化合物包含gRNA作为TM，以及(b)第二化合物，该第二化合物包含核酸酶作为TM。In embodiments, a composition is delivered to a cell, the composition comprising: (a) a first compound comprising a gRNA TM, and (b) a second compound that is or comprises a nuclease. In embodiments, a composition is delivered to a cell, the composition comprising: (a) a first compound comprising a nuclease, a CPP as a TM, and (b) a second molecule that is or comprises a gRNA. In embodiments, a composition is delivered to a cell, the composition comprising: (a) a first compound comprising a gRNA as a TM, and (b) a second compound comprising a nuclease as a TM.
感兴趣的遗传元件Genetic element of interest
在实施方案中，本文公开的化合物包含感兴趣的遗传元件作为TM。在实施方案中，感兴趣的遗传元件替换被核酸酶切割的基因组DNA序列。感兴趣的遗传元件的非限制性示例包括基因、单核苷酸多态性、启动子或终止子。In embodiments, the compounds disclosed herein comprise a genetic element of interest as a TM. In embodiments, the genetic element of interest replaces a genomic DNA sequence cleaved by a nuclease. Non-limiting examples of genetic elements of interest include genes, single nucleotide polymorphisms, promoters, or terminators.
核酸酶抑制剂Nuclease inhibitors
在实施方案中，本文公开的化合物包含核酸酶抑制剂作为TM。基因编辑的限制是潜在的脱靶编辑。核酸酶抑制剂的递送将限制脱靶编辑。在实施方案中，核酸酶抑制剂是多肽、多核苷酸或小分子。核酸酶抑制剂描述于美国公开号2020/087354、国际公开号2018/085288、美国公开号2018/0382741、国际公开号2019/089761、国际公开号2020/068304、国际公开号2020/041384和国际公开号2019/076651中，其各自全文以引用方式并入本文。In an embodiment, the compounds disclosed herein include nuclease inhibitors as TM. The limitation of gene editing is potential off-target editing. The delivery of nuclease inhibitors will limit off-target editing. In an embodiment, the nuclease inhibitor is a polypeptide, a polynucleotide or a small molecule. Nuclease inhibitors are described in U.S. Publication No. 2020/087354, International Publication No. 2018/085288, U.S. Publication No. 2018/0382741, International Publication No. 2019/089761, International Publication No. 2020/068304, International Publication No. 2020/041384 and International Publication No. 2019/076651, each of which is incorporated herein by reference in its entirety.
内体逃逸载体(EEV)Endosomal escape vector (EEV)
内体逃逸载体(EEV)可用于转运货物穿过细胞膜，例如，将货物递送到细胞的胞质溶胶或细胞核。货物可包含TM。EEV可包含细胞穿透肽(CPP)，例如环状细胞穿透肽(cCPP)。在实施方案中，EEV包含与环外肽(EP)缀合的cCPP。EP可互换地称为调节肽(MP)。EP可包含核定位信号(NLS)的序列。EP可与货物偶联。EP可与cCPP偶联。EP可与货物和cCPP偶联。EP、货物、cCPP或它们的组合之间的偶联可以是非共价的或共价的。EP可通过肽键附接到cCPP的N-末端。EP可通过肽键附接到cCPP的C-末端。EP可通过cCPP中氨基酸的侧链附接到cCPP。EP可通过赖氨酸的侧链附接到cCPP，赖氨酸可与cCPP中的谷氨酰胺的侧链缀合。EP可与寡核苷酸货物的5'或3'端缀合。EP可与接头偶联。环外肽可与接头的氨基基团缀合。EP可经由EP和cCPP的C-末端通过cCPP和/或EP上的侧链与接头偶联。例如，EP可包含末端赖氨酸，其然后可通过酰胺键与含有谷氨酰胺的cCPP偶联。当EP含有末端赖氨酸并且赖氨酸的侧链可用于附接cCPP时，C-末端或N-末端可附接到货物上的接头。Endosomal escape vectors (EEVs) can be used to transport cargo across the cell membrane, for example, to deliver cargo to the cytosol or nucleus of a cell. Cargo may include TM. EEV may include a cell penetrating peptide (CPP), such as a cyclic cell penetrating peptide (cCPP). In an embodiment, EEV includes a cCPP conjugated to an extracyclic peptide (EP). EP is interchangeably referred to as a regulatory peptide (MP). EP may include a sequence of a nuclear localization signal (NLS). EP may be coupled to cargo. EP may be coupled to cCPP. EP may be coupled to cargo and cCPP. The coupling between EP, cargo, cCPP, or a combination thereof may be non-covalent or covalent. EP may be attached to the N-terminus of cCPP by a peptide bond. EP may be attached to the C-terminus of cCPP by a peptide bond. EP may be attached to cCPP by the side chain of an amino acid in cCPP. EP may be attached to cCPP by the side chain of lysine, which may be conjugated to the side chain of glutamine in cCPP. The EP can be conjugated to the 5' or 3' end of the oligonucleotide cargo. The EP can be coupled to a linker. The exocyclic peptide can be conjugated to the amino group of the linker. The EP can be coupled to the linker via the C-terminus of the EP and the cCPP through the side chains on the cCPP and/or the EP. For example, the EP can contain a terminal lysine, which can then be coupled to a cCPP containing glutamine through an amide bond. When the EP contains a terminal lysine and the side chain of the lysine can be used to attach the cCPP, the C-terminus or N-terminus can be attached to the linker on the cargo.
环外肽Exocyclic peptide
环外肽(EP)可包含2至10个氨基酸残基，例如2、3、4、5、6、7、8、9或10个氨基酸残基，包括其间的所有范围和值。EP可包含6至9个氨基酸残基。EP可包含4至8个氨基酸残基。The exocyclic peptide (EP) may comprise 2 to 10 amino acid residues, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues, including all ranges and values therebetween. The EP may comprise 6 to 9 amino acid residues. The EP may comprise 4 to 8 amino acid residues.
环外肽中的每个氨基酸可以是天然的或非天然的氨基酸。术语“非天然氨基酸”是指一种有机化合物，它是天然氨基酸的同属种，因为它具有与天然氨基酸类似的结构，从而模拟天然氨基酸的结构和反应性。非天然氨基酸可以是经修饰的氨基酸和/或氨基酸类似物，其不是20种常见天然存在的氨基酸或稀有天然氨基酸硒代半胱氨酸或吡咯赖氨酸中的一种。非天然氨基酸也可以是天然氨基酸的D-异构体。合适的氨基酸的示例包括但不限于丙氨酸、别亮氨酸、精氨酸、瓜氨酸、天冬酰胺、天冬氨酸、半胱氨酸、谷氨酰胺、谷氨酸、甘氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、萘基丙氨酸、苯丙氨酸、脯氨酸、焦谷氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸、缬氨酸、它们的衍生物或它们的组合。这些和其他氨基酸连同它们在本文中使用的缩写列于表1中。例如，氨基酸可以是A、G、P、K、R、V、F、H、Nal或瓜氨酸。Each amino acid in the exocyclic peptide can be a natural or non-natural amino acid. The term "non-natural amino acid" refers to an organic compound that is a congener of a natural amino acid because it has a structure similar to a natural amino acid, thereby simulating the structure and reactivity of a natural amino acid. Non-natural amino acids can be modified amino acids and/or amino acid analogs that are not one of the 20 common naturally occurring amino acids or rare natural amino acids selenocysteine or pyrrolysine. Non-natural amino acids can also be D-isomers of natural amino acids. Examples of suitable amino acids include, but are not limited to, alanine, alloleucine, arginine, citrulline, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, naphthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, their derivatives or their combinations. These and other amino acids are listed in Table 1 together with the abbreviations they use in this article. For example, the amino acid can be A, G, P, K, R, V, F, H, NaI, or citrulline.
EP可包含至少一个带正电荷的氨基酸残基，例如至少一个赖氨酸残基和/或至少一个包含含有胍基或其质子化形式的侧链的胺酸残基。EP可包含1或2个氨基酸残基，其包含含有胍基或其质子化形式的侧链。包含含有胍基的侧链的氨基酸残基可以是精氨酸残基。质子化形式在整个公开内容中可指其盐。The EP may comprise at least one positively charged amino acid residue, such as at least one lysine residue and/or at least one amino acid residue comprising a side chain containing a guanidine group or a protonated form thereof. The EP may comprise 1 or 2 amino acid residues comprising a side chain containing a guanidine group or a protonated form thereof. The amino acid residue comprising a side chain containing a guanidine group may be an arginine residue. The protonated form may refer to a salt thereof throughout the disclosure.
EP可包含至少两个、至少三个或至少四个或更多个赖氨酸残基。EP可包含2、3或4个赖氨酸残基。每个赖氨酸残基的侧链上的氨基基团可被保护基团取代，该保护基团包括例如三氟乙酰基(-COCF3)、烯丙氧基羰基(Alloc)、1-(4,4-二甲基-2,6-二氧代亚环己基)乙基(Dde)或(4,4-二甲基-2,6-二氧代环己-1-亚基-3)-甲基丁基(ivDde)。每个赖氨酸残基的侧链上的氨基基团可被三氟乙酰基(-COCF3)取代。可包括保护基团以实现酰胺缀合。可在EP与cCPP缀合后去除保护基团。EP may contain at least two, at least three or at least four or more lysine residues. EP may contain 2, 3 or 4 lysine residues. The amino group on the side chain of each lysine residue may be substituted by a protecting group, which includes, for example, trifluoroacetyl (-COCF3 ), allyloxycarbonyl (Alloc), 1-(4,4-dimethyl-2,6-dioxocyclohexylene)ethyl (Dde) or (4,4-dimethyl-2,6-dioxocyclohexyl-1-ylidene-3)-methylbutyl (ivDde). The amino group on the side chain of each lysine residue may be substituted by a trifluoroacetyl (-COCF3 ). A protecting group may be included to achieve amide conjugation. The protecting group may be removed after the EP is conjugated to the cCPP.
EP可包含至少2个具有疏水侧链的氨基酸残基。具有疏水侧链的氨基酸残基可选自缬氨酸、脯氨酸、丙氨酸、亮氨酸、异亮氨酸和甲硫氨酸。具有疏水侧链的氨基酸残基可以是缬氨酸或脯氨酸。EP may contain at least 2 amino acid residues with hydrophobic side chains. The amino acid residue with hydrophobic side chains may be selected from valine, proline, alanine, leucine, isoleucine and methionine. The amino acid residue with hydrophobic side chains may be valine or proline.
EP可包含至少一个带正电荷的氨基酸残基，例如至少一个赖氨酸残基和/或至少一个精氨酸残基。EP可包含至少两个、至少三个或至少四个或更多个赖氨酸残基和/或精氨酸残基。EP may comprise at least one positively charged amino acid residue, such as at least one lysine residue and/or at least one arginine residue. EP may comprise at least two, at least three or at least four or more lysine residues and/or arginine residues.
EP可包含KK、KR、RR、HH、HK、HR、RH、KKK、KGK、KBK、KBR、KRK、KRR、RKK、RRR、KKH、KHK、HKK、HRR、HRH、HHR、HBH、HHH、HHHH(SEQ ID NO:1)、KHKK(SEQ ID NO:2)、KKHK(SEQ ID NO:3)、KKKH(SEQ ID NO:4)、KHKH(SEQ ID NO:5)、HKHK(SEQ ID NO:6)、KKKK(SEQ ID NO:7)、KKRK(SEQ ID NO:8)、KRKK(SEQ ID NO:9)、KRRK(SEQ ID NO:10)、RKKR(SEQ ID NO:11)、RRRR(SEQ ID NO:12)、KGKK(SEQ ID NO:13)、KKGK(SEQ ID NO:14)、HBHBH(SEQ ID NO:15)、HBKBH(SEQ ID NO:16)、RRRRR(SEQ ID NO:17)、KKKKK(SEQ ID NO:18)、KKKRK(SEQ ID NO:19)、RKKKK(SEQ ID NO:20)、KRKKK(SEQ ID NO:21)、KKRKK(SEQ ID NO:22)、KKKKR(SEQ IDNO:23)、KBKBK(SEQ ID NO:24)、RKKKKG(SEQ ID NO:25)、KRKKKG(SEQ ID NO:26)、KKRKKG(SEQ ID NO:27)、KKKKRG(SEQ ID NO:28)、RKKKKB(SEQ ID NO:29)、KRKKKB(SEQ ID NO:30)、KKRKKB(SEQ ID NO:31)、KKKKRB(SEQ ID NO:32)、KKKRKV(SEQ ID NO:33)、RRRRRR(SEQID NO:34)、HHHHHH(SEQ ID NO:35)、RHRHRH(SEQ ID NO:36)、HRHRHR(SEQ ID NO:37)、KRKRKR(SEQ ID NO:38)、RKRKRK(SEQ ID NO:39)、RBRBRB(SEQ ID NO:40)、KBKBKB(SEQ IDNO:41)、PKKKRKV(SEQ ID NO:42)、PGKKRKV(SEQ ID NO:43)、PKGKRKV(SEQ ID NO:44)、PKKGRKV(SEQ ID NO:45)、PKKKGKV(SEQ ID NO:46)、PKKKRGV(SEQ ID NO:47)或PKKKRKG(SEQ ID NO:48)，其中B是β-丙氨酸。EP中的氨基酸可具有D或L立体化学。EP can contain KK, KR, RR, HH, HK, HR, RH, KKK, KGK, KBK, KBR, KRK, KRR, RKK, RRR, KKH, KHK, HKK, HRR, HRH, HHR, HBH, HHH, HHHH (SEQ ID NO:1), KHKK (SEQ ID NO:2), KKHK (SEQ ID NO:3), KKKH (SEQ ID NO:4), KHKH (SEQ ID NO:5), HKHK (SEQ ID NO: 6), KKKK (SEQ ID NO: 7), KKRK (SEQ ID NO: 8), KRKK (SEQ ID NO: 9), KRRK (SEQ ID NO: 10), RKKR (SEQ ID NO: 11), RRRR ( SEQ ID NO:12), KGKK (SEQ ID NO:13), KKGK (SEQ ID NO:14), HBHBH (SEQ ID NO:15), HBKBH (SEQ ID NO:16), RRRRR (SEQ ID NO:17), KKKKK (SEQ ID NO:18), KKKRK (SEQ ID NO:19), RKKKK (SEQ ID NO:20), KRKKK (SEQ ID NO:21), KKRKK (SEQ ID NO:22), KKKKR (SEQ ID NO:23), KBKBK (SEQ ID NO:24), RKKKKG (SEQ ID NO:25), KRKKKG (SEQ ID NO:26), KKRKKG (SEQ ID NO: 27), KKKKRG (SEQ ID NO: 28), RKKKKB (SEQ ID NO: 29), KRKKKB (SEQ ID NO: 30), KKRKKB (SEQ ID NO: 31), KKKKRB (SEQ ID NO: 32), KKKRKV ( SEQ ID NO:33), RRRRRR (SEQ ID NO:34), HHHHHH (SEQ ID NO:35）、RHRHRH (SEQ ID NO:36）、HRHRHR (SEQ ID NO:37）、KRKRKR (SEQ ID NO:38）、RKRKRK (SEQ ID NO:39）、RBRBRB (SEQ ID NO:40）、 KBKBKB (SEQ ID NO: 41), PKKKRKV (SEQ ID NO: 42), PGKKRKV (SEQ ID NO: 43), PKGKRKV (SEQ ID NO: 44), PKKGRKV (SEQ ID NO: 45), PKKKGKV (SEQ ID NO: 46), PKKKRGV (SEQ ID NO: 47) or PKKKRKG (SEQ ID NO: 48), wherein B is β-alanine. The amino acids in EP can have D or L stereochemistry.
EP可包含KK、KR、RR、KKK、KGK、KBK、KBR、KRK、KRR、RKK、RRR、KKKK(SEQ ID NO:7)、KKRK(SEQ ID NO:8)、KRKK(SEQ ID NO:9)、KRRK(SEQ ID NO:10)、RKKR(SEQ ID NO:11)、RRRR(SEQ ID NO:12)、KGKK(SEQ ID NO:13)、KKGK(SEQ ID NO:14)、KKKKK(SEQ ID NO:18)、KKKRK(SEQ ID NO:19)、KBKBK(SEQ ID NO:24)、KKKRKV(SEQ ID NO:33)、PKKKRKV(SEQ IDNO:42)、PGKKRKV(SEQ ID NO:43)、PKGKRKV(SEQ ID NO:44)、PKKGRKV(SEQ ID NO:45)、PKKKGKV(SEQ ID NO:46)、PKKKRGV(SEQ ID NO:47)或PKKKRKG(SEQ ID NO:48)。EP可包含PKKKRKV(SEQ ID NO:42)、RR、RRR、RHR、RBR、RBRBR(SEQ ID NO:49)、RBHBR(SEQ ID NO:50)或HBRBH(SEQ ID NO:51)，其中B是β-丙氨酸。EP中的氨基酸可具有D或L立体化学。EP may contain KK, KR, RR, KKK, KGK, KBK, KBR, KRK, KRR, RKK, RRR, KKKK (SEQ ID NO:7), KKRK (SEQ ID NO:8), KRKK (SEQ ID NO:9 ), KRRK (SEQ ID NO: 10), RKKR (SEQ ID NO: 11), RRRR (SEQ ID NO: 12), KGKK (SEQ ID NO: 13), KKGK (SEQ ID NO: 14), KKKKK (SEQ ID NO:18), KKKRK (SEQ ID NO:19), KBKBK (SEQ ID NO:24), KKKRKV (SEQ ID NO:33), PKKKRKV (SEQ ID NO:42), PGKKRKV (SEQ ID NO:43), PKGKRKV (SEQ ID NO:44), PKKGRKV (SEQ ID NO:45), PKKKGKV (SEQ ID NO:46), PKKKRGV (SEQ ID NO:47) or PKKKRKG (SEQ ID NO:48). The EP may comprise PKKKRKV (SEQ ID NO:42), RR, RRR, RHR, RBR, RBRBR (SEQ ID NO:49 ), RBHBR (SEQ ID NO: 50) or HBRBH (SEQ ID NO: 51), wherein B is β-alanine. The amino acids in EP can have D or L stereochemistry.
EP可由KK、KR、RR、KKK、KGK、KBK、KBR、KRK、KRR、RKK、RRR、KKKK(SEQ ID NO:7)、KKRK(SEQ ID NO:8)、KRKK(SEQ ID NO:9)、KRRK(SEQ ID NO:10)、RKKR(SEQ ID NO:11)、RRRR(SEQ ID NO:12)、KGKK(SEQ ID NO:13)、KKGK(SEQ ID NO:14)、KKKKK(SEQ ID NO:18)、KKKRK(SEQ ID NO:19)、KBKBK(SEQ ID NO:24)、KKKRKV(SEQ ID NO:33)、PKKKRKV(SEQ IDNO:42)、PGKKRKV(SEQ ID NO:Z43)、PKGKRKV(SEQ ID NO:Z44)、PKKGRKV(SEQ ID NO:Z45)、PKKKGKV(SEQ ID NO:46)、PKKKRGV(SEQ ID NO:47)或PKKKRKG(SEQ ID NO:48)组成。EP可由PKKKRKV(SEQ ID NO:42)、RR、RRR、RHR、RBR、RBRBR(SEQ ID NO:49)、RBHBR(SEQ ID NO:50)或HBRBH(SEQ ID NO:51)组成，其中B是β-丙氨酸。EP中的氨基酸可具有D或L立体化学。EP can be composed of KK, KR, RR, KKK, KGK, KBK, KBR, KRK, KRR, RKK, RRR, KKKK (SEQ ID NO:7), KKRK (SEQ ID NO:8), KRKK (SEQ ID NO:9) , KRRK (SEQ ID NO: 10), RKKR (SEQ ID NO: 11), RRRR (SEQ ID NO: 12), KGKK (SEQ ID NO: 13), KKGK (SEQ ID NO: 14), KKKKK (SEQ ID NO:18), KKKRK (SEQ ID NO:19), KBKBK (SEQ ID NO:24), KKKRKV (SEQ ID NO:33), PKKKRKV (SEQ ID NO:42), PGKKRKV (SEQ ID NO:Z43), PKGKRKV (SEQ ID NO:Z44), PKKGRKV (SEQ ID NO:Z45), PKKKGKV (SEQ ID NO:46), PKKKRGV (SEQ ID NO:47) or PKKKRKG (SEQ ID NO:48). EP may be composed of PKKKRKV (SEQ ID NO:42), RR, RRR, RHR, RBR, RBRBR (SEQ ID NO: 49), RBHBR (SEQ ID NO: 50) or HBRBH (SEQ ID NO: 51), wherein B is β-alanine. The amino acids in EP can have D or L stereochemistry .
EP可包含本领域中鉴定为核定位序列(NLS)的氨基酸序列。EP可由本领域中鉴定为核定位序列(NLS)的氨基酸序列组成。EP可包含NLS，该NLS包含氨基酸序列PKKKRKV(SEQID NO:42)。EP可由NLS组成，该NLS包含氨基酸序列PKKKRKV(SEQ ID NO:42)。EP可包含NLS，该NLS包含选自NLSKRPAAIKKAGQAKKKK(SEQ ID NO:52)、PAAKRVKLD(SEQ ID NO:53)、RQRRNELKRSF(SEQ ID NO:54)、RMRKFKNKGKDTAELRRRRVEVSVELR(SEQ ID NO:Z55)、KAKKDEQILKRRNV(SEQ ID NO:56)、VSRKRPRP(SEQ ID NO:57)、PPKKARED(SEQ ID NO:58)、PQPKKKPL(SEQ ID NO:59)、SALIKKKKKMAP(SEQ ID NO:60)、DRLRR(SEQ ID NO:61)、PKQKKRK(SEQ ID NO:62)、RKLKKKIKKL(SEQ ID NO:63)、REKKKFLKRR(SEQ ID NO:64)、KRKGDEVDGVDEVAKKKSKK(SEQ ID NO:65)和RKCLQAGMNLEARKTKK(SEQ ID NO:66)的氨基酸序列。EP可由NLS组成，该NLS包含选自NLSKRPAAIKKAGQAKKKK(SEQ ID NO:52)、PAAKRVKLD(SEQID NO:53)、RQRRNELKRSF(SEQ ID NO:54)、RMRKFKNKGKDTAELRRRRVEVSVELR(SEQ ID NO:55)、KAKKDEQILKRRNV(SEQ ID NO:56)、VSRKRPRP(SEQ ID NO:57)、PPKKARED(SEQ ID NO:58)、PQPKKKPL(SEQ ID NO:59)、SALIKKKKKMAP(SEQ ID NO:60)、DRLRR(SEQ ID NO:61)、PKQKKRK(SEQ ID NO:62)、RKLKKKIKKL(SEQ ID NO:63)、REKKKFLKRR(SEQ ID NO:64)、KRKGDEVDGVDEVAKKKSKK(SEQ ID NO:65)和RKCLQAGMNLEARKTKK(SEQ ID NO:66)的氨基酸序列。The EP may comprise an amino acid sequence identified in the art as a nuclear localization sequence (NLS). The EP may consist of an amino acid sequence identified in the art as a nuclear localization sequence (NLS). The EP may comprise an NLS comprising the amino acid sequence PKKKRKV (SEQ ID NO: 42). The EP may consist of an NLS comprising the amino acid sequence PKKKRKV (SEQ ID NO: 42). EP可包含NLS，该NLS包含选自NLSKRPAAIKKAGQAKKKK(SEQ ID NO:52)、PAAKRVKLD(SEQ ID NO:53)、RQRRNELKRSF(SEQ ID NO:54)、RMRKFKNKGKDTAELRRRRVEVSVELR(SEQ ID NO:Z55)、KAKKDEQILKRRNV(SEQ ID NO:56)、VSRKRPRP(SEQ ID NO:57)、PPKKARED(SEQ ID NO:58)、PQPKKKPL(SEQ ID NO:59)、SALIKKKKKMAP(SEQ ID NO:60)、DRLRR(SEQ ID NO:61)、PKQKKRK(SEQ ID NO:62)、RKLKKKIKKL(SEQ ID NO:63)、REKKKFLKRR(SEQ ID NO:64)、KRKGDEVDGVDEVAKKKSKK(SEQ ID NO:65) and the amino acid sequence of RKCLQAGMNLEARKTKK (SEQ ID NO:66). EP可由NLS组成，该NLS包含选自NLSKRPAAIKKAGQAKKKK(SEQ ID NO:52)、PAAKRVKLD(SEQID NO:53)、RQRRNELKRSF(SEQ ID NO:54)、RMRKFKNKGKDTAELRRRRVEVSVELR(SEQ ID NO:55)、KAKKDEQILKRRNV(SEQ ID NO:56)、VSRKRPRP(SEQ ID NO:57)、PPKKARED(SEQ ID NO:58)、PQPKKKPL(SEQ ID NO:59)、SALIKKKKKMAP(SEQ ID NO:60)、DRLRR(SEQ ID NO:61)、PKQKKRK(SEQ ID NO:62)、RKLKKKIKKL(SEQ ID NO:63)、REKKKFLKRR(SEQ ID NO:64)、KRKGDEVDGVDEVAKKKSKK(SEQ ID NO:65) and the amino acid sequence of RKCLQAGMNLEARKTKK (SEQ ID NO:66).
所有环外序列还可含有N-末端乙酰基基团。因此，例如，EP可具有以下结构：Ac-PKKKRKV(SEQ ID NO:42)。All exocyclic sequences may also contain an N-terminal acetyl group. Thus, for example, EP may have the following structure: Ac-PKKKRKV (SEQ ID NO: 42).
细胞穿透肽(CPP)Cell Penetrating Peptides (CPP)
细胞穿透肽(CPP)可包含6至20个氨基酸残基。细胞穿透肽可以是环状细胞穿透肽(cCPP)。cCPP能够穿透细胞膜。环外肽(EP)可与cCPP缀合，并且所得构建体可称为内体逃逸载体(EEV)。cCPP可引导货物(例如，治疗性部分(TM)，诸如寡核苷酸、肽或小分子)穿透细胞膜。cCPP可将货物递送至细胞的胞质溶胶。cCPP可将货物递送至靶(例如，前mRNA)所在的细胞位置。为了将cCPP与货物(例如，肽、寡核苷酸或小分子)缀合，可替换cCPP上的至少一个键或孤对电子。The cell penetrating peptide (CPP) may comprise 6 to 20 amino acid residues. The cell penetrating peptide may be a cyclic cell penetrating peptide (cCPP). The cCPP is capable of penetrating the cell membrane. An exocyclic peptide (EP) may be conjugated to the cCPP, and the resulting construct may be referred to as an endosomal escape vector (EEV). The cCPP may guide cargo (e.g., a therapeutic moiety (TM), such as an oligonucleotide, a peptide, or a small molecule) to penetrate the cell membrane. The cCPP may deliver cargo to the cytosol of the cell. The cCPP may deliver cargo to the cellular location where the target (e.g., pre-mRNA) is located. In order to conjugate the cCPP to cargo (e.g., a peptide, an oligonucleotide, or a small molecule), at least one bond or lone pair of electrons on the cCPP may be replaced.
cCPP中氨基酸残基的总数在6至20个氨基酸残基的范围内，例如6、7、8、9、10、11、12、13、14、15、16、17、18、19或20个氨基酸残基，包括其间的所有范围和子范围。cCPP可包含6至13个氨基酸残基。本文公开的cCPP可包含6至10个氨基酸。以举例的方式，包含6-10个氨基酸残基的cCPP可具有根据式I-A至I-E中任一种的结构：The total number of amino acid residues in the cCPP is in the range of 6 to 20 amino acid residues, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid residues, including all ranges and subranges therebetween. The cCPP may comprise 6 to 13 amino acid residues. The cCPP disclosed herein may comprise 6 to 10 amino acids. By way of example, a cCPP comprising 6-10 amino acid residues may have a structure according to any one of Formulas I-A to I-E:
其中AA1、AA2、AA3、AA4、AA5、AA6、AA7、AA8、AA9和AA10是氨基酸残基。 Among them,AA1 ,AA2 ,AA3 ,AA4 ,AA5 ,AA6 ,AA7 ,AA8 ,AA9 andAA10 are amino acid residues.
cCPP可包含6至8个氨基酸。cCPP可包含8个氨基酸。cCPP may comprise 6 to 8 amino acids. cCPP may comprise 8 amino acids.
cCPP中的每个氨基酸可以是天然的或非天然的氨基酸。术语“非天然氨基酸”是指一种有机化合物，它是天然氨基酸的同属种，因为它具有与天然氨基酸类似的结构，从而模拟天然氨基酸的结构和反应性。非天然氨基酸可以是经修饰的氨基酸和/或氨基酸类似物，其不是20种常见天然存在的氨基酸或稀有天然氨基酸硒代半胱氨酸或吡咯赖氨酸中的一种。非天然氨基酸也可以是天然氨基酸的D-异构体。合适的氨基酸的示例包括但不限于丙氨酸、别亮氨酸、精氨酸、瓜氨酸、天冬酰胺、天冬氨酸、半胱氨酸、谷氨酰胺、谷氨酸、甘氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、萘基丙氨酸、苯丙氨酸、脯氨酸、焦谷氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸、缬氨酸、它们的衍生物或它们的组合。这些和其他氨基酸连同它们在本文中使用的缩写列于表1中。Each amino acid in cCPP can be a natural or non-natural amino acid. The term "non-natural amino acid" refers to an organic compound that is a congener of a natural amino acid because it has a structure similar to a natural amino acid, thereby simulating the structure and reactivity of a natural amino acid. Non-natural amino acids can be modified amino acids and/or amino acid analogs that are not one of the 20 common naturally occurring amino acids or rare natural amino acids selenocysteine or pyrrolysine. Non-natural amino acids can also be D-isomers of natural amino acids. Examples of suitable amino acids include, but are not limited to, alanine, alloleucine, arginine, citrulline, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, naphthylalanine, phenylalanine, proline, pyroglutamic acid, serine, threonine, tryptophan, tyrosine, valine, their derivatives, or combinations thereof. These and other amino acids are listed in Table 1 together with their abbreviations used herein.
表1.氨基酸缩写Table 1. Amino acid abbreviations
如本文所用，“聚乙二醇”和“PEG”可互换使用。“PEGm”和“PEGm”是或衍生自式HO(CO)-(CH2)n-(OCH2CH2)m-NH2的分子，其中n是1至5的任何整数，并且m是1至23的任何整数。在实施方案中，n是1或2。在实施方案中，n是1。在实施方案中，n是2。在实施方案中，n是1，并且m是2。在实施方案中，n是2，并且m是2。在实施方案中，n是1，并且m是4。在实施方案中，n是2，并且m是4。在实施方案中，n是1，并且m是12。在实施方案中，n是2，并且m是12。As used herein, "polyethylene glycol" and "PEG" are used interchangeably. "PEGm" and "PEGm " are or are derived from molecules of the formula HO(CO)-(CH2 )n -(OCH2 CH2 )m -NH2 , wherein n is any integer from 1 to 5, and m is any integer from 1 to 23. In embodiments, n is 1 or 2. In embodiments, n is 1. In embodiments, n is 2. In embodiments, n is 1 and m is 2. In embodiments, n is 2 and m is 2. In embodiments, n is 1 and m is 4. In embodiments, n is 2 and m is 4. In embodiments, n is 1 and m is 12. In embodiments, n is 2 and m is 12.
如本文所用，“miniPEGm”或“miniPEGm”是或衍生自式HO(CO)-(CH2)n-(OCH2CH2)m-NH2的分子，其中n是1，并且m是1至23的任何整数。例如，“miniPEG2”或“miniPEG2”是或衍生自(2-[2-[2-氨基乙氧基]乙氧基]乙酸)，并且“miniPEG4”或“miniPEG4”是或衍生自HO(CO)-(CH2)n-(OCH2CH2)m-NH2，其中n是1，并且m是4。As used herein, "miniPEGm" or "miniPEGm " is or is derived from a molecule of the formula HO(CO)-(CH2 )n -(OCH2 CH2 )m -NH2 , wherein n is 1 and m is any integer from 1 to 23. For example, "miniPEG2" or "miniPEG2 " is or is derived from (2-[2-[2-aminoethoxy]ethoxy]acetic acid), and "miniPEG4" or "miniPEG4 " is or is derived from HO(CO)-(CH2 )n -(OCH2 CH2 )m -NH2 , wherein n is 1 and m is 4.
cCPP可包含4至20个氨基酸，其中：(i)至少一个氨基酸具有包含胍基或其质子化形式的侧链；(ii)至少一个氨基酸不具有侧链或具有包含或其质子化形式的侧链；并且(iii)至少两个氨基酸独立地具有包含芳族或杂芳族基团的侧链。The cCPP may comprise 4 to 20 amino acids, wherein: (i) at least one amino acid has a side chain comprising a guanidine group or a protonated form thereof; (ii) at least one amino acid has no side chain or has a side chain comprising or a side chain thereof in a protonated form; and (iii) at least two amino acids independently have a side chain comprising an aromatic or heteroaromatic group.
至少两个氨基酸可不具有侧链或具有包含或其质子化形式的侧链。如本文所用，当不存在侧链时，氨基酸在连接胺和羧酸的碳原子上具有两个氢原子(例如，-CH2-)。At least two amino acids may have no side chains or have side chains comprising or a protonated form of its side chain. As used herein, when no side chain is present, an amino acid has two hydrogen atoms on the carbon atom connecting the amine and the carboxylic acid (eg,-CH2- ).
不具有侧链的氨基酸可以是甘氨酸或β-丙氨酸。The amino acid having no side chain may be glycine or β-alanine.
cCPP可包含形成cCPP的6至20个氨基酸残基，其中：(i)至少一个氨基酸可以是甘氨酸、β-丙氨酸或4-氨基丁酸残基；(ii)至少一个氨基酸可具有包含芳基或杂芳基基团的侧链；并且(iii)至少一个氨基酸具有包含胍基、或其质子化形式的侧链。The cCPP may comprise 6 to 20 amino acid residues forming a cCPP, wherein: (i) at least one amino acid may be a glycine, β-alanine or 4-aminobutyric acid residue; (ii) at least one amino acid may have a side chain comprising an aryl or heteroaryl group; and (iii) at least one amino acid may have a side chain comprising a guanidine group, or its protonated form.
cCPP可包含形成cCPP的6至20个氨基酸残基，其中：(i)至少两个氨基酸可独立地是甘氨酸、β-丙氨酸或4-氨基丁酸残基；(ii)至少一个氨基酸可具有包含芳基或杂芳基基团的侧链；并且(iii)至少一个氨基酸具有包含胍基、或其质子化形式的侧链。The cCPP may comprise 6 to 20 amino acid residues forming a cCPP, wherein: (i) at least two amino acids may independently be glycine, β-alanine or 4-aminobutyric acid residues; (ii) at least one amino acid may have a side chain comprising an aryl or heteroaryl group; and (iii) at least one amino acid may have a side chain comprising a guanidine group, or its protonated form.
cCPP可包含形成cCPP的6至20个氨基酸残基，其中：(i)至少三个氨基酸可独立地是甘氨酸、β-丙氨酸或4-氨基丁酸残基；(ii)至少一个氨基酸可具有包含芳族或杂芳族基团的侧链；并且(iii)至少一个氨基酸可具有包含胍基、或其质子化形式的侧链。The cCPP may comprise 6 to 20 amino acid residues forming a cCPP, wherein: (i) at least three amino acids may independently be glycine, β-alanine or 4-aminobutyric acid residues; (ii) at least one amino acid may have a side chain comprising an aromatic or heteroaromatic group; and (iii) at least one amino acid may have a side chain comprising a guanidine group, or its protonated form.
甘氨酸和相关的氨基酸残基Glycine and related amino acid residues
cCPP可包含(i)1、2、3、4、5或6个甘氨酸、β-丙氨酸、4-氨基丁酸残基或它们的组合。cCPP可包含(i)2个甘氨酸、β-丙氨酸、4-氨基丁酸残基或它们的组合。cCPP可包含(i)3个甘氨酸、β-丙氨酸、4-氨基丁酸残基或它们的组合。cCPP可包含(i)4个甘氨酸、β-丙氨酸、4-氨基丁酸残基或它们的组合。cCPP可包含(i)5个甘氨酸、β-丙氨酸、4-氨基丁酸残基或它们的组合。cCPP可包含(i)6个甘氨酸、β-丙氨酸、4-氨基丁酸残基或它们的组合。cCPP可包含(i)3、4或5个甘氨酸、β-丙氨酸、4-氨基丁酸残基或它们的组合。cCPP可包含(i)3或4个甘氨酸、β-丙氨酸、4-氨基丁酸残基或它们的组合。cCPP may contain (i) 1, 2, 3, 4, 5 or 6 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 2 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 3 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 4 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 5 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 6 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 3, 4 or 5 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 3 or 4 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof.
cCPP可包含(i)1、2、3、4、5或6个甘氨酸残基。cCPP可包含(i)2个甘氨酸残基。cCPP可包含(i)3个甘氨酸残基。cCPP可包含(i)4个甘氨酸残基。cCPP可包含(i)5个甘氨酸残基。cCPP可包含(i)6个甘氨酸残基。cCPP可包含(i)3、4或5个甘氨酸残基。cCPP可包含(i)3或4个甘氨酸残基。cCPP可包含(i)2或3个甘氨酸残基。cCPP可包含(i)1或2个甘氨酸残基。cCPP may comprise (i) 1, 2, 3, 4, 5 or 6 glycine residues. cCPP may comprise (i) 2 glycine residues. cCPP may comprise (i) 3 glycine residues. cCPP may comprise (i) 4 glycine residues. cCPP may comprise (i) 5 glycine residues. cCPP may comprise (i) 6 glycine residues. cCPP may comprise (i) 3, 4 or 5 glycine residues. cCPP may comprise (i) 3 or 4 glycine residues. cCPP may comprise (i) 2 or 3 glycine residues. cCPP may comprise (i) 1 or 2 glycine residues.
cCPP可包含(i)3、4、5或6个甘氨酸、β-丙氨酸、4-氨基丁酸残基或它们的组合。cCPP可包含(i)3个甘氨酸、β-丙氨酸、4-氨基丁酸残基或它们的组合。cCPP可包含(i)4个甘氨酸、β-丙氨酸、4-氨基丁酸残基或它们的组合。cCPP可包含(i)5个甘氨酸、β-丙氨酸、4-氨基丁酸残基或它们的组合。cCPP可包含(i)6个甘氨酸、β-丙氨酸、4-氨基丁酸残基或它们的组合。cCPP可包含(i)3、4或5个甘氨酸、β-丙氨酸、4-氨基丁酸残基或它们的组合。cCPP可包含(i)3或4个甘氨酸、β-丙氨酸、4-氨基丁酸残基或它们的组合。cCPP may contain (i) 3, 4, 5 or 6 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 3 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 4 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 5 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 6 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 3, 4 or 5 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof. cCPP may contain (i) 3 or 4 glycine, β-alanine, 4-aminobutyric acid residues or a combination thereof.
cCPP可包含至少三个甘氨酸残基。cCPP可包含(i)3、4、5或6个甘氨酸残基。cCPP可包含(i)3个甘氨酸残基。cCPP可包含(i)4个甘氨酸残基。cCPP可包含(i)5个甘氨酸残基。cCPP可包含(i)6个甘氨酸残基。cCPP可包含(i)3、4或5个甘氨酸残基。cCPP可包含(i)3或4个甘氨酸残基cCPP may comprise at least three glycine residues. cCPP may comprise (i) 3, 4, 5 or 6 glycine residues. cCPP may comprise (i) 3 glycine residues. cCPP may comprise (i) 4 glycine residues. cCPP may comprise (i) 5 glycine residues. cCPP may comprise (i) 6 glycine residues. cCPP may comprise (i) 3, 4 or 5 glycine residues. cCPP may comprise (i) 3 or 4 glycine residues
在实施方案中，cCPP中的甘氨酸、β-丙氨酸或4-氨基丁酸残基都不是邻接的。两个或三个甘氨酸、β-丙氨酸、或4-氨基丁酸残基可以是邻接的。两个甘氨酸、β-丙氨酸或4-氨基丁酸残基可以是邻接的。In an embodiment, none of the glycine, β-alanine, or 4-aminobutyric acid residues in the cCPP are contiguous. Two or three glycine, β-alanine, or 4-aminobutyric acid residues may be contiguous. Two glycine, β-alanine, or 4-aminobutyric acid residues may be contiguous.
在实施方案中，cCPP中没有一个甘氨酸残基是邻接的。cCPP中的每个甘氨酸残基可被不能是甘氨酸的氨基酸残基分开。两个或三个甘氨酸残基可以是邻接的。两个甘氨酸残基可以是邻接的。In an embodiment, no glycine residues in a cCPP are contiguous. Each glycine residue in a cCPP may be separated by an amino acid residue that cannot be glycine. Two or three glycine residues may be contiguous. Two glycine residues may be contiguous.
具有芳族或杂芳族基团的氨基酸侧链Amino acid side chains with aromatic or heteroaromatic groups
cCPP可包含(ii)2、3、4、5或6个独立地具有包含芳族或杂芳族基团的侧链的氨基酸残基。cCPP可包含(ii)2个独立地具有包含芳族或杂芳族基团的侧链的氨基酸残基。cCPP可包含(ii)3个独立地具有包含芳族或杂芳族基团的侧链的氨基酸残基。cCPP可包含(ii)4个独立地具有包含芳族或杂芳族基团的侧链的氨基酸残基。cCPP可包含(ii)5个独立地具有包含芳族或杂芳族基团的侧链的氨基酸残基。cCPP可包含(ii)6个独立地具有包含芳族或杂芳族基团的侧链的氨基酸残基。cCPP可包含(ii)2、3或4个独立地具有包含芳族或杂芳族基团的侧链的氨基酸残基。cCPP可包含(ii)2或3个独立地具有包含芳族或杂芳族基团的侧链的氨基酸残基。cCPP may comprise (ii) 2, 3, 4, 5 or 6 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 2 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 3 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 4 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 5 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 6 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 2, 3 or 4 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group. cCPP may comprise (ii) 2 or 3 amino acid residues independently having a side chain comprising an aromatic or heteroaromatic group.
cCPP可包含(ii)2、3、4、5或6个独立地具有包含芳族基团的侧链的氨基酸残基。cCPP可包含(ii)2个独立地具有包含芳族基团的侧链的氨基酸残基。cCPP可包含(ii)3个独立地具有包含芳族基团的侧链的氨基酸残基。cCPP可包含(ii)4个独立地具有包含芳族基团的侧链的氨基酸残基。cCPP可包含(ii)5个独立地具有包含芳族基团的侧链的氨基酸残基。cCPP可包含(ii)6个独立地具有包含芳族基团的侧链的氨基酸残基。cCPP可包含(ii)2、3或4个独立地具有包含芳族基团的侧链的氨基酸残基。cCPP可包含(ii)2或3个独立地具有包含芳族基团的侧链的氨基酸残基。cCPP may contain (ii) 2, 3, 4, 5 or 6 amino acid residues independently having a side chain containing an aromatic group. cCPP may contain (ii) 2 amino acid residues independently having a side chain containing an aromatic group. cCPP may contain (ii) 3 amino acid residues independently having a side chain containing an aromatic group. cCPP may contain (ii) 4 amino acid residues independently having a side chain containing an aromatic group. cCPP may contain (ii) 5 amino acid residues independently having a side chain containing an aromatic group. cCPP may contain (ii) 6 amino acid residues independently having a side chain containing an aromatic group. cCPP may contain (ii) 2, 3 or 4 amino acid residues independently having a side chain containing an aromatic group. cCPP may contain (ii) 2 or 3 amino acid residues independently having a side chain containing an aromatic group.
芳族基团可以是6至14元芳基。芳基可以是苯基、萘基或蒽基，它们各自任选地被取代。芳基可以是苯基或萘基，它们各自任选地被取代。杂芳族基团可以是具有1、2或3个选自N、O和S的杂原子的6至14元杂芳基。杂芳基可以是吡啶基、喹啉基或异喹啉基。The aromatic group may be a 6 to 14 membered aryl group. The aryl group may be a phenyl, naphthyl or anthracenyl group, each of which may be optionally substituted. The aryl group may be a phenyl or naphthyl group, each of which may be optionally substituted. The heteroaromatic group may be a 6 to 14 membered heteroaryl group having 1, 2 or 3 heteroatoms selected from N, O and S. The heteroaryl group may be a pyridyl, quinolyl or isoquinolyl group.
具有包含芳族或杂芳族基团的侧链的氨基酸残基可各自独立地是双(高萘基丙氨酸)、高萘基丙氨酸、萘基丙氨酸、苯基甘氨酸、双(高苯丙氨酸)、高苯丙氨酸、苯丙氨酸、色氨酸、3-(3-苯并噻吩基)-丙氨酸、3-(2-喹啉基)-丙氨酸、O-苄基丝氨酸、3-(4-(苄氧基)苯基)-丙氨酸、S-(4-甲基苄基)半胱氨酸、N-(萘-2-基)谷氨酰胺、3-(1,1'-联苯-4-基)-丙氨酸、3-(3-苯并噻吩基)-丙氨酸或酪氨酸，它们各自任选地被一个或多个取代基取代。具有包含芳族或杂芳族基团的侧链的氨基酸可各自独立地选自：The amino acid residues having side chains containing aromatic or heteroaromatic groups may each independently be bis(homonaphthylalanine), homonaphthylalanine, naphthylalanine, phenylglycine, bis(homophenylalanine), homophenylalanine, phenylalanine, tryptophan, 3-(3-benzothienyl)-alanine, 3-(2-quinolyl)-alanine, O-benzylserine, 3-(4-(benzyloxy)phenyl)-alanine, S-(4-methylbenzyl)cysteine, N-(naphthalene-2-yl)glutamine, 3-(1,1'-biphenyl-4-yl)-alanine, 3-(3-benzothienyl)-alanine or tyrosine, each of which is optionally substituted with one or more substituents. The amino acids having side chains containing aromatic or heteroaromatic groups may each independently be selected from:
其中N-末端的H和/或C-末端的H被肽键替换。 wherein the N-terminal H and/or the C-terminal H are replaced by a peptide bond.
具有包含芳族或杂芳族基团的侧链的氨基酸残基可各自独立地是苯丙氨酸、萘基丙氨酸、苯基甘氨酸、高苯丙氨酸、高萘基丙氨酸、双(高苯丙氨酸)、双-(高萘基丙氨酸)、色氨酸或酪氨酸的残基，它们各自任选地被一个或多个取代基取代。具有包含芳族基团的侧链的氨基酸残基可各自独立地是酪氨酸、苯丙氨酸、1-萘基丙氨酸、2-萘基丙氨酸、色氨酸、3-苯并噻吩基丙氨酸、4-苯基苯丙氨酸、3,4-二氟苯丙氨酸、4-三氟甲基苯丙氨酸、2,3,4,5,6-五氟苯丙氨酸、高苯丙氨酸、β-高苯丙氨酸、4-叔丁基-苯丙氨酸、4-吡啶基丙氨酸、3-吡啶基丙氨酸、4-甲基苯丙氨酸、4-氟苯丙氨酸、4-氯苯丙氨酸、3-(9-蒽基)-丙氨酸的残基。具有包含芳族基团的侧链的氨基酸残基可各自独立地是苯丙氨酸、萘基丙氨酸、苯基甘氨酸、高苯丙氨酸或高萘基丙氨酸的残基，它们各自任选地被一个或多个取代基取代。具有包含芳族基团的侧链的氨基酸残基可各自独立地是苯丙氨酸、萘基丙氨酸、高苯丙氨酸、高萘基丙氨酸、双(高萘基丙氨酸)或双(高萘基丙氨酸)的残基，它们各自任选地被一个或多个取代基取代。具有包含芳族基团的侧链的氨基酸残基可各自独立地是苯丙氨酸或萘基丙氨酸的残基，它们各自任选地被一个或多个取代基取代。至少一个具有包含芳族基团的侧链的氨基酸残基可以是苯丙氨酸的残基。至少两个具有包含芳族基团的侧链的氨基酸残基可以是苯丙氨酸的残基。每个具有包含芳族基团的侧链的氨基酸残基可以是苯丙氨酸的残基。The amino acid residues having side chains containing aromatic or heteroaromatic groups may each independently be residues of phenylalanine, naphthylalanine, phenylglycine, homophenylalanine, homonaphthylalanine, bis(homophenylalanine), bis-(homonaphthylalanine), tryptophan or tyrosine, each of which is optionally substituted with one or more substituents. The amino acid residues having side chains containing aromatic groups may each independently be residues of tyrosine, phenylalanine, 1-naphthylalanine, 2-naphthylalanine, tryptophan, 3-benzothienylalanine, 4-phenylphenylalanine, 3,4-difluorophenylalanine, 4-trifluoromethylphenylalanine, 2,3,4,5,6-pentafluorophenylalanine, homophenylalanine, β-homophenylalanine, 4-tert-butyl-phenylalanine, 4-pyridylalanine, 3-pyridylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, 3-(9-anthryl)-alanine. The amino acid residues having side chains containing aromatic groups may each independently be residues of phenylalanine, naphthylalanine, phenylglycine, homophenylalanine or homonaphthylalanine, each of which is optionally substituted with one or more substituents. The amino acid residues having side chains containing aromatic groups may each independently be residues of phenylalanine, naphthylalanine, homophenylalanine, homonaphthylalanine, bis(homonaphthylalanine) or bis(homonaphthylalanine), each of which is optionally substituted with one or more substituents. The amino acid residues having side chains containing aromatic groups may each independently be residues of phenylalanine or naphthylalanine, each of which is optionally substituted with one or more substituents. At least one amino acid residue having a side chain containing an aromatic group may be a residue of phenylalanine. At least two amino acid residues having a side chain containing an aromatic group may be a residue of phenylalanine. Each amino acid residue having a side chain containing an aromatic group may be a residue of phenylalanine.
在实施方案中，具有包含芳族或杂芳族基团的侧链的氨基酸没有一个是邻接的。两个具有包含芳族或杂芳族基团的侧链的氨基酸可以是邻接的。两个邻接的氨基酸可有相反的立体化学。两个邻接的氨基酸可具有相同的立体化学。三个具有包含芳族或杂芳族基团的侧链的氨基酸可以是邻接的。三个邻接的氨基酸可具有相同的立体化学。三个邻接的氨基酸可具有交替的立体化学。In an embodiment, none of the amino acids having side chains comprising aromatic or heteroaromatic groups are adjacent. Two amino acids having side chains comprising aromatic or heteroaromatic groups may be adjacent. Two adjacent amino acids may have opposite stereochemistry. Two adjacent amino acids may have the same stereochemistry. Three amino acids having side chains comprising aromatic or heteroaromatic groups may be adjacent. Three adjacent amino acids may have the same stereochemistry. Three adjacent amino acids may have alternating stereochemistry.
包含芳族或杂芳族基团的氨基酸残基可以是L-氨基酸。包含芳族或杂芳族基团的氨基酸残基可以是D-氨基酸。包含芳族或杂芳族基团的氨基酸残基可以是D-氨基酸和L-氨基酸的混合物。The amino acid residue containing an aromatic or heteroaromatic group can be an L-amino acid. The amino acid residue containing an aromatic or heteroaromatic group can be a D-amino acid. The amino acid residue containing an aromatic or heteroaromatic group can be a mixture of a D-amino acid and an L-amino acid.
任选的取代基可以是任何不显著降低(例如超过50％)cCPP的胞质递送效率的原子或基团，例如与不具有该取代基的其他相同序列相比。任选的取代基可以是疏水取代基或亲水取代基。任选的取代基可以是疏水取代基。取代基可增加疏水氨基酸的溶剂可及表面积(如本文所定义)。取代基可以是卤素、烷基、烯基、炔基、环烷基、环烯基、环炔基、杂环基、芳基、杂芳基、烷氧基、芳氧基、酰基、烷基氨基甲酰基、烷基羧酰胺基、烷氧羰基、烷硫基或芳硫基。取代基可以是卤素。The optional substituent may be any atom or group that does not significantly reduce (e.g., more than 50%) the cytoplasmic delivery efficiency of the cCPP, for example, compared to an otherwise identical sequence without the substituent. The optional substituent may be a hydrophobic substituent or a hydrophilic substituent. The optional substituent may be a hydrophobic substituent. The substituent may increase the solvent accessible surface area (as defined herein) of the hydrophobic amino acid. The substituent may be a halogen, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl, a cycloalkynyl, a heterocyclyl, an aryl, a heteroaryl, an alkoxy, an aryloxy, an acyl, an alkylcarbamoyl, an alkylcarboxamido, an alkoxycarbonyl, an alkylthio, or an arylthio. The substituent may be a halogen.
尽管不希望受理论束缚，但据信相对于具有较低疏水性值的氨基酸，具有较高疏水性值的芳族或杂芳族基团的氨基酸(即具有包含芳族或杂芳族基团的侧链的氨基酸)可改善cCPP的胞质递送效率。每个疏水氨基酸可独立地具有大于甘氨酸的疏水性值。每个疏水氨基酸可独立地是疏水性值大于丙氨酸的疏水氨基酸。每个疏水氨基酸可独立地具有大于或等于苯丙氨酸的疏水性值。疏水性可使用本领域已知的疏水性标度来测量。表2列出了各种氨基酸的疏水性值，如Eisenberg和Weiss(Proc.Natl.Acad.Sci.U.S.A.1984；81(1):140-144)、Engleman等人(Ann.Rev.of Biophys.Biophys.Chem.1986；1986(15):321-53)、Kyte和Doolittle(J.Mol.Biol.1982；157(1):105-132)、Hoop和Woods(Proc.Natl.Acad.Sci.U.S.A.1981；78(6):3824-3828)以及Janin(Nature.1979；277(5696):491-492)所报道，所述文献各自的全部内容以引用方式并入本文。疏水性可使用Engleman等人报道的疏水性标度来测量。Although not wishing to be bound by theory, it is believed that amino acids with aromatic or heteroaromatic groups (i.e., amino acids with side chains comprising aromatic or heteroaromatic groups) having higher hydrophobicity values can improve the cytoplasmic delivery efficiency of cCPPs relative to amino acids with lower hydrophobicity values. Each hydrophobic amino acid can independently have a hydrophobicity value greater than glycine. Each hydrophobic amino acid can independently be a hydrophobic amino acid having a hydrophobicity value greater than alanine. Each hydrophobic amino acid can independently have a hydrophobicity value greater than or equal to phenylalanine. Hydrophobicity can be measured using a hydrophobicity scale known in the art. Table 2 lists the hydrophobicity values of various amino acids as reported by Eisenberg and Weiss (Proc. Natl. Acad. Sci. U.S.A. 1984; 81(1): 140-144), Engleman et al. (Ann. Rev. of Biophys. Biophys. Chem. 1986; 1986(15): 321-53), Kyte and Doolittle (J. Mol. Biol. 1982; 157(1): 105-132), Hoop and Woods (Proc. Natl. Acad. Sci. U.S.A. 1981; 78(6): 3824-3828), and Janin (Nature. 1979; 277(5696): 491-492), the entire contents of each of which are incorporated herein by reference. Hydrophobicity can be measured using the hydrophobicity scale reported by Engleman et al.
表2.氨基酸疏水性Table 2. Amino Acid Hydrophobicity
可选择芳族或杂芳族基团的大小以改善cCPP的胞质递送效率。尽管不希望受理论束缚，但据信与具有较小疏水氨基酸的其他相同序列相比，氨基酸侧链上的较大芳族或杂芳族基团可改善胞质递送效率。疏水氨基酸的大小可根据疏水氨基酸的分子量、疏水氨基酸的位阻效应、侧链的溶剂可及表面积(SASA)或它们的组合来测量。疏水氨基酸的大小可根据疏水氨基酸的分子量来测量，并且较大的疏水氨基酸具有分子量为至少约90g/mol、或至少约130g/mol、或至少约141g/mol的侧链。氨基酸的大小可根据疏水侧链的SASA来测量。疏水氨基酸可具有SASA大于或等于丙氨酸、或大于或等于甘氨酸的侧链。较大的疏水氨基酸可具有SASA大于丙氨酸或大于甘氨酸的侧链。疏水氨基酸可具有SASA大于或等于约哌啶-2-羧酸、大于或等于约色氨酸、大于或等于约苯丙氨酸、或大于或等于约萘基丙氨酸的芳族或杂芳族基团。第一疏水氨基酸(AAH1)可具有SASA为至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约或至少约的侧链。第二疏水氨基酸(AAH2)可具有SASA为至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约或至少约的侧链。AAH1和AAH2的侧链可具有至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约大于约至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约至少约大于约至少约至少约至少约至少约或至少约的组合SASA。AAH2可以是疏水氨基酸残基，其侧链的SASA小于或等于AAH1的疏水侧链的SASA。以举例的方式而非限制，与具有Phe-Arg基序的其他方面相同的cCPP相比，具有Nal-Arg基序的cCPP可表现出改善的胞质递送效率；与具有Nal-Phe-Arg基序的其他方面相同的cCPP相比，具有Phe-Nal-Arg基序的cCPP可表现出改善的胞质递送效率；并且与具有nal-Phe-Arg基序的其他方面相同的cCPP相比，phe-Nal-Arg基序可表现出改善的胞质递送效率。The size of the aromatic or heteroaromatic group can be selected to improve the cytoplasmic delivery efficiency of the cCPP. Although it is not desired to be bound by theory, it is believed that a larger aromatic or heteroaromatic group on the amino acid side chain can improve the cytoplasmic delivery efficiency compared to other identical sequences with smaller hydrophobic amino acids. The size of the hydrophobic amino acid can be measured according to the molecular weight of the hydrophobic amino acid, the steric effect of the hydrophobic amino acid, the solvent accessible surface area (SASA) of the side chain, or a combination thereof. The size of the hydrophobic amino acid can be measured according to the molecular weight of the hydrophobic amino acid, and the larger hydrophobic amino acid has a side chain with a molecular weight of at least about 90 g/mol, or at least about 130 g/mol, or at least about 141 g/mol. The size of the amino acid can be measured according to the SASA of the hydrophobic side chain. The hydrophobic amino acid may have a side chain with a SASA greater than or equal to alanine, or greater than or equal to glycine. The larger hydrophobic amino acid may have a side chain with a SASA greater than alanine or greater than glycine. The hydrophobic amino acid may have an aromatic or heteroaromatic group with a SASA greater than or equal to about piperidine-2-carboxylic acid, greater than or equal to about tryptophan, greater than or equal to about phenylalanine, or greater than or equal to about naphthylalanine. The first hydrophobic amino acid (AAH1 ) may have a SASA of at least about At least about At least about At least about At least about At least about At least about At least about At least about At least about At least about At least about or at least about The second hydrophobic amino acid (AAH2 ) may have a SASA of at least about At least about At least about At least about At least about At least about At least about At least about At least about At least about At least about At least about or at least about The side chains of AAH1 and AAH2 may have at least about At least about At least about At least about At least about At least about At least about At least about At least about At least about At least about At least about At least about At least about At least about Greater than about At least about At least about At least about At least about At least about At least about At least about At least about At least about At least about At least about At least about At least about At least about Greater than about At least about At least about At least about At least about or at least about AAH2 may be a hydrophobic amino acid residue having a side chain SASA less than or equal to the SASA of the hydrophobic side chain of AAH1 . By way of example and not limitation, a cCPP having a Nal-Arg motif may exhibit improved cytoplasmic delivery efficiency compared to an otherwise identical cCPP having a Phe-Arg motif; a cCPP having a Phe-Nal-Arg motif may exhibit improved cytoplasmic delivery efficiency compared to an otherwise identical cCPP having a Nal-Phe-Arg motif; and a phe-Nal-Arg motif may exhibit improved cytoplasmic delivery efficiency compared to an otherwise identical cCPP having a nal-Phe-Arg motif.
如本文所用，“疏水表面积”或“SASA”是指氨基酸侧链的可被溶剂可及的表面积(报告为平方埃；)。SASA可使用由Shrake&Rupley(JMol Biol.79(2):351-71)开发的“滚球”算法来计算，其全文以引用方式并入本文用于所有目的。这种算法使用特定半径的溶剂“球体”来探测分子表面。球体的典型值是其近似于水分子的半径。As used herein, "hydrophobic surface area" or "SASA" refers to the solvent accessible surface area of amino acid side chains (reported as square angstroms; ). SASA can be calculated using the "rolling ball" algorithm developed by Shrake & Rupley (J Mol Biol. 79(2):351-71), which is incorporated herein by reference in its entirety for all purposes. This algorithm uses a solvent "sphere" of a specific radius to probe the molecular surface. Typical values for the sphere are This is approximately the radius of a water molecule.
某些侧链的SASA值示于下表3中。本文所述的SASA值基于下表3中列出的理论值，如Tien等人(PLOS ONE 8(11):e80635，可得自doi.org/10.1371/journal.pone.0080635)所报道，其全文以引用方式并入本文用于所有目的。The SASA values of certain side chains are shown below in Table 3. The SASA values described herein are based on the theoretical values listed in Table 3 below, as reported by Tien et al. (PLOS ONE 8(11):e80635, available at doi.org/10.1371/journal.pone.0080635), which is incorporated herein by reference in its entirety for all purposes.
表3.氨基酸SASA值Table 3. SASA values of amino acids
具有包含胍基、胍置换基团或其质子化形式的侧链的氨基酸残基Amino acid residues having a side chain containing a guanidine group, a guanidine replacement group or a protonated form thereof
如本文所用，胍是指以下结构：As used herein, guanidine refers to the following structure:
如本文所用，质子化形式的胍是指以下结构：As used herein, the protonated form of guanidine refers to the following structure:
胍置换基团是指氨基酸侧链上的官能团，其在生理pH或高于生理pH时将带正电荷，或者可再现胍基团的氢键给予和接受活性。Guanidine replacement groups are functional groups on the side chains of amino acids that are positively charged at or above physiological pH or that can reproduce guanidine. The hydrogen bond donating and accepting activity of the group.
胍置换基团促进细胞渗透和治疗剂的递送，同时降低与胍基或其质子化形式相关的毒性。cCPP可包含至少一个具有包含胍或胍置换基团的侧链的氨基酸。cCPP可包含至少两个具有包含胍或胍置换基团的侧链的氨基酸。cCPP可包含至少三个具有包含胍或胍置换基团的侧链的氨基酸The guanidine replacement group promotes cell penetration and delivery of therapeutic agents while reducing the toxicity associated with the guanidine group or its protonated form. The cCPP may include at least one The cCPP may contain at least two amino acids containing guanidine or guanidine The cCPP may contain at least three amino acids containing guanidine or guanidine Substitution groups on the side chains of amino acids
胍或胍基团可以是胍或胍的等排体。胍或胍置换基团的碱性可低于胍。Guanidine or guanidine The group can be guanidine or guanidine Guanidine or guanidine The displacing group may be less basic than guanidine.
如本文所用，胍置换基团是指或其质子化形式。As used herein, a guanidine replacement group refers to or its protonated form.
本公开涉及包含4至20个氨基酸残基的cCPP，其中：(i)至少一个氨基酸具有包含胍基或其质子化形式的侧链；(ii)至少一个氨基酸残基不具有侧链或具有包含或其质子化形式的侧链；并且(iii)至少两个氨基酸残基独立地具有包含芳族或杂芳族基团的侧链。The present disclosure relates to cCPPs comprising 4 to 20 amino acid residues, wherein: (i) at least one amino acid has a side chain comprising a guanidine group or a protonated form thereof; (ii) at least one amino acid residue does not have a side chain or has a side chain comprising or a side chain thereof in a protonated form; and (iii) at least two amino acid residues independently have a side chain comprising an aromatic or heteroaromatic group.
至少两个氨基酸残基可不具有侧链或具有包含或其质子化形式的侧链。如本文所用，当不存在侧链时，氨基酸残基在连接胺和羧酸的碳原子上具有两个氢原子(例如，-CH2-)。At least two amino acid residues may have no side chains or have or a protonated form of its side chain. As used herein, when no side chain is present, the amino acid residue has two hydrogen atoms on the carbon atom connecting the amine and the carboxylic acid (eg,-CH2- ).
cCPP可包含至少一个具有包含以下部分之一的侧链的氨基酸：The cCPP may comprise at least one amino acid having a side chain comprising one of the following moieties:
或其质子化形式。 or its protonated form.
cCPP可包含至少两个氨基酸，每个氨基酸独立地具有以下部分之一：The cCPP may comprise at least two amino acids, each of which independently has one of the following moieties:
或其质子化形式。至少两个氨基酸可具有包含选自以下的相同部分的侧链：或其质子化形式。至少一个氨基酸可具有包含或其质子化形式的侧链。至少两个氨基酸可具有包含或其质子化形式的侧链。一个、两个、三个或四个氨基酸可具有包含或其质子化形式的侧链。一个氨基酸可具有包含或其质子化形式的侧链。两个氨基酸可具有包含或其质子化形式的侧链。或其质子化形式可附接到氨基酸侧链的末端。可附接到氨基酸侧链的末端。 or a protonated form thereof. At least two amino acids may have a side chain comprising the same moiety selected from: or its protonated form. At least one amino acid may have or their protonated side chains. At least two amino acids may have or their protonated forms. One, two, three or four amino acids may have a or its protonated form. An amino acid may have a side chain comprising or their protonated side chains. The two amino acids may have or its protonated form. Or its protonated form may be attached to the terminus of the amino acid side chain. Can be attached to the terminal end of an amino acid side chain.
cCPP可包含(iii)2、3、4、5或6个独立地具有包含胍基、胍置换基团或其质子化形式的侧链的氨基酸残基。cCPP可包含(iii)2个独立地具有包含胍基、胍置换基团或其质子化形式的侧链的氨基酸残基。cCPP可包含(iii)3个独立地具有包含胍基、胍置换基团或其质子化形式的侧链的氨基酸残基。cCPP可包含(iii)4个独立地具有包含胍基、胍置换基团或其质子化形式的侧链的氨基酸残基。cCPP可包含(iii)5个独立地具有包含胍基、胍置换基团或其质子化形式的侧链的氨基酸残基。cCPP可包含(iii)6个独立地具有包含胍基、胍置换基团或其质子化形式的侧链的氨基酸残基。cCPP可包含(iii)2、3、4或5个独立地具有包含胍基、胍置换基团或其质子化形式的侧链的氨基酸残基。cCPP可包含(iii)2、3或4个独立地具有包含胍基、胍置换基团或其质子化形式的侧链的氨基酸残基。cCPP可包含(iii)2或3个独立地具有包含胍基、胍置换基团或其质子化形式的侧链的氨基酸残基。cCPP可包含(iii)至少一个具有包含胍基或其质子化形式的侧链的氨基酸残基。cCPP可包含(iii)两个具有包含胍基或其质子化形式的侧链的氨基酸残基。cCPP可包含(iii)三个具有包含胍基或其质子化形式的侧链的氨基酸残基。The cCPP may comprise (iii) 2, 3, 4, 5 or 6 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 2 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 3 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 4 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 5 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 6 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 2, 3, 4 or 5 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 2, 3 or 4 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) 2 or 3 amino acid residues independently having a side chain comprising a guanidine group, a guanidine replacement group or a protonated form thereof. The cCPP may comprise (iii) at least one amino acid residue having a side chain comprising a guanidine group or a protonated form thereof. The cCPP may comprise (iii) two amino acid residues having a side chain comprising a guanidine group or a protonated form thereof. The cCPP may comprise (iii) three amino acid residues having a side chain comprising a guanidine group or a protonated form thereof.
氨基酸残基可独立地具有包含不邻接的胍基、胍置换基团或其质子化形式的侧链。两个氨基酸残基可独立地具有包含可以是邻接的胍基、胍置换基团或其质子化形式的侧链。三个氨基酸残基可独立地具有包含可以是邻接的胍基、胍置换基团或其质子化形式的侧链。四个氨基酸残基可独立地具有包含可以是邻接的胍基、胍置换基团或其质子化形式的侧链。邻接的氨基酸残基可具有相同的立体化学。邻接的氨基酸可具有交替的立体化学。The amino acid residues may independently have side chains comprising non-adjacent guanidine groups, guanidine replacement groups or their protonated forms. Two amino acid residues may independently have side chains comprising guanidine groups, guanidine replacement groups or their protonated forms that may be adjacent. Three amino acid residues may independently have side chains comprising guanidine groups, guanidine replacement groups or their protonated forms that may be adjacent. Four amino acid residues may independently have side chains comprising guanidine groups, guanidine replacement groups or their protonated forms that may be adjacent. Adjacent amino acid residues may have the same stereochemistry. Adjacent amino acids may have alternating stereochemistry.
独立地具有包含胍基、胍置换基团或其质子化形式的侧链的氨基酸残基可以是L-氨基酸。独立地具有包含胍基、胍置换基团或其质子化形式的侧链的氨基酸残基可以是D-氨基酸。独立地具有包含胍基、胍置换基团或其质子化形式的侧链的氨基酸残基可以是L-氨基酸或D-氨基酸的混合物。The amino acid residue independently having a side chain comprising a guanidino group, a guanidine replacement group, or a protonated form thereof can be an L-amino acid. The amino acid residue independently having a side chain comprising a guanidino group, a guanidino replacement group, or a protonated form thereof can be a D-amino acid. The amino acid residue independently having a side chain comprising a guanidino group, a guanidino replacement group, or a protonated form thereof can be a mixture of L-amino acids or D-amino acids.
每个具有包含胍基或其质子化形式的侧链的氨基酸残基可独立地是精氨酸、高精氨酸、2-氨基-3-丙酸、2-氨基-4-胍基丁酸或其质子化形式的残基。每个具有包含胍基或其质子化形式的侧链的氨基酸残基可独立地是精氨酸残基或其质子化形式。Each amino acid residue having a side chain comprising a guanidine group or a protonated form thereof may independently be a residue of arginine, homoarginine, 2-amino-3-propionic acid, 2-amino-4-guanidinobutyric acid or a protonated form thereof. Each amino acid residue having a side chain comprising a guanidine group or a protonated form thereof may independently be an arginine residue or a protonated form thereof.
每个具有包含胍置换基团或其质子化形式的侧链的氨基酸可独立地是Each amino acid having a side chain containing a guanidine replacement group or a protonated form thereof may independently be
或其质子化形式。 or its protonated form.
不受理论的束缚，假设胍置换基团相对于精氨酸具有降低的碱度，并且在一些情况下在生理pH下不带电荷(例如，-N(H)C(O))，并且能够维持与质膜上的磷脂的二齿氢键相互作用，这被认为促进有效的膜结合和随后的内化。正电荷的去除也被认为降低了cCPP的毒性。Without being bound by theory, it is hypothesized that the guanidine replacement group has reduced basicity relative to arginine and, in some cases, is uncharged (e.g., -N(H)C(O)) at physiological pH and is able to maintain bidentate hydrogen bonding interactions with phospholipids on the plasma membrane, which is thought to promote efficient membrane binding and subsequent internalization. The removal of the positive charge is also thought to reduce the toxicity of cCPP.
本领域技术人员将理解，上述非天然芳族疏水氨基酸的N-末端/或C-末端在掺入本文公开的肽中后形成酰胺键。It will be understood by those skilled in the art that the N-terminus and/or C-terminus of the above-mentioned non-natural aromatic hydrophobic amino acids form an amide bond after incorporation into the peptides disclosed herein.
cCPP可包含具有包含芳族或杂芳族基团的侧链的第一氨基酸和具有包含芳族或杂芳族基团的侧链的第二氨基酸，其中第一甘氨酸的N-末端与具有包含芳族或杂芳族基团的侧链的第一氨基酸形成肽键，并且第一甘氨酸的C-末端与具有包含芳族或杂芳族基团的侧链的第二氨基酸形成肽键。尽管按照惯例，术语“第一氨基酸”通常是指肽序列的N-末端氨基酸，但如本文所用，“第一氨基酸”用于将所指氨基酸与cCPP中的另一氨基酸(例如，“第二个氨基酸”)区分开，使得术语“第一氨基酸”可以是或可指位于肽序列的N-末端的氨基酸。The cCPP may comprise a first amino acid having a side chain comprising an aromatic or heteroaromatic group and a second amino acid having a side chain comprising an aromatic or heteroaromatic group, wherein the N-terminus of the first glycine forms a peptide bond with the first amino acid having a side chain comprising an aromatic or heteroaromatic group, and the C-terminus of the first glycine forms a peptide bond with the second amino acid having a side chain comprising an aromatic or heteroaromatic group. Although by convention, the term "first amino acid" generally refers to the N-terminal amino acid of a peptide sequence, as used herein, "first amino acid" is used to distinguish the referred amino acid from another amino acid (e.g., "second amino acid") in the cCPP, such that the term "first amino acid" may be or may refer to an amino acid located at the N-terminus of the peptide sequence.
cCPP可包含：第二甘氨酸的N-末端与具有包含芳族或杂芳族基团的侧链的氨基酸形成肽键，并且第二甘氨酸的C-末端与具有包含胍基或其质子化形式的侧链的氨基酸形成肽键。The cCPP may comprise: the N-terminus of the second glycine forming a peptide bond with an amino acid having a side chain comprising an aromatic or heteroaromatic group, and the C-terminus of the second glycine forming a peptide bond with an amino acid having a side chain comprising a guanidine group or a protonated form thereof.
cCPP可包含具有包含胍基或其质子化形式的侧链的第一氨基酸，和具有包含胍基或其质子化形式的侧链的第二氨基酸，其中第三甘氨酸的N-末端与具有包含胍基或其质子化形式的侧链的第一氨基酸形成肽键，并且第三甘氨酸的C-末端与具有包含胍基或其质子化形式的侧链的第二氨基酸形成肽键。The cCPP may comprise a first amino acid having a side chain comprising a guanidine group or a protonated form thereof, and a second amino acid having a side chain comprising a guanidine group or a protonated form thereof, wherein the N-terminus of the third glycine forms a peptide bond with the first amino acid having a side chain comprising a guanidine group or a protonated form thereof, and the C-terminus of the third glycine forms a peptide bond with the second amino acid having a side chain comprising a guanidine group or a protonated form thereof.
cCPP可包含天冬酰胺、天冬氨酸、谷氨酰胺、谷氨酸或高谷氨酰胺的残基。cCPP可包含天冬酰胺的残基。cCPP可包含谷氨酰胺的残基。The cCPP may comprise residues of asparagine, aspartic acid, glutamine, glutamic acid or homoglutamine. The cCPP may comprise residues of asparagine. The cCPP may comprise residues of glutamine.
cCPP可包含酪氨酸、苯丙氨酸、1-萘基丙氨酸、2-萘基丙氨酸、色氨酸、3-苯并噻吩基丙氨酸、4-苯基苯丙氨酸、3,4-二氟苯丙氨酸、4-三氟甲基苯丙氨酸、2,3,4,5,6-五氟苯丙氨酸、高苯丙氨酸、β-高苯丙氨酸、4-叔丁基-苯丙氨酸、4-吡啶基丙氨酸、3-吡啶基丙氨酸、4-甲基苯丙氨酸、4-氟苯丙氨酸、4-氯苯丙氨酸、3-(9-蒽基)-丙氨酸的残基。cCPP may comprise residues of tyrosine, phenylalanine, 1-naphthylalanine, 2-naphthylalanine, tryptophan, 3-benzothienylalanine, 4-phenylphenylalanine, 3,4-difluorophenylalanine, 4-trifluoromethylphenylalanine, 2,3,4,5,6-pentafluorophenylalanine, homophenylalanine, β-homophenylalanine, 4-tert-butyl-phenylalanine, 4-pyridylalanine, 3-pyridylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, 3-(9-anthryl)-alanine.
尽管不希望受理论束缚，但据信cCPP中氨基酸的手性可影响胞质摄取效率。cCPP可包含至少一个D氨基酸。cCPP可包含一至十五个D氨基酸。cCPP可包含一至十个D氨基酸。cCPP可包含1、2、3或4个D氨基酸。cCPP可包含具有交替的D和L手性的2、3、4、5、6、7或8个邻接的氨基酸。cCPP可包含具有相同手性的三个邻接的氨基酸。cCPP可包含具有相同手性的两个邻接的氨基酸。至少两个氨基酸可具有相反的手性。具有相反手性的至少两个氨基酸可彼此相邻。至少三个氨基酸可具有相对于彼此交替的立体化学。相对于彼此具有交替手性的至少三个氨基酸可彼此相邻。至少四个氨基酸具有相对于彼此交替的立体化学。相对于彼此具有交替手性的至少四个氨基酸可彼此相邻。至少两个氨基酸可具有相同的手性。具有相同手性的至少两个氨基酸可彼此相邻。至少两个氨基酸具有相同的手性并且至少两个氨基酸具有相反的手性。具有相反手性的至少两个氨基酸可与具有相同手性的至少两个氨基酸相邻。因此，cCPP中的相邻氨基酸可具有以下序列中的任一种：D-L；L-D；D-L-L-D；L-D-D-L；L-D-L-L-D；D-L-D-D-L；D-L-L-D-L；或L-D-D-L-D。形成cCPP的氨基酸残基都可以是L-氨基酸。形成cCPP的氨基酸残基都可以是D-氨基酸。Although not wishing to be bound by theory, it is believed that the chirality of the amino acids in the cCPP can affect the efficiency of cytoplasmic uptake. The cCPP may comprise at least one D amino acid. The cCPP may comprise one to fifteen D amino acids. The cCPP may comprise one to ten D amino acids. The cCPP may comprise 1, 2, 3 or 4 D amino acids. The cCPP may comprise 2, 3, 4, 5, 6, 7 or 8 adjacent amino acids with alternating D and L chirality. The cCPP may comprise three adjacent amino acids with the same chirality. The cCPP may comprise two adjacent amino acids with the same chirality. At least two amino acids may have opposite chirality. At least two amino acids with opposite chirality may be adjacent to each other. At least three amino acids may have alternating stereochemistry relative to each other. At least three amino acids with alternating chirality relative to each other may be adjacent to each other. At least four amino acids have alternating stereochemistry relative to each other. At least four amino acids with alternating chirality relative to each other may be adjacent to each other. At least two amino acids may have the same chirality. At least two amino acids with the same chirality may be adjacent to each other. At least two amino acids have the same chirality and at least two amino acids have opposite chirality. At least two amino acids with opposite chirality may be adjacent to at least two amino acids with the same chirality. Thus, the adjacent amino acids in a cCPP may have any of the following sequences: D-L; L-D; D-L-L-D; L-D-D-L; L-D-L-L-D; D-L-D-D-L; D-L-L-D-L; or L-D-D-L-D. The amino acid residues that form the cCPP may all be L-amino acids. The amino acid residues that form the cCPP may all be D-amino acids.
至少两个氨基酸可具有不同的手性。具有不同手性的至少两个氨基酸可彼此相邻。至少三个氨基酸相对于相邻氨基酸可具有不同的手性。至少四个氨基酸相对于相邻氨基酸可具有不同的手性。至少两个氨基酸具有相同的手性并且至少两个氨基酸具有不同的手性。形成cCPP的一个或多个氨基酸残基可以是非手性的。cCPP可包含3、4或5个氨基酸的基序，其中具有相同手性的两个氨基酸可被非手性氨基酸分开。cCPP可包含下列序列：D-X-D；D-X-D-X；D-X-D-X-D；L-X-L；L-X-L-X；或L-X-L-X-L，其中X是非手性氨基酸。非手性氨基酸可以是甘氨酸。At least two amino acids may have different chirality. At least two amino acids with different chirality may be adjacent to each other. At least three amino acids may have different chirality relative to adjacent amino acids. At least four amino acids may have different chirality relative to adjacent amino acids. At least two amino acids have the same chirality and at least two amino acids have different chirality. One or more amino acid residues forming a cCPP may be achiral. The cCPP may comprise a motif of 3, 4 or 5 amino acids, wherein two amino acids with the same chirality may be separated by an achiral amino acid. The cCPP may comprise the following sequence: D-X-D; D-X-D-X; D-X-D-X-D; L-X-L; L-X-L-X; or L-X-L-X-L, wherein X is an achiral amino acid. The achiral amino acid may be glycine.
具有包含以下的侧链的氨基酸：Amino acids having side chains comprising:
或其质子化形式，可与具有包含芳族或杂芳族基团的侧链的氨基酸相邻。具有包含以下的侧链的氨基酸：或其质子化形式，可与至少一个具有包含胍或其质子化形式的侧链的氨基酸相邻。具有包含胍或其质子化形式的侧链的氨基酸可与具有包含芳族或杂芳族基团的侧链的氨基酸相邻。两个具有包含以下的侧链的氨基酸可彼此相邻：或其质子化形式。两个具有包含胍或其质子化形式的侧链的氨基酸彼此相邻。cCPP可包含至少两个具有可包含芳族或杂芳族基团的侧链的邻接氨基酸，和至少两个具有包含以下的侧链的不相邻氨基酸：或其质子化形式。cCPP可包含至少两个具有包含芳族或杂芳族基团的侧链的邻接氨基酸和至少两个具有包含或其质子化形式的侧链的不相邻氨基酸。相邻氨基酸可具有相同的手性。相邻氨基酸可具有相反的手性。氨基酸的其他组合可具有D和L氨基酸的任何排列，例如，在前述段落中描述的任何序列。 or its protonated form, adjacent to an amino acid having a side chain comprising an aromatic or heteroaromatic group. An amino acid having a side chain comprising: or a protonated form thereof, may be adjacent to at least one amino acid having a side chain comprising guanidine or a protonated form thereof. An amino acid having a side chain comprising guanidine or a protonated form thereof may be adjacent to an amino acid having a side chain comprising an aromatic or heteroaromatic group. Two amino acids having side chains comprising: or a protonated form thereof. Two amino acids having side chains comprising guanidine or a protonated form thereof are adjacent to each other. A cCPP may comprise at least two adjacent amino acids having side chains that may comprise an aromatic or heteroaromatic group, and at least two non-adjacent amino acids having side chains comprising: or a protonated form thereof. The cCPP may comprise at least two contiguous amino acids having side chains comprising an aromatic or heteroaromatic group and at least two amino acids having side chains comprising or non-adjacent amino acids with side chains in their protonated form. Adjacent amino acids may have the same chirality. Adjacent amino acids may have opposite chirality. Other combinations of amino acids may have any arrangement of D and L amino acids, for example, any of the sequences described in the preceding paragraphs.
至少两个具有包含以下的侧链的氨基酸：At least two amino acids having side chains comprising:
或其质子化形式，与至少两个具有包含胍基或其质子化形式的侧链的氨基酸交替。 or a protonated form thereof, alternating with at least two amino acids having a side chain comprising a guanidine group or a protonated form thereof.
cCPP可具有式(A)的结构：The cCPP may have the structure of formula (A):
或其质子化形式，其中：or a protonated form thereof, wherein:
R1、R2和R3各自独立地是H或氨基酸的芳族或杂芳族侧链；R1 , R2 and R3 are each independently H or an aromatic or heteroaromatic side chain of an amino acid;
R1、R2和R3中的至少一者是氨基酸的芳族或杂芳族侧链；At least one of R1 , R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid;
R4、R5、R6、R7独立地是H或氨基酸侧链；R4 , R5 , R6 , and R7 are independently H or an amino acid side chain;
R4、R5、R6、R7中的至少一者是3-胍基-2-氨基丙酸、4-胍基-2-氨基丁酸、精氨酸、高精氨酸、N-甲基精氨酸、N,N-二甲基精氨酸、2,3-二氨基丙酸、2,4-二氨基丁酸、赖氨酸、N-甲基赖氨酸、N,N-二甲基赖氨酸、N-乙基赖氨酸、N,N,N-三甲基赖氨酸、4-胍基苯丙氨酸、瓜氨酸、N,N-二甲基赖氨酸、β-高精氨酸、3-(1-哌啶基)丙氨酸的侧链；At least one of R4 , R5 , R6 , and R7 is a side chain of 3-guanidino-2-aminopropionic acid, 4-guanidino-2-aminobutyric acid, arginine, homoarginine, N-methylarginine, N,N-dimethylarginine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, lysine, N-methyllysine, N,N-dimethyllysine, N-ethyllysine, N,N,N-trimethyllysine, 4-guanidinophenylalanine, citrulline, N,N-dimethyllysine, β-homoarginine, or 3-(1-piperidinyl)alanine;
AASC是氨基酸侧链；并且AASC is an amino acid side chain; and
q是1、2、3或4。q is 1, 2, 3, or 4.
在实施方案中，式(A)的环肽不是FfΦRrRrQ(SEQ ID NO:67)。在实施方案中，式(A)的环肽是FfΦRrRrQ(SEQ ID NO:67)。In an embodiment, the cyclic peptide of formula (A) is not FfΦRrRrQ (SEQ ID NO: 67). In an embodiment, the cyclic peptide of formula (A) is FfΦRrRrQ (SEQ ID NO: 67).
cCPP可具有式(I)的结构：The cCPP may have the structure of formula (I):
或其质子化形式，其中：or a protonated form thereof, wherein:
R1、R2和R3可各自独立地是H或具有包含芳族基团的侧链的氨基酸残基；R1 , R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group;
R1、R2和R3中的至少一者是氨基酸的芳族或杂芳族侧链；At least one of R1 , R2 and R3 is an aromatic or heteroaromatic side chain of an amino acid;
R4和R7独立地是H或氨基酸侧链；R4 andR7 are independently H or an amino acid side chain;
AASC是氨基酸侧链；AASC is the amino acid side chain;
q是1、2、3或4；并且q is 1, 2, 3, or 4; and
每个m独立地是0、1、2或3的整数。Each m is independently an integer of 0, 1, 2 or 3.
R1、R2和R3可各自独立地是H、-亚烷基-芳基或-亚烷基-杂芳基。R1、R2和R3可各自独立地是H、-C1-3亚烷基-芳基或-C1-3亚烷基-杂芳基。R1、R2和R3可各自独立地是H或-亚烷基-芳基。R1、R2和R3可各自独立地是H或-C1-3亚烷基-芳基。C1-3亚烷基可以是亚甲基。芳基可以是6至14元芳基。杂芳基可以是具有一个或多个选自N、O和S的杂原子的6至14元杂芳基。芳基可选自苯基、萘基或蒽基。芳基可以是苯基或萘基。芳基可以是苯基。杂芳基可以是吡啶基、喹啉基和异喹啉基。R1、R2和R3可各自独立地是H、-C1-3亚烷基-Ph或-C1-3亚烷基-萘基。R1、R2和R3可各自独立地是H、-CH2Ph或-CH2萘基。R1、R2和R3可各自独立地是H或-CH2Ph。R1 , R2 and R3 may each independently be H, -alkylene-aryl or -alkylene-heteroaryl. R1 , R2 and R3 may each independently be H, -C1-3 alkylene-aryl or -C1-3 alkylene-heteroaryl. R1 , R2 and R3 may each independently be H or -alkylene-aryl.R 1 , R2 and R3 may each independently be H or -C1-3 alkylene-aryl. C1-3 alkylene may be a methylene group. The aryl group may be a 6 to 14-membered aryl group. The heteroaryl group may be a 6 to 14-membered heteroaryl group having one or more heteroatoms selected from N, O and S. The aryl group may be selected from phenyl, naphthyl or anthracenyl. The aryl group may be phenyl or naphthyl. The aryl group may be phenyl. The heteroaryl group may be pyridyl, quinolyl and isoquinolyl. R1 , R2 and R3 may each independently be H, -C1-3 alkylene-Ph or -C1-3 alkylene-naphthyl. R1 , R2 and R3 may each independently be H, -CH2 Ph or -CH2 naphthyl. R1 , R2 and R3 may each independently be H or -CH2 Ph.
R1、R2和R3可各自独立地是酪氨酸、苯丙氨酸、1-萘基丙氨酸、2-萘基丙氨酸、色氨酸、3-苯并噻吩基丙氨酸、4-苯基苯丙氨酸、3,4-二氟苯丙氨酸、4-三氟甲基苯丙氨酸、2,3,4,5,6-五氟苯丙氨酸、高苯丙氨酸、β-高苯丙氨酸、4-叔丁基-苯丙氨酸、4-吡啶基丙氨酸、3-吡啶基丙氨酸、4-甲基苯丙氨酸、4-氟苯丙氨酸、4-氯苯丙氨酸、3-(9-蒽基)-丙氨酸的侧链。R1 , R2 and R3 may each independently be a side chain of tyrosine, phenylalanine, 1-naphthylalanine, 2-naphthylalanine, tryptophan, 3-benzothienylalanine, 4-phenylphenylalanine, 3,4-difluorophenylalanine, 4-trifluoromethylphenylalanine, 2,3,4,5,6-pentafluorophenylalanine, homophenylalanine, β-homophenylalanine, 4-tert-butyl-phenylalanine, 4-pyridylalanine, 3-pyridylalanine, 4-methylphenylalanine, 4-fluorophenylalanine, 4-chlorophenylalanine, or 3-(9-anthryl)-alanine.
R1可以是酪氨酸的侧链。R1可以是苯丙氨酸的侧链。R1可以是1-萘基丙氨酸的侧链。R1可以是2-萘基丙氨酸的侧链。R1可以是色氨酸的侧链。R1可以是3-苯并噻吩基丙氨酸的侧链。R1可以是4-苯基苯丙氨酸的侧链。R1可以是3,4-二氟苯丙氨酸的侧链。R1可以是4-三氟甲基苯丙氨酸的侧链。R1可以是2,3,4,5,6-五氟苯丙氨酸的侧链。R1可以是高苯丙氨酸的侧链。R1可以是β-高苯丙氨酸的侧链。R1可以是4-叔丁基-苯丙氨酸的侧链。R1可以是4-吡啶基丙氨酸的侧链。R1可以是3-吡啶基丙氨酸的侧链。R1可以是4-甲基苯丙氨酸的侧链。R1可以是4-氟苯丙氨酸的侧链。R1可以是4-氯苯丙氨酸的侧链。R1可以是3-(9-蒽基)-丙氨酸的侧链。R1 may be a side chain of tyrosine.R1 may be a side chain of phenylalanine. R 1 may be a side chain of 1-naphthylalanine. R1 may be a side chain of 2-naphthylalanine. R1 may be a side chain of tryptophan.R 1 may be a side chain of 3-benzothienylalanine. R1 may be a side chain of 4-phenylphenylalanine. R 1 may be a side chain of 3,4-difluorophenylalanine. R 1maybe a side chain of 4-trifluoromethylphenylalanine.R 1 may be a side chain of 2,3,4,5,6-pentafluorophenylalanine. R1 may be a side chain of homophenylalanine. R1 may be a side chain of β-homophenylalanine.R 1 may be a side chain of 4-tert-butyl-phenylalanine.R 1 may be a side chain of 4-pyridylalanine.R 1 may be a side chain of 3-pyridylalanine.R 1 may be a side chain of 4-methylphenylalanine.R1 may be the side chain of 4-fluorophenylalanine.R1 may be the side chain of 4-chlorophenylalanine.R1 may be the side chain of 3-(9-anthryl)-alanine.
R2可以是酪氨酸的侧链。R2可以是苯丙氨酸的侧链。R2可以是1-萘基丙氨酸的侧链。R1可以是2-萘基丙氨酸的侧链。R2可以是色氨酸的侧链。R2可以是3-苯并噻吩基丙氨酸的侧链。R2可以是4-苯基苯丙氨酸的侧链。R2可以是3,4-二氟苯丙氨酸的侧链。R2可以是4-三氟甲基苯丙氨酸的侧链。R2可以是2,3,4,5,6-五氟苯丙氨酸的侧链。R2可以是高苯丙氨酸的侧链。R2可以是β-高苯丙氨酸的侧链。R2可以是4-叔丁基-苯丙氨酸的侧链。R2可以是4-吡啶基丙氨酸的侧链。R2可以是3-吡啶基丙氨酸的侧链。R2可以是4-甲基苯丙氨酸的侧链。R2可以是4-氟苯丙氨酸的侧链。R2可以是4-氯苯丙氨酸的侧链。R2可以是3-(9-蒽基)-丙氨酸的侧链。R2 may be a side chain of tyrosine.R2 may be a side chain of phenylalanine.R2 may be a side chain of 1-naphthylalanine.R1 may be a side chain of 2-naphthylalanine.R2 may be a side chain of tryptophan.R2 may be a side chain of 3-benzothienylalanine.R2 may be a side chain of 4-phenylphenylalanine.R2 may be a side chain of 3,4-difluorophenylalanine.R2 may be a side chain of 4-trifluoromethylphenylalanine.R2 may be a side chain of 2,3,4,5,6-pentafluorophenylalanine.R2 may be a side chain of homophenylalanine.R2 may be a side chain of β-homophenylalanine.R2 may be a side chain of 4-tert-butyl-phenylalanine.R2 may be a side chain of 4-pyridylalanine.R2 may be a side chain of 3-pyridylalanine.R2 may be a side chain of 4-methylphenylalanine.R2 may be the side chain of 4-fluorophenylalanine.R2 may be the side chain of 4-chlorophenylalanine.R2 may be the side chain of 3-(9-anthryl)-alanine.
R3可以是酪氨酸的侧链。R3可以是苯丙氨酸的侧链。R3可以是1-萘基丙氨酸的侧链。R3可以是2-萘基丙氨酸的侧链。R3可以是色氨酸的侧链。R3可以是3-苯并噻吩基丙氨酸的侧链。R3可以是4-苯基苯丙氨酸的侧链。R3可以是3,4-二氟苯丙氨酸的侧链。R3可以是4-三氟甲基苯丙氨酸的侧链。R3可以是2,3,4,5,6-五氟苯丙氨酸的侧链。R3可以是高苯丙氨酸的侧链。R3可以是β-高苯丙氨酸的侧链。R3可以是4-叔丁基-苯丙氨酸的侧链。R3可以是4-吡啶基丙氨酸的侧链。R3可以是3-吡啶基丙氨酸的侧链。R3可以是4-甲基苯丙氨酸的侧链。R3可以是4-氟苯丙氨酸的侧链。R3可以是4-氯苯丙氨酸的侧链。R3可以是3-(9-蒽基)-丙氨酸的侧链。R3 may be a side chain of tyrosine.R 3 may be a side chain of phenylalanine.R 3 may be a side chain of 1-naphthylalanine. R3 may be a side chain of 2-naphthylalanine. R3 may be a side chain of tryptophan.R 3 may be a side chain of 3-benzothienylalanine. R3 may be a side chain of 4-phenylphenylalanine. R 3 may be a side chain of 3,4-difluorophenylalanine. R 3maybe a side chain of 4-trifluoromethylphenylalanine.R 3 may be a side chain of 2,3,4,5,6-pentafluorophenylalanine. R3 may be a side chain of homophenylalanine. R3 may be a side chain of β-homophenylalanine.R 3 may be a side chain of 4-tert-butyl-phenylalanine.R 3 may be a side chain of 4-pyridylalanine.R 3 may be a side chain of 3-pyridylalanine.R 3 may be a side chain of 4-methylphenylalanine. R3 may be the side chain of 4-fluorophenylalanine. R3 may be the side chain of 4-chlorophenylalanine. R3 may be the side chain of 3-(9-anthryl)-alanine.
R4可以是H、-亚烷基-芳基、-亚烷基-杂芳基。R4可以是H、-C1-3亚烷基-芳基或-C1-3亚烷基-杂芳基。R4可以是H或-亚烷基-芳基。R4可以是H或-C1-3亚烷基-芳基。C1-3亚烷基可以是亚甲基。芳基可以是6至14元芳基。杂芳基可以是具有一个或多个选自N、O和S的杂原子的6至14元杂芳基。芳基可选自苯基、萘基或蒽基。芳基可以是苯基或萘基。芳基可以是苯基。杂芳基可以是吡啶基、喹啉基和异喹啉基。R4可以是H、-C1-3亚烷基-Ph或-C1-3亚烷基-萘基。R4可以是H或表1或表3中氨基酸的侧链。R4可以是H或具有包含芳族基团的侧链的氨基酸残基。R4可以是H、-CH2Ph或-CH2萘基。R4可以是H或-CH2Ph。R4 may be H, -alkylene-aryl, -alkylene-heteroaryl. R4 may be H, -C1-3 alkylene-aryl or -C1-3 alkylene-heteroaryl. R4 may be H or -alkylene-aryl.R 4 may be H or -C1-3 alkylene-aryl. C1-3 alkylene may be methylene. Aryl may be a 6- to 14-membered aryl. Heteroaryl may be a 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O and S. Aryl may be selected from phenyl, naphthyl or anthracenyl. Aryl may be phenyl or naphthyl. Aryl may be phenyl. Heteroaryl may be pyridyl, quinolyl and isoquinolyl.R 4 may be H, -C1-3 alkylene-Ph or -C1-3 alkylene-naphthyl. R4 may be H or a side chain of an amino acid in Table 1 or Table 3. R4 may be H or an amino acid residue having a side chain containing an aromatic group.R4 may be H,-CH2Ph or-CH2naphthyl .R4 may be H or-CH2Ph .
R5可以是H、-亚烷基-芳基、-亚烷基-杂芳基。R5可以是H、-C1-3亚烷基-芳基或-C1-3亚烷基-杂芳基。R5可以是H或-亚烷基-芳基。R5可以是H或-C1-3亚烷基-芳基。C1-3亚烷基可以是亚甲基。芳基可以是6至14元芳基。杂芳基可以是具有一个或多个选自N、O和S的杂原子的6至14元杂芳基。芳基可选自苯基、萘基或蒽基。芳基可以是苯基或萘基。芳基可以是苯基。杂芳基可以是吡啶基、喹啉基和异喹啉基。R5可以是H、-C1-3亚烷基-Ph或-C1-3亚烷基-萘基。R5可以是H或表1或表3中氨基酸的侧链。R4可以是H或具有包含芳族基团的侧链的氨基酸残基。R5可以是H、-CH2Ph或-CH2萘基。R4可以是H或-CH2Ph。R5 may be H, -alkylene-aryl, -alkylene-heteroaryl. R5 may be H, -C1-3 alkylene-aryl or -C1-3 alkylene-heteroaryl. R5 may be H or -alkylene-aryl. R5 may be H or -C1-3 alkylene-aryl. C1-3 alkylene may be methylene. Aryl may be a 6- to 14-membered aryl. Heteroaryl may be a 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O and S. Aryl may be selected from phenyl, naphthyl or anthracenyl. Aryl may be phenyl or naphthyl. Aryl may be phenyl. Heteroaryl may be pyridyl, quinolyl and isoquinolyl.R 5 may be H, -C1-3 alkylene-Ph or -C1-3 alkylene-naphthyl. R5 may be H or a side chain of an amino acid in Table 1 or Table 3. R4 may be H or an amino acid residue having a side chain containing an aromatic group. R5 may be H, -CH2 Ph or -CH2 naphthyl. R4 may be H or -CH2 Ph.
R6可以是H、-亚烷基-芳基、-亚烷基-杂芳基。R6可以是H、-C1-3亚烷基-芳基或-C1-3亚烷基-杂芳基。R6可以是H或-亚烷基-芳基。R6可以是H或-C1-3亚烷基-芳基。C1-3亚烷基可以是亚甲基。芳基可以是6至14元芳基。杂芳基可以是具有一个或多个选自N、O和S的杂原子的6至14元杂芳基。芳基可选自苯基、萘基或蒽基。芳基可以是苯基或萘基。芳基可以是苯基。杂芳基可以是吡啶基、喹啉基和异喹啉基。R6可以是H、-C1-3亚烷基-Ph或-C1-3亚烷基-萘基。R6可以是H或表1或表3中氨基酸的侧链。R6可以是H或具有包含芳族基团的侧链的氨基酸残基。R6可以是H、-CH2Ph或-CH2萘基。R6可以是H或-CH2Ph。R6 can be H, -alkylene-aryl, -alkylene-heteroaryl. R6 can be H, -C1-3 alkylene-aryl or -C1-3 alkylene-heteroaryl. R6 can be H or -alkylene-aryl.R 6 can be H or -C1-3 alkylene-aryl. C1-3 alkylene can be methylene. Aryl can be 6 to 14-membered aryl. Heteroaryl can be 6 to 14-membered heteroaryl having one or more heteroatoms selected from N, O and S. Aryl can be selected from phenyl, naphthyl or anthracenyl. Aryl can be phenyl or naphthyl. Aryl can be phenyl. Heteroaryl can be pyridyl, quinolyl and isoquinolyl. R6 can be H, -C1-3 alkylene-Ph or -C1-3 alkylene-naphthyl. R6 can be H or a side chain of an amino acid in Table 1 or Table 3. R6 can be H or an amino acid residue having a side chain containing an aromatic group.R6 may be H,-CH2Ph or-CH2naphthyl .R6 may be H or-CH2Ph .
R7可以是H、-亚烷基-芳基、-亚烷基-杂芳基。R7可以是H、-C1-3亚烷基-芳基或-C1-3亚烷基-杂芳基。R7可以是H或-亚烷基-芳基。R7可以是H或-C1-3亚烷基-芳基。C1-3亚烷基可以是亚甲基。芳基可以是6至14元芳基。杂芳基可以是具有一个或多个选自N、O和S的杂原子的6至14元杂芳基。芳基可选自苯基、萘基或蒽基。芳基可以是苯基或萘基。芳基可以是苯基。杂芳基可以是吡啶基、喹啉基和异喹啉基。R7可以是H、-C1-3亚烷基-Ph或-C1-3亚烷基-萘基。R7可以是H或表1或表3中氨基酸的侧链。R7可以是H或具有包含芳族基团的侧链的氨基酸残基。R7可以是H、-CH2Ph或-CH2萘基。R7可以是H或-CH2Ph。R7 may be H, -alkylene-aryl, -alkylene-heteroaryl. R7 may be H, -C1-3 alkylene-aryl or -C1-3 alkylene-heteroaryl. R7 may be H or -alkylene-aryl. R7 may be H or -C1-3 alkylene-aryl. C1-3 alkylene may be methylene. Aryl may be a 6- to 14-membered aryl. Heteroaryl may be a 6- to 14-membered heteroaryl having one or more heteroatoms selected from N, O and S. Aryl may be selected from phenyl, naphthyl or anthracenyl. Aryl may be phenyl or naphthyl. Aryl may be phenyl. Heteroaryl may be pyridyl, quinolyl and isoquinolyl.R 7 may be H, -C1-3 alkylene-Ph or -C1-3 alkylene-naphthyl. R7 may be H or a side chain of an amino acid in Table 1 or Table 3. R7 may be H or an amino acid residue having a side chain containing an aromatic group.R7 may be H,-CH2Ph or-CH2naphthyl .R7 may be H or-CH2Ph .
R1、R2、R3、R4、R5、R6和R7中的一者、两者或三者可以是-CH2Ph。R1、R2、R3、R4、R5、R6和R7中的一者可以是-CH2Ph。R1、R2、R3、R4、R5、R6和R7中的两者可以是-CH2Ph。R1、R2、R3、R4、R5、R6和R7中的三者可以是-CH2Ph。R1、R2、R3、R4、R5、R6和R7中的至少一者可以是-CH2Ph。R1、R2、R3、R4、R5、R6和R7中不超过四者可以是-CH2Ph。One, two or three of R1 , R2 , R3 , R4 , R5 , R6 and R7 may be -CH2 Ph. One of R1 , R 2 , R3 , R4 , R5 , R6 and R7 may be -CH2 Ph. Two ofR 1 , R2 , R3 , R4 , R5 , R6 and R7 may be -CH2 Ph. Three of R1 , R2 , R3 , R4 , R5 , R6 and R7 may be -CH2 Ph. At least one of R1 , R2 , R3 , R4, R5 , R6 and R7 may be -CH2 Ph. No more than four ofR1 ,R2 ,R3 ,R4 ,R5 ,R6 andR7 may be-CH2Ph .
R1、R2、R3和R4中的一者、两者或三者是-CH2Ph。R1、R2、R3和R4中的一者是-CH2Ph。R1、R2、R3和R4中的两者是-CH2Ph。R1、R2、R3和R4中的三者是-CH2Ph。R1、R2、R3和R4中的至少一者是-CH2Ph。One, two or three of R1 , R2 , R3 and R4 are -CH2 Ph. One of R1 , R2 , R3 and R4 is -CH2 Ph. Two of R 1 , R 2 , R 3 and R 4 are -CH 2Ph.ThreeofR1 , R2 , R3 and R4 are -CH2 Ph. At least one ofR 1 , R2 , R3 and R4 is -CH2 Ph.
R1、R2、R3、R4、R5、R6和R7中的一者、两者或三者可以是H。R1、R2、R3、R4、R5、R6和R7中的一者可以是H。R1、R2、R3、R4、R5、R6和R7中的两者是H。R1、R2、R3、R5、R6和R7中的三者可以是H。R1、R2、R3、R4、R5、R6和R7中的至少一者可以是H。R1、R2、R3、R4、R5、R6和R7中不超过三者可以是-CH2Ph。R1 , R2 , R3 , R4 , R5 , R6 , and R7 may be H. One, two, or three of R1 , R2 , R3 , R4 , R5 , R6 , and R7 may be H. Two ofR 1 , R 2, R3 , R 4, R5 , R6 , and R7 are H. Three ofR 1 , R2 , R3 , R4 , R5 , R6 , and R7 may be H. At least one of R1 , R2 , R3 , R4 , R5 , R6 ,and R7 may be H. No more than three of R1 , R2 , R3 , R4 , R5 , R6, and R7 may be -CH2 Ph.
R1、R2、R3和R4中的一者、两者或三者是H。R1、R2、R3和R4中的一者是H。R1、R2、R3和R4中的两者是H。R1、R2、R3和R4中的三者是H。R1、R2、R3和R4中的至少一者是H。One, two or three of R1 , R2 , R3 and R4 are H. One of R1 , R2 , R3 and R4 is H. Two of R1 , R2 , R3 and R4 are H. Three of R1 , R2 , R3 and R4 are H. At least one of R1 , R2 , R3 and R4 is H.
R4、R5、R6和R7中的至少一者可以是3-胍基-2-氨基丙酸的侧链。R4、R5、R6和R7中的至少一者可以是4-胍基-2-氨基丁酸的侧链。R4、R5、R6和R7中的至少一者可以是精氨酸的侧链。R4、R5、R6和R7中的至少一者可以是高精氨酸的侧链。R4、R5、R6和R7中的至少一者可以是N-甲基精氨酸的侧链。R4、R5、R6和R7中的至少一者可以是N,N-二甲基精氨酸的侧链。R4、R5、R6和R7中的至少一者可以是2,3-二氨基丙酸的侧链。R4、R5、R6和R7中的至少一者可以是2,4-二氨基丁酸、赖氨酸的侧链。R4、R5、R6和R7中的至少一者可以是N-甲基赖氨酸的侧链。R4、R5、R6和R7中的至少一者可以是N,N-二甲基赖氨酸的侧链。R4、R5、R6和R7中的至少一者可以是N-乙基赖氨酸的侧链。R4、R5、R6和R7中的至少一者可以是N,N,N-三甲基赖氨酸、4-胍基苯丙氨酸的侧链。R4、R5、R6和R7中的至少一者可以是瓜氨酸的侧链。R4、R5、R6和R7中的至少一者可以是N,N-二甲基赖氨酸、β-高精氨酸的侧链。R4、R5、R6和R7中的至少一者可以是3-(1-哌啶基)丙氨酸的侧链。At least one of R4 , R5 , R6 and R7 may be a side chain of 3-guanidino-2-aminopropionic acid. At least one of R4 , R5 , R6 and R7 may be a side chain of 4-guanidino-2-aminobutyric acid. At least one of R4 , R5 , R6 and R7 may be a side chain of arginine. At least one of R4 , R5 , R6 and R7 may be a side chain of homoarginine. At least one ofR 4 , R5 , R6 and R7 may be a side chain of N-methylarginine. At least one ofR 4 , R5 , R6 and R7 may be a side chain of N,N-dimethylarginine. At least one of R4 , R5 , R6 and R7 may be a side chain of 2,3-diaminopropionic acid. At least one of R4 , R5 , R6 and R7 may be a side chain of 2,4-diaminobutyric acid or lysine. At least one of R4 , R 5 , R6 and R7 may be a side chain of N-methyllysine. At least one ofR 4 , R5 , R6 and R7 may be a side chain of N,N-dimethyllysine. At least one of R4 , R5 , R6 and R7 may be a side chain of N-ethyllysine. At least one of R4 , R5 , R6 and R7 may be a side chain of N,N,N-trimethyllysine or 4-guanidinophenylalanine. At least one of R4 , R5, R6 and R7 may be a side chain of citrulline. At least one of R4 , R5 , R6 and R7 may be a side chain of N,N-dimethyllysine or β-homoarginine. At least one of R4 , R5 , R6 and R7 may be a side chain of 3-(1-piperidinyl)alanine.
R4、R5、R6和R7中的至少两者可以是3-胍基-2-氨基丙酸的侧链。R4、R5、R6和R7中的至少两者可以是4-胍基-2-氨基丁酸的侧链。R4、R5、R6和R7中的至少两者可以是精氨酸的侧链。R4、R5、R6和R7中的至少两者可以是高精氨酸的侧链。R4、R5、R6和R7中的至少两者可以是N-甲基精氨酸的侧链。R4、R5、R6和R7中的至少两者可以是N,N-二甲基精氨酸的侧链。R4、R5、R6和R7中的至少两者可以是2,3-二氨基丙酸的侧链。R4、R5、R6和R7中的至少两者可以是2,4-二氨基丁酸、赖氨酸的侧链。R4、R5、R6和R7中的至少两者可以是N-甲基赖氨酸的侧链。R4、R5、R6和R7中的至少两者可以是N,N-二甲基赖氨酸的侧链。R4、R5、R6和R7中的至少两者可以是N-乙基赖氨酸的侧链。R4、R5、R6和R7中的至少两者可以是N,N,N-三甲基赖氨酸、4-胍基苯丙氨酸的侧链。R4、R5、R6和R7中的至少两者可以是瓜氨酸的侧链。R4、R5、R6和R7中的至少两者可以是N,N-二甲基赖氨酸、β-高精氨酸的侧链。R4、R5、R6和R7中的至少两者可以是3-(1-哌啶基)丙氨酸的侧链。At least two of R4 , R5 , R6 and R7 may be a side chain of 3-guanidino-2-aminopropionic acid. At least two of R4 , R5 , R 6 and R7 may be a side chain of 4-guanidino-2-aminobutyric acid. At least two ofR 4 , R5 , R6 and R 7 may be a side chain of arginine. At least two ofR 4 , R5 , R6 and R7 may be a side chain of homoarginine. At least two ofR 4 , R 5 , R6 and R7 may be a side chain of N-methylarginine. At least two ofR4 , R 5 , R 6 and R 7 may be a side chain of N,N-dimethylarginine. At least two of R 4,R5,R6 andR 7may be a side chain of 2,3- diaminopropionic acid. At least two of R4 , R5 , R6 and R7 may be side chains of 2,4-diaminobutyric acid or lysine. At least two of R4 , R 5 , R6 and R7 may be side chains of N-methyllysine. At least two of R4 , R 5 , R 6 and R 7 may be side chains of N,N-dimethyllysine. At least two of R 4,R5,R6 and R 7 may be side chains of N-ethyllysine. At least two of R 4 , R 5 , R 6and R 7maybesidechains of N,N,N-trimethyllysine or 4-guanidinophenylalanine. At least two ofR 4 , R 5 , R 6 and R 7 may be side chains of citrulline. At least two of R 4,R 5,R 6andR7may be side chains of N,N-dimethyllysine or β-homoarginine. At least two of R4 , R5 , R6 and R7 may be a side chain of 3-(1-piperidinyl)alanine.
R4、R5、R6和R7中的至少三者可以是3-胍基-2-氨基丙酸的侧链。R4、R5、R6和R7中的至少三者可以是4-胍基-2-氨基丁酸的侧链。R4、R5、R6和R7中的至少三者可以是精氨酸的侧链。R4、R5、R6和R7中的至少三者可以是高精氨酸的侧链。R4、R5、R6和R7中的至少三者可以是N-甲基精氨酸的侧链。R4、R5、R6和R7中的至少三者可以是N,N-二甲基精氨酸的侧链。R4、R5、R6和R7中的至少三者可以是2,3-二氨基丙酸的侧链。R4、R5、R6和R7中的至少三者可以是2,4-二氨基丁酸、赖氨酸的侧链。R4、R5、R6和R7中的至少三者可以是N-甲基赖氨酸的侧链。R4、R5、R6和R7中的至少三者可以是N,N-二甲基赖氨酸的侧链。R4、R5、R6和R7中的至少三者可以是N-乙基赖氨酸的侧链。R4、R5、R6和R7中的至少三者可以是N,N,N-三甲基赖氨酸、4-胍基苯丙氨酸的侧链。R4、R5、R6和R7中的至少三者可以是瓜氨酸的侧链。R4、R5、R6和R7中的至少三者可以是N,N-二甲基赖氨酸、β-高精氨酸的侧链。R4、R5、R6和R7中的至少三者可以是3-(1-哌啶基)丙氨酸的侧链。At least three of R4 , R5 , R6 and R7 may be a side chain of 3-guanidino-2-aminopropionic acid. At least three of R4 , R5 , R6 and R7 may be a side chain of 4-guanidino-2-aminobutyric acid. At least three ofR 4 , R5 , R6 and R7 may be a side chain of arginine. At least three of R 4 , R5 , R6 and R7 may be a side chain of homoarginine. At least three ofR 4 , R5 , R6 and R 7 may be a side chain of N-methylarginine. At least three ofR 4 , R 5 , R 6 and R 7 may be a side chain of N,N-dimethylarginine. At least three of R 4,R 5, R 6andR7maybe a side chain of 2,3- diaminopropionic acid. At least three of R4 , R5 , R6 and R7 may be side chains of 2,4-diaminobutyric acid or lysine. At least three of R4 , R5 , R6 and R 7 may be side chains of N-methyllysine. At least three of R4 , R5 , R6 and R7 may be side chains of N,N-dimethyllysine. At least three of R4 , R5 , R 6 and R 7 may be side chains of N-ethyllysine. At least three of R 4 , R 5, R 6andR7maybe side chains of N,N,N-trimethyllysine or 4-guanidinophenylalanine. At least three of R4 , R5 , R6 and R 7 may be side chains of citrulline. At least three of R 4 , R 5 , R 6andR7maybeside chains of N,N-dimethyllysine or β-homoarginine. At least three of R4 , R5 , R6 and R7 may be a side chain of 3-(1-piperidinyl)alanine.
AASC可以是天冬酰胺、谷氨酰胺或高谷氨酰胺残基的侧链。AASC可以是谷氨酰胺残基的侧链。cCPP还可包含与AASC(例如，天冬酰胺、谷氨酰胺或高谷氨酰胺的残基)缀合的接头。因此，cCPP还可包含与天冬酰胺、谷氨酰胺或高谷氨酰胺残基缀合的接头。cCPP还可包含与谷氨酰胺残基缀合的接头。AASC can be a side chain of an asparagine, glutamine or homoglutamine residue. AASC can be a side chain of a glutamine residue. cCPP can also comprise a linker conjugated to AASC (e.g., a residue of asparagine, glutamine or homoglutamine). Thus, cCPP can also comprise a linker conjugated to an asparagine, glutamine or homoglutamine residue. cCPP can also comprise a linker conjugated to a glutamine residue.
q可以是1、2或3。q可以是1或2。q可以是1。q可以是2。q可以是3。q可以是4。q can be 1, 2, or 3. q can be 1 or 2. q can be 1. q can be 2. q can be 3. q can be 4.
m可以是1-3。m可以是1或2。m可以是0，m可以是1。m可以是2。m可以是3。m can be 1-3. m can be 1 or 2. m can be 0, m can be 1. m can be 2. m can be 3.
式(A)的cCPP可具有式(I)的结构：The cCPP of formula (A) may have the structure of formula (I):
或其质子化形式，其中AASC、R2、R3、R4、R7、m和q如本文所定义。 or a protonated form thereof, wherein AASC , R2 , R3 , R4 , R7 , m and q are as defined herein.
式(A)的cCPP可具有式(I-a)或式(I-b)的结构：The cCPP of formula (A) may have the structure of formula (I-a) or formula (I-b):
或其质子化形式，其中AASC、R1、R2、R3、R4和m如本文所定义。or a protonated form thereof, wherein AASC , R1 , R2 , R3 , R4 and m are as defined herein.
式(A)的cCPP可具有式(I-1)、(I-2)、(I-3)或(I-4)的结构：The cCPP of formula (A) may have the structure of formula (I-1), (I-2), (I-3) or (I-4):
或其质子化形式，其中AASC和m如本文所定义。式(A)的cCPP可具有式(I-5)或(I-6)的结构：or a protonated form thereof, wherein AASC and m are as defined herein. The cCPP of formula (A) may have the structure of formula (I-5) or (I-6):
或其质子化形式，其中AASC如本文所定义。 or a protonated form thereof, wherein AASC is as defined herein.
式(A)的cCPP可具有式(I-1)的结构：The cCPP of formula (A) may have the structure of formula (I-1):
或其质子化形式， or its protonated form,
其中AASC和m如本文所定义。wherein AA,SC and m are as defined herein.
式(A)的cCPP可具有式(I-2)的结构：The cCPP of formula (A) may have the structure of formula (I-2):
或其质子化形式，其中AASC和m如本文所定义。 or a protonated form thereof, wherein AA,SC and m are as defined herein.
式(A)的cCPP可具有式(I-3)的结构：The cCPP of formula (A) may have the structure of formula (I-3):
或其质子化形式，其中AASC和m如本文所定义。 or a protonated form thereof, wherein AA,SC and m are as defined herein.
式(A)的cCPP可具有式(I-4)的结构：The cCPP of formula (A) may have the structure of formula (I-4):
或其质子化形式，其中AASC和m如本文所定义。 or a protonated form thereof, wherein AA,SC and m are as defined herein.
式(A)的cCPP可具有式(I-5)的结构：The cCPP of formula (A) may have the structure of formula (I-5):
或其质子化形式， or its protonated form,
其中AASC和m如本文所定义。wherein AA,SC and m are as defined herein.
式(A)的cCPP可具有式(I-6)的结构：The cCPP of formula (A) may have the structure of formula (I-6):
或其质子化形式，其中AASC和m如本文所定义。 or a protonated form thereof, wherein AA,SC and m are as defined herein.
cCPP可包含以下序列之一：FGFGRGR(SEQ ID NO:68)；GfFGrGr(SEQ ID NO:69)、FfΦGRGR(SEQ ID NO:70)；FfFGRGR(SEQ ID NO:71)；或FfΦGrGr(SEQ ID NO:72)。cCPP可具有以下序列之一：FGFΦ(SEQ ID NO:73)；GfFGrGrQ(SEQ ID NO:74)、FfΦGRGRQ(SEQ IDNO:75)；FfFGRGRQ(SEQ ID NO:76)；或FfΦGrGrQ(SEQ ID NO:77)。The cCPP may comprise one of the following sequences: FGFGRGR (SEQ ID NO: 68); GfFGrGr (SEQ ID NO: 69), FfΦGRGR (SEQ ID NO: 70); FfFGRGR (SEQ ID NO: 71); or FfΦGrGr (SEQ ID NO: 72). The cCPP may have one of the following sequences: FGFΦ (SEQ ID NO: 73); GfFGrGrQ (SEQ ID NO: 74), FfΦGRGRQ (SEQ ID NO: 75); FfFGRGRQ (SEQ ID NO: 76); or FfΦGrGrQ (SEQ ID NO: 77).
本公开还涉及具有式(II)的结构的cCPP：The present disclosure also relates to cCPPs having the structure of formula (II):
其中：in:
AASC是氨基酸侧链；AASC is the amino acid side chain;
R1a、R1b和R1c各自独立地是6至14元芳基或6至14元杂芳基；R1a , R1b and R1c are each independently 6- to 14-membered aryl or 6- to 14-membered heteroaryl;
R2a、R2b、R2c和R2d独立地是氨基酸侧链；R2a , R2b , R2c and R2d are independently amino acid side chains;
R2a、R2b、R2c和R2d中的至少一者是或其质子化形式；At least one of R2a , R2b , R2c and R2d is or its protonated form;
R2a、R2b、R2c和R2d中的至少一者是胍或其质子化形式；At least one of R2a , R2b , R2c and R2d is guanidine or a protonated form thereof;
每个n”独立地是整数0、1、2、3、4或5；Each n" is independently an integer 0, 1, 2, 3, 4 or 5;
每个n'独立地是0、1、2或3的整数；并且Each n' is independently an integer of 0, 1, 2 or 3; and
如果n'是0，则R2a、R2b、R2b或R2d不存在。If n' is 0, then R2a , R2b , R2d or R2d is not present.
R2a、R2b、R2c和R2d中的至少两者可以是或其质子化形式。R2a、R2b、R2c和R2d中的两者或三者可以是或其质子化形式。R2a、R2b、R2c和R2d中的一者可以是或其质子化形式。R2a、R2b、R2c和R2d中的至少一者可以是或其质子化形式，并且R2a、R2b、R2c和R2d的其余部分可以是胍或其质子化形式。R2a、R2b、R2c和R2d中的至少两者可以是或其质子化形式，并且R2a、R2b、R2c和R2d的其余部分可以是胍或其质子化形式。At least two of R2a , R2b , R2c and R2d may be or a protonated form thereof. Two or three of R2a , R2b , R2c and R2d may be or a protonated form thereof. One of R2a , R2b , R2c and R2d may be or a protonated form thereof. At least one ofR 2a , R2b , R2c and R2d may be or a protonated form thereof, and the remaining parts of R2a , R2b , R2c and R2d may be guanidine or a protonated form thereof. At least two of R2a , R2b , R2c and R2d may be or a protonated form thereof, and the remaining parts of R2a , R2b , R2c and R2d may be guanidine or a protonated form thereof.
所有R2a、R2b、R2c和R2d可以是或其质子化形式。R2a、R2b、R2c和R2d中的至少一者可以是或其质子化形式，并且R2a、R2b、R2c和R2d的其余部分可以是胍或其质子化形式。R2a、R2b、R2c和R2d中的至少两者可以是或其质子化形式，并且R2a、R2b、R2c和R2d的其余部分是胍或其质子化形式。All of R2a , R2b , R2c and R2d may be or a protonated form thereof. At least one ofR 2a , R2b , R2c and R2d may be or a protonated form thereof, and the remaining parts of R2a , R2b , R2c and R2d may be guanidine or a protonated form thereof. At least two of R2a , R2b , R2c and R2d may be or a protonated form thereof, and the remainder of R2a , R2b , R2c and R2d are guanidine or a protonated form thereof.
R2a、R2b、R2c和R2d中的每一者可独立地是2,3-二氨基丙酸、2,4-二氨基丁酸、鸟氨酸、赖氨酸、甲基赖氨酸、二甲基赖氨酸、三甲基赖氨酸、高赖氨酸、丝氨酸、高丝氨酸、苏氨酸、别苏氨酸、组氨酸、1-甲基组氨酸、2-氨基丁酸、天冬氨酸、谷氨酸或高谷氨酸的侧链。Each of R2a , R2b , R2c and R2d may independently be a side chain of 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, ornithine, lysine, methyllysine, dimethyllysine, trimethyllysine, homolysine, serine, homoserine, threonine, allothreonine, histidine, 1-methylhistidine, 2-aminobutyric acid, aspartic acid, glutamic acid or homoglutamic acid.
AASC可以是其中t可以是0至5的整数。AASC可以是其中t可以是0至5的整数。t可以是1至5。t是2或3。t可以是2。t可以是3。AASC can be Where t can be an integer from 0 to 5. AASC can be Wherein t may be an integer from 0 to 5. t may be from 1 to 5. t is 2 or 3. t may be 2. t may be 3.
R1a、R1b和R1c可各自独立地是6至14元芳基。R1a、R1b和R1c可各自独立地是具有一个或多个选自N、O或S的杂原子的6至14元杂芳基。R1a、R1b和R1c可各自独立地选自苯基、萘基、蒽基、吡啶基、喹啉基或异喹啉基。R1a、R1b和R1c可各自独立地选自苯基、萘基或蒽基。R1a、R1b和R1c可各自独立地是苯基或萘基。R1a、R1b和R1c可各自独立地选自吡啶基、喹啉基或异喹啉基。R1a , R1b and R1c may each independently be a 6- to 14-membered aryl group. R1a , R1b and R1c may each independently be a 6- to 14-membered heteroaryl group having one or more heteroatoms selected from N, O or S. R1a , R1b and R1c may each independently be selected from phenyl, naphthyl, anthracenyl, pyridyl, quinolyl or isoquinolyl. R1a , R1b and R1c may each independently be selected from phenyl, naphthyl or anthracenyl. R1a , R1b and R1c may each independently be phenyl or naphthyl. R1a , R1b and R1c may each independently be selected from pyridyl, quinolyl or isoquinolyl.
每个n'可独立地是1或2。每个n'可以是1。每个n'可以是2。至少一个n'可以是0。至少一个n'可以是1。至少一个n'可以是2。至少一个n'可以是3。至少一个n'可以是4。至少一个n'可以是5。Each n' may independently be 1 or 2. Each n' may be 1. Each n' may be 2. At least one n' may be 0. At least one n' may be 1. At least one n' may be 2. At least one n' may be 3. At least one n' may be 4. At least one n' may be 5.
每个n"可独立地是1至3的整数。每个n"可独立地是2或3。每个n"可以是2。每个n"可以是3。至少一个n"可以是0。至少一个n"可以是1。至少一个n"可以是2。至少一个n"可以是3。Each n" may independently be an integer from 1 to 3. Each n" may independently be 2 or 3. Each n" may be 2. Each n" may be 3. At least one n" may be 0. At least one n" may be 1. At least one n" may be 2. At least one n" may be 3.
每个n"可独立地是1或2，并且每个n'可独立地是2或3。每个n"可以是1并且每个n'可独立地是2或3。每个n"可以是1并且每个n'可以是2。每个n"是1并且每个n'是3。Each n" may independently be 1 or 2, and each n' may independently be 2 or 3. Each n" may be 1 and each n' may independently be 2 or 3. Each n" may be 1 and each n' may be 2. Each n" is 1 and each n' is 3.
式(II)的cCPP可具有式(II-1)的结构：The cCPP of formula (II) may have the structure of formula (II-1):
其中R1a、R1b、R1c、R2a、R2b、R2c、R2d、AASC、n'和n”如本文所定义。wherein R1a ,R1b , R1c , R 2a , R2b , R2c , R2d , AASC , n′ and n″ are as defined herein.
式(II)的cCPP可具有式(IIa)的结构：The cCPP of formula (II) may have the structure of formula (IIa):
其中R1a、R1b、R1c、R2a、R2b、R2c、R2d、AASC-和n'如本文所定义。wherein R1a ,R1b , R1c , R 2a , R2b , R2c , R2d , AASC- and n′ are as defined herein.
式(II)的cCPP可具有式(IIb)的结构：The cCPP of formula (II) may have the structure of formula (IIb):
其中R2a、R2b、AASC-和n'如本文所定义。wherein R2a , R2b , AASC- and n′ are as defined herein.
cCPP可具有式(IIc)的结构：The cCPP may have the structure of formula (IIc):
或其质子化形式，其中： or a protonated form thereof, wherein:
AASC和n'如本文所定义。AA,SC and n' are as defined herein.
式(IIa)的cCPP具有以下结构之一：The cCPP of formula (IIa) has one of the following structures:
其中AASC和n如本文所定义。式(IIa)的cCPP具有以下结构之一： wherein AASC and n are as defined herein. The cCPP of formula (IIa) has one of the following structures:
其中AASC和n如本文所定义式(IIa)的cCPP具有以下结构之一： wherein AASC and n are as defined herein. The cCPP of formula (IIa) has one of the following structures:
其中AASC和n如本文所定义。式(II)的cCPP可具有以下结构： wherein AASC and n are as defined herein. The cCPP of formula (II) may have the following structure:
式(II)的cCPP可具有以下结构：The cCPP of formula (II) may have the following structure:
cCPP可具有式(III)的结构：The cCPP may have the structure of formula (III):
其中：in:
AASC是氨基酸侧链；AASC is the amino acid side chain;
R1a、R1b和R1c各自独立地是6至14元芳基或6至14元杂芳基；R1a , R1b and R1c are each independently 6- to 14-membered aryl or 6- to 14-membered heteroaryl;
R2a和R2c各自独立地是H、或其质子化形式；R2a and R2c are each independently H, or its protonated form;
R2b和R2d各自独立地是胍或其质子化形式；R2b and R2d are each independently guanidine or a protonated form thereof;
每个n"独立地是1至3的整数；Each n" is independently an integer from 1 to 3;
每个n'独立地是1至5的整数；并且Each n' is independently an integer from 1 to 5; and
每个p'独立地是0至5的整数。Each p' is independently an integer from 0 to 5.
式(III)的cCPP可具有式(III-1)的结构：The cCPP of formula (III) may have the structure of formula (III-1):
其中：in:
AASC、R1a、R1b、R1c、R2a、R2c、R2b、R2d、n'、n"和p'如本文所定义。AASC , R1a , R1b , R1c , R2a , R2c , R2b , R2d , n′, n″ and p′ are as defined herein.
式(III)的cCPP可具有式(IIIa)的结构：The cCPP of formula (III) may have the structure of formula (IIIa):
其中：in:
AASC、R2a、R2c、R2b、R2d、n'、n"和p'如本文所定义。AASC , R2a , R2c , R2b , R2d , n′, n″ and p′ are as defined herein.
在式(III)、(III-1)和(IIIa)中，Ra和Rc可以是H。Ra和Rc可以是H并且Rb和Rd可各自独立地是胍或其质子化形式。Ra可以是H。Rb可以是H。p'可以是0。Ra和Rc可以是H并且每个p'可以是0。In formula (III), (III-1) and (IIIa),Ra andRc may be H.Ra andRc may be H andRb andRd may each independently be guanidine or a protonated form thereof.Ra may be H.Rb may be H. p' may be 0.Ra andRc may be H and each p' may be 0.
在式(III)、(III-1)和(IIIa)中，Ra和Rc可以是H，Rb和Rd可各自独立地是胍或其质子化形式，n"可以是2或3，并且每个p'可以是0。In formula (III), (III-1) and (IIIa),Ra andRc may be H,Rb andRd may each independently be guanidine or a protonated form thereof, n" may be 2 or 3, and each p' may be 0.
p'可以是0。p'可以是1。p'可以是2。p'可以是3。p'可以是4。p'可以是5。p' can be 0. p' can be 1. p' can be 2. p' can be 3. p' can be 4. p' can be 5.
cCPP可具有以下结构：cCPP can have the following structure:
式(A)的cCPP可选自：The cCPP of formula (A) may be selected from:
式(A)的cCPP可选自：The cCPP of formula (A) may be selected from:
在实施方案中，cCPP选自：In an embodiment, the cCPP is selected from:
其中Φ＝L-萘基丙氨酸；-萘基丙氨酸；Ω＝L-正亮氨酸Where Φ = L-naphthylalanine; -naphthylalanine; Ω = L-norleucine
在实施方案中，cCPP不选自：In an embodiment, the cCPP is not selected from:
其中Φ＝L-萘基丙氨酸；-萘基丙氨酸；Ω＝L-正亮氨酸Where Φ = L-naphthylalanine; -naphthylalanine; Ω = L-norleucine
AASC可与接头缀合。AASC can be conjugated to a linker.
接头Connectors
本公开的cCPP可与接头缀合。接头可将货物连接至cCPP。接头可附接到cCPP的氨基酸的侧链，并且货物可附接在接头上的合适位置处。The cCPP of the present disclosure can be conjugated with a linker. The linker can connect the cargo to the cCPP. The linker can be attached to the side chain of the amino acid of the cCPP, and the cargo can be attached at a suitable position on the linker.
接头可以是任何合适的部分，其可将cCPP与一个或多个附加部分(例如环外肽(EP)和/或货物)缀合。在与cCPP和一个或多个附加部分缀合之前，接头具有两个或更多个官能团，它们各自能够独立地与cCPP和一个或多个附加部分形成共价键。如果货物是寡核苷酸，接头可共价结合到货物的5'端或货物的3'端。接头可共价结合到货物的5'端。接头可共价结合到货物的3'端。如果货物是肽，接头可共价结合到货物的N-末端或C-末端。接头可共价结合到寡核苷酸或肽货物的主链。接头可以是将本文所述的cCPP与货物诸如寡核苷酸、肽或小分子缀合的任何合适的部分。The linker can be any suitable part that can conjugate the cCPP to one or more additional parts (e.g., an exocyclic peptide (EP) and/or a cargo). Prior to conjugation with the cCPP and the one or more additional parts, the linker has two or more functional groups, each of which is capable of independently forming a covalent bond with the cCPP and the one or more additional parts. If the cargo is an oligonucleotide, the linker can be covalently bound to the 5' end of the cargo or the 3' end of the cargo. The linker can be covalently bound to the 5' end of the cargo. The linker can be covalently bound to the 3' end of the cargo. If the cargo is a peptide, the linker can be covalently bound to the N-terminus or C-terminus of the cargo. The linker can be covalently bound to the backbone of the oligonucleotide or peptide cargo. The linker can be any suitable part that conjugates the cCPP described herein to a cargo such as an oligonucleotide, a peptide, or a small molecule.
接头可包括烃接头。The connector may comprise a hydrocarbon connector.
接头可包含切割位点。切割位点可以是二硫化物或胱天蛋白酶切割位点(例如，Val-Cit-PABC)。The linker may comprise a cleavage site. The cleavage site may be a disulfide or a caspase cleavage site (e.g., Val-Cit-PABC).
接头可包含：(i)一个或多个D或L氨基酸，它们各自任选地被取代；(ii)任选取代的亚烷基；(iii)任选取代的亚烯基；(iv)任选取代的亚炔基；(v)任选取代的碳环基；(vi)任选取代的杂环基；(vii)一个或多个-(R1-J-R2)z"-亚基，其中R1和R2在每种情况下各自独立地选自亚烷基、亚烯基、亚炔基、碳环基和杂环基，每个J独立地是C、NR3、-NR3C(O)-、S和O，其中R3独立地选自H、烷基、烯基、炔基、碳环基和杂环基，它们各自任选地被取代，并且z"是1至50的整数；(viii)-(R1-J)z"-或-(J-R1)z"-，其中R1在每种情况下各自独立地是亚烷基、亚烯基、亚炔基、碳环基或杂环基，每个J独立地是C、NR3、-NR3C(O)-、S或O，其中R3是H、烷基、烯基、炔基、碳环基或杂环基，它们各自任选地被取代，并且z"是1至50的整数；或(ix)接头可包括(i)至(x)中的一者或多者。The linker may comprise: (i) one or more D or L amino acids, each of which is optionally substituted; (ii) optionally substituted alkylene; (iii) optionally substituted alkenylene; (iv) optionally substituted alkynylene; (v) optionally substituted carbocyclyl; (vi) optionally substituted heterocyclyl; (vii) one or more -(R1- JR2 )z"-subunits, wherein R1 and R2 are each independently selected from alkylene, alkenylene, alkynylene, carbocyclyl and heterocyclyl, each J is independently C, NR3 , -NR3 C(O)-, S and O, wherein R3 is independently selected from H, alkyl, alkenyl, alkynyl, carbocyclyl and heterocyclyl, each of which is optionally substituted, and z" is an integer from 1 to 50; (viii) -(R1- J)z"- or -(JR1 )z"-, wherein R1 is independently alkylene, alkenylene, alkynylene, carbocyclyl or heterocyclyl in each case, each J is independently C, NR3 , -NR3 C(O)-, S or O, wherein R3 is H, alkyl, alkenyl, alkynyl, carbocyclyl or heterocyclyl, each of which is optionally substituted, and z" is an integer from 1 to 50; or (ix) the linker may include one or more of (i) to (x).
接头可包含一个或多个D或L氨基酸和/或-(R1-J-R2)z"-，其中R1和R2在每种情况下各自独立地是亚烷基，每个J独立地是C、NR3、-NR3C(O)-、S和O，其中R4独立地选自H和烷基，并且z"是1至50的整数；或它们的组合。The linker may comprise one or more D or L amino acids and/or -(R1- JR2 )z"-, wherein R1 and R2 are each independently alkylene, each J is independently C, NR3 , -NR3 C(O)-, S and O, wherein R4 is independently selected from H and alkyl, and z" is an integer from 1 to 50; or a combination thereof.
接头可包含-(OCH2CH2)z'-(例如，作为间隔区)，其中z'是1至23的整数，例如2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22或23。“-(OCH2CH2)z'"也可称为聚乙二醇(PEG)。The linker can comprise -(OCH2CH2 )z'- (e.g., as a spacer), wherein z' is an integerfrom 1 to 23, e.g.,2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. "-(OCH2CH2 )z'" may also be referred to as polyethylene glycol (PEG).
接头可包含一个或多个氨基酸。接头可包含肽。接头可包含-(OCH2CH2)z'-和肽，其中z'是1至23的整数。肽可包含2至10个氨基酸。接头还可包含能够通过点击化学反应的官能团(FG)。FG可以是叠氮化物或炔，并且当货物与接头缀合时形成三唑。The linker may comprise one or more amino acids. The linker may comprise a peptide. The linker may comprise -(OCH2 CH2 )z'- and a peptide, wherein z' is an integer from 1 to 23. The peptide may comprise 2 to 10 amino acids. The linker may also comprise a functional group (FG) capable of reacting by click chemistry. The FG may be an azide or an alkyne, and a triazole is formed when the cargo is conjugated to the linker.
接头可包含(i)β丙氨酸残基和赖氨酸残基；(ii)-(J-R1)z"；或(iii)它们的组合。每个R1可独立地是亚烷基、亚烯基、亚炔基、碳环基或杂环基，每个J独立地是C、NR3、-NR3C(O)-、S或O，其中R3是H、烷基、烯基、炔基、碳环基或杂环基，它们各自任选地被取代，并且z"可以是1至50的整数。每个R1可以是亚烷基并且每个J可以是O。The linker may comprise (i) a β-alanine residue and a lysine residue; (ii) -(JR1 )z"; or (iii) a combination thereof. Each R1 may independently be an alkylene, alkenylene, alkynylene, carbocyclyl or heterocyclyl, each J is independently C, NR3 , -NR3 C(O)-, S or O, wherein R3 is H, alkyl, alkenyl, alkynyl, carbocyclyl or heterocyclyl, each of which is optionally substituted, and z" may be an integer from 1 to 50. Each R1 may be an alkylene and each J may be O.
接头可包含(i)β-丙氨酸、甘氨酸、赖氨酸、4-氨基丁酸、5-氨基戊酸、6-氨基己酸或它们的组合的残基；以及(ii)-(R1-J)z"-或-(J-R1)z"。每个R1可独立地是亚烷基、亚烯基、亚炔基、碳环基或杂环基，每个J独立地是C、NR3、-NR3C(O)-、S或O，其中R3是H、烷基、烯基、炔基、碳环基或杂环基，它们各自任选地被取代，并且z"可以是1至50的整数。每个R1可以是亚烷基并且每个J可以是O。接头可包含甘氨酸、β-丙氨酸、4-氨基丁酸、5-氨基戊酸、6-氨基己酸或它们的组合。The linker may comprise (i) the residue of β-alanine, glycine, lysine, 4-aminobutyric acid, 5-aminovaleric acid, 6-aminohexanoic acid, or a combination thereof; and (ii) -(R1- J)z"- or -(JR1 )z". Each R1 may independently be an alkylene, alkenylene, alkynylene, carbocyclyl, or heterocyclyl, each J is independently C, NR3 , -NR3 C(O)-, S, or O, wherein R3 is H, alkyl, alkenyl, alkynyl, carbocyclyl, or heterocyclyl, each of which is optionally substituted, and z" may be an integer from 1 to 50. Each R1 may be an alkylene and each J may be O. The linker may comprise glycine, β-alanine, 4-aminobutyric acid, 5-aminovaleric acid, 6-aminohexanoic acid, or a combination thereof.
接头可以是三价接头。接头可具有以下结构：其中A1、B1和C1可独立地是烃接头(例如，NRH-(CH2)n-COOH)、PEG接头(例如，NRH-(CH2O)n-COOH，其中R是H、甲基或乙基)或一个或多个氨基酸残基，并且Z独立地是保护基团。接头还可掺入切割位点，包括二硫化物[NH2-(CH2O)n-S-S-(CH2O)n-COOH]或胱天蛋白酶切割位点(Val-Cit-PABC)。The linker can be a trivalent linker. The linker can have the following structure: WhereinA1 ,B1 andC1 can independently be a hydrocarbon linker (e.g., NRH-(CH2 )n -COOH), a PEG linker (e.g., NRH-(CH2O )n -COOH, wherein R is H, methyl or ethyl) or one or more amino acid residues, and Z is independently a protecting group. The linker can also incorporate a cleavage site, including a disulfide [NH2- (CH2O )n -SS-(CH2O )n -COOH] or a caspase cleavage site (Val-Cit-PABC).
烃可以是甘氨酸或β-丙氨酸的残基。The hydrocarbon may be the residue of glycine or beta-alanine.
接头可以是二价的并且将cCPP连接到货物。接头可以是二价的并且将cCPP连接到环外肽(EP)。The linker may be bivalent and connect the cCPP to the cargo.The linker may be bivalent and connect the cCPP to the exocyclic peptide (EP).
接头可以是三价的并且将cCPP连接到货物和EP。The linker can be trivalent and connects the cCPP to the cargo and the EP.
接头可以是二价或三价C1-C50亚烷基，其中1-25个亚甲基基团任选地且独立地被-N(H)-、-N(C1-C4烷基)-、-N(环烷基)-、-O-、-C(O)-、-C(O)O-、-S-、-S(O)-、-S(O)2-、-S(O)2N(C1-C4烷基)-、-S(O)2N(环烷基)-、-N(H)C(O)-、-N(C1-C4烷基)C(O)-、-N(环烷基)C(O)-、-C(O)N(H)-、-C(O)N(C1-C4烷基)、-C(O)N(环烷基)、芳基、杂环基、杂芳基、环烷基或环烯基替换。接头可以是二价或三价C1-C50亚烷基，其中1-25个亚甲基基团任选地且独立地被-N(H)-、-O-、-C(O)N(H)-或它们的组合替换。The linker can be a divalent or trivalent C1 -C50 alkylene group in which 1-25 methylene groups are optionally and independently replaced by -N(H)-, -N(C1 -C4 alkyl)-, -N(cycloalkyl)-, -O-, -C(O)-, -C(O)O-, -S-, -S(O)-, -S(O)2 -, -S(O)2 N(C1 -C4 alkyl)-, -S(O)2 N(cycloalkyl)-, -N(H)C(O)-, -N(C1 -C4 alkyl)C(O)-, -N(cycloalkyl)C(O)-, -C(O)N(H)-, -C(O)N(C1 -C4 alkyl), -C(O)N(cycloalkyl), aryl, heterocyclyl, heteroaryl, cycloalkyl or cycloalkenyl. The linker may be a divalent or trivalent C1 -C50 alkylene group, wherein 1 to 25 methylene groups are optionally and independently replaced by -N(H)-, -O-, -C(O)N(H)-, or a combination thereof.
接头可具有以下结构：The joint can have the following structures:
其中：每个AA独立地是氨基酸残基；*是与AASC的附接点，并且AASC是cCPP的氨基酸残基的侧链；x是1-10的整数；y是1-5的整数；z是1-10的整数。X可以是1-5的整数。X可以是1-3的整数。X可以是1。Y可以是2-4的整数。Y可以是4。Z可以是1-5的整数。Z可以是1-3的整数。Z可以是1。每个AA可独立地选自甘氨酸、β-丙氨 wherein: each AA is independently an amino acid residue; * is the point of attachment to AASC , and AASC is the side chain of an amino acid residue of cCPP; x is an integer from 1 to 10; y is an integer from 1 to 5; z is an integer from 1 to 10. X may be an integer from 1 to 5. X may be an integer from 1 to 3. X may be 1. Y may be an integer from 2 to 4. Y may be 4. Z may be an integer from 1 to 5. Z may be an integer from 1 to 3. Z may be 1. Each AA may be independently selected from glycine, β-alanine,
酸、4-氨基丁酸、5-氨基戊酸和6-氨基己酸。Acid, 4-aminobutyric acid, 5-aminovaleric acid and 6-aminohexanoic acid.
cCPP可通过接头(“L”)附接到货物。接头可通过结合基团(“M”)与货物缀合。The cCPP can be attached to the cargo via a linker ("L"). The linker can be conjugated to the cargo via a binding group ("M").
接头可具有以下结构：The joint can have the following structures:
其中：x是1-10的整数；y是1-5的整数；z是1-10的整数；每个AA独立地是氨基酸残基；*是与AASC的附接点，并且AASC是cCPP的氨基酸残基的侧链；并且M是本文所定义的结合基团。 wherein: x is an integer from 1 to 10; y is an integer from 1 to 5; z is an integer from 1 to 10; each AA is independently an amino acid residue; * is the point of attachment to AASC , and AASC is the side chain of an amino acid residue of cCPP; and M is a binding group as defined herein.
接头可具有以下结构：The joint can have the following structures:
其中：x'是1-23的整数；y是1-5的整数；z'是1-23的整数；*是与AASC的附接点，并且AASC是cCPP的氨基酸残基的侧链；并且M是本文所定义的结合基团。wherein: x' is an integer from 1 to 23; y is an integer from 1 to 5; z' is an integer from 1 to 23; * is the point of attachment to AASC , and AASC is the side chain of an amino acid residue of cCPP; and M is a binding group as defined herein.
接头可具有以下结构：The joint can have the following structures:
其中：x'是1-23的整数；y是1-5的整数；并且z'是1-23的整数；*是与AASC的附接点，并且AASC是cCPP的氨基酸残基的侧链。wherein: x' is an integer from 1-23; y is an integer from 1-5; and z' is an integer from 1-23; * is the point of attachment to AASC , and AASC is the side chain of an amino acid residue of cCPP.
x可以是1-10的整数，例如1、2、3、4、5、6、7、8、9或10，包括其间的所有范围和子范围。x can be an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, including all ranges and subranges therebetween.
x'可以是1-23的整数，例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22或23，包括其间的所有范围和子范围。X'可以是5-15的整数。X'可以是9-13的整数。X'可以是1-5的整数。X'可以是1。X' may be an integer from 1 to 23, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, including all ranges and subranges therebetween. X' may be an integer from 5 to 15. X' may be an integer from 9 to 13. X' may be an integer from 1 to 5. X' may be 1.
y可以是1-5的整数，例如1、2、3、4或5，包括其间的所有范围和子范围。Y可以是2-5的整数。Y可以是3-5的整数。Y可以是3或4。Y可以是4或5。Y可以是3。Y可以是4。Y可以是5。Y may be an integer from 1 to 5, such as 1, 2, 3, 4 or 5, including all ranges and subranges therebetween. Y may be an integer from 2 to 5. Y may be an integer from 3 to 5. Y may be 3 or 4. Y may be 4 or 5. Y may be 3. Y may be 4. Y may be 5.
z可以是1-10的整数，例如1、2、3、4、5、6、7、8、9或10，包括其间的所有范围和子范围。z can be an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, including all ranges and subranges therebetween.
z'可以是1-23的整数，例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22或23，包括其间的所有范围和子范围。Z'可以是5-15的整数。Z'可以是9-13的整数。Z'可以是11。Z' can be an integer from 1 to 23, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23, including all ranges and subranges therebetween. Z' can be an integer from 5 to 15. Z' can be an integer from 9 to 13. Z' can be 11.
如上所讨论，接头或M(其中M是接头的一部分)可共价结合到货物上的任何合适的位置。接头或M(其中M是接头的一部分)可共价结合到寡核苷酸货物的3'端或寡核苷酸货物的5'端。接头或M(其中M是接头的一部分)可共价结合到肽货物的N-末端或C-末端。接头或M(其中M是接头的一部分)可共价结合到寡核苷酸或肽货物的主链。As discussed above, the linker or M (wherein M is part of a linker) can be covalently bound to any suitable position on the cargo. The linker or M (wherein M is part of a linker) can be covalently bound to the 3' end of the oligonucleotide cargo or the 5' end of the oligonucleotide cargo. The linker or M (wherein M is part of a linker) can be covalently bound to the N-terminus or C-terminus of the peptide cargo. The linker or M (wherein M is part of a linker) can be covalently bound to the backbone of the oligonucleotide or peptide cargo.
接头可结合到cCPP上的天冬氨酸、谷氨酸、谷氨酰胺、天冬酰胺或赖氨酸的侧链，或者谷氨酰胺或天冬酰胺的修饰侧链(例如，具有氨基基团的还原侧链)。接头可结合到cCPP上的赖氨酸的侧链。The linker can be attached to the side chain of aspartic acid, glutamic acid, glutamine, asparagine or lysine on the cCPP, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain with an amino group). The linker can be attached to the side chain of lysine on the cCPP.
接头可结合到肽货物上的天冬氨酸、谷氨酸、谷氨酰胺、天冬酰胺或赖氨酸的侧链，或者谷氨酰胺或天冬酰胺的修饰侧链(例如，具有氨基的还原侧链)。接头可结合到肽货物上的赖氨酸的侧链。The linker can be bound to the side chain of aspartic acid, glutamic acid, glutamine, asparagine or lysine on the peptide cargo, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain with an amino group). The linker can be bound to the side chain of lysine on the peptide cargo.
接头可具有以下结构：The joint can have the following structures:
其中in
M是将L与货物例如寡核苷酸缀合的基团；M is a group that conjugates L to a cargo, such as an oligonucleotide;
AAs是cCPP上的氨基酸的侧链或末端；AAs are the side chains or termini of the amino acids on the cCPP;
每个AAx独立地是氨基酸残基；Each AAx is independently an amino acid residue;
o是0-10的整数；并且o is an integer from 0 to 10; and
p是0至5的整数。p is an integer from 0 to 5.
接头可具有以下结构：The joint can have the following structures:
其中in
M是将L与货物例如寡核苷酸缀合的基团；M is a group that conjugates L to a cargo, such as an oligonucleotide;
AAs是cCPP上的氨基酸的侧链或末端；AAs are the side chains or termini of the amino acids on the cCPP;
每个AAx独立地是氨基酸残基；Each AAx is independently an amino acid residue;
o是0-10的整数；并且o is an integer from 0 to 10; and
p是0至5的整数。p is an integer from 0 to 5.
M可包括亚烷基、亚烯基、亚炔基、碳环基或杂环基，它们各自任选地被取代。M可选自：M may include alkylene, alkenylene, alkynylene, carbocyclic or heterocyclic groups, each of which is optionally substituted. M may be selected from:
其中R是烷基、烯基、炔基、碳环基或杂环基。 wherein R is an alkyl, alkenyl, alkynyl, carbocyclic or heterocyclic group.
M可选自：M can be selected from:
其中：R10是亚烷基、环烷基或其中a是0至10。Wherein: R10 is alkylene, cycloalkyl or Where a is 0 to 10.
M可以是R10可以是并且a是0至10。M可以是M can be R10 can be and a is 0 to 10. M can be
M可以是异双官能交联剂，例如其公开于Williams等人Curr.Protoc Nucleic Acid Chem.2010,42,4.41.1-4.41.20中，全文以引用方式并入本文。M may be a heterobifunctional cross-linker, such as It is disclosed in Williams et al. Curr. Protoc Nucleic Acid Chem. 2010, 42, 4.41.1-4.41.20, which is incorporated herein by reference in its entirety.
M可以是-C(O)-。M may be -C(O)-.
AAs可以是cCPP上的氨基酸的侧链或末端。AAs的非限制性示例包括天冬氨酸、谷氨酸、谷氨酰胺、天冬酰胺或赖氨酸，或谷氨酰胺或天冬酰胺的修饰侧链(例如，具有氨基基团的还原侧链)。AAs可以是如本文所定义的AASC。AAs can be side chains or ends of amino acids on cCPP. Non-limiting examples ofAAs include aspartic acid, glutamic acid, glutamine, asparagine or lysine, or modified side chains of glutamine or asparagine (e.g., reduced side chains with amino groups).AAs can beAAS as defined herein.
每个AAx独立地是天然或非天然氨基酸。一个或多个AAx可以是天然氨基酸。一个或多个AAx可以是非天然氨基酸。一个或多个AAx可以是β-氨基酸。β-氨基酸可以是β-丙氨酸。Each AAx is independently a natural or unnatural amino acid. One or more AAx can be a natural amino acid. One or more AAx can be an unnatural amino acid. One or more AAx can be a β-amino acid. The β-amino acid can be β-alanine.
o可以是0至10的整数，例如0、1、2、3、4、5、6、7、8、9和10。O可以是0、1、2或3。O可以是0。O可以是1。O可以是2。O可以是3。o may be an integer from 0 to 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. O may be 0, 1, 2, or 3. O may be 0. O may be 1. O may be 2. O may be 3.
p可以是0至5，例如0、1、2、3、4或5。P可以是0。P可以是1。P可以是2。P可以是3。P可以是4。P可以是5。p may be 0 to 5, for example 0, 1, 2, 3, 4 or 5. p may be 0. p may be 1. p may be 2. p may be 3. p may be 4. p may be 5.
接头可具有以下结构：The joint can have the following structures:
其中M、AAs、每个-(R1-J-R2)z"-、o和z"如本文所定义；r可以是0或1。wherein M, AAs , each -(R1- JR2 )z"-, o and z" are as defined herein; r may be 0 or 1.
r可以是0。R可以是1。r may be 0. R may be 1.
接头可具有以下结构：The joint can have the following structures:
其中M、AAs、o、p、q、r和z"各自可如本文所定义。wherein M, AAs , o, p, q, r and z" may each be as defined herein.
z"可以是1至50的整数，例如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和50，包括其间的所有范围和值。Z"可以是5-20的整数。Z"可以是10-15的整数。Z" may be an integer from 1 to 50, for example, 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 and 50, including all ranges and values therebetween. Z" may be an integer from 5-20. Z" may be an integer from 10-15.
接头可具有以下结构：The joint can have the following structures:
其中：in:
M、AAs和o如本文所定义。M,AAs and o are as defined herein.
合适接头的其他非限制性示例包括：Other non-limiting examples of suitable linkers include:
其中M和AAs如本文所定义。wherein M andAAs are as defined herein.
本文提供了包含cCPP和与前mRNA序列中的靶标互补的AC的化合物，该化合物还包含L，其中接头通过结合基团(M)与AC缀合，其中M是Provided herein are compounds comprising a cCPP and an AC complementary to a target in a pre-mRNA sequence, the compound further comprising L, wherein a linker is conjugated to AC via a binding group (M), wherein M is
本文提供了包含cCPP和货物的化合物，该货物包含与前mRNA序列中的靶标互补的反义化合物(AC)，例如反义寡核苷酸，其中该化合物还包含L，其中接头通过结合基团(M)与AC缀合，其中M选自：Provided herein are compounds comprising a cCPP and a cargo comprising an antisense compound (AC) complementary to a target in a pre-mRNA sequence, such as an antisense oligonucleotide, wherein the compound further comprises L, wherein a linker is conjugated to AC via a binding group (M), wherein M is selected from:
其中：R1是亚烷基、环烷基或其中t'是0至10，其中每个R独立地是烷基、烯基、炔基、碳环基或杂环基，其中R1是并且t'是2。 Wherein:R1 is alkylene, cycloalkyl or wherein t' is 0 to 10, wherein each R is independently alkyl, alkenyl, alkynyl, carbocyclyl or heterocyclyl, wherein R1 is And t' is 2.
接头可具有以下结构：The joint can have the following structures:
其中AAs如本文所定义，并且m'是0-10。whereinAAs are as defined herein, and m' is 0-10.
接头可具有下式：The linker may have the formula:
接头可具有下式：其中“碱基”是货物二氨基磷酸酯吗啉代寡聚物的3'端的核碱基。The linker may have the formula: wherein "base" is the nucleobase at the 3' end of the cargo phosphorodiamidate morpholino oligomer.
接头可具有下式：The linker may have the formula:
其中“碱基”对应于货物二氨基磷酸酯吗啉代寡聚物的3'端的核碱基。 where "base" corresponds to the nucleobase at the 3' end of the cargo phosphorodiamidate morpholino oligomer.
接头可具有下式：The linker may have the formula:
其中“碱基”是货物二氨基磷酸酯吗啉代寡聚物的3'端的核碱基。 wherein "base" is the nucleobase at the 3' end of the cargo phosphorodiamidate morpholino oligomer.
接头可具有下式：其中“碱基”是货物二氨基磷酸酯吗啉代寡聚物的3'端的核碱基。The linker may have the formula: wherein "base" is the nucleobase at the 3' end of the cargo phosphorodiamidate morpholino oligomer.
接头可具有下式：The linker may have the formula:
接头可在货物上的任何合适的位置处共价结合到货物。接头共价结合到货物的3'端或寡核苷酸货物的5'端。接头可共价结合到货物的主链。The linker can be covalently bound to the cargo at any suitable position on the cargo. The linker is covalently bound to the 3' end of the cargo or the 5' end of the oligonucleotide cargo. The linker can be covalently bound to the backbone of the cargo.
接头可结合到cCPP上的天冬氨酸、谷氨酸、谷氨酰胺、天冬酰胺或赖氨酸的侧链，或者谷氨酰胺或天冬酰胺的修饰侧链(例如，具有氨基基团的还原侧链)。接头可结合到cCPP上的赖氨酸的侧链。The linker can be attached to the side chain of aspartic acid, glutamic acid, glutamine, asparagine or lysine on the cCPP, or a modified side chain of glutamine or asparagine (e.g., a reduced side chain with an amino group). The linker can be attached to the side chain of lysine on the cCPP.
cCPP-接头缀合物cCPP-Linker Conjugate
cCPP可与本文所定义的接头缀合。接头可与如本文所定义的cCPP的AASC缀合。The cCPP may be conjugated to a linker as defined herein. The linker may be conjugated to the AASC of the cCPP as defined herein.
接头可包含-(OCH2CH2)z'-亚基(例如，作为间隔区)，其中z'是1至23的整数，例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22或23。“-(OCH2CH2)z'”也称为PEG。cCPP-接头缀合物可具有选自表4的结构：The linker may comprise a -(OCH2 CH2 )z ' -subunit (e.g., as a spacer), wherein z' is an integer from 1 to 23, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23. "-(OCH2 CH2 )z ' " is also known as PEG. The cCPP-linker conjugate may have a structure selected from Table 4:
表4：cCPP-接头缀合物和SEQ ID NOTable 4: cCPP-linker conjugates and SEQ ID NOs
接头可包含-(OCH2CH2)z'-亚基和肽亚基，其中z'是1至23的整数。肽亚基可包含2至10个氨基酸。cCPP-接头缀合物可具有选自表5的结构：The linker may comprise a -(OCH2 CH2 )z' -subunit and a peptide subunit, wherein z' is an integer from 1 to 23. The peptide subunit may comprise 2 to 10 amino acids. The cCPP-linker conjugate may have a structure selected from Table 5:
表5：cCPP-接头缀合物和SEQ ID NOTable5: cCPP-linker conjugates and SEQ ID NOs
提供了包含环状细胞穿透肽(cCPP)、接头和环外肽(EP)的EEV。EEV可具有式(B)的结构：Provided is an EEV comprising a cyclic cell penetrating peptide (cCPP), a linker and an exocyclic peptide (EP). The EEV may have a structure of formula (B):
或其质子化形式，其中： or a protonated form thereof, wherein:
R1、R2和R3各自独立地是H或氨基酸的芳族或杂芳族侧链；R1 , R2 and R3 are each independently H or an aromatic or heteroaromatic side chain of an amino acid;
R4和R7独立地是H或氨基酸侧链；R4 andR7 are independently H or an amino acid side chain;
EP是如本文所定义的环外肽；EP is an exocyclic peptide as defined herein;
每个m独立地是0-3的整数；Each m is independently an integer from 0 to 3;
n是0-2的整数；n is an integer from 0 to 2;
x'是1-20的整数；x' is an integer from 1 to 20;
y是1-5的整数；y is an integer from 1 to 5;
q是1-4；并且q is 1-4; and
z'是1-23的整数。z' is an integer from 1 to 23.
R1、R2、R3、R4、R7、EP、m、q、y、x'、z'如本文所述。R1 ,R2 ,R3 ,R4 ,R7 , EP, m, q, y, x', z' are as described herein.
n可以是0。n可以是1。n可以是2。n can be 0. n can be 1. n can be 2.
EEV可具有式(B-a)或(B-b)的结构：The EEV may have a structure of formula (B-a) or (B-b):
或其质子化形式，其中EP(示出为“PE”)、R1、R2、R3、R4、m和z'如上文在式(B)中所定义。 or a protonated form thereof, wherein EP (shown as "PE"),R1 ,R2 ,R3 ,R4 , m and z' are as defined above in formula (B).
EEV可具有式(B-c)的结构：The EEV may have the structure of formula (B-c):
或其质子化形式，其中EP、R1、R2、R3、R4和m如以上在式(B)中所定义；AA是如本文所定义的氨基酸；M如本文所定义；n是0-2的整数；x是1-10的整数；y是1-5的整数；z是1-10的整数。or a protonated form thereof, wherein EP,R1 ,R2 ,R3 ,R4 and m are as defined above in formula (B); AA is an amino acid as defined herein; M is as defined herein; n is an integer from 0 to 2; x is an integer from 1 to 10; y is an integer from 1 to 5; and z is an integer from 1 to 10.
EEV可具有式(B-1)、(B-2)、(B-3)或(B-4)的结构：The EEV may have a structure of formula (B-1), (B-2), (B-3) or (B-4):
或其质子化形式，其中EP如上文在式(B)中所定义。or a protonated form thereof, wherein EP is as defined above in formula (B).
EEV可包含式(B)并且可具有以下结构：Ac-PKKKRKVAEEA-K(环[FGFGRGRQ])-PEG12-OH(Ac-SEQ ID NO:132-K(环[SEQ ID NO:82])-PEG12-OH)或Ac-PK-KKR-KV-AEEA-K(环[GfFGrGrQ])-PEG12-OH(Ac-SEQ ID NO:133-K(环[SEQ ID NO:83])-PEG12-OH)。The EEV may comprise formula (B) and may have the following structure: Ac-PKKKRKVAEEA-K(cyclo[FGFGRGRQ])-PEG12 -OH(Ac-SEQ ID NO:132-K(cyclo[SEQ ID NO:82])-PEG12 -OH) or Ac-PK-KKR-KV-AEEA-K(cyclo[GfFGrGrQ])-PEG12- OH(Ac-SEQ ID NO:133-K(cyclo[SEQ ID NO:83])-PEG12 -OH).
EEV可包含下式的cCPP：The EEV may comprise a cCPP of the formula:
EEV可包含下式：Ac-PKKKRKV-miniPEG2-Lys(环(FfFGRGRQ)-miniPEG2-K(N3)(Ac-SEQ ID NO:42-PEG2-Lys(环(SEQ ID NO:81)-PEG2-K(N3))。The EEV may comprise the formula: Ac-PKKKRKV-miniPEG2-Lys(cyclo(FfFGRGRQ)-miniPEG2-K(N3)(Ac-SEQ ID NO:42-PEG2 -Lys(cyclo(SEQ ID NO:81)-PEG2 -K(N3 )).
EEV可以是：An EEV can be:
EEV可以是EEV can be
EEV可以是Ac-P-K(Tfa)-K(Tfa)-K(Tfa)-R-K(Tfa)-V-miniPEG2-K(环(Ff-Nal-GrGrQ)-PEG12-OH(Ac-SEQ ID NO:134-miniPEG2-K(环(SEQ ID NO:135)-PEG12-OH)。EEV can be Ac-PK(Tfa)-K(Tfa)-K(Tfa)-RK(Tfa)-V-miniPEG2 -K(cyclo(Ff-Nal-GrGrQ)-PEG12 -OH(Ac-SEQ ID NO: 134-miniPEG2 -K (cyclo(SEQ ID NO: 135)-PEG12 -OH).
EEV可以是EEV can be
EEV可以是Ac-P-K-K-K-R-K-V-miniPEG2-K(环(Ff-Nal-GrGrQ)-PEG12-OH(Ac-SEQID NO:42-PEG2-K(环(SEQ ID NO:135)-PEG12-OH)。The EEV can be Ac-PKKKRKV-miniPEG2 -K(cyclo(Ff-Nal-GrGrQ)-PEG12 -OH(Ac-SEQ ID NO:42-PEG2 -K(cyclo(SEQ ID NO:135)-PEG12 -OH).
EEV可以是EEV can be
EEV可以是EEV can be
EEV可以是EEV can be
EEV可以是EEV can be
EEV可以是EEV can be
EEV可以是EEV can be
EEV可以是：An EEV can be:
EEV可以是EEV can be
EEV可以是EEV can be
EEV可以是EEV can be
EEV可以是EEV can be
EEV可选自EEV can be selected from
EEV可选自：EEV can be selected from:
Ac-PKKKRKV-Lys(环[FfΦGrGrQ])-PEG12-K(N3)-NH2Ac-PKKKRKV-Lys(cyclo[FfΦGrGrQ])-PEG12 -K(N3 )-NH2
(Ac-SEQ ID NO:42-Lys(环[SEQ ID NO:80])-PEG12-K(N3)-NH2)(Ac-SEQ ID NO:42-Lys(cyclo[SEQ ID NO:80])-PEG12 -K(N3 )-NH2 )
Ac-PKKKRKV-miniPEG2-Lys(环[FfΦGrGrQ])-miniPEG2-K(N3)-NH2Ac-PKKKRKV-miniPEG2 -Lys(cyclo[FfΦGrGrQ])-miniPEG2 -K(N3 )-NH2
(Ac-SEQ ID NO:42-miniPEG2-Lys(环[SEQ ID NO:80])-miniPEG2-K(N3)-NH2)(Ac-SEQ ID NO:42-miniPEG2 -Lys(cyclo[SEQ ID NO:80])-miniPEG2 -K(N3 )-NH2 )
Ac-PKKKRKV-miniPEG2-Lys(环[FGFGRGRQ])-miniPEG2-K(N3)-NH2Ac-PKKKRKV-miniPEG2 -Lys(cyclo[FGFGRGRQ])-miniPEG2 -K(N3 )-NH2
(Ac-SEQ ID NO:42-miniPEG2-Lys(环[SEQ ID NO:82])-miniPEG2-K(N3)-NH2)(Ac-SEQ ID NO:42-miniPEG2 -Lys(cyclo[SEQ ID NO:82])-miniPEG2 -K(N3 )-NH2 )
Ac-KR-PEG2-K(环[FGFGRGRQ])-PEG2-K(N3)-NH2Ac-KR-PEG2 -K(cyclo[FGFGRGRQ])-PEG2 -K(N3 )-NH2
(Ac-KR-PEG2-K(环[SEQ ID NO:82])-PEG2-K(N3)-NH2)(Ac-KR-PEG2 -K(cyclo[SEQ ID NO:82])-PEG2 -K(N3 )-NH2 )
Ac-PKKKGKV-PEG2-K(环[FGFGRGRQ])-PEG2-K(N3)-NH2Ac-PKKKGKV-PEG2 -K(cyclo[FGFGRGRQ])-PEG2 -K(N3 )-NH2
(Ac-SEQ ID NO:46-PEG2-K(环[SEQ ID NO:82])-PEG2-K(N3)-NH2)(Ac-SEQ ID NO:46-PEG2 -K(cyclo[SEQ ID NO:82])-PEG2 -K(N3 )-NH2 )
Ac-PKKKRKG-PEG2-K(环[FGFGRGRQ])-PEG2-K(N3)-NH2Ac-PKKKRKG-PEG2 -K(cyclo[FGFGRGRQ])-PEG2 -K(N3 )-NH2
(Ac-SEQ ID NO:48-PEG2-K(环[SEQ ID NO:82])-PEG2-K(N3)-NH2)(Ac-SEQ ID NO:48-PEG2 -K(cyclo[SEQ ID NO:82])-PEG2 -K(N3 )-NH2 )
Ac-KKKRK-PEG2-K(环[FGFGRGRQ])-PEG2-K(N3)-NH2Ac-KKKRK-PEG2 -K(cyclo[FGFGRGRQ])-PEG2 -K(N3 )-NH2
(Ac-SEQ ID NO:19-PEG2-K(环[SEQ ID NO:82])-PEG2-K(N3)-NH2)(Ac-SEQ ID NO:19-PEG2 -K(cyclo[SEQ ID NO:82])-PEG2 -K(N3 )-NH2 )
Ac-PKKKRKV-miniPEG2-Lys(环[FFΦGRGRQ])-miniPEG2-K(N3)-NH2Ac-PKKKRKV-miniPEG2 -Lys(cyclo[FFΦGRGRQ])-miniPEG2 -K(N3 )-NH2
(Ac-SEQ ID NO:42mini-PEG2-Lys(环[SEQ ID NO:80])-miniPEG2-K(N3)-NH2)(Ac-SEQ ID NO:42mini-PEG2 -Lys(cyclo[SEQ ID NO:80])-miniPEG2 -K(N3 )-NH2 )
Ac-PKKKRKV-miniPEG2-Lys(环[βhFfΦGrGrQ])-miniPEG2-K(N3)-NH2Ac-PKKKRKV-miniPEG2 -Lys(cyclo[βhFfΦGrGrQ])-miniPEG2 -K(N3 )-NH2
(Ac-SEQ ID NO:42-miniPEG2-Lys(环[SEQ ID NO:142])-miniPEG2-K(N3)-NH2)(Ac-SEQ ID NO:42-miniPEG2 -Lys(cyclo[SEQ ID NO:142])-miniPEG2 -K(N3 )-NH2 )
Ac-PKKKRKV-miniPEG2-Lys(环[FfΦSrSrQ])-miniPEG2-K(N3)-NH2Ac-PKKKRKV-miniPEG2 -Lys(cyclo[FfΦSrSrQ])-miniPEG2 -K(N3 )-NH2
(Ac-SEQ ID NO:42-miniPEG2-Lys(环[SEQ ID NO:143])-miniPEG2-K(N3)-NH2)。(Ac-SEQ ID NO:42-miniPEG2- Lys(cyclo[SEQ ID NO:143])-miniPEG2- K(N3 )-NH2 ).
EEV可选自：EEV can be selected from:
Ac-PKKKRKV-miniPEG2-Lys(环(GfFGrGrQ])-PEG12-OHAc-PKKKRKV-miniPEG2 -Lys(cyclo(GfFGrGrQ])-PEG12 -OH
(Ac-SEQ ID NO:42-miniPEG2-Lys(环(SEQ ID NO:133])-PEG12-OH)(Ac-SEQ ID NO:42-miniPEG2 -Lys(cyclo(SEQ ID NO:133])-PEG12 -OH)
Ac-PKKKRKV-miniPEG2-Lys(环[FGFKRKRQ])-PEG12-OHAc-PKKKRKV-miniPEG2 -Lys(cyclo[FGFKRKRQ])-PEG12 -OH
(Ac-SEQ ID NO:42-miniPEG2-Lys(环[SEQ ID NO:144])-PEG12-OH)(Ac-SEQ ID NO:42-miniPEG2 -Lys(cyclo[SEQ ID NO:144])-PEG12 -OH)
Ac-PKKKRKV-miniPEG2-Lys(环[FGFRGRGQ])-PEG12-OHAc-PKKKRKV-miniPEG2 -Lys(cyclo[FGFRGRGQ])-PEG12 -OH
(Ac-SEQ ID NO:42-miniPEG2-Lys(环[SEQ ID NO:145])-PEG12-OH)(Ac-SEQ ID NO:42-miniPEG2 -Lys(cyclo[SEQ ID NO:145])-PEG12 -OH)
Ac-PKKKRKV-miniPEG2-Lys(环[FGFGRGRGRQ])-PEG12-OHAc-PKKKRKV-miniPEG2 -Lys(cyclo[FGFGRGRQ])-PEG12 -OH
(Ac-SEQ ID NO:42-miniPEG2-Lys(环[SEQ ID NO:146])-PEG12-OH)(Ac-SEQ ID NO:42-miniPEG2 -Lys(cyclo[SEQ ID NO:146])-PEG12 -OH)
Ac-PKKKRKV-miniPEG2-Lys(环[FGFGRrRQ])-PEG12-OHAc-PKKKRKV-miniPEG2 -Lys(cyclo[FGFGRrRQ])-PEG12 -OH
(Ac-SEQ ID NO:42-miniPEG2-Lys(环[SEQ ID NO:147])-PEG12-OH)(Ac-SEQ ID NO:42-miniPEG2 -Lys(cyclo[SEQ ID NO:147])-PEG12 -OH)
Ac-PKKKRKV-miniPEG2-Lys(环[FGFGRRRQ])-PEG12-OHAc-PKKKRKV-miniPEG2 -Lys(cyclo[FGFGRRRQ])-PEG12 -OH
(Ac-SEQ ID NO:42-miniPEG2-Lys(环[SEQ ID NO:84])-PEG12-OH)，以及(Ac-SEQ ID NO:42-miniPEG2 -Lys(cyclo[SEQ ID NO:84])-PEG12 -OH), and
Ac-PKKKRKV-miniPEG2-Lys(环[FGFRRRRQ])-PEG12-OHAc-PKKKRKV-miniPEG2 -Lys(cyclo[FGFRRRRQ])-PEG12 -OH
(Ac-SEQ ID NO:42-miniPEG2-Lys(环[SEQ ID NO:85])-PEG12-OH)。(Ac-SEQ ID NO:42-miniPEG2- Lys(cyclo[SEQ ID NO:85])-PEG12 -OH).
EEV可选自：EEV can be selected from:
Ac-K-K-K-R-K-G-miniPEG2-K(环[FGFGRGRQ])-PEG12-OHAc-KKKRKG-miniPEG2 -K(cyclo[FGFGRGRQ])-PEG12 -OH
(Ac-SEQ ID NO:148-miniPEG2-K(环[SEQ ID NO:82])-PEG12-OH)(Ac-SEQ ID NO:148-miniPEG2 -K(cyclo[SEQ ID NO:82])-PEG12 -OH)
Ac-K-K-K-R-K-miniPEG2-K(环[FGFGRGRQ])-PEG12-OHAc-KKKRK-miniPEG2 -K(cyclo[FGFGRGRQ])-PEG12 -OH
(Ac-SEQ ID NO:19-miniPEG2-K(环[SEQ ID NO:82])-PEG12-OH)(Ac-SEQ ID NO:19-miniPEG2 -K(cyclo[SEQ ID NO:82])-PEG12 -OH)
Ac-K-K-R-K-K-PEG4-K(环[FGFGRGRQ])-PEG12-OHAc-KKRKK-PEG4 -K(cyclo[FGFGRGRQ])-PEG12 -OH
(Ac-SEQ ID NO:22-PEG4-K(环[SEQ ID NO:82])-PEG12-OH)(Ac-SEQ ID NO:22-PEG4 -K(cyclo[SEQ ID NO:82])-PEG12 -OH)
Ac-K-R-K-K-K-PEG4-K(环[FGFGRGRQ])-PEG12-OHAc-KRKKK-PEG4 -K(cyclo[FGFGRGRQ])-PEG12 -OH
(Ac-SEQ ID NO:21-PEG4-K(环[SEQ ID NO:82])-PEG12-OH)(Ac-SEQ ID NO:21-PEG4 -K(cyclo[SEQ ID NO:82])-PEG12 -OH)
Ac-K-K-K-K-R-PEG4-K(环[FGFGRGRQ])-PEG12-OHAc-KKKKR-PEG4 -K(cyclo[FGFGRGRQ])-PEG12 -OH
(Ac-SEQ ID NO:23-PEG4-K(环[SEQ ID NO:82])-PEG12-OH)(Ac-SEQ ID NO:23-PEG4 -K(cyclo[SEQ ID NO:82])-PEG12 -OH)
Ac-R-K-K-K-K-PEG4-K(环[FGFGRGRQ])-PEG12-OHAc-RKKKK-PEG4 -K(cyclo[FGFGRGRQ])-PEG12 -OH
(Ac-SEQ ID NO:20-PEG4-K(环[SEQ ID NO:82])-PEG12-OH)，以及(Ac-SEQ ID NO:20-PEG4 -K(cyclo[SEQ ID NO:82])-PEG12 -OH), and
Ac-K-K-K-R-K-PEG4-K(环[FGFGRGRQ])-PEG12-OHAc-KKKRK-PEG4 -K(cyclo[FGFGRGRQ])-PEG12 -OH
(Ac-SEQ ID NO:19-PEG4-K(环[SEQ ID NO:82])-PEG12-OH)。(Ac-SEQ ID NO:19-PEG4 -K(cyclo[SEQ ID NO:82])-PEG12 -OH).
EEV可选自：EEV can be selected from:
Ac-PKKKRKV-PEG2-K(环[FGFGRGRQ])-PEG2-K(N3)-NH2Ac-PKKKRKV-PEG2 -K(cyclo[FGFGRGRQ])-PEG2 -K(N3 )-NH2
(Ac-SEQ ID NO:42-PEG2-K(环[SEQ ID NO:82])-PEG2-K(N3)-NH2)(Ac-SEQ ID NO:42-PEG2 -K(cyclo[SEQ ID NO:82])-PEG2 -K(N3 )-NH2 )
Ac-PKKKRKV-PEG2-K(环[FGFGRGRQ])-PEG12-OHAc-PKKKRKV-PEG2 -K(cyclo[FGFGRGRQ])-PEG12 -OH
(Ac-SEQ ID NO:42-PEG2-K(环[SEQ ID NO:82])-PEG12-OH)(Ac-SEQ ID NO:42-PEG2 -K(cyclo[SEQ ID NO:82])-PEG12 -OH)
Ac-PKKKRKV-PEG2-K(环[GfFGrGrQ])-PEG2-K(N3)-NH2Ac-PKKKRKV-PEG2 -K(cyclo[GfFGrGrQ])-PEG2 -K(N3 )-NH2
(Ac-SEQ ID NO:42-PEG2-K(环[SEQ ID NO:133])-PEG2-K(N3)-NH2)，以及(Ac-SEQ ID NO:42-PEG2 -K(cyclo[SEQ ID NO:133])-PEG2 -K(N3 )-NH2 ), and
Ac-PKKKRKV-PEG2-K(环[GfFGrGrQ])-PEG12-OHAc-PKKKRKV-PEG2 -K(cyclo[GfFGrGrQ])-PEG12 -OH
(Ac-SEQ ID NO:42-PEG2-K(环[SEQ ID NO:133])-PEG12-OH)。(Ac-SEQ ID NO:42-PEG2 -K(cyclo[SEQ ID NO:133])-PEG12 -OH).
货物可以是蛋白质并且EEV可以选自：The cargo may be a protein and the EEV may be selected from:
Ac-PKKKRKV-PEG2-K(环[FfΦGrGrQ])-PEG12-OHAc-PKKKRKV-PEG2 -K(cyclo[FfΦGrGrQ])-PEG12 -OH
(Ac-SEQ ID NO:42-PEG2-K(环[SEQ ID NO:80])-PEG12-OH)(Ac-SEQ ID NO:42-PEG2 -K(cyclo[SEQ ID NO:80])-PEG12 -OH)
Ac-PKKKRKV-PEG2-K(环[FfΦCit-r-Cit-rQ])-PEG12-OHAc-PKKKRKV-PEG2 -K(cyclo[FfΦCit-r-Cit-rQ])-PEG12 -OH
(Ac-SEQ ID NO:42-PEG2-K(环[SEQ ID NO:79])-PEG12-OH)(Ac-SEQ ID NO:42-PEG2 -K(cyclo[SEQ ID NO:79])-PEG12 -OH)
Ac-PKKKRKV-PEG2-K(环[FfFGRGRQ])-PEG12-OHAc-PKKKRKV-PEG2 -K(cyclo[FfFGRGRQ])-PEG12 -OH
(Ac-SEQ ID NO:42-PEG2-K(环[SEQ ID NO:81])-PEG12-OH)(Ac-SEQ ID NO:42-PEG2 -K(cyclo[SEQ ID NO:81])-PEG12 -OH)
Ac-PKKKRKV-PEG2-K(环[FGFGRGRQ])-PEG12-OHAc-PKKKRKV-PEG2 -K(cyclo[FGFGRGRQ])-PEG12 -OH
(Ac-SEQ ID NO:42-PEG2-K(环[SEQ ID NO:82])-PEG12-OH)(Ac-SEQ ID NO:42-PEG2 -K(cyclo[SEQ ID NO:82])-PEG12 -OH)
Ac-PKKKRKV-PEG2-K(环[GfFGrGrQ])-PEG12-OHAc-PKKKRKV-PEG2 -K(cyclo[GfFGrGrQ])-PEG12 -OH
(Ac-SEQ ID NO:42-PEG2-K(环[SEQ ID NO:133])-PEG12-OH)(Ac-SEQ ID NO:42-PEG2 -K(cyclo[SEQ ID NO:133])-PEG12 -OH)
Ac-PKKKRKV-PEG2-K(环[FGFGRRRQ])-PEG12-OHAc-PKKKRKV-PEG2 -K(cyclo[FGFGRRRQ])-PEG12 -OH
(Ac-SEQ ID NO:42-PEG2-K(环[SEQ ID NO:84])-PEG12-OH)Ac-PKKKRKV-PEG2-K(环[FGFRRRRQ])-PEG12-OH(Ac-SEQ ID NO:42-PEG2-K(环[SEQ ID NO:85])-PEG12-OH)Ac-rr-PEG2-K(环[FfΦGrGrQ])-PEG12-OH(Ac-SEQ ID NO:42-PEG2 -K(cyclo[SEQ ID NO:84])-PEG12 -OH)Ac-PKKKRKV-PEG2 -K(cyclo[FGFRRRRQ])-PEG12 -OH(Ac-SEQ ID NO:42-PEG2 -K(cyclo[SEQ ID NO:85])-PEG12 -OH)Ac-rr-PEG2 -K(cyclo[FfΦGrGrQ])-PEG12 -OH
(Ac-rr-PEG2-K(环[SEQ ID NO:80])-PEG12-OH)(Ac-rr-PEG2 -K(cyclo[SEQ ID NO:80])-PEG12 -OH)
Ac-rr-PEG2-K(环[FfΦCit-r-Cit-rQ])-PEG12-OHAc-rr-PEG2 -K(cyclo[FfΦCit-r-Cit-rQ])-PEG12 -OH
(Ac-rr-PEG2-K(环[SEQ ID NO:79])-PEG12-OH)(Ac-rr-PEG2 -K(cyclo[SEQ ID NO:79])-PEG12 -OH)
Ac-rr-PEG2-K(环[FfF-GRGRQ])-PEG12-OHAc-rr-PEG2 -K(cyclo[FfF-GRGRQ])-PEG12 -OH
(Ac-rr-PEG2-K(环[SEQ ID NO:81])-PEG12-OH)(Ac-rr-PEG2 -K(cyclo[SEQ ID NO:81])-PEG12 -OH)
Ac-rr-PEG2-K(环[FGFGRGRQ])-PEG12-OHAc-rr-PEG2 -K(cyclo[FGFGRGRQ])-PEG12 -OH
(Ac-rr-PEG2-K(环[SEQ ID NO:82])-PEG12-OH)(Ac-rr-PEG2 -K(cyclo[SEQ ID NO:82])-PEG12 -OH)
Ac-rr-PEG2-K(环[GfFGrGrQ])-PEG12-OHAc-rr-PEG2 -K(cyclo[GfFGrGrQ])-PEG12 -OH
(Ac-rr-PEG2-K(环[SEQ ID NO:133])-PEG12-OH)(Ac-rr-PEG2 -K(cyclo[SEQ ID NO:133])-PEG12 -OH)
Ac-rr-PEG2-K(环[FGFGRRRQ])-PEG12-OHAc-rr-PEG2 -K(cyclo[FGFGRRRQ])-PEG12 -OH
(Ac-rr-PEG2-K(环[SEQ ID NO:84])-PEG12-OH)(Ac-rr-PEG2 -K(cyclo[SEQ ID NO:84])-PEG12 -OH)
Ac-rr-PEG2-K(环[FGFRRRRQ])-PEG12-OHAc-rr-PEG2 -K(cyclo[FGFRRRRQ])-PEG12 -OH
(Ac-rr-PEG2-K(环[SEQ ID NO:85])-PEG12-OH)(Ac-rr-PEG2 -K(cyclo[SEQ ID NO:85])-PEG12 -OH)
Ac-rrr-PEG2-K(环[FfΦGrGrQ])-PEG12-OHAc-rrr-PEG2 -K(cyclo[FfΦGrGrQ])-PEG12 -OH
(Ac-rrr-PEG2-K(环[SEQ ID NO:80])-PEG12-OH)(Ac-rrr-PEG2 -K(cyclo[SEQ ID NO:80])-PEG12 -OH)
Ac-rrr-PEG2-K(环[FfΦCit-r-Cit-rQ])-PEG12-OHAc-rrr-PEG2 -K(cyclo[FfΦCit-r-Cit-rQ])-PEG12 -OH
(Ac-rrr-PEG2-K(环[SEQ ID NO:79])-PEG12-OH)(Ac-rrr-PEG2 -K(cyclo[SEQ ID NO:79])-PEG12 -OH)
Ac-rrr-PEG2-K(环[FfFGRGRQ])-PEG12-OHAc-rrr-PEG2 -K(cyclo[FfFGRGRQ])-PEG12 -OH
(Ac-rrr-PEG2-K(环[SEQ ID NO:81])-PEG12-OH)(Ac-rrr-PEG2 -K(cyclo[SEQ ID NO:81])-PEG12 -OH)
Ac-rrr-PEG2-K(环[FGFGRGRQ])-PEG12-OHAc-rrr-PEG2 -K(cyclo[FGFGRGRQ])-PEG12 -OH
(Ac-rrr-PEG2-K(环[SEQ ID NO:82])-PEG12-OH)(Ac-rrr-PEG2 -K(cyclo[SEQ ID NO:82])-PEG12 -OH)
Ac-rrr-PEG2-K(环[GfFGrGrQ])-PEG12-OHAc-rrr-PEG2 -K(cyclo[GfFGrGrQ])-PEG12 -OH
(Ac-rrr-PEG2-K(环[SEQ ID NO:133])-PEG12-OH)(Ac-rrr-PEG2 -K(cyclo[SEQ ID NO:133])-PEG12 -OH)
Ac-rrr-PEG2-K(环[FGFGRRRQ])-PEG12-OHAc-rrr-PEG2 -K(cyclo[FGFGRRRQ])-PEG12 -OH
(Ac-rrr-PEG2-K(环[SEQ ID NO:84])-PEG12-OH)(Ac-rrr-PEG2 -K(cyclo[SEQ ID NO:84])-PEG12 -OH)
Ac-rrr-PEG2-K(环[FGFRRRRQ])-PEG12-OHAc-rrr-PEG2 -K(cyclo[FGFRRRRQ])-PEG12 -OH
(Ac-rrr-PEG2-K(环[SEQ ID NO:85])-PEG12-OH)(Ac-rrr-PEG2 -K(cyclo[SEQ ID NO:85])-PEG12 -OH)
Ac-rhr-PEG2-K(环[FfΦGrGrQ])-PEG12-OHAc-rhr-PEG2 -K(cyclo[FfΦGrGrQ])-PEG12 -OH
(Ac-rhr-PEG2-K(环[SEQ ID NO:80])-PEG12-OH)(Ac-rhr-PEG2 -K(cyclo[SEQ ID NO:80])-PEG12 -OH)
Ac-rhr-PEG2-K(环[FfΦCit-r-Cit-rQ])-PEG12-OHAc-rhr-PEG2 -K(cyclo[FfΦCit-r-Cit-rQ])-PEG12 -OH
(Ac-rhr-PEG2-K(环[SEQ ID NO:79])-PEG12-OH)(Ac-rhr-PEG2 -K(cyclo[SEQ ID NO:79])-PEG12 -OH)
Ac-rhr-PEG2-K(环[FfFGRGRQ])-PEG12-OHAc-rhr-PEG2 -K(cyclo[FfFGRGRQ])-PEG12 -OH
(Ac-rhr-PEG2-K(环[SEQ ID NO:81])-PEG12-OH)(Ac-rhr-PEG2 -K(cyclo[SEQ ID NO:81])-PEG12 -OH)
Ac-rhr-PEG2-K(环[FGFGRGRQ])-PEG12-OHAc-rhr-PEG2 -K(cyclo[FGFGRGRQ])-PEG12 -OH
(Ac-rhr-PEG2-K(环[SEQ ID NO:82])-PEG12-OH)(Ac-rhr-PEG2 -K(cyclo[SEQ ID NO:82])-PEG12 -OH)
Ac-rhr-PEG2-K(环[GfFGrGrQ])-PEG12-OHAc-rhr-PEG2 -K(cyclo[GfFGrGrQ])-PEG12 -OH
(Ac-rhr-PEG2-K(环[SEQ ID NO:133])-PEG12-OH)(Ac-rhr-PEG2 -K(cyclo[SEQ ID NO:133])-PEG12 -OH)
Ac-rhr-PEG2-K(环[FGFGRRRQ])-PEG12-OHAc-rhr-PEG2 -K(cyclo[FGFGRRRQ])-PEG12 -OH
(Ac-rhr-PEG2-K(环[SEQ ID NO:84])-PEG12-OH)(Ac-rhr-PEG2 -K(cyclo[SEQ ID NO:84])-PEG12 -OH)
Ac-rhr-PEG2-K(环[FGFRRRRQ])-PEG12-OHAc-rhr-PEG2 -K(cyclo[FGFRRRRQ])-PEG12 -OH
(Ac-rhr-PEG2-K(环[SEQ ID NO:85])-PEG12-OH)(Ac-rhr-PEG2 -K(cyclo[SEQ ID NO:85])-PEG12 -OH)
Ac-rbr-PEG2-K(环[FfΦGrGrQ])-PEG12-OHAc-rbr-PEG2 -K(cyclo[FfΦGrGrQ])-PEG12 -OH
(Ac-rbr-PEG2-K(环[SEQ ID NO:80])-PEG12-OH)(Ac-rbr-PEG2 -K(cyclo[SEQ ID NO:80])-PEG12 -OH)
Ac-rbr-PEG2-K(环[FfΦCit-r-Cit-rQ])-PEG12-OHAc-rbr-PEG2 -K(cyclo[FfΦCit-r-Cit-rQ])-PEG12 -OH
(Ac-rbr-PEG2-K(环[SEQ ID NO:79])-PEG12-OH)(Ac-rbr-PEG2 -K(cyclo[SEQ ID NO:79])-PEG12 -OH)
Ac-rbr-PEG2-K(环[FfFGRGRQ])-PEG12-OHAc-rbr-PEG2 -K(cyclo[FfFGRGRQ])-PEG12 -OH
(Ac-rbr-PEG2-K(环[SEQ ID NO:81])-PEG12-OH)(Ac-rbr-PEG2 -K(cyclo[SEQ ID NO:81])-PEG12 -OH)
Ac-rbr-PEG2-K(环[FGFGRGRQ])-PEG12-OHAc-rbr-PEG2 -K(cyclo[FGFGRGRQ])-PEG12 -OH
(Ac-rbr-PEG2-K(环[SEQ ID NO:82])-PEG12-OH)(Ac-rbr-PEG2 -K(cyclo[SEQ ID NO:82])-PEG12 -OH)
Ac-rbr-PEG2-K(环[GfFGrGrQ])-PEG12-OHAc-rbr-PEG2 -K(cyclo[GfFGrGrQ])-PEG12 -OH
(Ac-rbr-PEG2-K(环[SEQ ID NO:133])-PEG12-OH)(Ac-rbr-PEG2 -K(cyclo[SEQ ID NO:133])-PEG12 -OH)
Ac-rbr-PEG2-K(环[FGFGRRRQ])-PEG12-OHAc-rbr-PEG2 -K(cyclo[FGFGRRRQ])-PEG12 -OH
(Ac-rbr-PEG2-K(环[SEQ ID NO:84])-PEG12-OH)(Ac-rbr-PEG2 -K(cyclo[SEQ ID NO:84])-PEG12 -OH)
Ac-rbr-PEG2-K(环[FGFRRRRQ])-PEG12-OHAc-rbr-PEG2 -K(cyclo[FGFRRRRQ])-PEG12 -OH
(Ac-rbr-PEG2-K(环[SEQ ID NO:85])-PEG12-OH)(Ac-rbr-PEG2 -K(cyclo[SEQ ID NO:85])-PEG12 -OH)
Ac-rbrbr-PEG2-K(环[FfΦGrGrQ])-PEG12-OHAc-rbrbr-PEG2 -K(cyclo[FfΦGrGrQ])-PEG12 -OH
(Ac-SEQ ID NO:138-PEG2-K(环[SEQ ID NO:80])-PEG12-OH)Ac-rbrbr-PEG2-K(环[FfΦCit-r-Cit-rQ])-PEG12-OH(Ac-SEQ ID NO:138-PEG2 -K(cyclo[SEQ ID NO:80])-PEG12 -OH)Ac-rbrbr-PEG2 -K(cyclo[FfΦCit-r-Cit-rQ])-PEG12 -OH
(Ac-SEQ ID NO:138-PEG2-K(环[SEQ ID NO:79])-PEG12-OH)Ac-rbrbr-PEG2-K(环[FfFGRGRQ])-PEG12-OH(Ac-SEQ ID NO:138-PEG2 -K(cyclo[SEQ ID NO:79])-PEG12 -OH)Ac-rbrbr-PEG2 -K(cyclo[FfFGRGRQ])-PEG12 -OH
(Ac-SEQ ID NO:138-PEG2-K(环[SEQ ID NO:81])-PEG12-OH)Ac-rbrbr-PEG2-K(环[FGFGRGRQ])-PEG12-OH(Ac-SEQ ID NO:138-PEG2 -K(cyclo[SEQ ID NO:81])-PEG12 -OH)Ac-rbrbr-PEG2 -K(cyclo[FGFGRGRQ])-PEG12 -OH
(Ac-SEQ ID NO:138-PEG2-K(环[SEQ ID NO:82])-PEG12-OH)Ac-rbrbr-PEG2-K(环[GfFGrGrQ])-PEG12-OH(Ac-SEQ ID NO:138-PEG2 -K(cyclo[SEQ ID NO:82])-PEG12 -OH)Ac-rbrbr-PEG2 -K(cyclo[GfFGrGrQ])-PEG12 -OH
(Ac-SEQ ID NO:138-PEG2-K(环[SEQ ID NO:133])-PEG12-OH)Ac-rbrbr-PEG2-K(环[FGFGRRRQ])-PEG12-OH(Ac-SEQ ID NO:138-PEG2 -K(cyclo[SEQ ID NO:133])-PEG12 -OH)Ac-rbrbr-PEG2 -K(cyclo[FGFGRRRQ])-PEG12 -OH
(Ac-SEQ ID NO:138-PEG2-K(环[SEQ ID NO:84])-PEG12-OH)Ac-rbrbr-PEG2-K(环[FGFRRRRQ])-PEG12-OH(Ac-SEQ ID NO:138-PEG2 -K(cyclo[SEQ ID NO:84])-PEG12 -OH)Ac-rbrbr-PEG2 -K(cyclo[FGFRRRRQ])-PEG12 -OH
(Ac-SEQ ID NO:138-PEG2-K(环[SEQ ID NO:85])-PEG12-OH)Ac-rbhbr-PEG2-K(环[FfΦGrGrQ])-PEG12-OH(Ac-SEQ ID NO:138-PEG2 -K(cyclo[SEQ ID NO:85])-PEG12 -OH)Ac-rbhbr-PEG2 -K(cyclo[FfΦGrGrQ])-PEG12 -OH
(Ac-SEQ ID NO:149-PEG2-K(环[SEQ ID NO:80])-PEG12-OH)Ac-rbhbr-PEG2-K(环[FfΦCit-r-Cit-rQ])-PEG12-OH(Ac-SEQ ID NO:149-PEG2 -K(cyclo[SEQ ID NO:80])-PEG12 -OH)Ac-rbhbr-PEG2 -K(cyclo[FfΦCit-r-Cit-rQ])-PEG12 -OH
(Ac-SEQ ID NO:149-PEG2-K(环[SEQ ID NO:79])-PEG12-OH)Ac-rbhbr-PEG2-K(环[FfFGRGRQ])-PEG12-OH(Ac-SEQ ID NO:149-PEG2 -K(cyclo[SEQ ID NO:79])-PEG12 -OH)Ac-rbhbr-PEG2 -K(cyclo[FfFGRGRQ])-PEG12 -OH
(Ac-SEQ ID NO:149-PEG2-K(环[SEQ ID NO:81])-PEG12-OH)Ac-rbhbr-PEG2-K(环[FGFGRGRQ])-PEG12-OH(Ac-SEQ ID NO:149-PEG2 -K(cyclo[SEQ ID NO:81])-PEG12 -OH)Ac-rbhbr-PEG2 -K(cyclo[FGFGRGRQ])-PEG12 -OH
(Ac-SEQ ID NO:149-PEG2-K(环[SEQ ID NO:82])-PEG12-OH)Ac-rbhbr-PEG2-K(环[GfFGrGrQ])-PEG12-OH(Ac-SEQ ID NO:149-PEG2 -K(cyclo[SEQ ID NO:82])-PEG12 -OH)Ac-rbhbr-PEG2 -K(cyclo[GfFGrGrQ])-PEG12 -OH
(Ac-SEQ ID NO:149-PEG2-K(环[SEQ ID NO:133])-PEG12-OH)Ac-rbhbr-PEG2-K(环[FGFGRRRQ])-PEG12-OH(Ac-SEQ ID NO:149-PEG2 -K(cyclo[SEQ ID NO:133])-PEG12 -OH)Ac-rbhbr-PEG2 -K(cyclo[FGFGRRRQ])-PEG12 -OH
(Ac-SEQ ID NO:149-PEG2-K(环[SEQ ID NO:84])-PEG12-OH)Ac-rbhbr-PEG2-K(环[FGFRRRRQ])-PEG12-OH(Ac-SEQ ID NO:149-PEG2 -K(cyclo[SEQ ID NO:84])-PEG12 -OH)Ac-rbhbr-PEG2 -K(cyclo[FGFRRRRQ])-PEG12 -OH
(Ac-SEQ ID NO:149-PEG2-K(环[SEQ ID NO:85])-PEG12-OH)Ac-hbrbh-PEG2-K(环[FfΦGrGrQ])-PEG12-OH(Ac-SEQ ID NO:149-PEG2 -K(cyclo[SEQ ID NO:85])-PEG12 -OH)Ac-hbrbh-PEG2 -K(cyclo[FfΦGrGrQ])-PEG12 -OH
(Ac-SEQ ID NO:141-PEG2-K(环[SEQ ID NO:80])-PEG12-OH)Ac-hbrbh-PEG2-K(环[FfΦCit-r-Cit-rQ])-PEG12-OH(Ac-SEQ ID NO:141-PEG2 -K(cyclo[SEQ ID NO:80])-PEG12 -OH)Ac-hbrbh-PEG2 -K(cyclo[FfΦCit-r-Cit-rQ])-PEG12 -OH
(Ac-SEQ ID NO:141-PEG2-K(环[SEQ ID NO:79])-PEG12-OH)Ac-hbrbh-PEG2-K(环[FfFGRGRQ])-PEG12-OH(Ac-SEQ ID NO:141-PEG2 -K(cyclo[SEQ ID NO:79])-PEG12 -OH)Ac-hbrbh-PEG2 -K(cyclo[FfFGRGRQ])-PEG12 -OH
(Ac-SEQ ID NO:141-PEG2-K(环[SEQ ID NO:81])-PEG12-OH)Ac-hbrbh-PEG2-K(环[FGFGRGRQ])-PEG12-OH(Ac-SEQ ID NO:141-PEG2 -K(cyclo[SEQ ID NO:81])-PEG12 -OH)Ac-hbrbh-PEG2 -K(cyclo[FGFGRGRQ])-PEG12 -OH
(Ac-SEQ ID NO:141-PEG2-K(环[SEQ ID NO:82])-PEG12-OH)(Ac-SEQ ID NO:141-PEG2 -K(cyclo[SEQ ID NO:82])-PEG12 -OH)
Ac-hbrbh-PEG2-K(环[GfFGrGrQ])-PEG12-OHAc-hbrbh-PEG2 -K(cyclo[GfFGrGrQ])-PEG12 -OH
(Ac-SEQ ID NO:141-PEG2-K(环[SEQ ID NO:133])-PEG12-OH)(Ac-SEQ ID NO:141-PEG2 -K(cyclo[SEQ ID NO:133])-PEG12 -OH)
Ac-hbrbh-PEG2-K(环[FGFGRRRQ])-PEG12-OHAc-hbrbh-PEG2 -K(cyclo[FGFGRRRQ])-PEG12 -OH
(Ac-SEQ ID NO:141-PEG2-K(环[SEQ ID NO:84])-PEG12-OH)(Ac-SEQ ID NO:141-PEG2 -K(cyclo[SEQ ID NO:84])-PEG12 -OH)
Ac-hbrbh-PEG2-K(环[FGFRRRRQ])-PEG12-OHAc-hbrbh-PEG2 -K(cyclo[FGFRRRRQ])-PEG12 -OH
(Ac-SEQ ID NO:141-PEG2-K(环[SEQ ID NO:85])-PEG12-OH)，(Ac-SEQ ID NO:141-PEG2 -K(cyclo[SEQ ID NO:85])-PEG12 -OH),
其中b是β-丙氨酸，并且环外序列可以是D或L立体化学。wherein b is β-alanine and the exocyclic sequence can be of D or L stereochemistry.
货物goods
细胞穿透肽(CPP)，诸如环状细胞穿透肽(例如，cCPP)，可与货物缀合。如本文所用，“货物”是期望递送到细胞中的化合物或部分。货物可与接头的末端羰基基团缀合。环肽的至少一个原子可被货物替换或者至少一个孤对可形成的与货物键。货物可通过接头与cCPP缀合。货物可通过接头与AASC缀合。cCPP的至少一个原子可被治疗性部分替换或者cCPP的至少一个孤对形成与治疗性部分的键。cCPP的氨基酸侧链上的羟基基团可被与货物的键替换。cCPP的谷氨酰胺侧链上的羟基基团可被与货物的键替换。货物可通过接头与cCPP缀合。货物可通过接头与AASC缀合。Cell penetrating peptides (CPPs), such as cyclic cell penetrating peptides (e.g., cCPPs), can be conjugated to cargo. As used herein, "cargo" is a compound or moiety that is desired to be delivered to a cell. Cargo can be conjugated to the terminal carbonyl group of a linker. At least one atom of the cyclic peptide can be replaced by cargo or at least one lone pair can form a bond with cargo. Cargo can be conjugated to cCPP through a linker. Cargo can be conjugated to AASC through a linker. At least one atom of cCPP can be replaced by a therapeutic moiety or at least one lone pair of cCPP can form a bond with a therapeutic moiety. The hydroxyl group on the amino acid side chain of cCPP can be replaced by a bond with cargo. The hydroxyl group on the glutamine side chain of cCPP can be replaced by a bond with cargo. Cargo can be conjugated to cCPP through a linker. Cargo can be conjugated to AASC through a linker.
在实施方案中，氨基酸侧链包含与接头或货物缀合的化学反应性基团。化学反应性基团可包括胺基团、羧酸、酰胺、羟基基团、巯基基团、胍基、酚基团、硫醚基团、咪唑基团或吲哚基团。在实施方案中，与货物缀合的cCPP的氨基酸包括赖氨酸、精氨酸、天冬氨酸、谷氨酸、天冬酰胺、谷氨酰胺、高谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、精氨酸、酪氨酸、甲硫氨酸、组氨酸或色氨酸。In an embodiment, the amino acid side chain comprises a chemically reactive group that is conjugated to a linker or cargo. The chemically reactive group may include an amine group, a carboxylic acid, an amide, a hydroxyl group, a sulfhydryl group, a guanidinium group, a phenolic group, a thioether group, an imidazole group, or an indole group. In an embodiment, the amino acid of the cCPP conjugated to the cargo comprises lysine, arginine, aspartic acid, glutamic acid, asparagine, glutamine, homoglutamine, serine, threonine, tyrosine, cysteine, arginine, tyrosine, methionine, histidine, or tryptophan.
货物可包含一个或多个可检测部分、一个或多个治疗性部分(TM)、一个或多个靶向部分或它们的任何组合。在实施方案中，货物包含TM。在实施方案中，TM包含反义化合物(AC)。在实施方案中，AC结合靶基因转录物的剪接元件(SE)的至少一部分或与靶基因转录物的SE足够接近以调节靶基因转录物的剪接。在实施方案中，AC结合靶IRF-5、DPMK或DUX4基因转录物的SE的至少一部分。在实施方案中，AC足够接近地结合靶IRF-5、DPMK或DUX4基因转录物的SE，以调节靶IRF-5、DPMK或DUX4基因转录物的剪接。Goods may include one or more detectable moieties, one or more therapeutic moieties (TM), one or more targeting moieties, or any combination thereof. In an embodiment, goods include TM. In an embodiment, TM includes an antisense compound (AC). In an embodiment, AC binds to at least a portion of a splicing element (SE) of a target gene transcript or is sufficiently close to the SE of a target gene transcript to regulate the splicing of a target gene transcript. In an embodiment, AC binds to at least a portion of the SE of a target IRF-5, DPMK, or DUX4 gene transcript. In an embodiment, AC binds to the SE of a target IRF-5, DPMK, or DUX4 gene transcript sufficiently close to regulate the splicing of a target IRF-5, DPMK, or DUX4 gene transcript.
与货物部分缀合的环状细胞穿透肽(cCPP)Cyclic cell penetrating peptide (cCPP) conjugated to cargo moiety
环状细胞穿透肽(cCPP)可与货物部分缀合。Circular cell penetrating peptides (cCPPs) can be conjugated to cargo moieties.
货物部分可在末端羰基处与接头缀合以提供以下结构：The cargo moiety can be conjugated to a linker at the terminal carbonyl group to provide the following structure:
其中： in:
EP是环外肽，并且M、AASC、货物、x'、y和z'如上文所定义，*是与AASC的附接点。x'可以是1。y可以是4。z'可以是11。-(OCH2CH-2)x'-和/或-(OCH2CH-2)z'-可独立地被一个或多个氨基酸替换，包括例如甘氨酸、β-丙氨酸、4-氨基丁酸、5-氨基戊酸、6-氨基己酸或它们的组合。EP is an exocyclic peptide, and M, AASC , cargo, x', y and z' are as defined above, * is the point of attachment to AASC . x' can be 1. y can be 4. z' can be 11. -(OCH2 CH-2 )x' - and/or -(OCH2 CH-2 )z' - can be independently replaced by one or more amino acids, including, for example, glycine, β-alanine, 4-aminobutyric acid, 5-aminovaleric acid, 6-aminohexanoic acid, or a combination thereof.
内体逃逸载体(EEV)可包含环状细胞穿透肽(cCPP)、环外肽(EP)和接头，并且可与货物缀合以形成包含式(C)的结构的EEV-缀合物：An endosomal escape vector (EEV) may comprise a cyclic cell penetrating peptide (cCPP), an exocyclic peptide (EP), and a linker, and may be conjugated to a cargo to form an EEV-conjugate comprising a structure of formula (C):
或其质子化形式，or its protonated form,
其中：in:
R1、R2和R3可各自独立地是H或具有包含芳族基团的侧链的氨基酸残基；R1 , R2 and R3 may each independently be H or an amino acid residue having a side chain containing an aromatic group;
R4是H或氨基酸侧链；R4 is H or an amino acid side chain;
EP是如本文所定义的环外肽；EP is an exocyclic peptide as defined herein;
货物是如本文所定义的部分；Goods are Parts as defined herein;
每个m独立地是0-3的整数；Each m is independently an integer from 0 to 3;
n是0-2的整数；n is an integer from 0 to 2;
x'是2-20的整数；x' is an integer from 2 to 20;
y是1-5的整数；y is an integer from 1 to 5;
q是1-4的整数；并且q is an integer from 1 to 4; and
z'是2-20的整数。z' is an integer from 2 to 20.
R1、R2、R3，R4、EP、货物、m、n、x'、y、q和z'如本文所定义。R1 , R2 , R3 , R4 , EP, cargo, m, n, x', y, q and z' are as defined herein.
EEV可与货物缀合，并且EEV-缀合物可包含式(C-a)或(C-b)的结构：The EEV may be conjugated to a cargo, and the EEV-conjugate may comprise a structure of formula (C-a) or (C-b):
或其质子化形式，其中EP、m和z如上文在式(C)中所定义。 or a protonated form thereof, wherein EP, m and z are as defined above in formula (C).
EEV可与货物缀合，并且EEV-缀合物可包含式(C-c)的结构：The EEV can be conjugated to a cargo, and the EEV-conjugate can comprise a structure of formula (C-c):
或其质子化形式，其中EP、R1、R2、R3、R4和m如上文在式(III)中所定义；AA可以是如本文所定义的氨基酸；n可以是0-2的整数；x可以是1-10的整数；y可以是1-5的整数；z可以是1-10的整数。or a protonated form thereof, wherein EP, R1 , R2 , R3 , R4 and m are as defined above in formula (III); AA may be an amino acid as defined herein; n may be an integer from 0 to 2; x may be an integer from 1 to 10; y may be an integer from 1 to 5; and z may be an integer from 1 to 10.
EEV可与寡核苷酸货物缀合，并且EEV-寡核苷酸缀合物可包含式(C-1)、(C-2)、(C-3)或(C-4)的结构：The EEV may be conjugated to an oligonucleotide cargo, and the EEV-oligonucleotide conjugate may comprise a structure of formula (C-1), (C-2), (C-3), or (C-4):
EEV可与寡核苷酸货物缀合，并且EEV-缀合物可包含以下结构：EEV can be conjugated to an oligonucleotide cargo, and the EEV-conjugate can comprise the following structure:
胞质递送效率Cytoplasmic delivery efficiency
对环状细胞穿透肽(cCPP)的修饰可提高胞质递送效率。通过将具有经修饰的序列的cCPP的胞质递送效率与对照序列进行比较，可测量改善的胞质摄取效率。调控序列不包括修饰序列中的特定置换氨基酸残基(包括但不限于精氨酸、苯丙氨酸和/或甘氨酸)，但在其他方面是相同的。Modification of cyclic cell penetrating peptides (cCPPs) can improve cytoplasmic delivery efficiency. By comparing the cytoplasmic delivery efficiency of cCPPs with modified sequences with control sequences, improved cytoplasmic uptake efficiency can be measured. The regulatory sequence does not include specific replacement amino acid residues (including but not limited to arginine, phenylalanine and/or glycine) in the modified sequence, but is otherwise identical.
如本文所用，胞质递送效率是指cCPP穿过细胞膜并进入细胞的胞质溶胶的能力。cCPP的胞质递送效率不一定依赖于受体或细胞类型。胞质递送效率可指绝对胞质递送效率或相对胞质递送效率。As used herein, cytoplasmic delivery efficiency refers to the ability of a cCPP to cross the cell membrane and enter the cytosol of a cell. The cytoplasmic delivery efficiency of a cCPP is not necessarily dependent on receptors or cell types. The cytoplasmic delivery efficiency may refer to an absolute cytoplasmic delivery efficiency or a relative cytoplasmic delivery efficiency.
绝对胞质递送效率是生长培养基中cCPP(或cCPP-货物缀合物)的胞质浓度与cCPP(或cCPP-货物缀合物)的浓度的比率。相对胞质溶胶递送效率是指与对照cCPP在胞质溶胶中的浓度相比cCPP在胞质溶胶中的浓度。定量可通过荧光标记cCPP(例如，用FITC染料)并使用本领域熟知的技术测量荧光强度来实现。The absolute cytoplasmic delivery efficiency is the ratio of the cytoplasmic concentration of the cCPP (or cCPP-cargo conjugate) to the concentration of the cCPP (or cCPP-cargo conjugate) in the growth medium. The relative cytosol delivery efficiency refers to the concentration of the cCPP in the cytosol compared to the concentration of the control cCPP in the cytosol. Quantification can be achieved by fluorescently labeling the cCPP (e.g., with a FITC dye) and measuring the fluorescence intensity using techniques well known in the art.
通过比较(i)被细胞类型(例如，HeLa细胞)内化的本发明cCPP的量与(ii)被相同细胞类型内化的对照cCPP的量来确定相对胞质递送效率。为了测量相对胞质递送效率，可将细胞类型在cCPP的存在下孵育指定的时间段(例如，30分钟、1小时、2小时等)，之后使用本领域已知的方法例如荧光显微术定量被细胞内化的cCPP的量。单独地，将相同浓度的对照cCPP在该细胞类型的存在下孵育相同的时间段，并且对被细胞内化的对照cCPP的量进行定量。The relative cytoplasmic delivery efficiency is determined by comparing (i) the amount of the cCPP of the invention internalized by a cell type (e.g., HeLa cells) with (ii) the amount of the control cCPP internalized by the same cell type. To measure the relative cytoplasmic delivery efficiency, the cell type can be incubated in the presence of the cCPP for a specified period of time (e.g., 30 minutes, 1 hour, 2 hours, etc.), followed by quantification of the amount of the cCPP internalized by the cell using methods known in the art, such as fluorescence microscopy. Separately, the same concentration of the control cCPP is incubated in the presence of the cell type for the same period of time, and the amount of the control cCPP internalized by the cell is quantified.
相对胞质递送效率可通过测量具有经修饰的序列的cCPP对胞内靶标的IC50并将具有经修饰的序列的cCPP对对照序列的IC50(如本文所述)进行比较来确定。Relative cytoplasmic delivery efficiency can be determined by measuring theIC50 of a cCPP with a modified sequence against an intracellular target and comparing theIC50 of the cCPP with a modified sequence to a control sequence (as described herein).
与环(FfФRrRrQ,SEQ ID NO:150)相比，cCPP的相对胞质递送效率可在约50％至约450％的范围内，例如约60％、约70％、约80％、约90％、约100％、约110％、约120％、约130％、约140％、约150％、约160％、约170％、约180％、约190％、约200％、约210％、约220％、约230％、约240％、约250％、约260％、约270％、约280％、约290％、约300％、约310％、约320％、约330％、约340％、约350％、约360％、约370％、约380％、约390％、约400％、约410％、约420％、约430％、约440％、约450％、约460％、约470％、约480％、约490％、约500％、约510％、约520％、约530％、约540％、约550％、约560％、约570％、约580％或约590％，包括其间的所有范围和值。与包含环(FfФRrRrQ,SEQ ID NO:150)的环肽相比，cCPP的相对胞质递送效率可提高大于约600％。Compared to loop (FfФRrRrQ, SEQ ID NO: 150), the relative cytoplasmic delivery efficiency of cCPP can be in the range of about 50% to about 450%, for example, about 60%, about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%, about 280%, about 290%, about 300% %, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490%, about 500%, about 510%, about 520%, about 530%, about 540%, about 550%, about 560%, about 570%, about 580% or about 590%, including all ranges and values therebetween. Compared with the cyclic peptide comprising the ring (FfФRrRrQ, SEQ ID NO: 150), the relative cytoplasmic delivery efficiency of the cCPP can be increased by greater than about 600%.
绝对胞质递送效力为约40％至约100％，例如约45％、约50％、约55％、约60％、约65％、约70％、约75％、约80％、约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％、约99％，包括其间的所有值和子范围。The absolute cytoplasmic delivery efficacy is about 40% to about 100%, for example, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, including all values and subranges therebetween.
与其他相同的序列相比，本公开的cCPP可将胞质递送效率提高约1.1倍至约30倍，例如，约1.2、约1.3、约1.4、约1.5、约1.6、约1.7、约1.8、约1.9、约2.0、约2.5、约3.0、约3.5、约4.0、约4.5、约5.0、约5.5、约6.0、约6.5、约7.0、约7.5、约8.0、约8.5、约9.0、约10、约10.5、约11.0、约11.5、约12.0、约12.5、约13.0、约13.5、约14.0、约14.5、约15.0、约15.5、约16.0、约16.5、约17.0、约17.5、约18.0、约18.5、约19.0、约19.5、约20、约20.5、约21.0、约21.5、约22.0、约22.5、约23.0、约23.5、约24.0、约24.5、约25.0、约25.5、约26.0、约26.5、约27.0、约27.5、约28.0、约28.5、约29.0、或约29.5倍，包括其间的所有值和子范围。Compared to other identical sequences, the cCPPs of the present disclosure can improve the cytoplasmic delivery efficiency by about 1.1-fold to about 30-fold, for example, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 10, about 10.5, about 11.0, about 11.5, about 12.0, about 12.5, about 13.0, about 13.5, about 14. about 14.0, about 14.5, about 15.0, about 15.5, about 16.0, about 16.5, about 17.0, about 17.5, about 18.0, about 18.5, about 19.0, about 19.5, about 20, about 20.5, about 21.0, about 21.5, about 22.0, about 22.5, about 23.0, about 23.5, about 24.0, about 24.5, about 25.0, about 25.5, about 26.0, about 26.5, about 27.0, about 27.5, about 28.0, about 28.5, about 29.0, or about 29.5 times, including all values and subranges therebetween.
可检测部分Detectable part
在实施方案中，本文公开的化合物包含可检测部分。在实施方案中，可检测部分在细胞穿透肽的氨基基团、羧酸根基团或任何氨基酸的侧链处(例如，在CPP中的氨基基团、羧酸根基团或任何氨基酸的侧链处)附接到细胞穿透肽。在实施方案中，治疗性部分包含可检测部分。可检测部分可包括任何可检测标记。合适的可检测标记的示例包括但不限于UV-Vis标记、近红外标记、发光基团、磷光基团、磁自旋共振标记、光敏剂、光可切割部分、螯合中心、重原子、放射性同位素、同位素可检测自旋共振标记、顺磁性部分、发色团或它们的任何组合。在实施方案中，标记在不添加其他试剂的情况下是可检测的。In an embodiment, the compound disclosed herein comprises a detectable portion. In an embodiment, the detectable portion is attached to the cell penetrating peptide at the amino group, carboxylate group or the side chain of any amino acid of the cell penetrating peptide (for example, the amino group, carboxylate group or the side chain of any amino acid in CPP). In an embodiment, the therapeutic moiety comprises a detectable portion. The detectable portion may include any detectable label. The example of a suitable detectable label includes but is not limited to UV-Vis labeling, near infrared labeling, luminescent group, phosphorescent group, magnetic spin resonance labeling, photosensitizer, photocleavable portion, chelating center, heavy atom, radioisotope, isotope detectable spin resonance labeling, paramagnetic moiety, chromophore or any combination thereof. In an embodiment, the labeling is detectable without adding other reagents.
在实施方案中，可检测部分是生物相容的可检测部分，使得化合物可适用于多种生物应用。如本文所用，“生物相容的”和“生物学上相容的”通常是指连同其任何代谢物或降解产物一起通常对细胞和组织无毒并且当细胞和组织在它们的存在下孵育(例如，培养)时不对细胞和组织造成任何显著不良作用的化合物。In embodiments, the detectable moiety is a biocompatible detectable moiety, making the compound suitable for use in a variety of biological applications. As used herein, "biocompatible" and "biologically compatible" generally refer to compounds that, together with any metabolites or degradation products thereof, are generally non-toxic to cells and tissues and do not cause any significant adverse effects to cells and tissues when cells and tissues are incubated (e.g., cultured) in their presence.
可检测部分可含有发光体，诸如荧光标记或近红外标记。合适的发光体的示例包括但不限于金属卟啉；苯并卟啉；氮杂苯并卟啉；萘卟啉；酞菁；多环芳族烃，诸如二萘嵌苯二亚胺、芘；偶氮染料；呫吨染料；二吡咯亚甲基硼、氮杂二吡咯亚甲基硼、花青染料、金属配体络合物诸如联吡啶、联吡啶类、菲咯啉、香豆素以及钌和铱的乙酰丙酮化物；吖啶、嗪衍生物，诸如二苯并嗪；氮杂轮烯、方酸；8-羟基喹啉、聚甲炔、发光纳米粒子，诸如量子点、纳米晶体；喹诺酮；铽络合物；无机磷光体；离子载体，诸如冠醚附属或衍生的染料；或它们的组合。合适的发光体的具体示例包括但不限于八乙基卟啉Pd(II)；八乙基卟啉Pt(II)；四苯基卟啉Pd(II)；四苯基卟啉Pt(II)；内消旋-四苯基卟啉四苯并卟吩Pd(II)；内消旋-四苯基甲基苯并卟啉Pt(II)；八乙基卟啉酮Pd(II)；八乙基卟啉酮Pt(II)；内消旋-四(五氟苯基)卟啉Pd(II)；内消旋-四(五氟苯基)卟啉Pt(II)；三(4,7-二苯基-1,10-菲咯啉)Ru(II)(Ru(dpp)3)；三(1,10-菲咯啉)Ru(II)(Ru(phen)3)，三(2,2'-联吡啶)氯化钌(II)六水合物(Ru(bpy)3)；赤藓红B；荧光素；异硫氰酸荧光素(FITC)；曙红；((N-甲基-苯并咪唑-2-基)-7-(二乙氨基)-香豆素)铱(III)；The detectable moiety may contain a luminophore, such as a fluorescent label or a near infrared label. Examples of suitable luminophores include, but are not limited to, metalloporphyrins; benzoporphyrins; azabenzoporphyrins; naphthoporphyrins; phthalocyanines; polycyclic aromatic hydrocarbons, such as perylene diimide, pyrene; azo dyes; xanthene dyes; dipyrromethene boron, azadipyrromethene boron, cyanine dyes, metal ligand complexes such as bipyridine, bipyridines, phenanthrolines, coumarins, and acetylacetonates of ruthenium and iridium; acridine, Oxazine derivatives, such as dibenzo azine; azacyclic olefins, squaric acid; 8-hydroxyquinoline, polymethine, luminescent nanoparticles, such as quantum dots, nanocrystals; quinolone; terbium complex; inorganic phosphor; ion carrier, such as crown ether-attached or -derived dye; or a combination thereof. Specific examples of suitable luminophores include, but are not limited to, octaethylporphyrin Pd(II); octaethylporphyrin Pt(II); tetraphenylporphyrin Pd(II); tetraphenylporphyrin Pt(II); meso-tetraphenylporphyrin tetrabenzoporphine Pd(II); meso-tetraphenylmethylbenzoporphyrin Pt(II); octaethylporphyrinone Pd(II); octaethylporphyrinone Pt(II); meso-tetrakis(pentafluorophenyl)porphyrin Pd(II); meso-tetrakis(pentafluorophenyl)porphyrin Pt(II); tris(4,7-diphenyl-1,10-phenanthroline)Ru(II)(Ru(dpp)3 ); tris(1,10-phenanthroline)Ru(II)(Ru(phen)3 ), tris(2,2'-bipyridyl)ruthenium(II) chloride hexahydrate (Ru(bpy)3 ); erythrosine B; fluorescein; fluorescein isothiocyanate (FITC); eosin; ((N-methyl-benzimidazol-2-yl)-7-(diethylamino)-coumarin) iridium (III);
苯并噻唑)((苯并噻唑-2-基)-7-(二乙氨基)-香豆素))-2-(乙酰丙酮化物)；Lumogen染料；Macroflex荧光红；Macrolex荧光黄；得克萨斯红；罗丹明B；罗丹明6G；硫罗丹明；间甲酚；百里酚蓝；二甲苯酚蓝；甲酚红；氯酚蓝；溴甲酚绿；溴甲酚红；溴百里酚蓝；Cy2；Cy3；Cy5；Cy5.5；Cy7；4-硝基苯酚；茜素；酚酞；邻甲酚酞；氯酚红；钙镁试剂；溴二甲苯酚；酚红；中性红；硝嗪；3,4,5,6-四溴酚酞；刚果红；荧光素；曙红；2',7'-二氯荧光素；5(6)-羧基荧光素；羧基萘并荧光素；8-羟基芘-136-三磺酸；半萘并罗丹荧(semi-naphthorhodafluor)；半萘并荧光素；三(4,7-二苯基-1,10-菲咯啉)二氯化钌(II)；(4,7-二苯基-1,10-菲咯啉)钌(II)四苯基硼；八乙基卟啉铂(II)；二烷基碳菁；双十八烷基环氧碳菁；芴基甲氧基碳酰氯；7-氨基-4-甲基香豆素(Amc)；绿色荧光蛋白(GFP)；以及它们的衍生物或组合。benzothiazole)((benzothiazol-2-yl)-7-(diethylamino)-coumarin))-2-(acetylacetonate); Lumogen dyes; Macroflex fluorescent red; Macrolex fluorescent yellow; Texas Red; Rhodamine B; Rhodamine 6G; Sulforhodamine; m-cresol; thymol blue; xylenol blue; cresol red; chlorophenol blue; bromocresol green; bromocresol red; bromothymol blue; Cy2; Cy3; Cy5; Cy5.5; Cy7; 4-nitrophenol; alizarin; phenolphthalein; o-cresolphthalein; chlorophenol red; calcium magnesium reagent; bromoxylenol; phenol red; neutral red; nitrazine; 3,4,5,6-tetrabromophenolphthalein; Congo red; fluorescent fluorescein; eosin; 2',7'-dichlorofluorescein; 5(6)-carboxyfluorescein; carboxynaphthofluorescein; 8-hydroxypyrene-136-trisulfonic acid; semi-naphthorhodafluor; semi-naphthofluorescein; tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II)dichloride; (4,7-diphenyl-1,10-phenanthroline)ruthenium(II)tetraphenylboron; octaethylporphyrin platinum(II); dialkylcarbocyanine; dioctadecylepoxycarbocyanine; fluorenylmethoxyphosgene; 7-amino-4-methylcoumarin (Amc); green fluorescent protein (GFP); and derivatives or combinations thereof.
在一些示例中，可检测部分可包括罗丹明B(Rho)、异硫氰酸荧光素(FITC)、7-氨基-4-甲基香豆素(Amc)、绿色荧光蛋白(GFP)或它们的衍生物或组合。In some examples, the detectable moiety can include rhodamine B (Rho), fluorescein isothiocyanate (FITC), 7-amino-4-methylcoumarin (Amc), green fluorescent protein (GFP), or derivatives or combinations thereof.
制备方法Preparation method
本文所述的化合物可以有机合成领域技术人员已知的多种方式或本领域技术人员所理解的其变化形式制备。本文所述的化合物可由容易获得的起始材料制备。最佳反应条件可随所用的特定反应物或溶剂而变化，但此类条件可由本领域技术人员确定。The compounds described herein can be prepared in a variety of ways known to those skilled in the art of organic synthesis or variations thereof as understood by those skilled in the art. The compounds described herein can be prepared from readily available starting materials. Optimal reaction conditions may vary with the specific reactants or solvents used, but such conditions can be determined by those skilled in the art.
本文所述化合物的变化形式包括如针对每种化合物所述的各种成分的添加、减去或移动。类似地，当分子中存在一个或多个手性中心时，分子的手性可改变。另外，化合物合成可涉及各种化学基团的保护和脱保护。保护和脱保护的使用以及适当的保护基团的选择可由本领域技术人员确定。保护基团的化学性质可见于例如Wuts和Greene,ProtectiveGroups in Organic Synthesis，第4版，Wiley&Sons,2006，其全文以引用方式并入本文。The variation of compounds described herein includes the addition, subtraction or movement of various components as described for each compound. Similarly, when there are one or more chiral centers in a molecule, the chirality of the molecule can be changed. In addition, compound synthesis can involve the protection and deprotection of various chemical groups. The use of protection and deprotection and the selection of appropriate protecting groups can be determined by those skilled in the art. The chemical properties of protecting groups can be found in, for example, Wuts and Greene, Protective Groups in Organic Synthesis, 4th edition, Wiley & Sons, 2006, which is incorporated herein by reference in its entirety.
用于制备所公开的化合物和组合物的起始材料和试剂可购自商业供应商诸如Aldrich Chemical Co.(Milwaukee,WI)、Acros Organics(Morris Plains,NJ)、FisherScientific(Pittsburgh,PA)、Sigma(St.Louis,MO)、Pfizer(New York,NY)、GlaxoSmithKline(Raleigh,NC)、Merck(Whitehouse Station,NJ)、Johnson&Johnson(NewBrunswick,NJ)、Aventis(Bridgewater,NJ)、AstraZeneca(Wilmington,DE)、Novartis(Basel,Switzerland)、Wyeth(Madison,NJ)、Bristol-Myers-Squibb(New York,NY)、Roche(Basel,Switzerland)、Lilly(Indianapolis,IN)、Abbott(Abbott Park,IL)、ScheringPlough(Kenilworth,NJ)、或Boehringer Ingelheim(Ingelheim,Germany)，或者通过本领域技术人员已知的方法按照参考文献中所述的程序制备，该参考文献诸如Fieser andFieser's Reagents for Organic Synthesis，第1-17卷(John Wiley and Sons,1991)；Rodd's Chemistry of Carbon Compounds，第1-5卷和补充版(Elsevier SciencePublishers,1989)；Organic Reactions，第1-40卷(John Wiley and Sons,1991)；March'sAdvanced Organic Chemistry,(John Wiley and Sons，第4版)；以及Larock'sComprehensive Organic Transformations(VCH Publishers Inc.,1989)。其他材料，诸如本文公开的药物载剂可从商业来源获得。The starting materials and reagents used to prepare the disclosed compounds and compositions can be purchased from commercial suppliers such as Aldrich Chemical Co. (Milwaukee, WI), Acros Organics (Morris Plains, NJ), Fisher Scientific (Pittsburgh, PA), Sigma (St. Louis, MO), Pfizer (New York, NY), GlaxoSmithKline (Raleigh, NC), Merck (Whitehouse Station, NJ), Johnson & Johnson (New Brunswick, NJ), Aventis (Bridgewater, NJ), AstraZeneca (Wilmington, DE), Novartis (Basel, Switzerland), Wyeth (Madison, NJ), Bristol-Myers-Squibb (New York, NY), Roche (Basel, Switzerland), Lilly (Indianapolis, IN), Abbott (Abbott Park, IL), Schering Plough (Kenilworth, NJ), or Boehringer Ingelheim (Ingelheim, Germany), or by methods known to those skilled in the art following the procedures described in references such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Supplementary Editions (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced Organic Chemistry, (John Wiley and Sons, 4th Edition); and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989). Other materials, such as the pharmaceutical carriers disclosed herein, can be obtained from commercial sources.
制备本文所述化合物的反应可在溶剂中进行，该溶剂可由有机合成领域的技术人员选择。在进行反应的条件(例如，温度和压力)下，溶剂可基本上不与起始材料(反应物)、中间体或产物反应。反应可在一种溶剂或多于一种溶剂的混合物中进行。产物或中间体形成可根据本领域已知的任何合适的方法进行监测。例如，产物形成可通过光谱手段诸如核磁共振光谱法(例如，1H或13C)、红外光谱法、分光光度测定(例如，UV-可见光)或质谱法，或通过色谱法诸如高效液相色谱法(HPLC)或薄层色谱法来监测。The reaction for preparing the compounds described herein can be carried out in a solvent, which can be selected by a person skilled in the art of organic synthesis. Under the conditions (e.g., temperature and pressure) under which the reaction is carried out, the solvent may not react substantially with the starting material (reactant), intermediate or product. The reaction can be carried out in a solvent or a mixture of more than one solvent. The formation of the product or intermediate can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectral means such as nuclear magnetic resonance spectroscopy (e.g.,1 H or13 C), infrared spectroscopy, spectrophotometry (e.g., UV-visible light) or mass spectrometry, or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.
所公开的化合物可通过固相肽合成来制备，其中氨基酸α-N-末端被酸或碱保护基团保护。此类保护基团应具有对肽连接形成条件稳定的特性，同时可容易地去除而不破坏生长的肽链或使其中所含的任何手性中心外消旋化。合适的保护基团是9-芴基甲氧羰基(Fmoc)、叔丁氧羰基(Boc)、苄氧羰基(Cbz)、联苯基异丙氧羰基、叔戊氧羰基、异冰片基氧羰基、α,α-二甲基-3,5-二甲氧基苄氧羰基、邻硝基苯基亚磺酰基、2-氰基-叔丁氧羰基等。9-芴基甲氧羰基(Fmoc)保护基团对于所公开化合物的合成是特别优选的。对于侧链氨基基团如赖氨酸和精氨酸，其他优选的侧链保护基团是2,2,5,7,8-五甲基色满-6-磺酰基(pmc)、硝基、对甲苯磺酰基、4-甲氧基苯磺酰基、Cbz、Boc和金刚烷氧基羰基；对于酪氨酸是苄基、邻溴苄氧基-羰基、2,6-二氯苄基、异丙基、叔丁基(t-Bu)、环己基、环戊基和乙酰基(Ac)；对于丝氨酸是叔丁基、苄基和四氢吡喃基；对于组氨酸是三苯甲基、苄基、Cbz、对甲苯磺酰基和2,4-二硝基苯基；对于色氨酸是甲酰基；对于天冬氨酸和谷氨酸是苄基和叔丁基，并且对于半胱氨酸是三苯基甲基(三苯甲基)。The disclosed compounds can be prepared by solid phase peptide synthesis, wherein the amino acid α-N-terminus is protected by an acid or base protecting group. Such protecting groups should have the property of being stable to the peptide connection forming conditions, while being easily removable without destroying the growing peptide chain or racemizing any chiral center contained therein. Suitable protecting groups are 9-fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, tert-pentyloxycarbonyl, isobornyloxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfinyl, 2-cyano-tert-butyloxycarbonyl, and the like. The 9-fluorenylmethoxycarbonyl (Fmoc) protecting group is particularly preferred for the synthesis of the disclosed compounds. For side chain amino groups such as lysine and arginine, other preferred side chain protecting groups are 2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc), nitro, p-toluenesulfonyl, 4-methoxybenzenesulfonyl, Cbz, Boc and adamantyloxycarbonyl; for tyrosine, benzyl, o-bromobenzyloxy-carbonyl, 2,6-dichlorobenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopentyl and acetyl (Ac); for serine, t-butyl, benzyl and tetrahydropyranyl; for histidine, trityl, benzyl, Cbz, p-toluenesulfonyl and 2,4-dinitrophenyl; for tryptophan, formyl; for aspartic acid and glutamic acid, benzyl and t-butyl, and for cysteine, triphenylmethyl (trityl).
在固相肽合成方法中，将α-C-末端氨基酸附接到合适的固体支持物或树脂上。可用于上述合成的合适固体支持物是对逐步缩合-脱保护反应的试剂和反应条件呈惰性以及不溶于所用介质的那些材料。用于合成α-C-末端羧基肽的固体支持物是可购自AppliedBiosystems(Foster City,Calif.)的4-羟甲基苯氧基甲基-共聚(苯乙烯-1％二乙烯基苯)或4-(2',4'-二甲氧基苯基-Fmoc-氨基甲基)苯氧基乙酰胺基乙基树脂。α-C-末端氨基酸通过N,N'-二环己基碳二亚胺(DCC)、N,N'-二异丙基碳二亚胺(DIC)或O-苯并三唑-1-基-N,N,N',N'-四甲基脲六氟磷酸盐(HBTU)，在有或没有4-二甲基氨基吡啶(DMAP)、1-羟基苯并三唑(HOBT)、苯并三唑-1-基氧基-三(二甲基氨基)六氟磷酸盐(BOP)或双(2-氧代-3-唑烷基)氯化膦(BOPCl)的情况下，在10℃至50℃的温度下在溶剂诸如二氯甲烷或DMF中介导偶联约1至约24小时而偶联到树脂上。当固体支持物是4-(2',4'-二甲氧基苯基-Fmoc-氨基甲基)苯氧基-乙酰氨基乙基树脂时，在如上所述与α-C-末端氨基酸偶联之前，用仲胺(优选地哌啶)切割Fmoc基团。一种用于与脱保护的4-(2',4'-二甲氧基苯基-Fmoc-氨基甲基)苯氧基-乙酰氨基乙基树脂偶联的方法是在DMF中的O-苯并三唑-1-基-N,N,N',N'-四甲基脲六氟磷酸盐(HBTU，1当量)和1-羟基苯并三唑(HOBT，1当量)。可在自动多肽合成仪中进行连续的受保护氨基酸的偶联。在一个示例中，用Fmoc保护生长肽链的氨基酸中的α-N-末端。从生长肽的α-N-末端侧去除Fmoc保护基团是通过用仲胺(优选地哌啶)处理完成的。然后以约3倍摩尔过量引入每个受保护的氨基酸，并且优选地在DMF中进行偶联。偶联剂可以是O-苯并三唑-1-基-N,N,N',N'-四甲基脲六氟磷酸盐(HBTU，1当量)和1-羟基苯并三唑(HOBT，1当量)。在固相合成结束时，将多肽从树脂上去除并脱保护，连续地或在单一操作中进行。通过用包含茴香硫醚、水、乙二硫醇和三氟乙酸的切割试剂处理树脂结合的多肽，可在单一操作中完成多肽的去除和脱保护。在多肽的α-C-末端是烷基酰胺的情况下，通过用烷基胺氨解来切割树脂。另选地，可通过酯交换(例如，用甲醇)、接着氨解或通过直接转酰胺基来去除肽。受保护的肽可在此时纯化或直接用于下一步骤。侧链保护基团的去除可使用上述切割混合物来完成。完全脱保护的肽可通过使用任何或所有以下类型的一系列色谱步骤来纯化：在弱碱性树脂(乙酸盐形式)上的离子交换；在未衍生的聚苯乙烯-二乙烯基苯(例如，Amberlite XAD)上的疏水吸附色谱法；硅胶吸附色谱法；羧甲基纤维素上的离子交换色谱法；分配色谱法(例如，在Sephadex G-25、LH-20上)或逆流分布；高效液相色谱法(HPLC)，特别是在辛基-十八烷基甲硅烷基-二氧化硅键合相柱填料上的反相HPLC。In the solid phase peptide synthesis method, the α-C-terminal amino acid is attached to a suitable solid support or resin. Suitable solid supports that can be used for the above synthesis are those materials that are inert to the reagents and reaction conditions of the stepwise condensation-deprotection reaction and are insoluble in the medium used. The solid support used for the synthesis of α-C-terminal carboxyl peptides is 4-hydroxymethylphenoxymethyl-co-(styrene-1% divinylbenzene) or 4-(2',4'-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resin, which can be purchased from Applied Biosystems (Foster City, Calif.). The α-C-terminal amino acid was quantified by N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), or O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) in the presence or absence of 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBT), benzotriazol-1-yloxy-tris(dimethylamino)pyridine (DMAP), and 1-hydroxybenzotriazole (HOBT). Hexafluorophosphate (BOP) or bis(2-oxo-3- In the case of oxazolidinyl) phosphine chloride (BOPCl), the coupling is mediated in a solvent such as dichloromethane or DMF at a temperature of 10°C to 50°C for about 1 to about 24 hours to couple to the resin. When the solid support is 4-(2', 4'-dimethoxyphenyl-Fmoc-aminomethyl) phenoxy-acetamidoethyl resin, the Fmoc group is cleaved with a secondary amine (preferably piperidine) before coupling with the α-C-terminal amino acid as described above. A method for coupling with deprotected 4-(2', 4'-dimethoxyphenyl-Fmoc-aminomethyl) phenoxy-acetamidoethyl resin is O-benzotriazole-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, 1 equivalent) and 1-hydroxybenzotriazole (HOBT, 1 equivalent) in DMF. The coupling of consecutive protected amino acids can be performed in an automatic peptide synthesizer. In one example, the α-N-terminus in the amino acid of the growing peptide chain is protected with Fmoc. Removal of the Fmoc protecting group from the α-N-terminal side of the growing peptide is accomplished by treatment with a secondary amine (preferably piperidine). Each protected amino acid is then introduced in about a 3-fold molar excess, and coupling is preferably performed in DMF. The coupling agent can be O-benzotriazole-1-yl-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU, 1 equivalent) and 1-hydroxybenzotriazole (HOBT, 1 equivalent). At the end of solid phase synthesis, the polypeptide is removed from the resin and deprotected, either continuously or in a single operation. The removal and deprotection of the polypeptide can be accomplished in a single operation by treating the resin-bound polypeptide with a cutting reagent comprising thioanisole, water, ethanedithiol and trifluoroacetic acid. In the case where the α-C-terminus of the polypeptide is an alkylamide, the resin is cut by aminolysis with an alkylamine. Alternatively, the peptide can be removed by transesterification (e.g., with methanol), followed by aminolysis or by direct transamidation. The protected peptide can be purified at this time or used directly in the next step. The removal of side chain protecting groups can be accomplished using the above-described cleavage mixture. The fully deprotected peptide can be purified by a series of chromatographic steps using any or all of the following types: ion exchange on a weakly basic resin (acetate form); hydrophobic adsorption chromatography on underivatized polystyrene-divinylbenzene (e.g., Amberlite XAD); silica gel adsorption chromatography; ion exchange chromatography on carboxymethyl cellulose; partition chromatography (e.g., on Sephadex G-25, LH-20) or countercurrent distribution; high performance liquid chromatography (HPLC), particularly reversed phase HPLC on octyl-octadecylsilyl-silica bonded phase column packing.
上述聚合物诸如PEG基团可以在任何合适的条件下附接到寡核苷酸，诸如AC。可使用本领域已知的任何方法，包括经由酰化、还原烷基化、迈克尔加成、硫醇烷基化或通过PEG部分上的反应性基团(例如，醛、氨基、酯、硫醇、α-卤代乙酰基、马来酰亚胺基或肼基)与AC上的反应性基团(例如，醛、氨基、酯、硫醇、α-卤代乙酰基、马来酰亚胺基或肼基)的其他化学选择性缀合/连接方法。可用于将水溶性聚合物连接到一种或多种蛋白质的活化基团包括但不限于砜、马来酰亚胺、巯基、硫醇、三氟甲磺酸根、三氟乙磺酸根、azidirine、环氧乙烷、5-吡啶基和α-卤代酰基(例如，α-碘乙酸、α-溴乙酸、α-氯乙酸)。如果通过还原烷基化附接到AC，则所选择的聚合物应具有单一反应性醛，从而控制聚合度。参见例如Kinstler等人，Adv.Drug.Delivery Rev.(2002),54:477-485；Roberts等人，Adv.Drug Delivery Rev.(2002),54:459-476；以及Zalipsky等人，Adv.Drug Delivery Rev.(1995),16:157-182。The above polymers, such as PEG groups, can be attached to oligonucleotides, such as AC, under any suitable conditions. Any method known in the art can be used, including other chemical selective conjugation/linking methods via acylation, reductive alkylation, Michael addition, thiol alkylation, or through reactive groups on the PEG portion (e.g., aldehyde, amino, ester, thiol, α-haloacetyl, maleimido, or hydrazine) and reactive groups on AC (e.g., aldehyde, amino, ester, thiol, α-haloacetyl, maleimido, or hydrazine). Activated groups that can be used to attach water-soluble polymers to one or more proteins include, but are not limited to, sulfone, maleimide, sulfhydryl, thiol, triflate, trifluoroethanesulfonate, azidirine, ethylene oxide, 5-pyridyl, and α-haloacyl (e.g., α-iodoacetic acid, α-bromoacetic acid, α-chloroacetic acid). If attached to AC by reductive alkylation, the polymer selected should have a single reactive aldehyde to control the degree of polymerization. See, e.g., Kinstler et al., Adv. Drug. Delivery Rev. (2002), 54:477-485; Roberts et al., Adv. Drug Delivery Rev. (2002), 54:459-476; and Zalipsky et al., Adv. Drug Delivery Rev. (1995), 16:157-182.
为了将AC或接头直接共价连接到CPP，CPP的合适当氨基酸残基可以与有机衍生剂反应，所述有机衍生剂能够与氨基酸的所选侧链或N-末端或C-末端反应。肽或缀合物部分上的反应性基团包括例如醛、氨基、酯、硫醇、α-卤代乙酰基、马来酰亚胺基或肼基。衍生剂包括例如马来酰亚胺苯甲酰基磺基琥珀酰亚胺酯(通过半胱氨酸残基缀合)、N-羟基琥珀酰亚胺(通过赖氨酸残基)、戊二醛、琥珀酸酐或本领域已知的其他试剂。In order to covalently link the AC or linker directly to the CPP, the appropriate amino acid residues of the CPP can be reacted with an organic derivatizing agent that is capable of reacting with the selected side chain or N-terminus or C-terminus of the amino acid. Reactive groups on the peptide or conjugate moiety include, for example, aldehydes, amino groups, esters, thiols, α-haloacetyl groups, maleimido groups, or hydrazines. Derivatizing agents include, for example, maleimidobenzoyl sulfosuccinimide esters (conjugated via cysteine residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, or other reagents known in the art.
制备AC和将AC与线性CPP缀合的方法一般描述于美国公开号2018/0298383中，该公开以引用方式并入本文用于所有目的。所述方法可应用于本文公开的环状CPP。Methods for preparing AC and conjugating AC to linear CPPs are generally described in U.S. Publication No. 2018/0298383, which is incorporated herein by reference for all purposes. The methods can be applied to the cyclic CPPs disclosed herein.
合成方案提供于图5A至图5D和图6中。Synthetic schemes are provided in FIGS. 5A-5D and 6 .
包含CPP和可用于与AC缀合的反应性基团的化合物的非限制性示例示于表6中。还示出了示例性接头基团。反应性基团的示例包括四氟苯基酯(TFP)、游离羧酸(COOH)和叠氮化物(N3)。在表6中，n是0至20的整数；Pipa6是AcRXRRBRRXRYQFLIRXRBRXRB，其中B是β-丙氨酸，并且X是氨基己酸；Dap是2,3-二氨基丙酸；NLS是核定位序列；βA是β丙氨酸；-ss-是二硫化物；PABC是聚(A)结合蛋白C-末端结构域；Cx是长度x的烷基链，其中x是某一数字；并且BCN是双环[6.1.0]壬炔。Non-limiting examples of compounds comprising CPP and reactive groups that can be used for conjugation with AC are shown in Table 6. Exemplary linker groups are also shown. Examples of reactive groups include tetrafluorophenyl esters (TFP), free carboxylic acids (COOH), and azides (N3 ). In Table 6, n is an integer from 0 to 20; Pipa6 is AcRXRRBRRXRYQFLIRXRBRXRB, where B is β-alanine and X is aminocaproic acid; Dap is 2,3-diaminopropionic acid; NLS is a nuclear localization sequence; βA is β-alanine; -ss- is a disulfide; PABC is a poly(A) binding protein C-terminal domain; Cx is an alkyl chain of length x, where x is a number; and BCN is a bicyclo[6.1.0]nonyne.
表6.包含CPP和反应性基团的化合物Table 6. Compounds containing CPP and reactive groups
在实施方案中，CPP具有可用于与AC缀合的游离羧酸基团。在实施方案中，EEV具有可用于与AC缀合的游离羧酸基团。In embodiments, the CPP has a free carboxylic acid group available for conjugation to AC. In embodiments, the EEV has a free carboxylic acid group available for conjugation to AC.
下面的结构是用于与包含叠氮化物的化合物进行点击反应的经3'环辛炔修饰的PMO：The structure below is a 3' cyclooctyne modified PMO for click reactions with azide containing compounds:
CPP和接头经由酰胺键与AC的3'端缀合的示例性方案如下所示。An exemplary scheme for conjugation of a CPP and a linker to the 3' end of AC via an amide bond is shown below.
CPP和接头经由应变促进的叠氮化物-炔环加成与经3'-环辛炔修饰的PMO缀合的示例性方案如下所示：An exemplary scheme for the conjugation of CPPs and linkers via strain-promoted azide-alkyne cycloaddition with 3'-cyclooctyne-modified PMOs is shown below:
用于将AC和CPP与含有聚乙二醇部分的附加接头连接的缀合化学的示例如下所示：An example of a conjugation chemistry for linking AC and CPP with an additional linker containing a polyethylene glycol moiety is shown below:
将CPP-接头经由应变促进的叠氮化物-炔环加成(点击化学)与经5'-环辛炔修饰的PMO缀合的示例如下所示：An example of conjugation of a CPP-linker via strain-promoted azide-alkyne cycloaddition (click chemistry) to a 5'-cyclooctyne-modified PMO is shown below:
合成寡聚反义化合物的方法是本领域已知的。本公开不受合成AC的方法的限制。在实施方案中，本文提供了具有反应性磷基团的化合物，所述反应性磷基团可用于形成核苷间连接，包括例如磷酸二酯和硫代磷酸酯核苷间连接。前体或反义化合物的制备和/或纯化方法不是对本文提供的组合物或方法的限制。DNA、RNA和反义化合物的合成和纯化方法是本领域技术人员众所周知的。Methods for synthesizing oligomeric antisense compounds are known in the art. The present disclosure is not limited by the methods for synthesizing AC. In embodiments, compounds with reactive phosphorus groups are provided herein that can be used to form internucleoside linkages, including, for example, phosphodiester and thiophosphate internucleoside linkages. The preparation and/or purification methods of precursors or antisense compounds are not limitations of the compositions or methods provided herein. The synthesis and purification methods of DNA, RNA, and antisense compounds are well known to those skilled in the art.
经修饰的和未修饰的核苷的寡聚化可根据DNA(Protocols forOligonucleotides and Analogs，编者Agrawal(1993),Humana Press)和/或RNA(Scaringe,Methods(2001),23,206-217；Gait等人，Applications of Chemicallysynthesized RNA in RNA:Protein Interactions，编者Smith(1998),1-36；Gallo等人，Tetrahedron(2001),57,5707-5713)的文献程序常规地进行。Oligomerization of modified and unmodified nucleosides can be routinely performed according to literature procedures for DNA (Protocols for Oligonucleotides and Analogs, ed. Agrawal (1993), Humana Press) and/or RNA (Scaringe, Methods (2001), 23, 206-217; Gait et al., Applications of Chemicallysynthesized RNA in RNA: Protein Interactions, ed. Smith (1998), 1-36; Gallo et al., Tetrahedron (2001), 57, 5707-5713).
本文提供的反义化合物可通过众所周知的固相合成技术便利地且常规地制备。用于此类合成的设备由若干个供应商出售，包括例如Applied Biosystems(Foster City,CA)。可以附加地或另选地使用本领域已知的用于此类合成的任何其他手段。使用类似技术制备寡核苷酸诸如硫代磷酸酯和烷基化衍生物是众所周知的。本公开不受反义化合物合成方法的限制。Antisense compounds provided herein can be conveniently and routinely prepared by well-known solid phase synthesis techniques. Equipment for such synthesis is sold by several suppliers, including, for example, Applied Biosystems (Foster City, CA). Any other means known in the art for such synthesis can be used additionally or alternatively. It is well known that oligonucleotides such as phosphorothioates and alkylated derivatives are prepared using similar techniques. The present disclosure is not limited by the antisense compound synthesis method.
寡核苷酸纯化和分析的方法是本领域技术人员已知的。分析方法包括毛细管电泳(CE)和电喷雾质谱法。此类合成和分析方法可以在多孔板中进行。本发明的方法不受寡聚物纯化方法的限制。Methods for oligonucleotide purification and analysis are known to those skilled in the art. Analytical methods include capillary electrophoresis (CE) and electrospray mass spectrometry. Such synthesis and analytical methods can be performed in multiwell plates. The method of the present invention is not limited by the oligomer purification method.
疾病disease
在一些实施方案中，可通过施用包含一种或多种本文所述化合物的组合物来治疗、预防或改善各种疾病或病症。在实施方案中，用本公开的组合物治疗、预防或改善的疾病与剪接、蛋白质表达和/或蛋白质活性的调节异常相关。In some embodiments, various diseases or conditions can be treated, prevented or ameliorated by administering a composition comprising one or more compounds described herein. In embodiments, the disease treated, prevented or ameliorated with the compositions of the present disclosure is associated with abnormal regulation of splicing, protein expression and/or protein activity.
在实施方案中，本文公开的化合物用于治疗、预防或改善疾病或病症。可使用本公开的化合物治疗、预防或调节的例示性疾病或病症可包括但不限于癌症，包括例如急性髓系白血病、B细胞白血病/淋巴瘤、膀胱癌、乳腺癌、慢性淋巴细胞白血病、结肠癌、结直肠癌、杜氏肌肉营养不良症、食管鳞状细胞癌、范科尼贫血、胃癌、恶性胶质瘤、肝细胞癌、肺癌、林奇综合征、套细胞淋巴瘤、黑素瘤、鼻咽癌、成神经细胞瘤、卵巢癌、胰腺导管腺癌、增生性病症、前列腺癌和小肠神经内分泌癌；心血管病症，包括例如动脉粥样硬化、心脏肥大、扩张型心肌病、高血压、局部缺血/再灌注损伤、血栓形成(深静脉)和血栓形成(静脉)；先天性异常，包括小眼畸形、苗勒氏发育不全、骨脆性(成骨不全症)和佝偻病；内分泌疾病，包括新生儿糖尿病和2型糖尿病；血液病，包括血小板无力症、α-地中海贫血和β-地中海贫血；免疫病症，包括IPEX综合征、鼻息肉、严重联合免疫缺陷病、系统性红斑狼疮和维斯科特-奥尔德里奇综合征；肺部病症，包括肺纤维化；肌骨骼病症，包括肌纤维化、面肩胛肱肌营养不良、眼咽肌营养不良、强直性肌营养不良和眼咽型肌营养不良；神经病症，包括阿尔茨海默病、肌萎缩性侧索硬化症、焦虑症、法布里病、脆性X综合征、弗里德里希共济失调、亨廷顿病、异染性脑白质营养不良、伪缺乏症、神经精神疾病、帕金森病和自杀行为；应激；泽尔韦格综合征；糖原贮积病，诸如庞贝氏症；以及它们的组合。In an embodiment, the compounds disclosed herein are used to treat, prevent or improve a disease or condition. Exemplary diseases or conditions that can be treated, prevented or regulated using the compounds disclosed herein may include, but are not limited to, cancer, including, for example, acute myeloid leukemia, B-cell leukemia/lymphoma, bladder cancer, breast cancer, chronic lymphocytic leukemia, colon cancer, colorectal cancer, Duchenne muscular dystrophy, esophageal squamous cell carcinoma, Fanconi anemia, gastric cancer, malignant glioma, hepatocellular carcinoma, lung cancer, Lynch syndrome, mantle cell lymphoma, melanoma, nasopharyngeal carcinoma, neuroblastoma, ovarian cancer, pancreatic ductal adenocarcinoma, proliferative disorders, prostate cancer, and small intestinal neuroendocrine cancer; cardiovascular disorders, including, for example, atherosclerosis, cardiac hypertrophy, dilated cardiomyopathy, hypertension, ischemia/reperfusion injury, thrombosis (deep vein) and thrombosis (venous); congenital anomalies, including microphthalmia, Müllerian dysplasia, bone fragility (osteogenesis imperfecta) and rickets endocrine disorders, including neonatal diabetes and type 2 diabetes; hematological disorders, including asthenia thrombocytopenia, alpha-thalassemia, and beta-thalassemia; immune disorders, including IPEX syndrome, nasal polyposis, severe combined immunodeficiency, systemic lupus erythematosus, and Wiskott-Aldrich syndrome; pulmonary disorders, including pulmonary fibrosis; musculoskeletal disorders, including myofibrosis, facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, myotonic dystrophy, and oculopharyngeal muscular dystrophy; neurological disorders, including Alzheimer's disease, amyotrophic lateral sclerosis, anxiety disorders, Fabry disease, fragile X syndrome, Friedrich's ataxia, Huntington's disease, metachromatic leukodystrophy, pseudoasthenia, neuropsychiatric disorders, Parkinson's disease, and suicidal behavior; stress; Zellweger syndrome; glycogen storage diseases, such as Pompe disease; and combinations thereof.
在实施方案中，本文公开的化合物用于治疗、预防或改善与异常基因转录、剪接和/或翻译相关的疾病。在实施方案中，本文公开的化合物用于治疗、预防或改善与异常IRF-5、GYS1和/或DUX4转录、剪接和/或翻译相关的疾病。在实施方案中，本文公开的化合物用于治疗、预防或改善与以下相关的疾病：IRF-5、GYS1和/或DUX4上调；IRF-5、GYS1和/或DUX4多态性；突变体IRF-5、GYS1和/或DUX4的积累；或它们的组合。In an embodiment, the compounds disclosed herein are used to treat, prevent or improve diseases associated with abnormal gene transcription, splicing and/or translation. In an embodiment, the compounds disclosed herein are used to treat, prevent or improve diseases associated with abnormal IRF-5, GYS1 and/or DUX4 transcription, splicing and/or translation. In an embodiment, the compounds disclosed herein are used to treat, prevent or improve diseases associated with: IRF-5, GYS1 and/or DUX4 upregulation; IRF-5, GYS1 and/or DUX4 polymorphisms; accumulation of mutant IRF-5, GYS1 and/or DUX4; or combinations thereof.
糖原贮积病Glycogen storage disease
糖原合成和降解是涉及许多不同酶促反应的多步骤过程。例如，α-葡糖苷酶(GAA)通过切割α-1,4和α-1,6糖苷键催化糖原的水解，从而允许葡萄糖被释放到细胞质中。在不存在GAA的情况下，糖原在各种组织(主要是心肌和骨骼肌)中的溶酶体内积累。由该蛋白质的缺乏引起的病症被称为糖原贮积病(GSD)(Douillard-Guilloux等人，Hum.Mol.Genet.(2010),19(4):684-96)。Glycogen synthesis and degradation is a multi-step process involving many different enzymatic reactions. For example, α-glucosidase (GAA) catalyzes the hydrolysis of glycogen by cutting α-1,4 and α-1,6 glycosidic bonds, allowing glucose to be released into the cytoplasm. In the absence of GAA, glycogen accumulates in lysosomes in various tissues (mainly myocardial and skeletal muscles). The disease caused by the deficiency of this protein is called glycogen storage disease (GSD) (Douillard-Guilloux et al., Hum. Mol. Genet. (2010), 19 (4): 684-96).
GSD是糖原代谢的遗传代谢病症。糖原贮积病有超过12种类型，其基于酶缺乏和受影响的组织(主要是肝脏或肌肉)进行分类。0型GSD是由于糖原合酶的缺乏。I型是由于缺乏葡萄糖-6-磷酸酶a。II型是由于缺乏α-葡糖苷酶(GAA)。III型是由于缺乏糖原脱支酶(GDE)。IV型是由于缺乏糖原分支活性。V型是由于缺乏糖原磷酸化酶的肌同工型(由PYGM编码)。VI型是由于缺乏糖原磷酸化酶的肝脏同工型(由PYGL编码)。几乎没有关于其余GSD的信息，并且若干种以前的GSD已被分类到其他病症中。糖原贮积病的列表提供于表7中(Ellingwood S.等人，(2018),J.Endocrinol.238(3):R131-R141.doi:10.1530/JOE-18-0120)。GSD is a genetic metabolic disorder of glycogen metabolism. Glycogen storage disease has more than 12 types, which are classified based on enzyme deficiency and affected tissues (mainly liver or muscle). Type 0 GSD is due to the lack of glycogen synthase. Type I is due to the lack of glucose-6-phosphatase a. Type II is due to the lack of α-glucosidase (GAA). Type III is due to the lack of glycogen debranching enzyme (GDE). Type IV is due to the lack of glycogen branching activity. Type V is due to the lack of muscle isoforms of glycogen phosphorylase (encoded by PYGM). Type VI is due to the lack of liver isoforms of glycogen phosphorylase (encoded by PYGL). There is almost no information about the rest of GSD, and several previous GSDs have been classified into other diseases. The list of glycogen storage diseases is provided in Table 7 (Ellingwood S. et al., (2018), J.Endocrinol.238 (3): R131-R141.doi: 10.1530 / JOE-18-0120).
表7：糖原贮积病Table 7: Glycogen storage diseases
*OMIM(在线人类孟德尔遗传)；#在早期来源中，GSD XI型与乳酸脱氢酶A(OMIM612933)缺乏相关。*OMIM (Online Mendelian Inheritance in Man); #In early sources, GSD type XI was associated with deficiency of lactate dehydrogenase A (OMIM 612933).
糖原贮积病II型(GSDII)或庞贝氏症是由编码酸性葡糖苷酶α(GAA)的基因中的突变引起的常染色体隐性溶酶体贮积病，所述突变导致对于复合糖、糖原的分解必不可少的GAA蛋白的缺失或缺乏。通常，身体使用GAA分解复杂碳水化合物糖原并将其转化为葡萄糖。无法实现适当的分解和糖原代谢异常导致糖原在身体细胞中，特别是在心肌细胞、平滑肌细胞和骨骼肌细胞中的过度积累，这可导致正常组织和器官功能的损伤和退化。庞贝氏症患者会经历严重的肌相关问题，包括全身的进行性肌无力，尤其是在腿、躯干和横膈膜中。随着病症的发展，呼吸问题可能会导致呼吸衰竭。迄今为止，已经在GAA中鉴定出超过300种致病性突变。在美国和欧洲，普遍估计总共5,000至10,000名患者患有庞贝氏症；然而，新生儿筛查的出现表明该疾病诊断不足。Glycogen storage disease type II (GSDII) or Pompe disease is an autosomal recessive lysosomal storage disease caused by mutations in the gene encoding acid glucosidase alpha (GAA), which results in the absence or deficiency of the GAA protein essential for the breakdown of the complex sugar, glycogen. Normally, the body uses GAA to break down the complex carbohydrate glycogen and convert it into glucose. The inability to achieve proper breakdown and abnormal glycogen metabolism lead to excessive accumulation of glycogen in body cells, especially in cardiomyocytes, smooth muscle cells, and skeletal muscle cells, which can lead to damage and degradation of normal tissue and organ function. Pompe disease patients experience severe muscle-related problems, including progressive muscle weakness throughout the body, especially in the legs, trunk, and diaphragm. As the disease progresses, breathing problems may lead to respiratory failure. To date, more than 300 pathogenic mutations have been identified in GAA. In the United States and Europe, it is generally estimated that a total of 5,000 to 10,000 patients suffer from Pompe disease; however, the advent of newborn screening indicates that the disease is underdiagnosed.
基于症状的发病年龄和严重程度，庞贝氏症通常被分类为婴儿型庞贝氏症(IOPD)或晚发型庞贝氏症(LOPD)。IOPD的特征在于严重的肌无力和异常减少的肌张力，并且通常在生命的前几个月内显现。如果不进行治疗，由于进行性心力衰竭、呼吸窘迫或由喂养困难导致的营养不良，IOPD通常是致命的。LOPD存在于儿童期、青春期或成人期。患有LOPD的患者通常具有较轻微的症状，诸如活动能力降低和呼吸问题。具有LOPD的患者会经历进行性行走困难和呼吸下降。LOPD的初始症状可能是微妙的，并且多年来未被注意到。Pompe disease is usually classified as infantile Pompe disease (IOPD) or late-onset Pompe disease (LOPD) based on the age of onset and the severity of symptoms. IOPD is characterized by severe muscle weakness and abnormally reduced muscle tone, and usually manifests in the first few months of life. If not treated, IOPD is usually fatal due to progressive heart failure, respiratory distress or malnutrition caused by feeding difficulties. LOPD is present in childhood, adolescence or adulthood. Patients with LOPD usually have milder symptoms, such as reduced mobility and breathing problems. Patients with LOPD can experience progressive walking difficulties and respiratory decline. The initial symptoms of LOPD may be subtle and have not been noticed for many years.
在提交时，目前唯一批准的用于庞贝氏症的疗法是阿葡糖苷酶α(在美国为Lumizyme，在其他地区为Myozyme)和阿伐糖苷酶α-ngpt(在美国为Nexviazyme)，它们都是经由IV输注递送的酶替代疗法(ERT)的形式。尽管用ERT治疗庞贝氏症的婴儿患者已显示出改善的生存率，但ERT不是治愈性的，并且在长期观察研究中，许多患者持续具有增加的心肌病和心力衰竭两者的风险。这些患者还会经历残余肌无力，包括吞咽困难和伴随的增加的误吸风险。ERT改善骨骼肌肌病和呼吸功能障碍的能力特别有限，主要是由于其不能穿透受该病影响的关键组织、在细胞溶胶中缺乏活性以及潜在的免疫原性。尽管可获得ERT，但在IOPD或LOPD或的患者中仍然存在显著未满足的医疗需求。At the time of submission, the only currently approved therapies for Pompe disease are alglucosidase alfa (Lumizyme in the United States and Myozyme in other regions) and avaglycosidase alfa-ngpt (Nexviazyme in the United States), both of which are forms of enzyme replacement therapy (ERT) delivered via IV infusion. Although infantile patients with Pompe disease treated with ERT have shown improved survival, ERT is not curative, and in long-term observational studies, many patients continue to have an increased risk of both cardiomyopathy and heart failure. These patients also experience residual muscle weakness, including dysphagia and an accompanying increased risk of aspiration. The ability of ERT to improve skeletal muscle myopathy and respiratory dysfunction is particularly limited, primarily due to its inability to penetrate key tissues affected by the disease, lack of activity in the cytosol, and potential immunogenicity. Despite the availability of ERT, there is still a significant unmet medical need in patients with IOPD or LOPD or.
GAA通过切割α-1,4和α-1,6糖苷键催化糖原的水解，从而允许葡萄糖被释放到细胞质中。在不存在GAA的情况下，糖原在各种组织(主要是心肌和骨骼肌)中的溶酶体内积累。由该蛋白质的缺乏引起的病症被称为糖原贮积病(GSD)(Douillard-Guilloux(2010)Hum.Mol.Genet.19(4):684-96)。GAA catalyzes the hydrolysis of glycogen by cleaving α-1,4 and α-1,6 glycosidic bonds, allowing glucose to be released into the cytoplasm. In the absence of GAA, glycogen accumulates in lysosomes in various tissues (mainly cardiac and skeletal muscle). The condition caused by the deficiency of this protein is called glycogen storage disease (GSD) (Douillard-Guilloux (2010) Hum. Mol. Genet. 19 (4): 684-96).
可治疗糖原贮积病的一种方式是通过下调糖原合成，例如通过下调糖原合酶的表达和/或活性。糖原合酶有两种主要的同功酶，GYS1和GYS2。GYS1在骨骼肌和心肌中遍在表达(NCBI参考2997)。GYS2主要在肝脏和脂肪组织中表达(NCBI基因参考2998)。GYS1用于分解摄入的葡萄糖，为肌肉提供糖原能量储备。相比之下，GYS2用于维持血糖水平。GYS1和GYS2的mRNA的比对显示，两种同工酶的54％共享71％的同源性。One way in which glycogen storage diseases can be treated is by downregulating glycogen synthesis, for example by downregulating the expression and/or activity of glycogen synthase. There are two major isozymes of glycogen synthase, GYS1 and GYS2. GYS1 is ubiquitously expressed in skeletal and cardiac muscle (NCBI reference 2997). GYS2 is primarily expressed in the liver and adipose tissue (NCBI gene reference 2998). GYS1 is used to break down ingested glucose to provide muscle with glycogen energy reserves. In contrast, GYS2 is used to maintain blood glucose levels. An alignment of the mRNAs of GYS1 and GYS2 showed that 54% of the two isozymes shared 71% homology.
已证明，下调糖原合酶(GYS1)表达导致糖原积累的逆转(Douillard-Guilloux等人，Hum.Mol.Genet.(2010),19(4):684-96)。已对GYS1的结构和作用机制进行了综述(Palm,D.C.等人，FEBS(2013),280(1),2-27；以及Baskaran S.等人，Proc.Natl.Acad.Sci.USA(2010)107,17563-17568)。由于GYS1和GYS1的功能差异，重要的是选择性靶向GYS1进行下调。It has been demonstrated that downregulation of glycogen synthase (GYS1) expression results in reversal of glycogen accumulation (Douillard-Guilloux et al., Hum. Mol. Genet. (2010), 19(4): 684-96). The structure and mechanism of action of GYS1 have been reviewed (Palm, D.C. et al., FEBS (2013), 280(1), 2-27; and Baskaran S. et al., Proc. Natl. Acad. Sci. USA (2010) 107, 17563-17568). Due to the functional differences between GYS1 and GYS1, it is important to selectively target GYS1 for downregulation.
在实施方案中，提供了一种用于治疗糖原贮积病的方法。在实施方案中，该方法包括施用下调糖原合成的化合物。在实施方案中，该方法包括施用下调糖原合酶的表达的化合物。在实施方案中，该方法包括施用下调糖原合酶的肌形式(GYS1)的表达的化合物。在实施方案中，所述化合物包含AC。AC可以是任何AC并且具有如本文别处所述的任何AC特征。在实施方案中，AC可结合到如本文别处所述的靶转录物的SE或SRE的至少一部分。在实施方案中，AC可结合到如本文别处所述的靶转录物的SE或SRE附近。在实施方案中，AC是ASO。在实施方案中，ASO是PMO。AC可结合到如本文别处所述的GYS1靶转录物的任何剪接元件。In an embodiment, a method for treating glycogen storage disease is provided. In an embodiment, the method comprises administering a compound that downregulates glycogen synthesis. In an embodiment, the method comprises administering a compound that downregulates the expression of glycogen synthase. In an embodiment, the method comprises administering a compound that downregulates the expression of the muscle form of glycogen synthase (GYS1). In an embodiment, the compound comprises an AC. The AC can be any AC and has any AC characteristics as described elsewhere herein. In an embodiment, the AC can bind to at least a portion of a SE or SRE of a target transcript as described elsewhere herein. In an embodiment, the AC can bind to the vicinity of a SE or SRE of a target transcript as described elsewhere herein. In an embodiment, the AC is an ASO. In an embodiment, the ASO is a PMO. The AC can bind to any splicing element of a GYS1 target transcript as described elsewhere herein.
在实施方案中，提供了一种用于治疗糖原贮积病的方法。在实施方案中，提供了一种用于治疗与肌肉组织中糖原积累相关的糖原贮积病的方法。在实施方案中，提供了一种用于治疗与心肌组织中糖原积累相关的糖原贮积病的方法。在实施方案中，提供了一种用于治疗与骨骼肌组织中糖原积累相关的糖原贮积病的方法。在实施方案中，提供了一种用于治疗II型糖原贮积病的方法。在实施方案中，提供了一种用于治疗庞贝氏症的方法。在实施方案中，提供了一种用于治疗安德森氏症的方法。在实施方案中，提供了一种用于治疗麦卡德尔病的方法。在实施方案中，提供了一种用于治疗拉福拉病的方法。在实施方案中，提供了一种用于治疗塔里乌病(Tariu disease)的方法。In embodiments, a method for treating glycogen storage disease is provided. In embodiments, a method for treating glycogen storage disease associated with glycogen accumulation in muscle tissue is provided. In embodiments, a method for treating glycogen storage disease associated with glycogen accumulation in myocardial tissue is provided. In embodiments, a method for treating glycogen storage disease associated with glycogen accumulation in skeletal muscle tissue is provided. In embodiments, a method for treating type II glycogen storage disease is provided. In embodiments, a method for treating Pompe disease is provided. In embodiments, a method for treating Anderson disease is provided. In embodiments, a method for treating McArdle disease is provided. In embodiments, a method for treating Lafora disease is provided. In embodiments, a method for treating Tariu disease is provided.
在实施方案中，GYS1由编码同种型1或同种型2的核苷酸序列编码。核苷酸序列可通过在线NCBI数据库公开获得(同种型1＝NM_002103.5；同种型2＝NM_001161587.2)。在实施方案中，编码GYS1的核苷酸序列与编码同种型1或同种型2的核苷酸序列的区别在于一个或多个核酸。在实施方案中，编码GYS1的核苷酸序列的区别在于一种或多种多态性(例如，单核苷酸多态性或SNP)。在实施方案中，编码GYS1的核苷酸序列与编码同种型1或同种型2的核苷酸序列共享小于100％的序列同一性，在实施方案中，GYS1由与编码同种型1或同种型2的核酸序列具有至少约80％、约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％同一性的核苷酸序列编码。在实施方案中，GYS1由与编码同种型1或同种型2的核酸序列具有80％至100％、90％至100％、95％至100％、或99％至100％同一性的核苷酸序列编码。In an embodiment, GYS1 is encoded by a nucleotide sequence encoding isoform 1 or isoform 2. The nucleotide sequences are publicly available through the online NCBI database (isoform 1 = NM_002103.5; isoform 2 = NM_001161587.2). In an embodiment, the nucleotide sequence encoding GYS1 differs from the nucleotide sequence encoding isoform 1 or isoform 2 by one or more nucleic acids. In an embodiment, the nucleotide sequence encoding GYS1 differs by one or more polymorphisms (e.g., single nucleotide polymorphisms or SNPs). In embodiments, the nucleotide sequence encoding GYS1 shares less than 100% sequence identity with the nucleotide sequence encoding isoform 1 or isoform 2, in embodiments, GYS1 is encoded by a nucleotide sequence having at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identity with the nucleic acid sequence encoding isoform 1 or isoform 2. In embodiments, GYS1 is encoded by a nucleotide sequence having 80% to 100%, 90% to 100%, 95% to 100%, or 99% to 100% identity with the nucleic acid sequence encoding isoform 1 or isoform 2.
在实施方案中，所述方法包括施用诱导GYS1靶转录物中一个或多个外显子的外显子跳跃的化合物。在实施方案中，所述方法包括施用包含反义化合物(AC)的化合物，所述反义化合物诱导GYS1靶转录物中一个或多个外显子的跳跃。在实施方案中，AC与GYS1转录物的靶核苷酸序列的杂交导致靶转录物中一个或多个外显子的包含或跳跃。在实施方案中，一个或多个外显子的跳跃或包含诱导GYS1靶转录物中的移码。在实施方案中，移码得到编码具有降低活性的糖原合酶的GYS1转录物。在实施方案中，移码得到截短的或非功能性的糖原合酶。在实施方案中，移码导致在GYS1转录物中引入提前终止密码子。在实施方案中，引入提前终止密码子导致GYS1 mRNA转录物通过无义介导的衰变而降解。In embodiments, the method comprises administering a compound that induces exon skipping of one or more exons in a GYS1 target transcript. In embodiments, the method comprises administering a compound comprising an antisense compound (AC) that induces skipping of one or more exons in a GYS1 target transcript. In embodiments, hybridization of AC with a target nucleotide sequence of a GYS1 transcript results in inclusion or skipping of one or more exons in the target transcript. In embodiments, skipping or inclusion of one or more exons induces a frameshift in a GYS1 target transcript. In embodiments, the frameshift results in a GYS1 transcript encoding a glycogen synthase with reduced activity. In embodiments, the frameshift results in a truncated or non-functional glycogen synthase. In embodiments, the frameshift results in the introduction of a premature stop codon in a GYS1 transcript. In embodiments, the introduction of a premature stop codon results in degradation of the GYS1 mRNA transcript by nonsense-mediated decay.
在实施方案中，化合物包含诱导人和/或小鼠GYS1的外显子2、3、4、5、6、7、8、9、10、11、12、13、14或15中的一者或多者的跳跃的反义化合物(AC)。在实施方案中，化合物包含诱导一个或多个外显子跳跃以产生框外移码的AC，从而导致GYS1靶转录物被降解(例如，无义介导的衰变)或被翻译成具有降低活性或无活性的GYS1蛋白。在实施方案中，化合物包含诱导外显子2、5、6、7、8、10、12和/或14中的一者或多者的跳跃以产生框外移码的AC。在实施方案中，化合物包含诱导一个或多个外显子的跳跃以在GYS1靶转录物中产生框内缺失的AC。在实施方案中，化合物包含诱导外显子3、4、9、11、13和/或15中的一者或多者的跳跃的AC。在实施方案中，化合物包含诱导外显子3、4、9、11、13和/或15中的一者或多者的跳跃以在GYS1靶转录物中产生框内缺失的AC。In an embodiment, the compound comprises an antisense compound (AC) that induces the skipping of one or more of exons 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of human and/or mouse GYS1. In an embodiment, the compound comprises an AC that induces the skipping of one or more exons to produce an out-of-frame frameshift, thereby causing the GYS1 target transcript to be degraded (e.g., nonsense-mediated decay) or translated into a GYS1 protein with reduced activity or no activity. In an embodiment, the compound comprises an AC that induces the skipping of one or more of exons 2, 5, 6, 7, 8, 10, 12, and/or 14 to produce an out-of-frame frameshift. In an embodiment, the compound comprises an AC that induces the skipping of one or more exons to produce an in-frame deletion in the GYS1 target transcript. In an embodiment, the compound comprises an AC that induces the skipping of one or more exons 3, 4, 9, 11, 13, and/or 15. In embodiments, the compound comprises an AC that induces skipping of one or more of exons 3, 4, 9, 11, 13, and/or 15 to generate an in-frame deletion in the GYS1 target transcript.
在实施方案中，化合物包含结合一个或多个外显子/内含子和/或内含子/外显子连接以诱导外显子跳跃的AC。在实施方案中，AC化合物包含表8中的下列序列中的任一个，其中大写字母表示外显子核苷酸，小写字母表示内含子核苷酸。在表8中，SEQ ID NO:151-247被设计成诱导外显子跳跃以产生移码改变。在实施方案中，移码改变得到提前终止密码子。在实施方案中，移码改变导致GYS1靶转录物的无义介导的衰变。在表8中，SEQ ID NO:249-318被设计成诱导外显子跳跃以产生框内缺失。表8中列出的AC被设计成结合包含外显子、外显子/内含子连接和/或内含子/外显子连接的靶核苷酸序列。In an embodiment, the compound comprises an AC that binds to one or more exons/introns and/or intron/exon junctions to induce exon skipping. In an embodiment, the AC compound comprises any one of the following sequences in Table 8, wherein capital letters represent exon nucleotides and lowercase letters represent intron nucleotides. In Table 8, SEQ ID NOs: 151-247 are designed to induce exon skipping to produce frameshift changes. In an embodiment, frameshift changes result in premature termination codons. In an embodiment, frameshift changes result in nonsense-mediated decay of GYS1 target transcripts. In Table 8, SEQ ID NOs: 249-318 are designed to induce exon skipping to produce in-frame deletions. The ACs listed in Table 8 are designed to bind to target nucleotide sequences comprising exons, exon/intron junctions and/or intron/exon junctions.
表8：靶向GYS1的各种AC序列Table 8: Various AC sequences targeting GYS1
在一些实施方案中，AC包含来自美国申请号16/867,261和/或Clayton等人，Molecular Therapy-Nucleic Acids(2014)3,e206的PMO序列，如表9中列出的那些，或其一部分。In some embodiments, the AC comprises a PMO sequence from U.S. Application No. 16/867,261 and/or Clayton et al., Molecular Therapy-Nucleic Acids (2014) 3, e206, such as those listed in Table 9, or a portion thereof.
PMO序列被设计成诱导外显子跳跃，从而导致移码改变。在实施方案中，移码改变得到提前终止密码子，其导致GYS1靶转录物的无义介导的衰变。SEQ ID NO:321-327被设计成结合包含GYS1靶转录物的内含子/外显子和/或外显子/内含子连接的靶核苷酸序列。SEQID NO:319和327被设计成结合包含靶GYS1转录物的内含子序列的靶核苷酸序列。The PMO sequence is designed to induce exon skipping, resulting in a frameshift change. In embodiments, the frameshift change results in a premature stop codon, which results in nonsense-mediated decay of the GYS1 target transcript. SEQ ID NOs: 321-327 are designed to bind to target nucleotide sequences comprising intron/exon and/or exon/intron junctions of the GYS1 target transcript. SEQ ID NOs: 319 and 327 are designed to bind to target nucleotide sequences comprising intron sequences of the target GYS1 transcript.
表9：靶向GYS1的各种AC序列Table 9: Various AC sequences targeting GYS1
在实施方案中，AC包含表8和/或表9中任一序列的10个或更多个、15个或更多个、或20个或更多个连续碱基。在实施方案中，AC包含表8和/或表9中任一序列的25个或更少个、20个或更少个、或15个或更少个连续碱基。在实施方案中，AC包含表8和/或表9中任一序列的10个至25个、10个至20个、或10个至15个连续碱基。在实施方案中，AC包含表8和/或表9中任一序列的15个至25个或15个至20个连续碱基。在实施方案中，AC包含表8和/或表9中任一序列的20-25个连续碱基。In embodiments, AC comprises 10 or more, 15 or more, or 20 or more consecutive bases of any sequence in Table 8 and/or Table 9. In embodiments, AC comprises 25 or less, 20 or less, or 15 or less consecutive bases of any sequence in Table 8 and/or Table 9. In embodiments, AC comprises 10 to 25, 10 to 20, or 10 to 15 consecutive bases of any sequence in Table 8 and/or Table 9. In embodiments, AC comprises 15 to 25 or 15 to 20 consecutive bases of any sequence in Table 8 and/or Table 9. In embodiments, AC comprises 20-25 consecutive bases of any sequence in Table 8 and/or Table 9.
在实施方案中，将使用小鼠GYS1的小鼠模型用于研究诱导GYS1靶转录物中的外显子跳跃的化合物的作用。小鼠和人GYS1二在19号染色体上具有97％同源性。另外，小鼠和人GYS1都具有16个外显子和相同的剪接模式，得到737个氨基酸长的全长蛋白质。In an embodiment, a mouse model using mouse GYS1 is used to study the effects of compounds that induce exon skipping in the GYS1 target transcript. Mouse and human GYS1 share 97% homology on chromosome 19. In addition, both mouse and human GYS1 have 16 exons and identical splicing patterns, resulting in a full-length protein of 737 amino acids.
在实施方案中，化合物包含通过靶向其起始密码子而诱导人和/或小鼠GYS1下调的反义化合物(AC)。此类序列的示例包括表10中的那些。In an embodiment, the compound comprises an antisense compound (AC) that induces downregulation of human and/or mouse GYS1 by targeting its start codon. Examples of such sequences include those in Table 10.
表10：靶向GYS1的各种AC序列Table 10: Various AC sequences targeting GYS1
干扰素调节因子5(IRF-5)Interferon regulatory factor 5 (IRF-5)
在实施方案中，提供了用于调节干扰素调节因子5(IRF-5)的活性的化合物。IRF-5是IRF转录因子家族的成员，其在单核细胞、巨噬细胞、B细胞和树突状细胞中高度表达，并且其表达可以在其他类型的细胞中被I型干扰素诱导(Almuttaqi和Udalova,FEBS J.(2018),286:1624-1637)。IRF-5参与先天和适应性免疫、抗病毒防御、促炎性细胞因子的产生、巨噬细胞极化、细胞生长调节以及分化和凋亡。In embodiments, there is provided a compound for regulating the activity of interferon regulatory factor 5 (IRF-5). IRF-5 is a member of the IRF transcription factor family, which is highly expressed in monocytes, macrophages, B cells and dendritic cells, and its expression can be induced by type I interferon in other types of cells (Almuttaqi and Udalova, FEBS J. (2018), 286: 1624-1637). IRF-5 is involved in innate and adaptive immunity, antiviral defense, the production of proinflammatory cytokines, macrophage polarization, cell growth regulation and differentiation and apoptosis.
异常IRF-5表达与多种疾病相关。此外，增加的IRF5 mRNA水平与疾病病理高度相关。例如，IRF-5的上调可导致IFN的产生增加，这与多种炎性疾病的发展有关，所述炎性疾病包括自身免疫疾病、感染性疾病、癌症、肥胖症、神经病理性疼痛、心血管疾病(例如，动脉粥样硬化)和代谢功能障碍(Banga等人，Sci.Adv.(2020),6:eaay1057)。另外，与较高的IRF-5表达相关的IRF-5基因多态性与对炎性和自身免疫性疾病的易感性相关，所述炎性和自身免疫性疾病包括类风湿性关节炎(RA)、炎性肠病(IBD)、多发性硬化症(MS)、炎性肠病(IBD)、系统性红斑狼疮(SLE)和舍格伦综合征(Almuttaqi和Udalova(2018)FEBS J.286:1624-1637；Thompson等人，Front.Immunol.,2018,9:2622；Ban等人，InternationalImmunology(2018),30,11:529-536；Chehimi等人，J.Clin.Med.(2017),6,712,doi.org/10.3390/jcm6070068)。此外，IRF-5参与I型干扰素和Toll样受体信号传导通路，并且是细胞因子表达的下游调节剂(Krisjansdottir等人，J.Med.Genet.(2008),45:362-369)。Abnormal IRF-5 expression is associated with a variety of diseases. In addition, increased IRF5 mRNA levels are highly correlated with disease pathology. For example, the upregulation of IRF-5 can lead to increased production of IFN, which is related to the development of a variety of inflammatory diseases, including autoimmune diseases, infectious diseases, cancer, obesity, neuropathic pain, cardiovascular diseases (e.g., atherosclerosis) and metabolic dysfunction (Banga et al., Sci. Adv. (2020), 6: eaay1057). In addition, IRF-5 gene polymorphisms associated with higher IRF-5 expression are associated with susceptibility to inflammatory and autoimmune diseases, including rheumatoid arthritis (RA), inflammatory bowel disease (IBD), multiple sclerosis (MS), inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), and Sjögren's syndrome (Almuttaqi and Udalova (2018) FEBS J. 286: 1624-1637; Thompson et al., Front. Immunol., 2018, 9: 2622; Ban et al., International Immunology (2018), 30, 11: 529-536; Chehimi et al., J. Clin. Med. (2017), 6, 712, doi.org/10.3390/jcm6070068). Furthermore, IRF-5 is involved in type I interferon and Toll-like receptor signaling pathways and is a downstream regulator of cytokine expression (Krisjansdottir et al., J. Med. Genet. (2008), 45:362-369).
IRF-5以由三个另选的非编码5'外显子和至少九个选择性剪接的mRNA生成的多种同种型存在。IRF-5同种型的序列可例如通过在线NCBI数据库公开获得。同种型显示出细胞类型特异性表达、亚细胞定位和功能。一些同种型与自身免疫疾病的风险相关。例如，同种型2与IRF-5的过表达和对自身免疫疾病诸如系统性红斑狼疮的易感性有关。另外，导致更高mRNA表达的多态性(包括编码IRF-5的基因中的单核苷酸多态性)与许多自身免疫疾病相关(Krausgruber等人，Nat.Immunol.(2010),12(3):231-238；Kozyrev等人，Arthritis andRheumatology(2007),56(4):1234-1241)。IRF-5 exists in multiple isoforms generated by three alternative non-coding 5' exons and at least nine alternatively spliced mRNAs. The sequence of IRF-5 isoforms can be publicly obtained, for example, through the online NCBI database. Isoforms show cell type-specific expression, subcellular localization and function. Some isoforms are associated with the risk of autoimmune diseases. For example, isoform 2 is related to the overexpression of IRF-5 and the susceptibility to autoimmune diseases such as systemic lupus erythematosus. In addition, polymorphisms (including single nucleotide polymorphisms in genes encoding IRF-5) that lead to higher mRNA expression are associated with many autoimmune diseases (Krausgruber et al., Nat. Immunol. (2010), 12 (3): 231-238; Kozyrev et al., Arthritis and Rheumatology (2007), 56 (4): 1234-1241).
已对IRF-5活化、作用机制、信号传导通路和调控元件进行了综述(Song等人，J.Clin.Invest.(2020),130(12):6700-6717；Almutaqqi和Udalova FEBS J.(2018),286:1624-1637；Banga等人，Sci.Adv.(2020),6:eaay1057；Thompson等人，Front.Immunol.(2018),9:2622)。IRF-5 activation, mechanism of action, signaling pathways, and regulatory elements have been reviewed (Song et al., J. Clin. Invest. (2020), 130(12):6700-6717; Almutaqqi and Udalova FEBS J. (2018), 286:1624-1637; Banga et al., Sci. Adv. (2020), 6:eaay1057; Thompson et al., Front. Immunol. (2018), 9:2622).
编码IRF-5的基因包含9个外显子(外显子1、外显子2、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8和外显子9)。外显子1位于5'-非翻译区(5'-UTR)，并且具有三个变体，外显子1A、外显子1B、外显子1C和外显子1D。主要同种型包含外显子1A。外显子1B与IRF-5超活化和疾病进展相关。引入供体剪接位点的单核苷酸多态性(SNP)(例如，rs2004640)可导致增加的外显子1B转录物表达和减少的外显子1C衍生的转录物表达。其他SNP(例如，rs2280714)也与升高的IRF-5表达相关(Kozyrev等人，Arthritis andRheumatology(2007),56(4):1234-1241)。The gene encoding IRF-5 comprises 9 exons (exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8 and exon 9). Exon 1 is located in the 5'-untranslated region (5'-UTR) and has three variants, exon 1A, exon 1B, exon 1C and exon 1D. The major isoform comprises exon 1A. Exon 1B is associated with IRF-5 hyperactivation and disease progression. The single nucleotide polymorphism (SNP) (e.g., rs2004640) introduced into the donor splice site can lead to increased exon 1B transcript expression and reduced exon 1C derived transcript expression. Other SNPs (eg, rs2280714) are also associated with elevated IRF-5 expression (Kozyrev et al., Arthritis and Rheumatology (2007), 56(4): 1234-1241).
下面提供了IRF-5的六个同种型。The six isoforms of IRF-5 are provided below.
人干扰素调节因子5(IRF-5)(同种型1)Human interferon regulatory factor 5 (IRF-5) (isoform 1)
人干扰素调节因子5(IRF-5)(同种型2)Human interferon regulatory factor 5 (IRF-5) (isoform 2)
人干扰素调节因子5(IRF-5)(同种型3)Human interferon regulatory factor 5 (IRF-5) (isoform 3)
人干扰素调节因子5(IRF-5)(同种型4)Human interferon regulatory factor 5 (IRF-5) (isoform 4)
人干扰素调节因子5(IRF-5)(同种型5)Human interferon regulatory factor 5 (IRF-5) (isoform 5)
人干扰素调节因子5(IRF-5)(同种型6)Human interferon regulatory factor 5 (IRF-5) (isoform 6)
在实施方案中，IRF-5由编码IRF-5同种型1、IRF-5同种型2、IRF-5同种型3、IRF-5同种型4、IRF-5同种型5或IRF-5同种型6的核苷酸序列编码。在实施方案中，编码IRF-5的核苷酸序列与编码IRF-5同种型1、IRF-5同种型2、IRF-5同种型3、IRF-5同种型4、IRF-5同种型5或IRF-5同种型6的核苷酸序列的区别在于一个或多个核酸。在实施方案中，编码IRF-5的核苷酸序列的区别在于一种或多种多态性(例如，单核苷酸多态性或SNP)。在实施方案中，编码IRF-5的核苷酸序列与编码IRF-5同种型1、IRF-5同种型2、IRF-5同种型3、IRF-5同种型4、IRF-5同种型5或IRF-5同种型6的核苷酸序列共享小于100％的序列同一性。在实施方案中，IRF-5由与编码IRF-5同种型1、IRF-5同种型2、IRF-5同种型3、IRF-5同种型4、IRF-5同种型5或IRF-5同种型6的核酸序列具有至少约80％、约85％、约90％、约91％、约92％、约93％、约94％、约95％、约96％、约97％、约98％或约99％同一性的核苷酸序列编码。在实施方案中，IRF-5由与编码IRF-5同种型1、IRF-5同种型2、IRF-5同种型3、IRF-5同种型4、IRF-5同种型5或IRF-5同种型6的核酸序列具有80％至100％、90％至100％、95％至100％、或99％至100％同一性的核苷酸序列编码。In embodiments, IRF-5 is encoded by a nucleotide sequence encoding IRF-5 isoform 1, IRF-5 isoform 2, IRF-5 isoform 3, IRF-5 isoform 4, IRF-5 isoform 5, or IRF-5 isoform 6. In embodiments, the nucleotide sequence encoding IRF-5 differs from the nucleotide sequence encoding IRF-5 isoform 1, IRF-5 isoform 2, IRF-5 isoform 3, IRF-5 isoform 4, IRF-5 isoform 5, or IRF-5 isoform 6 by one or more nucleic acids. In embodiments, the nucleotide sequence encoding IRF-5 differs by one or more polymorphisms (e.g., single nucleotide polymorphisms or SNPs). In embodiments, the nucleotide sequence encoding IRF-5 shares less than 100% sequence identity with a nucleotide sequence encoding IRF-5 isoform 1, IRF-5 isoform 2, IRF-5 isoform 3, IRF-5 isoform 4, IRF-5 isoform 5, or IRF-5 isoform 6. In embodiments, IRF-5 is encoded by a nucleotide sequence that is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to a nucleic acid sequence encoding IRF-5 isoform 1, IRF-5 isoform 2, IRF-5 isoform 3, IRF-5 isoform 4, IRF-5 isoform 5, or IRF-5 isoform 6. In an embodiment, IRF-5 is encoded by a nucleotide sequence that is 80% to 100%, 90% to 100%, 95% to 100%, or 99% to 100% identical to a nucleic acid sequence encoding IRF-5 isoform 1, IRF-5 isoform 2, IRF-5 isoform 3, IRF-5 isoform 4, IRF-5 isoform 5, or IRF-5 isoform 6.
IRF-5已被证明能够影响炎性巨噬细胞表型(Almuttaqi和Udalova,FEBS J.(2018),286:1624-1637)。巨噬细胞可分类为M1(经典活化的巨噬细胞)或M2(替代性活化的巨噬细胞)，并且可根据组织微环境相互转化。有三类替代性活化的巨噬细胞(M2a、M2b和M2c)。在正常组织中，M1与M2巨噬细胞的比率受到高度调节。M1和M2巨噬细胞之间的不平衡可导致诸如哮喘、慢性肺病、动脉粥样硬化或类风湿性关节炎中的破骨细胞生成的病状。IRF-5是促炎M1巨噬细胞极化的主要调节因子(Weiss等人Mediators of Inflammation(2013)Dx.doi.org/10.1155/2013/245804)。IRF-5 has been shown to affect inflammatory macrophage phenotype (Almuttaqi and Udalova, FEBS J. (2018), 286: 1624-1637). Macrophages can be classified as M1 (classically activated macrophages) or M2 (alternatively activated macrophages), and can be transformed into each other according to the tissue microenvironment. There are three types of alternatively activated macrophages (M2a, M2b and M2c). In normal tissues, the ratio of M1 to M2 macrophages is highly regulated. An imbalance between M1 and M2 macrophages can lead to pathologies such as osteoclastogenesis in asthma, chronic lung disease, atherosclerosis or rheumatoid arthritis. IRF-5 is a major regulator of proinflammatory M1 macrophage polarization (Weiss et al. Mediators of Inflammation (2013) Dx.doi.org/10.1155/2013/245804).
)。天然单核细胞或募集的巨噬细胞暴露于Th1细胞因子IFN-γ、TNF或LPS会促进M1发育，其分泌促炎细胞因子诸如TNF、IL-1β、IL-6、IL-12、IL-23，并且促进Th1淋巴细胞的发育。单核细胞暴露于IL-4和IL-13会促进M2a表型，其表达促进Th2细胞、嗜酸性粒细胞和嗜碱性粒细胞生长的趋化因子。M2b巨噬细胞由LPS、免疫复合物、凋亡细胞和IL-1Ra的组合诱导。M2b巨噬细胞分泌高水平的IL-10和促炎细胞因子TNF和IL-6并且表达iNOS。M2c巨噬细胞由IL-10、TGF-β和糖皮质激素的组合诱导并且分泌IL-10和TGF-β，其促进Th2淋巴细胞的发育(Duque和Descoteaux.(2014)Front.Immunol.5:491.Doi:10.3389/fimmu.2014.00491)。). Exposure of natural monocytes or recruited macrophages to the Th1 cytokines IFN-γ, TNF or LPS promotes M1 development, which secretes proinflammatory cytokines such as TNF, IL-1β, IL-6, IL-12, IL-23, and promotes the development of Th1 lymphocytes. Exposure of monocytes to IL-4 and IL-13 promotes the M2a phenotype, which expresses chemokines that promote the growth of Th2 cells, eosinophils and basophils. M2b macrophages are induced by a combination of LPS, immune complexes, apoptotic cells and IL-1Ra. M2b macrophages secrete high levels of IL-10 and the proinflammatory cytokines TNF and IL-6 and express iNOS. M2c macrophages are induced by a combination of IL-10, TGF-β, and glucocorticoids and secrete IL-10 and TGF-β, which promote the development of Th2 lymphocytes (Duque and Descoteaux. (2014) Front. Immunol. 5:491. Doi: 10.3389/fimmu.2014.00491).
IRF-5在巨噬细胞中的表达由炎性刺激可逆地诱导，并且有助于巨噬细胞极化。IRF-5上调M1巨噬细胞的表达并下调M2巨噬细胞的表达(Krausgruber等人，Nat.Immunol.(2010),12(3):231-238)。IRF-5 expression in macrophages is reversibly induced by inflammatory stimuli and contributes to macrophage polarization. IRF-5 upregulates expression in M1 macrophages and downregulates expression in M2 macrophages (Krausgruber et al., Nat. Immunol. (2010), 12(3):231-238).
在实施方案中，提供了一种用于治疗炎性疾病的方法。在实施方案中，疾病与IRF-5的异常表达相关。在实施方案中，疾病与IRF-5过表达相关。在实施方案中，所述方法包括施用下调IRF-5表达的化合物。在实施方案中，所述化合物包含AC。AC可以是任何AC并且具有如本文别处所述的任何AC特征。在实施方案中，AC可结合到如本文别处所述的靶转录物的SE或SRE的至少一部分。在实施方案中，AC可结合到如本文别处所述的靶转录物的SE或SRE附近。在实施方案中，AC是ASO。在实施方案中，ASO是PMO。AC可结合到如本文别处所述的IRF-5靶转录物的任何剪接元件(SE)。In embodiments, a method for treating an inflammatory disease is provided. In embodiments, the disease is associated with abnormal expression of IRF-5. In embodiments, the disease is associated with overexpression of IRF-5. In embodiments, the method comprises administering a compound that downregulates IRF-5 expression. In embodiments, the compound comprises AC. AC can be any AC and has any AC features as described elsewhere herein. In embodiments, AC may bind to at least a portion of a SE or SRE of a target transcript as described elsewhere herein. In embodiments, AC may bind to a SE or SRE near a target transcript as described elsewhere herein. In embodiments, AC is an ASO. In embodiments, ASO is a PMO. AC may bind to any splicing element (SE) of an IRF-5 target transcript as described elsewhere herein.
在实施方案中，所述方法包括施用诱导IRF-5mRNA转录物中一个或多个外显子的外显子跳跃的化合物。在实施方案中，所述方法包括施用包含反义化合物(AC)的化合物，所述反义化合物诱导IRF-5靶转录物中一个或多个外显子的跳跃。在实施方案中，AC与包含IRF-5靶转录物的至少一部分的靶核苷酸序列的杂交导致mRNA转录物中一个或多个外显子的包含或跳跃。在实施方案中，一个或多个外显子的跳跃或包含诱导IRF-5靶转录物中的移码。在实施方案中，移码得到编码具有降低活性的蛋白质的IRF-5靶转录物。在实施方案中，移码得到截短的或非功能性的IRF-5。在实施方案中，移码导致在IRF-5mRNA转录物中引入提前终止密码子。在实施方案中，移码导致IRF-5mRNA转录物通过无义介导的衰变而降解。在实施方案中，化合物包含诱导人和/或小鼠IRF-5的外显子2、3、4、5、6、7和/或8中的一者或多者的跳跃的反义化合物(AC)。在实施方案中，化合物包含诱导一个或多个外显子跳跃以产生框外移码的AC，从而导致IRF-5靶转录物被降解(例如，无义介导的衰变)或被翻译成具有降低活性或无活性的IRF-5蛋白。在实施方案中，化合物包含诱导外显子3、4、5和/或8中的一者或多者的跳跃以产生框外移码的AC。In embodiments, the method includes administering a compound that induces exon skipping of one or more exons in an IRF-5 mRNA transcript. In embodiments, the method includes administering a compound comprising an antisense compound (AC), which induces skipping of one or more exons in an IRF-5 target transcript. In embodiments, hybridization of AC with a target nucleotide sequence comprising at least a portion of an IRF-5 target transcript results in inclusion or skipping of one or more exons in an mRNA transcript. In embodiments, skipping or inclusion of one or more exons induces frameshifting in an IRF-5 target transcript. In embodiments, frameshifting results in encoding an IRF-5 target transcript of a protein with reduced activity. In embodiments, frameshifting results in truncated or non-functional IRF-5. In embodiments, frameshifting results in the introduction of an early termination codon in an IRF-5 mRNA transcript. In embodiments, frameshifting results in degradation of an IRF-5 mRNA transcript by nonsense-mediated decay. In an embodiment, the compound comprises an antisense compound (AC) that induces the skipping of one or more of exons 2, 3, 4, 5, 6, 7, and/or 8 of human and/or mouse IRF-5. In an embodiment, the compound comprises an AC that induces skipping of one or more exons to produce an out-of-frame frameshift, thereby causing the IRF-5 target transcript to be degraded (e.g., nonsense-mediated decay) or translated into an IRF-5 protein with reduced activity or no activity. In an embodiment, the compound comprises an AC that induces skipping of one or more of exons 3, 4, 5, and/or 8 to produce an out-of-frame frameshift.
在实施方案中，AC包含表11中的SEQ ID NO:157-161中的任一中。在实施方案中，AC包含表11中任一序列的10个至25个、10个至20个、或10个至15个连续碱基。在实施方案中，SEQ ID NO:340、365、369或其片段诱导外显子4的跳跃以在外显子5中产生提前终止密码子。在实施方案中，SEQ ID NO:340、365、369或其片段诱导外显子4的外显子跳跃，从而导致IRF-5靶转录物的无义介导的衰变。在实施方案中，SEQ ID NO:340和365或其片段诱导外显子4的跳跃以产生提前终止密码子。在实施方案中，SEQ ID NO:366至368或其片段诱导外显子5的外显子跳跃，从而导致外显子6中的提前终止密码子。在实施方案中，SEQ ID NO:366-368或其片段诱导外显子5的外显子跳跃，从而导致IRF-5靶转录物的无义介导的衰变。In an embodiment, AC comprises any one of SEQ ID NOs: 157-161 in Table 11. In an embodiment, AC comprises 10 to 25, 10 to 20, or 10 to 15 consecutive bases of any sequence in Table 11. In an embodiment, SEQ ID NOs: 340, 365, 369, or a fragment thereof induces exon skipping of exon 4 to produce an early stop codon in exon 5. In an embodiment, SEQ ID NOs: 340, 365, 369, or a fragment thereof induces exon skipping of exon 4, thereby resulting in nonsense-mediated decay of the IRF-5 target transcript. In an embodiment, SEQ ID NOs: 340 and 365, or a fragment thereof, induces exon skipping of exon 4 to produce an early stop codon. In an embodiment, SEQ ID NOs: 366 to 368, or a fragment thereof, induces exon skipping of exon 5, thereby resulting in an early stop codon in exon 6. In embodiments, SEQ ID NOs: 366-368 or fragments thereof induce exon skipping of exon 5, thereby resulting in nonsense-mediated decay of the IRF-5 target transcript.
表11：诱导外显子跳跃的AC序列Table 11: AC sequences that induce exon skipping
在实施方案中，化合物包含诱导一个或多个外显子的跳跃以在IRF-5靶转录物中产生框内缺失的AC。在实施方案中，化合物包含诱导外显子6和/或7中的一者或多者的跳跃以在IRF-5靶转录物中产生框内缺失的AC。In embodiments, the compound comprises an AC that induces skipping of one or more exons to generate an in-frame deletion in an IRF-5 target transcript. In embodiments, the compound comprises an AC that induces skipping of one or more of exons 6 and/or 7 to generate an in-frame deletion in an IRF-5 target transcript.
在实施方案中，提供了一种用于治疗与IRF-5相关的疾病或病症的方法。在实施方案中，疾病或病症与IRF-5遗传变异相关。在实施方案中，疾病或病症与IRF-5基因中的遗传突变相关。在实施方案中，IRF-5中的遗传突变导致IRF-5过表达。在实施方案中，遗传突变导致替代性同种型表达。在实施方案中，疾病或病症与IRF-5过表达相关。在实施方案中，疾病或病症与IRF-5同种型表达相关。In embodiments, a method for treating a disease or condition associated with IRF-5 is provided. In embodiments, the disease or condition is associated with an IRF-5 genetic variation. In embodiments, the disease or condition is associated with a genetic mutation in an IRF-5 gene. In embodiments, a genetic mutation in IRF-5 results in overexpression of IRF-5. In embodiments, a genetic mutation results in expression of alternative isoforms. In embodiments, the disease or condition is associated with overexpression of IRF-5. In embodiments, the disease or condition is associated with expression of an IRF-5 isoform.
在实施方案中，提供了一种用于治疗患者的炎症、自身抗体产生、炎症细胞浸润、胶原沉积或炎性细胞因子产生的方法。In an embodiment, a method for treating inflammation, autoantibody production, inflammatory cell infiltration, collagen deposition, or inflammatory cytokine production in a patient is provided.
IRF-5参与多种疾病已有记载(参见例如Graham等人，Nat Genet.(2006),38(5):550-5；Rueda等人，Arthritis Rheum.(2006),54(12):3815-9；Henrique da Mota,ClinRheumatol.(2015),34(9):1495-501；Sigurdsson等人，Hum Mol Genet.(2008),17(6):872-81；Peng，等人，Nephrology(Carlton)(2010),15(7):710-3；Ishimura等人，J ClinImmunol.(2011),31(6):946-51；Summers等人，J Rheumatol.(2008),35(11):2106-18；Ni等人，Inflammation(2019),2(5):1821-1829；Dideberg等人，Hum Mol Genet.(2007),16(24):3008-16；Lim等人，J.Dig.Dis.(2015),16(4):205-16；Nordal等人，Ann.Rheum.Dis.(2012),71(7):1197-202；Rebora,Int.J.Dermatol.(2016),55(4):408-16；Zhao等人，Rheumatol.Int.(2017),37(8):1303-131；Carmona等人，PLoS One(2013),8(1):e54419；Flesch等人，Tissue Antigens(2011),78(1):65-8；Heijde等人，Arthritis Rheum.(2007),56(12):3989-94；Hafler等人，Genes Immun.(2009),10(1):68-76；Balasa等人，Eur.Cytokine Netw.(2012),23(4):166-72；Byre等人，Mucosal Immunol.(2017),10(3):716-726；Wang等人，Gene(2012),10,504(2):220-5；Pimenta等人，Mol.Cancer(2015),14(1):32；Rambod等人，Clin Rheumatol.(2018),37(10):2661-2665；Davi等人，JRheumatol.(2011),38(4):769-74；Zimmerman等人，Kidney 360(2020),1(3):179-190；Pandey等人，Mucosal.Immunol.(2019),12(4):874-887；Masuda等人，Nat.Commun.(2014),5:3771；Alzaid等人，JCI Insight(2016),1(20):e88689；Senevirante等人，Circulation(2017),136(12):1140-1154；Cevik等人，J.Biol.Chem.(2017),292(52):21676-21689；Sharif等人，Ann.Rheum.Dis.(2012),71(7):1197-1202；以及Yang等人，J Pediatr.Surg.(2017),52(12):1984-1988)。IRF-5 has been implicated in a variety of diseases (see, e.g., Graham et al., Nat Genet. (2006), 38(5):550-5; Rueda et al., Arthritis Rheum. (2006), 54(12):3815-9; Henrique da Mota, Clin Rheumatol. (2015), 34(9):1495-501; Sigurdsson et al., Hum Mol Genet. (2008), 17(6):872-81; Peng et al., Nephrology (Carlton) (2010), 15(7):710-3; Ishimura et al., J Clin Immunol. (2011), 31(6):946-51; Summers et al., J Clin Immunol. (2012), 31(6):950-9; Rheumatol. (2008), 35(11):2106-18; Ni et al., Inflammation (2019), 2(5):1821-1829; Dideberg et al., Hum Mol Genet. (2007), 16(24):3008-16; Lim et al., J. Dig. Dis. (2015), 16(4):205-16; Nordal et al., Ann. Rheum. Dis. (2012), 71(7):1197-202; Rebora, Int. J. Dermatol. (2016), 55(4):408-16; Zhao et al., Rheumatol. Int. (2017), 37(8):1303-131; Carmona et al., PLoS One (2013), 8(1):e54419; Flesch et al., Tissue Antigens (2011), 78(1): 65-8; Heijde et al., Arthritis Rheum. (2007), 56(12): 3989-94; Hafler et al., Genes Immun. (2009), 10(1): 68-76; Balasa et al., Eur. Cytokine Netw. (2012), 23(4): 166-72; Byre et al., Mucosal Immunol. (2017), 10(3): 716-726; Wang et al., Gene (2012), 10, 504(2): 220-5; Pimenta et al., Mol. Cancer (2015), 14(1): 32; Rambod et al., Clin Rheumatol. (2018), 37(10): 2661-2665; Davi et al., J Rheumatol. (2011), 38(4): 769-74; Zimmerman et al., Kidney 360 (2020), 1(3): 179-190; Pandey et al., Mucosal. Immunol. (2019), 12(4): 874-887; Masuda et al., Nat. Commun. (2014), 5: 3771; Alzaid et al., JCI Insight (2016), 1(20): e88689; Senevirante et al., Circulation (2017), 136(12): 1140-1154; Cevik et al., J. Biol. Chem. (2017), 292(52): 21676-21689; Sharif et al., Ann. Rheum. Dis. (2012), 71(7): 1197-1202; and Yang et al., J Pediatr. Surg. (2017), 52(12): 1984-1988).
在实施方案中，提供了使用本文公开的一种或多种化合物下调患者的IRF-5表达的方法。在实施方案中，巨噬细胞中的IRF-5表达降低。在实施方案中，库普弗细胞中的IRF-5表达降低。在实施方案中，胃肠道中的IRF-5表达降低。在实施方案中，IRF-5在肝脏中的表达降低。在实施方案中，IRF-5在肺部中的表达降低。在实施方案中，IRF-5在肾脏中的表达降低。在实施方案中，IRF-5在关节中的表达降低。在实施方案中，IRF-5在中枢神经系统中的表达降低。In embodiments, methods are provided for down-regulating IRF-5 expression in a patient using one or more compounds disclosed herein. In embodiments, IRF-5 expression in macrophages is reduced. In embodiments, IRF-5 expression in Kupffer cells is reduced. In embodiments, IRF-5 expression in the gastrointestinal tract is reduced. In embodiments, IRF-5 expression in the liver is reduced. In embodiments, IRF-5 expression in the lungs is reduced. In embodiments, IRF-5 expression in the kidneys is reduced. In embodiments, IRF-5 expression in the joints is reduced. In embodiments, IRF-5 expression in the central nervous system is reduced.
在实施方案中，本文公开的化合物用于治疗与IRF-5相关的疾病。与IRF-5相关的疾病的示例包括但不限于炎性肠病(IBD)、溃疡性结肠炎、克罗恩氏病、系统性红斑狼疮(SLE)、类风湿性关节炎、原发性胆汁性肝硬化、系统性硬化症、舍格伦综合征、多发性硬化症、硬皮病、间质性肺病(SSc-ILD)、多囊性肾病(PKD)、慢性肾病(CKD)、非酒精性脂肪性肝炎(NASH)、肝纤维化、哮喘、重症哮喘以及它们的组合。在实施方案中，本文公开的化合物用于减少患者的炎症、硬化症、纤维化、蛋白尿、关节炎症、自身抗体产生、炎性细胞浸润、胶原沉积、炎性细胞因子产生或它们的组合。在实施方案中，本文公开的化合物用于减少胃肠道中的炎症、腹泻、疼痛、疲劳、腹部痉挛、便血、肠道炎症、胃肠道上皮屏障破坏、生态失调、排便频率增加、肛门括约肌的里急后重或疼痛性痉挛、便秘、意外的体重减轻或它们的组合。In an embodiment, compounds disclosed herein are used to treat the disease related to IRF-5. The example of the disease related to IRF-5 includes but is not limited to inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease, systemic lupus erythematosus (SLE), rheumatoid arthritis, primary biliary cirrhosis, systemic sclerosis, Sjögren's syndrome, multiple sclerosis, scleroderma, interstitial lung disease (SSc-ILD), polycystic kidney disease (PKD), chronic kidney disease (CKD), nonalcoholic fatty hepatitis (NASH), liver fibrosis, asthma, severe asthma and their combination. In an embodiment, compounds disclosed herein are used to reduce inflammation, sclerosis, fibrosis, proteinuria, joint inflammation, autoantibody production, inflammatory cell infiltration, collagen deposition, inflammatory cytokine production or their combination in patients. In an embodiment, the compounds disclosed herein are used to reduce inflammation in the gastrointestinal tract, diarrhea, pain, fatigue, abdominal cramping, blood in the stool, intestinal inflammation, gastrointestinal epithelial barrier disruption, dysbiosis, increased frequency of bowel movements, tenesmus or painful spasms of the anal sphincter, constipation, unintentional weight loss, or a combination thereof.
在实施方案中，本文公开的化合物用于治疗炎性疾病。“炎性疾病”是指其中先天或适应性免疫应答的激活是临床状况的主要原因的疾病。炎性疾病包括但不限于寻常痤疮、哮喘、COPD、自身免疫疾病、乳糜泻、慢性(斑块)前列腺炎、肾小球肾炎、超敏反应、炎性肠病(IBD、克罗恩氏病、溃疡性结肠炎)、盆腔炎、再灌注损伤、类风湿性关节炎、结节病、移植排斥、血管炎、间质性膀胱炎、动脉粥样硬化、变态反应(1、2和3型超敏反应、花粉症)、炎性肌病如系统性硬化症，并且包括皮肌炎、多肌炎、包涵体肌炎、契-东综合征、慢性肉芽肿病、维生素A缺乏症、癌症(实体瘤、胆囊癌)、牙周炎、肉芽肿性炎症(肺结核、麻风病、结节病和梅毒)、纤维素性炎症、化脓性炎症、浆液性炎症、溃疡性炎症和缺血性心脏病、I型糖尿病、糖尿病性肾病以及它们的组合。In an embodiment, the compounds disclosed herein are used to treat inflammatory diseases. "Inflammatory diseases" refers to diseases in which activation of the innate or adaptive immune response is the primary cause of the clinical condition. Inflammatory diseases include, but are not limited to, acne vulgaris, asthma, COPD, autoimmune diseases, celiac disease, chronic (plaque) prostatitis, glomerulonephritis, hypersensitivity reactions, inflammatory bowel disease (IBD, Crohn's disease, ulcerative colitis), pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis, sarcoidosis, transplant rejection, vasculitis, interstitial cystitis, atherosclerosis, allergies (type 1, 2 and 3 hypersensitivity reactions, hay fever), inflammatory myopathies such as systemic sclerosis, and include dermatomyositis, polymyositis, inclusion body myositis, Chi-Hung syndrome, chronic granulomatous disease, vitamin A deficiency, cancer (solid tumors, gallbladder cancer), periodontitis, granulomatous inflammation (tuberculosis, leprosy, sarcoidosis and syphilis), fibrinous inflammation, suppurative inflammation, serous inflammation, ulcerative inflammation and ischemic heart disease, type I diabetes, diabetic nephropathy, and combinations thereof.
在实施方案中，本文公开的化合物用于治疗自身免疫疾病。“自身免疫疾病”是指患者的免疫系统攻击患者自身组织的疾病或病症。自身免疫疾病或病症的示例包括但不限于炎症反应，诸如炎症性皮肤病，包括牛皮癣和皮炎(例如特应性皮炎)；系统性硬皮病和硬化症；与炎性肠病相关的反应(诸如克罗恩氏病和溃疡性结肠炎)；呼吸窘迫综合征(包括成人呼吸窘迫综合征；ARDS)；皮炎；脑膜炎；脑炎；葡萄膜炎；结肠炎；肾小球肾炎；过敏性病症，诸如湿疹和哮喘以及其他涉及T细胞浸润和慢性炎症反应的疾病；动脉粥样硬化；白细胞粘附不足；类风湿性关节炎；系统性红斑狼疮(SLE)(包括但不限于狼疮性肾炎、皮肤狼疮)；系统性硬化症(硬皮病)；糖尿病(例如I型糖尿病或胰岛素依赖性糖尿病)；多发性硬化症；雷诺综合征；自身免疫性甲状腺炎；桥本氏甲状腺炎；过敏性脑脊髓炎；舍格伦综合征；幼年型糖尿病；以及与细胞因子和T淋巴细胞介导的急性和迟发型超敏反应相关的免疫应答，其通常存在于肺结核、结节病、多肌炎、皮肌炎中；肉芽肿病和血管炎；原发性胆汁性肝硬化；恶性贫血(艾迪森病)；自身免疫性胃炎；自身免疫性肝炎；涉及白细胞渗出的疾病；中枢神经系统(CNS)炎性病症；白癜风；多器官损伤综合征；溶血性贫血(包括但不限于冷沉球蛋白血症或库姆斯阳性贫血)；重症肌无力；抗原-抗体复合物介导的疾病；抗肾小球基底膜疾病；抗磷脂综合征；过敏性神经炎；格雷夫斯病；兰伯特-伊顿肌无力综合征；大疱性类天疱疮；天疱疮；自身免疫性多内分泌腺病；赖特氏病；僵人综合征；白塞病；巨细胞动脉炎；免疫复合物性肾炎；IgA肾病；IgM多神经病；免疫性血小板减少性紫癜(ITP)或自身免疫性血小板减少症；自身免疫性脑脊髓炎；非酒精性脂肪性肝炎(NASH)；强直性脊柱炎；肺纤维化；或它们的组合。In an embodiment, the compounds disclosed herein are used to treat autoimmune diseases. "Autoimmune disease" refers to a disease or condition in which a patient's immune system attacks the patient's own tissues. Examples of autoimmune diseases or conditions include, but are not limited to, inflammatory reactions, such as inflammatory skin diseases, including psoriasis and dermatitis (e.g., atopic dermatitis); systemic scleroderma and sclerosis; reactions associated with inflammatory bowel disease (such as Crohn's disease and ulcerative colitis); respiratory distress syndrome (including adult respiratory distress syndrome; ARDS); dermatitis; meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergic conditions, such as eczema and asthma, and other diseases involving T cell infiltration and chronic inflammatory reactions ; atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis; systemic lupus erythematosus (SLE) (including but not limited to lupus nephritis, cutaneous lupus); systemic sclerosis (scleroderma); diabetes (e.g., type I diabetes or insulin-dependent diabetes); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; Hashimoto's thyroiditis; allergic encephalomyelitis; Sjögren's syndrome; juvenile diabetes; and immune-related acute and delayed hypersensitivity reactions mediated by cytokines and T lymphocytes. responses, which are commonly found in tuberculosis, sarcoidosis, polymyositis, dermatomyositis; granulomatous diseases and vasculitis; primary biliary cirrhosis; pernicious anemia (Addison's disease); autoimmune gastritis; autoimmune hepatitis; diseases involving leukocytic infiltration; inflammatory disorders of the central nervous system (CNS); vitiligo; multiple organ injury syndrome; hemolytic anemias (including but not limited to cryoglobulinemia or Coombs-positive anemia); myasthenia gravis; antigen-antibody complex-mediated diseases; anti-glomerular basement membrane disease; anti-phosphodiesterase inhibitors; lipid syndrome; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; bullous pemphigoid; pemphigus; autoimmune polyendocrine disease; Reiter's disease; stiff-man syndrome; Behcet's disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathy; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia; autoimmune encephalomyelitis; nonalcoholic steatohepatitis (NASH); ankylosing spondylitis; pulmonary fibrosis; or a combination thereof.
在实施方案中，本文公开的化合物用于治疗自身免疫疾病，诸如系统性红斑狼疮(SLE)、系统性硬化症(硬皮病)、多肌炎/皮肌炎、克罗恩氏病、溃疡性结肠炎、类风湿性关节炎、舍格伦综合征、自身免疫性脑脊髓炎、非酒精性脂肪性肝炎(NASH)、结节病、白塞病、重症肌无力、狼疮性肾炎、炎性肠病(IBD)、强直性脊柱炎、原发性胆汁性肝硬化、结肠炎、肺纤维化、抗磷脂综合征或牛皮癣。In an embodiment, the compounds disclosed herein are used to treat an autoimmune disease such as systemic lupus erythematosus (SLE), systemic sclerosis (scleroderma), polymyositis/dermatomyositis, Crohn's disease, ulcerative colitis, rheumatoid arthritis, Sjögren's syndrome, autoimmune encephalomyelitis, nonalcoholic steatohepatitis (NASH), sarcoidosis, Behcet's disease, myasthenia gravis, lupus nephritis, inflammatory bowel disease (IBD), ankylosing spondylitis, primary biliary cirrhosis, colitis, pulmonary fibrosis, antiphospholipid syndrome, or psoriasis.
在实施方案中，本文公开的化合物用于治疗心血管疾病。在实施方案中，心血管疾病与炎症相关。在实施方案中，心血管疾病包括系统性硬皮病。在实施方案中，心血管疾病包括动脉瘤；心绞痛；动脉粥样硬化；脑血管意外(卒中)；脑血管疾病；充血性心力衰竭；冠状动脉疾病；心肌梗死(心脏病发作)；外周血管疾病；或它们的组合。在实施方案中，心血管疾病包括动脉粥样硬化。In embodiments, the compounds disclosed herein are used to treat cardiovascular disease. In embodiments, cardiovascular disease is associated with inflammation. In embodiments, cardiovascular disease comprises systemic scleroderma. In embodiments, cardiovascular disease comprises aneurysm; angina pectoris; atherosclerosis; cerebrovascular accident (stroke); cerebrovascular disease; congestive heart failure; coronary artery disease; myocardial infarction (heart attack); peripheral vascular disease; or a combination thereof. In embodiments, cardiovascular disease comprises atherosclerosis.
在实施方案中，本文公开的化合物用于治疗胃肠疾病。在实施方案中，胃肠疾病包括克罗恩氏病、原发性胆汁性肝硬化、硬化性胆管炎、溃疡性结肠炎、炎性肠病、舍格伦综合征或它们的组合。In an embodiment, the compounds disclosed herein are used to treat a gastrointestinal disease. In an embodiment, the gastrointestinal disease comprises Crohn's disease, primary biliary cirrhosis, sclerosing cholangitis, ulcerative colitis, inflammatory bowel disease, Sjogren's syndrome, or a combination thereof.
在实施方案中，本文公开的化合物用于治疗泌尿系统疾病。在实施方案中，泌尿系统疾病包括系统性红斑狼疮、系统性硬皮病或它们的组合。In an embodiment, the compounds disclosed herein are used to treat a urological disease. In an embodiment, the urological disease comprises systemic lupus erythematosus, systemic scleroderma, or a combination thereof.
在实施方案中，本文公开的化合物用于治疗遗传性、家族性或先天性疾病。在实施方案中，遗传性、家族性或先天性疾病包括克罗恩氏病、原发性胆汁性肝硬化、系统性硬皮病、系统性红斑狼疮、溃疡性结肠炎、牛皮癣、炎性肠病或它们的组合。In an embodiment, the compounds disclosed herein are used to treat a hereditary, familial or congenital disease. In an embodiment, the hereditary, familial or congenital disease comprises Crohn's disease, primary biliary cirrhosis, systemic scleroderma, systemic lupus erythematosus, ulcerative colitis, psoriasis, inflammatory bowel disease, or a combination thereof.
在实施方案中，本文公开的化合物用于治疗内分泌系统疾病。在实施方案中，内分泌系统疾病包括甲状腺腺癌、原发性胆汁性肝硬化、硬化性胆管炎、甲状腺功能减退或它们的组合。In an embodiment, the compounds disclosed herein are used to treat endocrine system diseases. In an embodiment, endocrine system diseases comprise thyroid adenocarcinoma, primary biliary cirrhosis, sclerosing cholangitis, hypothyroidism, or a combination thereof.
在实施方案中，本文公开的化合物用于治疗细胞增殖病症。在实施方案中，细胞增殖病症包括原发性胆汁性肝硬化、甲状腺腺癌、瘤或它们的组合。In an embodiment, the compounds disclosed herein are used to treat a cell proliferative disorder. In an embodiment, the cell proliferative disorder comprises primary biliary cirrhosis, thyroid adenocarcinoma, neoplasm, or a combination thereof.
在实施方案中，本文公开的化合物用于治疗免疫系统疾病。在实施方案中，免疫系统疾病包括舍格伦综合征、炎性肠病、牛皮癣、肌炎、系统性硬皮病、自身免疫疾病、系统性红斑狼疮、类风湿性关节炎、克罗恩氏病、溃疡性结肠炎、强直性脊柱炎或它们的组合。In an embodiment, the compounds disclosed herein are used to treat immune system diseases. In an embodiment, immune system diseases include Sjögren's syndrome, inflammatory bowel disease, psoriasis, myositis, systemic sclerosis, autoimmune diseases, systemic lupus erythematosus, rheumatoid arthritis, Crohn's disease, ulcerative colitis, ankylosing spondylitis, or a combination thereof.
在实施方案中，本文公开的化合物用于治疗血液病。在实施方案中，血液病包括系统性红斑狼疮。In an embodiment, the compounds disclosed herein are used to treat a blood disorder. In an embodiment, the blood disorder comprises systemic lupus erythematosus.
在实施方案中，本文公开的化合物用于治疗肌骨骼或结缔组织疾病。在实施方案中，肌骨骼或结缔组织疾病包括肌炎、系统性硬皮病、系统性红斑狼疮、类风湿性关节炎、强直性脊柱炎、青少年特发性脊柱侧凸或它们的组合。In an embodiment, the compounds disclosed herein are used to treat a musculoskeletal or connective tissue disease. In an embodiment, the musculoskeletal or connective tissue disease comprises myositis, systemic sclerosis, systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, juvenile idiopathic scoliosis, or a combination thereof.
在实施方案中，本文公开的化合物用于治疗神经炎症性疾病。在实施方案中，神经炎症性疾病或病症包括由于创伤性脑损伤引起的炎症、急性播散性脑脊髓炎(ADEM)、自身免疫性脑炎、急性视神经炎(AON)、慢性脑膜炎、抗髓鞘少突胶质细胞糖蛋白(MOG)病、横贯性脊髓炎、视神经脊髓炎(NMO)、阿尔茨海默病、帕金森病、多发性硬化症(MS)或它们的组合。In an embodiment, the compounds disclosed herein are used to treat a neuroinflammatory disease. In an embodiment, a neuroinflammatory disease or condition comprises inflammation due to traumatic brain injury, acute disseminated encephalomyelitis (ADEM), autoimmune encephalitis, acute optic neuritis (AON), chronic meningitis, anti-myelin oligodendrocyte glycoprotein (MOG) disease, transverse myelitis, neuromyelitis optica (NMO), Alzheimer's disease, Parkinson's disease, multiple sclerosis (MS), or a combination thereof.
在实施方案中，本文公开的化合物用于治疗由于微生物诸如病毒、细菌、真菌、寄生生物或它们的组合的感染引起的炎症。In an embodiment, the compounds disclosed herein are used to treat inflammation due to infection with microorganisms such as viruses, bacteria, fungi, parasites, or combinations thereof.
在实施方案中，本文公开的化合物用于治疗与纤维化相关的疾病，其在本文中被称为纤维化疾病。“纤维化”是指例如由于损伤、刺激或慢性炎症引起的纤维结缔组织的病理性形成，并且包括组织中超过正常量的成纤维细胞积累和胶原沉积。“纤维化疾病”是指与病理性纤维化相关的疾病。纤维化疾病的示例包括但不限于特发性肺纤维化；硬皮病；皮肤硬皮病；肺部硬皮病；胶原血管病(例如，狼疮；类风湿性关节炎；硬皮病)；遗传性肺纤维化(例如，赫曼斯基-普德拉克综合征)；放射线肺炎；哮喘；哮喘气道重塑；化疗诱导的肺纤维化(例如，博来霉素、甲氨蝶呤或环磷酰胺诱导)；放射性纤维化；戈谢病；间质性肺病；腹膜后纤维化；骨髓纤维化；间质或肺血管疾病；与药物暴露相关的纤维化或间质性肺病；与暴露相关的间质性肺病，诸如石棉肺、硅肺病和颗粒暴露；慢性超敏性肺炎；粘连；肠或腹腔粘连；心脏纤维化；肾纤维化；硬化症；非酒精性脂肪性肝炎(NASH)诱导的纤维化；或它们的组合。在实施方案中，纤维化疾病包括非酒精性脂肪性肝炎NASH。In an embodiment, the compounds disclosed herein are used to treat diseases associated with fibrosis, which are referred to herein as fibrotic diseases. "Fibrosis" refers to the pathological formation of fibrous connective tissue, such as due to injury, irritation, or chronic inflammation, and includes the accumulation of fibroblasts and collagen deposition in tissues in excess of normal amounts. "Fibrotic diseases" refers to diseases associated with pathological fibrosis. Examples of fibrotic diseases include, but are not limited to, idiopathic pulmonary fibrosis; scleroderma; cutaneous scleroderma; pulmonary scleroderma; collagen vascular disease (e.g., lupus; rheumatoid arthritis; scleroderma); hereditary pulmonary fibrosis (e.g., Hermansky-Pudlak syndrome); radiation pneumonitis; asthma; asthmatic airway remodeling; chemotherapy-induced pulmonary fibrosis (e.g., bleomycin, methotrexate, or cyclophosphamide-induced); radiation fibrosis; Gaucher disease; interstitial lung disease; retroperitoneal fibrosis; myelofibrosis; interstitial or pulmonary vascular disease; fibrosis or interstitial lung disease associated with drug exposure; interstitial lung disease associated with exposure, such as asbestosis, silicosis, and particle exposure; chronic hypersensitivity pneumonitis; adhesions; intestinal or peritoneal adhesions; cardiac fibrosis; renal fibrosis; sclerosis; fibrosis induced by nonalcoholic steatohepatitis (NASH); or a combination thereof. In embodiments, fibrotic diseases include nonalcoholic steatohepatitis NASH.
在实施方案中，本文公开的化合物用于治疗呼吸系统或胸部疾病，诸如系统性硬皮病。在实施方案中，本文公开的化合物用于治疗皮肤系统疾病，诸如牛皮癣或系统性硬皮病。在实施方案中，本文公开的化合物用于治疗视觉系统疾病，诸如舍格伦综合征或系统性硬皮病。在实施方案中，本文公开的化合物用于治疗与嗜酸性粒细胞计数、肾小球滤过率、收缩压、白细胞的嗜酸性粒细胞百分比或它们的组合相关的病症。在实施方案中，本文公开的化合物用于治疗溃疡疾病或口腔溃疡。In an embodiment, the compounds disclosed herein are used to treat respiratory or chest diseases, such as systemic scleroderma. In an embodiment, the compounds disclosed herein are used to treat skin system diseases, such as psoriasis or systemic scleroderma. In an embodiment, the compounds disclosed herein are used to treat visual system diseases, such as Sjögren's syndrome or systemic scleroderma. In an embodiment, the compounds disclosed herein are used to treat disorders associated with eosinophil count, glomerular filtration rate, systolic blood pressure, eosinophil percentage of white blood cells, or a combination thereof. In an embodiment, the compounds disclosed herein are used to treat ulcerative diseases or oral ulcers.
炎性肠病(IBD)Inflammatory bowel disease (IBD)
炎性肠病(IBD)是指以胃肠(GI)道的慢性炎症为特征的两种病症：克罗恩氏病和溃疡性结肠炎。IBD的常见症状包括持续性腹泻、腹痛、直肠出血/便血、体重减轻和疲劳。2015年，估计有1.3％的美国成人(3百万人)报告被诊断为IBD(克罗恩氏病或溃疡性结肠炎)。IBD与肠巨噬细胞中由IRF-5促进的炎性巨噬细胞表型相关。Inflammatory bowel disease (IBD) refers to two conditions characterized by chronic inflammation of the gastrointestinal (GI) tract: Crohn's disease and ulcerative colitis. Common symptoms of IBD include persistent diarrhea, abdominal pain, rectal bleeding/blood in stool, weight loss, and fatigue. In 2015, an estimated 1.3% of U.S. adults (3 million people) reported being diagnosed with IBD (Crohn's disease or ulcerative colitis). IBD is associated with an inflammatory macrophage phenotype in intestinal macrophages that is promoted by IRF-5.
类风湿性关节炎(RA)Rheumatoid arthritis (RA)
类风湿性关节炎(RA)是影响全世界0.5％至1％人口的自身免疫疾病。它会引起关节疼痛和遍及患者身体的损伤。RA的治疗通常包括使用减缓病情并防止关节变形的药物，称为改善病情的抗风湿药物(DMARD)和靶向免疫系统的触发引起关节和组织损伤的炎症的部分的生物制剂(抗体)。IRF-5多态性已被鉴定为RA的风险因素。降低的IRF-5水平与降低的疾病表型相关。TLR3和TLR7的IRF-5活化促进炎性细胞因子和趋化因子产生。Rheumatoid arthritis (RA) is an autoimmune disease that affects 0.5% to 1% of the world's population. It causes joint pain and damage throughout the patient's body. Treatment of RA typically includes the use of drugs that slow the disease and prevent joint deformation, called disease-modifying antirheumatic drugs (DMARDs) and biological agents (antibodies) that target the part of the immune system that triggers inflammation that causes joint and tissue damage. IRF-5 polymorphisms have been identified as risk factors for RA. Reduced IRF-5 levels are associated with reduced disease phenotypes. IRF-5 activation of TLR3 and TLR7 promotes the production of inflammatory cytokines and chemokines.
舍格伦综合征(SS)Sjögren's syndrome (SS)
舍格伦综合征(SS)是通过眼干和口干鉴定的免疫病症。该病症通常伴随有其他免疫系统病症，诸如类风湿性关节炎(RA)和系统性红斑狼疮(SLE)。该疾病主要影响年龄在40-60岁之间的女性。在美国，原发性SS的患病率估计为每10,000个居民2至10个。现有的SS疗法包括治疗眼干和口干的症状。尚没有疾病改善疗法。在多项研究中，IRF-5rs2004640T等位基因和CGGGG插入/缺失与SS相关。Sjögren's syndrome (SS) is an immune disorder identified by dry eyes and dry mouth. The disease is usually accompanied by other immune system disorders, such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). The disease mainly affects women aged between 40-60 years old. In the U.S., the prevalence of primary SS is estimated to be 2 to 10 per 10,000 residents. Existing SS therapies include treating the symptoms of dry eyes and dry mouth. There is no disease-modifying therapy. In multiple studies, IRF-5rs2004640T allele and CGGGG insertion/deletion are associated with SS.
多发性硬化症(MS)Multiple Sclerosis (MS)
多发性硬化症(MS)是使中枢神经系统(脑和脊髓)衰弱的疾病。在MS中，免疫系统攻击覆盖神经纤维的保护鞘(髓磷脂)并且引起患者的脑和身体之间的通信问题。多发性硬化症引起广泛的神经症状，包括感觉或运动麻痹、视觉障碍、共济失调、协调受损、疼痛、认知功能障碍和疲劳。目前的估计表明在美国有300,000至400,000个个体受到影响，并且在全世界有超过2百万个个体受到影响。MS的治疗通常限于皮质类固醇和血浆置换疗法。Multiple sclerosis (MS) is a disease that weakens the central nervous system (brain and spinal cord). In MS, the immune system attacks the protective sheath (myelin) that covers nerve fibers and causes communication problems between the patient's brain and body. Multiple sclerosis causes extensive neurological symptoms, including sensory or motor paralysis, visual impairment, ataxia, impaired coordination, pain, cognitive dysfunction and fatigue. Current estimates show that 300,000 to 400,000 individuals are affected in the U.S., and more than 2 million individuals are affected in the world. The treatment of MS is usually limited to corticosteroids and plasma exchange therapy.
两种单核苷酸多态性(SNP)(rs4728142、rs3807306)和位于IRF-5基因的启动子和第一内含子中的5bp插入-缺失多态性与MS高度相关。Kristjansdottir等人(2008)“Interferon regulatory factor 5(IRF-5)gene variants are associated withmultiple sclerosis in three distinct populations,”J.Med.Genet.45(6):362-369。Two single nucleotide polymorphisms (SNPs) (rs4728142, rs3807306) and a 5 bp insertion-deletion polymorphism located in the promoter and first intron of the IRF-5 gene are highly associated with MS. Kristjansdottir et al. (2008) "Interferon regulatory factor 5 (IRF-5) gene variants are associated with multiple sclerosis in three distinct populations," J. Med. Genet. 45 (6): 362-369.
硬皮病或系统性硬化症(SSc)Scleroderma or systemic sclerosis (SSc)
硬皮病是与皮肤和内部器官的广泛纤维化、小血管病变和产生自身抗体的免疫失调相关的慢性结缔组织疾病。Sharif等人(2012)“IRF-5polymorphism predictsprognosis in patients with systemic sclerosis,”Ann.Rheum.Dis.71(7):1197-1202。Scleroderma is a chronic connective tissue disease associated with extensive fibrosis of the skin and internal organs, small vessel disease, and immune dysregulation producing autoantibodies. Sharif et al. (2012) "IRF-5 polymorphism predicts prognosis in patients with systemic sclerosis," Ann. Rheum. Dis. 71(7): 1197-1202.
IRF-5变体rs4728142与SSc患者的较长生存期和较低IRF-5转录物水平相关，并且预示SSc患者的较长生存期和较温和的间质性肺病(ILD)。不具有IRF-5rs4728142拷贝的患者增加了IRF-5表达水平并且经历了更严重的ILD和更短的生存期。在系统性硬化症(SS)的全基因组关联研究(GWAS)中鉴定了另外的单核苷酸多态性(rs10488631和rs12537284)。Sharif等人(2012)“IRF-5polymorphism predicts prognosis in patients withsystemic sclerosis,”Ann Rheum Dis.71(7):1197-202。IRF-5 variant rs4728142 is associated with a longer survival period and lower IRF-5 transcript levels in SSc patients, and predicts a longer survival period and milder interstitial lung disease (ILD) in SSc patients. Patients without IRF-5rs4728142 copies have increased IRF-5 expression levels and experienced more severe ILD and shorter survival periods. Other single nucleotide polymorphisms (rs10488631 and rs12537284) were identified in a genome-wide association study (GWAS) of systemic sclerosis (SS). Sharif et al. (2012) "IRF-5 polymorphism predicts prognosis in patients with systemic sclerosis," Ann Rheum Dis.71 (7): 1197-202.
双同源盒4(DUX4)基因Double homeobox 4 (DUX4) gene
面肩胛肱型肌营养不良(FSHD)是第三种最常见的遗传性肌营养不良的形式。它是由骨骼肌中转录因子双同源盒(DUX4)的不完全抑制引起的。肌原细胞中的DUX4过表达诱导不同的毒性级联反应，包括氧化应激反应的增加、无义介导的衰变抑制和肌生成的抑制(Bouwman等人，Curr.Opin.Neurol.(2020),33(5):635-640)。Facioscapulohumeral muscular dystrophy (FSHD) is the third most common form of inherited muscular dystrophy. It is caused by incomplete inhibition of the transcription factor double homeobox (DUX4) in skeletal muscle. Overexpression of DUX4 in myogenic cells induces different toxicity cascades, including increased oxidative stress, inhibition of nonsense-mediated decay, and inhibition of myogenesis (Bouwman et al., Curr. Opin. Neurol. (2020), 33(5): 635-640).
DUX4基因位于4号染色体末端附近的称为D4Z4的区域。所述区域包含11至超过100个重复区段，每个区段约3,300个DNA碱基(3.3kb)长。D4Z4区中的每个重复区段含有DUX4基因的拷贝。最接近染色体末端的拷贝称为DUX4，而其他拷贝称为“DUX4样”或DUX4L。The DUX4 gene is located in a region called D4Z4 near the end of chromosome 4. The region contains 11 to more than 100 repeat segments, each about 3,300 DNA bases (3.3 kb) long. Each repeat segment in the D4Z4 region contains a copy of the DUX4 gene. The copy closest to the end of the chromosome is called DUX4, while the other copies are called "DUX4-like" or DUX4L.
DUXc也已被鉴定为在FSHD中被上调(Ansseau等人，PLoS One.(2009),4(10):e7482,doi:10.1371/journal.pone.0007482)。DUXc已被定位于D4Z4区的42kb着丝粒序列。DUX4c编码47kb与DUX4相同的蛋白质，羧基端区域除外。DUXc has also been identified as being upregulated in FSHD (Ansseau et al., PLoS One. (2009), 4(10):e7482, doi:10.1371/journal.pone.0007482). DUXc has been localized to a 42 kb centromeric sequence in the D4Z4 region. DUX4c encodes a 47 kb protein identical to DUX4, except for the carboxyl-terminal region.
FSHD的特征在于位于4号染色体的亚端粒区域中的D4Z4阵列的收缩，从而导致DUX4转录因子的异常表达和数百个基因的错误调控(Marsollier等人，(Int.J.Mol.Sci.(2018),19,1347,doi:10.3390/ijms19051347)。FSHD is characterized by the contraction of the D4Z4 array located in the subtelomeric region of chromosome 4, resulting in aberrant expression of the DUX4 transcription factor and misregulation of hundreds of genes (Marsollier et al., (Int. J. Mol. Sci. (2018), 19, 1347, doi: 10.3390/ijms19051347).
人DUX4基因有四种变体，其核苷酸序列可通过NCBI数据库公开获得：变体1(NM_001306068.3)、变体2(NM_001293798.3)、变体3(NR_137167.1)和变体4(NM_001363820.2)。DUX4变体1和变体2都编码全长DUX4(DUX4-fl)。全长DUX4的过表达与FSHD相关。变体1和变体2之间的区别在于变体2与变体1相比在3'UTR中缺乏替代区段。与变体1相比，DUX4变体3在3'端具有多个差异，包括不同的3'末端。该变体被表示为非编码的，因为使用5'-最期望的翻译起始密码子使得该转录物成为无义介导的mRNA衰变(NMD)的候选物。与变体1相比，变体4缺少大部分编码区。与同种型DUX4-fl相比，所得截短的DUX4同种型(DUX4-s)具有更短且不同的C末端。DUX4-s蛋白已被证明对细胞无毒。There are four variants of the human DUX4 gene, whose nucleotide sequences are publicly available through the NCBI database: variant 1 (NM_001306068.3), variant 2 (NM_001293798.3), variant 3 (NR_137167.1) and variant 4 (NM_001363820.2). Both DUX4 variant 1 and variant 2 encode full-length DUX4 (DUX4-fl). Overexpression of full-length DUX4 is associated with FSHD. The difference between variant 1 and variant 2 is that variant 2 lacks an alternative segment in the 3'UTR compared to variant 1. Compared to variant 1, DUX4 variant 3 has multiple differences at the 3' end, including a different 3' end. This variant is represented as non-coding because the use of the 5'-most expected translation start codon makes the transcript a candidate for nonsense-mediated mRNA decay (NMD). Compared to variant 1, variant 4 lacks most of the coding region. The resulting truncated DUX4 isoform (DUX4-s) has a shorter and different C-terminus compared to the isoform DUX4-fl. The DUX4-s protein has been shown to be non-toxic to cells.
DUX4包含三个外显子。外显子一是DUX4蛋白的编码外显子，外显子2和3是未翻译的。全长DUX4蛋白包含两个DNA结合结构域和C-末端反式激活结构域。DUX4的截短同种型包含两个蛋白结合结构域，但不包含C-末端反式激活结构域。第一外显子包含两个5'剪接位点。根据所使用的5'ss，产生编码全长或截短的DUX4蛋白的转录物。为了产生全长同种型，使用位于第一外显子3'端的第一5'ss。为了产生截短的同种型，使用位于外显子1内并且比第一5'ss更接近转录物的5'端的第二5'ss。DUX4 contains three exons. Exon one is the coding exon for the DUX4 protein, and exons 2 and 3 are untranslated. The full-length DUX4 protein contains two DNA-binding domains and a C-terminal transactivation domain. The truncated isoform of DUX4 contains two protein-binding domains but does not contain a C-terminal transactivation domain. The first exon contains two 5' splice sites. Depending on the 5'ss used, transcripts encoding full-length or truncated DUX4 proteins are produced. To produce the full-length isoform, the first 5'ss located at the 3' end of the first exon is used. To produce the truncated isoform, the second 5'ss located within exon 1 and closer to the 5' end of the transcript than the first 5'ss is used.
变体1、2和4共享最后一个外显子。变体1、2和4的序列如下所示。The last exon is shared by variants 1, 2 and 4. The sequences of variants 1, 2 and 4 are shown below.
变体1(DUX4-fl2)：Variant 1 (DUX4-fl2):
变体2(DUX4-fl1)：Variant 2 (DUX4-fl1):
变体4(DUX4-s)：Variant 4 (DUX4-s):
由于FSHD是由获得功能突变引起的，因此DUX4和/或DUX4c抑制是有前景的治疗策略。除此之外或另选地，由于DUX4-s已被证明是无毒的，因此通过上调DUX4-s的表达来下调DUX4-fl的表达是可能的治疗策略。然而，DUX4的许多高度同源的拷贝可存在于人类基因组中，并且D4Z4重复片段是极其富含GC的，使得DUX4和DUX4c成为困难的靶标。此时，尚没有预防或延缓FSHD患者的病情进展的疗法(Bouwman等人，Curr.Opin.Neurol.(2020),33(5):635-640)。Since FSHD is caused by gain-of-function mutations, DUX4 and/or DUX4c inhibition is a promising therapeutic strategy. In addition or alternatively, since DUX4-s has been shown to be non-toxic, downregulating the expression of DUX4-fl by upregulating the expression of DUX4-s is a possible therapeutic strategy. However, many highly homologous copies of DUX4 may exist in the human genome, and the D4Z4 repeat fragment is extremely GC-rich, making DUX4 and DUX4c difficult targets. At this time, there is no therapy to prevent or delay the progression of FSHD patients (Bouwman et al., Curr. Opin. Neurol. (2020), 33 (5): 635-640).
美国专利号10,907,157和加拿大专利号2999192描述了使用反义剂和RNA干扰剂来降低DUX4或DUX4c的表达。公布PCT US2017/019422已使用小核RNA来诱导DUX4的外显子跳跃，从而导致DUX4-s的表达。靶向DUX4的各种SE的二氨基磷酸酯吗啉代寡聚物已显示出能够改变DUX4下游基因的表达(Marsollier等人，Human molecular genetics(2016),25(8),1468-1478；以及Lu-Nguyen等人，Hum Mol Genet.(2021),30(15):1398-1412)。U.S. Patent No. 10,907,157 and Canadian Patent No. 2999192 describe the use of antisense agents and RNA interference agents to reduce the expression of DUX4 or DUX4c. PCT US2017/019422 has been published to use small nuclear RNA to induce exon skipping of DUX4, resulting in the expression of DUX4-s. Phosphorodiamidate morpholino oligomers targeting various SEs of DUX4 have been shown to be able to alter the expression of DUX4 downstream genes (Marsollier et al., Human molecular genetics (2016), 25 (8), 1468-1478; and Lu-Nguyen et al., Hum Mol Genet. (2021), 30 (15): 1398-1412).
本文提供了用于调节DUX4和/或DUX4c表达的组合物和方法。在实施方案中，提供了用于治疗FSHD的化合物。在实施方案中，提供了诱导DUX4转录物中外显子跳跃的化合物，从而导致DUX4-s而不是DUX4-fl的表达。在实施方案中，该化合物包含至少一种AC和至少一种CPP。Provided herein are compositions and methods for modulating the expression of DUX4 and/or DUX4c. In embodiments, compounds for treating FSHD are provided. In embodiments, compounds are provided that induce exon skipping in the DUX4 transcript, thereby resulting in the expression of DUX4-s instead of DUX4-fl. In embodiments, the compound comprises at least one AC and at least one CPP.
在实施方案中，AC与包含DUX4转录物的剪接元件的至少一部分的靶核苷酸序列杂交。在实施方案中，AC与包含DUX4转录物的至少一部分的靶核苷酸序列杂交并且诱导外显子跳跃以产生编码DUX4-s的转录物。在实施方案中，外显子跳跃上调DUX4-s的表达。在实施方案中，外显子跳跃下调DUX4-fl的表达。In embodiments, AC hybridizes to a target nucleotide sequence comprising at least a portion of a splicing element of a DUX4 transcript. In embodiments, AC hybridizes to a target nucleotide sequence comprising at least a portion of a DUX4 transcript and induces exon skipping to produce a transcript encoding DUX4-s. In embodiments, exon skipping upregulates expression of DUX4-s. In embodiments, exon skipping downregulates expression of DUX4-fl.
在实施方案中，提供了诱导DUX4靶转录物的选择性剪接的化合物和方法。在实施方案中，提供了将DUX4的剪接转移到第二个5'ss以产生编码截短的DUX4蛋白的转录物的化合物和方法。在实施方案中，提供了下调全长DUX4 mRNA转录物和/或蛋白质的产生的化合物和方法。在实施方案中，提供了上调截短的DUX4 mRNA转录物和/或蛋白质的产生的化合物和方法。在实施方案中，所述化合物包含AC。AC可以是任何AC并且具有如本文别处所述的任何AC特征。在实施方案中，AC是ASO。在实施方案中，ASO是PMO。AC可结合到如本文别处所述的DUX4靶转录物的任何剪接元件。In embodiments, compounds and methods are provided that induce alternative splicing of DUX4 target transcripts. In embodiments, compounds and methods are provided that shift splicing of DUX4 to a second 5'ss to produce a transcript encoding a truncated DUX4 protein. In embodiments, compounds and methods are provided that downregulate the production of full-length DUX4 mRNA transcripts and/or proteins. In embodiments, compounds and methods are provided that upregulate the production of truncated DUX4 mRNA transcripts and/or proteins. In embodiments, the compound comprises an AC. The AC can be any AC and has any AC characteristics as described elsewhere herein. In embodiments, the AC is an ASO. In embodiments, the ASO is a PMO. The AC may bind to any splicing element of a DUX4 target transcript as described elsewhere herein.
在实施方案中，AC包含公布PCT US2017/019422(美国专利号11,180,755)中的小核RNA的任何部分。在实施方案中，AC包含表12中的序列的任何部分。In an embodiment, AC comprises any portion of the small nuclear RNA published in PCT US2017/019422 (U.S. Patent No. 11,180,755). In an embodiment, AC comprises any portion of the sequence in Table 12.
表12：靶向DUX4的各种AC序列Table 12: Various AC sequences targeting DUX4
在实施方案中，AC可包含表12中任一序列的10个或更多个、15个或更多个、或20个或更多个连续碱基。在实施方案中，AC可包含表12中任一序列的25个或更少个、20个或更少个、或15个或更少个连续碱基。在实施方案中，AC可包含表12中任一序列的10-25个、10-20个或10-15个连续碱基。在实施方案中，AC可包含表12中任一序列的15-25个或10-20个连续碱基。在实施方案中，AC可包含表12中任一序列的20-25个连续碱基。In embodiments, AC may comprise 10 or more, 15 or more, or 20 or more consecutive bases of any sequence in Table 12. In embodiments, AC may comprise 25 or less, 20 or less, or 15 or less consecutive bases of any sequence in Table 12. In embodiments, AC may comprise 10-25, 10-20, or 10-15 consecutive bases of any sequence in Table 12. In embodiments, AC may comprise 15-25 or 10-20 consecutive bases of any sequence in Table 12. In embodiments, AC may comprise 20-25 consecutive bases of any sequence in Table 12.
治疗方法Treatment
本公开提供了一种治疗对其有需要的患者的疾病的方法，该方法包括施用本文公开的化合物。在实施方案中，疾病是本公开中提供的任何疾病。在实施方案中，治疗疾病的方法包括向患者施用本文公开的化合物，从而治疗疾病。在实施方案中，治疗与IRF-5、GYS1或DUX4相关的疾病的方法包括向患者施用本文公开的化合物，从而治疗疾病。The present disclosure provides a method for treating a disease in a patient in need thereof, the method comprising administering a compound disclosed herein. In an embodiment, the disease is any disease provided in the present disclosure. In an embodiment, the method for treating a disease comprises administering a compound disclosed herein to a patient, thereby treating the disease. In an embodiment, the method for treating a disease associated with IRF-5, GYS1 or DUX4 comprises administering a compound disclosed herein to a patient, thereby treating the disease.
在实施方案中，患者被鉴定为患有与IRF-5、GYS1或DUX4相关的疾病或具有患所述疾病的风险。在实施方案中，疾病或病症与IRF-5、GYS1或DUX4遗传变异相关。在实施方案中，疾病或病症与IRF-5基因、GYS1基因或DUX4基因的遗传突变相关。在实施方案中，遗传突变导致IRF-5、GYS1或DUX4(例如，DUX4-fl)的过表达。在实施方案中，遗传突变导致IRF-5、GYS1或DUX4的另选同种型的表达。在实施方案中，疾病或病症与IRF-5、GYS1或DUX4(例如，DUX4-fl)的过表达相关。In embodiments, the patient is identified as having a disease associated with IRF-5, GYS1 or DUX4 or having a risk of suffering from the disease. In embodiments, the disease or condition is associated with IRF-5, GYS1 or DUX4 genetic variation. In embodiments, the disease or condition is associated with a genetic mutation of the IRF-5 gene, the GYS1 gene or the DUX4 gene. In embodiments, the genetic mutation results in overexpression of IRF-5, GYS1 or DUX4 (e.g., DUX4-fl). In embodiments, the genetic mutation results in the expression of an alternative isoform of IRF-5, GYS1 or DUX4. In embodiments, the disease or condition is associated with overexpression of IRF-5, GYS1 or DUX4 (e.g., DUX4-fl).
在各种实施方案中，治疗是指部分或完全减轻、改善、缓解、抑制患者的一种或多种症状，延迟所述症状的发作，降低所述症状的严重程度和/或发生率。In various embodiments, treatment refers to partially or completely alleviating, ameliorating, alleviating, inhibiting, delaying the onset of, reducing the severity and/or incidence of one or more symptoms of a patient.
在实施方案中，提供了一种通过下调靶蛋白的表达来治疗疾病或病症的方法。在实施方案中，靶蛋白的表达通过诱导外显子跳跃来下调。在实施方案中，外显子跳跃诱导导致靶蛋白的表达或活性降低的移码。在实施方案中，外显子跳跃导致提前终止密码子和靶转录物的降解。在实施方案中，治疗导致蛋白质同种型的表达降低。In embodiments, a method of treating a disease or condition by downregulating the expression of a target protein is provided. In embodiments, the expression of a target protein is downregulated by inducing exon skipping. In embodiments, exon skipping induces a frameshift that results in reduced expression or activity of the target protein. In embodiments, exon skipping results in premature stop codons and degradation of the target transcript. In embodiments, treatment results in reduced expression of a protein isoform.
在实施方案中，治疗调节对其有需要的患者的IRF-5活性。在实施方案中，治疗调节患者的细胞中的IRF-5活性。在实施方案中，治疗调节患者的免疫细胞中的IRF-5活性。在实施方案中，免疫细胞是单核细胞、淋巴细胞或树突状细胞。在实施方案中，淋巴细胞是B-淋巴细胞。在实施方案中，单核细胞是巨噬细胞。在实施方案中，巨噬细胞是组织驻留巨噬细胞。在实施方案中，巨噬细胞是单核细胞衍生的巨噬细胞。在实施方案中，巨噬细胞是库普弗细胞、肾小球内系膜细胞、肺泡巨噬细胞、窦组织细胞、霍夫鲍尔细胞、小神经胶质细胞或朗格汉斯细胞。在实施方案中，免疫细胞是库普弗细胞。In embodiments, treatment modulates IRF-5 activity in patients in need thereof. In embodiments, treatment modulates IRF-5 activity in cells of patients. In embodiments, treatment modulates IRF-5 activity in immune cells of patients. In embodiments, immune cells are monocytes, lymphocytes, or dendritic cells. In embodiments, lymphocytes are B-lymphocytes. In embodiments, monocytes are macrophages. In embodiments, macrophages are tissue-resident macrophages. In embodiments, macrophages are monocyte-derived macrophages. In embodiments, macrophages are Kupffer cells, glomerular mesangial cells, alveolar macrophages, sinus tissue cells, Hofbauer cells, microglia, or Langerhans cells. In embodiments, immune cells are Kupffer cells.
在实施方案中，治疗调节对其有需要的患者的DUX4活性。在实施方案中，治疗调节患者的细胞中的DUX4活性。在实施方案中，治疗调节患者的肌细胞中的DUX4活性。在实施方案中，肌细胞是骨骼肌细胞。In embodiments, treatment modulates DUX4 activity in a patient in need thereof. In embodiments, treatment modulates DUX4 activity in a cell of a patient. In embodiments, treatment modulates DUX4 activity in a muscle cell of a patient. In embodiments, the muscle cell is a skeletal muscle cell.
在实施方案中，与治疗前的患者、未治疗的患有类似疾病的一个或多个对照个体中靶蛋白的平均水平和/或活性相比，或与使用未与本文公开的CPP缀合的治疗性部分的治疗相比，根据本公开的治疗导致患者的IRF-5、DUX4-fl或GYS1活性和/或表达降低5％至10％、5％至20％、5％至30％、5％至40％、5％至50％、5％至60％、5％至70％、5％至80％、5％至90％或5％至100％。在实施方案中，与治疗前的患者、未治疗的患有类似疾病的一个或多个对照个体中靶蛋白的平均水平和/或活性相比，或与使用未与本文公开的CPP缀合的治疗性部分的治疗相比，根据本公开的治疗导致患者的IRF-5、DUX4-fl或GYS1活性和/或表达降低10％至20％、10％至30％、10％至40％、10％至50％、10％至60％、10％至70％、10％至80％、10％至90％或10％至100％。在实施方案中，与治疗前的患者、未治疗的患有类似疾病的一个或多个对照个体中靶蛋白的平均水平和/或活性相比，或与使用未与本文公开的CPP缀合的治疗性部分的治疗相比，根据本公开的治疗导致患者的IRF-5、DUX4-fl或GYS1活性和/或表达降低20％至30％、20％至40％、20％至50％、20％至60％、20％至70％、20％至80％、20％至90％或20％至100％。在实施方案中，与治疗前的患者、未治疗的患有类似疾病的一个或多个对照个体中靶蛋白的平均水平和/或活性相比，或与使用未与本文公开的CPP缀合的治疗性部分的治疗相比，根据本公开的治疗导致患者的IRF-5、DUX4-fl或GYS1活性和/或表达降低30％至40％、30％至50％、30％至60％、30％至70％、30％至80％、30％至90％或30％至100％。在实施方案中，与治疗前的患者、未治疗的患有类似疾病的一个或多个对照个体中靶蛋白的平均水平和/或活性相比，或与使用未与本文公开的CPP缀合的治疗性部分的治疗相比，根据本公开的治疗导致患者的IRF-5、DUX4-fl或GYS1活性和/或表达降低40％至50％、40％至60％、40％至70％、40％至80％、40％至90％或40％至100％。在实施方案中，与治疗前的患者、未治疗的患有类似疾病的一个或多个对照个体中靶蛋白的平均水平和/或活性相比，或与使用未与本文公开的CPP缀合的治疗性部分的治疗相比，根据本公开的治疗导致患者的IRF-5、DUX4-fl或GYS1活性和/或表达降低50％至60％、50％至70％、50％至80％、50％至90％或50％至100％。在实施方案中，与治疗前的患者、未治疗的患有类似疾病的一个或多个对照个体中靶蛋白的平均水平和/或活性相比，或与使用未与本文公开的CPP缀合的治疗性部分的治疗相比，根据本公开的治疗导致患者的IRF-5、DUX4-fl或GYS1活性和/或表达降低60％至70％、60％至80％、60％至90％或60％至100％。在实施方案中，与治疗前的患者、未治疗的患有类似疾病的一个或多个对照个体中靶蛋白的平均水平和/或活性相比，或与使用未与本文公开的CPP缀合的治疗性部分的治疗相比，根据本公开的治疗导致患者的IRF-5、DUX4-fl或GYS1活性和/或表达降低70％至80％、70％至90％或70％至100％。在实施方案中，与治疗前的患者、未治疗的患有类似疾病的一个或多个对照个体中靶蛋白的平均水平和/或活性相比，或与使用未与本文公开的CPP缀合的治疗性部分的治疗相比，根据本公开的治疗导致患者的IRF-5、DUX4-fl或GYS1活性和/或表达降低80％至90％或80％至100％。在实施方案中，与治疗前的患者、未治疗的患有类似疾病的一个或多个对照个体中靶蛋白的平均水平和/或活性相比，或与使用未与本文公开的CPP缀合的治疗性部分的治疗相比，根据本公开的治疗导致患者的IRF-5、DUX4-fl或GYS1活性和/或表达的降低90％至100％。In embodiments, treatment in accordance with the present disclosure results in a 5% to 10%, 5% to 20%, 5% to 30%, 5% to 40%, 5% to 50%, 5% to 60%, 5% to 70%, 5% to 80%, 5% to 90%, or 5% to 100% decrease in IRF-5, DUX4-fl, or GYS1 activity and/or expression in a patient compared to the average level and/or activity of the target protein in the patient before treatment, in one or more untreated control individuals with a similar disease, or compared to treatment with a therapeutic moiety not conjugated to a CPP disclosed herein. In embodiments, treatment in accordance with the present disclosure results in a 10% to 20%, 10% to 30%, 10% to 40%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, or 10% to 100% decrease in IRF-5, DUX4-fl, or GYS1 activity and/or expression in a patient compared to the average level and/or activity of the target protein in the patient before treatment, in one or more untreated control individuals with a similar disease, or compared to treatment with a therapeutic moiety not conjugated to a CPP disclosed herein. In embodiments, treatment in accordance with the present disclosure results in a 20% to 30%, 20% to 40%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, or 20% to 100% decrease in IRF-5, DUX4-fl, or GYS1 activity and/or expression in a patient compared to the average level and/or activity of the target protein in the patient before treatment, in one or more untreated control individuals with a similar disease, or compared to treatment with a therapeutic moiety not conjugated to a CPP disclosed herein. In embodiments, treatment in accordance with the present disclosure results in a 30% to 40%, 30% to 50%, 30% to 60%, 30% to 70%, 30% to 80%, 30% to 90%, or 30% to 100% reduction in IRF-5, DUX4-fl, or GYS1 activity and/or expression in a patient compared to the average level and/or activity of the target protein in the patient before treatment, in one or more untreated control individuals with a similar disease, or compared to treatment with a therapeutic moiety not conjugated to a CPP disclosed herein. In embodiments, treatment according to the present disclosure results in a 40% to 50% reduction in IRF-5, DUX4-fl or GYS1 activity and/or expression in a patient compared to the average level and/or activity of the target protein in the patient before treatment, in one or more untreated control individuals with a similar disease, or compared to treatment with a therapeutic moiety not conjugated to a CPP disclosed herein. In embodiments, treatment according to the present disclosure results in a 50% to 60% reduction in IRF-5, DUX4-fl or GYS1 activity and/or expression in a patient compared to the average level and/or activity of the target protein in the patient before treatment, in one or more untreated control individuals with a similar disease, or compared to treatment with a therapeutic moiety not conjugated to a CPP disclosed herein. In embodiments, treatment according to the present disclosure results in a 60% to 70%, 60% to 80%, 60% to 90%, or 60% to 100% reduction in IRF-5, DUX4-fl, or GYS1 activity and/or expression in a patient compared to the average level and/or activity of the target protein in the patient before treatment, in one or more untreated control individuals with a similar disease, or compared to treatment with a therapeutic moiety not conjugated to a CPP disclosed herein. In embodiments, treatment according to the present disclosure results in a 70% to 80%, 70% to 90%, or 70% to 100% reduction in IRF-5, DUX4-fl, or GYS1 activity and/or expression in a patient compared to the average level and/or activity of the target protein in the patient before treatment, in one or more untreated control individuals with a similar disease, or compared to treatment with a therapeutic moiety not conjugated to a CPP disclosed herein. In embodiments, treatment according to the present disclosure results in a 80% to 90% or 80% to 100% reduction in IRF-5, DUX4-fl or GYS1 activity and/or expression in a patient compared to the average level and/or activity of the target protein in the patient before treatment, in one or more untreated control individuals with a similar disease, or compared to treatment with a therapeutic moiety not conjugated to a CPP disclosed herein. In embodiments, treatment according to the present disclosure results in a 90% to 100% reduction in IRF-5, DUX4-fl or GYS1 activity and/or expression in a patient compared to the average level and/or activity of the target protein in the patient before treatment, in one or more untreated control individuals with a similar disease, or compared to treatment with a therapeutic moiety not conjugated to a CPP disclosed herein.
如本文所用的术语“改善”、“增加”、“减少”、“降低”等表示相对于对照的值。在实施方案中，合适的对照是基线测量，诸如在开始本文所述的治疗之前同一个体中的测量，或在不存在本文所述的治疗的对照个体(或多个对照个体)中的测量。“对照个体”是患有相同疾病的个体，其与被治疗的个体的年龄和/或性别大致相同(以确保被治疗的个体和对照个体的疾病阶段是相当的)。As used herein, the terms "improve", "increase", "decrease", "lower" and the like refer to values relative to a control. In embodiments, a suitable control is a baseline measurement, such as a measurement in the same individual prior to starting a treatment described herein, or a measurement in a control individual (or multiple control individuals) in the absence of a treatment described herein. A "control individual" is an individual with the same disease who is approximately the same age and/or sex as the individual being treated (to ensure that the disease stage of the individual being treated and the control individual is comparable).
被治疗的个体(也称为“患者”或“受试者”)是患有疾病或具有发展疾病的可能性的个体(胎儿、婴儿、儿童、青少年或成人)。个体可能患有由异常基因表达或异常基因剪接介导的疾病。在各种实施方案中，患有疾病的个体的野生型靶蛋白表达或活性水平可低于未患该疾病的个体的正常蛋白表达或活性水平的约1％-99％。在实施方案中，该范围包括但不限于低于正常胸苷磷酸化酶表达或活性水平的约80％-99％、低于约65％-80％、低于约50％-65％、低于约30％-50％、低于约25％-30％、低于约20％-25％、低于约15％-20％、低于约10％-15％、低于约5％-10％、低于约1％-5％。在实施方案中，个体的靶蛋白表达或活性水平可以比正常野生型靶蛋白表达或活性水平高1％-500％。在实施方案中，该范围包括但不限于高约1％-10％、约10％-50％、约50％-100％、约100％-200％、约200％-300％、约300％-400％、约400％-500％或约500％-1000％。The treated individual (also referred to as a "patient" or "subject") is an individual (fetus, infant, child, adolescent or adult) who suffers from a disease or has the possibility of developing a disease. An individual may suffer from a disease mediated by abnormal gene expression or abnormal gene splicing. In various embodiments, the wild-type target protein expression or activity level of an individual suffering from a disease may be lower than about 1%-99% of the normal protein expression or activity level of an individual not suffering from the disease. In embodiments, the range includes but is not limited to about 80%-99% lower than normal thymidine phosphorylase expression or activity level, lower than about 65%-80%, lower than about 50%-65%, lower than about 30%-50%, lower than about 25%-30%, lower than about 20%-25%, lower than about 15%-20%, lower than about 10%-15%, lower than about 5%-10%, lower than about 1%-5%. In embodiments, the target protein expression or activity level of an individual may be 1%-500% higher than the normal wild-type target protein expression or activity level. In embodiments, the range includes, but is not limited to, about 1%-10% higher, about 10%-50% higher, about 50%-100% higher, about 100%-200% higher, about 200%-300% higher, about 300%-400% higher, about 400%-500% higher, or about 500%-1000% higher.
在实施方案中，个体是最近被诊断患有疾病的患者。通常，早期治疗(在确诊后尽可能快地开始治疗)减少了疾病的影响并且增加了治疗的有益效果。In embodiments, the individual is a patient who has recently been diagnosed with a disease. Typically, early treatment (starting treatment as soon as possible after diagnosis) reduces the impact of the disease and increases the beneficial effects of treatment.
组合物和施用方法Compositions and methods of administration
在实施方案中，提供了包含一种或多种本文所述化合物的组合物。In embodiments, compositions comprising one or more compounds described herein are provided.
在实施方案中，提供了所公开的化合物的药学上可接受的盐和/或前药。药学上可接受的盐包括根据化合物上发现的特定取代基用酸或碱制备的所公开的化合物的盐。在其中本文公开的化合物具有足够的碱性或酸性以形成稳定的无毒酸或碱盐的条件下，以盐的形式施用化合物可以是适当的。药学上可接受的碱加成盐的示例包括钠盐、钾盐、钙盐、铵盐或镁盐。生理上可接受的酸加成盐的示例包括盐酸、氢溴酸、硝酸、磷酸、碳酸、硫酸和有机酸如乙酸、丙酸、苯甲酸、琥珀酸、富马酸、扁桃酸、草酸、柠檬酸、酒石酸、丙二酸、抗坏血酸、α-酮戊二酸、α-糖磷酸、马来酸、甲苯磺酸、甲磺酸等。因此，本文公开了盐酸盐、硝酸盐、磷酸盐、碳酸盐、碳酸氢盐、硫酸盐、乙酸盐、丙酸盐、苯甲酸盐、琥珀酸盐、富马酸盐、扁桃酸盐、草酸盐、柠檬酸盐、酒石酸盐、丙二酸盐、抗坏血酸盐、α-酮戊二酸盐、α-糖磷酸盐、马来酸盐、甲苯磺酸盐和甲磺酸盐。化合物的药学上可接受的盐可使用本领域熟知的标准方法获得，例如，通过使足够碱性的化合物诸如胺与提供生理上可接受的阴离子的合适的酸反应。还可制备羧酸的碱金属(例如钠、钾或锂)或碱土金属(例如钙)盐。In an embodiment, a pharmaceutically acceptable salt and/or prodrug of the disclosed compound is provided. Pharmaceutically acceptable salts include salts of the disclosed compound prepared with acid or base according to the specific substituent found on the compound. Under conditions where the compound disclosed herein has sufficient alkalinity or acidity to form a stable non-toxic acid or base salt, it may be appropriate to administer the compound in the form of a salt. Examples of pharmaceutically acceptable base addition salts include sodium salts, potassium salts, calcium salts, ammonium salts, or magnesium salts. Examples of physiologically acceptable acid addition salts include hydrochloric acid, hydrobromic acid, nitric acid, phosphoric acid, carbonic acid, sulfuric acid, and organic acids such as acetic acid, propionic acid, benzoic acid, succinic acid, fumaric acid, mandelic acid, oxalic acid, citric acid, tartaric acid, malonic acid, ascorbic acid, α-ketoglutaric acid, α-sugar phosphate, maleic acid, toluenesulfonic acid, methanesulfonic acid, etc. Thus, disclosed herein are hydrochlorides, nitrates, phosphates, carbonates, bicarbonates, sulfates, acetates, propionates, benzoates, succinates, fumarates, mandelates, oxalates, citrates, tartrates, malonates, ascorbates, α-ketoglutarate, α-sugar phosphates, maleates, toluenesulfonates, and mesylates. Pharmaceutically acceptable salts of compounds can be obtained using standard methods well known in the art, for example, by reacting sufficiently basic compounds such as amines with suitable acids providing physiologically acceptable anions. Alkali metal (e.g., sodium, potassium, or lithium) or alkaline earth metal (e.g., calcium) salts of carboxylic acids can also be prepared.
所公开的化合物和含有它们的组合物的体内应用可通过本领域技术人员目前或预期已知的任何合适的方法和技术来实现。例如，所公开的化合物可被配制成生理学上或药学上可接受的形式，并通过本领域已知的任何合适的途径施用，包括例如口服和肠胃外施用途径。如本文所用，术语肠胃外包括皮下、皮内、静脉内、肌内、腹膜内、胸骨内和鞘内施用，诸如通过注射。所公开的化合物或组合物的施用可以是单次施用，或以连续或不同的间隔施用，如本领域技术人员可容易地确定的。The in vivo application of the disclosed compounds and compositions containing them can be achieved by any suitable method and technology currently or expectedly known to those skilled in the art. For example, the disclosed compounds can be formulated into physiologically or pharmaceutically acceptable forms and administered by any suitable route known in the art, including, for example, oral and parenteral routes of administration. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, intrasternal and intrathecal administration, such as by injection. The administration of the disclosed compounds or compositions can be a single administration, or administered at continuous or different intervals, as can be easily determined by those skilled in the art.
本文公开的化合物和包含它们的组合物也可利用脂质体技术、缓释胶囊、可植入泵和可生物降解容器来施用。这些递送方法可有利地在延长的时间段内提供均匀的剂量。化合物还可以其盐衍生物形式或结晶形式施用。Compounds disclosed herein and compositions comprising them can also be administered using liposome technology, sustained-release capsules, implantable pumps, and biodegradable containers. These delivery methods can advantageously provide uniform dosages over extended time periods. Compounds can also be administered in the form of their salt derivatives or in crystalline form.
本文公开的化合物可根据用于制备药学上可接受的组合物的已知方法配制。在本领域技术人员熟知且容易获得的许多来源中详细描述了制剂。例如，Remington'sPharmaceutical Science,E.W.Martin(1995)描述了可与所公开的方法结合使用的制剂。通常，本文公开的化合物可被配制成使得有效量的化合物与合适的载剂组合，以便促进化合物的有效施用。所用的组合物还可以是各种形式。这些剂型包括例如固体、半固体和液体剂型，诸如片剂、丸剂、粉剂、液体溶液或悬浮液、栓剂、可注射和可输注溶液、以及喷雾剂。形式取决于预期的施用方式和治疗应用。组合物还可包含本领域技术人员已知的常规药学上可接受的载剂和稀释剂。与化合物一起使用的载剂或稀释剂的示例包括乙醇、二甲亚砜、甘油、氧化铝、淀粉、盐水和等效载剂和稀释剂。为了提供用于期望的治疗性治疗的此类剂量的施用，基于包含载剂或稀释剂的总组合物的重量，本文公开的组合物可有利地包含总共约0.1重量％至100重量％之间的主题化合物中的一种或多种。The compounds disclosed herein can be formulated according to known methods for preparing pharmaceutically acceptable compositions. Preparations are described in detail in many sources that are well known and easily available to those skilled in the art. For example, Remington's Pharmaceutical Science, E.W.Martin (1995) describes preparations that can be used in combination with the disclosed methods. Generally, the compounds disclosed herein can be formulated so that an effective amount of the compound is combined with a suitable carrier to facilitate the effective administration of the compound. The composition used can also be in various forms. These dosage forms include, for example, solid, semisolid and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspensions, suppositories, injectable and infusible solutions, and sprays. The form depends on the intended mode of administration and therapeutic application. The composition may also contain conventional pharmaceutically acceptable carriers and diluents known to those skilled in the art. Examples of carriers or diluents used with the compound include ethanol, dimethyl sulfoxide, glycerol, aluminum oxide, starch, saline, and equivalent carriers and diluents. To provide for administration of such dosages for the desired therapeutic treatment, the compositions disclosed herein may advantageously contain a total of between about 0.1 wt % to 100 wt % of one or more of the subject compounds, based on the weight of the total composition including carrier or diluent.
适于施用的制剂包括例如无菌注射水溶液，其可含有抗氧化剂、缓冲剂、抑菌剂和使制剂与预期接受者的血液等渗的溶质；以及水性和非水性无菌悬浮液，其可包含悬浮剂和增稠剂。制剂可存在于单位剂量或多剂量容器例如密封的安瓿和小瓶中，并且可储存在冷冻干燥(冻干)条件下，在使用前仅需要无菌液体载剂(例如注射用水)的条件。临时注射溶液和悬浮液可由无菌粉末、颗粒、片剂等制备。应当理解，除了以上特别提及的成分外，本文公开的组合物还可包含本领域中关于所讨论的制剂类型的其他常规剂。Preparations suitable for administration include, for example, sterile injection aqueous solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that make the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. The preparation may be present in unit dose or multi-dose containers such as sealed ampoules and vials, and may be stored under freeze-dried (lyophilized) conditions, requiring only sterile liquid carriers (e.g., water for injection) before use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, tablets, etc. It should be understood that, in addition to the ingredients specifically mentioned above, the compositions disclosed herein may also include other conventional agents in the art for the type of preparation discussed.
本文公开的化合物和包含它们的组合物可通过与细胞直接接触或经由载剂手段递送至细胞。用于将化合物和组合物递送至细胞的载剂手段是本领域已知的，并且包括例如将组合物包封在脂质体部分中。用于将本文公开的化合物和组合物递送至细胞的另一手段包括将化合物附接到靶向递送至靶细胞的蛋白质或核酸。美国专利号6,960,648和美国申请公开号20030032594和20020120100公开了可与另一种组合物偶联并允许该组合物跨生物膜易位的氨基酸序列。美国申请公开号20020035243也描述了用于跨细胞膜转运生物部分以用于细胞内递送的组合物。也可将化合物掺入聚合物中，该聚合物的示例包括用于颅内肿瘤的聚(D-L丙交酯-共-乙交酯)聚合物；摩尔比为20:80的聚[双(对羧基苯氧基)丙烷：癸二酸](如GLIADEL中所用)；软骨素；甲壳质；和壳聚糖。Compounds disclosed herein and compositions comprising them can be delivered to cells by direct contact with cells or via carrier means. Carrier means for delivering compounds and compositions to cells are known in the art, and include, for example, encapsulating the composition in a liposome portion. Another means for delivering compounds disclosed herein and compositions to cells includes attaching the compound to a protein or nucleic acid targeted for delivery to a target cell. U.S. Patent No. 6,960,648 and U.S. Application Publication Nos. 20030032594 and 20020120100 disclose amino acid sequences that can be coupled to another composition and allow the composition to translocate across biological membranes. U.S. Application Publication No. 20020035243 also describes compositions for transcellular membrane transport of biological parts for intracellular delivery. The compounds can also be incorporated into polymers, examples of which include poly(D-L lactide-co-glycolide) polymers for intracranial tumors; poly[bis(p-carboxyphenoxy)propane:sebacic acid] at a molar ratio of 20:80 (as used in GLIADEL); chondroitin; chitin; and chitosan.
本文公开的化合物和组合物，包括其药学上可接受的盐或前药，可通过输注或注射静脉内、肌内或腹膜内施用。活性剂或其盐的溶液可在水中制备，任选地与无毒表面活性剂混合。分散体也可在甘油、液体聚乙二醇、三醋精以及它们的混合物中和在油中制备。在普通的储存和使用条件下，这些制剂可含有防腐剂以防止微生物的生长。The compounds and compositions disclosed herein, including pharmaceutically acceptable salts or prodrugs thereof, can be administered intravenously, intramuscularly or intraperitoneally by infusion or injection. Solutions of the active agent or its salt can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, triacetin and mixtures thereof and in oil. Under ordinary storage and use conditions, these preparations may contain preservatives to prevent the growth of microorganisms.
适于注射或输注的药物剂型可包括包含活性成分的无菌水溶液或分散体或无菌粉末，其适于临时制备任选地包封在脂质体中的无菌可注射或可输注溶液或分散体。最终剂型应当是无菌的、流体的并且在制造和储存条件下是稳定的。液体载剂或媒介物可以是溶剂或液体分散介质，包括例如水、乙醇、多元醇(例如，甘油、丙二醇、液体聚乙二醇等)、植物油、无毒甘油酯以及它们的合适混合物。适当的流动性可例如通过形成脂质体、在分散体的情况下通过维持所需的粒度或通过使用表面活性剂来维持。任选地，可通过各种其他抗细菌剂和抗真菌剂，例如对羟基苯甲酸酯、氯丁醇、苯酚、山梨酸、硫柳汞等来防止微生物的作用。在许多情况下，期望包含等渗剂，例如糖、缓冲液或氯化钠。通过包含延迟吸收的药剂，例如单硬脂酸铝和明胶，可实现可注射组合物的延长吸收。The pharmaceutical dosage form suitable for injection or infusion may include a sterile aqueous solution or dispersion or sterile powder containing active ingredients, which is suitable for temporary preparation of sterile injectable or infusible solutions or dispersions optionally encapsulated in liposomes. The final dosage form should be sterile, fluid and stable under manufacturing and storage conditions. The liquid carrier or vehicle can be a solvent or liquid dispersion medium, including, for example, water, ethanol, polyols (for example, glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, non-toxic glycerides and their suitable mixtures. Suitable fluidity can be maintained, for example, by forming liposomes, by maintaining the required particle size in the case of dispersions, or by using a surfactant. Optionally, various other antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc., can be used to prevent the effect of microorganisms. In many cases, it is desirable to include isotonic agents, such as sugar, buffer or sodium chloride. By including a medicament for delayed absorption, such as aluminum monostearate and gelatin, the extended absorption of injectable compositions can be achieved.
通过将所需量的本文公开的化合物和/或药剂与上文列举的各种其他成分掺入适当溶剂中，根据需要，随后过滤灭菌来制备无菌可注射溶液。就用于制备无菌可注射溶液的无菌粉末而言，制备方法包括真空干燥和冷冻干燥技术，其产生活性成分加上存在于先前无菌过滤的溶液中的任何附加的期望成分的粉末。Sterile injectable solutions are prepared by incorporating the desired amount of the compounds disclosed herein and/or agents with the various other ingredients listed above in an appropriate solvent, as required, followed by filtered sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze drying techniques, which produce a powder of the active ingredient plus any additional desired ingredient present in a previously sterile-filtered solution.
对于局部施用，本文公开的化合物和药剂可作为液体或固体形式应用。然而，通常期望将它们作为与皮肤病学可接受的载剂组合的组合物局部施用于皮肤，该载剂可以是固体或液体。本文公开的化合物和药剂以及组合物可局部施用于患者的皮肤以减小恶性或良性生长物的大小(并且可包括完全去除)，或治疗感染部位。本文公开的化合物和药剂可直接应用于生长或感染部位。在实施方案中，化合物和药剂以诸如软膏、霜剂、洗剂、溶液剂、酊剂等制剂应用于生长或感染部位。For topical application, compounds and medicaments disclosed herein can be applied as liquid or solid forms. However, it is generally desirable to apply them topically to the skin as a composition combined with a dermatologically acceptable carrier, which can be solid or liquid. Compounds and medicaments disclosed herein and compositions can be topically applied to the patient's skin to reduce the size of malignant or benign growths (and may include complete removal), or treat infection sites. Compounds and medicaments disclosed herein can be directly applied to growth or infection sites. In embodiments, compounds and medicaments are applied to growth or infection sites with preparations such as ointments, creams, lotions, solutions, tinctures, etc.
有用的固体载剂包括精细分散的固体，诸如滑石、粘土、微晶纤维素、二氧化硅、氧化铝等。有用的液体载剂包括水、醇或二醇或水-醇/二醇混合物，其中化合物可以有效水平溶解或分散，任选地借助于无毒表面活性剂。可添加佐剂诸如香料和附加的抗微生物剂以改善针对给定用途的特性。所得液体组合物可从吸收垫应用，用于浸渍绷带和其他敷料，或使用例如泵型或气溶胶喷雾器喷雾到受影响区域上。Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silicon dioxide, aluminum oxide, and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol mixtures, in which the compound can be dissolved or dispersed at effective levels, optionally with the aid of a nontoxic surfactant. Adjuvants such as fragrances and additional antimicrobial agents may be added to improve the properties for a given use. The resulting liquid composition may be applied from an absorbent pad, used to impregnate bandages and other dressings, or sprayed onto the affected area using, for example, a pump-type or aerosol sprayer.
增稠剂诸如合成聚合物、脂肪酸、脂肪酸盐和酯、脂肪醇、改性纤维素或改性矿物材料也可与液体载剂一起使用以形成可涂抹的糊剂、凝胶、软膏、肥皂等，用于直接应用于使用者的皮肤。Thickening agents such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with the liquid carrier to form spreadable pastes, gels, ointments, soaps and the like for application directly to the user's skin.
本文公开的化合物和药剂以及药物组合物的有用剂量可通过比较它们在动物模型中的体外活性和体内活性来确定。将小鼠和其他动物中的有效剂量外推至人的方法是本领域已知的。Useful dosages of the compounds and agents and pharmaceutical compositions disclosed herein can be determined by comparing their in vitro activity and in vivo activity in animal models. Methods for extrapolating effective dosages in mice and other animals to humans are known in the art.
组合物施用的剂量范围是大到足以产生影响症状或障碍的期望效果的剂量范围。剂量不应大到引起不良副作用，诸如不希望的交叉反应、过敏反应等。通常，剂量将随患者的年龄、状况、性别和疾病程度而变化，并且可由本领域技术人员确定。在任何禁忌症的情况下，剂量可由个别医师调整。剂量可变化，并且可每天一次或多次剂量施用，持续一天或几天。The dosage range of the composition administration is a dosage range large enough to produce the desired effect affecting the symptom or disorder. The dosage should not be large enough to cause adverse side effects, such as undesirable cross-reactions, allergic reactions, etc. Usually, the dosage will vary with the age, condition, sex and degree of disease of the patient, and can be determined by those skilled in the art. In the case of any contraindications, the dosage can be adjusted by individual physicians. The dosage can vary and can be administered once or multiple times a day for one or more days.
还公开了包含本文公开的化合物与药学上可接受的载剂的组合的药物组合物。在实施方案中，该药物组合物适于口服、局部或肠胃外施用。施用于患者、具体地人的剂量应当足以在合理的时间范围内在患者中实现治疗反应，而没有致死毒性，并且不会引起超过可接受水平的副作用或发病率。本领域技术人员将认识到，剂量将取决于多种因素，包括患者的状况(健康)、患者的体重、同时治疗的种类(如果有的话)、治疗频率、治疗比率以及病理状况的严重程度和阶段。Also disclosed are pharmaceutical compositions comprising a combination of a compound disclosed herein and a pharmaceutically acceptable carrier. In an embodiment, the pharmaceutical composition is suitable for oral, topical or parenteral administration. The dosage applied to a patient, specifically a human, should be sufficient to achieve a therapeutic response in the patient within a reasonable time frame without lethal toxicity, and without causing side effects or morbidity exceeding an acceptable level. Those skilled in the art will recognize that the dosage will depend on a variety of factors, including the patient's condition (health), the patient's weight, the type of simultaneous treatment (if any), the frequency of treatment, the treatment ratio, and the severity and stage of the pathological condition.
还公开了在一个或多个容器中包含本文公开的化合物的试剂盒。所公开的试剂盒可任选地包括药学上可接受的载剂和/或稀释剂。在实施方案中，试剂盒包括一种或多种如本文所述的其他组分、助剂或佐剂。在另一个实施方案中，试剂盒包括一种或多种抗癌剂，诸如本文所述的那些药剂。在实施方案中，试剂盒包括描述如何施用试剂盒的化合物或组合物的说明书或包装材料。试剂盒的容器可以是任何合适的材料，例如玻璃、塑料、金属等，并且可以是任何合适的尺寸、形状或构型。在实施方案中，本文公开的化合物和/或药剂作为固体(诸如片剂、丸剂或粉末形式)提供在试剂盒中。在另一个实施方案中，本文公开的化合物和/或药剂作为液体或溶液提供在试剂盒中。在实施方案中，试剂盒包括含有液体或溶液形式的本文公开的化合物和/或药剂的安瓿或注射器。Also disclosed is a kit comprising a compound disclosed herein in one or more containers. The disclosed kit may optionally include a pharmaceutically acceptable carrier and/or diluent. In an embodiment, the kit includes one or more other components, auxiliary agents or adjuvants as described herein. In another embodiment, the kit includes one or more anticancer agents, such as those described herein. In an embodiment, the kit includes instructions or packaging materials describing how to administer the compound or composition of the kit. The container of the kit can be any suitable material, such as glass, plastic, metal, etc., and can be any suitable size, shape or configuration. In an embodiment, the compound disclosed herein and/or the medicament are provided in the kit as a solid (such as a tablet, pill or powder form). In another embodiment, the compound disclosed herein and/or the medicament are provided in the kit as a liquid or solution. In an embodiment, the kit includes an ampoule or syringe containing a compound disclosed herein and/or the medicament in the form of a liquid or solution.
某些定义Some definitions
如在说明书和所附权利要求书中所使用的，单数形式“一个”、“一种”和“该”包括复数指示物，除非上下文另外明确指出。因此，例如，提及“一种组合物”包括两种或更多种此类组合物的混合物，提及“一种药剂”包括两种或更多种此类药剂的混合物，提及“该组分”包括两种或更多种此类组分的混合物，等等。As used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" includes mixtures of two or more such compositions, reference to "an agent" includes mixtures of two or more such agents, reference to "the component" includes mixtures of two or more such components, and so forth.
术语“约”当紧接在数值之前时意指范围(例如，该值的加或减10％)。例如，“约50”可意指45至55，“约25,000”可意指22,500至27,500等。除非本公开的上下文另外指出，或者与此类解释不一致。例如，在诸如“约49、约50、约55、…”的数值列表中，“约50”意指延伸至小于前后值之间一半间隔的范围，例如，大于49.5至小于52.5。此外，短语“小于约”值或“大于约”值应当根据本文提供的术语“约”的定义来理解。类似地，术语“约”当在一系列数值或数值范围(例如，“约10、20、30”或“约10-30”)之前时分别指该系列中的所有数值或该范围的端点。The term "about" means a range (e.g., plus or minus 10% of the value) when it is immediately before a numerical value. For example, "about 50" may mean 45 to 55, "about 25,000" may mean 22,500 to 27,500, etc. Unless otherwise indicated by the context of the present disclosure, or inconsistent with such an explanation. For example, in a numerical list such as "about 49, about 50, about 55, ... ", "about 50" means a range extending to less than half the interval between the preceding and following values, for example, greater than 49.5 to less than 52.5. In addition, the phrase "less than about" value or "greater than about" value should be understood according to the definition of the term "about" provided herein. Similarly, the term "about" refers to all numerical values in the series or the endpoints of the range when it is before a series of numerical values or numerical ranges (e.g., "about 10, 20, 30" or "about 10-30"), respectively.
如本文所用，“细胞穿透肽”或“CPP”是指促进将货物例如治疗性部分(TM)递送到细胞中的肽。在实施方案中，CPP是环状的并且表示为“cCPP”。在实施方案中，cCPP能够引导治疗性部分穿透细胞膜。在实施方案中，cCPP将治疗性部分递送到细胞的胞质溶胶。在实施方案中，cCPP将反义化合物(AC)递送到前mRNA所在的细胞位置。As used herein, "cell penetrating peptide" or "CPP" refers to a peptide that facilitates the delivery of a cargo, such as a therapeutic moiety (TM), into a cell. In embodiments, the CPP is cyclic and is denoted "cCPP". In embodiments, the cCPP is capable of directing the therapeutic moiety to penetrate a cell membrane. In embodiments, the cCPP delivers the therapeutic moiety to the cytosol of a cell. In embodiments, the cCPP delivers an antisense compound (AC) to the cellular location of the pre-mRNA.
如本文所用，术语“内体逃逸载体”(EEV)是指通过化学连接(即，共价键或非共价相互作用)与接头和/或环外肽(EP)缀合的cCPP。EEV可以是式(B)的EEV。As used herein, the term "endosomal escape vector" (EEV) refers to a cCPP conjugated to a linker and/or an exocyclic peptide (EP) by chemical linkage (ie, covalent bond or non-covalent interaction). The EEV may be an EEV of formula (B).
如本文所用，术语“EEV-缀合物”是指通过化学连接(即，共价键或非共价相互作用)与货物缀合的本文定义的内体逃逸载体。货物可以是可通过EEV递送到细胞中的治疗性部分(例如，寡核苷酸、肽或小分子)。EEV-缀合物可以是式(C)的EEV-缀合物。As used herein, the term "EEV-conjugate" refers to an endosomal escape vector as defined herein that is conjugated to a cargo by chemical linkage (i.e., covalent bond or non-covalent interaction). The cargo may be a therapeutic moiety (e.g., an oligonucleotide, peptide, or small molecule) that can be delivered into a cell by an EEV. The EEV-conjugate may be an EEV-conjugate of formula (C).
如本文所用，术语“环外肽”(EP)和“调节肽”(MP)可互换使用，是指通过肽键连接的两个或更多个氨基酸残基，其可与本文公开的环状细胞穿透肽(cCPP)缀合。当与本文公开的环肽缀合时，EP可改变化合物的组织分布和/或保留。典型地，EP包含至少一个带正电荷的氨基酸残基，例如至少一个赖氨酸残基和/或至少一个精氨酸残基。本文描述了EP的非限制性示例。EP可以是在本领域中被鉴定为“核定位序列”(NLS)的肽。核定位序列的非限制性示例包括SV40病毒大T抗原的核定位序列，其最小功能单元是七个氨基酸的序列PKKKRKV(SEQ ID NO:42)、具有序列NLSKRPAAIKKAGQAKKKK(SEQ ID NO:52)的双分型核质蛋白NLS、具有氨基酸序列PAAKRVKLD(SEQ ID NO:53)或RQRRNELKRSF(SEQ ID NO:54)的c-myc核定位序列、来自输入蛋白-α的IBB域的序列RMRKFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV(SEQID NO:50)、肌瘤T蛋白的序列VSRKRPRP(SEQ ID NO:57)和PPKKARED(SEQ ID NO:58)、人p53的序列PQPKKKPL(SEQ ID NO:59)、小鼠c-abl IV的序列SALIKKKKKMAP(SEQ ID NO:60)、流感病毒NS1的序列DRLRR(SEQ ID NO:61)和PKQKKRK(SEQ ID NO:62)、肝炎病毒δ抗原的序列RKLKKKIKKL(SEQ ID NO:63)和小鼠Mxl蛋白的序列REKKKFLKRR(SEQ ID NO:64)、人聚(ADP-核糖)聚合酶的序列KRKGDEVDGVDEVAKKKSKK(SEQ ID NO:65)和类固醇激素受体(人)糖皮质激素的序列RKCLQAGMNLEARKTKK(SEQ ID NO:66)。国际公开号2001/038547描述了NLS的附加示例并且全文以引用方式并入本文。As used herein, the terms "exocyclic peptide" (EP) and "modulatory peptide" (MP) are used interchangeably and refer to two or more amino acid residues connected by a peptide bond, which can be conjugated to the cyclic cell penetrating peptide (cCPP) disclosed herein. When conjugated to the cyclic peptide disclosed herein, the EP can change the tissue distribution and/or retention of the compound. Typically, the EP comprises at least one positively charged amino acid residue, such as at least one lysine residue and/or at least one arginine residue. Non-limiting examples of EP are described herein. The EP can be a peptide identified in the art as a "nuclear localization sequence" (NLS). Non-limiting examples of nuclear localization sequences include the nuclear localization sequence of the SV40 virus large T antigen, whose minimum functional unit is the seven amino acid sequence PKKKRKV (SEQ ID NO:42), the bipartite nucleoplasmic protein NLS having the sequence NLSKRPAA IKKAGQAKKKK (SEQ ID NO:52), the c-myc nuclear localization sequence having the amino acid sequence PAAKRVKLD (SEQ ID NO:53) or RQRRNELKRSF (SEQ ID NO:54), the sequence RMRKFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO:50) from the IBB domain of importin-α, the sequences VSRKRPRP (SEQ ID NO:57) and PPKKARED (SEQ ID NO:58) of myoma T protein, the sequence PQPKKKPL (SEQ ID NO:59) of human p53, the sequence SALIKKKKKMAP (SEQ ID NO:60) of mouse c-abl IV. NO:60), the sequences DRLRR (SEQ ID NO:61) and PKQKKRK (SEQ ID NO:62) of influenza virus NS1, the sequence RKLKKKIKKL (SEQ ID NO:63) of the hepatitis virus delta antigen, and the sequence REKKKFLKRR (SEQ ID NO:64) of the mouse Mxl protein, the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO:65) of human poly (ADP-ribose) polymerase, and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO:66) of the steroid hormone receptor (human) glucocorticoid. Additional examples of NLSs are described in International Publication No. 2001/038547 and are incorporated herein by reference in their entirety.
如本文所用，“接头”或“L”是指将一个或多个部分(例如，环外肽(EP)和货物，例如寡核苷酸、肽或小分子)与环状细胞穿透肽(cCPP)共价结合的部分。接头可包含天然或非天然氨基酸或多肽。接头可以是含有两个或更多个适于将cCPP结合到货物部分从而形成本文公开的化合物的适当官能团的合成化合物。接头可包含聚乙二醇(PEG)部分。接头可包含一个或多个氨基酸。cCPP可经由接头与货物共价结合。As used herein, "linker" or "L" refers to a moiety that covalently binds one or more moieties (e.g., an exocyclic peptide (EP) and a cargo, such as an oligonucleotide, peptide, or small molecule) to a cyclic cell penetrating peptide (cCPP). The linker may comprise a natural or non-natural amino acid or polypeptide. The linker may be a synthetic compound containing two or more suitable functional groups suitable for binding the cCPP to the cargo moiety to form a compound disclosed herein. The linker may comprise a polyethylene glycol (PEG) moiety. The linker may comprise one or more amino acids. The cCPP may be covalently bound to the cargo via a linker.
术语“肽”、“蛋白质”和“多肽”可互换使用，是指包含通过一个氨基酸的羧基基团与另一个氨基酸的α氨基基团连接的两个或更多个氨基酸的天然或合成分子。两个或更多个氨基酸残基可通过一个氨基酸的羧基基团与α氨基基团连接。多肽的两个或更多个氨基酸可通过肽键接合。多肽可包括肽主链修饰，其中两个或更多个氨基酸通过除肽键以外的键共价连接。多肽可包括一种或多种非天然氨基酸、氨基酸类似物或能够整合到多肽中的其他合成分子。术语多肽包括天然存在的和人工存在的氨基酸。术语多肽包括例如包含约2至约100个氨基酸残基的肽以及包含超过约100个氨基酸残基或超过约1000个氨基酸残基的蛋白质，包括但不限于治疗性蛋白质，诸如抗体、酶、受体、可溶性蛋白质等。The terms "peptide", "protein" and "polypeptide" are used interchangeably and refer to natural or synthetic molecules comprising two or more amino acids connected by the carboxyl group of one amino acid to the alpha amino group of another amino acid. Two or more amino acid residues can be connected to the alpha amino group through the carboxyl group of one amino acid. Two or more amino acids of a polypeptide can be joined by peptide bonds. A polypeptide may include a peptide backbone modification in which two or more amino acids are covalently linked by a bond other than a peptide bond. A polypeptide may include one or more non-natural amino acids, amino acid analogs, or other synthetic molecules that can be incorporated into a polypeptide. The term polypeptide includes naturally occurring and artificially occurring amino acids. The term polypeptide includes, for example, peptides comprising about 2 to about 100 amino acid residues and proteins comprising more than about 100 amino acid residues or more than about 1000 amino acid residues, including but not limited to therapeutic proteins, such as antibodies, enzymes, receptors, soluble proteins, etc.
如本文所用，术语“邻接”是指通过共价键连接的两个氨基酸。例如，在代表性环状细胞穿透肽(cCPP)诸如AA1/AA2、AA2/AA3、AA3/AA4和AA5/AA1的情况下举例说明了邻接氨基酸对。As used herein, the term "adjacent" refers to two amino acids linked by a covalent bond. For example, in representative cyclic cell penetrating peptides (cCPPs) such as Contiguous amino acid pairs are exemplified in the cases of AA1 /AA2 , AA2 /AA3 , AA3 /AA4 and AA5 /AA1 .
如本文所用，化学物质的残基是指存在于特定产物中的化学物质的衍生物。为了形成产物，该物质的至少一个原子被与另一部分的键取代，使得产物含有该化学物质的衍生物或残基。例如，本文所述的环状细胞穿透肽(cCPP)具有通过形成一个或多个肽键而掺入其中的氨基酸(例如，精氨酸)。掺入cCPP中的氨基酸可称为残基，或简单地称为氨基酸。因此，精氨酸或精氨酸残基是指As used herein, a residue of a chemical substance refers to a derivative of a chemical substance present in a particular product. To form the product, at least one atom of the substance is replaced by a bond to another moiety, such that the product contains a derivative or residue of the chemical substance. For example, the cyclic cell penetrating peptides (cCPPs) described herein have an amino acid (e.g., arginine) incorporated therein by forming one or more peptide bonds. The amino acid incorporated into the cCPP can be referred to as a residue, or simply an amino acid. Thus, arginine or an arginine residue refers to
术语“其质子化形式”是指氨基酸的质子化形式。例如，精氨酸侧链上的胍基可被质子化以形成胍基团。质子化形式的精氨酸的结构是The term "protonated form thereof" refers to the protonated form of an amino acid. For example, the guanidine group on the side chain of arginine can be protonated to form guanidine. The structure of the protonated form of arginine is
如本文所用，术语“手性”是指具有多于一种在原子的三维空间排列上不同的立体异构体的分子，其中一种立体异构体是另一种立体异构体的不可重叠的镜像。除了甘氨酸外，氨基酸具有与羧基基团相邻的手性碳原子。术语“对映体”是指手性的立体异构体。手性分子可以是具有“D”和“L”对映体的氨基酸残基。没有手性中心的分子，诸如甘氨酸，可被称为“非手性的”。As used herein, the term "chiral" refers to molecules having more than one stereoisomer that differs in the three-dimensional spatial arrangement of the atoms, wherein one stereoisomer is a non-superimposable mirror image of another stereoisomer. With the exception of glycine, amino acids have chiral carbon atoms adjacent to the carboxyl group. The term "enantiomer" refers to a chiral stereoisomer. A chiral molecule can be an amino acid residue having "D" and "L" enantiomers. Molecules without chiral centers, such as glycine, can be referred to as "achiral".
如本文所用，术语“疏水的”是指不溶于水或在水中溶解度极小的部分。通常，中性部分和/或非极性部分，或者主要是中性和/或非极性的部分是疏水的。疏水性可通过本文公开的方法之一来测量。As used herein, the term "hydrophobic" refers to a portion that is insoluble or has minimal solubility in water. Typically, a neutral portion and/or a non-polar portion, or a predominantly neutral and/or non-polar portion is hydrophobic. Hydrophobicity can be measured by one of the methods disclosed herein.
如本文所用，“芳族”是指具有4n+2π电子的不饱和环分子，其中n是任何整数。术语“非芳族”是指不属于芳族定义的任何不饱和环分子。As used herein, "aromatic" refers to an unsaturated ring molecule having 4n+2π electrons, where n is any integer. The term "non-aromatic" refers to any unsaturated ring molecule that does not fall within the definition of aromatic.
“烷基”、“烷基链”或“烷基基团”是指具有一至四十个碳原子且通过单键与分子的其余部分连接的完全饱和的直链或支链烃链基团。包括包含1至40个碳原子的任何数量的烷基。包含至多40个碳原子的烷基是C1-C40烷基，包含至多10个碳原子的烷基是C1-C10烷基，包含至多6个碳原子的烷基是C1-C6烷基，并且包含至多5个碳原子的烷基是C1-C5烷基。C1-C5烷基包括C5烷基、C4烷基、C3烷基、C2烷基和C1烷基(即，甲基)。C1-C6烷基包括上文对于C1-C5烷基所述的所有部分，但也包括C6烷基。C1-C10烷基包括上文对于C1-C5烷基和C1-C6烷基所述的所有部分，但也包括C7、C8、C9和C10烷基。类似地，C1-C12烷基包括所有前述部分，但也包括C11和C12烷基。C1-C12烷基的非限制性示例包括甲基、乙基、正丙基、异丙基、仲丙基、正丁基、异丁基、仲丁基、叔丁基、正戊基、叔戊基、正己基、正庚基、正辛基、正壬基、正癸基、正十一烷基和正十二烷基。除非在说明书中另有具体说明，否则烷基基团可任选地被取代。"Alkyl", "alkyl chain" or "alkyl group" refers to a fully saturated straight or branched hydrocarbon chain radical having from one to forty carbon atoms and connected to the rest of the molecule by a single bond. Any number of alkyl groups containing from 1 to 40 carbon atoms are included. Alkyl groups containing up to 40 carbon atoms are C1 -C40 alkyl groups, alkyl groups containing up to 10 carbon atoms are C1 -C10 alkyl groups, alkyl groups containing up to 6 carbon atoms are C1 -C6 alkyl groups, and alkyl groups containing up to 5 carbon atoms are C1 -C5 alkyl groups. C1 -C5 alkyl groups include C5 alkyl groups, C4 alkyl groups, C3 alkyl groups, C2 alkyl groups, and C1 alkyl groups (i.e., methyl groups). C1 -C6 alkyl groups include all of the moieties described above for C1 -C5 alkyl groups, but also include C6 alkyl groups. C1 -C10 alkyl includes all moieties described above for C1 -C5 alkyl and C1 -C6 alkyl, but also includes C7 , C8 , C9 and C10 alkyl. Similarly, C1 -C12 alkyl includes all of the aforementioned moieties, but also includes C11 and C12 alkyl. Non-limiting examples of C1 -C12 alkyl include methyl, ethyl, n-propyl, isopropyl, sec-propyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, tert-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl and n-dodecyl. Unless otherwise specifically stated in the specification, the alkyl group may be optionally substituted.
“亚烷基”、“亚烷基链”或“亚烷基基团”是指具有一至四十个碳原子的完全饱和的直链或支链二价烃链基团。C2-C40亚烷基的非限制性示例包括亚乙基、亚丙基、正亚丁基、亚乙烯基、亚丙烯基、正亚丁烯基、亚丙炔基、正亚丁炔基等。除非在说明书中另有具体说明，否则亚烷基链可任选地被取代。"Alkylene", "alkylene chain" or "alkylene group" refers to a fully saturated straight or branched divalent hydrocarbon chain group having one to forty carbon atoms. Non-limiting examples ofC2 -C40 alkylene include ethylene, propylene, n-butylene, vinylene, propenylene, n-butylene, propynylene, n-butylene, etc. Unless otherwise specifically stated in the specification, the alkylene chain may be optionally substituted.
“烯基”、“烯基链”或“烯基基团”是指具有二至四十个碳原子并具有一个或多个碳-碳双键的直链或支链烃链基团。每个烯基基团通过单键与分子的其余部分附接。包括包含2至40个碳原子的任何数量的烯基基团。包含至多40个碳原子的烯基是C2-C40烯基，包含至多10个碳原子的烯基是C2-C10烯基，包含至多6个碳原子的烯基是C2-C6烯基，并且包含至多5个碳原子的烯基是C2-C5烯基。C2-C5烯基包括C5烯基、C4烯基、C3烯基和C2烯基。C2-C6烯基包括上文关于C2-C5烯基所述的所有部分，但也包括C6烯基。C2-C10烯基包括上文对于C2-C5烯基和C2-C6烯基所述的所有部分，但也包括C7、C8、C9和C10烯基。类似地，C2-C12烯基包括所有前述部分，但也包括C11和C12烯基。C2-C12烯基的非限制性示例包括乙烯基(ethenyl/vinyl))、1-丙烯基、2-丙烯基(烯丙基)、异丙烯基、2-甲基-1-丙烯基、1-丁烯基、2-丁烯基、3-丁烯基、1-戊烯基、2-戊烯基、3-戊烯基、4-己烯基、1-己烯基、2-己烯基、3-己烯基、4-己烯基、5-己烯基、1-庚烯基、2-庚烯基、3-庚烯基、4-庚烯基、5-庚烯基、6-庚烯基、1-辛烯基、2-辛烯基、3-辛烯基、4-辛烯基、5-辛烯基、6-辛烯基、7-辛烯基、1-壬烯基、2-壬烯基、3-壬烯基、4-壬烯基、5-壬烯基、6-壬烯基、7-壬烯基、8-壬烯基、1-癸烯基、2-癸烯基、3-癸烯基、4-癸烯基、5-癸烯基、6-癸烯基、7-癸烯基、8-癸烯基、9-癸烯基、1-十一烯基、2-十一烯基、3-十一烯基、4-十一烯基、5-十一烯基、6-十一烯基、7-十一烯基、8-十一烯基、9-十一烯基、10-十一烯基、1-十二烯基、2-十二烯基、3-十二烯基、4-十二烯基、5-十二烯基、6-十二烯基、7-十二烯基、8-十二烯基、9-十二烯基、10-十二烯基和11-十二烯基。除非在说明书中另有具体说明，否则烷基基团可任选地被取代。"Alkenyl", "alkenyl chain" or "alkenyl group" refers to a straight or branched hydrocarbon chain group having from two to forty carbon atoms and having one or more carbon-carbon double bonds. Each alkenyl group is attached to the rest of the molecule by a single bond. Any number of alkenyl groups containing from 2 to 40 carbon atoms are included. Alkenyl groups containing up to 40 carbon atoms areC2 -C40 alkenyl, alkenyl groups containing up to 10 carbon atoms areC2 -C10 alkenyl, alkenyl groups containing up to 6 carbon atoms areC2 -C6 alkenyl, and alkenyl groups containing up to 5 carbon atoms areC2 -C5 alkenyl.C2 -C5 alkenyl includesC5 alkenyl,C4 alkenyl,C3 alkenyl andC2 alkenyl.C2 -C6 alkenyl includes all of the moieties described above forC2 -C5 alkenyl, but also includesC6 alkenyl.C2 -C10 alkenyl includes all of the moieties described above for C2-C5 alkenyl andC2 -C6 alkenyl, but also includesC7 ,C8 ,C9 , andC10 alkenyl. Similarly,C2 -C12 alkenyl includes all of the aforementioned moieties, but also includesC11 andC12 alkenyl. Non-limiting examples ofalkenyl include ethenyl (vinyl), 1-propenyl, 2-propenyl (allyl), isopropenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-hexenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 4-heptenyl, 5-heptenyl, 6-heptenyl, 1-octenyl, 2-octenyl, 3-octenyl, 4-octenyl, 5-octenyl, 6-octenyl, 7-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 4-nonenyl, 5-nonenyl, 6-nonenyl, 7-nonenyl, 8-nonenyl, 1-decenyl, 2-decenyl, 3-decenyl, 4-decenyl, 5-decenyl, 6-decenyl, 7-decenyl, 8-decenyl, 9-decenyl, 1-undecenyl, 2-undecenyl, 3-undecenyl, 4-undecenyl, 5-undecenyl, 6-undecenyl, 7-undecenyl, 8-undecenyl, 9-undecenyl, 10-undecenyl, 1-dodecenyl, 2-dodecenyl, 3-dodecenyl, 4-dodecenyl, 5-dodecenyl, 6-dodecenyl, 7-dodecenyl, 8-dodecenyl, 9-dodecenyl, 10-dodecenyl and 11-dodecenyl. Unless otherwise specifically stated in the specification, the alkyl group may be optionally substituted.
“亚烯基”、“亚烯基链”或“亚烯基基团”是指具有二至四十个碳原子并且具有一个或多个碳-碳双键的直链或支链二价烃链基团。C2-C40亚烯基的非限制性示例包括乙烯、丙烯、丁烯等。除非在说明书中另有具体说明，否则亚烯基链可以是任选的。"Alkenylene", "alkenylene chain" or "alkenylene group" refers to a straight or branched divalent hydrocarbon chain group having two to forty carbon atoms and having one or more carbon-carbon double bonds. Non-limiting examples ofC2 -C40 alkenylene include ethylene, propylene, butene, etc. Unless specifically stated otherwise in the specification, the alkenylene chain may be optional.
“烷氧基”或“烷氧基基团”是指基团-OR，其中R是如本文所定义的烷基、烯基、炔基、环烷基或杂环基。除非在说明书中另有具体说明，否则烷氧基基团可任选地被取代。"Alkoxy" or "alkoxy group" refers to a group -OR, wherein R is alkyl, alkenyl, alkynyl, cycloalkyl or heterocyclyl as defined herein. Unless stated otherwise specifically in the specification, an alkoxy group may be optionally substituted.
“酰基”或“酰基基团”是指基团-C(O)R，其中R是氢、烷基、烯基、炔基、碳环基或杂环基，如本文所定义。除非在说明书中另有具体说明，否则酰基可任选地被取代。"Acyl" or "acyl group" refers to the group -C(O)R, wherein R is hydrogen, alkyl, alkenyl, alkynyl, carbocyclyl or heterocyclyl, as defined herein. Unless stated otherwise specifically in the specification, an acyl group may be optionally substituted.
“烷基氨基甲酰基”或“烷基氨基甲酰基基团”是指基团-O-C(O)-NRaRb，其中Ra和Rb相同或不同并且独立地是如本文所定义的烷基、烯基、炔基、芳基、杂芳基，或RaRb可合一起形成如本文所定义的环烷基基团或杂环基基团。除非在说明书中另有具体说明，否则烷基氨基甲酰基基团可任选地被取代。"Alkylcarbamoyl" or "alkylcarbamoyl group" refers to the group -OC(O)-NRaRb, wherein Ra and Rbarethesame or different and are independently alkyl,alkenyl , alkynyl, aryl, heteroaryl as defined herein, orRaRb may be taken together to form a cycloalkyl group or a heterocyclyl group as defined herein. Unless stated otherwise specifically in the specification, an alkylcarbamoyl group may be optionally substituted.
“烷基羧酰胺基”或“烷基羧酰胺基基团”是指基团-C(O)-NRaRb，其中Ra和Rb相同或不同并且独立地是如本文所定义的烷基、烯基、炔基、芳基、杂芳基、环烷基、环烯基、环炔基或杂环基基团，或RaRb可合一起形成如本文所定义的环烷基基团。除非在说明书中另有具体说明，否则烷基羧酰胺基基团可任选地被取代。"Alkylcarboxamido" or "alkylcarboxamido group" refers to the group -C(O)-NRaRb , whereinRa andRb are the same or different and are independently alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkynyl or heterocyclyl groups as defined herein, orRaRb may be takentogether to form acycloalkyl group as defined herein. Unless otherwise specifically stated in the specification, an alkylcarboxamido group may be optionally substituted.
“芳基”是指包含氢、6至18个碳原子和至少一个芳环的烃环体系基团。为了本发明的目的，芳基基团可以是单环、双环、三环或四环环体系，其可包括稠环或桥环体系。芳基包括但不限于衍生自醋蒽烯(aceanthrylene)、苊烯(acenaphthylene)、醋菲烯(acephenanthrylene)、蒽、薁(azulene)、苯、屈(chrysene)、荧蒽(fluoranthene)、芴、不对称引达省(as-indacene)、对称引达省(s-indacene)、茚满、茚、萘、非那烯(phenalene)、菲、七曜烯(pleiadene)、芘和苯并菲的芳基基团。除非在说明书中另有具体说明，否则术语“芳基”意指包括任选取代的芳基基团。"Aryl" refers to a hydrocarbon ring system radical comprising hydrogen, 6 to 18 carbon atoms and at least one aromatic ring. For purposes of the present invention, an aryl group may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include a fused ring or a bridged ring system. Aryl includes, but is not limited to, aryl groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, pleiadene, pyrene and triphenylene. Unless otherwise specifically stated in the specification, the term "aryl" is intended to include an optionally substituted aryl group.
“杂芳基”是指包含氢原子、一至十三个碳原子、一至六个选自氮、氧和硫的杂原子和至少一个芳环的5至20元环体系基团。为了本发明的目的，杂芳基基团可以是单环、双环、三环或四环环体系，其可包括稠环或桥环体系；并且杂芳基基团中的氮、碳或硫原子可任选地被氧化；氮原子可任选地被季铵化。示例包括但不限于氮杂环庚烯基、吖啶基、苯并咪唑基、苯并噻唑基、苯并吲哚基、苯并二氧杂环戊烯基、苯并呋喃基、苯并唑基、苯并噻唑基、苯并噻二唑基、苯并[b][1,4]二氧杂环庚烯基、1,4-苯并二烷基、苯并萘并呋喃基、苯并唑基、苯并二氧杂环戊烯基、苯并二氧杂环己烯基、苯并吡喃基、苯并吡喃酮基、苯并呋喃基、苯并呋喃酮基、苯并噻吩基(苯并苯硫基)、苯并三唑基、苯并[4,6]咪唑并[1,2-a]吡啶基、咔唑基、噌啉基、二苯并呋喃基、二苯并噻吩基、呋喃基、呋喃酮基、异噻唑基、咪唑基、吲唑基、吲哚基、吲唑基、异吲哚基、吲哚啉基、异吲哚啉基、异喹啉基、中氮茚基、异唑基、萘啶基、二唑基、2-氧代氮杂环庚烯基、唑基、环氧乙烷基、1-氧吡啶基、1-氧化嘧啶基、1-氧化吡嗪基、1-氧化哒嗪基、1-苯基-1H-吡咯基、吩嗪基、吩噻嗪基、吩嗪基、酞嗪基、蝶啶基、嘌呤基、吡咯基、吡唑基、吡啶基、吡嗪基、嘧啶基、哒嗪基、喹唑啉基、喹喔啉基、喹啉基、奎宁环基、异喹啉基、四氢喹啉基、噻唑基、噻二唑基、三唑基、四唑基、三嗪基和苯硫基(即噻吩基)。除非在说明书中另有具体说明，否则杂芳基基团可任选地被取代。"Heteroaryl" refers to a 5- to 20-membered ring system radical containing hydrogen atoms, one to thirteen carbon atoms, one to six heteroatoms selected from nitrogen, oxygen and sulfur, and at least one aromatic ring. For the purposes of the present invention, the heteroaryl group can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heteroaryl group can be optionally oxidized; the nitrogen atom can be optionally quaternized. Examples include, but are not limited to, azepanyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzo oxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxadiazole Alkyl, benzonaphthofuranyl, benzo oxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyrone, benzofuranyl, benzofuranone, benzothiophenyl (benzophenylthio), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanone, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolyl, isoindolyl, isoquinolyl, indolizine, isothiazolyl, Azolyl, naphthyridinyl, Oxazolyl, 2-oxoazepinene, oxazolyl, oxirane, 1-oxypyridyl, 1-oxypyrimidyl, 1-oxypyrazinyl, 1-oxypyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenazinyl The heteroaryl groups include thiophenyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, and thiophenyl (i.e., thienyl). Unless otherwise specifically stated in the specification, heteroaryl groups may be optionally substituted.
本文所用的术语“取代的”意指其中至少一个原子被非氢原子替换的任何上述基团(即，烷基、烯基、炔基、环烷基、环烯基、环炔基、杂环基、芳基、杂芳基、烷氧基、芳氧基、酰基、烷基氨基甲酰基、烷基羧酰胺基、烷氧羰基、烷硫基或芳硫基)，该非氢原子诸如但不限于：卤素原子，诸如F、Cl、Br和I；基团诸如羟基基团、烷氧基基团和酯基团等中的氧原子；基团诸如硫醇基团、硫代烷基基团、砜基团、磺酰基基团和亚砜基团中的硫原子；基团诸如胺、酰胺、烷基胺、二烷基胺、芳基胺、烷基芳基胺、二芳基胺、N-氧化物、酰亚胺和烯胺中的氮原子；基团诸如三烷基甲硅烷基基团、二烷基芳基甲硅烷基基团、烷基二芳基甲硅烷基基团和三芳基甲硅烷基基团中的硅原子；以及各种其他基团中的其他杂原子。“取代的”还意指其中一个或多个原子被与杂原子(诸如氧代、羰基、羧基和酯基团中的氧)的高阶键(例如双键或三键)替换的任何上述基团；以及基团诸如亚胺、肟、腙和腈中的氮。例如，“取代的”包括其中一个或多个原子被-NRgRh、-NRgC(＝O)Rh、-NRgC(＝O)NRgRh、-NRgC(＝O)ORh、-NRgSO2Rh、-OC(＝O)NRgRh、-ORg、-SRg、-SORg、-SO2Rg、-OSO2Rg、-SO2ORg、＝NSO2Rg和-SO2NRgRh替换的任何上述基团。“取代的”还意指其中一个或多个氢原子被-C(＝O)Rg、-C(＝O)ORg、-C(＝O)NRgRh、-CH2SO2Rg、-CH2SO2NRgRh替换的任何上述基团。在上文中，Rg和Rh相同或不同，并且独立地是氢、烷基、烯基、炔基、烷氧基、烷基氨基、硫代烷基、芳基、芳烷基、环烷基、环烯基、环炔基、环烷基烷基、卤代烷基、卤代烯基、卤代炔基、杂环基、N-杂环基、杂环基烷基、杂芳基、N-杂芳基和/或杂芳基烷基。“取代的”还意指其中一个或多个原子被氨基、氰基、羟基、亚氨基、硝基、氧代、硫代、卤代、烷基、烯基、炔基、烷氧基、烷基氨基、硫代烷基、芳基、芳烷基、环烷基、环烯基、环炔基、环烷基烷基、卤代烷基、卤代烯基、卤代炔基、杂环基、N-杂环基、杂环基烷基、杂芳基、N-杂芳基和/或杂芳基烷基替换的任何上述基团。“取代的”还可意指侧链上的一个或多个原子被烷基、烯基、炔基、酰基、烷基羧酰胺基、烷氧羰基、碳环基、杂环基、芳基或杂芳基替换的氨基酸。此外，前述取代基中的每一者还可任选地被上述取代基中的一者或多者取代。As used herein, the term "substituted" means any of the above groups (i.e., alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkoxy, aryloxy, acyl, alkylcarbamoyl, alkylcarboxamido, alkoxycarbonyl, alkylthio, or arylthio) in which at least one atom is replaced by a non-hydrogen atom, such as, but not limited to, halogen atoms such as F, Cl, Br, and I; oxygen atoms in groups such as hydroxyl groups, alkoxy groups, and ester groups; sulfur atoms in groups such as thiol groups, thioalkyl groups, sulfone groups, sulfonyl groups, and sulfoxide groups; nitrogen atoms in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines, N-oxides, imides, and enamines; silicon atoms in groups such as trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, and triarylsilyl groups; and other heteroatoms in various other groups. "Substituted" also means any of the above groups wherein one or more atoms are replaced by a higher order bond (e.g., a double or triple bond) to a heteroatom (such as oxo, carbonyl, carboxyl, and oxygen in ester groups); and nitrogen in groups such as imines, oximes, hydrazones, and nitriles. For example, "substituted" includes any of the abovegroups wherein oneormore atoms are replacedby-NRgRh,-NRgC (=O)Rh ,-NRgC( =O)NRgRh , -NRgC(=O)ORh , -NRgSO2Rh, -OC( =O )NRgRh ,-ORg, -SRg ,-SORg ,-SO2Rg ,-OSO2Rg ,-SO2ORg , =NSO2Rg,and-SO2NRgRh . "Substituted"also means any of the above groups wherein one or more hydrogen atoms are replaced by-C (=O)Rg , -C(=O)ORg , -C(=O)NRgRh, -CH2SO2Rg,-CH2SO2NRgRh . In the above,Rg andRh are the same or different and are independently hydrogen, alkyl,alkenyl , alkynyl, alkoxy, alkylamino, thioalkyl,aryl , aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkyl. "Substituted" also means any of the above groups in which one or more atoms are replaced by amino, cyano, hydroxy, imino, nitro, oxo, thio, halo, alkyl, alkenyl, alkynyl, alkoxy, alkylamino, thioalkyl, aryl, aralkyl, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkylalkyl, haloalkyl, haloalkenyl, haloalkynyl, heterocyclyl, N-heterocyclyl, heterocyclylalkyl, heteroaryl, N-heteroaryl and/or heteroarylalkyl. "Substituted" also means amino acids in which one or more atoms on the side chain are replaced by alkyl, alkenyl, alkynyl, acyl, alkylcarboxamido, alkoxycarbonyl, carbocyclyl, heterocyclyl, aryl or heteroaryl. In addition, each of the aforementioned substituents may also be optionally substituted by one or more of the above substituents.
如本文所用，符号(下文中可称为“附接点键”)表示作为两个化学实体之间的附接点的键，其中一个化学实体被描述为与附接点附接，另一个未被描述为与附接点附接。例如，表示化学实体“XY”经由附接点键与另一化学实体键合。此外，与未描述的化学实体的具体附接点可通过推断来指定。例如，化合物CH3-R3，其中R3是H或推断当R3是“XY”时，附接点键是与R3被描述为与CH3键合的键相同的键。As used herein, the symbol (hereinafter may be referred to as a "point of attachment bond") refers to a bond that is a point of attachment between two chemical entities, one of which is described as being attached to the point of attachment and the other is not described as being attached to the point of attachment. For example, Indicates that the chemical entity "XY" is bonded to another chemical entity via a point of attachment bond. In addition, specific points of attachment to undescribed chemical entities may be assigned by inference. For example, the compoundCH3 -R3 , whereR3 is H or It is inferred that whenR3 is "XY", the point of attachment bond is the same bond asR3 is described as being bonded toCH3 .
如本文所用，“受试者”是指个体。因此，“受试者”可包括驯养动物(例如，猫、狗等)、家畜(例如，牛、马、猪、绵羊、山羊等)、实验动物(例如，小鼠、兔、大鼠、豚鼠等)和鸟。“受试者”还可包括哺乳动物，诸如灵长类动物或人。因此，受试者可以是人或兽医患者。术语“患者”是指在临床医生(例如，医师)的治疗下的受试者。As used herein, "subject" refers to an individual. Thus, "subject" may include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mice, rabbits, rats, guinea pigs, etc.), and birds. "Subject" may also include mammals, such as primates or humans. Thus, the subject may be a human or veterinary patient. The term "patient" refers to a subject under the treatment of a clinician (e.g., physician).
术语“抑制”是指活性、反应、病症、疾病或其他生物参数的降低。这可包括但不限于活性、反应、病症或疾病的完全消除。这还可包括，例如，与天然或对照水平相比，活性、反应、病症或疾病减少10％。因此，减少可以是与天然或对照水平相比10％、20％、30％、40％、50％、60％、70％、80％、90％、100％或其间的任何量的减少。The term "inhibit" refers to a reduction in an activity, response, condition, disease or other biological parameter. This may include, but is not limited to, complete elimination of an activity, response, condition or disease. This may also include, for example, a 10% reduction in an activity, response, condition or disease compared to a natural or control level. Thus, the reduction may be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% reduction or any amount therebetween compared to a natural or control level.
“减少”或该词的其他形式，诸如“减少(reducing)”或“减少(reduction)”，意指事件或特征(例如，肿瘤生长)的降低。应当理解，这通常与一些标准或预期值有关，换句话讲，它是相对的，但并不总是需要参考标准或相对值。例如，“减少肿瘤生长”意指相对于标准或对照(例如，未治疗的肿瘤)减少肿瘤的生长速率。"Reduce" or other forms of the word, such as "reducing" or "reduction", means a decrease in an event or characteristic (e.g., tumor growth). It should be understood that this is usually related to some standard or expected value, in other words, it is relative, but does not always require reference to a standard or relative value. For example, "reduce tumor growth" means reducing the growth rate of a tumor relative to a standard or control (e.g., an untreated tumor).
术语“治疗”是指旨在治愈、改善、稳定或预防疾病、病理状况或障碍而对患者进行的医学管理。该术语包括积极治疗，即专门针对改善疾病、病理状况或障碍的治疗，并且还包括病因治疗，即针对消除相关疾病、病理状况或障碍的病因的治疗。此外，该术语包括姑息治疗，即旨在缓解症状而非治愈疾病、病理状况或障碍的治疗；预防性治疗，即旨在最小化或部分或完全抑制相关疾病、病理状况或障碍的发展的治疗；和支持性治疗，即用于补充另一种旨在改善相关疾病、病理状况或障碍的特定疗法的治疗。The term "treatment" refers to the medical management of a patient with the intent to cure, ameliorate, stabilize or prevent a disease, pathological condition or disorder. The term includes active treatment, i.e., treatment specifically directed toward amelioration of a disease, pathological condition or disorder, and also includes causal treatment, i.e., treatment directed toward eliminating the cause of the disease, pathological condition or disorder in question. In addition, the term includes palliative treatment, i.e., treatment directed toward relieving symptoms rather than curing the disease, pathological condition or disorder; preventive treatment, i.e., treatment directed toward minimizing or partially or completely inhibiting the development of the disease, pathological condition or disorder in question; and supportive treatment, i.e., treatment used to supplement another specific therapy directed toward amelioration of the disease, pathological condition or disorder in question.
术语“治疗有效”是指所用组合物的量足以改善疾病或障碍的一种或多种病因或症状。此类改善仅需要减少或改变，而不必消除。The term "therapeutically effective" means that the amount of the composition used is sufficient to improve one or more causes or symptoms of the disease or disorder. Such improvement only needs to be reduced or altered, not eliminated.
术语“药学上可接受的”是指在合理的医学判断范围内适用于与人和动物的组织接触而没有过度的毒性、刺激、过敏反应或其他问题或并发症的与合理的效益/风险比相称的那些化合物、材料、组合物和/或剂型。The term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio.
术语“载剂”意指化合物、组合物、物质或结构，当与化合物或组合物组合时，其有助于或促进化合物或组合物的制备、储存、施用、递送、有效性、选择性或用于其预期用途或目的的任何其他特征。例如，可选择载剂以使活性成分的任何降解最小化并使受试者中的任何不良副作用最小化。The term "carrier" means a compound, composition, substance, or structure that, when combined with a compound or composition, aids or promotes the preparation, storage, administration, delivery, effectiveness, selectivity, or any other characteristic of the compound or composition for its intended use or purpose. For example, the carrier may be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.
如本文所用，术语“药学上可接受的载剂”是指适于施用于患者的载剂。药学上可接受的载剂是指无菌水性或非水性溶液、分散体、悬浮液或乳液，以及用于在临近使用前重构成无菌可注射溶液或分散体的无菌粉末。合适的水性和非水性载剂、稀释剂、溶剂或媒介物的示例包括水、乙醇、多元醇(诸如甘油、丙二醇、聚乙二醇等)、羧甲基纤维素及其合适的混合物、植物油(诸如橄榄油)和可注射有机酯诸如油酸乙酯。适当的流动性可例如通过使用包衣材料诸如卵磷脂、在分散体的情况下通过维持所需的粒度以及通过使用表面活性剂来维持。这些组合物还可含有佐剂，诸如防腐剂、润湿剂、乳化剂和分散剂。可通过包含各种抗细菌剂和抗真菌剂诸如对羟基苯甲酸酯、氯丁醇、苯酚、山梨酸等来确保防止微生物的作用。还可期望包括等渗剂，诸如糖、氯化钠等。可注射制剂可例如通过经细菌截留过滤器过滤或通过掺入无菌固体组合物形式的灭菌剂来灭菌，该无菌固体组合物可在临近使用前溶解或分散在无菌水或其他无菌可注射介质中。合适的惰性载剂可包括糖诸如乳糖。As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier suitable for application to a patient. A pharmaceutically acceptable carrier refers to a sterile aqueous or non-aqueous solution, dispersion, suspension or emulsion, and a sterile powder for reconstitution into a sterile injectable solution or dispersion just before use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained, for example, by using a coating material such as lecithin, by maintaining the desired particle size in the case of a dispersion, and by using a surfactant. These compositions may also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. It can be ensured that the action of microorganisms is prevented by including various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. It may also be desirable to include isotonic agents such as sugars, sodium chloride, etc. The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium immediately prior to use. Suitable inert carriers may include sugars such as lactose.
术语“药学上可接受的盐”包括通过使用作碱的活性化合物与无机酸或有机酸反应以形成盐而获得的那些，例如盐酸、硫酸、磷酸、甲磺酸、樟脑磺酸、草酸、马来酸、琥珀酸、柠檬酸、甲酸、氢溴酸、苯甲酸、酒石酸、富马酸、水杨酸、扁桃酸、碳酸等的盐。本领域技术人员将进一步认识到，酸加成盐可通过化合物与适当的无机酸或有机酸经由多种已知方法中的任一种反应来制备。术语“药学上可接受的盐”还包括通过使用作酸的活性化合物与无机碱或有机碱反应以形成盐而获得的那些，例如乙二胺、N-甲基-葡糖胺、赖氨酸、精氨酸、鸟氨酸、胆碱、N,N'-二苄基乙二胺、氯普鲁卡因、二乙醇胺、普鲁卡因、N-苄基苯乙胺、二乙胺、哌嗪、三-(羟甲基)-氨基甲烷、四甲基氢氧化铵、三乙胺、二苄胺、二苯羟甲胺(ephenamine)、脱氢枞胺、N-乙基哌啶、苄胺、四甲基铵、四乙基铵、甲胺、二甲胺、三甲胺、乙胺、碱性氨基酸等的盐。无机或金属盐的非限制性示例包括锂盐、钠盐、钙盐、钾盐、镁盐等。The term "pharmaceutically acceptable salts" includes those obtained by reacting the active compound using as a base with an inorganic or organic acid to form a salt, such as hydrochloric acid, sulfuric acid, phosphoric acid, methanesulfonic acid, camphorsulfonic acid, oxalic acid, maleic acid, succinic acid, citric acid, formic acid, hydrobromic acid, benzoic acid, tartaric acid, fumaric acid, salicylic acid, mandelic acid, carbonic acid, etc. Those skilled in the art will further recognize that acid addition salts can be prepared by reacting the compound with an appropriate inorganic or organic acid via any of a variety of known methods. The term "pharmaceutically acceptable salt" also includes those obtained by reacting the active compound using as an acid with an inorganic base or an organic base to form a salt, such as salts of ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris-(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids, etc. Non-limiting examples of inorganic or metal salts include lithium salts, sodium salts, calcium salts, potassium salts, magnesium salts, etc.
如本文所用，术语“肠胃外施用”是指通过注射或输注施用。肠胃外施用包括但不限于皮下施用、静脉内施用或肌内施用。As used herein, the term "parenteral administration" refers to administration by injection or infusion. Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, or intramuscular administration.
如本文所用，术语“皮下施用”是指在皮肤下方施用。“静脉内施用”意指施用到静脉中。As used herein, the term "subcutaneous administration" means administration beneath the skin. "Intravenous administration" means administration into a vein.
如本文所用，术语“剂量”是指在单次施用中提供的药剂的指定量。在实施方案中，剂量可以两个或更多个大丸剂、片剂或注射剂施用。在需要皮下施用的实施方案中，期望剂量需要不易通过单次注射施用的体积。在此类实施方案中，可以使用两个或更多个注射剂以达到期望的剂量。在实施方案中，剂量可以在两个或更多个注射剂中施用以减少病人的注射部位反应。As used herein, the term "dose" refers to a specified amount of a medicament provided in a single administration. In embodiments, a dose can be administered in two or more boluses, tablets, or injections. In embodiments requiring subcutaneous administration, the desired dose requires a volume that is not easily administered by a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In embodiments, a dose may be administered in two or more injections to reduce injection site reactions in patients.
如本文所用，术语“剂量单位”是指在其中提供药剂的形式。在实施方案中，剂量单位是包含冻干的反义寡核苷酸的小瓶。在实施方案中，剂量单位是包含重构的反义寡核苷酸的小瓶。As used herein, the term "dosage unit" refers to the form in which a pharmaceutical agent is provided. In an embodiment, the dosage unit is a vial containing a lyophilized antisense oligonucleotide. In an embodiment, the dosage unit is a vial containing a reconstituted antisense oligonucleotide.
术语“治疗性部分”(TM)是指可用于治疗疾病或病症的至少一种症状的化合物，并且可包括但不限于治疗性多肽、寡核苷酸、小分子和可用于治疗疾病或病症的至少一种症状的其他药剂。在实施方案中，治疗性部分调节靶蛋白的表达或活性。在实施方案中，治疗性部分调节剪接。在实施方案中，治疗性部分诱导靶mRNA转录物中的外显子跳跃。在实施方案中，治疗性部分下调靶蛋白的表达或活性。在实施方案中，治疗性部分通过诱导靶转录物中的外显子跳跃来下调靶蛋白的表达或活性。The term "therapeutic moiety" (TM) refers to a compound that can be used to treat at least one symptom of a disease or disorder, and can include, but is not limited to, therapeutic polypeptides, oligonucleotides, small molecules, and other agents that can be used to treat at least one symptom of a disease or disorder. In embodiments, the therapeutic moiety modulates the expression or activity of a target protein. In embodiments, the therapeutic moiety modulates splicing. In embodiments, the therapeutic moiety induces exon skipping in a target mRNA transcript. In embodiments, the therapeutic moiety downregulates the expression or activity of a target protein. In embodiments, the therapeutic moiety downregulates the expression or activity of a target protein by inducing exon skipping in a target transcript.
术语“调节(modulate)”、“调节(modulating)”和“调节(modulation)”是指当与调节前的表达、功能或活性水平相比时对表达、功能或活性的扰动。调节可包括表达、功能或活性的增加(刺激或诱导)或降低(抑制或减少)。在实施方案中，本文公开的化合物包含下调靶蛋白的表达、功能和/或活性的治疗性部分(TM)。在实施方案中，本文公开的化合物包含上调靶蛋白的表达、功能和/或活性的治疗性部分。The terms "modulate", "modulating" and "modulation" refer to a disturbance in expression, function or activity when compared to the level of expression, function or activity before modulation. Modulation may include an increase (stimulation or induction) or decrease (inhibition or reduction) in expression, function or activity. In an embodiment, the compounds disclosed herein comprise a therapeutic portion (TM) that down-regulates the expression, function and/or activity of a target protein. In an embodiment, the compounds disclosed herein comprise a therapeutic portion that up-regulates the expression, function and/or activity of a target protein.
“氨基酸”是指包含氨基基团和羧酸基团并且具有通式的有机化合物，其中R可以是任何有机基团。氨基酸可以是天然存在的氨基酸或非天然存在的氨基酸。氨基酸可以是蛋白氨基酸或非蛋白氨基酸。氨基酸可以是L-氨基酸或D-氨基酸。术语“氨基酸侧链”或“侧链”是指与天然或非天然α-氨基酸的α-碳结合的特征性取代基(“R”)。氨基酸可经由肽键掺入多肽中。"Amino acid" refers to an amino group and a carboxylic acid group and having the general formula An organic compound wherein R can be any organic group. An amino acid can be a naturally occurring amino acid or a non-naturally occurring amino acid. An amino acid can be a proteinogenic amino acid or a non-proteinogenic amino acid. An amino acid can be an L-amino acid or a D-amino acid. The term "amino acid side chain" or "side chain" refers to a characteristic substituent ("R") bound to the α-carbon of a natural or non-natural α-amino acid. An amino acid can be incorporated into a polypeptide via a peptide bond.
如本文所用，术语“序列同一性”是指两个寡核苷酸或多肽序列之间的分别是相同的且处于相同相对位置的核酸或氨基酸的百分比。因此，一个序列与另一个序列相比具有一定百分比的序列同一性。对于序列比较，通常将一个序列作为参考序列，将测试序列与其进行比较。本领域普通技术人员将理解，如果两个序列在对应的位置含有相同的残基，则它们通常被认为是“基本上相同的”。在实施方案中，序列之间的序列同一性可使用尼德曼-翁施算法来确定(Needleman和Wunsch,1970,J.Mol.Biol.48:443-453)，该算法在EMBOSS软件包的Needle程序实现(EMBOSS：欧洲分子生物学开放软件套件，Rice等人，Trends Genet.(2000),16:276-277)中实现，采用在提交日期时存在的版本。所用的参数是空位开放罚分10、空位延伸罚分0.5和EBLOSUM62(BLOSUM62的EMBOSS版本)取代矩阵。标记为“最长同一性”的Needle的输出(使用-nobrief选项获得)用作同一性百分比并且如下计算：(相同残基×100)/(比对长度-比对空位总数)As used herein, the term "sequence identity" refers to the percentage of nucleic acids or amino acids that are identical and in the same relative position, respectively, between two oligonucleotides or polypeptide sequences. Therefore, a sequence has a certain percentage of sequence identity compared to another sequence. For sequence comparison, a sequence is usually used as a reference sequence to compare a test sequence with it. It will be appreciated by those of ordinary skill in the art that if two sequences contain identical residues in corresponding positions, they are generally considered to be "substantially identical". In an embodiment, the sequence identity between sequences can be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J.Mol.Biol.48:443-453), which is implemented in the Needle program of the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., Trends Genet. (2000), 16:276-277), using the version present at the time of submission date. The parameters used are gap opening penalty 10, gap extension penalty 0.5 and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle marked as "longest identity" (obtained using -nobrief option) is used as identity percentage and is calculated as follows: (same residue × 100) / (total number of alignment gaps in length-alignment)
在其他实施方案中，序列同一性可使用史密斯-沃特曼算法确定，采用在提交日期时存在的版本。In other embodiments, sequence identity may be determined using the Smith-Waterman algorithm, using the version existing on the submission date.
如本文所用，“序列同源性”是指两个多肽序列之间的同源且处于相同相对位置的氨基酸的百分比。因此，一个多肽序列与另一个多肽序列相比具有一定百分比的序列同源性。如本领域普通技术人员将理解的那样，如果两个序列在对应的位置含有同源残基，则它们通常被认为是“基本上同源的”。同源残基可以是相同的残基。另选地，同源残基可以是具有适当相似的结构和/或功能特征的不同残基。例如，如本领域普通技术人员众所周知的那样，某些氨基酸通常被分类为“疏水性”或“亲水性”氨基酸，和/或具有“极性”或“非极性”侧链，并且一个氨基酸取代另一个相同类型的氨基酸通常可被认为是“同源”取代。As used herein, "sequence homology" refers to the percentage of amino acids that are homologous and in the same relative position between two polypeptide sequences. Thus, one polypeptide sequence has a certain percentage of sequence homology compared to another polypeptide sequence. As will be appreciated by those of ordinary skill in the art, if two sequences contain homologous residues at corresponding positions, they are generally considered to be "substantially homologous". Homologous residues can be identical residues. Alternatively, homologous residues can be different residues with appropriately similar structural and/or functional characteristics. For example, as is well known to those of ordinary skill in the art, certain amino acids are generally classified as "hydrophobic" or "hydrophilic" amino acids, and/or have "polar" or "non-polar" side chains, and the substitution of one amino acid for another of the same type can generally be considered a "homologous" substitution.
如本领域众所周知的那样，氨基酸序列可使用多种算法中的任一种来比较，包括可在商用计算机程序中获得的那些，诸如BLASTP、gapped BLAST和PSI-BLAST，其在提交日期时存在。此类程序描述于Altschul等人，J.Mol.Biol.,(1990),215(3):403-410；Altschul等人，Nucleic Acids Res.(1997),25:3389-3402；Baxevanis等人，Bioinformatics A Practical Guide to the Analysis of Genes and Proteins,Wiley,1998；以及Misener等人，(编),Bioinformatics Methods and Protocols(Methods inMolecular Biology,Vol.132),Humana Press,1999。除了鉴定同源序列之外，上述程序通常会提供同源性程度的指示。As is well known in the art, amino acid sequences can be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTP, gapped BLAST, and PSI-BLAST, which exist as of the date of submission. Such programs are described in Altschul et al., J. Mol. Biol., (1990), 215(3):403-410; Altschul et al., Nucleic Acids Res. (1997), 25:3389-3402; Baxevanis et al., Bioinformatics A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener et al., (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying homologous sequences, the above programs typically provide an indication of the degree of homology.
如本文所用，“细胞靶向部分”是指特异性地结合靶细胞表面上的分子诸如受体的分子或大分子。在实施方案中，细胞表面分子仅在靶细胞的表面上表达。在实施方案中，细胞表面分子也存在于一种或多种非靶细胞的表面上，但细胞表面分子表达的量在靶细胞的表面上更高。细胞靶向部分的示例包括但不限于抗体、肽、蛋白质、适体或小分子。As used herein, "cell targeting moiety" refers to a molecule or macromolecule that specifically binds to a molecule such as a receptor on the surface of a target cell. In an embodiment, the cell surface molecule is expressed only on the surface of the target cell. In an embodiment, the cell surface molecule is also present on the surface of one or more non-target cells, but the amount of expression of the cell surface molecule is higher on the surface of the target cell. Examples of cell targeting moieties include, but are not limited to, antibodies, peptides, proteins, aptamers, or small molecules.
如本文所用，术语“反义化合物”和“AC”可互换使用，是指与其(该AC)杂交的靶核酸分子至少部分地互补的聚合核酸结构。AC可以是短的(在实施方案中，少于50个碱基)多核苷酸或多核苷酸同系物，其包含与靶序列互补的序列。在实施方案中，AC是包含与靶前mRNA链中的靶序列互补的序列的多核苷酸或多核苷酸同系物。AC可由天然核酸、合成核酸、核酸同系物或它们的任何组合形成。在实施方案中，AC包括寡核苷。在实施方案中，AC包括反义寡核苷酸。在实施方案中，AC包含缀合基团。AC的非限制性示例包括但不限于引物、探针、反义寡核苷酸、外部引导序列(EGS)寡核苷酸、siRNA、寡核苷酸、寡核苷、寡核苷酸类似物、寡核苷酸模拟物和这些的嵌合组合。因此，这些化合物可以单链、双链、环状、支链或发夹的形式引入，并且可含有结构元件诸如内部或末端凸起或环。寡聚双链化合物可以是杂交形成双链化合物的两条链，或者是具有足够自身互补性以允许杂交和形成完全或部分双链化合物的单链。在实施方案中，AC调节(增加、减少或改变)靶核酸的表达。As used herein, the terms "antisense compound" and "AC" are used interchangeably and refer to a polymeric nucleic acid structure that is at least partially complementary to a target nucleic acid molecule hybridized with it (the AC). AC can be a short (in an embodiment, less than 50 bases) polynucleotide or polynucleotide homologue comprising a sequence complementary to a target sequence. In an embodiment, AC is a polynucleotide or polynucleotide homologue comprising a sequence complementary to a target sequence in a target pre-mRNA chain. AC can be formed by natural nucleic acids, synthetic nucleic acids, nucleic acid homologues, or any combination thereof. In an embodiment, AC includes oligonucleosides. In an embodiment, AC includes antisense oligonucleotides. In an embodiment, AC includes a conjugated group. Non-limiting examples of AC include, but are not limited to, primers, probes, antisense oligonucleotides, external guide sequence (EGS) oligonucleotides, siRNA, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, and chimeric combinations of these. Therefore, these compounds can be introduced in the form of single-stranded, double-stranded, circular, branched, or hairpins, and can contain structural elements such as internal or terminal protrusions or loops. An oligomeric double-stranded compound may be two strands that hybridize to form a double-stranded compound, or a single strand that has sufficient self-complementarity to allow hybridization and formation of a fully or partially double-stranded compound. In embodiments, the AC modulates (increases, decreases or alters) expression of a target nucleic acid.
如本文所用，术语“靶向”是指治疗性部分例如反义化合物与靶核酸分子或靶核酸分子的区域的缔合。在实施方案中，治疗性部分包括能够在生理条件下与靶核酸杂交的反义化合物。在实施方案中，反义化合物靶向靶核酸内的特定部分或位点，例如靶核酸的具有至少一个可鉴定的结构、功能或特征的部分，诸如特定的外显子或内含子，或外显子或内含子内的选定核碱基或基序，诸如剪接元件或顺式作用剪接调控元件。As used herein, the term "targeting" refers to the association of a therapeutic moiety, such as an antisense compound, with a target nucleic acid molecule or a region of a target nucleic acid molecule. In embodiments, the therapeutic moiety comprises an antisense compound that is capable of hybridizing with a target nucleic acid under physiological conditions. In embodiments, the antisense compound targets a specific portion or site within a target nucleic acid, such as a portion of a target nucleic acid having at least one identifiable structure, function, or feature, such as a specific exon or intron, or a selected nucleobase or motif within an exon or intron, such as a splicing element or a cis-acting splicing regulatory element.
如本文所用，术语“靶核酸序列”和“靶核苷酸序列”是指与治疗性部分诸如反义化合物结合或杂交的核酸序列或核苷酸序列。靶核酸包括但不限于靶转录物的一部分、靶RNA(包括但不限于前mRNA和mRNA或其部分)、来源于此类RNA的靶cDNA的一部分以及靶非翻译RNA(诸如miRNA)的一部分。例如，在实施方案中，靶核酸可以是靶细胞基因(或从此类基因转录的mRNA)的一部分，其表达与特定病症或疾病状态相关。术语“部分”是指核酸的限定数量的连续(即，连接)核苷酸。As used herein, the terms "target nucleic acid sequence" and "target nucleotide sequence" refer to a nucleic acid sequence or nucleotide sequence that is bound or hybridized to a therapeutic moiety such as an antisense compound. Target nucleic acids include, but are not limited to, a portion of a target transcript, a target RNA (including but not limited to pre-mRNA and mRNA or portions thereof), a portion of a target cDNA derived from such RNA, and a portion of a target non-translated RNA (such as miRNA). For example, in embodiments, a target nucleic acid can be a portion of a target cell gene (or an mRNA transcribed from such a gene), the expression of which is associated with a particular disorder or disease state. The term "portion" refers to a defined number of consecutive (i.e., connected) nucleotides of a nucleic acid.
如本文所用，术语“转录物”或“基因转录物”指从DNA转录的RNA分子，并且包括但不限于mRNA、前mRNA和部分加工的RNA。As used herein, the term "transcript" or "gene transcript" refers to an RNA molecule transcribed from DNA, and includes, but is not limited to, mRNA, pre-mRNA, and partially processed RNA.
术语“靶转录物”和“靶RNA”是指与治疗性部分结合的前mRNA或mRNA转录物。靶转录物可包含靶核苷酸序列。在一个实施方案中，靶转录物包含剪接位点。The terms "target transcript" and "target RNA" refer to a pre-mRNA or mRNA transcript to which a therapeutic moiety is bound. The target transcript may comprise a target nucleotide sequence. In one embodiment, the target transcript comprises a splice site.
术语“靶基因”和“感兴趣的基因”是指需要或想要调节其表达和/或活性的基因。靶基因可以被转录成包括靶核苷酸序列的靶转录物。靶转录物可被翻译成感兴趣的蛋白质。The terms "target gene" and "gene of interest" refer to a gene whose expression and/or activity needs or is desired to be regulated. A target gene can be transcribed into a target transcript comprising a target nucleotide sequence. The target transcript can be translated into a protein of interest.
术语“靶蛋白”是指由靶转录物(例如，靶mRNA)编码的多肽或蛋白质。The term "target protein" refers to a polypeptide or protein encoded by a target transcript (eg, target mRNA).
如本文所用，术语“mRNA”是指编码蛋白质的RNA分子，并且包括前mRNA和成熟mRNA。“前mRNA”指紧接在DNA转录后新合成的真核mRNA分子。在实施方案中，前mRNA用5'帽加帽，用3'多聚A尾修饰，并且/或者剪接以产生成熟mRNA序列。在实施方案中，前mRNA包含一个或多个内含子。在一个实施方案中，前mRNA经历称为剪接的过程以去除内含子和连接外显子。在实施方案中，前mRNA包含一个或多个剪接元件或剪接调控元件。在实施方案中，前mRNA包含聚腺苷酸化位点。As used herein, the term "mRNA" refers to an RNA molecule encoding a protein, and includes pre-mRNA and mature mRNA." Pre-mRNA" refers to a newly synthesized eukaryotic mRNA molecule immediately after DNA transcription. In an embodiment, the pre-mRNA is capped with a 5' cap, modified with a 3' poly A tail, and/or spliced to produce a mature mRNA sequence. In an embodiment, the pre-mRNA includes one or more introns. In one embodiment, the pre-mRNA undergoes a process called splicing to remove introns and connect exons. In an embodiment, the pre-mRNA includes one or more splicing elements or splicing regulatory elements. In an embodiment, the pre-mRNA includes a polyadenylation site.
如本文所用，术语“表达”、“基因表达”、“基因的表达”等是指基因中编码的信息在细胞中转化成功能性基因产物(诸如多肽或非编码RNA)的所有功能和步骤。非编码RNA的示例包括转移RNA(tRNA)和核糖体RNA。多肽的基因表达包括基因的转录以形成前mRNA，前mRNA的加工以形成成熟mRNA，成熟mRNA从细胞核易位到细胞质，成熟mRNA翻译成多肽，以及编码多肽的组装。表达包括部分表达。例如，基因的表达在本文中可称为基因转录物的生成。成熟mRNA的翻译在本文中可称为成熟mRNA的表达。As used herein, the terms "expression", "gene expression", "expression of a gene", etc. refer to all functions and steps of converting the information encoded in a gene into a functional gene product (such as a polypeptide or non-coding RNA) in a cell. Examples of non-coding RNA include transfer RNA (tRNA) and ribosomal RNA. Gene expression of a polypeptide includes transcription of a gene to form pre-mRNA, processing of pre-mRNA to form mature mRNA, translocation of mature mRNA from the nucleus to the cytoplasm, translation of mature mRNA into a polypeptide, and assembly of the encoded polypeptide. Expression includes partial expression. For example, expression of a gene may be referred to herein as the generation of a gene transcript. Translation of mature mRNA may be referred to herein as expression of mature mRNA.
如本文所用，“基因表达的调节”等是指与基因表达相关的一个或多个过程的调节。例如，基因表达的修饰可包括基因转录、RNA加工、RNA从细胞核到细胞质的易位和mRNA翻译成蛋白质中的一者或多者的修饰。As used herein, "regulation of gene expression" and the like refer to the regulation of one or more processes associated with gene expression. For example, modification of gene expression may include modification of one or more of gene transcription, RNA processing, translocation of RNA from the nucleus to the cytoplasm, and translation of mRNA into protein.
如本文所用，术语“基因”是指包含与基因产物的表达相关的5'启动子区以及与基因产物的表达相关的任何内含子区和外显子区和3'非翻译区(“UTR”)的核酸序列。As used herein, the term "gene" refers to a nucleic acid sequence comprising a 5' promoter region associated with expression of a gene product as well as any intronic and exonic regions and a 3' untranslated region ("UTR") associated with expression of the gene product.
术语“免疫细胞”是指造血来源的并且在免疫应答中起作用的细胞。免疫细胞包括但不限于淋巴细胞(例如，B细胞和T细胞)、天然杀伤(NK)细胞和骨髓细胞。术语“骨髓细胞”包括单核细胞、巨噬细胞和粒细胞(例如，嗜碱性粒细胞、嗜中性粒细胞、嗜酸性粒细胞和肥大细胞)。单核细胞是在血液中循环1-3天的淋巴细胞，在这段时间后，它们迁移到组织中并分化为巨噬细胞或炎性树突状细胞或死亡。如本文所用的术语“巨噬细胞”包括胚胎来源的巨噬细胞(其也可称为驻留组织巨噬细胞)和来源于已从血流迁移到体内组织中的单核细胞的巨噬细胞(其可称为单核细胞来源的巨噬细胞)。根据巨噬细胞所处的组织，其被称为库普弗细胞(肝)、肾小球内系膜细胞(肾)、肺泡巨噬细胞(肺)、窦组织细胞(淋巴结)、霍夫鲍尔细胞(胎盘)、小神经胶质细胞(脑和脊髓)或朗格汉斯细胞(皮肤)等。The term "immune cell" refers to a cell of hematopoietic origin and that works in an immune response. Immune cells include, but are not limited to, lymphocytes (e.g., B cells and T cells), natural killer (NK) cells and bone marrow cells. The term "bone marrow cells" includes monocytes, macrophages and granulocytes (e.g., basophils, neutrophils, eosinophils and mast cells). Monocytes are lymphocytes that circulate in the blood for 1-3 days, and after this period of time, they migrate to tissues and differentiate into macrophages or inflammatory dendritic cells or die. As used herein, the term "macrophage" includes macrophages of embryonic origin (which may also be referred to as resident tissue macrophages) and macrophages derived from mononuclear cells that have migrated from the bloodstream to tissues in vivo (which may be referred to as macrophages of mononuclear cell origin). Depending on the tissue in which they are located, macrophages are called Kupffer cells (liver), mesangial cells (kidney), alveolar macrophages (lung), sinus histiocytes (lymph nodes), Hofbauer cells (placenta), microglia (brain and spinal cord), or Langerhans cells (skin).
如本文所用，关于AC和剪接调控元件的“接近”意指AC结合在剪接调控元件的约25、约20、约15、约10、约5、约4、约3、约2或约1个核苷酸内的核酸序列，所述剪接调控元件包括例如5'剪接位点(5'ss)、分支点序列(BPS)、聚嘧啶(Py)束或3'剪接位点(3'ss)。As used herein, "proximity" with respect to AC and a splicing regulatory element means that AC binds to a nucleic acid sequence within about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 nucleotides of a splicing regulatory element, including, for example, a 5' splice site (5'ss), a branch point sequence (BPS), a polypyrimidine (Py) tract, or a 3' splice site (3'ss).
如本文所用，“剪接调控元件(SRE)”和“剪接元件(SE)”可互换使用，并且是指转录物内的发生剪接或促进、抑制或改变剪接的任何核苷酸序列。剪接元件的示例包括末端茎环序列(TLS)、分支点序列(BPS)、聚嘧啶序列(Py)、5'剪接位点(5'ss)、3'剪接位点(3'ss)和顺式调控元件，诸如内含子剪接沉默子(ISS)序列、内含子剪接增强子(ISE)序列、外显子剪接增强子(ESE)序列、外显子剪接沉默子(ESS)序列和包含外显子/内含子连接的序列。As used herein, "splicing regulatory element (SRE)" and "splicing element (SE)" are used interchangeably and refer to any nucleotide sequence within a transcript at which splicing occurs or promotes, inhibits or alters splicing. Examples of splicing elements include terminal stem-loop sequences (TLS), branch point sequences (BPS), polypyrimidine sequences (Py), 5' splice sites (5'ss), 3' splice sites (3'ss), and cis-regulatory elements such as intronic splicing silencer (ISS) sequences, intronic splicing enhancer (ISE) sequences, exonic splicing enhancer (ESE) sequences, exonic splicing silencer (ESS) sequences, and sequences comprising exon/intron junctions.
如本文所用，术语“剪接”是指转录后前mRNA的修饰，其中去除内含子并连接外显子。剪接发生在由大RNA-蛋白质复合物催化的一系列反应中，所述大RNA-蛋白质复合物包含五个小核核糖核蛋白(snRNP)，称为剪接体。剪接调控元件包括3'剪接位点、5'剪接位点和分支位点。5'剪接位点被U1 snRNP结合，随后被U6 snRNP结合。RNA结合蛋白SF1结合分支点序列，但随后被U2 snRNP置换(参见例如Ward和Cooper(2011)“The pathobiology ofsplicing,”J.Pathol.220(2):152-163)。As used herein, the term "splicing" refers to the modification of pre-mRNA after transcription, wherein introns are removed and exons are connected. Splicing occurs in a series of reactions catalyzed by a large RNA-protein complex comprising five small nuclear ribonucleoproteins (snRNPs), referred to as spliceosomes. Splicing regulatory elements include 3' splice sites, 5' splice sites, and branch sites. The 5' splice site is bound by U1 snRNP and subsequently by U6 snRNP. RNA binding protein SF1 binds to the branch point sequence, but is subsequently displaced by U2 snRNP (see, e.g., Ward and Cooper (2011) "The pathobiology of splicing," J. Pathol. 220 (2): 152-163).
如本文所用，“剪接位点”是指前mRNA分子中外显子与内含子之间的连接。“隐蔽剪接位点”是通常不使用的剪接位点，但是可以在常用剪接位点被阻断或不可用时或者在突变导致正常非活性位点变成活性剪接位点时使用。“异常剪接位点”是由天然DNA和mRNA中的突变产生的剪接位点。“靶向剪接位点”的反义化合物是指与包含剪接位点的靶核苷酸序列的至少一部分杂交的化合物，或与剪接位点附近的内含子或外显子杂交从而调节mRNA的剪接的化合物。靶向剪接位点可以是常见剪接位点、隐蔽剪接位点或异常剪接位点。As used herein, "splice site" refers to the connection between exons and introns in a pre-mRNA molecule. "Hidden splice site" is a splice site that is not usually used, but can be used when a common splice site is blocked or unavailable or when a mutation causes a normal inactive site to become an active splice site. "Abnormal splice site" is a splice site produced by a mutation in natural DNA and mRNA. The antisense compound of "targeted splice site" refers to a compound hybridized with at least a portion of a target nucleotide sequence comprising a splice site, or a compound hybridized with an intron or exon near a splice site to regulate the splicing of mRNA. The targeted splice site can be a common splice site, a hidden splice site, or an abnormal splice site.
如本文所用，“剪接供体位点”可与术语“5'剪接位点”互换使用，是指直接围绕内含子5'端的外显子-内含子边界的核苷酸序列。术语“剪接受体位点”可与术语“3'剪接位点”互换使用，是指直接围绕内含子3'端的内含子-外显子边界的核酸序列。已经表征了许多剪接供体和受体位点(参见例如Ohshima等人(1987)“Signals for the selection of asplice site in pre-mRNA:computer analysis of splice junction sequences andlike sequences,”J.Mol.Biol.,195:247-259(1987))。As used herein, "splice donor site" is used interchangeably with the term "5' splice site" and refers to the nucleotide sequence of the exon-intron boundary immediately surrounding the 5' end of an intron. The term "splice acceptor site" is used interchangeably with the term "3' splice site" and refers to the nucleotide sequence of the intron-exon boundary immediately surrounding the 3' end of an intron. Many splice donor and acceptor sites have been characterized (see, e.g., Ohshima et al. (1987) "Signals for the selection of asplice site in pre-mRNA: computer analysis of splice junction sequences andlike sequences," J. Mol. Biol., 195: 247-259 (1987)).
如本文所用，术语"寡核苷酸"是指包含多个连接的核苷酸或核苷的寡聚化合物。寡核苷酸的一个或多个核苷酸可被修饰。寡核苷酸可包括核糖核酸(RNA)或脱氧核糖核酸(DNA)。寡核苷酸可由天然和/或经修饰的核碱基、糖和共价核苷间连接组成，并且可进一步包括非核酸缀合物。As used herein, the term "oligonucleotide" refers to an oligomeric compound comprising a plurality of nucleotides or nucleosides connected. One or more nucleotides of an oligonucleotide may be modified. An oligonucleotide may include ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). An oligonucleotide may be composed of natural and/or modified nucleobases, sugars, and covalent nucleoside connections, and may further include non-nucleic acid conjugates.
如本文所用，术语“核苷”是指包含核碱基和糖的葡基胺。核苷包括但不限于天然核苷、脱碱基核苷、经修饰的核苷和具有模拟碱基和/或糖基的核苷。“天然核苷”或“未修饰的核苷”是包含天然核碱基和天然糖的核苷。天然核苷包括RNA和DNA核苷。As used herein, the term "nucleoside" refers to a glycosylamine comprising a nucleobase and a sugar. Nucleosides include, but are not limited to, natural nucleosides, abasic nucleosides, modified nucleosides, and nucleosides with simulated bases and/or sugar groups. "Natural nucleosides" or "unmodified nucleosides" are nucleosides comprising natural nucleobases and natural sugars. Natural nucleosides include RNA and DNA nucleosides.
如本文所用，术语“天然糖”是指核苷的糖，其未对其在RNA(2'-OH)或DNA(2'-H)中天然存在的形式进行修饰。As used herein, the term "natural sugar" refers to the sugar of a nucleoside that is not modified from its naturally occurring form in RNA (2'-OH) or DNA (2'-H).
如本文所用，术语“核苷酸”是指具有与糖共价连接的磷酸基团的核苷。核苷酸可用多种取代基中的任一种取代基修饰。As used herein, the term "nucleotide" refers to a nucleoside having a phosphate group covalently linked to a sugar. Nucleotides can be modified with any of a variety of substituents.
如本文所用，术语“核碱基”是指核苷或核苷酸的碱基部分。核碱基可包含能够与另一核酸的碱基氢键合的任何原子或原子团。天然核碱基是未对其在RNA或DNA中天然存在的形式进行修饰的核碱基。As used herein, the term "nucleobase" refers to the base portion of a nucleoside or nucleotide. A nucleobase may comprise any atom or group of atoms capable of hydrogen bonding to a base of another nucleic acid. A natural nucleobase is a nucleobase that has not been modified from its naturally occurring form in RNA or DNA.
如本文所用，术语“杂环碱基部分”是指包含杂环的核碱基。As used herein, the term "heterocyclic base moiety" refers to a nucleobase comprising a heterocyclic ring.
如本文所用，“核苷间连接”是指相邻核苷之间的共价连接。As used herein, "internucleoside linkage" refers to a covalent linkage between adjacent nucleosides.
如本文所用，“天然核苷间连接”是指3'至5'磷酸二酯连接。As used herein, "natural internucleoside linkage" refers to a 3' to 5' phosphodiester linkage.
如本文所用，术语“经修饰的核苷间连接”是指除天然存在的核苷间连接之外的核苷或核苷酸之间的任何连接。As used herein, the term "modified internucleoside linkage" refers to any linkage between nucleosides or nucleotides other than naturally occurring internucleoside linkages.
如本文所用，术语“嵌合反义化合物”是指具有至少一个糖、核碱基和/或核苷间连接的反义化合物，其与相同寡聚化合物内的其他糖、核碱基和核苷间连接相比以不同方式修饰。糖、核碱基和核苷间连接的其余部分可以独立地被修饰或未被修饰。一般来讲，嵌合寡聚化合物将具有经修饰的核苷，该经修饰的核苷可以位于分离的位置或在将限定特定基序的区域中聚集在一起。修饰和/或模拟基团的任何组合可包括如本文所述的嵌合寡聚化合物。As used herein, the term "chimeric antisense compound" refers to an antisense compound having at least one sugar, core base and/or internucleoside connection that is modified in a different manner than other sugars, core bases and internucleoside connections within the same oligomeric compound. The remainder of the sugar, core base and internucleoside connection can be independently modified or unmodified. Generally speaking, a chimeric oligomeric compound will have modified nucleosides that can be located in a separated position or gathered together in a region that will define a specific motif. Any combination of modifications and/or mimetic groups may include chimeric oligomeric compounds as described herein.
如本文所用，术语“混合主链反义寡核苷酸”是指其中反义寡核苷酸的至少一个核苷间连接不同于反义寡核苷酸的至少一个其他核苷间连接的反义寡核苷酸。As used herein, the term "mixed-backbone antisense oligonucleotide" refers to an antisense oligonucleotide in which at least one internucleoside linkage of the antisense oligonucleotide is different from at least one other internucleoside linkage of the antisense oligonucleotide.
如本文所用，术语“核碱基互补性”是指能够与另一个核碱基进行碱基配对的核碱基。例如，在DNA中，腺嘌呤(A)与胸腺嘧啶(T)互补。例如，在RNA中，腺嘌呤(A)与尿嘧啶(U)互补。在实施方案中，互补核碱基是指反义化合物的能够与其靶核酸的核碱基进行碱基配对的核碱基。例如，如果反义化合物的特定位置处的核碱基能够与靶核酸的特定位置处的核碱基氢键合，那么寡核苷酸与靶核酸之间的氢键合位置被认为在该核碱基对处是互补的。As used herein, the term "nucleobase complementarity" refers to a nucleobase that can base pair with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In an embodiment, a complementary nucleobase refers to a nucleobase that can base pair with a nucleobase of its target nucleic acid of an antisense compound. For example, if the nucleobase at the specific position of an antisense compound can hydrogen bond with the nucleobase at the specific position of a target nucleic acid, the hydrogen bonding position between an oligonucleotide and a target nucleic acid is considered to be complementary at the nucleobase pair.
如本文所用，术语“非互补核碱基”是指彼此不形成氢键或以其他方式支持杂交的一对核碱基。As used herein, the term "non-complementary nucleobases" refers to a pair of nucleobases that do not hydrogen bond with each other or otherwise support hybridization.
如本文所用，术语“互补”是指寡聚化合物通过核碱基互补性与另一寡聚化合物或核酸杂交的能力。在实施方案中，当每个分子中足够数量的对应位置被核碱基占据时，反义化合物与其靶标彼此互补，所述核碱基可以彼此键合以允许反义化合物与靶标之间的稳定缔合。本领域技术人员认识到，在不消除寡聚化合物保持缔合的能力的情况下，包含错配是可能的。因此，本文所述的反义化合物可包含最多约20％的错配的核苷酸(即，不是与靶标的对应核苷酸互补的核碱基)。在实施方案中，反义化合物含有不超过约15％，例如不超过约10％，例如不超过5％的错配或无错配。其余的核苷酸是核碱基互补的或以其他方式不破坏杂交(例如，通用碱基)。本领域普通技术人员将认识到，本文提供的化合物与靶核酸至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％核碱基互补。As used herein, the term "complementary" refers to the ability of an oligomeric compound to hybridize with another oligomeric compound or nucleic acid by core base complementarity. In an embodiment, when a sufficient number of corresponding positions in each molecule are occupied by core bases, antisense compounds and their targets are complementary to each other, and the core bases can be bonded to each other to allow the stable association between antisense compounds and targets. It is recognized by those skilled in the art that it is possible to include mispairing without eliminating the ability of oligomeric compounds to maintain association. Therefore, antisense compounds as described herein may include up to about 20% of the nucleotides of mispairing (that is, not the core bases complementary to the corresponding nucleotides of the target). In an embodiment, antisense compounds contain no more than about 15%, for example, no more than about 10%, for example, no more than 5% mispairing or no mispairing. The remaining nucleotides are core base complementary or do not otherwise destroy hybridization (for example, universal bases). One of ordinary skill in the art will recognize that the compounds provided herein are at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% nucleobase complementary to a target nucleic acid.
如本文所用，“杂交”意指互补寡聚化合物(例如，反义化合物及其靶核酸)的配对。尽管不限于特定机制，但最常见的配对机制涉及互补核苷或核苷酸碱基(核碱基)之间的氢键合，其可以是沃森-克里克、胡斯坦或反向胡斯坦氢键合。例如，天然碱基腺嘌呤是与通过形成氢键进行配对的天然核碱基胸腺嘧啶和尿嘧啶互补的核碱基。天然碱基鸟嘌呤是与天然碱基胞嘧啶和5-甲基胞嘧啶互补的核碱基。杂交可在不同情况下发生。As used herein, "hybridization" means the pairing of complementary oligomeric compounds (e.g., antisense compounds and their target nucleic acids). Although not limited to a particular mechanism, the most common pairing mechanism involves hydrogen bonding between complementary nucleosides or nucleotide bases (nucleobases), which can be Watson-Crick, Hoostein or reverse Hoostein hydrogen bonding. For example, the natural base adenine is a nucleobase complementary to the natural nucleobases thymine and uracil that are paired by forming hydrogen bonds. The natural base guanine is a nucleobase complementary to the natural bases cytosine and 5-methylcytosine. Hybridization can occur in different situations.
如本文所用，术语“特异性杂交”是指寡聚化合物与一个核酸位点杂交的亲和力大于其与另一个核酸位点杂交的亲和力的能力。在实施方案中，反义寡核苷酸与多于一个靶位点特异性杂交。在实施方案中，寡聚化合物在严格杂交条件下与其靶标特异性杂交。As used herein, the term "specific hybridization" refers to the ability of an oligomeric compound to hybridize to one nucleic acid site with an affinity greater than its affinity to hybridize to another nucleic acid site. In embodiments, antisense oligonucleotides specifically hybridize to more than one target site. In embodiments, an oligomeric compound specifically hybridizes to its target under stringent hybridization conditions.
在核酸杂交的上下文中，“严格杂交条件”和“严格杂交洗涤条件”是序列依赖性的，并且在不同的环境参数下是不同的。核酸杂交的广泛指导可见于Tijssen,LaboratoryTechniques in Biochemistry and Molecular Biology-Hybridization with NucleicAcid Probes第I部分第2章“Overview of principles of hybridization and thestrategy of nucleic acid probe assays”Elsevier,New York(1993)。通常，高度严格的杂交和洗涤条件被选择为在限定的离子强度和pH下比特定序列的热解链温度(Tm)低约5℃。Tm是50％的靶序列与完全匹配的探针杂交时的温度(在限定的离子强度和pH下)。极严格条件被选择为等于特定探针的Tm。用于在Southern或Northern印迹中在过滤器上杂交具有超过100个互补残基的互补核苷酸序列的严格杂交条件的示例是在42℃处50％甲酰胺与1mg肝素，其中杂交过夜进行。高严格性洗涤条件的示例是在72℃处用0.15M NaCl洗涤约15分钟。严格洗涤条件的示例是在65℃处0.2x SSC洗涤15分钟(关于SSC缓冲液的描述，参见Sambrook和Russel,Molecular Cloning:A laboratory Manual,3rd ed.,Cold SpringHarbor Laboratory Press,2001)。通常，在高严格性洗涤之前进行低严格性洗涤以去除背景探针信号。用于例如超过100个核苷酸的双链体的中等严格性洗涤的示例是在45℃处1xSSC洗涤15分钟。用于例如超过100个核苷酸的双链体的低严格性洗涤的示例是在40℃处4-6xSSC洗涤15分钟。对于短探针(例如，约10至50个核苷酸)，严格条件通常涉及在pH 7.0至8.3下小于约1.0M Na离子、通常约0.01M至1.0M Na离子浓度(或其他盐)的盐浓度，并且温度通常为至少约30℃。严格条件还可通过添加去稳定剂诸如甲酰胺来实现。In the context of nucleic acid hybridization, "stringent hybridization conditions" and "stringent hybridization wash conditions" are sequence-dependent and are different under different environmental parameters. An extensive guide to nucleic acid hybridization can be found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes Part I Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays" Elsevier, New York (1993). Typically, highly stringent hybridization and wash conditions are selected to be about 5°C lower than the thermal melting point (Tm) of the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the Tm of a specific probe. An example of stringent hybridization conditions for hybridizing complementary nucleotide sequences having more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide with 1 mg heparin at 42°C, where hybridization is performed overnight. An example of a high stringency washing condition is a 0.15M NaCl wash at 72°C for about 15 minutes. An example of a stringent washing condition is a 0.2x SSC wash at 65°C for 15 minutes (for a description of SSC buffer, see Sambrook and Russel, Molecular Cloning: A laboratory Manual,3 ed., Cold Spring Harbor Laboratory Press, 2001). Typically, a low stringency wash is performed before a high stringency wash to remove background probe signals. An example of a medium stringency wash for a duplex of, for example, more than 100 nucleotides is a 1xSSC wash at 45°C for 15 minutes. An example of a low stringency wash for a duplex of, for example, more than 100 nucleotides is a 4-6xSSC wash at 40°C for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve a salt concentration of less than about 1.0 M Na ion, typically about 0.01 M to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30° C. Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide.
如本文所用，术语“经2'-修饰的”或“2'-取代的”是指在2'位包含除H或OH之外的取代基的糖。经2'-修饰的单体包括但不限于BNA和具有2'-取代基的单体(例如，核苷和核苷酸)，所述取代基诸如烯丙基、氨基、叠氮基、硫代、O-烯丙基、O-C1-C10烷基、-OCF3、O-(CH2)2-O-CH3、2'-O(CH2)2SCH3、O-(CH2)2-O-N(Rm)(Rn)或O-CH2-C(＝O)-N(Rm)(Rn)，其中每个Rm和Rn独立地是H或取代或未取代的C1-C10烷基。As used herein, the term "2'-modified" or "2'-substituted" refers to a sugar containing a substituent other than H or OH at the 2' position. 2'-modified monomers include, but are not limited to, BNA and monomers (e.g., nucleosides and nucleotides) having 2'-substituents such as allyl, amino, azido, thio, O-allyl, O-C1-C10 alkyl, -OCF3, O-(CH2)2-O-CH3, 2'-O(CH2)2SCH3, O-(CH2)2-O-N(Rm)(Rn) or O-CH2-C(=O)-N(Rm)(Rn), wherein each Rm and Rn are independently H or substituted or unsubstituted C1-C10 alkyl.
如本文所用，术语“MOE”是指2'-O-甲氧基乙基取代基。As used herein, the term "MOE" refers to a 2'-0-methoxyethyl substituent.
如本文所用，术语“经高亲和力修饰的核苷酸”是指具有至少一个经修饰的核碱基、核苷间连接或糖部分的核苷酸，使得修饰增加包含经修饰的核苷酸的反义化合物对靶核酸的亲和力。高亲和力修饰包括但不限于BNA、LNA和2'-MOE。As used herein, the term "high affinity modified nucleotide" refers to a nucleotide having at least one modified nucleobase, internucleoside linkage, or sugar moiety such that the modification increases the affinity of the antisense compound comprising the modified nucleotide for the target nucleic acid. High affinity modifications include, but are not limited to, BNA, LNA, and 2'-MOE.
如本文所用，术语“模拟物”是指取代AC中的糖、核碱基和/或核苷间连接的基团。通常，使用模拟物代替糖或糖-核苷间连接组合，并且维持核碱基以与所选靶标杂交。糖模拟物的代表性示例包括但不限于环己烯基或吗啉基。糖-核苷间连接组合的模拟物的代表性示例包括但不限于通过不带电荷的非手性连接进行连接的肽核酸(PNA)和吗啉基团。在一些情况下，使用模拟物代替核碱基。代表性核碱基模拟物是本领域众所周知的，包括但不限于三环吩嗪类似物和通用碱基(Berger等人，Nuc Acid Res.2000,28:2911-14，以引用方式并入本文)。糖、核苷和核碱基模拟物的合成方法是本领域技术人员众所周知的。As used herein, the term "mimetic" refers to a group that replaces the sugar, nucleobase, and/or internucleoside linkage in AC. Typically, a mimetic is used to replace a sugar or a sugar-nucleoside linkage combination, and the nucleobase is maintained to hybridize with a selected target. Representative examples of sugar mimics include, but are not limited to, cyclohexenyl or morpholinyl. Representative examples of mimics of sugar-nucleoside linkage combinations include, but are not limited to, peptide nucleic acids (PNAs) and morpholino groups connected by uncharged achiral linkages. In some cases, mimics are used to replace nucleobases. Representative nucleobase mimics are well known in the art and include, but are not limited to, tricyclic phenoxy groups. Oxazine analogs and universal bases (Berger et al., Nuc Acid Res. 2000, 28:2911-14, incorporated herein by reference). Methods for the synthesis of sugar, nucleoside and nucleobase mimetics are well known to those skilled in the art.
如本文所用，术语“双环核苷”或“BNA”是指其中核苷的呋喃糖部分包含连接呋喃糖环上的两个原子的桥从而形成二环环系的核苷。BNA包括但不限于α-L-LNA、β-D-LNA、ENA、氧氨基BNA(2'-O-N(CH3)-CH2-4')和氨氧基BNA(2'-N(CH3)-O-CH2-4')。As used herein, the term "bicyclic nucleoside" or "BNA" refers to a nucleoside in which the furanose portion of the nucleoside comprises a bridge connecting two atoms on the furanose ring to form a bicyclic ring system. BNAs include, but are not limited to, α-L-LNA, β-D-LNA, ENA, oxyamino BNA (2'-O-N (CH3)-CH2-4') and aminooxy BNA (2'-N (CH3)-O-CH2-4').
如本文所用，术语“4'至2'二环核苷”是指其中连接呋喃糖环的两个原子的桥将呋喃糖环的4'碳原子和2'碳原子桥接从而形成二环环系的BNA。As used herein, the term "4' to 2' bicyclic nucleoside" refers to a BNA in which the bridge connecting the two atoms of the furanose ring bridges the 4' carbon atom and the 2' carbon atom of the furanose ring to form a bicyclic ring system.
如本文所用，“锁核酸”或“LNA”是指经修饰使得核糖基糖环的2'-羟基基团经由亚甲基基团连接到糖环的4'碳原子从而形成2'-C,4'-C-氧亚甲基连接的核苷酸。LNA包括但不限于α-L-LNA和β-D-LNA。As used herein, "locked nucleic acid" or "LNA" refers to a nucleotide modified so that the 2'-hydroxyl group of the ribosyl sugar ring is linked to the 4' carbon atom of the sugar ring via a methylene group to form a 2'-C,4'-C-oxymethylene linkage. LNA includes but is not limited to α-L-LNA and β-D-LNA.
如本文所用，术语“帽结构”或“末端帽部分”是指已在AC的任一端掺入的化学修饰。As used herein, the term "cap structure" or "terminal cap moiety" refers to a chemical modification that has been incorporated at either end of an AC.
本说明书中提及的所有出版物、专利和专利申请指示本发明所属领域的技术人员的技术水平。所有出版物、专利和专利申请均以引用方式并入本文，其程度如同每个单独的出版物或专利申请被具体地且单独地指出以引用方式并入一样。All publications, patents and patent applications mentioned in this specification are indicative of the technical levels of those skilled in the art to which the invention pertains. All publications, patents and patent applications are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
实施例Example
实施例1.细胞穿透肽-反义化合物缀合物的构建Example 1. Construction of cell penetrating peptide-antisense compound conjugate
被设计成结合IRF-5和/或GYS1并阻断其表达的SEQ ID NO:155至333和/或340中任一者的反义化合物(AC)被构建为具有C6-硫醇5'修饰的二氨基磷酸酯吗啉代寡聚物(PMO)。被设计成生成DUX4的无毒同种型的SEQ IS NO:344至364中任一者的反义化合物被构建为具有C6-巯醇5'修饰的二氨基磷酸酯吗啉代寡聚物(PMO)。Antisense compounds (AC) of any one of SEQ ID NOs: 155 to 333 and/or 340 designed to bind to IRF-5 and/or GYS1 and block their expression are constructed as phosphorodiamidate morpholino oligomers (PMOs) with a C6-thiol 5' modification. Antisense compounds of any one of SEQ ID NOs: 344 to 364 designed to generate non-toxic isoforms of DUX4 are constructed as phosphorodiamidate morpholino oligomers (PMOs) with a C6-thiol 5' modification.
配制包含CCP的EEV。使用Fmoc化学配制细胞穿透肽并与AC缀合，例如，如2021年12月21日由Entrada Therapeutics,Inc.提交的名称为“COMPOSITIONS FOR DELIVERY OFANTISENSE COMPOUNDS”的国际申请号PCT/US20/66459中所述，该国际申请的公开内容全文据此并入本文。在实施方案中，cCPP包含氨基酸序列FfΦRrRrQ(SEQ ID NO:78)。在实施方案中，EEV包含具有序列KKKRKV(SEQ ID NO:33)的环外肽。在实施方案中，EEV包含KKKRKV-PEG2-K-(环(FfΦRrRrQ))-PEG12-K(N3)(SEQ ID NO:33-PEG2-K-(环(SEQ ID NO:78))-PEG12-K(N3))。在实施方案中，使用点击化学将AC化合物与EEV缀合。在实施方案中，化合物包含KKKRKV-PEG2-K-(环(FfΦRrRrQ))-PEG12-K-接头-3'-AC-5'(SEQ ID NO:33-PEG2-K-(环(SEQ ID NO:78))-PEG12-K-接头-3'-AC-5')，其中接头包括应变促进的叠氮化物与环辛炔之间点击反应的产物。接头还可包括其他基团，诸如碳链、PEG链、氨基甲酸酯、脲等。Formulate EEVs containing CCPs. Cell penetrating peptides are formulated using Fmoc chemistry and conjugated to AC, for example, as described in International Application No. PCT/US20/66459, entitled "COMPOSITIONS FOR DELIVERY OF ANTISENSE COMPOUNDS," filed by Entrada Therapeutics, Inc. on December 21, 2021, the disclosure of which is hereby incorporated herein in its entirety. In an embodiment, the cCPP comprises the amino acid sequence FfΦRrRrQ (SEQ ID NO: 78). In an embodiment, the EEV comprises an extracyclic peptide having the sequence KKKRKV (SEQ ID NO: 33). In an embodiment, the EEV comprises KKKRKV-PEG2 -K-(cyclo(FfΦRrRrQ))-PEG12 -K(N3 )(SEQ ID NO: 33-PEG2 -K-(cyclo(SEQ ID NO: 78))-PEG12 -K(N3 )). In an embodiment, the AC compound is conjugated to EEV using click chemistry. In an embodiment, the compound comprises KKKRKV-PEG2 -K-(cyclo(FfΦRrRrQ))-PEG12 -K-linker-3'-AC-5' (SEQ ID NO: 33-PEG2 -K-(cyclo(SEQ ID NO: 78))-PEG12 -K-linker-3'-AC-5'), wherein the linker comprises the product of a strain-promoted click reaction between an azide and a cyclooctyne. The linker may also comprise other groups such as a carbon chain, a PEG chain, a carbamate, a urea, and the like.
实施例2.经由外显子跳跃敲低GYS1表达Example 2. Knockdown of GYS1 expression via exon skipping
EEV-PMO用于诱导外显子6的外显子跳跃，导致GYS1靶转录物的提前终止密码子和无义介导的衰变。所用的EEV是(Ac-PKKKRKV-PEG2-K(环[Ff-Nal-GrGrQ])-PEG2-K(N3)-NH2)(SEQ ID NO:42-PEG2-K(环[SEQ ID NO:135])-PEG2-K(N3)-NH2))。PMO序列是TCACTGTCTGGCTCA CATACC CATA(SEQ ID NO:327)。使用叠氮化物-炔点击化学将PMO和EEV缀合。EEV-PMO was used to induce exon skipping of exon 6, resulting in premature stop codons and nonsense-mediated decay of the GYS1 target transcript. The EEV used was (Ac-PKKKRKV-PEG2 -K(cyclo[Ff-Nal-GrGrQ])-PEG2 -K(N3 )-NH2 )(SEQ ID NO:42-PEG2 -K(cyclo[SEQ ID NO:135])-PEG2 -K(N3 )-NH2 )). The PMO sequence was TCACTGTCTGGCTCA CATACC CATA (SEQ ID NO:327). PMO and EEV were conjugated using azide-alkyne click chemistry.
当与GAA单敲除小鼠相比时，GYS1/GAA双敲除小鼠表现出心脏和骨骼肌中糖原量的显著减少、溶酶体肿胀和自噬积聚的显著降低。这些细胞级变化导致心脏肥大校正、葡萄糖代谢正常化和肌萎缩校正。尽管不存在GAA，但GYS1的消除可发挥重要作用，对iGAA敲除小鼠(GAA-/-)注射单次IV剂量的13.5mg/kg的EEV-PMO、27mg/kg的EEV-PMO、27mg/kg的PMO或阴性对照(媒介物)。注射后一周测量GYS1 mRNA和蛋白水平。在13.5mg/kg EEV-PMO的IV剂量后一周、两周、四周和八周，还评估了GYS1的水平。When compared with GAA single knockout mice, GYS1/GAA double knockout mice showed a significant reduction in the amount of glycogen in heart and skeletal muscle, a significant reduction in lysosomal swelling and autophagy accumulation. These cellular changes lead to correction of cardiac hypertrophy, normalization of glucose metabolism and correction of amyotrophy. Despite the absence of GAA, the elimination of GYS1 can play an important role, and iGAA knockout mice (GAA-/- ) were injected with a single IV dose of 13.5mg/kg of EEV-PMO, 27mg/kg of EEV-PMO, 27mg/kg of PMO or negative control (vehicle). GYS1 mRNA and protein levels were measured one week after injection. The level of GYS1 was also evaluated one week, two weeks, four weeks and eight weeks after the IV dose of 13.5mg/kg EEV-PMO.
图7A至图7D示出了在EEV-PMO组而不是仅PMO组中的横膈膜和心肌中GYS1表达的显著敲低。这种药效学结果是值得注意的，因为这是以非常低的剂量施用的单剂量实验，并且表明GYS1是可寻址的靶标。另外，在心脏、横膈膜、四头肌和三头肌中注射时，GYS1蛋白水平和mRNA持续长达八周(图8A至图8D和图9A至图9D)。蛋白水平是相对于总蛋白而言的。mRNA水平是相对于小鼠β-肌动蛋白和小鼠GAPDH(两种对照管家基因)而言的。Figures 7A to 7D show significant knockdown of GYS1 expression in the diaphragm and myocardium in the EEV-PMO group rather than only the PMO group. This pharmacodynamic result is noteworthy because this is a single-dose experiment administered at a very low dose and shows that GYS1 is an addressable target. In addition, when injected in the heart, diaphragm, quadriceps and triceps, GYS1 protein levels and mRNA continued for up to eight weeks (Figures 8A to 8D and Figures 9A to 9D). Protein levels are relative to total protein. MRNA levels are relative to mouse β-actin and mouse GAPDH (two control housekeeping genes).
实施例3.经由外显子跳跃敲低IRF5表达Example 3. Knockdown of IRF5 expression via exon skipping
使用四种EEV-PMO缀合物诱导外显子4的外显子跳跃，以引入提前终止密码子，从而导致IRF-5靶转录物的无义介导的衰变。四种缀合物各自的PMO序列是5'-AGA ACG TAATCA TCA GTG GGT TGG C-3'(SEQ ID NO:340)。所用的EEV是Ac-PKKKRKV-miniPEG2-K(环[FGFGRGRQ])-PEG12-OH(EEV#1，1120)(Ac-SEQ ID NO:42-miniPEG2-K(环[SEQ ID NO:82])-PEG12-OH)；Ac-PKKKRKV-miniPEG2-K(环[Ff-Nal-GrGrQ])-PEG12-OH(EEV#2，1113)(Ac-SEQ ID NO:42-miniPEG2-K(环[SEQ ID NO:135])-PEG12-OH)；Ac-PKKKRKV-miniPEG2-K(环[FGFGRRRQ])-PEG12-OH(EEV#4；1184)(Ac-SEQ ID NO:42-miniPEG2-K(环[SEQ ID NO:84])-PEG12-OH)；以及1185：Ac-PKKKRKV-miniPEG2-K(环[FGFRRRRQ])-PEG12-OH(EEV#4，1185)(Ac-SEQ ID NO:42-miniPEG2-K(环[SEQ ID NO:85])-PEG12-OH)。使用酰胺缀合化学将EEV与PMO缀合。Four EEV-PMO conjugates were used to induce exon skipping of exon 4 to introduce a premature stop codon, leading to nonsense-mediated decay of the IRF-5 target transcript. The PMO sequence for each of the four conjugates was 5'-AGA ACG TAATCA TCA GTG GGT TGG C-3' (SEQ ID NO: 340). The EEVs used were Ac-PKKKRKV-miniPEG2 -K (cyclo[FGFGRGRQ])-PEG12-OH (EEV #1, 1120) (Ac-SEQ ID NO:42-miniPEG2 -K (cyclo[SEQ ID NO:82])-PEG12 -OH); Ac-PKKKRKV-miniPEG2 -K (cyclo[Ff-Nal-GrGrQ])-PEG12 -OH ( EEV #2, 1113) (Ac-SEQ ID NO: 42-miniPEG2 -K (cyclo [SEQ ID NO: 135]) -PEG12 -OH );2 -OH); and 1185: Ac-PKKKRKV-miniPEG2 -K(cyclo[FGFRRRRQ])-PEG12 -OH (EEV#4, 1185) (Ac-SEQ ID NO:42-miniPEG2 -K(cyclo[SEQ ID NO:85])-PEG12 -OH). EEV was conjugated to PMO using amide conjugation chemistry.
对于体内研究，在第0天和第3天用两个剂量的EEV#1-PMO处理野生型小鼠。在第7天收集样品用于qPCR以测量mRNA水平。对于体外研究，如下处理小鼠巨噬细胞：用EEV#1-PMO处理，或用2μM EEV-PMOs#1-4预处理4小时，随后用R848(一种咪唑并喹啉化合物，其是toll样受体(TLR)7/8的特异性激活剂)刺激过夜。处理后24小时，收获细胞并通过Western印迹进行评价。For in vivo studies, wild-type mice were treated with two doses of EEV#1-PMO on days 0 and 3. Samples were collected on day 7 for qPCR to measure mRNA levels. For in vitro studies, mouse macrophages were treated as follows: treated with EEV#1-PMO, or pretreated with 2 μM EEV-PMOs#1-4 for 4 hours, followed by stimulation overnight with R848, an imidazoquinoline compound that is a specific activator of toll-like receptors (TLR) 7/8. 24 hours after treatment, cells were harvested and evaluated by Western blotting.
在用EEV#1-PMO处理后，在肝脏(图10A)、小肠(图10B)和胫骨前肌(图10C)中观察到IRF5水平的显著敲低。在所有组织中，敲低是剂量依赖性的。另外，在30μM、10μM和3μM的剂量下，用EEV#1-PMO处理的小鼠巨噬细胞具有IRF5蛋白水平的统计学显著降低(图11A)。然而，当与EEV#1-PMO相比时，用EEV#2-PMO、EEV#3-PMO和EEV#4-PMO预处理再用R848刺激的小鼠巨噬细胞在相对效力方面具有显著改善，如通过IRF-5蛋白表达所测量(图11B)。mRNA水平是相对于被设定为100％的媒介物对照而言的。After treatment with EEV#1-PMO, significant knockdown of IRF5 levels was observed in the liver (Figure 10A), small intestine (Figure 10B) and tibialis anterior muscle (Figure 10C). In all tissues, knockdown was dose-dependent. In addition, at a dose of 30 μM, 10 μM and 3 μM, mouse macrophages treated with EEV#1-PMO had a statistically significant reduction in IRF5 protein levels (Figure 11A). However, when compared with EEV#1-PMO, mouse macrophages pretreated with EEV#2-PMO, EEV#3-PMO and EEV#4-PMO and then stimulated with R848 had significant improvements in relative efficacy, as measured by IRF-5 protein expression (Figure 11B). mRNA levels are relative to the vehicle control set to 100%.
实施例4.经由外显子跳跃体外敲低GYS1Example 4. In vitro knockdown of GYS1 via exon skipping
评估了PMO 220和PMO-EEV 220-814在小鼠细胞系中敲低mGYS1水平的有效性。PMO被设计成诱导小鼠GYS1的外显子6的外显子跳跃，以引入提前终止密码子，从而导致IRF-5靶转录物的无义介导的衰变。构建体220-814是与具有Ac-PKKKRKV-PEG2-K(环[FfΦCit-r-Cit-rQ])-PEG12-K(N3)-NH2(EEV 814)(Ac-SEQ ID NO:42-PEG2-K(环[SEQ ID NO:79])-PEG12-K(N3)-NH2)序列的EEV缀合的PMO 220(5'-TCACTGTCTGGCTCACATACCCATA-3'；SEQ IDNO:327)。使用点击化学将PMO与EEV缀合。The effectiveness of PMO 220 and PMO-EEV 220-814 in knocking down mGYS1 levels in mouse cell lines was evaluated. PMO was designed to induce exon skipping of exon 6 of mouse GYS1 to introduce a premature stop codon, resulting in nonsense-mediated decay of the IRF-5 target transcript. Construct 220-814 is PMO 220 (5'-TCACTGTCTGGCTCACATACCCATA-3'; SEQ ID NO:327 ) conjugated to EEV having the sequence Ac-PKKKRKV-PEG2 -K(cyclo[FfΦCit-r-Cit-rQ])-PEG12 -K(N3 )-NH2 (EEV 814) (Ac-SEQ ID NO: 42-PEG2 -K(cyclo[SEQ ID NO: 79])-PEG 12 -K(N3 )-NH2 ) PMO was conjugated to EEV using click chemistry.
野生型小鼠成肌细胞系C2C12和野生型小鼠成纤维细胞系3T3用不同浓度的PMO220和PMO-EEV 220-814经由Endoporter转染(6μL/ml；6μM)进行处理。处理后两天，评价细胞系的GYS1 mRNA水平。Wild-type mouse myoblast cell line C2C12 and wild-type mouse fibroblast cell line 3T3 were treated with different concentrations of PMO220 and PMO-EEV 220-814 via Endoporter transfection (6 μL/ml; 6 μM). Two days after treatment, the cell lines were evaluated for GYS1 mRNA levels.
PMO 220和PMO-EEV 220-814均显示出C2C12成肌细胞系中GYS1mRNA水平的降低(图12和图13A)。PMO 220还显示出3T3成纤维细胞系中GYS1 mRNA水平的降低(图13B)。Both PMO 220 and PMO-EEV 220-814 showed a decrease in GYS1 mRNA levels in the C2C12 myoblast cell line (Figures 12 and 13A). PMO 220 also showed a decrease in GYS1 mRNA levels in the 3T3 fibroblast cell line (Figure 13B).
实施例5.靶向GYS1的PMO-EEV构建体的体内评价Example 5. In vivo evaluation of PMO-EEV constructs targeting GYS1
使用庞贝氏症模拟小鼠模型确定PMO-EEV构建体(EEV-PMO 220-814)在调节GYS1水平和Pompe表型方面的体内有效性。PMO-EEV 220是与EEV 814(Ac-PKKKRKV-PEG2-K(环[FfΦCit-r-Cit-rQ])-PEG12-K(N3)-NH2)(Ac-SEQ ID NO:42-PEG2-K(环[SEQ ID NO:79])-PEG12-K(N3)-NH2)缀合的PMO 220(5'-TCACTGTCTGGCTCACATACCCATA-3'；SEQ ID NO:327)。The in vivo effectiveness of the PMO-EEV construct (EEV-PMO 220-814) in modulating GYS1 levels and the Pompe phenotype was determined using a Pompe disease mimetic mouse model. PMO-EEV 220 is PMO 220 (5'-TCACTGTCTGGCTCACATACCCATA-3'; SEQ ID NO: 327) conjugated to EEV 814 (Ac-PKKKRKV-PEG2 -K(cyclo[FfΦCit-r-Cit-rQ])-PEG12 -K(N3 )-NH2 )(Ac-SEQ ID NO:42 -PEG2 -K(cyclo[SEQ ID NO: 79])-PEG 12 -K(N3 )-NH2 ).
将野生型B6-129小鼠和酸性α-葡糖苷酶(GAA)敲除小鼠(GAA-/-)经由静脉内注射用单剂量的27mpk PMO 220、20mpk PMO-EEV 220-814或40mpk PMO-EEV 220-814进行处理。处理后一周，处死小鼠，收集组织用于蛋白质印迹和RT-qPCR分析。使用点击化学将PMO与EEV缀合。Wild-type B6-129 mice and acid α-glucosidase (GAA) knockout mice (GAA-/- ) were treated with a single dose of 27 mpk PMO 220, 20 mpk PMO-EEV 220-814 or 40 mpk PMO-EEV 220-814 via intravenous injection. One week after treatment, mice were sacrificed and tissues were collected for western blot and RT-qPCR analysis. PMO was conjugated to EEV using click chemistry.
在用PMO-EEV 220-814处理后观察到心肌和骨骼肌中小鼠GYS1mRNA的有效敲低，但在仅用PMO 220处理后未观察到(图14A至图14D)。Efficient knockdown of mouse GYS1 mRNA in cardiac and skeletal muscle was observed following treatment with PMO-EEV 220-814, but not following treatment with PMO 220 alone ( FIGS. 14A-14D ).
GYS2是肝脏中最普遍的GYS蛋白。PMO 220和PMO-EEV 220-814治疗在低于30mpk的治疗水平下不影响GYS2 mRNA的水平(图15)，这指示选择性GYS1靶接合。40mpk 220 814处理组在肝脏中显示出较高的GYS2 mRNA水平。这可能是由于GYS1与GYS2之间的反馈回路，其中GYS1的下调导致GYS2的上调。GYS2 is the most prevalent GYS protein in the liver. PMO 220 and PMO-EEV 220-814 treatment did not affect the level of GYS2 mRNA at treatment levels below 30 mpk (Figure 15), indicating selective GYS1 target engagement. The 40 mpk 220 814 treatment group showed higher GYS2 mRNA levels in the liver. This may be due to a feedback loop between GYS1 and GYS2, in which downregulation of GYS1 leads to upregulation of GYS2.
使用对GYS1非特异性的GYS抗体测量四头肌和三头肌中的GYS1水平(图25A至图25B)。在用EEV-PMO构建体处理后，观察到四头肌中的GYS1蛋白减少(图25A)。由于上样中的凝胶不一致，不能得出三头肌中GYS1蛋白水平的结论(图25B)。GYS1 levels in the quadriceps and triceps were measured using a GYS antibody that is non-specific for GYS1 (FIG. 25A-B). After treatment with the EEV-PMO construct, a decrease in GYS1 protein was observed in the quadriceps (FIG. 25A). Due to the inconsistency of the gel in the loading, no conclusions could be drawn on the GYS1 protein levels in the triceps (FIG. 25B).
还将GYS1特异性抗体用于测量横膈膜、心脏和三头肌中的GYS1蛋白水平(图26)。在用EEV-PMO构建体处理后，观察到横膈膜和心脏中GYS1减少的明显趋势。三头肌中的GYS21水平没有明显降低。值得注意的是，未处理动物的蛋白水平存在大的波动，并且使用GYS1特异性抗体在肝脏中未观察到GYS1信号，这可能是由于肝脏中的低GYS1表达。实施例6.靶向GYS1的第二PMO-EEV构建体的体内评价A GYS1-specific antibody was also used to measure GYS1 protein levels in the diaphragm, heart, and triceps (Figure 26). A clear trend toward a decrease in GYS1 in the diaphragm and heart was observed following treatment with the EEV-PMO construct. There was no significant decrease in GYS21 levels in the triceps. Of note, there were large fluctuations in protein levels in untreated animals, and no GYS1 signal was observed in the liver using the GYS1-specific antibody, which may be due to low GYS1 expression in the liver. Example 6. In vivo evaluation of a second PMO-EEV construct targeting GYS1
使用庞贝氏症模拟小鼠模型确定PMO-EEV构建体220-1055的体内有效性。构建体220-1055是与EEV 1055(Ac-PKKKRKV-K(环[FfΦGrGrQ])-PEG2-K(N3)-NH2)(Ac-SEQ ID NO:42-K(SEQ ID NO:77)-PEG2-K(N3)-NH2)缀合的PMO 220(5'-TCACTGTCTGGCTCACATACCCATA-3'；SEQ ID NO:327)。使用点击化学将PMO与EEV缀合。The in vivo effectiveness of the PMO-EEV construct 220-1055 was determined using a Pompe disease mimetic mouse model. Construct 220-1055 is PMO 220 (5'-TCACTGTCTGGCTCACATACCCATA-3'; SEQ ID NO: 327) conjugated to EEV 1055 (Ac-PKKKRKV-K(cyclo[FfΦGrGrQ])-PEG2 -K(N3 )-NH2 )(Ac-SEQ ID NO: 42-K(SEQ ID NO: 77)-PEG2 -K(N3 )-NH2 ). PMO was conjugated to EEV using click chemistry.
使用与实施例5相同的敲除小鼠模型。经由静脉内注射用单剂量的15mpk PMO220、10mpk PMO-EEV 220-1055或20mpk PMO-EEV 220-1055处理小鼠。处理后一周、2周、4周或8周处死小鼠。收获组织用于蛋白质印迹、RT-qPCR和糖原贮存分析。在2周和8周处死之前，将小鼠禁食过夜。The same knockout mouse model as in Example 5 was used. Mice were treated with a single dose of 15 mpk PMO220, 10 mpk PMO-EEV 220-1055, or 20 mpk PMO-EEV 220-1055 via intravenous injection. Mice were sacrificed one, two, four, or eight weeks after treatment. Tissues were harvested for Western blot, RT-qPCR, and glycogen storage analysis. Mice were fasted overnight prior to sacrifice at 2 and 8 weeks.
在用PMO-EEV 220-1055处理后1周、2周和4周，在心脏中观察到Gys1 mRNA水平的降低(图16A)。在用PMO-EEV 220-1055处理后8周，在心脏中观察到GYS1 mRNA水平的轻微降低(图16A)。在用PMO-EEV 220-1055处理后1周、2周、4周和8周观察到横膈膜(图16B)、四头肌(图16C)和三头肌(图16D)中GYS1 mRNA水平的降低。A decrease in GYS1 mRNA levels was observed in the heart at 1, 2, and 4 weeks after treatment with PMO-EEV 220-1055 ( FIG. 16A ). A slight decrease in GYS1 mRNA levels was observed in the heart at 8 weeks after treatment with PMO-EEV 220-1055 ( FIG. 16A ). A decrease in GYS1 mRNA levels was observed in the diaphragm ( FIG. 16B ), quadriceps ( FIG. 16C ), and triceps ( FIG. 16D ) at 1, 2, 4, and 8 weeks after treatment with PMO-EEV 220-1055.
与RNA水平类似，在PMO-EEV 220-1055处理后2周和4周，心脏(图17A)、横膈膜(图17B)、三头肌(图17C)和四头肌(图17D)均显示出GYS1蛋白水平的大大降低。GYS1的降低似乎随时间推移而变强，直至治疗后4周，然后变弱。Similar to RNA levels, heart (Figure 17A), diaphragm (Figure 17B), triceps (Figure 17C), and quadriceps (Figure 17D) muscles all showed a significant reduction in GYS1 protein levels at 2 and 4 weeks after PMO-EEV 220-1055 treatment. The reduction in GYS1 appeared to become stronger over time until 4 weeks after treatment, and then weakened.
在用PMO-EEV 220-1055处理后1周至8周，心脏(图18A)、横膈膜(图18B)、三头肌(图18C)和四头肌(图18D)中的药物暴露降低。然而，对于分析的所有肌肉组织，可在注射后8周检测到药物暴露。Drug exposure decreased in the heart ( FIG. 18A ), diaphragm ( FIG. 18B ), triceps ( FIG. 18C ), and quadriceps ( FIG. 18D ) muscles from 1 to 8 weeks after treatment with PMO-EEV 220-1055. However, drug exposure was detectable at 8 weeks post-injection for all muscle tissues analyzed.
使用AMPLEX red葡萄糖/葡萄糖氧化酶测定试剂盒(得自ThermoFischer,Waltham,MA)测定所选组织中的糖原贮存水平。通过从来自用α-淀粉葡萄糖苷酶消化的相同样品的葡萄糖水平中减去葡萄糖水平来确定糖原水平。Glycogen storage levels in selected tissues were determined using the AMPLEX red glucose/glucose oxidase assay kit (available from ThermoFischer, Waltham, MA).Glycogen levels were determined by subtracting glucose levels from those from the same sample digested with alpha-amyloglucosidase.
在用PMO-EEV 220-1055处理后1周、2周和4周，在心脏、横膈膜、三头肌和四头肌中观察到糖原的轻微但非显著的减少。处理后8周进行类似的观察。糖原水平降低的缺乏可能是由于当处理小鼠时糖原贮存表型未完全发育。例如，野生型小鼠和GAA敲除小鼠的比较显示，心脏、横膈膜、四头肌和三头肌中的糖原贮存水平随年龄增长而增加。这一发现与文献一致(Raben,N.等人，Journal of Biological Chemistry(1998),273(30),pg.19086)。如果当处理小鼠时糖原表型仍在发育，则在测试时小鼠可能未准确地反映庞贝氏症模型。A slight but non-significant reduction in glycogen was observed in the heart, diaphragm, triceps and quadriceps at 1 week, 2 weeks and 4 weeks after treatment with PMO-EEV 220-1055. Similar observations were made 8 weeks after treatment. The lack of reduced glycogen levels may be due to the incomplete development of the glycogen storage phenotype when the mice were treated. For example, a comparison of wild-type mice and GAA knockout mice showed that the glycogen storage levels in the heart, diaphragm, quadriceps and triceps increased with age. This finding is consistent with the literature (Raben, N. et al., Journal of Biological Chemistry (1998), 273 (30), pg. 19086). If the glycogen phenotype is still developing when the mice are treated, the mice may not accurately reflect the Pompe disease model when tested.
实施例7.靶向GYS1的第三PMO-EEV构建体的体内评价Example 7. In vivo evaluation of the third PMO-EEV construct targeting GYS1
使用庞贝氏症模拟小鼠模型确定PMO-EEV构建体220-1120的体内有效性。构建体220-1120是与EEV 1120Ac-PKKKRKV-AEEA-Lys(环[FGFGRGRQ]-PEG12-OH)(Ac-SEQ ID NO:42-AEEA-Lys(环[SEQ ID NO:82]-PEG12-OH))缀合的PMO 220(5'-TCACTGTCTGGCTCACATACCCATA-3'；SEQ ID NO:327)。使用酰胺缀合化学将PMO与EEV缀合。The in vivo effectiveness of the PMO-EEV construct 220-1120 was determined using a Pompe disease mimetic mouse model. Construct 220-1120 is PMO 220 (5'-TCACTGTCTGGCTCACATACCCATA-3'; SEQ ID NO: 327) conjugated to EEV 1120Ac-PKKKRKV-AEEA-Lys(cyclo[FGFGRGRQ]-PEG12 -OH)(Ac-SEQ ID NO: 42-AEEA-Lys(cyclo[SEQ ID NO: 82]-PEG12 -OH)). PMO was conjugated to EEV using amide conjugation chemistry.
使用与实施例5相同的小鼠模型。将野生型小鼠和GAA敲除小鼠经由静脉内注射用单一剂量的40mpk PMO 220、5mpk PMO-EEV 220-1120、10mpk PMO-EEV 220-1120、20mpkPMO-EEV 220-1120或40mpk PMO-EEV 220-1120处理。处理后2周处死小鼠。收获组织用于蛋白质印迹分析、RT-qPCR和糖原贮存。在处死之前，将小鼠禁食过夜。The same mouse model as in Example 5 was used. Wild-type mice and GAA knockout mice were treated with a single dose of 40 mpk PMO 220, 5 mpk PMO-EEV 220-1120, 10 mpk PMO-EEV 220-1120, 20 mpk PMO-EEV 220-1120, or 40 mpk PMO-EEV 220-1120 via intravenous injection. Mice were sacrificed 2 weeks after treatment. Tissues were harvested for Western blot analysis, RT-qPCR, and glycogen storage. Prior to sacrifice, mice were fasted overnight.
在用PMO-EEV 220-1120处理的小鼠的三头肌(C)和四头肌(D)中观察到GYS1 mRNA(图19)和蛋白水平(图20)的剂量依赖性敲低。在心脏(A)和横膈膜(B)中观察到GYS1 mRNA和蛋白水平的适度敲低，可能是因为测试的小鼠数量有限。Dose-dependent knockdown of GYS1 mRNA (Figure 19) and protein levels (Figure 20) was observed in triceps (C) and quadriceps (D) of mice treated with PMO-EEV 220-1120. Moderate knockdown of GYS1 mRNA and protein levels was observed in the heart (A) and diaphragm (B), likely due to the limited number of mice tested.
进行类似研究，但小鼠接受多剂量的PMO-EEV 220-1120。简而言之，将GAA敲除小鼠经由静脉内注射每两周给予7mpk PMO 220或10mpk PMO-EEV 220-1120，持续8周(PMO的总剂量＝35mpk；PMO-EEV的总剂量＝50mpk)。第一次处理后十周，处死小鼠。收获组织用于蛋白质印迹、RT-qPCR和糖原贮存分析。在第一次处理前、第三次处理后和最后一次处理后，探查小鼠的握力、挂线时间和心脏功能(经由超声心动图)。A similar study was performed, but mice received multiple doses of PMO-EEV 220-1120. Briefly, GAA knockout mice were given 7 mpk PMO 220 or 10 mpk PMO-EEV 220-1120 via intravenous injection every two weeks for 8 weeks (total dose of PMO = 35 mpk; total dose of PMO-EEV = 50 mpk). Ten weeks after the first treatment, mice were sacrificed. Tissues were harvested for Western blot, RT-qPCR, and glycogen storage analysis. Grip strength, wire hanging time, and cardiac function (via echocardiography) were investigated before the first treatment, after the third treatment, and after the last treatment.
在心肌和骨骼肌中均观察到稳健的GYS1 mRNA敲低(图21A至图21C)。GYS1和GYS2的mRNA水平在肝脏中不受影响(图22A至图22B)。Robust knockdown of GYS1 mRNA was observed in both cardiac and skeletal muscle (FIG. 21A-21C). GYS1 and GYS2 mRNA levels were not affected in the liver (FIG. 22A-22B).
在心脏和四头肌中观察到稳健的糖原合酶活性敲低。然而，重复剂量的EEV-PMO220-1120未减少骨骼和肌肉组织中的组织糖原贮存。另外，与未处理的小鼠相比，在处理的小鼠中未观察到握力或前肢悬挂时间的显著变化。Robust knockdown of glycogen synthase activity was observed in the heart and quadriceps. However, repeated doses of EEV-PMO220-1120 did not reduce tissue glycogen storage in bone and muscle tissue. In addition, no significant changes in grip strength or forelimb hanging time were observed in treated mice compared to untreated mice.
实施例8：使用两个剂量的EEV-PMO小鼠研究的IFR-5消融Example 8: IFR-5 Ablation Using Two Doses of EEV-PMO Mouse Studies
使用两剂量小鼠研究来研究PM-EEV 278-1120的有效性。在第零天和第三天，给予小鼠40毫克/千克(mpk)或20mpk的PMO 278(AGA ACG TAA TCA TCA GTG GGT TGG C；SEQ IDNO:340)或EEV-PMO化合物278-1120。PMO-EEV 278-1120包含与EEV 1120(Ac-PKKKRKV-PEG2-K(环[FGFGRGRQ])-PEG12-OH)(Ac-SEQ ID NO:42-PEG2-K(环[SEQ ID NO:82])-PEG12-OH)缀合的PMO 278。第二次给药后五天，处死小鼠，收集血液和组织。A two-dose mouse study was used to investigate the effectiveness of PM-EEV 278-1120. On days zero and three, mice were given 40 milligrams per kilogram (mpk) or 20 mpk of PMO 278 (AGA ACG TAA TCA TCA GTG GGT TGG C; SEQ ID NO: 340) or EEV-PMO compound 278-1120. PMO-EEV 278-1120 contained PMO 278 conjugated to EEV 1120 (Ac-PKKKRKV-PEG2 -K(cyclo[FGFGRGRQ])-PEG12 -OH)(Ac-SEQ ID NO: 42-PEG2 -K(cyclo[SEQ ID NO: 82])-PEG12 -OH). Five days after the second dose, mice were sacrificed and blood and tissues were collected.
图23A至图23C示出了处理后各种组织中的IRF-5表达水平。在小鼠TiA和肝脏组织中观察到IRF-5表达敲低，但在小肠组织中未观察到。Figures 23A to 23C show the expression levels of IRF-5 in various tissues after treatment. IRF-5 expression knockdown was observed in mouse TiA and liver tissues, but not in small intestine tissues.
实施例9：使用单剂量的EEV-PMO小鼠研究的IFR-5消融Example 9: IFR-5 ablation using a single dose of EEV-PMO mouse studies
使用单剂量小鼠研究来研究PM-EEV 278-1120的有效性。PMO-EEV 278-1120是与EEV 1120(Ac-PKKKRKV-PEG2-K(环[FGFGRGRQ])-PEG12-OH)(Ac-SEQ ID NO:42-PEG2-K(环[SEQ ID NO:82])-PEG12-OH)缀合的PMO 278(AGA ACG TAA TCA TCA GTG GGT TGG C；SEQID NO:340)。在第零天，给予小鼠80毫克/千克(mpk)PMO 278；经由IV的40mpk或80mpk的PMO-EEV 278-1120；皮下(SC)120mpk PMO-EEV 278-1120。第二次给药后七天，处死小鼠，收集血液和组织。A single-dose mouse study was used to investigate the effectiveness of PM-EEV 278-1120. PMO-EEV 278-1120 is PMO 278 (AGA ACG TAATCA TCA GTG GGT TGG C; SEQ ID NO: 340) conjugated to EEV 1120 (Ac-PKKKRKV-PEG2 -K(cyclo[FGFGRGRQ])-PEG12 -OH)(Ac-SEQ ID NO:42-PEG 2 -K(cyclo[SEQ ID NO:82])-PEG12 -OH). On day zero, mice were given 80 milligrams/kilogram (mpk) PMO 278; 40 mpk or 80 mpk PMO-EEV 278-1120 via IV; 120 mpk PMO-EEV 278-1120 subcutaneously (SC). Seven days after the second dose, mice were sacrificed and blood and tissues were collected.
图24示出了在80mpk PMO 278的情况下肝脏(A)、肾脏(B)和胫骨前肌(C)组织中的IRF-5表达水平；A是80mpk PMO，B是经由IV递送的40mpk PMO-EEV 278-1120；C是经由IV递送的80mpk PMO-EEV278-1120；并且D是皮下递送的120mpk PMO-EEV 278-1120。单剂量施用40mpk和80mpk的278-1120，小鼠肝脏组织中的IRF-5蛋白表达显著降低，分别对应于40％和53％的降低(A)。肾组织中的IRF-5水平与所检查的其他组织相比较低。数据显示出波动，可能是由于难以定量条带强度与背景。另外，由于样品在凝胶上运行不好，观察到胫骨前肌组织数据的波动(数据未示出)。总的来说，数据显示出肾脏与肝脏中类似的趋势；单剂量施用后IRF-5蛋白水平显著降低。Figure 24 shows the expression level of IRF-5 in liver (A), kidney (B) and tibialis anterior muscle (C) tissues in the case of 80mpk PMO 278; A is 80mpk PMO, B is 40mpk PMO-EEV 278-1120 delivered via IV; C is 80mpk PMO-EEV278-1120 delivered via IV; and D is 120mpk PMO-EEV 278-1120 delivered subcutaneously. Single doses of 40mpk and 80mpk of 278-1120 significantly reduced IRF-5 protein expression in mouse liver tissue, corresponding to a 40% and 53% decrease, respectively (A). IRF-5 levels in kidney tissue are lower than other tissues examined. The data show fluctuations, probably due to the difficulty in quantifying band intensity and background. In addition, fluctuations in tibialis anterior muscle tissue data were observed due to poor performance of the samples on the gel (data not shown). Overall, the data showed a similar trend in the kidney as in the liver; IRF-5 protein levels were significantly reduced after a single dose.
实施例10：靶向IRF-5的各种EEV-PMO的体外外显子跳跃的评价Example 10: Evaluation of exon skipping of various EEV-PMOs targeting IRF-5 in vitro
在用两种EEV-PMO化合物277-1120和278-1120处理后，使用未刺激的RAW 264.7单核细胞/巨噬细胞评价IRF-5表达和外显子跳跃。PMO-EEV 277-1120是通过酰胺缀合化学与EEV 1120Ac-PKKKRKV-AEEA-Lys-(环[FGFGRGRQ])-PEG12-OH(Ac-SEQ ID NO:42-AEEA-Lys-环(SEQ ID NO:82)-PEG12-OH)缀合的PMO序列ACG TAA TCA TCA GTG GGT TGG CTC T(SEQID NO:365)。PMO-EEV 278-1120是通过酰胺缀合化学与EEV 1120Ac-PKKKRKV-AEEA-Lys-(环[FGFGRGRQ])-PEG12-OH(Ac-SEQ ID NO:42-AEEA-Lys-环(SEQ ID NO:82)-PEG12-OH)缀合的PMO序列AGA ACG TAA TCA TCA GTG GGT TGG C(SEQ ID NO:340)。简而言之，将150K个细胞/孔接种在24孔板的0.5ml DMEM中。4小时后，向细胞中添加EEV-PMO化合物，得到500μL的总体积。然后将细胞孵育24小时。孵育后，收集细胞培养基用于细胞因子、IL6和TNF-α检测。提取RNA并用于IRF-5转录物定量。使用蛋白裂解物测量IRF-5蛋白水平变化。IRF-5表达水平相对于β-微管蛋白确定。Unstimulated RAW 264.7 monocytes/macrophages were used to evaluate IRF-5 expression and exon skipping after treatment with two EEV-PMO compounds, 277-1120 and 278-1120. PMO-EEV 277-1120 is the PMO sequence ACG TAA TCA TCA GTG GGT TGG CTC T (SEQ ID NO: 365) conjugated to EEV 1120 Ac-PKKKRKV-AEEA-Lys-(cyclo[FGFGRGRQ])-PEG12-OH (Ac-SEQ ID NO: 42-AEEA-Lys-cyclo(SEQ ID NO: 82)-PEG12 -OH) via amide conjugation chemistry. PMO-EEV 278-1120 is a PMO sequence AGA ACG TAA TCA TCA GTG GGT TGG C (SEQ ID NO: 340) conjugated to EEV 1120Ac-PKKKRKV-AEEA-Lys-(cyclo[FGFGRGRQ])-PEG12-OH (Ac-SEQ ID NO: 42-AEEA-Lys-cyclo(SEQ ID NO: 82)-PEG12 -OH) via amide conjugation chemistry. Briefly, 150K cells/well were seeded in 0.5 ml DMEM in a 24-well plate. After 4 hours, EEV-PMO compounds were added to the cells to give a total volume of 500 μL. The cells were then incubated for 24 hours. After incubation, cell culture medium was collected for cytokine, IL6, and TNF-α detection. RNA was extracted and used for IRF-5 transcript quantification. Protein lysates were used to measure changes in IRF-5 protein levels. IRF-5 expression levels were determined relative to β-tubulin.
对于外显子跳跃研究，如上所述处理细胞。在用EEV-PMO化合物孵育后，用新鲜培养基洗涤细胞，然后孵育过夜。在第二次孵育后，收获RNA并使用检测IRF-5基因中外显子5跳跃的引物进行RT-PCR。For exon skipping studies, cells were treated as described above. After incubation with EEV-PMO compounds, cells were washed with fresh medium and then incubated overnight. After the second incubation, RNA was harvested and RT-PCR was performed using primers that detect exon 5 skipping in the IRF-5 gene.
277-1120和278-1120均显示出RAW 264.7小鼠巨噬细胞/单核细胞中的靶接合，并且以剂量依赖性方式显著降低了IRF-5蛋白水平(图27A)。化合物277-1120在30μM下显著消耗IRF-5蛋白水平约80％，在10μM下显著消耗约50％，并且在3.3μM的较低剂量下未观察到显著变化。化合物278-1120对IRF-5消耗的影响比277-1120强。化合物278-1120在30μM下使IRF-5蛋白水平降低约80％，在10μM下降低约65％。即使在3.3μM的较低剂量下，278-1120也具有约40％的IRF-5蛋白消耗。Both 277-1120 and 278-1120 showed target engagement in RAW 264.7 mouse macrophages/monocytes, and significantly reduced IRF-5 protein levels in a dose-dependent manner (Figure 27A). Compound 277-1120 significantly consumed IRF-5 protein levels by about 80% at 30 μM, consumed about 50% at 10 μM, and no significant changes were observed at a lower dose of 3.3 μM. The effect of compound 278-1120 on IRF-5 consumption is stronger than 277-1120. Compound 278-1120 reduced IRF-5 protein levels by about 80% at 30 μM and by about 65% at 10 μM. Even at a lower dose of 3.3 μM, 278-1120 had about 40% IRF-5 protein consumption.
EEV-PMO化合物0278-1120在暴露后30分钟就诱导部分外显子跳跃，效力随着暴露时间的增加而增加(图27B)。EEV-PMO compound 0278-1120 induced partial exon skipping as early as 30 minutes after exposure, and the potency increased with increasing exposure time ( FIG. 27B ).
对另外的EEV-PMO化合物进行类似的实验，其中PMO是PMO 278(AGA ACG TAA TCATCA GTG GGT TGG C；SEQ ID NO:340)。使用酰胺缀合化学将PMO 278与EEV缀合，各种EEV包括Ac-PKKKRKV-PEG2-K(环[FGFGRGRQ])-PEG12-OH(EEV#1，1120，Ac-SEQ ID NO:42-PEG2-K(环[SEQ ID NO:82])-PEG12-OH)；Ac-PKKKRKV-PEG2-K(环[Ff-Nal-GrGrQ])-PEG12-OH(EEV#2，1113，Ac-SEQ ID NO:42-PEG2-K(环[SEQ ID NO:135])-PEG12-OH)；Ac-PKKKRKV-PEG2-K(环[FGFGRRRQ])-PEG12-OH(EEV#3；1184，Ac-SEQ ID NO:42-PEG2-K(环[SEQ ID NO:84])-PEG12-OH)；以及Ac-PKKKRKV-PEG2-K(环[FGFRRRRQ])-PEG12-OH(EEV#4，1185，Ac-SEQ ID NO:42-PEG2-K(环[SEQ ID NO:85])-PEG12-OH)。Similar experiments were performed for an additional EEV-PMO compound, wherein the PMO was PMO 278 (AGA ACG TAA TCATCA GTG GGT TGG C; SEQ ID NO: 340). PMO 278 was conjugated to EEVs using amide conjugation chemistry, with various EEVs including Ac-PKKKRKV-PEG2 -K(cyclo[FGFGRGRQ])-PEG12 -OH (EEV #1, 1120, Ac-SEQ ID NO:42-PEG2 -K(cyclo[SEQ ID NO:82])-PEG12 -OH); Ac-PKKKRKV-PEG2 -K(cyclo[Ff-Nal-GrGrQ])-PEG12 -OH (EEV #2, 1113, Ac-SEQ ID NO:42-PEG2 -K(cyclo[SEQ ID NO:135])-PEG12 -OH); Ac-PKKKRKV-PEG2 -K(cyclo[FGFGRRRQ])-PEG12 -OH (EEV #3; 1184, Ac-SEQ ID NO:42-PEG2 -K(cyclo[SEQ ID NO: NO:84])-PEG12 -OH); and Ac-PKKKRKV-PEG2 -K(cyclo[FGFRRRRQ])-PEG12 -OH (EEV#4, 1185, Ac-SEQ ID NO:42-PEG2 -K(cyclo[SEQ ID NO:85])-PEG12 -OH).
使用如上所述的类似方法，不同的是用EEV-PMO化合物预处理细胞，然后用R848刺激过夜。R484是Toll样受体激动剂，并引起IRF-5表达的诱导。总处理时间为24小时。A similar approach as described above was used, except that cells were pretreated with EEV-PMO compounds and then stimulated overnight with R848. R484 is a Toll-like receptor agonist and causes induction of IRF-5 expression. The total treatment time was 24 hours.
R848显著增加RAW264.7细胞中的IRF-5蛋白表达。当与用R848刺激的细胞相比时，所有测试浓度的所有EEV-PMO处理的样品显示出IRF-5蛋白表达的显著降低(图28A)。当与用R848刺激的细胞中的IRF-5水平相比时，EEV-PMO化合物278-1113、278-1184和278-1185的效力平均是278-1120的5倍，在低至2μM的浓度下IRF-5蛋白减少约80％。R848 significantly increased IRF-5 protein expression in RAW264.7 cells. When compared to cells stimulated with R848, all EEV-PMO treated samples at all tested concentrations showed a significant reduction in IRF-5 protein expression (Figure 28A). When compared to IRF-5 levels in cells stimulated with R848, the potency of EEV-PMO compounds 278-1113, 278-1184, and 278-1185 was an average of 5 times that of 278-1120, with an approximately 80% reduction in IRF-5 protein at concentrations as low as 2 μM.
EEV-PMO化合物278-1113、278-1184和278-1185在5μM下表现出比278-1120更高的外显子跳跃(图12B)。在278-1113、278-1184和278-1185之间未观察到外显子跳跃的显著差异。EEV-PMO compounds 278-1113, 278-1184, and 278-1185 exhibited higher exon skipping than 278-1120 at 5 μM ( FIG. 12B ). No significant difference in exon skipping was observed between 278-1113, 278-1184, and 278-1185.
实施例11：人THP1细胞中各种EEV-PMO化合物的评价Example 11: Evaluation of various EEV-PMO compounds in human THP1 cells
在用各种PMO化合物和各种EEV-PMO化合物处理后，使用人THP1细胞评价IRF-5表达和外显子跳跃。所测试的PMO化合物包含344(TTGGCAACATCCTCTGCAGCTGAAG；SEQ ID NO:366，Hs-IRF-5-E4N6)；345(GCAACATCCTCTGCAGCTG；SEQ ID NO:367，Hs-IRF-5-E4N3)；346(TCAGGCTTGGCAACATCCTCTGCAG；SEQ ID NO:368，Hs-IRF-5-E5P0；IRF5-E4N3(TAATCATCAGTGGGTTGGCTCTCTG，SEQ ID NO:369)；278(AGA ACG TAA TCA TCA GTG GGT TGGC；SEQ ID NO:340，Hs-IRF-5-E4P3)，以及277(ACG TAA TCA TCA GTG GGT TGG CTC T；SEQID NO:365，Hs-IRF-5-E4PO)。包括PMO 344、345和346的EEV-PMO化合物经由酰胺缀合化学分别与EEV 1120(Ac-PKKKRKV-AEEA-Lys-(环[FGFGRGRQ])-PEG12-OH)(Ac-SEQ ID NO:42—AEEA-Lys-环(SEQ ID NO:82)-PEG12-OH)缀合。Human THP1 cells were used to evaluate IRF-5 expression and exon skipping after treatment with various PMO compounds and various EEV-PMO compounds.所测试的PMO化合物包含344(TTGGCAACATCCTCTGCAGCTGAAG；SEQ ID NO:366，Hs-IRF-5-E4N6)；345(GCAACATCCTCTGCAGCTG；SEQ ID NO:367，Hs-IRF-5-E4N3)；346(TCAGGCTTGGCAACATCCTCTGCAG；SEQ ID NO:368，Hs-IRF-5-E5P0；IRF5-E4N3(TAATCATCAGTGGGTTGGCTCTCTG，SEQ ID NO:369)；278(AGA ACG TAA TCA TCA GTG GGT TGGC；SEQ ID NO:340，Hs-IRF-5-E4P3)，以及277(ACG TAA TCA TCA GTG GGT TGG CTC T；SEQID NO:365，Hs-IRF-5-E4PO)。包括PMO EEV-PMO compounds 344, 345 and 346 were conjugated to EEV 1120 (Ac-PKKKRKV-AEEA-Lys-(cyclo[FGFGRGRQ])-PEG12-OH) (Ac-SEQ ID NO:42-AEEA-Lys-cyclo(SEQ ID NO:82)-PEG12 -OH), respectively, via amide conjugation chemistry.
简而言之，对于仅PMO的研究，使用核转染方法将PMO化合物转染到THP1细胞中。在核转染后将细胞铺板在含有PMA的培养基中，并在收获前孵育24小时。收获RNA，并使用检测IRF-5基因中外显子4和外显子5跳跃的引物进行RT-PCR。Briefly, for PMO-only studies, PMO compounds were transfected into THP1 cells using a nucleofection method. Cells were plated in medium containing PMA after nucleofection and incubated for 24 h before harvesting. RNA was harvested and RT-PCR was performed using primers that detect exon 4 and exon 5 skipping in the IRF-5 gene.
简而言之，对于EEV-PMO研究，THP1细胞通过PMA分化过夜。然后用各种EEV-PMO缀合物处理细胞，并在收获前孵育24小时。收获RNA，并使用检测IRF-5基因中外显子5跳跃的引物进行RT-PCR。Briefly, for EEV-PMO studies, THP1 cells were differentiated overnight by PMA. Cells were then treated with various EEV-PMO conjugates and incubated for 24 h before harvesting. RNA was harvested and RT-PCR was performed using primers that detect exon 5 skipping in the IRF-5 gene.
图29A示出了用各种PMO化合物处理后的外显子4和外显子5跳跃水平。在小鼠细胞中效果良好的PMO化合物不一定翻译成人细胞。例如，对于Hs-IRF-5-E4P3(PMO 278)和Hs-IRF-5-E4PO(PMO 277)，观察到低水平的外显子跳跃。对于Hs-IRF-5-E5N6(PMO 344)、Hs-IRF-5-E5N3(PMO 345)和Hs-IRF-5-E5P0(PMO 346)，观察到外显子跳跃。FIG. 29A shows the level of exon 4 and exon 5 skipping after treatment with various PMO compounds. PMO compounds that work well in mouse cells do not necessarily translate into human cells. For example, low levels of exon skipping were observed for Hs-IRF-5-E4P3 (PMO 278) and Hs-IRF-5-E4PO (PMO 277). Exon skipping was observed for Hs-IRF-5-E5N6 (PMO 344), Hs-IRF-5-E5N3 (PMO 345), and Hs-IRF-5-E5PO (PMO 346).
图29B示出了用各种PMO-EEV化合物处理后的外显子5跳跃水平。结果表明，EEV-PMO缀合物可诱导THP1细胞中的外显子跳跃和靶基因的下调。Figure 29B shows the level of exon 5 skipping after treatment with various PMO-EEV compounds. The results show that EEV-PMO conjugates can induce exon skipping and downregulation of target genes in THP1 cells.
本文已经描述了多个实施方案。然而，应当理解，在不脱离本发明的实质和范围的情况下，可以进行各种修改。因此，其他实施方案也在以下权利要求书的范围内。A number of embodiments have been described herein. However, it should be understood that various modifications may be made without departing from the spirit and scope of the invention. Therefore, other embodiments are also within the scope of the following claims.
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US63/186,664 | 2021-05-10 | ||
| US63/210,882 | 2021-06-15 | ||
| US63/210,866 | 2021-06-15 | ||
| US63/239,671 | 2021-09-01 | ||
| US63/298,587 | 2022-01-11 | ||
| US63/318,201 | 2022-03-09 | ||
| US63/321,921 | 2022-03-21 | ||
| US202263362295P | 2022-03-31 | 2022-03-31 | |
| US63/362,295 | 2022-03-31 | ||
| PCT/US2022/028357WO2022240760A2 (en) | 2021-05-10 | 2022-05-09 | COMPOSITIONS AND METHODS FOR MODULATING mRNA SPLICING |
| Publication Number | Publication Date |
|---|---|
| CN117915957Atrue CN117915957A (en) | 2024-04-19 |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202280044682.9APendingCN117897176A (en) | 2021-05-10 | 2022-05-09 | Compositions and methods for modulating tissue distribution of intracellular therapeutic agents |
| CN202280041112.4APendingCN117915957A (en) | 2021-05-10 | 2022-05-09 | Compositions and methods for modulating mRNA splicing |
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202280044682.9APendingCN117897176A (en) | 2021-05-10 | 2022-05-09 | Compositions and methods for modulating tissue distribution of intracellular therapeutic agents |
| Country | Link |
|---|---|
| CN (2) | CN117897176A (en) |
| Publication number | Publication date |
|---|---|
| CN117897176A (en) | 2024-04-16 |
| Publication | Publication Date | Title |
|---|---|---|
| US20230020092A1 (en) | Compositions for delivery of antisense compounds | |
| US20240247259A1 (en) | Compositions and methods for modulating gene expression | |
| AU2017200365B2 (en) | Compositions and methods for modulating apolipoprotein c-iii expression | |
| JP2021192609A (en) | Rna modulating oligonucleotides for treating duchenne and becker muscular dystrophy with modified characteristics | |
| JP6851201B2 (en) | Antisense oligonucleotides useful in the treatment of Pompe disease | |
| US20250051780A1 (en) | COMPOSITIONS AND METHODS FOR MODULATING mRNA SPLICING | |
| US20240417429A1 (en) | Compositions and methods for skipping exon 45 in duchenne muscular dystrophy | |
| JP2019500899A (en) | Cellular RNA tracking and manipulation through nuclear delivery of CRISPR / Cas9 | |
| EP4359006A1 (en) | Antisense compounds and methods for targeting cug repeats | |
| AU2010239779A1 (en) | Oligonucleotide comprising an inosine for treating DMD | |
| KR20240012425A (en) | Compositions and methods for intracellular therapeutics | |
| US20250129365A1 (en) | Compounds and methods for skipping exon 44 in duchenne muscular dystrophy | |
| TW202039848A (en) | Myostatin signal inhibitor | |
| US20250051393A1 (en) | Compositions and methods for modulating interferon regulatory factor-5 (irf-5) activity | |
| CN117915957A (en) | Compositions and methods for modulating mRNA splicing | |
| JP6928025B2 (en) | RNA-regulated oligonucleotides with improved features for the treatment of Duchenne and Becker muscular dystrophy | |
| US20250011774A1 (en) | Antisense compounds and methods for targeting cug repeats | |
| RU2847465C2 (en) | Compositions and methods of skipping exon 45 in duchenne muscular dystrophy | |
| OA21931A (en) | Antisense compounds and methods for targeting CUG repeats. | |
| CN117957022A (en) | Antisense compounds and methods for targeting CUG repeat sequences | |
| CN118284434A (en) | Compositions and methods for skipping exon 45 in duchenne muscular dystrophy | |
| CN118215504A (en) | Compounds and methods for skipping exon 44 in duchenne muscular dystrophy | |
| KR20250039397A (en) | Hybrid oligonucleotides | |
| WO2025072246A1 (en) | Compounds and methods for skipping exon 50 in duchenne muscular dystrophy |
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |