Compositions and methods for treating microsatellite DNA expansion disorders

Abstract

The present disclosure provides single-stranded or double-stranded interfering RNA molecules (e.g., siRNAs) targeting the MutS homolog 3 (MSH3) gene. The interfering RNA molecules may include specific patterns of nucleoside and internucleoside linkage modifications, as may pharmaceutical compositions containing them. The siRNA molecules may be branched siRNA molecules, such as bi-, tri-, or tetra-branched siRNA molecules. The disclosed siRNA molecules may further feature a 5' phosphorus stabilizing moiety and / or a hydrophobic moiety. Additionally, the present disclosure provides methods for delivering the siRNA molecules of the present disclosure to the central nervous system of a subject, such as a subject identified as having Huntington's disease.

Description

[Technical field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated herein by reference in its entirety. The XML copy was created on May 15, 2023, has the filename "51436-034WO2_Sequence_Listing_5_15_23" and is 1,089,536 bytes in size.

[0002] The present disclosure relates to small interfering RNA (siRNA) molecules that target RNA transcripts (e.g., mRNA) of the MutS homolog 3 (MSH3) gene and compositions containing the same. The disclosure further relates to methods for silencing MSH3 by delivering MSH3-targeting siRNA molecules to a target tissue in a subject in need of treatment, and to methods for treating diseases that may benefit from silencing MSH3 (e.g., diseases caused by pathological microsatellite DNA expansions, including Huntington's disease and myotonic dystrophy type 1, among others). [Background technology]

[0003] MSH3 (MutS homolog 3) encodes a key protein of the DNA mismatch repair system that is believed to play a role in the age of onset and rate of progression of diseases caused by pathological microsatellite DNA expansions, including Huntington's disease and myotonic dystrophy type 1. Recent studies have shown that patients with loss-of-function MSH3 mutations develop Huntington's disease later in life compared to patients with normal forms of the gene. Currently, there are no treatments that can alter the course of Huntington's disease. Thus, there is a need for therapeutic agents that can selectively reduce MSH3 activity in a manner that provides an effective treatment for Huntington's disease or other MSH3-related diseases or disorders. Summary of the Invention

[0004] The present disclosure provides compositions and methods for reducing expression of MSH3 (MutS homolog 3) by small interfering RNA (siRNA)-mediated silencing of the transcript of MSH3, which offers the advantage of being highly selective for MSH3 over other genes.

[0005] The siRNA molecules of the present disclosure can be used to silence MSH3 gene, thereby preventing the translation of corresponding mRNA transcripts and reducing the expression of MSH3 protein.Since the expansion of DNA microsatellite repeats to pathological length depends on the function of MSH3, such reduction in MSH3 level prevents the onset or progression of disease.The siRNA molecules of the present disclosure can be directly delivered to the subject who needs to silence MSH3, for example, by intrathecal injection, intracerebroventricular injection, intrastriatal injection, intraparenchymal injection, intracisternal injection by catheter insertion, intravenous injection, subcutaneous injection, or intramuscular injection.

[0006] In one aspect, the present disclosure provides an siRNA molecule comprising an antisense strand and a sense strand having complementarity to the antisense strand, the antisense strand having sufficient complementarity to hybridize to a region within an MSH3 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-408. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within an MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 5, 17-20, 33, 42, 44, 102, 103, 105, 106, 108, 109, 113, 126, 129, 130, 158, 161, 166, 177, 183, 193, 194, 196, 197, 202, 209, 217, 231, 271, 273, 291, 294, 305, 307, 317, 318, and 362. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within an MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 5, 17-20, 42, 44, 126, 129, 130, 158, 177, 183, 193, 194, 196, 197, 202, 209, 231, 271, 273, 291, 294, 305, 307, 317, 318, and 362. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within an MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-24, 40-84, 100, 118-139, 173-181, 193-205, 220-222, 240-242, 249-308, 319-330, 336-377, 384-386, and 398-408. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within an MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 18-20, 102, 105, 106, 108, 109, 113, 183, 194, and 196. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within an MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 18-20, 183, 194, and 196.

[0007] The antisense strand may be, for example, 10 to 50 nucleotides in length (e.g., 10 to 45 nucleotides in length, 10 to 40 nucleotides in length, 10 to 35 nucleotides in length, 10 to 30 nucleotides in length, 10 to 29 nucleotides in length, 10 to 28 nucleotides in length, 10 to 27 nucleotides in length, 10 to 26 nucleotides in length, 10 to 25 nucleotides in length, 10 to 24 nucleotides in length, 10 to 23 nucleotides in length, 10 to 22 nucleotides in length, 10 to 21 nucleotides in length, or 10 to 20 nucleotides in length). In some embodiments, the antisense strand is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more nucleotides in length.

[0008] In some embodiments, the antisense strand has at least 70% complementarity to a region within an MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-408. In some embodiments, the region within an MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 5, 17-20, 33, 42, 44, 102, 103, 105, 106, 108, 109, 113, 126, 129, 130, 158, 161, 166, 177, 183, 193, 194, 196, 197, 202, 209, 217, 231, 271, 273, 291, 294, 305, 307, 317, 318, and 362. In some embodiments, the region within the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 5, 17-20, 42, 44, 126, 129, 130, 158, 177, 183, 193, 194, 196, 197, 202, 209, 231, 271, 273, 291, 294, 305, 307, 317, 318, and 362. In some embodiments, the region within the MSH3 transcript has the nucleic acid sequence of any one of SEQ ID NOs: 1-24, 40-84, 100, 118-139, 173-181, 193-205, 220-222, 240-242, 249-308, 319-330, 336-377, 384-386, and 398-408. In some embodiments, the region within the MSH3 transcript has the nucleic acid sequence of any one of SEQ ID NOs: 18-20, 102, 105, 106, 108, 109, 113, 183, 194, and 196. In some embodiments, the region within the MSH3 transcript has the nucleic acid sequence of any one of SEQ ID NOs: 18-20, 183, 194, and 196.

[0009] In some embodiments, the antisense strand has at least 75% complementarity to a region within an MSH3 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-408. For example, the antisense strand can have at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to a region within an MSH3 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-408. In some embodiments, the region within the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 5, 17-20, 33, 42, 44, 102, 103, 105, 106, 108, 109, 113, 126, 129, 130, 158, 161, 166, 177, 183, 193, 194, 196, 197, 202, 209, 217, 231, 271, 273, 291, 294, 305, 307, 317, 318, and 362. In some embodiments, the region within the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 5, 17-20, 42, 44, 126, 129, 130, 158, 177, 183, 193, 194, 196, 197, 202, 209, 231, 271, 273, 291, 294, 305, 307, 317, 318, and 362. In some embodiments, the region within the MSH3 transcript has the nucleic acid sequence of any one of SEQ ID NOs: 1-24, 40-84, 100, 118-139, 173-181, 193-205, 220-222, 240-242, 249-308, 319-330, 336-377, 384-386, and 398-408. In some embodiments, the region within the MSH3 transcript has the nucleic acid sequence of any one of SEQ ID NOs: 18-20, 102, 105, 106, 108, 109, 113, 183, 194, and 196. In some embodiments, the region within the MSH3 transcript has the nucleic acid sequence of any one of SEQ ID NOs: 18-20, 183, 194, and 196.

[0010] In some embodiments, the antisense strand has at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or 30 contiguous nucleotides that are completely complementary to a contiguous polynucleotide segment of equal length within a region of an MSH3 RNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-408. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 5, 17-20, 33, 42, 44, 102, 103, 105, 106, 108, 109, 113, 126, 129, 130, 158, 161, 166, 177, 183, 193, 194, 196, 197, 202, 209, 217, 231, 271, 273, 291, 294, 305, 307, 317, 318, and 362. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 5, 17-20, 42, 44, 126, 129, 130, 158, 177, 183, 193, 194, 196, 197, 202, 209, 231, 271, 273, 291, 294, 305, 307, 317, 318, and 362. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 1-24, 40-84, 100, 118-139, 173-181, 193-205, 220-222, 240-242, 249-308, 319-330, 336-377, 384-386, and 398-408. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 18-20, 102, 105, 106, 108, 109, 113, 183, 194, and 196. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 18-20, 183, 194, and 196.

[0011] In some embodiments, the antisense strand has 10 to 30 contiguous nucleotides (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides) that are completely complementary to a contiguous polynucleotide segment of equal length within a region of an MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-408. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 5, 17-20, 33, 42, 44, 102, 103, 105, 106, 108, 109, 113, 126, 129, 130, 158, 161, 166, 177, 183, 193, 194, 196, 197, 202, 209, 217, 231, 271, 273, 291, 294, 305, 307, 317, 318, and 362. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 5, 17-20, 42, 44, 126, 129, 130, 158, 177, 183, 193, 194, 196, 197, 202, 209, 231, 271, 273, 291, 294, 305, 307, 317, 318, and 362. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 1-24, 40-84, 100, 118-139, 173-181, 193-205, 220-222, 240-242, 249-308, 319-330, 336-377, 384-386, and 398-408. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 18-20, 102, 105, 106, 108, 109, 113, 183, 194, and 196. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 18-20, 183, 194, and 196.

[0012] In some embodiments, the antisense strand has 12 to 30 contiguous nucleotides (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides) that are completely complementary to a contiguous polynucleotide segment of equal length within a region of an MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-408. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 5, 17-20, 33, 42, 44, 102, 103, 105, 106, 108, 109, 113, 126, 129, 130, 158, 161, 166, 177, 183, 193, 194, 196, 197, 202, 209, 217, 231, 271, 273, 291, 294, 305, 307, 317, 318, and 362. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 5, 17-20, 42, 44, 126, 129, 130, 158, 177, 183, 193, 194, 196, 197, 202, 209, 231, 271, 273, 291, 294, 305, 307, 317, 318, and 362. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 1-24, 40-84, 100, 118-139, 173-181, 193-205, 220-222, 240-242, 249-308, 319-330, 336-377, 384-386, and 398-408. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 18-20, 102, 105, 106, 108, 109, 113, 183, 194, and 196. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 18-20, 183, 194, and 196.

[0013] In some embodiments, the antisense strand has 15 to 30 contiguous nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides) that are completely complementary to a contiguous polynucleotide segment of equal length within a region of an MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-408. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 5, 17-20, 33, 42, 44, 102, 103, 105, 106, 108, 109, 113, 126, 129, 130, 158, 161, 166, 177, 183, 193, 194, 196, 197, 202, 209, 217, 231, 271, 273, 291, 294, 305, 307, 317, 318, and 362. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 5, 17-20, 42, 44, 126, 129, 130, 158, 177, 183, 193, 194, 196, 197, 202, 209, 231, 271, 273, 291, 294, 305, 307, 317, 318, and 362. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 1-24, 40-84, 100, 118-139, 173-181, 193-205, 220-222, 240-242, 249-308, 319-330, 336-377, 384-386, and 398-408. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 18-20, 102, 105, 106, 108, 109, 113, 183, 194, and 196. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 18-20, 183, 194, and 196.

[0014] In some embodiments, the antisense strand has 18 to 30 contiguous nucleotides (e.g., 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous nucleotides) that are completely complementary to a contiguous polynucleotide segment of equal length within a region of an MSH3 RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-408. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 5, 17-20, 33, 42, 44, 102, 103, 105, 106, 108, 109, 113, 126, 129, 130, 158, 161, 166, 177, 183, 193, 194, 196, 197, 202, 209, 217, 231, 271, 273, 291, 294, 305, 307, 317, 318, and 362. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 5, 17-20, 42, 44, 126, 129, 130, 158, 177, 183, 193, 194, 196, 197, 202, 209, 231, 271, 273, 291, 294, 305, 307, 317, 318, and 362. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 1-24, 40-84, 100, 118-139, 173-181, 193-205, 220-222, 240-242, 249-308, 319-330, 336-377, 384-386, and 398-408. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 18-20, 102, 105, 106, 108, 109, 113, 183, 194, and 196. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 18-20, 183, 194, and 196.

[0015] In some embodiments, the antisense strand has 18 to 25 contiguous nucleotides (e.g., 18, 19, 20, 21, 22, 23, 24, or 25 contiguous nucleotides) that are completely complementary to a contiguous polynucleotide segment of equal length within a region of an MSH3 RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-408. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 5, 17-20, 33, 42, 44, 102, 103, 105, 106, 108, 109, 113, 126, 129, 130, 158, 161, 166, 177, 183, 193, 194, 196, 197, 202, 209, 217, 231, 271, 273, 291, 294, 305, 307, 317, 318, and 362. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 5, 17-20, 42, 44, 126, 129, 130, 158, 177, 183, 193, 194, 196, 197, 202, 209, 231, 271, 273, 291, 294, 305, 307, 317, 318, and 362. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 1-24, 40-84, 100, 118-139, 173-181, 193-205, 220-222, 240-242, 249-308, 319-330, 336-377, 384-386, and 398-408. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 18-20, 102, 105, 106, 108, 109, 113, 183, 194, and 196. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 18-20, 183, 194, and 196.

[0016] In some embodiments, the antisense strand has 18-21 contiguous nucleotides (e.g., 18, 19, 20, or 21 contiguous nucleotides) that are completely complementary to a contiguous polynucleotide segment of equal length within a region of an MSH3 RNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-408. In some embodiments, the region of an MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 5, 17-20, 33, 42, 44, 102, 103, 105, 106, 108, 109, 113, 126, 129, 130, 158, 161, 166, 177, 183, 193, 194, 196, 197, 202, 209, 217, 231, 271, 273, 291, 294, 305, 307, 317, 318, and 362. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 5, 17-20, 42, 44, 126, 129, 130, 158, 177, 183, 193, 194, 196, 197, 202, 209, 231, 271, 273, 291, 294, 305, 307, 317, 318, and 362. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 1-24, 40-84, 100, 118-139, 173-181, 193-205, 220-222, 240-242, 249-308, 319-330, 336-377, 384-386, and 398-408. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 18-20, 102, 105, 106, 108, 109, 113, 183, 194, and 196. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 18-20, 183, 194, and 196.

[0017] In some embodiments, the antisense strand has 21 contiguous nucleotides that are fully complementary to a contiguous polynucleotide segment of equal length within a region of an MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-408. In some embodiments, the region of an MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 5, 17-20, 33, 42, 44, 102, 103, 105, 106, 108, 109, 113, 126, 129, 130, 158, 161, 166, 177, 183, 193, 194, 196, 197, 202, 209, 217, 231, 271, 273, 291, 294, 305, 307, 317, 318, and 362. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 5, 17-20, 42, 44, 126, 129, 130, 158, 177, 183, 193, 194, 196, 197, 202, 209, 231, 271, 273, 291, 294, 305, 307, 317, 318, and 362. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 1-24, 40-84, 100, 118-139, 173-181, 193-205, 220-222, 240-242, 249-308, 319-330, 336-377, 384-386, and 398-408. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 18-20, 102, 105, 106, 108, 109, 113, 183, 194, and 196. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 18-20, 183, 194, and 196.

[0018] In some embodiments, the antisense strand has 9 or fewer nucleotide mismatches to a region of 21 contiguous nucleobases of an MSH3 RNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-408, and optionally the antisense strand contains 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or only 1 mismatch to a region of an MSH3 RNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-408. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 5, 17-20, 33, 42, 44, 102, 103, 105, 106, 108, 109, 113, 126, 129, 130, 158, 161, 166, 177, 183, 193, 194, 196, 197, 202, 209, 217, 231, 271, 273, 291, 294, 305, 307, 317, 318, and 362. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 5, 17-20, 42, 44, 126, 129, 130, 158, 177, 183, 193, 194, 196, 197, 202, 209, 231, 271, 273, 291, 294, 305, 307, 317, 318, and 362. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 1-24, 40-84, 100, 118-139, 173-181, 193-205, 220-222, 240-242, 249-308, 319-330, 336-377, 384-386, and 398-408. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 18-20, 102, 105, 106, 108, 109, 113, 183, 194, and 196. In some embodiments, the region of the MSH3 mRNA transcript has the nucleic acid sequence of any one of SEQ ID NOs: 18-20, 183, 194, and 196.

[0019] In some embodiments, the antisense strand has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to any one of the nucleic acid sequences of SEQ ID NOs: 817-1224. In some embodiments, the antisense strand has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to any one of the nucleic acid sequences of SEQ ID NOs: 821, 833-836, 849, 858, 860, 918, 919, 921, 922, 924, 925, 929, 942, 945, 946, 974, 977, 982, 993, 999, 1009, 1010, 1012, 1013, 1018, 1025, 1033, 1047, 1050, 1060, 1070, 1080, 1090, 2000, 2010, 2012, 2013, 2018, 2025, 2033, 2047, 2050, 2060, 2070, 2080, 2090, 3000, 3014, 3016, 087, 1089, 1107, 1110, 1121, 1123, 1133, and 1134. In some embodiments, the antisense strand has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to any one of the nucleic acid sequences of SEQ ID NOs: 821, 833-836, 858, 860, 942, 945, 946, 974, 993, 999, 1009, 1010, 1012, 1013, 1018, 1025, 1047, 1087, 1089, 1107, 1110, 1121, 1123, 1133, and 1134. In some embodiments, the antisense strand has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to any one of the nucleic acid sequences of SEQ ID NOs: 817-840, 856-900, 916, 934-955, 989-997, 1009-1021, 1036-1038, 1056-1058, 1065-1124, 1135-1146, 1152-1193, 1200-1202, and 1214-1224.In some embodiments, the antisense strand has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 834-836, 918, 921, 922, 924, 925, 999, 1010, and 1012. In some embodiments, the antisense strand has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 834-836, 999, 1010, and 1012.

[0020] In some embodiments, the antisense strand has a nucleic acid sequence that is at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 817-1224. In some embodiments, the antisense strand has a nucleic acid sequence that is at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to any one of the nucleic acid sequences set forth in SEQ ID NOs: 821, 833-836, 849, 858, 860, 918, 919, 921, 922, 924, 925, 929, 942, 945, 946, 974, 977, 982, 993, 999, 1009, 1010, 1012, 1013, 1018, 1025, 1033, 1047, 1087, 1089, 1107, 1110, 1121, 1123, 1133, and 1134. In some embodiments, the antisense strand has a nucleic acid sequence that is at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to any one of the nucleic acid sequences set forth in SEQ ID NOs: 821, 833-836, 858, 860, 942, 945, 946, 974, 993, 999, 1009, 1010, 1012, 1013, 1018, 1025, 1047, 1087, 1089, 1107, 1110, 1121, 1123, 1133, and 1134. In some embodiments, the antisense strand has a nucleic acid sequence that is at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to any one of the nucleic acid sequences of SEQ ID NOs: 817-840, 856-900, 916, 934-955, 989-997, 1009-1021, 1036-1038, 1056-1058, 1065-1124, 1135-1146, 1152-1193, 1200-1202, and 1214-1224.In some embodiments, the antisense strand has a nucleic acid sequence that is at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to any one of the nucleic acid sequences of SEQ ID NOs: 834-836, 918, 921, 922, 924, 925, 999, 1010, and 1012. In some embodiments, the antisense strand has a nucleic acid sequence that is at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to any one of the nucleic acid sequences of SEQ ID NOs: 834-836, 999, 1010, and 1012.

[0021] In some embodiments, the antisense strand has a nucleic acid sequence that is at least 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 817-1224, and optionally the antisense strand has a nucleic acid sequence that is at least 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of any one of SEQ ID NOs: 817-1224. In some embodiments, the antisense strand has a nucleic acid sequence that is at least 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical) to any one of the nucleic acid sequences set forth in SEQ ID NOs: 821, 833-836, 849, 858, 860, 918, 919, 921, 922, 924, 925, 929, 942, 945, 946, 974, 977, 982, 993, 999, 1009, 1010, 1012, 1013, 1018, 1025, 1033, 1047, 1087, 1089, 1107, 1110, 1121, 1123, 1133, and 1134. In some embodiments, the antisense strand has a nucleic acid sequence that is at least 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical) to any one of the nucleic acid sequences set forth in SEQ ID NOs: 821, 833-836, 858, 860, 942, 945, 946, 974, 993, 999, 1009, 1010, 1012, 1013, 1018, 1025, 1047, 1087, 1089, 1107, 1110, 1121, 1123, 1133, and 1134. In some embodiments, the antisense strand has a nucleic acid sequence that is at least 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical) to any one of the nucleic acid sequences of SEQ ID NOs: 817-840, 856-900, 916, 934-955, 989-997, 1009-1021, 1036-1038, 1056-1058, 1065-1124, 1135-1146, 1152-1193, 1200-1202, and 1214-1224. In some embodiments, the antisense strand has a nucleic acid sequence that is at least 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical) to any one of the nucleic acid sequences of SEQ ID NOs: 834-836, 918, 921, 922, 924, 925, 999, 1010, and 1012.In some embodiments, the antisense strand has a nucleic acid sequence that is at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 834-836, 999, 1010, and 1012.

[0022] In some embodiments, the antisense strand has the nucleic acid sequence of any one of SEQ ID NOs: 817-1224. In some embodiments, the antisense strand has the nucleic acid sequence of any one of SEQ ID NOs: 821, 833-836, 849, 858, 860, 918, 919, 921, 922, 924, 925, 929, 942, 945, 946, 974, 977, 982, 993, 999, 1009, 1010, 1012, 1013, 1018, 1025, 1033, 1047, 1087, 1089, 1107, 1110, 1121, 1123, 1133, and 1134. In some embodiments, the antisense strand has the nucleic acid sequence of any one of SEQ ID NOs: 821, 833-836, 858, 860, 942, 945, 946, 974, 993, 999, 1009, 1010, 1012, 1013, 1018, 1025, 1047, 1087, 1089, 1107, 1110, 1121, 1123, 1133, and 1134. In some embodiments, the antisense strand has any one of the nucleic acid sequences of SEQ ID NOs: 817-840, 856-900, 916, 934-955, 989-997, 1009-1021, 1036-1038, 1056-1058, 1065-1124, 1135-1146, 1152-1193, 1200-1202, and 1214-1224. In some embodiments, the antisense strand has any one of the nucleic acid sequences of SEQ ID NOs: 834-836, 918, 921, 922, 924, 925, 999, 1010, and 1012. In some embodiments, the antisense strand has any one of the nucleic acid sequences of SEQ ID NOs: 834-836, 999, 1010, and 1012.

[0023] In some embodiments, the sense strand has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 409-816. In some embodiments, the sense strand has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to any one of the nucleic acid sequences of SEQ ID NOs: 413, 425-428, 441, 450, 452, 510, 511, 513, 514, 516, 517, 521, 534, 537, 538, 566, 569, 574, 585, 591, 601, 602, 604, 605, 610, 617, 625, 639, 679, 681, 699, 702, 713, 715, 725, 726, and 770. In some embodiments, the sense strand has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to any one of the nucleic acid sequences of SEQ ID NOs: 413, 425-428, 450, 452, 534, 537, 538, 566, 585, 591, 601, 602, 604, 605, 610, 617, 639, 679, 681, 699, 702, 713, 715, 725, 726, and 770. In some embodiments, the sense strand has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to any one of the nucleic acid sequences of SEQ ID NOs: 409-432, 448-492, 508, 526-547, 581-589, 601-613, 628-630, 648-650, 657-716, 727-738, 744-785, 792-794, and 806-816.In some embodiments, the sense strand has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 426-428, 510, 513, 514, 516, 517, 521, 591, 602, and 604. In some embodiments, the sense strand has a nucleic acid sequence that is at least 85% identical (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 426-428, 591, 602, and 604.

[0024] In some embodiments, the sense strand has a nucleic acid sequence that is at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 409-816. In some embodiments, the sense strand has a nucleic acid sequence that is at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to any one of the nucleic acid sequences set forth in SEQ ID NOs: 413, 425-428, 441, 450, 452, 510, 511, 513, 514, 516, 517, 521, 534, 537, 538, 566, 569, 574, 585, 591, 601, 602, 604, 605, 610, 617, 625, 639, 679, 681, 699, 702, 713, 715, 725, 726, and 770. In some embodiments, the sense strand has a nucleic acid sequence that is at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to any one of the nucleic acid sequences of SEQ ID NOs: 413, 425-428, 450, 452, 534, 537, 538, 566, 585, 591, 601, 602, 604, 605, 610, 617, 639, 679, 681, 699, 702, 713, 715, 725, 726, and 770. In some embodiments, the sense strand has a nucleic acid sequence that is at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to any one of the nucleic acid sequences of SEQ ID NOs: 409-432, 448-492, 508, 526-547, 581-589, 601-613, 628-630, 648-650, 657-716, 727-738, 744-785, 792-794, and 806-816. In some embodiments, the sense strand has a nucleic acid sequence that is at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 426-428, 510, 513, 514, 516, 517, 521, 591, 602, and 604.In some embodiments, the sense strand has a nucleic acid sequence that is at least 90% identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 426-428, 591, 602, and 604.

[0025] In some embodiments, the sense strand has a nucleic acid sequence that is at least 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 409-816, and optionally the sense strand has a nucleic acid sequence that is at least 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of any one of SEQ ID NOs: 409-816. In some embodiments, the sense strand has a nucleic acid sequence that is at least 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical) to any one of the nucleic acid sequences set forth in SEQ ID NOs: 413, 425-428, 441, 450, 452, 510, 511, 513, 514, 516, 517, 521, 534, 537, 538, 566, 569, 574, 585, 591, 601, 602, 604, 605, 610, 617, 625, 639, 679, 681, 699, 702, 713, 715, 725, 726, and 770. In some embodiments, the sense strand has a nucleic acid sequence that is at least 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical) to any one of the nucleic acid sequences set forth in SEQ ID NOs: 413, 425-428, 450, 452, 534, 537, 538, 566, 585, 591, 601, 602, 604, 605, 610, 617, 639, 679, 681, 699, 702, 713, 715, 725, 726, and 770. In some embodiments, the sense strand has a nucleic acid sequence that is at least 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical) to any one of the nucleic acid sequences of SEQ ID NOs: 409-432, 448-492, 508, 526-547, 581-589, 601-613, 628-630, 648-650, 657-716, 727-738, 744-785, 792-794, and 806-816. In some embodiments, the sense strand has a nucleic acid sequence that is at least 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical) to any one of the nucleic acid sequences set forth in SEQ ID NOs: 426-428, 510, 513, 514, 516, 517, 521, 591, 602, and 604.In some embodiments, the sense strand has a nucleic acid sequence that is at least 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, or 100% identical) to any one of the nucleic acid sequences of SEQ ID NOs: 426-428, 591, 602, and 604.

[0026] In some embodiments, the siRNA molecule has a sense strand having the nucleic acid sequence of any one of SEQ ID NOs: 409-816. In some embodiments, the sense strand has the nucleic acid sequence of any one of SEQ ID NOs: 413, 425-428, 441, 450, 452, 510, 511, 513, 514, 516, 517, 521, 534, 537, 538, 566, 569, 574, 585, 591, 601, 602, 604, 605, 610, 617, 625, 639, 679, 681, 699, 702, 713, 715, 725, 726, and 770. In some embodiments, the sense strand has a nucleic acid sequence of any one of SEQ ID NOs: 413, 425-428, 450, 452, 534, 537, 538, 566, 585, 591, 601, 602, 604, 605, 610, 617, 639, 679, 681, 699, 702, 713, 715, 725, 726, and 770. In some embodiments, the siRNA molecule has a sense strand having a nucleic acid sequence of any one of SEQ ID NOs: 409-432, 448-492, 508, 526-547, 581-589, 601-613, 628-630, 648-650, 657-716, 727-738, 744-785, 792-794, and 806-816. In some embodiments, the sense strand has the nucleic acid sequence of any one of SEQ ID NOs: 426-428, 510, 513, 514, 516, 517, 521, 591, 602, and 604. In some embodiments, the sense strand has the nucleic acid sequence of any one of SEQ ID NOs: 426-428, 591, 602, and 604.

[0027] In some embodiments, the antisense strand has a structure represented by Formula I, which, in the 5' to 3' direction, is: AB-(A') j -CP 2 -DP1 -(C'-P 1 ) k -C' Formula I; where A is of the formula CP 1 -DP 1 Represented by; Each A' has the formula CP 2 -DP 2 Represented by; B is the formula CP 2 -DP 2 -DP 2 -DP 2 Represented by; each C is a 2'-O-methyl (2'-O-Me) ribonucleoside; each C' is independently a 2'-O-Me ribonucleoside or a 2'-fluoro (2'-F) ribonucleoside; each D is a 2'-F ribonucleoside; Each P 1 is a phosphorothioate internucleoside linkage; Each P 2 is a phosphodiester internucleoside linkage; j is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); k is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7).

[0028] In some embodiments, the antisense strand has a structure represented by Formula A1, which, in the 5' to 3' direction, is: ASBSAOBOBOBOAOBOAOBOA-OBOAOBOAOBSASASASBSA Formula A1; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0029] In some embodiments, the antisense strand has a structure represented by Formula II, which, in the 5' to 3' direction, is: AB-(A') j -CP 2 -DP 1 -(CP 1 ) k -C' Formula II; where A is of the formula CP 1 -DP 1 Represented by; Each A' has the formula CP 2 -DP 2 Represented by; B is the formula CP 2 -DP 2 -DP 2 -DP 2 Represented by; each C is a 2'-O-methyl (2'-O-Me) ribonucleoside; each C' is independently a 2'-O-Me ribonucleoside or a 2'-fluoro (2'-F) ribonucleoside; each D is a 2'-F ribonucleoside; Each P 1 is a phosphorothioate internucleoside linkage; Each P 2 is a phosphodiester internucleoside linkage; j is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); k is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7).

[0030] In some embodiments, the antisense strand has a structure represented by Formula A2, which, in the 5' to 3' direction, is: ASBSAOBOBOBOAOBOAOBOA-OBOAAOBOAOBSASASASASA Formula A2; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0031] In some embodiments, the sense strand has a structure represented by Formula III, which, in the 5' to 3' direction, is: E-(A') m -F Formula III; In the formula, E is a compound represented by the formula (CP 1 ) 2 Represented by; F is the formula (CP 2 ) 3 -DP 1 -CP 1 -C, (CP 2 ) 3 -DP 2 -CP 2 -C, (CP 2 ) 3 -DP 1 -CP 1 -D, or (CP 2 ) 3 -DP 2 -CP 2 - represented by D; A', C, D, P 1 , and P 2 is as defined in formula II; m is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7).

[0032] In some embodiments, the sense strand has a structure represented by formula S1, which, in the 5' to 3' direction, is: ASASAOBOAOBOAOBOAOBOA-OAOAOBBSASA Formula S1; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0033] In some embodiments, the sense strand has a structure represented by formula S2, which, in the 5' to 3' direction, is: ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOA Formula S2; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0034] In some embodiments, the sense strand has a structure represented by formula S3, which, in the 5' to 3' direction, is: ASASAOBOAOBOAOBOAOBOA-OAOAOBSASB formula S3; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0035] In some embodiments, the sense strand has a structure represented by formula S4, which, in the 5' to 3' direction, is: ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOB Equation S4; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0036] In some embodiments, the antisense strand has a structure represented by Formula IV, which, in the 5' to 3' direction, is: A-(A') j -CP2 -B-(CP 1 ) k -C' Formula IV; where A is of the formula CP 1 -DP 1 Represented by; Each A' has the formula CP 2 -DP 2 Represented by; B is the formula DP 1 -CP 1 -DP 1 Represented by; each C is a 2'-O-Me ribonucleoside; each C' is independently a 2'-O-Me ribonucleoside or a 2'-F ribonucleoside; each D is a 2'-F ribonucleoside; Each P 1 is a phosphorothioate internucleoside linkage; Each P 2 is a phosphodiester internucleoside linkage; j is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); k is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7).

[0037] In some embodiments, the antisense strand has a structure represented by Formula A3, which, in the 5' to 3' direction, is: ASBSAOBOAOBOAOBOAOBOA-OBOAOBOAOBSASASASA formula A3; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0038] In some embodiments, the sense strand has a structure represented by Formula V, where V is, in the 5' to 3' direction, E-(A')m -CP 2 -F formula V; In the formula, E is a compound represented by the formula (CP 1 ) 2 Represented by; F is the formula DP 1 -CP 1 -C, D.P. 2 -CP 2 -C, D.P. 1 -CP 1 -D, or DP 2 -CP 2 - represented by D; A', C, D, P 1 , and P 2 is as defined in formula IV; m is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7).

[0039] In some embodiments, the sense strand has a structure represented by formula S5, which, in the 5' to 3' direction, is: ASASAOBOAOBOAOBOAOBOA-OBOAOBSASA Formula S5; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0040] In some embodiments, the sense strand has a structure represented by formula S6, which, in the 5' to 3' direction, is: ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOA Formula S6; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0041] In some embodiments, the sense strand has a structure represented by formula S7, which, in the 5' to 3' direction, is: ASASAOBOAOBOAOBOAOBOA-OBOAOBSASB Formula S7; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0042] In some embodiments, the sense strand has a structure represented by formula S8, which, in the 5' to 3' direction, is: ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOB formula S8; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0043] In some embodiments, the antisense strand has a structure represented by Formula VI, which, in the 5' to 3' direction, is: A.B. j -EB k -EFG l -DP 1 -C' Formula VI; where A is of the formula CP 1 -DP 1 Represented by; Each B is a function of the formula CP 2 Represented by; each C is a 2'-O-Me ribonucleoside; each C' is independently a 2'-O-Me ribonucleoside or a 2'-F ribonucleoside; each D is a 2'-F ribonucleoside; Each E is a function of the formula DP 2 -CP2 Represented by; F is the formula DP 1 -CP 1 Represented by; Each G has the formula CP 1 Represented by; Each P 1 is a phosphorothioate internucleoside linkage; Each P 2 is a phosphodiester internucleoside linkage; j is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); k is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); l is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7).

[0044] In some embodiments, the antisense strand has a structure represented by Formula A4, which, in the 5' to 3' direction, is: ASBSAOAOAOBOAOAOAOAOA-OAOAOBOAOBSASASASBSA formula A4; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0045] In some embodiments, the sense strand has a structure represented by Formula VII, which, in the 5' to 3' direction, is: HB m -I n -A'-B o -HC Formula VII; where A' is a compound of the formula CP 2 -DP 2 Represented by; Each H is a function of the formula (CP 1 ) 2 Represented by; Each I is independently represented by the formula (DP 2) is represented as; B, C, D, P 1 , and P 2 is as defined in formula VI; m is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); n is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); o is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7).

[0046] In some embodiments, the sense strand has a structure represented by formula S9, which, in the 5' to 3' direction, is: ASASAOAOAOBOBOBOAOBOA-OAOAOAASASA Formula S9; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0047] In some embodiments, the antisense strand also has a 5' phosphorus stabilizing moiety at the 5' end of the antisense strand.

[0048] In some embodiments, the sense strand also has a 5' phosphorus stabilizing moiety at the 5' end of the sense strand.

[0049] In some embodiments, each 5' phosphorus stabilizing moiety is independently represented by any one of formulas IX, X, XI, XII, XIII, XIV, XV, or XVI.

[0050] [ka] wherein Nuc represents a nucleobase selected from the group consisting of adenine, uracil, guanine, thymine, and cytosine, and R represents optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, phenyl, benzyl, or hydrogen.

[0051] In some embodiments, the nucleobase is adenine, uracil, guanine, thymine, or cytosine.

[0052] In some embodiments, the 5' phosphorus stabilizing moiety is an (E)-vinyl phosphonate of formula XI.

[0053] In some embodiments, the siRNA molecule also has a hydrophobic portion at the 5' or 3' end of the siRNA molecule.

[0054] In some embodiments, the hydrophobic moiety is selected from the group consisting of cholesterol, vitamin D, or tocopherol.

[0055] In some embodiments, the length of the sense strand is between 12 and 30 nucleotides (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides).

[0056] In some embodiments, the siRNA molecule is a branched siRNA molecule.

[0057] In some embodiments, the branched siRNA molecule is biantennary, triantennary, or tetraantennary.

[0058] In some embodiments, the siRNA molecule is biantennary, and optionally the biantennary siRNA molecule is represented by any one of Formulas XVII, XVIII, or XIX: [ka] wherein each RNA is independently a siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

[0059] In some embodiments, the siRNA molecule is tri-antennary, and optionally the tri-antennary siRNA molecule is represented by any one of Formulas XX, XXI, XXII, or XXIII: [ka] wherein each RNA is independently a siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

[0060] In some embodiments, the siRNA molecule is tetraantennary, and optionally the tetraantennary siRNA molecule is represented by any one of Formulas XXIV, XXV, XXVI, XXVII, or XXVIII: [ka] wherein each RNA is independently a siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

[0061] In some embodiments, the linker is selected from the group consisting of one or more consecutive subunits of ethylene glycol, alkyl, carbohydrate, block copolymer, peptide, RNA, and DNA.

[0062] In some embodiments, the one or more consecutive subunits are between 2 and 20 consecutive subunits (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive subunits).

[0063] In some embodiments of any of the siRNA molecules described herein, the antisense strand has sufficient complementarity to hybridize to a region in the MSH3 mRNA transcript that is within the open reading frame of the MSH3 mRNA transcript, hi some embodiments, the antisense strand has sufficient complementarity to hybridize to a region in the MSH3 mRNA transcript that is within the 3' untranslated region.

[0064] In some embodiments of any of the siRNA molecules described herein, the antisense strand has sufficient complementarity to hybridize to a region within exon 1 of the MSH3 mRNA transcript. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 1-4.

[0065] In some embodiments of any of the siRNA molecules described herein, the antisense strand has sufficient complementarity to hybridize to a region within exon 2 of the MSH3 mRNA transcript. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 5-20. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 5-15.

[0066] In some embodiments of any of the siRNA molecules described herein, the antisense strand has sufficient complementarity to hybridize to a region within exon 3 of the MSH3 mRNA transcript. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 16-47. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 21-47.

[0067] In some embodiments of any of the siRNA molecules described herein, the antisense strand has sufficient complementarity to hybridize to a region within exon 4 of the MSH3 mRNA transcript. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 48-84.

[0068] In some embodiments of any of the siRNA molecules described herein, the antisense strand has sufficient complementarity to hybridize to a region within exon 5 of the MSH3 mRNA transcript. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 85-103. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 85-100.

[0069] In some embodiments of any of the siRNA molecules described herein, the antisense strand has sufficient complementarity to hybridize to a region within exon 6 of the MSH3 mRNA transcript. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 101-120. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 104-120.

[0070] In some embodiments of any of the siRNA molecules described herein, the antisense strand has sufficient complementarity to hybridize to a region within exon 7 of the MSH3 mRNA transcript. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 121-133.

[0071] In some embodiments of any of the siRNA molecules described herein, the antisense strand has sufficient complementarity to hybridize to a region within exon 8 of the MSH3 mRNA transcript. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 134-139.

[0072] In some embodiments of any of the siRNA molecules described herein, the antisense strand has sufficient complementarity to hybridize to a region within exon 9 of an MSH3 mRNA transcript. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within an MSH3 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 140-162. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within an MSH3 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 140-154.

[0073] In some embodiments of any of the siRNA molecules described herein, the antisense strand has sufficient complementarity to hybridize to a region within exon 10 of the MSH3 mRNA transcript. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 155-187. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 163-182.

[0074] In some embodiments of any of the siRNA molecules described herein, the antisense strand has sufficient complementarity to hybridize to a region within exon 11 of the MSH3 mRNA transcript. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 183-200. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 188-192.

[0075] In some embodiments of any of the siRNA molecules described herein, the antisense strand has sufficient complementarity to hybridize to a region within exon 12 of the MSH3 mRNA transcript. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 193-205.

[0076] In some embodiments of any of the siRNA molecules described herein, the antisense strand has sufficient complementarity to hybridize to a region within exon 13 of the MSH3 mRNA transcript. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 206-223. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 206-222.

[0077] In some embodiments of any of the siRNA molecules described herein, the antisense strand has sufficient complementarity to hybridize to a region within exon 14 of the MSH3 mRNA transcript. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 223-242. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 224-242.

[0078] In some embodiments of any of the siRNA molecules described herein, the antisense strand has sufficient complementarity to hybridize to a region within exon 15 of the MSH3 mRNA transcript. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 243-262. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 243-255.

[0079] In some embodiments of any of the siRNA molecules described herein, the antisense strand has sufficient complementarity to hybridize to a region within exon 16 of the MSH3 mRNA transcript. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 256-276. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 263-268.

[0080] In some embodiments of any of the siRNA molecules described herein, the antisense strand has sufficient complementarity to hybridize to a region within exon 17 of the MSH3 mRNA transcript. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 269-296. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 277-289.

[0081] In some embodiments of any of the siRNA molecules described herein, the antisense strand has sufficient complementarity to hybridize to a region within exon 18 of an MSH3 mRNA transcript. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within an MSH3 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 290-302. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within an MSH3 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 297-302.

[0082] In some embodiments of any of the siRNA molecules described herein, the antisense strand has sufficient complementarity to hybridize to a region within exon 19 of the MSH3 mRNA transcript. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 303-304.

[0083] In some embodiments of any of the siRNA molecules described herein, the antisense strand has sufficient complementarity to hybridize to a region within exon 20 of the MSH3 mRNA transcript. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 303-324. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 305-318.

[0084] In some embodiments of any of the siRNA molecules described herein, the antisense strand has sufficient complementarity to hybridize to a region within exon 21 of the MSH3 mRNA transcript. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 319-339. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 325-336.

[0085] In some embodiments of any of the siRNA molecules described herein, the antisense strand has sufficient complementarity to hybridize to a region within exon 22 of the MSH3 mRNA transcript. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 337-344. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 340-344.

[0086] In some embodiments of any of the siRNA molecules described herein, the antisense strand has sufficient complementarity to hybridize to a region within exon 23 of the MSH3 mRNA transcript. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 345-358.

[0087] In some embodiments of any of the siRNA molecules described herein, the antisense strand has sufficient complementarity to hybridize to a region within exon 24 of the MSH3 mRNA transcript. In some embodiments, the antisense strand has sufficient complementarity to hybridize to a region within the MSH3 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 359-408.

[0088] In a further aspect, the present disclosure provides a pharmaceutical composition comprising an siRNA molecule of any of the preceding aspects or embodiments of the present disclosure and a pharma- ceutically acceptable excipient, carrier, or diluent.

[0089] In a further aspect, the present disclosure provides a method for delivering siRNA molecules to a subject diagnosed with microsatellite repeat expansion disease by administering a therapeutically effective amount of the siRNA molecule or pharmaceutical composition of any of the above aspects or embodiments of the present disclosure to the subject.In some embodiments, the microsatellite repeat expansion disease is Huntington's disease.In some embodiments, the microsatellite repeat expansion disease is spinocerebellar ataxia.In some embodiments, the microsatellite repeat expansion disease is fragile X syndrome.In some embodiments, the microsatellite repeat expansion disease is myotonic dystrophy.

[0090] In a further aspect, the present disclosure provides a method of treating a microsatellite repeat expansion disease in a subject in need thereof by administering to the subject a therapeutically effective amount of the siRNA molecule or pharmaceutical composition of any of the above aspects or embodiments of the present disclosure. In some embodiments, the microsatellite repeat expansion disease is Huntington's disease.

[0091] In another aspect, the present disclosure provides a method of reducing MSH3 expression in a subject in need thereof by administering a therapeutically effective amount of a siRNA or pharmaceutical composition of any of the above aspects or embodiments of the present disclosure.

[0092] In some embodiments, the siRNA molecule or pharmaceutical composition is administered to the subject by intracerebroventricular, intrastriatal, intraparenchymal or intrathecal injection.In some embodiments, the siRNA molecule or pharmaceutical composition is administered to the subject by intravenous, intramuscular or subcutaneous injection.

[0093] In some embodiments, the subject is a human.

[0094] In another aspect, the present disclosure provides a kit comprising an siRNA molecule or pharmaceutical composition of any of the above aspects or embodiments of the present disclosure and a package insert instructing a user of the kit to perform a method of any of the above aspects or embodiments of the present disclosure. [Brief description of the drawings]

[0095] [Figure 1] 1 is a graph showing the reduction of MSH3 protein in FVB mice administered siRNA molecules of the present disclosure. siRNA identifiers are shown in Table 6 below. Protein was quantified in the motor cortex 28 days after di-siRNA treatment at a dose level of 1 nmol or 5 nmol. [Diagram 2]Figure 1 is a graph showing the reduction of MSH3 protein in FVB mice administered siRNA molecules of the present disclosure. The siRNA identifiers are shown in Table 6 below. Protein was quantified in the striatum 28 days after di-siRNA treatment at a dose level of 1 nmol or 5 nmol. [Diagram 3] Figure 1 is a graph showing the reduction of MSH3 protein in FVB mice administered siRNA molecules of the present disclosure. The siRNA identifiers are shown in Table 6 below. Protein was quantified in the hippocampus 28 days after di-siRNA treatment at a dose level of 1 nmol or 5 nmol. [Figure 4] 1 is a graph showing the reduction of MSH3 mRNA in FVB mice administered siRNA molecules of the present disclosure. siRNA identifiers are shown in Table 6 below. mRNA was quantified in the motor cortex 28 days after di-siRNA treatment at dose levels of 1 nmol or 5 nmol. [Diagram 5] 1 is a graph showing the reduction of MSH3 mRNA in FVB mice administered siRNA molecules of the present disclosure. siRNA identifiers are shown in Table 6 below. mRNA was quantified in the striatum 28 days after di-siRNA treatment at dose levels of 1 nmol or 5 nmol. [Figure 6] 1 is a graph showing the reduction of MSH3 mRNA in FVB mice administered siRNA molecules of the present disclosure. siRNA identifiers are shown in Table 6 below. mRNA was quantified in the hippocampus 28 days after di-siRNA treatment at dose levels of 1 nmol or 5 nmol. [Figure 7] 1 is a graph showing the reduction of MSH3 protein in FVB mice administered siRNA molecules of the present disclosure. siRNA identifiers are shown in Table 6 below. Protein was quantified in the motor cortex, striatum, and hippocampus 28 days after di-siRNA treatment at a dose level of 5 nmol. [Figure 8] 1 is a graph showing the reduction of MSH3 mRNA in FVB mice administered siRNA molecules of the present disclosure. siRNA identifiers are shown in Table 6 below. a mRNA was quantified in the motor cortex, striatum, and hippocampus after 28 days of di-siRNA treatment at a dose level of 5 nmol. [Figure 9]1 is a graph showing the reduction of MSH3 protein in FVB mice administered siRNA molecules of the present disclosure. siRNA identifiers are shown in Table 6 below. Protein was quantified in the motor cortex, striatum, and hippocampus 28 days after di-siRNA treatment at dose levels of 0.5 nmol, 1 nmol, 2.5 nmol, 5 nmol, and 20 nmol. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0096] definition Unless otherwise defined herein, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. Unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. The use of "or" means "and / or" unless otherwise stated. The use of the term "including" as well as other forms such as "include" and "included" are not limiting.

[0097] As used herein, the term "nucleic acid" refers to an RNA or DNA molecule composed of a chain of ribonucleotides or deoxyribonucleotides, respectively.

[0098] As used herein, the term "therapeutic nucleic acid" refers to a nucleic acid molecule (e.g., a ribonucleic acid) that has partial or complete complementarity with, interacts with, and mediates the silencing of expression of a disease-associated target mRNA.

[0099] As used herein, the term "carrier nucleic acid" refers to a nucleic acid molecule (e.g., a ribonucleic acid) that has sequence complementarity with and hybridizes to a therapeutic nucleic acid. As used herein, the term "3' end" refers to the end of a nucleic acid that contains an unmodified hydroxyl group at the 3' carbon of the ribose ring.

[0100] As used herein, the term "nucleoside" refers to a molecule that consists of a heterocyclic base and its sugar.

[0101] As used herein, the term "nucleotide" refers to a nucleoside having a phosphate group or variant thereof at its 3' or 5' sugar hydroxyl group. Examples of phosphate group variants include, but are not limited to, saturated alkyl phosphonates, unsaturated alkenyl phosphonates, phosphorothioates, and phosphoramidites.

[0102] In the context of the present invention, the term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages, as well as oligonucleotides having non-naturally occurring (e.g., modified) portions that function similarly. Such modified or substituted oligonucleotides are often preferred over native forms due to desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid targets, and increased stability in the presence of nucleases.

[0103] As used herein, the term "siRNA" refers to small interfering RNA duplexes that induce the RNA interference (RNAi) pathway. siRNA molecules may be of various lengths (generally 10-30 base pairs) and may contain various degrees of complementarity to their target mRNA. The term "siRNA" includes duplexes of two separate strands, and single strands that optionally form a hairpin structure containing a double-stranded region.

[0104] As used herein, the term "antisense strand" refers to the strand of the siRNA duplex that contains a degree of complementarity to a target gene.

[0105] As used herein, the term "sense strand" refers to the siRNA duplex that contains complementarity to the antisense strand.

[0106] The term "interfering RNA molecule" refers to an RNA molecule, such as a small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), or antisense oligonucleotide (ASO), that inhibits the endogenous function of a target RNA transcript.

[0107] As used herein, the terms "express" and "expression" refer to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' capping, and / or 3' end processing); and (3) translation of the RNA into a polypeptide or protein. In the context of a gene that encodes a protein product, "gene expression" and like terms are used interchangeably with "protein expression" and like terms. Changes in the expression of a gene or protein of interest in a patient can be detected, for example, by changes in the amount or concentration of mRNA encoding the corresponding protein in a sample obtained from the patient (e.g., assessed using RNA detection methods described herein or known in the art, such as quantitative polymerase chain reaction (qPCR) and RNA-seq techniques), changes in the amount or concentration of the corresponding protein (e.g., assessed using protein detection methods described herein or known in the art, such as enzyme-linked immunosorbent assay (ELISA), among others), and / or changes in the activity of the corresponding protein (e.g., in the case of an enzyme, assessed using enzyme activity assays described herein or known in the art). As used herein, a cell is considered to "express" a gene or protein of interest if one or more, or all of the above events are detectable within the cell or in the medium in which the cell resides. For example, a gene or protein of interest is considered to be "expressed" by a cell, or population of cells, if it is possible to detect: (i) production of a corresponding RNA transcript, such as an mRNA template, by the cell, or population of cells (e.g., using the RNA detection procedures described herein); (ii) processing of the RNA transcript (e.g., splicing, editing, 5' capping, and / or 3' end processing, using the RNA detection procedures described herein); (iii) translation of the RNA template into a protein product (e.g., using the protein detection procedures described herein); and / or (iv) post-translational modification of the protein product (e.g., using the protein detection procedures described herein).

[0108] As used herein, the terms "target," "targeting," and "targeted" in the context of siRNA design refer to generating an antisense strand such that it anneals within a region within an mRNA transcript of interest in a manner that reduces translation of the mRNA into a protein product.

[0109] As used herein, the terms "chemically modified nucleotide," "nucleotide analog," "altered nucleotide," and "modified nucleotide" refer to non-standard nucleotides, including non-naturally occurring ribonucleotides or deoxyribonucleotides. Exemplary nucleotide analogs are modified at any position such that certain chemical properties of the nucleotide are altered while retaining the ability of the nucleotide analog to perform its intended function.

[0110] As used herein, the term "metabolically stabilized" refers to an RNA molecule that contains chemically modified ribonucleotides to reduce the metabolic rate of the RNA molecule administered to a subject. Exemplary modifications include 2'-hydroxy to 2'-O-methoxy or 2'-fluoro, and phosphodiester to phosphorothioate.

[0111] As used herein, the term "phosphorothioate" refers to the phosphate group of a nucleotide that is modified by replacing one or more of the oxygens of the phosphate group with sulfur.

[0112] As used herein, "internucleoside linkage," "internucleoside bond," and like terms refer to the bond between nucleosides in a nucleic acid molecule.

[0113] As used herein, the term "antagomir" refers to a nucleic acid that can function as an inhibitor of miRNA activity.

[0114] As used herein, the term "gapmer" refers to a chimeric antisense nucleic acid that contains a central block of deoxynucleotide monomers of sufficient length to induce RNase H cleavage. The deoxynucleotide block is flanked by ribonucleotide monomers or ribonucleotide monomers containing modifications.

[0115] As used herein, the term "mixmer" refers to a nucleic acid composed of a mixture of locked nucleic acid (LNA) and DNA.

[0116] As used herein, the term "guide RNA" refers to a nucleic acid that has sequence complementarity to a specific sequence in a genome immediately upstream or one base pair upstream of a protospacer adjacent motif (PAM) sequence used in the CRISPR / Cas9 gene editing system. Alternatively, "guide RNA" may refer to a nucleic acid that has sequence complementarity (e.g., is antisense) to a specific messenger RNA (mRNA) sequence. In this context, a guide RNA may also have sequence complementarity to a "passenger RNA" sequence of equal or shorter length that is identical or substantially identical to the sequence of the mRNA to which the guide RNA hybridizes.

[0117] As used herein, the term "branched siRNA" refers to a compound that contains two or more double-stranded siRNA molecules that are covalently linked to each other.Branched siRNA molecules can be "bi-branched", also referred to herein as "di-siRNA", where this siRNA molecule comprises two siRNA molecules that are covalently linked to each other, for example, via a linker.Branched siRNA molecules can be "tri-branched", also referred to herein as "tri-siRNA", where this siRNA molecule comprises three siRNA molecules that are covalently linked to each other, for example, via a linker.Branched siRNA molecules can be "four-branched", also referred to herein as "tetra-siRNA", where this siRNA molecule comprises four siRNA molecules that are covalently linked to each other, for example, via a linker.

[0118] As used herein, the term "branch point moiety" refers to the chemical moiety of the branched siRNA structure of the present disclosure, which can be covalently attached to the 5'-end or 3'-end of the antisense or sense strand of siRNA molecule, and can support the attachment of additional single-stranded or double-stranded siRNA molecules.Non-limiting examples of branch point moieties suitable for use with the disclosed methods and compositions include, for example, phosphoramidite, tosylated solketal, 1,3-diaminopropanol, pentaerythritol, and any one of the branch point moieties described in US Patent No. 10,478,503.

[0119] The term "phosphate moiety" as used herein refers to a terminal phosphate group, including phosphate and modified phosphates. The phosphate moiety may be located at either terminus, but is preferably at the 5'-terminal nucleoside. In one aspect, the terminal phosphate has the formula -OP(=O)(OH)OH and is unmodified. In another aspect, the terminal phosphate is modified such that one or more of the O and OH groups are replaced with H, O, S, N(R'), or an alkyl, amino protecting group, or unsubstituted or substituted alkyl where R' is H. In some embodiments, the 5' and or 3' terminal groups can each independently comprise 1-3 phosphate moieties that are unmodified (diphosphate or triphosphate) or modified.

[0120] As used herein, the term "5' phosphorus stabilizing moiety" refers to a terminal phosphate group, including phosphate as well as modified phosphates (e.g., phosphorothioates, phosphodiesters, phosphonates). The phosphate moiety may be located at either terminus, but is preferably at the 5' terminal nucleoside. In one aspect, the terminal phosphate has the formula -OP(=O)(OH)OH and is unmodified. In another aspect, the terminal phosphate is modified such that one or more of the O and OH groups are replaced with H, O, S, N(R'), or an alkyl where R' is H, an amino protecting group, or an unsubstituted or substituted alkyl. In some embodiments, the 5' and or 3' terminal groups can each independently comprise one to three phosphate moieties that are unmodified (diphosphate or triphosphate) or modified.

[0121] The phosphate group of the nucleotide may also be modified, for example, by replacing one or more oxygens of the phosphate group with sulfur (e.g., phosphorothioate) or by other substitutions that allow the nucleotide to perform its intended function, as described, for example, in Eckstein, Antisense Nucleic Acid Drug Dev. 10:117-21, 2000; Rusckowski et al., Antisense Nucleic Acid Drug Dev. 10:333-45, 2000; Stein, Antisense Nucleic Acid Drug Dev. 11:317-25, 2001; Vorobjev et al., Antisense Nucleic Acid Drug Dev. 11:77-85, 2001; and US5,684,143. Certain of the above-referenced modifications (e.g., phosphate group modifications) preferably reduce, for example, the hydrolysis rate of polynucleotides that contain the analog in vivo or in vitro.

[0122] As used herein, the term "complementary" refers to two nucleotides that form a canonical Watson-Crick base pair. For the avoidance of doubt, in the context of this disclosure, Watson-Crick base pairs include adenine-thymine, adenine-uracil, and cytosine-guanine base pairs. In this context, proper Watson-Crick base pairs are referred to as "matches," while the respective unpaired and improperly paired nucleotides are referred to as "mismatches." Alignment for purposes of determining percent nucleic acid sequence complementarity can be accomplished in a variety of ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software.

[0123] "Percent sequence complementarity" to a reference polynucleotide sequence is defined as the percentage of nucleic acids in a candidate sequence that are complementary to nucleic acids in the reference polynucleotide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence complementarity. A given nucleotide is considered to be "complementary" to a reference nucleotide as described herein if the two nucleotides form a standard Watson-Crick base pair. For the avoidance of doubt, in the context of the present disclosure, Watson-Crick base pairs include adenine-thymine, adenine-uracil, and cytosine-guanine base pairs. In this context, a proper Watson-Crick base pair is referred to as a "match," while each unpaired nucleotide and an improperly paired nucleotide are referred to as a "mismatch." Alignment for purposes of determining percent nucleic acid sequence complementarity can be accomplished in a variety of ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum complementarity over the entire length of the sequences being compared. By way of illustration, the percent sequence complementarity of a given nucleic acid sequence A to a given nucleic acid sequence B (which can alternatively be referred to as a given nucleic acid sequence A having a certain percent complementarity to a given nucleic acid sequence B) is calculated as follows: 100×(fraction X / Y) where X is the number of complementary base pairs in a program alignment of A and B (e.g., as performed by computer software such as BLAST) and Y is the total number of nucleic acids in B. It is understood that if the length of nucleic acid sequence A is not equal to the length of nucleic acid sequence B, then the percent sequence complementarity to B is not equal to the percent sequence complementarity of B to A. As used herein, a query nucleic acid sequence is considered to be "fully complementary" to a reference nucleic acid sequence if the query nucleic acid sequence has 100% sequence complementarity to the reference nucleic acid sequence.

[0124] "Percent sequence identity" with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to those in a reference polynucleotide or polypeptide sequence after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity. Alignment for the purpose of determining percent nucleic acid or amino acid sequence identity can be achieved in a variety of ways that are within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to obtain maximum alignment over the full length of the sequences being compared. For example, percent sequence identity values ​​can be generated using the sequence comparison computer program BLAST. By way of illustration, the percent sequence identity of a given nucleic acid or amino acid sequence A to, with, or against a given nucleic acid or amino acid sequence B (which may alternatively be expressed as a given nucleic acid or amino acid sequence A having a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence B) is calculated as follows: 100×(fraction X / Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program's alignment of A with B, and Y is the total number of nucleic acids in B. It will be recognized that if the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, then the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

[0125] The term "sufficiently complementary to hybridize" as used herein refers to a nucleic acid sequence or a portion thereof that does not necessarily have to be fully complementary (e.g., 100% complementary) to a target region or nucleic acid sequence or a portion thereof, which has one or more nucleotide mismatches with the target region but can still hybridize to the target region under specified conditions. For example, a nucleic acid may be, for example, 95% complementary, 90% complementary, 85% complementary, 80% complementary, 75% complementary, 70% complementary, 65% complementary, 60% complementary, 55% complementary, 50% complementary, or less, but still form sufficient base pairs with the target to hybridize over its entire length. A sequence having sufficient complementarity to hybridize to a target region or nucleic acid region within a particular exon includes a target region that is entirely contained within the particular exon, or a target region that includes one or more nucleotides within the particular exon but spans an exon-exon junction.

[0126] Nucleic acid "hybridization" or "annealing" occurs when one or more nucleoside residues in a polynucleotide base pair with one or more complementary nucleosides to form a stable duplex. Base pairing is usually caused by hydrogen bonding events. Hybridization includes Watson-Crick base pairs formed from natural and / or modified nucleobases. Hybridization may also include non-Watson-Crick base pairs, such as wobble base pairs (guanosine-uracil, hypoxanthine-uracil, hypoxanthine-adenine, and hypoxanthine-cytosine) and Hoogsteen base pairs. Nucleic acids do not need to be 100% complementary to undergo hybridization. For example, one nucleic acid can be, e.g., 95% complementary, 90% complementary, 85% complementary, 80% complementary, 75% complementary, 70% complementary, 65% complementary, 60% complementary, 55% complementary, 50% complementary, or less, to another nucleic acid, yet the two nucleic acids can form sufficient base pairs with each other to hybridize.

[0127] A "stable duplex" formed upon annealing / hybridization of one nucleic acid to another is a duplex structure that is not denatured by harsh washing. Exemplary stringent washing conditions are well known in the art and include temperatures about 5°C below the melting temperatures of the individual strands of the duplex and low monovalent salt concentrations, such as monovalent salt concentrations (e.g., NaCl concentrations) of less than 0.2M (0.2M, 0.19M, 0.18M, ​​0.17M, 0.16M, 0.15M, 0.14M, 0.13M, 0.12M, 0.11M, 0.1M, 0.09M, 0.08M, 0.07M, 0.06M, 0.05M, 0.04M, 0.03M, 0.02M, 0.01M, or lower).

[0128] The term "gene silencing" refers to the suppression of gene expression, e.g., endogenous gene expression of MSH3, which may be mediated through processes affecting transcription and / or post-transcriptional mechanisms. In some embodiments, gene silencing occurs when an RNAi molecule initiates the inhibition or degradation of mRNA transcribed from a gene of interest in a sequence-specific manner by RNA interference, thereby preventing translation of the gene's product.

[0129] As used herein, the term "overactive disease driver gene" refers to a gene with increased activity and / or expression that contributes to or causes a disease state in a subject (e.g., a human). The disease state may be caused or exacerbated by the overactive disease driver gene directly, or by an intermediary gene(s).

[0130] As used herein, the term "ethylene glycol chain" refers to a group of the formula ((CH 2 OH) 2 ) refers to a carbon chain having

[0131] As used herein, "alkyl" refers to a saturated hydrocarbon group. Alkyl groups can be acyclic or cyclic and, if unsubstituted, contain only C and H. When naming an alkyl residue with a specific number of carbons, all geometric isomers with that number of carbons are intended to be encompassed and described. Thus, for example, "butyl" is meant to include n-butyl, sec-butyl, and iso-butyl. Examples of alkyl include ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like. In some embodiments, alkyl can be substituted. Suitable substituents that can be introduced into alkyl groups include, for example, hydroxy, alkoxy, amino, alkylamino, and halo, among others.

[0132] As used herein, "alkenyl" refers to an acyclic or cyclic unsaturated hydrocarbon group having at least one site of olefinic unsaturation (i.e., having at least one moiety of the formula C=C). Alkenyl groups, when unsubstituted, contain only C and H. When an alkenyl residue having a specific number of carbons is named, all geometric isomers having that number of carbons are intended to be encompassed and described. Thus, for example, "butenyl" is meant to include n-butenyl, sec-butenyl, and iso-butenyl. Examples of alkenyl include -CH=CH 2 , -CH 2 -CH=CH 2 , and -CH 2 -CH=CH-CH=CH 2 In some embodiments, the alkenyl can be substituted. Suitable substituents that can be introduced into the alkenyl group include, for example, hydroxy, alkoxy, amino, alkylamino, and halo, among others.

[0133] As used herein, "alkynyl" refers to an acyclic or cyclic unsaturated hydrocarbon group having at least one site of acetylenic unsaturation (i.e., having at least one moiety of the formula C≡C). Alkynyl groups, when unsubstituted, contain only C and H. When an alkynyl residue having a specific number of carbons is named, all geometric isomers having that number of carbons are intended to be encompassed and described. Thus, for example, "pentynyl" is meant to include n-pentynyl, sec-pentynyl, iso-pentynyl, and tert-pentynyl. Examples of alkynyl include -C≡CH and -C≡C-CH 3 In some embodiments, alkynyl can be substituted. Suitable substituents that can be introduced into alkynyl groups include, for example, hydroxy, alkoxy, amino, alkylamino, and halo, among others.

[0134] As used herein, the term "phenyl" refers to a monocyclic arene having one hydrogen atom removed from a carbon atom of the ring. A phenyl group can be unsubstituted or substituted with one or more suitable substituents, which substitute for the H of the phenyl group.

[0135] As used herein, the term "benzyl" refers to the monovalent radical obtained when the hydrogen atom attached to the methyl group of toluene is removed. A benzyl group is generally a phenyl-CH 2 -. A benzyl group can be unsubstituted or substituted with one or more suitable substituents. For example, the substituents can be H and / or methylene (-CH) on the phenyl component. 2 -) may replace H in the component.

[0136] As used herein, the term "amide" refers to an alkyl, alkenyl, alkynyl, or aromatic group bound to an aminocarbonyl functional group.

[0137] As used herein, the term “triazole” refers to a compound of formula (C) having a five-membered ring of two carbons and three nitrogens, the positions of which can vary to give rise to multiple isomers. 2 H 3 N 3 ) refers to a heterocyclic compound having the formula:

[0138] As used herein, the term "end group" refers to the group at which a carbon chain or nucleic acid ends.

[0139] As used herein, "amino acid" refers to a molecule that contains an amine and a carboxyl functional group as well as a side chain characteristic of an amino acid.

[0140] In some embodiments, the amino acid is selected from the group of proteinogenic amino acids. In some embodiments, the amino acid is an L-amino acid or a D-amino acid. In some embodiments, the amino acid is a synthetic amino acid (e.g., a beta-amino acid).

[0141] As used herein, the term "lipophilic amino acid" refers to an amino acid that contains a hydrophobic moiety (eg, an alkyl chain or an aromatic ring).

[0142] As used herein, the term "delivery target" refers to an organ or part of the body to which it is desired to deliver a branched oligonucleotide composition.

[0143] As used herein, the term "between X and Y" is inclusive of values ​​of X and Y. For example, "between X and Y" refers to a range of values ​​between a value of X and a value of Y, as well as a value of X and a value of Y.

[0144] As used herein, the terms "subject" and "patient" are used interchangeably and refer to an organism, such as a mammal (e.g., a human), that has a neurodegenerative disease or disorder (e.g., a microsatellite repeat expansion disorder, Huntington's disease, myotonic dystrophy, or spinocerebellar ataxia) and / or has a gain-of-function MSH3 variant allele.

[0145] As used herein, the term "MSH3" refers to a gene encoding MutS homolog 3, including any native MSH3 gene from any source. MSH3 is a DNA mismatch repair protein that heterodimerizes with another mismatch repair protein, MutS homolog 2 (MSH2), to form the complex MutSβ. MutSβ corrects insertion / deletion loops and base-base mispairings in microsatellites during DNA synthesis or post-transcriptional DNA strand reannealing. The term encompasses "full-length" unprocessed MSH3, and any form of MSH3 that results from processing in the cell. The term also encompasses naturally occurring variants of MSH3, such as splice variants or allelic variants. The nucleic acid sequence of an exemplary MSH3 gene is shown in European Nucleotide Archive (ENA) Accession No. J04810.1. The amino acid sequence of an exemplary protein encoded by the MSH3 gene is shown in UNIPROT™ Accession No. P20585.

[0146] As used herein, the terms "microsatellite repeat expansion disease," "microsatellite repeat expansion disease," "nucleotide repeat expansion disease," and "nucleotide repeat expansion disease" are used interchangeably to refer to any disease or disorder caused by instability and expansion of specific microsatellites. A "microsatellite" is a coding or non-coding DNA sequence that contains tandemly repeated units of base pairs. Exemplary microsatellite repeat expansion diseases include, but are not limited to, fragile X syndrome, fragile XE syndrome, fragile X associated tremor / ataxia syndrome, fragile X primary ovarian failure, progressive myoclonus epilepsy type 1 / Unverricht-Lundborg disease, spinocerebellar ataxia (SCA) 12, neuronal intranuclear inclusion disease, glutaminase deficiency, Huntington's disease, SCA1, SCA2, SCA3, SCA6, SCA7, SCA17, dentatorubral-pallidal syndrome, and dendritic cell-associated fibrosis. These include Luysian atrophy, spinal-bulbar muscular atrophy, oculopharyngeal muscular dystrophy, Huntington's disease type 2, amyotrophic lateral sclerosis, myotonic dystrophy type 2 (DM2), Friedrich ataxia, Fuchs corneal endothelial dystrophy, SCA10, SCA31, SCA36, SCA37, cerebellar ataxia, neuropathy, and vestibular loss of reflexes syndrome (CANVAS), benign adult familial myoclonic epilepsy, SCA8, and myotonic dystrophy type 1 (DM1). Other microsatellite repeat expansion disorders are described in Rodriguez et al., Neurobiology of Disease, 130:104515,2019, the disclosure of which is incorporated herein by reference.

[0147] As used herein, the terms "treat", "treated" and "treating" refer to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent, ameliorate or delay (alleviate) an undesirable physiological condition, disorder or disease, or to obtain a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes include, but are not limited to, reducing the patient's dependency on drug therapy; alleviating symptoms; reducing the severity of the condition, disorder or disease; stabilizing (i.e., not worsening) the condition, disorder or disease, delaying or slowing the progression of the condition, disorder or disease; improving or remitigating (whether partial or complete) the condition, disorder or disease, whether detectable or undetectable; improving at least one measurable physical, cognitive, or behavioral (e.g., depressive behavior or apathy) parameter, not necessarily recognized by the patient; or improving or ameliorating the condition, disorder, or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.

[0148] As used herein, the terms "benefit" and "response" are used interchangeably in the context of a subject undergoing therapy for the treatment of a microsatellite repeat expansion disease, such as, for example, Huntington's disease or myotonic dystrophy type 1. For example, clinical benefit in the context of a subject having Huntington's disease administered an siRNA molecule or siRNA composition of the present disclosure includes, but is not limited to, a reduction in involuntary movements, memory loss, mood swings, or symptoms of anxiety and depression, and / or a reduction in wild-type MSH3 transcripts, mutant MSH3 transcripts, variant MSH3 transcripts, splice isoforms of MSH3 transcripts, and / or overexpressed MSH3 transcripts; and / or a cessation or reduction in the length of microsatellite repeats in the HTT gene.

[0149] The present disclosure provides compositions of small interfering RNA (siRNA) molecules having sequence homology with MutS homolog 3 (MSH3) gene, and methods for administering siRNA molecules to subjects. Furthermore, the siRNA molecules described herein may be configured as branched siRNA structures, such as bi-, tri-, and tetra-antennary siRNA structures, and may further include specific patterns of chemical modifications (e.g., 2' ribose modifications or internucleoside linkage modifications) to improve resistance to nuclease enzymes, toxicity profiles, and physicochemical properties (e.g., thermal stability). Small interfering RNA molecules are short double-stranded RNA molecules. They can mediate RNA interference (RNAi) by degrading mRNAs with complementary nucleotide sequences, thus preventing the translation of target genes.

[0150] The siRNA molecules of the present disclosure may, for example, exhibit potent gene-specific suppression of MSH3 relative to other genes in the MutS family (eg, MSH2, MSH4, MSH5, and MSH6) and other human genes.

[0151] The siRNA molecules of the present disclosure can feature an antisense strand having a nucleic acid sequence that is complementary to a region within an MSH3 mRNA transcript having a nucleic acid sequence of any one of SEQ ID NOs: 1-408. The degree of complementarity of the antisense strand to the region of the MSH3 mRNA transcript can be sufficient for the antisense strand to anneal over the entire length of the region of the MSH3 mRNA transcript. For example, the antisense strand can have a nucleic acid sequence that is at least 60% complementary (e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to a region of the MSH3 mRNA transcript. In some embodiments, the region of the MSH3 RNA transcript has the sequence of any one of SEQ ID NOs: 5, 17-20, 33, 42, 44, 102, 103, 105, 106, 108, 109, 113, 126, 129, 130, 158, 161, 166, 177, 183, 193, 194, 196, 197, 202, 209, 217, 231, 271, 273, 291, 294, 305, 307, 317, 318, and 362. In some embodiments, the region of the MSH3 RNA transcript has the sequence of any one of SEQ ID NOs: 5, 17-20, 42, 44, 126, 129, 130, 158, 177, 183, 193, 194, 196, 197, 202, 209, 231, 271, 273, 291, 294, 305, 307, 317, 318, and 362.

[0152] In some embodiments, the siRNA molecules of the present disclosure are characterized by an antisense strand having a nucleic acid sequence of any one of SEQ ID NOs: 817-1224, or a nucleic acid sequence that is at least 60% identical thereto. For example, siRNA molecules of the present disclosure can be characterized as having an antisense strand having a nucleic acid sequence that is at least 60% identical (e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 817-1224. In some embodiments, the nucleic acid sequence is the nucleotide sequence of any one of SEQ ID NOs: 821, 833-836, 849, 858, 860, 918, 919, 921, 922, 924, 925, 929, 942, 945, 946, 974, 977, 982, 993, 999, 1009, 1010, 1012, 1013, 1018, 1025, 1033, 1047, 1087, 1089, 1107, 1110, 1121, 1123, 1133, and 1134. In some embodiments, the nucleic acid sequence is any one of SEQ ID NOs: 821, 833-836, 858, 860, 942, 945, 946, 974, 993, 999, 1009, 1010, 1012, 1013, 1018, 1025, 1047, 1087, 1089, 1107, 1110, 1121, 1123, 1133, and 1134.

[0153] In some embodiments, the siRNA molecules of the present disclosure are characterized by a sense strand having a nucleic acid sequence of any one of SEQ ID NOs: 409-816, or a nucleic acid sequence that is at least 60% identical thereto. For example, siRNA molecules of the present disclosure can be characterized as having a sense strand having a nucleic acid sequence that is at least 60% identical (e.g., 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical) to the nucleic acid sequence of any one of SEQ ID NOs: 409-816. In some embodiments, the nucleic acid sequence is the nucleotide sequence of any one of SEQ ID NOs: 413, 425-428, 441, 450, 452, 510, 511, 513, 514, 516, 517, 521, 534, 537, 538, 566, 569, 574, 585, 591, 601, 602, 604, 605, 610, 617, 625, 639, 679, 681, 699, 702, 713, 715, 725, 726, and 770. In some embodiments, the nucleic acid sequence is any one of SEQ ID NOs: 413, 425-428, 450, 452, 534, 537, 538, 566, 585, 591, 601, 602, 604, 605, 610, 617, 639, 679, 681, 699, 702, 713, 715, 725, 726, and 770.

[0154] Exemplary siRNA molecules of the disclosure are shown below in Table 1A, which summarizes the antisense strand, the sense strand, and the corresponding region of the MSH3 mRNA transcript targeted by each antisense strand.

[0155] [Table 1-1] [Table 1-2] [Table 1-3]

Table 1-4

Table 1-5

Table 1-6

Table 1-7

Table 1-8

Table 1-9

Table 1-10

Table 1-11

Table 1-12

Table 1-13

Table 1-14

Table 1-15

Table 1-16

Table 1-17

Table 1-18

Table 1-19

Table 1-20

Table 1-21

Table 1-22

Table 1-23

Table 1-24

Table 1-25

Table 1-26

Table 1-27

Table 1-28

Table 1-29

Table 1-30

Table 1-31

Table 1-32

Table 1-33

Table 1-34

Table 1-35

Table 1-36

Table 1-37

[0156] In some embodiments, the siRNA molecules of the present disclosure have sufficient complementarity to hybridize to a region within an MSH3 RNA transcript that is within the open reading frame of the MSH3 RNA transcript, hi some embodiments, the siRNA molecules of the present disclosure have sufficient complementarity to hybridize to a region within an MSH3 mRNA transcript that is within the 3' untranslated region.

[0157] In some embodiments, the siRNA molecules of the present disclosure have sufficient complementarity to hybridize to a region within exon 1 of the MSH3 mRNA transcript. In some embodiments, the siRNA molecules of the present disclosure have sufficient complementarity to hybridize to a region within exon 2 of the MSH3 mRNA transcript. In some embodiments, the siRNA molecules of the present disclosure have sufficient complementarity to hybridize to a region within exon 3 of the MSH3 mRNA transcript. In some embodiments, the siRNA molecules of the present disclosure have sufficient complementarity to hybridize to a region within exon 4 of the MSH3 mRNA transcript. In some embodiments, the siRNA molecules of the present disclosure have sufficient complementarity to hybridize to a region within exon 5 of the MSH3 mRNA transcript. In some embodiments, the siRNA molecules of the present disclosure have sufficient complementarity to hybridize to a region within exon 6 of the MSH3 mRNA transcript. In some embodiments, the siRNA molecules of the present disclosure have sufficient complementarity to hybridize to a region within exon 7 of the MSH3 mRNA transcript. In some embodiments, the siRNA molecule of the present disclosure has sufficient complementarity to hybridize to a region within exon 8 of the MSH3 mRNA transcript. In some embodiments, the siRNA molecule of the present disclosure has sufficient complementarity to hybridize to a region within exon 9 of the MSH3 mRNA transcript. In some embodiments, the siRNA molecule of the present disclosure has sufficient complementarity to hybridize to a region within exon 10 of the MSH3 mRNA transcript. In some embodiments, the siRNA molecule of the present disclosure has sufficient complementarity to hybridize to a region within exon 11 of the MSH3 mRNA transcript. In some embodiments, the siRNA molecule of the present disclosure has sufficient complementarity to hybridize to a region within exon 12 of the MSH3 mRNA transcript. In some embodiments, the siRNA molecule of the present disclosure has sufficient complementarity to hybridize to a region within exon 13 of the MSH3 mRNA transcript. In some embodiments, the siRNA molecule of the present disclosure has sufficient complementarity to hybridize to a region within exon 14 of the MSH3 mRNA transcript.In some embodiments, the siRNA molecule of the present disclosure has sufficient complementarity to hybridize to a region within exon 15 of the MSH3 mRNA transcript. In some embodiments, the siRNA molecule of the present disclosure has sufficient complementarity to hybridize to a region within exon 16 of the MSH3 mRNA transcript. In some embodiments, the siRNA molecule of the present disclosure has sufficient complementarity to hybridize to a region within exon 17 of the MSH3 mRNA transcript. In some embodiments, the siRNA molecule of the present disclosure has sufficient complementarity to hybridize to a region within exon 18 of the MSH3 mRNA transcript. In some embodiments, the siRNA molecule of the present disclosure has sufficient complementarity to hybridize to a region within exon 19 of the MSH3 mRNA transcript. In some embodiments, the siRNA molecule of the present disclosure has sufficient complementarity to hybridize to a region within exon 20 of the MSH3 mRNA transcript. In some embodiments, the siRNA molecule of the present disclosure has sufficient complementarity to hybridize to a region within exon 21 of the MSH3 mRNA transcript. In some embodiments, the siRNA molecules of the present disclosure have sufficient complementarity to hybridize to a region within exon 22 of the MSH3 mRNA transcript. In some embodiments, the siRNA molecules of the present disclosure have sufficient complementarity to hybridize to a region within exon 23 of the MSH3 mRNA transcript. In some embodiments, the siRNA molecules of the present disclosure have sufficient complementarity to hybridize to a region within exon 24 of the MSH3 mRNA transcript.

[0158] Table 1B below discloses the location in the gene in which each target region is contained (eg, the open reading frame or 3' untranslated region) and the exon in which each target region is contained.

[0159] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6]

[0160] Structure of siRNA The siRNA molecule of the present disclosure may be in the form of a single-stranded (ss) or double-stranded (ds) oligonucleotide structure. In some embodiments, the siRNA molecule may be a biantennary, triantennary, or tetraantennary molecule. In addition, the siRNA molecule of the present disclosure may contain one or more phosphodiester internucleoside linkages and / or their analogs, such as phosphorothioate internucleoside linkages. The siRNA molecule of the present disclosure may further contain chemically modified nucleosides with 2' sugar modifications.

[0161] The simplest siRNA consists of a ribonucleic acid containing a ss- or ds-structure, formed by a first strand (i.e., antisense strand) and, in the case of ds-siRNA, together with a second strand (i.e., sense strand). The first strand comprises a stretch of consecutive nucleotides that is at least partially complementary to the target nucleic acid. The second strand also comprises a stretch of consecutive nucleotides, the second stretch being at least partially identical to the target nucleic acid. The first strand and said second strand can hybridize to each other to form a double-stranded structure. Hybridization typically occurs by Watson-Crick base pairing.

[0162] Depending on the sequence of the first and second strand, hybridization or base pairing is not necessarily complete or perfect, which means that the first and second strands are not 100% base paired due to mismatch.One or more mismatches can also exist in the double strand, without necessarily affecting the RNAi activity of siRNA.

[0163] The first strand comprises a stretch of contiguous nucleotides essentially complementary to the target nucleic acid. Typically, the target nucleic acid sequence is ss-RNA, preferably mRNA, according to the mode of action of the interfering ribonucleic acid. Such hybridization most likely occurs via Watson-Crick base pairing, but is not necessarily limited thereto. The extent to which the first strand has a complementary stretch of contiguous nucleotides to the target nucleic acid sequence can be 80% to 100%, for example, 80%, 85%, 90%, 95%, or 100% complementary.

[0164] The siRNA molecules described herein may employ modifications to the nucleobases, phosphate backbone, ribose core, 5' and 3' ends, and branching, where multiple strands of the siRNA may be covalently linked.

[0165] Length of the small interfering RNA molecule Within the scope of the present invention, any length known in the art and previously unknown can be employed for the present invention. As described herein, the potential length of the antisense strand of the siRNA molecule of the present disclosure is between 10 and 30 nucleotides (e.g., 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides), between 15 and 25 nucleotides (e.g., 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, or 25 nucleotides), or between 18 and 23 nucleotides (e.g., 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, or 23 nucleotides). In some embodiments, the antisense strand is 20 nucleotides. In some embodiments, the antisense strand is 21 nucleotides. In some embodiments, the antisense strand is 22 nucleotides. In some embodiments, the antisense strand is 23 nucleotides. In some embodiments, the antisense strand is 24 nucleotides. In some embodiments, the antisense strand is 25 nucleotides. In some embodiments, the antisense strand is 26 nucleotides. In some embodiments, the antisense strand is 27 nucleotides. In some embodiments, the antisense strand is 28 nucleotides. In some embodiments, the antisense strand is 29 nucleotides. In some embodiments, the antisense strand is 30 nucleotides.

[0166] In some embodiments, the sense strand of the siRNA molecules of the present disclosure is between 12-30 nucleotides (e.g., 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides), or between 14-23 nucleotides (e.g., 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, or 23 nucleotides). In some embodiments, the sense strand is 15 nucleotides in length. In some embodiments, the sense strand is 16 nucleotides in length. In some embodiments, the sense strand is 17 nucleotides in length. In some embodiments, the sense strand is 18 nucleotides in length. In some embodiments, the sense strand is 19 nucleotides in length. In some embodiments, the sense strand is 20 nucleotides in length. In some embodiments, the sense strand is 21 nucleotides in length. In some embodiments, the sense strand is 22 nucleotides in length. In some embodiments, the sense strand is 23 nucleotides in length. In some embodiments, the sense strand is 24 nucleotides in length. In some embodiments, the sense strand is 25 nucleotides in length. In some embodiments, the sense strand is 26 nucleotides in length. In some embodiments, the sense strand is 27 nucleotides in length. In some embodiments, the sense strand is 28 nucleotides in length. In some embodiments, the sense strand is 29 nucleotides in length. In some embodiments, the sense strand is 30 nucleotides in length.

[0167] 2' sugar modification The present disclosure may include ss-siRNA and ds-siRNA molecular compositions that include at least one (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or more) nucleosides with a 2' sugar modification. Possible 2'-modifications include all possible orientations of OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl can be substituted or unsubstituted C1-C10 alkyl or C2-C10 alkenyl and alkynyl. In some embodiments, the modification includes a 2'-O-methyl (2'-O-Me) modification. Other potential sugar substituents include C1-C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH, or 2'-O-methyl. 3 , OCN, Cl, Br, CN, CF 3 , OCF 3 , SOCH 3 , S.O. 2 CH 3 , O.N.O. 2 , NO 2 , N 3 , N.H. 2 , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, groups for improving the pharmacokinetic properties of oligonucleotides, or groups for improving the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. In some embodiments, the modification is 2'-methoxyethoxy (2'-O-CH 2 CH 2 OCH 3 , also known as 2'-O-(2-methoxyethyl) or 2'-MOE). In some embodiments, the modification includes 2'-dimethylaminooxyethoxy, O(CH 2 ) 2 ON(CH 3 ) 2groups, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylamino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CH 2 OCH 2 N(CH 3 ) 2 Other potential sugar substituents include, for example, aminopropoxy (-OCH 2 CH 2 CH 2 NH 2 ), allyl (-CH 2 -CH=CH 2 ), -O-allyl (-O-CH 2 -CH=CH 2 ), and fluoro (F). The 2'-sugar substituent can be at the arabino (up) or ribo (down) position. In some embodiments, the 2'-arabino modification is 2'-F. Similar modifications may be made at other positions in the siRNA molecule, particularly at the 3' position of the sugar in the 3' terminal nucleoside or 2'-5' linked oligonucleotides and the 5' position of the 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0168] Nucleobase Modifications The siRNA molecules of the present disclosure may also comprise nucleosides or other substitute or mimic monomer subunits that comprise nucleobases (often referred to in the art simply as "bases" or "heterocyclic base moieties").Nucleobases are other moieties that can be modified or substituted in a wide variety of ways, and such modified and / or substituted nucleobases can be in accordance with the present invention.As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases, also referred to herein as heterocyclic base moieties, include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3) uracil and cytosine, and other alkynyl derivatives of pyrimidine bases. Derivatives include 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaguanine and 3-deazaguanine. Nucleobases may also include those in which the purine or pyrimidine base is replaced by other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone.Further nucleobases include those disclosed in US3,687,808, those disclosed in Kroschwitz, JI, ed. The Concise Encyclopedia of Polymer Science and Engineering, New York, John Wiley & Sons, 1990, pp. 858-859; those disclosed in Englisch et al., Angewandte Chemie, International Edition 30:613, 1991; and those disclosed in Sanghvi, YS, Chapter 16, Antisense Research and Applications, CRC Press, Gait, MJ ed., 1993, pp. 289-302. The siRNA molecules of the present disclosure can also contain polycyclic heterocyclic compounds instead of one or more heterocyclic base moieties. Many tricyclic heterocyclic compounds have been reported. These compounds are routinely used in antisense applications to increase the binding properties of modified strand to target strand.

[0169] Representative cytosine analogs that form three hydrogen bonds with guanosine in the second strand include 1,3-diazaphenoxazin-2-one (Kurchavov et al., Nucleosides and Nucleotides,16:1837-46, 1997), 1,3-diazaphenothiazin-2-one (Lin et al. Am. Chem. Soc.,117:3873-4, 1995), and 6,7,8,9-tetrafluoro-1,3-diazaphenoxazin-2-one (Wang et al., Tetrahedron Lett.,39:8385-8, 1998). These base modifications have been shown to hybridize with complementary guanine when incorporated into oligonucleotides, and guanine has also been shown to hybridize with adenine, improving the thermal stability of the helix through extended stacking interactions (see also US10 / 155,920 and US10 / 013,295, both of which are incorporated herein by reference in their entireties). Further helix stabilizing properties have been observed when cytosine analogs / substitutes bear aminoethoxy moieties attached to a rigid 1,3-diazaphenoxazin-2-one scaffold (Lin et al., Am. Chem. Soc., 120:8531-2,1998).

[0170] Internucleoside bond modification Another variable in the design of the present invention is the internucleoside bond that constitutes the phosphate backbone of the siRNA molecule.Natural RNA phosphate backbone may be employed herein, but derivatives thereof may be used to improve the desired properties of the siRNA molecule.Of particular importance in this disclosure, but not limited to, is to protect part or the entire siRNA molecule from hydrolysis.One example of the modification that reduces the rate of hydrolysis is phosphorothioate.Any part or the entire backbone may contain phosphate substitution (e.g., phosphorothioate). For example, the internucleoside bond may be between 0 and 100% phosphorothioate, e.g., between 0 and 100%, 10 and 100%, 20 and 100%, 30 and 100%, 40 and 100%, 50 and 100%, 60 and 100%, 70 and 100%, 80 and 100%, 90 and 100%, 0 and 90%, 0 and 80%, 0 and 70%, 0 and 60%, 0 and 50%, 0 and 40%, 0 and 30%, 0 and 20%, 0 and 10%, 10 and 90%, 20 and 80%, 30 and 70%, 40% and 60%, 10 and 40%, 20 and 50%, 30 and 60%, 40 and 70%, 50 and 80%, or 60 and 90% phosphorothioate bond. Similarly, the internucleoside bond may be between 0 and 100% phosphodiester bonds, for example, between 0 and 100%, 10 and 100%, 20 and 100%, 30 and 100%, 40 and 100%, 50 and 100%, 60 and 100%, 70 and 100%, 80 and 100%, 90 and 100%, 0 and 90%, 0 and 80%, 0 and 70%, 0 and 60%, 0 and 50%, 0 and 40%, 0 and 30%, 0 and 20%, 0 and 10%, 10 and 90%, 20 and 80%, 30 and 70%, 40% and 60%, 10 and 40%, 20 and 50%, 30 and 60%, 40 and 70%, 50 and 80%, or 60 and 90% phosphodiester bonds.

[0171] Specific examples of some potential siRNA molecules useful in the present invention include oligonucleotides that contain modified, for example, non-naturally occurring internucleoside linkages.As defined herein, oligonucleotides with modified internucleoside linkages include internucleoside linkages that retain phosphorus atoms and internucleoside linkages that do not have phosphorus atoms.For the purposes of this specification and as sometimes referred to in the art, modified oligonucleotides that do not have phosphorus atoms in their internucleoside backbone can also be considered to be oligonucleosides.Preferred phosphorus-containing modified internucleoside linkages are phosphorothioate internucleoside linkages. In some embodiments, modified oligonucleotide backbones comprising a phosphorus atom therein include, for example, phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thinophosphoramidates, thinoalkyl phosphonates, thinoalkyl phosphotriesters, selenophosphates, boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs thereof, as well as those having inverted polarity where one or more internucleotide linkages are 3'-3', 5'-5', or 2'-2' linkages.Exemplary United States patents describing the preparation of phosphorus-containing linkages include, but are not limited to, U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; Same No. 5,321,131; Same No. 5,399,676; Same No. 5,405,939; Same No. 5,453,496; Same No. 5,455,233; Same No. 5,466,677; Same No. 5,476, No. 925; No. 5,519,126; No. 5,536,821; No. 5,541,316; No. 5,550,111; No. 5,563,253; No. 5,571,799; No. 5, No. 587,361; No. 5,625,050; No. 6,028,188; No. 6,124,445; No. 6,160,109; No. 6,169,170; No. 6,172,209 ; Same No. 6,239,265; Same No. 6,277,603; Same No. 6,326,199; Same No. 6,346,614; Same No. 6,444,423; Same No. 6,531,590; Same No. 6,534, Nos. 6,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Patent No. RE39464, the entire contents of each of which are incorporated herein by reference.

[0172] In some embodiments, modified oligonucleotide backbones that do not contain a phosphorus atom therein have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages, including morpholino linkages (formed in part from the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; riboacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and N, O, S and CH 2 Non-limiting examples of U.S. patents that teach the preparation of non-phosphorus backbones include, but are not limited to, U.S. Patent Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,64,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, 5,512,111, and 5,512,112. Nos. 5,489,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, and 5,677,439, the entire contents of each of which are incorporated herein by reference.

[0173] Modification patterns of siRNA molecules The following section provides a set of exemplary scaffolds into which the siRNA molecules of the present disclosure can be incorporated.

[0174] In some embodiments of the present disclosure, the siRNA may contain an antisense strand that includes a region represented by Formula I, where Formula I is in the 5' to 3' direction: AB-(A') j -CP 2 -DP 1 -(C'-P 1 ) k -C' Formula I; where A is of the formula CP 1 -DP 1 Each A' is represented by the formula CP 2 -DP 2 B is represented by the formula CP 2 -DP 2 -DP 2 -DP 2 each C is a 2'-O-methyl (2'-O-Me) ribonucleoside; each C' is independently a 2'-O-Me ribonucleoside or a 2'-fluoro (2'-F) ribonucleoside; each D is a 2'-F ribonucleoside; each P 1 is a phosphorothioate internucleoside linkage; each P 2 is a phosphodiester internucleoside linkage; j is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); and k is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, j is 4. In some embodiments, k is 4. In some embodiments, j is 4 and k is 4. The antisense is complementary (e.g., fully or partially complementary) to a target nucleic acid sequence.

[0175] In some embodiments, the antisense strand comprises a structure represented by Formula A1, which, in the 5' to 3' direction, is: ASBSAOBOBOBOAOBOAOBOA-OBOAOBOAOBSASASASBSA Formula A1; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0176] In some embodiments of the present disclosure, the siRNA may contain an antisense strand that includes a region represented by Formula II, which in the 5' to 3' direction is: AB-(A') j -CP 2 -DP 1 -(CP 1 ) k -C' Formula II; where A is of the formula CP 1 -DP 1 Each A' is represented by the formula CP 2 -DP 2 B is represented by the formula CP 2 -DP 2 -DP 2 -DP 2 each C is a 2'-O-methyl (2'-O-Me) ribonucleoside; each C' is independently a 2'-O-Me ribonucleoside or a 2'-fluoro (2'-F) ribonucleoside; each D is a 2'-F ribonucleoside; each P 1 is a phosphorothioate internucleoside linkage; each P 2 is a phosphodiester internucleoside linkage; j is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); and k is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, j is 4. In some embodiments, k is 4. In some embodiments, j is 4 and k is 4. The antisense is complementary (e.g., fully or partially complementary) to a target nucleic acid sequence.

[0177] In some embodiments, the antisense strand comprises a structure represented by Formula A2, which, in the 5' to 3' direction, is: ASBSAOBOBOBOAOBOAOBOA-OBOAAOBOAOBSASASASASA Formula A2; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0178] In some embodiments of the present disclosure, the sense strand comprises a structure represented by Formula III, which, in the 5' to 3' direction, is: E-(A') m -F Formula III; In the formula, E is the formula (CP 1 ) 2 F is represented by the formula (CP 2 ) 3 -DP 1 -CP 1 -C, (CP 2 ) 3 -DP 2 -CP 2 -C, (CP 2 ) 3 -DP 1 -CP 1 -D, or (CP 2 ) 3 -DP 2 -CP 2 -D; A', C, D, P 1 , and P 2 is as defined in Formula I; and m is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, m is 4. The sense strand is complementary (e.g., completely or partially complementary) to the antisense strand.

[0179] In some embodiments of the present disclosure, the sense strand comprises a structure represented by formula S1, which, in the 5' to 3' direction, is: ASASAOBOAOBOAOBOAOBOA-OAOAOBBSASA Formula S1; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0180] In some embodiments of the present disclosure, the sense strand comprises a structure represented by formula S2, which, in the 5' to 3' direction, is: ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOA Formula S2; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0181] In some embodiments of the present disclosure, the sense strand comprises a structure represented by formula S3, which, in the 5' to 3' direction, is: ASASAOBOAOBOAOBOAOBOA-OAOAOBSASB formula S3; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0182] In some embodiments of the present disclosure, the sense strand comprises a structure represented by formula S4, which, in the 5' to 3' direction, is: ASASAOBOAOBOAOBOAOBOA-OAOAOBOAOB Equation S4; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0183] In some embodiments of the present disclosure, the siRNA may contain an antisense strand that includes a region represented by Formula IV, where Formula IV is, in the 5' to 3' direction, A-(A') j -CP 2 -B-(CP 1 ) k -C' Formula IV; where A is of the formula CP 1 -DP 1 Each A' is represented by the formula CP 2 -DP 2 B is represented by the formula DP 1 -CP 1 -DP 1 each C is a 2'-O-Me ribonucleoside; each C' is independently a 2'-O-Me ribonucleoside or a 2'-F ribonucleoside; each D is a 2'-F ribonucleoside; each P 1 is a phosphorothioate internucleoside linkage; each P 2 is a phosphodiester internucleoside linkage; j is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); and k is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, j is 6. In some embodiments, k is 4. In some embodiments, j is 6 and k is 4. The antisense is complementary (e.g., fully or partially complementary) to a target nucleic acid.

[0184] In some embodiments, the antisense strand comprises a structure represented by Formula A3, which, in the 5' to 3' direction, is: ASBSAOBOAOBOAOBOAOBOA-OBOAOBOAOBSASASASA formula A3; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0185] In some embodiments of the present disclosure, the siRNA of the present disclosure can have a sense strand represented by Formula V, where Formula V is, in the 5' to 3' direction, E-(A') m -CP 2 -F formula V; In the formula, E is the formula (CP 1 ) 2 F is represented by the formula DP 1 -CP 1 -C, D.P. 2 -CP 2 -C, D.P. 1 -CP 1 -D, or DP 2 -CP 2 -D; A', C, D, P 1 , and P 2 is as defined in formula IV; and m is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, m is 5. The sense strand is complementary (e.g., fully or partially complementary) to the antisense strand.

[0186] In some embodiments of the present disclosure, the sense strand comprises a structure represented by formula S5, which, in the 5' to 3' direction, is: ASASAOBOAOBOAOBOAOBOA-OBOAOBSASA Formula S5; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0187] In some embodiments of the present disclosure, the sense strand comprises a structure represented by formula S6, which, in the 5' to 3' direction, is: ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOA Formula S6; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage. In some embodiments of the present disclosure, the sense strand comprises a structure represented by formula S7, which, in the 5' to 3' direction, is: ASASAOBOAOBOAOBOAOBOA-OBOAOBSASB Formula S7; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0188] In some embodiments of the present disclosure, the sense strand comprises a structure represented by formula S8, which, in the 5' to 3' direction, is: ASASAOBOAOBOAOBOAOBOA-OBOAOBOAOB formula S8; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0189] In some embodiments of the present disclosure, the siRNA may contain an antisense strand that includes a region represented by Formula VI, where Formula VI is, in the 5' to 3' direction, A.B. j -EB k -EFG l -DP 1 -C' Formula VI; where A is of the formula CP 1 -DP 1 Each B is represented by the formula CP 2 each C is a 2'-O-Me ribonucleoside; each C' is independently a 2'-O-Me ribonucleoside or a 2'-F ribonucleoside; each D is a 2'-F ribonucleoside; each E is represented by the formula DP 2 -CP 2 F is represented by the formula DP 1 -CP 1 Each G is represented by the formula CP 1 Each P 1 is a phosphorothioate internucleoside linkage; each P 2 is a phosphodiester internucleoside linkage; j is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); k is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); and l is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, j is 3. In some embodiments, k is 6. In some embodiments, l is 2. In some embodiments, j is 3, k is 6, and l is 2. The antisense strand is complementary (e.g., fully or partially complementary) to a target nucleic acid.

[0190] In some embodiments, the antisense strand comprises a structure represented by formula A4, which, in the 5' to 3' direction, is: ASBSAOAOAOBOAOAOAOAOA-OAOAOBOAOBSASASASBSA formula A4; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0191] In some embodiments of the present disclosure, the siRNA may contain a sense strand that includes a region represented by Formula VII, which in the 5' to 3' direction is: HB m -I n -A'-B o -HC Formula VII; where A' is a compound of the formula CP 2 -DP 2 Each H is represented by the formula (CP 1 ) 2 Each I is represented by the formula (DP 2 ) are represented by B, C, D, P 1 , and P 2 is as defined in formula VI; m is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); n is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7); and o is an integer from 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7). In some embodiments, m is 3. In some embodiments, n is 3. In some embodiments, o is 3. In some embodiments, m is 3, n is 3, and o is 3. The sense strand is complementary (e.g., fully or partially complementary) to the antisense strand.

[0192] In some embodiments of the present disclosure, the sense strand comprises a structure represented by formula S9, which, in the 5' to 3' direction, is: ASASAOAOAOBOBOBOAOBOA-OAOAOAASASA Formula S9; wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester internucleoside linkage, and S represents a phosphorothioate internucleoside linkage.

[0193] In some embodiments of the present disclosure, the siRNA may contain an antisense strand comprising a region represented by Formula VIII: Z-((AP-) n(BP-) m ) q ; Formula VIII wherein Z is a 5' phosphorus stabilizing moiety; each A is a 2'-O-methyl (2'-O-Me) ribonucleoside; each B is a 2'-fluoro-ribonucleoside; each P is independently an internucleoside linkage selected from a phosphodiester linkage and a phosphorothioate linkage; n is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5); m is an integer from 1 to 5 (e.g., 1, 2, 3, 4, or 5); and q is an integer between 1 and 30 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30).

[0194] Methods for synthesizing siRNA The siRNA molecules of the present disclosure can be synthesized by standard methods well known in the art, for example, by use of an automated DNA synthesizer such as those commercially available from Biosearch, Applied Biosystems, Inc., as discussed further below.

[0195] siRNA agent can be prepared by using liquid phase organic synthesis or solid phase organic synthesis or both.Organic synthesis has the advantage that it is easy to prepare the oligonucleotide that contains non-natural nucleotide or modified nucleotide.The siRNA molecule of the present disclosure can be prepared by using liquid phase organic synthesis or solid phase organic synthesis or both.

[0196] Furthermore, it is contemplated that any siRNA agent disclosed herein can be further optimized by systematically adding or removing linked nucleosides to generate longer or shorter sequences.Furthermore, such optimized sequences can be adjusted by introducing modified nucleosides and / or modified internucleoside linkages, as described herein or known in the art, including, for example, alternative nucleosides, alternative sugar moieties, and / or alternative internucleoside linkages, as known in the art and / or discussed herein, to further optimize the molecule (e.g., increase serum stability or circulating half-life, increase thermal stability, enhance transmembrane delivery, and / or target to specific locations or cell types).

[0197] Divalent cations The siRNA molecules of the present disclosure are those in which the oxyanion moiety is Ba 2+ , B.E. 2+ , Ca 2+ , Cu 2+ , Mg 2+ , Mn 2+ , Ni 2+ , or Zn 2+ The nucleic acid may include one or more phosphodiester internucleoside linkages, such as phosphorothioate internucleoside linkages, electrostatically neutralized by ionic bonding with a divalent metal cation, such as, for example, a divalent metal cation ...

[0198] The siRNA molecules of the present disclosure may be fused to one or more divalent cations (e.g., Ba 2+ , B.E. 2+ , Ca 2+ , Cu 2+ , Mg 2+ , Mn 2+ , Ni 2+ , or Zn 2+ , , or combinations thereof). Divalent cations, due to their positive charge, are usually reactive with negatively charged atoms (e.g., oxyanions from phosphate or phosphorothioate groups, which have unit or partial negative charges).

[0199] The one or more divalent cations can have an ionic radius, measured in the form of a crystal lattice, of about 30 picometers to about 150 picometers (e.g., about 30 picometers to about 140 picometers, about 40 picometers to about 130 picometers, about 50 picometers to about 120 picometers, about 60 picometers to about 110 picometers, about 60 picometers to about 100 picometers, or about 60 picometers to about 90 picometers). The calculated crystal radii of the divalent cations disclosed by RD Shannon, Acta Crystallographica A. 32:751-767, 1976, are incorporated herein by reference.

[0200] The degree of saturation of the cationic binding sites of the siRNA molecule with one or more divalent cations can be in the range of about 10% to about 100% (e.g., about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, or about 90% to about 100%).

[0201] In some embodiments, the antisense strand of the siRNA molecule may have a length of 10 to 30 nucleotides and may be ionically bound to a total of 10 to 30 divalent cations. For example, the molar ratio of antisense strand nucleotides to divalent cations in the siRNA molecule may be 1:3 to 3:1 (e.g., 1:3, 1.1:3, 1.2:3, 1.3:3, 1.4:3, 1.5:3, 1.6:3, 1.7:3, 1.8:3, 1.9:3, 2:3, 2.1:3, 2.2:3, 2.3:3, 2.4:3, 2.5:3, 2.6:3, 2.7:3, 2.8:3, 2.9:3, 1:1, 3:2.9, 3:2.8, 3:2.7, 3:2.6, 3:2.5, 3:2.4, 3:2.3, 3:2.2, 3:2.1, 3:2, 3:1.9, 3:1.8, 3:1.7, 3:1.6, 3:1.5, 3:1.4, 3:1.3, 3:1.2, 3:1.1, or 3:1).

[0202] In some embodiments, the sense strand of the siRNA molecule may have a length of 10-30 nucleotides and may be ionically bound to a total of 10-30 divalent cations. For example, the molar ratio of sense strand nucleotides to divalent cations in the siRNA molecule may be 1:3 to 3:1 (e.g., 1:3, 1.1:3, 1.2:3, 1.3:3, 1.4:3, 1.5:3, 1.6:3, 1.7:3, 1.8:3, 1.9:3, 2:3, 2.1:3, 2.2:3, 2.3:3, 2.4:3, 2.5:3, 2.6:3, 2.7:3, 2.8:3, 2.9:3, 3.0:3, 3.1:3, 3.2:3, 3.3:3, 3.4:3, 3.5:3, 3.6:3, 3.7:3, 3.8:3, 3.9 ...8:3, 3.9:3, 3.1:3, 3.2:3, 3.3:3, 3.4:3, 3.5:3, 3.6:3, 3.7:3, 3.8:3, 3.9:3, 3.1:3, 3.2:3, 3.3:3, 3.4:3, 3.5: 3:1, 3:1.8, 3:1.7, 3:1.6, 3:1.5, 3:1.4, 3:1.3, 3:1.2, 3:1.1, or 3:1).

[0203] The siRNA molecules of the present disclosure can be combined with one or more divalent cations in a specific molar ratio. The specific molar ratio of the siRNA molecule to the divalent cation can be related to the cytotoxicity advantage provided by the divalent cation. For example, the molar ratio of siRNA molecules to divalent cations can range from 1:10 to 1:50 (e.g., 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, 1:38, 1:39, 1:40, 1:41, 1:42, 1:43. 1:44, 1:45, 1:46, 1:47, 1:48, 1:49, or 1:50). In some embodiments, the molar ratio of siRNA molecules to divalent cations may range from 1:18 to 1:38 (e.g., 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:31, 1:32, 1:33, 1:34, 1:35, 1:36, 1:37, or 1:38). In some embodiments, the molar ratio of siRNA molecules to divalent cations may range from 1:20 to 1:25 (e.g., 1:20, 1:21, 1:22, 1:23, 1:24, or 1:25). In some embodiments, the molar ratio of siRNA molecules to divalent cations may be 1:20. In some embodiments, the molar ratio of siRNA molecules to divalent cations may be 1:25.

[0204] The siRNA molecules of the present disclosure can be combined with one or more divalent cations, and the divalent cations are present at a particular concentration or concentration range. The concentration of the divalent cations can be related to the toxic advantage provided by the divalent cations. For example, the concentration of divalent cations may range from 20 mM to 150 mM (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 1 9, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150 mM). In some embodiments, the concentration of divalent cations is between 20 mM and 100 mM (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 mM).In some embodiments, the concentration of divalent cations can be between 35 mM and 75 mM (e.g., 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 mM). In some embodiments, the concentration of divalent cations can be between 40 mM and 70 mM (e.g., 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 mM).

[0205] siRNA molecule may comprise one or more atoms with negative charge, and divalent cation may comprise positive charge.In some embodiments, siRNA molecule and divalent cation are present in an amount that makes the negative charge and positive charge present in the composition a certain ratio.Methods for determining the ratio of negative charge and positive charge are well known in the art, and are shown, for example, in Furst et al., Electrophoresis., 37:2685-2691, 2016, the disclosure of which is incorporated herein by reference. In some embodiments, the ratio of negative to positive charges is between 0.75 and 7.5 (e.g., 0.76, 0.77, 0.78, 0.79, 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 ... .4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5). In some embodiments, the ratio of negative to positive charges is between 1.0 and 2.0 (e.g., between 1.0 and 1.9, between 1.0 and 1.8, between 1.0 and 1.7, between 1.0 and 1.6, between 1.0 and 1.5, between 1.0 and 1.4, between 1.0 and 1.3, between 1.0 and 1.2, between 1.0 and 1.1, between 1.1 and 2.0, between 1.2 and 2.0, between 1.3 and 2.0, between 1.4 and 2.0, between 1.5 and 2.0, between 1.6 and 2.0, between 1.7 and 2.0, between 1.8 and 2.0, or between 1.9 and 2.0). In some embodiments, the ratio of negative charges to positive charges is between 0.75 and 6.5 (e.g., between 0.75 and 5.5, between 0.75 and 4.5, between 0.75 and 3.5, between 0.75 and 2.5, between 0.75 and 1.5, or between 0.75 and 1).In some embodiments, the ratio of negative to positive charges is between 1 and 7.5 (e.g., between 1.5 and 7.5, between 2.5 and 7.5, between 3.5 and 7.5, between 4.5 and 7.5, between 5.5 and 7.5, or between 6.5 and 7.5).

[0206] 5' phosphorus stabilizing moiety To further protect the siRNA molecule of the present disclosure from degradation, 5'-phosphorus stabilizing moiety can be adopted. 5'-phosphorus stabilizing moiety replaces 5'-phosphate to prevent hydrolysis of phosphate. Hydrolysis of 5'-phosphate prevents binding to RISC, which is a necessary step of gene silencing. Any substitution of phosphate that does not prevent binding to RISC is contemplated in the present disclosure. In some embodiments, the substitution of 5'-phosphate is also stable to hydrolysis in vivo. Each strand of siRNA molecule may independently and optionally adopt any suitable 5'-phosphorus stabilizing moiety. [ka]

[0207] Some exemplary end caps are illustrated in Formulae IX-XVI. Nuc in Formulae IX-XVI represents a nucleobase or nucleobase derivative or substitute as described herein. X in Formulae IX-XVI represents a 2'-modification as described herein. Some embodiments employ hydroxy as in Formula IX, phosphate as in Formula X, vinyl phosphonate as in Formulae XI and XIV, 5'-methyl substituted phosphate as in Formulae XII, XIII, and XVI, methylene phosphonate as in Formula XV, or vinyl 5'-vinyl phosphonate as shown in Formula XI as the 5'-phosphorus stabilizing moiety.

[0208] hydrophobic part The present disclosure further provides siRNA molecules with one or more hydrophobic moieties attached.Hydrophobic moieties can be covalently attached to the 5'-end or 3'-end of the siRNA molecules of the present disclosure.Non-limiting examples of the hydrophobic moieties suitable for use in the siRNA molecules of the present disclosure can include cholesterol, vitamin D, tocopherol, phosphatidylcholine (PC), docohexaenoic acid, docosanoic acid, PC-docosanoic acid, eicosapentaenoic acid, lithocholic acid, or any combination of the above hydrophobic moieties and PC.

[0209] siRNA branching The siRNA molecules of the present disclosure may be branched. For example, the siRNA molecules of the present disclosure may have one of several branching patterns as described herein.

[0210] According to the present disclosure, the siRNA molecule disclosed herein may be a branched siRNA molecule. The siRNA molecule may be unbranched or bi-, tri- or tetra-branched, connected via a linker. Each main branch may be further branched to allow 2, 3, 4, 5, 6, 7 or 8 separate RNA single or double strands. The branching points on the linker may originate from the same atom or may originate from separate atoms along the linker. Some exemplary embodiments are listed in Table 2.

[0211] [Table 3]

[0212] In some embodiments, the siRNA molecule is a branched siRNA molecule. In some embodiments, the branched siRNA molecule is biantennary, triantennary, or tetraantennary. In some embodiments, the biantennary siRNA molecule is represented by any one of formulas XVII-XIX, where each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branch point moiety (e.g., phosphoramidite, tosylated solketal, 1,3-diaminopropanol, pentaerythritol, or any one of the branch point moieties described in US10,478,503).

[0213] In some embodiments, the three-branched siRNA molecule is represented by any one of formulas XX-XXIII, wherein each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

[0214] In some embodiments, the four-branched siRNA molecule is represented by any one of Formulas XXIV-XXVIII, wherein each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branch point moiety.

[0215] Linker The multiple strands of the siRNA described herein may be covalently linked by a linker. This branching effect, among other things, improves cell permeability, allowing better access to cells (e.g., neurons or glial cells) in the CNS. Any linker moiety that is not incompatible with the siRNA of the present invention may be employed. Linkers include ethylene glycol chains of 2-10 subunits (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 subunits), alkyl chains, carbohydrate chains, block copolymers, peptides, RNA, DNA, and others. In some embodiments, the carbon or oxygen atoms of the linker are optionally replaced with nitrogen atoms, have hydroxyl substituents, or have oxo substituents. In some embodiments, the linker is a polyethylene glycol (PEG) linker. PEG linkers suitable for use in the disclosed compositions and methods include linear or non-linear PEG linkers. Examples of non-linear PEG linkers include branched PEG, linear forked PEG, or branched forked PEG.

[0216] PEG linkers of various weights may be used in the disclosed compositions and methods. For example, the PEG linker may have a weight between 5 and 500 daltons. In some embodiments, a PEG linker having a weight between 500 and 1,000 daltons may be used. In some embodiments, a PEG linker having a weight between 1,000 and 10,000 daltons may be used. In some embodiments, a PEG linker having a weight between 200 and 20,000 daltons may be used. In some embodiments, the linker is covalently attached to the sense strand of the siRNA. In some embodiments, the linker is covalently attached to the antisense strand of the siRNA. In some embodiments, the PEG linker is a triethylene glycol (TrEG) linker. In some embodiments, the PEG linker is a tetraethylene glycol (TEG) linker.

[0217] In some embodiments, the linker is an alkyl chain linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is an RNA linker. In some embodiments, the linker is a DNA linker.

[0218] The linker may covalently link two, three, four, or five unique siRNA strands. The linker may be covalently linked to any portion of the siRNA oligomer. In some embodiments, the linker is attached to the 3' end of the nucleoside of each siRNA strand. In some embodiments, the linker is attached to the 5' end of the nucleoside of each siRNA strand. In some embodiments, the linker is attached to the nucleoside of the siRNA strand (e.g., the sense strand or the antisense strand) via a covalent bond forming moiety. In some embodiments, the covalent bond forming moiety is selected from the group consisting of alkyl, ester, amide, carbonate, carbamate, triazole, urea, formacetal, phosphonate, phosphate, and phosphate derivatives (e.g., phosphorothioate, phosphoramidate, etc.).

[0219] In some embodiments, the linker has the structure of formula L1. [ka]

[0220] In some embodiments, the linker has the structure of formula L2: [ka]

[0221] In some embodiments, the linker has the structure of formula L3. [ka]

[0222] In some embodiments, the linker has the structure of formula L4. [ka]

[0223] In some embodiments, the linker has the structure of formula L5. [ka]

[0224] In some embodiments, the linker has the structure of formula L6. [ka]

[0225] In some embodiments, the linker has the structure of formula L7, as shown below: [ka]

[0226] In some embodiments, the linker has the structure of formula L8. [ka]

[0227] In some embodiments, the linker has the structure of formula L9. [ka]

[0228] In some embodiments, the selection of a linker for use in one or more of the branched siRNA molecules disclosed herein may be based on the hydrophobicity of the linker, for example, to achieve a desired hydrophobicity for one or more of the branched siRNA molecules of the present disclosure.For example, a linker containing an alkyl chain may be used to increase the hydrophobicity of the branched siRNA molecule compared to a branched siRNA molecule having a less hydrophobic or hydrophilic linker.

[0229] The siRNA agents disclosed herein can be synthesized and / or modified by methods well established in the art, such as those described in Beaucage, SL et al. (eds.), Current Protocols in Nucleic Acid Chemistry, John Wiley & Sons, Inc., New York, NY, 2000, which is incorporated herein by reference.

[0230] Treatment method The MSH3-targeting siRNA molecule of the present disclosure can be delivered to a subject to treat microsatellite repeat expansion disease and / or reduce the phenotype associated with the disease. For example, the siRNA molecule can be delivered to a subject to treat Huntington's disease and / or reduce the phenotype associated with Huntington's disease (e.g., movement disorders such as chorea and dystonia, cognitive disorders, and psychiatric disorders such as depression and anxiety). Alternatively, the MSH3-targeting siRNA molecule of the present disclosure can be delivered to a subject to treat myotonic dystrophy type 1 and / or reduce the phenotype associated with myotonic dystrophy type 1 (e.g., muscle wasting and weakness). As a further alternative, the MSH-targeting siRNA molecule of the present disclosure can be delivered to a subject to treat and reduce the effects of spinocerebellar ataxia or any one of the many syndromes caused by DNA microsatellite repeat expansion. Exemplary microsatellite repeat expansion diseases are described herein and discussed in Rodriguez et al., Neurobiology of Disease, 130:104515, 2019, the disclosure of which is incorporated herein by reference. Additionally, the siRNA molecules of the present disclosure can also be used to treat Huntington's disease, myotonic dystrophy, spinocerebellar ataxia, or another microsatellite repeat expansion disease by delivery to a subject having a variant of the MSH3 gene, where siRNA-mediated gene silencing of the MSH3 variant gene reduces expression levels of the MSH3 transcript.

[0231] The present disclosure provides a method of treating a subject by MSH3 gene silencing with one or more siRNA molecules described herein. The gene silencing can be performed in a subject to silence wild-type MSH3 transcripts, mutant MSH3 transcripts, splice isoforms of MSH3 transcripts, and / or those MSH3 transcripts that are overexpressed compared to healthy subjects. The method can include delivering the siRNA molecule of the present disclosure or a pharmaceutical composition containing the same to the CNS or affected tissue of a subject (e.g., a human) by any suitable route of administration (e.g., intracerebroventricular, intrathecal, intrastriatal, intracisternal injection by catheter insertion, intraparenchymal, intravenous, subcutaneous, or intramuscular injection). The active compound can be administered at any suitable dose. The actual dosage of the composition of the present disclosure administered to a patient can be determined by physical and physiological factors such as body weight, severity of the condition, prior or concurrent therapeutic interventions, idiopathic disease of the patient, and route of administration. Depending on the dosage and route of administration, the preferred dosage and / or the number of times of administration of the effective amount may vary depending on the response of the subject.The practitioner responsible for administration will in any case determine the concentration of active ingredient(s) in the composition and the appropriate dose(s) for each subject.Administration can be carried out any suitable number of times per day for the period required.The subject may be an adult or a child with or without co-morbidities.

[0232] Selecting a target The subject that can be treated with the siRNA molecules disclosed herein is, for example, a subject that needs treatment for microsatellite repeat expansion disease, such as Huntington's disease, and / or any other medical risk(s) associated with DNA microsatellite repeat expansion or gain-of-function mutation in MSH3 gene. The subject that can be treated with the siRNA molecules disclosed herein can include, for example, humans, monkeys, rats, mice, pigs, and other mammals that contain at least one orthologous copy of MSH3 gene. The subject can be an adult or a child with or without comorbidities.

[0233] Pharmaceutical Compositions The siRNA molecule of the present disclosure can be formulated in a pharmaceutical composition for administering to a subject in a biologically compatible form suitable for in vivo administration.Therefore, the present disclosure provides a pharmaceutical composition that contains the siRNA molecule of the present disclosure mixed with suitable diluent, carrier or excipient.The siRNA molecule can be, for example, directly administered to the CNS or diseased tissue of a subject (for example, by intracerebroventricular, intrastriatal, intrathecal injection, intracisternal injection by catheter insertion, intraparenchymal injection, intravenous injection, subcutaneous injection or intramuscular injection).

[0234] Conventional procedures and ingredients for selecting and preparing suitable formulations are described, for example, in Remington, JP The Science and Practice of Pharmacy, Easton, PA. Mack Publishers, 2012, 22 nd ed. and the National Formulary, United States Pharmacopeial, 2015, USP 38 NF 33).

[0235] Under normal storage and use conditions, the pharmaceutical composition may contain preservatives, for example, to prevent the growth of microorganisms.The pharmaceutical composition includes sterile aqueous solutions, sterile dispersions, or powders for extemporaneous preparation of sterile solutions or dispersions.In all cases, the articles can be sterilized using techniques known in the art and can be fluidized to the extent that they can be easily administered to the subject in need of treatment.

[0236] Pharmaceutical compositions may be administered to a subject, e.g., a human subject, alone or in combination with pharma- ceutically acceptable carriers, as provided herein, the proportions of which may be determined by the solubility and / or chemical properties of the compound, the chosen route of administration, and standard pharmaceutical practice.

[0237] Dosing regimen A physician of ordinary skill in the art can easily determine the effective amount of siRNA molecules to be administered to a mammalian subject (e.g., a human) in need of administration. For example, a physician can begin to prescribe a dose of one of the siRNA molecules of the present disclosure at a level lower than that required to achieve a desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. Alternatively, a physician can begin a treatment regimen by administering one of the siRNA molecules of the present disclosure at a high dose, and then administer progressively lower doses until the minimum dosage at which a therapeutic effect (e.g., reduced expression of a target gene sequence) is reached. In general, a suitable daily dose of one of the siRNA molecules of the present disclosure is the amount of the lowest dose of the siRNA molecule effective to produce a therapeutic effect. The ss- or ds-siRNA molecules of the present disclosure can be administered by injection, for example, intrathecal, intracerebroventricular, intracisternal injection by catheter insertion, intraparenchymal, intravenous, subcutaneous, or intramuscular injection. The daily dosage of the therapeutic composition of the siRNA molecule of the present disclosure can be administered as a single dose, or as two, three, four, five, six or more doses administered separately at appropriate intervals throughout the day, week, month or year, optionally in unit dosage form.The siRNA molecule of the present disclosure can be administered alone, but can also be administered in pharmaceutical formulations in combination with excipients, carriers and optionally additional therapeutic agents.

[0238] Route of administration The disclosed methods contemplate any route of administration tolerated by the therapeutic composition, some embodiments of the methods include by intrathecal injection, intracerebroventricular injection, intrastriatal injection, intraparenchymal injection, or by intracisternal injection via catheter insertion.

[0239] Intrathecal injection is a direct injection into the spinal column or subarachnoid space. By injecting directly into the CSF of the spinal column, the siRNA molecules of the present disclosure have direct access to cells (e.g., neurons and glial cells) in the spinal column and have a route to bypass the blood-brain barrier and access cells in the brain.

[0240] Intracerebroventricular (ICV) injection is a method of injection directly into the CSF in the ventricles of the brain. Like intrathecal injection, ICV is an injection method that bypasses the blood-brain barrier. The use of ICV offers the advantage of accessing cells in the brain and spinal column without the risk of the therapeutic agent being degraded in the blood.

[0241] Intrastriatal injection is a direct injection into the striatum (or corpus striatum), a subcortical basal ganglia region of the brain. Injection into the striatum bypasses the blood-brain barrier, avoiding the pharmacokinetic challenges of injection into the bloodstream, and allows direct access to brain cells.

[0242] Intraparenchymal administration is direct injection into the parenchyma (e.g., brain parenchyma). Injection into the brain parenchyma allows for direct injection into the brain region affected by the disease or disorder, bypassing the blood-brain barrier.

[0243] Intracisternal injection by catheterization is a direct injection into the cisterna magna, an area of ​​the brain located between the cerebellum and the dorsal aspect of the medulla oblongata. Injection into the cisterna magna provides more direct delivery to cells in the cerebellum, brainstem, and spinal cord.

[0244] In some embodiments of the methods described herein, the therapeutic composition can be delivered to the subject by systemic administration, for example, intravenous, intramuscular, or subcutaneous administration.

[0245] Intravenous (IV) injection is a method of injection directly into the bloodstream of a subject. IV administration may be in the form of a bolus dose, by continuous infusion, or any other method permitted by the therapeutic composition.

[0246] An intramuscular (IM) injection is an injection into a muscle of the subject, such as the deltoid or gluteal muscle. IM may allow for rapid absorption of the therapeutic composition.

[0247] Subcutaneous injections are injections into the subcutaneous tissue. Absorption of compositions delivered subcutaneously may be slower than IV or IM injections, which may be beneficial for compositions that require continuous absorption. EXAMPLES

[0248] The following examples are presented to provide one of ordinary skill in the art with an illustration of how the compositions and methods described herein can be used, made, and evaluated, and are intended to be purely illustrative of the disclosure and are not intended to limit the scope of what the inventors regard as their disclosure.

[0249] Example 1. Knockdown of MSH3 the purpose This example describes the results of a series of experiments performed to examine the ability of siRNA molecules complementary to specific regions within the human MSH3 mRNA transcript to result in down-regulation of the MSH3 gene.

[0250] Materials and Methods MeWo cells (metastatic melanoma cells, ATCC catalogue number HTB-65) were seeded at 30,000 cells per well in 96-well tissue culture plates in modified Eagle's medium alpha (MEM alpha) containing 6% fetal bovine serum without antibiotics or antimycotics. Equivalent amounts of siRNA were added to the cells at 4 μM or 1 μM in serum-free Opti-MEM, 3% fetal bovine serum, and final assay conditions of 2 μM or 0.5 μM siRNA. Non-siRNA treated cells exposed to the same media composition (untreated) served as controls. Cells were incubated at 37°C and 5% CO 2Cells were grown in a standard cell culture incubator at 4°C for 30 min. After 72 h of siRNA treatment, all cells were lysed, genomic DNA was digested, reverse transcription was performed on the resulting RNA, and quantitative polymerase chain reaction (qPCR) was performed on the resulting cDNA using the TaqMan fast Advanced Cells-to-CT kit (Thermo). Exon-spanning MSH3-specific primers / probes (Thermo) were multiplexed with primers / probes for a housekeeping gene (ATP5B, Thermo) and all reactions were performed in a Quantstudio 7 Flex (Thermo). MSH3 mRNA expression was calculated relative to untreated control cells using the ΔΔCt method. Biological and technical (qPCR) replicates were performed for each condition and all results are expressed as the average of all technical and biological replicates as well as residual mRNA expression as a percentage (%) of untreated control cells.

[0251] result Cells were treated with siRNA molecules of the present disclosure having antisense and sense strands as shown in Tables 3 and 4 below. The knockdown efficiency of the siRNA molecules was measured as a percentage (%) of the remaining mRNA expression level at 2 μM and 0.5 μM compared to untreated. The knockdown results are shown in Table 3 (2 μM) and Table 4 (0.5 μM).

[0252] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6] [Table 4-7] [Table 4-8] [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7] [Table 5-8]

[0253] Example 2. Determination of IC50 of MSH3-targeting siRNA molecules the purpose This example describes the results of a series of experiments performed to determine the IC50 of siRNA molecules complementary to specific regions within human MSH3 mRNA.

[0254] Materials and Methods MeWo cells (metastatic melanoma cells, ATCC catalog number HTB-65) were actively transfected at concentrations ranging from 1 fM to 100 nM using 0.05% RNAiMax as the transfection reagent. Non-siRNA treated cells (untreated) exposed to the same media composition were used as controls. Cells were incubated at 37 °C and 5% CO 2Cells were grown in a standard cell culture incubator at 4°C for 30 min. After 72 h of siRNA treatment, all cells were lysed, genomic DNA was digested, reverse transcription was performed on the resulting RNA, and quantitative polymerase chain reaction (qPCR) was performed on the resulting cDNA using the TaqMan fast Advanced Cells-to-CT kit (Thermo). Exon-spanning MSH3-specific primers / probes (Thermo) were multiplexed with primers / probes for a housekeeping gene (ATP5B, Thermo) and all reactions were performed in a Quantstudio 7 Flex (Thermo). MSH3 mRNA expression was calculated relative to untreated control cells using the ΔΔCt method. Biological and technical (qPCR) replicates were performed for each condition and all results are expressed as the average of all technical and biological replicates as well as residual mRNA expression as a percentage (%) of untreated control cells.

[0255] result Cells were treated with siRNA molecules of the present disclosure having antisense and sense strands as shown below in Table 5. IC50s were calculated and the knockdown results are shown in Table 5.

[0256] [Table 6]

[0257] Example 3. In vivo inhibition of MSH3 gene expression in mice using di-siRNA sequence variants In vivo administration of di-siRNA Twelve siRNA sequences targeting MSH3 were synthesized as di-siRNAs having the structure of Formula XVII. The test substances are shown in Table 6 below.

[0258] [Table 7]

[0259] Each di-siRNA was formulated in PBS. A 20 nmol dose of siRNA was formulated as a divalent cation salt. In vivo studies were performed in FVB / NJ female mice by intracerebroventricular (ICV) administration in groups of 8 animals. Stereotaxic injections were performed on day 1, and bilateral ICV injections (5 μL per side, 10 μL total) of test substances or PBS control were performed at 0.5-2 μL / min after needle placement at the following coordinates from bregma: -0.45 mm or -0.25 mm anterior-posterior, + / -1 mm medial-lateral and -2.5 mm dorsal. Dose levels of 0.5 nmol, 1 nmol, 2.5 nmol, 5 nmol, and / or 20 nmol were used. 28 days after injection, animals were perfused with cold 1x PBS and brains were harvested and sliced. Tissue punch samples of fixed diameter and thickness were taken from different brain regions (motor cortex, hippocampus and striatum) and snap frozen on dry ice.

[0260] Protein expression analysis To assess MSH3 protein expression levels, punch samples of mouse brain tissue were homogenized in Cell Lysis Buffer containing a protease / phosphatase inhibitor cocktail (Cell Signaling Technologies) using a TissueLyser II. Tissue homogenates were centrifuged at 1000 rpm for 5 min to remove air bubbles. Total protein content was normalized to 0.125 mg / mL in 16 μL using BCA (Pierce).

[0261] For MSH3 protein expression analysis, Western blots were performed using the Jess (Protein Simple) system and reagents recommended by the manufacturer. A 12-230 kD separation cartridge was used with Fluorescent Master Mix 1 and an anti-mouse detection module (both from Protein Simple). Samples were probed for total mouse MSH3 expression (MABE324 antibody, EMD Millipore, dilution 1:100) and normalized to the multiplexed housekeeping protein reference mouse actin (MAB8929, R&D Systems, dilution 1:20,000). Comparative analysis was performed for quantification of MSH3 relative to samples taken from PBS-treated mice. The results are shown in Figures 1, 2, 3, 7, and 9.

[0262] RNA expression analysis To assess MSH3 mRNA expression levels, total RNA was extracted from punch samples of mouse brain tissue using phenol:chloroform extraction. This was done by first disrupting tissue samples in TRIzol reagent (Invitrogen) using a TissueLyser II (Qiagen) and adding chloroform to the homogenized samples at a 5:1 TRIzol:chloroform ratio. The tubes were then vigorously shaken and spun at 12,000×g for 15 min, and the resulting upper aqueous phase containing total RNA was carefully removed and added to a clean tube. An equal volume of 70% ethanol was added to each sample and mixed gently. Samples were further purified using Qiagen RNeasy column purification according to the standard kit protocol. Samples were eluted in 40 μL of RNase-free water. After elution, RNA was analyzed on a TapeStation 4200 Bioanalyzer (Agilent) to assess concentration and quality. All samples were normalized for total RNA. cDNA synthesis was performed using the Applied Biosystems High-Capacity cDNA Reverse Transcription Kit in a reaction volume of 20 μL. After RT-PCR, nuclease-free water was added to the cDNA to bring the final sample volume to 50 μL.

[0263] For Htt gene expression analysis, qPCR was performed using TaqMan reagents on a QuantStudio7 Real-Time PCR instrument (Life Technologies). Samples were probed for total mouse MSH3 expression (Assay ID Mm00487756_m1) and normalized to the multiplexed housekeeping gene reference mouse TBP (Assay ID Mm01277042_m1). Comparative analysis was performed for quantification of MSH3 relative to samples taken from PBS-treated mice (ΔΔCt). Results are shown in Figures 1, 4, 5, 6, and 8.

[0264] Statistical evaluation Statistical significance between the PBS control and each treatment group was determined using two-way ANOVA and Dunnett's multiple comparison test, except for FIG. 9, where Sidak's multiple comparison test was used.

[0265] Example 4. Generation of MSH3-targeting siRNA molecules The siRNA molecules of the present disclosure can be synthesized by standard methods well known in the art, for example, by use of an automated DNA synthesizer such as those commercially available from Biosearch, Applied Biosystems, Inc., as discussed further below.

[0266] siRNA agents can be prepared using liquid phase organic synthesis or solid phase organic synthesis, or both. Organic synthesis has the advantage that oligonucleotides containing non-natural or modified nucleotides can be easily prepared. Specific examples of siRNA molecules are shown in Table 1A above, along with the nucleotide sequences of sense and antisense strands, and MutS homolog 3 (MSH3) mRNA target sequence. Those skilled in the art will recognize that antisense (AS) strands can be annealed to corresponding sense (S) strands to generate ds-siRNA molecules. Alternatively, those skilled in the art can use only antisense strands to induce ss-siRNA molecules.

[0267] Example 5. Optimization of MSH3-targeting siRNA molecules It is contemplated that in any of the small interfering RNA (siRNA) agents disclosed herein, the efficacy or biophysical properties of the molecule can be further optimized by modification of the siRNA (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, and / or targeting to a specific location or cell type). Such optimization can be achieved by systematically adding or removing linked nucleosides to generate longer or shorter sequences. Further optimization of the siRNA can include, for example, the incorporation of one or more alternative nucleosides, alternative 2' sugar moieties, and / or alternative internucleoside linkages. Furthermore, such optimized siRNA molecules can include the introduction of hydrophobic and / or stabilizing moieties at the 5' and / or 3' ends.

[0268] Optimizing siRNA with alternative nucleosides Optimization of the siRNA molecules of the present disclosure may include one or more of the following nucleoside modifications: 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C=C-CH3) uracil and cytosine, and other alkynyl derivatives of pyrimidine bases. , 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, and / or 7-deazaguanine and 3-deazaguanine. siRNA molecules can also include nucleobases in which the purine or pyrimidine base is replaced by other heterocycles, such as 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine, and / or 2-pyridone. Further optimization of the siRNA molecules of the present disclosure may also include nucleobases disclosed in US 3,687,808; Kroschwitz, JI, ed. The Concise Encyclopedia of Polymer Science and Engineering, New York, John Wiley & Sons, 1990, pp. 858-859; Englisch et al., Angewandte Chemie, International Edition 30:613, 1991; and Sanghvi, YS, Chapter 16, Antisense Research and Applications, CRC Press, Gait, MJ ed., 1993, pp. 289-302.

[0269] Optimizing siRNAs through alternative sugar modifications Optimization of the siRNA molecules of the present disclosure may include one or more of the following 2' sugar modifications: 2'-O-methyl (2'-O-Me), 2'-methoxyethoxy (2'-O-CH 2 CH 2 OCH 3 , also known as 2'-O-(2-methoxyethyl) or 2'-MOE), 2'-dimethylaminooxyethoxy, i.e., O(CH 2 ) 2 ON(CH 3 ) 2 The group, also known as 2'-DMAOE, and / or 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylamino-ethoxy-ethyl or 2'-DMAEOE), i.e., 2'-O-CH 2 OCH 2 N(CH 3 ) 2 Other possible 2' modifications that can optimize the siRNA molecules of the present disclosure include all possible orientations of OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl include all possible orientations of substituted or unsubstituted C1-C10 alkyl or C2-C10 alkenyl and alkynyl. Other potential sugar substituents include, for example, aminopropoxy (-OCH 2 CH 2 CH 2 NH 2 ), allyl (-CH 2 -CH=CH 2 ), -O-allyl (-O-CH 2 -CH=CH 2), and fluoro (F). The 2'-sugar substituent can be at the arabino (up) or ribo (down) position. In some embodiments, the 2'-arabino modification is 2'-F. Similar modifications may be made at other positions in the siRNA molecule, particularly at the 3' position of the sugar in the 3' terminal nucleoside or 2'-5' linked oligonucleotides and the 5' position of the 5' terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.

[0270] Optimization of siRNAs with alternative internucleoside linkages Optimization of the siRNA molecules of the present disclosure may include one or more of the following internucleoside modifications: phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thinophosphoramidates, thinoalkyl phosphonates, thinoalkyl phosphotriesters, selenophosphates, boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs of these, as well as those with inverted polarity where one or more internucleotide linkages are 3'-3', 5'-5', or 2'-2' linkages.

[0271] Optimization of siRNA using hydrophobic moieties The optimization of the siRNA molecule of the present disclosure may include covalently linked hydrophobic moiety at 5'-end or 3'-end.The non-limiting examples of the hydrophobic moiety suitable for use in the siRNA molecule of the present disclosure may include cholesterol, vitamin D, tocopherol, phosphatidylcholine (PC), docohexaenoic acid, docosanoic acid, PC-docosanoic acid, eicosapentaenoic acid, lithocholic acid, or any combination of the above hydrophobic moiety and PC.

[0272] Optimization of siRNA using stabilizing molecules The optimization of the siRNA molecules of the present disclosure may include a 5'-phosphorus stabilizing moiety that protects the siRNA molecules from degradation. The 5'-phosphorus stabilizing moiety replaces the 5'-phosphate to prevent hydrolysis of the phosphate. Hydrolysis of the 5'-phosphate prevents binding to RISC, which is a necessary step of gene silencing. Any substitution of the phosphate that does not prevent binding to RISC is contemplated in the present disclosure. In some embodiments, the substitution of the 5'-phosphate is also stable to hydrolysis in vivo. Each siRNA strand may independently and optionally employ any suitable 5'-phosphorus stabilizing moiety. Non-limiting examples of 5' stabilizing moieties suitable for use in the siRNA molecules of the present disclosure include those shown in formulas IX to XVI above.

[0273] Optimization of siRNA using branched siRNA The optimization of the siRNA molecule of the present disclosure may include the incorporation of branching patterns, such as bi-branched, tri-branched, or tetra-branched siRNAs connected via linker.Each main branch may be further branched to allow 2, 3, 4, 5, 6, 7, or 8 separate RNA single strands or double strands.The branching points on the linker may originate from the same atom, or may originate from separate atoms along the linker.Some exemplary embodiments are listed in Table 2 above.

[0274] The siRNA compositions of the present disclosure can be optimized to be in the form of a biantennary siRNA molecule represented by any one of formulas XVII-XIX, a triantennary siRNA molecule represented by any one of formulas XX-XXIII, and / or a tetraantennary siRNA molecule represented by any one of formulas XXIV-XXVIII, wherein each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branch point moiety (e.g., a phosphoramidite, a tosylated solketal, 1,3-diaminopropanol, pentaerythritol, or any one of the branch point moieties described in US 10,478,503).

[0275] Example 6. Preparation and administration of MSH3-targeting siRNA molecules The siRNA molecule of the present disclosure can be formulated in a pharmaceutical composition for administration to a subject in a biologically compatible form suitable for in vivo administration.For example, the siRNA molecule of the present disclosure can be administered with a suitable diluent, carrier, or excipient, and can further include a preservative, for example, to prevent microbial growth.The procedure and ingredients for selecting and preparing suitable formulations can be found, for example, in Remington, JP The Science and Practice of Pharmacy, Easton, PA. Mack Publishers, 2012, 22 nd ed. and the National Formulary, United States Pharmacopeial, 2015, USP 38 NF 33).

[0276] The method of the present disclosure contemplates any administration route that can be tolerated by the siRNA composition of the present disclosure to a subject.Non-limiting examples of siRNA injection into the CNS include intrathecal, intracerebroventricular, intrastriatal, intraparenchymal, or intracisternal injection by catheter insertion.Examples of systemic administration include intravenous, intramuscular, and subcutaneous injection.A physician of ordinary skill in the art can easily determine effective administration route.

[0277] Example 7. Methods for treating microsatellite repeat expansion diseases using siRNA molecules targeting MSH3 A subject in need of treatment for a microsatellite repeat expansion disease, such as Huntington's disease, is treated with a dose of the siRNA molecule or siRNA composition of the present disclosure formulated as a salt, at a frequency determined by the physician. A physician of ordinary skill in the art can easily determine an effective amount of the siRNA molecule to be administered to a mammalian subject (e.g., a human) in need of administration. For example, a physician can begin prescribing a dose of one of the siRNA molecules of the present disclosure at a level lower than that required to achieve a desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. Alternatively, a physician can begin a treatment regimen by administering one of the siRNA molecules of the present disclosure at a high dose, and then administer decreasing doses until a minimum dose that produces a therapeutic effect (e.g., a reduction in the expression of MSH3 mRNA or a suitable biomarker) is achieved. In general, a suitable daily dose of one of the siRNA molecules of the present disclosure is the amount of the minimum dose that is effective to produce a therapeutic effect. The ss-siRNA molecule or ds-siRNA molecule of the present disclosure can be administered by injection, for example, intrathecal, intracerebroventricular, intrastriatal, intraparenchymal, intravenous, intramuscular, or intracisternal injection by catheter insertion.The daily dose of the therapeutic composition of one of the siRNA molecules of the present disclosure can be administered as a single dose, or as two, three, four, five, six or more doses administered separately at appropriate intervals throughout the day, week, month, or year, optionally in unit dosage form.Any of the siRNA molecules of the present disclosure can be administered alone, but can also be administered in pharmaceutical formulations in combination with excipients, carriers, and optionally additional therapeutic agents.The dosage and frequency are determined based on the height, weight, age, sex, and other disorders of the subject.

[0278] The siRNA molecule of the present disclosure is selected by a physician considering its compatibility with a subject.Can select single-stranded or double-stranded siRNA molecules (e.g., unbranched siRNA, bibranched siRNA, tri-branched siRNA, tetra-branched siRNA).Selected siRNA molecule can have antisense strand and sense strand with the most suitable sequence and RNA modification (e.g., natural and non-natural internucleoside bond, modified sugar, 5' phosphorus stabilizing portion, hydrophobic portion, and / or branched structure) for that patient.

[0279] The siRNA molecules are delivered by the route most appropriate for the patient and condition (e.g., injection, e.g., intrathecal, intracerebroventricular, intrastriatal, intraparenchymal, intravenous, intramuscular, or intracisternal injection via catheterization) at a rate that can be tolerated by the patient until the subject reaches the maximum tolerated dose or until symptoms are sufficiently alleviated.

[0280] Other embodiments All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0281] While the invention has been described in connection with specific embodiments thereof, it will be understood that the invention is capable of further modifications, and that this application is intended to cover any variations, uses, or adaptations of the invention which are generally in accordance with the principles of the invention, including departures therefrom within known or customary practice in the art to which the invention pertains, and which are applicable to the essential features described above and fall within the scope of the claims.

[0282] Other embodiments are within the scope of the claims.

Claims

1. A small interfering RNA (siRNA) molecule comprising an antisense strand and a sense strand complementary to the antisense strand, wherein the antisense strand is 10 to 30 nucleotides in length and has sufficient complementarity to hybridize to a region in a MutS homolog 3 (MSH3) mRNA transcript having one nucleic acid sequence of sequence numbers 1 to 408.

2. The aforementioned antisense chain, (i) Having at least 70% complementarity to a region of 21 consecutive nucleic acid bases within the MSH3 mRNA transcript having one nucleic acid sequence of any one of sequence numbers 1 to 408, and / or (ii) comprising at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or 30 consecutive nucleotides that are completely complementary to the consecutive polynucleotide segments of equal length within the region of the MSH3 RNA transcript having the nucleic acid sequence of any one of sequence numbers 1 to 408, The siRNA molecule according to claim 1.

3. The aforementioned antisense chain, (i) 18 to 21 consecutive nucleotides that are completely complementary to a contiguous polynucleotide segment of equal length within the region of the MSH3 RNA transcript having one nucleic acid sequence of any one of sequence numbers 1 to 408, and / or (ii) Nine or fewer nucleotide mismatches in a region of 21 consecutive nucleic acid bases of the MSH3 RNA transcript having one nucleic acid sequence of any one of sequence numbers 1 to 408. The siRNA molecule according to claim 1, comprising:

4. The region of the MSH3 RNA transcript is (i) Any one nucleic acid sequence of sequence numbers 1-24, 40-84, 100, 118-139, 173-181, 193-205, 220-222, 240-242, 249-308, 319-330, 336-377, 384-386, and 398-408, or (ii) The nucleic acid sequence described in any one of SEQ ID NOs: 5, 17-20, 33, 42, 44, 102, 103, 105, 106, 108, 109, 113, 126, 129, 130, 158, 161, 166, 177, 183, 193, 194, 196, 197, 202, 209, 217, 231, 271, 273, 291, 294, 305, 307, 317, 318, and 362 A siRNA molecule according to claim 1, having the following characteristics.

5. The siRNA molecule according to claim 1, wherein the antisense strand has a nucleic acid sequence that is at least 85% identical to any one of the nucleic acid sequences of sequence numbers 817 to 1224.

6. The nucleic acid sequence is (i) Any one of sequence numbers 817-840, 856-900, 916, 934-955, 989-997, 1009-1021, 1036-1038, 1056-1058, 1065-1124, 1135-1146, 1152-1193, 1200-1202, and 1214-1224, or (ii) Any one of sequence numbers 821, 833-836, 849, 858, 860, 918, 919, 921, 922, 924, 925, 929, 942, 945, 946, 974, 977, 982, 993, 999, 1009, 1010, 1012, 1013, 1018, 1025, 1033, 1047, 1087, 1089, 1107, 1110, 1121, 1123, 1133, and 1134 The siRNA molecule according to claim 5.

7. The siRNA molecule according to claim 1, wherein the sense strand has a nucleic acid sequence that is at least 85% identical to any one of the nucleic acid sequences of sequence numbers 409 to 816.

8. The nucleic acid sequence is (i) Any one of sequence numbers 409-432, 448-492, 508, 526-547, 581-589, 601-613, 628-630, 648-650, 657-716, 727-738, 744-785, 792-794, and 806-816, or (ii) Any one of sequence numbers 413, 425-428, 441, 450, 452, 510, 511, 513, 514, 516, 517, 521, 534, 537, 538, 566, 569, 574, 585, 591, 601, 602, 604, 605, 610, 617, 625, 639, 679, 681, 699, 702, 713, 715, 725, 726, and 770 The siRNA molecule according to claim 7.

9. The aforementioned antisense chain, (i) It has a structure represented by formula I, where formula I is as follows in the 5' to 3' direction: A - B - (A') ｊ - C - P ２ - D - P １ -(C' - P １ ) ｋ - C' Formula I; In the formula, A is equal to formula C - P １ -D-P １ It is represented by; Each A' is of the formula C-P ２ -D-P ２ and is represented by; B is given by equation C - P ２ -D-P ２ -D-P ２ -D-P ２ It is represented by; Each C is a 2'-O-methyl (2'-O-Me) ribonucleoside; Each C' is independently a 2'-O-Me ribonucleoside or a 2'-fluoro(2'-F) ribonucleoside; Each D is a 2'-F ribonucleoside; Each P １ This is a phosphorothioate nucleoside bond; Each P ２ This is a phosphodiester nucleoside interbonding; j is an integer between 1 and 7; k is an integer from 1 to 7. (ii) Having a structure represented by equation II, where equation II is as follows in the 5' to 3' direction: A - B - (A') ｊ - C - P ２ - D - P １ -(C - P １ ) ｋ - C' Formula II; In the formula, A is equal to formula C - P １ -D-P １ It is represented by; Each A' is given by formula C - P ２ -D-P ２ It is represented by; B is given by equation C - P ２ -D-P ２ -D-P ２ -D-P ２ It is represented by; Each C is a 2'-O-methyl (2'-O-Me) ribonucleoside; Each C' is independently a 2'-O-Me ribonucleoside or a 2'-fluoro(2'-F) ribonucleoside; Each D is a 2'-F ribonucleoside; Each P １ This is a phosphorothioate nucleoside bond; Each P ２ This is a phosphodiester nucleoside interbonding; j is an integer between 1 and 7; k is an integer from 1 to 7. (iii) Having a structure represented by formula IV, where formula IV is as follows in the 5' to 3' direction: A-(A') ｊ -C-P ２ -B-(C-P １ ) ｋ -C' Formula IV; In the formula, A is equal to formula C - P １ -D-P １ It is represented by; Each A' is given by formula C - P ２ -D-P ２ It is represented by; B is given by equation D - P １ -C-P １ -D-P １ It is represented by; Each C is a 2'-O-Me ribonucleoside; Each C' is independently a 2'-O-Me ribonucleoside or a 2'-F ribonucleoside; Each D is a 2'-F ribonucleoside; Each P １ This is a phosphorothioate nucleoside bond; Each P ２ This is a phosphodiester nucleoside interbonding; j is an integer between 1 and 7; k is an integer from 1 to 7, or (iv) Having a structure represented by formula VI, where formula VI is as follows in the 5' to 3' direction: A-B ｊ -E-B ｋ -E-F-G ｌ -D-P １ -C' formula VI; In the formula, A is equal to formula C - P １ -D-P １ It is represented by; Each B is given by formula C - P ２ It is represented by; Each C is a 2'-O-Me ribonucleoside; Each C' is independently a 2'-O-Me ribonucleoside or a 2'-F ribonucleoside; Each D is a 2'-F ribonucleoside; Each E is given by equation D - P ２ -C-P ２ It is represented by; F is given by equation D - P １ -C-P １ It is represented by; Each G is given by formula C - P １ It is represented by; Each P １ This is a phosphorothioate nucleoside bond; Each P ２ This is a phosphodiester nucleoside interbonding; j is an integer between 1 and 7; k is an integer between 1 and 7; The siRNA molecule according to claim 1, wherein l is an integer from 1 to 7.

10. The antisense chain includes a structure represented by any one of formulas A1 to A4, wherein formulas A1 to A4 are as follows in the 5' to 3' directions: A-S-B-S-A-O-B-O-B-O-B-O-A-O-BO-A-O-BO-A-O-BO-O-A-O-B-O-A-O-B-S-A-S-A-S-A-S-B-S-A Formula A1; A-S-B-S-A-O-B-O-B-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-O-A-O-B-S-A-S-A-S-A-S-A-S-A-S-A Form A2; A-S-B-S-A-O-B-O-A-O-B-O-A-O-BO-A-O-BO-A-O-BO-O-A-O-B-O-A-O-B-S-A-S-B-S-A-S-A-S-A Formula A3; and A-S-B-S-A-O-A-O-A-O-BO-A-O-A-O-A-O-A-O-A-O-A-O-A-O-B-O-A-O-B-S-A-S-A-S-A-S-B-S-A Formula A4; The siRNA molecule according to claim 9, wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester nucleoside bond, and S represents a phosphorothioate nucleoside bond.

11. The aforementioned sense chain, (i) It has a structure represented by formula III, and formula III is as follows in the 5' to 3' direction: E-(A') ｍ -F Formula III; In the formula, E is given by (C - P １ ) ２ It is represented by; F is represented by the formula (C-P ２ ), ３ -D-P １ -C-P １ -C, (C-P ２ ), ３ -D-P ２ -C-P ２ -C, (C-P ２ ), ３ -D-P １ -C-P １ -D, or (C-P ２ ), ３ -D-P ２ -C-P ２ -D; A', C, D, P １ , and P ２ This is defined by equation II; m is an integer from 1 to 7, or (ii) The structure includes the structure represented by equation V, where equation V is as follows in the 5' to 3' direction: E-(A') ｍ -C-P ２ -F type V; In the formula, E is given by (C - P １ ) ２ It is represented by; F is represented by the formula D - P １ - C - P １ - C, D - P ２ - C - P ２ - C, D - P １ - C - P １ - D, or D - P ２ - C - P ２ - D; A', C, D, P １ , and P ２ This is defined by formula IV; m is an integer from 1 to 7, or (iii) Having a structure represented by formula VII, where formula VII is as follows in the 5' to 3' direction: H-B ｍ -I ｎ -A'-B ｏ -H-C formula VII; where A' is represented by the formula C-P ２ -D-P ２ ; Each H is given by equation (C - P １ ) ２ It is represented by; Each I is independent of the equation (D - P ２ ) is represented as; B, C, D, P １ , and P ２ This is defined by formula VI; m is an integer between 1 and 7; n is an integer between 1 and 7; The siRNA molecule according to claim 1, wherein o is an integer from 1 to 7.

12. The sense chain includes a structure represented by any one of formulas S1 to S9, wherein formulas S1 to S9 are as follows in the 5' to 3' directions: A-S-A-S-A-O-BO-O-A-O-BO-A-O-BO-A-O-BO-O-A-O-A-O-A-O-B-S-A-S-A Formula S1; A-S-A-S-A-O-BO-O-A-O-BO-A-O-BO-A-O-BO-O-A-O-A-O-A-O-B-O-A-O-A Formula S2; A-S-A-S-A-O-BO-O-A-O-BO-A-O-BO-A-O-BO-O-A-O-A-O-A-O-B-S-A-SB Formula S3; A-S-A-S-A-O-BO-O-A-O-BO-A-O-BO-A-O-BO-O-A-O-A-O-A-O-B-O-A-O-B Formula S4; A-S-A-S-A-O-BO-O-A-O-BO-A-O-BO-A-O-BO-O-A-O-BO-O-A-O-B-S-A-SA Formula S5; A-S-A-S-A-O-BO-O-A-O-BO-A-O-BO-A-O-BO-O-A-O-BO-O-A-O-B-O-A-O-A Formula S6; A-S-A-S-A-O-B-O-A-O-BO-A-O-BO-A-O-BO-O-A-O-B-O-A-O-B-S-A-SB Formula S7; A-S-A-S-A-O-BO-O-A-O-BO-A-O-BO-A-O-BO-O-A-O-BO-O-A-O-B-O-A-O-B Formula S8; and A-S-A-S-A-O-A-O-A-O-B-O-BO-O-BO-A-O-BO-O-A-O-A-O-A-O-A-S-A-S-A Formula S9; The siRNA molecule according to claim 11, wherein A represents a 2'-O-Me ribonucleoside, B represents a 2'-F ribonucleoside, O represents a phosphodiester nucleoside bond, and S represents a phosphorothioate nucleoside bond.

13. (i) The antisense chain further comprises a 5' phosphorus-stabilizing moiety at the 5' end of the antisense chain, and / or (ii) The sense chain further includes a 5' phosphorus-stabilizing moiety at the 5' end of the sense chain, The siRNA molecule according to claim 1.

14. (i) Each 5'-phosphorus stabilization part is independently represented by one of the equations IX to XVI, 【Chemistry 1】 In the formula, Nuc represents a nucleic acid base selected from the group consisting of adenine, uracil, guanine, thymine, and cytosine, and R represents optionally a substituted alkyl, optionally a substituted alkenyl, optionally a substituted alkynyl, phenyl, benzyl, or hydrogen, or (ii) The 5'-phosphorus stabilizing portion is an (E)-vinylphosphonate represented by formula XI, The siRNA molecule according to claim 13.

15. The siRNA molecule according to claim 1, wherein the length of the sense strand is 12 to 30 nucleotides.

16. The aforementioned siRNA molecule (i) A bifurcated siRNA molecule represented by any one of formulas XVII to XIX, 【Chemistry 2】 (ii) A tribranched siRNA molecule represented by any one of the formulas XX to XXIII, 【Transformation 3】 Or, (iii) A tetrabranched siRNA molecule represented by any one of the formulas XXIV to XXVIII 【Chemistry 4】 And, The siRNA molecule according to claim 15, wherein each RNA is independently an siRNA molecule, L is a linker, and each X independently represents a branching point.

17. The siRNA molecule according to claim 16, wherein the linker is selected from the group consisting of 2 to 20 consecutive subunits from ethylene glycol, alkyl, carbohydrate, block copolymer, peptide, RNA, and DNA.

18. A pharmaceutical composition comprising the siRNA molecule described in claim 1 and a pharmaceutically acceptable excipient, carrier, or diluent.

19. An siRNA molecule according to any one of claims 1 to 17 or a pharmaceutical composition according to claim 18 for use in delivering an siRNA molecule to a subject diagnosed with a microsatellite repeat extension disorder.

20. An siRNA molecule according to any one of claims 1 to 17 or a pharmaceutical composition according to claim 18 for use in treating a microsatellite repeat extension disorder in a subject requiring treatment for the microsatellite repeat extension disorder, wherein the microsatellite repeat extension disorder is (i) Huntington's disease, (ii) Spinocerebellar ataxia, (iii) Fragile X syndrome, or (iv) Myotonic dystrophy The siRNA molecule or pharmaceutical composition described above.

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