WO2025082057A1 - Inhibitor for inhibiting expression of inhibin beta e gene and use thereof - Google Patents

Abstract

A dsRNA and double-stranded RNAi agent for inhibiting the expression of INHBE and a use thereof. The dsRNA and the double-stranded RNAi agent can be used for inhibiting the expression of INHBE to prevent and treat a metabolic disorder (such as a metabolic syndrome).

Description

Translated fromChinese

抑制抑制素Beta E基因表达的抑制剂及其用途Inhibitors for inhibiting the expression of inhibin Beta E gene and uses thereof

相关申请的交叉引用CROSS-REFERENCE TO RELATED APPLICATIONS

本公开要求申请号为202311333351.X，申请日为2023年10月16日的中国专利申请以及申请日为2023年10月16日的美国临时申请US63/590,562的优先权，该中国专利申请的全部内容在此引入本公开作为参考。This disclosure claims priority to Chinese patent application number 202311333351.X, filed on October 16, 2023, and U.S. provisional application US63/590,562, filed on October 16, 2023, the entire contents of which are incorporated herein by reference.

技术领域Technical Field

本公开涉及一种抑制抑制素Beta E基因(INHBE)表达的抑制剂，例如RNAi剂或RNA，及其药物组合物，以及用于预防、治疗和/或抑制与INHBE基因相关的疾病和病症的方法。The present disclosure relates to an inhibitor of the expression of the inhibin Beta E gene (INHBE), such as an RNAi agent or RNA, and a pharmaceutical composition thereof, as well as a method for preventing, treating and/or inhibiting diseases and conditions associated with the INHBE gene.

背景技术Background Art

代谢综合征是一种以腹部肥胖、胰岛素抵抗、高血压、高血脂症为特征的病理状态。代谢综合征可引起机体代谢紊乱，不仅是心血管疾病、糖尿病及肾脏疾病的危险因素，还增加了癌症及全因死亡风险。全世界约有20％～25％的成年人受到代谢综合征的困扰。因肥胖造成的代谢综合征在儿童及青年中的发病率逐年上升，这与高卡路里和低纤维的饮食结构，以及久坐不动的不良习惯有关。除对各疾病分组进行药物管理之外，目前对于代谢综合征的治疗方法主要包括饮食及生活方式的改变，但临床上大多数患者依从性较差。Metabolic syndrome is a pathological state characterized by abdominal obesity, insulin resistance, hypertension, and hyperlipidemia. Metabolic syndrome can cause metabolic disorders in the body. It is not only a risk factor for cardiovascular disease, diabetes, and kidney disease, but also increases the risk of cancer and all-cause mortality. About 20% to 25% of adults worldwide suffer from metabolic syndrome. The incidence of metabolic syndrome caused by obesity in children and young people has increased year by year, which is related to a high-calorie and low-fiber diet and a sedentary lifestyle. In addition to drug management for each disease group, the current treatment methods for metabolic syndrome mainly include changes in diet and lifestyle, but most patients have poor compliance in clinical practice.

抑制素Beta E基因(inhibin beta E Gene，INHBE)编码一种名为激活素E(Activin E)的分泌蛋白。INHBE的mRNA主要在肝脏中表达，其参与肝细胞的生长和分化调节。有研究报告(Hashimoto O.et al.，Life sciences.2009；85(13–14):534–40)指出，胰岛素刺激肝细胞中INHBE的表达，并且饮食诱导的肥胖小鼠的肝脏中INHBE mRNA上调，这表明INHBE与葡萄糖代谢有关。进一步的研究(Sugiyama M.et al.，PLoS One，2018；13(3)：e0194798)表明，激活素E是诱发胰岛素抵抗的推定因素，INHBE的基因表达与人类的胰岛素抵抗和体重指数(BMI)呈正相关性。此外，有文献指出(Akbari,P.et al.，Nat Commun 13，4844(2022).)，INHBE是肝脏表达的脂肪储存负调节因子，阻断INHBE可能有益于治疗与脂肪分布相关的代谢疾病。The inhibin beta E gene (INHBE) encodes a secretory protein called activin E. INHBE mRNA is mainly expressed in the liver, where it is involved in regulating the growth and differentiation of hepatocytes. A study report (Hashimoto O. et al., Life sciences. 2009; 85(13–14): 534–40) pointed out that insulin stimulates the expression of INHBE in hepatocytes, and INHBE mRNA is upregulated in the liver of diet-induced obese mice, indicating that INHBE is related to glucose metabolism. Further studies (Sugiyama M. et al., PLoS One, 2018; 13(3): e0194798) showed that activin E is a putative factor inducing insulin resistance, and the gene expression of INHBE is positively correlated with insulin resistance and body mass index (BMI) in humans. In addition, some literature points out (Akbari, P. et al., Nat Commun 13, 4844 (2022).) that INHBE is a negative regulator of fat storage expressed in the liver, and blocking INHBE may be beneficial in treating metabolic diseases related to fat distribution.

WO2023003922A1公开了与INHBE靶点相关的抑制代谢紊乱相关靶基因表达的双链核糖核酸(dsRNA)剂。WO2023044094A1公开了INHBE调节剂组合物及其使用方法及其使用方法。然而，仍需针对INHBE靶点开发具有更好疗效的RNAi药物。WO2023003922A1 discloses a double-stranded ribonucleic acid (dsRNA) agent related to the INHBE target for inhibiting the expression of target genes related to metabolic disorders. WO2023044094A1 discloses an INHBE modulator composition and a method of using the same and a method of using the same. However, there is still a need to develop RNAi drugs with better efficacy for the INHBE target.

发明内容Summary of the invention

本公开提供一种抑制抑制素Beta E基因(INHBE)表达的抑制剂，例如RNAi或RNA，及其药物组合物，及其在制备用于预防、治疗、和/或抑制相关疾病例如代谢综合征的药物中的用途。The present invention provides an inhibitor for inhibiting the expression of inhibin Beta E gene (INHBE), such as RNAi or RNA, and a pharmaceutical composition thereof, and use thereof in the preparation of a drug for preventing, treating, and/or inhibiting related diseases such as metabolic syndrome.

本公开的RNAi剂和RNA被设计为靶向INHBE基因，包括在其他哺乳动物物种的直系同源物中保守的基因部分。RNAi剂通常包含形成双链体、双链RNA(本文称之为“dsRNA”)的正义链和反义链。包含dsRNA的RNAi剂在本文中也被称为“dsRNAi”剂。The RNAi agents and RNAs disclosed herein are designed to target the INHBE gene, including portions of the gene that are conserved in orthologs in other mammalian species. RNAi agents generally comprise duplex-forming, double-stranded RNA (referred to herein as "dsRNA") comprises a sense strand and an antisense strand. RNAi agents comprising dsRNA are also referred to herein as "dsRNAi" agents.

无意受到理论的限制，本公开的RNAi剂和RNA以及这些RNAi剂和RNA中的特定靶位和/或修饰方式赋予了改进的功效、稳定性、效力、持久性和/或安全性。例如但不限于，在一些实施方式中，本公开的RNAi剂和/或RNA表现出：(1)改善的功效和/或效力，例如通过与靶基因mRNA更强的杂交(例如，由反义链/靶mRNA双链体的Tm增加所确定的，例如反义链的理论Tm的增加)；和/或，(2)改善的安全性，例如，通过降低脱靶效应，例如，通过降低或减弱与脱靶RNA的杂交(例如，由反义链和脱靶RNA形成的双链体的Tm降低所确定的)。Without intending to be limited by theory, the RNAi agents and RNAs disclosed herein and the specific targets and/or modifications in these RNAi agents and RNAs impart improved efficacy, stability, potency, durability and/or safety. For example, but not limited to, in some embodiments, the RNAi agents and/or RNAs disclosed herein exhibit: (1) improved efficacy and/or potency, for example, by stronger hybridization with target gene mRNA (e.g., determined by an increase in the Tm of the antisense strand/target mRNA duplex, such as an increase in the theoretical Tm of the antisense strand); and/or, (2) improved safety, for example, by reducing off-target effects, for example, by reducing or attenuating hybridization with off-target RNAs (e.g., determined by a decrease in the Tm of the duplex formed by the antisense strand and the off-target RNA).

本公开的RNAi剂的使用能使得哺乳动物中的INHBE靶基因的mRNA靶向降解。本发明人已经证实，本公开的RNAi剂可以引发RNA诱导的沉默复合物(RISC)介导的INHBE RNA转录物的切割，从而导致INHBE靶基因的表达的显著抑制。在某些实施方式中，本公开的RNAi剂比先前的靶向相同基因的RNAi剂更有效(例如更有效力)和/或更具特异性(例如更安全，更少的脱靶效应)。在某些实施方式中，通过选择RNAi剂靶向INHBE mRNA中的特定位点，和/或包括RNA修饰(例如，非规范碱基配对配对核苷酸、修饰核苷酸、化学修饰)以增加功效、效力、特异性和/或安全性。在一些这样的实施方式中，RNAi剂包含至少一种修饰的核苷酸，例如非规范碱基配对核苷酸。包含这些RNAi剂的方法和组合物可用于治疗患有INHBE相关疾病或病症例如代谢病症的受试者。因此，本公开提供了用于治疗、预防或抑制受试者中的代谢病症的方法，所述受试者将受益于使用本公开的RNAi剂和组合物抑制或减少INHBE表达。The use of the RNAi agents disclosed herein enables targeted degradation of the mRNA of the INHBE target gene in mammals. The inventors have demonstrated that the RNAi agents disclosed herein can trigger RNA-induced silencing complex (RISC)-mediated cleavage of INHBE RNA transcripts, resulting in significant inhibition of the expression of the INHBE target gene. In certain embodiments, the RNAi agents disclosed herein are more effective (e.g., more potent) and/or more specific (e.g., safer, less off-target effects) than previous RNAi agents targeting the same gene. In certain embodiments, the RNAi agent is targeted to a specific site in the INHBE mRNA by selecting the RNAi agent, and/or includes RNA modifications (e.g., non-canonical base pairing nucleotides, modified nucleotides, chemical modifications) to increase efficacy, potency, specificity and/or safety. In some such embodiments, the RNAi agent comprises at least one modified nucleotide, such as a non-canonical base pairing nucleotide. Methods and compositions comprising these RNAi agents can be used to treat subjects suffering from INHBE-related diseases or disorders, such as metabolic disorders. Thus, the present disclosure provides methods for treating, preventing, or inhibiting a metabolic disorder in a subject who would benefit from inhibiting or reducing INHBE expression using the RNAi agents and compositions of the present disclosure.

一方面，本公开提供了一种用于抑制细胞中抑制素亚基beta E(INHBE)表达的双链核糖核酸干扰(dsRNAi)剂，其中所述dsRNAi剂包含形成双链RNA(dsRNA)区域的正义链和反义链，所述反义链包含与INHBE mRNA互补的区域，其中所述互补区域包含至少15个连续的核苷酸。在某些实施方式中，所述dsRNAi剂包含至少一个非规范碱基配对核苷酸，如下文进一步描述。In one aspect, the present disclosure provides a double-stranded RNA interference (dsRNAi) agent for inhibiting the expression of inhibin subunit beta E (INHBE) in a cell, wherein the dsRNAi agent comprises a sense strand and an antisense strand forming a double-stranded RNA (dsRNA) region, the antisense strand comprising a region complementary to INHBE mRNA, wherein the complementary region comprises at least 15 consecutive nucleotides. In certain embodiments, the dsRNAi agent comprises at least one non-canonical base-pairing nucleotide, as further described below.

在一些实施方式中，所述dsRNAi剂包含一、二、三、四、五或更多个非规范碱基配对核苷酸。非规范碱基配对核苷酸和其他的修饰核苷酸可以存在于互补区域、dsRNA区域或dsRNAi剂中的任何其他位置。非规范碱基配对核苷酸和其他的修饰核苷酸可以包含在反义链、正义链或这两者上。在一些实施方式中，除了非规范碱基配对核苷酸之外，dsRNAi剂还包含至少一种其他的修饰核苷酸；此类其他的修饰核苷酸可以在或可以不在与非规范碱基配对核苷酸相同的寡核苷酸链上。In some embodiments, the dsRNAi agent comprises one, two, three, four, five or more non-canonical base pairing nucleotides. Non-canonical base pairing nucleotides and other modified nucleotides may be present in any other position in the complementary region, dsRNA region or dsRNAi agent. Non-canonical base pairing nucleotides and other modified nucleotides may be included in the antisense strand, sense strand or both. In some embodiments, in addition to non-canonical base pairing nucleotides, the dsRNAi agent also comprises at least one other modified nucleotide; Such other modified nucleotides may or may not be on the same oligonucleotide chain as the non-canonical base pairing nucleotides.

在一些实施方式中，所述dsRNAi剂或RNA与具有相同核苷酸序列而没有非规范碱基配对和/或修饰核苷酸的dsRNAi剂或RNA相比，具有增加的功效、效力、特异性和/或安全性，和/或减少的脱靶效应。在一些这样的实施方式中，与具有相同核苷酸序列但没有非规范碱基配对和/或修饰的核苷酸的dsRNAi剂或RNA相比，所述dsRNAi剂或RNA的解链温度(Tm)的改变至少在2℃以上。In some embodiments, the dsRNAi agent or RNA has increased efficacy, potency, specificity and/or safety, and/or reduced off-target effects compared to a dsRNAi agent or RNA having the same nucleotide sequence but without non-canonical base pairing and/or modified nucleotides. In some such embodiments, the melting temperature (Tm) of the dsRNAi agent or RNA is altered by at least 2°C compared to a dsRNAi agent or RNA having the same nucleotide sequence but without non-canonical base pairing and/or modified nucleotides.

在一些实施方式中，本公开的dsRNAi剂或RNA包含非规范碱基配对核苷酸和/或修饰的核苷酸，其使Tm改变至少2℃，例如2℃、超过2℃、3℃、4℃、5℃或更多。在一些此类的实施方式中，使用本文所述的公式或算法来计算Tm。In some embodiments, the dsRNAi agents or RNAs of the present disclosure comprise non-canonical base-paired nucleotides and/or modified nucleotides that alter the Tm by at least 2° C., e.g., 2° C., more than 2° C., 3° C., 4° C., 5° C., or more. In some such embodiments, the Tm is calculated using the formulas or algorithms described herein.

在一些实施方式中，本公开的dsRNAi剂或RNA在寡核苷酸链(例如反义链)从5'端到3'端的方向上的第1-11位中，包含至少一个非规范碱基配对核苷酸和/或经修饰的核苷酸。In some embodiments, the dsRNAi agent or RNA of the present disclosure comprises at least one non-canonical base-pairing nucleotide and/or modified nucleotide in positions 1-11 in the direction from the 5' end to the 3' end of the oligonucleotide strand (eg, the antisense strand).

在一些实施方式中，本公开的dsRNAi剂或RNA在寡核苷酸链(例如反义链)从5'端到3'端的方向上的第12-21位中，包含至少一个非规范碱基配对核苷酸和/或经修饰的核苷酸。在一些实施方式中，在序列片段N₁N₂G₀N₃N₄中，N₁、N₂、N₃和N₄各自独立地为包含腺嘌呤(A)、胞嘧啶(C)、鸟嘌呤(G)、胸腺嘧啶(T)或尿嘧啶(U)为碱基的核苷酸，G₀是包含鸟嘌呤为碱基的核苷酸，当N₁、N₂、N₃和N₄中的至少三个碱基是腺嘌呤或尿嘧啶时，序列中G₀的鸟嘌呤被次黄嘌呤(I)取代。在一些此类的实施方式中，N₁、N₂、N₃、N₄和G₀中的至少一个还具有修饰的糖基和/或修饰的核苷酸间键合。In some embodiments, the dsRNAi agent or RNA of the present disclosure comprises at least one non-canonical_base -pairing nucleotide and/or modified nucleotide in positions 12-21 of the oligonucleotide strand (e.g., antisense strand) from the₅ ' end to the_3' end. In some embodiments, in the sequence fragment_N1N2G0N3N4 ,_N1 ,_N2 ,_N3 , and_N4 are each independently In some embodiments,_N1 , N2,_N3 , N4 and G0 are nucleotides containing adenine (A), cytosine (C), guanine (G), thymine (T) or uracil (U) as bases,_G0 is a nucleotide containing guanine as base, and when at least three bases among N1,_N2 , N3 and_N4 are adenine or uracil, the guanine of_G0 in the sequence is replaced by hypoxanthine (I). In some such embodiments, at least one of_N1 ,_N2 ,_N3 ,_N4 and_G0 further has a modified sugar group and/or a modified internucleotide bond.

在一些实施方式中，本公开的dsRNAi剂或RNA在寡核苷酸链从5'端到3'端的方向上的第6、7或8位中，包含至少一个非规范碱基配对核苷酸和/或修饰的核苷酸，如果第2-8位中的至少四个核苷酸是A或U，并且第6-8位中的至少一个核苷酸是G，则第6、7和/或8位的G被非规范碱基配对核苷酸替换。在一些实施方式中，本公开的dsRNAi剂或RNA在寡核苷酸链(例如反义链和/或正义链)从5'端到3'端的方向上的第6、7或8位中，如果第2-8位的核苷酸中，腺嘌呤和尿嘧啶的数量在4个以上，并且第6-8位的核苷酸中的碱基至少一个是鸟嘌呤，则第6、7和/或8位的鸟嘌呤被非规范碱基(例如次黄嘌呤)替换。In some embodiments, the dsRNAi agent or RNA of the present disclosure comprises at least one non-canonical base-pairing nucleotide and/or modified nucleotide in the 6th, 7th or 8th position in the direction from the 5' end to the 3' end of the oligonucleotide chain, and if at least four nucleotides in positions 2-8 are A or U, and at least one nucleotide in positions 6-8 is G, then the G in positions 6, 7 and/or 8 is replaced by a non-canonical base-pairing nucleotide. In some embodiments, the dsRNAi agent or RNA of the present disclosure comprises at least one non-canonical base-pairing nucleotide and/or modified nucleotide in the 6th, 7th or 8th position in the direction from the 5' end to the 3' end of the oligonucleotide chain (e.g., the antisense chain and/or the sense chain), if the number of adenine and uracil in the nucleotides in positions 2-8 is more than 4, and at least one base in the nucleotides in positions 6-8 is guanine, then the guanine in positions 6, 7 and/or 8 is replaced by a non-canonical base (e.g., hypoxanthine).

在一些此类实施方式中，本公开的dsRNAi剂或RNA包含与表1、表2a或表2b中所示的任一序列相差不超过3个核苷酸的至少15个连续核苷酸。在一些此类实施方式中，所述反义链包含与表1、表2a或表2b中所示的任一序列相差不超过3个核苷酸的至少15个连续核苷酸，并且所述正义链包含至少15个与所述反义链互补的核苷酸。在一些此类实施方式中，所述正义链包含与表1、表2a或表2b中所示的任一序列相差不超过3个核苷酸的至少15个连续核苷酸，并且所述反义链包含至少15个与所述正义链互补的核苷酸。在一些此类实施方式中，所述反义链和/或所述正义链与表1、表2a或表2b中所示的任一序列相差不超过1、2、3个核苷酸。在一些实施方式中，所述dsRNAi剂包含dsRNA，所述dsRNA包含表1、表2a或表2b中任一种双链体序列，例如IN-001至IN-488和INI-001至INI-308中的任一种，又例如IN-489至IN-546中的任一种。在本申请中，应当理解的是“相差”既可以表示核苷酸序列长度的差异，也可以表示核苷酸的差异，也可以表示两者的组合。In some such embodiments, the dsRNAi agent or RNA of the present disclosure comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from any of the sequences shown in Table 1, Table 2a, or Table 2b. In some such embodiments, the antisense strand comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from any of the sequences shown in Table 1, Table 2a, or Table 2b, and the sense strand comprises at least 15 nucleotides that are complementary to the antisense strand. In some such embodiments, the sense strand comprises at least 15 consecutive nucleotides that differ by no more than 3 nucleotides from any of the sequences shown in Table 1, Table 2a, or Table 2b, and the antisense strand comprises at least 15 nucleotides that are complementary to the sense strand. In some such embodiments, the antisense strand and/or the sense strand differ by no more than 1, 2, or 3 nucleotides from any of the sequences shown in Table 1, Table 2a, or Table 2b. In some embodiments, the dsRNAi agent comprises a dsRNA, and the dsRNA comprises any duplex sequence in Table 1, Table 2a or Table 2b, such as any one of IN-001 to IN-488 and INI-001 to INI-308, and any one of IN-489 to IN-546. In the present application, it should be understood that "difference" can represent the difference in nucleotide sequence length, the difference in nucleotides, or a combination of the two.

示例性的非规范碱基配对核苷酸中的碱基包括但不限于，选自肌苷(I)、黄苷(X)、7-甲基鸟苷(m7G)、N6-甲基腺苷(m6A)、二氢尿苷、5-甲基胞嘧啶(m5C)、假尿苷(Ψ)和N1-甲基假尿苷(m1Ψ)中的碱基。在某些实施方式中，非规范碱基配对核苷酸是肌苷酸(I)。在一些实施方式中，本公开的dsRNAi剂或RNA(例如正义链和/或反义链)中的至少一个鸟嘌呤(G)被次黄嘌呤(I)替代。其他的修饰核苷酸的实例在文中另述。Exemplary bases in non-canonical base pairing nucleotides include, but are not limited to, bases selected from inosine (I), xanthosine (X), 7-methylguanosine (m7G), N6-methyladenosine (m6A), dihydrouridine, 5-methylcytosine (m5C), pseudouridine (Ψ) and N1-methylpseudouridine (m1Ψ). In certain embodiments, the non-canonical base pairing nucleotide is inosinic acid (I). In some embodiments, at least one guanine (G) in a dsRNAi agent or RNA (e.g., a sense strand and/or an antisense strand) of the present disclosure is replaced by hypoxanthine (I). Examples of other modified nucleotides are described elsewhere herein.

另一方面，本公开提供了一种dsRNA，其包含正义链和反义链，其中，所述反义链包含与表1、表2a或表2b中所示的任一序列相差不超过3个(0、1、2或3个)核苷酸的至少15个连续核苷酸，所述正义链具有至少15个、16个、17个、18个、19个、20个或21个与所述反义链互补的核苷酸。On the other hand, the present disclosure provides a dsRNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 consecutive nucleotides that differ from any one of the sequences shown in Table 1, Table 2a or Table 2b by no more than 3 (0, 1, 2 or 3) nucleotides, and the sense strand has at least 15, 16, 17, 18, 19, 20 or 21 nucleotides that are complementary to the antisense strand.

在一些实施方式中，所述反义链包含与表1、表2a或表2b中所示的任一序列相差0、1、2或3个核苷酸的至少15个、16个、17个、18个、19个、20个或21个连续核苷酸。In some embodiments, the antisense strand comprises at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides that differ from any of the sequences shown in Table 1, Table 2a or Table 2b by 0, 1, 2 or 3 nucleotides.

在一些实施方式中，所述正义链序列与所述反义链序列至少基本上互补。在一些优选的实施方式中，所述正义链序列与所述反义链序列完全互补(即，100％互补)。In some embodiments, the sense strand sequence is at least substantially complementary to the antisense strand sequence. In some preferred embodiments, the sense strand sequence is completely complementary to the antisense strand sequence (ie, 100% complementary).

在一些实施方式中，所述正义链和反义链的长度各自独立地为17-25个核苷酸；优选地，所述正义链和反义链的长度各自独立地为19-23个核苷酸；更优选地，所述正义链和反义链的长度各自独立地为19-21个核苷酸。In some embodiments, the length of the sense strand and the antisense strand is each independently 17-25 nucleotides; preferably, the length of the sense strand and the antisense strand is each independently 19-23 nucleotides; more preferably, the length of the sense strand and the antisense strand is each independently 19-21 nucleotides.

在一些实施方式中，所述正义链长度为19个核苷酸，包括以下项中的任一项：In some embodiments, the sense strand is 19 nucleotides in length and includes any one of the following:

由SEQ ID NO 1621:GGGUCAAGCACAGCU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1621: GGGUCAAGCACAGCU;

由SEQ ID NO 1622:AAGCACAGCUAUCCA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1622:AAGCACAGCUAUCCA;

由SEQ ID NO 1623:CAGCUAUCCAUCAGA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1623:CAGCUAUCCAUCAGA;

由SEQ ID NO 1624:AUCCAUCAGAUGAUC所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1624: AUCCAUCAGAUGAUC;

由SEQ ID NO 1625:UCAGAUGAUCUACUU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1625:UCAGAUGAUCUACUU;

由SEQ ID NO 1626:UGAUCUACUUUCAGC所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1626:UGAUCUACUUUCAGC;

由SEQ ID NO 1627:UGAGUCCCAGACAAU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1627:UGAGUCCCAGACAAU;

由SEQ ID NO 1628:AGCCAAGCAGCAAAU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1628: AGCCAAGCAGCAAAU;

由SEQ ID NO 1629:GUCGUCCCAGAAUAA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1629: GUCGUCCCAGAAUAA;

由SEQ ID NO 1630:AUCAGCUUUGCUACU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1630: AUCAGCUUUGCUACU;

由SEQ ID NO 1631:UGCUACUGUCACAGA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1631:UGCUACUGUCACAGA;

由SEQ ID NO 1632:UUCCUGGCACUCUUU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1632:UUCCUGGCACUCUUU;

由SEQ ID NO 1633:UCUUUGCUUGAGGAU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1633: UCUUUGCUUGAGGAU;

由SEQ ID NO 1634:UGGUGUCCUGAAACU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1634:UGGUGUCCUGAAACU;

由SEQ ID NO 1635:CCUGAAACUGCAACU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1635:CCUGAAACUGCAACU;

由SEQ ID NO 1636:AGGCAACAGCACAGU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1636:AGGCAACAGCACAGU;

由SEQ ID NO 1637:CCUAGAGCUUAAGAU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1637:CCUAGAGCUUAAGAU;

由SEQ ID NO 1638:CUUAAGAUCCGAGCC所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1638:CUUAAGAUCCGAGCC;

由SEQ ID NO 1639:ACCAUUACGUAGACU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1639:ACCAUUACGUAGACU;

由SEQ ID NO 1640:CUCAAAGCCAACAAU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1640: CUCAAAGCCAACAAU;

由SEQ ID NO 1641:CUCUACCUGGAUCAU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1641:CUCUACCUGGAUCAU;

由SEQ ID NO 1642:GAUCAUAAUGGCAAU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1642:GAUCAUAAUGGCAAU;

由SEQ ID NO 1643:AAGAGACCAAGAUGA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1643: AAGAGACCAAGAUGA;

由SEQ ID NO 1644:CUGGCAAUAUGACUC所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1644:CUGGCAAUAUGACUC;

由SEQ ID NO 1645:UGGGCACUUUCUUGU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1645:UGGGCACUUUCUUGU;

由SEQ ID NO 1646:UUCUUGUCUGAGACU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1646: UUCUUGUCUGAGACU;

由SEQ ID NO 1647:UGAGACUCUGGCUUA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1647:UGAGACUCUGGCUUA;

由SEQ ID NO 1648:UUGGGAGAUGGGUAA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1648: UUGGGAGAUGGGUAA;

由SEQ ID NO 1649:GAUGGGUAAAGCGUU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1649:GAUGGGUAAAGCGUU;

由SEQ ID NO 1650:AAAGCGUUUCUUCUA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1650:AAAGCGUUUCUUCUA;

由SEQ ID NO 1651:CUUCUAAAGGGGUCU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1651:CUUCUAAAGGGGUCU;

由SEQ ID NO 1652:UACCCAGAAAGCAUG所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1652: UACCCAGAAAGCAUG;

由SEQ ID NO 1653:AAGUCCUGUGAGAAG所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1653: AAGUCCUGUGAGAAG;

由SEQ ID NO 1654:GAAGGCAGAGAAAAA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1654:GAAGGCAGAGAAAAA;

由SEQ ID NO 1655:CAGAGAAAAAUUACU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1655:CAGAGAAAAAUUACU;

由SEQ ID NO 1656:UCCCAAGAUGAGAAA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1656:UCCCAAGAUGAGAAA;

由SEQ ID NO 1657:GAGGAAGCAGAUAGA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1657:GAGGAAGCAGAUAGA;

由SEQ ID NO 1658:CUGUUGAGGUACCUU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1658: CUGUUGAGGUACCUU;

由SEQ ID NO 1659:AGGUACCUUAAGGGA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1659:AGGUACCUUAAGGGA;

由SEQ ID NO 1660:CAGGAGUCAGGAAAA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1660: CAGGAGUCAGGAAAA;

由SEQ ID NO 1661:GGGAGACAAGCAUUU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1661: GGGAGACAAGCAUUU;

由SEQ ID NO 1662:ACAAGCAUUUAUACU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1662:ACAAGCAUUUAUACU;

由SEQ ID NO 1663:CAUUUAUACUUUCUU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1663: CAUUUAUACUUUCUU;

由SEQ ID NO 1664:AAAGAAAAUCAACAA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1664:AAAGAAAAUCAACAA;

由SEQ ID NO 1665:CAAAUGUGAGUCAUA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1665: CAAAUGUGAGUCAUA;

由SEQ ID NO 1666:GUCAUAAAGAAGGGU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1666: GUCAUAAAGAAGGGU;

由SEQ ID NO 1667:AGAGCAACAGUUCUU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1667:AGAGCAACAGUUCUU;

由SEQ ID NO 1668:GGGUGUCCACAAAGU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1668: GGGUGUCCACAAAGU;

由SEQ ID NO 1669:UCCACAAAGUCAAAG所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1669: UCCACAAAGUCAAAG;

由SEQ ID NO 1670:AAAGUCAAAGCUAUU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1670:AAAGUCAAAGCUAUU;

由SEQ ID NO 1671:AAAGCUAUUUUCAUA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1671:AAAGCUAUUUUCAUA;

由SEQ ID NO 1672:AUUUUCAUAAUAAUA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1672:AUUUUCAUAAUAAUA;

由SEQ ID NO 1673:AUAAUACUAACAUGU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1673:AUAAUACUAACAUGU;

由SEQ ID NO 1674:ACUAACAUGUUAUUU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1674:ACUAACAUGUUAUUU;

由SEQ ID NO 1675:AUGUUAUUUGCCUUU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1675: AUGUUAUUUGCCUUU;

由SEQ ID NO 1676:AUUUGCCUUUUGAAU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1676:AUUUGCCUUUUGAAU;

由SEQ ID NO 1677:UUUUGAAUUCUCAUU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1677: UUUUGAAUUCUCAUU;

由SEQ ID NO 1678:UUCUCAUUAUCUUAA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1678: UUCUCAUUAUCUUAA;

由SEQ ID NO 1679:UUAUCUUAAAAUUGU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1679:UUAUCUUAAAAUUGU;

由SEQ ID NO 1680:GUGUGACAUGUGAUU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1680: GUGUGACAUGUGAUU;

由SEQ ID NO 1681:AUGUGAUUACAUCAU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1681: AUGUGAUUACAUCAU;

由SEQ ID NO 1682:UUACAUCAUCUUUCU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1682: UUACAUCAUCUUUCU;

由SEQ ID NO 1683:AUCUUUCUGACAUCA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1683:AUCUUUCUGACAUCA;

由SEQ ID NO 1684:UCUGACAUCAUUGUU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1684: UCUGACAUCAUUGUU;

由SEQ ID NO 1685:GUUAAUGGAAUGUGU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1685: GUUAAUGGAAUGUGU;

由SEQ ID NO 1686:CAGCUUUGCUACUGU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1686: CAGCUUUGCUACUGU;

由SEQ ID NO 1687:AGACUCUGGCUUAUU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1687:AGACUCUGGCUUAUU;

由SEQ ID NO 1688:GCAACAGUUCUUCAA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1688: GCAACAGUUCUUCAA;

由SEQ ID NO 1689:AAAGCUAUUUUCAUA所示的连续15个核苷酸。15 consecutive nucleotides shown by SEQ ID NO 1689:AAAGCUAUUUUCAUA.

在一些实施方式中，所述反义链长度为21个核苷酸，包括以下项中的任一项：In some embodiments, the antisense strand is 21 nucleotides in length and includes any one of the following:

由SEQ ID NO 1690:AGCUGUGCUUGACCC所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1690:AGCUGUGCUUGACCC;

由SEQ ID NO 1691:UGGAUAGCUGUGCUU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1691:UGGAUAGCUGUGCUU;

由SEQ ID NO 1692:UCUGAUGGAUAGCUG所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1692:UCUGAUGGAUAGCUG;

由SEQ ID NO 1693:GAUCAUCUGAUGGAU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1693:GAUCAUCUGAUGGAU;

由SEQ ID NO 1694:AAGUAGAUCAUCUGA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1694: AAGUAGAUCAUCUGA;

由SEQ ID NO 1695:AAGUAGAUCAUCUGA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1695: AAGUAGAUCAUCUGA;

由SEQ ID NO 1696:AUUGUCUGGGACUCA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1696:AUUGUCUGGGACUCA;

由SEQ ID NO 1697:AUUUGCUGCUUGGCU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1697:AUUUGCUGCUUGGCU;

由SEQ ID NO 1698:UUAUUCUGGGACGAC所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1698: UUAUUCUGGGACGAC;

由SEQ ID NO 1699:AGUAGCAAAGCUGAU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1699:AGUAGCAAAGCUGAU;

由SEQ ID NO 1700:UCUGUGACAGUAGCA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1700:UCUGUGACAGUAGCA;

由SEQ ID NO 1701:UCUGUGACAGUAGCA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1701:UCUGUGACAGUAGCA;

由SEQ ID NO 1702:AUCCUCAAGCAAAGA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1702:AUCCUCAAGCAAAGA;

由SEQ ID NO 1703:AGUUUCAGGACACCA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1703:AGUUUCAGGACACCA;

由SEQ ID NO 1704:AGUUGCAGUUUCAGG所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1704:AGUUGCAGUUUCAGG;

由SEQ ID NO 1705:ACUGUGCUGUUGCCU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1705:ACUGUGCUGUUGCCU;

由SEQ ID NO 1706:AUCUUAAGCUCUAGG所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1706:AUCUUAAGCUCUAGG;

由SEQ ID NO 1707:GGCUCGGAUCUUAAG所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1707: GGCUCGGAUCUUAAG;

由SEQ ID NO 1708:AGUCUACGUAAUGGU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1708: AGUCUACGUAAUGGU;

由SEQ ID NO 1709:AUUGUUGGCUUUGAG所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1709:AUUGUUGGCUUUGAG;

由SEQ ID NO 1710:AUGAUCCAGGUAGAG所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1710: AUGAUCCAGGUAGAG;

由SEQ ID NO 1711:AUUGCCAUUAUGAUC所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1711:AUUGCCAUUAUGAUC;

由SEQ ID NO 1712:UCAUCUUGGUCUCUU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1712:UCAUCUUGGUCUCUU;

由SEQ ID NO 1713:AGUCAUAUUGCCAGG所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1713:AGUCAUAUUGCCAGG;

由SEQ ID NO 1714:ACAAGAAAGUGCCCA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1714: ACAAGAAAGUGCCCA;

由SEQ ID NO 1715:AGUCUCAGACAAGAA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1715:AGUCUCAGACAAGAA;

由SEQ ID NO 1716:UAAGCCAGAGUCUCA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1716:UAAGCCAGAGUCUCA;

由SEQ ID NO 1717:UUACCCAUCUCCCAA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1717: UUACCCAUCUCCCAA;

由SEQ ID NO 1718:AACGCUUUACCCAUC所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1718:AACGCUUUACCCAUC;

由SEQ ID NO 1719:UAGAAGAAACGCUUU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1719:UAGAAGAAACGCUUU;

由SEQ ID NO 1720:AGACCCCUUUAGAAG所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1720:AGACCCCUUUAGAAG;

由SEQ ID NO 1721:CAUGCUUUCUGGGUA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1721: CAUGCUUUCUGGGUA;

由SEQ ID NO 1722:CUUCUCACAGGACUU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1722:CUUCUCACAGGACUU;

由SEQ ID NO 1723:UUUUUCUCUGCCUUC所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1723: UUUUUCUCUGCCUUC;

由SEQ ID NO 1724:AGUAAUUUUUCUCUG所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1724:AGUAAUUUUUUCUCUG;

由SEQ ID NO 1725:UUUCUCAUCUUGGGA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1725: UUUCUCAUCUUGGGA;

由SEQ ID NO 1726:UCUAUCUGCUUCCUC所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1726:UCUAUCUGCUUCCUC;

由SEQ ID NO 1727:AAGGUACCUCAACAG所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1727: AAGGUACCUCAACAG;

由SEQ ID NO 1728:UCCCUUAAGGUACCU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1728: UCCCUUAAGGUACCU;

由SEQ ID NO 1729:UUUUCCUGACUCCUG所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1729:UUUUCCUGACUCCUG;

由SEQ ID NO 1730:AAAUGCUUGUCUCCC所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1730:AAAUGCUUGUCUCCC;

由SEQ ID NO 1731:AGUAUAAAUGCUUGU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1731:AGUAUAAAUGCUUGU;

由SEQ ID NO 1732:AAGAAAGUAUAAAUG所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1732: AAGAAAGUAUAAAUG;

由SEQ ID NO 1733:UUGUUGAUUUUCUUU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1733: UUGUUGAUUUUCUUU;

由SEQ ID NO 1734:UAUGACUCACAUUUG所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1734:UAUGACUCACAUUUG;

由SEQ ID NO 1735:ACCCUUCUUUAUGAC所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1735:ACCCUUCUUUAUGAC;

由SEQ ID NO 1736:AAGAACUGUUGCUCU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1736:AAGAACUGUUGCUCU;

由SEQ ID NO 1737:ACUUUGUGGACACCC所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1737: ACUUUGUGGACACCC;

由SEQ ID NO 1738:CUUUGACUUUGUGGA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1738:CUUUGACUUUGUGGA;

由SEQ ID NO 1739:AAUAGCUUUGACUUU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1739: AAUAGCUUUGACUUU;

由SEQ ID NO 1740:UAUGAAAAUAGCUUU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1740:UAUGAAAAUAGCUUU;

由SEQ ID NO 1741:UAUUAUUAUGAAAAU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1741: UAUUAUUAUGAAAAU;

由SEQ ID NO 1742:ACAUGUUAGUAUUAU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1742: ACAUGUUAGUAUUAU;

由SEQ ID NO 1743:AAAUAACAUGUUAGU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1743:AAAUAACAUGUUUAGU;

由SEQ ID NO 1744:AAAGGCAAAUAACAU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1744:AAAGGCAAAUAACAU;

由SEQ ID NO 1745:AUUCAAAAGGCAAAU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1745:AUUCAAAAGGCAAAU;

由SEQ ID NO 1746:AAUGAGAAUUCAAAA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1746: AAUGAGAAUUCAAAA;

由SEQ ID NO 1747:UUAAGAUAAUGAGAA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1747: UUAAGAUAAUGAGAA;

由SEQ ID NO 1748:ACAAUUUUAAGAUAA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1748: ACAAUUUUUAAGAUAA;

由SEQ ID NO 1749:AAUCACAUGUCACAC所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1749: AAUCAAUGUCACAC;

由SEQ ID NO 1750:AUGAUGUAAUCACAU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1750:AUGAUGUAAUCACAU;

由SEQ ID NO 1751:AGAAAGAUGAUGUAA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1751:AGAAAGAUGAUGUAA;

由SEQ ID NO 1752:UGAUGUCAGAAAGAU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1752: UGAUGUCAGAAAGAU;

由SEQ ID NO 1753:AACAAUGAUGUCAGA所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1753:AACAAUGAUGUCAGA;

由SEQ ID NO 1754:ACACAUUCCAUUAAC所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1754:ACACAUUCCAUUAAC;

由SEQ ID NO 1755:ACAGUAGCAAAGCUG所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1755:ACAGUAGCAAAGCUG;

由SEQ ID NO 1756:AAUAAGCCAGAGUCU所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1756:AAUAAGCCAGAGUCU;

由SEQ ID NO 1757:UUGAAGAACUGUUGC所示的连续15个核苷酸；15 consecutive nucleotides represented by SEQ ID NO 1757:UUGAAGAACUGUUGC;

由SEQ ID NO 1758:UAUGAAAAUAGCUUU所示的连续15个核苷酸。15 consecutive nucleotides shown by SEQ ID NO 1758:UAUGAAAAUAGCUUU.

在一些实施方式中，本公开的正义链源自人INHBE的mRNA序列(如Gene ID:83729，NM_031479.5(SEQ ID NO:1619))。可替代地，本公开的正义链是来自人INHBE的mRNA序列的片段。在一些实施方式中，本公开的正义链源自食蟹猴INHBE的mRNA序列(如Gene ID:102127493，XM_005571319.3(SEQ ID NO:1620))。可替代地，本公开的正义链是来自食蟹猴INHBE的mRNA序列的片段。In some embodiments, the sense strand of the present disclosure is derived from the mRNA sequence of human INHBE (such as Gene ID: 83729, NM_031479.5 (SEQ ID NO: 1619)). Alternatively, the sense strand of the present disclosure is a fragment of the mRNA sequence from human INHBE. In some embodiments, the sense strand of the present disclosure is derived from the mRNA sequence of cynomolgus monkey INHBE (such as Gene ID: 102127493, XM_005571319.3 (SEQ ID NO: 1620)). Alternatively, the sense strand of the present disclosure is a fragment of the mRNA sequence from cynomolgus monkey INHBE.

在一些实施方式中，所述dsRNA包含表1、表2a或表2b中的任一个反义链或正义链序列。在一些实施方式中，所述dsRNA包含如SEQ ID NO：1-1590所示的任一正义链或反义链。在一些实施方式中，所述dsRNA包含如SEQ ID NO：1759-1874所示的任一正义链或反义链。In some embodiments, the dsRNA comprises any one of the antisense or sense strand sequences in Table 1, Table 2a, or Table 2b. In some embodiments, the dsRNA comprises any one of the sense or antisense strands as shown in SEQ ID NO: 1-1590. In some embodiments, the dsRNA comprises any one of the sense or antisense strands as shown in SEQ ID NO: 1759-1874.

在一些实施方式中，所述dsRNA包含表1、表2a或表2b中的双链体序列所示的反义链序列和正义链序列。在一些实施方式中，所述dsRNA包含任一选自IN-001至IN-488和INI-001至INI-308的双链体。在一些实施方式中，所述dsRNA包含任一选自IN-489至IN-546的双链体。In some embodiments, the dsRNA comprises an antisense strand sequence and a sense strand sequence as shown in the duplex sequence in Table 1, Table 2a or Table 2b. In some embodiments, the dsRNA comprises any duplex selected from IN-001 to IN-488 and INI-001 to INI-308. In some embodiments, the dsRNA comprises any duplex selected from IN-489 to IN-546.

在一些实施方式中，所述正义链和/或所述反义链中的至少一个核苷酸为经修饰的核苷酸。在一些此类实施方式中，所述修饰核苷酸是非规范碱基配对核苷酸。In some embodiments, at least one nucleotide in the sense strand and/or the antisense strand is a modified nucleotide. In some such embodiments, the modified nucleotide is a non-canonical base pairing nucleotide.

在一些实施方式中，所述正义链和/或所述反义链中的基本所有的核苷酸为经修饰的核苷酸。在一些此类实施方式中，所述修饰核苷酸中的至少一个是非规范碱基配对核苷酸。In some embodiments, substantially all nucleotides in the sense strand and/or the antisense strand are modified nucleotides. In some such embodiments, at least one of the modified nucleotides is a non-canonical base pairing nucleotide.

在一些实施方式中，所述正义链和/或所述反义链中的所有的核苷酸为经修饰的核苷酸。在一些此类实施方式中，所述修饰核苷酸中的至少一个是非规范碱基配对核苷酸。In some embodiments, all nucleotides in the sense strand and/or the antisense strand are modified nucleotides. In some such embodiments, at least one of the modified nucleotides is a non-canonical base pairing nucleotide.

在一些实施方式中，本公开所述经修饰的核苷酸选自：2’-O-甲基修饰核苷酸、2’-氟修饰核苷酸、2’-脱氧核苷酸、2’-甲氧基乙基修饰核苷酸、2’-氨基修饰核苷酸、2’-烷基修饰核苷酸、2’-烷氧基修饰核苷酸、2’-F-阿糖核苷酸、硫代磷酸酯修饰核苷酸、脱碱基核苷酸、吗啉代核苷酸、锁定核苷酸(locked nucleic acid)、倒置核苷酸和次黄嘌呤碱基置换核苷酸。In some embodiments, the modified nucleotides disclosed herein are selected from: 2’-O-methyl modified nucleotides, 2’-fluoro modified nucleotides, 2’-deoxy nucleotides, 2’-methoxyethyl modified nucleotides, 2’-amino modified nucleotides, 2’-alkyl modified nucleotides, 2’-alkoxy modified nucleotides, 2’-F-arabino nucleotides, phosphorothioate modified nucleotides, abasic nucleotides, morpholino nucleotides, locked nucleotides (locked nucleic acid), inverted nucleotides and inosine base substituted nucleotides.

在一些实施方式中，本公开所述经修饰的核苷酸选自：2’-O-甲基修饰核苷酸、2’-氟修饰核苷酸、2’-脱氧核苷酸、硫代磷酸酯修饰核苷酸和倒置核苷酸。在一些优选实施方式中，倒置核苷酸选自：倒置A核苷酸、倒置dA核苷酸、倒置dT核苷酸、倒置C核苷酸和倒置U核苷酸。In some embodiments, the modified nucleotides of the present disclosure are selected from: 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxy nucleotides, phosphorothioate modified nucleotides and inverted nucleotides. In some preferred embodiments, the inverted nucleotides are selected from: inverted A nucleotides, inverted dA nucleotides, inverted dT nucleotides, inverted C nucleotides and inverted U nucleotides.

在一些实施方式中，本公开所述修饰的核苷酸包括以下任一种或其组合：In some embodiments, the modified nucleotides disclosed herein include any one or a combination of the following:

(1)按照5’端到3’端的方向，所述反义链第2、4、12、14位的核苷酸为2’-氟修饰的核苷酸，其余位置的核苷酸为2’-O-甲基修饰的核苷酸；(1) From the 5' end to the 3' end, the nucleotides at positions 2, 4, 12, and 14 of the antisense strand are 2'-fluorine-modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl-modified nucleotides;

(2)按照5’端到3’端的方向，所述正义链第7、8、9位的核苷酸为2’-氟修饰的核苷酸；(2) From the 5' end to the 3' end, the nucleotides at positions 7, 8, and 9 of the sense strand are 2'-fluorinated nucleotides;

(3)按照5’端到3’端的方向，所述反义链第2-8位中的鸟嘌呤(G中的碱基)替换为次黄嘌呤(肌苷或肌苷酸I的碱基)。(3) From the 5' end to the 3' end, the guanine (the base in G) in positions 2-8 of the antisense strand is replaced with hypoxanthine (the base of inosine or inosinic acid I).

在一些此类实施方式中，所述反义链第2-8位中的次黄嘌呤替换的修饰核苷酸进一步满足下述特征中的一种或其组合：In some such embodiments, the modified nucleotides substituted with hypoxanthine at positions 2-8 of the antisense strand are further The step meets one or a combination of the following characteristics:

(i)按照5’端到3’端的方向，所述反义链第6-8位中的鸟嘌呤替换为次黄嘌呤(I)；(i) from the 5' end to the 3' end, the guanine in positions 6-8 of the antisense strand is replaced by hypoxanthine (I);

(ii)按照5’端到3’端的方向，所述反义链的序列片段N₁N₂GN₃N₄中的鸟嘌呤替换为次黄嘌呤(I)，条件是N₁、N₂、N₃和N₄中的至少三个碱基是腺嘌呤或尿嘧啶；(ii) in the direction from the 5' end to the 3' end, the guanine in the sequence_{fragmentN1N2N3N4} of the antisense strand_is replaced by hypoxanthine (I), provided that at least three bases among_N1 ,_N2 ,_N3 and_N4 are adenine or uracil_;

(iii)按照5’端到3’端的方向，所述反义链第2-8位中的腺嘌呤和尿嘧啶的数量在4个以上；和/或，(iii) the number of adenine and uracil in positions 2 to 8 of the antisense strand is greater than 4 in the direction from the 5' end to the 3' end; and/or

(iv)所述反义链中的至少一个核苷酸中的碱基鸟嘌呤替换为次黄嘌呤，其中该次黄嘌呤的替换使得包含该反义链的dsRNA与相应的未经次黄嘌呤替换的dsRNA之间的解离温度存在至少2℃、2℃或大于2℃的差异。(iv) the base guanine in at least one nucleotide in the antisense strand is replaced by hypoxanthine, wherein the replacement of hypoxanthine results in a difference of at least 2°C, 2°C or greater than 2°C in the dissociation temperature between the dsRNA comprising the antisense strand and the corresponding dsRNA without hypoxanthine replacement.

在一些实施方式中，所述反义链包含选自SEQ ID NO：86、182、212、350、376、402、440、460、462、464、466、468、470、472、474、476、478、480、482、484、486、488、490、492、494、496、498、500、502、504、506、508、510、512、514、516、518、520、522、524、526、528、530、532、534、536、538、540、542、544、546、548、550、552、554、556、558、560、562、564、566、568、570、572、574、576、578、580、582、584、586、588、590、592、594、596、598、600、602、604、606、608、610、612、614、616、618、620、622、624、626、628、630、632、634、636、638、640、642、644、646、648、650、652、654、656、658、660、662、664、666、668、670、672、674、676、678、680、682、684、686、688、690、692、694、696、698、700、702、704、706、708、710、712、714、716、718、720、722、724、726、728、730、732、734、736、738、740、742、744、746、748、750、752、754、756、758、760、762、764、766、768、770、772、774、776、778、780、782、784、786、788、790、792、794、796、798、800、802、804、806、808、810、812、814、816、818、820、822、824、826、828、830、832、834、836、838、840、842、844、846、848、850、852、854、856、858、860、862、864、866、868、870、872、874、876、878、880、882、884、886、888、890、892、894、896、898、900、902、904、906、908、910、912、914、916、918、920、922、924、926、928、930、932、934、936、938、940、942、944、946、948、950、952、954、956、958、960、962、964、966、968、970、972、974、1760、1762、1764、1766、1768、1770、1772、1774、1776、1778、1780、1782、1784、1786、1788、1790、1792、1794、1796、1798、1800、1802、1804、1806、1808、1810、1812、1814、1816、1818、1820、1822、1824、1826、1828、1830、1832、1834、1836、1838、1840、1842、1844、1846、1848、1850、1852、1854、1856、1858、1860、1862、1864、1866、1868、1870、1872或者1874的完全连续的核苷酸。In some embodiments, the antisense strand comprises a member selected from the group consisting of SEQ ID NOs: 86, 182, 212, 350, 376, 402, 440, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 517, 8. 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604 ,606,608,610,612,614,616,618,620,622,624,626,628,630,632,634,636,638,640,642,644,646,6 48, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 676, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706, 708, 710, 712, 714, 716, 718, 720, 722, 724, 726, 728, 730, 732, 734 ,736,738,740,742,744,746,748,750,752,754,756,758,760,762,764,766,768,770,772,774,776,77 8. 780, 782, 784, 786, 788, 790, 792, 794, 796, 798, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826, 828, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850, 852, 854, 856, 858, 860, 862, 864 ,866,868,870,872,874,876,878,880,882,884,886,888,890,892,894,896,898,900,902,904,906,90 8. 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 9 52, 954, 956, 958, 960, 962, 964, 966, 968, 970, 972, 974, 1760, 1762, 1764, 1766, 1768, 1770, 1772, 1774 ,1776,1778,1780,1782,1784,1786,1788,1790,1792,1794,1796,1798,1800,1802,1804,1806,1808,1 810, 1812, 1814, 1816, 1818, 1820, 1822, 1824, 1826, 1828, 1830, 1832, 1834, 1836, 1838, 1840, 1842, 1844, 1846, 1848, 1850, 1852, 1854, 1856, 1858, 1860, 1862, 1864, 1866, 1868, 1870, 1872 or 1874 of completely consecutive nucleotides.

在一些实施方式中，所述正义链包含选自SEQ ID NO：85、181、211、349、375、401、439、459、461、463、465、467、469、471、473、475、477、479、481、483、485、487、489、491、493、495、497、499、501、503、505、507、509、511、513、515、517、519、521、523、525、527、529、531、533、535、537、539、541、543、545、547、549、551、553、555、557、559、561、563、565、567、569、571、573、575、577、579、581、583、585、587、589、591、593、595、597、599、601、603、605、607、609、611、613、615、617、619、621、623、625、627、629、631、633、635、637、639、641、643、645、647、649、651、653、655、657、659、661、663、665、667、669、671、673、675、677、679、681、683、685、687、689、691、693、695、697、699、701、703、705、707、709、711、713、715、717、719、721、723、725、727、729、731、733、735、737、739、741、743、745、747、749、751、753、755、757、759、761、763、765、767、769、771、773、775、777、779、781、783、785、787、789、791、793、795、797、799、801、803、805、807、809、811、813、815、817、819、821、823、825、827、829、831、833、835、837、839、841、843、845、847、849、851、853、855、857、859、861、863、865、867、869、871、873、875、877、879、881、883、885、887、889、891、893、895、897、899、901、903、905、907、909、911、913、915、917、919、921、923、925、927、929、931、933、935、937、939、941、943、945、947、949、951、953、955、957、959、961、963、965、967、969、971、973、1759、1761、1763、1765、1767、1769、1771、1773、1775、1777、1779、1781、1783、1785、1787、1789、1791、1793、1795、1797、1799、1801、1803、1805、1807、1809、1811、1813、1815、1817、1819、1821、1823、1825、1827、1829、1831、1833、1835、1837、1839、1841、1843、1845、1847、1849、1851、1853、1855、1857、1859、1861、1863、1865、1867、1869、1871或者1873的完全连续的核苷酸。In some embodiments, the sense strand comprises a polypeptide selected from the group consisting of SEQ ID NOs: 85, 181, 211, 349, 375, 401, 439, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 520, 521, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548 21, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559 ,561,563,565,567,569,571,573,575,577,579,581,583,585,587,589,591,593,595,597,5 99, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 63 7. 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699, 701, 703, 705, 707, 709, 711, 713, 71 5. 717, 719, 721, 723, 725, 727, 729, 731, 733, 735, 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 771, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, 827, 829, 831, 833, 835, 837, 839, 841, 843, 845, 8 47, 849, 851, 853, 855, 857, 859, 861, 863, 865, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 8 93, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 93 9, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 1759, 1761, 1763, 1765, 17 67, 1769, 1771, 1773, 1775, 1777, 1779, 1781, 1783, 1785, 1787, 1789, 1791, 1793, 1795, 1797, 1799, 1801, 1803, 1805, 1807, 1809, 1811, 1813, 1815, 1817, 1819, 1821, 1823, 1825, 1827, 1829, 1831, 1833, 1835, 1837, 1839, 1841, 1843, 1845, 1847, 1849, 1851, 1853, 1855, 1857, 1859, 1861, 1863, 1865, 1867, 1869, 1871 or 1873 of completely consecutive nucleotides.

在一些实施方式中，所述dsRNA包含选自如下的正义链序列和反义链序列：SEQ ID NO：85和86；SEQ ID NO：181和182；SEQ ID NO：211和212；SEQ ID NO：349和350；SEQ ID NO：375和376；SEQ ID NO：401和402；SEQ ID NO：439和440；SEQ ID NO:459和460；SEQ ID NO:461和462；SEQ ID NO:463和464；SEQ ID NO:465和466；SEQ ID NO:467和468；SEQ ID NO:469和470；SEQ ID NO:471和472；SEQ ID NO:473和474；SEQ ID NO:475和476；SEQ ID NO:477和478；SEQ ID NO:479和480；SEQ ID NO:481和482；SEQ ID NO:483和484；SEQ ID NO:485和486；SEQ ID NO:487和488；SEQ ID NO:489和490；SEQ ID NO:491和492；SEQ ID NO:493和494；SEQ ID NO:495和496；SEQ ID NO:497和498；SEQ ID NO:499和500；SEQ ID NO：501和502；SEQ ID NO：503和504；SEQ ID NO：505和506；SEQ ID NO：507和508；SEQ ID NO：509和510；SEQ ID NO：511和512；SEQ ID NO：513和514；SEQ ID NO：515和516；SEQ ID NO：517和518；SEQ ID NO：519和520；SEQ ID NO：521和522；SEQ ID NO：523和524；SEQ ID NO：525和526；SEQ ID NO：527和528；SEQ ID NO：529和530；SEQ ID NO：531和532；SEQ ID NO：533和534；SEQ ID NO：535和536；SEQ ID NO：537和538；SEQ ID NO：539和540；SEQ ID NO：541和542；SEQ ID NO：543和544；SEQ ID NO：545和546；SEQ ID NO：547和548；SEQ ID NO：549和550；SEQ ID NO：551和552；SEQ ID NO：553和554；SEQ ID NO：555和556；SEQ ID NO：557和558；SEQ ID NO：559和560；SEQ ID NO：561和562；SEQ ID NO：563和564；SEQ ID NO：565和566；SEQ ID NO：567和568；SEQ ID NO：569和570；SEQ ID NO：571和572；SEQ ID NO：573和574；SEQ ID NO：575和576；SEQ ID NO：577和578；SEQ ID NO：579和580；SEQ ID NO：581和582；SEQ ID NO：583和584；SEQ ID NO：585和586；SEQ ID NO：587和588；SEQ ID NO：589和590；SEQ ID NO：591和592；SEQ ID NO：593和594；SEQ ID NO：595和596；SEQ ID NO：597和598；SEQ ID NO：599和600；SEQ ID NO：601和602；SEQ ID NO：603和604；SEQ ID NO：605和606；SEQ ID NO：607和608；SEQ ID NO：609和610；SEQ ID NO：611和612；SEQ ID NO：613和614；SEQ ID NO：615和616；SEQ ID NO：617和618；SEQ ID NO：619和620；SEQ ID NO：621和622；SEQ ID NO：623和624；SEQ ID NO：625和626；SEQ ID NO：627和628；SEQ ID NO：629和630；SEQ ID NO：631和632；SEQ ID NO：633和634；SEQ ID NO：635和636；SEQ ID NO：637和638；SEQ ID NO：639和640；SEQ ID NO：641和642；SEQ ID NO：643和644；SEQ ID NO：645和646；SEQ ID NO：647和648；SEQ ID NO：649和650；SEQ ID NO：651和652；SEQ ID NO：653和654；SEQ ID NO：655和656；SEQ ID NO：657和658；SEQ ID NO：659和660；SEQ ID NO：661和662；SEQ ID NO：663和664；SEQ ID NO：665和666；SEQ ID NO：667和668；SEQ ID NO：669和670；SEQ ID NO：671和672；SEQ ID NO：673和674；SEQ ID NO：675和676；SEQ ID NO：677和678；SEQ ID NO：679和680；SEQ ID NO：681和682；SEQ ID NO：683和684；SEQ ID NO：685和686；SEQ ID NO：687和688；SEQ ID NO：689和690；SEQ ID NO：691和692；SEQ ID NO：693和694；SEQ ID NO：695和696；SEQ ID NO：697和698；SEQ ID NO：699和700；SEQ ID NO：701和702；SEQ ID NO：703和704；SEQ ID NO：705和706；SEQ ID NO：707和708；SEQ ID NO：709和710；SEQ ID NO：711和712；SEQ ID NO：713和714；SEQ ID NO：715和716；SEQ ID NO：717和718；SEQ ID NO：719和720；SEQ ID NO：721和722；SEQ ID NO：723和724；SEQ ID NO：725和726；SEQ ID NO：727和728；SEQ ID NO：729和730；SEQ ID NO：731和732；SEQ ID NO：733和734；SEQ ID NO：735和736；SEQ ID NO：737和738；SEQ ID NO：739和740；SEQ ID NO：741和742；SEQ ID NO：743和744；SEQ ID NO：745和746；SEQ ID NO：747和748；SEQ ID NO：749和750；SEQ ID NO：751和752；SEQ ID NO：753和754；SEQ ID NO：755和756；SEQ ID NO：757和758；SEQ ID NO：759和760；SEQ ID NO：761和762；SEQ ID NO：763和764；SEQ ID NO：765和766；SEQ ID NO：767和768；SEQ ID NO：769和770；SEQ ID NO：771和772；SEQ ID NO：773和774；SEQ ID NO：775和776；SEQ ID NO：777和778；SEQ ID NO：779和780；SEQ ID NO：781和782；SEQ ID NO：783和784；SEQ ID NO：785和786；SEQ ID NO：787和788；SEQ ID NO：789和790；SEQ ID NO：791和792；SEQ ID NO：793和794；SEQ ID NO：795和796；SEQ ID NO：797和798；SEQ ID NO：799和800；SEQ ID NO：801和802；SEQ ID NO：803和804；SEQ ID NO：805和806；SEQ ID NO：807和808；SEQ ID NO：809和810；SEQ ID NO：811和812；SEQ ID NO：813和814；SEQ ID NO：815和816；SEQ ID NO：817和818；SEQ ID NO：819和820；SEQ ID NO：821和822；SEQ ID NO：823和824；SEQ ID NO：825和826；SEQ ID NO：827和828；SEQ ID NO：829和830；SEQ ID NO：831和832；SEQ ID NO：833和834；SEQ ID NO：835和836；SEQ ID NO：837和838；SEQ ID NO：839和840；SEQ ID NO：841和842；SEQ ID NO：843和844；SEQ ID NO：845和846；SEQ ID NO：847和848；SEQ ID NO：849和850；SEQ ID NO：851和852；SEQ ID NO：853和854；SEQ ID NO：855和856；SEQ ID NO：857和858；SEQ ID NO:859和860；SEQ ID NO:861和862；SEQ ID NO:863和864；SEQ ID NO:865和866；SEQ ID NO:867和868；SEQ ID NO:869和870；SEQ ID NO:871和872；SEQ ID NO:873和874；SEQ ID NO:875和876；SEQ ID NO:877和878；SEQ ID NO:879和880；SEQ ID NO:881和882；SEQ ID NO:883和884；SEQ ID NO:885和886；SEQ ID NO:887和888；SEQ ID NO:889和890；SEQ ID NO:891和892；SEQ ID NO:893和894；SEQ ID NO:895和896；SEQ ID NO:897和898；SEQ ID NO:899和900；SEQ ID NO:901和902；SEQ ID NO:903和904；SEQ ID NO:905和906；SEQ ID NO:907和908；SEQ ID NO:909和910；SEQ ID NO:911和912；SEQ ID NO:913和914；SEQ ID NO:915和916；SEQ ID NO:917和918；SEQ ID NO:919和920；SEQ ID NO:921和922；SEQ ID NO:923和924；SEQ ID NO:925和926；SEQ ID NO:927和928；SEQ ID NO:929和930；SEQ ID NO:931和932；SEQ ID NO:933和934；SEQ ID NO:935和936；SEQ ID NO:937和938；SEQ ID NO:939和940；SEQ ID NO:941和942；SEQ ID NO:943和944；SEQ ID NO:945和946；SEQ ID NO:947和948；SEQ ID NO:949和950；SEQ ID NO:951和952；SEQ ID NO:953和954；SEQ ID NO:955和956；SEQ ID NO:957和958；SEQ ID NO:959和960；SEQ ID NO:961和962；SEQ ID NO:963和964；SEQ ID NO:965和966；SEQ ID NO:967和968；SEQ ID NO:969和970；SEQ ID NO:971和972；SEQ ID NO:973和974；SEQ ID NO:1759和1760；SEQ ID NO:1761和1762；SEQ ID NO:1763和1764；SEQ ID NO:1765和1766；SEQ ID NO:1767和1768；SEQ ID NO:1769和1770；SEQ ID NO:1771和1772；SEQ ID NO:1773和1774；SEQ ID NO:1775和1776；SEQ ID NO:1777和1778；SEQ ID NO:1779和1780；SEQ ID NO:1781和1782；SEQ ID NO:1783和1784；SEQ ID NO:1785和1786；SEQ ID NO:1787和1788；SEQ ID NO:1789和1790；SEQ ID NO:1791和1792；SEQ ID NO:1793和1794；SEQ ID NO:1795和1796；SEQ ID NO:1797和1798；SEQ ID NO:1799和1800；SEQ ID NO:1801和1802；SEQ ID NO:1803和1804；SEQ ID NO:1805和1806；SEQ ID NO:1807和1808；SEQ ID NO:1809和1810；SEQ ID NO:1811和1812；SEQ ID NO:1813和1814；SEQ ID NO:1815和1816；SEQ ID NO:1817和1818；SEQ ID NO:1819和1820；SEQ ID NO:1821和1822；SEQ ID NO:1823和1824；SEQ ID NO:1825和1826；SEQ ID NO:1827和1828；SEQ ID NO:1829和1830；SEQ ID NO:1831和1832；SEQ ID NO:1833和1834；SEQ ID NO:1835和1836；SEQ ID NO:1837和1838；SEQ ID NO:1839和1840；SEQ ID NO:1841和1842；SEQ ID NO:1843和1844；SEQ ID NO:1845和1846；SEQ ID NO:1847和1848；SEQ ID NO:1849和1850；SEQ ID NO:1851和1852；SEQ ID NO:1853和1854；SEQ ID NO:1855和1856；SEQ ID NO:1857和1858；SEQ ID NO:1859和1860；SEQ ID NO:1861和1862；SEQ ID NO:1863和1864；SEQ ID NO:1865和1866；SEQ ID NO:1867和1868；SEQ ID NO:1869和1870；SEQ ID NO:1871和1872；或者，SEQ ID NO:1873和1874。In some embodiments, the dsRNA comprises a sense strand sequence and an antisense strand sequence selected from the group consisting of SEQ ID NOs: 85 and 86; SEQ ID NOs: 181 and 182; SEQ ID NOs: 211 and 212; SEQ ID NOs: 349 and 350; SEQ ID NOs: 375 and 376; SEQ ID NOs: 401 and 402; SEQ ID NOs: 439 and 440; SEQ ID NOs: 459 and 460; SEQ ID NOs: 461 and 462; SEQ ID NOs: 463 and 464; SEQ ID NOs: 465 and 466; SEQ ID NOs: 467 and 468; SEQ ID NOs: 469 and 470; SEQ ID NOs: 471 and 472; SEQ ID NOs: 473 and 474; SEQ ID NOs: 475 and 476; SEQ ID NOs: 477 and 478; SEQ ID NOs: 479 and 480; SEQ ID NOs: 481 and 482; SEQ ID NOs: 483 and 484; SEQ ID NOs: 485 and 486; SEQ ID NOs: 487 and 488; SEQ ID NOs: 489 and 490; SEQ ID NOs:481 and 482; SEQ ID NOs:483 and 484; SEQ ID NOs:485 and 486; SEQ ID NOs:487 and 488; SEQ ID NOs:489 and 490; SEQ ID NOs:491 and 492; SEQ ID NOs:493 and 494; SEQ ID NOs:495 and 496; SEQ ID NOs:497 and 498; SEQ ID NOs:499 and 500; SEQ ID NOs:501 and 502; SEQ ID NOs:503 and 504; SEQ ID NOs:505 and 506; SEQ ID NOs:507 and 508; SEQ ID NOs:509 and 510; SEQ ID NOs:511 and 512; SEQ ID NOs:513 and 514; SEQ ID NOs:515 and 516; SEQ ID NOs:517 and 518; SEQ ID NOs:519 and 520; SEQ ID NOs:521 and 522; SEQ ID NOs: 521 and 522; SEQ ID NOs: 523 and 524; SEQ ID NOs: 525 and 526; SEQ ID NOs: 527 and 528; SEQ ID NOs: 529 and 530; SEQ ID NOs: 531 and 532; SEQ ID NOs: 533 and 534; SEQ ID NOs: 535 and 536; SEQ ID NOs: 537 and 538; SEQ ID NOs: 539 and 540; SEQ ID NOs: 541 and 542; SEQ ID NOs: 543 and 544; SEQ ID NOs: 545 and 546; SEQ ID NOs: 547 and 548; SEQ ID NOs: 549 and 550; SEQ ID NOs: 551 and 552; SEQ ID NOs: 553 and 554; SEQ ID NOs: 555 and 556; SEQ ID NOs: 557 and 558; SEQ ID NOs: 559 and 560; SEQ ID NOs: 561 and 562; SEQ ID NOs: 563 and 564; SEQ ID NOs: 565 and 566; SEQ ID NOs: 567 and 568; SEQ ID NOs: 569 and 570; SEQ ID NOs: 561 and 562; SEQ ID NOs: 563 and 564; SEQ ID NOs: 565 and 566; SEQ ID NOs: 567 and 568; SEQ ID NOs: 569 and 570; SEQ ID NOs: 571 and 572; SEQ ID NOs: 573 and 574; SEQ ID NOs: 575 and 576; SEQ ID NOs: 577 and 578; SEQ ID NOs: 579 and 580; SEQ ID NOs: 581 and 582; SEQ ID NOs: 583 and 584; SEQ ID NOs: 585 and 586; SEQ ID NOs: 587 and 588; SEQ ID NOs: 589 and 590; SEQ ID NOs: 591 and 592; SEQ ID NOs: 593 and 594; SEQ ID NOs: 595 and 596; SEQ ID NOs: 597 and 598; SEQ ID NOs: 599 and 600; SEQ ID NOs: 601 and 602; SEQ ID NOs: 601 and 602; SEQ ID NOs: 603 and 604; SEQ ID NOs: 605 and 606; SEQ ID NOs: 607 and 608; SEQ ID NOs: 609 and 610; SEQ ID NOs: 611 and 612; SEQ ID NOs: 613 and 614; SEQ ID NOs: 615 and 616; SEQ ID NOs: 617 and 618; SEQ ID NOs: 619 and 620; SEQ ID NOs: 621 and 622; SEQ ID NOs: 623 and 624; SEQ ID NOs: 625 and 626; SEQ ID NOs: 627 and 628; SEQ ID NOs: 629 and 630; SEQ ID NOs: 631 and 632; SEQ ID NOs: 633 and 634; SEQ ID NOs: 635 and 636; SEQ ID NOs: 637 and 638; SEQ ID NOs: 639 and 640; SEQ ID NOs: 641 and 642; SEQ ID NO: 641 and 642; SEQ ID NO: 643 and 644; SEQ ID NO: 645 and 646; SEQ ID NO: 647 and 648; SEQ ID NO: 649 and 650; SEQ ID NO: 651 and 652; SEQ ID NO: 653 and 654; SEQ ID NO: SEQ ID NOs: 651 and 652; SEQ ID NOs: 653 and 654; SEQ ID NOs: 655 and 656; SEQ ID NOs: 657 and 658; SEQ ID NOs: 659 and 660; SEQ ID NOs: 661 and 662; SEQ ID NOs: 663 and 664; SEQ ID NOs: 665 and 666; SEQ ID NOs: 667 and 668; SEQ ID NOs: 669 and 670; SEQ ID NOs: 671 and 672; SEQ ID NOs: 673 and 674; SEQ ID NOs: 675 and 676; SEQ ID NOs: 677 and 678; SEQ ID NOs: 679 and 680; SEQ ID NOs: 681 and 682; SEQ ID NOs: 683 and 684; SEQ ID NOs: 685 and 686; SEQ ID NOs: 687 and 688; SEQ ID NOs: 689 and 690; SEQ ID NOs: 691 and 692; SEQ ID NOs: 693 and 694; SEQ ID NOs: SEQ ID NOs: 695 and 696; SEQ ID NOs: 697 and 698; SEQ ID NOs: 699 and 700; SEQ ID NOs: 701 and 702; SEQ ID NOs: 703 and 704; SEQ ID NOs: 705 and 706; SEQ ID NOs: 707 and 708; SEQ ID NOs: 709 and 710; SEQ ID NOs: 711 and 712; SEQ ID NOs: 713 and 714; SEQ ID NOs: 715 and 716; SEQ ID NOs: 717 and 718; SEQ ID NOs: 719 and 720; SEQ ID NOs: 721 and 722; SEQ ID NOs: 723 and 724; SEQ ID NOs: 725 and 726; SEQ ID NOs: 727 and 728; SEQ ID NOs: 729 and 730; SEQ ID NOs: 731 and 732; SEQ ID NOs: 733 and 734; SEQ ID NOs: 735 and 736; SEQ ID NOs: 735 and 736; SEQ ID NOs: 737 and 738; SEQ ID NOs: 739 and 740; SEQ ID NOs: 741 and 742; SEQ ID NOs: 743 and 744; SEQ ID NOs: 745 and 746; SEQ ID NOs: 747 and 748; SEQ ID NOs: 749 and 750; SEQ ID NOs: 751 and 752; SEQ ID NOs: 753 and 754; SEQ ID NOs: 755 and 756; SEQ ID NOs: 757 and 758; SEQ ID NOs: 759 and 760; SEQ ID NOs: 761 and 762; SEQ ID NOs: 763 and 764; SEQ ID NOs: 765 and 766; SEQ ID NOs: 767 and 768; SEQ ID NOs: 769 and 770; SEQ ID NOs: 771 and 772; SEQ ID NOs: 773 and 774; SEQ ID NOs: 775 and 776; SEQ ID NOs: 775 and 776; SEQ ID NOs: 777 and 778; SEQ ID NOs: 779 and 780; SEQ ID NOs: 781 and 782; SEQ ID NOs: 783 and 784; SEQ ID NOs: 785 and 786; SEQ ID NOs: 787 and 788; SEQ ID NOs: 789 and 790; SEQ ID NOs: 791 and 792; SEQ ID NOs: 793 and 794; SEQ ID NOs: 795 and 796; SEQ ID NOs: 797 and 798; SEQ ID NOs: 799 and 800; SEQ ID NOs: 801 and 802; SEQ ID NOs: 803 and 804; SEQ ID NOs: 805 and 806; SEQ ID NOs: 807 and 808; SEQ ID NOs: 809 and 810; SEQ ID NOs: 811 and 812; SEQ ID NOs: 813 and 814; SEQ ID NOs: SEQ ID NOs: 815 and 816; SEQ ID NOs: 817 and 818; SEQ ID NOs: 819 and 820; SEQ ID NOs: 821 and 822; SEQ ID NOs: 823 and 824; SEQ ID NOs: 825 and 826; SEQ ID NOs: 827 and 828; SEQ ID NOs: 829 and 830; SEQ ID NOs: 831 and 832; SEQ ID NOs: 833 and 834; SEQ ID NOs: 835 and 836; SEQ ID NOs: 837 and 838; SEQ ID NOs: 839 and 840; SEQ ID NOs: 841 and 842; SEQ ID NOs: 843 and 844; SEQ ID NOs: 845 and 846; SEQ ID NOs: 847 and 848; SEQ ID NOs: 849 and 850; SEQ ID NOs: 851 and 852; SEQ ID NOs: 853 and 854; SEQ ID NOs: 855 and 856; SEQ ID NOs:855 and 856; SEQ ID NOs:857 and 858; SEQ ID NOs:859 and 860; SEQ ID NOs:861 and 862; SEQ ID NOs:863 and 864; SEQ ID NOs:865 and 866; SEQ ID NOs:867 and 868; SEQ ID NOs:869 and 870; SEQ ID NOs:871 and 872; SEQ ID NOs:873 and 874; SEQ ID NOs:875 and 876; SEQ ID NOs:877 and 878; SEQ ID NOs:879 and 880; SEQ ID NOs:881 and 882; SEQ ID NOs:883 and 884; SEQ ID NOs:885 and 886; SEQ ID NOs:887 and 888; SEQ ID NOs:889 and 890; SEQ ID NOs:891 and 892; SEQ ID NOs:893 and 894; SEQ ID NOs:895 and 896; SEQ ID NOs:895 and 896; SEQ ID NOs:897 and 898; SEQ ID NOs:899 and 900; SEQ ID NOs:901 and 902; SEQ ID NOs:903 and 904; SEQ ID NOs:905 and 906; SEQ ID NOs:907 and 908; SEQ ID NOs:909 and 910; SEQ ID NOs:911 and 912; SEQ ID NOs:913 and 914; SEQ ID NOs:915 and 916; SEQ ID NOs:917 and 918; SEQ ID NOs:919 and 920; SEQ ID NOs:921 and 922; SEQ ID NOs:923 and 924; SEQ ID NOs:925 and 926; SEQ ID NOs:927 and 928; SEQ ID NOs:929 and 930; SEQ ID NOs:931 and 932; SEQ ID NOs:933 and 934; SEQ ID NOs:935 and 936; SEQ ID NOs:935 and 936; SEQ ID NOs:937 and 938; SEQ ID NOs:939 and 940; SEQ ID NOs:941 and 942; SEQ ID NOs:943 and 944; SEQ ID NOs:945 and 946; SEQ ID NOs:947 and 948; SEQ ID NOs:949 and 950; SEQ ID NOs:951 and 952; SEQ ID NOs:953 and 954; SEQ ID NOs:955 and 956; SEQ ID NOs:957 and 958; SEQ ID NOs:959 and 960; SEQ ID NOs:961 and 962; SEQ ID NOs:963 and 964; SEQ ID NOs:965 and 966; SEQ ID NOs:967 and 968; SEQ ID NOs: 969 and 970; SEQ ID NOs: 971 and 972; SEQ ID NOs: 973 and 974; SEQ ID NOs: 1759 and 1760; SEQ ID NOs: 1761 and 1762; SEQ ID NOs: 1763 and 1764; SEQ ID NOs: 1765 and 1766; SEQ ID NOs: 1767 and 1768; SEQ ID NOs: 1769 and 1770; SEQ ID NOs: 1771 and 1772; SEQ ID NOs: 1773 and 1774; SEQ ID NOs: 1775 and 1776; SEQ ID NOs: 1777 and 1778; SEQ ID NOs: 1779 and 1780; SEQ ID NOs: 1781 and 1782; SEQ ID NOs: 1783 and 1784; SEQ ID NOs: 1785 and 1786; SEQ ID NOs: 1787 and 1788; SEQ ID NOs: 1787 and 1788; SEQ ID NOs: 1789 and 1790; SEQ ID NOs: 1791 and 1792; SEQ ID NOs: 1793 and 1794; SEQ ID NOs: 1795 and 1796; SEQ ID NOs: 1797 and 1798; SEQ ID NOs: 1799 and 1800; SEQ ID NOs: 1801 and 1802; SEQ ID NOs: 1803 and 1804; SEQ ID NOs: 1805 and 1806; SEQ ID NOs: 1807 and 1808; SEQ ID NOs: 1809 and 1810; SEQ ID NOs: 1811 and 1812; SEQ ID NOs: 1813 and 1814; SEQ ID NOs: 1815 and 1816; SEQ ID NOs: 1817 and 1818; SEQ ID NOs: 1819 and 1820; SEQ ID NOs: 1821 and 1822; SEQ ID NOs: 1823 and 1824; SEQ ID NOs: 1825 and 1826; SEQ ID NOs: 1827 and 1828; SEQ ID NOs: 1829 and 1830; SEQ ID NOs: 1821 and 1822; SEQ ID NOs: 1823 and 1824; SEQ ID NOs: 1825 and 1826; SEQ ID NOs: 1827 and 1828; SEQ ID NOs: 1829 and 1830; SEQ ID NOs: 1831 and 1832; SEQ ID NOs: 1833 and 1834; SEQ ID NOs: 1835 and 1836; SEQ ID NOs: 1837 and 1838; SEQ ID NOs: 1839 and 1840; SEQ ID NOs: 1841 and 1842; SEQ ID NOs: 1843 and 1844; SEQ ID NOs: 1845 and 1846; SEQ ID NOs: 1847 and 1848; SEQ ID NOs: 1849 and 1850; SEQ ID NOs: 1851 and 1852; SEQ ID NOs: 1853 and 1854; SEQ ID NOs: 1855 and 1856; NO:1855 and 1856; SEQ ID NO:1857 and 1858; SEQ ID NO:1859 and 1860; SEQ ID NO:1861 and 1862; SEQ ID NO:1863 and 1864; SEQ ID NO:1865 and 1866; SEQ ID NO:1867 and 1868; SEQ ID NO:1869 and 1870; SEQ ID NO:1871 and 1872; or, SEQ ID NO:1873 and 1874.

在一些实施方式中，所述双链RNA包含选自IN-001至IN-488和INI-001至INI-308中的任一双链体的正义链和反义链。在一些实施方式中，所述双链RNA包含选自IN-489至IN-546中的任一双链体的正义链和反义链。In some embodiments, the double-stranded RNA comprises a sense strand and an antisense strand of any duplex selected from IN-001 to IN-488 and INI-001 to INI-308. In some embodiments, the double-stranded RNA comprises a sense strand and an antisense strand of any duplex selected from IN-489 to IN-546.

在一些实施方式中，本公开的反义寡核苷酸与靶mRNA(例如INHBE mRNA)基本上互补，并且包含与本文提供的任一种正义链寡核苷酸或正义链寡核苷酸的部分基于全长至少约85％互补的连续核苷酸序列，例如约85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或约99％互补。In some embodiments, the antisense oligonucleotides of the present invention are substantially complementary to the target mRNA (e.g., INHBE mRNA) and comprise a contiguous nucleotide sequence that is at least about 85% complementary to any one of the sense strand oligonucleotides or a portion of the sense strand oligonucleotide provided herein based on the full length, for example, about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or about 99% complementary.

在一些实施方式中，本公开的反义寡核苷酸与本文提供的任一种正义链寡核苷酸基本上互补，并且包含与本文提供的任一种正义链寡核苷酸或正义链寡核苷酸的部分基于全长至少约85％互补的连续核苷酸序列，例如约85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或约99％互补。In some embodiments, the antisense oligonucleotides of the present disclosure are substantially complementary to any of the sense strand oligonucleotides provided herein and comprise a contiguous nucleotide sequence that is at least about 85% complementary to any of the sense strand oligonucleotides or a portion of a sense strand oligonucleotide provided herein based on the full length, e.g., about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or about 99% complementary.

在一些实施方式中，本公开的dsRNA包括一正义链，所述正义链与一反义寡核苷酸基本上互补，相对地，所述反义寡核苷酸与靶mRNA互补，例如INHBE mRNA，其中，所述正义链与任一种所述反义寡核苷酸或反义寡核苷酸的部分基于全长至少约85％互补，例如约85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或约99％互补。In some embodiments, the dsRNA of the present disclosure includes a sense strand that is substantially complementary to an antisense oligonucleotide, which is in turn complementary to a target mRNA, such as INHBE mRNA, wherein the sense strand is at least about 85% complementary to any one of the antisense oligonucleotides or a portion of the antisense oligonucleotide based on the total length, such as about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or about 99% complementary.

在一些实施方式中，所述dsRNA的双链区域的长度等于或至少为10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30或更多个核苷酸对。In some embodiments, the length of the double-stranded region of the dsRNA is equal to or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotide pairs.

在一些实施方式中，所述dsRNA的反义链的长度等于或至少为14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30或更多个核苷酸。In some embodiments, the antisense strand of the dsRNA is equal to or at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides in length.

在一些实施方式中，所述dsRNA的正义链的长度等于或至少为10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30或更多个核苷酸。In some embodiments, the sense strand of the dsRNA is equal to or at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides in length.

在一些实施方式中，所述dsRNA的正义链和反义链的长度独立地为15至30个核苷酸。In some embodiments, the sense strand and antisense strand of the dsRNA are independently 15 to 30 nucleotides in length. Glycoside acid.

在一些实施方式中，所述dsRNA的正义链和反义链的长度独立地为19至25个核苷酸。In some embodiments, the sense and antisense strands of the dsRNA are independently 19 to 25 nucleotides in length.

在一些实施方式中，所述dsRNA的正义链和反义链的长度独立地为21至23个核苷酸。In some embodiments, the sense and antisense strands of the dsRNA are independently 21 to 23 nucleotides in length.

在一些实施方式中，所述dsRNA的正义链的长度为21个核苷酸，并且所述反义链的长度为23个核苷酸，其中所述链形成21个连续碱基对的双链区域，在3'-端具有2个核苷酸长的单链突出端。In some embodiments, the sense strand of the dsRNA is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, wherein the strands form a double-stranded region of 21 consecutive base pairs with a 2 nucleotide long single-stranded overhang at the 3'-end.

另一方面，本公开提供了一种RNAi剂(例如dsRNAi剂)，其包含前述任意的dsRNA，并且任选地包含靶向配体。所述靶向配体通常与所述dsRNA缀合，并起到将所述RNAi剂靶向细胞的作用。On the other hand, the present disclosure provides a kind of RNAi agent (such as dsRNAi agent), it comprises any of the aforementioned dsRNA, and optionally comprises a targeting ligand.The targeting ligand is usually conjugated with the dsRNA, and plays the role of targeting the RNAi agent to cells.

在一些实施方式中，所述双链RNAi剂包含任一前述的dsRNA，并且还包含至少一个靶向配体，所述dsRNA的正义链与所述靶向配体缀合。在一些优选实施方式中，所述正义链的3’端与所述靶向配体缀合。In some embodiments, the double-stranded RNAi agent comprises any of the aforementioned dsRNAs, and further comprises at least one targeting ligand, the sense strand of the dsRNA being conjugated to the targeting ligand. In some preferred embodiments, the 3' end of the sense strand is conjugated to the targeting ligand.

在一些实施方式中，本公开的靶向配体特异性地靶向至肝细胞表面的去唾液酸糖蛋白受体(asialoglycoprotein receptors，ASGPR)。优选地，所述靶向配体包含N-乙酰基-半乳糖胺(GalNAc)，或，所述靶向配体是GalNAc衍生物。更优选地，所述靶向配体是WO2022266753A1中公开的任一的靶向配体(targeting moiety)。除非明显矛盾，将WO2022266753A1全文引入本申请作为参考。In some embodiments, the targeting ligand disclosed herein specifically targets asialoglycoprotein receptors (ASGPR) on the surface of hepatocytes. Preferably, the targeting ligand comprises N-acetyl-galactosamine (GalNAc), or the targeting ligand is a GalNAc derivative. More preferably, the targeting ligand is any targeting moiety disclosed in WO2022266753A1. Unless otherwise clearly contradictory, the entire text of WO2022266753A1 is incorporated herein by reference.

在一些实施方式中，所述双链RNAi剂的结构选自式1～式33，其中R²为所述dsRNA。按本领域常识，R²通过所述正义链的3’端或5’端与靶向配体缀合形成dsRNA剂；优选地，通过所述正义链的3’端与所述靶向配体缀合。In some embodiments, the structure of the double-stranded RNAi agent is selected from Formula 1 to Formula 33, wherein R² is the dsRNA. According to common knowledge in the art, R² is conjugated to the targeting ligand through the 3' end or 5' end of the sense strand to form a dsRNA agent; preferably, it is conjugated to the targeting ligand through the 3' end of the sense strand.

表A1.本申请双链RNAi剂的结构








Table A1. Structure of the double-stranded RNAi agent of the present application

另一方面，本公开还提供了一种包含本公开的双链RNAi剂的细胞、载体、宿主细胞、和药物组合物。In another aspect, the present disclosure also provides a cell, a vector, a host cell, and a pharmaceutical composition comprising the double-stranded RNAi agent of the present disclosure.

在一些实施方式中，所述药物组合物，其包含前述任意的双链RNAi剂或其药学上可接受的盐，以及药学上可接受的载体。可将本公开的药物组合物实际用于对各种相应疾病或病症的预防和/或治疗，如下文进一步所述。In some embodiments, the pharmaceutical composition comprises any of the aforementioned double-stranded RNAi agents or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. The pharmaceutical composition of the present disclosure can be used in practice for the prevention and/or treatment of various corresponding diseases or conditions, as further described below.

在一些实施方式中，所述药物组合物被配制成用于通过注射或输注施用，例如用于静脉内、皮下、腹膜内或肌内施用。在一些实施方式中，所述药物组合物被配制成用于皮下施用。在一些实施方式中，所述药物组合物被配制成用于静脉内施用。In some embodiments, the pharmaceutical composition is formulated for administration by injection or infusion, such as intravenous, subcutaneous, intraperitoneal, or intramuscular administration. In some embodiments, the pharmaceutical composition is formulated for subcutaneous administration. In some embodiments, the pharmaceutical composition is formulated for intravenous administration.

在一些实施方式中，所述药物组合物的载体是非缓冲溶液或缓冲溶液。典型的非缓冲溶液是盐水或水，缓冲溶液包括乙酸盐、柠檬酸盐、醇溶谷蛋白、碳酸盐和磷酸盐中的一种或多种。优选的缓冲溶液是磷酸盐缓冲盐水(PBS)。In some embodiments, the carrier of the pharmaceutical composition is a non-buffered solution or a buffered solution. Typical non-buffered solutions are saline or water, and buffered solutions include one or more of acetate, citrate, prolamin, carbonate, and phosphate. The preferred buffered solution is phosphate buffered saline (PBS).

在本公开的另一方面，还提供了抑制细胞中INHBE表达的方法。这些方法包括：使所述细胞与本公开的双链RNAi剂或RNA接触，使得INHBE基因的mRNA转录物降解，由此抑制所述细胞中INHBE基因的表达。In another aspect of the present disclosure, methods for inhibiting INHBE expression in cells are also provided. These methods include: contacting the cells with the double-stranded RNAi agent or RNA of the present disclosure, so that the mRNA transcript of the INHBE gene is degraded, thereby inhibiting the expression of the INHBE gene in the cells.

在一些实施方式中，所述细胞在受试者体内。在一些实施方式中，所述细胞是肝细胞。在一些实施方式中，所述细胞是脂肪细胞。在一些实施方式中，所述受试者是人。In some embodiments, the cell is in a subject. In some embodiments, the cell is a hepatocyte. In some embodiments, the cell is a fat cell. In some embodiments, the subject is a human.

在一些实施方式中，INHBE表达被抑制至少约30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％或95％。In some embodiments, INHBE expression is inhibited by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

另一方面，本公开还提供了治疗具有由INHBE表达介导的病症(例如INHBE相关疾病或病症)的受试者的方法。所述方法包括向受试者给与治疗有效量的本公开的双链RNAi剂，从而抑制细胞中INHBE基因的表达。On the other hand, the present disclosure also provides a method for treating a subject having a condition mediated by INHBE expression (e.g., an INHBE-related disease or condition). The method comprises administering a therapeutically effective amount of a double-stranded RNAi agent of the present disclosure to the subject, thereby inhibiting the expression of the INHBE gene in the cell.

在一些实施方式中，所述受试者是人。In some embodiments, the subject is a human.

在一些实施方式中，所述受试者患有代谢紊乱。In some embodiments, the subject suffers from a metabolic disorder.

在一些实施方式中，所述代谢紊乱是代谢综合征、2型糖尿病、肥胖、前驱糖尿病、高甘油三酯血症(elevated triglyceride level)、脂肪代谢障碍(lipodystrophy)、肝脏炎症、脂肪肝、高胆固醇血症、与肝酶升高相关的疾病(elevated liver enzyme)、非酒精性脂肪性肝炎、心血管疾病、肾脏疾病。所述代谢综合征包括但不限于腹部肥胖、胰岛素抵抗、高血压、异常血脂症(dyslipidemia)和高血脂症中的一种或多种。In some embodiments, the metabolic disorder is metabolic syndrome, type 2 diabetes, obesity, prediabetes, Hypertriglyceridemia, lipodystrophy, liver inflammation, fatty liver, hypercholesterolemia, elevated liver enzymes, nonalcoholic steatohepatitis, cardiovascular disease, kidney disease. The metabolic syndrome includes, but is not limited to, one or more of abdominal obesity, insulin resistance, hypertension, dyslipidemia, and hyperlipidemia.

在一些实施方式中，抑制细胞中INHBE基因的表达，使受试者血清中INHBE基因表达的蛋白水平较未施用所述dsRNAi剂前降低至少50％、60％、70％、80％、90％或95％。In some embodiments, the expression of the INHBE gene in the cell is inhibited so that the protein level of the INHBE gene expression in the serum of the subject is reduced by at least 50%, 60%, 70%, 80%, 90% or 95% compared with before the administration of the dsRNAi agent.

在一些实施方式中，将双链RNAi剂以约0.10mg/kg至约50mg/kg的剂量施用于受试者，例如是以约0.01mg/kg至约10mg/kg(例如3mg/kg、5mg/kg或9mg/kg)、约0.5mg/kg至约50mg/kg、约10mg/kg至约30mg/kg、约10mg/kg至约20mg/kg、约15mg/kg至约20mg/kg、约15mg/kg至约25mg/kg、约15mg/kg至约30mg/kg、或约20mg/kg至约30mg/kg的剂量给予的。In some embodiments, the double-stranded RNAi agent is administered to a subject at a dose of about 0.10 mg/kg to about 50 mg/kg, for example, at a dose of about 0.01 mg/kg to about 10 mg/kg (e.g., 3 mg/kg, 5 mg/kg, or 9 mg/kg), about 0.5 mg/kg to about 50 mg/kg, about 10 mg/kg to about 30 mg/kg, about 10 mg/kg to about 20 mg/kg, about 15 mg/kg to about 20 mg/kg, about 15 mg/kg to about 25 mg/kg, about 15 mg/kg to about 30 mg/kg, or about 20 mg/kg to about 30 mg/kg.

在一些实施方式中，所述方法进一步包括确定来自受试者的样品中的INHBE水平。在一些实施方式中，在向受试者施用dsRNAi剂之前、期间和/或之后测定来自受试者的样品中的INHBE水平。可以使用任何合适的样品，例如但不限于血液样品、血清样品或肝组织样品。In some embodiments, the method further comprises determining the level of INHBE in a sample from the subject. In some embodiments, the level of INHBE in a sample from the subject is determined before, during, and/or after administering the dsRNAi agent to the subject. Any suitable sample can be used, such as, but not limited to, a blood sample, a serum sample, or a liver tissue sample.

在一些实施方式中，所述方法进一步包括向受试者施用另外的治疗剂以治疗代谢紊乱。所述治疗剂包括但不限于选自胰岛素、胰高血糖素样肽1(GLP-1)激动剂、葡萄糖依赖性促胰岛素多肽(GIP)受体激动剂、胰高血糖素受体激动剂、磺酰脲、司格列奈、双胍、噻唑烷二酮、α-葡萄糖苷酶抑制剂、SGLT2抑制剂、DPP-4抑制剂、HMG-CoA还原酶抑制剂、他汀类药物以及任何前述药物的组合。In some embodiments, the method further comprises administering to the subject an additional therapeutic agent to treat the metabolic disorder. The therapeutic agent includes, but is not limited to, selected from insulin, glucagon-like peptide 1 (GLP-1) agonists, glucose-dependent insulinotropic polypeptide (GIP) receptor agonists, glucagon receptor agonists, sulfonylureas, seglininides, biguanides, thiazolidinediones, alpha-glucosidase inhibitors, SGLT2 inhibitors, DPP-4 inhibitors, HMG-CoA reductase inhibitors, statins, and any combination of the foregoing drugs.

在一些实施方式中，本公开的双链RNAi剂可以与另外的治疗剂同时或依次给予。在一些实施方式中，双链RNAi剂在给予另外的治疗剂，例如标准治疗药剂之前或之后给予。In some embodiments, the double-stranded RNAi agent of the present disclosure can be administered simultaneously or sequentially with an additional therapeutic agent. In some embodiments, the double-stranded RNAi agent is administered before or after administration of an additional therapeutic agent, such as a standard therapeutic agent.

附图说明BRIEF DESCRIPTION OF THE DRAWINGS

图1-图5b显示使用rt-PCR法在HEP3B中检测dsRNA对人源INHBE的敲低活性(全曲线)的结果。Figures 1 to 5b show the results of detecting the knockdown activity of dsRNA against human INHBE in HEP3B using the rt-PCR method (full curve).

图6显示使用报告基因法检测dsRNA对人源及鼠源INHBE的敲低活性(单点)的结果。FIG6 shows the results of detecting the knockdown activity of dsRNA against human and mouse INHBE (single point) using the reporter gene method.

图7-8显示使用报告基因法检测dsRNA对人源INHBE的敲低活性(3点)的结果。Figures 7-8 show the results of detecting the knockdown activity of dsRNA against human INHBE (3 points) using the reporter gene method.

图9显示使用报告基因法检测dsRNA对鼠源INHBE的敲低活性(3点)的结果。FIG. 9 shows the results of detecting the knockdown activity of dsRNA against mouse INHBE using the reporter gene method (3 points).

图10-图15显示使用报告基因法检测dsRNA对人源INHBE的敲低活性(全曲线)的结果。FIG. 10 to FIG. 15 show the results of detecting the knockdown activity of dsRNA against human INHBE (full curve) using a reporter gene method.

图16显示使用报告基因法检测dsRNA对鼠源INHBE的敲低活性(全曲线)的结果。FIG. 16 shows the results of detecting the knockdown activity of dsRNA against mouse INHBE using a reporter gene method (full curve).

图17-19和图21显示使用报告基因法检测dsRNA对人源INHBE的敲低活性(3点)的结果。Figures 17 to 19 and Figure 21 show the results of detecting the knockdown activity of dsRNA against human INHBE (3 points) using the reporter gene method.

图20和图22显示使用报告基因法检测dsRNA对人源INHBE的敲低活性(2点)的结果。FIG. 20 and FIG. 22 show the results of detecting the knockdown activity of dsRNA against human INHBE (2 points) using the reporter gene method.

图23和图24显示使用报告基因法检测dsRNA对人源INHBE的敲低活性(全曲线)的结果。FIG. 23 and FIG. 24 show the results of detecting the knockdown activity of dsRNA against human INHBE (full curve) using the reporter gene method.

图25为siRNA在血清稳定性检测结果。FIG. 25 shows the results of siRNA stability test in serum.

图26为siRNA在人肝S9稳定性检测结果。FIG. 26 shows the results of siRNA stability test in human liver S9.

图27示出了给药不同时间后每组小鼠(3只)肝脏中siRNA含量变化。FIG. 27 shows the changes in siRNA content in the liver of each group of mice (3 mice) after administration at different time points.

图28示出的是以每个RNAi在给药7天后小鼠(3只)肝组织中的含量进行标准化后的不同siRNA在小鼠肝脏中的含量百分比。FIG. 28 shows the percentage of different siRNAs in mouse liver after being normalized by the content of each RNAi in the liver tissue of mice (3 mice) 7 days after administration.

具体实施方式DETAILED DESCRIPTION

本公开提供了RNAi剂和组合物，其引发RNA诱导的沉默复合物(RISC)介导的靶基因抑制素亚基beta E(INHBE)的RNA转录物的切割。该基因可以在细胞内，例如脂肪细胞和/或肝实质细胞(hepatocyte)或其他肝脏细胞(或称肝细胞，liver cell)，例如受试者(例如人)体内的细胞。这些RNAi剂和组合物的使用使得哺乳动物中的INHBE mRNA能够靶向降解。作为INHBE表达的抑制剂，本公开的RNAi剂和组合物可用于预防、治疗和/或抑制INHBE相关疾病或病症，例如代谢综合征和相关病症。The present disclosure provides RNAi agents and compositions that trigger RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of the target gene inhibin subunit beta E (INHBE). The gene can be within a cell, such as an adipocyte and/or hepatocyte or other liver cell (or liver cell), such as a cell in a subject (e.g., a human). The use of these RNAi agents and compositions enables targeted degradation of INHBE mRNA in mammals. As inhibitors of INHBE expression, the RNAi agents and compositions of the present disclosure can be used to prevent, treat and/or inhibit INHBE-related diseases or disorders, such as metabolic syndrome and related disorders.

因此，本公开提供了用于治疗、预防或抑制INHBE相关疾病或病症的方法，所述疾病或病症例如但不限于代谢病症，例如代谢综合征；碳水化合物紊乱，例如2型糖尿病、前驱糖尿病；脂质代谢障碍，例如高脂血症、高血压、脂肪营养不良；肾脏疾病；心血管疾病；或，体重失调，例如肥胖、超重；使用RNAi组合物，其引发RNA诱导的沉默复合物(RISC)介导的抑制素亚基beta E(INHBE)靶基因的RNA转录物的切割。Thus, the present disclosure provides methods for treating, preventing or inhibiting INHBE-related diseases or conditions, such as, but not limited to, metabolic disorders, e.g., metabolic syndrome; carbohydrate disorders, e.g., type 2 diabetes, prediabetes; lipid metabolism disorders, e.g., hyperlipidemia, hypertension, lipodystrophy; kidney disease; cardiovascular disease; or, weight disorders, e.g., obesity, overweight; using RNAi compositions that trigger RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of inhibin subunit beta E (INHBE) target genes.

本公开的RNAi剂包含反义RNA链，其具有长度最多约30个核苷酸的区域，例如长度为19-30、19-29、19-28、19-27、19-26、19-25、19-24、19-23、19-22、19-21、19-20、20-30、20-29、20-28、20-27、20-26、20-25、20-24、20-23、20-22、20-21、21-30、21-29、21-28、21-27、21-26、21-25、21-24、21-23、21-22、15、15-16、15-17、15-20、15-21、15-22、15-23、15-24或至少15个核苷酸，所述区域与INHBE靶基因的转录物mRNA的至少一部分基本上互补。The RNAi agents of the present disclosure comprise an antisense RNA strand having a region of up to about 30 nucleotides in length, such as 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23 , 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, 21-22, 15, 15-16, 15-17, 15-20, 15-21, 15-22, 15-23, 15-24 or at least 15 nucleotides, wherein the region is substantially complementary to at least a portion of the transcript mRNA of the INHBE target gene.

在某些实施方式中，本公开的双链RNAi剂的一条链或两条链的长度最多为66个核苷酸，例如36-66、26-36、25-36、31-60、22-43、或27-53个核苷酸，具有至少15个连续核苷酸的区域，所述区域与INHBE靶基因的转录物mRNA的至少一部分基本上互补。在一些实施方式中，具有更长的反义链的该些RNAi剂可以，例如，包括一长度在20-60个核苷酸的第二RNA链(正义链)，其中，该正义链和反义链形成一15-30个连续核苷酸的双链区域(双链体)。In certain embodiments, one or both strands of the double-stranded RNAi agent disclosed herein are up to 66 nucleotides in length, such as 36-66, 26-36, 25-36, 31-60, 22-43, or 27-53 nucleotides, with a region of at least 15 consecutive nucleotides that is substantially complementary to at least a portion of the transcript mRNA of the INHBE target gene. In some embodiments, the RNAi agents with longer antisense strands can, for example, include a second RNA strand (sense strand) of 20-60 nucleotides in length, wherein the sense strand and the antisense strand form a double-stranded region (duplex) of 15-30 consecutive nucleotides.

本公开的RNAi剂的使用使得哺乳动物中的INHBE mRNA能够靶向降解。本发明人已经证实，本公开的RNAi剂可以引发RNA诱导的沉默复合物(RISC)介导的INHBE RNA转录物的切割，从而导致INHBE靶基因的表达的显著抑制。在某些实施方式中，本公开的RNAi剂比先前的靶向相同基因的RNAi剂更有效(例如更有效力)和/或更具特异性(例如更安全，更少的脱靶效应)。在某些实施方式中，通过选择RNAi剂靶向INHBE mRNA中的特定位点，和/或包括RNA修饰(例如，非规范碱基配对核苷酸、修饰核苷酸、化学修饰)以增加功效、效力、特异性和/或安全性。在一些这样的实施方式中，RNAi剂包含至少一种修饰的核苷酸，例如非规范碱基配对核苷酸。包含这些RNAi剂的方法和组合物可用于治疗患有INHBE相关疾病或病症例如代谢病症的受试者。因此，本公开提供了用于治疗、预防或抑制受试者中的代谢病症的方法，所述受试者将受益于使用本公开的RNAi剂和组合物抑制或减少INHBE表达。The use of the RNAi agents disclosed herein enables targeted degradation of INHBE mRNA in mammals. The inventors have demonstrated that the RNAi agents disclosed herein can trigger RNA-induced silencing complex (RISC)-mediated cleavage of INHBE RNA transcripts, resulting in significant inhibition of expression of the INHBE target gene. In certain embodiments, the RNAi agents disclosed herein are more effective (e.g., more potent) and/or more specific (e.g., safer, fewer off-target effects) than previous RNAi agents targeting the same gene. In certain embodiments, the RNAi agent is selected to target a specific site in INHBE mRNA, and/or includes RNA modifications (e.g., non-canonical base-pairing nucleotides, modified nucleotides, chemical modifications) to increase efficacy, potency, specificity, and/or safety. In some such embodiments, the RNAi agent comprises at least one modified nucleotide, such as a non-canonical base-pairing nucleotide. Methods and compositions comprising these RNAi agents can be used to treat subjects suffering from INHBE-related diseases or disorders, such as metabolic disorders. Thus, the present disclosure provides methods for treating, preventing, or inhibiting a metabolic disorder in a subject who would benefit from inhibiting or reducing INHBE expression using the RNAi agents and compositions of the present disclosure.

本公开还提供了用于预防患有病症的受试者的至少一种症状的方法，其收益于INHBE表达的抑制或减少。以下的详细说明将公开如何制备和使用RNAi剂及其组合物以抑制INHBE靶基因的表达，以及用于治疗将受益于抑制和/或减少INHBE靶基因表达的受试者的组合物、用途和方法，例如易患或诊断患有代谢紊乱的受试者。The present disclosure also provides methods for preventing at least one symptom of a subject with a disorder that would benefit from inhibition or reduction of INHBE expression. The following detailed description will disclose how to prepare and use RNAi agents and compositions thereof to inhibit the expression of INHBE target genes, as well as compositions, uses and methods for treating subjects that would benefit from inhibition and/or reduction of INHBE target gene expression, such as subjects susceptible to or diagnosed with metabolic disorders.

定义definition

为了对本发明的说明书中所使用的术语提供清楚且一致的理解，在下文中提供一些定义。此外，除了特殊说明，本发明所用的全部技术和科学术语具有同本发明所属领域中普通技术人员通常所理解的相同的含义。In order to provide a clear and consistent understanding of the terms used in the specification of the present invention, some definitions are provided below. In addition, unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by ordinary technicians in the field to which the present invention belongs.

当在权利要求和/或说明书中与术语“包括”结合使用时，词语“一”的使用可以表示“一个”，但它也与“一个或多个”，“至少一个”和“一个或多于一个”的含义一致。类似地，词语“另一个”可以表示至少第二个或者更多个。When used in conjunction with the term "comprising" in the claims and/or the specification, the use of the word "a" can mean "one", but it is also consistent with the meaning of "one or more", "at least one" and "one or more than one". Similarly, the word "another" can mean at least a second or more.

术语“或”在本文中用于表示术语“和/或”，并且可与术语“和/或”互换使用，除非上下文清楚地另有说明。例如，“正义链或反义链”被理解为“正义链或反义链，或正义链和反义链”。The term "or" is used herein to represent the term "and/or", and can be used interchangeably with the term "and/or", unless the context clearly indicates otherwise. For example, "sense strand or antisense strand" is understood to mean "sense strand or antisense strand, or sense strand and antisense strand".

如本说明书和权利要求书中所使用的，词语“包含”(以及任何形式的包含，例如“包含”和“包含”)、“具有”(以及任何形式的具有，例如“有”和“具有”)、“包括”(以及任何形式的包含，例如“包含”和“包括”)和“包含”(以及任何形式的包含，例如“含有”和“包含”)，是包容性的或开放式的，并且不排除额外的、未叙述的要素或过程步骤。As used in this specification and claims, the words "comprising" (and any forms of inclusion, such as "includes" and "including"), "having" (and any forms of having, such as "have" and "having"), "including" (and any forms of inclusion, such as "includes" and "including"), and "including" (and any forms of inclusion, such as "containing" and "including"), are inclusive or open-ended, and do not exclude additional, unrecited elements or process steps.

术语“约”或“大约”用于表示该值包括在确定该值中所用的仪器和方法带来的误差。术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值，包括但不限于±5％、±2％、±1％和±0.1％，因为这些变化适于进行所公开的方法。当“约”出现在一系列数字或范围之前时，应理解“约”可以修饰该系列或范围中的每个数字。The term "about" or "approximately" is used to indicate that the value includes the error caused by the instruments and methods used in determining the value. The term "about" when used in conjunction with a numerical value is meant to encompass numerical values within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value, including but not limited to ±5%, ±2%, ±1% and ±0.1%, as these variations are suitable for performing the disclosed methods. When "about" appears before a series of numbers or ranges, it should be understood that "about" can modify each number in the series or range.

在一个数字或一系列数字之前的术语“至少”、“不少于”或“或更多”应理解为包括与术语“至少”相邻的数字，以及从上下文中可以清楚地看出的逻辑上可以包含的所有后续数字或整数。例如，核酸分子中核苷酸的数量必须是整数。例如，“17个核苷酸的核酸分子中的至少15个核苷酸”意指具有指定的性质的15、16或17个核苷酸。当“至少”出现在一系列数字或范围之前时，应理解“至少”可以修饰该系列或范围中的每个数字。The terms "at least", "not less than" or "or more" preceding a number or a series of numbers should be understood to include the number adjacent to the term "at least", as well as all subsequent numbers or integers that can be logically included as can be clearly seen from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 15 nucleotides in a nucleic acid molecule of 17 nucleotides" means 15, 16 or 17 nucleotides having the specified properties. When "at least" appears before a series of numbers or ranges, it should be understood that "at least" can modify each number in the series or range.

如本文所使用的，“不超过”或“或更少”被理解为与该短语相邻的值以及逻辑上较低的数值或整数，如根据上下文合乎逻辑，至零。例如，具有“不超过2个核苷酸”突出端的双链体具有2、1或0个核苷酸突出端。当“不超过”出现在一系列数字或范围之前时，应理解“不超过”可以修饰该系列或范围中的每个数字。如本文所用，范围包括上限和下限。As used herein, "no more than" or "or less" is understood to mean the value adjacent to the phrase and a logically lower numerical value or integer, such as is logical from the context, to zero. For example, a duplex having "no more than 2 nucleotides" overhang has 2, 1, or 0 nucleotide overhangs. When "no more than" appears before a series of numbers or ranges, it is understood that "no more than" can modify each number in the series or range. As used herein, a range includes an upper limit and a lower limit.

术语“抑制素Beta E”和术语“INHBE”可以互换使用，也称为“抑制素亚基Beta E链”、“抑制素Beta E”、“抑制素βE”、“激活素βE”、“激活素Beta E”和“MGC4638”。人INHBE mRNA转录物的序列可以在例如GenBank登录号NM_031479.5中找到。小鼠INHBE mRNA的序列可以在例如GenBank登录号NM_008382.3(SEQ ID NO:1875)中找到。食蟹猴INHBE mRNA的预测序列可以在例如GenBank登录号XM_005571319.3中找到。INHBE mRNA序列的其他示例可通过公开数据库轻松获得，例如GenBank、UniProt、OMIM和Macaca基因组计划网站。有关INHBE的更多信息，例如，可访问www.ncbi.nlm.nih.gov/gene/？term＝INHBE。截至提交本申请之日，上述每个GenBank登录号和基因数据库号的全部内容通过引用并入本文。The term "inhibin Beta E" and the term "INHBE" may be used interchangeably, also referred to as "inhibin subunit Beta E chain," "inhibin Beta E," "inhibin βE," "activin βE," "activin Beta E," and "MGC4638." The sequence of the human INHBE mRNA transcript can be found, for example, in GenBank Accession No. NM_031479.5. The sequence of the mouse INHBE mRNA can be found, for example, in GenBank Accession No. NM_008382.3 (SEQ ID NO: 1875). The predicted sequence of the cynomolgus monkey INHBE mRNA can be found, for example, in GenBank Accession No. XM_005571319.3. Other examples of INHBE mRNA sequences are readily available through public databases, such as GenBank, UniProt, OMIM, and the Macaca Genome Project website. For more information on INHBE, for example, visit www.ncbi.nlm.nih.gov/gene/?term=INHBE. As of the date of filing this application, each of the above-mentioned GenBank Accession Numbers and Gene Database Numbers are incorporated herein by reference in their entirety.

术语“靶序列”或“靶核酸”或“靶mRNA”是指在靶基因的转录过程中形成的mRNA分子的核苷酸序列的连续部分，包括作为初级转录产物的RNA加工的产物的mRNA。在一个实施方式中，序列的靶部分至少足够长以充当在靶基因转录过程中形成的mRNA分子的核苷酸序列部分处或附近进行RNAi定向切割的底物。在一种实施方式中，靶序列位于靶基因的蛋白质编码区内。在另一个实施方式中，靶序列位于靶基因的3’UTR内。靶核酸可以是细胞基因(或从该基因转录的mRNA)，其表达与特定病症或疾病状态相关。靶序列的长度可以为约19-36个核苷酸，例如，长度为约19-30个核苷酸，例如长度为19-30、19-29、19-28、19-27、19-26、19-25、19-24、19-23、19-22、19-21、19-20、20-30、20-29、20-28、20-27、20-26、20-25、20-24、20-23、20-22、20-21、21-30、21-29、21-28、21-27、21-26、21-25、21-24、21-23或21-22个核苷酸。在某些实施方案中，靶序列的长度为15-30个核苷酸，例如长度为15-23个核苷酸，例如长度为15-30、15-29、15-28、15-27、15-26、15-25、15-24、15-23、15-22、15-21、15-20、17-30、17-29、18-28、17-27、16-26、15-25、15-24、16-23、16-22、16-21、16-30、16-29、16-28、16-27、16-26、16-25、16-24、17-25、17-23或15-17个核苷酸。在某些实施方式中，靶序列的长度为17-25个核苷酸，19-21个核苷酸，19-23个核苷酸，或21-23个核苷酸。上述范围和长度中间的范围和长度也被认为是本公开的一部分。The term "target sequence" or "target nucleic acid" or "target mRNA" refers to a continuous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a target gene, including mRNA that is a product of RNA processing of a primary transcription product. In one embodiment, the target portion of the sequence is at least long enough to serve as a substrate for RNAi-directed cleavage at or near the nucleotide sequence portion of the mRNA molecule formed during the transcription of the target gene. In one embodiment, the target sequence is located within the protein coding region of the target gene. In another embodiment, the target sequence is located within the 3'UTR of the target gene. The target nucleic acid can be a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a specific disorder or disease state. The target sequence can be about 19-36 nucleotides in length, for example, about 19-30 nucleotides in length, for example, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. In certain embodiments, the target sequence is 15-30 nucleotides in length, e.g., 15-23 nucleotides in length, e.g., 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 17-30, 17-29, 18-28, 17-27, 16-26, 15-25, 15-24, 16-23, 16-22, 16-21, 16-30, 16-29, 16-28, 16-27, 16-26, 16-25, 16-24, 17-25, 17-23, or 15-17 nucleotides in length. In certain embodiments, the target sequence is 17-25 nucleotides, 19-21 nucleotides, 19-23 nucleotides, or 21-23 nucleotides in length. Ranges and lengths intermediate to the above ranges and lengths are also considered part of the present disclosure.

如本文所用，术语“包含序列的链”是指寡核苷酸，其包含使用标准核苷酸命名法所指的序列以描述的核苷酸链。As used herein, the term "strand comprising a sequence" refers to an oligonucleotide comprising a chain of nucleotides described by the sequence referred to using standard nucleotide nomenclature.

术语“siRNA”、“RNAi剂”、“siRNA剂”和“RNA干扰剂”在本文中可互换使用，是指含有RNA并且通过RNA诱导的沉默复合物(RISC)途径介导RNA转录物靶向切割的生物活性剂。RNAi剂通过称为RNA干扰的过程引导mRNA序列的特异性降解。RNAi剂调节(例如抑制)细胞中基因的表达，例如受试者(例如哺乳动物受试者，例如人)体内的细胞。在一些实施方式中，在本公开的组合物、用途和方法中使用的RNAi剂包含本公开的双链RNA(dsRNA)或双链体，并且在本文中可以被称为“双链RNAi剂”、“dsRNAi剂”或“dsRNA剂”。The terms "siRNA", "RNAi agent", "siRNA agent" and "RNA interference agent" are used interchangeably herein and refer to a biologically active agent that contains RNA and mediates targeted cleavage of RNA transcripts through an RNA-induced silencing complex (RISC) pathway. RNAi agents direct the specific degradation of mRNA sequences through a process known as RNA interference. RNAi agents regulate (e.g., inhibit) the expression of genes in cells, such as cells in a subject (e.g., a mammalian subject, such as a human). In some embodiments, the RNAi agent used in the compositions, uses and methods of the present disclosure comprises a double-stranded RNA (dsRNA) or duplex of the present disclosure, and may be referred to herein as a "double-stranded RNAi agent", "dsRNAi agent" or "dsRNA agent".

在某些实施方式中，本公开的dsRNAi剂包括双链RNA剂，当将其引入细胞中时，其被称为Dicer的核酸内切酶加工成短干扰RNA。短干扰RNA被整合到RISC中，一个或多个解旋酶解开RNA双链体，使互补的反义链能够引导目标识别。与靶mRNA结合后，RISC中的一种或多种核酸内切酶会切割靶mRNA以诱导沉默。因此，在其他实施方式中，siRNA剂涉及细胞内产生的单链RNA并且其促进RISC复合物的形成以实现靶基因的沉默。在一些这样的实施方式中，RNAi剂是单链siRNA(ssRNAi)，其可以被引入细胞或生物体中以抑制靶mRNA。单链RNAi剂与RISC核酸内切酶，Argonaute 2结合，然后切割目标mRNA。ssRNAi剂通常长度在15-30个核苷酸，并且可以被化学修饰。本文描述的任何反义寡核苷酸可以用作本文描述的ssRNAi剂。在一些实施方式中，ssRNAi剂包含至少一种非规范碱基配对核苷酸。在一些实施方式中，ssRNAi剂包含至少一种修饰的核苷酸。In certain embodiments, the dsRNAi agents of the present disclosure include double-stranded RNA agents that, when introduced into a cell, are processed into short interfering RNAs by an endonuclease called Dicer. Short interfering RNAs are integrated into RISC, and one or more helicases unwind the RNA duplexes, enabling the complementary antisense strand to guide target recognition. After binding to the target mRNA, one or more endonucleases in RISC cleave the target mRNA to induce silencing. Therefore, in other embodiments, siRNA agents involve single-stranded RNA produced within the cell and which promotes the formation of the RISC complex to achieve silencing of the target gene. In some such embodiments, the RNAi agent is a single-stranded siRNA (ssRNAi), which can be introduced into a cell or organism to inhibit the target mRNA. The single-stranded RNAi agent binds to the RISC endonuclease, Argonaute 2, and then cleaves the target mRNA. ssRNAi agents are typically 15-30 nucleotides in length and can be chemically modified. Any antisense oligonucleotide described herein can be used as a ssRNAi agent described herein. In some embodiments, the ssRNAi agent comprises at least one non-canonical base-pairing nucleotide. In some embodiments, the ssRNAi agent comprises at least one modified nucleotide.

术语“双链RNA”或“dsRNA”是指核糖核酸分子的复合物，其是具有包含两条反平行且基本上互补的核酸链的双链体结构，相对于靶RNA具有“有义”(或“正义”)和“反义”方向。在本公开的一些实施方案中，dsRNA通过本文称为RNA干扰或RNAi的转录后基因沉默机制触发靶RNA例如mRNA的降解。一般而言，dsRNA分子每条链的大部分核苷酸是核糖核苷酸，但如本文详细描述的，每条或两条链还可以包括一个或多个非核糖核苷酸，例如脱氧核糖核苷酸或修饰的核苷酸。dsRNA分子的每条链可具有12-40个核苷酸范围内的长度。举例来说，每条链可以在14-40个核苷酸长度、17-37个核苷酸长度、25-37个核苷酸长度、17-25个核苷酸长度、17-22个核苷酸长度、19-25个核苷酸长度、19-23个核苷酸长度、21-23个核苷酸长度之间，正义链和反义链可以是相等的长度或不等的长度，对此没有限制。The term "double-stranded RNA" or "dsRNA" refers to a complex of ribonucleic acid molecules, which is a duplex structure comprising two antiparallel and substantially complementary nucleic acid chains, with a "sense" (or "justice") and "antisense" orientation relative to the target RNA. In some embodiments of the present disclosure, dsRNA triggers the degradation of target RNA, such as mRNA, by a post-transcriptional gene silencing mechanism referred to herein as RNA interference or RNAi. In general, most of the nucleotides of each chain of a dsRNA molecule are ribonucleotides, but as described in detail herein, each or both chains may also include one or more non-ribonucleotides, such as deoxyribonucleotides or modified nucleotides. Each chain of a dsRNA molecule may have a length in the range of 12-40 nucleotides. For example, each strand can be between 14-40 nucleotides in length, 17-37 nucleotides in length, 25-37 nucleotides in length, 17-25 nucleotides in length, 17-22 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 21-23 nucleotides in length, and the sense strand and antisense strand can be equal length or unequal length without limitation.

术语“反义链”是指一种iRNA(例如dsRNA)的包括与靶序列(例如INHBE mRNA)基本上互补的区域的链。如在此所使用，术语“互补区域”是指反义链上与一个序列基本上互补的区域。在互补区域不完全与靶序列互补的情况，可在分子的内部或末端区域有错配。通常，最耐受的错配存在于末端区域内，例如在dsRNA的5'-和/或3'-末端的5、4、3或2个核苷酸内部。dsRNA的反义链和正义链可以具有相同或不同的长度，所述的这些均是本领域已知的。The term "antisense strand" refers to the strand of an iRNA (e.g., dsRNA) that includes a region that is substantially complementary to a target sequence (e.g., INHBE mRNA). As used herein, the term "complementary region" refers to a region on the antisense strand that is substantially complementary to a sequence. In the case where the complementary region is not completely complementary to the target sequence, there may be mismatches in the interior or terminal regions of the molecule. Typically, the most tolerated mismatches occur in the terminal regions, such as at the 5', 5'- and/or 3'-ends of the dsRNA. The antisense and sense strands of a dsRNA can be of the same or different lengths, as is known in the art.

当第一序列被称为相对于第二序列“基本上互补”时，这两个序列可以是完全互补的(即，在一个或两个核苷酸序列的整个长度上互补)，或者它们可以在最多可达30个碱基对的杂交时形成一个或更多，但一般不超过5、4、3或2个错配碱基对，同时保留在适当条件下杂交的能力(在与其应用相关的条件下，如抑制基因表达，如生理条件)。应当注意，当两个寡核苷酸被设计为在杂交时形成一个或多个单链突出端时，就确定互补性而言，此类突出端不应被视为错配。例如，包含一个长度为21个核苷酸的寡核苷酸和另一个长度为23个核苷酸的寡核苷酸的dsRNA，其中较长的寡核苷酸包含与较短的寡核苷酸完全互补的21个核苷酸的序列，就本文所述目的而言，可称为"完全互补"。When a first sequence is referred to as being "substantially complementary" to a second sequence, the two sequences may be fully complementary (i.e., complementary over the entire length of one or both nucleotide sequences), or they may form one or more, but generally no more than 5, 4, 3, or 2, mismatched base pairs upon hybridization of up to 30 base pairs, while retaining the ability to hybridize under appropriate conditions (under conditions relevant to their application, such as inhibition of gene expression, such as physiological conditions). It should be noted that when two oligonucleotides are designed to form one or more single-stranded overhangs upon hybridization, such overhangs should not be considered mismatches for purposes of determining complementarity. For example, a dsRNA comprising one oligonucleotide of 21 nucleotides in length and another oligonucleotide of 23 nucleotides in length, wherein the longer oligonucleotide comprises a 21 nucleotide sequence that is fully complementary to the shorter oligonucleotide, may be referred to as "fully complementary" for purposes described herein.

术语“正义链”是指dsRNA的一条链，其包含与反义链区域基本上互补的区域。The term "sense strand" refers to the strand of a dsRNA that comprises a region that is substantially complementary to a region of the antisense strand.

如在此所使用，并且除非另外指明，当用来描述与第二核苷酸序列相关的第一核苷酸序列时，术语“互补”是指包含该第一核苷酸序列的寡核苷酸或多核苷酸在某些条件下与包含该第二核苷酸序列的寡核苷酸或多核苷酸杂交并且形成双链体结构的能力。这类条件可以例如是严格条件，其中严格条件可以包括：400mM NaCl，40mM PIPES，pH 6.4，1mM EDTA，50℃或70℃持续12-16小时。其他条件，例如生物体内可能遇到的生理相关条件，也可适用。例如，互补序列足以使核酸的相关功能得以进行，例如RNAi。技术人员可以根据杂交核苷酸的最终用途，确定最适合测试两个序列互补性的条件集。As used herein, and unless otherwise indicated, the term "complementary" when used to describe a first nucleotide sequence associated with a second nucleotide sequence refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under certain conditions. Such conditions may, for example, be stringent conditions, wherein stringent conditions may include: 400 mM NaCl, 40 mM PIPES, pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours. Other conditions, such as physiologically relevant conditions that may be encountered in an organism, may also be applicable. For example, complementary sequences are sufficient to enable the relevant function of the nucleic acid to be performed, such as RNAi. A skilled person can determine the set of conditions that is most suitable for testing the complementarity of two sequences based on the ultimate use of the hybridizing nucleotides.

本文中的术语“互补”、“完全互补”和“基本上互补”可用于描述dsRNA的正义链和反义链之间的碱基匹配，或两个寡核酸或多核苷酸之间的碱基匹配，例如一dsRNA试剂的反义链和一靶序列的碱基匹配，其含义可从使用该术语的上下文进行理解。The terms "complementary", "fully complementary" and "substantially complementary" herein may be used to describe base matching between the sense and antisense strands of a dsRNA, or base matching between two oligonucleotides or polynucleotides, such as the antisense strand of a dsRNA agent and a target sequence, and their meanings are understood from the context in which the terms are used.

术语“解链温度”(melting temperature)或“Tm”在本文中用于指50％的双链RNA(dsRNA)分子打开或变性(即50％的双链RNA分子被分离成单链，50％的互补寡核苷酸链不互相杂交)。对于给定的寡核苷酸，其相应的Tm值可以通过使用本领域已知的任何可接受的公式或软件计算来获得。例如但不限于，可以使用来自Integrated DNA Technologies(IDT)(Coralville,Iowa,USA)的OligoAnalyzer^TM工具来计算Tm；使用网站http://insilico.ehu.es/tm.php？formula＝basic上的Tm计算工具等等。术语“ΔTm”是指两种不同寡核苷酸(例如，未修饰的寡核苷酸和具有相同序列的包含一种或多种修饰的核苷酸的寡核苷酸)之间的Tm(例如，计算的Tm)差异。在某些实施方式中，ΔTm用于指本公开的两个dsRNA区域或双链体之间的解链温度差异，其中dsRNA区域或双链体之一包含至少一个修饰的核苷酸(例如非规范碱基配对的核苷酸)进行的核苷酸取代。在一些实施方式中，ΔTm用于指两个寡核苷酸(例如，两个反义链、两个正义链)之间解链温度的差异，其中寡核苷酸之一包含至少一个被非规范碱基配对核苷酸取代的核苷酸。The term "melting temperature" or "Tm" is used herein to refer to the opening or denaturation of 50% of double-stranded RNA (dsRNA) molecules (i.e., 50% of the double-stranded RNA molecules are separated into single strands and 50% of the complementary oligonucleotide chains do not hybridize with each other). For a given oligonucleotide, its corresponding Tm value can be obtained by calculating using any acceptable formula or software known in the art. For example, but not limited to, the OligoAnalyzer^TM tool from Integrated DNA Technologies (IDT) (Coralville, Iowa, USA) can be used to calculate Tm; using the Tm calculation tool on the website http://insilico.ehu.es/tm.php?formula=basic, etc. The term "ΔTm" refers to the Tm (e.g., calculated Tm) difference between two different oligonucleotides (e.g., an unmodified oligonucleotide and an oligonucleotide having the same sequence containing one or more modified nucleotides). In certain embodiments, ΔTm is used to refer to the difference in melting temperature between two dsRNA regions or duplexes of the present disclosure, wherein one of the dsRNA regions or duplexes comprises a nucleotide substitution by at least one modified nucleotide (e.g., a non-canonical base-pairing nucleotide). In some embodiments, ΔTm is used to refer to the difference in melting temperature between two oligonucleotides (e.g., two antisense strands, two sense strands), wherein one of the oligonucleotides comprises at least one nucleotide substituted by a non-canonical base-pairing nucleotide.

“G”、“C”、“A”、“T”和“U”通常分别代表含有鸟嘌呤、胞嘧啶、腺嘌呤、胸腺嘧啶(也被称为5-甲基尿嘧啶)和尿嘧啶作为碱基的核苷酸。然而，应当理解，术语“核糖核苷酸”或“核苷酸”还可以指修饰的核苷酸，如下文进一步详述。G、C、A、T和U在本文中被称为“规范”核苷酸，规范核苷酸的定义如表A2所示。相应地，鸟嘌呤、胞嘧啶、腺嘌呤、胸腺嘧啶和尿嘧啶在本文中被称为“规范”碱基，其是用于RNA或DNA构建的常用碱基。在本文中，在仅针对碱基基团或碱基部分进行表述或描述且不引起歧义的情况下，也可使用“G”代表鸟嘌呤、“C”代表胞嘧啶、“A”代表腺嘌呤、“T”代表胸腺嘧啶以及“U”代表尿嘧啶。对于规范碱基或规范核苷酸，在大多数情况下，A与U(DNA中的T)配对，G与C配对，遵循Watson-Crick碱基对规则(本文称为“规范碱基配对”)。"G", "C", "A", "T" and "U" generally represent nucleotides containing guanine, cytosine, adenine, thymine (also known as 5-methyluracil) and uracil as bases, respectively. However, it should be understood that the term "ribonucleotide" or "nucleotide" can also refer to modified nucleotides, as described in further detail below. G, C, A, T and U are referred to as "standard" nucleotides in this article, and the definition of standard nucleotides is shown in Table A2. Accordingly, guanine, cytosine, adenine, thymine and uracil are referred to as "standard" bases in this article, which are common bases for RNA or DNA construction. In this article, when only base groups or base parts are stated or described and no ambiguity is caused, "G" can also be used to represent guanine, "C" represents cytosine, "A" represents adenine, "T" represents thymine and "U" represents uracil. For canonical bases or canonical nucleotides, in most cases, A pairs with U (T in DNA) and G pairs with C, following the Watson-Crick base pairing rules (referred to herein as "canonical base pairing").

表A2.规范核苷酸的定义
Table A2. Definition of canonical nucleotides

不遵循Watson-Crick碱基对规则的“非规范”碱基配对也是可能的。例如，G-U摆动碱基配对经常发生，并且具有与Watson-Crick碱基对相当的配对强度(例如热力学稳定性)。因此，本公开的dsRNAi剂的核苷酸序列中任何位置的腺嘌呤和胞嘧啶可以分别被鸟嘌呤和尿嘧啶替代，以与靶序列形成G-U摆动碱基配对。"Non-canonical" base pairing that does not follow the Watson-Crick base pairing rules is also possible. For example, G-U wobble base pairing occurs frequently and has a pairing strength (e.g., thermodynamic stability) comparable to Watson-Crick base pairs. Therefore, adenine and cytosine at any position in the nucleotide sequence of the dsRNAi agent of the present disclosure can be replaced by guanine and uracil, respectively, to form G-U wobble base pairing with the target sequence.

某些修饰的核苷酸同样表现出非规范碱基配对。此类修饰的核苷酸具有与规范核苷酸不同的碱基配对特征。例如，以次黄嘌呤作为碱基的修饰核苷酸(例如肌苷酸)可以与含有腺嘌呤、胞嘧啶或尿嘧啶的核苷酸进行碱基配对。因此，本公开的dsRNAi剂的核苷酸序列中，含有尿嘧啶、鸟嘌呤或腺嘌呤的核苷酸可以被含有碱基次黄嘌呤的修饰核苷酸(例如肌苷酸)替换。在某些实施方式中，本公开的dsRNAi剂中的一个或多个规范核苷酸被具有不同碱基配对特征的修饰核苷酸替换；此类替换部分在本文中被称为“非规范碱基配对核苷酸”。本公开的非规范碱基配对核苷酸包括能够进行非Watson-Crick或摆动碱基配对的核苷酸和/或与其替代的规范核苷酸相比具有不同碱基配对特征的修饰核苷酸。含有此类非规范碱基配对核苷酸的序列适用于本公开的RNAi剂、组合物和方法。Certain modified nucleotides also exhibit non-canonical base pairing. Such modified nucleotides have base pairing characteristics different from canonical nucleotides. For example, modified nucleotides (e.g., inosinic acid) with hypoxanthine as a base can be base paired with nucleotides containing adenine, cytosine, or uracil. Therefore, in the nucleotide sequence of the dsRNAi agent of the present disclosure, nucleotides containing uracil, guanine, or adenine can be replaced by modified nucleotides (e.g., inosinic acid) containing the base hypoxanthine. In certain embodiments, one or more canonical nucleotides in the dsRNAi agent of the present disclosure are replaced by modified nucleotides with different base pairing characteristics; such replacement parts are referred to herein as "non-canonical base pairing nucleotides". The non-canonical base pairing nucleotides of the present disclosure include nucleotides capable of non-Watson-Crick or wobble base pairing and/or modified nucleotides with different base pairing characteristics compared to the canonical nucleotides replaced by them. Sequences containing such non-canonical base pairing nucleotides are suitable for RNAi agents, compositions, and methods of the present disclosure.

应当理解，非规范碱基配对核苷酸(例如，具有与它们所取代的规范核苷酸不同的碱基配对特征的修饰核苷酸)不仅可以在它们形成的碱基对方面不同，而且可以在碱基配对的强度或稳定性方面不同。非规范碱基配对可能比规范碱基配对更强或更弱。例如，m1Ψ(从U修改而来)与A的配对比U与A的配对更强，并且m1Ψ-G配对甚至比m1Ψ-A配对更强。因此，通过用非规范碱基配对核苷酸替换规范核苷酸，可以改变碱基配对强度，从而改变反义链双链体的Tm和/或反义链与靶RNA(例如INHBE mRNA)之间杂交的Tm。因此，在一些实施方式中，规范核苷酸被非规范碱基配对核苷酸取代，从而改变寡核苷酸的Tm(例如，改变计算的寡核苷酸的Tm、改变所得双链RNA分子的Tm，例如与靶mRNA和/或有义链杂交的反义链形成的双链体的Tm)。不希望受理论限制，通过用非规范碱基配对核苷酸替换至少一种规范核苷酸来改变寡核苷酸的Tm，可以调控dsRNAi的功效、效力、特异性、安全性和/或脱靶效应。例如但不限于，可以通过增加与所需靶mRNA的配对强度和/或减少与脱靶mRNA的配对来增加dsRNAi剂的功效或效力。类似地，可以通过增加与所需靶mRNA的配对强度和/或降低与脱靶mRNA的配对强度来减少不期望的脱靶效应。因此，在一些实施方式中，与不包含至少一种非规范碱基配对核苷酸的类似RNAi剂相比，本公开的RNAi剂具有改善的功效、效力、特异性和/或安全性。It should be understood that non-canonical base pairing nucleotides (e.g., modified nucleotides having different base pairing characteristics than the canonical nucleotides they replace) can differ not only in the base pairs they form, but also in the strength or stability of the base pairing. Non-canonical base pairing can be stronger or weaker than canonical base pairing. For example, m1Ψ (modified from U) pairs more strongly with A than U pairs with A, and m1Ψ-G pairs even more strongly than m1Ψ-A pairs. Thus, by replacing canonical nucleotides with non-canonical base pairing nucleotides, the base pairing strength can be altered, thereby altering the Tm of the antisense strand duplex and/or the Tm of hybridization between the antisense strand and the target RNA (e.g., INHBE mRNA). Thus, in some embodiments, canonical nucleotides are replaced with non-canonical base pairing nucleotides, thereby altering the Tm of an oligonucleotide (e.g., altering the calculated Tm of an oligonucleotide, altering the Tm of a resulting double-stranded RNA molecule, such as the Tm of a duplex formed by the antisense strand hybridized with a target mRNA and/or a sense strand). Without wishing to be limited by theory, by replacing at least one canonical nucleotide with non-canonical base pairing nucleotides to change the Tm of the oligonucleotide, the efficacy, effectiveness, specificity, safety and/or off-target effects of dsRNAi can be regulated and controlled. For example, but not limited to, the efficacy or effectiveness of dsRNAi agents can be increased by increasing the pairing strength with the desired target mRNA and/or reducing the pairing with the mRNA that misses the target. Similarly, undesirable off-target effects can be reduced by increasing the pairing strength with the desired target mRNA and/or reducing the pairing strength with the mRNA that misses the target. Therefore, in some embodiments, compared with similar RNAi agents that do not include at least one non-canonical base pairing nucleotide, RNAi agents of the present disclosure have improved efficacy, effectiveness, specificity and/or safety.

在本公开的dsRNAi剂、反义链和正义链的某些实施方式中，至少一个规范核苷酸被非规范碱基配对核苷酸替换。在一些此类实施方式中，一个核苷酸被非规范碱基配对核苷酸替代，即dsRNAi剂、反义链或正义链包含一个非规范碱基配对核苷酸。在一些实施方式中，两个、三个、四个、五个或更多个核苷酸被非规范碱基配对核苷酸替代，即dsRNAi剂、反义链或正义链包含两个、三个、四个、五个或更多个非规范碱基配对核苷酸。在一些实施方式中，寡核苷酸或dsRNAi剂中的5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或更多的核苷酸是修饰的核苷酸，例如非规范碱基配对核苷酸。在一些实施方式中，dsRNAi剂中，例如正义链和/或反义链中的所有核苷酸，都是修饰的核苷酸，例如非规范碱基配对核苷酸。在一些实施方式中，dsRNAi剂中，例如正义链和/或反义链中的至少一个核苷酸，是修饰的核苷酸，例如非规范碱基配对核苷酸。In certain embodiments of the dsRNAi agents, antisense strands, and sense strands of the present disclosure, at least one canonical nucleotide is replaced by a non-canonical base-pairing nucleotide. In some such embodiments, one nucleotide is replaced by a non-canonical base-pairing nucleotide, i.e., the dsRNAi agent, antisense strand, or sense strand comprises one non-canonical base-pairing nucleotide. In some embodiments, two, three, four, five, or more nucleotides are replaced by non-canonical base-pairing nucleotides, i.e., the dsRNAi agent, antisense strand, or sense strand comprises two, three, four, five, or more non-canonical base-pairing nucleotides. In some embodiments, 5%, 10%, 15%, 20%, 25%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 10 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the nucleotides are modified nucleotides, such as non-canonical base pairing nucleotides. In some embodiments, all nucleotides in the dsRNAi agent, such as the sense strand and/or the antisense strand, are modified nucleotides, such as non-canonical base pairing nucleotides. In some embodiments, at least one nucleotide in the dsRNAi agent, such as the sense strand and/or the antisense strand, is a modified nucleotide, such as a non-canonical base pairing nucleotide.

因此，在某些实施方式中，本公开的RNAi剂包含至少一种非规范碱基配对核苷酸，即，反义链和/或正义链中的至少一个核苷酸被非规范碱基配对核苷酸替换。在一些此类实施方式中，至少一种非规范碱基配对核苷酸存在于反义链上。在一些此类实施方式中，至少一种非规范碱基配对核苷酸存在于正义链上。在一些此类实施方式中，至少一种非规范碱基配对核苷酸同时存在于反义链和正义链上。在一些实施方式中，至少一种非规范碱基配对核苷酸存在于互补性区域中，即，寡核苷酸中与靶序列基本上互补的区域，例如，本公开的反义链中与靶序列(例如，INHBE mRNA)互补的区域。Thus, in certain embodiments, the RNAi agents of the present disclosure comprise at least one non-canonical base pairing nucleotide, i.e., at least one nucleotide in the antisense strand and/or the sense strand is replaced by a non-canonical base pairing nucleotide. In some such embodiments, at least one non-canonical base pairing nucleotide is present on the antisense strand. In some such embodiments, at least one non-canonical base pairing nucleotide is present on the sense strand. In some such embodiments, at least one non-canonical base pairing nucleotide is present on both the antisense strand and the sense strand. In some embodiments, at least one non-canonical base pairing nucleotide is present in a complementary region, i.e., a region in an oligonucleotide that is substantially complementary to a target sequence, e.g., a region in an antisense strand of the present disclosure that is complementary to a target sequence (e.g., INHBE mRNA).

在某些实施方式中，用非规范碱基配对核苷酸替换至少一个规范核苷酸改变了寡核苷酸或dsRNA双链体的解链温度(Tm)。在一些实施方式中，Tm改变至少2℃(即，ΔTm为至少约2℃)。在一些实施方式中，ΔTm为约2℃。在一些实施方式中，ΔTm超过2℃。在一些实施方式中，ΔTm为约2.5℃、3℃、3.5℃、4℃、4.5℃或5℃。In certain embodiments, replacing at least one canonical nucleotide with a non-canonical base pairing nucleotide changes the melting temperature (Tm) of the oligonucleotide or dsRNA duplex. In some embodiments, the Tm changes by at least 2°C (i.e., the ΔTm is at least about 2°C). In some embodiments, the ΔTm is about 2°C. In some embodiments, the ΔTm exceeds 2°C. In some embodiments, the ΔTm is about 2.5°C, 3°C, 3.5°C, 4°C, 4.5°C, or 5°C.

在某些实施方式中，至少一种非规范碱基配对核苷酸存在于反义链从5'端到3'端方向的第1-11位处，例如在第1、2、3、4、5、6、7、8、9、10和/或11位处。In certain embodiments, at least one non-canonical base pairing nucleotide is present at positions 1-11, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and/or 11, from the 5' to the 3' direction of the antisense strand.

在某些实施方式中，至少一种非规范碱基配对核苷酸存在于反义链从5'端到3'端方向的第12-21位处，例如在第12、13、14、15、16、17、18、19、20和/或21位处。In certain embodiments, at least one non-canonical base pairing nucleotide is present at positions 12-21, such as 12, 13, 14, 15, 16, 17, 18, 19, 20 and/or 21, from the 5' to the 3' direction of the antisense strand.

在本公开的寡核苷酸和dsRNAi剂的某些实施方式中，从5'端到3'端的方向，如果反义链的第2-8位中的至少四个核苷酸是A或U并且第6-8位的核苷酸中的至少一个是G，则第6、7和/或8位处的G被非规范碱基配对核苷酸替换。In certain embodiments of the oligonucleotides and dsRNAi agents of the present disclosure, in the direction from the 5' end to the 3' end, if at least four nucleotides in positions 2-8 of the antisense strand are A or U and at least one of the nucleotides in positions 6-8 is G, then the G at positions 6, 7 and/or 8 is replaced by a non-canonical base pairing nucleotide.

在本公开的寡核苷酸和dsRNAi剂的某些实施方式中，序列5′-N₁N₂G₀N₃N₄ -3′中，当N₁、N₂、N₃和N₄中的至少三个碱基是腺嘌呤或尿嘧啶时，G₀中的鸟嘌呤被非规范碱基例如次黄嘌呤替换，其中，N₁、N₂、N₃和N₄各自独立地为包含腺嘌呤(A)、胞嘧啶(C)、鸟嘌呤(G)或尿嘧啶(U)的核苷酸，G₀是包含鸟嘌呤的核苷酸。In certain embodiments of the oligonucleotides and dsRNAi agents of the present disclosure, in the sequence 5′-N₁ N₂ G₀ N₃ N₄ -3′, when at least three bases among N₁ , N₂ , N₃ and N₄ are adenine or uracil, the guanine in G₀ is replaced by a non-canonical base, such as hypoxanthine, wherein N₁ , N₂ , N₃ and N₄ are each independently a nucleotide comprising adenine (A), cytosine (C), guanine (G) or uracil (U), and G₀ is a nucleotide comprising guanine.

应当理解，本文公开的或本领域已知的所有非规范碱基配对核苷酸或非规范碱基都适合用于本公开的RNAi剂、组合物和方法。非规范碱基配对核苷酸(或核苷)或非规范碱基的非限制性实例如表B中所示定义。在一些实施方式中，非规范碱基配对核苷(或核苷酸)或非规范碱基是表B中所示的修饰基团。在一些实施方式中，非规范碱基是次黄嘌呤。在一些实施方式中，非规范碱基配对核苷是肌苷。在一些实施方式中，非规范碱基配对核苷酸是能够摆动配对的规范核苷酸。前述的组合也涵盖在内。It should be understood that all non-canonical base pairing nucleotides or non-canonical bases disclosed herein or known in the art are suitable for use in the RNAi agents, compositions and methods disclosed herein. Non-limiting examples of non-canonical base pairing nucleotides (or nucleosides) or non-canonical bases are defined as shown in Table B. In some embodiments, non-canonical base pairing nucleosides (or nucleotides) or non-canonical bases are the modifying groups shown in Table B. In some embodiments, the non-canonical base is hypoxanthine. In some embodiments, the non-canonical base pairing nucleosides are inosine. In some embodiments, the non-canonical base pairing nucleotides are canonical nucleotides that can swing pairing. The aforementioned combinations are also encompassed.

表B根据某些实施方式的非规范碱基配对核苷(或核苷酸)、非规范碱基的示例

Table B Non-canonical base-paired nucleosides (or nucleotides), examples of non-canonical bases according to certain embodiments

术语“经修饰的核苷酸”是指任意的独立地具有经修饰的糖部分、经修饰的核苷酸间键联和/或经修饰的核碱基的核苷酸。因此，该术语“经修饰的核苷酸”涵盖核苷间键联、糖部分或核碱基的例如一个官能团或原子的取代、添加或去除。适用于本公开的修饰包括在此披露的或本领域中已知的所有类型的修饰。在一些实施方式中，修饰的核苷酸是本文定义的非规范碱基配对核苷酸，即，具有与其取代的规范核苷酸不同的配对特征的核苷酸。在其他实施方式中，修饰的核苷酸可以不具有不同的配对特征，但仍然可以拥有本公开的RNAi剂、组合物及方法所期望的或有利的特征，例如稳定性、抗降解性(例如，抗核酸酶)、可制造性等。在某些实施方式中，本公开的RNAi剂除了包含至少一个非规范碱基配对核苷酸之外，还包含至少一个修饰的核苷酸，即，除了包含至少一个非规范碱基配对核苷酸之外，在反义链和/或正义链中还包含至少一个另外的核苷酸被额外的修饰核苷酸(其可以是也可以不是非规范碱基配对核苷酸)替代。在一些此类实施方式中，至少一个另外的修饰核苷酸存在于反义链上。在一些这样的实施方式中，至少一个另外的修饰的核苷酸存在于正义链上。在一些此类实施方式中，至少一个另外的修饰核苷酸存在于反义链和正义链上。在一些这样的实施方式中，至少一个另外的修饰核苷酸与至少一个非规范碱基配对核苷酸存在于相同的链上。在其他实施方式中，至少一个另外的修饰核苷酸与至少一个非规范碱基配对核苷酸不存在于相同的链上，即，存在于另一条链上。在一些实施方式中，至少一个另外的修饰核苷酸存在于互补性区域中，即，寡核苷酸中与靶序列基本上互补的区域，例如，本公开的反义链中与靶序列mRNA(例如，INHBE mRNA)互补的区域。The term "modified nucleotide" refers to any independently modified sugar moiety, modified internucleotide linkage and/or modified nucleobase nucleotide. Therefore, the term "modified nucleotide" encompasses substitution, addition or removal of, for example, a functional group or atom of a nucleoside linkage, a sugar moiety or a nucleobase. Modifications applicable to the present disclosure include all types of modifications disclosed herein or known in the art. In some embodiments, the modified nucleotide is a non-canonical base pairing nucleotide defined herein, that is, a nucleotide having a pairing feature different from the canonical nucleotide replaced by it. In other embodiments, the modified nucleotide may not have different pairing features, but may still have the desired or advantageous features of the RNAi agent, composition and method disclosed herein, such as stability, degradation resistance (e.g., anti-nuclease), manufacturability, etc. In certain embodiments, the RNAi agent of the present disclosure comprises at least one modified nucleotide in addition to at least one non-canonical base pairing nucleotide, i.e., in addition to at least one non-canonical base pairing nucleotide, at least one additional nucleotide in the antisense strand and/or sense strand is replaced by an additional modified nucleotide (which may or may not be a non-canonical base pairing nucleotide). In some such embodiments, at least one additional modified nucleotide is present on the antisense strand. In some such embodiments, at least one additional modified nucleotide is present on the sense strand. In some such embodiments, at least one additional modified nucleotide is present on both the antisense strand and the sense strand. In some such embodiments, at least one additional modified nucleotide is present on the same strand as the at least one non-canonical base pairing nucleotide. In other embodiments, at least one additional modified nucleotide is not present on the same strand as the at least one non-canonical base pairing nucleotide, i.e., is present on another strand. In some embodiments, at least one additional modified nucleotide is present in a complementary region, i.e., a region in an oligonucleotide that is substantially complementary to a target sequence, e.g., a region in an antisense strand of the present disclosure that is complementary to a target sequence mRNA (e.g., INHBE mRNA).

在本公开中，常见修饰核苷酸和相关部分的非限制性实例如表C中所示定义。在一些实施方式中，修饰核苷酸是表C中所示的修饰核苷酸。在一些实施方式中，本公开的RNAi剂的至少一个另外的修饰核苷酸是表C中所示的修饰核苷酸。In the present disclosure, non-limiting examples of common modified nucleotides and related moieties are defined as shown in Table C. In some embodiments, the modified nucleotide is a modified nucleotide shown in Table C. In some embodiments, at least one additional modified nucleotide of the RNAi agent of the present disclosure is a modified nucleotide shown in Table C.

表C.一些实施方式中的修饰核苷酸的定义。
Table C. Definition of modified nucleotides in some embodiments.

其中，倒置核苷酸的修饰模式在一些文献中又称反转碱基，是指具有由正常的5'至3'键联反转的键联的那些碱基(即5'至5'键联或3'至3'键联)。Among them, the modification pattern of inverted nucleotides is also called inverted bases in some literatures, which refers to those bases with linkages reversed from the normal 5' to 3' linkages (ie, 5' to 5' linkages or 3' to 3' linkages).

在一些实施方式中，包含脱氧核苷酸可以被认为构成修饰的核苷酸。In some embodiments, the inclusion of deoxynucleotides may be considered to constitute modified nucleotides.

应当理解，本文公开的或本领域已知的所有类型的修饰都适用于本公开的RNAi剂、组合物和方法。It should be understood that all types of modifications disclosed herein or known in the art are applicable to the RNAi agents, compositions and methods of the present disclosure.

本公开所用的术语“衍生物”应理解为是结构上类似，在一些细微结构上不同的另一种化合物。The term "derivative" as used in the present disclosure should be understood as another compound that is similar in structure but different in some minor structures.

术语“抑制”和诸如此类的表述是指降低或有效地停止，可以与“减少”、“沉默”、“下调”、“遏制”和其他类似术语交替使用，并且包括任何水平的抑制。作为非限制性实例，本文的“抑制”是指降低或有效地降低受试者的代谢紊乱或相关疾病的发作或进展，包括在疾病的一个或多个方面(例如症状、组织特征、细胞活性、炎症活性或免疫活性等)的减少、检测不到恶化。The term "inhibit" and the like refers to reducing or effectively stopping, and can be used interchangeably with "reduce", "silence", "downregulate", "suppress" and other similar terms, and includes any level of inhibition. As a non-limiting example, "inhibit" herein refers to reducing or effectively reducing the onset or progression of a metabolic disorder or related disease in a subject, including a reduction in one or more aspects of the disease (e.g., symptoms, tissue characteristics, cell activity, inflammatory activity or immune activity, etc.), and no detectable deterioration.

如本文所用，基因(例如INHBE)的“抑制表达”是指与适当参考(例如参考细胞、细胞群体、样本或受试者)相比，由所述基因编码的RNA转录物(例如INHBE mRNA)或蛋白质的量或水平降低和/或所述基因在细胞、细胞群体、样品或受试者中的活性量或水平降低。如本文所用，“抑制INHBE表达”是指与适当参考(例如参考细胞、细胞群体、样品或受试者)相比，细胞、细胞群体、样品或受试者中INHBE mRNA和/或激活素E的量或水平降低，如抑制至少约5％、至少约10％、至少约15％、至少约20％、至少约25％、至少约30％、至少约35％、至少约40％、至少约45％、至少约50％、至少约55％、至少约60％、至少约65％、至少约70％、至少约75％、至少约80％、至少约85％、至少约90％、至少约91％、至少约92％、至少约93％、至少约94％、至少约95％、至少约96％、至少约97％、至少约98％、或至少约99％。As used herein, "inhibited expression" of a gene (e.g., INHBE) means that the amount or level of RNA transcript (e.g., INHBE mRNA) or protein encoded by the gene is reduced and/or the amount or level of activity of the gene in a cell, cell population, sample or subject is reduced compared to an appropriate reference (e.g., a reference cell, cell population, sample or subject). As used herein, "inhibiting INHBE expression" refers to a decrease in the amount or level of INHBE mRNA and/or activin E in a cell, cell population, sample or subject, as compared to an appropriate reference (e.g., a reference cell, cell population, sample or subject), such as an inhibition of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

如本文所用，短语“使细胞与RNAi剂接触”(例如dsRNAi剂)，包括通过任何可能的方式接触细胞。使细胞与RNAi剂接触包括使细胞在体外与RNAi剂接触或使细胞在体内与RNAi剂接触。该接触可以直接或间接进行。因此，例如，可将RNAi剂与细胞物理接触，或者可替代地，可将RNAi剂置于将允许或导致其随后其与细胞接触的情况中。体外接触细胞可以通过例如将细胞与RNAi剂一起温育来进行。体内接触细胞可以例如通过将RNAi剂注射到细胞所在的组织中或附近，或通过将RNAi剂注射到另一区域(例如血流或皮下空间)中来进行，使得RNAi剂随后到达待接触细胞所在的组织。例如，RNAi剂可以含有或偶联至靶向配体，例如GalNAc，其将RNAi剂引导至感兴趣的位点，例如肝脏。在其他实施方式中，RNAi剂可以含有或偶联至一种或多种C22烃链和一种或多种GalNAc衍生物。在其他实施方式中，RNAi剂含有或偶联至一种或多种C22烃链，并且不含有或未偶联至一种或多种GalNAc衍生物。体外和体内接触方法的组合也是可能的。例如，细胞还可以在体外与本公开的RNAi剂接触并且随后移植到受试者中。在某些实施方式中，使细胞与RNAi剂接触包括促进或影响细胞的摄取或吸收。RNAi剂的吸收或摄取可以通过无辅助扩散或活性细胞过程，或者通过辅助剂或装置来发生。将RNAi剂引入细胞可以在体外或体内进行。例如，对于体内引入，可以将RNAi剂注射到组织部位或全身施用。体外引入细胞包括本领域已知的方法，例如电穿孔和脂转染。As used herein, the phrase "contacting a cell with an RNAi agent" (e.g., a dsRNAi agent) includes contacting a cell by any possible means. Contacting a cell with an RNAi agent includes contacting a cell with an RNAi agent in vitro or contacting a cell with an RNAi agent in vivo. The contact can be performed directly or indirectly. Therefore, for example, the RNAi agent can be physically contacted with the cell, or alternatively, the RNAi agent can be placed in a situation where it will allow or cause it to subsequently contact the cell. Contacting cells in vitro can be performed by, for example, incubating the cell with the RNAi agent. Contacting cells in vivo can be performed, for example, by injecting the RNAi agent into or near the tissue where the cell is located, or by injecting the RNAi agent into another area (e.g., bloodstream or subcutaneous space) so that the RNAi agent subsequently reaches the tissue where the cell is to be contacted. For example, the RNAi agent can contain or be coupled to a targeting ligand, such as GalNAc, which guides the RNAi agent to a site of interest, such as the liver. In other embodiments, the RNAi agent can contain or be coupled to one or more C22 hydrocarbon chains and one or more GalNAc derivatives. In other embodiments, the RNAi agent contains or is coupled to one or more C22 hydrocarbon chains, and does not contain or is not coupled to one or more GalNAc derivatives. A combination of in vitro and in vivo contact methods is also possible. For example, cells can also be contacted with RNAi agents disclosed in vitro and subsequently transplanted into a subject. In certain embodiments, contacting cells with RNAi agents includes promoting or affecting the uptake or absorption of cells. The absorption or uptake of RNAi agents can occur by unassisted diffusion or active cell processes, or by adjuvants or devices. Introducing RNAi agents into cells can be performed in vitro or in vivo. For example, for in vivo introduction, RNAi agents can be injected into tissue sites or systemically administered. In vitro introduction of cells includes methods known in the art, such as electroporation and lipofection.

“受试者”包括任何人或非人动物。术语“非人动物”包括所有脊椎动物，例如哺乳动物和非哺乳动物，诸如非人灵长类动物、绵羊、狗、猫、马、牛、鸡、两栖动物、爬行动物等。在某些实施方式中，所述受试者是人。"Subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. In certain embodiments, the subject is a human.

在一些实施方式中，任何疾病或病症的“治疗”是指改善至少一种疾病或病症。在某些实施方式中，“治疗”是指改善至少一种身体参数，其可以或可以不被患者察觉。在某些实施方式中，“治疗”是指在物理上(例如，明显症状的稳定)、在生理上(例如，物理参数的稳定)或两方面上的抑制疾病或病症。在一些实施方式中，“治疗”是指改善有需要的受试者的生活质量或减少疾病的症状或副作用。“治疗有效量”是指dsRNA或dsRNAi剂当单独或与其它治疗药物组合给予细胞、组织或受试者时，足以实现疾病的治疗或预防的dsRNA或dsRNAi剂的量。“治疗有效量”将根据化合物或RNAi剂、疾病及其严重程度、以及患有待治疗或预防的疾病的受试者的年龄、体重等而变化。本文所用的术语“治疗有效量”是指足以预防、治疗、抑制、减少、改善或消除疾病或病症的一种或多种原因、症状或并发症的化合物或组合物的量，所述疾病或病症例如，代谢综合征。术语“有效量”和“治疗有效量”在本文中可互换使用。当对个体施用单独给予的活性成分时，治疗有效剂量仅是指该成分。当组合施用时，治疗有效剂量是指引起治疗效果的活性成分的综合量，不论是组合、依次给予还是同时给予。治疗剂的有效量将导致诊断标准或参数提高至少10％，通常至少20％，优选至少约30％，更优选至少40％，最优选至少50％。In some embodiments, "treatment" of any disease or condition refers to the improvement of at least one disease or condition. In certain embodiments, "treatment" refers to the improvement of at least one physical parameter, which may or may not be perceived by the patient. In certain embodiments, "treatment" refers to the inhibition of a disease or condition physically (e.g., stabilization of overt symptoms), physiologically (e.g., stabilization of a physical parameter), or both. In some embodiments, "treatment" refers to improving the quality of life of a subject in need or reducing the symptoms or side effects of a disease. "Therapeutically effective amount" refers to the amount of a dsRNA or dsRNAi agent sufficient to achieve treatment or prevention of a disease when the dsRNA or dsRNAi agent is administered alone or in combination with other therapeutic agents to a cell, tissue, or subject. The "therapeutically effective amount" will vary depending on the compound or RNAi agent, the disease and its severity, and the age, weight, etc. of the subject suffering from the disease to be treated or prevented. The term "therapeutically effective amount" as used herein refers to an amount sufficient to prevent, treat, inhibit, reduce, improve, or eliminate a disease or condition. The term "effective amount" and "therapeutically effective amount" are used interchangeably herein. When a single active ingredient is administered to an individual, the therapeutically effective dose refers only to that ingredient. When administered in combination, the therapeutically effective dose refers to the combined amount of active ingredients that cause the therapeutic effect, whether in combination, administered sequentially or simultaneously. The effective amount of the therapeutic agent will result in an increase of at least 10%, typically at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50% in the diagnostic criteria or parameters.

在一些实施方式中，任何疾病或病症的“预防”或“防止”，是指至少降低获得疾病或病症的风险(或易感性)的可能性(即，使得至少一种疾病的临床症状不会在可能接触或易患该疾病但尚未经历或表现出该疾病症状的患者中出现)。当使用在关于可从INHBE表达的降低而受益的疾病、障碍或其病状时，意指降低受试者将发展出与此种疾病、障碍或病状有关的症状的可能性，如，代谢综合征。没有发展一种疾病、障碍或病症，或者减少发展与该疾病、障碍或病症相关的症状(例如在临床上接受的比例上，针对该疾病或病症减少至少约10％)，或表现出症状延迟(如延迟数天、周、月或年)被认为是有效的预防。In some embodiments, "prevention" or "prevention" of any disease or condition refers to at least reducing the likelihood of the risk (or susceptibility) of acquiring the disease or condition (i.e., so that at least one clinical symptom of the disease does not occur in a patient who may be exposed to or susceptible to the disease but does not yet experience or show symptoms of the disease). When used in reference to a disease, disorder, or condition that would benefit from a reduction in INHBE expression, it means reducing the likelihood that a subject will develop symptoms associated with such a disease, disorder, or condition, such as metabolic syndrome. Failure to develop a disease, disorder, or condition, or a reduction in the development of symptoms associated with the disease, disorder, or condition (e.g., a reduction of at least about 10% on a clinically accepted scale for the disease or condition), or a delay in the onset of symptoms (e.g., a delay of days, weeks, months, or years) is considered effective prevention.

本文中，所用短语“药学上可接受的”是指在合理的医学判断范围内适合与人类受试者和动物受试者的组织接触的那些化合物、材料、组合物或剂型，其无过度毒性、刺激、过敏反应或其他问题或并发症，与合理的效益/风险比相称。As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human and animal subjects without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit/risk ratio.

术语“药学上可接受的载体”是指药学上可接受的材料、组合物或媒介物，如液体或固体填充剂、稀释剂、赋形剂、制造助剂(例如润滑剂、滑石镁、硬脂酸钙或硬脂酸锌、或硬脂酸)、或溶剂封装材料(涉及将化合物或dsRNAi剂从身体的一个器官或部分携带或运输到身体的另一个器官或部分)。The term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., a lubricant, magnesium talc, calcium or zinc stearate, or stearic acid), or solvent encapsulating material (involved in carrying or transporting a compound or dsRNAi agent from one organ or part of the body to another organ or part of the body).

二、RNA干扰2. RNA Interference

本公开提供了通过RNA干扰(RNAi)过程抑制代谢病症相关靶基因INHBE的表达的RNA(siRNA或RNAi)剂。在一些实施方式中，RNAi剂包含用于抑制细胞(例如脂肪细胞和/或肝脏细胞，例如肝实质细胞)中INHBE基因表达的双链核糖核酸(dsRNA)分子。包含dsRNA分子的siRNA或RNA剂在本文中也被称为“dsRNAi”剂。在某些实施方案中，所述细胞存在于受试者体内。在一些实施方案中，受试者是哺乳动物，例如人。在一些实施方案中，所述受试者已经患有或易患代谢类病症(例如代谢综合征)、碳水化合物病症(例如2型糖尿病、前驱糖尿病)、脂质代谢障碍(例如高脂血症、高血压、脂肪代谢障碍)、肾脏疾病、心血管疾病，和/或体重失调(例如肥胖、超重)。dsRNAi剂包括具有互补区域的反义链，该互补区域与INHBE基因的表达中形成的mRNA的至少一部分互补。在一些实施方式中，互补区域的长度为约19-30个核苷酸(例如，长度为约30、29、28、27、26、25、24、23、22、21、20或19个核苷酸)。在一些实施方式中，互补区域的长度为约15-30个核苷酸(例如，长度为约30、29、28、27、26、25、24、23、22、21、20、19、18、17、16、或15个核苷酸)。在某些实施方式中，dsRNAi剂包含至少一种修饰的核苷酸，如本文所述。在某些实施方式中，dsRNAi剂包含至少一个非规范碱基配对核苷酸，如本文所述。The present disclosure provides RNA (siRNA or RNAi) agents that inhibit the expression of metabolic disorder-related target gene INHBE by RNA interference (RNAi) process. In some embodiments, the RNAi agent comprises a double-stranded ribonucleic acid (dsRNA) molecule for inhibiting the expression of INHBE gene in cells (e.g., adipocytes and/or liver cells, e.g., hepatocytes). The siRNA or RNA agent comprising a dsRNA molecule is also referred to herein as a "dsRNAi" agent. In certain embodiments, the cell is present in a subject. In some embodiments, the subject is a mammal, e.g., a human. In some embodiments, the subject has suffered from or is susceptible to metabolic disorders (e.g., metabolic syndrome), carbohydrate disorders (e.g., type 2 diabetes, prediabetes), lipid metabolism disorders (e.g., hyperlipidemia, hypertension, lipodystrophy), kidney disease, cardiovascular disease, and/or weight disorders (e.g., obesity, overweight). The dsRNAi agent includes an antisense strand having a complementary region, which is complementary to at least a portion of the mRNA formed in the expression of the INHBE gene. In some embodiments, the length of the complementary region is about 19-30 nucleotides (e.g., a length of about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, or 19 nucleotides). In some embodiments, the length of the complementary region is about 15-30 nucleotides (e.g., a length of about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, or 15 nucleotides). In certain embodiments, the dsRNAi agent comprises at least one modified nucleotide, as described herein. In certain embodiments, the dsRNAi agent comprises at least one non-canonical base pairing nucleotide, as described herein.

根据本公开的方法，表达靶基因(例如，人、灵长类、非灵长类或小鼠INHBE基因)的细胞与抑制靶基因表达的siRNA剂接触。在一些实施方式中，靶基因的表达被抑制至少约50％。靶基因表达的抑制可以使用任何合适的方法来确定，例如但不限于，通过基于PCR或分支DNA(bDNA)的方法，或通过基于蛋白质的方法，例如通过免疫荧光分析，使用例如蛋白质印迹或流式细胞术技术测定。在某些实施方案中，表达的抑制通过本文实施例中提供的rt-PCR方法(例如，下文的实施例中所述，使用例如10nM浓度的siRNA在合适的生物体细胞系中)测定。在某些实施方案中，使用动物模型来测定体内表达的抑制，例如通过在表达人类基因的啮齿动物(例如表达INHBE基因的小鼠)中的人类基因的敲低来确定。在一些这样的实施方式中，siRNA以单剂量(例如3mg/kg、5mg/kg、6mg/kg或9mg/kg)被施用与受试者(例如，动物模型)。According to the methods disclosed herein, cells expressing a target gene (e.g., human, primate, non-primate or mouse INHBE gene) are contacted with a siRNA agent that inhibits target gene expression. In some embodiments, the expression of the target gene is inhibited by at least about 50%. The inhibition of target gene expression can be determined using any suitable method, such as, but not limited to, by a PCR-based or branched DNA (bDNA) method, or by a protein-based method, such as by immunofluorescence analysis, using, for example, Western blotting or flow cytometry techniques. In certain embodiments, the inhibition of expression is determined by the rt-PCR method provided in the examples herein (e.g., as described in the examples below, using, for example, 10 nM concentrations of siRNA in a suitable organism cell line). In certain embodiments, animal models are used to measure In some embodiments, the siRNA is administered to a subject (e.g., an animal model) at a single dose (e.g., 3 mg/kg, 5 mg/kg, 6 mg/kg, or 9 mg/kg).

dsRNA包括两条RNA链，这两条RNA链是互补的并且在dsRNA将被使用的条件下(例如在生理条件下)杂交形成双链体结构。dsRNA的一条链(反义链)包括与靶序列基本上互补并且通常完全互补的互补区域。靶序列可源自INHBE基因表达期间形成的mRNA序列。另一条链(正义链)包含与反义链互补的区域，使得两条链在合适的条件下组合时，它们将杂交并形成双链体结构。如本文别处所述和本领域已知的，dsRNA的互补序列可以包含在单个核酸分子的自互补区域中，而不是在单独的寡核苷酸上。一般而言，双链体结构的长度为15至30个碱基对，例如长度为15-29、15-28、15-27、15-26、15-25、15-24、15-23、15-22、15-21、15-20、15-19、15-18、15-17、18-30、18-29、18-28、18-27、18-26、18-25、18-24、18-23、18-22、18-21、18-20、19-30、19-29、19-28、19-27、19-26、19-25、19-24、19-23、19-22、19-21、19-20、20-30、20-29、20-28、20-27、20-26、20-25、20-24、20-23、20-22、20-21、21-30、21-29、21-28、21-27、21-26、21-25、21-24、21-23或21-22个碱基对。在某些实施方案中，双链体结构的长度是17至25个碱基对，例如长度为17-23、17-25、18-25、18-24、18-23、18-22、18-21、18-20、19-25、19-24、19-23、19-22、19-21、19-20、20-25、20-24、20-23、20-22、20-21、21-25、21-24、21-23、21-22、22-25、22-24、22-23、23-25、23-24或24-25个碱基对，例如长度为19-21个碱基对。上述范围和长度中间的范围和长度也被认为是本公开的一部分。dsRNA includes two RNA chains, which are complementary and hybridize to form a duplex structure under the conditions (e.g., under physiological conditions) under which the dsRNA will be used. One chain (the antisense chain) of the dsRNA includes a complementary region that is substantially complementary to the target sequence and is usually completely complementary. The target sequence may be derived from an mRNA sequence formed during the expression of the INHBE gene. The other chain (the positive chain) includes a region complementary to the antisense chain so that when the two chains are combined under suitable conditions, they will hybridize and form a duplex structure. As described elsewhere herein and known in the art, the complementary sequence of the dsRNA may be contained in a self-complementary region of a single nucleic acid molecule, rather than on a separate oligonucleotide. Generally, the duplex structure is 15 to 30 base pairs in length, for example, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. In certain embodiments, the length of the duplex structure is 17 to 25 base pairs, such as 17-23, 17-25, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-25, 20-24, 20-23, 20-22, 20-21, 21-25, 21-24, 21-23, 21-22, 22-25, 22-24, 22-23, 23-25, 23-24 or 24-25 base pairs in length, such as 19-21 base pairs in length. Ranges and lengths intermediate to the above ranges and lengths are also considered part of the present disclosure.

类似地，与靶序列互补的区域长度为15至30个核苷酸，例如长度为15-29、15-28、15-27、15-26、15-25、15-24、15-23、15-22、15-21、15-20、15-19、15-18、15-17、18-30、18-29、18-28、18-27、18-26、18-25、18-24、18-23、18-22、18-21、18-20、19-30、19-29、19-28、19-27、19-26、19-25、19-24、19-23、19-22、19-21、19-20、20-30、20-29、20-28、20-27、20-26、20-25、20-24,20-23、20-22、20-21、21-30、21-29、21-28、21-27、21-26、21-25、21-24、21-23或21-22个核苷酸，例如长度为19-23个核苷酸或长度为21-23个核苷酸。上述范围和长度中间的范围和长度也被认为是本公开的一部分。Similarly, the region complementary to the target sequence is 15 to 30 nucleotides in length, for example, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 21-23 nucleotides in length or 21-23 nucleotides in length. Ranges and lengths intermediate to the above ranges and lengths are also considered to be part of the present disclosure.

在一些实施方案中，双链体结构的长度为19至30个碱基对。类似地，与靶序列互补的区域的长度为19至30个核苷酸。In some embodiments, the length of the duplex structure is 19 to 30 base pairs. Similarly, the length of the region complementary to the target sequence is 19 to 30 nucleotides.

在一些实施方案中，双链体结构的长度为15至23个碱基对。类似地，与靶序列互补的区域的长度为15至23个核苷酸。In some embodiments, the length of the duplex structure is 15 to 23 base pairs. Similarly, the length of the region complementary to the target sequence is 15 to 23 nucleotides.

在一些实施方案中，dsRNA的长度为约19至约23个核苷酸对，或约25至约30个核苷酸。在一些实施方案中，dsRNA的长度为约15至约23个核苷酸、或约17至约23个核苷酸、或约17至约25个核苷酸、或约19至约21个核苷酸。In some embodiments, the length of the dsRNA is about 19 to about 23 nucleotide pairs, or about 25 to about 30 nucleotides. In some embodiments, the length of the dsRNA is about 15 to about 23 nucleotides, or about 17 to about 23 nucleotides, or about 17 to about 25 nucleotides, or about 19 to about 21 nucleotides.

双链体区域是dsRNA的主要功能部分，例如，约15至约30个碱基对、或约17至约30个碱基对、或约19至约30个碱基对的双链体区域，例如约15-23、15-25、17-25、17-23、19-30、19-29、19-28、19-27、19-26、19-25、19-24、19-23、19-22、19-21、19-20、20-30、20-29、20-28、20-27、20 -26、20-25、20-24、20-23、20-22、20-21、21-30、21-29、21-28、21-27、21-26、21-25、21-24、21-23或21-22个碱基对的双链体区域。因此，在一个实施方案中，在dsRNA能被加工成功能性双链体(例如15-30个碱基对或至少15个碱基对)的程度上，其中该功能性双链体可靶向所需进行切割的RNA，dsRNA可以是具有大于30个碱基对的双链体区域的RNA分子或RNA分子复合物。因此，在一个实施方案中，dsRNA是miRNA。在另一个实施方案中，dsRNA不是天然存在的miRNA。在另一个实施方案中，可用于靶向INHBE基因表达的siRNA剂不是通过较大的dsRNA的切割在靶细胞中产生的。The duplex region is the major functional portion of the dsRNA, e.g., a duplex region of about 15 to about 30 base pairs, or about 17 to about 30 base pairs, or about 19 to about 30 base pairs, e.g., about 15-23, 15-25, 17-25, 17-23, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-28 In one embodiment, the dsRNA can be processed into a functional duplex (e.g., 15-30 base pairs or at least 15 base pairs), wherein the functional duplex can target the RNA required for cleavage, and the dsRNA can be an RNA molecule or RNA molecule complex having a duplex region of greater than 30 base pairs. Therefore, in one embodiment, the dsRNA is a miRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In another embodiment, the siRNA agent that can be used to target the expression of the INHBE gene is not produced in the target cell by cleavage of a larger dsRNA.

如本文所述的dsRNA还可包括一个或多个单链核苷酸突出端，例如1-4、2-4、1-3、2-3、1、2、3或4个核苷酸。具有至少一个核苷酸突出端的dsRNA相对于其平端对应物具有优异的抑制特性。核苷酸突出端可包含核苷酸/核苷类似物或由其组成，所述核苷酸/核苷类似物包括脱氧核苷酸/核苷。突出端可以位于正义链、反义链或其任意组合上。此外，突出端的核苷酸可以存在于dsRNA的反义链或有义链的5'-末端、3'-末端或两端上。DsRNA as described herein may also include one or more single-stranded nucleotide overhangs, such as 1-4, 2-4, 1-3, 2-3, 1, 2, 3 or 4 nucleotides. The dsRNA with at least one nucleotide overhang has excellent inhibitory properties relative to its flat-ended counterpart. The nucleotide overhangs may include or consist of nucleotides/nucleoside analogs, which include deoxynucleotides/nucleosides. The overhangs may be located on a sense strand, an antisense strand or any combination thereof. In addition, the nucleotides of the overhangs may be present in the 5'-end, 3'-end or both ends of the antisense strand or sense strand of the dsRNA.

“平的”或“平端”意指在dsRNA的末端不存在未配对的核苷酸，即，没有核苷酸突出端。“平端”dsRNA在其整个长度上是双链的，即，在分子的任一端都没有核苷酸突出端。本公开的dsRNAi剂包括在一端没有核苷酸突出端的dsRNA(即，具有一个突出端和一个平端的试剂)或在任一端没有核苷酸突出端的dsRNA。在一些实施方案中，此类寡核苷酸在其整个长度上是双链的。"Flat" or "flat-ended" means that there are no unpaired nucleotides at the ends of the dsRNA, i.e., there are no nucleotide overhangs. "Flat-ended" dsRNAs are double-stranded over their entire length, i.e., there are no nucleotide overhangs at either end of the molecule. The dsRNAi agents of the present disclosure include dsRNAs without nucleotide overhangs at one end (i.e., agents with one overhang and one flat end) or dsRNAs without nucleotide overhangs at either end. In some embodiments, such oligonucleotides are double-stranded over their entire length.

dsRNA可以通过本领域已知的标准方法合成。本公开的双链RNAi化合物可以使用两步程序来制备。首先，分别制备双链RNA分子的各个链，然后对其进行退火。siRNA化合物的各个链可以使用溶液相或固相有机合成或两者来制备。有机合成的优点是可以容易地制备包含非天然或修饰的核苷酸的寡核苷酸链。类似地，本公开的单链寡核苷酸可以使用溶液相或固相有机合成或两者来制备。dsRNA can be synthesized by standard methods known in the art. Double-stranded RNAi compounds of the present disclosure can be prepared using a two-step procedure. First, each chain of the double-stranded RNA molecule is prepared separately and then annealed. Each chain of the siRNA compound can be prepared using solution phase or solid phase organic synthesis or both. The advantage of organic synthesis is that oligonucleotide chains containing non-natural or modified nucleotides can be easily prepared. Similarly, single-stranded oligonucleotides of the present disclosure can be prepared using solution phase or solid phase organic synthesis or both.

在一个方面，本公开的dsRNA包括至少两个核苷酸序列，正义序列和反义序列。在一些实施方案中，正义链选自表1、表2a和表2b中任一项提供的序列组，并且正义链的相应反义链选自表1、表2a和表2b中任一项提供的序列。在这个方面，两个序列之一与两个序列中的另一个互补，其中一个序列与相关靶基因表达中产生的mRNA序列基本上互补。因此，在这个方面，dsRNA将包括两个寡核苷酸，其中一个寡核苷酸被描述为表1、表2a和表2b中的任一个正义链，并且第二个寡核苷酸被描述为表1、表2a和表2b中的任一个正义链的相应反义链。In one aspect, dsRNA of the present disclosure comprises at least two nucleotide sequences, sense sequence and antisense sequence.In some embodiments, sense strand is selected from the sequence group provided in any one of Table 1, Table 2a and Table 2b, and the corresponding antisense strand of sense strand is selected from the sequence provided in any one of Table 1, Table 2a and Table 2b.In this respect, one of two sequences is complementary to another one of two sequences, and one of them is substantially complementary to the mRNA sequence produced in the expression of relevant target genes.Therefore, in this respect, dsRNA will comprise two oligonucleotides, one of which is described as any one sense strand in Table 1, Table 2a and Table 2b, and second oligonucleotide is described as the corresponding antisense strand of any one sense strand in Table 1, Table 2a and Table 2b.

在一些实施方案中，正义链或反义链选自双链体IN-043、IN-091、IN-106、IN-176、IN-189、IN-202和IN-221中任一种的正义链或反义链。In some embodiments, the sense strand or antisense strand is selected from the sense strand or antisense strand of any one of duplexes IN-043, IN-091, IN-106, IN-176, IN-189, IN-202, and IN-221.

应当理解，虽然例如表1中的序列是未被修饰的或缀合的序列，但是本公开的siRNA的RNA，例如本公开的dsRNA，可以包含序表1、表2a和表2b中列出的任一的未修饰的、未缀合的、经修饰的、或缀合的序列。换言之，本公开涵盖表1、表2a和表2b的dsRNA，其是未修饰的、未缀合的、修饰的或缀合的，如本文所述。It should be understood that, although, for example, the sequences in Table 1 are unmodified or conjugated sequences, the RNA of the siRNA disclosed herein, such as the dsRNA disclosed herein, may comprise any unmodified, unconjugated, modified, or conjugated sequence listed in Table 1, Table 2a, and Table 2b. In other words, the present disclosure encompasses the dsRNA of Table 1, Table 2a, and Table 2b, which are unmodified, unconjugated, modified, or conjugated, as described herein.

本领域技术人员熟知，具有约20至23个碱基对(例如21个碱基对)的双链体结构的dsRNA被认为在诱导RNA干扰方面特别有效(Elbashir等人，EMBO 2001，20：6877-6888))。然而，其他人发现更短或更长的RNA双链体结构也可以是有效的(Chu和Rana(2007)RNA 14：1714-1719；Kim等人(2005)Nat Biotech 23：222-226)。在一些实施方案中，所述dsRNA可包括长度至少为21个核苷酸的至少一条链。可以合理地预期，与上述dsRNA相比，表1、表2a和表2b中的dsRNA的一端或两端减去仅几个核苷酸后的较短双链体，也可以具有类似的效果。因此，具有源自表1、表2a和表2b中的任一种的至少12、13、14、15、19、20或更多个连续核苷酸的序列的dsRNA，并且其抑制INHBE基因表达能力与包含完整序列的dsRNA的差异不超过约5％、10％、15％、20％、25％或30％的dsRNA，被认为在涵盖本公开的范围内。It is well known to those skilled in the art that dsRNAs having a duplex structure of about 20 to 23 base pairs (e.g., 21 base pairs) are considered to be particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20: 6877-6888). However, others have found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14: 1714-1719; Kim et al. (2005) Nat Biotech 23: 222-226). In some embodiments, the dsRNA may include at least one strand of at least 21 nucleotides in length. It is reasonable to expect that shorter duplexes of the dsRNAs in Tables 1, 2a, and 2b, minus only a few nucleotides at one or both ends, compared to the above dsRNAs, can also have similar effects. Therefore, dsRNAs having a sequence of at least 12, 13, 14, 15, 19, 20 or more consecutive nucleotides derived from any one of Table 1, Table 2a, and Table 2b, and whose ability to inhibit INHBE gene expression differs by no more than about 5%, 10%, 15%, 20%, 25% or 30% from a dsRNA comprising the entire sequence, are considered to be within the scope of the present disclosure.

三、RNAi剂的修饰3. Modification of RNAi Agents

在某些实施方案中，本公开的dsRNAi剂的RNA是未修饰的，并且不包含例如本领域已知的和本文所述的化学修饰或缀合。在其他实施方案中，本公开的dsRNAi剂的RNA(例如dsRNA)被化学修饰以增强稳定性或提供其他有益特性。在某些实施方案中，本公开的RNA的基本上所有核苷酸都被修饰。在其他实施方案中，RNA的所有核苷酸或RNA的基本上所有核苷酸都被修饰。在一些实施方案中，RNA的链(例如寡核苷酸、反义链、正义链或dsRNA)中存在不超过5、4、3、2或1个未修饰的核苷酸。In certain embodiments, the RNA of the dsRNAi agent of the present disclosure is unmodified and does not contain chemical modifications or conjugations such as those known in the art and described herein. In other embodiments, the RNA (e.g., dsRNA) of the dsRNAi agent of the present disclosure is chemically modified to enhance stability or provide other beneficial properties. In certain embodiments, substantially all nucleotides of the RNA of the present disclosure are modified. In other embodiments, all nucleotides of the RNA Or substantially all nucleotides of the RNA are modified. In some embodiments, no more than 5, 4, 3, 2, or 1 unmodified nucleotides are present in a strand of the RNA (e.g., an oligonucleotide, an antisense strand, a sense strand, or a dsRNA).

在一些实施方案中，本公开的dsRNAi剂包含至少一种本文所述的核酸修饰。例如，至少一种修饰选自由修饰的核苷间键合、修饰的核碱基、修饰的糖及其任何组合组成的组。无限制地，这样的修饰可以存在于本公开的dsRNAi剂中的任何地方。在某些实施方案中，dsRNAi剂包含至少一种非规范碱基配对核苷酸。不受限制地，此类核苷酸可以存在于本公开的dsRNAi剂中的任何地方。在一些实施方案中，至少一种修饰的核苷酸和/或非规范碱基配对核苷酸改变寡核苷酸的解链温度，例如，ΔTm为至少2℃，例如约2℃、超过2℃、约2-5℃、约3℃、约4℃或约5℃。In some embodiments, the dsRNAi agent of the present disclosure comprises at least one nucleic acid modification described herein. For example, at least one modification is selected from the group consisting of modified internucleoside bonds, modified core bases, modified sugars, and any combination thereof. Without limitation, such modifications may be present anywhere in the dsRNAi agent of the present disclosure. In certain embodiments, the dsRNAi agent comprises at least one non-canonical base pairing nucleotide. Without limitation, such nucleotides may be present anywhere in the dsRNAi agent of the present disclosure. In some embodiments, at least one modified nucleotide and/or non-canonical base pairing nucleotide changes the melting temperature of the oligonucleotide, for example, ΔTm is at least 2°C, for example, about 2°C, more than 2°C, about 2-5°C, about 3°C, about 4°C, or about 5°C.

在一实施方案中，本公开的dsRNA剂包含一种或多种靶向配体，例如一种或多种GalNAc衍生物，并且包含至少一种本文所述的另外的核酸修饰。例如，dsRNAi剂可包含至少一种选自由修饰的核苷间键合、修饰的核碱基、修饰的糖及其任何组合的修饰。无限制地，这样的修饰可以存在于本公开的dsRNAi剂中的任何地方。例如，修饰可以存在于RNA分子之一中。修饰包括例如末端修饰，例如5’-端修饰(磷酸化、缀合、反向连接)或3’-端修饰(缀合、DNA核苷酸、反向连接等)；碱基修饰，例如，用稳定碱基、去稳定碱基或与扩展碱基配对的碱基替换，去除碱基(脱碱基核苷酸)或缀合碱基；糖修饰(例如，在2'-位或4'-位)或糖替换；或主链修饰，包括磷酸二酯键的修饰或替换。可用于本文所述实施例的RNAi剂的具体实例包括但不限于含有修饰主链或不含天然核苷间键合的RNA。具有修饰主链的RNA包括主链中不具有磷原子的RNA等。在一些实施方案中，修饰的RNAi剂的核苷间主链中具有磷原子。In one embodiment, the dsRNA agent of the present disclosure comprises one or more targeting ligands, such as one or more GalNAc derivatives, and comprises at least one additional nucleic acid modification described herein. For example, the dsRNAi agent may include at least one modification selected from modified internucleoside bonds, modified nucleobases, modified sugars, and any combination thereof. Without limitation, such modifications may be present anywhere in the dsRNAi agent of the present disclosure. For example, the modification may be present in one of the RNA molecules. Modifications include, for example, terminal modifications, such as 5'-end modifications (phosphorylation, conjugation, reverse connection) or 3'-end modifications (conjugation, DNA nucleotides, reverse connection, etc.); base modifications, for example, replacement of bases with stable bases, destabilizing bases, or bases paired with extended bases, removal of bases (absaccharide nucleotides) or conjugation of bases; sugar modifications (e.g., at 2'-position or 4'-position) or sugar replacement; or backbone modifications, including modification or replacement of phosphodiester bonds. Specific examples of RNAi agents that can be used in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or free of natural internucleoside bonds. RNAs with modified backbones include RNAs without phosphorus atoms in the backbone, etc. In some embodiments, the modified RNAi agent has a phosphorus atom in the internucleoside backbone.

在一些实施方案中，主链修饰是指核苷间连键或主链包括但不限于硫代磷酸酯基团、手性硫代磷酸酯、硫代磷酸酯、二硫代磷酸酯、磷酸三酯、氨基烷基-磷酸三酯、手性膦酸酯、次膦酸酯、氨基磷酸酯、硫代烷基膦酸酯、硫代烷基磷酸三酯、吗啉代连接，其中相邻的核苷单元对是3’-5’至5’-3’或2’-5’至5’-2’连接。In some embodiments, backbone modifications refer to internucleoside linkages or backbones including but not limited to phosphorothioate groups, chiral phosphorothioates, phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, chiral phosphonates, phosphinates, phosphoramidates, thioalkylphosphonates, thioalkylphosphotriesters, morpholino linkages, wherein adjacent pairs of nucleoside units are 3'-5' to 5'-3' or 2'-5' to 5'-2' linked.

在一些实施方案中，dsRNA的正义链可含有1、2、3、4、5或6个硫代磷酸酯连键(硫代磷酸酯修饰核苷酸)，dsRNA的反义链可含有1、2、3、4、5或6个硫代磷酸酯连键(硫代磷酸酯修饰核苷酸)。在一些实施方案中，dsRNA的正义链可含有1或2个硫代磷酸酯连键，siRNA的反义链可含有1、2、3或4个硫代磷酸酯连键。In some embodiments, the sense strand of the dsRNA may contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages (phosphorothioate modified nucleotides), and the antisense strand of the dsRNA may contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages (phosphorothioate modified nucleotides). In some embodiments, the sense strand of the dsRNA may contain 1 or 2 phosphorothioate linkages, and the antisense strand of the siRNA may contain 1, 2, 3, or 4 phosphorothioate linkages.

在一些实施方案中，dsRNA的正义链含有2个硫代磷酸酯核苷间连键。在一些实施方案中，硫代磷酸酯核苷间连键位于从正义链的5’端开始1-3位的核苷酸之间。在一些实施方案中，硫代磷酸酯核苷间连键位于从正义链的3’端开始1-3位的核苷酸之间。在一些实施方案中，一个硫代磷酸酯核苷间连键位于正义链的5’端，和另一个硫代磷酸酯连键位于正义链的3’末端。在一些实施方案中，dsRNA的正义链含有1个硫代磷酸酯核苷间连键。在一些实施方案中，硫代磷酸酯核苷间连键位于从正义链的5’端开始1-2位的核苷酸之间。在一些实施方案中，硫代磷酸酯核苷间连键位于从正义链的5’端开始2-3位的核苷酸之间。在一些实施方案中，靶向配体通过硫代磷酸酯连键连接于正义链。In some embodiments, the sense strand of the dsRNA contains 2 phosphorothioate nucleoside interlinkages. In some embodiments, the phosphorothioate nucleoside interlinkage is located between the nucleotides at positions 1-3 from the 5' end of the sense strand. In some embodiments, the phosphorothioate nucleoside interlinkage is located between the nucleotides at positions 1-3 from the 3' end of the sense strand. In some embodiments, one phosphorothioate nucleoside interlinkage is located at the 5' end of the sense strand, and another phosphorothioate interlinkage is located at the 3' end of the sense strand. In some embodiments, the sense strand of the dsRNA contains 1 phosphorothioate nucleoside interlinkage. In some embodiments, the phosphorothioate nucleoside interlinkage is located between the nucleotides at positions 1-2 from the 5' end of the sense strand. In some embodiments, the phosphorothioate nucleoside interlinkage is located between the nucleotides at positions 2-3 from the 5' end of the sense strand. In some embodiments, the targeting ligand is connected to the sense strand via a phosphorothioate linkage.

在一些实施方案中，dsRNA的反义链含有4个硫代磷酸酯核苷间连键。在一些实施方案中，4个硫代磷酸酯核苷间连键位于从反义链的5’端开始1-3位的核苷酸之间和从3’端开始1-3位的核苷酸之间。在一些实施方案中，dsRNA的反义链含有3个硫代磷酸酯核苷间连键。在一些实施方案中，3个硫代磷酸酯核苷间连键分别位于从反义链的5’端开始1-2位的核苷酸之间和从3’端开始1-3位的核苷酸之间。在一些实施方案中，3个硫代磷酸酯核苷间连键分别位于从反义链的5’端开始1-3位的核苷酸之间和从3’端开始1-2位的核苷酸之间。在一些实施方案中，dsRNA的反义链含有2个硫代磷酸酯核苷间连键。在一些实施方案中，2个硫代磷酸酯核苷间连键位于从反义链的5’端开始1-2位的核苷酸之间和从3’端开始1-2位的核苷酸之间。In some embodiments, the antisense strand of the dsRNA contains 4 phosphorothioate internucleoside linkages. In some embodiments, the 4 phosphorothioate internucleoside linkages are located between the nucleotides at positions 1-3 from the 5' end of the antisense strand and between the nucleotides at positions 1-3 from the 3' end. In some embodiments, the antisense strand of the dsRNA contains 3 phosphorothioate internucleoside linkages. In some embodiments, the 3 phosphorothioate internucleoside linkages are respectively located between the nucleotides at positions 1-2 from the 5' end of the antisense strand and between the nucleotides at positions 1-3 from the 3' end. In some embodiments, the 3 phosphorothioate internucleoside linkages are respectively located between the nucleotides at positions 1-3 from the 5' end of the antisense strand and between the nucleotides at positions 1-2 from the 3' end. In some embodiments, the antisense strand of the dsRNA contains 2 phosphorothioate internucleoside linkages. In some embodiments, the 2 phosphorothioate internucleoside linkages are located between the nucleotides at positions 1-2 from the 5' end of the antisense strand. between the nucleotides at positions 1 and 2 from the 3' end.

在一些实施方案中，dsRNA的反义链包含表1、2a或2b中任一个反义链序列的核苷酸(从5’端→3’端)序列。在一些实施方案中，dsRNA的正义链包含表1、2a或2b中任一个正义链的核苷酸(从5’端→3’端)序列。在一些实施方案中，dsRNA的反义链包含表1、2a或2b中任一个反义链的核苷酸(从5’端→3’端)序列，和正义链包含表1、2a或2b中任一个正义链的核苷酸(从5’端→3’端)序列。In some embodiments, the antisense strand of the dsRNA comprises a nucleotide sequence (from 5' end → 3' end) of any antisense strand sequence in Table 1, 2a or 2b. In some embodiments, the sense strand of the dsRNA comprises a nucleotide sequence (from 5' end → 3' end) of any sense strand in Table 1, 2a or 2b. In some embodiments, the antisense strand of the dsRNA comprises a nucleotide sequence (from 5' end → 3' end) of any antisense strand in Table 1, 2a or 2b, and the sense strand comprises a nucleotide sequence (from 5' end → 3' end) of any sense strand in Table 1, 2a or 2b.

在一些实施方案中，本公开所述修饰的核苷酸选自：2’-O-甲基修饰核苷酸、2’-氟修饰核苷酸、2’-脱氧核苷酸、2’-甲氧基乙基修饰核苷酸、2’-氨基修饰核苷酸、2’-烷基修饰核苷酸、2’-烷氧基修饰核苷酸、2’-F-阿糖核苷酸、硫代磷酸酯修饰核苷酸、脱碱基核苷酸、吗啉代核苷酸、锁定核苷酸、倒置核苷酸和次黄嘌呤碱基置换核苷酸。In some embodiments, the modified nucleotides disclosed herein are selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy nucleotides, 2'-methoxyethyl modified nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 2'-alkoxy modified nucleotides, 2'-F-arabino nucleotides, phosphorothioate modified nucleotides, abasic nucleotides, morpholino nucleotides, locked nucleotides, inverted nucleotides, and inosine base substituted nucleotides.

在一些实施方案中，本公开所述修饰的核苷酸选自：2’-O-甲基修饰核苷酸、2’-氟修饰核苷酸、2’-脱氧核苷酸、硫代磷酸酯修饰核苷酸和倒置核苷酸。在一些优选实施方式中，倒置核苷酸选自：倒置A核苷酸、倒置dA核苷酸、倒置dT核苷酸、倒置C核苷酸和倒置U核苷酸。In some embodiments, the modified nucleotides disclosed herein are selected from: 2'-O-methyl modified nucleotides, 2'-fluorine modified nucleotides, 2'-deoxynucleotides, phosphorothioate modified nucleotides and inverted nucleotides. In some preferred embodiments, the inverted nucleotides are selected from: inverted A nucleotides, inverted dA nucleotides, inverted dT nucleotides, inverted C nucleotides and inverted U nucleotides.

示例性修饰的核苷酸或核碱基包括但不限于：合成的和天然的核苷或核碱基，例如肌苷、黄嘌呤、次黄嘌呤、水粉蕈素(nebularine)、异鸟苷、结核菌素、2-(卤代)腺嘌呤、2-(烷基)腺嘌呤、2-(丙基)腺嘌呤、2-(氨基)腺嘌呤、2-(氨基烷基)腺嘌呤、2-(氨基丙基)腺嘌呤、2-(甲硫基)-N6-(异戊烯基)腺嘌呤、6-(烷基)腺嘌呤、6-(甲基)腺嘌呤、7-(脱氮杂)腺嘌呤、8-(烯基)腺嘌呤、8-(烷基)腺嘌呤、8-(炔基)腺嘌呤、8-(氨基)腺嘌呤、8-(卤代)腺嘌呤、8-(羟基)腺嘌呤，8-(硫代烷基)腺嘌呤，8-(硫醇)腺嘌呤，N6-(异戊基)腺嘌呤，N6-(甲基)腺嘌呤，N6,N6-(二甲基)腺嘌呤，2-(烷基)鸟嘌呤，2-(丙基)鸟嘌呤、6-(烷基)鸟嘌呤、6-(甲基)鸟嘌呤、7-(烷基)鸟嘌呤、7-(甲基)鸟嘌呤、7-(脱氮杂)鸟嘌呤、8-(烷基)鸟嘌呤、8-(烯基)鸟嘌呤、8-(炔基)鸟嘌呤、8-(氨基)鸟嘌呤、8-(卤代)鸟嘌呤、8-(羟基)鸟嘌呤、8-(硫烷基)鸟嘌呤、8-(硫醇)鸟嘌呤、N-(甲基)鸟嘌呤、2-(硫代)胞嘧啶、3-(脱氮杂)-5-(氮杂)胞嘧啶、3-(烷基)胞嘧啶、3-(甲基)胞嘧啶、5-(烷基)胞嘧啶、5-(炔基)胞嘧啶、5-(卤代)胞嘧啶、5-(甲基)胞嘧啶、5-(丙烯基)胞嘧啶、5-(丙炔基)胞嘧啶、5-(三氟甲基)胞嘧啶、6--(偶氮)胞嘧啶、N4-(乙酰基)胞嘧啶、3-(3-氨基-3-羧丙基)尿嘧啶、2-(硫代)尿嘧啶、5-(甲基)-2-(硫代)尿嘧啶、5-(甲基氨基甲基)-2-(硫代)尿嘧啶、4-(硫代)尿嘧啶、5-(甲基)-4-(硫代)尿嘧啶、5-(甲氨基甲基)-4-(硫代)尿嘧啶、5-(甲基)-2,4-(二硫代)尿嘧啶、5-(甲氨基甲基)-2,4-(二硫代)尿嘧啶、5-(2-氨基丙基)尿嘧啶、5-(烷基)尿嘧啶、5-(炔基)尿嘧啶、5-(烯丙氨基)尿嘧啶、5-(氨基烯丙基)尿嘧啶、5-(氨基烷基)尿嘧啶、5-(胍烷基)尿嘧啶、5-(1,3-二唑-1-烷基)尿嘧啶、5-(氰基烷基)尿嘧啶、5-(二烷基氨基烷基)尿嘧啶、5-(二甲基氨基烷基)尿嘧啶、5-(卤代)尿嘧啶、5-(甲氧基)尿嘧啶、尿嘧啶-5-氧基乙酸、5-(甲氧基羰基甲基)-2-(硫代)尿嘧啶、5-(甲氧基羰基甲基)尿嘧啶、5-(丙烯基)尿嘧啶、5-(丙炔基)尿嘧啶、5-(三氟甲基)尿嘧啶、6-(偶氮)尿嘧啶、二氢尿嘧啶、N3-(甲基)尿嘧啶、5-尿嘧啶(即假尿嘧啶)、2-(硫代)假尿嘧啶、4-(硫代)假尿嘧啶、2,4-(二硫代)假尿嘧啶、5-(烷基)假尿嘧啶、5-(甲基)假尿嘧啶、5-(烷基)-2-(硫代)假尿嘧啶、5-(甲基)-2-(硫代)假尿嘧啶、5-(烷基)-4-(硫代)假尿嘧啶、5-(甲基)-4-(硫代)假尿嘧啶、5-(烷基)-2,4-(二硫代)假尿嘧啶、5-(甲基)-2,4-(二硫代)假尿嘧啶、I-取代的假尿嘧啶、I-取代的2(硫代)-假尿嘧啶、I-取代的4-(硫代)假尿嘧啶、I-取代的2,4-(二硫代)假尿嘧啶、1-(氨基羰基乙烯基)-假尿嘧啶、1-(氨基羰基乙烯基)-2(硫代)假尿嘧啶、1-(氨基羰基乙烯基)-4-(硫代)假尿嘧啶、1-(氨基羰基乙烯基)-2,4-(二硫代)假尿嘧啶、1-(氨基烷基氨基羰基乙烯基)-假尿嘧啶、1-(氨基烷基氨基羰基乙烯基)-2(硫代)-假尿嘧啶、1-(氨基烷基氨基羰基乙烯基)-4-(硫代)假尿嘧啶、1-(氨基烷基氨基羰基乙烯基)-2,4-(二硫代)假尿嘧啶、1,3-(二氮杂)-2-(氧代)-吩噁嗪-1-基、1-(氮杂)-2-(硫代)-3-(氮杂)-吩噁嗪-1-基、1,3-(二氮杂)-2-(氧代)-吩噻嗪-1-基、1-(氮杂)-2-(硫代)-3-(氮杂)-吩噻嗪-1-基、7-取代的1,3-(二氮杂)-2-(氧代)-吩噁嗪-1-基、7-取代的1-(氮杂)-2-(硫代)-3-(氮杂)-吩噁嗪-1-基、7-取代的1,3-(二氮杂)-2-(氧代)-吩噻嗪-1-基、7-取代的1(氮杂)-2-(硫代)-3-(氮杂)-吩噻嗪-1-基、7-(氨基烷基羟基)-1、3-(二氮杂)-2-(氧代)-吩噁嗪-1-基、7-(氨基烷基羟基)-1-(氮杂)-2-(硫代)-3-(氮杂)-吩噁嗪-1-基、7-(氨基烷基羟基)-1,3-(二氮杂)-2-(氧代)-吩噻嗪-1-基、7-(氨基烷基羟基)-1-(氮杂)-2-(硫代)-3-(氮杂)-吩噻嗪-1-基、7-(胍烷基羟基)-1,3-(二氮杂)-2-(氧代)-吩噁嗪-1-基、7-(胍烷基羟基)-1-(氮杂)-2-(硫代)-3-(氮杂)-吩噁嗪-1-基、7-(胍烷基羟基)-1,3-(二氮杂)-2-(氧代)吩噻嗪-1-基、7-(胍烷基羟基)-1-(氮杂)-2-(硫代)-3-(氮杂)-吩噻嗪-1-基、1,3,5-(三氮杂)-2,6-(二氧杂)-萘、肌苷基、2-氮杂肌苷基、7-脱氮肌苷基、硝基咪唑基、硝基吡唑基、硝基苯并咪唑基、硝基吲唑基、氨基吲哚基、吡咯并嘧啶基、3-(甲基)异喹诺酮基、5-(甲基)异喹诺酮基、3-(甲基)-7-(丙炔基)异喹诺酮基、7-(氮杂)吲哚基、6-(甲基)-7-(氮杂)吲哚基、咪唑并吡啶基、9-(甲基)-咪唑并吡啶基、吡咯并吡嗪基、异喹诺酮基、7-(丙炔基)异喹啉基、丙炔基-7-(氮杂)吲哚基、2,4,5-(三甲基)苯基、4-(甲基)吲哚基、4,6-(二甲基)吲哚基、苯基、萘基、蒽基、菲基、芘基、芪基、并四苯基、并五苯基、二氟甲苯基、4-(氟)-6-(甲基)苯并咪唑、4-(甲基)苯并咪唑、6-(偶氮)胸腺嘧啶、2-吡啶酮、5-硝基吲哚、3-硝基吡咯、6-(氮杂)嘧啶、2-(氨基)嘌呤、2,6-(二氨基)嘌呤、5-取代嘧啶、N2-取代的嘌呤、N6-取代的嘌呤、O6-取代的嘌呤、取代的1,2,4-三唑、吡咯并嘧啶-2-酮-3-基、6-苯基-吡咯并-嘧啶-2-酮-3-基、对位取代的-6-苯基-吡咯并-嘧啶-2-酮-3-基、邻位取代的-6-苯基-吡咯并-嘧啶-2-酮-3-基、双邻位取代的-6-苯基-吡咯-嘧啶-2-酮-3-基、对-(氨基烷基羟基)-6-苯基-吡咯并-嘧啶-2-酮-3-基、邻-(氨基烷基羟基)-6-苯基-吡咯并-嘧啶-2-酮-3-基、双-邻-(氨基烷基羟基)-6-苯基-吡咯并嘧啶-2-酮-3-基、吡啶并嘧啶-3-基，2-氧代-7-氨基-吡啶并嘧啶-3-基，2-氧代-吡啶并嘧啶-3-基，或其任何O-烷基化或N-烷基化衍生物。或者，任何上述核苷酸或核碱基的取代或修饰的类似物可用于本公开的RNAi剂、组合物和方法中。Exemplary modified nucleotides or nucleobases include, but are not limited to, synthetic and natural nucleosides or nucleobases such as inosine, xanthine, hypoxanthine, nebularine, isoguanosine, tuberculin, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, 2-(methylthio)-N6-(isopentenyl)adenine, 6-(alkyl)adenine, 6-(methyl)adenine, 7-(deaza)adenine, ) adenine, 8-(alkenyl) adenine, 8-(alkyl) adenine, 8-(alkynyl) adenine, 8-(amino) adenine, 8-(halogenated) adenine, 8-(hydroxy) adenine, 8-(thioalkyl) adenine, 8-(thiol) adenine, N6-(isopentyl) adenine, N6-(methyl) adenine, N6,N6-(dimethyl) adenine, 2-(alkyl) guanine, 2-(propyl) guanine, 6-(alkyl) guanine, 6-(methyl) guanine, 7-(alkyl) guanine, 7-(methyl) guanine, 7 -(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8-(hydroxy)guanine, 8-(sulfanyl)guanine, 8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl) Cytosine, 5-(propenyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N4-(acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil, 2-(thio)uracil, 5-(methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil, 4-(thio)uracil, 5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(methyl)-2,4-( dithio) uracil, 5-(methylaminomethyl)-2,4-(dithio) uracil, 5-(2-aminopropyl) uracil, 5-(alkyl) uracil, 5-(alkynyl) uracil, 5-(allylamino) uracil, 5-(aminoallyl) uracil, 5-(aminoalkyl) uracil, 5-(guanidinyl) uracil, 5-(1,3-oxadiazol-1-alkyl) uracil, 5-(cyanoalkyl) uracil, 5-(dialkylaminoalkyl) uracil, 5-(dimethylaminoalkyl) uracil, 5-(halogenated) uracil, 5 -(Methoxy)uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonylmethyl)uracil, 5-(propenyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N3-(methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudouracil, 4-(thio)pseudouracil, 2,4-(dithio)pseudouracil, 5-(alkyl)pseudouracil, 5-(methyl)pseudouracil uracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5-(methyl)-4-(thio)pseudouracil, 5-(alkyl)-2,4-(dithio)pseudouracil, 5-(methyl)-2,4-(dithio)pseudouracil, 1-substituted pseudouracil, 1-substituted 2(thio)-pseudouracil, 1-substituted 4-(thio)pseudouracil, 1-substituted 2,4-(dithio)pseudouracil, 1-(aminocarbonyl)- uracil, 1-(aminoalkylaminocarbonylvinyl)-pseudouracil, 1-(aminoalkylaminocarbonylvinyl)-pseudouracil, 1-(aminoalkylaminocarbonylvinyl)-pseudouracil, 1-(aminoalkylaminocarbonylvinyl)-2(thio)-pseudouracil, 1-(aminoalkylaminocarbonylvinyl)-4-(thio)pseudouracil, 1-(aminoalkylaminocarbonylvinyl)-2,4-(dithio)pseudouracil, 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 1,3-(diaza)-2-(oxo)-phenothiazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2-( thio)-3-(aza)-phenoxazine-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenothiazine-1-yl, 7-substituted 1(aza)-2-(thio)-3-(aza)-phenothiazine-1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazine-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazine -1-yl, 7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenothiazin-1-yl, 7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 7-(guanidinylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidinylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, yl, 7-(guanidinylhydroxy)-1,3-(diaza)-2-(oxo)phenothiazin-1-yl, 7-(guanidinylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenothiazin-1-yl, 1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosinyl, 2-azainosinyl, 7-deazainosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrole pyrimidinyl, 3-(methyl)isoquinolone, 5-(methyl)isoquinolone, 3-(methyl)-7-(propynyl)isoquinolone, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidazopyridinyl, 9-(methyl)-imidazopyridinyl, pyrrolopyrazinyl, isoquinolone, 7-(propynyl)isoquinolyl, propynyl-7-(aza)indolyl, 2,4,5-(trimethyl)phenyl, 4-( methyl) indolyl, 4,6-(dimethyl) indolyl, phenyl, naphthyl, anthracenyl, phenanthrenyl, pyrenyl, stilbenyl, tetraphenylene, pentacene, difluorotolyl, 4-(fluoro)-6-(methyl) benzimidazole, 4-(methyl) benzimidazole, 6-(azo) thymine, 2-pyridone, 5-nitroindole, 3-nitropyrrole, 6-(aza) pyrimidine, 2-(amino) purine, 2,6-(diamino) purine, 5-substituted pyrimidine, N2- Substituted purine, N6-substituted purine, O6-substituted purine, substituted 1,2,4-triazole, pyrrolopyrimidin-2-one-3-yl, 6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, para-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, di-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-one-3-yl, para-(amino) alkylhydroxy)-6-phenyl-pyrrolo-pyrimidine-2-one-3-yl, o-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidine-2-one-3-yl, bis-o-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidine-2-one-3-yl, pyridopyrimidine-3-yl, 2-oxo-7-amino-pyridopyrimidine-3-yl, 2-oxo-pyridopyrimidine-3-yl, or any O-alkylated or N-alkylated derivative thereof. Alternatively, substituted or modified analogs of any of the above nucleotides or nucleobases can be used in the RNAi agents, compositions and methods of the present disclosure.

在一些实施方案中，本公开所述修饰的核苷酸包括以下任一种或其组合：In some embodiments, the modified nucleotides disclosed herein include any one or a combination of the following:

(1)按照5’端到3’端的方向，所述反义链第2、4、12、14位的核苷酸为2’-氟修饰的核苷酸，其余位置的核苷酸为2’-O-甲基修饰的核苷酸；(1) From the 5' end to the 3' end, the nucleotides at positions 2, 4, 12, and 14 of the antisense strand are 2'-fluorine-modified nucleotides, and the nucleotides at the remaining positions are 2'-O-methyl-modified nucleotides;

(2)按照5’端到3’端的方向，所述正义链第7、8、9位的核苷酸为2’-氟修饰的核苷酸；(2) From the 5' end to the 3' end, the nucleotides at positions 7, 8, and 9 of the sense strand are 2'-fluorinated nucleotides;

(3)按照5’端到3’端的方向，所述反义链第2-8位中G替换为I；或者，按照5’端到3’端的方向，所述反义链第2-8位中的鸟嘌呤(G中的碱基)替换为次黄嘌呤(肌苷或肌苷酸I的碱基)。(3) From the 5' end to the 3' end, the G in positions 2-8 of the antisense strand is replaced by I; or, from the 5' end to the 3' end, the guanine (the base in G) in positions 2-8 of the antisense strand is replaced by hypoxanthine (the base of inosine or inosinic acid I).

在一些此类实施方式中，所述反义链第2-8位中的次黄嘌呤替换的修饰核苷酸进一步满足下述特征中的一种或其组合物：In some such embodiments, the modified nucleotides substituted with hypoxanthine in positions 2-8 of the antisense strand further satisfy one of the following characteristics or a combination thereof:

(i)按照5’端到3’端的方向，所述反义链第6-8位中的鸟嘌呤替换为次黄嘌呤(I)；(i) from the 5' end to the 3' end, the guanine in positions 6-8 of the antisense strand is replaced by hypoxanthine (I);

(ii)按照5’端到3’端的方向，所述反义链的序列N₁N₂GN₃N₄中的鸟嘌呤替换为次黄嘌呤(I)，如果N₁、N₂、N₃和N₄中的至少三个碱基是腺嘌呤或尿嘧啶；(ii) in the 5' to₃ ' direction, the guanine in the sequence_N1N2GN3N4 of the antisense strand_is replaced by hypoxanthine (I), if at least three bases_inN1 ,_N2 ,_N3 and_N4 are adenine or uracil;

(iii)按照5’端到3’端的方向，所述反义链第2-8位中的腺嘌呤和尿嘧啶的数量在4个以上；和/或，(iii) the number of adenine and uracil in positions 2 to 8 of the antisense strand is greater than 4 in the direction from the 5' end to the 3' end; and/or

(iv)所述反义链中的至少一个核苷酸中的鸟嘌呤替换为次黄嘌呤，当次黄嘌呤的替换导致了包含该反义链的该dsRNA相比未经次黄嘌呤替换的dsRNA的至少2℃、2℃或大于2℃的解离温度的差异。(iv) guanine in at least one nucleotide in the antisense strand is replaced by hypoxanthine, when the replacement of hypoxanthine results in a difference in the dissociation temperature of the dsRNA comprising the antisense strand compared to the dsRNA without hypoxanthine replacement of at least 2°C, 2°C or more than 2°C.

在一些实施方案中，dsRNAi剂进一步包含位于反义链5'-端的磷酸盐或磷酸盐模拟物。在一些实施方案中，磷酸盐模拟物是5’-乙烯基磷酸酯(VP)。在一些实施方案中，dsRNAi剂的反义链的5'-端不包含5'-乙烯基磷酸酯(VP)。In some embodiments, the dsRNAi agent further comprises a phosphate or phosphate mimic located at the 5'-end of the antisense strand. In some embodiments, the phosphate mimic is 5'-vinyl phosphate (VP). In some embodiments, the 5'-end of the antisense strand of the dsRNAi agent does not comprise 5'-vinyl phosphate (VP).

本公开的RNAi试剂的末端可以被修饰，这种修饰可以在一端或两端。例如，dsRNA的3'和/或5'端可以缀合至其他功能性分子实体，例如标记部分，例如荧光团(例如芘、TAMRA、荧光素、Cy3或Cy5染料)或保护基团(基于例如，硫、硅、硼或酯)。功能性分子实体可以通过磷酸基团和/或连接基连接至糖。接头的末端原子可以连接或取代糖的磷酸基团或C-3'、C-5'、O、N、S或C基团的连接原子。或者，接头可以连接或取代核苷酸替代物(例如，PNA)的末端原子。当连接基/磷酸官能分子实体-连接基/磷酸阵列插入双链寡聚化合物的两条链之间时，该阵列可以替代发夹型寡聚化合物中的发夹环。在一些实施方案中，末端修饰也可用于监测分布，并且在这种情况下，要添加的优选基团包括荧光团，例如荧光素或Alexa染料，例如Alexa 488。在一些实施方案中，末端修饰也可用于增强摄取，对此有用的非限制性的修饰包括靶向配体。The ends of the RNAi reagents disclosed herein can be modified, and this modification can be at one or both ends. For example, the 3' and/or 5' ends of dsRNA can be conjugated to other functional molecular entities, such as labeling moieties, such as fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protective groups (based on, for example, sulfur, silicon, boron or esters). Functional molecular entities can be connected to sugars through phosphate groups and/or linkers. The terminal atoms of the linker can connect or replace the phosphate groups of sugars or the connecting atoms of C-3', C-5', O, N, S or C groups. Alternatively, the linker can connect or replace the terminal atoms of nucleotide substitutes (e.g., PNA). When the linker/phosphate functional molecular entity-linker/phosphate array is inserted between the two chains of a double-stranded oligomeric compound, the array can replace the hairpin loop in the hairpin type oligomeric compound. In some embodiments, terminal modifications can also be used to monitor distribution, and in this case, the preferred groups to be added include fluorophores, such as fluorescein or Alexa dyes, such as Alexa 488. In some embodiments, terminal modifications may also be used to enhance uptake, non-limiting modifications useful for this include targeting ligands.

本公开还涵盖了所述dsRNA剂的各种盐、混合盐和游离酸形式。在一些实施方案中，dsRNA剂是游离酸形式。在一些实施方案中，dsRNAi剂是盐形式。在一个实施方案中，dsRNAi剂是钠盐形式。按本领域常识，当本公开的dsRNAi剂呈钠盐形式时，钠离子作为磷酸二酯和/或硫代磷酸酯基团的抗衡离子存在于试剂中。The present disclosure also encompasses various salts, mixed salts, and free acid forms of the dsRNA agent. In some embodiments, the dsRNA agent is in free acid form. In some embodiments, the dsRNAi agent is in salt form. In one embodiment, the dsRNAi agent is in sodium salt form. According to common knowledge in the art, when the dsRNAi agent of the present disclosure is in sodium salt form, sodium ions are present in the reagent as counterions of phosphodiester and/or phosphorothioate groups.

在某些实施方案中，本公开的dsRNAi剂通过共价连接一个或多个缀合物基团而被进一步修饰。一般而言，缀合物基团改变所连接的本公开的dsRNA剂的一种或多种性质，包括但不限于药效学、药代动力学、结合、吸收、细胞分布、细胞摄取、电荷和清除。缀合基团通常用于化学领域，并且直接或通过任选的连接部分或连接基团连接至母体化合物。缀合物基团的优选包括但不限于聚胺、聚酰胺、聚乙二醇、硫醚、聚醚、胆固醇、硫代胆固醇、胆酸部分、叶酸、脂质、磷脂、生物素、吩嗪、菲啶、蒽醌、金刚烷、吖啶、荧光素、罗丹明、香豆素和染料。In certain embodiments, the dsRNAi agent of the present disclosure is further modified by covalently linking one or more conjugate groups. In general, the conjugate group changes one or more properties of the dsRNA agent of the present disclosure connected, including but not limited to pharmacodynamics, pharmacokinetics, combination, absorption, cell distribution, cellular uptake, charge and removal. Conjugate groups are commonly used in the field of chemistry, and are directly or by optional linking moieties or linking groups connected to the parent compound. Conjugate groups preferably include but are not limited to polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, bile acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin and dyes.

在一些实施方案中，本公开的靶向配体包含N-乙酰基-半乳糖胺(GalNAc)，或者是GalNAc衍生物，例如L96(参见siRNA药物Inclisiran)。在一些实施方案中，所述靶向配体是WO2022266753A1中公开的任一的靶向配体(targeting moiety)。除非明显矛盾，将WO2022266753A1全文引入本申请作为参考。In some embodiments, the targeting ligand disclosed herein comprises N-acetyl-galactosamine (GalNAc), or a GalNAc derivative, such as L96 (see siRNA drug Inclisiran). In some embodiments, the targeting ligand is any targeting ligand disclosed in WO2022266753A1. Unless otherwise clearly contradictory, the entire text of WO2022266753A1 is incorporated into this application as a reference.

在一些实施方案中，dsRNAi剂的结构选自式1～式33，其中R²为dsRNA。按本领域常识，R²通过dsRNA的正义链的3’端或5’端与靶向配体缀合形成dsRNAi剂。In some embodiments, the structure of the dsRNAi agent is selected from Formula 1 to Formula 33, wherein^R2 is dsRNA. According to common knowledge in the art,^R2 is conjugated to a targeting ligand through the 3' end or 5' end of the sense strand of the dsRNA to form a dsRNAi agent.

四、RNAi剂的递送与用途IV. Delivery and Use of RNAi Agents

可以以多种方式将本公开的RNAi剂递送至细胞，例如受试者(例如患有代谢病症的受试者)体内的细胞。举例来说，递送可以通过使细胞与本公开的RNAi剂在体外或体内接触来进行。体内递送还可以通过向受试者施用包含RNAi剂(例如dsRNA)的组合物(例如药物组合物)来直接进行。或者，体内递送可通过施用编码并引导RNAi剂表达的一种或多种载体来间接进行。The RNAi agents of the present disclosure can be delivered to cells, such as cells in a subject (e.g., a subject with a metabolic disorder) in a variety of ways. For example, delivery can be performed by contacting cells with the RNAi agents of the present disclosure in vitro or in vivo. In vivo delivery can also be performed directly by administering a composition (e.g., a pharmaceutical composition) comprising an RNAi agent (e.g., dsRNA) to the subject. Alternatively, in vivo delivery can be performed indirectly by administering one or more vectors that encode and guide the expression of the RNAi agent.

在一个实施方案中，细胞是肝脏细胞(liver cells)，例如肝实质细胞(hepatocytes)。在一个实施方案中，细胞是脂肪细胞。在某些实施方案中，RNAi剂被器官中存在的一种或多种组织或细胞(例如肝脏、脂肪组织)类型吸收。In one embodiment, the cells are liver cells, such as hepatocytes. In one embodiment, the cells are adipocytes. In certain embodiments, the RNAi agent is taken up by one or more tissue or cell types present in an organ (e.g., liver, adipose tissue).

本公开的另一方面涉及降低受试者中INHBE基因的表达和/或活性的方法，包括向受试者施用本公开的dsRNAi剂。在一些实施方案中，所述方法包括向受试者给与治疗有效量的本公开的dsRNAi剂，从而抑制或减少受试者(例如受试者中的细胞)中INHBE基因的表达。在一些实施方案中，所述方法包括使细胞与本公开的双链RNAi剂接触，使得INHBE基因的表达在细胞中被抑制或减少。在一些此类实施方案中，使得靶基因例如INHBE基因的mRNA转录物在所述受试者或细胞中被降解，由此抑制或减少INHBE基因在所述受试者或细胞中的表达。Another aspect of the present disclosure relates to a method for reducing the expression and/or activity of an INHBE gene in a subject, comprising administering a dsRNAi agent of the present disclosure to the subject. In some embodiments, the method comprises administering a therapeutically effective amount of a dsRNAi agent of the present disclosure to the subject, thereby inhibiting or reducing the expression of an INHBE gene in the subject (e.g., a cell in the subject). In some embodiments, the method comprises contacting a cell with a double-stranded RNAi agent of the present disclosure, The expression of the INHBE gene is inhibited or reduced in the cell. In some such embodiments, the mRNA transcript of the target gene, such as the INHBE gene, is degraded in the subject or cell, thereby inhibiting or reducing the expression of the INHBE gene in the subject or cell.

另一个方面，本公开涉及治疗患有代谢病症或处于患有代谢病症的风险或处于发展代谢病症的风险的受试者的方法，包括向受试者施用治疗有效量的本公开的dsRNAi剂，从而治疗受试者。In another aspect, the disclosure relates to a method of treating a subject having or at risk of having or developing a metabolic disorder, comprising administering to the subject a therapeutically effective amount of a dsRNAi agent of the disclosure, thereby treating the subject.

另一方面，本公开涉及在需要的受试者中治疗或预防代谢紊乱的方法，包括向受试者施用治疗有效量的本公开的dsRNAi剂，以使得所述代谢紊乱得到治疗或预防。In another aspect, the present disclosure relates to a method of treating or preventing a metabolic disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a dsRNAi agent of the present disclosure, such that the metabolic disorder is treated or prevented.

另一方面，本公开涉及在需要的受试者中治疗或预防INHBE相关的疾病或病症的方法，包括向受试者施用治疗有效量的dsRNAi剂，以使得INHBE相关的疾病或病症得到治疗或预防。术语“INHBE相关疾病或病症”包括由INHBE基因表达或蛋白质产生所引起、介导或相关的任何疾病或病症，并且包括将受益于INHBE基因表达或蛋白质活性的减少或由该减少得以改善的任何疾病或病症。INHBE相关疾病或病症的实例包括但不限于，代谢紊乱、代谢综合征、2型糖尿病、肥胖、前驱糖尿病、甘油三酯水平升高(高甘油三酯血症)、脂肪代谢障碍、肝脏炎症、脂肪肝、高胆固醇血症、与肝酶升高相关的疾病、非酒精性脂肪性肝炎、心血管疾病、肾脏疾病、腹部肥胖、胰岛素抵抗、高血压、高脂血症(hyperlipidemia)、心脏代谢紊乱以及与INHBE表达相关的癌症。In another aspect, the present disclosure relates to a method for treating or preventing an INHBE-related disease or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a dsRNAi agent such that an INHBE-related disease or condition is treated or prevented. The term "INHBE-related disease or condition" includes any disease or condition caused, mediated, or associated with INHBE gene expression or protein production, and includes any disease or condition that would benefit from or be improved by a reduction in INHBE gene expression or protein activity. Examples of INHBE-related diseases or conditions include, but are not limited to, metabolic disorders, metabolic syndrome, type 2 diabetes, obesity, prediabetes, elevated triglyceride levels (hypertriglyceridemia), lipodystrophy, liver inflammation, fatty liver, hypercholesterolemia, diseases associated with elevated liver enzymes, nonalcoholic steatohepatitis, cardiovascular disease, kidney disease, abdominal obesity, insulin resistance, hypertension, hyperlipidemia, cardiometabolic disorders, and cancers associated with INHBE expression.

在一些实施方案中，所述受试者是人。In some embodiments, the subject is a human.

在一些实施方案中，所述受试者患有代谢紊乱。In some embodiments, the subject suffers from a metabolic disorder.

在一些实施方案中，所述代谢紊乱是代谢综合征、2型糖尿病、肥胖、前驱糖尿病、高甘油三酯血症、脂肪代谢障碍、肝脏炎症、脂肪肝、高胆固醇血症、与肝酶升高相关的疾病病、非酒精性脂肪性肝炎、心血管疾病和肾脏疾病中的一种或多种。在一些实施方案中，所述代谢综合征包括但不限于腹部肥胖、胰岛素抵抗、高血压和异常血脂症中的一种或多种。In some embodiments, the metabolic disorder is one or more of metabolic syndrome, type 2 diabetes, obesity, prediabetes, hypertriglyceridemia, dyslipidemia, liver inflammation, fatty liver, hypercholesterolemia, diseases associated with elevated liver enzymes, nonalcoholic steatohepatitis, cardiovascular disease, and kidney disease. In some embodiments, the metabolic syndrome includes, but is not limited to, one or more of abdominal obesity, insulin resistance, hypertension, and dyslipidemia.

在一些实施方案中，所述INHBE相关的疾病或病症是代谢紊乱、代谢综合征、2型糖尿病、肥胖、前驱糖尿病、高甘油三酯血症、脂肪代谢障碍、肝脏炎症、脂肪肝、高胆固醇血症、与肝酶升高相关的疾病、非酒精性脂肪性肝炎、心血管疾病、肾脏疾病、腹部肥胖、胰岛素抵抗、高血压、异常血脂症、心脏代谢紊乱以及与INHBE表达相关的癌症中的一种或多种。In some embodiments, the INHBE-associated disease or condition is one or more of a metabolic disorder, metabolic syndrome, type 2 diabetes, obesity, prediabetes, hypertriglyceridemia, lipodystrophy, liver inflammation, fatty liver, hypercholesterolemia, a disease associated with elevated liver enzymes, nonalcoholic steatohepatitis, cardiovascular disease, kidney disease, abdominal obesity, insulin resistance, hypertension, dyslipidemia, a cardiometabolic disorder, and a cancer associated with INHBE expression.

非限制的代谢紊乱的实例包括碳水化合物紊乱，例如糖尿病、1型糖尿病、2型糖尿病、半乳糖血症、遗传性果糖不耐受、果糖1,6-二磷酸酶缺乏症、糖原贮积病、先天性糖基化病症、胰岛素抵抗、胰岛素不足、高胰岛素血症、糖耐量受损(IGT)、糖原代谢异常、氨基酸代谢紊乱，例如枫糖浆尿病(MSUD)、同型半胱氨酸尿症；有机酸代谢障碍，例如甲基丙二酸尿症、3-甲基戊二酸尿症-巴特综合征、戊二酸尿症、2-羟基戊二酸尿症-D型和L型；脂肪酸β-氧化障碍，例如中链酰基辅酶A脱氢酶缺乏症(MCAD)、长链3-羟酰基辅酶A脱氢酶缺乏症(LCHAD)、极长链酰基辅酶A脱氢酶缺乏症(VLCAD)；脂质代谢紊乱，例如GMI神经节苷脂贮积症、泰-萨克斯病、桑德霍夫病、法布里病、戈谢病、尼曼-匹克病、克拉伯病、粘脂贮积病、粘多糖贮积病；脂质分布和/或储存紊乱，例如脂肪营养不良、线粒体病症，例如线粒体心肌病、利氏病、线粒体脑病、乳酸性酸中毒、中风样发作(MELAS)；肌阵挛性癫痫伴破碎红纤维(MERRF)；神经病；共济失调；色素性视网膜炎(NARP)；巴特综合征；和过氧化物酶体疾病，例如泽韦格综合征(脑肝肾综合征)、X-连锁肾上腺脑白质营养不良、雷夫叙姆病。Non-limiting examples of metabolic disorders include carbohydrate disorders, such as diabetes, type 1 diabetes, type 2 diabetes, galactosemia, hereditary fructose intolerance, fructose 1,6-bisphosphatase deficiency, glycogen storage diseases, inborn errors of glycosylation, insulin resistance, insulin insufficiency, hyperinsulinemia, impaired glucose tolerance (IGT), dysglycogen metabolism, amino acid metabolism disorders, such as maple syrup urine disease (MSUD), homocystinuria; organic acid metabolism disorders, such as methylmalonic aciduria, 3-methylglutaric aciduria-Bartter syndrome, glutaric aciduria, 2-hydroxyglutaric aciduria-type D and L; fatty acid β-oxidation disorders, such as medium chain acyl-CoA dehydrogenase deficiency (MCAD), long chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LC-ADHD), acetyl-CoA dehydrogenase deficiency (ACED ... disorders of lipid distribution and/or storage, such as lipodystrophy, mitochondrial disorders, such as mitochondrial cardiomyopathy, Leigh's disease, mitochondrial encephalopathy, lactic acidosis, stroke-like episodes (MELAS); myoclonic epilepsy with ragged red fibers (MERRF); neuropathy; ataxia; retinitis pigmentosa (NARP); Bartter syndrome; and peroxisomal diseases, such as Zellweger syndrome (cerebrohepatorenal syndrome), X-linked adrenoleukodystrophy, and Refsum disease.

在一些实施方案中，代谢紊乱与身体脂肪分布相关，并且包括但不限于代谢综合征、2型糖尿病、高脂血症或血脂异常(低密度脂蛋白胆固醇(LDL-C)的循环水平高或改变)、高脂血症或血脂异常(低密度脂蛋白胆固醇(LDL-C)、甘油三酯、极低密度脂蛋白胆固醇(VLDL-C)、载脂蛋白B或其他脂质成分的循环水平高或改变)、肥胖(特别是腹部肥胖)、脂肪营养不良(例如无法在局部体脂贮藏脂肪(部分脂肪营养不良)或全身贮藏脂肪(脂肪萎缩))、胰岛素抵抗或在禁食或代谢挑战期间胰岛素水平升高或改变、肝脏脂肪沉积或脂肪肝身体脂肪分布及其并发症(例如肝硬化、肝纤维化或肝脏炎症)、非酒精性脂肪性肝炎、其他类型的肝脏炎症、肝酶水平增高或升高或改变或其他标志物提示的肝损伤、肝脏中的炎症或脂肪沉积、升高的血压和/或高血压、升高的血糖或葡萄糖或高血糖、代谢综合征、冠状动脉疾病和其他动脉粥样硬化病症及其并发症。在一些实施方案中，代谢紊乱与身体脂肪分布相关，其特征是腰部周围脂肪堆积较多(例如腹部脂肪较多或腰围较大)和/或臀部周围脂肪堆积较少(例如臀股脂肪较低或或臀围较小)，导致更大的腰臀比(WHR)，以及更高的心脏代谢风险，与体重指数(BMI)无关。In some embodiments, metabolic disorders are associated with body fat distribution and include, but are not limited to, metabolic syndrome, Type 2 diabetes, hyperlipidemia or dyslipidemia (high or altered circulating levels of low-density lipoprotein cholesterol (LDL-C)), hyperlipidemia or dyslipidemia (high or altered circulating levels of low-density lipoprotein cholesterol (LDL-C), triglycerides, very low-density lipoprotein cholesterol (VLDL-C), apolipoprotein B or other lipid components), obesity (especially abdominal obesity), lipodystrophy (such as the inability to store fat locally in body fat (partial lipodystrophy) or throughout the body (lipoatrophy)), insulin resistance or increased or altered insulin levels during fasting or metabolic challenges, liver fat deposits or fatty liver body fat distribution and its complications (such as cirrhosis, liver fibrosis or liver inflammation), non-alcoholic steatohepatitis, other types of liver inflammation, increased or increased or altered liver enzyme levels or other markers of liver damage, inflammation or fat deposits in the liver, increased blood pressure and/or hypertension, increased blood sugar or glucose or hyperglycemia, metabolic syndrome, coronary artery disease and other atherosclerotic conditions and their complications. In some embodiments, the metabolic disorder is associated with body fat distribution characterized by greater fat accumulation around the waist (e.g., more abdominal fat or larger waist circumference) and/or less fat accumulation around the hips (e.g., lower buttock fat or smaller hip circumference), resulting in a larger waist-to-hip ratio (WHR), and higher cardiometabolic risk, independent of body mass index (BMI).

在一个实施方案中，代谢紊乱是代谢综合征。本文中所用术语“代谢综合征”是指包括表现出营养过剩、久坐生活方式、遗传因素、年龄增长和由此产生的过度肥胖的集合的病症。代谢综合征包括腹部肥胖、胰岛素抵抗、血脂异常和血压升高，并且代谢综合征与其他合并症相关，包括血栓前状态、促炎症状态、非酒精性脂肪肝和生殖障碍。代谢综合征大约增加一倍的心血管疾病风险以及5倍的2型糖尿病发生风险。腹部肥胖(例如腰围过大(腰臀比高))、高血压、胰岛素抵抗和血脂异常是代谢综合征及其各个组成部分(例如向心性肥胖、空腹血糖(FBG)/前驱糖尿病/糖尿病、高胆固醇血症、高甘油三酯血症和高血压)中的核心部分。In one embodiment, metabolic disorder is metabolic syndrome.Term used herein " metabolic syndrome " refers to the disease that comprises the collection that shows overnutrition, sedentary lifestyle, genetic factors, age increase and the resulting excessive obesity.Metabolic syndrome comprises abdominal obesity, insulin resistance, dyslipidemia and elevated blood pressure, and metabolic syndrome is relevant with other comorbidities, comprises prothrombotic state, pro-inflammatory state, non-alcoholic fatty liver and reproductive disorder.Metabolic syndrome approximately doubles the risk of cardiovascular disease and 5 times the risk of type 2 diabetes.Abdominal obesity (such as waist circumference is too large (high waist-to-hip ratio)), hypertension, insulin resistance and dyslipidemia are the core parts in metabolic syndrome and its various components (such as central obesity, fasting blood glucose (FBG)/prediabetes/diabetes, hypercholesterolemia, hypertriglyceridemia and hypertension).

在一实施方案中，代谢紊乱是碳水化合物紊乱。在一个实施方案中，碳水化合物紊乱是糖尿病。如本文所用，术语“糖尿病”是指以高血糖(葡萄糖)水平为特征的代谢紊乱的集合，其由胰岛素分泌或作用或两者的缺陷引起。两种最常见的糖尿病类型，即1型糖尿病(也称为“I型糖尿病”)和2型糖尿病(也称为“II型糖尿病”)，都是由于身体无法调节胰岛素而导致的。胰岛素是胰腺响应血液中血糖(葡萄糖)水平升高而释放的一种激素。In one embodiment, the metabolic disorder is a carbohydrate disorder. In one embodiment, the carbohydrate disorder is diabetes. As used herein, the term "diabetes" refers to a collection of metabolic disorders characterized by high blood sugar (glucose) levels, which are caused by defects in insulin secretion or action or both. The two most common types of diabetes, type 1 diabetes (also referred to as "type I diabetes") and type 2 diabetes (also referred to as "type II diabetes"), are caused by the body's inability to regulate insulin. Insulin is a hormone released by the pancreas in response to increased blood sugar (glucose) levels in the blood.

本文使用的术语“1型糖尿病”是指当胰腺产生太少胰岛素而无法适当调节血糖水平时发生的慢性疾病。1型糖尿病也称为胰岛素依赖型糖尿病、IDDM和青少年型糖尿病。患有1型糖尿病(胰岛素依赖型糖尿病)的人通常产生很少的胰岛素或根本不产生胰岛素。1型糖尿病可能是由于胰腺β细胞的进行性自身免疫破坏以及随后的胰岛素缺乏所致。1型糖尿病患者必须定期注射胰岛素。The term "type 1 diabetes" as used herein refers to a chronic disease that occurs when the pancreas produces too little insulin to properly regulate blood sugar levels. Type 1 diabetes is also known as insulin-dependent diabetes, IDDM, and juvenile diabetes. People with type 1 diabetes (insulin-dependent diabetes) typically produce little or no insulin. Type 1 diabetes may be due to progressive autoimmune destruction of pancreatic beta cells and subsequent insulin deficiency. People with type 1 diabetes must take regular insulin injections.

在2型糖尿病(也称为非胰岛素依赖型糖尿病，NDDM)中，胰腺继续产生胰岛素，有时甚至高于正常水平，然而身体对其作用产生抵抗，导致相对胰岛素缺乏。肥胖是2型糖尿病的危险因素，大多数患有这种疾病的人都是肥胖的。In type 2 diabetes (also known as non-insulin-dependent diabetes mellitus, NDDM), the pancreas continues to produce insulin, sometimes even above normal levels, yet the body becomes resistant to its effects, resulting in relative insulin deficiency. Obesity is a risk factor for type 2 diabetes, and most people with the disease are obese.

在一些实施方案中，糖尿病包括前驱糖尿病。“前驱糖尿病”是指一种或多种早期糖尿病病症，包括葡萄糖利用受损、空腹血糖水平异常或受损、葡萄糖耐量受损、胰岛素敏感性受损和胰岛素抵抗。前驱糖尿病是2型糖尿病、心血管疾病和死亡的主要危险因素。开发治疗干预措施，通过有效治疗前驱糖尿病以预防2型糖尿病的发展，已受到广泛的关注。In some embodiments, diabetes includes prediabetes. "Prediabetes" refers to one or more early diabetic conditions, including impaired glucose utilization, abnormal or impaired fasting blood glucose levels, impaired glucose tolerance, impaired insulin sensitivity, and insulin resistance. Prediabetes is a major risk factor for type 2 diabetes, cardiovascular disease, and death. The development of therapeutic interventions to prevent the development of type 2 diabetes by effectively treating prediabetes has received widespread attention.

糖尿病可以通过进行葡萄糖耐量测试来诊断。临床上，糖尿病常分为几个基本类别。这些类别的主要实例包括自身免疫性糖尿病、非胰岛素依赖型糖尿病(2型NDDM或NIDDM)、胰岛素依赖型糖尿病(1型IDDM)、非自身免疫性糖尿病和青少年发病的成年型糖尿病(MODY)。另一分类，通常称为继发性糖尿病，是指由某些可识别的病症引起的糖尿病，这些病症导致或促使糖尿病综合征的发展。第二类别的实例包括但不限于，由胰腺疾病、激素异常引起的糖尿病、药物或化学物质诱发的糖尿病、由胰岛素受体异常引起的糖尿病、与遗传综合征相关的糖尿病和其他原因的糖尿病。Diabetes can be diagnosed by performing a glucose tolerance test. Clinically, diabetes is often divided into several basic categories. Major examples of these categories include autoimmune diabetes, non-insulin-dependent diabetes mellitus (type 2 NDDM or NIDDM), insulin-dependent diabetes mellitus (type 1 IDDM), non-autoimmune diabetes, and maturity-onset diabetes of the young (MODY). Another category, often called secondary diabetes, refers to diabetes caused by certain identifiable conditions. Diabetes caused by these conditions leads to or contributes to the development of diabetic syndromes. Examples of the second category include, but are not limited to, diabetes caused by pancreatic disease, hormonal abnormalities, diabetes induced by drugs or chemicals, diabetes caused by insulin receptor abnormalities, diabetes associated with genetic syndromes, and other causes of diabetes.

在一个实施方案中，代谢紊乱是脂质代谢紊乱。如本文所用的“脂质代谢紊乱”或“脂质代谢病症”是指与脂质代谢紊乱相关或由脂质代谢紊乱引起的任何病症。该术语还包括可导致高脂血症或表征为血液中任何或所有脂质和/或脂蛋白水平异常升高的症状的任何病症、疾病或症状。该术语可指遗传性疾病，例如家族性高甘油三酯血症、1型家族性部分性脂肪营养不良(FPLDI)，或继发或获得性疾病，例如由于疾病、病症或症状(例如，肾功能衰竭)、饮食或摄入某些药物(例如，由于治疗，例如，AIDS或HIV，而使用高效抗逆转录病毒疗法(HAART)从而诱发或获得的疾病)而诱发或获得的疾病。该术语还指脂肪分布和/或贮藏的紊乱，例如脂肪营养不良。In one embodiment, metabolic disorder is lipid metabolism disorder. " lipid metabolism disorder " or " lipid metabolism disorder " as used herein refers to any disease related to lipid metabolism disorder or caused by lipid metabolism disorder. The term also includes any disease, disease or symptom that can cause hyperlipidemia or be characterized as any or all lipids and/or lipoprotein levels in blood abnormally increased. The term may refer to hereditary diseases, such as familial hypertriglyceridemia, 1 type familial partial lipodystrophy (FPLDI), or secondary or acquired diseases, such as due to disease, disease or symptom (for example, renal failure), diet or intake of certain drugs (for example, due to treatment, for example, AIDS or HIV, and using highly effective antiretroviral therapy (HAART) to induce or obtain the disease) and induce or obtain the disease. The term also refers to the disorder of fat distribution and/or storage, such as lipodystrophy.

脂质代谢病症的另外的实例包括但不限于，动脉粥样硬化、血脂异常、高甘油三酯血症(包括药物诱发的高甘油三酯血症、利尿剂诱发的高甘油三酯血症、酒精诱发的高甘油三酯血症、β-肾上腺素能阻断剂诱发的高甘油三酯血症、雌激素诱发的高甘油三酯血症、糖皮质激素诱发的高甘油三酯血症、维生素A类诱发的高甘油三酯血症、西咪替丁诱发的高甘油三酯血症和家族性高甘油三酯血症)、与高甘油三酯血症相关的急性胰腺炎、乳糜微粒综合征、家族性乳糜微粒血症、Apo-E缺乏或抵抗、LPL缺乏或活性减退、高脂血症(包括家族性联合高脂血症)、高胆固醇血症、脂肪营养不良、与高胆固醇血症相关的痛风、黄瘤病(皮下胆固醇沉积)、异质性LPL缺乏症高脂血症、高LDL和异质性LPL缺乏症高脂血症、脂肪肝病或非酒精性脂肪性肝炎(NASH)。Additional examples of lipid metabolism disorders include, but are not limited to, atherosclerosis, dyslipidemia, hypertriglyceridemia (including drug-induced hypertriglyceridemia, diuretic-induced hypertriglyceridemia, alcohol-induced hypertriglyceridemia, beta-adrenergic blocking agent-induced hypertriglyceridemia, estrogen-induced hypertriglyceridemia, glucocorticoid-induced hypertriglyceridemia, vitamin A-induced hypertriglyceridemia, cimetidine-induced hypertriglyceridemia, and familial hypertriglyceridemia. hyperlipidemia, hyperlipidemia (including familial combined hyperlipidemia), hypercholesterolemia, lipodystrophy, gout associated with hypercholesterolemia, xanthomatosis (subcutaneous cholesterol deposits), heterogeneous LPL deficiency hyperlipidemia, high LDL and heterogeneous LPL deficiency hyperlipidemia, fatty liver disease, or nonalcoholic steatohepatitis (NASH).

在一个实施方案中，代谢紊乱是心血管疾病。所述心血管疾病可包括但不限于冠状动脉疾病(也称为缺血性心脏病)、高血压、与冠状动脉疾病相关的炎症、再狭窄、外周血管疾病或中风。In one embodiment, the metabolic disorder is a cardiovascular disease. The cardiovascular disease may include, but is not limited to, coronary artery disease (also known as ischemic heart disease), hypertension, inflammation associated with coronary artery disease, restenosis, peripheral vascular disease, or stroke.

在一个实施方案中，代谢紊乱是肾脏疾病。所述肾脏疾病可包括但不限于慢性肾脏疾病、糖尿病肾病或痛风。In one embodiment, the metabolic disorder is a kidney disease. The kidney disease may include, but is not limited to, chronic kidney disease, diabetic nephropathy, or gout.

在一个实施方案中，代谢紊乱是与体重相关的紊乱。体重紊乱可包括但不限于肥胖、代谢低下状态、甲状腺功能减退、尿毒症和与体重增加(包括体重快速增加)、体重减轻、持续体重减轻或体重减轻后体重恢复风险。In one embodiment, the metabolic disorder is a disorder related to weight. Weight disorders may include, but are not limited to, obesity, hypometabolic states, hypothyroidism, uremia, and risk of weight gain (including rapid weight gain), weight loss, sustained weight loss, or weight regain after weight loss.

在一个实施方案中，代谢紊乱是血糖紊乱。血糖紊乱可包括但不限于糖尿病、高血压和与胰岛素抵抗相关的多囊卵巢综合征(PCOS)。In one embodiment, the metabolic disorder is a blood sugar disorder. Blood sugar disorders may include, but are not limited to, diabetes, hypertension, and polycystic ovary syndrome (PCOS) associated with insulin resistance.

其他示例性代谢病症包括但不限于肾移植、肾病综合征、库欣综合征、肢端肥大症、系统性红斑狼疮、球蛋白异常血症、脂肪营养不良、1型糖原增多症和艾迪生氏病。Other exemplary metabolic disorders include, but are not limited to, renal transplantation, nephrotic syndrome, Cushing's syndrome, acromegaly, systemic lupus erythematosus, dysglobulinemia, lipodystrophy, glycogenotype 1, and Addison's disease.

在一个实施方案中，代谢紊乱是原发性高血压。“原发性高血压”可能是环境或遗传原因的结果(例如，没有明显的潜在医学原因的结果)。在另一个实施方案中，代谢病症是继发性高血压。“继发性高血压”具有可识别的潜在疾病，其可以是多种病因的，包括肾脏、血管和内分泌原因，例如肾实质疾病(例如多囊肾、肾小球或间质疾病)、肾血管疾病(例如肾动脉狭窄、纤维肌性发育不良)、内分泌疾病(例如肾上腺皮质类固醇或盐皮质激素过多、嗜铬细胞瘤、甲状腺功能亢进或甲状腺功能减退、生长激素过多、甲状旁腺功能亢进)、主动脉缩窄或使用口服避孕药。In one embodiment, the metabolic disorder is essential hypertension."Essential hypertension" may be the result of environmental or genetic causes (e.g., the result without obvious underlying medical causes). In another embodiment, the metabolic disorder is secondary hypertension."Secondary hypertension" has an identifiable underlying disease, which may be of multiple etiologies, including kidney, blood vessels and endocrine causes, such as renal parenchymal disease (e.g., polycystic kidney, glomerular or interstitial disease), renal vascular disease (e.g., renal artery stenosis, fibromuscular dysplasia), endocrine disease (e.g., adrenal cortical steroids or mineralocorticoids are excessive, pheochromocytoma, hyperthyroidism or hypothyroidism, growth hormone is excessive, hyperparathyroidism), coarctation of the aorta or use of oral contraceptives.

在一个实施方案中，代谢紊乱是难治性高血压。“难治性高血压”是指尽管同时使用三种不同类别的抗高血压药(其中之一是噻嗪类利尿剂)，但血压仍高于目标(例如，收缩压高于130毫米汞柱或舒张压高于90毫米汞柱)。使用四种或更多种药物控制血压的受试者也被认为患有难治性高血压。In one embodiment, the metabolic disorder is resistant hypertension. "Resistant hypertension" refers to blood pressure that is above target (e.g., systolic blood pressure above 130 mmHg or diastolic blood pressure above 90 mmHg) despite the simultaneous use of three different classes of antihypertensive drugs (one of which is a thiazide diuretic). Subjects who use four or more drugs to control their blood pressure are also considered to have resistant hypertension.

在本公开的一些实施方案中，受试者或细胞中INHBE基因的表达使受试者血清中的INHBE蛋白水平降低至少50％、至少60％、至少70％、至少80％、至少90％、或至少95％。In some embodiments of the present disclosure, the expression of the INHBE gene in the subject or cell causes the serum of the subject to The level of INHBE protein in the patient's tissues is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%.

在一些实施方案中，抑制细胞中INHBE基因的表达，使受试者血清中INHBE基因表达的蛋白水平降低至少30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％或95％。In some embodiments, the expression of the INHBE gene in a cell is inhibited, and the protein level of the INHBE gene expression in the serum of the subject is reduced by at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.

在本公开的一些实施方案中，以约0.01mg/kg至约50mg/kg的剂量、或以约0.10mg/kg至约50mg/kg的剂量向受试者施用dsRNAi剂，例如但不限于，约0.01mg/kg至约10mg/kg、约0.5mg/kg至约50mg/kg、约10mg/kg至约30mg/kg、约10mg/kg至约20mg/kg、约15mg/kg至约20mg/kg、约15mg/kg至约25mg/kg、约15mg/kg至约30mg/kg、或约20mg/kg至约30mg/kg的剂量。In some embodiments of the present disclosure, the dsRNAi agent is administered to a subject at a dose of about 0.01 mg/kg to about 50 mg/kg, or at a dose of about 0.10 mg/kg to about 50 mg/kg, for example, but not limited to, a dose of about 0.01 mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 50 mg/kg, about 10 mg/kg to about 30 mg/kg, about 10 mg/kg to about 20 mg/kg, about 15 mg/kg to about 20 mg/kg, about 15 mg/kg to about 25 mg/kg, about 15 mg/kg to about 30 mg/kg, or about 20 mg/kg to about 30 mg/kg.

在本公开的一些实施方案中，所述方法进一步包括确定来自受试者的样品中的INHBE水平，例如，在血液、血清、肝脏组织或脂肪组织样品中。可以在向受试者施用dsRNAi剂之前、期间和/或之后测定来自受试者的样品中的INHBE水平(例如，监测疗效或治疗效率，监测治疗前、治疗中或治疗后的INHBE mRNA和/或蛋白质水平等)。In some embodiments of the present disclosure, the method further comprises determining the level of INHBE in a sample from the subject, for example, in a blood, serum, liver tissue or adipose tissue sample. The level of INHBE in a sample from the subject can be determined before, during and/or after administration of a dsRNAi agent to the subject (e.g., monitoring efficacy or treatment efficiency, monitoring INHBE mRNA and/or protein levels before, during or after treatment, etc.).

在本公开的一些实施方案中，所述方法进一步包括向受试者施用另外的治疗剂以治疗代谢紊乱。所述治疗剂包括但不限于选自胰岛素、胰高血糖素样肽1激动剂、葡萄糖依赖性促胰岛素多肽(GIP)受体激动剂、胰高血糖素受体激动剂、磺酰脲、司格列奈、双胍、噻唑烷二酮、α-葡萄糖苷酶抑制剂、SGLT2抑制剂、DPP-4抑制剂、HMG-CoA还原酶抑制剂、他汀类药物以及任何前述药物的组合。In some embodiments of the present disclosure, the method further comprises administering to the subject an additional therapeutic agent to treat a metabolic disorder. The therapeutic agent includes, but is not limited to, selected from insulin, glucagon-like peptide 1 agonists, glucose-dependent insulinotropic polypeptide (GIP) receptor agonists, glucagon receptor agonists, sulfonylureas, seglininides, biguanides, thiazolidinediones, alpha-glucosidase inhibitors, SGLT2 inhibitors, DPP-4 inhibitors, HMG-CoA reductase inhibitors, statins, and any combination of the foregoing drugs.

在一些实施方案中，本公开的dsRNAi剂通过注射或者通过输注施用。在一个实施方案中，双链RNAi剂经皮下施用。在一个实施方案中，双链RNAi剂通过肌内施用。在一个实施方案中，双链RNAi剂通过静脉内施用。在一个实施方式中，双链RNAi剂通过肺部全身给药施用，例如鼻内施用或口腔吸入施用。In some embodiments, the dsRNAi agent of the present disclosure is administered by injection or by infusion. In one embodiment, the double-stranded RNAi agent is administered subcutaneously. In one embodiment, the double-stranded RNAi agent is administered intramuscularly. In one embodiment, the double-stranded RNAi agent is administered intravenously. In one embodiment, the double-stranded RNAi agent is administered by systemic administration to the lungs, such as intranasal administration or oral inhalation administration.

在一些实施方案中，药物组合物其包含本公开的dsRNAi剂或其药学上可接受的盐，以及药学上可接受的载体。可将本公开的药物组合物实际用于对各种相应疾病或病症的预防和/或治疗。可接受的载体(或赋形剂)是除活性药用成分(API，治疗性产物，例如本公开的dsRNA剂)以外有意包含在药物递送系统中的物质。载体或赋形剂不以或不旨在以预期剂量发挥治疗作用。载体或赋形剂可起以下作用：a)助于药物递送系统在制备期间的处理；b)保护、支持或增强API的稳定性、生物利用度或患者可接受性；c)助于产物鉴定；和/或d)增强API在储存或使用期间的整体安全性、有效性或递送方面的任何其它属性。In some embodiments, the pharmaceutical composition comprises a dsRNAi agent of the present disclosure or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. The pharmaceutical composition of the present disclosure can be actually used for the prevention and/or treatment of various corresponding diseases or conditions. An acceptable carrier (or excipient) is a substance that is intentionally included in a drug delivery system in addition to an active pharmaceutical ingredient (API, therapeutic product, such as a dsRNA agent of the present disclosure). The carrier or excipient does not or is not intended to exert a therapeutic effect at the expected dose. The carrier or excipient may play the following roles: a) assists in the handling of the drug delivery system during preparation; b) protects, supports or enhances the stability, bioavailability or patient acceptability of the API; c) assists in product identification; and/or d) enhances any other properties of the overall safety, effectiveness or delivery of the API during storage or use.

载体或赋形剂包括但不限于以下组分：吸收增强剂、抗粘剂、消泡剂、抗氧化剂、粘合剂、缓冲剂、载体、包衣剂、着色剂、递送增强剂、递送聚合物、去污剂、右旋糖酐、右旋糖、稀释剂、崩解剂、乳化剂、膨胀剂、填充剂、调味剂、助流剂、湿润剂、润滑剂、油类、聚合物、防腐剂、盐水、盐类、溶剂、糖类、表面活性剂、悬浮剂、持续释放基质、甜味剂、增稠剂、张度剂、媒介物、防水剂和润湿剂。Carriers or excipients include, but are not limited to, the following components: absorption enhancers, anti-adherents, anti-foaming agents, antioxidants, binders, buffers, carriers, coatings, colorants, delivery enhancers, delivery polymers, detergents, dextran, dextrose, diluents, disintegrants, emulsifiers, expanders, fillers, flavoring agents, glidants, wetting agents, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, surfactants, suspending agents, sustained release matrices, sweeteners, thickeners, tonicity agents, vehicles, waterproofing agents, and wetting agents.

在一些实施方案中，所述药物组合物的载体是非缓冲溶液或缓冲溶液。典型的非缓冲溶液是盐水或水，缓冲溶液包括乙酸盐、柠檬酸盐、醇溶谷蛋白、碳酸盐和磷酸盐中的一种或多种。在一些实施方案中，缓冲溶液是磷酸盐缓冲盐水(PBS)。In some embodiments, the carrier of the pharmaceutical composition is a non-buffered solution or a buffered solution. Typical non-buffered solutions are saline or water, and buffered solutions include one or more of acetate, citrate, prolamin, carbonate, and phosphate. In some embodiments, the buffered solution is phosphate buffered saline (PBS).

本公开包括所述特定实施方式的所有组合。本公开的进一步实施方式及可应用性的完整范畴将自下文所提供的详细描述变得显而易见。然而，应理解，尽管详细描述及特定实施例指示本公开的优选实施方式，但仅以说明的方式提供这些描述及实施例，因为本公开的精神及范畴内的各种改变及修改将自此详细描述对熟悉此项技术者变得显而易见。出于所有目的，包括引文在内的本文所引用的所有公开物、专利及专利申请将以引用的方式全部并入本文。The present disclosure includes all combinations of the specific embodiments described. Further embodiments and the full scope of applicability of the present disclosure will become apparent from the detailed description provided below. However, it should be understood that although the detailed description and specific examples indicate preferred embodiments of the present disclosure, these descriptions and examples are provided by way of illustration only, as various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art from this detailed description. All publications, patents, and patent applications cited herein, including citations, are hereby incorporated by reference in their entirety for all purposes.

本公开的化合物可以通过本领域技术人员所熟知的多种合成方法来制备，包括下面列举的具体实施方式、其与其他方法的结合所形成的实施方式以及本领域技术上人员所熟知的等同替换方式，优选的实施方式包括但不限于本公开的实施例。The compounds disclosed herein can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the examples disclosed herein.

实施例Example

通过参考以下实施例将更容易理解本发明，所述实施例用于说明本发明，而不应被解释为以任何方式限制本发明的范围。The present invention will be more readily understood by reference to the following examples, which are provided to illustrate the present invention and are not to be construed as limiting the scope of the present invention in any way.

除非另有定义或上下文另有明确规定，本发明使用的所有技术和科学术语具有与本发明所属领域的普通技术人员通常理解的相同的含义。应当理解，与本发明所述类似或等同的任何方法和材料可用于本发明的实践或测试。除非另有说明，否则本发明中所使用的材料和仪器均常规商购所得。Unless otherwise defined or the context clearly dictates otherwise, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. It should be understood that any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. Unless otherwise specified, the materials and instruments used in the present invention are conventionally commercially available.

实施例1、siRNA合成Example 1. siRNA Synthesis

1.1靶序列筛选1.1 Target sequence screening

siRNA根据INHBE全mRNA序列设计，所有序列均来源于NCBI基因数据库(https://www.ncbi.nlm.nih.gov/gene/)。所有siRNA设计时保证与人(Gene ID:83729)及食蟹猴(Gene ID:102127493)序列完全一致。siRNA was designed based on the full mRNA sequence of INHBE, and all sequences were derived from the NCBI gene database (https://www.ncbi.nlm.nih.gov/gene/). All siRNAs were designed to be completely consistent with the sequences of humans (Gene ID: 83729) and cynomolgus monkeys (Gene ID: 102127493).

对全序列进行扫描后获得所有潜在19个核苷酸长的siRNA序列，并同时比对食蟹猴的序列，保证对食蟹猴序列的匹配。所有人/食蟹猴结合序列通过BLAST比对人源全转录组mRNA序列，并去除所有可能存在脱靶作用的siRNA。通过siRNA理性设计原则对所有siRNA的活性进行评估，去除理论活性较低的分子。After scanning the entire sequence, all potential 19-nucleotide-long siRNA sequences were obtained, and the cynomolgus monkey sequence was compared to ensure the match of the cynomolgus monkey sequence. All human/cynomolgus monkey combined sequences were compared to the human full transcriptome mRNA sequence by BLAST, and all siRNAs that may have off-target effects were removed. The activity of all siRNAs was evaluated using the siRNA rational design principle to remove molecules with low theoretical activity.

1.2 siRNA合成1.2 siRNA synthesis

针对INHBE在不同区域设计siRNA(siRNA序列如表1、表2a和表2b所示)，交由苏州贝信生物科技有限公司进行合成并退火后获得。其中，在具有平端的siRNA序列(例如IN-151至IN-230)的反义链3’端添加两个额外的互补核苷酸或2个dT(即dTdT)后进行合成，以用于测定体外活性。siRNAs were designed for different regions of INHBE (siRNA sequences are shown in Table 1, Table 2a and Table 2b), and synthesized and annealed by Suzhou Beixin Biotechnology Co., Ltd. Among them, two additional complementary nucleotides or 2 dTs (i.e., dTdT) were added to the 3' end of the antisense strand of the siRNA sequences with blunt ends (e.g., IN-151 to IN-230) before synthesis for in vitro activity determination.

表1：未修饰的INHBE dsRNA剂的正义链序列和反义链序列





Table 1: Sense and antisense strand sequences of unmodified INHBE dsRNA agents

表2a：修饰的INHBE dsRNA试剂的正义链序列和反义链序列















Table 2a: Sense and antisense strand sequences of modified INHBE dsRNA reagents

表2b：修饰的INHBE dsRNA试剂的正义链序列和反义链序列



Table 2b: Sense and antisense strand sequences of modified INHBE dsRNA reagents

1.3 RNAi合成1.3 RNAi synthesis

使用式1-式33中的GalNAc偶联固载(CPG或PS)，并依照原始序列设计全修饰siRNA，并交由苏州贝信生物科技有限公司进行合成并退火后获得。The GalNAc-coupled immobilized carrier (CPG or PS) in Formula 1-Formula 33 was used, and the fully modified siRNA was designed according to the original sequence, and then delivered to Suzhou Beixin Biotechnology Co., Ltd. for synthesis and annealing to obtain it.

其中，式1-式33中的靶向配体的合成参考WO2022266753A1。将表1、表2a和表2b中的dsRNA与靶向配体连接形成式5化合物的RNAi的命名为“双链体编号-L5”。The synthesis of the targeting ligands in Formula 1 to Formula 33 refers to WO2022266753A1. The RNAi of the compound of Formula 5 formed by connecting the dsRNA in Table 1, Table 2a and Table 2b with the targeting ligand is named "Duplex No.-L5".

实施例2、siRNA体外活性检测方法Example 2: siRNA in vitro activity detection method

2.1、荧光定量PCR2.1 Fluorescence quantitative PCR

2.1.1细胞培养及转染2.1.1 Cell culture and transfection

Hep3B细胞使用添加有10％胎牛血清(FBS,Gibco)和双抗(Gibco)的MEM培养基(Gibco)于5％CO₂，37℃进行培养，在细胞生长接近完全覆盖培养瓶后，使用胰酶消化后重悬。重悬后的细胞调整密度并以1.5×10⁴/孔接种至96孔板中，悬浮加入siRNA转染复合物。siRNA转染复合物为含有0.3μL/孔脂质体RNAi Max(Thermo)的Opti-MEM(Gibco)与siRNA 1:1混合后所得。细胞培养一定时间后按照使用说明使用Dynabeads^TMmRNA分离试剂盒(Thermo)抽提获得全mRNA，最后使用20μL RNase free H₂O于80℃ 5分钟洗脱mRNA，迅速在磁力架上转出15μL上清液。加热过程由BIO-RAD T100 Thermal Cycler PCR仪完成。Hep3B cells were cultured in MEM medium (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and double antibody (Gibco) at 5% CO₂ and 37°C. When the cells grew to almost completely cover the culture flask, they were digested with trypsin and resuspended. The resuspended cells were adjusted to a density and seeded into a 96-well plate at 1.5×10⁴ /well, and siRNA transfection complex was added to the suspension. The siRNA transfection complex was obtained by mixing Opti-MEM (Gibco) containing 0.3μL/well liposome RNAi Max (Thermo) with siRNA at a ratio of 1:1. After the cells were cultured for a certain period of time, the Dynabeads^TM mRNA isolation kit (Thermo) was used according to the instructions to extract the total mRNA. Finally, 20μL RNase free H₂ O was used to elute the mRNA at 80°C for 5 minutes, and 15μL of the supernatant was quickly transferred on the magnetic stand. The heating process was completed by a BIO-RAD T100 Thermal Cycler PCR instrument.

2.1.2反转录与荧光定量PCR2.1.2 Reverse transcription and fluorescence quantitative PCR

使用一步法进行cDNA合成及荧光定量PCR。使用ΔΔCt法对INHBE及GAPDH(甘油醛-3-磷酸脱氢酶)的相对mRNA水平进行检测。向上述实施例2.1.1中转移出的上清液中加入按照使用说明(HiScript II One Step RT-PCR Kit，Vazyme)加入One Step mix buffer，One Step Enzyme mix，目标基因的正、反向引物进行反应。One-step method was used for cDNA synthesis and fluorescence quantitative PCR. The relative mRNA levels of INHBE and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were detected using the ΔΔCt method. One Step mix buffer, One Step Enzyme mix, and the forward and reverse primers of the target gene were added to the supernatant transferred from the above Example 2.1.1 according to the instructions (HiScript II One Step RT-PCR Kit, Vazyme) for reaction.

使用仪器为LightCycler 480 II(Roche)。反应条件为：(1)反转录50℃，3分钟(2)预变性95℃，30秒；(3)变性95℃，10秒，退火延伸60℃，30秒。步骤(3)持续40个循环。所得结果使用空白对照(Blank)进行标准化后获得相对mRNA水平及敲低效率。如需运算IC50则使用Graphpad Prism进行四参数拟合后获得。The instrument used was LightCycler 480 II (Roche). The reaction conditions were: (1) reverse transcription at 50°C for 3 minutes (2) pre-denaturation at 95°C for 30 seconds; (3) denaturation at 95°C for 10 seconds, annealing and extension at 60°C for 30 seconds. Step (3) lasted for 40 cycles. The results were normalized with the blank control to obtain the relative mRNA level and knockdown efficiency. If IC50 needs to be calculated, it can be obtained by four-parameter fitting using Graphpad Prism.

其中，NC、NCm和基于WO2023003922A1的IN-Ref1至IN-Ref9、IN-Ref1m,、IN-Ref2m和IN-Ref8m的序列如下表3所示。IN-Ref1m-L96为IN-Ref1m的dsRNA与L96连接形成的RNAi，L96的结构与制备参见WO2014089313A1。Among them, the sequences of NC, NCM and IN-Ref1 to IN-Ref9, IN-Ref1m, IN-Ref2m and IN-Ref8m based on WO2023003922A1 are shown in Table 3. IN-Ref1m-L96 is an RNAi formed by connecting the dsRNA of IN-Ref1m and L96. The structure and preparation of L96 refer to WO2014089313A1.

表3：NC、NCm及其他参考序列

Table 3: NC, NCm and other reference sequences

测试结果见图1至图5b。表4示出了siRNA的IC50测定结果及最大抑制率。“NC”表示阴性对照。“NA”表示不适用。其中siRNA ID的双链体编号中小数点后数字或字母代表不同的测定批次。其中，批次1、批次2和批次3均涉及人源INHBE，其余批次也均涉及人源INHBE。表5a示出了以IN-Ref8为标准，通过IC50值进行标准化后获得的相对活性。表5b示出了不同浓度下IN-Ref8和IN-Ref8m的抑制率。The test results are shown in Figures 1 to 5b. Table 4 shows the IC50 determination results and maximum inhibition rate of siRNA. "NC" indicates negative control. "NA" indicates not applicable. The numbers or letters after the decimal point in the duplex number of the siRNA ID represent different batches of determination. Among them, batch 1, batch 2 and batch 3 all involve human INHBE, and the remaining batches also involve human INHBE. Table 5a shows the relative activity obtained after standardization by IC50 value with IN-Ref8 as the standard. Table 5b shows the inhibition rate of IN-Ref8 and IN-Ref8m at different concentrations.

表4
Table 4

表5a

Table 5a

表5b
Table 5b

2.2报告基因法2.2 Reporter gene method

SK-Hep-1-PsiCheck-INHBE细胞含有INHBE全长mRNA序列及荧光素酶基因串联表达系统。使用添加有10％FBS(Gibco)、1ug/ml嘌呤霉素以及双抗(Gibco)的MEM培养基(Gibco)于5％CO₂，37℃进行培养，在细胞生长接近完全覆盖培养瓶后，使用胰酶消化后重悬。重悬后的细胞调整密度并以1.5×10⁴/孔接种至96孔板中，同时加入siRNA转染复合物。siRNA转染复合物为含有0.3μL/孔脂质体RNAi Max(Thermo)的Opti-MEM(Gibco)与siRNA 1:1混合后所得。细胞培养一定时间后裂解并加入Renilla底物(Vazyme)进行荧光活性检测。SK-Hep-1-PsiCheck-INHBE cells contain the full-length INHBE mRNA sequence and the luciferase gene tandem expression system. MEM medium (Gibco) supplemented with 10% FBS (Gibco), 1ug/ml puromycin and double antibody (Gibco) was used for culture at 5% CO₂ and 37°C. After the cells grew to almost completely cover the culture flask, they were digested with trypsin and resuspended. The resuspended cells were adjusted to a density and seeded into a 96-well plate at 1.5×10⁴ /well, and siRNA transfection complex was added at the same time. The siRNA transfection complex was obtained by mixing Opti-MEM (Gibco) containing 0.3μL/well liposome RNAi Max (Thermo) with siRNA at a ratio of 1:1. After a certain period of cell culture, the cells were lysed and Renilla substrate (Vazyme) was added for fluorescence activity detection.

测试结果见图6～图22、图23和图24。图9和图16为鼠源INHBE中测得，其余均为人源INHBE中测得。表6和表8示出了siRNA的IC50测定结果(通过Graphpad Prism进行四参数拟合)以及标准化至空白对照组的抑制率，其中siRNA ID的双链体编号中小数点后数字或字母代表不同的测定批次。其中，批次4和批次5均涉及人源INHBE，批次6涉及鼠源INHBE，其余批次均涉及人源INHBE。表7示出的是以IN-Ref8为标准在相同浓度下的抑制率进行标准化后获得的IC50值。表9示出了不同浓度下IN-Ref8和IN-Ref8m的抑制率。The test results are shown in Figures 6 to 22, 23 and 24. Figures 9 and 16 were measured in mouse INHBE, and the rest were measured in human INHBE. Tables 6 and 8 show the IC50 determination results of siRNA (four-parameter fitting by Graphpad Prism) and the inhibition rate standardized to the blank control group, where the decimal numbers or letters after the decimal point in the duplex number of the siRNA ID represent different measurement batches. Among them, batches 4 and 5 both involve human INHBE, batch 6 involves mouse INHBE, and the remaining batches all involve human INHBE. Table 7 shows the IC50 values obtained after standardization of the inhibition rate at the same concentration using IN-Ref8 as the standard. Table 9 shows the inhibition rates of IN-Ref8 and IN-Ref8m at different concentrations.

表6

Table 6

表7
Table 7

表8
Table 8

表9
Table 9

实施例3：siRNA体外稳定性检测方法Example 3: siRNA in vitro stability detection method

3.1、Stem-loop PCR3.1. Stem-loop PCR

Stem-loop法已广泛运用于检测siRNA及miRNA的绝对浓度(Curr Protoc Mol Biol.2011 Jul；Chapter 15:Unit 15.10.)。针对不同的siRNA设计相应的茎-环引物，并使用该引物替代寡dT进行反转录后，使用荧光定量PCR的方式应用标准曲线换算进行siRNA浓度的测定。反转录使用HiScript III 1st Strand cDNA Synthesis Kit(Vazyme)，过程为：(1)样品预处理：1)85℃、5分钟；2)60℃、5分钟；(2)反转录：根据试剂盒所述条件配置反应体系并完成反转录；(3)使用SYBR green(TIANGEN)试剂盒所述条件配置反应体系并完成进行荧光定量PCR。反转录过程由BIO-RAD T100 Thermal Cycler PCR仪完成，荧光定量PCR由罗氏LC480 II完成。The stem-loop method has been widely used to detect the absolute concentration of siRNA and miRNA (Curr Protoc Mol Biol. 2011 Jul; Chapter 15:Unit 15.10.). Corresponding stem-loop primers were designed for different siRNAs, and the primers were used to replace oligo dT for reverse transcription. Then, the concentration of siRNA was determined by fluorescence quantitative PCR using standard curve conversion. Reverse transcription was performed using HiScript III 1st Strand cDNA Synthesis Kit (Vazyme), and the process was as follows: (1) Sample pretreatment: 1) 85°C, 5 minutes; 2) 60°C, 5 minutes; (2) Reverse transcription: Prepare the reaction system according to the conditions described in the kit and complete the reverse transcription; (3) Prepare the reaction system using the conditions described in the SYBR green (TIANGEN) kit and complete the fluorescence quantitative PCR. The reverse transcription process was completed by BIO-RAD T100 Thermal Cycler PCR instrument, and the fluorescence quantitative PCR was completed by Roche LC480 II.

3.1.1血清稳定性3.1.1 Serum stability

(1)将siRNA稀释至2uM，使用FBS(Gibco，胎牛血清)将siRNA稀释至100nM，FBS占比≥90％，标记为零点；(2)从零点吸取10μL加入到新的样品管中标记相应时间点，放入37℃孵育不同时间；(3)根据孵育时间取出相应样品，投入液氮中快速冷冻，随后将样品保存于-80℃，使用前混匀样品，使用零点组样品梯度稀释作为标准曲线，使用3.1 Stem-loop PCR方法进行检测。(1) Dilute siRNA to 2uM, and use FBS (Gibco, fetal bovine serum) to dilute siRNA to 100nM, with FBS accounting for ≥90%, marked as zero point; (2) Pipette 10μL from zero point into a new sample tube, mark the corresponding time point, and incubate at 37℃ for different time periods; (3) Take out the corresponding samples according to the incubation time, quickly freeze them in liquid nitrogen, and then store the samples at -80℃. Mix the samples before use, use the gradient dilution of the zero point group samples as the standard curve, and use the 3.1 Stem-loop PCR method for detection.

将不同浓度的标准品PCR得到的Ct值使用GraphPad prism软件进行线性拟合，得到标准品浓度与Ct值相对应的标准曲线，再将各孵育时间点样品的Ct值通过标准曲线回算得到样品中siRNA浓度。将各时间点样品浓度标准化到0h样品浓度，计算得到各时间点相对于0h的样品siRNA残余浓度百分比。图25为siRNA在血清稳定性检测结果。The Ct values obtained by PCR of standards with different concentrations were linearly fitted using GraphPad prism software to obtain a standard curve corresponding to the standard concentration and Ct value. The Ct values of the samples at each incubation time point were then back-calculated using the standard curve to obtain the siRNA concentration in the sample. The sample concentration at each time point was standardized to the sample concentration at 0h, and the percentage of residual siRNA concentration of the sample at each time point relative to 0h was calculated. Figure 25 shows the results of siRNA stability test in serum.

3.1.2人肝S9稳定性3.1.2 Stability of human liver S9

(1)使用人肝S9(BioIVT)稀释siRNA；(2)放入37℃孵育不同时间。(3)根据孵育时间取出相应样品，投入到液氮中快速冷冻。随后将样品保存于-80℃备用。依照3.1 Stem-loop PCR方法进行检测。(1) Use human liver S9 (BioIVT) to dilute siRNA; (2) Incubate at 37℃ for different time periods. (3) Take out the corresponding samples according to the incubation time and quickly freeze them in liquid nitrogen. Then store the samples at -80℃ for later use. Perform the test according to 3.1 Stem-loop PCR method.

将不同浓度的标准品PCR得到的Ct值使用GraphPad prism软件进行线性拟合，得到标准品浓度与Ct值相对应的标准曲线，再将各孵育时间点样品的Ct值通过标准曲线回算得到样品中siRNA浓度。将各时间点样品浓度标准化到0h样品浓度，计算得到各时间点相对于0h的样品siRNA残余浓度百分比。图26为siRNA在人肝S9稳定性检测结果。The Ct values obtained by PCR of standards with different concentrations were linearly fitted using GraphPad prism software to obtain a standard curve corresponding to the standard concentration and Ct value. The Ct values of the samples at each incubation time point were then back-calculated using the standard curve to obtain the siRNA concentration in the sample. The sample concentration at each time point was standardized to the sample concentration at 0h, and the percentage of residual siRNA concentration of the sample at each time point relative to 0h was calculated. Figure 26 shows the results of siRNA stability test in human liver S9.

实施例4：肝脏中siRNA药物浓度检测Example 4: Detection of siRNA drug concentration in the liver

小鼠以3mg/kg剂量皮下给药后，不同时间取肝脏组织。肝脏组织称重，并于4℃条件下加入PBS，使用匀浆仪(上海净信JXFSTPRP-64)匀浆。使用Stem-loop PCR方法进行肝匀浆中siRNA含量的检测。根据标准曲线回算后得到给药组小鼠肝组织中siRNA浓度。图27示出了给药不同时间后每组小鼠(3只)肝脏中siRNA含量变化。图28示出的以每个RNAi在给药7天后小鼠(3只)肝组织中的含量进行标准化后的不同siRNA在小鼠肝脏中的含量百分比。After mice were subcutaneously administered 3 mg/kg, liver tissues were collected at different times. The liver tissues were weighed and added with PBS at 4°C and homogenized using a homogenizer (Shanghai Jingxin JXFSTPRP-64). The siRNA content in the liver homogenate was detected using the Stem-loop PCR method. The siRNA content in the liver tissue of the mice in the drug group was obtained by back-calculation based on the standard curve. siRNA concentration. Figure 27 shows the changes in siRNA content in the liver of each group of mice (3 mice) after administration at different times. Figure 28 shows the percentage of different siRNA content in the liver of mice after standardization with the content of each RNAi in the liver tissue of mice (3 mice) 7 days after administration.

实施例5：INHBE siRNA在健康食蟹猴中的体内药效Example 5: In vivo efficacy of INHBE siRNA in healthy cynomolgus monkeys

为了评估siRNA对INHBE的体内活性，使用健康食蟹猴进行体内活性检测。siRNA偶联物(RNAi)，使用生理盐水进行稀释后于第1天按照实验设计以9mg/kg的剂量进行皮下注射。使用空白的生理盐水作为阴性对照。分别于在给药前1天及给药后第14、28、42、56、70、天时采集肝穿刺样品。使用FastPure Cell/Tissue Total RNA Isolation Kit V2(Vazyme)试剂盒提取总RNA。按照使用说明(HiScript II One Step RT-PCR Kit，Vazyme)进行荧光定量PCR测定INHBE mRNA水平。荧光定量PCR使用仪器为LightCycler 480 II(Roche)。In order to evaluate the in vivo activity of siRNA against INHBE, healthy cynomolgus monkeys were used for in vivo activity detection. siRNA conjugates (RNAi) were diluted with saline and injected subcutaneously at a dose of 9 mg/kg on the first day according to the experimental design. Blank saline was used as a negative control. Liver puncture samples were collected 1 day before administration and on days 14, 28, 42, 56, and 70 after administration. Total RNA was extracted using the FastPure Cell/Tissue Total RNA Isolation Kit V2 (Vazyme). Fluorescence quantitative PCR was performed according to the instructions (HiScript II One Step RT-PCR Kit, Vazyme) to determine the level of INHBE mRNA. The instrument used for fluorescence quantitative PCR was LightCycler 480 II (Roche).

首先将所得测试结果使用内参基因(GAPDH)进行标准化后获得相对mRNA水平，对于各动物的INHBE mRNA水平进行个体标准化。对于个体标准化，在一个时间点分别对于各动物的INHBE mRNA水平的平均值除以该动物的治疗前表达水平的平均值以确定“标准化至治疗前”的相对表达量。表10示出了以治疗前每只动物自身肝组织中mRNA水平为标准，各动物进行标准化后，每组动物相对表达水平的均值，“vehicle”表示空白对照。First, the test results were standardized using the internal reference gene (GAPDH) to obtain relative mRNA levels, and the INHBE mRNA levels of each animal were individually standardized. For individual standardization, the mean value of the INHBE mRNA level of each animal at a time point was divided by the mean value of the expression level of the animal before treatment to determine the relative expression level "normalized to before treatment". Table 10 shows the mean value of the relative expression level of each group of animals after each animal was standardized using the mRNA level in the liver tissue of each animal before treatment as the standard, and "vehicle" represents the blank control.

表10
Table 10

尽管参照本发明的实施例详细描述了本发明，但提供这些实施例是为了说明而不是限制本发明。根据本发明原理能够得到的其它实施例均属于本发明权利要求所界定的范畴。Although the present invention has been described in detail with reference to the embodiments of the present invention, these embodiments are provided to illustrate rather than limit the present invention. Other embodiments that can be obtained according to the principles of the present invention all belong to the scope defined by the claims of the present invention.

Claims (31)

Translated fromChinese

一种dsRNA，其包含正义链和反义链，所述反义链包含与表1、表2a或表2b中所示的任一序列相差0、1、2或3个核苷酸的至少15个连续核苷酸，所述正义链与所述反义链具有至少15个、16个、17个、18个、19个、20个或21个核苷酸互补。A dsRNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises at least 15 consecutive nucleotides that differ from any one of the sequences shown in Table 1, Table 2a or Table 2b by 0, 1, 2 or 3 nucleotides, and the sense strand has at least 15, 16, 17, 18, 19, 20 or 21 nucleotides complementary to the antisense strand.如权利要求1所述的dsRNA，其特征在于，所述反义链包含与表1、表2a或表2b中所示的任一序列相差0、1、2或3个核苷酸的至少15个、16个、17个、18个、19个、20个或21个连续核苷酸。The dsRNA of claim 1, wherein the antisense strand comprises at least 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides that differ from any of the sequences shown in Table 1, Table 2a or Table 2b by 0, 1, 2 or 3 nucleotides.如权利要求1或2所述的dsRNA，其特征在于，所述反义链和正义链的长度各自独立地为17-25个核苷酸。The dsRNA according to claim 1 or 2, characterized in that the length of the antisense strand and the sense strand are each independently 17-25 nucleotides.如权利要求1至3中任一项所述的dsRNA，其特征在于，其包含表1、表2a或表2b中的任一个反义链或正义链序列。The dsRNA according to any one of claims 1 to 3, characterized in that it comprises any antisense strand or sense strand sequence in Table 1, Table 2a or Table 2b.如权利要求1至4中任一项所述的dsRNA，其特征在于，其包含表1、表2a或表2b中的双链体序列所示的反义链序列和正义链序列。The dsRNA according to any one of claims 1 to 4, characterized in that it comprises an antisense strand sequence and a sense strand sequence shown in the duplex sequence in Table 1, Table 2a or Table 2b.如权利要求1至5中任一项所述的dsRNA，其特征在于，所述正义链和所述反义链中的至少一个核苷酸为经修饰的核苷酸。The dsRNA according to any one of claims 1 to 5, characterized in that at least one nucleotide in the sense strand and the antisense strand is a modified nucleotide.如权利要求6所述的dsRNA，其特征在于，所述经修饰的核苷酸选自：2’-O-甲基修饰核苷酸、2’-氟修饰核苷酸、2’-脱氧核苷酸、2’-氟，2’-脱氧修饰核苷酸、2’-甲氧基乙基修饰核苷酸、2’-氨基修饰核苷酸、2’-烷基修饰核苷酸、2’-烷氧基修饰核苷酸、2’-F-阿糖核苷酸、硫代磷酸酯修饰核苷酸、脱碱基核苷酸、吗啉代核苷酸、锁定核苷酸、倒置核苷酸和次黄嘌呤碱基置换核苷酸。The dsRNA of claim 6, wherein the modified nucleotide is selected from the group consisting of 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy nucleotides, 2'-fluoro, 2'-deoxy modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, 2'-alkoxy modified nucleotides, 2'-F-arabino nucleotides, phosphorothioate modified nucleotides, abasic nucleotides, morpholino nucleotides, locked nucleotides, inverted nucleotides, and inosine base substituted nucleotides.如权利要求1所述的dsRNA，其特征在于，所述反义链包含选自SEQ ID NO：86、182、212、350、376、402、440、476、478、502、504、506、508、510、512、514、516、518、520、522、524、526、528、530、532、534、536、538、540、542、544、546、548、550、552、554、556、558、560、562、564、566、568、570、572、574、576、578、580、582、584、586、588、590、592、594、596、598、600、602、604、606、608、610、612、614、616、618、620、622、624、626、628、630、632、634、636、638、640、642、644、646、648、650、652、654、656、658、660、662、664、666、668、670、672、674、676、678、680、682、684、686、688、690、692、694、696、698、700、702、704、706、708、710、712、714、716、718、720、722、724、726、728、730、732、734、736、738、740、742、744、746、748、750、752、754、756、758、760、762、764、766、768、770、772、774、776、778、780、782、784、786、788、790、792、794、796、798、800、802、804、806、808、810、812、814、816、818、820、822、824、826、828、830、832、834、836、838、840、842、844、846、848、850、852、854、856、858、970、972、974、1766、1770、1772、1774、1776、1778、1786、1802、1806、1812、1814、1816、1818、1820、1840、1842、1844、1846、1848、1850、1852、1854、1856、1858、1860、1862、1864、1866、1868、1870、1872或1874的完全连续的核苷酸。The dsRNA of claim 1, wherein the antisense strand comprises a moiety selected from the group consisting of SEQ ID NOs: 86, 182, 212, 350, 376, 402, 440, 476, 478, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, 634, 636, 638, 640, 642, 644, 6 46, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, 668, 670, 672, 674, 67 6, 678, 680, 682, 684, 686, 688, 690, 692, 694, 696, 698, 700, 702, 704, 706 ,708,710,712,714,716,718,720,722,724,726,728,730,732,734,736 ,738,740,742,744,746,748,750,752,754,756,758,760,762,764,766, 768, 770, 772, 774, 776, 778, 780, 782, 784, 786, 788, 790, 792, 794, 796, 7 98, 800, 802, 804, 806, 808, 810, 812, 814, 816, 818, 820, 822, 824, 826, 82 8, 830, 832, 834, 836, 838, 840, 842, 844, 846, 848, 850, 852, 854, 856, 858, 970, 972, 974, 1766, 1770, 1772, 1774, 1776, 1778, 1786, 1802, 1806, 1812, 1814, 1816, 1818, 1820, 1840, 1842, 1844, 1846, 1848, 1850, 1852, 1854, 1856, 1858, 1860, 1862, 1864, 1866, 1868, 1870, 1872, or 1874 of completely consecutive nucleotides.如权利要求1所述的dsRNA，其特征在于，所述正义链包含选自SEQ ID NO：85、181、211、349、375、401、439、475、477、501、503、505、507、509、511、513、515、517、519、521、523、525、527、529、531、533、535、537、539、541、543、545、547、549、551、553、555、557、559、561、563、565、567、569、571、573、575、577、579、581、583、585、587、589、591、593、595、597、599、601、603、605、607、609、611、613、615、617、619、621、623、625、627、629、631、633、635、637、639、641、643、645、647、649、651、653、655、657、659、661、663、665、667、669、671、673、675、677、679、681、683、685、687、689、691、693、695、697、699、701、703、705、707、709、711、713、715、717、719、721、723、725、727、729、731、733、735、737、739、741、743、745、747、749、751、753、755、757、759、761、763、765、767、769、771、773、775、777、779、781、783、785、787、789、791、793、795、797、799、801、803、805、807、809、811、813、815、817、819、821、823、825、827、829、831、833、835、837、839、841、843、845、847、849、851、853、855、857、969、971、973、1765、1769、1771、1773、1775、1777、1785、1801、1805、1811、1813、1815、1817、1819、1839、1841、1843、1845、1847、1849、1851、1853、1855、1857、1859、1861、1863、1865、1867、1869、1871或1873的完全连续的核苷酸。The dsRNA of claim 1, wherein the sense strand comprises a member selected from the group consisting of SEQ ID NOs: 85, 181, 211, 349, 375, 401, 439, 475, 477, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592 73, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, 667, 669, 671, 673, 675, 677, 679, 681, 683, 685, 687, 689, 691, 693, 695, 697, 699 ,701,703,705,707,709,711,713,715,717,719,721,723,725,727,729,731,733,73 5. 737, 739, 741, 743, 745, 747, 749, 751, 753, 755, 757, 759, 761, 763, 765, 767, 769, 7 71, 773, 775, 777, 779, 781, 783, 785, 787, 789, 791, 793, 795, 797, 799, 801, 803, 805, 807, 809, 811, 813, 815, 817, 819, 821, 823, 825, 827, 829, 831, 833, 835, 837, 839, 841 ,843,845,847,849,851,853,855,857,969,971,973,1765,1769,1771,1773,1775,1 completely consecutive nucleotides of 777, 1785, 1801, 1805, 1811, 1813, 1815, 1817, 1819, 1839, 1841, 1843, 1845, 1847, 1849, 1851, 1853, 1855, 1857, 1859, 1861, 1863, 1865, 1867, 1869, 1871 or 1873.如权利要求1所述的dsRNA，其特征在于，其包含选自如下的正义链序列和反义链序列：SEQ ID NO：85和86；SEQ ID NO：181和182；SEQ ID NO：211和212；SEQ ID NO：349和350；SEQ ID NO：375和376；SEQ ID NO：401和402；SEQ ID NO：439和440；SEQ ID NO：475和476；SEQ ID NO：477和478；SEQ ID NO：501和502；SEQ ID NO：503和504；SEQ ID NO：505和506；SEQ ID NO：507和508；SEQ ID NO：509和510；SEQ ID NO：511和512；SEQ ID NO：513和514；SEQ ID NO：515和516；SEQ ID NO：517和518；SEQ ID NO：519和520；SEQ ID NO：521和522；SEQ ID NO：523和524；SEQ ID NO：525和526；SEQ ID NO：527和528；SEQ ID NO：529和530；SEQ ID NO：531和532；SEQ ID NO：533和534；SEQ ID NO：535和536；SEQ ID NO：537和538；SEQ ID NO：539和540；SEQ ID NO：541和542；SEQ ID NO：543和544；SEQ ID NO：545和546；SEQ ID NO：547和548；SEQ ID NO：549和550；SEQ ID NO：551和552；SEQ ID NO：553和554；SEQ ID NO：555和556；SEQ ID NO：557和558；SEQ ID NO：559和560；SEQ ID NO：561和562；SEQ ID NO：563和564；SEQ ID NO：565和566；SEQ ID NO：567和568；SEQ ID NO：569和570；SEQ ID NO：571和572；SEQ ID NO：573和574；SEQ ID NO：575和576；SEQ ID NO：577和578；SEQ ID NO：579和580；SEQ ID NO：581和582；SEQ ID NO：583和584；SEQ ID NO：585和586；SEQ ID NO：587和588；SEQ ID NO：589和590；SEQ ID NO：591和592；SEQ ID NO：593和594；SEQ ID NO：595和596；SEQ ID NO：597和598；SEQ ID NO：599和600；SEQ ID NO：601和602；SEQ ID NO：603和604；SEQ ID NO：605和606；SEQ ID NO：607和608；SEQ ID NO：609和610；SEQ ID NO：611和612；SEQ ID NO：613和614；SEQ ID NO：615和616；SEQ ID NO：617和618；SEQ ID NO：619和620；SEQ ID NO：621和622；SEQ ID NO：623和624；SEQ ID NO：625和626；SEQ ID NO：627和628；SEQ ID NO：629和630；SEQ ID NO：631和632；SEQ ID NO：633和634；SEQ ID NO：635和636；SEQ ID NO：637和638；SEQ ID NO：639和640；SEQ ID NO：641和642；SEQ ID NO：643和644；SEQ ID NO：645和646；SEQ ID NO：647和648；SEQ ID NO：649和650；SEQ ID NO：651和652；SEQ ID NO：653和654；SEQ ID NO：655和656；SEQ ID NO：657和658；SEQ ID NO：659和660；SEQ ID NO：661和662；SEQ ID NO：663和664；SEQ ID NO：665和666；SEQ ID NO：667和668；SEQ ID NO：669和670；SEQ ID NO：671和672；SEQ ID NO：673和674；SEQ ID NO：675和676；SEQ ID NO：677和678；SEQ ID NO：679和680；SEQ ID NO：681和682；SEQ ID NO：683和684；SEQ ID NO：685和686；SEQ ID NO：687和688；SEQ ID NO：689和690；SEQ ID NO：691和692；SEQ ID NO：693和694；SEQ ID NO：695和696；SEQ ID NO：697和698；SEQ ID NO：699和700；SEQ ID NO：701和702；SEQ ID NO：703和704；SEQ ID NO：705和706；SEQ ID NO：707和708；SEQ ID NO：709和710；SEQ ID NO：711和712；SEQ ID NO：713和714；SEQ ID NO：715和716；SEQ ID NO：717和718；SEQ ID NO：719和720；SEQ ID NO：721和722；SEQ ID NO：723和724；SEQ ID NO：725和726；SEQ ID NO：727和728；SEQ ID NO：729和730；SEQ ID NO：731和732；SEQ ID NO：733和734；SEQ ID NO：735和736；SEQ ID NO：737和738；SEQ ID NO：739和740；SEQ ID NO：741和742；SEQ ID NO：743和744；SEQ ID NO：745和746；SEQ ID NO：747和748；SEQ ID NO：749和750；SEQ ID NO：751和752；SEQ ID NO：753和754；SEQ ID NO：755和756；SEQ ID NO：757和758；SEQ ID NO：759和760；SEQ ID NO：761和762；SEQ ID NO：763和764；SEQ ID NO：765和766；SEQ ID NO：767和768；SEQ ID NO：769和770；SEQ ID NO：771和772；SEQ ID NO：773和774；SEQ ID NO：775和776；SEQ ID NO：777和778；SEQ ID NO：779和780；SEQ ID NO：781和782；SEQ ID NO：783和784；SEQ ID NO：785和786；SEQ ID NO：787和788；SEQ ID NO：789和790；SEQ ID NO：791和792；SEQ ID NO：793和794；SEQ ID NO：795和796；SEQ ID NO：797和798；SEQ ID NO：799和800；SEQ ID NO：801和802；SEQ ID NO：803和804；SEQ ID NO：805和806；SEQ ID NO：807和808；SEQ ID NO：809和810；SEQ ID NO：811和812；SEQ ID NO：813和814；SEQ ID NO：815和816；SEQ ID NO：817和818；SEQ ID NO：819和820；SEQ ID NO：821和822；SEQ ID NO：823和824；SEQ ID NO：825和826；SEQ ID NO：827和828；SEQ ID NO：829和830；SEQ ID NO：831和832；SEQ ID NO：833和834；SEQ ID NO：835和836；SEQ ID NO：837和838；SEQ ID NO：839和840；SEQ ID NO：841和842；SEQ ID NO：843和844；SEQ ID NO：845和846；SEQ ID NO：847和848；SEQ ID NO：849和850；SEQ ID NO：851和852；SEQ ID NO：853和854；SEQ ID NO：855和856；SEQ ID NO：857和858；SEQ ID NO：969和970；SEQ ID NO：971和972；SEQ ID NO：973和974；SEQ ID NO：1765和1766；SEQ ID NO：1769和1770；SEQ ID NO：1771和1772；SEQ ID NO：1773和1774；SEQ ID NO：1775和1776；SEQ ID NO：1777和1778；SEQ ID NO：1785和1786；SEQ ID NO：1801和1802；SEQ ID NO：1805和1806；SEQ ID NO：1811和1812；SEQ ID NO：1813和1814；SEQ ID NO：1815和1816；SEQ ID NO：1817和1818；SEQ ID NO：1819和1820；SEQ ID NO：1839和1840；SEQ ID NO：1841和1842；SEQ ID NO：1843和1844；SEQ ID NO：1845和1846；SEQ ID NO：1847和1848；SEQ ID NO：1849和1850；SEQ ID NO：1851和1852；SEQ ID NO：1853和1854；SEQ ID NO：1855和1856；SEQ ID NO：1857和1858；SEQ ID NO：1859和1860；SEQ ID NO：1861和1862；SEQ ID NO：1863和1864；SEQ ID NO：1865和1866；SEQ ID NO：1867和1868；SEQ ID NO：1869和1870；SEQ ID NO：1871和1872；或者，SEQ ID NO：1873和1874。The dsRNA of claim 1, characterized in that it comprises a sense strand sequence and an antisense strand sequence selected from the group consisting of SEQ ID NOs: 85 and 86; SEQ ID NOs: 181 and 182; SEQ ID NOs: 211 and 212; SEQ ID NOs: 349 and 350; SEQ ID NOs: 375 and 376; SEQ ID NOs: 401 and 402; SEQ ID NOs: 439 and 440; SEQ ID NOs: 475 and 476; SEQ ID NOs: 477 and 478; SEQ ID NOs: 501 and 502; SEQ ID NOs: 503 and 504; SEQ ID NOs: 505 and 506; SEQ ID NOs: 507 and 508; SEQ ID NOs: 509 and 510; SEQ ID NOs: 511 and 512; SEQ ID NOs: 513 and 514; SEQ ID NOs: 515 and 516; SEQ ID NOs: 517 and 518; SEQ ID NOs: 519 and 520; SEQ ID NOs: 521 and 522; SEQ ID NOs: 523 and 524; SEQ ID NOs: 525 and 526; SEQ ID NOs: 527 and 528; SEQ ID NOs: 529 and 530 SEQ ID NOs: 519 and 520; SEQ ID NOs: 521 and 522; SEQ ID NOs: 523 and 524; SEQ ID NOs: 525 and 526; SEQ ID NOs: 527 and 528; SEQ ID NOs: 529 and 530; SEQ ID NOs: 531 and 532; SEQ ID NOs: 533 and 534; SEQ ID NOs: 535 and 536; SEQ ID NOs: 537 and 538; SEQ ID NOs: 539 and 540; SEQ ID NOs: 541 and 542; SEQ ID NOs: 543 and 544; SEQ ID NOs: 545 and 546; SEQ ID NOs: 547 and 548; SEQ ID NOs: 549 and 550; SEQ ID NOs: 551 and 552; SEQ ID NOs: 553 and 554; SEQ ID NOs: 555 and 556; SEQ ID NOs: 557 and 558; SEQ ID NOs: 559 and 560; SEQ ID NOs: 559 and 560; SEQ ID NOs: 561 and 562; SEQ ID NOs: 563 and 564; SEQ ID NOs: 565 and 566; SEQ ID NOs: 567 and 568; SEQ ID NOs: 569 and 570; SEQ ID NOs: 571 and 572; SEQ ID NOs: 573 and 574; SEQ ID NOs: 575 and 576; SEQ ID NOs: 577 and 578; SEQ ID NOs: 579 and 580; SEQ ID NOs: 581 and 582; SEQ ID NOs: 583 and 584; SEQ ID NOs: 585 and 586; SEQ ID NOs: 587 and 588; SEQ ID NOs: 589 and 590; SEQ ID NOs: 591 and 592; SEQ ID NOs: 593 and 594; SEQ ID NOs: 595 and 596; SEQ ID NOs: 597 and 598; SEQ ID NOs: SEQ ID NOs: 599 and 600; SEQ ID NOs: 601 and 602; SEQ ID NOs: 603 and 604; SEQ ID NOs: 605 and 606; SEQ ID NOs: 607 and 608; SEQ ID NOs: 609 and 610; SEQ ID NOs: 611 and 612; SEQ ID NOs: 613 and 614; SEQ ID NOs: 615 and 616; SEQ ID NOs: 617 and 618; SEQ ID NOs: 619 and 620; SEQ ID NOs: 621 and 622; SEQ ID NOs: 623 and 624; SEQ ID NOs: 625 and 626; SEQ ID NOs: 627 and 628; SEQ ID NOs: 629 and 630; SEQ ID NOs: 631 and 632; SEQ ID NOs: 633 and 634; SEQ ID NOs: 635 and 636; SEQ ID NOs: 637 and 638; SEQ ID NOs: 639 and 640; SEQ ID NOs: 639 and 640; SEQ ID NOs: 641 and 642; SEQ ID NOs: 643 and 644; SEQ ID NOs: 645 and 646; SEQ ID NOs: 647 and 648; SEQ ID NOs: 649 and 650; SEQ ID NOs: 651 and 652; SEQ ID NOs: 653 and 654; SEQ ID NOs: 655 and 656; SEQ ID NOs: 657 and 658; SEQ ID NOs: 659 and 660; SEQ ID NOs: 661 and 662; SEQ ID NOs: 663 and 664; SEQ ID NOs: 665 and 666; SEQ ID NOs: 667 and 668; SEQ ID NOs: 669 and 670; SEQ ID NOs: 671 and 672; SEQ ID NOs: 673 and 674; SEQ ID NOs: 675 and 676; SEQ ID NOs: 677 and 678; SEQ ID NOs: 680 and 681; SEQ ID NOs: 679 and 680; SEQ ID NOs: 681 and 682; SEQ ID NOs: 683 and 684; SEQ ID NOs: 685 and 686; SEQ ID NOs: 687 and 688; SEQ ID NOs: 689 and 690; SEQ ID NOs: 691 and 692; SEQ ID NOs: 693 and 694; SEQ ID NOs: 695 and 696; SEQ ID NOs: 697 and 698; SEQ ID NOs: 699 and 700; SEQ ID NOs: 701 and 702; SEQ ID NOs: 703 and 704; SEQ ID NOs: 705 and 706; SEQ ID NOs: 707 and 708; SEQ ID NOs: 709 and 710; SEQ ID NOs: 711 and 712; SEQ ID NOs: 713 and 714; SEQ ID NOs: 715 and 716; SEQ ID NOs: 717 and 718; SEQ ID NOs: 719 and 720; SEQ ID NOs: 721 and 722; SEQ ID NOs: 723 and 724; SEQ ID NOs: 725 and 726; SEQ ID NOs: 727 and 728; SEQ ID NOs: 729 and 730; SEQ ID NOs: 731 and 732; SEQ ID NOs: 733 and 734; SEQ ID NOs: 735 and 736; SEQ ID NOs: 737 and 738; SEQ ID NOs: 739 and 740; SEQ ID NOs: 741 and 742; SEQ ID NOs: 743 and 744; SEQ ID NOs: 745 and 746; SEQ ID NOs: 747 and 748; SEQ ID NOs: 749 and 750; SEQ ID NOs: 751 and 752; SEQ ID NOs: 753 and 754; SEQ ID NOs: 751 and 752; SEQ ID NOs: 753 and 754; SEQ ID NOs: 755 and 756; SEQ ID NOs: 757 and 758; SEQ ID NOs: 759 and 760; SEQ ID NOs: 761 and 762; SEQ ID NOs: 763 and 764; SEQ ID NOs: 765 and 766; SEQ ID NOs: 767 and 768; SEQ ID NOs: 769 and 770; SEQ ID NOs: 771 and 772; SEQ ID NOs: 773 and 774; SEQ ID NOs: 775 and 776; SEQ ID NOs: 777 and 778; SEQ ID NOs: 779 and 780; SEQ ID NOs: 781 and 782; SEQ ID NOs: 783 and 784; SEQ ID NOs: 785 and 786; SEQ ID NOs: 787 and 788; SEQ ID NOs: 789 and 790; SEQ ID NOs: 791 and 792; SEQ ID NOs: 793 and 794; SEQ ID NOs: 795 and 796; SEQ ID NOs: 797 and 798; SEQ ID NOs: 793 and 794; SEQ ID NOs: 795 and 796; SEQ ID NOs: 797 and 798; SEQ ID NOs: 799 and 800; SEQ ID NOs: 801 and 802; SEQ ID NOs: 803 and 804; SEQ ID NOs: 805 and 806; SEQ ID NOs: 807 and 808; SEQ ID NOs: 809 and 810; SEQ ID NOs: 811 and 812; SEQ ID NOs: 813 and 814; SEQ ID NOs: 815 and 816; SEQ ID NOs: 817 and 818; SEQ ID NOs: 819 and 820; SEQ ID NOs: 821 and 822; SEQ ID NOs: 823 and 824; SEQ ID NOs: 825 and 826; SEQ ID NOs: 827 and 828; SEQ ID NOs: 829 and 830; SEQ ID NOs: 831 and 832; SEQ ID NOs: 833 and 834; SEQ ID NOs: 833 and 834; SEQ ID NOs: 835 and 836; SEQ ID NOs: 837 and 838; SEQ ID NOs: 839 and 840; SEQ ID NOs: 841 and 842; SEQ ID NOs: 843 and 844; SEQ ID NOs: 845 and 846; SEQ ID NOs: 847 and 848; SEQ ID NOs: 849 and 850; SEQ ID NOs: 851 and 852; SEQ ID NOs: 853 and 854; SEQ ID NOs: 855 and 856; SEQ ID NOs: 857 and 858; SEQ ID NOs: 969 and 970; SEQ ID NOs: 971 and 972; SEQ ID NOs: 973 and 974; SEQ ID NOs: 1765 and 1766; SEQ ID NOs: 1769 and 1770; SEQ ID NOs: 1771 and 1772; SEQ ID NOs: SEQ ID NOs: 1773 and 1774; SEQ ID NOs: 1775 and 1776; SEQ ID NOs: 1777 and 1778; SEQ ID NOs: 1785 and 1786; SEQ ID NOs: 1801 and 1802; SEQ ID NOs: 1805 and 1806; SEQ ID NOs: 1811 and 1812; SEQ ID NOs: 1813 and 1814; SEQ ID NOs: 1815 and 1816; SEQ ID NOs: 1817 and 1818; SEQ ID NOs: 1819 and 1820; SEQ ID NOs: 1839 and 1840; SEQ ID NOs: 1841 and 1842; SEQ ID NOs: 1843 and 1844; SEQ ID NOs: 1845 and 1846; SEQ ID NOs: 1847 and 1848; SEQ ID NOs: 1849 and 1850; SEQ ID NOs: 1860 and 1861; SEQ ID NOs: 1862 and 1863; SEQ ID NOs: 1864 and 1865; SEQ ID NOs: 1866 and 1867; SEQ ID NOs: 1868 and 1869; SEQ ID NOs: 1870 and 1871; SEQ ID NOs: 1872 and 1873; NO: 1851 and 1852; SEQ ID NO: 1853 and 1854; SEQ ID NO: 1855 and 1856; SEQ ID NO: 1857 and 1858; SEQ ID NO: 1859 and 1860; SEQ ID NO: 1861 and 1862; SEQ ID NO: 1863 and 1864; SEQ ID NO: 1865 and 1866; SEQ ID NO: 1867 and 1868; SEQ ID NO: 1869 and 1870; SEQ ID NO: 1871 and 1872; or, SEQ ID NO: 1873 and 1874.一种双链RNAi剂，其包含如权利要求1-10任一项所述的dsRNA，并且任选地包含靶向配体。A double-stranded RNAi agent comprising the dsRNA according to any one of claims 1 to 10, and optionally comprising a targeting ligand.如权利要求11所述的双链RNAi剂，其特征在于，所述双链RNAi剂包含权利要求1-10任一项所述的dsRNA，以及至少一个靶向配体，其中，所述dsRNA的正义链的与所述靶向配体缀合。The double-stranded RNAi agent according to claim 11, characterized in that the double-stranded RNAi agent comprises the dsRNA according to any one of claims 1 to 10, and at least one targeting ligand, wherein the sense strand of the dsRNA is conjugated to the targeting ligand.如权利要求11所述的双链RNAi剂，其特征在于，所述靶向配体包含N-乙酰基-半乳糖胺(GalNAc)，或，所述靶向配体是GalNAc衍生物。The double-stranded RNAi agent according to claim 11, characterized in that the targeting ligand comprises N-acetyl-galactosamine (GalNAc), or the targeting ligand is a GalNAc derivative.如权利要求11所述的双链RNAi剂，其特征在于，所述双链RNAi剂的结构选自表A1，其中R²为权利要求1至10中任一项所述的dsRNA。The double-stranded RNAi agent according to claim 11, characterized in that the structure of the double-stranded RNAi agent is selected from Table A1, wherein^R2 is the dsRNA according to any one of claims 1 to 10.一种抑制细胞中抑制素亚基beta E(INHBE)表达的双链核糖核酸干扰(dsRNAi)剂，其中所述dsRNAi剂包含形成双链RNA(dsRNA)区域的正义链和反义链，所述反义链包含与INHBE mRNA互补的区域，其中所述互补区域包含至少15个连续的核苷酸，其中所述dsRNAi剂包含至少一个非规范碱基配对核苷酸。A double-stranded RNA interference (dsRNAi) agent that inhibits the expression of inhibin subunit beta E (INHBE) in a cell, wherein the dsRNAi agent comprises a sense strand and an antisense strand that form a double-stranded RNA (dsRNA) region, the antisense strand comprises a region complementary to INHBE mRNA, wherein the complementary region comprises at least 15 consecutive nucleotides, wherein the dsRNAi agent comprises at least one non-canonical base-pairing nucleotide.根据权利要求15所述的dsRNAi剂，其中，从5'端到3'端的方向上的第1-11位中，所述dsRNAi剂包含至少一个非规范碱基配对核苷酸；和/或在第12-21位中，包含至少一个非规范碱基配对核苷酸。The dsRNAi agent according to claim 15, wherein the dsRNAi agent comprises at least one non-canonical base-pairing nucleotide in positions 1-11 in the direction from the 5' end to the 3' end; and/or comprises at least one non-canonical base-pairing nucleotide in positions 12-21.根据权利要求15或16所述的dsRNAi剂，其中，所述dsRNAi剂包含一、二、三、四、五或更多个非规范碱基配对核苷酸。The dsRNAi agent of claim 15 or 16, wherein the dsRNAi agent comprises one, two, three, four, five or more non-canonical base pairing nucleotides.根据权利要求15至17中任一项所述的dsRNAi剂，其中，对于从5'端到3'端的方向上的第1-11位或者第12-21位中的序列片段N₁N₂G₀N₃N₄，其中N₁、N₂、N₃和N₄各自独立地为包含腺嘌呤(A)、胞嘧啶(C)、鸟嘌呤(G)、胸腺嘧啶(T)或尿嘧啶(U)为碱基的核苷酸，G₀是包含鸟嘌呤为碱基的核苷酸，当N₁、N₂、N₃和N₄中的至少三个碱基是腺嘌呤或尿嘧啶时，序列中G₀的鸟嘌呤被次黄嘌呤(I)取代。The dsRNAi agent according to any one of claims 15 to 17, wherein, for the sequence fragment N1N2G0N3N4 in positions 1-11_or positions 12-21 in the direction from the 5' end_to the₃ ' end_, wherein_N1 ,_N2 ,_N3 and_N4 are each independently a nucleotide comprising adenine (A), cytosine (C), guanine (G), thymine (T)_or uracil (U) as a base,_G0 is a nucleotide comprising guanine as a base, and when at least three bases of_N1 ,_N2 ,_N3 and_N4 are adenine or uracil, the guanine of_G0 in the sequence is substituted by hypoxanthine (I).根据权利要求18所述的dsRNAi剂，其中，N₁、N₂、N₃、N₄和G₀中的至少一个还具有修饰的糖基和/或修饰的核苷酸间键合。The dsRNAi agent according to claim 18, wherein at least one of_N1 ,_N2 ,_N3 ,_N4 and_G0 further has a modified sugar group and/or a modified internucleotide linkage.根据权利要求15至19中任一项所述的dsRNAi剂，其中，所述dsRNAi剂或RNA在寡核苷酸链从5'端到3'端的方向上的第6、7或8位中，包含至少一个非规范碱基配对核苷酸和/或经修饰的核苷酸。The dsRNAi agent according to any one of claims 15 to 19, wherein the dsRNAi agent or RNA comprises at least one non-canonical base pairing nucleotide and/or modified nucleotide in the 6th, 7th or 8th position in the direction from the 5' end to the 3' end of the oligonucleotide chain.根据权利要求15至20中任一项所述的dsRNAi剂，其中，当从5'端到3'端的方向上的第2-8位中的至少四个核苷酸是A或U，并且第6-8位中的至少一个核苷酸是G，则第6、7和/或8位的G被非规范碱基配对核苷酸替换。The dsRNAi agent according to any one of claims 15 to 20, wherein when at least four nucleotides in positions 2-8 in the direction from the 5' end to the 3' end are A or U, and at least one nucleotide in positions 6-8 is G, then the G at positions 6, 7 and/or 8 is replaced by a non-canonical base pairing nucleotide.根据权利要求15至21中任一项所述的dsRNAi剂，其中，所述非规范碱基配对核苷酸中的碱基包括选自肌苷(I)、黄苷(X)、7-甲基鸟苷(m7G)、N6-甲基腺苷(m6A)、二氢尿苷、5-甲基胞嘧啶(m5C)、假尿苷(Ψ)和N1-甲基假尿苷(m1Ψ)中的碱基。The dsRNAi agent according to any one of claims 15 to 21, wherein the base in the non-canonical base pairing nucleotide includes a base selected from inosine (I), xanthosine (X), 7-methylguanosine (m7G), N6-methyladenosine (m6A), dihydrouridine, 5-methylcytosine (m5C), pseudouridine (Ψ) and N1-methylpseudouridine (m1Ψ).一种药物组合物，其特征在于，其包含如权利要求11-22中任一项所述的双链RNAi剂或其药学上可接受的盐，以及药学上可接受的载体。A pharmaceutical composition, characterized in that it comprises the double-stranded RNAi agent or a pharmaceutically acceptable salt thereof according to any one of claims 11 to 22, and a pharmaceutically acceptable carrier.如权利要求23所述的药物组合物，其特征在于，所述药物组合物被配制成用于皮下施用或静脉内施用。The pharmaceutical composition of claim 23, wherein the pharmaceutical composition is formulated for subcutaneous administration or intravenous administration.如权利要求23或24所述的药物组合物，其特征在于，所述载体是非缓冲溶液或缓冲溶液。The pharmaceutical composition according to claim 23 or 24, characterized in that the carrier is a non-buffered solution or a buffered solution.一种抑制细胞中INHBE表达的方法，其特征在于，使所述细胞与如权利要求11-22中任一项所述的双链RNAi剂接触，使得INHBE基因的mRNA转录物降解，由此抑制所述细胞中INHBE基因的表达。A method for inhibiting the expression of INHBE in a cell, characterized in that the cell is contacted with the double-stranded RNAi agent according to any one of claims 11 to 22, so that the mRNA transcript of the INHBE gene is degraded, thereby inhibiting the expression of the INHBE gene in the cell.如权利要求26所述的方法，其特征在于，所述细胞是肝细胞。The method of claim 26, wherein the cells are hepatocytes.如权利要求26或27所述的方法，其特征在于，INHBE表达被抑制至少约30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％或95％。The method of claim 26 or 27, wherein INHBE expression is inhibited by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.如权利要求11-22中任一项所述的双链RNAi剂用于制备治疗具有由INHBE表达介导的失调的受试者的药物的用途。Use of a double-stranded RNAi agent as described in any one of claims 11 to 22 for the preparation of a medicament for treating a subject having a disorder mediated by INHBE expression.如权利要求29所述的用途，其特征在于，所述受试者患有代谢紊乱。The use according to claim 29, wherein the subject suffers from a metabolic disorder.如权利要求30所述的用途，其特征在于，所述代谢紊乱是代谢综合征、2型糖尿病、肥胖、高甘油三酯血症、脂肪代谢障碍、肝脏炎症、脂肪肝、高胆固醇血症、与肝酶升高相关的肝病、非酒精性脂肪性肝炎、心血管疾病、肾脏疾病；可选地，所述代谢综合征包括腹部肥胖、胰岛素抵抗、异常血脂症和高血压中的一种或多种。The use as claimed in claim 30, characterized in that the metabolic disorder is metabolic syndrome, type 2 diabetes, obesity, hypertriglyceridemia, dyslipidemia, liver inflammation, fatty liver, hypercholesterolemia, liver disease associated with elevated liver enzymes, non-alcoholic fatty hepatitis, cardiovascular disease, kidney disease; optionally, the metabolic syndrome includes one or more of abdominal obesity, insulin resistance, dyslipidemia and hypertension.