CN118853661A - Methods, compositions and uses of editing RNA - Google Patents

Abstract

The present application relates to a single stranded antisense oligonucleotide (ANTISENSE OLIGONUCLEOTIDE, ASO) for targeted RNA editing, wherein the ASO comprises a targeting region (SPECIFICITY DOMAIN, SD) and a helper binding region (Binding Helper Domain, BHD), wherein the SD is complementarily paired with a targeted RNA region and the BHD is complementarily paired with a non-targeted RNA region. The application also provides methods of editing RNA, compositions and uses thereof.

Description

Translated fromChinese

编辑RNA的方法、组合物及其应用Methods, compositions and uses of editing RNA

技术领域Technical Field

本申请涉及生物医药领域，具体的涉及一种用于靶向RNA编辑的单链反义寡核苷酸(Antisense Oligonucleotide，ASO)、一种编辑RNA的方法、组合物及其应用。The present application relates to the field of biomedicine, and specifically to a single-stranded antisense oligonucleotide (ASO) for targeted RNA editing, a method for editing RNA, a composition and applications thereof.

背景技术Background Art

靶向RNA编辑以可逆和可调的方式操作遗传信息，不会对基因组造成永久性的改变，因此它与DNA编辑相比更为安全，在治疗应用中具有一定优势。RNA编辑主要包含腺苷(A)到肌苷(I)的转化和胞苷(C)到尿苷(U)的转化，分别通过腺苷脱氨酶(ADAR)和胞苷脱氨酶发生。研究最广泛的RNA编辑系统是ADAR介导的A-to-I的编辑，其中ADAR成员ADAR1(异构体p110和p150)和ADAR2已被设计用于A-to-I RNA编辑。ADAR是一种多结构域蛋白，其通过识别结构域识别特定的dsRNA序列和/或构象，并使用催化结构域通过核碱基的脱氨基化将A变为I。在翻译过程中A被读取为鸟苷(G)并与C配对，完成蛋白质序列的重新编码。Targeted RNA editing manipulates genetic information in a reversible and adjustable manner without causing permanent changes to the genome, so it is safer than DNA editing and has certain advantages in therapeutic applications. RNA editing mainly includes the conversion of adenosine (A) to inosine (I) and the conversion of cytidine (C) to uridine (U), which occurs through adenosine deaminase (ADAR) and cytidine deaminase, respectively. The most widely studied RNA editing system is ADAR-mediated A-to-I editing, in which ADAR members ADAR1 (isoforms p110 and p150) and ADAR2 have been designed for A-to-I RNA editing. ADAR is a multidomain protein that recognizes specific dsRNA sequences and/or conformations through a recognition domain and uses a catalytic domain to convert A to I through deamination of the nucleobase. During translation, A is read as guanosine (G) and paired with C to complete the recoding of the protein sequence.

一些RNA编辑工具已经被设计用于靶向A-to-I的转换。通过基因工程的方法将ADAR蛋白或其催化结构域与噬菌体λN肽、SNAP标签、MS2或dCas13b蛋白融合(PMID：34569891)，并设计一个向导RNA(guide RNA，gRNA)来将ADAR融合蛋白引导至靶位点，从而获得RNA的定向编辑。也有研究显示，过表达ADAR1或ADAR2蛋白与含有R/G基序(ADAR招募区)的gRNA一起可实现靶向RNA编辑。Some RNA editing tools have been designed for targeted A-to-I conversion. ADAR protein or its catalytic domain is fused with bacteriophage λN peptide, SNAP tag, MS2 or dCas13b protein by genetic engineering (PMID: 34569891), and a guide RNA (gRNA) is designed to guide the ADAR fusion protein to the target site, thereby obtaining directed editing of RNA. Studies have also shown that overexpression of ADAR1 or ADAR2 protein together with gRNA containing R/G motif (ADAR recruitment region) can achieve targeted RNA editing.

研究人员进一步利用内源ADAR蛋白进行RNA编辑，无需外源蛋白，仅需引入一段gRNA即可完成内源ADAR蛋白的招募和对目标RNA的编辑，此种编辑方法包括两大类。The researchers further used endogenous ADAR proteins for RNA editing. No exogenous proteins were required. Only a gRNA was needed to recruit endogenous ADAR proteins and edit the target RNA. This editing method includes two major categories.

第一类gRNA包含一段靶向区和一段招募区，靶向区用于与包括目标腺苷的目标RNA区域互补，招募区用于招募天然存在于细胞中的ADAR蛋白并对靶向区进行RNA编辑。WO2016/097212(ProQR Therapeutics)描述了同时具有靶向区和招募区的寡核苷酸结构，其中招募区为单链分子内的茎环结构，靶向区包含部分化学修饰。WO2017/050306描述了与上述结构类似的RNA编辑工具，并在随后的申请CN112752844A中报道了基于上述工具进一步开发的名为“RESTORE”的RNA编辑技术。所述RESTORE优化了招募区序列，并在整条gRNA序列上分布了密集的化学修饰，但是需要在IFN-γ存在的前提下(诱导ADAR1 p150亚型的表达)才能有较高的编辑效率，而IFN-γ是决定自身免疫发展和严重程度的关键因子，这使之在医学领域的应用大打折扣。WO2022078569A1进一步公开了名为“CLUSTER”的RNA编辑方法，所述CLUSTER的gRNA在RESTORE招募区和靶向区的基础上，增加了3-10个募集序列簇，以提高RNA编辑的序列靶向性和灵活性。WO2022026928(ADARx Pharmaceuticals)也描述了由靶向区和招募区组成的gRNA，其中招募区由双链RNA互补形成的分子间结构，靶向区和招募区包含大量化学修饰，以达到较高编辑效率。WO2022147573A1(MALI Prashant et al)公开了一种环化的gRNA策略，通过共价闭环结构保护cadRNA不受核酸外切酶的影响，其中所述cadRNA通过招募区募集内源ADAR，从而实现高效且持久的RNA编辑。The first type of gRNA includes a targeting region and a recruitment region, the targeting region is used to complement the target RNA region including the target adenosine, and the recruitment region is used to recruit the ADAR protein naturally present in the cell and perform RNA editing on the targeting region. WO2016/097212 (ProQR Therapeutics) describes an oligonucleotide structure having both a targeting region and a recruitment region, wherein the recruitment region is a stem-loop structure within a single-stranded molecule, and the targeting region includes a partial chemical modification. WO2017/050306 describes an RNA editing tool similar to the above structure, and in the subsequent application CN112752844A, an RNA editing technology named "RESTORE" further developed based on the above tool was reported. The RESTORE optimizes the recruitment region sequence and distributes dense chemical modifications on the entire gRNA sequence, but it requires the presence of IFN-γ (inducing the expression of the ADAR1 p150 subtype) to have a higher editing efficiency, and IFN-γ is a key factor in determining the development and severity of autoimmunity, which greatly reduces its application in the medical field. WO2022078569A1 further discloses an RNA editing method named "CLUSTER", in which the gRNA of CLUSTER adds 3-10 recruitment sequence clusters based on the RESTORE recruitment region and the targeting region to improve the sequence targeting and flexibility of RNA editing. WO2022026928 (ADARx Pharmaceuticals) also describes a gRNA consisting of a targeting region and a recruitment region, wherein the recruitment region is an intermolecular structure formed by the complementarity of double-stranded RNA, and the targeting region and the recruitment region contain a large number of chemical modifications to achieve a higher editing efficiency. WO2022147573A1 (MALI Prashant et al) discloses a cyclized gRNA strategy that protects cadRNA from the influence of nucleases through a covalent closed loop structure, wherein the cadRNA recruits endogenous ADARs through the recruitment region, thereby achieving efficient and durable RNA editing.

第二类gRNA是一段仅包含靶向区的单链反义寡核苷酸。WO2017/220751(ProQRTherapeutics)公开的单链反义寡核苷酸AON包含18-50个核苷酸，所述AON具有一个或多个复杂修饰的核苷酸，且5’和3’以硫代磷酸酯键连接，但编辑效率不高，申请PCT/EP2017/071912(ProQR Therapeutics)进一步通过特殊化学修饰以提高编辑。WO/2020/074001(博雅辑因)记载了称为“LEAPER”的RNA编辑方法，LEAPER使用的gRNA包含60-200个与目标RNA互补配对的线性核苷酸dRNA，所述dRNA的5’和3’包含甲基化和/或硫代磷酸酯化修饰，针对目标腺苷产生较高效的编辑，但其对于治疗应用通常过长。HK40062258A(博雅辑因)在LEAPER的基础上，进一步开发了LEAPER 2.0，该方法将dRNA环化，保护dRNA不受核酸外切酶的影响，以实现持久编辑。The second type of gRNA is a single-stranded antisense oligonucleotide that only contains the targeting region. The single-stranded antisense oligonucleotide AON disclosed in WO2017/220751 (ProQR Therapeutics) contains 18-50 nucleotides, and the AON has one or more complex modified nucleotides, and the 5' and 3' are connected by thiophosphate bonds, but the editing efficiency is not high. The application PCT/EP2017/071912 (ProQR Therapeutics) further improves the editing through special chemical modifications. WO/2020/074001 (Boya Gene) records an RNA editing method called "LEAPER". The gRNA used by LEAPER contains 60-200 linear nucleotide dRNAs that are complementary to the target RNA. The 5' and 3' of the dRNA contain methylation and/or thiophosphate modifications, which produce more efficient editing for the target adenosine, but it is usually too long for therapeutic applications. HK40062258A (BoYaGene) further developed LEAPER 2.0 based on LEAPER, which circularizes dRNA and protects it from nuclease exonucleases to achieve persistent editing.

但是，以上RNA编辑系统都具有一定局限性。针对第一类RNA编辑系统：首先，外源编辑蛋白和gRNA的异位表达最理想的体内递送方法是病毒载体，腺相关病毒AAV是较为理想的病毒载体，但是其负载量难以同时容纳编辑蛋白和gRNA；其次，非人类来源蛋白质的异位表达具有引发免疫原性的潜在风险，并且可能被适应性免疫系统损害编辑活性；再次，ADAR的过表达极易影响其本身的内源基因编辑，造成内稳态的紊乱，具有致病风险。最后，ADAR的过表达会引起转录组全局的脱靶现象，引发致癌风险。针对第二类RNA编辑系统：使用包含一段靶向区和一段招募区的gRNA或者一段仅包含靶向区的单链反义寡核苷酸虽然可以募集内源ADAR以避免上述问题，但是仍存在编辑效率低的问题。However, the above RNA editing systems all have certain limitations. For the first type of RNA editing system: First, the most ideal in vivo delivery method for the ectopic expression of exogenous editing proteins and gRNA is viral vectors. Adeno-associated virus AAV is a more ideal viral vector, but its load capacity is difficult to accommodate both editing proteins and gRNA; secondly, the ectopic expression of non-human proteins has the potential risk of causing immunogenicity, and the editing activity may be impaired by the adaptive immune system; thirdly, the overexpression of ADAR can easily affect its own endogenous gene editing, causing homeostasis disorders and posing a pathogenic risk. Finally, the overexpression of ADAR can cause global off-target phenomena in the transcriptome, causing carcinogenic risks. For the second type of RNA editing system: Although the use of gRNA containing a targeting region and a recruitment region or a single-stranded antisense oligonucleotide containing only a targeting region can recruit endogenous ADAR to avoid the above problems, there is still a problem of low editing efficiency.

因此，亟待对募集内源ADAR的RNA编辑系统进行进一步改进，提高单链反义寡核苷酸与目标RNA的结合稳定性，从而提高RNA编辑系统的编辑效率。Therefore, it is urgent to further improve the RNA editing system that recruits endogenous ADARs to enhance the binding stability between single-stranded antisense oligonucleotides and target RNA, thereby improving the editing efficiency of the RNA editing system.

发明内容Summary of the invention

本申请提供了一种用于靶向RNA编辑的单链反义寡核苷酸(AntisenseOligonucleotide，ASO)。本申请提供的ASO包含靶向区(Specificity Domain，SD)和辅助结合区(Binding Helper Domain，BHD)，能够与目标RNA形成双链复合物。所述ASO可以通过细胞中存在的ADAR酶对靶向RNA区域中存在的靶腺苷进行脱氨基化，所述BHD利于ASO与目标RNA的稳定结合，提高编辑效率。本申请还提供使用所述ASO编辑RNA的方法，组合物及其应用。The present application provides a single-stranded antisense oligonucleotide (AntisenseOligonucleotide, ASO) for targeted RNA editing. The ASO provided in the present application comprises a targeting region (Specificity Domain, SD) and an auxiliary binding region (Binding Helper Domain, BHD), which can form a double-stranded complex with the target RNA. The ASO can deaminize the target adenosine present in the targeted RNA region by the ADAR enzyme present in the cell, and the BHD is conducive to the stable binding of the ASO to the target RNA, thereby improving the editing efficiency. The present application also provides methods, compositions and applications thereof for editing RNA using the ASO.

一方面，本申请提供了一种用于靶向RNA编辑的单链反义寡核苷酸(AntisenseOligonucleotide，ASO)，其中所述ASO包含靶向区(Specificity Domain，SD)和辅助结合区(Binding Helper Domain，BHD)，其中所述SD与靶向RNA区域互补配对，所述BHD与非靶向RNA区域互补配对，所述ASO能够与目标RNA形成双链复合物。On the one hand, the present application provides a single-stranded antisense oligonucleotide (Antisense Oligonucleotide, ASO) for targeted RNA editing, wherein the ASO comprises a targeting region (Specificity Domain, SD) and an auxiliary binding region (Binding Helper Domain, BHD), wherein the SD is complementary to the targeting RNA region, the BHD is complementary to the non-targeting RNA region, and the ASO can form a double-stranded complex with the target RNA.

在某些实施方式中，所述ASO中，所述靶向RNA区域与非靶向RNA区域非连续。In certain embodiments, in the ASO, the targeting RNA region is non-contiguous with the non-targeting RNA region.

在某些实施方式中，所述靶向RNA区域和所述非靶向RNA区域间隔的长度为4-1000nt。In certain embodiments, the length of the interval between the targeting RNA region and the non-targeting RNA region is 4-1000 nt.

在某些实施方式中，所述靶向RNA区域和所述非靶向RNA区域间隔的长度为4-200nt。In certain embodiments, the length of the interval between the targeting RNA region and the non-targeting RNA region is 4-200 nt.

在某些实施方式中，所述靶向RNA区域和所述非靶向RNA区域间隔的长度为200-500nt。In certain embodiments, the length of the interval between the targeting RNA region and the non-targeting RNA region is 200-500 nt.

在某些实施方式中，所述靶向RNA区域和所述非靶向RNA区域间隔的长度为500-1000nt。In certain embodiments, the length of the interval between the targeting RNA region and the non-targeting RNA region is 500-1000 nt.

在某些实施方式中，所述靶向RNA区域和所述非靶向RNA区域间隔的长度为4nt、8nt、11nt、12nt、24nt、25nt、36nt、237nt、340nt、343nt、494nt、681nt。In certain embodiments, the length of the interval between the targeting RNA region and the non-targeting RNA region is 4nt, 8nt, 11nt, 12nt, 24nt, 25nt, 36nt, 237nt, 340nt, 343nt, 494nt, or 681nt.

在某些实施方式中，所述BHD与非靶向RNA区域完全互补配对。In certain embodiments, the BHD is fully complementary to a non-targeting RNA region.

在某些实施方式中，所述BHD与非靶向RNA区域互补配对，存在一个或多个错配、摆动、缺失、和/或凸起。In certain embodiments, the BHD is complementary to a non-target RNA region and has one or more mismatches, wobbles, deletions, and/or bulges.

在某些实施方式中，所述BHD具有选自下组中的一种或者多种修饰：硫代磷酸酯化修饰、2’-OMe、2’-F、LNA、UNA。In certain embodiments, the BHD has one or more modifications selected from the group consisting of phosphorothioate modification, 2’-OMe, 2’-F, LNA, and UNA.

在某些实施方式中，所述BHD具有硫代磷酸酯化修饰、2’-OMe、LNA、2’-MOE、肌苷替代修饰。In certain embodiments, the BHD has phosphorothioate modification, 2'-OMe, LNA, 2'-MOE, inosine substitution modification.

在某些实施方式中，所述BHD的长度为8-40nt。In certain embodiments, the BHD is 8-40 nt in length.

在某些实施方式中，所述BHD的长度为15nt、20nt、25nt。In certain embodiments, the length of the BHD is 15 nt, 20 nt, or 25 nt.

在某些实施方式中，所述ASO包含一个或者多个BHD。In certain embodiments, the ASO comprises one or more BHDs.

在某些实施方式中，所述一个或者多个BHD不形成分子内二级结构。In certain embodiments, the one or more BHDs do not form intramolecular secondary structures.

在某些实施方式中，所述一个或者多个BHD形成分子内二级结构。In certain embodiments, the one or more BHDs form an intramolecular secondary structure.

在某些实施方式中，所述多个BHD不形成分子间二级结构。In certain embodiments, the plurality of BHDs do not form intermolecular secondary structures.

在某些实施方式中，所述多个BHD形成分子间二级结构。In certain embodiments, the plurality of BHDs form an intermolecular secondary structure.

在某些实施方式中，所述BHD与SD不形成分子间二级结构。In certain embodiments, the BHD and SD do not form an intermolecular secondary structure.

在某些实施方式中，所述BHD与SD形成分子间二级结构。In certain embodiments, the BHD forms an intermolecular secondary structure with SD.

在某些实施方式中，所述BHD在所述SD的5’端。In certain embodiments, the BHD is at the 5' end of the SD.

在某些实施方式中，所述BHD在所述SD的3’端。In certain embodiments, the BHD is at the 3' end of the SD.

在某些实施方式中，所述多个BHD在所述SD的5’端和/或3’端。In certain embodiments, the plurality of BHDs are located at the 5' end and/or 3' end of the SD.

在某些实施方式中，所述BHD与SD直接连接。In certain embodiments, the BHD is directly connected to the SD.

在某些实施方式中，所述BHD与SD间接连接。In certain embodiments, the BHD is indirectly linked to the SD.

在某些实施方式中，所述BHD与SD通过Linker间接连接。In some embodiments, the BHD and SD are indirectly connected via a Linker.

在某些实施方式中，所述Linker包括常规(磷酸二酯)或修饰(如硫代磷酸)的一个或多个核苷酸、寡肽或任何其他化学连接子。In certain embodiments, the linker comprises one or more conventional (phosphodiester) or modified (such as phosphorothioate) nucleotides, oligopeptides or any other chemical linkers.

在某些实施方式中，所述Linker选自下组中的一种或者多种：AAA、AAAC、AACAA、AAAACAAAA、PEG2、C6。In certain embodiments, the linker is selected from one or more of the following groups: AAA, AAAC, AACAA, AAAACAAAA, PEG2, and C6.

在某些实施方式中，所述Linker为AAA。In certain embodiments, the Linker is AAA.

在某些实施方式中，所述Linker为AAAC。In certain embodiments, the Linker is AAAC.

在某些实施方式中，所述Linker为AACAA。In certain embodiments, the linker is AACAA.

在某些实施方式中，所述Linker为AAAACAAAA。In certain embodiments, the Linker is AAAAAAAA.

在某些实施方式中，所述Linker为PEG2。In certain embodiments, the linker is PEG2.

在某些实施方式中，所述Linker为C6。In certain embodiments, the linker is C6.

在某些实施方式中，所述BHD增加ASO与目标RNA形成双链复合物的稳定性。In certain embodiments, the BHD increases the stability of the double-stranded complex formed between the ASO and the target RNA.

在某些实施方式中，所述SD与靶向RNA区域完全互补配对或在一个或多个错配、摆动、缺失、和/或凸起。In certain embodiments, the SD is fully complementary to the target RNA region or has one or more mismatches, wobbles, deletions, and/or bulges.

在某些实施方式中，所述SD具有选自下组修饰的一种或者多种：糖修饰、碱基修饰。In certain embodiments, the SD has one or more modifications selected from the group consisting of sugar modification and base modification.

在某些实施方式中，所述SD具有选自下组中的一种或者多种修饰：硫代磷酸酯化修饰、2’-OMe、2’-F、LNA、UNA。In certain embodiments, the SD has one or more modifications selected from the following group: phosphorothioate modification, 2’-OMe, 2’-F, LNA, UNA.

在某些实施方式中，所述SD具有硫代磷酸酯化修饰。In certain embodiments, the SD has a phosphorothioate modification.

在某些实施方式中，所述SD具有选自下组修饰的一种或者多种：2’-OMe、2’-F、LNA、UNA、2’-FANA、DNA碱基替代、肌苷替代修饰。In certain embodiments, the SD has one or more modifications selected from the following group: 2’-OMe, 2’-F, LNA, UNA, 2’-FANA, DNA base substitution, and inosine substitution modification.

在某些实施方式中，所述SD具有2’-OMe修饰。In certain embodiments, the SD has a 2'-OMe modification.

在某些实施方式中，所述SD具有2’-F修饰。In certain embodiments, the SD has a 2'-F modification.

在某些实施方式中，所述SD的长度为20-50nt。In certain embodiments, the SD is 20-50 nt in length.

在某些实施方式中，所述SD的长度为30nt。In certain embodiments, the SD is 30 nt in length.

在某些实施方式中，所述靶向RNA区域包含靶腺苷。In certain embodiments, the targeting RNA region comprises a target adenosine.

在某些实施方式中，所述靶向RNA区域的长度为15-60nt。In certain embodiments, the targeting RNA region is 15-60 nt in length.

在某些实施方式中，所述靶向RNA区域的长度为30nt。In certain embodiments, the targeting RNA region is 30 nt in length.

在某些实施方式中，所述非靶向RNA区域不包含靶腺苷。In certain embodiments, the non-targeting RNA region does not contain a target adenosine.

在某些实施方式中，所述非靶向RNA区域的长度为5-50nt。In certain embodiments, the non-targeting RNA region is 5-50 nt in length.

在某些实施方式中，所述非靶向RNA区域的长度为15nt，20nt，25nt。In certain embodiments, the length of the non-targeting RNA region is 15 nt, 20 nt, or 25 nt.

在某些实施方式中，所述ASO通过细胞中存在的ADAR酶对靶向RNA区域中存在的靶腺苷进行脱氨基化。In certain embodiments, the ASO deaminates a target adenosine present in a targeted RNA region by an ADAR enzyme present in the cell.

在某些实施方式中，所述目标RNA选自下组中的一种或者多种：pre-mRNA、mRNA、rRNA、tRNA、lnc-RNA、snRNA、snoRNA。In certain embodiments, the target RNA is selected from one or more of the following groups: pre-mRNA, mRNA, rRNA, tRNA, lnc-RNA, snRNA, and snoRNA.

另一方面，本申请提供了一种编辑RNA的方法，其包含使用所述的ASO。On the other hand, the present application provides a method for editing RNA, which comprises using the ASO.

在某些实施方式中，所述方法包含以下步骤：In certain embodiments, the method comprises the following steps:

(1)提供所述的ASO；(1) providing the ASO;

(2)允许细胞摄取所述ASO；(2) allowing cells to take up the ASO;

(3)允许所述ASO与目标RNA结合；(3) allowing the ASO to bind to the target RNA;

(4)允许细胞内ADAR酶将靶向RNA区域中的靶腺苷脱氨基成为肌苷；(4) allowing intracellular ADAR enzymes to deaminate target adenosine in the targeted RNA region to inosine;

(5)鉴定靶向RNA区域中肌苷的存在。(5) Identify the presence of inosine in the targeted RNA region.

在某些实施方式中，所述方法能够招募内源脱氨基酶对特定核苷酸位点进行脱氨基反应。In certain embodiments, the method is capable of recruiting endogenous deaminases to perform deamination reactions on specific nucleotide sites.

另一方面，本申请提供了一种或者多种分离的核酸分子，其编码所述的ASO、或ASO包含的SD、或ASO包含的BHD。In another aspect, the present application provides one or more isolated nucleic acid molecules encoding the ASO, or the SD contained in the ASO, or the BHD contained in the ASO.

另一方面，本申请提供了表达载体，其表达所述的一种或者多种分离的核酸分子。In another aspect, the present application provides an expression vector that expresses one or more isolated nucleic acid molecules.

另一方面，本申请提供了递送载体，其递送所述的ASO。In another aspect, the present application provides a delivery vehicle that delivers the ASO.

另一方面，本申请提供了细胞，其包含所述的ASO、所述的一种或者多种分离的核酸分子所述的表达载体、和/或所述的递送载体。In another aspect, the present application provides a cell comprising the ASO, the one or more isolated nucleic acid molecules, the expression vector, and/or the delivery vector.

在某些实施方式中，所述细胞为真核细胞。In certain embodiments, the cell is a eukaryotic cell.

在某些实施方式中，所述细胞是人类细胞或小鼠细胞。In certain embodiments, the cell is a human cell or a mouse cell.

在某些实施方式中，所述细胞是肝细胞。In certain embodiments, the cell is a hepatocyte.

在某些实施方式中，所述细胞是神经细胞。In certain embodiments, the cell is a neural cell.

另一方面，本申请提供了药物组合物，其包含所述的ASO，所述的一种或者多种分离的核酸分子，所述的表达载体，所述的递送载体，所述的细胞，和/或药学上可接受的载体。On the other hand, the present application provides a pharmaceutical composition comprising the ASO, the one or more isolated nucleic acid molecules, the expression vector, the delivery vector, the cell, and/or a pharmaceutically acceptable carrier.

另一方面，本申请提供了所述的ASO，所述的分离的核酸分子，所述的表达载体，所述的递送载体，所述的细胞，所述的药物组合物，其用于预防和/或治疗疾病和/或病症。On the other hand, the present application provides the ASO, the isolated nucleic acid molecule, the expression vector, the delivery vector, the cell, and the pharmaceutical composition for preventing and/or treating diseases and/or disorders.

另一方面，本申请提供了所述的ASO，所述的一种或者多种分离的核酸分子，所述的表达载体，所述的递送载体，所述的细胞，所述的药物组合物在制备预防和/或治疗疾病和/或病症的药物中的用途。On the other hand, the present application provides the use of the ASO, the one or more isolated nucleic acid molecules, the expression vector, the delivery vector, the cell, and the pharmaceutical composition in the preparation of a drug for preventing and/or treating a disease and/or condition.

另一方面，本申请提供了一种预防和/或治疗疾病和/或病症，其包括向有需要的受试者施用有效量的所述的ASO，所述的一种或者多种分离的核酸分子，所述的表达载体，所述的递送载体，所述的细胞，所述的药物组合物。On the other hand, the present application provides a method for preventing and/or treating a disease and/or condition, comprising administering an effective amount of the ASO, the one or more isolated nucleic acid molecules, the expression vector, the delivery vector, the cell, or the pharmaceutical composition to a subject in need thereof.

本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。Those skilled in the art can easily perceive other aspects and advantages of the present application from the detailed description below. In the detailed description below, only exemplary embodiments of the present application are shown and described. As will be appreciated by those skilled in the art, the content of the present application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which the present application relates. Accordingly, the description in the drawings and specification of the present application is merely exemplary and not restrictive.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明如下：The specific features of the invention involved in this application are shown in the attached claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments and drawings described in detail below. The drawings are briefly described as follows:

图1显示的是本申请所述GAPDH基因目标CDS区域及其对应的ASO序列。FIG. 1 shows the target CDS region of the GAPDH gene and its corresponding ASO sequence described in the present application.

图2显示的是本申请所述Sanger测序展示针对GAPDH目标CDS区域的ASO的编辑效率。FIG. 2 shows the editing efficiency of ASOs targeting the GAPDH target CDS region as shown by Sanger sequencing described in the present application.

图3显示的是本申请所述GAPDH基因目标3’UTR区域及其对应的ASO序列。FIG3 shows the target 3′UTR region of the GAPDH gene and its corresponding ASO sequence described in the present application.

图4显示的是本申请所述Sanger测序显示针对GAPDH目标3’UTR区域的ASO的编辑效率。FIG. 4 shows the editing efficiency of ASO targeting the 3′UTR region of GAPDH as shown by Sanger sequencing described in the present application.

图5显示的是本申请所述OGT基因目标3’UTR区域及其对应的ASO序列。FIG. 5 shows the target 3′UTR region of the OGT gene described in the present application and its corresponding ASO sequence.

图6显示的是本申请所述Sanger测序显示针对OGT基因目标3’UTR区域的ASO的编辑效率。FIG6 shows the editing efficiency of ASO targeting the 3′UTR region of the OGT gene as shown by Sanger sequencing described in the present application.

图7显示的是本申请所述Sanger测序展示针对GAPDH目标CDS区域的ASO的编辑效率。FIG. 7 shows the editing efficiency of ASOs targeting the GAPDH target CDS region as shown by Sanger sequencing described in the present application.

图8显示的是本申请所述Sanger测序展示针对GAPDH目标3’-UTR区域的ASO的编辑效率。FIG8 shows the editing efficiency of ASOs targeting the 3′-UTR region of GAPDH as shown by Sanger sequencing described in the present application.

图9显示的是本申请所述Sanger测序显示针对OGT基因目标3’UTR区域的ASO的编辑效率。FIG9 shows the editing efficiency of ASO targeting the 3′UTR region of the OGT gene as shown by Sanger sequencing described in the present application.

具体实施方式DETAILED DESCRIPTION

以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。The following is an explanation of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

术语定义Definition of terms

在本申请中，术语“反义寡核苷酸”，也称“Antisense Oligonucleotide”，简写为“ASO”，通常指是人工合成的单链或双链形态的寡核苷酸，可以通过改造ASO，改变其结构、结合位点等。本申请中，ASO可以是单链形式，其作用可以是与目标RNA的特定序列互补配对并进行编辑。本申请中，ASO可以包含辅助结合区(Binding Helper Domain，BHD)和靶向区(Specificity Domain，SD)。例如，ASO可以与目标RNA形成双链复合物，然后招募内源脱氨基酶对特定核苷酸位点进行脱氨基反应。In the present application, the term "antisense oligonucleotide", also known as "Antisense Oligonucleotide", abbreviated as "ASO", generally refers to an artificially synthesized single-stranded or double-stranded oligonucleotide, and its structure, binding site, etc. can be changed by modifying ASO. In the present application, ASO can be in a single-stranded form, and its function can be to complement and edit a specific sequence of the target RNA. In the present application, ASO can include a binding helper domain (Binding Helper Domain, BHD) and a targeting domain (Specificity Domain, SD). For example, ASO can form a double-stranded complex with the target RNA, and then recruit endogenous deaminase to perform a deamination reaction on a specific nucleotide site.

在本申请中，术语“目标RNA”，也称“靶RNA”或者“Target RNA”，在本申请中可以互换地使用，通常指包含靶位点的RNA。目标RNA可以是pre-mRNA，mRNA，rRNA，tRNA，lncRNA，sRNA等多种类型。某些核苷酸位点的突变会导致目标RNA产生不同类型的功能差异，例如：RNA的异常剪接、可变剪接，蛋白质的截短、延伸、错误折叠等。在本请中，目标RNA可以包含靶向RNA区域和非靶向RNA区域。In this application, the term "target RNA", also known as "target RNA" or "Target RNA", can be used interchangeably in this application, and generally refers to RNA containing a target site. The target RNA can be pre-mRNA, mRNA, rRNA, tRNA, lncRNA, sRNA and other types. Mutations in certain nucleotide sites can cause different types of functional differences in the target RNA, such as abnormal splicing and alternative splicing of RNA, truncation, extension, misfolding of proteins, etc. In this application, the target RNA can include a targeted RNA region and a non-targeted RNA region.

在本申请中，术语“靶位点”，也称“编辑位点”，在本申请中可以互换地使用，通常指指RNA编辑中，ASO定向编辑位点。本申请中，靶位点可以是腺苷。In this application, the term "target site", also known as "editing site", can be used interchangeably in this application, generally referring to the ASO-directed editing site in RNA editing. In this application, the target site can be adenosine.

在本申请中，术语“靶向区”，也称“Specificity Domain”，简写为“SD”，通常指ASO中的序列，其结合的目标RNA中的序列包含靶位点。本申请中，SD可以与靶向RNA区域互补配对。本申请中，可以对SD进行修饰。In the present application, the term "targeting region", also known as "Specificity Domain", abbreviated as "SD", generally refers to a sequence in an ASO that binds to a sequence in a target RNA that contains a target site. In the present application, the SD may be complementary to a region of a target RNA. In the present application, the SD may be modified.

在本申请中，术语“辅助结合区”，也称“Binding Helper Domain”，简写为“BHD”，通常指ASO中的序列，其结合的目标RNA中的序列不包含靶位点。本申请中，BHD增强ASO与目标RNA的结合稳定性。本申请中，BHD可以与非靶向RNA区域互补配对。本申请中，可以对BHD进行修饰。本申请中，BHD可以与SD直接连接，也可以与SD间接连接。In the present application, the term "auxiliary binding region", also known as "Binding Helper Domain", abbreviated as "BHD", generally refers to a sequence in an ASO, and the sequence in the target RNA to which it binds does not contain a target site. In the present application, BHD enhances the binding stability of ASO and target RNA. In the present application, BHD can be complementary paired with a non-target RNA region. In the present application, BHD can be modified. In the present application, BHD can be directly connected to SD or indirectly connected to SD.

在本申请中，术语“靶向RNA区域”，也称“编辑RNA区域”，在本申请中可以互换地使用，通常指目标RNA中的序列，其包含靶位点，该段序列是SD能够结合的序列。本申请中，靶向RNA区域可以与靶向区(Specificity Domain，SD)互补配对。例如，靶向RNA区域的靶位点可以是腺苷。In the present application, the term "targeting RNA region", also known as "editing RNA region", can be used interchangeably in the present application, generally referring to a sequence in the target RNA, which contains a target site, and the sequence is a sequence that the SD can bind to. In the present application, the targeting RNA region can be complementary to the targeting region (Specificity Domain, SD). For example, the target site of the targeting RNA region can be adenosine.

在本申请中，术语“非靶向RNA区域”，也称“非编辑RNA区域”，在本申请中可以互换地使用，通常指目标RNA中的不包含靶位点的一段RNA序列，该段序列是BHD能够结合的序列。本申请中，非靶向RNA区域可以与辅助结合区(Binding Helper Domain，BHD)互补配对。本申请中，非靶向RNA区域可以与靶向RNA区域连续，也可以与靶向RNA区域非连续。In the present application, the term "non-targeting RNA region", also known as "non-editing RNA region", can be used interchangeably in the present application, generally referring to a section of RNA sequence in the target RNA that does not contain the target site, which is a sequence that BHD can bind to. In the present application, the non-targeting RNA region can be complementary to the auxiliary binding region (Binding Helper Domain, BHD). In the present application, the non-targeting RNA region can be continuous with the targeted RNA region, or it can be non-continuous with the targeted RNA region.

在本申请中，术语“修饰”，通常是指对天然的或人工合成的成分进行改造。修饰可以包括对碱基的修饰、对核苷的修饰、对糖的修饰、核苷酸间键联修饰；可以包括化学修饰和非化学修饰。本申请中，所述修饰可以包括LNA、UNA、2’-MOE、2’-OMe、2’-F、硫代磷酸酯化修饰、DNA碱基替代、肌苷替代修饰等。LNA指核糖环被—个连2’-O原子和4’-C原子的亚甲基桥“锁定”的修饰。UNA指核糖环缺乏C2’与C3’化学键的修饰。2’-MOE指核糖核苷酸的2’-羟基上的氢替换为甲氧基乙基。2’-OMe是指将核糖核苷酸的2’-羟基上的氢替换为甲氧基。2’-F将核糖核苷酸的2’-羟基上的氢替换为氟。硫代磷酸酯化修饰将原有磷酸键中一个非桥连的氧原子用硫原子来置换。DNA碱基替代修饰将ASO中的RNA碱基替换为DNA碱基。肌苷替代修饰将ASO中的鸟苷碱基替换为肌苷碱基。In the present application, the term "modification" generally refers to the modification of natural or synthetic components. Modifications may include modifications to bases, modifications to nucleosides, modifications to sugars, modifications to internucleotide linkages; and may include chemical modifications and non-chemical modifications. In the present application, the modifications may include LNA, UNA, 2'-MOE, 2'-OMe, 2'-F, phosphorothioate modification, DNA base substitution, inosine substitution modification, etc. LNA refers to a modification in which the ribose ring is "locked" by a methylene bridge connecting the 2'-O atom and the 4'-C atom. UNA refers to a modification in which the ribose ring lacks a chemical bond between C2' and C3'. 2'-MOE refers to the replacement of the hydrogen on the 2'-hydroxyl group of a ribonucleotide with a methoxyethyl group. 2'-OMe refers to the replacement of the hydrogen on the 2'-hydroxyl group of a ribonucleotide with a methoxy group. 2'-F replaces the hydrogen on the 2'-hydroxyl group of a ribonucleotide with a fluorine group. The phosphorothioate modification replaces a non-bridging oxygen atom in the original phosphate bond with a sulfur atom. DNA base substitution modification replaces RNA bases in the ASO with DNA bases. Inosine substitution modification replaces guanosine bases in the ASO with inosine bases.

在本申请中，术语“互补配对”与“互补”可以互换地使用，通常指核酸分子的核苷酸单位间的Watson-Crick或Hoogsteen碱基配对。本申请中，碱基配对可以指A-T，C-G，T*A/T(指质子化的T能和A-T碱基对中的A配对)，C*G/C(指质子化的C能和G-C碱基对中的G配对)。本申请中，互补配对可以是完全互补配对，也可以在核酸分子之间存在一个或者多个凸起、摆动、缺失、和/或错配。本申请中，ASO可以通过互补配对与目标RNA形成双链复合物。In the present application, the terms "complementary pairing" and "complementary" can be used interchangeably, generally referring to Watson-Crick or Hoogsteen base pairing between nucleotide units of nucleic acid molecules. In the present application, base pairing can refer to A-T, C-G, T*A/T (referring to the protonated T energy and the A in the A-T base pair), C*G/C (referring to the protonated C energy and the G in the G-C base pair). In the present application, complementary pairing can be a complete complementary pairing, or there can be one or more protrusions, swings, deletions, and/or mismatches between nucleic acid molecules. In the present application, ASO can form a double-stranded complex with the target RNA through complementary pairing.

在本申请中，术语“凸起”通常指突起区域的上下游一个碱基均与靶向RNA区域互补配对且靶向RNA区域的对应两个碱基是连续的。在本申请中，术语“摆动”通常指互补链上的摇摆碱基配对为G-U配对。在本申请中，术语“缺失”通常指缺失区域的上下游碱基是连续的，在缺失区域的靶向RNA区域有对应数量的碱基。In the present application, the term "protrusion" generally refers to that one base upstream and downstream of the protrusion region is complementary paired with the target RNA region and the corresponding two bases in the target RNA region are continuous. In the present application, the term "swing" generally refers to that the swing base pairing on the complementary chain is G-U pairing. In the present application, the term "deletion" generally refers to that the upstream and downstream bases of the deletion region are continuous, and there is a corresponding number of bases in the target RNA region of the deletion region.

在本申请中，术语“错配”通常指双链RNA复合物中相对的核苷酸并非是根据Watson-Crick碱基配对法则形成完美的碱基对。错配的核苷酸可以为A-A、A-G、A-C、U-U、U-G、U-C、G-G、G-A、G-U、C-A、C-C、C-U等。In the present application, the term "mismatch" generally refers to the fact that the relative nucleotides in the double-stranded RNA complex do not form a perfect base pair according to the Watson-Crick base pairing rules. The mismatched nucleotides can be A-A, A-G, A-C, U-U, U-G, U-C, G-G, G-A, G-U, C-A, C-C, C-U, etc.

在本申请中，术语“完全互补配对”，通常指核酸分子的核苷酸单位间仅存在严格的Watson-Crick或Hoogsteen碱基配对。完全互补配对不存在凸起、摆动、缺失、和/或错配。In the present application, the term "completely complementary pairing" generally refers to the presence of only strict Watson-Crick or Hoogsteen base pairing between nucleotide units of a nucleic acid molecule. Completely complementary pairing does not have bulges, wobbles, deletions, and/or mismatches.

在本申请中，术语“二级结构”，通常指核酸分子不完全互补配对形成的结构。不完全互补配对可能引发凸起、摆动、缺失、和/或错配。DNA分子的二级结构可以指DNA的螺旋构像。RNA分子的二级结构可以指螺旋构像、环、茎、和/或臂。本申请中，二级结构可以指RNA由于分子内碱基作用形成的二级结构，也可以指RNA由于分子间碱基作用形成的二级结构。环可以指没有配对的碱基突出形成的二级结构，茎可以指互补的碱基之间配对形成的局部A-型双螺旋二级结构，臂可以指紧靠着茎又不属于环的非配对核苷酸二级结构。In the present application, the term "secondary structure" generally refers to the structure formed by incomplete complementary pairing of nucleic acid molecules. Incomplete complementary pairing may cause protrusions, swings, deletions, and/or mismatches. The secondary structure of a DNA molecule may refer to the helical conformation of the DNA. The secondary structure of an RNA molecule may refer to a helical conformation, loops, stems, and/or arms. In the present application, the secondary structure may refer to the secondary structure formed by the action of bases within the molecule, or the secondary structure formed by the action of bases between molecules. The loop may refer to a secondary structure formed by protrusions of unpaired bases, the stem may refer to a local A-type double helix secondary structure formed by pairing between complementary bases, and the arm may refer to a non-paired nucleotide secondary structure that is close to the stem and does not belong to the loop.

在本申请中，术语“分离的核酸分子”通常是指任何长度的分离形式的核苷酸，脱氧核糖核苷酸或核糖核苷酸，或从其天然环境分离的或人工合成的类似物。As used herein, the term "isolated nucleic acid molecule" generally refers to nucleotides of any length in isolated form, either deoxyribonucleotides or ribonucleotides, or analogs thereof, separated from their natural environment or artificially synthesized.

在本申请中，术语“表达载体”，通常指包含表达元件(如启动子、RBS、终止子等)使核酸序列能够在细胞内表达的载体，或它的一种或多种具有相关表达元件的转录物。本申请中，所述表达载体可以表达ASO的全部或者部分序列。本申请中，表达载体可以表达ASO中的SD，例如所述表达载体所表达的SD序列还未经化学修饰。本申请中，表达载体可以表达ASO中的BHD，例如所述表达载体所表达的BHD序列还未经化学修饰。In the present application, the term "expression vector" generally refers to a vector containing expression elements (such as promoters, RBS, terminators, etc.) that enable a nucleic acid sequence to be expressed in a cell, or one or more transcripts thereof with relevant expression elements. In the present application, the expression vector can express all or part of the sequence of an ASO. In the present application, the expression vector can express the SD in an ASO, for example, the SD sequence expressed by the expression vector has not been chemically modified. In the present application, the expression vector can express the BHD in an ASO, for example, the BHD sequence expressed by the expression vector has not been chemically modified.

在本申请中，术语“递送载体”，通常指将一种或者多种核苷酸递送到细胞的载体。所述载体可以包括病毒载体，非病毒载体。病毒载体包括DNA病毒和RNA病毒，例如逆转录病毒(Retrovirus，RV)、慢病毒(Lentivirus，LV)载体，腺病毒(Adenovirus，AdV)载体和腺相关病毒(Adeno-associated virus，AAV)载体等。非病毒载体可以包括脂质体、分子偶联受体、聚合物、复合载体以及纳米粒子载体等。本申请中，所述递送载体可以将本申请所述的ASO或者分离的核酸分子递送到细胞内。In the present application, the term "delivery vector" generally refers to a vector that delivers one or more nucleotides to a cell. The vector may include a viral vector and a non-viral vector. Viral vectors include DNA viruses and RNA viruses, such as retrovirus (RV), lentivirus (LV) vectors, adenovirus (AdV) vectors and adeno-associated virus (AAV) vectors, etc. Non-viral vectors may include liposomes, molecular coupled receptors, polymers, composite vectors, and nanoparticle vectors, etc. In the present application, the delivery vector may deliver the ASO or isolated nucleic acid molecules described in the present application into cells.

在本申请中，细胞摄取表达载体或递送载体的方法包括但不限于电穿孔、脂质转染、核转染、显微注射、病毒感染、脂质体、外泌体，聚合阳离子或脂质、核酸缀合物、纳米颗粒，微泡、基因枪和裸核苷酸。In the present application, methods for cellular uptake of expression vectors or delivery vectors include, but are not limited to, electroporation, lipofection, nucleofection, microinjection, viral infection, liposomes, exosomes, polymeric cations or lipids, nucleic acid conjugates, nanoparticles, microbubbles, gene guns, and naked nucleotides.

本申请中，术语“细胞”通常包括原核细胞和真核细胞。可以在细胞中转染核酸，可以在原核细胞中繁衍质粒，可以在真核细胞中表达核酸、编码多肽。例如，细胞可以包括ASO，SD，BHD，分离的核酸分子，表达载体，递送载体。细胞可以是来自以下任何器官的细胞：例如皮肤、肺、心脏、肾脏、肝脏、胰腺、肠、肌肉、腺体、眼睛、脑、血液等。例如，细胞可以是人类细胞或小鼠细胞。例如，细胞可以是免疫细胞。例如，免疫细胞可以是T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞、外周血单个核细胞、胚胎干细胞、淋巴祖细胞和/或多能干细胞。在本申请中，术语“药物组合物”通常指适合施用于个体的化学或生物组合物。例如，所述个体可以是哺乳动物。例如，所述药物组合物可以包括所述ASO，所述分离的核酸分子，所述载体和/或所述细胞，以及可选的药学上可接受的载体。In the present application, the term "cell" generally includes prokaryotic cells and eukaryotic cells. Nucleic acids can be transfected in cells, plasmids can be propagated in prokaryotic cells, and nucleic acids and encoded polypeptides can be expressed in eukaryotic cells. For example, cells can include ASO, SD, BHD, isolated nucleic acid molecules, expression vectors, and delivery vectors. Cells can be cells from any of the following organs: for example, skin, lungs, heart, kidneys, liver, pancreas, intestines, muscles, glands, eyes, brain, blood, etc. For example, cells can be human cells or mouse cells. For example, cells can be immune cells. For example, immune cells can be T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes, peripheral blood mononuclear cells, embryonic stem cells, lymphoid progenitor cells, and/or pluripotent stem cells. In the present application, the term "pharmaceutical composition" generally refers to a chemical or biological composition suitable for administration to an individual. For example, the individual can be a mammal. For example, the pharmaceutical composition may include the ASO, the isolated nucleic acid molecule, the vector and/or the cell, and optionally a pharmaceutically acceptable carrier.

在本申请中，术语“药学上可接受的载体”，通常是指药学上可以掺入给予患者的药物组合物中，而不会引起任何不良的生物效应或以有害的方式与药物组合物中的任何其他成分相互作用的材料和/或成分。例如，液体或固体填充剂，稀释剂，赋形剂，溶剂或包囊材料。可充当药学上可接受的载剂的材料的一些实例包括：糖，诸如乳糖、葡萄糖和蔗糖；淀粉，诸如玉米淀粉和马铃薯淀粉；纤维素及其衍生物，诸如羧甲基纤维素钠、乙基纤维素和乙酸纤维素；粉状黄芪胶；麦芽；明胶；滑石；赋形剂，诸如可可脂和栓剂蜡；油，诸如花生油、棉籽油、红花油、芝麻油、橄榄油、玉米油和大豆油；二醇，诸如丙二醇；多元醇，诸如甘油、山梨醇、甘露醇和聚乙二醇；酯，诸如油酸乙酯和月桂酸乙酯；琼脂；缓冲剂，诸如氢氧化镁和氢氧化铝；海藻酸；无热原水；等渗盐水；林格氏溶液；乙醇；pH缓冲溶液；聚酯、聚碳酸酯和/或聚酸酐；以及在药物配制品中采用的其他无毒相容的物质。药学上可接受的载体包括药学上可接受的盐，其中术语“药学上可接受的盐”包括使用相对无毒性的酸或碱制备的活性化合物的盐，这取决于本文所述化合物上发现的特定取代基。In this application, the term "pharmaceutically acceptable carrier" generally refers to materials and/or ingredients that can be incorporated into a pharmaceutical composition to be administered to a patient without causing any adverse biological effect or interacting in a deleterious manner with any other ingredients in the pharmaceutical composition, such as liquid or solid fillers, diluents, excipients, solvents or encapsulating materials. Some examples of materials that can serve as pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; pH buffered solutions; polyesters, polycarbonates, and/or polyanhydrides; and other nontoxic, compatible substances employed in pharmaceutical formulations. Pharmaceutically acceptable carriers include pharmaceutically acceptable salts, wherein the term "pharmaceutically acceptable salts" includes salts of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein.

在本申请中，术语“治疗”通常是指：(1)预防可能易患疾病、病症和/或病状、但尚未诊断出患病的患者出现该疾病、病症或病状；(2)抑制该疾病、病症或病状，亦即遏制其发展；以及(3)缓解该疾病、病症或病状，亦即使得该疾病、病症和/或病状和/或与该疾病、病症和/或病状相关联的症状消退。As used herein, the term "treat" generally refers to: (1) preventing a disease, disorder, and/or condition from occurring in a patient who may be susceptible to the disease, but has not yet been diagnosed with the disease; (2) inhibiting the disease, disorder, or condition, i.e., curbing its development; and (3) alleviating the disease, disorder, or condition, i.e., causing the disease, disorder, and/or condition and/or symptoms associated with the disease, disorder, and/or condition to subside.

在本申请中，术语“受试者”通常指人类或非人类动物，包括但不限于猫、狗、马、猪、奶牛、羊、兔、小鼠、大鼠或猴。In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat or monkey.

在本申请中，术语“RNA编辑”，通常指在基因组编码的RNA序列中引入变化，从而导致RNA突变的共转录或转录后修饰过程。双链RNA(dsRNA)中的腺苷编辑为肌苷(A-到-I)，由作用于RNA(ADAR)酶家族的腺苷脱氨酶催化，是哺乳动物中常见的RNA编辑类型。在脊椎动物中，先前已对三种ADAR蛋白ADAR1、ADAR2和ADAR3的家族进行了表征。ADAR1和ADAR2(ADAR)催化所有当前已知的A-到-I编辑位点。ADAR3没有已知的脱氨酶活性。肌苷(I)模拟鸟苷(G)，因此ADAR蛋白在转录物中引入了虚拟的A到G取代。这种变化可以导致特定的氨基酸取代、可变剪接、miRNA介导的基因沉默、转录物定位或稳定性或表达的变化。In this application, the term "RNA editing" generally refers to the co-transcriptional or post-transcriptional modification process that introduces changes in the RNA sequence encoded by the genome, resulting in RNA mutations. Adenosine editing in double-stranded RNA (dsRNA) is catalyzed by adenosine deaminases acting on RNA (ADAR) enzyme family and is a common type of RNA editing in mammals. In vertebrates, a family of three ADAR proteins ADAR1, ADAR2 and ADAR3 has been previously characterized. ADAR1 and ADAR2 (ADAR) catalyze all currently known A-to-I editing sites. ADAR3 has no known deaminase activity. Inosine (I) mimics guanosine (G), so ADAR proteins introduce virtual A to G substitutions in transcripts. This change can lead to specific amino acid substitutions, alternative splicing, miRNA-mediated gene silencing, changes in transcript localization or stability or expression.

在本申请中，RNA编辑不仅可以用于动物细胞，例如哺乳动物细胞，还可以用于编辑植物或真菌的RNA，例如，在具有内源表达的脱氨酶(如ADAR)的植物或真菌中。本申请的方法可用于产生优化的基因工程植物和真菌。In the present application, RNA editing can be used not only in animal cells, such as mammalian cells, but also in editing RNA of plants or fungi, for example, in plants or fungi with endogenously expressed deaminases (such as ADARs). The method of the present application can be used to produce optimized genetically engineered plants and fungi.

在本申请中，术语“腺嘌呤”，“鸟嘌呤”，“胞嘧啶”，“胸腺嘧啶”，“尿嘧啶”和“次黄嘌呤”是指核碱基本身。术语“腺苷”，“鸟苷”，“胞苷”，“胸苷”，“尿苷”和“肌苷”是指与核糖或脱氧核糖的糖部分连接的核碱基。术语“核苷”是指与核糖或脱氧核糖连接的核碱基。In this application, the terms "adenine", "guanine", "cytosine", "thymine", "uracil" and "hypoxanthine" refer to the nucleobases themselves. The terms "adenosine", "guanosine", "cytidine", "thymidine", "uridine" and "inosine" refer to the nucleobases linked to the sugar moiety of ribose or deoxyribose. The term "nucleoside" refers to the nucleobase linked to ribose or deoxyribose.

在本申请中，术语“核苷酸”是指各自的核碱基-核糖基-磷酸酯或核碱基-脱氧核糖基-磷酸酯。本申请中，术语“腺苷”和“腺嘌呤”(缩写“A”)，“鸟苷”和“鸟嘌呤”In this application, the term "nucleotide" refers to the respective nucleobase-ribosyl-phosphate or nucleobase-deoxyribosyl-phosphate. In this application, the terms "adenosine" and "adenine" (abbreviated "A"), "guanosine" and "guanine"

(缩写“G”)，“胞苷”和“胞嘧啶”(缩写“C”)，“尿苷”和“尿嘧啶”(缩写“U”)，“胸苷”和“胸腺嘧啶”(缩写“T”)，“次黄嘌呤”和“肌苷”(缩写“I”)，可互换使用。(abbreviated "G"), "cytidine" and "cytosine" (abbreviated "C"), "uridine" and "uracil" (abbreviated "U"), "thymidine" and "thymine" (abbreviated "T"), and "hypoxanthine" and "inosine" (abbreviated "I") are used interchangeably.

在本申请中，术语“体外”是指在人造环境中发生的事件，例如在试管或反应容器中，在细胞培养物中等，而不是在生物体(例如，动物、植物和/或微生物)内。In this application, the term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, in cell culture, etc., rather than within an organism (eg, animal, plant and/or microorganism).

在本申请中，术语“体内”是指在生物体(例如，动物、植物和/或微生物)内发生的事件。In this application, the term "in vivo" refers to events that occur within an organism (eg, an animal, a plant, and/or a microorganism).

在本申请中，术语“和/或”应理解为意指可选项中的任一项或可选项的两项。In the present application, the term "and/or" should be understood to mean either one of the alternatives or both of the alternatives.

在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。在某些情形中，“包含”也涵盖仅包括指定的组分的情况。例如，包含也表示为也表示“由……组成”的含义。In this application, the term "comprising" generally means including the features explicitly specified, but not excluding other elements. In some cases, "comprising" also covers the situation of only including the specified components. For example, comprising is also expressed as also meaning "consisting of..."

在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below a specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

在本申请中，术语“包括”通常是指包含、总括、含有或包涵的含义。在某些情况下，也表示“为”、“由……组成”的含义。In this application, the term "include" generally means to include, encompass, contain or encompass. In some cases, it also means "for", "consisting of...".

发明详述DETAILED DESCRIPTION OF THE INVENTION

用于靶向RNA编辑的单链反义寡核苷酸Single-stranded antisense oligonucleotides for targeted RNA editing

一方面，本申请提供一种用于靶向RNA编辑的单链反义寡核苷酸(AntisenseOligonucleotide，ASO)，其中所述ASO包含靶向区(Specificity Domain，SD)和辅助结合区(Binding Helper Domain，BHD)，其中所述SD与靶向RNA区域互补配对，所述BHD与非靶向RNA区域互补配对，所述ASO能够与目标RNA形成双链复合物。On the one hand, the present application provides a single-stranded antisense oligonucleotide (Antisense Oligonucleotide, ASO) for targeted RNA editing, wherein the ASO comprises a targeting region (Specificity Domain, SD) and an auxiliary binding region (Binding Helper Domain, BHD), wherein the SD is complementary to the targeted RNA region, the BHD is complementary to the non-targeted RNA region, and the ASO can form a double-stranded complex with the target RNA.

本申请中，所述的靶向RNA区域与非靶向RNA区域非连续。In the present application, the targeting RNA region is not continuous with the non-targeting RNA region.

例如，所述靶向RNA区域和所述非靶向RNA区域间隔的长度可以为4-1000nt。例如，所述靶向RNA区域和所述非靶向RNA区域间隔的长度可以为4-200nt。例如，所述靶向RNA区域和所述非靶向RNA区域间隔的长度可以为200-500nt。例如，所述靶向RNA区域和所述非靶向RNA区域间隔的长度可以为500-1000nt。例如，所述靶向RNA区域和所述非靶向RNA区域间隔的长度可以为4-200nt。例如，所述靶向RNA区域和所述非靶向RNA区域间隔的长度可以为4nt、8nt、11nt、12nt、24nt、25nt、36nt、237nt、340nt、343nt、494nt、681nt nt。For example, the length of the target RNA region and the non-target RNA region interval can be 4-1000nt. For example, the length of the target RNA region and the non-target RNA region interval can be 4-200nt. For example, the length of the target RNA region and the non-target RNA region interval can be 200-500nt. For example, the length of the target RNA region and the non-target RNA region interval can be 500-1000nt. For example, the length of the target RNA region and the non-target RNA region interval can be 4-200nt. For example, the length of the target RNA region and the non-target RNA region interval can be 4nt, 8nt, 11nt, 12nt, 24nt, 25nt, 36nt, 237nt, 340nt, 343nt, 494nt, 681nt nt.

本申请中，所述的ASO可以通过细胞中存在的ADAR酶对靶向RNA区域中存在的靶腺苷进行脱氨基化。In the present application, the ASO can deaminize the target adenosine present in the target RNA region by the ADAR enzyme present in the cell.

本申请中，所述的目标RNA选自下组中的一种或者多种：pre-mRNA、mRNA、rRNA、tRNA、lnc-RNA、snRNA、snoRNA。In the present application, the target RNA is selected from one or more of the following groups: pre-mRNA, mRNA, rRNA, tRNA, lnc-RNA, snRNA, and snoRNA.

本申请中，所述的ASO可以具有一种或者多种修饰。In the present application, the ASO may have one or more modifications.

例如，所述ASO中的BHD可以包含一种或者多种修饰。例如，所述ASO中的SD可以包含一种或者多种修饰。For example, the BHD in the ASO may comprise one or more modifications. For example, the SD in the ASO may comprise one or more modifications.

例如，所述修饰可以是糖修饰和/或碱基修饰。For example, the modification may be a sugar modification and/or a base modification.

例如，所述ASO可以包含同位素修饰。例如，ASO可以包含一种或多种元素(例如，氢、碳、氮等)的同位素修饰。例如，ASO可以包含一个或多个同位素修饰。For example, the ASO may comprise an isotopic modification. For example, the ASO may comprise an isotopic modification of one or more elements (e.g., hydrogen, carbon, nitrogen, etc.). For example, the ASO may comprise one or more isotopic modifications.

例如，所述ASO中，被修饰的可以是核碱基。经修饰的核碱基可以具有核碱基的至少一种功能。例如，能够进行碱基互补配对。例如，经修饰的核碱基可以是A、T、C、G或U。例如，可以对碱基进行替代修饰，所述替代修饰可以是DNA碱基替代修饰和/或核苷类似物替代修饰(如肌苷)。例如，所述ASO可以包含一个或多个经修饰的核碱基。例如，所述ASO中，经修饰的核碱基占比可以是约5％-100％、约10％-100％、20-100％、30％-100％、40％-100％、50％-80％、50％-85％、50％-90％、50％-95％、60％-80％、60％-85％、60％-90％、60％-95％、60％-100％、65％-80％、65％-85％、65％-90％、65％-95％、65％-100％、70％-80％、70％-85％、70％-90％、70％-95％、70％-100％、75％-80％、75％-85％、75％-90％、75％-95％、75％-100％、80％-85％、80％-90％、80％-95％、80％-100％、85％-90％、85％-95％、85％-100％、90％-95％、90％-100％，10％、20％、30％、40％、50％、60％、65％、70％、75％、80％、85％、90％、95％或100％等。For example, in the ASO, the modified nucleobase may be a nucleobase. The modified nucleobase may have at least one function of a nucleobase. For example, it may be capable of base complementary pairing. For example, the modified nucleobase may be A, T, C, G or U. For example, the base may be subjected to substitution modification, and the substitution modification may be a DNA base substitution modification and/or a nucleoside analog substitution modification (such as inosine). For example, the ASO may contain one or more modified nucleobases. For example, in the ASO, the modified nucleobase ratio can be about 5%-100%, about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, 70%-80%, 70%-85%, 70%-85%, 70%-90%, 70%-95%, 70%-100%, 70%-80%, 70%-85%, 70%-80%, 70%-85%, 70%-90%, 70%-85%, 70%-90%, 70%-85%, 70%-80%, 70%-85%, 70%-90%, 70%-85%, 70%-90%, 70%-85%, 70%-80%, 70%-85%, 70%-90%, 70%-85%, 70%-90%, 70%-85%, 70%-80%, 70%-85%, 70%-90%, 70%-95%, 70%-100%, 70%-80%, 70%-85 ... 0%-90%, 70%-95%, 70%-100%, 75%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, etc.

例如，所述ASO中，被修饰的可以是核糖。经修饰的糖可以具有未经修饰的核糖的空间排列、电子特性、或一些其他物理化学特性。例如，经修饰的糖是经取代的核糖或脱氧核糖。例如，经修饰的糖包含2’-修饰，所述2’-修饰可以是2’-MOE、2’-OR，例如2’-OMe(其中R表示烷基)、2’-F。例如，经修饰的糖可以包含LNA修饰。例如，经修饰的糖可以包含UNA修饰。例如，所述ASO可以包含一个或多个经修饰的糖。例如，所述ASO中，经修饰的糖占比可以是约5％-100％、约10％-100％、20-100％、30％-100％、40％-100％、50％-80％、50％-85％、50％-90％、50％-95％、60％-80％、60％-85％、60％-90％、60％-95％、60％-100％、65％-80％、65％-85％、65％-90％、65％-95％、65％-100％、70％-80％、70％-85％、70％-90％、70％-95％、70％-100％、75％-80％、75％-85％、75％-90％、75％-95％、75％-100％、80％-85％、80％-90％、80％-95％、80％-100％、85％-90％、85％-95％、85％-100％、90％-95％、90％-100％，10％、20％、30％、40％、50％、60％、65％、70％、75％、80％、85％、90％、95％或100％等。For example, in the ASO, the modified sugar may be ribose. The modified sugar may have the spatial arrangement, electronic properties, or some other physicochemical properties of the unmodified ribose. For example, the modified sugar is a substituted ribose or deoxyribose. For example, the modified sugar comprises a 2'-modification, and the 2'-modification may be 2'-MOE, 2'-OR, such as 2'-OMe (wherein R represents an alkyl group), 2'-F. For example, the modified sugar may comprise an LNA modification. For example, the modified sugar may comprise a UNA modification. For example, the ASO may comprise one or more modified sugars. For example, in the ASO, the modified sugar ratio can be about 5%-100%, about 10%-100%, 20-100%, 30%-100%, 40%-100%, 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, 70%-80%, 70%-85%, 70%-90%, 70%-95%, 70%-100%, 70%-80%, 70%-85%, 70%-9 ...5%, 70%-100%, 70%-80%, 70%-85%, 70%-85%, 70%-90%, 70%-85%, 70%-95%, 70%-100%, 70%-80%, 70%-85%, 70%-85%, 70%-90%, 70%-95%, 70%-1 %-90%, 70%-95%, 70%-100%, 75%-80%, 75%-85%, 75%-90%, 75%-95%, 75%-100%, 80%-85%, 80%-90%, 80%-95%, 80%-100%, 85%-90%, 85%-95%, 85%-100%, 90%-95%, 90%-100%, 10%, 20%, 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, etc.

辅助结合区(Binding Helper Domain，BHD)Binding Helper Domain (BHD)

另一方面，本申请提供的ASO包含BHD，BHD结合的目标RNA中的序列不包含靶位点。On the other hand, the ASO provided by the present application comprises a BHD, and the sequence in the target RNA bound by the BHD does not comprise a target site.

本申请中，BHD可以增加ASO与目标RNA形成双链复合物的稳定性。本申请中，BHD可以不具有招募功能。In the present application, BHD can increase the stability of the double-stranded complex formed by ASO and target RNA. In the present application, BHD may not have a recruitment function.

例如，稳定性的提高可以体现为ASO编辑效率的提高。例如，ASO的编辑效率与不含BHD的ASO相比，可以提高约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％。For example, the improved stability can be reflected in an improved editing efficiency of the ASO. For example, the editing efficiency of the ASO can be improved by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% compared to an ASO without BHD.

本申请中，BHD可以与非靶向RNA区域完全互补配对。In the present application, the BHD can be fully complementary to the non-targeting RNA region.

本申请中，BHD可以与非靶向RNA区域互补配对，且存在一个或多个错配、摆动、缺失、和/或凸起。In the present application, the BHD may be complementary to a non-target RNA region and may have one or more mismatches, wobbles, deletions, and/or bulges.

例如，所述互补配对原则为Watson-Crick或Hoogsteen碱基配对。例如，所述互补配对可以指A-T，C-G，T*A/T(指质子化的T能和A-T碱基对中的A配对)，C*G/C(指质子化的C能和G-C碱基对中的G配对)。例如，可以存在1-10个(例如，1-2、1-3、1-4、1-5、1-6、1-7、1-8、1-9、1-10、2-3、2-4、2-5、2-6、2-7、2-8、2-9、2-10、3-4、3-5、3-6、3-7、3-8、3-9、3-10，0、1、2、3、4、5、6、7、8、9或10个等)碱基错配。例如，可以存在1-10个(例如，1-2、1-3、1-4、1-5、1-6、1-7、1-8、1-9、1-10、2-3、2-4、2-5、2-6、2-7、2-8、2-9、2-10、3-4、3-5、3-6、3-7、3-8、3-9、3-10，0、1、2、3、4、5、6、7、8、9或10个等)摆动。例如，可以存在1-10个(例如，1-2、1-3、1-4、1-5、1-6、1-7、1-8、1-9、1-10、2-3、2-4、2-5、2-6、2-7、2-8、2-9、2-10、3-4、3-5、3-6、3-7、3-8、3-9、3-10，0、1、2、3、4、5、6、7、8、9或10个等)缺失。例如，可以存在1-10个(例如，1-2、1-3、1-4、1-5、1-6、1-7、1-8、1-9、1-10、2-3、2-4、2-5、2-6、2-7、2-8、2-9、2-10、3-4、3-5、3-6、3-7、3-8、3-9、3-10，0、1、2、3、4、5、6、7、8、9或10个等)凸起。例如，所述BHD与所述非靶向RNA区域之间的互补性为约50％-100％(例如，约50％-80％、50％-85％、50％-90％、50％-95％、60％-80％、60％-85％、60％-90％、60％-95％、60％-100％、65％-80％、65％-85％、65％-90％、65％-95％、65％-100％、70％-80％、70％-85％、70％-90％、70％-95％、70％-100％、75％-80％、75％-85％、75％-90％、75％-95％、75％-100％、80％-85％、80％-90％、80％-95％、80％-100％、85％-90％、85％-95％、85％-100％、90％-95％、90％-100％，50％、60％、65％、70％、75％、80％、85％、90％、95％或100％等)。例如，互补性为至少约60％。在一些实施例中，互补性为至少约65％。例如，互补性为至少约70％。例如，互补性为至少约75％。例如，互补性为至少约80％。例如，互补性为至少约85％。例如，互补性为至少约90％。例如，互补性为至少约95％。For example, the complementary pairing principle is Watson-Crick or Hoogsteen base pairing. For example, the complementary pairing can refer to A-T, C-G, T*A/T (referring to protonated T can pair with A in A-T base pairing), C*G/C (referring to protonated C can pair with G in G-C base pairing). For example, there can be 1-10 (for example, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 etc.) base mismatches. For example, there may be 1-10 (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, etc.) wiggles. For example, there can be 1-10 (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 etc.) deletions. For example, there may be 1-10 (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, etc.) protrusions. For example, the complementarity between the BHD and the non-targeting RNA region is about 50%-100% (e.g., about 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, 70%-80%, 70%-85%, 70%-90%). In some embodiments, the complementarity is at least about 60%. In some embodiments, the complementarity is at least about 65%. In some embodiments, the complementarity is at least about 70%. In some embodiments, the complementarity is at least about 75%. In some embodiments, the complementarity is at least about 80%. In some embodiments, the complementarity is at least about 85%. In some embodiments, the complementarity is at least about 90%. In some embodiments, the complementarity is at least about 100%. In some embodiments, the complementarity is at least about 150%. In some embodiments, the complementarity is at least about 100%. In some embodiments, the complementarity is at least about 150%. In some embodiments, the complementarity is at least about 100%. In some embodiments, the complementarity is at least about 150%. In some embodiments, the complementarity is at least about 100%. In some embodiments, the complementarity is at least about 150%. In some embodiments, the complementarity is at least about 150%. In some embodiments, the complementarity is at least about 150%. For example, the complementarity is at least about 95%.

本申请中，BHD可以具有选自下组修饰的一种或者多种：糖修饰、碱基修饰。In the present application, BHD may have one or more modifications selected from the following group: sugar modification, base modification.

例如，所述BHD可以具有选自下组中的一种或者多种修饰：硫代磷酸酯化修饰、2’-OMe、2’-F、LNA、UNA、2’-MOE、肌苷替代修饰。For example, the BHD may have one or more modifications selected from the following group: phosphorothioate modification, 2'-OMe, 2'-F, LNA, UNA, 2'-MOE, and inosine substitution modification.

例如，所述BHD可以具有硫代磷酸酯化修饰、2’-OMe、LNA修饰。例如，所述BHD可以具有硫代磷酸酯化修饰、2’-OMe修饰。For example, the BHD may have a phosphorothioate modification, a 2'-OMe modification, or a LNA modification. For example, the BHD may have a phosphorothioate modification, a 2'-OMe modification.

本申请中，所述的BHD的长度可以为8-40nt。In the present application, the length of the BHD can be 8-40 nt.

例如，所述BHD的长度可以为8-15nt、8-25nt、8-35nt、15-25nt、15-35nt、15-40nt、25-35nt、25-40nt、35-40nt。例如，所述BHD的长度可以为15nt、20nt、25nt。For example, the length of the BHD may be 8-15 nt, 8-25 nt, 8-35 nt, 15-25 nt, 15-35 nt, 15-40 nt, 25-35 nt, 25-40 nt, 35-40 nt. For example, the length of the BHD may be 15 nt, 20 nt, 25 nt.

本申请中，所述的ASO可以包含一个或者多个BHD。In the present application, the ASO may include one or more BHDs.

例如，可以存在1-10个(例如，1-2、1-3、1-4、1-5、1-6、1-7、1-8、1-9、1-10、2-3、2-4、2-5、2-6、2-7、2-8、2-9、2-10、3-4、3-5、3-6、3-7、3-8、3-9、3-10，0、1、2、3、4、5、6、7、8、9或10个等)BHD。例如，多个BHD的长度可以不同，多个BHD与SD的相对位置可以不同。For example, there may be 1-10 (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, etc.) BHDs. For example, the lengths of the multiple BHDs may be different, and the relative positions of the multiple BHDs to the SD may be different.

本申请中，所述的一个或者多个BHD可以不形成分子内二级结构。In the present application, the one or more BHDs may not form a secondary structure within the molecule.

本申请中，所述的一个或者多个BHD可以形成分子内二级结构。In the present application, the one or more BHDs may form a secondary structure within the molecule.

本申请中，所述的多个BHD可以不形成分子间二级结构。In the present application, the multiple BHDs may not form an intermolecular secondary structure.

本申请中，所述的多个BHD可以形成分子间二级结构。In the present application, the multiple BHDs can form an intermolecular secondary structure.

本申请中，所述的一个或者多个BHD可以与SD不形成分子间二级结构。In the present application, the one or more BHDs may not form an intermolecular secondary structure with SD.

本申请中，所述的一个或者多个BHD可以与SD形成分子间二级结构。In the present application, the one or more BHDs can form an intermolecular secondary structure with SD.

例如，可以存在1-10个(例如，1-2、1-3、1-4、1-5、1-6、1-7、1-8、1-9、1-10、2-3、2-4、2-5、2-6、2-7、2-8、2-9、2-10、3-4、3-5、3-6、3-7、3-8、3-9、3-10，0、1、2、3、4、5、6、7、8、9或10个等)分子内二级结构。例如，可以存在1-10个(例如，1-2、1-3、1-4、1-5、1-6、1-7、1-8、1-9、1-10、2-3、2-4、2-5、2-6、2-7、2-8、2-9、2-10、3-4、3-5、3-6、3-7、3-8、3-9、3-10，0、1、2、3、4、5、6、7、8、9或10个等)分子间二级结构。For example, there may be 1-10 (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, etc.) intramolecular secondary structures. For example, there may be 1-10 (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, etc.) intermolecular secondary structures.

例如，二级结构可以指核酸分子不完全互补配对形成的结构。不完全互补配对可能引发凸起、摆动、缺失、和/或错配。例如，二级结构可以指RNA由于分子内碱基作用形成的二级结构，也可以指RNA由于分子间碱基作用形成的二级结构。环可以指没有配对的碱基突出形成的二级结构，茎可以指互补的碱基之间配对形成的局部A-型双螺旋二级结构，臂可以指紧靠着茎又不属于环的非配对核苷酸二级结构。For example, secondary structure can refer to the structure formed by incomplete complementary pairing of nucleic acid molecules. Incomplete complementary pairing may cause protrusions, swings, deletions, and/or mismatches. For example, secondary structure can refer to the secondary structure formed by RNA due to the action of intramolecular bases, and can also refer to the secondary structure formed by RNA due to the action of intermolecular bases. Loops can refer to secondary structures formed by protruding bases without pairing, stems can refer to local A-type double helix secondary structures formed by pairing between complementary bases, and arms can refer to non-paired nucleotide secondary structures that are close to stems and do not belong to loops.

本申请中，所述的BHD与SD的连接顺序不受限制。In the present application, the connection order of the BHD and SD is not limited.

例如，所述BHD可以在所述SD的5’端。例如，所述BHD可以在所述SD的3’端。例如，所述多个BHD可以在所述SD的5’端。例如，所述多个BHD可以在所述SD的3’端。例如，所述多个BHD可以在所述SD的5’端和3’端。For example, the BHD may be at the 5' end of the SD. For example, the BHD may be at the 3' end of the SD. For example, the multiple BHDs may be at the 5' end of the SD. For example, the multiple BHDs may be at the 3' end of the SD. For example, the multiple BHDs may be at both the 5' end and the 3' end of the SD.

本申请中，所述的BHD与SD的连接方式不受限制。In this application, the connection method between the BHD and SD is not limited.

例如，所述BHD可以与SD直接连接。例如，所述BHD可以与SD间接连接。例如，所述间接连接可以通过一个或者多个Linker连接，多个Linker可以是相同种类，也可以是不同种类。例如，所述Linker可以包括常规(磷酸二酯)或修饰(如硫代磷酸)的一个或多个核苷酸、寡肽或任何其他化学连接子。例如，所述Linker选自下组中的一种或者多种：AAA、AAAC、AACAA、AAAACAAAA、PEG2、C6。例如，所述Linker可以是AAA。例如，所述Linker可以是AAAC。例如，所述Linker可以是AACAA。例如，所述Linker可以是AAAACAAAA。例如，所述Linker可以是PEG2。例如，所述Linker可以是C6。例如，多个BHD与SD连接时，可以有一个或者多个Linker，Linker的种类可以相同或者不同。For example, the BHD can be directly connected to the SD. For example, the BHD can be indirectly connected to the SD. For example, the indirect connection can be connected through one or more Linkers, and the multiple Linkers can be of the same type or of different types. For example, the Linker can include one or more nucleotides, oligopeptides, or any other chemical linkers that are conventional (phosphodiester) or modified (such as thiophosphate). For example, the Linker is selected from one or more of the following groups: AAA, AAAC, AACAA, AAAACAAAA, PEG2, C6. For example, the Linker can be AAA. For example, the Linker can be AAAC. For example, the Linker can be AACAA. For example, the Linker can be AAAACAAAA. For example, the Linker can be PEG2. For example, the Linker can be C6. For example, when multiple BHDs are connected to SD, there can be one or more Linkers, and the types of Linkers can be the same or different.

靶向区(Specificity Domain，SD)Targeting domain (Specificity Domain, SD)

本申请提供的ASO包含SD，SD结合的目标RNA中的序列包含靶位点。ASO通过SD识别并结合目标RNA。The ASO provided in the present application comprises SD, and the sequence in the target RNA bound by SD comprises the target site. The ASO recognizes and binds to the target RNA through SD.

本申请中，所述的SD可以与靶向RNA区域完全互补配对。In the present application, the SD can be fully complementary to the target RNA region.

本申请中，所述的SD可以与靶向RNA区域互补配对，且存在一个或多个错配、摆动、缺失、和/或凸起。In the present application, the SD may be complementary to the target RNA region and may have one or more mismatches, wobbles, deletions, and/or protrusions.

例如，所述互补配对原则为Watson-Crick或Hoogsteen碱基配对。例如，所述互补配对可以指A-T，C-G，T*A/T(指质子化的T能和A-T碱基对中的A配对)，C*G/C(指质子化的C能和G-C碱基对中的G配对)。例如，可以存在1-10个(例如，1-2、1-3、1-4、1-5、1-6、1-7、1-8、1-9、1-10、2-3、2-4、2-5、2-6、2-7、2-8、2-9、2-10、3-4、3-5、3-6、3-7、3-8、3-9、3-10，0、1、2、3、4、5、6、7、8、9或10个等)碱基错配。例如，可以存在1-10个(例如，1-2、1-3、1-4、1-5、1-6、1-7、1-8、1-9、1-10、2-3、2-4、2-5、2-6、2-7、2-8、2-9、2-10、3-4、3-5、3-6、3-7、3-8、3-9、3-10，0、1、2、3、4、5、6、7、8、9或10个等)摆动。例如，可以存在1-10个(例如，1-2、1-3、1-4、1-5、1-6、1-7、1-8、1-9、1-10、2-3、2-4、2-5、2-6、2-7、2-8、2-9、2-10、3-4、3-5、3-6、3-7、3-8、3-9、3-10，0、1、2、3、4、5、6、7、8、9或10个等)缺失。例如，可以存在1-10个(例如，1-2、1-3、1-4、1-5、1-6、1-7、1-8、1-9、1-10、2-3、2-4、2-5、2-6、2-7、2-8、2-9、2-10、3-4、3-5、3-6、3-7、3-8、3-9、3-10，0、1、2、3、4、5、6、7、8、9或10个等)凸起。例如，所述SD与所述靶向RNA区域之间的互补性为约50％-100％(例如，约50％-80％、50％-85％、50％-90％、50％-95％、60％-80％、60％-85％、60％-90％、60％-95％、60％-100％、65％-80％、65％-85％、65％-90％、65％-95％、65％-100％、70％-80％、70％-85％、70％-90％、70％-95％、70％-100％、75％-80％、75％-85％、75％-90％、75％-95％、75％-100％、80％-85％、80％-90％、80％-95％、80％-100％、85％-90％、85％-95％、85％-100％、90％-95％、90％-100％，50％、60％、65％、70％、75％、80％、85％、90％、95％或100％等)。例如，互补性为至少约60％。在一些实施例中，互补性为至少约65％。例如，互补性为至少约70％。例如，互补性为至少约75％。例如，互补性为至少约80％。例如，互补性为至少约85％。例如，互补性为至少约90％。例如，互补性为至少约95％。For example, the complementary pairing principle is Watson-Crick or Hoogsteen base pairing. For example, the complementary pairing can refer to A-T, C-G, T*A/T (referring to protonated T can pair with A in A-T base pairing), C*G/C (referring to protonated C can pair with G in G-C base pairing). For example, there can be 1-10 (for example, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 etc.) base mismatches. For example, there may be 1-10 (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, etc.) wiggles. For example, there can be 1-10 (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 etc.) deletions. For example, there may be 1-10 (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, etc.) protrusions. For example, the complementarity between the SD and the targeting RNA region is about 50%-100% (e.g., about 50%-80%, 50%-85%, 50%-90%, 50%-95%, 60%-80%, 60%-85%, 60%-90%, 60%-95%, 60%-100%, 65%-80%, 65%-85%, 65%-90%, 65%-95%, 65%-100%, 70%-80%, 70%-85%, 70%-90%, In some embodiments, the complementarity is at least about 60%. In some embodiments, the complementarity is at least about 65%. In some embodiments, the complementarity is at least about 70%. In some embodiments, the complementarity is at least about 75%. In some embodiments, the complementarity is at least about 80%. In some embodiments, the complementarity is at least about 85%. In some embodiments, the complementarity is at least about 90%. In some embodiments, the complementarity is at least about 90%. In some embodiments, the complementarity is at least about 100%. In some embodiments, the complementarity is at least about 150%. In some embodiments, the complementarity is at least about 100%. In some embodiments, the complementarity is at least about 150%. In some embodiments, the complementarity is at least about 100%. In some embodiments, the complementarity is at least about 150%. In some embodiments, the complementarity is at least about 100%. In some embodiments, the complementarity is at least about 150%. In some embodiments, the complementarity is at least about 150%. For example, the complementarity is at least about 95%.

本申请中，所述的SD可以具有选自下组修饰的一种或者多种：糖修饰、碱基修饰。In the present application, the SD may have one or more modifications selected from the following group: sugar modification, base modification.

例如，所述SD可以具有选自下组中的一种或者多种修饰：硫代磷酸酯化修饰、2’-OMe、2’-F、LNA、UNA、2’-MOE、2’-FANA、DNA碱基替代修饰和肌苷替代修饰。例如，所述SD具有硫代磷酸酯化修饰。例如，所述SD可以具有2’-OMe修饰。例如，所述SD可以具有2’-F修饰。例如，所述SD可以具有硫代磷酸酯化修饰和2’-OMe修饰。例如，所述SD可以具有硫代磷酸酯化修饰和2’-F修饰。For example, the SD may have one or more modifications selected from the group consisting of phosphorothioate modification, 2'-OMe, 2'-F, LNA, UNA, 2'-MOE, 2'-FANA, DNA base substitution modification, and inosine substitution modification. For example, the SD has a phosphorothioate modification. For example, the SD may have a 2'-OMe modification. For example, the SD may have a 2'-F modification. For example, the SD may have a phosphorothioate modification and a 2'-OMe modification. For example, the SD may have a phosphorothioate modification and a 2'-F modification.

本申请中，所述SD的长度可以为20-50nt。In the present application, the length of the SD may be 20-50 nt.

例如，所述SD的长度可以为20-25nt、20-30nt、20-40nt、20-45nt、30-35nt、30-40nt、30-45nt、30-50nt、40-45nt、45-50nt。例如，所述SD的长度可以为30nt。For example, the length of the SD may be 20-25 nt, 20-30 nt, 20-40 nt, 20-45 nt, 30-35 nt, 30-40 nt, 30-45 nt, 30-50 nt, 40-45 nt, 45-50 nt. For example, the length of the SD may be 30 nt.

靶向RNA区域和非靶向RNA区域Targeted RNA region and non-targeted RNA region

本申请中，靶向RNA区域包含靶腺苷。In the present application, the targeted RNA region comprises a target adenosine.

本申请中，靶向RNA区域的长度可以为15-60nt，其包含与SD结合的序列，还可以包含错配、凸起、缺失、和/或摆动的序列。In the present application, the length of the targeting RNA region can be 15-60 nt, which includes a sequence that binds to SD and may also include mismatches, protrusions, deletions, and/or wobble sequences.

例如，所述靶向RNA区域的长度可以为15-20nt、15-30nt、15-40nt、15-50nt、25-30nt、25-40nt、25-50nt、25-60nt、35-40nt、35-50nt、35-60nt、45-50nt、45-60nt。例如，所述靶向RNA区域的长度可以为30nt。For example, the length of the targeting RNA region can be 15-20nt, 15-30nt, 15-40nt, 15-50nt, 25-30nt, 25-40nt, 25-50nt, 25-60nt, 35-40nt, 35-50nt, 35-60nt, 45-50nt, 45-60nt. For example, the length of the targeting RNA region can be 30nt.

本申请中，所述的非靶向RNA区域不包含靶腺苷，其包含与BHD结合的序列，还可以包含错配、凸起、缺失、和/或摆动的序列。In the present application, the non-targeting RNA region does not contain a target adenosine, but contains a sequence that binds to BHD, and may also contain a mismatch, a protrusion, a deletion, and/or a wobble sequence.

本申请中，所述的非靶向RNA区域的长度为5-50nt。In the present application, the length of the non-targeting RNA region is 5-50 nt.

例如，所述非靶向RNA区域的长度可以为5-10nt、5-20nt、5-30nt、5-40nt、15-20nt、15-30nt、15-40nt、15-50nt、25-30nt、25-40nt、25-50nt、35-40nt、35-50nt、45-50nt。例如，所述非靶向RNA区域的长度可以为15nt，20nt，25nt。For example, the length of the non-targeting RNA region can be 5-10nt, 5-20nt, 5-30nt, 5-40nt, 15-20nt, 15-30nt, 15-40nt, 15-50nt, 25-30nt, 25-40nt, 25-50nt, 35-40nt, 35-50nt, 45-50nt. For example, the length of the non-targeting RNA region can be 15nt, 20nt, 25nt.

编辑RNA的方法Methods for editing RNA

另一方面，本申请提供一种编辑RNA的方法，其可以包含使用所述的ASO。On the other hand, the present application provides a method for editing RNA, which may include using the ASO.

本申请中，所述的方法可以包括以下步骤：In this application, the method may include the following steps:

(1)提供所述的ASO；(1) providing the ASO;

(2)允许细胞摄取所述ASO；(2) allowing cells to take up the ASO;

(3)允许所述ASO与目标RNA结合；(3) allowing the ASO to bind to the target RNA;

(4)允许细胞内ADAR酶将靶向RNA区域中的靶腺苷脱氨基成为肌苷；(4) allowing intracellular ADAR enzymes to deaminate target adenosine in the targeted RNA region to inosine;

(5)鉴定靶向RNA区域中肌苷的存在。(5) Identify the presence of inosine in the targeted RNA region.

本申请中，所述的方法能够招募内源脱氨基酶对特定核苷酸位点进行脱氨基反应。In the present application, the method described is capable of recruiting endogenous deaminase to perform deamination reaction on specific nucleotide sites.

本申请中，所述的方法可以提高编辑效率。例如，所述编辑效率与不使用所述ASO相比，可以提高约10％、20％、30％、40％、50％、60％、70％、80％、90％、100％。In the present application, the method described can improve editing efficiency. For example, the editing efficiency can be improved by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% compared to not using the ASO.

例如，ADAR是ADAR1和/或ADAR2。例如，ADAR是选自下组的一种或多种ADAR：hADAR1、hADAR2、小鼠ADAR1、小鼠ADAR2。For example, the ADAR is ADAR1 and/or ADAR2. For example, the ADAR is one or more ADARs selected from the group consisting of hADAR1, hADAR2, mouse ADAR1, mouse ADAR2.

例如，RNA编辑方法不仅可以用于动物细胞，例如，哺乳动物细胞，还可以用于编辑植物或真菌的RNA，例如，在具有内源表达的ADAR的植物或真菌中，可以使用本身提供的RNA编辑方法产生具有改善性质的基因工程植物和真菌。For example, RNA editing methods can be used not only in animal cells, e.g., mammalian cells, but can also be used to edit RNA in plants or fungi. For example, in plants or fungi with endogenously expressed ADARs, the RNA editing methods provided by themselves can be used to produce genetically engineered plants and fungi with improved properties.

一种或者多种分离的核酸分子One or more isolated nucleic acid molecules

另一方面，本申请提供一种或者多种分离的核酸分子，可以编码本申请所述的ASO，可以编码ASO中的SD，也可以编码ASO中的BHD，其编码的ASO、SD、BHD都是未经修饰的。可以对一种或者多种分离的核酸分子编码出的序列进行进一步修饰。可以使用修饰或者未修饰的编码后的序列进行RNA编辑。可以将编码的SD与一个或者多个编码的BHD进行直接连接或者间接连接，制备ASO。On the other hand, the present application provides one or more isolated nucleic acid molecules that can encode the ASO described in the present application, can encode the SD in the ASO, and can also encode the BHD in the ASO, and the encoded ASO, SD, and BHD are all unmodified. The sequence encoded by one or more isolated nucleic acid molecules can be further modified. The modified or unmodified encoded sequence can be used for RNA editing. The encoded SD can be directly or indirectly connected to one or more encoded BHDs to prepare an ASO.

载体Carrier

另一方面，本申请提供一种表达载体，可以表达本申请所述的ASO、SD、BHD。表达载体可以在克隆载体基本骨架的基础上增加表达元件(如启动子、RBS、终止子等)，然后表达本申请中的ASO、SD、BHD。例如，所述表达载体可以是质粒。可以将表达载体表达的ASO进行进一步修饰；可以将表达载体表达的SD与一个或者多个BHD进行直接连接或者间接连接，制备ASO，然后对合成的ASO进行或者不进行修饰。表达载体表达的ASO、SD、BHD可以用于RNA编辑。On the other hand, the present application provides an expression vector that can express the ASO, SD, and BHD described in the present application. The expression vector can add expression elements (such as promoters, RBS, terminators, etc.) on the basis of the basic skeleton of the cloning vector, and then express the ASO, SD, and BHD in the present application. For example, the expression vector can be a plasmid. The ASO expressed by the expression vector can be further modified; the SD expressed by the expression vector can be directly or indirectly connected to one or more BHDs to prepare the ASO, and then the synthesized ASO may be modified or not. The ASO, SD, and BHD expressed by the expression vector can be used for RNA editing.

另一方面，本申请提供一种递送载体，可以将本申请中的ASO递送到细胞。例如，所述载体可以包括病毒载体，非病毒载体。病毒载体可以包括慢病毒(Lentivirus，LV)载体，腺病毒(Adenovirus，AdV)载体和腺相关病毒(Adeno-associated virus，AAV)载体等。例如，非病毒载体可以包括脂质体、分子偶联受体、聚合物、复合载体以及纳米粒子载体等。可以使用递送载体将ASO递送到细胞进行RNA编辑。On the other hand, the present application provides a delivery vector that can deliver the ASO in the present application to cells. For example, the vector may include a viral vector, a non-viral vector. Viral vectors may include lentivirus (LV) vectors, adenovirus (AdV) vectors, and adeno-associated virus (AAV) vectors, etc. For example, non-viral vectors may include liposomes, molecular coupled receptors, polymers, composite vectors, and nanoparticle vectors, etc. A delivery vector may be used to deliver ASO to cells for RNA editing.

细胞cell

另一方面，本申请提供一种细胞，可以包含本申请所述的ASO、一种或者多种分离的核酸分子、表达载体、递送载体。On the other hand, the present application provides a cell, which may contain the ASO described in the present application, one or more isolated nucleic acid molecules, expression vectors, and delivery vectors.

例如，细胞用于质粒繁殖，然后表达ASO、SD、BHD。例如，可以向细胞转染ASO，可以通过内吞的方式使细胞获得ASO，可以使用递送载体向细胞递送ASO，进行RNA编辑。For example, cells are used for plasmid propagation and then express ASO, SD, BHD. For example, ASO can be transfected into cells, ASO can be acquired by cells through endocytosis, and ASO can be delivered to cells using delivery vectors for RNA editing.

例如，细胞是具有内源表达的ADAR的细胞。例如，对细胞操作可以在体内进行。例如，可以将细胞从体内分离、或者培养细胞系，在体外进行操作。For example, the cell is a cell with endogenously expressed ADAR. For example, the cell manipulation can be performed in vivo. For example, the cell can be isolated from the body or a cell line can be cultured and manipulated in vitro.

药物组合物Pharmaceutical composition

另一方面，本申请提供一种药物组合物，可以包含本申请所述的ASO、一种或者多种分离的核酸分子、表达载体、递送载体、细胞，和/或药学上可接受的载体。On the other hand, the present application provides a pharmaceutical composition, which may include the ASO described in the present application, one or more isolated nucleic acid molecules, expression vectors, delivery vectors, cells, and/or pharmaceutically acceptable carriers.

例如，药物组合物可以通过在一段时间内通过静脉内输注施用。例如，药物组合物可以通过皮下递送来施用。例如，药物组合物可以定期重复施用，诸如每周、每两周、每一个月，两个月、三个月、四个月定期施用一次。例如，每周或每两周给药持续三个月后，可将给药每月一次持续六个月或一年或更长时间。For example, the pharmaceutical composition can be administered by intravenous infusion over a period of time. For example, the pharmaceutical composition can be administered by subcutaneous delivery. For example, the pharmaceutical composition can be administered repeatedly on a regular basis, such as once a week, every two weeks, every month, two months, three months, four months. For example, after weekly or biweekly administration for three months, administration can be continued monthly for six months or a year or more.

应用application

另一方面，本申请提供了所述的ASO，所述的分离的核酸分子，所述的表达载体，所述的递送载体，所述的细胞，所述的药物组合物，其用于预防和/或治疗疾病和/或病症。On the other hand, the present application provides the ASO, the isolated nucleic acid molecule, the expression vector, the delivery vector, the cell, and the pharmaceutical composition for preventing and/or treating diseases and/or disorders.

另一方面，本申请提供了所述的ASO，所述的一种或者多种分离的核酸分子，所述的表达载体，所述的递送载体，所述的细胞，所述的药物组合物在制备预防和/或治疗疾病和/或病症的药物中的用途。On the other hand, the present application provides the use of the ASO, the one or more isolated nucleic acid molecules, the expression vector, the delivery vector, the cell, and the pharmaceutical composition in the preparation of a drug for preventing and/or treating a disease and/or condition.

另一方面，本申请提供了一种预防和/或治疗疾病和/或病症，其包括向有需要的受试者施用有效量的所述的ASO，所述的一种或者多种分离的核酸分子，所述的表达载体，所述的递送载体，所述的细胞，所述的药物组合物。On the other hand, the present application provides a method for preventing and/or treating a disease and/or condition, comprising administering an effective amount of the ASO, the one or more isolated nucleic acid molecules, the expression vector, the delivery vector, the cell, or the pharmaceutical composition to a subject in need thereof.

例如，所述疾病和/或病症可以是单基因或多基因突变相关疾病。例如，基因可以是GAPDH、OGT、ATP7B、FGFR、TP53、APC、SERPINA1、MECP2、SCN1A。For example, the disease and/or disorder can be a single gene or multiple gene mutation-related disease. For example, the gene can be GAPDH, OGT, ATP7B, FGFR, TP53, APC, SERPINA1, MECP2, SCN1A.

本申请还提供以下的实施方式：The present application also provides the following implementation modes:

1.一种用于靶向RNA编辑的单链反义寡核苷酸(Antisense Oligonucleotide，ASO)，其中所述ASO包含靶向区(Specificity Domain，SD)和辅助结合区(Binding HelperDomain，BHD)，其中所述SD与靶向RNA区域互补配对，所述BHD与非靶向RNA区域互补配对，所述ASO能够与目标RNA形成双链复合物。1. A single-stranded antisense oligonucleotide (ASO) for targeted RNA editing, wherein the ASO comprises a targeting region (Specificity Domain, SD) and an auxiliary binding region (Binding Helper Domain, BHD), wherein the SD is complementary to the targeting RNA region, the BHD is complementary to the non-targeting RNA region, and the ASO can form a double-stranded complex with the target RNA.

2.根据实施方式1所述的ASO，其中所述靶向RNA区域与非靶向RNA区域非连续。2. The ASO of embodiment 1, wherein the targeting RNA region is non-contiguous with the non-targeting RNA region.

3.根据实施方式1-2中任一项所述的ASO，其中所述靶向RNA区域和所述非靶向RNA区域间隔的长度为4-1000nt。3. The ASO according to any one of embodiments 1-2, wherein the length of the interval between the targeting RNA region and the non-targeting RNA region is 4-1000 nt.

4.根据实施方式1-3中任一项所述的ASO，其中所述靶向RNA区域和所述非靶向RNA区域间隔的长度为4-200nt。4. The ASO according to any one of embodiments 1-3, wherein the length of the interval between the targeting RNA region and the non-targeting RNA region is 4-200 nt.

5.根据实施方式1-3中任一项所述的ASO，其中所述靶向RNA区域和所述非靶向RNA区域间隔的长度为200-500nt。5. The ASO according to any one of embodiments 1-3, wherein the length of the interval between the targeting RNA region and the non-targeting RNA region is 200-500 nt.

6.根据实施方式1-3中任一项所述的ASO，其中所述靶向RNA区域和所述非靶向RNA区域间隔的长度为500-1000nt。6. The ASO of any one of embodiments 1-3, wherein the length of the interval between the targeting RNA region and the non-targeting RNA region is 500-1000 nt.

7.根据实施方式1-3中任一项所述的ASO，其中所述靶向RNA区域和所述非靶向RNA区域间隔的长度为4nt、8nt、11nt、12nt、24nt、25nt、36nt、237nt、340nt、343nt、494nt、681nt。7. The ASO according to any one of embodiments 1-3, wherein the length of the interval between the targeting RNA region and the non-targeting RNA region is 4nt, 8nt, 11nt, 12nt, 24nt, 25nt, 36nt, 237nt, 340nt, 343nt, 494nt, or 681nt.

8.根据实施方式1-7中任一项所述的ASO，其中所述BHD与非靶向RNA区域完全互补配对。8. The ASO of any one of embodiments 1-7, wherein the BHD is fully complementary to the non-targeting RNA region.

9.根据实施方式1-7中任一项所述的ASO，其中所述BHD与非靶向RNA区域互补配对，且存在一个或多个错配、摆动、缺失、和/或凸起。9. The ASO of any one of embodiments 1-7, wherein the BHD is complementary to a non-targeting RNA region and has one or more mismatches, wobbles, deletions, and/or bulges.

10.根据实施方式1-9中任一项所述的ASO，其中所述BHD具有选自下组修饰的一种或者多种：糖修饰、碱基修饰。10. The ASO according to any one of embodiments 1-9, wherein the BHD has one or more modifications selected from the group consisting of: sugar modification, base modification.

11.根据实施方式1-10中任一项所述的ASO，其中所述BHD具有选自下组中的一种或者多种修饰：硫代磷酸酯化修饰、2’-OMe、2’-F、LNA、UNA、2’-MOE、肌苷替代修饰。11. An ASO according to any one of embodiments 1-10, wherein the BHD has one or more modifications selected from the following group: phosphorothioate modification, 2’-OMe, 2’-F, LNA, UNA, 2’-MOE, and inosine substitution modification.

12.根据实施方式1-11中任一项所述的ASO，其中所述BHD具有硫代磷酸酯化修饰、2’-OMe、LNA修饰。12. The ASO of any one of embodiments 1-11, wherein the BHD has phosphorothioate modification, 2’-OMe, or LNA modification.

13.根据实施方式1-12中任一项所述的ASO，其中所述BHD的长度为8-40nt。13. The ASO of any one of embodiments 1-12, wherein the BHD is 8-40 nt in length.

14.根据实施方式1-13中任一项所述的ASO，其中所述BHD的长度为15nt、20nt、25nt。14. The ASO according to any one of embodiments 1-13, wherein the BHD is 15 nt, 20 nt, or 25 nt in length.

15.根据实施方式1-14中任一项所述的ASO，其中所述ASO包含一个或者多个BHD。15. The ASO of any one of embodiments 1-14, wherein the ASO comprises one or more BHDs.

16.根据实施方式15所述的ASO，其中所述一个或者多个BHD不形成分子内二级结构。16. The ASO of embodiment 15, wherein the one or more BHDs do not form intramolecular secondary structures.

17.根据实施方式15所述的ASO，其中所述多个BHD不形成分子间二级结构。17. The ASO of embodiment 15, wherein the plurality of BHDs do not form intermolecular secondary structures.

18.根据实施方式1-17中任一项所述的ASO，其中所述BHD与SD不形成分子间二级结构。18. The ASO according to any one of embodiments 1-17, wherein the BHD and SD do not form an intermolecular secondary structure.

19.根据实施方式1-18中任一项所述的ASO，其中所述BHD在所述SD的5’端。19. The ASO of any one of embodiments 1-18, wherein the BHD is at the 5' end of the SD.

20.根据实施方式1-18中任一项所述的ASO，其中所述BHD在所述SD的3’端。20. The ASO of any one of embodiments 1-18, wherein the BHD is at the 3’ end of the SD.

21.根据实施方式1-18中任一项所述的ASO，其中所述多个BHD在所述SD的5’端和/或3’端。21. The ASO of any one of embodiments 1-18, wherein the multiple BHDs are at the 5' end and/or 3' end of the SD.

22.根据实施方式1-21中任一项所述的ASO，其中所述BHD与SD直接连接。22. The ASO of any one of embodiments 1-21, wherein the BHD is directly connected to the SD.

23.根据实施方式1-21中任一项所述的ASO，其中所述BHD与SD间接连接。23. The ASO of any one of embodiments 1-21, wherein the BHD is indirectly linked to the SD.

24.根据实施方式23所述的ASO，其中所述BHD与SD通过Linker间接连接。24. The ASO according to embodiment 23, wherein the BHD and SD are indirectly connected via a Linker.

25.根据实施方式23-24中任一项所述的ASO，其中所述Linker包括常规(磷酸二酯)或修饰(如硫代磷酸)的一个或多个核苷酸、寡肽或任何其他化学连接子。25. The ASO according to any one of embodiments 23-24, wherein the linker comprises one or more nucleotides, oligopeptides or any other chemical linkers, either conventional (phosphodiester) or modified (such as phosphorothioate).

26.根据实施方式23-25中任一项所述的ASO，其中所述Linker选自下组中的一种或者多种：AAA、AAAC、AACAA、AAAACAAAA、PEG2、C6。26. The ASO according to any one of embodiments 23-25, wherein the linker is selected from one or more of the following groups: AAA, AAAC, AACAA, AAAACAAAA, PEG2, C6.

27.根据实施方式22-26中任一项所述的ASO，其中所述Linker为AAA。27. The ASO of any one of embodiments 22-26, wherein the Linker is AAA.

28.根据实施方式22-26中任一项所述的ASO，其中所述Linker为PEG2。28. The ASO according to any one of embodiments 22-26, wherein the linker is PEG2.

29.根据实施方式22-26中任一项所述的ASO，其中所述Linker为C6。29. The ASO of any one of embodiments 22-26, wherein the linker is C6.

30.根据实施方式1-29中任一项所述的ASO，其中所述BHD增加ASO与目标RNA形成双链复合物的稳定性。30. The ASO of any one of embodiments 1-29, wherein the BHD increases the stability of a double-stranded complex formed by the ASO and the target RNA.

31.根据实施方式1-30中任一项所述的ASO，其中所述SD与靶向RNA区域完全互补配对或存在一个或多个错配、摆动、缺失、和/或凸起。31. The ASO according to any one of embodiments 1-30, wherein the SD is fully complementary to the targeting RNA region or has one or more mismatches, wobbles, deletions, and/or protrusions.

32.根据实施方式1-31中任一项所述的ASO，其中所述SD具有选自下组修饰的一种或者多种：糖修饰、碱基修饰。32. The ASO according to any one of embodiments 1-31, wherein the SD has one or more modifications selected from the following group: sugar modification, base modification.

33.根据实施方式1-32中任一项所述的ASO，其中所述SD具有选自下组中的一种或者多种修饰：硫代磷酸酯化修饰、2’-OMe、2’-F、LNA、UNA、2’-MOE、2’-FANA、DNA碱基替代修饰，肌苷替代修饰。33. An ASO according to any one of embodiments 1-32, wherein the SD has one or more modifications selected from the following group: phosphorothioate modification, 2’-OMe, 2’-F, LNA, UNA, 2’-MOE, 2’-FANA, DNA base substitution modification, and inosine substitution modification.

34.根据实施方式1-33中任一项所述的ASO，其中所述SD具有硫代磷酸酯化修饰。34. The ASO of any one of embodiments 1-33, wherein the SD has a phosphorothioate modification.

35.根据实施方式1-34中任一项所述的ASO，其中所述SD具有2’-OMe修饰。35. The ASO of any one of embodiments 1-34, wherein the SD has a 2’-OMe modification.

36.根据实施方式1-34中任一项所述的ASO，其中所述SD具有2’-F修饰。36. The ASO of any one of embodiments 1-34, wherein the SD has a 2’-F modification.

37.根据实施方式1-36中任一项所述的ASO，其中所述SD的长度为20-50nt。37. The ASO of any one of embodiments 1-36, wherein the SD has a length of 20-50 nt.

38.根据实施方式1-37中任一项所述的ASO，其中所述SD的长度为30nt。38. The ASO of any one of embodiments 1-37, wherein the SD has a length of 30 nt.

39.根据实施方式1-38中任一项所述的ASO，其中所述靶向RNA区域包含靶腺苷。39. The ASO of any one of embodiments 1-38, wherein the targeting RNA region comprises a target adenosine.

40.根据实施方式1-39中任一项所述的ASO，其中所述靶向RNA区域的长度为15-60nt。40. The ASO of any one of embodiments 1-39, wherein the targeting RNA region is 15-60 nt in length.

41.根据实施方式1-40中任一项所述的ASO，其中所述靶向RNA区域的长度为30nt。41. The ASO of any one of embodiments 1-40, wherein the targeting RNA region is 30 nt in length.

42.根据实施方式1-41中任一项所述的ASO，其中所述非靶向RNA区域不包含靶腺苷。42. The ASO of any one of embodiments 1-41, wherein the non-targeting RNA region does not comprise a target adenosine.

43.根据实施方式1-42中任一项所述的ASO，其中所述非靶向RNA区域的长度为5-50nt。43. The ASO of any one of embodiments 1-42, wherein the non-targeting RNA region is 5-50 nt in length.

44.根据实施方式1-43中任一项所述的ASO，其中所述非靶向RNA区域的长度为15nt，20nt，25nt。44. The ASO of any one of embodiments 1-43, wherein the length of the non-targeting RNA region is 15 nt, 20 nt, or 25 nt.

45.根据实施方式1-44中任一项所述的ASO，其中所述ASO通过细胞中存在的ADAR酶对靶向RNA区域中存在的靶腺苷进行脱氨基化。45. The ASO of any one of embodiments 1-44, wherein the ASO deaminates a target adenosine present in the targeting RNA region by an ADAR enzyme present in the cell.

46.根据实施方式1-45中任一项所述的ASO，其中所述目标RNA选自下组中的一种或者多种：pre-mRNA、mRNA、rRNA、tRNA、lnc-RNA、snRNA、snoRNA。46. The ASO of any one of embodiments 1-45, wherein the target RNA is selected from one or more of the following groups: pre-mRNA, mRNA, rRNA, tRNA, lnc-RNA, snRNA, snoRNA.

47.编辑RNA的方法，其包含使用实施方式1-46中任一项所述的ASO。47. A method of editing RNA comprising using the ASO of any one of embodiments 1-46.

48.根据实施方式47所述的方法，其中所述方法包括以下步骤：48. The method of embodiment 47, wherein the method comprises the following steps:

(1)提供实施方式1-46中任一项所述的ASO；(1) providing an ASO as described in any one of embodiments 1-46;

(2)允许细胞摄取所述ASO；(2) allowing cells to take up the ASO;

(3)允许所述ASO与目标RNA结合；(3) allowing the ASO to bind to the target RNA;

(4)允许细胞内ADAR酶将靶向RNA区域中的靶腺苷脱氨基成为肌苷；(4) allowing intracellular ADAR enzymes to deaminate target adenosine in the targeted RNA region to inosine;

(5)鉴定靶向RNA区域中肌苷的存在。(5) Identify the presence of inosine in the targeted RNA region.

49.根据实施方式47-48所述的方法，其中所述方法能够招募内源脱氨基酶对特定核苷酸位点进行脱氨基反应。49. A method according to embodiments 47-48, wherein the method is capable of recruiting endogenous deaminases to perform deamination reactions on specific nucleotide sites.

50.一种或者多种分离的核酸分子，其编码实施方式1-46中任一项所述的ASO、或ASO包含的SD、或ASO包含的BHD。50. One or more isolated nucleic acid molecules encoding the ASO, or the SD comprised by the ASO, or the BHD comprised by the ASO according to any one of embodiments 1-46.

51.表达载体，其表达实施方式50所述的一种或者多种分离的核酸分子。51. An expression vector that expresses one or more isolated nucleic acid molecules of embodiment 50.

52.递送载体，其递送实施方式1-46中任一项所述的ASO。52. A delivery vehicle that delivers the ASO of any one of embodiments 1-46.

53.细胞，其包含实施方式1-46中任一项所述的ASO、实施方式50所述的一种或者多种分离的核酸分子、实施方式51所述的表达载体、和/或实施方式52所述的递送载体。53. A cell comprising the ASO of any one of embodiments 1-46, the one or more isolated nucleic acid molecules of embodiment 50, the expression vector of embodiment 51, and/or the delivery vector of embodiment 52.

54.根据实施方式53所述的细胞，其中所述细胞为真核细胞。54. A cell according to embodiment 53, wherein the cell is a eukaryotic cell.

55.根据实施方式53-54中任一项所述的细胞，其中所述细胞是人类细胞或小鼠细胞。55. A cell according to any one of embodiments 53-54, wherein the cell is a human cell or a mouse cell.

56.根据实施方式53-55中任一项所述的细胞，其中所述细胞是肝脏细胞。56. A cell according to any one of embodiments 53-55, wherein the cell is a liver cell.

57.根据实施方式53-56中任一项所述的细胞，其中所述细胞是神经细胞。57. A cell according to any one of embodiments 53-56, wherein the cell is a neural cell.

58.药物组合物，其包含实施方式1-46中任一项所述的ASO，实施方式50所述的一种或者多种分离的核酸分子，实施方式51所述的表达载体、实施方式52所述的递送载体，实施方式53-57中任一项所述的细胞，和/或药学上可接受的载体。58. A pharmaceutical composition comprising the ASO of any one of embodiments 1-46, one or more isolated nucleic acid molecules of embodiment 50, the expression vector of embodiment 51, the delivery vector of embodiment 52, the cell of any one of embodiments 53-57, and/or a pharmaceutically acceptable carrier.

59.实施方式1-46中任一项所述的ASO，实施方式50所述的一种或者多种分离的核酸分子，实施方式51所述的表达载体、实施方式52所述的递送载体，实施方式53-57中任一项所述的细胞，实施方式58所述的药物组合物，其用于预防和/或治疗疾病和/或病症。59. The ASO of any one of embodiments 1-46, the one or more isolated nucleic acid molecules of embodiment 50, the expression vector of embodiment 51, the delivery vector of embodiment 52, the cell of any one of embodiments 53-57, the pharmaceutical composition of embodiment 58, for preventing and/or treating a disease and/or condition.

60.实施方式1-46中任一项所述的ASO，实施方式50所述的一种或者多种分离的核酸分子，实施方式51所述的表达载体、实施方式52所述的递送载体，实施方式53-57中任一项所述的细胞，实施方式58所述的药物组合物在制备预防和/或治疗疾病和/或病症的药物中的用途。60. Use of the ASO of any one of embodiments 1-46, the one or more isolated nucleic acid molecules of embodiment 50, the expression vector of embodiment 51, the delivery vector of embodiment 52, the cell of any one of embodiments 53-57, and the pharmaceutical composition of embodiment 58 in the preparation of a medicament for preventing and/or treating a disease and/or condition.

61.一种预防和/或治疗疾病和/或病症，其包括向有需要的受试者施用有效量的实施方式1-46中任一项所述的ASO，实施方式50所述的一种或者多种分离的核酸分子，实施方式51所述的表达载体、实施方式52所述的递送载体，实施方式53-57中任一项所述的细胞，实施方式58所述的药物组合物。61. A method for preventing and/or treating a disease and/or condition, comprising administering to a subject in need thereof an effective amount of the ASO of any one of embodiments 1-46, one or more isolated nucleic acid molecules of embodiment 50, the expression vector of embodiment 51, the delivery vector of embodiment 52, the cell of any one of embodiments 53-57, or the pharmaceutical composition of embodiment 58.

不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的ASO、其制备方法和用途等，而不用于限制本申请发明的范围。Without intending to be bound by any theory, the following examples are merely intended to illustrate the ASO of the present application, its preparation method and use, etc., and are not intended to limit the scope of the present invention.

实施例Example

材料和方法Materials and methods

引物Primers

实施例1和4中用到的引物序列：Primer sequences used in Examples 1 and 4:

GAPDH-primer-F1(SEQ ID NO:18)：TCATCATCTCTGCCCCCTCTGAPDH-primer-F1 (SEQ ID NO:18): TCATCATCTCTGCCCCCTCT

GAPDH-primer-R1(SEQ ID NO:19)：GGCAGGGATGATGTTCTGGAGAPDH-primer-R1 (SEQ ID NO:19): GGCAGGGATGATGTTCTGGA

实施例2和5中用到的引物序列：Primer sequences used in Examples 2 and 5:

GAPDH-primer-F2(SEQ ID NO:20)：GCTGGCATTGCCCTCAACGAGAPDH-primer-F2 (SEQ ID NO:20): GCTGGCATTGCCCTCAACGA

GAPDH-primer-R2(SEQ ID NO:21)：ACATGACAAGGTGCGGCTCCGAPDH-primer-R2 (SEQ ID NO:21):ACATGACAAGGTGCGGCTCC

实施例3和6中用到的引物序列：Primer sequences used in Examples 3 and 6:

OGT-primer-F(SEQ ID NO:22)：TGGCAACAAACCTGACCACAOGT-primer-F (SEQ ID NO:22): TGGCAACAAACCTGACCACA

OGT-primer-R3(SEQ ID NO:23)：ATCTGGTCGCCGCAAAATTCOGT-primer-R3 (SEQ ID NO:23):ATCTGGTCGCCGCAAAATTC

基因NCBI号：Gene NCBI number:

GAPDH mRNA：NM_002046.7GAPDH mRNA: NM_002046.7

OGT mRNA：NM_181673.3OGT mRNA: NM_181673.3

ADAR1 CDS：NM_015840.4ADAR1 CDS: NM_015840.4

人源ADAR1表达质粒构建Construction of human ADAR1 expression plasmid

通过PCR扩增人源ADAR1全长序列，并在其两端加上pcDNA3.1载体NheI和EcoRI酶切位点两端同源臂，利用NheI和EcoRI双酶切把pcDNA3.1载体线性化，并用同源重组的方法把ADAR1片段连接到载体。The full-length sequence of human ADAR1 was amplified by PCR, and homologous arms at both ends of the NheI and EcoRI restriction sites of the pcDNA3.1 vector were added to it. The pcDNA3.1 vector was linearized by double restriction digestion with NheI and EcoRI, and the ADAR1 fragment was connected to the vector by homologous recombination.

细胞培养Cell culture

1.细胞传代：1. Cell passaging:

(1)紫外照射超净工作台消毒30分钟；(1) Disinfect the clean bench with ultraviolet light for 30 minutes;

(2)PBS润洗待传细胞，加入适量0.25％胰酶，待细胞变圆后加入含有血清的完全培养基终止消化；(2) Rinse the cells to be passaged with PBS, add an appropriate amount of 0.25% trypsin, and add complete medium containing serum to terminate digestion after the cells become round;

(3)把消化后的细胞悬液转移到15mL离心管中，室温200g离心2分钟；(3) Transfer the digested cell suspension to a 15 mL centrifuge tube and centrifuge at 200 g for 2 minutes at room temperature;

(4)吸弃上清，加入新鲜的培养基重悬细胞；(4) Aspirate and discard the supernatant, and add fresh culture medium to resuspend the cells;

(5)取20ul细胞悬液和20ul台盼蓝染色液并混匀，取20μL使用血球计数板进行细胞计数；(5) Take 20ul of cell suspension and 20ul of trypan blue staining solution and mix them well. Take 20μL and use a hemocytometer to count the cells.

(6)取适量细胞接种到细胞培养板，放入细胞培养箱培养。(6) Take an appropriate amount of cells and inoculate them into a cell culture plate, and place it in a cell culture incubator for culture.

2.细胞转染实验步骤：2. Cell transfection experimental steps:

(1)细胞生长24小时可达到70％汇合度时则可进行细胞转染实验；(1) When cells reach 70% confluence after 24 hours of growth, cell transfection experiments can be performed;

(2)根据转染试剂lipofectamine 3000提供的说明书配制转染试剂混合液，室温静置15分钟；(2) Prepare the transfection reagent mixture according to the instructions provided by the transfection reagent lipofectamine 3000 and let it stand at room temperature for 15 minutes;

(3)把转染液加入到细胞中，轻轻混匀后放入培养箱。(3) Add the transfection solution to the cells, mix gently, and place in the incubator.

ADAR1过表达HEK293细胞株构建Construction of HEK293 cell line overexpressing ADAR1

按照上述步骤把ADAR1表达载体转染到HEK293细胞，48小时后加入筛选药物G418，每两天换一次液，待未转染组已无活细胞完成稳转ADAR1的HEK293细胞株筛选。According to the above steps, the ADAR1 expression vector was transfected into HEK293 cells. After 48 hours, the screening drug G418 was added and the solution was changed every two days. The screening of HEK293 cell lines stably transfected with ADAR1 was completed when there were no living cells in the non-transfected group.

ASO编辑水平验证ASO editorial level verification

将4.5×10⁵个ADAR1稳定过表达的HER293细胞或3×10⁵个Hela细胞接种到12孔板的每个孔中，24小时后，待细胞长满80％-90％即可转染ASO。使用lipo8000(碧云天)转染ASO，每孔转染40pmol。48小时后收集细胞，使用RNA分离试剂盒(诺唯赞)提取RNA，随后进行DNase I(Thermo)消化，逆转录(Promega)，并使用相应引物和DNA Taq酶(GeneStar)对目标位点进行PCR扩增。获得DNA产物使用琼脂糖凝胶分析，根据大小进行凝胶纯化(ZYMO)，随后送至金唯智公司进行Sanger测序。通过测量各自位点的G和A的高度，并将G高度除以G和A峰高度之和，来量化A-to-I的编辑产量。如果使用反向引物进行测序，则相应处理C和T的峰高度。4.5×10⁵ HER293 cells or 3×10⁵ Hela cells with stable overexpression of ADAR1 were seeded into each well of a 12-well plate. After 24 hours, the cells were transfected with ASO when they were 80%-90% full. ASO was transfected using lipo8000 (Biyuntian) with 40 pmol per well. Cells were collected 48 hours later, RNA was extracted using an RNA isolation kit (Novozyme), followed by DNase I (Thermo) digestion, reverse transcription (Promega), and PCR amplification of the target site using the corresponding primers and DNA Taq enzyme (GeneStar). The DNA products were analyzed using agarose gels, gel purified according to size (ZYMO), and then sent to GeneWeichi for Sanger sequencing. The A-to-I editing yield was quantified by measuring the height of G and A at each site and dividing the G height by the sum of the G and A peak heights. If sequencing is performed using a reverse primer, the peak heights of C and T are processed accordingly.

ASO序列ASO sequence

表1本申请使用的ASO序列Table 1 ASO sequences used in this application

实施例1使用不同的ASO编辑GAPDH基因CDS区域特定腺苷Example 1 Editing specific adenosine in the CDS region of the GAPDH gene using different ASOs

在人源ADAR1过表达HEK293细胞株中对RNA编辑进行考察，考察使用不同结构ASOSEQ-1至SEQ-9(图1)，是否可以将内源GAPDH mRNA的CDS区域特定UAG三联体中间的A碱基脱氨基化成I(其随后被读成G)。如图1所示：SEQ-1只包含SD区域；SEQ-2至SEQ-4同时包含SD和下游BHD，中间分别以linker三个腺苷、PEG2、C6相连；SEQ-5同时包含SD和下游BHD，中间无linker；SEQ-6至SEQ-7同时包含SD和上游BHD，中间以linker三个腺苷相连；SEQ-8和SEQ-9同时包含SD、下游BHD和上游BHD，其中SEQ-8的BHD和SD之间以三个腺苷相连，SEQ-9不含linker。所述SEQ-1至SEQ-9的SD区域序列和长度相同(30nt)；SEQ-2至SEQ-5下游BHD区域序列和长度相同(15nt)；SEQ-6和SEQ-7上游BHD区域分别长25nt和20nt；SEQ-8和SEQ-9上游和下游BHD长15bp。RNA editing was investigated in human ADAR1 overexpressing HEK293 cell lines to investigate whether the A base in the middle of the specific UAG triplet in the CDS region of endogenous GAPDH mRNA could be deaminated to I (which is subsequently read as G) using different structures ASOs SEQ-1 to SEQ-9 (Figure 1). As shown in Figure 1: SEQ-1 contains only the SD region; SEQ-2 to SEQ-4 contain SD and downstream BHD at the same time, connected by three adenosines, PEG2, and C6 in the middle; SEQ-5 contains SD and downstream BHD at the same time, without a linker in the middle; SEQ-6 to SEQ-7 contain SD and upstream BHD at the same time, connected by three adenosines in the middle; SEQ-8 and SEQ-9 contain SD, downstream BHD, and upstream BHD at the same time, where the BHD and SD of SEQ-8 are connected by three adenosines, and SEQ-9 does not contain a linker. The SD region sequences and lengths of SEQ-1 to SEQ-9 are the same (30nt); the downstream BHD region sequences and lengths of SEQ-2 to SEQ-5 are the same (15nt); the upstream BHD regions of SEQ-6 and SEQ-7 are 25nt and 20nt long, respectively; the upstream and downstream BHDs of SEQ-8 and SEQ-9 are 15bp long.

实验结果如图所示：The experimental results are shown in the figure:

图2显示，转染同时包含SD和下游BHD结构的SEQ-2至SEQ-4和仅包含SD结构的SEQ-1都观察到目标腺苷处A到G的转变，但是SEQ-1在特定位点的编辑水平仅为18％，SEQ-2至SEQ-4对应的编辑水平分别为40％、36％和35％，编辑效率显著提高。另外，转染不含特殊linker、但同时包含SD和下游BHD的SEQ-5编辑效率为34％，同样能提高编辑效率。Figure 2 shows that the conversion of A to G at the target adenosine was observed in SEQ-2 to SEQ-4 transfected with both SD and downstream BHD structures and SEQ-1 containing only SD structure, but the editing level of SEQ-1 at the specific site was only 18%, and the corresponding editing levels of SEQ-2 to SEQ-4 were 40%, 36% and 35%, respectively, and the editing efficiency was significantly improved. In addition, the editing efficiency of SEQ-5 transfected without special linker but containing SD and downstream BHD was 34%, which can also improve the editing efficiency.

以上结果说明：The above results show that:

下游BHD结构能够有效帮助SD提高编辑效率，且SD和BHD之间以不同形式的linker相连或无linker都能帮助SD提高编辑。The downstream BHD structure can effectively help SD improve editing efficiency, and the connection between SD and BHD with different forms of linkers or without linkers can help SD improve editing.

实验结果如图所示：The experimental results are shown in the figure:

图2显示，转染同时具有SD和上游BHD的SEQ-6(BHD为25nt)和SEQ-7(BHD为20nt)编辑水平分别为29％和22％，与SEQ-1相比显著提高。FIG2 shows that the editing levels of SEQ-6 (BHD is 25 nt) and SEQ-7 (BHD is 20 nt) transfected with both SD and upstream BHD were 29% and 22%, respectively, which were significantly increased compared with SEQ-1.

以上结果说明：The above results show that:

不同长度的上游BHD结构都能够有效帮助SD提高编辑效率。Upstream BHD structures of different lengths can effectively help SD improve editing efficiency.

实验结果如图所示：The experimental results are shown in the figure:

图2显示，转染同时具有SD、下游BHD和上游BHD的SEQ-8和SEQ-9(不含linker)编辑效率分别为29％和24％，相对SEQ-1显著提高。FIG2 shows that the editing efficiencies of SEQ-8 and SEQ-9 (without linker) transfected with SD, downstream BHD, and upstream BHD were 29% and 24%, respectively, which were significantly improved compared with SEQ-1.

以上结果说明：The above results show that:

同时具有上游BHD和下游BHD(含有或不含有linker)能够帮助SD提高编辑。Having both upstream BHD and downstream BHD (with or without linker) can help SD improve editing.

实施例2使用不同的ASO编辑GAPDH基因3’UTR区域特定腺苷Example 2 Editing specific adenosine in the 3'UTR region of the GAPDH gene using different ASOs

在人源ADAR1过表达HEK293细胞株中对RNA编辑进行考察，考察使用不同结构ASOSEQ10-SEQ21(图3)，是否可以将内源GAPDH mRNA的3’UTR区域特定UAG三联体中间的A碱基脱氨基化成I(其随后被读成G)。RNA editing was investigated in a human ADAR1-overexpressing HEK293 cell line to examine whether the A base in the middle of a specific UAG triplet in the 3’UTR region of endogenous GAPDH mRNA could be deaminated to I (which is subsequently read as G) using different structures ASOSEQ10-SEQ21 ( FIG. 3 ).

图3显示，SEQ-10只包含SD区域；SEQ-11至SEQ-13同时包含SD和下游BHD，中间分别以linker三个腺苷、PEG2、C6相连；SEQ-14同时包含SD和下游BHD，中间无linker；SEQ-15至SEQ-18同时包含SD和上游BHD，中间以linker三个腺苷相连；SEQ-19同时包含SD和上游BHD，中间无linker；SEQ-20和SEQ-21同时包含SD、下游BHD和上游BHD，其中SEQ-20的BHD和SD之间以三个腺苷相连，SEQ-21不含linker。所述SEQ-10至SEQ-21的SD区域序列和长度相同(30nt)；SEQ-11至SEQ-14下游BHD区域序列和长度相同(15nt)；SEQ-15和SEQ-16上游BHD区域长25nt，SEQ-17和SEQ-18上游BHD区域长20nt，其中SEQ-16和SEQ-19的上游BHD序列相同；SEQ-20和SEQ-21上游和下游BHD长15bp。Figure 3 shows that SEQ-10 only contains the SD region; SEQ-11 to SEQ-13 contain SD and downstream BHD at the same time, which are connected by three adenosines, PEG2, and C6 in the middle, respectively; SEQ-14 contains SD and downstream BHD at the same time, with no linker in the middle; SEQ-15 to SEQ-18 contain SD and upstream BHD at the same time, which are connected by three adenosines in the middle; SEQ-19 contains SD and upstream BHD at the same time, with no linker in the middle; SEQ-20 and SEQ-21 contain SD, downstream BHD, and upstream BHD at the same time, among which the BHD and SD of SEQ-20 are connected by three adenosines, and SEQ-21 does not contain a linker. The SD region sequence and length of SEQ-10 to SEQ-21 are the same (30nt); the downstream BHD region sequence and length of SEQ-11 to SEQ-14 are the same (15nt); the upstream BHD region of SEQ-15 and SEQ-16 is 25nt long, and the upstream BHD region of SEQ-17 and SEQ-18 is 20nt long, among which the upstream BHD sequence of SEQ-16 and SEQ-19 is the same; the upstream and downstream BHD of SEQ-20 and SEQ-21 are 15bp long.

实验结果如图所示：The experimental results are shown in the figure:

图4显示，转染同时包含SD和下游BHD结构的SEQ-11至SEQ-13和仅包含SD结构的SEQ-10都观察到目标腺苷处A到G的转变，但是SEQ-10在特定位点的编辑水平仅为18％，SEQ-11至SEQ-13对应的编辑水平分别为39％、24％和27％，编辑效率显著提高。另外，转染不含特殊linker、但同时包含SD和BHD的SEQ-14编辑效率为40％，同样能提高编辑效率。Figure 4 shows that the conversion of A to G at the target adenosine was observed in SEQ-11 to SEQ-13 transfected with both SD and downstream BHD structures and SEQ-10 containing only SD structure, but the editing level of SEQ-10 at a specific site was only 18%, and the corresponding editing levels of SEQ-11 to SEQ-13 were 39%, 24% and 27%, respectively, and the editing efficiency was significantly improved. In addition, the editing efficiency of SEQ-14 transfected without a special linker but containing both SD and BHD was 40%, which can also improve the editing efficiency.

以上结果说明：The above results show that:

下游BHD结构能够有效帮助SD提高编辑效率，且SD和BHD之间以不同形式的linker相连或无linker都能帮助SD提高编辑。The downstream BHD structure can effectively help SD improve editing efficiency, and the connection between SD and BHD with different forms of linkers or without linkers can help SD improve editing.

实验结果如图所示：The experimental results are shown in the figure:

图4显示，转染同时具有SD和上游BHD(25nt)的SEQ-15和SEQ-16编辑水平分别为20％和30％，转染同时具有SD和上游BHD(20nt)的SEQ-18编辑水平为22％，转染与SEQ-10相比显著提高；另外，转染不含特殊linker、但同时包含SD和上游BHD的SEQ-19编辑效率为20％，同样能提高编辑效率。Figure 4 shows that the editing levels of SEQ-15 and SEQ-16 transfected with both SD and upstream BHD (25nt) were 20% and 30%, respectively, and the editing level of SEQ-18 transfected with both SD and upstream BHD (20nt) was 22%, which was significantly improved compared with SEQ-10; in addition, the editing efficiency of SEQ-19 transfected without a special linker but with both SD and upstream BHD was 20%, which also improved the editing efficiency.

以上结果说明：The above results show that:

不同长度的上游BHD结构(有linker或无linker)能够有效帮助SD提高编辑效率。Upstream BHD structures of different lengths (with or without linker) can effectively help SD improve editing efficiency.

实验结果如图所示：The experimental results are shown in the figure:

图4显示，转染同时具有SD、下游BHD和上游BHD的SEQ-20和SEQ-21(不含linker)编辑效率分别为35％和21％，相对SEQ-10明显提高。FIG4 shows that the editing efficiencies of SEQ-20 and SEQ-21 (without linker) transfected with SD, downstream BHD, and upstream BHD were 35% and 21%, respectively, which were significantly improved compared with SEQ-10.

以上结果说明：The above results show that:

同时具有上游BHD和下游BHD(有linker或无linker)能够帮助SD提高编辑。Having both upstream BHD and downstream BHD (with or without linker) can help SD improve editing.

实施例3使用不同的ASO编辑N乙酰葡糖胺转移酶基因(OGT)3'-UTR区域特定腺苷Example 3 Editing of specific adenosine in the 3'-UTR region of the N-acetylglucosamine transferase gene (OGT) using different ASOs

在人源ADAR1过表达HEK293细胞株中对RNA编辑进行考察，考察使用不同结构ASOSEQ-22至SEQ-29(图5)，是否可以将内源OGT基因3'-UTR区域的UAC三联体中间的A脱氨基化成I(其随后被读成G)。RNA editing was investigated in a human ADAR1-overexpressing HEK293 cell line to examine whether the A in the middle of the UAC triplet in the 3'-UTR region of the endogenous OGT gene could be deaminated to I (which was subsequently read as G) using different structures of ASOSEQ-22 to SEQ-29 ( FIG. 5 ).

图5显示，SEQ-22只包含SD区域；SEQ-23至SEQ-25同时包含SD和下游BHD，中间分别以linker三个腺苷、PEG2、C6相连；SEQ-26同时包含SD和下游BHD，中间无linker；SEQ-27至SEQ-28同时包含SD和上游BHD，SD中间以linker三个腺苷相连；SEQ-29同时包含SD、下游BHD和上游BHD，中间分别以三个腺苷相连。所述SEQ-22至SEQ-29的SD区域序列和长度相同(30nt)；SEQ-13至SEQ-26下游BHD区域序列和长度相同(15nt)；SEQ-27上游BHD区域长25nt，SEQ-28上游BHD区域长20nt；SEQ-29上游和下游BHD长15bp。Figure 5 shows that SEQ-22 only contains the SD region; SEQ-23 to SEQ-25 contain SD and downstream BHD at the same time, and are connected by three adenosines, PEG2, and C6 in the middle; SEQ-26 contains SD and downstream BHD at the same time, and there is no linker in the middle; SEQ-27 to SEQ-28 contain SD and upstream BHD at the same time, and SD is connected by three adenosines in the middle of the linker; SEQ-29 contains SD, downstream BHD, and upstream BHD at the same time, and are connected by three adenosines in the middle. The SD region sequence and length of SEQ-22 to SEQ-29 are the same (30nt); the downstream BHD region sequence and length of SEQ-13 to SEQ-26 are the same (15nt); the upstream BHD region of SEQ-27 is 25nt long, and the upstream BHD region of SEQ-28 is 20nt long; the upstream and downstream BHD of SEQ-29 are 15bp long.

实验结果如图所示：The experimental results are shown in the figure:

图6显示，转染同时包含SD和下游BHD结构的SEQ-23至SEQ-25和仅包含SD结构的SEQ-22都观察到目标腺苷处A到G的转变，但是SEQ-22在特定位点的编辑水平仅为20％，SEQ-23至SEQ-25对应的编辑水平分别为28％、38％和44％，编辑效率显著提高。另外，转染不含特殊linker、但同时包含SD和BHD的SEQ-26编辑效率为26％，同样能提高编辑效率。Figure 6 shows that the conversion of A to G at the target adenosine was observed in SEQ-23 to SEQ-25 transfected with both SD and downstream BHD structures and SEQ-22 containing only SD structure, but the editing level of SEQ-22 at a specific site was only 20%, and the corresponding editing levels of SEQ-23 to SEQ-25 were 28%, 38% and 44%, respectively, and the editing efficiency was significantly improved. In addition, the editing efficiency of SEQ-26 transfected without a special linker but containing both SD and BHD was 26%, which can also improve the editing efficiency.

以上结果说明：The above results show that:

下游BHD结构能够有效帮助SD提高编辑效率，且SD和BHD之间以不同形式的linker相连或无linker都能帮助SD提高编辑。The downstream BHD structure can effectively help SD improve editing efficiency, and the connection between SD and BHD with different forms of linkers or without linkers can help SD improve editing.

实验结果如图所示：The experimental results are shown in the figure:

图6显示，转染同时具有SD和上游BHD(25nt)的SEQ-27编辑水平为24％，转染同时具有SD和上游BHD(20nt)的SEQ-28编辑水平为21％，与SEQ-22相比显著提高。FIG6 shows that the editing level of SEQ-27 transfected with both SD and upstream BHD (25nt) was 24%, and the editing level of SEQ-28 transfected with both SD and upstream BHD (20nt) was 21%, which were significantly improved compared with SEQ-22.

以上结果说明：The above results show that:

不同长度的上游BHD结构能够有效帮助SD提高编辑效率。Upstream BHD structures of different lengths can effectively help SD improve editing efficiency.

实施例4在Hela细胞中使用不同的ASO编辑GAPDH基因CDS区域特定腺苷Example 4 Editing of specific adenosine in the CDS region of the GAPDH gene using different ASOs in Hela cells

在Hela细胞株中利用内源表达的ADAR脱氨酶对RNA编辑进行考察，考察使用不同结构ASO SEQ-1至SEQ-8(图1)，是否可以将内源GAPDH mRNA的CDS区域特定UAG三联体中间的A碱基脱氨基化成I(其随后被读成G)。SEQ-1至SEQ-8的结构描述见实施例1。RNA editing was investigated using endogenously expressed ADAR deaminase in Hela cell lines to investigate whether the A base in the middle of a specific UAG triplet in the CDS region of endogenous GAPDH mRNA could be deaminated to I (which is then read as G) using different structural ASOs SEQ-1 to SEQ-8 ( FIG. 1 ). The structural descriptions of SEQ-1 to SEQ-8 are shown in Example 1.

实验结果如图所示：The experimental results are shown in the figure:

图7显示，转染同时包含SD和下游BHD结构的SEQ-2和仅包含SD结构的SEQ-1都观察到目标腺苷处A到G的转变，但是SEQ-1在特定位点的编辑水平仅为17％，SEQ-2对应的编辑水平为35％，编辑效率显著提高。另外，转染不含特殊linker、但同时包含SD和下游BHD的SEQ-5编辑效率为23％，同样能提高编辑效率。Figure 7 shows that the conversion of A to G at the target adenosine was observed in both SEQ-2 transfected with both SD and downstream BHD structures and SEQ-1 containing only SD structure, but the editing level of SEQ-1 at the specific site was only 17%, while the corresponding editing level of SEQ-2 was 35%, and the editing efficiency was significantly improved. In addition, the editing efficiency of SEQ-5 transfected without special linker but containing both SD and downstream BHD was 23%, which can also improve the editing efficiency.

以上结果说明：The above results show that:

下游BHD结构能够有效帮助SD提高编辑效率，且SD和BHD之间以linker相连或无linker都能帮助SD提高编辑。The downstream BHD structure can effectively help SD improve editing efficiency, and whether SD and BHD are connected with a linker or without a linker can help SD improve editing.

实验结果如图所示：The experimental results are shown in the figure:

图7显示，转染同时具有SD和上游BHD的SEQ-6(BHD为25nt)的编辑水平为27％，与SEQ-1相比显著提高。FIG. 7 shows that the editing level of SEQ-6 (BHD is 25 nt) transfected with both SD and upstream BHD was 27%, which was significantly increased compared with SEQ-1.

以上结果说明：The above results show that:

上游BHD结构能够有效帮助SD提高编辑效率。The upstream BHD structure can effectively help SD improve editing efficiency.

实验结果如图所示：The experimental results are shown in the figure:

图7显示，转染同时具有SD、下游BHD和上游BHD的SEQ-8编辑效率为31％，相对SEQ-1显著提高。FIG7 shows that the editing efficiency of SEQ-8 transfected with SD, downstream BHD and upstream BHD was 31%, which was significantly improved compared with SEQ-1.

以上结果说明：The above results show that:

同时具有上游BHD和下游BHD能够帮助SD提高编辑。Having both upstream BHD and downstream BHD can help SD improve editing.

实施例5在Hela细胞中使用不同的ASO编辑GAPDH基因3’UTR区域特定腺苷Example 5 Editing of specific adenosine in the 3'UTR region of the GAPDH gene using different ASOs in Hela cells

在Hela细胞株中利用内源表达的ADAR脱氨酶对RNA编辑进行考察，考察使用不同结构ASO SEQ-10至SEQ-21(图3)，是否可以将内源GAPDH mRNA的3’UTR区域特定UAG三联体中间的A碱基脱氨基化成I(其随后被读成G)。SEQ-10至SEQ-21的结构描述见实施例2。RNA editing was investigated using endogenously expressed ADAR deaminase in Hela cell lines to investigate whether the A base in the middle of a specific UAG triplet in the 3'UTR region of endogenous GAPDH mRNA could be deaminated to I (which is then read as G) using different structural ASOs SEQ-10 to SEQ-21 ( FIG. 3 ). The structural descriptions of SEQ-10 to SEQ-21 are shown in Example 2.

实验结果如图所示：The experimental results are shown in the figure:

图8显示，转染同时包含SD和下游BHD结构的SEQ-11至SEQ-13和仅包含SD结构的SEQ-10都观察到目标腺苷处A到G的转变，但是SEQ-10在特定位点的编辑水平仅为10％，SEQ-11至SEQ-13对应的编辑水平分别为30％、26％和20％，编辑效率显著提高。另外，转染不含特殊linker、但同时包含SD和BHD的SEQ-14编辑效率为29％，同样能提高编辑效率。Figure 8 shows that the conversion of A to G at the target adenosine was observed in SEQ-11 to SEQ-13 transfected with both SD and downstream BHD structures and SEQ-10 containing only SD structure, but the editing level of SEQ-10 at a specific site was only 10%, and the corresponding editing levels of SEQ-11 to SEQ-13 were 30%, 26% and 20%, respectively, and the editing efficiency was significantly improved. In addition, the editing efficiency of SEQ-14 transfected without a special linker but containing both SD and BHD was 29%, which can also improve the editing efficiency.

以上结果说明：The above results show that:

下游BHD结构能够有效帮助SD提高编辑效率，且SD和BHD之间以不同形式的linker相连或无linker都能帮助SD提高编辑。The downstream BHD structure can effectively help SD improve editing efficiency, and the connection between SD and BHD with different forms of linkers or without linkers can help SD improve editing.

实验结果如图所示：The experimental results are shown in the figure:

图8显示，转染同时具有SD和上游BHD(25nt)的SEQ-15和SEQ-16编辑水平分别为17％和35％，转染同时具有SD和上游BHD(20nt)的SEQ-17和SEQ-18编辑水平分别为16％和22％，与SEQ-10相比显著提高；另外，转染不含特殊linker、但同时包含SD和上游BHD的SEQ-19编辑效率为26％，同样能提高编辑效率。Figure 8 shows that the editing levels of SEQ-15 and SEQ-16 transfected with both SD and upstream BHD (25nt) were 17% and 35%, respectively, and the editing levels of SEQ-17 and SEQ-18 transfected with both SD and upstream BHD (20nt) were 16% and 22%, respectively, which were significantly improved compared with SEQ-10; in addition, the editing efficiency of SEQ-19 transfected without a special linker but with both SD and upstream BHD was 26%, which also improved the editing efficiency.

以上结果说明：The above results show that:

不同长度的上游BHD结构(有linker或无linker)能够有效帮助SD提高编辑效率。Upstream BHD structures of different lengths (with or without linker) can effectively help SD improve editing efficiency.

实验结果如图所示：The experimental results are shown in the figure:

图8显示，转染同时具有SD、下游BHD和上游BHD的SEQ-20和SEQ-21(不含linker)编辑效率分别为18％和20％，相对SEQ-10明显提高。FIG8 shows that the editing efficiencies of SEQ-20 and SEQ-21 (without linker) transfected with SD, downstream BHD, and upstream BHD were 18% and 20%, respectively, which were significantly improved compared with SEQ-10.

以上结果说明：The above results show that:

同时具有上游BHD和下游BHD(有linker或无linker)能够帮助SD提高编辑。Having both upstream BHD and downstream BHD (with or without linker) can help SD improve editing.

实施例6在Hela细胞中使用不同的ASO编辑N乙酰葡糖胺转移酶基因(OGT)3'-UTR区域特定腺苷Example 6 Editing of specific adenosine in the 3'-UTR region of the N-acetylglucosamine transferase gene (OGT) using different ASOs in Hela cells

在Hela细胞株中利用内源表达的ADAR脱氨酶对RNA编辑进行考察，考察使用不同结构ASO SEQ-22至SEQ-29(图5)，是否可以将内源OGT基因3'-UTR区域的UAC三联体中间的A脱氨基化成I(其随后被读成G)。SEQ-22至SEQ-29的结构描述见实施例3。RNA editing was investigated using endogenously expressed ADAR deaminase in Hela cell lines to investigate whether ASOs of different structures SEQ-22 to SEQ-29 ( FIG. 5 ) could deaminate the A in the middle of the UAC triplet in the 3′-UTR region of the endogenous OGT gene to I (which is then read as G). The structures of SEQ-22 to SEQ-29 are described in Example 3.

实验结果如图所示：The experimental results are shown in the figure:

图9显示，转染同时包含SD和下游BHD结构的SEQ-23至SEQ-24和仅包含SD结构的SEQ-22都观察到目标腺苷处A到G的转变，但是SEQ-22在特定位点的编辑水平仅为20％，SEQ-23和SEQ-24对应的编辑水平分别为39％和39％，编辑效率显著提高。另外，转染不含特殊linker、但同时包含SD和BHD的SEQ-26编辑效率为34％，同样能提高编辑效率。Figure 9 shows that the conversion of A to G at the target adenosine was observed in SEQ-23 to SEQ-24 transfected with both SD and downstream BHD structures and SEQ-22 containing only SD structure, but the editing level of SEQ-22 at a specific site was only 20%, while the corresponding editing levels of SEQ-23 and SEQ-24 were 39% and 39%, respectively, and the editing efficiency was significantly improved. In addition, the editing efficiency of SEQ-26 transfected without a special linker but containing both SD and BHD was 34%, which can also improve the editing efficiency.

以上结果说明：The above results show that:

下游BHD结构能够有效帮助SD提高编辑效率，且SD和BHD之间以不同形式的linker相连或无linker都能帮助SD提高编辑。The downstream BHD structure can effectively help SD improve editing efficiency, and the connection between SD and BHD with different forms of linkers or without linkers can help SD improve editing.

实验结果如图所示：The experimental results are shown in the figure:

图9显示，转染同时具有SD和上游BHD(25nt)的SEQ-27编辑水平为43％，转染同时具有SD和上游BHD(20nt)的SEQ-28编辑水平为29％，与SEQ-22相比显著提高。FIG9 shows that the editing level of SEQ-27 transfected with both SD and upstream BHD (25nt) was 43%, and the editing level of SEQ-28 transfected with both SD and upstream BHD (20nt) was 29%, which were significantly improved compared with SEQ-22.

以上结果说明：The above results show that:

不同长度的上游BHD结构能够有效帮助SD提高编辑效率。Upstream BHD structures of different lengths can effectively help SD improve editing efficiency.

实验结果如图所示：The experimental results are shown in the figure:

转染同时具有SD、下游BHD和上游BHD的SEQ-29编辑效率为35％，相对SEQ-22明显提高。The editing efficiency of SEQ-29 transfected with SD, downstream BHD and upstream BHD was 35%, which was significantly improved compared with SEQ-22.

以上结果说明：The above results show that:

同时具有上游BHD和下游BHD能够帮助SD提高编辑。Having both upstream BHD and downstream BHD can help SD improve editing.

Claims (10)

Translated fromChinese

1.一种用于靶向RNA编辑的单链反义寡核苷酸(Antisense Oligonucleotide，ASO)，其中所述ASO包含靶向区(Specificity Domain，SD)和辅助结合区(Binding Helper1. A single-stranded antisense oligonucleotide (ASO) for targeted RNA editing, wherein the ASO comprises a targeting domain (SD) and a binding helper domain (Binding HelperDomain，BHD)，其中所述SD与靶向RNA区域互补配对，所述BHD与非靶向RNADomain, BHD, wherein the SD is complementary to the targeting RNA region, and the BHD is complementary to the non-targeting RNA区域互补配对，所述ASO能够与目标RNA形成双链复合物，所述BHD增加ASO与目标RNA形成双链复合物的稳定性。The ASO can form a double-stranded complex with the target RNA through complementary pairing of the regions, and the BHD increases the stability of the double-stranded complex formed by the ASO and the target RNA.2.根据权利要求1所述的ASO，其中所述靶向RNA区域与非靶向RNA区域非连续，所述靶向RNA区域和所述非靶向RNA区域间隔的长度为4-1000nt。2. The ASO according to claim 1, wherein the targeting RNA region is non-contiguous with the non-targeting RNA region, and the length of the interval between the targeting RNA region and the non-targeting RNA region is 4-1000 nt.3.根据权利要求1-2中任一项所述的ASO，其中所述BHD与非靶向RNA区域完全互补配对。3. The ASO according to any one of claims 1-2, wherein the BHD is fully complementary to the non-targeting RNA region.4.根据权利要求1-3中任一项所述的ASO，其中所述BHD具有选自下组中的一种或者多种修饰：硫代磷酸酯化修饰、2’-OMe、2’-F、LNA、UNA、2’-MOE、肌苷替代修饰。4. The ASO according to any one of claims 1-3, wherein the BHD has one or more modifications selected from the group consisting of phosphorothioate modification, 2'-OMe, 2'-F, LNA, UNA, 2'-MOE, and inosine substitution modification.5.根据权利要求1-4中任一项所述的ASO，其中所述一个或者多个BHD之间，或者一个或者多个BHD与SD之间，不形成分子间二级结构和/或分子内二级结构。5. The ASO according to any one of claims 1 to 4, wherein no intermolecular secondary structure and/or intramolecular secondary structure is formed between the one or more BHDs, or between the one or more BHDs and the SD.6.根据权利要求1-5中任一项所述的ASO，其中所述一个和/或多个BHD在所述SD的5’端和/或3’端。6. The ASO according to any one of claims 1-5, wherein the one and/or multiple BHDs are at the 5' end and/or 3' end of the SD.7.根据权利要求1-6中任一项所述的ASO，其中所述BHD与SD直接连接或者间接连接。7. The ASO according to any one of claims 1 to 6, wherein the BHD is directly or indirectly connected to the SD.8.根据权利要求1-7中任一项所述的ASO，其中所述SD具有选自下组中的一种或者多种修饰：硫代磷酸酯化修饰、2’-OMe、2’-F、LNA、UNA、2’-MOE、2’-FANA、DNA碱基替代修饰，肌苷替代修饰。8. The ASO according to any one of claims 1-7, wherein the SD has one or more modifications selected from the group consisting of phosphorothioate modification, 2'-OMe, 2'-F, LNA, UNA, 2'-MOE, 2'-FANA, DNA base substitution modification, and inosine substitution modification.9.编辑RNA的方法，其包含使用权利要求1-8中任一项所述的ASO。9. A method for editing RNA, comprising using the ASO of any one of claims 1-8.10.药物组合物，其包含权利要求1-9中任一项所述的ASO，和/或药学上可接受的载体。10. A pharmaceutical composition comprising the ASO according to any one of claims 1 to 9, and/or a pharmaceutically acceptable carrier.