Modified double-stranded RNA molecule

Abstract

A modified double-stranded RNA molecule, comprising a sense strand and an antisense strand. Each strand comprises 19-25 nucleotides. The sense strand comprises: at the 5' end, 3-9 consecutive nucleotides containing 2'-modified ribose; at the 3' end, two consecutive nucleotides containing 2'-modified ribose, and in a region opposite to a seed region in the antisense strand, 0-7 nucleotides containing 2'-modified ribose, wherein the 2'-modification is selected from 2'-halo and 2'-alkoxy, and three consecutive nucleotides at the 5' end are linked by means of phosphorothioate bonds; and the antisense strand comprises: in position 1 of the 5' end, a nucleotide containing 2'-modified ribose; in position 2 of the 5' end, a nucleotide containing 2'-halogenated ribose; in the seed region, 1-7 nucleotides containing 2'-modified ribose; in a region adjacent to the 3' side of the seed region, 0 or 2 consecutive nucleotides containing 2'-modified ribose; and at the 3' end, 2-11 consecutive nucleotides comprising 2'-modified ribose, wherein the 2'-modification is selected from 2'-halo, 2'-alkoxy, and 2'-alkoxy-alkyl, three consecutive nucleotides at the 5' end are linked by means of phosphorothioate bonds, and three consecutive nucleotides at the 3' end are linked by means of phosphorothioate bonds.

Description

Modified double-stranded RNA molecules

[0001] Cross-reference to related applications

[0002] This application claims priority to Chinese Patent Application No. 202411368120.7, filed September 27, 2024, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application relates to a modified double-stranded RNA molecule, in particular a small interfering RNA (siRNA) molecule, with specific chemical modifications on its nucleotide chains, which has good stability and target gene silencing specificity. The present application also relates to the use of the double-stranded RNA molecule in inhibiting or reducing the expression of a target gene, in particular for use in inhibiting or reducing the expression of a target gene associated with a disease in a subject. BACKGROUND

[0004] RNA interference (RNAi) is an intrinsic post-transcriptional gene regulation mechanism that organisms can use to defend against the invasion of foreign nucleic acids. Both small interfering RNA (siRNA) and microRNA (miRNA) can trigger RNAi, which knocks down the expression of target genes in a sequence-specific manner by binding to target mRNA and interfering with its expression. Compared with miRNA, siRNA has higher target gene knockdown specificity and higher knockdown efficiency accordingly.

[0005] siRNA is a type of double-stranded RNA (dsRNA) that contains a guide strand (also known as an antisense strand) and a passenger strand (also known as a sense strand), and is usually composed of a 19-21 bp core double strand and two nucleotides overhangs at the 3’ end of each strand, where the 2-8th nucleotides of the guide strand are the seed region. Dicer enzyme can form siRNA by cutting longer exogenous or endogenous dsRNA. siRNA enters the cell through endocytosis, is wrapped in endosomes and lysosomal compartments, and is gradually released into the cytoplasm to form an RNA-induced silencing complex (RISC) with proteins such as AGO and TRBP. During RISC formation, siRNA unwinds to form a single strand, which is the thermodynamically unstable strand, i.e., the guide strand, and becomes part of RISC. RISC searches for and binds to mRNA that is 100% complementary to the guide strand, and cuts the mRNA at the cleavage site (e.g., between the nucleotide paired with the 10th nucleotide of the guide strand and the nucleotide paired with the 11th nucleotide of the guide strand), achieving the effect of silencing the expression of a specific sequence. When the cut mRNA is released from RISC, RISC begins to search for the next mRNA target molecule.

[0006] Since the discovery of synthetic siRNA that can specifically inhibit the expression of endogenous and exogenous genes in mammalian cells by the team of Thomas Tuschl in 2001, siRNA has entered the field of drug development as a potential gene therapy. In theory, sequence-specific silencing mediated by siRNA can knock down the expression of any gene, especially disease-related genes, without being subject to factors such as target distribution in cells and spatial conformation, and has a wider potential application range than antibodies and other small molecule drugs.

[0007] However, siRNA molecules also have some obvious disadvantages, such as unsatisfactory molecular stability and pharmacokinetic properties, and may cause certain off-target effects, which must be modified to improve the drug properties. Specifically, in unmodified siRNA, the phosphodiester bond connecting each nucleotide is easily attacked by RNase and phosphatase, causing rapid degradation of siRNA into small fragments, and the 2' hydroxyl group on the ribose will also be attacked by nucleases, causing hydrolysis. About 50% of unmodified siRNA entering the body will be degraded within 1 minute, and the target gene silencing effect is very limited (Layzer JM, McCaffrey AP, Tanner AK, Huang Z, Kay MA, Sullenger BA. In vivo activity of nuclease-resistant siRNAs. RNA. 2004 May; 10(5): 766-71). In addition, in theory, only when the guide strand in RISC perfectly pairs with the mRNA target molecule will it cause the cleavage and degradation of the mRNA target molecule. However, the guide strand of siRNA can also cause off-target effects due to base pairing between the seed region and mRNA, and in some cases, the companion strand can enter RISC, causing off-target effects mediated by the companion strand, which has certain potential toxicity to the organism.

[0008] These problems can be alleviated by introducing modifications to the 2' position of ribose and the end of the backbone phosphate bond, as well as certain delivery systems (Tang Q, Khvorova A. RNAi-based drug design: considerations and future directions. Nat Rev Drug Discov. 2024 May; 23(5): 341-364).

[0009] For example, to avoid metabolic degradation during tissue distribution and intracellular retention, i.e., to reduce hydrolysis mediated by 2'-hydroxyl on ribose, as well as degradation by endonucleases and exonucleases, modifications can be made to the 2' position of ribose and the phosphodiester bond. For example, the 2'-hydroxyl of ribose can be replaced with, for example, 2'-methoxy (2'-OMe), 2'-fluoro (2'-F), 2'-methoxyethyl (2'-MOE), and the like, and the non-bridging oxygen in the phosphodiester bond can be replaced with sulfur to form a phosphorothioate bond (PS). These modifications are compatible with RISC and greatly improve the nuclease resistance of siRNA. The steric hindrance of 2'-OMe is greater, which can provide higher stability, but the impact on siRNA activity often depends partly on the sequence and the position, for example, when it is at the 2nd and 14th positions of the guide strand, it can reduce the silencing activity (Foster DJ, Brown CR, Shaikh S, Trapp C, Schlegel MK, Qian K, Sehgal A, Rajeev KG, Jadhav V, Manoharan M, Kuchimanchi S, Maier MA, Milstein S. Advanced siRNA Designs Further Improve In Vivo Performance of GalNAc-siRNA Conjugates. Mol Ther. 2018 Mar 7;26(3):708-717). The 5' terminal nucleoside of the guide strand is usually not involved in target mRNA reactions, and when modified by 2'-OMe, it can be better loaded into the MID domain of Ago2 (Schirle NT, MacRae IJ. The crystal structure of human Argonaute2. Science. 2012 May 25;336(6084):1037-40). When the 2-5 nucleotides of the seed region of the guide strand of siRNA are modified by 2'-OMe, the off-target effect is inhibited, and when the 6-8 nucleotides are modified by 2'-OMe, the target and off-target silencing effects are promoted (Kobayashi Y, Tian S, Ui-Tei K. The siRNA Off-Target Effect Is Determined by Base-Pairing Stabilities of Two Different Regions with Opposite Effects. Genes (Basel). 2022 Feb 9;13(2):319).2'-F has a small footprint and can be used in all positions, it also enhances hydrophobicity and improves cellular uptake and potency of siRNA (Manoharan M, Akinc A, Pandey RK, Qin J, Hadwiger P, John M, Mills K, Charisse K, Maier MA, Nechev L, Greene EM, Pallan PS, Rozners E, Rajeev KG, Egli M. Unique gene-silencing and structural properties of 2'-fluoro-modified siRNAs. Angew Chem Int Ed Engl. 2011 Mar 1;50(10):2284-8; Blidner RA, Hammer RP, Lopez MJ, Robinson SO, Monroe WT. Fully 2'-deoxy-2'-fluoro substituted nucleic acids induce RNA interference in mammalian cell culture. Chem Biol Drug Des. 2007 Aug;70(2):113-22. doi: 10.1111 / j.1747-0285.2007.00542.x. PMID: 17683372). However, there are concerns about the introduction of this unnatural modification, 2'-F, as its metabolite can potentially bind to mitochondrial DNA and cause toxicity. Introducing two PS at the 5' end can improve resistance to exonucleases by several orders of magnitude, and PS also needs to be introduced at the 3' end to improve the stability of siRNA, as the protrusion of two nucleotides makes it more vulnerable to exonucleases. A phosphor-modified 5' terminal nucleotide, such as a 5'-(E)-vinyl phosphonate (5'-VP) nucleotide, can not only resist phosphatase action, but also facilitate RISC loading (Tang Q, Khvorova A. RNAi-based drug design: considerations and future directions. Nat Rev Drug Discov. 2024 May;23(5):341-364).

[0010] In the past 20 years, there has been great progress in the modification of siRNA, and the siRNA products of companies such as Alnylam have undergone multiple iterations. Initially, only individual nucleotides were modified, and now the mainstream direction is almost to modify all nucleotides, and operations such as replacing ribonucleotides with deoxyribonucleotides, locked nucleic acids (UNA), and glycerol nucleic acids (GNA) are added. Although there are certain concerns about 2’-F modification, there are still a large number of 2’-F modifications in new siRNA products (Hu B, Zhong L, Weng Y, Peng L, Huang Y, Zhao Y, Liang XJ. Therapeutic siRNA: state of the art. Signal Transduct Target Ther. 2020 Jun 19; 5(1): 101).

[0011] The citation of any document herein is not intended as an admission that such document is prior art with respect to the present application. SUMMARY

[0012] The inventors of the present application enhance the asymmetry of the 5’ end of the sense strand and the antisense strand by arranging different types of modified bases, thereby improving the silencing efficiency of siRNA. In addition, the current common siRNA modification combination is to modify the whole chain, which is high in cost, and often causes immunogenicity or toxicity due to the increase of modified bases. The inventors of the present application found that only the nucleotides at specific positions in the siRNA molecule need to be modified, which can increase the stability of siRNA and the silencing efficiency of target genes, and the effect is even better than the prior art, such as the RNA modification technology of Alnylam, and because only a limited number of ribose nucleotides containing 2’-F modification are contained, the potential harm of this non-natural modification metabolite to the organism is reduced to a low level. The siRNA modification mode of the present application can also reduce off-target effects, including off-target effects caused by the partial sequence of the antisense strand pairing with mRNA and off-target effects caused by the sense strand entering RISC.

[0013] Thus, in a first aspect, the application provides a modified double-stranded RNA molecule comprising a sense strand and an antisense strand, wherein the sense strand can comprise at least 3 consecutive 2'-position-modified ribose-containing nucleotides at the 5' end, at least 2 consecutive 2'-position-modified ribose-containing nucleotides at the 3' end, and the 3 consecutive nucleotides at the 5' end can be linked by phosphorothioate bonds, and wherein the antisense strand can comprise a 2'-halogenated ribose-containing nucleotide at the 2nd position from the 5' end, at least 2 consecutive 2'-position-modified ribose-containing nucleotides at the 3' end, the 3 consecutive nucleotides at the 5' end can be linked by phosphorothioate bonds, and the 3 consecutive nucleotides at the 3' end can be linked by phosphorothioate bonds. The 2'-position modification can be selected from the group consisting of 2'-halogenated, 2'-alkoxy, and 2'-alkoxy-alkyl.

[0014] In the modified double-stranded RNA molecule of the application, the sense strand can comprise 3-9 consecutive 2'-position-modified ribose-containing nucleotides at the 5' end, 2 consecutive 2'-position-modified ribose-containing nucleotides at the 3' end, and 0-7 2'-position-modified ribose-containing nucleotides in the region opposite to the seed region of the antisense strand, wherein the 2'-position modification is selected from the group consisting of 2'-halogenated, 2'-alkoxy, and 2'-alkoxy-alkyl, and the 3 consecutive nucleotides at the 5' end can be linked by phosphorothioate bonds; and the antisense strand can comprise a 2'-position-modified ribose-containing nucleotide at the 1st position from the 5' end, a 2'-halogenated ribose-containing nucleotide at the 2nd position from the 5' end, 1-7 2'-position-modified ribose-containing nucleotides in the seed region, 0 or 2 consecutive 2'-position-modified ribose-containing nucleotides in the region adjacent to the 3' side of the seed region, and 2-11 consecutive 2'-position-modified ribose-containing nucleotides at the 3' end, wherein the 2'-position modification is selected from the group consisting of 2'-halogenated, 2'-alkoxy, and 2'-alkoxy-alkyl, and the 3 consecutive nucleotides at the 5' end can be linked by phosphorothioate bonds, and the 3 consecutive nucleotides at the 3' end can be linked by phosphorothioate bonds.

[0015] The 2'-position-modified ribose-containing nucleotides comprised in the seed region of the antisense strand can be located at the 4 positions close to the 5' end, the 3 positions close to the 3' end, or throughout the seed region.

[0016] 2'-halo can be 2'-fluoro. 2'-alkoxy can be 2'-alkoxy containing 1-3 carbons, such as 2'-methoxy, 2'-ethoxy, or 2'-propoxy. In some embodiments, 2'-alkoxy can be 2'-methoxy. The alkoxy of 2'-alkoxy-alkyl can be alkoxy containing 1-3 carbons, such as methoxy, ethoxy, and propoxy, and the alkyl can be alkyl containing 1-3 carbons, such as methyl, ethyl, and propyl. 2'-alkoxy-alkyl can be, for example, 2'-methoxyethyl, 2'-methoxymethyl, 2'-methoxypropyl, and the like. In some embodiments, 2'-alkoxy-alkyl can be 2'-methoxyethyl.

[0017] In some embodiments, the sense strand can comprise 5 consecutive 2'-positionally modified ribose-containing nucleotides at the 5' terminus, the region opposite the seed region of the antisense strand can comprise 0 2'-positionally modified ribose-containing nucleotides, the antisense strand can comprise 4 2'-positionally modified ribose-containing nucleotides at the seed region, 0 2'-positionally modified ribose-containing nucleotides at the region immediately 3' of the seed region, and 2 consecutive 2'-positionally modified ribose-containing nucleotides at the 3' terminus. In some embodiments, the sense strand can comprise 5 consecutive 2'-methoxyribose-containing nucleotides at the 5' terminus, 2 consecutive 2'-methoxyribose-containing nucleotides at the 3' terminus, and the antisense strand can comprise a 2'-fluororibose-containing nucleotide at position 2, 2'-methoxyribose-containing nucleotides at positions 1, 3, 4, 5, and 2 consecutive 2'-methoxyribose-containing nucleotides at the 3' terminus. In some embodiments, the sense strand can comprise 5 consecutive 2'-methoxyribose-containing nucleotides at the 5' terminus, 2 consecutive 2'-methoxyribose-containing nucleotides at the 3' terminus, and the antisense strand can comprise a 2'-fluororibose-containing nucleotide at position 2, 2'-methoxyribose-containing nucleotides at positions 1, 6, 7, 8, and 2 consecutive 2'-methoxyribose-containing nucleotides at the 3' terminus.

[0018] In some embodiments, the sense strand can comprise 3 consecutive 2' position modified ribose-containing nucleotides at the 5' terminus, 1 2' position modified ribose-containing nucleotide at the region opposite the seed region of the antisense strand, the antisense strand can comprise 1 2' position modified ribose-containing nucleotide at the seed region, 2 consecutive 2' position modified ribose-containing nucleotides at the region 3' adjacent to the seed region, and 2 consecutive 2' position modified ribose-containing nucleotides at the 3' terminus. In some embodiments, the sense strand can comprise 3 consecutive 2'-methoxyribose-containing nucleotides at the 5' terminus, 2 consecutive 2'-methoxyribose-containing nucleotides at the 3' terminus, 1 2'-methoxyribose-containing nucleotide at the region opposite the seed region of the antisense strand, the antisense strand can comprise a 2'-methoxyribose-containing nucleotide at position 1, a 2'-fluoro-ribose-containing nucleotide at position 2, 2 consecutive 2'-methoxyethylribose-containing nucleotides at the region 3' adjacent to the seed region (e.g., positions 9 and 10), and 2 consecutive 2'-methoxyribose-containing nucleotides at the 3' terminus.

[0019] In some embodiments, the sense strand can comprise 6 consecutive 2' position modified ribose-containing nucleotides at the 5' terminus, 1 2' position modified ribose-containing nucleotide at the region opposite the seed region of the antisense strand, the antisense strand can comprise 4 2' position modified ribose-containing nucleotides at the seed region, 2 consecutive 2' position modified ribose-containing nucleotides at the region 3' adjacent to the seed region, and 2 consecutive 2' position modified ribose-containing nucleotides at the 3' terminus. In some embodiments, the sense strand can comprise 5 consecutive 2'-methoxyribose-containing nucleotides followed by 1 2'-fluoro-ribose-containing nucleotide at the 5' terminus, 2 consecutive 2'-methoxyribose-containing nucleotides at the 3' terminus, 1 2'-methoxyribose-containing nucleotide at the region opposite the seed region of the antisense strand, the antisense strand can comprise 2'-fluoro-ribose-containing nucleotides at positions 2 and 4, 2'-methoxy-ribose-containing nucleotides at positions 1, 3, and 5, 2 consecutive 2'-methoxyethylribose-containing nucleotides at the region 3' adjacent to the seed region (e.g., positions 9 and 10), and 2 consecutive 2'-methoxyribose-containing nucleotides at the 3' terminus.

[0020] In some embodiments, the sense strand can comprise 9 contiguous 2' position modified ribose-containing nucleotides at the 5' terminus, 7 2' position modified ribose- containing nucleotides at the region opposite the seed region of the antisense strand, the antisense strand can comprise 6 2' position modified ribose-containing nucleotides at the seed region, 2 contiguous 2' position modified ribose-containing nucleotides at the region immediately 3' of the seed region, and 11 contiguous 2' position modified ribose- containing nucleotides at the 3' terminus. In some embodiments, the sense strand can comprise 6 contiguous 2'-methoxyribose-containing nucleotides followed by 3 2'-fluoro-ribose- containing nucleotides at the 5' terminus, 8 contiguous 2'-methoxyribose-containing nucleotides at the 3' terminus, 7 2'-methoxy or 2'-fluoro ribose-containing nucleotides at the region opposite the seed region of the antisense strand, the antisense strand can comprise a 2'- methoxyethyl ribose-containing nucleotide at position 1, 2'-fluoro ribose-containing nucleotides at positions 2 and 4, 2'-methoxy or ribose-containing nucleotides at positions 3, 5, 7, and 8, 2 contiguous 2'-methoxyethyl ribose-containing nucleotides at the region immediately 3' of the seed region (e.g., positions 9 and 10), and 7 contiguous 2'- methoxyribose-containing nucleotides followed by 1 2'-fluoro ribose-containing nucleotide and 2 contiguous 2'-methoxyribose-containing nucleotides at the 3' terminus.

[0021] In some embodiments, the sense strand can comprise 6 contiguous 2' position modified ribose-containing nucleotides at the 5' terminus, 1 2' position modified ribose- containing nucleotide at the region opposite the seed region of the antisense strand, the antisense strand can comprise 4 2' position modified ribose-containing nucleotides at the seed region, 2 contiguous 2' position modified ribose-containing nucleotides at the region immediately 3' of the seed region, and 2 contiguous 2' position modified ribose-containing nucleotides at the 3' terminus. In some embodiments, the sense strand can comprise 6 contiguous 2'-methoxyribose-containing nucleotides at the 5' terminus, 2 contiguous 2'-methoxyribose-containing nucleotides at the 3' terminus, 1 2'- methoxyribose-containing nucleotide at the region opposite the seed region of the antisense strand, the antisense strand can comprise 2'-methoxyribose-containing nucleotides at positions 1, 6, 7, and 8, a 2'-fluoro ribose-containing nucleotide at position 2, 2 contiguous 2'-methoxyethyl ribose-containing nucleotides at the region immediately 3' of the seed region (e.g., positions 9 and 10), and 2 contiguous 2'-methoxyribose-containing nucleotides at the 3' terminus.

[0022] In some embodiments, the sense strand can comprise 8 consecutive 2' position modified ribose-containing nucleotides at the 5' end, 0 2' position modified ribose-containing nucleotides in the region opposite the seed region of the antisense strand, 7 2' position modified ribose-containing nucleotides in the seed region of the antisense strand, 0 2' position modified ribose-containing nucleotides in the region immediately 3' to the seed region, and 2 consecutive 2' position modified ribose-containing nucleotides at the 3' end. In some embodiments, the sense strand can comprise 8 consecutive 2'-methoxyribose-containing nucleotides at the 5' end, 2 consecutive 2'-methoxyribose-containing nucleotides at the 3' end, and the antisense strand can comprise 2'-methoxyribose-containing nucleotides at positions 1, 3, 4, 6, 7, and 8, 2'-fluoro-ribose-containing nucleotides at positions 2 and 5, and 2 consecutive 2'-methoxyribose-containing nucleotides at the 3' end.

[0023] The 3-9 consecutive nucleotides at the 5' end of the sense strand can comprise ribose containing a 2' position modification selected from the group consisting of 2'-halo, and 2'-alkoxy. The 2 consecutive nucleotides at the 3' end of the sense strand can comprise ribose containing a 2'-alkoxy modification. The 2' position modified ribose-containing nucleotides in the region of the sense strand opposite the seed region of the antisense strand can comprise ribose containing a 2'-alkoxy modification. The nucleotide at position 1 of the 5' end of the antisense strand can comprise ribose containing a 2'-halo, 2'-alkoxy, or 2'-alkoxy-alkyl. The 2' position modified ribose-containing nucleotides in the seed region of the antisense strand can comprise ribose containing a modification selected from the group consisting of 2'-halo, and 2'-alkoxy. The 2-11 consecutive nucleotides at the 3' end of the antisense strand can comprise ribose containing a modification selected from the group consisting of 2'-alkoxy and 2'-halo. The 2' position modified ribose-containing nucleotides in the region of the antisense strand immediately 3' to the seed region can comprise ribose containing a 2'-alkoxyalkyl modification.

[0024] The 2' position modified ribose-containing nucleotides in the region of the antisense strand immediately 3' to the seed region can comprise ribose containing a 2' position modification having a steric bulk greater than the 2' position modifications of the other 2' position modified ribose-containing nucleotides in the antisense strand, or a steric bulk that is the largest 2' position modification of the nucleotides in the antisense strand, or a steric bulk that is the largest 2' position modification of the nucleotides in the sense and antisense strands. In some embodiments, the 2' position modified ribose-containing nucleotides in the region of the antisense strand immediately 3' to the seed region can comprise ribose containing a 2'-methoxyethyl, and the other 2' position modified ribose-containing nucleotides in the antisense strand can comprise ribose containing a 2'-methoxy or 2'-fluoro.

[0025] The double-stranded RNA molecule can be a small interfering RNA (siRNA).

[0026] The length of the sense strand and the antisense strand can be the same or different. In some embodiments, the length of the sense strand and the antisense strand is the same. The length of the sense strand and the antisense strand can each be 19-25, for example, can be 19, 20, 21, 22, 23, 24, or 25. In some embodiments, the length of the sense strand and the antisense strand can be 21. The sense strand and the antisense strand can each comprise a 1-5 nucleotide overhang at its 3’ end, for example, a 2 nucleotide overhang.

[0027] In some embodiments, the length of the sense strand and the antisense strand can be 21, the sense strand comprises a nucleotide comprising a 2’ position modified ribose at positions 1, 2, 3, 4, 5, 20, and 21, and the antisense strand comprises a nucleotide comprising a 2’ position modified ribose at positions 1, 2, 3, 4, 5, 20, and 21. In some embodiments, the sense strand comprises a nucleotide comprising a 2’-methoxyribose at positions 1, 2, 3, 4, 5, 20, and 21, and the antisense strand comprises a nucleotide comprising a 2’-methoxyribose at positions 1, 3, 4, 5, 20, and 21, and a nucleotide comprising a 2’-fluoro-ribose at position 2. In some embodiments, the sense strand and the antisense strand can comprise the nucleotide sequences set forth in SEQ ID NOs: 3 and 4, respectively.

[0028] In some embodiments, the length of the sense strand and the antisense strand can be 21, the sense strand comprises a nucleotide comprising a 2’ position modified ribose at positions 1, 2, 3, 4, 5, 20, and 21, and the antisense strand comprises a nucleotide comprising a 2’ position modified ribose at positions 1, 2, 6, 7, 8, 20, and 21. In some embodiments, the sense strand comprises a nucleotide comprising a 2’-methoxyribose at positions 1, 2, 3, 4, 5, 20, and 21, and the antisense strand comprises a nucleotide comprising a 2’-methoxyribose at positions 1, 6, 7, 8, 20, and 21, and a nucleotide comprising a 2’-fluoro-ribose at position 2. In some embodiments, the sense strand and the antisense strand can comprise the nucleotide sequences set forth in SEQ ID NOs: 7 and 8, SEQ ID NOs: 9 and 10, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 16, SEQ ID NOs: 19 and 20, SEQ ID NOs: 21 and 22, SEQ ID NOs: 25 and 26, SEQ ID NOs: 27 and 28, SEQ ID NOs: 33 and 34, SEQ ID NOs: 43 and 44, SEQ ID NOs: 47 and 48, or SEQ ID NOs: 49 and 50, respectively.

[0029] In some embodiments, the sense and antisense strands can be 21 in length, the sense strand comprises 2'-position modified ribose-containing nucleotides at positions 1, 2, 3, 14, 20, and 21, and the antisense strand comprises 2'-position modified ribose-containing nucleotides at positions 1, 2, 9, 10, 20, and 21. In some embodiments, the sense strand comprises 2'-methoxyribose-containing nucleotides at positions 1, 2, 3, 14, 20, and 21, and the antisense strand comprises 2'-methoxyribose-containing nucleotides at positions 1, 20, and 21, 2'-fluoro-modified nucleotide at position 2, and 2'-methoxy-ethylribose-containing nucleotides at positions 9 and 10. In some embodiments, the sense and antisense strands can comprise the nucleotide sequences set forth in SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 6, SEQ ID NO: 13 and 14, SEQ ID NO: 17 and 18, SEQ ID NO: 19 and 20, SEQ ID NO: 27 and 28, or SEQ ID NO: 51 and 52, respectively.

[0030] In some embodiments, the sense and antisense strands can be 21 in length, the sense strand comprises 2'-position modified ribose-containing nucleotides at positions 1, 2, 3, 14, 20, and 21, and the antisense strand comprises 2'-position modified ribose-containing nucleotides at positions 1, 2, 9, 10, 20, and 21. In some embodiments, the sense strand comprises 2'-methoxyribose-containing nucleotides at positions 1, 2, 3, 14, 20, and 21, and the antisense strand comprises 2'-methoxyribose-containing nucleotides at positions 1, 20, and 21, 2'-fluoro-modified nucleotide at position 2, and 2'-methoxy-ethylribose-containing nucleotides at positions 9 and 10. In some embodiments, the sense and antisense strands can comprise the nucleotide sequences set forth in SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 6, SEQ ID NO: 13 and 14, SEQ ID NO: 17 and 18, SEQ ID NO: 19 and 20, SEQ ID NO: 27 and 28, or SEQ ID NO: 51 and 52, respectively.

[0031] In some embodiments, the sense and antisense strands can be 21 in length, the sense strand comprising a nucleotide comprising 2' position modified ribose at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, and the antisense strand comprising a nucleotide comprising 2' position modified ribose at positions 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21. In some embodiments, the sense strand comprises a nucleotide comprising 2'-methoxyribose at positions 1, 2, 3, 4, 5, 6, 14, 15, 16, 17, 18, 19, 20, and 21, and a nucleotide comprising 2'-fluororibose at positions 7, 8, 9, 12, and 13, and the antisense strand comprises a nucleotide comprising 2'-methoxyribose at positions 3, 5, 7, 8, 11, 12, 13, 15, 16, 17, 18, 19, 20, and 21, a nucleotide comprising 2'-fluororibose at positions 2, 4, and 14, and a nucleotide comprising 2'-methoxyethylribose at positions 1, 9, and 10. In some embodiments, the sense and antisense strands can comprise the nucleotide sequences set forth in SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 21 and 22, SEQ ID NOs: 23 and 24, SEQ ID NOs: 29 and 30, SEQ ID NOs: 35 and 36, SEQ ID NOs: 37 and 38, SEQ ID NOs: 39 and 40, SEQ ID NOs: 41 and 42, SEQ ID NOs: 43 and 44, SEQ ID NOs: 45 and 46, SEQ ID NOs: 49 and 50, or SEQ ID NOs: 51 and 52, respectively.

[0032] In some embodiments, the sense and antisense strands can be 21 in length, the sense strand comprises 2'-position modified ribose-containing nucleotides at positions 1, 2, 3, 4, 5, 6, 14, 20, and 21, and the antisense strand comprises 2'-position modified ribose-containing nucleotides at positions 1, 2, 6, 7, 8, 9, 10, 20, and 21. In some embodiments, the sense strand comprises 2'-methoxyribose-containing nucleotides at positions 1, 2, 3, 4, 5, 6, 14, 20, and 21, and the antisense strand comprises 2'-methoxyribose-containing nucleotides at positions 1, 6, 7, 8, 20, and 21, 2'-fluororibose-containing nucleotides at position 2, and 2'-methoxyethylribose-containing nucleotides at positions 9 and 10. In some embodiments, the sense and antisense strands can comprise the nucleotide sequences set forth in SEQ ID NOs: 3 and 4, respectively.

[0033] In some embodiments, the sense and antisense strands can be 21 in length, the sense strand comprises 2'-position modified ribose-containing nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 20, and 21, and the antisense strand comprises 2'-position modified ribose-containing nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 20, and 21. In some embodiments, the sense strand comprises 2'-methoxyribose-containing nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 20, and 21, and the antisense strand comprises 2'-methoxyribose-containing nucleotides at positions 1, 3, 4, 6, 7, 8, 20, and 21, 2'-fluororibose-containing nucleotides at positions 2 and 5. In some embodiments, the sense and antisense strands can comprise the nucleotide sequences set forth in SEQ ID NOs: 31 and 32, respectively.

[0034] The double-stranded RNA molecules of the present application can also comprise a delivery agent. For example, a delivery agent can be comprised at the 3' end of the sense strand. The sense strand can comprise the delivery agent at the 3' end by coupling, linker attachment, or the like. When the sense strand comprises the delivery agent at the 3' end by a linker, the linker can be a cleavable linker, such as a linker that is cleavable under certain conditions, such as pH conditions, or the like.

[0035] The delivery can target a cell or tissue, in particular a specific cell or tissue, including but not limited to a saccharide molecule, a protein, a lipid, or a nucleic acid molecule, etc. For example, the delivery can be a ligand of a surface receptor of a target cell or a target tissue, such as a ligand of ASGPR like GalNac, a ligand of glucagon-like peptide-1 (GLP1) receptor like GLP1. For example, the delivery can be a lipid, such as docosanoic acid (DCA), phosphatidylcholine, cholesterol, 2'-O-hexadecyl (C16) lipid, such as docosanoic acid-phosphatidylcholine. For example, the delivery can be a recombinant protein, such as an antibody or an antigen-binding fragment thereof, Centyrin.

[0036] The double-stranded RNA molecule of the application can be hydrophobic.

[0037] The sense strand can also be coupled with a hydrophobic substance. For example, the sense strand can be coupled with a hydrophobic substance, such as a C16 lipid, on the ribose, such as the 2' position of the ribose.

[0038] In a second aspect, the application provides a composition, which can comprise the double-stranded RNA molecule of the application, and a carrier. The composition can be a pharmaceutical composition, which can comprise the double-stranded RNA molecule of the application, and a pharmaceutically acceptable carrier, such as an excipient.

[0039] The pharmaceutical composition can further comprise a delivery carrier, such as a lipid nanoparticle, a polypeptide nanoparticle, an exosome, etc. In some embodiments, the delivery carrier can be a lipid nanoparticle, such as a lipid nanoparticle (LNP). The double-stranded RNA molecule in the pharmaceutical composition can be encapsulated in the delivery carrier.

[0040] The pharmaceutical composition can further comprise a delivery, which is capable of targeting a cell or tissue, in particular a specific cell or tissue. The delivery can be linked or coupled with the double-stranded RNA molecule, to direct the double-stranded RNA molecule to the cell or tissue.

[0041] In a third aspect, the application provides a method for inhibiting or reducing the expression of a target gene in a sample, comprising contacting the double-stranded RNA molecule or the composition of the application with the sample. The sample can comprise a cell, a tissue, etc. The sample can comprise the target gene, or an mRNA transcribed from the target gene. The target gene can be capable of expressing an mRNA.

[0042] The method can further comprise introducing the double-stranded RNA molecule of the application into the cell. For example, the method can comprise introducing the double-stranded RNA molecule of the application into the cell by transfection, electroporation, etc. Or the method can comprise allowing the double-stranded RNA molecule of the application to enter the cell via endocytosis of the cell.

[0043] In a fourth aspect, the present application provides a method for inhibiting or reducing the expression of a target gene in a subject, comprising administering to the subject a therapeutically effective amount of a double-stranded RNA molecule or a composition of the present application. The method can comprise local administration, or systemic administration. Local administration can comprise injection, for example, into the cerebrospinal fluid, into the skin, etc. Systemic administration can comprise intravenous injection.

[0044] The target gene can be a gene associated with a disease. The target gene can be capable of expressing an mRNA.

[0045] The present application also provides the use of a double-stranded RNA molecule or a composition of the present application in the manufacture of a medicament for inhibiting or reducing the expression of a target gene.

[0046] In a fifth aspect, the present application provides a method for modifying a double-stranded RNA molecule. The method can improve the stability of the double-stranded RNA molecule. In some embodiments, the double-stranded RNA molecule is an siRNA molecule, and the method can improve the silencing efficiency of the siRNA molecule on a target gene, and / or reduce off-target effects.

[0047] In particular, the method can comprise modifying the double-stranded RNA molecule such that the sense strand comprises 3-9 consecutive 2'-position-modified ribose-containing nucleotides at the 5' end, 2 consecutive 2'-position-modified ribose-containing nucleotides at the 3' end, and 0-7 2'-position-modified ribose-containing nucleotides in the region opposite to the seed region of the antisense strand, wherein the 2'-position modification is selected from 2'-halo, 2'-alkoxy, and 2'-alkoxy-alkyl, and the 3 consecutive nucleotides at the 5' end are linked by phosphorothioate bond; the antisense strand comprises a 2'-position-modified ribose-containing nucleotide at the 1st position at the 5' end, a 2'-halo ribose-containing nucleotide at the 2nd position, 1-7 2'-position-modified ribose-containing nucleotides in the seed region, 0 or 2 consecutive 2'-position-modified ribose-containing nucleotides in the region adjacent to the 3' side of the seed region, and 2-11 consecutive 2'-position-modified ribose-containing nucleotides at the 3' end, wherein the 2'-position modification is selected from 2'-halo, 2'-alkoxy, and 2'-alkoxy-alkyl, and the 3 consecutive nucleotides at the 5' end and the 3 consecutive nucleotides at the 3' end are linked by phosphorothioate bond. The method can comprise making the 2'-position-modified ribose-containing nucleotides in the region adjacent to the 3' side of the seed region of the antisense strand have a 2'-position modification with a steric bulk larger than that of the 2'-position modifications of the other 2'-position-modified ribose-containing nucleotides in the antisense strand, or a 2'-position modification with the largest steric bulk in the antisense strand, or a 2'-position modification with the largest steric bulk in the sense strand and the antisense strand.

[0048] In some embodiments, the method can comprise modifying the double stranded RNA molecule such that i) the sense strand comprises 5 consecutive 2' position modified ribose containing nucleotides at the 5' terminus, the region opposite the seed region of the antisense strand comprises 0 2' position modified ribose containing nucleotides, the antisense strand comprises 4 2' position modified ribose containing nucleotides at the seed region, 0 2' position modified ribose containing nucleotides at the region 3' adjacent to the seed region, and 2 consecutive 2' position modified ribose containing nucleotides at the 3' terminus, ii) the sense strand comprises 3 consecutive 2' position modified ribose containing nucleotides at the 5' terminus, 1 2' position modified ribose containing nucleotide at the region opposite the seed region of the antisense strand, the antisense strand comprises 1 2' position modified ribose containing nucleotide at the seed region, 2 consecutive 2' position modified ribose containing nucleotides at the region 3' adjacent to the seed region, and 2 consecutive 2' position modified ribose containing nucleotides at the 3' terminus, iii) the sense strand comprises 6 consecutive 2' position modified ribose containing nucleotides at the 5' terminus, 1 2' position modified ribose containing nucleotide at the region opposite the seed region of the antisense strand, the antisense strand comprises 4 2' position modified ribose containing nucleotides at the seed region, 2 consecutive 2' position modified ribose containing nucleotides at the region 3' adjacent to the seed region, and 2 consecutive 2' position modified ribose containing nucleotides at the 3' terminus, iv) the sense strand comprises 9 consecutive 2' position modified ribose containing nucleotides at the 5' terminus, 7 2' position modified ribose containing nucleotides at the region opposite the seed region of the antisense strand, the antisense strand comprises 6 2' position modified ribose containing nucleotides at the seed region, 2 consecutive 2' position modified ribose containing nucleotides at the region 3' adjacent to the seed region, and 11 consecutive 2' position modified ribose containing nucleotides at the 3' terminus, or v) the sense strand comprises 8 consecutive 2' position modified ribose containing nucleotides at the 5' terminus, 0 2' position modified ribose containing nucleotides at the region opposite the seed region of the antisense strand, the antisense strand comprises 7 2' position modified ribose containing nucleotides at the seed region, 0 2' position modified ribose containing nucleotides at the region 3' adjacent to the seed region, and 2 consecutive 2' position modified ribose containing nucleotides at the 3' terminus.

[0049] In some embodiments, the method can comprise modifying the double stranded RNA molecule, wherein the double stranded RNA molecule comprises a sense strand and an antisense strand, and each of the sense strand and the antisense strand comprises 21 nucleotides, such that i) the sense strand comprises 2'-methoxyribose-containing nucleotides at positions 1, 2, 3, 4, 5, 20, and 21, and the antisense strand comprises 2'-methoxyribose-containing nucleotides at positions 1, 3, 4, 5, 20, and 21, and a 2'-fluoro-ribose-containing nucleotide at position 2, ii) the sense strand comprises 2'-methoxyribose-containing nucleotides at positions 1, 2, 3, 4, 5, 20, and 21, and the antisense strand comprises 2'-methoxyribose-containing nucleotides at positions 1, 6, 7, 8, 20, and 21, a 2'-fluoro-ribose-containing nucleotide at position 2, iii) the sense strand comprises 2'-methoxyribose-containing nucleotides at positions 1, 2, 3, 14, 20, and 21, and the antisense strand comprises 2'-methoxyribose-containing nucleotides at positions 1, 20, and 21, a 2'-fluoro-modified nucleotide at position 2, 2'-methoxy-ethyl-ribose-containing nucleotides at positions 9 and 10, iv) the sense strand comprises 2'-methoxyribose-containing nucleotides at positions 1, 2, 3, 4, 5, 14, 20, and 21, a 2'-fluoro-ribose-containing nucleotide at position 6, and the antisense strand comprises 2'-methoxyribose-containing nucleotides at positions 1, 3, 5, 20, and 21, 2'-fluoro-ribose-containing nucleotides at positions 2 and 4, 2'-methoxy-ethyl-ribose-containing nucleotides at positions 9 and 10, v) the sense strand comprises 2'-methoxyribose-containing nucleotides at positions 1, 2, 3, 4, 5, 6, 14, 15, 16, 17, 18, 19, 20, and 21, 2'-fluoro-ribose-containing nucleotides at positions 7, 8, 9, 12, and 13, and the antisense strand comprises 2'-methoxyribose-containing nucleotides at positions 3, 5, 7, 8, 11, 12, 13, 15, 16, 17, 18, 19, 20, and 21, 2'-fluoro-ribose-containing nucleotides at positions 2, 4, and 14, 2'-methoxy-ethyl-ribose-containing nucleotides at positions 1, 9, and 10, vi) the sense strand comprises 2'-methoxyribose-containing nucleotides at positions 1, 2, 3, 4, 5, 6, 14, 20, and 21, and the antisense strand comprises 2'-methoxyribose-containing nucleotides at positions 1, 6, 7, 8, 20, and 21, a 2'-fluoro-ribose-containing nucleotide at position 2, 2'-methoxy-ethyl-ribose-containing nucleotides at positions 9 and 10, or vii) the sense strand comprises 2'-methoxyribose-containing nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 20, and 21, and the antisense strand comprises 2'-methoxyribose-containing nucleotides at positions 1, 3, 4, 6, 7, 8, 20, and 21, 2'-fluoro-ribose-containing nucleotides at positions 2, and 5.

[0050] Other features and advantages of the presently disclosed application will be apparent from the following detailed description and examples, which should not be construed as limiting. All documents, Genbank Accession Numbers, patents, and published patent applications cited herein are hereby incorporated by reference. BRIEF DESCRIPTION OF DRAWINGS

[0051] The following detailed description is presented in connection with an example embodiment, and is not intended to limit the application to the specific embodiments described. The description is presented as an illustration of the application.

[0052] Figure 1 shows seven exemplary modification patterns of a double-stranded RNA molecule in the present application. DETAILED DESCRIPTION

[0053] As used herein, the terms "a", "an", and "the" include singular and plural referents unless the context clearly dictates otherwise. The following descriptions of terms are provided for clarity, and are not intended to be limiting, unless otherwise indicated.

[0054] As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0055] The term "or" means a single element of an enumerated list of alternatives, unless the context clearly dictates otherwise.

[0056] The term "comprising" or "including" means including but not limited to, such that other elements, integers or steps can be added. In the present document, when the term "comprising" or "including" is used, combinations of the recited elements, integers or steps are also contemplated, unless otherwise indicated. The term "consisting of" or "consisting only of means containing only the recited elements, integers or steps, and no other elements, integers or steps.

[0057] The 5' end or 5' terminus of a nucleic acid molecule can be a terminal end having a free phosphate group, and the 3' end or 3' terminus can be a terminal end having a free hydroxyl group. The 5' side refers to the side closer to the 5' end, and the 3' side refers to the side closer to the 3' end.

[0058] "Complementary", "pairing", "complementary pairing" or "base pairing" as used herein means that two nucleotides or two bases can pair with each other according to the base complementary principle of A-T, A-U, C-G. When one nucleotide sequence is "complementary" to another nucleotide sequence, it can mean that the two nucleotide sequences are 100% complementary to each other, or it can mean that the two nucleotide sequences have high complementarity, for example, more than 90% complementary. The two strands that are "complementary" to each other are the "complementary strands" of each other.

[0059] "Nucleotide" is the basic component of nucleic acid, which contains a nitrogenous base, a ribose and one or more phosphate groups. There are five kinds of nitrogenous bases, namely adenine (A), guanine (G), cytosine (C), thymine (T) and uracil (U). A nucleotide can contain a hydroxyl group at the 5' position and a phosphate group at the 3' position, or a hydroxyl group at the 3' position and a phosphate group at the 5' position. Two nucleotides can be connected together by reacting the groups at the 3' and 5' positions to form a phosphodiester bond.

[0060] "2' position modification" mentioned in this application refers to modification of the 2' position of ribose, for example, replacing the 2' position hydroxyl group with other groups that are not easily attacked by nucleases to cause RNA hydrolysis, such as alkoxy, halo, alkyl, etc. Accordingly, 2'-halo, 2'-alkoxy, and 2'-alkoxy-alkyl refer to the group at the 2' position of ribose.

[0061] "Thiophosphodiester bond" or "thiophosphodiester bond" refers to a bond obtained by replacing oxygen in the phosphodiester bond connecting two nucleotides with sulfur, which is divided into Rp conformation and Sp conformation

[0062] "Seed region" as referred to herein refers to the region containing the 2nd-8th nucleotides from the 5' end of the antisense strand. The region "opposite to the seed region of the antisense strand" in the sense strand refers to the region in the sense strand that is opposite to or complementary to the seed region of the antisense strand when the sense strand and the antisense strand are complementary to each other to form a double strand.

[0063] "Sense strand" as used herein refers to the strand in a double-stranded RNA whose sequence is the same as that of the mRNA target molecule or part of the sequence, while "antisense strand" refers to the strand that is reverse complementary to the "sense strand" or the mRNA target molecule sequence or part of the sequence.

[0064] "Overhang" or "nucleotide overhang" in a double-stranded RNA refers to the nucleotides in the sense strand or the antisense strand that are not complementary to the other strand to form a double strand and protrude out of the paired double strand.

[0065] The "delivery agent" herein refers to a substance that can direct the double-stranded RNA molecule to the target cell or tissue when coupled or linked with the double-stranded RNA molecule of the present application, such as GalNac that can bind to ASGPR on hepatocytes or liver tissue, GLP1 that can bind to GLP1 receptor in pancreas, and antibodies or antigen-binding fragments thereof that can bind to molecules on cells or tissues, etc. The delivery agent can also function to facilitate cellular endocytosis, prolong the blood half-life of the double-stranded RNA molecule, etc.

[0066] The "delivery vehicle" refers to a substance that delays degradation of the double-stranded RNA molecule of the present application from various enzymes in the body, such as lipid nanoparticles, polypeptide nanoparticles, exosomes, etc. that can encapsulate the double-stranded RNA molecule of the present application so that it can reach the target cell or tissue. The delivery vehicle can also function to facilitate cellular endocytosis, prolong the blood half-life of the double-stranded RNA molecule, etc.

[0067] The "region adjacent to the 3' side of the seed region" herein refers to a region that is immediately next to the 3' side of the seed region without a gap. The 3' side refers to the direction close to the 3' end.

[0068] The "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, e.g., non-human primates, sheep, dog, cat, cow, horse, chicken, amphibians, and reptiles, although mammals, e.g., non-human primates, sheep, dog, cat, cow, and horse, are preferred.

[0069] The "therapeutically effective amount" refers to an amount of the double-stranded RNA molecule of the present application that is sufficient to prevent or slow down the symptoms associated with the disease or disorder. The therapeutically effective amount is related to the disease being treated, where the person skilled in the art can easily determine the actual effective amount.

[0070] Small interfering RNA (siRNA) is a hot candidate for gene therapy drugs. In theory, siRNA-mediated sequence-specific silencing can knock down the expression of any gene, particularly the expression of a gene associated with a disease, without being subject to factors such as the distribution location of the target gene and its product in the cell, the spatial conformation, etc., thereby having a wider potential application range than antibodies and other small molecule drugs. In addition, siRNA-mediated gene silencing requires 100% matching of the mRNA target sequence in theory, thereby having higher sequence specificity. Using this feature, siRNA can be used for research on single nucleotide polymorphism (SNP), etc., and for specifically silencing the gene product that causes disease due to mutation so that the normal gene product can be expressed.

[0071] However, it is well known that RNA molecules are extremely susceptible to degradation, even double-stranded RNA molecules are not immune. The ribose 2' position of ribonucleotides is a hydroxyl group, which is susceptible to attack by nucleases, leading to degradation of the RNA strand. The phosphodiester bonds connecting the nucleotides are also susceptible to attack by RNases and phosphatases, leading to rapid degradation of siRNA into small fragments. Thus, in the manipulation and application of RNA, an extremely stringent enzyme-free environment is required. Studies have shown that without any protection, about 50% of siRNA will be degraded within 1 minute of entering the body, which results in very limited actual target gene silencing efficiency.

[0072] This problem can be solved by modifying the ribonucleotides. Ribonucleotides contain nitrogenous bases, ribose, and one or more phosphate groups, any of which or multiple of which can be modified, see, e.g., Hu B, Zhong L, Weng Y, Peng L, Huang Y, Zhao Y, Liang XJ. Therapeutic siRNA: state of the art. Signal Transduct Target Ther. 2020 Jun 19; 5(1): 101. In particular, the phosphodiester bonds in the nucleotides, especially the phosphodiester bonds at the ends of the RNA strand, can be modified, for example, replaced by phosphorothioate bonds, and the hydroxyl group at the 2' position of ribose can be replaced by more stable alkyl, halo, alkoxy, etc. At the same time, wrapping the RNA molecule in some delivery vehicles can also protect the RNA molecule to some extent from attack by various enzymes before it takes effect.

[0073] In addition, siRNA also has some off-target problems, including off-target effects caused by base pairing between the seed region of the antisense strand of siRNA and mRNA, and off-target effects caused by base pairing between the sense strand entering RISC and mRNA, which can cause certain toxicity to the organism. Studies have shown that asymmetric design of the two strands of siRNA, such as designing the antisense strand to be longer than the sense strand, making the 3' end have a longer overhang, using 5'-morpholino analogs at the 5' end of the sense strand, 5'-O-methylation of the sense strand, etc., can reduce the probability of the sense strand entering RISC. In addition, modification of the nucleotides at the 5' end of the antisense strand can promote the loading of the antisense strand into RISC. Moreover, ribose modification of the seed region of the antisense strand, such as modification of the 2' position of ribose at positions 2-5 of the seed region, can reduce the off-target effects caused by base pairing between the seed region and mRNA.

[0074] 2'-methoxy (2'-OMe), and 2'-fluoro (2'-F) are the two earliest and most commonly used 2' position modification groups for ribose. In addition to increasing the resistance of the RNA strand to various enzymes, they can also increase the hydrophobicity of the RNA molecule, prolong its blood half-life, improve endocytosis, etc. The 2'-F has a small spatial volume and can be used in all positions without affecting the overall conformation of the RNA molecule. However, 2'-F is a non-natural modification, and its metabolites can potentially bind to mitochondrial DNA, causing mitochondrial swelling in hepatocytes and some muscle cells, which has potential toxicity.

[0075] In the past 20 years, modified siRNA products have undergone several iterations. The most mainstream products almost all modify all nucleotides of the sense and antisense strands, and use a large number of 2'-F modifications.

[0076] The inventors of the present application have explored various combinations of modifications in order to provide a stable double-stranded RNA molecule with as few artificial modifications as possible. It has been found that modifying the nucleotides at specific positions in the siRNA molecule can increase the stability of the siRNA and improve the target gene silencing efficiency, and the effect is even better than that of the prior art, such as the RNA modification technology of Alnylam. The siRNA modification mode of the present application can also reduce off-target effects, including off-target effects caused by the partial sequence of the antisense strand pairing with mRNA, and off-target effects caused by the sense strand entering RISC. In addition, the inventors of the present application have only used a limited number of ribose nucleotides containing 2'-F modification, so that the potential harm of the metabolites of this non-natural modification to the organism is reduced to a low level.

[0077] Specifically, the inventors of the present application have realized the increase in stability of the siRNA molecule and the improvement in target gene silencing efficiency by modifying the phosphorothioate bond at the 5' end of the sense strand, and the 5' end and 3' end of the antisense strand, and modifying the 2' position of the nucleotides at the 5' end and 3' end of the sense and antisense strands, and the seed region of the antisense strand. In particular, 2'-F modification is used at the 2nd position at the 5' end of the antisense strand, and a nucleic acid 2' position modification with a larger spatial volume is optionally introduced in the region adjacent to the 3' side of the seed region of the antisense strand.

[0078] The modified double-stranded RNA molecules of the present application comprise a sense strand and an antisense strand, wherein the sense strand can comprise at least 3 consecutive 2' position modified ribose-containing nucleotides at the 5' end, at least 2 consecutive 2' position modified ribose-containing nucleotides at the 3' end, and the 3 consecutive nucleotides at the 5' end can be linked by phosphorothioate bonds, and wherein the antisense strand can comprise a 2'-halogenated ribose-containing nucleotide at the 2nd position from the 5' end, at least 2 consecutive 2' position modified ribose-containing nucleotides at the 3' end, the 3 consecutive nucleotides at the 5' end can be linked by phosphorothioate bonds, and the 3 consecutive nucleotides at the 3' end can be linked by phosphorothioate bonds. The 2' position modification can be selected from the group consisting of 2'-halogenated, 2'-alkoxy, and 2'-alkoxy-alkyl.

[0079] In some embodiments, the sense strand can comprise 3-9 consecutive 2' position modified ribose-containing nucleotides at the 5' end, 2 consecutive 2' position modified ribose-containing nucleotides at the 3' end, and 0-7 2' position modified ribose-containing nucleotides in the region opposite to the seed region of the antisense strand, wherein the 2' position modification can be selected from the group consisting of 2'-halogenated, 2'-alkoxy, and 2'-alkoxy-alkyl, and the 3 consecutive nucleotides at the 5' end can be linked by phosphorothioate bonds; and the antisense strand can comprise a 2' position modified ribose-containing nucleotide at the 1st position from the 5' end, a 2'-halogenated ribose-containing nucleotide at the 2nd position, 1-7 2' position modified ribose-containing nucleotides in the seed region, 0 or 2 consecutive 2' position modified ribose-containing nucleotides in the region adjacent to the 3' side of the seed region, and 2-11 consecutive 2' position modified ribose-containing nucleotides at the 3' end, wherein the 2' position modification is selected from the group consisting of 2'-halogenated, 2'-alkoxy, and 2'-alkoxy-alkyl, and the 3 consecutive nucleotides at the 5' end can be linked by phosphorothioate bonds, and the 3 consecutive nucleotides at the 3' end can be linked by phosphorothioate bonds.

[0080] The 7 exemplary modification combinations of the present application are shown in Figure 1, which, when applied to a variety of siRNA molecules, achieved the improvement of the target gene expression silencing efficiency of siRNA. The improvement of siRNA silencing efficiency by the modification combinations of the present application can be partially dependent on the sequence of siRNA, and the results showed that the 7 exemplary modification combinations had particular preferences for some siRNAs. However, in general, the modification combinations of the present application, particularly the 7 exemplary modification combinations, can be used to improve the stability and target gene silencing efficiency of various siRNAs.

[0081] In particular, the 1st exemplary modification combination in FIG. 1 can be employed when the siRNA sense strand and the antisense strand comprise the sequences of SEQ ID NOs: 3 and 4, respectively. The 2nd exemplary modification combination in FIG. 1 can be employed when the siRNA sense strand and the antisense strand comprise the sequences of SEQ ID NOs: 7 and 8, SEQ ID NOs: 9 and 10, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 16, SEQ ID NOs: 19 and 20, SEQ ID NOs: 21 and 22, SEQ ID NOs: 25 and 26, SEQ ID NOs: 27 and 28, SEQ ID NOs: 33 and 34, SEQ ID NOs: 43 and 44, SEQ ID NOs: 47 and 48, or SEQ ID NOs: 49 and 50, respectively. The 3rd exemplary modification combination in FIG. 1 can be employed when the siRNA sense strand and the antisense strand comprise the sequences of SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, SEQ ID NOs: 13 and 14, SEQ ID NOs: 17 and 18, SEQ ID NOs: 19 and 20, SEQ ID NOs: 27 and 28, or SEQ ID NOs: 51 and 52, respectively. The 4th exemplary modification combination in FIG. 1 can be employed when the siRNA sense strand and the antisense strand comprise the sequences of SEQ ID NOs: 3 and 4, SEQ ID NOs: 31 and 32, or SEQ ID NOs: 51 and 52, respectively. The 5th exemplary modification combination in FIG. 1 can be employed when the siRNA sense strand and the antisense strand comprise the sequences of SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, SEQ ID NOs: 11 and 12, SEQ ID NOs: 13 and 14, SEQ ID NOs: 21 and 22, SEQ ID NOs: 23 and 24, SEQ ID NOs: 29 and 30, SEQ ID NOs: 35 and 36, SEQ ID NOs: 37 and 38, SEQ ID NOs: 39 and 40, SEQ ID NOs: 41 and 42, SEQ ID NOs: 43 and 44, SEQ ID NOs: 45 and 46, SEQ ID NOs: 49 and 50, or SEQ ID NOs: 51 and 52, respectively. The 6th exemplary modification combination in FIG. 1 can be employed when the siRNA sense strand and the antisense strand comprise the sequences of SEQ ID NOs: 3 and 4, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 16, or SEQ ID NOs: 27 and 28, respectively. The 7th exemplary modification combination in FIG. 1 can be employed when the siRNA sense strand and the antisense strand comprise the sequences of SEQ ID NOs: 31 and 32, respectively.

[0082] Of the seven exemplary modification combinations, the second and fifth modification combinations are most applicable to the sequences. In particular, the second modification combination, in which additional modifications are made only to a few nucleotides at the 5' and 3' ends of both strands and a few nucleotides 3' of the seed region of the antisense strand, is almost as effective as the most effective. It is also notable that only one nucleotide in the second combination contains a 2'-fluoro modification.

[0083] When the siRNA contains a sequence other than those described above, the appropriate modification combination can be selected based on the similarity of the sequence to the sequences described above. In particular, when the siRNA contains a sequence other than those described above, any one of the seven exemplary modification combinations described above can be selected. The effectiveness of using different modification combinations can be determined based on the tests disclosed herein.

[0084] Methods for synthesizing the modified double-stranded RNA molecules of the present application are known to those skilled in the art. RNA strands can be synthesized using nucleotides that have been modified for nitrogenous bases, ribose, and phosphate groups, for example, by solid phase synthesis. During the oxidation step of the RNA synthesis cycle, the phosphite triester can be converted to the phosphorothioate by adding a sulfhydrylating reagent (0.05 M 5-N-[(dimethylamino)methylene]amino-3H-1,2,4- dithiazole-3-thione in 60:40 (v / v) pyridine / acetonitrile) to the solid phase synthesis support.

[0085] The siRNA molecules can be administered locally or systemically to a subject. When administered systemically, the siRNA accumulates in the liver and kidneys, which are the primary clearance organs for the siRNA. The high blood flow rate, discontinuous endothelium of the liver, and the natural filtration function of the kidneys cause most of the injected compound to be eliminated before sufficient systemic accumulation is achieved.

[0086] Thus, when the target tissue for the siRNA molecule is not the liver or the kidneys, local administration can be considered. In addition, the siRNA can be delivered more rapidly and more specifically to the target tissue by means of targeted delivery vehicles, such as GLP1 targeting the GLP1 receptor of the pancreas, antibodies targeting various tissues, and the like. When the siRNA targets the liver, the efficiency of delivery can also be enhanced by means of ligands for the ASGPR, such as GalNac.

[0087] The antisense strand plays the primary role in the silencing of the target gene expression, and thus the delivery vehicle is attached or conjugated to the sense strand, particularly the 3' end of the sense strand. The delivery vehicle can also serve to increase the hydrophobicity of the siRNA molecule, to increase its blood half-life, and the like. The delivery vehicle can be attached to the sense strand via a cleavable linker, such as a phosphodiester bond that becomes unstable under acidic conditions, to ensure stability while not interfering with RISC loading, and the like.

[0088] Local administration of siRNA has shown strong efficacy in the treatment of central nervous system, eye, lung, and skin-related diseases. Direct delivery of compounds to relatively closed or easily accessible tissues can enhance retention and ensure sufficient compound supply for cellular uptake. The molecular properties of siRNA, such as hydrophobicity and molecular size, can also reduce clearance and promote uptake. Hydrophobic conjugates can significantly improve the retention of siRNA in the skin after local injection and support functional modulation of disease pathways in rodents and human skin in vitro. The skin is the largest and most accessible human tissue for local administration of siRNA (transdermal and intradermal). Treating local skin diseases by increasing the hydrophobicity of the scaffold can reduce the extent of systemic exposure of siRNA. Hydrophilic conjugates limit local retention and cellular uptake, resulting in more systemic absorption.

[0089] siRNA molecules can also be further protected when delivered in vivo with the help of delivery vehicles. For example, siRNA molecules can be wrapped in delivery vehicles, such as lipid nanoparticles, polypeptide nanoparticles, exosomes, which can avoid the attack of various enzymes in the environment on the molecules before reaching the target tissue, and also reduce the metabolic clearance rate. The most popular delivery vehicle is lipid nanoparticles (LNP), which has been applied in a variety of clinical RNA therapeutic molecules.

[0090] Examples

[0091] Example 1. Synthesis and purification of chemically modified double-stranded siRNA

[0092] Based on the naked sequences of the 26 siRNAs in Table 1, siRNA molecules containing the naked sequences were synthesized, and siRNA molecules with 7 chemical modification combinations in Figure 1 were synthesized.

[0093] Specifically, modification combination one (GS1) shown in Figure 1 contains 2'-methoxy (2'-OMe) modification on the ribose of nucleotides 1, 2, 3, 4, 5, 20, and 21 of the sense strand (SS), and contains 2 consecutive thiosulfate modifications at the 5' end; contains 2'-methoxy modification on the ribose of nucleotides 1, 3, 4, 5, 20, and 21 of the antisense strand (AS), contains 2'-fluoro (2'-F) modification on the ribose of nucleotide 2, and contains 2 consecutive thiosulfate modifications at the 5' end and the 3' end, respectively.

[0094] In modification set two (GS2), the sense strand (SS) contains 2'-methoxy (2'- OMe) modifications on the ribose of nucleotides 1, 2, 3, 4, 5, 20, and 21, and two consecutive phosphorothioate modifications at the 5' end; the antisense strand (AS) contains 2'-methoxy modifications on the ribose of nucleotides 1, 6, 7, 8, 20, and 21, a 2'-fluoro (2'-F) modification on the ribose of nucleotide 2, and two consecutive phosphorothioate modifications at the 5' end and 3' end, respectively.

[0095] In modification set three (GS3), the sense strand (SS) contains 2'-methoxy (2'- OMe) modifications on the ribose of nucleotides 1, 2, 3, 14, 20, and 21, and two consecutive phosphorothioate modifications at the 5' end; the antisense strand (AS) contains 2'-methoxy modifications on the ribose of nucleotides 1, 20, and 21, a 2'-fluoro (2'-F) modification on the ribose of nucleotide 2, 2'-methoxyethyl (2'-MOE) modifications on the ribose of nucleotides 9 and 10, and two consecutive phosphorothioate modifications at the 5' end and 3' end, respectively.

[0096] In modification set four (GS4), the sense strand (SS) contains 2'-methoxy (2'- OMe) modifications on the ribose of nucleotides 1, 2, 3, 4, 5, 14, 20, and 21, a 2'-fluoro (2'-F) modification on the ribose of nucleotide 6, and two consecutive phosphorothioate modifications at the 5' end; the antisense strand (AS) contains 2'-methoxy modifications on the ribose of nucleotides 1, 3, 5, 20, and 21, 2'-fluoro (2'-F) modifications on the ribose of nucleotides 2 and 4, 2'-methoxyethyl (2'-MOE) modifications on the ribose of nucleotides 9 and 10, and two consecutive phosphorothioate modifications at the 5' end and 3' end, respectively.

[0097] Table 1. Naked sequences of double-stranded siRNAs

[0098] In Modified Set Five (GS5), the sense strand (SS) comprises 2'-methoxy (2'-OMe) modifications at the ribose of nucleotides at positions 1, 2, 3, 4, 5, 6, 14, 15, 16, 17, 18, 19, 20, and 21, 2'-fluoro (2'-F) modifications at the ribose of nucleotides at positions 7, 8, 9, 12, and 13, and 2 consecutive phosphorothioate modifications at the 5' end; the antisense strand (AS) comprises 2'-methoxy modifications at the ribose of nucleotides at positions 3, 5, 7, 8, 11, 12, 13, 15, 16, 17, 18, 19, 20, and 21, 2'-fluoro (2'-F) modifications at the ribose of nucleotides at positions 2, 4, and 14, 2'-methoxyethyl (2'-MOE) modifications at the ribose of nucleotides at positions 1, 9, and 10, and 2 consecutive phosphorothioate modifications at the 5' end and 3' end, respectively.

[0099] In Modified Set Six (GS6), the sense strand (SS) comprises 2'-methoxy (2'-OMe) modifications at the ribose of nucleotides at positions 1, 2, 3, 4, 5, 6, 14, 20, and 21, and 2 consecutive phosphorothioate modifications at the 5' end; the antisense strand (AS) comprises 2'-methoxy modifications at the ribose of nucleotides at positions 1, 6, 7, 8, 20, and 21, 2'-fluoro (2'-F) modification at the ribose of nucleotide at position 2, 2'-methoxyethyl (2'-MOE) modifications at the ribose of nucleotides at positions 9 and 10, and 2 consecutive phosphorothioate modifications at the 5' end and 3' end, respectively.

[0100] In Modified Set Seven (GS7), the sense strand (SS) comprises 2'-methoxy (2'-OMe) modifications at the ribose of nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 20, and 21, and 2 consecutive phosphorothioate modifications at the 5' end; the antisense strand (AS) comprises 2'-O-methylation modifications at the ribose of nucleotides at positions 1, 3, 4, 6, 7, 8, 20, and 21, 2'-fluoro (2'-F) modifications at the ribose of nucleotides at positions 2, and 5, and 2 consecutive phosphorothioate modifications at the 5' end and 3' end, respectively.

[0101] Specifically, the synthesis of the sense strand and the antisense strand of the siRNA is performed from the 3' end to the 5' end using the solid phase synthesis method, using 2'-O-TBDMS phosphoramidite (A / G / C / U) monomers, 2'-methoxy phosphoramidite (A / G / C / U) monomers, 2'-fluoro phosphoramidite (A / G / C / U) monomers, and 2'-methoxyethyl phosphoramidite (A / G / C / U) monomers dissolved in acetonitrile as needed.

[0102] The reagent and phosphoramidite solutions are connected to the YB 192 synthesizer, the solid phase synthesis support is inserted into the synthesis plate, and the synthesis plate is placed into the synthesis instrument cavity. The synthesis support is washed twice with acetonitrile, and the YB 192 software is started to start the synthesis. The synthesis is completed by repeating the synthesis cycle of deprotection-coupling-oxidation-capping. In particular, in the deprotection step, a trityl removal reagent (3% trichloroacetic acid in dichloromethane) is added to the solid phase synthesis support to remove the 5'-dimethoxytrityl (DMT) protecting group, and the synthesis support is washed with acetonitrile; in the coupling step, the required phosphoramidite solution and activator solution (0.25M 5-(ethylthio)-1H-tetrazole (ETT) in acetonitrile) are added to the solid phase synthesis support, incubated, so that the added phosphoramidite monomer is coupled with the 5'-hydroxyl group that becomes free in the deprotection step, and the synthesis support is washed with acetonitrile; in the oxidation step, an oxidation reagent (0.025M iodine in 70:20:10 (v / v / v) tetrahydrofuran / pyridine / water) or a thiolation reagent (0.05M 5-N-[(dimethylamino)methylene]amino-3H-1,2,4-dithiazole-3-thione in 60:40 (v / v) pyridine / acetonitrile) is added to the solid phase synthesis support to convert the phosphite triester to a phosphotriester or a phosphorothioate; in the capping step, capping reagents A (70:30 (v / v) acetonitrile / acetic anhydride) and B (20% 1-methylimidazole / acetonitrile) are added to the solid phase synthesis support to terminate any unreacted oligonucleotide chains, and the support is washed with acetonitrile. After the final reaction cycle, the synthesis support is washed with a diethylamine solution (20% DEA in acetonitrile) to remove the 2-cyanoethyl protecting group on the 3' position phosphite / phosphate group. The synthesis support is washed with acetonitrile and dried under argon.

[0103] The synthesis column is removed from the synthesis plate, and the solid phase synthesis support in the synthesis column is transferred to a 2 mL ammonia lysis tube. 1.5 mL of RNA ammonia lysis solution is added to the ammonia lysis tube, the cap is tightened, and the tube is heated at 65°C for 1.5 h. After that, the ammonia lysis tube is placed in a -20°C refrigerator to cool, the support is transferred from the ammonia lysis tube, and the ammonia lysis tube is placed in a centrifuge to chase ammonia and dry.

[0104] Deprotection reagent is added to the ammonia lysis tube to remove the 2' protection group TBDMS, the cap is tightened, the primer is shaken to completely dissolve, and the tube is heated at 65°C for 2.0 h.

[0105] After the deprotection of the RNA, alcohol precipitation reagent is added, mixed, placed in a -20°C refrigerator to cool for 6 h, and then placed in a refrigerated centrifuge to centrifuge, the supernatant is poured out, and the ammonia lysis tube is placed in a centrifuge to dry.

[0106] The synthesized RNA was purified by RP-HPLC using a C18 column (Water Xbridge BEH C18, 5 μm, 10 x 100 mm) (Agilent 1200 HPLC), and the purified RNA was detected by MS to ensure the correctness of the target sequence, and then the purity of the RNA was analyzed by HPLC to ensure that the single-stranded purity was more than 90%.

[0107] The A260 was measured using a microplate reader, and according to the Lambert-Beer law, the absorbance A260 and the extinction coefficient E were introduced to calculate the sub-packaging volume of equal nmol of sense and antisense strands, which were mixed in a centrifuge tube, and then vacuum centrifugation was performed at 40°C to dry, PBS buffer was added for annealing, and the annealing condition was denaturation at 95°C for 10 min and slow cooling to room temperature.

[0108] Example 2. Knockdown efficiency test of double-stranded siRNA

[0109] A dual-luciferase reporter system was used to detect the knockdown efficiency of the double-stranded siRNA obtained in Example 1.

[0110] Specifically, a PsiCHECK TM -2 vector containing a Renilla luciferase reporter gene and a firefly luciferase reporter gene was used, and the target gene (i.e., the gene to be knocked down) was added downstream of the Renilla luciferase reporter gene by homologous recombination, so that the target gene was expressed in fusion with the Renilla luciferase reporter gene.

[0111] In the experiment, the cell group transfected only with the above constructed plasmid vector was used as a positive control, and the negative control group was a cell group without any treatment, which was used to eliminate the background signal. The experimental group was a cell group transfected with the above constructed plasmid vector and siRNA. By comparing the change in the ratio of the main reporter gene (Renilla luciferase) to the internal reference reporter gene (firefly luciferase) in the positive control group and the experimental group, the silencing efficiency of the siRNA was evaluated. For each siRNA, three groups of repeats were set for the group without chemical modification combination, the group with chemical modification combination, or the group with Anlylam modification. The Anlylam modification specifically included that the nucleotides at positions 1, 2, 3, 4, 6, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 of the sense strand contained 2'-methoxy modification, the nucleotides at positions 5, 7, 8, 9 contained 2'-fluoro modification, the last three consecutive nucleotides at the 5' end were connected by a phosphorothioate bond, the nucleotides at positions 1, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21 of the antisense strand contained 2'-methoxy modification, the nucleotides at positions 2, 6, 14, 16 contained 2'-fluoro modification, the last three consecutive nucleotides at the 5' end were connected by a phosphorothioate bond, and the last three consecutive nucleotides at the 3' end were connected by a phosphorothioate bond.

[0112] Specifically, 293T cells (ATCC, CRL-3216) were cultured with DMEM medium (Gibco, 10569010) added with 10% fetal bovine serum (FBS, Thermo Fisher, A5669701), and the cells were subcultured every 2-3 days. When the cells grew to a confluence of 80%-90%, the culture medium in the culture dish was discarded, the culture dish containing the cells was washed once with 5 mL of PBS, and then 1 mL of 0.25% trypsin (Gbico, 25200072) was added to the culture dish containing the cells, which was placed in a 37°C incubator for about 1-2 min for digestion. 5 mL of 10% FBS DMEM medium was added to the culture dish to terminate the digestion, and after blowing and stirring, the added medium in the dish was transferred to a 15 mL centrifuge tube and centrifuged at 1000 rpm for 5 min. The supernatant after centrifugation was discarded, and the cell pellet was resuspended with 5 mL of 10% FBS DMEM medium, and after mixing, a small amount of cell suspension was taken for cell counting, subculture and plating, and the cells were cultured in a 37°C / 5% CO2 incubator.

[0113] The cells were plated in a 96-well plate, 25,000 cells per well, and the medium volume per well was 75 μl.

[0114] For each siRNA, two 1.5 mL RNase-free centrifuge tubes (referred to as A tube and B tube) were prepared. 17.5 μL of opti-MEM (Gibco, 31985062) was added to the A tube and the B tube, respectively, and then 0.2625 pmol of siRNA and 0.035 pmol of the above constructed plasmid vector (only the above plasmid vector was added for the positive control group) were added to the A tube, and 1.75 μL of Lipofectamine 2000 transfection reagent (Invitrogen, 11668019) was added to the B tube. Then, the siRNA / plasmid vector in the A tube was transferred to the B tube, mixed gently by blowing, incubated at room temperature for 5 min, and then the mixture in the B tube was added to the corresponding well of the 96-well plate, 10 μL per well, so that the final concentration of siRNA was 1 nM per well, and the final amount of plasmid vector added was 0.01 pmol per well. The cells were cultured in a 37°C, 5% CO2 incubator for 24 h.

[0115] The 96-well plate was taken out of the incubator, and the cell state was observed under a microscope. If the cell state was not contaminated, the dual luciferase detection could be performed. Specifically, 75 μl of Luciferase Reagent (this reagent facilitates cell lysis and also provides the substrate for firefly luciferase), shake at 300 rpm for 10 min on a horizontal shaker, then incubate in the dark for another 10 min. After the cells are fully lysed, detect the firefly luciferase luminescence signal using a microplate reader. Then, add an equal volume (75 μΐ) of Dual-Glo® Stop & Glo® Reagent (this reagent quenches the light produced by the firefly luciferase reaction by at least 10,000-fold and also provides the substrate for Renilla luciferase) to each well of the above 96-well plate, shake at 300 rpm for 10 min on a horizontal shaker, then incubate in the dark for another 10 min, and detect the Renilla luciferase luminescence signal using a microplate reader. Stop& Reagent (this reagent quenches the light produced by the firefly luciferase reaction by at least 10,000-fold and also provides the substrate for Renilla luciferase), shake at 300 rpm for 10 min on a horizontal shaker, then incubate in the dark for another 10 min, and detect the Renilla luciferase luminescence signal using a microplate reader.

[0116] Calculate the ratio of Renilla luciferase to firefly luciferase signal in each well, and normalize the ratio of each experimental group using the ratio of the control group to calculate the knockdown rate of siRNA in each experimental group.

[0117] Table 2 shows the data of siRNAs with the modification combination of the present application and showing better silencing efficiency than naked sequences. It can be seen that most of these siRNAs have higher silencing efficiency than their counterparts modified by the Anylam modification pattern, indicating the superiority of the modification pattern of the present application.

[0118] Table 2. Silencing efficiency of siRNA on target gene expression

[0119] Example 3. IC of double-stranded siRNA 50 Test

[0120] The probe-based qPCR method was used to detect the IC50 of the double-stranded siRNA obtained in Example 1 and the approved drug Vutrisiran.

[0121] Specifically, HEPG2 cells (ATCC; HB-8065) were cultured with EMEM medium (ATCC; 30-2003) added with 10% fetal bovine serum (FBS, Thermo Fisher, A5669701), and the cells were subcultured every 2-3 days. When the cells grew to 80%-90% confluence, the culture medium in the culture dish was discarded, the culture dish containing the cells was washed once with 5 mL of PBS, and then 1 mL of 0.25% trypsin (Gbico, 25200072) was added to the culture dish containing the cells, and the dish was incubated in a 37°C incubator for 1-2 min. 5 mL of EMEM medium containing 10% FBS was added to the dish to terminate the digestion, and the medium added to the dish was transferred to a 15 mL centrifuge tube after being mixed by blowing and sucking. The supernatant was discarded after centrifugation at 1000 rpm for 5 min, and the cell pellet was resuspended with 5 mL of EMEM medium containing 10% FBS. After mixing, a small amount of cell suspension was taken for cell counting, and the cells were subcultured and cultured.

[0122] The day before the transfection experiment, the cells were plated according to the number of viable cells, 4W cells per well of a 48-well plate, and 270 μL of complete medium was added. After incubation in the incubator for 24 h, the nucleic acid was transfected using the commercial transfection reagent RNAiMAX (THERMO; 13778075). During the cell digestion and centrifugation interval, the samples were prepared, and for each condition, two duplicate wells were prepared with 2.5 reactions of Mix, with a maximum final concentration of 100 nM, and a 5-fold gradient dilution was used, with a total of 10 concentration points. Taking siRNA with a final concentration of 100 nM as an example, two sterile, DNase / RNase-free 1.5 ml centrifuge tubes were prepared, defined as tubes A and B. 80 μL of Opti-MEM and 20 μL of siRNA (siRNA stock concentration of 10 μM) were added to tube A to dilute to the required solution concentration. Tube B was added with 37.5 μL of pre-mixed RNAiMAX reagent (35.25 μL of Opti-MEM plus 2.25 μL of RNAiMAX). 37.5 μL of liquid was transferred from tube A to tube B (the final volume of tube A after transfer to tube B was 75 μL), and the mixture was mixed gently by blowing and then incubated at room temperature for 10 min. Then the siRNA-RNAiMAX complex was added to the 48-well plate, 30 uL per well (if concerned that the addition of the complex will cause uneven distribution of the cells, the cells can be shaken again after addition), and the cell plate was incubated in a 37°C / 5% CO2 incubator for 48 h.

[0123] 48h later, the 48-well plate was taken out of the incubator, and the cell status was observed under a microscope. If the cell status was good and the confluence was more than 80%, RNA extraction and subsequent qPCR detection could be performed. RNA extraction (NORWEGIAN; RC112-01) and qPCR detection (NORWEGIAN; QN222-02) were performed according to the kit instructions. The knockdown efficiency of siRNA at each concentration point was calculated by detecting the ct value and using the ΔΔCt method.

[0124] Table 4 shows that the GS5, GS3, GS6, GS2, GS4, GS1 modification combination of the present application shows lower IC50 values than Vutrisiran, indicating the superiority of the modification mode of the present application.

[0125] Table 3. GS1-7 modification of human TTR target and sequence of Vutrisiran

[0126] Table 4. IC of GS1-7 and Vutrisiran 50 Data pairs

[0127] Although the present application has been described in connection with one or more embodiments, it will be understood that the application is not limited to such embodiments. The description herein is intended to cover all variants and equivalents included within the spirit and scope of the claims appended hereto. All documents cited herein are hereby incorporated by reference in their entirety.

Claims

1. A modified double-stranded RNA molecule comprising a sense strand and an antisense strand, each strand comprising 19-25 nucleotides, wherein the sense strand comprises 3-9 consecutive 2'-position-modified ribose-containing nucleotides at the 5' end, 2 consecutive 2'-position-modified ribose-containing nucleotides at the 3' end, and 0-7 2'-position-modified ribose-containing nucleotides in the region opposite the seed region of the antisense strand, wherein the 2'-position modification is selected from the group consisting of 2'-halo, 2'-alkoxy, and 2'-alkoxy-alkyl, and the 3 consecutive nucleotides at the 5' end are linked by phosphorothioate linkages, wherein the antisense strand comprises a 2'-position-modified ribose-containing nucleotide at position 1 of the 5' end, a 2'-halo ribose-containing nucleotide at position 2, 1-7 2'-position-modified ribose-containing nucleotides in the seed region, 0 or 2 consecutive 2'-position-modified ribose-containing nucleotides in the region adjacent the 3' side of the seed region, and 2-11 consecutive 2'-position-modified ribose-containing nucleotides at the 3' end, wherein the 2'-position modification is selected from the group consisting of 2'-halo, 2'-alkoxy, and 2'-alkoxy-alkyl, and the 3 consecutive nucleotides at the 5' end are linked by phosphorothioate linkages and the 3 consecutive nucleotides at the 3' end are linked by phosphorothioate linkages.

2. The double-stranded RNA molecule of claim 1, wherein the 2'-position-modified ribose- containing nucleotide in the region adjacent the 3' side of the seed region of the antisense strand comprises a 2'-position modification having a steric bulk greater than the 2'-position modifications of the other 2'-position-modified ribose-containing nucleotides in the antisense strand.

3. The double-stranded RNA molecule of claim 1, wherein the 3-9 consecutive nucleotides at the 5' end of the sense strand comprise ribose having a 2'-position modification selected from the group consisting of 2'-halo and 2'-alkoxy; wherein the 2 consecutive nucleotides at the 3' end of the sense strand comprise ribose having a 2'-alkoxy modification; wherein the 2'-position-modified ribose-containing nucleotides in the region of the sense strand opposite the seed region of the antisense strand comprise ribose having a 2'-position modification selected from the group consisting of 2'-halo and 2'-alkoxy; wherein the nucleotide at position 1 of the 5' end of the antisense strand comprises ribose having a 2'-alkoxy or 2'-alkoxy-alkyl modification; wherein the 2'-position-modified ribose-containing nucleotides in the seed region of the antisense strand comprise ribose having a 2'-position modification selected from the group consisting of 2'-halo and 2'-alkoxy; wherein the 2-7 consecutive nucleotides at the 3' end of the antisense strand comprise ribose having a 2'-position modification selected from the group consisting of 2'-alkoxy and 2'-halo; or wherein the 2'-position-modified ribose-containing nucleotide in the region adjacent the 3' side of the seed region of the antisense strand comprises ribose having a 2'-alkoxy-alkyl modification.

4. The double-stranded RNA molecule of claim 1, wherein the 2'-halo is 2'-fluoro, the 2'-alkoxy is 2'-methoxy, and the 2'-alkoxy-alkyl is 2'-methoxyethyl.

5. The double-stranded RNA molecule of claim 1, which is a small interfering RNA (siRNA) molecule.

6. The double-stranded RNA molecule of claim 1, wherein each of the sense strand and the antisense strand comprises a 2-nucleotide overhang at the 3' end. ​ ​ 7. The double-stranded RNA molecule of claim 1, wherein the antisense strand comprises 2'-position modified ribose-containing nucleotides at 4 positions near the 5' end and / or at 3 positions near the 3' end of the seed region.

8. The double-stranded RNA molecule of claim 1, wherein, i) the sense strand comprises 5 consecutive 2'-position modified ribose-containing nucleotides at the 5' end, 0 2'-position modified ribose-containing nucleotides in the region opposite the seed region of the antisense strand, 4 2'-position modified ribose-containing nucleotides in the seed region of the antisense strand, 0 2'-position modified ribose-containing nucleotides in the region immediately 3' of the seed region, and 2 consecutive 2'-position modified ribose-containing nucleotides at the 3' end, ii) the sense strand comprises 3 consecutive 2'-position modified ribose-containing nucleotides at the 5' end, 1 2'-position modified ribose-containing nucleotide in the region opposite the seed region of the antisense strand, 1 2'-position modified ribose-containing nucleotide in the seed region of the antisense strand, 2 consecutive 2'-position modified ribose-containing nucleotides in the region immediately 3' of the seed region, and 2 consecutive 2'-position modified ribose-containing nucleotides at the 3' end, iii) the sense strand comprises 6 consecutive 2'-position modified ribose-containing nucleotides at the 5' end, 1 2'-position modified ribose-containing nucleotide in the region opposite the seed region of the antisense strand, 4 2'-position modified ribose-containing nucleotides in the seed region of the antisense strand, 2 consecutive 2'-position modified ribose-containing nucleotides in the region immediately 3' of the seed region, and 2 consecutive 2'-position modified ribose-containing nucleotides at the 3' end, iv) the sense strand comprises 9 consecutive 2'-position modified ribose-containing nucleotides at the 5' end, 7 2'-position modified ribose-containing nucleotides in the region opposite the seed region of the antisense strand, 6 2'-position modified ribose-containing nucleotides in the seed region of the antisense strand, 2 consecutive 2'-position modified ribose-containing nucleotides in the region immediately 3' of the seed region, and 7 consecutive 2'-position modified ribose-containing nucleotides at the 3' end, or v) the sense strand comprises 8 consecutive 2'-position modified ribose-containing nucleotides at the 5' end, 0 2'-position modified ribose-containing nucleotides in the region opposite the seed region of the antisense strand, 7 2'-position modified ribose-containing nucleotides in the seed region of the antisense strand, 0 2'-position modified ribose-containing nucleotides in the region immediately 3' of the seed region, and 2 consecutive 2'-position modified ribose-containing nucleotides at the 3' end.

9. The double-stranded RNA molecule of claim 8, wherein the sense strand and the antisense strand each comprise 21 nucleotides, wherein, ​ ​ ​ ​ ​ i) the sense strand comprises a 2'-methoxyribose containing nucleotide at positions 1, 2, 3, 4, 5, 6, 14, 15, 16, 17, 18, 19, 20, and 21, a 2'-fluororibose containing nucleotide at positions 7, 8, 9, 12, and 13, and the antisense strand comprises a 2'-methoxyribose containing nucleotide at positions 3, 5, 7, 8, 11, 12, 13, 15, 16, 17, 18, 19, 20, and 21, a 2'-fluororibose containing nucleotide at positions 2, 4, and 14, and a 2'-methoxyethylribose containing nucleotide at positions 1, 9, and 10, ii) the sense strand comprises a 2'-methoxyribose containing nucleotide at positions 1, 2, 3, 4, 5, 20, and 21, and the antisense strand comprises a 2'-methoxyribose containing nucleotide at positions 1, 6, 7, 8, 20, and 21, a 2'-fluororibose containing nucleotide at position 2, iii) the sense strand comprises a 2'-methoxyribose containing nucleotide at positions 1, 2, 3, 14, 20, and 21, and the antisense strand comprises a 2'-methoxyribose containing nucleotide at positions 1, 20, and 21, a 2'-fluoromodified nucleotide at position 2, and a 2'-methoxyethylribose containing nucleotide at positions 9 and 10, iv) the sense strand comprises a 2'-methoxyribose containing nucleotide at positions 1, 2, 3, 4, 5, 6, 14, 20, and 21, and the antisense strand comprises a 2'-methoxyribose containing nucleotide at positions 1, 6, 7, 8, 20, and 21, a 2'-fluororibose containing nucleotide at position 2, and a 2'-methoxyethylribose containing nucleotide at positions 9 and 10, v) the sense strand comprises a 2'-methoxyribose containing nucleotide at positions 1, 2, 3, 4, 5, 14, 20, and 21, and a 2'-fluororibose containing nucleotide at position 6, and the antisense strand comprises a 2'-methoxyribose containing nucleotide at positions 1, 3, 5, 20, and 21, a 2'-fluororibose containing nucleotide at positions 2 and 4, and a 2'-methoxyethylribose containing nucleotide at positions 9 and 10, vi) the sense strand comprises a 2'-methoxyribose containing nucleotide at positions 1, 2, 3, 4, 5, 20, and 21, and the antisense strand comprises a 2'-methoxyribose containing nucleotide at positions 1, 3, 4, 5, 20, and 21, and a 2'-fluororibose containing nucleotide at position 2, or vii) the sense strand comprises a 2'-methoxyribose containing nucleotide at positions 1, 2, 3, 4, 5, 6, 7, 8, 20, and 21, and the antisense strand comprises a 2'-methoxyribose containing nucleotide at positions 1, 3, 4, 6, 7, 8, 20, and 21, a 2'-fluororibose containing nucleotide at positions 2 and 5.

10. The double stranded RNA molecule of claim 9, wherein, i) the sense strand comprises a 2’-methoxyribose containing nucleotide at positions 1, 2, 3, 4, 5, 6, 14, 15, 16, 17, 18, 19, 20, and 21, a 2’-fluoro-ribose containing nucleotide at positions 7, 8, 9, 12, and 13, and the antisense strand comprises a 2’-methoxyribose containing nucleotide at positions 3, 5, 7, 8, 11, 12, 13, 15, 16, 17, 18, 19, 20, and 21, a 2’-fluoro-ribose containing nucleotide at positions 2, 4, and 14, and a 2’- methoxy-ethylribose containing nucleotide at positions 1, 9, and 10, wherein the sense and antisense strands comprise the nucleotide sequences set forth in SEQ ID NO: 1 and 2, SEQ ID NO: 3 and 4, SEQ ID NO: 11 and 12, SEQ ID NO: 13 and 14, SEQ ID NO: 21 and 22, SEQ ID NO: 23 and 24, SEQ ID NO: 29 and 30, SEQ ID NO: 35 and 36, SEQ ID NO: 37 and 38, SEQ ID NO: 39 and 40, SEQ ID NO: 41 and 42, SEQ ID NO: 43 and 44, SEQ ID NO: 45 and 46, SEQ ID NO: 49 and 50, or SEQ ID NO: 51 and 52, respectively, ii) the sense strand comprises a 2’-methoxyribose containing nucleotide at positions 1, 2, 3, 4, 5, 20, and 21, and the antisense strand comprises a 2’-methoxyribose containing nucleotide at positions 1, 6, 7, 8, 20, and 21, and a 2’-fluoro-ribose containing nucleotide at position 2, wherein the sense and antisense strands comprise the nucleotide sequences set forth in SEQ ID NO: 7 and 8, SEQ ID NO: 9 and 10, SEQ ID NO: 13 and 14, SEQ ID NO: 15 and 16, SEQ ID NO: 19 and 20, SEQ ID NO: 21 and 22, SEQ ID NO: 25 and 26, SEQ ID NO: 27 and 28, SEQ ID NO: 33 and 34, SEQ ID NO: 43 and 44, SEQ ID NO: 47 and 48, or SEQ ID NO: 49 and 50, respectively, iii) the sense strand comprises 2’-methoxyribose-containing nucleotides at positions 1, 2, 3, 14, 20, and 21, and the antisense strand comprises 2’-methoxyribose-containing nucleotides at positions 1, 20, and 21, a 2’-fluoro-modified nucleotide at position 2, and 2’-methoxyethylribose-containing nucleotides at positions 9 and 10, wherein the sense and antisense strands comprise the nucleotide sequences set forth in SEQ ID NOs: 3 and 4, SEQ ID NOs: 5 and 6, SEQ ID NOs: 13 and 14, SEQ ID NOs: 17 and 18, SEQ ID NOs: 19 and 20, SEQ ID NOs: 27 and 28, or SEQ ID NOs: 51 and 52, respectively, iv) the sense strand comprises 2’-methoxyribose-containing nucleotides at positions 1, 2, 3, 4, 5, 6, 14, 20, and 21, and the antisense strand comprises 2’-methoxyribose-containing nucleotides at positions 1, 6, 7, 8, 20, and 21, a 2’-fluoro-ribose-containing nucleotide at position 2, and 2’-methoxyethylribose-containing nucleotides at positions 9 and 10, wherein the sense and antisense strands comprise the nucleotide sequences set forth in SEQ ID NOs: 3 and 4, SEQ ID NOs: 13 and 14, SEQ ID NOs: 15 and 16, or SEQ ID NOs: 27 and 28, respectively, v) the sense strand comprises 2’-methoxyribose-containing nucleotides at positions 1, 2, 3, 4, 5, 14, 20, and 21, and a 2’-fluoro-ribose-containing nucleotide at position 6, and the antisense strand comprises 2’-methoxyribose-containing nucleotides at positions 1, 3, 5, 20, and 21, 2’-fluoro-ribose-containing nucleotides at positions 2 and 4, and 2’-methoxyethylribose-containing nucleotides at positions 9 and 10, wherein the sense and antisense strands comprise the nucleotide sequences set forth in SEQ ID NOs: 3 and 4, SEQ ID NOs: 31 and 32, or SEQ ID NOs: 51 and 52, respectively, vi) the sense strand comprises 2’-methoxyribose-containing nucleotides at positions 1, 2, 3, 4, 5, 20, and 21, and the antisense strand comprises 2’-methoxyribose-containing nucleotides at positions 1, 3, 4, 5, 20, and 21, and a 2’-fluoro-ribose-containing nucleotide at position 2, wherein the sense and antisense strands comprise the nucleotide sequences set forth in SEQ ID NOs: 3 and 4, respectively, vii) the sense strand comprises 2’-methoxyribose-containing nucleotides at positions 1, 2, 3, 4, 5, 6, 7, 8, 20, and 21, and the antisense strand comprises 2’-methoxyribose-containing nucleotides at positions 1, 3, 4, 6, 7, 8, 20, and 21, and 2’-fluoro-ribose-containing nucleotides at positions 2 and 5, wherein the sense and antisense strands comprise the nucleotide sequences set forth in SEQ ID NOs: 31 and 32, respectively.

11. The double stranded RNA molecule of claim 1, wherein the sense strand further comprises a delivery at the 3’ end.

12. A composition comprising the double-stranded RNA molecule of any one of claims 1-11.

13. The composition of claim 12, further comprising a delivery vehicle.

14. A method for inhibiting or reducing expression of a target gene in a sample, comprising contacting the composition of claim 12 with the sample.

15. Use of the RNA molecule of claim 11 in the manufacture of a medicament for inhibiting or reducing expression of a target gene.

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