Modified short interfering nucleic acid (siNA) molecules and uses thereof

Abstract

Short interfering nucleic acid (siNA) molecules, compositions, and methods containing modified nucleotides and their uses are described.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119 to Provisional Application No. 63 / 421,946, filed November 2, 2022, and Provisional Application No. 63 / 591,984, filed October 20, 2023, the disclosures of which are incorporated herein by reference in their entireties.

[0002] Short interfering nucleic acid (siNA) molecules, compositions, and methods containing modified nucleotides and their uses are described. [Background technology]

[0003] RNA interference (RNAi) is a biological response to double-stranded RNA that mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids and regulates the expression of protein-coding genes. Short interfering nucleic acids (siNAs), such as siRNAs, have been developed for RNAi therapy to treat various diseases. For example, RNAi therapy has been proposed for the treatment of metabolic diseases, neurodegenerative diseases, cancer, and pathogenic infectious diseases (see, e.g., Rondindone, Biotechniques, 2018, 40(4S), doi.org / 10.2144 / 000112163; Boudreau and Davidson, Curr Top Dev Biol, 2006, 75:73-92; Chalbatani et al., Int J Nanomedicine, 2019, 14:3111-3128; Arbuthnot, Drug News Perspect, 2010, 23(6):341-50; and Chernikov et al., Front. Pharmacol., 2019, doi.org / 10.3389 / fphar.2019.00444, each of which is incorporated by reference in its entirety). However, the major limitations of RNAi therapy are the ability to effectively deliver siRNA to target cells and the degradation of siRNA.

[0004] The present disclosure provides siNA molecules containing modified nucleobases to improve the delivery and stability of siNA molecules. The siNA molecules of the present disclosure provide optimized combinations and numbers of modified nucleotides, nucleotide lengths, designs (e.g., blunt ends or overhangs, internucleoside linkages, conjugates), and modification patterns to improve the delivery and stability of siNA molecules. Summary of the Invention

[0005] Described herein are short interfering nucleic acid (siNA) molecules containing novel modified nucleobase monomers, phosphate mimetics, and / or other modifications. Also described herein are methods of using the disclosed siNA molecules to treat various diseases and conditions.

[0006] In a first aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: [ka] and a nucleotide having a structure selected from wherein B is a nucleobase selected from adenine, guanine, cytosine, thymine and uracil, an aryl, a heteroaryl, or H; TIFF2025535539000003.tif8161 provides oligonucleotides that exhibit phosphodiester linkages, phosphorothioate linkages, mesyl phosphoramidate linkages, or H. For example, the oligonucleotides may be [ka] [ka] [ka] wherein B is a nucleobase, aryl, heteroaryl, or H; TIFF2025535539000007.tif8161 represents a phosphodiester bond, a phosphorothioate bond, a mesyl phosphoramidate bond, or H.

[0007] In some embodiments, the oligonucleotide is [ka] [ka] wherein B is a nucleobase, aryl, heteroaryl, or H; TIFF2025535539000010.tif8161 represents a phosphodiester bond, a phosphorothioate bond, a mesyl phosphoramidate bond, or H.

[0008] In a second aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: [ka] and at least two, at least three, at least four, or at least five nucleotide analogs having structures independently selected from: wherein B is a nucleobase, aryl, heteroaryl, or H; TIFF2025535539000012.tif8161 provides short interfering nucleic acids (siNAs), where * represents a phosphodiester bond, a phosphorothioate bond, or H, and * represents a chiral center (e.g., R or S isomer). For example, oligonucleotides can be [ka] and a nucleotide analog having the structure wherein B is a nucleobase, aryl, heteroaryl, or H; TIFF2025535539000014.tif8161 provides oligonucleotides where H represents a phosphodiester bond, a phosphorothioate bond, or H, and * represents a chiral center (e.g., R or S isomer).

[0009] In a third aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: [ka] wherein B is a nucleobase, aryl, heteroaryl, or H; TIFF2025535539000016.tif8161 provides oligonucleotides exhibiting phosphodiester, phosphorothioate, or mesyl phosphoramidate linkages.

[0010] In a fourth aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: [ka] wherein each B is independently selected from a nucleobase, an aryl, a heteroaryl, and H; TIFF2025535539000018.tif8161 provides oligonucleotides exhibiting phosphodiester, phosphorothioate, or mesyl phosphoramidate linkages.

[0011] In some embodiments, the oligonucleotide is selected from a short interfering nucleic acid (siNA), an antisense oligonucleotide (ASO), a steric blocker, a short hairpin RNA (shRNA), and an mRNA.

[0012] In a fifth aspect, the present disclosure provides a short interfering nucleic acid (siNA) comprising a sense strand and an antisense strand, wherein the sense strand, the antisense strand, or both, comprise: [ka] [ka] at least one, at least two, at least three, at least four, or at least five nucleotides independently selected from: [ka] at least one, at least two, at least three, at least four, or at least five nucleotide analogues independently selected from wherein B is a nucleobase, aryl, heteroaryl, or H; TIFF2025535539000022.tif8161 provides short interfering nucleic acids (siNAs), where H represents a phosphodiester bond, a phosphorothioate bond, or H, and * represents a chiral center (e.g., R or S isomer).

[0013] In a sixth aspect, the present disclosure provides a method for producing a cellular membrane comprising: (a) a sense strand comprising a first nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to an RNA corresponding to a target gene, wherein the first nucleotide sequence is (i) 15 to 30 nucleotides in length, and (ii) a sense strand comprising 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least one modified nucleotide is a 2'-O-methyl nucleotide and the nucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and / or 19 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides, or at least one modified nucleotide is a 2'-O-methyl nucleotide and at least one modified nucleotide is a 2'-fluoro nucleotide; and an antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to RNA corresponding to the target gene, wherein the second nucleotide sequence is (iii) 15 to 30 nucleotides in length, and (iv) an antisense strand comprising 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least one modified nucleotide is a 2'-O-methyl nucleotide and at least one modified nucleotide is a 2'-fluoro nucleotide; or (b) a sense strand comprising a first nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to an RNA corresponding to the target gene, wherein the first nucleotide sequence is (i) 15 to 30 nucleotides in length, and (ii) a sense strand comprising 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least one modified nucleotide is a 2'-O-methyl nucleotide and at least one modified nucleotide is a 2'-fluoro nucleotide; and an antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to RNA corresponding to the target gene, wherein the second nucleotide sequence is (iii) 15 to 30 nucleotides in length, and (iv) an antisense strand comprising 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least one modified nucleotide is a 2'-O-methyl nucleotide and the nucleotides at positions 2, 5, 6, 7, 8, 10, 14, 16, 17, and / or 18 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides; The sense strand and / or the antisense strand are [ka] at least one, at least two, at least three, at least four, or at least five nucleotides independently selected from [ka] at least one, at least two, at least three, at least four, or at least five nucleotide analogues independently selected from wherein B is a nucleobase, aryl, heteroaryl, or H; TIFF2025535539000025.tif8161 provides oligonucleotides where H represents a phosphodiester bond, a phosphorothioate bond, or H, and * represents a chiral center (e.g., R or S isomer).

[0014] In some embodiments, the antisense strand of the siNA is [ka] wherein R y is the nucleobase and R 15 is H or CH3, TIFF2025535539000027.tif8161 represents a phosphodiester bond, a phosphorothioate bond, or a mesyl phosphoramidate bond.

[0015] In some embodiments, the antisense strand of the siRNA comprises a 5′ stabilized end cap selected from the group consisting of Formula (1) to Formula (16), Formula (9X) to Formula (12X), Formula (16X), Formula (9Y) to Formula (12Y), Formula (16Y), Formula (21) to Formula (36), Formula 36X, Formula (41) to (56), Formula (49X) to (52X), Formula (49Y) to (52Y), Formula 56X, Formula 56Y, Formula (61), Formula (62), and Formula (63), wherein R x is a nucleobase, aryl, heteroaryl, or H.

[0016] In some embodiments, the antisense strand of the siNA comprises a 5′ stabilized endcap selected from the group consisting of Formula (71)-(86), Formula (79X)-(82X), Formula (79Y)-(82Y), Formula 86X, Formula 86X′, Formula 86Y, and Formula 86Y′, wherein R x is a nucleobase, aryl, heteroaryl, or H.

[0017] In some embodiments, the antisense strand of the siNA comprises a 5' stabilized end cap selected from the group consisting of formulas (1A) to (15A), (1A-1) to (7A-1), (1A-2) to (7A-2), (1A-3) to (7A-3), (1A-4) to (7A-4), (9B) to (12B), (9AX) to (12AX), (9AY) to (12AY), (9BX) to (12BX), and (9BY) to (12BY).

[0018] In some embodiments, the antisense strand of the siNA molecule comprises a 5' stabilized end cap selected from the group consisting of formulas (21A) to (35A), formulas (29B) to (32B), formulas (29AX) to (32AX), formulas (29AY) to (32AY), formulas (29BX) to (32BX), and formulas (29BY) to (32BY).

[0019] In some embodiments, the antisense strand of the siRNA comprises a 5' stabilized end cap selected from the group consisting of Formulas (71A) to (86A), Formulas (79XA) to (82XA), Formulas (79YA) to (82YA), Formula (86XA), Formula (86X'A), Formula (86Y), and Formula (86Y').

[0020] In a seventh aspect, the present disclosure provides a short interfering nucleic acid (siNA) comprising a sense strand and an antisense strand, wherein the antisense strand comprises: [ka] wherein each B is independently selected from a nucleobase, an aryl, a heteroaryl, and H; TIFF2025535539000029.tif8161 provides short interfering nucleic acids (siNAs) that display phosphodiester, phosphorothioate, or mesyl phosphoramidate linkages.

[0021] In some embodiments, the sense strand of the siNA, the antisense strand of the siNA, or both [ka] at least one, at least two, at least three, at least four, or at least five nucleotides independently selected from: [ka] wherein B is a nucleobase, aryl, heteroaryl, or H; TIFF2025535539000032.tif8161 represents a phosphodiester bond, a phosphorothioate bond, or H, and * represents a chiral center (e.g., R or S isomer). In some embodiments, the sense strand, the antisense strand, or both, each independently, comprise one or more phosphorothioate internucleoside linkages. In some embodiments, the siNA further comprises a phosphorylation blocker.

[0022] In some embodiments, the sense strand of the siNA comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more phosphorothioate internucleoside linkages. In some embodiments, at least one phosphorothioate internucleoside linkage in the sense strand is between the nucleotides 1 and 2 from the 5' end of the sense strand, and / or at least one phosphorothioate internucleoside linkage in the sense strand is between the nucleotides 2 and 3 from the 5' end of the sense strand.

[0023] In some embodiments, the antisense strand of the siNA further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more phosphorothioate internucleoside linkages. In some embodiments, at least one phosphorothioate internucleoside linkage in the antisense strand is between nucleotides 1 and 2 from the 5' end of the antisense strand, at least one phosphorothioate internucleoside linkage in the antisense strand is between nucleotides 2 and 3 from the 5' end of the antisense strand, at least one phosphorothioate internucleoside linkage in the antisense strand is between nucleotides 1 and 2 from the 3' end of the antisense strand, and / or at least one phosphorothioate internucleoside linkage is between nucleotides 2 and 3 from the 3' end of the antisense strand.

[0024] In some embodiments, the sense strand of the siNA further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mesyl phosphoramidate internucleoside linkages. In some embodiments, at least one mesyl phosphoramidate internucleoside linkage in the sense strand is between the nucleotides 1 and 2 from the 5' end of the sense strand, and at least one mesyl phosphoramidate internucleoside linkage is between the nucleotides 2 and 3 from the 5' end of the sense strand.

[0025] In some embodiments, the antisense strand of the siNA further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mesyl phosphoramidate internucleoside linkages. In some embodiments, at least one mesyl phosphoramidate internucleoside linkage in the antisense strand is between nucleotides 1 and 2 from the 5' end of the antisense strand, at least one mesyl phosphoramidate internucleoside linkage in the antisense strand is between nucleotides 2 and 3 from the 5' end of the antisense strand, at least one mesyl phosphoramidate internucleoside linkage in the antisense strand is between nucleotides 1 and 2 from the 3' end of the antisense strand, and / or at least one mesyl phosphoramidate internucleoside linkage is between nucleotides 2 and 3 from the 3' end of the antisense strand.

[0026] In some embodiments, the sense strand of the siNA, the antisense strand of the siNA, or both, each independently: [ka] (In the formula, R x is a nucleobase, aryl, heteroaryl, or H), [ka] (In the formula, R y are nucleobases), [ka] (In the formula, R y are nucleobases), or combinations thereof.

[0027] In some embodiments, the siNA further comprises a galactosamine. In some embodiments, the galactosamine is N-acetylgalactosamine (GalNAc) of formula (VI): [ka] wherein m is 1, 2, 3, 4, or 5; each n is independently 1 or 2; p is 0 or 1; each R is independently H; each Y is independently selected from -OP(=O)(SH)-, -OP(=O)(O)-, -OP(=O)(OH)-, and -OP(S)S-; Z is H or a second protecting group; any L is a linker, or L and Y together are a linker; and A is H, OH, a third protecting group, an activating group, or an oligonucleotide. In some embodiments, the galactosamine is N-acetylgalactosamine (GalNAc) of formula (VII): [ka] In the formula, R z is OH or SH, and each n is independently 1 or 2.

[0028] In some embodiments, at least one end of the siNA is blunt-ended, at least one end of the siNA comprises an overhang, the overhang comprising at least one nucleotide, or both ends of the siNA comprise overhangs, the overhang comprising at least one nucleotide.

[0029] In some embodiments, the target gene of the siNA is a viral gene, and the gene is derived from a DNA virus, a double-stranded DNA (dsDNA) virus, a hepadnavirus, a hepatitis B virus (HBV), or a gene from any of HBV genotypes A to J, or the target gene is selected from the S gene or X gene of HBV.

[0030] In a seventh aspect, the present disclosure provides a siNA shown in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, Table 10, Table 11, Table 12, Table 13, Table 14, Table 15, Table 16, Table 17, or Table 18.

[0031] The present disclosure provides a composition comprising a siNA according to any one of the siNAs disclosed herein and a pharmaceutically acceptable excipient. In some embodiments, the composition comprises two, three, four, five, six, seven, eight, nine, ten, or more of the siNAs disclosed herein. In some embodiments, the composition comprises an additional therapeutic agent. In some embodiments, the additional therapeutic agent is selected from a nucleotide analog, a nucleoside analog, a capsid assembly modulator (CAM), a recombinant interferon, an entry inhibitor, a small molecule immune modulator, and an oligonucleotide therapy. In some embodiments, the oligonucleotide therapy is an additional siNA, an antisense oligonucleotide (ASO), a NAP, or STOPS™.

[0032] The present disclosure provides methods for treating a disease in a subject in need thereof, comprising administering to the subject a siNA disclosed herein or a composition comprising a siNA disclosed herein. The present disclosure further provides uses of the disclosed siNAs and compositions for treating a disease in a subject. The present disclosure further provides siNAs and compositions for use in treating a disease in a subject.

[0033] In some embodiments of the methods and uses of the present disclosure, the disease is a viral disease, optionally caused by a DNA virus or a double-stranded DNA (dsDNA) virus. In some embodiments, the dsDNA virus is a hepadnavirus. In some embodiments, the hepadnavirus is hepatitis B virus (HBV), optionally wherein the HBV is selected from HBV genotypes A-J. In some embodiments, the methods and uses may further comprise administering an additional HBV therapeutic agent. In some embodiments, the siNA or composition and the additional HBV therapeutic agent are administered simultaneously or sequentially. In some embodiments, the additional HBV therapeutic agent is selected from a nucleotide analog, a nucleoside analog, a capsid assembly modulator (CAM), a recombinant interferon, an entry inhibitor, a small molecule immune modulator, and an oligonucleotide therapy. In some embodiments, the viral disease is a disease caused by a coronavirus, optionally wherein the coronavirus is SARS-CoV-2.

[0034] In some embodiments of the methods and uses of the present disclosure, the disease is a liver disease. In some embodiments, the liver disease is non-alcoholic fatty liver disease (NAFLD) or hepatocellular carcinoma (HCC). In some embodiments, the NAFLD is non-alcoholic steatohepatitis (NASH). Some embodiments may further comprise administering to the subject a drug for treating liver disease. In some embodiments, the drug for treating liver disease is selected from a peroxisome proliferator-activated receptor (PPAR) agonist, a farnesoid X receptor (FXR) agonist, a lipid-altering agent, and an incretin-based therapy. In some embodiments, (i) the PPAR agonist is selected from a PPARα agonist, a dual PPARα / δ agonist, a PPARγ agonist, and a dual PPARα / γ agonist, (ii) the lipid-altering agent is aramchol, or (iii) the incretin-based therapy is a glucagon-like peptide 1 (GLP-1) receptor agonist or a dipeptidyl peptidase 4 (DPP-4) inhibitor. In some embodiments, the siNA or composition and the liver disease therapeutic agent are administered simultaneously or sequentially.

[0035] In some embodiments of the methods and uses of the present disclosure, the siNA or composition is administered at a dose of at least 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, or 15 mg / kg.

[0036] In some embodiments of the disclosed methods and uses, the siNA or composition may be administered in an amount of 0.5 mg / kg to 50 mg / kg, 0.5 mg / kg to 40 mg / kg, 0.5 mg / kg to 30 mg / kg, 1 mg / kg to 50 mg / kg, 1 mg / kg to 40 mg / kg, 1 mg / kg to 30 mg / kg, 1 mg / kg to 20 mg / kg, 3 mg / kg to 50 mg / kg, 3 mg / kg to 40 mg / kg, 3 mg / kg to 30 mg / kg, 3 ... It is administered at a dose of 15mg / kg, 3mg / kg to 10mg / kg, 4mg / kg to 50mg / kg, 4mg / kg to 40mg / kg, 4mg / kg to 30mg / kg, 4mg / kg to 20mg / kg, 4mg / kg to 15mg / kg, 4mg / kg to 10mg / kg, 5mg / kg to 50mg / kg, 5mg / kg to 40mg / kg, 5mg / kg to 30mg / kg, 5mg / kg to 20mg / kg, 5mg / kg to 15mg / kg, or 5mg / kg to 10mg / kg.

[0037] In some embodiments of the methods and uses of the disclosure, the siNA or composition is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0038] In some embodiments of the methods and uses of the present disclosure, the siNA or composition is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per day, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per week, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per month.

[0039] In some embodiments of the methods and uses of the disclosure, the siNA or composition is administered at least once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days.

[0040] In some embodiments of the disclosed methods and uses, the siNA or composition is administered for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days, or for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 51, 52, 53, 54, or 55 weeks.

[0041] In some embodiments of the methods and uses of the present disclosure, the siNA or composition is administered at a single dose of 5 mg / kg or 10 mg / kg, three doses of 10 mg / kg once a week, three doses of 10 mg / kg once every three days, or five doses of 10 mg / kg once every three days.

[0042] In some embodiments of the disclosed methods and uses, the siNA or composition is administered in six doses of 1 mg / kg to 15 mg / kg, 1 mg / kg to 10 mg / kg, 2 mg / kg to 15 mg / kg, 2 mg / kg to 10 mg / kg, 3 mg / kg to 15 mg / kg, or 3 mg / kg to 10 mg / kg, where the first and second doses are optionally administered at least 3 days apart, the second and third doses are optionally administered at least 4 days apart, and the third and fourth doses, the fourth and fifth doses, or / and the fifth and sixth doses are optionally administered at least 7 days apart.

[0043] In some embodiments of the methods and uses of the present disclosure, the siNA or composition is administered in a particle or viral vector, and the viral vector is optionally selected from adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpes simplex virus, lentivirus, measles virus, picornavirus, poxvirus, retrovirus, and rhabdovirus vectors. In some embodiments, the viral vector is a recombinant viral vector. In some embodiments, the viral vector is selected from AAVrh.74, AAVrh.10, AAVrh.20, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13.

[0044] In some embodiments of the methods and uses of the present disclosure, the siNA or composition is administered systemically or locally.

[0045] In some embodiments of the methods and uses of the present disclosure, the siNA or composition is administered intravenously, subcutaneously, or intramuscularly.

[0046] In an eighth aspect, the present disclosure provides a siNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises: [ka] wherein B is a nucleobase, aryl, heteroaryl, or H; TIFF2025535539000039.tif8161 is a phosphodiester bond, a phosphorothioate bond, a mesyl phosphoramidate bond, or H. For example, the nucleotide is [ka] In some embodiments, the modified nucleotide is the last or penultimate nucleotide at the 3' end of the antisense strand. In some embodiments, the siNA is resistant to nuclease activity compared to a siNA of the same sequence that lacks the modified nucleotide in the 3' overhang.

[0047] In a seventh aspect, the present disclosure provides a method for manufacturing a semiconductor device, comprising: [ka] [ka] [ka] [ka] The present invention provides a phosphoramidite having the structure:

[0048] The foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the present disclosure as claimed. Other objects, advantages, and novel features will become readily apparent to those skilled in the art from the following brief description of the drawings and detailed description of the present disclosure. [Brief explanation of the drawings]

[0049] [Figure 1] An exemplary siNA molecule is shown. [Figure 2] An exemplary siNA molecule is shown. [Figure 3A] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 3B] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 3C] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 3D]An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 3E] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 3F] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 3G] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 3H] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 3I] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 3J] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 4A] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 4B] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 4C] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 4D] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 4E] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 4F] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 4G] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 4H] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 4I] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 4J] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 4K] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 4L] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 4M] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 4N] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 4O] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 4P] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 4Q] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 4R] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 4S] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 4T] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 4U] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 4V] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 4W] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 4X] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 4Y] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 4Z] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 4AA] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 4AB] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 5A] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 5B] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 5C] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 5D] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 5E] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 5F] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 6A] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 6B] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 6C] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 6D] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 6E] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 6F] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 6G] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 6H] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 6I] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 6J] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 6K] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 7A] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 7B] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 7C] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 7D] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 8A] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 8B] An exemplary double-stranded siNA molecule is shown, where G3 represents the GalNAc-conjugated moiety. [Figure 9]Figure 1 shows the in vivo activity of ds-siNAs containing a 5' vinylphosphonate moiety and modified unlocked nucleotides on the antisense strand. Activity was determined by measuring serum HBsAg levels measured by ELISA. Error bars represent the standard error of the mean. [Figure 10] Figure 1 shows the effect of 5'-cyclopropyl nucleotides on the stability of siNA in mouse liver homogenate. [Figure 11] Figure 1 shows the in vivo activity of ds-siNA containing 5'-cyclopropyl nucleotides on the antisense strand. Activity was determined by measuring serum HBsAg levels measured by ELISA. Error bars represent the standard error of the mean. [Figure 12] Figure 1 shows the in vivo activity of ds-siNA containing 3OH and unlocked modified nucleotides on the antisense strand. Activity was determined by measuring serum HBsAg levels measured by ELISA. Error bars represent the standard error of the mean. [Figure 13] Figure 1 shows the in vivo activity of ds-siNAs containing a 5' end cap on the antisense strand. Activity was determined by measuring serum HBsAg levels measured by ELISA. Error bars represent the standard error of the mean. [Figure 14A] 1 shows in vitro stability measured in mouse liver homogenate. [Figure 14B] Figure 1 shows the in vivo activity of ds-siNA analogs as determined by measuring serum HBsAg levels by ELISA. Error bars represent the standard error of the mean. [Figure 15A] 1 shows the effect of xylo-modification on the stability of siNA in mouse liver homogenate. [Figure 15B] Figure 1 shows the in vivo activity of ds-siNAs containing xylo-modifications as determined by measuring serum HBsAg levels by ELISA. Error bars represent the standard error of the mean. [Figure 16A]Figure 1 shows the in vivo activity of ds-siNAs containing 2'F modifications along the antisense strand. Activity was determined by measuring serum HBsAg levels measured by ELISA. Error bars represent the standard error of the mean. [Figure 16B] 1 shows the effect of 2'F modifications on the stability of siNA in mouse liver homogenate. [Figure 17A] Figure 1 shows a comparison of the in vivo activity of ds-siNA containing 2'F modifications along the antisense strand with the HBV therapeutic Vir-2218. Activity was measured by measuring serum HBsAg levels as determined by ELISA. Error bars represent the standard error of the mean. [Figure 17B] Figure 1 shows a comparison of the in vivo activity of ds-siNA containing 2'F modifications along the antisense strand with the HBV therapeutic Vir-2218. Activity was measured by measuring alanine aminotransferase (ALT) levels as determined by ELISA. Error bars represent the standard error of the mean. [Figure 18A] Figure 1 shows a comparison of the in vivo activity of ds-siNA analogs with the HBV treatment Vir-2218. Activity was determined by measuring serum HBsAg levels as measured by ELISA. Error bars represent the standard error of the mean. [Figure 18B] Figure 1 shows a comparison of the in vivo activity of ds-siNA analogs with the HBV treatment Vir-2218. Activity was determined by measuring HBeAg levels as measured by ELISA. Error bars represent the standard error of the mean. [Figure 18C] Figure 1 shows a comparison of the in vivo activity of ds-siNA analogs with the HBV treatment Vir-2218. Activity was determined by measuring alanine aminotransferase (ALT) levels as measured by ELISA. Error bars represent the standard error of the mean. [Figure 19] Figure 1 shows the in vivo activity of ds-siNA and Roch / Discerna administered at different concentrations to uninfected mice. Activity was determined by measuring serum ALT levels measured by ELISA. Error bars represent the standard error of the mean. [Figure 20A]Figure 1 shows the in vivo activity of ganciclovir, denvir, and 3ocp-modified ds-siNA. Activity was determined by measuring serum ALT levels measured by ELISA. Error bars represent the standard error of the mean. [Figure 20B] Figure 1 shows the in vivo activity of ganciclovir, denvir, and 3ocp-modified ds-siNA. Activity was determined by measuring serum HBsAg levels measured by ELISA. Error bars represent the standard error of the mean. [Figure 21A] Figure 1 shows the effect of xylo-modification on the stability of siNA in mouse liver homogenate. [Figure 21B] Figure 1 shows the effect of xylo-modification on the stability of siNA in mouse liver homogenate. [Figure 22A] Figure 1 shows the in vivo activity of xylo-modified ds-siNA. Activity was measured by measuring serum HBsAg levels as determined by ELISA. Error bars represent the standard error of the mean. [Figure 22B] Figure 1 shows the in vivo activity of xylo-modified ds-siNA. Activity was measured by measuring serum ALT levels as determined by ELISA. Error bars represent the standard error of the mean. [Figure 23A] 1 shows the effect of stereospecific PS binding on siNA stability in mouse liver homogenate. [Figure 23B] 1 shows the effect of stereospecific PS binding on siNA stability in mouse liver homogenate. [Figure 24] Figure 1 shows the in vivo activity of ds-siNA containing stereospecific PS linkages. Activity was determined by measuring serum HBsAg levels measured by ELISA. Error bars represent the standard error of the mean. [Figure 25] Figure 1 shows the in vivo activity of ds-siNA containing denavir (S)-modified nucleotides on the antisense strand. Activity was determined by measuring serum HBsAg levels measured by ELISA. Error bars represent the standard error of the mean. DETAILED DESCRIPTION OF THE INVENTION

[0050] Disclosed herein are oligonucleotide molecules (including short interfering nucleic acids or "siNAs") that comprise novel modified nucleotide monomers and dimers that contain unique chemical moieties and / or other modifications. Further disclosed herein are methods of using the disclosed oligonucleotide and siNA molecules to treat various diseases and conditions.

[0051] Generally, the siNA molecules described herein may be double-stranded siNA (ds-siNA) molecules. The siNA molecules described herein may contain modified nucleotides selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides. The siNA molecules described herein may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more phosphorothioate internucleoside linkages. The siNA molecules described herein may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mesyl phosphoramidate internucleoside linkages. The siNA molecules described herein may contain at least one phosphorylation blocker. The siNA molecules described herein may contain a 5' stabilized end cap. The siNA molecules described herein may contain galactosamine. The siNA molecules described herein may contain one or more blunt ends. The siNA molecules described herein may contain one or more overhangs.

[0052] For example, the disclosure provides modified nucleotides comprising the following structure: [ka] wherein B is a nucleobase, aryl, heteroaryl, or H; TIFF2025535539000046.tif8161 represents a phosphodiester bond, a phosphorothioate bond, a mesyl phosphoramidate bond, or H. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof. In some embodiments, the modified nucleotide is [ka] In some embodiments, the modified nucleotide may comprise the structure: [ka] The structure may include:

[0053] The present disclosure also provides [ka] wherein B is a nucleobase, aryl, heteroaryl, or H; TIFF2025535539000050.tif8161 represents a phosphodiester bond, a phosphorothioate bond, a mesyl phosphoramidate bond, or H. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof. In some embodiments, the modified nucleotide is [ka] In some embodiments, the modified nucleotide may comprise the structure: [ka] The structure may include:

[0054] The present disclosure also provides [ka] wherein B is a nucleobase, aryl, heteroaryl, or H; TIFF2025535539000054.tif8161 represents a phosphodiester bond, a phosphorothioate bond, a mesyl phosphoramidate bond, or H. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof. In some embodiments, the modified nucleotide is [ka] where A is adenine and G is guanine.

[0055] The present disclosure also provides [ka] wherein B is a nucleobase, aryl, heteroaryl, or H; TIFF2025535539000057.tif8161 represents a phosphodiester bond, a phosphorothioate bond, a mesyl phosphoramidate bond, or H. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0056] The present disclosure also provides [ka] wherein B is a nucleobase, aryl, heteroaryl, or H; TIFF2025535539000059.tif8161 represents a phosphodiester bond, a phosphorothioate bond, a mesyl phosphoramidate bond, or H. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0057] The present disclosure also provides [ka] wherein B is a nucleobase, aryl, heteroaryl, or H; TIFF2025535539000061.tif8161 represents a phosphodiester bond, a phosphorothioate bond, a mesyl phosphoramidate bond, or H. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof. In some embodiments, the modified nucleotide is [ka] The structure may include:

[0058] The present disclosure also provides [ka] wherein B is a nucleobase, aryl, heteroaryl, or H; TIFF2025535539000064.tif8161 represents a phosphodiester bond, a phosphorothioate bond, a mesyl phosphoramidate bond, or H. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof. In some embodiments, the modified nucleotide is [ka] The structure may include:

[0059] The present disclosure also provides [ka] wherein B is a nucleobase, aryl, heteroaryl, or H; TIFF2025535539000067.tif8161 represents a phosphodiester bond, a phosphorothioate bond, a mesyl phosphoramidate bond, or H. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof. In some embodiments, the modified nucleotide is [ka] The structure may include:

[0060] The present disclosure also provides [ka] wherein B is a nucleobase, aryl, heteroaryl, or H; TIFF2025535539000070.tif8161 represents a phosphodiester bond, a phosphorothioate bond, a mesyl phosphoramidate bond, or H. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof. In some embodiments, the modified nucleotide is [ka] The structure may include:

[0061] The present disclosure also provides [ka] wherein B is a nucleobase, aryl, heteroaryl, or H; TIFF2025535539000073.tif8161 represents a phosphodiester bond, a phosphorothioate bond, a mesyl phosphoramidate bond, or H. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof. In some embodiments, the modified nucleotide is [ka] The structure may include:

[0062] The present disclosure also provides [ka] wherein B is a nucleobase, aryl, heteroaryl, or H; TIFF2025535539000076.tif8161 represents a phosphodiester bond, a phosphorothioate bond, a mesyl phosphoramidate bond, or H. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof. In some embodiments, the modified nucleotide is [ka] The structure may include:

[0063] The present disclosure also provides [ka] wherein B is a nucleobase, aryl, heteroaryl, or H. TIFF2025535539000079.tif8161 represents a phosphodiester bond, a phosphorothioate bond, or H, and * represents a chiral center (e.g., R or S isomer). In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0064] The present disclosure further provides modified nucleotides comprising the following structure:

[0065] In a third aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: [ka] wherein B is a nucleobase, aryl, heteroaryl, or H; TIFF2025535539000081.tif8161 provides oligonucleotides exhibiting phosphodiester, phosphorothioate, or mesyl phosphoramidate linkages.

[0066] The present disclosure further provides modified nucleotides comprising the following structure:

[0067] [ka] and [ka] Further provided are structures of modified nucleotides, including the structure x is a nucleobase, aryl, heteroaryl, or H. In some embodiments, the modified nucleotide is [ka] and R y is a nucleobase. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0068] The present disclosure also provides oligonucleotides comprising a structure that can function as a stabilizing endcap at the 5' end of the antisense strand of any of the disclosed siNAs. The disclosed 5' stabilizing endcaps include: [ka] wherein each B is independently selected from a nucleobase, an aryl, a heteroaryl, and H; TIFF2025535539000086.tif8161 represents a phosphodiester bond, a phosphorothioate bond, or a mesyl phosphoramidate bond. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0069] The present disclosure also provides structures that can function as stabilizing endcaps at the 5' end of the antisense strand of any of the disclosed siNAs. The disclosed 5' stabilizing endcaps include: [ka] The structures may include, but are not limited to, y is the nucleobase and R 15 is H or CH3, TIFF2025535539000088.tif8161 represents a phosphodiester bond, a phosphorothioate bond, or a mesyl phosphoramidate bond. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof. In some embodiments, the 5' stabilized end cap is [ka] [ka] [ka] [ka] In this formula, R 15 is H or CH3, TIFF2025535539000093.tif8161 represents a phosphodiester bond, a phosphorothioate bond, or a mesyl phosphoramidate bond.

[0070] The disclosed short interfering nucleic acid (siNA) molecules can include at least one, at least two, at least three, at least four, or at least five of the aforementioned modified nucleotides and / or one of the aforementioned 5' stabilized end caps at the 5' end of the antisense strand. Indeed, the disclosed short interfering nucleic acid (siNA) molecules can include (a) a sense strand comprising a first nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to an RNA corresponding to a target gene, wherein the first nucleotide sequence is (i) 15 to 30 nucleotides in length, and (ii) a sense strand comprising 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least one modified nucleotide is a 2'-O-methyl nucleotide and the nucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and / or 19 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides, or at least one modified nucleotide is a 2'-O-methyl nucleotide and at least one modified nucleotide is a 2'-fluoro nucleotide; and an antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to RNA corresponding to the target gene, wherein the second nucleotide sequence is (iii) 15 to 30 nucleotides in length, and (iv) an antisense strand comprising 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least one modified nucleotide is a 2'-O-methyl nucleotide and at least one modified nucleotide is a 2'-fluoro nucleotide; or (b) a sense strand comprising a first nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to an RNA corresponding to the target gene, wherein the first nucleotide sequence is (i) 15 to 30 nucleotides in length, and (ii) a sense strand comprising 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least one modified nucleotide is a 2'-O-methyl nucleotide and at least one modified nucleotide is a 2'-fluoro nucleotide; and an antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to RNA corresponding to the target gene, wherein the second nucleotide sequence is (iii) 15 to 30 nucleotides in length, and (iv) an antisense strand comprising 15 or more modified nucleotides and / or nucleotide analogs independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least one modified nucleotide is a 2'-O-methyl nucleotide and the nucleotides at positions 2, 5, 6, 7, 8, 10, 14, 16, 17, and / or 18 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides; or (c) a sense strand comprising a first nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to an RNA corresponding to the target gene, wherein the first nucleotide sequence is (v) is 15 to 30 nucleotides in length; and (vi) a sense strand comprising 15 or more modified nucleotides and / or nucleotide analogs independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least one modified nucleotide and / or nucleotide analog is a 2'-O-methyl nucleotide and the nucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and / or 19 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides, or at least one modified nucleotide is a 2'-O-methyl nucleotide and at least one modified nucleotide is a 2'-fluoro nucleotide; and an antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to RNA corresponding to the target gene, wherein the second nucleotide sequence is (vii) 15 to 30 nucleotides in length, and (viii) an antisense strand comprising 15 or more modified nucleotides and / or nucleotide analogs independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least one modified nucleotide is a 2'-O-methyl nucleotide and the nucleotides at positions 2, 5, 6, 7, 8, 10, 14, 16, 17, and / or 18 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides; The sense strand and / or the antisense strand may be [ka] [ka] at least one, at least two, at least three, at least four, or at least five modified nucleotide(s) and / or nucleotide analog(s) selected from: [ka] wherein B is a nucleobase, aryl, heteroaryl, or H; TIFF2025535539000097.tif8161 represents a phosphodiester bond, a phosphorothioate bond, or H, and * represents a chiral center (e.g., R or S isomer).

[0071] Furthermore, siNAs of the present disclosure may each independently comprise a sense strand and / or an antisense strand comprising one or more phosphorothioate internucleoside linkages, one or more mesyl phosphoramidate internucleoside linkages, or a combination thereof. siNAs may also comprise a phosphorylation blocker, galactosamine, and / or a 5'-stabilizing end cap (other than those described above). siNAs may also be conjugated to a targeting moiety, such as galactosamine.

[0072] The present disclosure also provides a siNA comprising a sense strand and an antisense strand, wherein the antisense strand is [ka] wherein B is a nucleobase, aryl, heteroaryl, or H; TIFF2025535539000099.tif8161 represents a phosphodiester bond, a phosphorothioate bond, a mesyl phosphoramidate bond, or H. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof. In some embodiments, the disclosed nucleotide is [ka] In some embodiments, the modified nucleotide is the last or penultimate nucleotide at the 3' end of the antisense strand. In some embodiments, the siNA is resistant to nuclease activity compared to a siNA of the same sequence that does not have a modified nucleotide in the 3' overhang.

[0073] Further disclosed herein are compositions comprising two or more of the siNA molecules described herein.

[0074] Further disclosed herein are compositions comprising any of the described siNA molecules and a pharmaceutically acceptable carrier or diluent. Such compositions may also include an additional therapeutic agent, or may be administered in conjunction with (simultaneously or sequentially) an additional therapeutic agent.

[0075] Further disclosed herein are compositions comprising two or more of the siNA molecules described herein for use as a pharmaceutical.

[0076] Further disclosed herein are compositions comprising any of the described siNA molecules and a pharmaceutically acceptable carrier or diluent for use as a pharmaceutical. Such pharmaceuticals may also include, or be administered in conjunction with (simultaneously or sequentially) an additional therapeutic agent.

[0077] Also disclosed herein is a method for treating a disease in a subject in need thereof, comprising administering to the subject any of the siNA molecules (or combinations thereof) or compositions / medicines described herein.

[0078] Further disclosed herein is the use of any of the siNA molecules described herein (or a combination thereof) in the manufacture of a medicament for treating a disease.

[0079] Short interfering nucleic acid (siNA) molecules As described above, the present disclosure provides siNA molecules containing modified nucleotides. Any of the siNA molecules described herein may be double-stranded siNA (ds-siNA) molecules. The terms "siNA molecule" and "ds-siNA molecule" may be used interchangeably. In some embodiments, a ds-siNA molecule comprises a sense strand and an antisense strand.

[0080] For purposes of this disclosure, a siNA molecule disclosed herein may generally comprise (a) at least one phosphorylation blocker, conjugation moiety, and / or 5'-stabilized endcap; and (b) a short interfering nucleic acid (siNA). In some embodiments, the phosphorylation blocker is a phosphorylation blocker disclosed herein. In some embodiments, the conjugation moiety is galactosamine disclosed herein. In some embodiments, the 5'-stabilized endcap is a 5'-stabilized endcap disclosed herein.

[0081] The siNA may comprise any of the first nucleotide, second nucleotide, sense strand, or antisense strand sequences disclosed herein. The siNA may comprise 5 to 100, 5 to 90, 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 30, 10 to 25, 15 to 100, 15 to 90, 15 to 80, 15 to 70, 15 to 60, 15 to 50, 15 to 30, or 15 to 25 nucleotides. The siNA may contain at least 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides. The siNA may contain 50, 45, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, or 19 nucleotides or less. The nucleotides may be modified nucleotides. The nucleotides may be nucleotide analogs. The siNA may be single-stranded (ss-siNA). The siNA may be double-stranded (ds-siNA).

[0082] ds-siNAs are composed of (a) a sense strand containing 15-30, 15-25, 15-24, 15-23, 15-22, 15-21, 17-30, 17-25, 17-24, 17-23, 17-22, 17-21, 18-30, 18-25, 18-24, 18-23, 18-22, 18-21, 19-30, 19-25, 19-24, 19-23, 19-22, 19-21, 20-25, 20-24, 20-23, 21-25, 21-24, or 21-23 nucleotides; b) an antisense strand comprising 15 to 30, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 17 to 30, 17 to 25, 17 to 24, 17 to 23, 17 to 22, 17 to 21, 18 to 30, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 19 to 30, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 20 to 25, 20 to 24, 20 to 23, 21 to 25, 21 to 24, or 21 to 23 nucleotides. The ds-siNA may comprise (a) a sense strand comprising about 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides and (b) an antisense strand comprising about 15, 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides. The ds-siNA may comprise (a) a sense strand comprising about 19 nucleotides and (b) an antisense strand comprising about 21 nucleotides. The ds-siNA may comprise (a) a sense strand comprising about 21 nucleotides and (b) an antisense strand comprising about 23 nucleotides.

[0083] Any of the siNA molecules disclosed herein may further comprise one or more linkers independently selected from a phosphodiester (PO) linker, a phosphorothioate (PS) linker, a phosphorodithioate linker, a mesyl phosphoramidate (Ms), and a PS-mimetic linker. In some embodiments, the PS-mimetic linker is a sulfur linker. In some embodiments, the linker is an internucleoside linker. Alternatively, or in addition, the linker may connect a nucleotide of the siNA molecule to at least one phosphorylation blocker, a conjugation moiety, or a 5'-stabilized endcap. In some embodiments, the linker connects the conjugation moiety to the phosphorylation blocker or the 5'-stabilized endcap.

[0084] An exemplary siNA molecule of the present disclosure is shown in Figure 1. As shown in Figure 1, the exemplary siNA molecule includes a sense strand (101) and an antisense strand (102). The sense strand (101) may include a first oligonucleotide sequence (103). The first oligonucleotide sequence (103) may include one or more phosphorothioate internucleoside linkages (109). The phosphorothioate internucleoside linkages (109) may be located between nucleotides at the 5' or 3' end of the first oligonucleotide sequence (103). The phosphorothioate internucleoside linkages (109) may be located between the first three nucleotides from the 5' end of the first oligonucleotide sequence (103). The first oligonucleotide sequence (103) may include one or more 2'-fluoronucleotides (110). The first oligonucleotide sequence (103) may contain one or more 2'-O-methyl nucleotides (111). The first oligonucleotide sequence (103) may contain 15 or more modified nucleotides independently selected from 2'-fluoronucleotides (110) and 2'-O-methyl nucleotides (111). The sense strand (101) may further contain a phosphorylation blocker (105). The sense strand (101) may further contain a galactosamine (106). The antisense strand (102) may contain a second oligonucleotide sequence (104). The second oligonucleotide sequence (104) may contain one or more phosphorothioate internucleoside linkages (109). The phosphorothioate internucleoside linkages (109) may be located between nucleotides at the 5' or 3' end of the second oligonucleotide sequence (104). The phosphorothioate internucleoside linkage (109) may be between the first three nucleotides from the 5' end of the second oligonucleotide sequence (104). The phosphorothioate internucleoside linkage (109) may be between the first three nucleotides from the 3' end of the second oligonucleotide sequence (104). The second oligonucleotide sequence (104) may contain one or more 2'-fluoro nucleotides (110).The second oligonucleotide sequence (104) may comprise one or more 2'-O-methyl nucleotides (111). The second oligonucleotide sequence (104) may comprise 15 or more modified nucleotides independently selected from 2'-fluoro nucleotides (110) and 2'-O-methyl nucleotides (111). The antisense strand (102) may further comprise a 5' stabilized end cap (107). The siNA may further comprise one or more blunt ends. Alternatively, or in addition, one end of the siNA may comprise an overhang (108). The overhang (108) may be part of the sense strand (101). The overhang (108) may be part of the antisense strand (102). The overhang (108) may be different from the first nucleotide sequence (103). The overhang (108) may be different from the second nucleotide sequence (104). The overhang (108) may be part of the first nucleotide sequence (103). The overhang (108) may be part of the second nucleotide sequence (104). The overhang (108) may comprise one or more nucleotides. The overhang (108) may comprise one or more deoxyribonucleotides. The overhang (108) may comprise one or more modified nucleotides. The overhang (108) may comprise one or more modified ribonucleotides. The sense strand (101) may be shorter than the antisense strand (102). The sense strand (101) may be the same length as the antisense strand (102). The sense strand (101) may be longer than the antisense strand (102).

[0085] An exemplary siNA molecule of the present disclosure is shown in Figure 2. As shown in Figure 2, the exemplary siNA molecule includes a sense strand (201) and an antisense strand (202). The sense strand (201) may include a first oligonucleotide sequence (203). The first oligonucleotide sequence (203) may include one or more phosphorothioate internucleoside linkages (209). The phosphorothioate internucleoside linkages (209) may be located between nucleotides at the 5' or 3' end of the first oligonucleotide sequence (203). The phosphorothioate internucleoside linkages (209) may be located between the first three nucleotides from the 5' end of the first oligonucleotide sequence (203). The first oligonucleotide sequence (203) may include one or more 2'-fluoronucleotides (210). The first oligonucleotide sequence (203) may contain one or more 2'-O-methyl nucleotides (211). The first oligonucleotide sequence (203) may contain 15 or more modified nucleotides independently selected from 2'-fluoro nucleotides (210) and 2'-O-methyl nucleotides (211). The sense strand (201) may further contain a phosphorylation blocker (205). The sense strand (201) may further contain a galactosamine (206). The antisense strand (202) may contain a second oligonucleotide sequence (204). The second oligonucleotide sequence (204) may contain one or more phosphorothioate internucleoside linkages (209). The phosphorothioate internucleoside linkages (209) may be located between nucleotides at the 5' or 3' end of the second oligonucleotide sequence (204). The phosphorothioate internucleoside linkage (209) may be between the first three nucleotides from the 5' end of the second oligonucleotide sequence (204). The phosphorothioate internucleoside linkage (209) may be between the first three nucleotides from the 3' end of the second oligonucleotide sequence (204). The second oligonucleotide sequence (204) may contain one or more 2'-fluoro nucleotides (210).The second oligonucleotide sequence (204) may comprise one or more 2'-O-methyl nucleotides (211). The second oligonucleotide sequence (204) may comprise 15 or more modified nucleotides independently selected from 2'-fluoro nucleotides (210) and 2'-O-methyl nucleotides (211). The antisense strand (202) may further comprise a 5' stabilized end cap (207). The siNA may further comprise one or more overhangs (208). The overhang (208) may be part of the sense strand (201). The overhang (208) may be part of the antisense strand (202). The overhang (208) may be different from the first nucleotide sequence (203). The overhang (208) may be different from the second nucleotide sequence (204). The overhang (208) may be part of the first nucleotide sequence (203). The overhang (208) may be part of the second nucleotide sequence (204). The overhang (208) may be adjacent to the 3' end of the first nucleotide sequence (203). The overhang (208) may be adjacent to the 5' end of the first nucleotide sequence (203). The overhang (208) may be adjacent to the 3' end of the second nucleotide sequence (204). The overhang (208) may be adjacent to the 5' end of the second nucleotide sequence (204). The overhang (208) may comprise one or more nucleotides. The overhang (208) may comprise one or more deoxyribonucleotides. The overhang (208) may comprise a TT sequence. The overhang (208) may comprise one or more modified nucleotides. The overhang (208) may comprise one or more modified nucleotides disclosed herein (e.g., 2'-fluoro nucleotides, 2'-O-methyl nucleotides, 2'-fluoro nucleotide mimics, 2'-O-methyl nucleotide mimics, or nucleotides containing modified nucleobases). The overhang (208) may comprise one or more modified ribonucleotides. The sense strand (201) may be shorter than the antisense strand (202).The sense strand (201) may be the same length as the antisense strand (202). The sense strand (201) may be longer than the antisense strand (202).

[0086] Figures 3A-3J, 4A-4AB, 5A-F, 6A-K, 7A-D, and 8A-B show exemplary ds-siNA modification patterns. As shown in Figures 3A-3J, an exemplary ds-siNA molecule may have the formula: 5'-A n 1 B n 2 A n 3 B n 4 A n 5 B n 6 A n 7 B n 8 A n 9 -3' 3'-C q 1 A q 2 B q 3 A q 4 B q 5 A q 6 B q 7 A q 8 B q 9 A q 10 B q 11 A q 12 -5' During the ceremony, the top strand is a sense strand comprising a first nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to an RNA corresponding to a target gene, the first nucleotide sequence comprising 15 to 30 nucleotides; the lower strand is an antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to an RNA corresponding to a target gene, the second nucleotide sequence comprising 15 to 30 nucleotides; each A is independently a 2'-O-methyl nucleotide, or a nucleotide containing a 5' stabilized end cap or phosphorylation blocker; B is a 2'-fluoronucleotide; C represents an overhanging nucleotide, which is a 2'-O-methyl nucleotide, a deoxynucleotide, or uracil; n 1 = 1 to 6 nucleotides in length, each n 2 , n 6 , n 8 , q 3 , q 5 , q 7 , q 9 , q 11 , and q 12 are independently 0 to 1 nucleotides in length, each n 3 and n 4 are independently 1 to 3 nucleotides in length, n 5 is 1 to 10 nucleotides in length, n 7 is 0 to 4 nucleotides in length, each n 9 , q 1 , and q 2 are independently 0 to 2 nucleotides in length; q 4 is 0 to 3 nucleotides in length, q 6 is 0 to 5 nucleotides in length, q 8 is 2 to 7 nucleotides in length, and q 10 but are 2 to 11 nucleotides in length.

[0087] The ds-siNA may further comprise a conjugation moiety. The conjugation moiety may comprise any of the galactosamines disclosed herein. The ds-siNA may further comprise (i) phosphorothioate internucleoside linkages between nucleotides at positions 1 and 2 and positions 2 and 3 from the 5' end of the sense strand, and (ii) the ds-siNA may further comprise a 5'-stabilizing endcap. The 5'-stabilizing endcap may be vinyl phosphonate. The 5'-stabilizing endcap may be attached to the 5' end of the antisense strand. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the sense strand is further modified to contain a 5'-stabilizing endcap. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the antisense strand is further modified to contain a 5'-stabilizing endcap. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the sense strand is further modified to contain a 5'-stabilizing endcap. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the sense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 3' end of the sense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the antisense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 3' end of the antisense strand is further modified to contain a phosphorylation blocker. Exemplary ds-siNA molecules may have the following formula: 5'-A 2-4 B1A 1-3 B 2-3 A 2-10 B 0-1 A 0-4 B 0-1 A 0-2 -3' 3'-C2A 0-2 B 0-1 A 0-3 B 0-1 A 0-5 B 0-1 A 2-7 B1A 2-11 B1A1-5' During the ceremony, the top strand is a sense strand comprising a first nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to an RNA corresponding to a target gene, the first nucleotide sequence comprising 15 to 30 nucleotides; the lower strand is an antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to an RNA corresponding to a target gene, the second nucleotide sequence comprising 15 to 30 nucleotides; each A is independently a 2'-O-methyl nucleotide, or a nucleotide containing a 5' stabilized end cap or phosphorylation blocker; B is a 2'-fluoronucleotide; C represents an overhanging nucleotide, which is a 2'-O-methyl nucleotide, a deoxynucleotide, or uracil.

[0088] The ds-siNA may further comprise a conjugation moiety. The conjugation moiety may comprise any of the galactosamines disclosed herein. The ds-siNA may further comprise (i) phosphorothioate internucleoside linkages between nucleotides at positions 1 and 2 and positions 2 and 3 from the 5' end of the sense strand, and (ii) the ds-siNA may further comprise a 5'-stabilizing endcap. The 5'-stabilizing endcap may be a vinyl phosphonate. The vinyl phosphonate may be a deuterated vinyl phosphonate. The deuterated vinyl phosphonate may be a mono-deuterated vinyl phosphonate. The deuterated vinyl phosphonate may be a mono-di-deuterated vinyl phosphonate. The 5'-stabilizing endcap may be attached to the 5'-end of the antisense strand. The 5'-stabilizing endcap may be attached to the 3'-end of the antisense strand. The 5'-stabilizing endcap may be attached to the 5'-end of the sense strand. The 5'-stabilizing endcap may be attached to the 3'-end of the sense strand. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the sense strand is further modified to contain a 5'-stabilized endcap. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the antisense strand is further modified to contain a 5'-stabilized endcap. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the sense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 3' end of the sense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the antisense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 3' end of the antisense strand is further modified to contain a phosphorylation blocker.

[0089] Exemplary ds-siNAs shown in Figures 3A-3J, 4A-4AB, 5A-F, 6A-K, 7A-D, and 8A-B comprise (i) a sense strand containing 19-21 nucleotides and (ii) an antisense strand containing 21-23 nucleotides. The ds-siNA may optionally further comprise (iii) a conjugation moiety (e.g., GalNAc, shown as G3 in the figures) attached to the 3' or 5' end of the sense or antisense strand. The ds-siNA may comprise a two-nucleotide overhang consisting of nucleotides 20 and 21 from the 5' end of the antisense strand. The ds-siNA may comprise a two-nucleotide overhang consisting of nucleotides 22 and 23 from the 5' end of the antisense strand. The ds-siNA may further comprise 1, 2, 3, 4, 5, 6, or more phosphorothioate (ps) internucleoside linkages or mesyl phosphoramidate internucleoside linkages (Ms). At least one phosphorothioate internucleoside linkage or mesyl phosphoramidate internucleoside linkage (Ms) may be located between nucleotides 1 and 2 or 2 and 3 from the 5' end of the sense strand. At least one phosphorothioate internucleoside linkage or mesyl phosphoramidate internucleoside linkage (Ms) may be located between nucleotides 1 and 2 or 2 and 3 from the 5' end of the antisense strand. At least one phosphorothioate internucleoside linkage or mesyl phosphoramidate internucleoside linkage (Ms) may be present between nucleotides 19 and 20, 20 and 21, 21 and 22, or 22 and 23 from the 5' end of the antisense strand. As shown in Figures 3A-3J, 4A-4AB, 5A-F, 6A-K, 7A-D, and 8A-B, four to six nucleotides in the sense strand may be 2'-fluoro nucleotides. As shown in Figures 3A-3J, 4A-4AB, 5A-F, 6A-K, 7A-D, and 8A-B, two to five nucleotides in the antisense strand may be 2'-fluoro nucleotides.As shown in Figures 3A-3J, 4A-4D, 4P, 4R-4AB, 5A-F, 6A-K, 7A-D, and 8A-B, 13-15 nucleotides in the sense strand can be 2'-O-methyl nucleotides. As shown in Figures 3A-3J, 4E, 4F, 4O, 4R-X, 5A-F, 6A-K, 7A-D, and 8A-B, 14-19 nucleotides in the antisense strand can be 2'-O-methyl nucleotides. As shown in Figures 4E and 4G-4J, up to eight nucleotides in the sense strand (i.e., 1, 2, 3, 4, 5, 6, 7, and 8) can be 2'-O-cyclopropane (2'-ocp). As shown in Figures 4A, 4B, 4G, 4H, 4K, 4L, 4R, 4S, 4V, 4X, 4Y, 4AA, 5B, and 5D, up to 11 nucleotides (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11) in the antisense strand may be 2'-octyl nucleotides. As shown in Figures 4F, 4K-4N, and 4Q, 9-15 nucleotides in the sense strand may be 2'-OMe-cyclopropane (2'-octyl nucleotides). As shown in Figures 4J, 4M, 4N, 4P, 4Q, 4T, 4U, 4W, 4Z, 4AB, and 5C, 1-15 nucleotides in the sense strand may be 2'-octyl nucleotides. As shown in Figures 5A-C, E, and F, 1 position from the 5' end of the antisense strand may be vmX. As shown in Figures 6A-6G, one or two nucleotides in the antisense strand can be xylonucleotides, i.e., 2'-OMe-3'-xylo or 2'-F-3'-xylonucleotides. As shown in Figures 7A-D, one nucleotide in the antisense strand can be modified with ganciclovir or denavir. As shown in Figures 8A-B, one nucleotide on the antisense strand can be 3'-ocp. As shown in Figures 3A-3J, 4A-4AB, 5A-F, 6A-K, 7A-D, and 8A-B, ds-siNA does not contain base pairs between 2'-fluoro nucleotides on the sense and antisense strands. In some embodiments, the 2'-O-methyl, 2'-ocp, or 2'-omcp nucleotide at position 1 from the 5' end of the sense strand is further modified to contain a 5'-stabilizing endcap.In some embodiments, the 2'-O-methyl, 2'-ocp, or 2'-omcp nucleotide at position 1 from the 5' end of the antisense strand is further modified to contain a 5'-stabilized endcap. In some embodiments, the 2'-O-methyl, 2'-ocp, or 2'-omcp nucleotide at position 1 from the 5' end of the sense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl, 2'-ocp, or 2'-omcp nucleotide at position 1 from the 3' end of the sense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl, 2'-ocp, or 2'-omcp nucleotide at position 1 from the 5' end of the antisense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl, 2'-ocp, or 2'-omcp nucleotide at position 1 from the 3' end of the antisense strand is further modified to contain a phosphorylation blocker.

[0090] ds-siNA is a 19-nucleotide sense strand in which 2'-fluoro nucleotides are located at positions 3, 7 to 9, 12, and 17 from the 5' end of the sense strand and 2'-O-methyl nucleotides are located at positions 1, 2, 4 to 6, 10, 11, 13 to 16, 18, and 19 from the 5' end of the sense strand (Figure 3A); and a 19-nucleotide sense strand in which 2'-fluoro nucleotides are located at positions 3, 7, 8, and 17 from the 5' end of the sense strand and 2'-O-methyl nucleotides are located at positions 1, 2, 4 to 6, 9 from the 5' end of the sense strand (Figure 3B). a sense strand (Figure 3B) consisting of 19 nucleotides, where the 2'-fluoro nucleotides are at positions 3, 7-9, 12, and 17 from the 5' end of the sense strand, and the 2'-O-methyl nucleotides are at positions 1, 2, 4-6, 10, 11, 13-16, 18, and 19 from the 5' end of the sense strand; and a sense strand (Figure 3C) consisting of 19 nucleotides, where the 2'-fluoro nucleotides are at positions 5 and 7-9 from the 5' end of the sense strand, and the 2'-O-methyl nucleotides are at positions 1, 2, 4-6, 10, 11, 13-16, 18, and 19 from the 5' end of the sense strand. a sense strand (Figure 3D-F) consisting of 21 nucleotides, with 2'-fluoro nucleotides at positions 5, 9-11, 14, and 19 from the 5' end of the sense strand, and 2'-O-methyl nucleotides at positions 1-4, 6-8, 12, 13, 15-18, 20, and 21 from the 5' end of the sense strand; and a sense strand (Figure 3G) consisting of 21 nucleotides, with 2'-fluoro nucleotides at positions 7 and 9-11 from the 5' end of the sense strand, and 2'-O-methyl nucleotides at positions a sense strand (Figure 3H) consisting of 21 nucleotides, where the 2'-fluoro nucleotides are at positions 1 to 6, 8, and 12 to 21 from the 5' end of the sense strand; and 2'-O-methyl nucleotides are at positions 1, 2, 4 to 6, 10, 11, 13 to 16, 18, and 19 from the 5' end of the sense strand; and a sense strand (Figure 3I) consisting of 21 nucleotides, where the 2'-fluoro nucleotides are at positions 3, 7 to 9, 12, and 17 from the 5' end of the sense strand.2'-O-methyl nucleotides may be present in the sense strand (Figure 3J) at positions 1, 2, 4-6, 10, 11, 13-16, 18, and 19 from the 5' end of the sense strand.

[0091] ds-siNA is an antisense strand consisting of 21 nucleotides, in which the nucleotides at positions 2 and 14 from the 5' end of the antisense strand are 2'-fluoro nucleotides, and the nucleotides at positions 1, 3 to 13, and 15 to 21 are 2'-O-methyl nucleotides (Figures 3A and B), and an antisense strand consisting of 21 nucleotides, in which the nucleotides in the antisense strand contain an alternating 1:3 modification pattern, in which one nucleotide is a 2'-fluoro nucleotide and three nucleotides are 2'-O-methyl nucleotides (Figures 3A and B). and an antisense strand (Figures C and D) consisting of 21 nucleotides, where the nucleotides in the antisense strand contain an alternating 1:2 modification pattern, with one nucleotide being a 2'-fluoro nucleotide and two nucleotides being 2'-O-methyl nucleotides (Figure 3E). The antisense strand consists of 21 nucleotides, where the 2'-fluoro nucleotides are at positions 2, 6, 14, and 16 from the 5' end of the antisense strand, and the 2'-O-methyl nucleotides are at positions 1, 2, 3, 4, and 5 from the 5' end of the antisense strand. an antisense strand (Figure 3F) consisting of 23 nucleotides, with 2'-fluoro nucleotides at positions 2 and 14 from the 5' end of the antisense strand, and 2'-O-methyl nucleotides at positions 1, 3 to 13, and 15 to 23 from the 5' end of the antisense strand; and an antisense strand (Figure 3G) consisting of 23 nucleotides, with 2'-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand. The antisense strand (Figure 3H) has 23 nucleotides in positions 1, 3 to 5, 7 to 13, 15, and 17 to 23 from the 5' end of the antisense strand, and the 2'-fluoro nucleotides are located at positions 2, 7, and 14 from the 5' end of the antisense strand. The antisense strand (Figure 3I) has 23 nucleotides in positions 1, 3 to 6, 8 to 13, and 15 to 23 from the 5' end of the antisense strand, and the 2'-fluoro nucleotides are located at positionsThe antisense strand may comprise 2'-O-methyl nucleotides at positions 2, 14, and 16 from the 5' end of the antisense strand, and 2'-O-methyl nucleotides at positions 1, 3-13, 15, and 17-23 from the 5' end of the antisense strand (Figure 3J).

[0092] As shown in Figures 3A-G, I, and J, the ds-siNA may further comprise a conjugated moiety attached to the 3' end of the sense strand. As shown in Figures 3A-J, the ds-siNA may further comprise phosphorothioate internucleoside linkages between nucleotides at positions 1 and 2 and positions 2 and 3 from the 5' end of the sense strand. As shown in Figures A-F, I, and J, the ds-siNA may further comprise phosphorothioate internucleoside linkages between nucleotides at positions 1 and 2, positions 2 and 3, positions 19 and 20, and positions 20 and 21 from the 5' end of the antisense strand. As shown in Figures G and H, the ds-siNA may further comprise phosphorothioate internucleoside linkages between nucleotides at positions 1 and 2, positions 2 and 3, positions 21 and 22, and positions 22 and 23 from the 5' end of the antisense strand.

[0093] Optionally, the nucleotides at positions 22 and 23 from the 5' end of the antisense strand in Figures 3G and 3H may be unlocked nucleotides. The ds-siNA may optionally include a vinyl phosphonate attached to the 5' end of the antisense strand (Figure 3H), although in some embodiments, a 5' end cap as disclosed herein may also be suitable.

[0094] In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the sense strand is further modified to contain a 5'-stabilized endcap. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the antisense strand is further modified to contain a 5'-stabilized endcap. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the sense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 3' end of the sense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the antisense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 3' end of the antisense strand is further modified to contain a phosphorylation blocker.

[0095] The ds-siNAs were classified into two groups: a sense strand consisting of 19 nucleotides, in which 2'-fluoro nucleotides were located at positions 5 and 7-9 from the 5' end of the sense strand, and 2'-O-methyl nucleotides were located at positions 1-4, 6, and 10-19 from the 5' end of the sense strand (Figure 4A-D, P, and R-AB); and a sense strand consisting of 19 nucleotides, in which 2'-fluoro nucleotides were located at positions 5 and 7-9 from the 5' end of the sense strand, and 2'-O-methyl nucleotides were located at positions 2, 4, 11, 13, 15, 17, and 19 from the 5' end of the sense strand, and 2'-ocp nucleotides were located at positions 1, 3, 6, 10, 12, 14, 16, and 18 from the 5' end of the sense strand (Figure 4E, G). 4F and K-N) or a sense strand consisting of 19 nucleotides, where the 2'-fluoro nucleotides are at positions 5 and 7-9 from the 5' end of the sense strand, the 2'-O-methyl nucleotides are at positions 2, 4, 11, 13, 15, and 17 from the 5' end of the sense strand, and the 2'-omcp nucleotides are at positions 1, 3, 6, 10, 12, 14, 16, 18, and 19 from the 5' end of the sense strand (Figure 4F and K-N) or a sense strand consisting of 19 nucleotides, where the 2'-fluoro nucleotides are at positions 5 and 7-9 from the 5' end of the sense strand, and the 2'-omcp nucleotides are at positions 1-4, 6, and 10-19 from the 5' end of the sense strand (Figure 4O and Q).

[0096] The ds-siNA consisted of an antisense strand consisting of 21 nucleotides, with 2'-fluoro nucleotides at positions 2, 5, 8, 14, and 17 from the 5' end of the antisense strand, 2'-O-methyl nucleotides at positions 3, 4, 6, 9, 11, 13, 16, 19, and 21 from the 5' end of the antisense strand, and 2'-ocp nucleotides at positions 1, 7, 10, 12, 15, 18, and 20 from the 5' end of the antisense strand (Figure 4A, G, and K). The antisense strand (Figure 4B, H, and L) consisted of 21 nucleotides, with 2'-fluoro nucleotides at positions 2, 5, 8, 14, and 17 from the 5' end of the antisense strand, 2'-O-methyl nucleotides at positions 1, 3, 4, 6, 7, 9, 11, 13, 16, 19, and 21 from the 5' end of the antisense strand, and 2'-ocp nucleotides at positions 10, 12, 15, 18, and 20 from the 5' end of the antisense strand. The antisense strand (Fig. 4C, I, and M) consisted of 21 nucleotides, with 2'-fluoronucleotides at positions 2, 5, 8, 14, and 17 from the 5' end of the antisense strand, and 2'-O-methyl nucleotides at positions 1, 3, 4, 6, 7, 10, 12, 15, 18, and 20 from the 5' end of the antisense strand (Fig. 4C, I, and M). an antisense strand consisting of 21 nucleotides, with 2'-fluoro nucleotides at positions 2, 5, 8, 14, and 17 from the 5' end of the antisense strand and 2'-O-methyl nucleotides at positions 1, 3, 4, 6, 7, 9-13, 15, 16, and 18-21 from the 5' end of the antisense strand (Figure 4D and J); and an antisense strand consisting of 21 nucleotides, with 2'-fluoro nucleotides at positions 2, 5, 8, 14, and 17 from the 5' end of the antisense strand and 2'-O-methyl nucleotides at positions 1, 3, 4, 6, 7, 9-13, 15, 16, and 18-21 from the 5' end of the antisense strand (Figure 4E, F, and O).an antisense strand consisting of 21 nucleotides, in which 2'-fluoro nucleotides are located at positions 2, 5, 8, 14, and 17 from the 5' end of the antisense strand, 2'-O-methyl nucleotides are located at positions 1, 3, 4, 6, 7, 11, 13, 16, 19, and 21 from the 5' end of the antisense strand, and 2'-omcp nucleotides are located at positions 9, 10, 12, 15, 18, and 20 from the 5' end of the antisense strand (Figure 4N); and an antisense strand consisting of 21 nucleotides, in which 2'-fluoro nucleotides are located at positions 2, 5, 8, 14, and 17 from the 5' end of the antisense strand, 2'-O-methyl nucleotides are located at positions 1, 3, 4, 6, 7, 11, 13, 16, 19, and 21 from the 5' end of the antisense strand, and 2'-omcp nucleotides are located at positions 9, 10, 12, 15, 18, and 20 from the 5' end of the antisense strand (Figure 4N). The antisense strand (Fig. 4P and Q) consisted of 21 nucleotides, with 2'-fluoronucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand, 2'-O-methylnucleotides at positions 1, 3, 4, 6, 7, 9-12, 15, 16, and 18-21 from the 5' end of the antisense strand, and 2'-oct nucleotides at positions 1, 3, 4, 6, 7, 9-12, 15, 16, and 18-21 from the 5' end of the antisense strand. , and an antisense strand (Figure 4R) consisting of 21 nucleotides, in which 2'-fluoro nucleotides are located at positions 2, 6, 14, and 16 from the 5' end of the antisense strand, 2'-O-methyl nucleotides are located at positions 3 to 5, 7 to 13, 15, and 17 to 21 from the 5' end of the antisense strand, and 2'-ocp nucleotide is located at position 1 from the 5' end of the antisense strand; and an antisense strand (Figure 4S) consisting of 21 nucleotides, in which 2'-fluoro nucleotides are located at positions 2, 6, 14, and 16 from the 5' end of the antisense strand, 2'-O-methyl nucleotides are located at positions 3 to 5, 7 to 13, 15, and 17 to 21 from the 5' end of the antisense strand, and 2'-ocp nucleotide is located at position 1 from the 5' end of the antisense strand. The antisense strand (Figure 4T) consisted of 21 nucleotides, with 2'-fluoronucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand, 2'-O-methyl nucleotides at positions 1, 3 to 5, 7 to 13, 15, and 17 to 20 from the 5' end of the antisense strand, and 2'-omcp nucleotides at position 21 from the 5' end of the antisense strand (Figure 4T).The antisense strand (Figure 4U) consists of 21 nucleotides, with 2'-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand, 2'-O-methyl nucleotides at positions 3, 7, 13, 15, and 17, 20 from the 5' end of the antisense strand, and 2'-ocp nucleotides at positions 1 and 21 from the 5' end of the antisense strand (Figure 4V). ) and an antisense strand consisting of 21 nucleotides, where 2'-fluoro nucleotides are located at positions 2, 6, 14, and 16 from the 5' end of the antisense strand, 2'-O-methyl nucleotides are located at positions 3-5, 7-13, 15, and 17-20 from the 5' end of the antisense strand, and 2'-omcp nucleotides are located at positions 1 and 21 from the 5' end of the antisense strand (Figure 4W). an antisense strand (Figure 4X) consisting of 21 nucleotides, with 2'-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand, and 2'-O-methyl nucleotides at positions 1, 3-5, 7, 8, 10, 12, 18, and 20 from the 5' end of the antisense strand; and an antisense strand consisting of 21 nucleotides, with 2'-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand, and 2'-O-methyl nucleotides at positions 1, 3-5, 7, 8, 10, 12, 18, and 20 from the 5' end of the antisense strand. and the antisense strand (Figure 4Y) consisted of 21 nucleotides, with 2'-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand, 2'-O-methyl nucleotides at positions 1, 3 to 5, 7, 8, 10, 12, 18, and 20 from the 5' end of the antisense strand, and 2'-omcp nucleotides at positions 9, 11, 13, 15, 17, 19, and 21 from the 5' end of the antisense strand.and 21 positions from the 5' end of the antisense strand (Figure 4Z), and an antisense strand consisting of 21 nucleotides, where 2'-fluoro nucleotides are at positions 2, 6, 14, and 16 from the 5' end of the antisense strand, 2'-O-methyl nucleotides are at positions 4, 8, 10, 12, 18, and 20 from the 5' end of the antisense strand, and 2'-ocp nucleotides are at positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 from the 5' end of the antisense strand. and an antisense strand (Figure 4AA) consisting of 21 nucleotides, wherein 2'-fluoro nucleotides are located at positions 2, 6, 14, and 16 from the 5' end of the antisense strand, 2'-O-methyl nucleotides are located at positions 4, 8, 10, 12, 18, and 20 from the 5' end of the antisense strand, and 2'-omcp nucleotides are located at positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 from the 5' end of the antisense strand (Figure 4AB).

[0097] Optionally, the ds-siNA may further comprise a conjugated moiety attached to the 3'-end of the sense strand. The ds-siNA may further comprise (i) phosphorothioate internucleoside linkages between nucleotides at positions 1 and 2, 2 and 3, and 20 and 21 from the 5'-end of the sense strand, and (ii) phosphorothioate internucleoside linkages between nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 from the 5'-end of the antisense strand. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5'-end of the sense strand is further modified to contain a 5'-stabilizing endcap. In some embodiments, the 2'-ocp nucleotide at position 1 from the 5'-end of the antisense strand is further modified to contain a 5'-stabilizing endcap. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5'-end of the sense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 3' end of the sense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-ocp nucleotide at the 1 position from the 5' end of the antisense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 3' end of the antisense strand is further modified to contain a phosphorylation blocker.

[0098] The ds-siNA may comprise a sense strand consisting of 19 nucleotides, in which 2'-fluoro nucleotides are located at positions 5 and 7-9 from the 5' end of the sense strand, and 2'-O-methyl nucleotides are located at positions 1-4, 6, and 10-19 from the 5' end of the sense strand (Figures 5A-C, E, and F), or a sense strand consisting of 19 nucleotides, in which 2'-fluoro nucleotides are located at positions 3, 7-9, 12, and 17 from the 5' end of the sense strand, and 2'-O-methyl nucleotides are located at positions 1, 2, 4-6, 10, 11, 13-16, 18, and 19 from the 5' end of the sense strand (Figure 5D).

[0099] The ds-siNA was composed of an antisense strand consisting of 21 nucleotides, with 2'-fluoro nucleotides at positions 2, 5, 8, 14, and 17 from the 5' end of the antisense strand, 2'-O-methyl nucleotides at positions 3, 4, 6, 7, 9-13, 15, 16, and 18-21 from the 5' end of the antisense strand, and a vmX nucleotide at position 1 from the 5' end of the antisense strand (Figure 5A, E, and F). an antisense strand consisting of nucleotides, wherein 2'-fluoro nucleotides are located at positions 2, 5, 8, 14, and 17 from the 5' end of the antisense strand; 2'-O-methyl nucleotides are located at positions 3, 4, 6, 7, 9-13, 15, 16, and 18-20 from the 5' end of the antisense strand; a 2'-ocp nucleotide is located at position 21 from the 5' end of the antisense strand; and a vmX nucleotide is located at position 1 from the 5' end of the antisense strand. and an antisense strand consisting of 21 nucleotides, with 2'-fluoro nucleotides at positions 2, 5, 8, 14, and 17 from the 5' end of the antisense strand, 2'-O-methyl nucleotides at positions 3, 4, 6, 7, 9-13, 15, 16, and 18-20 from the 5' end of the antisense strand, 2'-omcp nucleotides at position 21 from the 5' end of the antisense strand, and vmX nucleotides at the 5' end of the antisense strand. an antisense strand having 2'-fluoro nucleotides at positions 2, 7, and 14 from the 5' end of the antisense strand, 2'-O-methyl nucleotides at positions 1, 3-6, 8-13, and 15-20 from the 5' end of the antisense strand, and a 2'-ocp nucleotide at position 21 from the 5' end of the antisense strand (Figure 5D).

[0100] Optionally, the ds-siNA may further comprise a conjugated moiety attached to the 3'-end of the sense strand. The ds-siNA may further comprise (i) phosphorothioate internucleoside linkages between nucleotides at positions 1 and 2, 2 and 3, and 20 and 21 from the 5'-end of the sense strand, and (ii) phosphorothioate internucleoside linkages between nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 from the 5'-end of the antisense strand. In some embodiments, the phosphorothioate internucleoside linkages may be S enantiomers. In some embodiments, the phosphorothioate internucleoside linkages may be R enantiomers.

[0101] In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the sense strand is further modified to contain a 5'-stabilized endcap. In some embodiments, the vmX nucleotide at position 1 from the 5' end of the antisense strand is further modified to contain a 5'-stabilized endcap. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the sense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 3' end of the sense strand is further modified to contain a phosphorylation blocker. In some embodiments, the vmX nucleotide at position 1 from the 5' end of the antisense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 3' end of the antisense strand is further modified to contain a phosphorylation blocker.

[0102] The ds-siNA may comprise a sense strand consisting of 19 nucleotides, in which 2'-fluoro nucleotides are located at positions 3, 7 to 9, 12, and 17 from the 5' end of the sense strand, and 2'-O-methyl nucleotides are located at positions 1, 2, 4 to 6, 10, 11, 13 to 16, 18, and 19 from the 5' end of the sense strand (Figures 6A to 6G), or a sense strand consisting of 19 nucleotides, in which 2'-fluoro nucleotides are located at positions 5, 7 to 9, and 12 from the 5' end of the sense strand, and 2'-O-methyl nucleotides are located at positions 1 to 4, 6, and 10 to 19 from the 5' end of the sense strand (Figures 6H to 6K).

[0103] ds-siNA is an antisense strand consisting of 21 nucleotides, with 2'-fluoro nucleotides at positions 2, 7, and 14 from the 5' end of the antisense strand, 2'-O-methyl nucleotides at positions 1, 3, 4, 6, 8-13, and 15-21 from the 5' end of the antisense strand, and xylosinate at position 5 from the 5' end of the antisense strand (Figure 6A); and an antisense strand consisting of 21 nucleotides, with 2'-fluoro nucleotides at positions 2, 7, and 14 from the 5' end of the antisense strand. an antisense strand (Figure 6B) consisting of 21 nucleotides, with 2'-fluoronucleotides at positions 2 and 14 from the 5' end of the antisense strand, 2'-O-methylnucleotides at positions 1, 3 to 5, 8 to 13, and 15 to 21 from the 5' end of the antisense strand, and xylonucleotides at position 6 from the 5' end of the antisense strand; and an antisense strand consisting of 21 nucleotides, with 2'-fluoronucleotides at positions 2 and 14 from the 5' end of the antisense strand, 2'-O-methylnucleotides at positions 1, 3 to 6, 8 to 13, and 15 to 21 from the 5' end of the antisense strand, and xylonucleotides at position 7 from the 5' end of the antisense strand. and an antisense strand (Figure 6C) consisting of 21 nucleotides, with 2'-fluoro nucleotides at positions 2, 7, and 14 from the 5' end of the antisense strand, with 2'-O-methyl nucleotides at positions 1, 3-6, 9-13, and 15-21 from the 5' end of the antisense strand, and with xylose nucleotides at position 8 from the 5' end of the antisense strand (Figure 6D). an antisense strand (Figure 6E) consisting of 21 nucleotides, with 2'-fluoronucleotides at positions 2, 7, and 14 from the 5' end of the antisense strand, 2'-O-methyl nucleotides at positions 1, 3 to 6, 8 to 13, 15 to 19, and 21 from the 5' end of the antisense strand, and xylonucleotides at position 20 from the 5' end of the antisense strand; and an antisense strand consisting of 21 nucleotides, with 2'-fluoronucleotides at positions 2, 7, and 14 from the 5' end of the antisense strand, and 2'-O-methyl nucleotides at positions 1, 3 to 5, 8 to 13, 15 to 19, and 21 from the 5' end of the antisense strand, and xylonucleotides at positionsThe antisense strand (Figure 6F) consisted of 21 nucleotides, with 2'-fluoronucleotides at positions 2, 7, and 14 from the 5' end of the antisense strand, 2'-O-methylnucleotides at positions 1, 3 to 6, 9 to 13, 15 to 19, and 21 from the 5' end of the antisense strand, and xylosinic nucleotides at positions 8 and 20 from the 5' end of the antisense strand (Figure 6G). an antisense strand (Figure 6H) consisting of 21 nucleotides, in which 2'-fluoro nucleotides are located at positions 2, 6, 14, and 16 from the 5' end of the antisense strand, 2'-O-methyl nucleotides are located at positions 1, 3-5, 7-13, 15, and 17-20 from the 5' end of the antisense strand, and xylosinic nucleotides are located at position 21 from the 5' end of the antisense strand; and an antisense strand consisting of 21 nucleotides, in which 2'-fluoro nucleotides are located at positions 2, 6, 14, and 16 from the 5' end of the antisense strand, and 2'- The antisense strand (Figure 6I) consists of 21 nucleotides, with 2'-fluoronucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand, and 2'-O-methylnucleotides at positions 3 to 5, 7 to 13, 15, and 17 to 21 from the 5' end of the antisense strand, and xylonucleotides at position 1 from the 5' end of the antisense strand. The antisense strand may comprise an antisense strand in which 2'-fluoronucleotides are located at positions 1 and 21 from the 5' end of the antisense strand (Figure 6J), or an antisense strand consisting of 21 nucleotides, with 2'-fluoronucleotides located at positions 2, 6, 14, and 16 from the 5' end of the antisense strand, 2'-O-methyl nucleotides located at positions 1, 3-5, 7-13, 15, 17-19, and 21 from the 5' end of the antisense strand, and a xylosinic nucleotide located at position 20 from the 5' end of the antisense strand (Figure 6K).

[0104] In some embodiments, the xylonucleotide can be a 2'-OMe-3'-xylonucleotide. In some embodiments, the xylonucleotide can be a 2'-F-3'-xylonucleotide. Optionally, the ds-siNA can further comprise a conjugated moiety attached to the 3'-end of the sense strand. The ds-siNA can further comprise (i) phosphorothioate internucleoside linkages between nucleotides at positions 1 and 2, 2 and 3, and 20 and 21 from the 5'-end of the sense strand, and (ii) phosphorothioate internucleoside linkages between nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 from the 5'-end of the antisense strand. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5'-end of the sense strand is further modified to contain a 5'-stabilizing endcap. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the antisense strand is further modified to contain a 5'-stabilized endcap. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the sense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 3' end of the sense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the antisense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 3' end of the antisense strand is further modified to contain a phosphorylation blocker.

[0105] The ds-siNA may comprise a sense strand consisting of 19 nucleotides, with 2'-fluoro nucleotides at positions 3, 7-9, 12, and 17 from the 5' end of the sense strand and 2'-O-methyl nucleotides at positions 1, 2, 4-6, 10, 11, 13-16, 18, and 19 from the 5' end of the sense strand (Figures 7A-D).

[0106] ds-siNA has an antisense strand consisting of 21 nucleotides, with 2'-fluoro nucleotides at positions 2, 7, and 14 from the 5' end of the antisense strand, 2'-O-methyl nucleotides at positions 1, 3-5, 8-13, and 15-21 from the 5' end of the antisense strand, and an acyclic ganciclovir nucleotide analog at position 6 from the 5' end of the antisense strand (Figure 7A), and an antisense strand consisting of 21 nucleotides, with 2'-fluoro nucleotides at positions 2, 7, and 14 from the 5' end of the antisense strand, 2'-O-methyl nucleotides at positions 1, 3-5, 8-13, and 15-21 from the 5' end of the antisense strand, and an acyclic ganciclovir nucleotide analog at position 6 from the 5' end of the antisense strand (Figure 7B). 7B), and an antisense strand consisting of 21 nucleotides, where 2'-fluoro nucleotides are located at positions 2, 7, and 14 from the 5' end of the antisense strand, 2'-O-methyl nucleotides are located at positions 1, 3-6, 9-13, and 15-21 from the 5' end of the antisense strand, and a ganciclovir nucleotide is located at position 8 from the 5' end of the antisense strand (Figure 7C), or an antisense strand consisting of 21 nucleotides, where 2'-fluoro nucleotides are located at positions 2, 7, and 14 from the 5' end of the antisense strand, 2'-O-methyl nucleotides are located at positions 1, 3-6, 9-13, and 15-21 from the 5' end of the antisense strand, and a ganciclovir nucleotide is located at position 8 from the 5' end of the antisense strand (Figure 7D).

[0107] In some embodiments, the acyclic ganciclovir nucleotide analog is an S enantiomer. In some embodiments, the acyclic ganciclovir nucleotide analog is an R enantiomer. In some embodiments, the denavir nucleotide is an S antimer. In some embodiments, the denavir nucleotide is an R antimer.

[0108] Optionally, the ds-siNA may further comprise a conjugated moiety attached to the 3'-end of the sense strand. The ds-siNA may further comprise (i) phosphorothioate internucleoside linkages between nucleotides at positions 1 and 2, 2 and 3, and 20 and 21 from the 5'-end of the sense strand, and (ii) phosphorothioate internucleoside linkages between nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 from the 5'-end of the antisense strand. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5'-end of the sense strand is further modified to contain a 5'-stabilizing endcap. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5'-end of the antisense strand is further modified to contain a 5'-stabilizing endcap. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5'-end of the sense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 3' end of the sense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the antisense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 3' end of the antisense strand is further modified to contain a phosphorylation blocker.

[0109] The ds-siNA may comprise a sense strand consisting of 19 nucleotides, with 2'-fluoro nucleotides at positions 3, 7-9, 12, and 17 from the 5' end of the sense strand and 2'-O-methyl nucleotides at positions 1, 2, 4-6, 10, 11, 13-16, 18, and 19 from the 5' end of the sense strand (Figures 8A and B).

[0110] The ds-siNA may comprise an antisense strand consisting of 21 nucleotides, in which 2'-fluoro nucleotides are located at positions 2, 7, and 14 from the 5' end of the antisense strand, 2'-O-methyl nucleotides are located at positions 1, 3-5, 8-13, and 15-21 from the 5' end of the antisense strand, and a denavir 3'-ocp nucleotide is located at position 6 from the 5' end of the antisense strand (Figure 8A), or an antisense strand consisting of 21 nucleotides, in which 2'-fluoro nucleotides are located at positions 2, 7, and 14 from the 5' end of the antisense strand, 2'-O-methyl nucleotides are located at positions 1, 3-6, 9-13, and 15-21 from the 5' end of the antisense strand, and a 3'-ocp nucleotide is located at position 8 from the 5' end of the antisense strand (Figure 8B).

[0111] Optionally, the ds-siNA may further comprise a conjugated moiety attached to the 3'-end of the sense strand. The ds-siNA may further comprise (i) phosphorothioate internucleoside linkages between nucleotides at positions 1 and 2, 2 and 3, and 20 and 21 from the 5'-end of the sense strand, and (ii) phosphorothioate internucleoside linkages between nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 from the 5'-end of the antisense strand. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5'-end of the sense strand is further modified to contain a 5'-stabilizing endcap. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5'-end of the antisense strand is further modified to contain a 5'-stabilizing endcap. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5'-end of the sense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 3' end of the sense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the antisense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 3' end of the antisense strand is further modified to contain a phosphorylation blocker.

[0112] In some embodiments, the nucleotide at position 1 from the 5' end of the sense strand is a 5' vinylphosphonate dimer moiety (e.g., an enantiomer of either PP2O or PP2OH), a d2vd3 nucleotide, a d2vd3U nucleotide, an omeco-d3 nucleotide, an omeco-d3U nucleotide, a 4h nucleotide, a 4hU nucleotide, a v-mun nucleotide, a c2o-4h nucleotide, an omeco-munb nucleotide, a d2vm nucleotide, or a d2vmA nucleotide, a d2vd3U nucleotide, an omeco-d3U nucleotide, a 4hU nucleotide, a v-mun nucleotide, a c2o-4h nucleotide, an omeco-munb nucleotide, or a d2vmA nucleotide. In some embodiments, the nucleotide at position 1 from the 5' end of the antisense strand is a 5' vinylphosphonate dimer moiety (e.g., an enantiomer of either PP2O or PP2OH), a d2vd3 nucleotide, a d2vd3U nucleotide, an omeco-d3 nucleotide, an omeco-d3U nucleotide, a 4h nucleotide, a 4hU nucleotide, a v-mun nucleotide, a c2o-4h nucleotide, an omeco-munb nucleotide, a d2vm nucleotide, or a d2vmA nucleotide. In some embodiments, the nucleotide at position 1 from the 3' end of the sense strand is a 5' vinylphosphonate dimer moiety (e.g., an enantiomer of either PP2O or PP2OH), a d2vd3 nucleotide, a d2vd3U nucleotide, an omeco-d3 nucleotide, an omeco-d3U nucleotide, a 4h nucleotide, a 4hU nucleotide, a v-mun nucleotide, a c2o-4h nucleotide, an omeco-munb nucleotide, a d2vm nucleotide, or a d2vmA nucleotide.In some embodiments, the nucleotide at position 1 from the 3' end of the antisense strand is a 5' vinylphosphonate dimer moiety (e.g., either an enantiomer of PP2O or PP2OH), a d2vd3 nucleotide, a d2vd3U nucleotide, an omeco-d3 nucleotide, an omeco-d3U nucleotide, a 4h nucleotide, a 4hU nucleotide, a v-mun nucleotide, a c2o-4h nucleotide, an omeco-munb nucleotide, a d2vm nucleotide, or a d2vmA nucleotide. In some embodiments, at least one, two, three, four, or more 2'-fluoro nucleotides on the sense strand or antisense strand are 2'-fluoro nucleotide mimics. In some embodiments, at least one, two, three, four, or more 2'-fluoro nucleotides on the sense strand are 3',4'-secoF, 3',4'-secoFA, fB, fN, f(4nh)Q, f4P, f2P, or fX nucleotides. In some embodiments, at least 1, 2, 3, 4, or more 2'-fluoro nucleotides in the antisense strand are 3',4'-secoF, 3',4'-secoFA, fB, fN, f(4nh)Q, f4P, f2P, or fX nucleotides. In some embodiments, at least 1, 2, 3, 4, or more 2'-O-methyl nucleotides on the sense or antisense strand are 2'-O-methyl nucleotide mimics. In some embodiments, at least 1, 2, 3, 4, or more nucleotides on the sense or antisense strand are 2'-ocp, 2'-ocmp, 3'-ocp, 3'-omcp, 5cp, 5mcp, mun12, moe, 3m, L-2'-OMe, tn2o, tn, 2'-OMe-3'-xylo, or 2'-F-3'-xylo nucleotides. In some embodiments, one or more nucleotides in the sense strand and / or antisense strand may be 3',4'-seco modified nucleotides in which the bond between the 3' and 4' positions of the furanose ring is broken (e.g., 3'4'-secoOBz, 3'4'-secoF, or mun34). In some embodiments, the sense strand and / or antisense strand may also include one or more nucleotide analogs (e.g., An1 and An2).

[0113] siNA sense strand Any of the siNA molecules described herein can comprise a sense strand. The sense strand can comprise a first nucleotide sequence. The first nucleotide sequence can be 15-30, 15-25, 15-23, 17-23, 19-23, or 19-21 nucleotides in length. In some embodiments, the first nucleotide sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the first nucleotide sequence is at least 19 nucleotides in length. In some embodiments, the first nucleotide sequence is at least 21 nucleotides in length.

[0114] In some embodiments, the sense strand is the same length as the first nucleotide sequence. In some embodiments, the sense strand is longer than the first nucleotide sequence. In some embodiments, the sense strand may further comprise 1, 2, 3, 4, 5, or more nucleotides than the first nucleotide sequence. In some embodiments, the sense strand may further comprise deoxyribonucleic acid (DNA). In some embodiments, the DNA is thymine (T). In some embodiments, the sense strand may further comprise a TT sequence. In some embodiments, the sense strand may further comprise one or more modified nucleotides adjacent to the first nucleotide sequence. In some embodiments, the one or more modified nucleotides are independently selected from any of the modified nucleotides disclosed herein (e.g., 2'-fluoro nucleotides, 2'-O-methyl nucleotides, 2'-fluoro nucleotide mimics, 2'-O-methyl nucleotide mimics, 2'-ocp nucleotides, 2'-omcp nucleotides, or nucleotides comprising modified nucleobases).

[0115] In some embodiments, the first nucleotide sequence comprises 15, 16, 17, 18, 19, 20, 21, 22, 23, or more modified nucleotides independently selected from 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-ocp nucleotides, 2'-omcp nucleotides, 3'-ocp nucleotides, 2'-OMe-3'-xylonucleotides, 2'-F-3'-xylonucleotides, vmX nucleotides, ganciclovir nucleotides (interchangeably referred to herein as "acyclic ganciclovir nucleotide analogs"), and denavir nucleotides. In some embodiments, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the nucleotides in the first nucleotide sequence are modified nucleotides independently selected from 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-ocp nucleotides, 2'-omcp nucleotides, 3'-ocp nucleotides, 2'-OMe-3'-xylonucleotides, 2'-F-3'-xylonucleotides, vmX nucleotides, ganciclovir nucleotides, and denavir nucleotides. In some embodiments, 100% of the nucleotides in the first nucleotide sequence are modified nucleotides independently selected from 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-ocp nucleotides, 2'-omcp nucleotides, 3'-ocp nucleotides, 2'-OMe-3'-xylonucleotides, 2'-F-3'-xylonucleotides, vmX nucleotides, ganciclovir nucleotides, and denavir nucleotides. In some embodiments, the 2'-O-methyl nucleotide is a 2'-O-methyl nucleotide mimic. In some embodiments, the 2'-fluoro nucleotide is a 2'-fluoro nucleotide mimic.

[0116] In some embodiments, about 15-30, 15-25, 15-24, 15-23, 15-22, 15-21, 17-30, 17-25, 17-24, 17-23, 17-22, 17-21, 18-30, 18-25, 18-24, 18-23, 18-22, 18-21, 19-30, 19-25, 19-24, 19-23, 19-22, 19-21, 20-25, 20-24, 20-23, 21-25, 21-24, or 21-23 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, one of the modified nucleotides of the first nucleotide sequence is or is not a 2'-O-methyl nucleotide. In some embodiments, about 2-20 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, about 5-25 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, about 10-25 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, about 12-25 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 12 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 13 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 14 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 15 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 16 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides.In some embodiments, at least about 17 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 18 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 19 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 21 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 20 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 19 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 18 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 17 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 16 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 15 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 14 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 13 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least one modified nucleotide of the first nucleotide sequence is a 2'-O-methyl pyrimidine. In some embodiments, at least 5, 6, 7, 8, 9, or 10 modified nucleotides of the first nucleotide sequence are 2'-O-methylpyrimidines.In some embodiments, at least one modified nucleotide of the first nucleotide sequence is a 2'-O-methyl purine. In some embodiments, at least 5, 6, 7, 8, 9, or 10 modified nucleotides of the first nucleotide sequence are 2'-O-methyl purines. In some embodiments, the 2'-O-methyl nucleotides are 2'-O-methyl nucleotide mimics.

[0117] In some embodiments, 2 to 15 modified nucleotides of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 2 to 10 modified nucleotides of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 2 to 6 modified nucleotides of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 1 to 6, 1 to 5, 1 to 4, or 1 to 3 modified nucleotides of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least 1, 2, 3, 4, 5, or 6 modified nucleotides of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least one modified nucleotide of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, at least two modified nucleotides of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least three modified nucleotides of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least four modified nucleotides of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least five modified nucleotides in the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least six modified nucleotides in the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 10, 9, 8, 7, 6, 5, 4, 3, or fewer modified nucleotides in the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 10 or fewer modified nucleotides in the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 7 or fewer modified nucleotides in the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 6 or fewer modified nucleotides in the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 5 or fewer modified nucleotides in the first nucleotide sequence are 2'-fluoro nucleotides.In some embodiments, no more than four modified nucleotides in the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, no more than three modified nucleotides in the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, no more than two modified nucleotides in the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least one modified nucleotide in the first nucleotide sequence is a 2'-fluoro pyrimidine. In some embodiments, 1, 2, 3, 4, 5, or 6 modified nucleotides in the first nucleotide sequence are 2'-fluoro pyrimidines. In some embodiments, at least one modified nucleotide in the first nucleotide sequence is a 2'-fluoro purine. In some embodiments, 1, 2, 3, 4, 5, or 6 modified nucleotides in the first nucleotide sequence are 2'-fluoro purines. In some embodiments, the 2'-fluoro nucleotides are 2'-fluoro nucleotide mimics.

[0118] In some embodiments, the nucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and / or 19 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least two nucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and / or 19 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least three nucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and / or 19 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least four nucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and / or 19 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least five nucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and / or 19 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and / or 19 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotide at position 3 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 7 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 8 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 9 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 12 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 17 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the 2'-fluoro nucleotide is a 2'-fluoro nucleotide mimic.

[0119] In some embodiments, at least 1, 2, 3, 4, 5, 6, or 7 nucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and / or 19 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and / or 19 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least two nucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and / or 19 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least three nucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and / or 19 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotide at position 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and / or 19 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 3 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 5 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 7 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 8 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 9 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 10 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 11 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 12 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide.In some embodiments, the nucleotide at position 14 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 17 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 19 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at positions 3, 7, 8, 9, 12, and / or 17 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at positions 3, 7, 8, and / or 17 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at positions 3, 7, 8, 9, 12, and / or 17 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at positions 5, 7, 8, and / or 9 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotides at positions 5, 9, 10, 11, 12, and / or 19 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the 2'-fluoro nucleotides are 2'-fluoro nucleotide mimics.

[0120] In some embodiments, the 2'-fluoro or 2'-O-methyl nucleotide mimic is a nucleotide mimic of formula (V): [ka] In the formula, R x are independently a nucleobase, aryl, heteroaryl, or H, and Q 1 and Q 2 are independently S or O, and R 5 are independently -OCD3, -F, or -OCH3, and R 6 and R 7is independently H, D, or CD3. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0121] In some embodiments, the 2'-fluoro or 2'-O-methyl nucleotide mimic is a nucleotide mimic of Formula (16) through Formula (20): [ka] In the formula, R x is independently a nucleobase, aryl, heteroaryl, or H; R 2 is F or —OCH 3 . In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0122] In some embodiments, the sense strand, the antisense strand, or both, each independently comprise: [ka] [ka] wherein B is a nucleobase, aryl, heteroaryl, or H; TIFF2025535539000105.tif8161 represents a phosphodiester bond, a phosphorothioate bond, a mesyl phosphoramidate bond, or H. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0123] In some embodiments, the sense strand, the antisense strand, or both, each independently comprise: [ka] and R may additionally comprise at least one, at least two, at least three, at least four, or at least five, or more modified nucleotides having the chemical structure x is a nucleobase, aryl, heteroaryl, or H, and R y is a nucleobase, TIFF2025535539000107.tif8161 represents a phosphodiester bond, a phosphorothioate bond, a mesyl phosphoramidate bond, or H. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0124] In some embodiments, the sense strand, the antisense strand, or both, each independently comprise: [ka] and optionally, at least one, at least two, at least three, at least four, or at least five or more modified nucleotides having the chemical structure: y is a nucleobase, TIFF2025535539000109.tif8161 represents a phosphodiester bond, a phosphorothioate bond, a mesyl phosphoramidate bond, or H. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0125] For purposes of this disclosure, the modified nucleotide or nucleotide analogue may be at any position in the sense strand, hi some embodiments, the modified nucleotide or nucleotide analogue may be at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 of the sense strand relative to the 5' end.

[0126] In some embodiments, the first nucleotide sequence comprises, consists of, or consists essentially of ribonucleic acid (RNA). In some embodiments, the first nucleotide sequence comprises, consists of, or consists essentially of modified RNA. In some embodiments, the modified RNA is selected from 2'-O-methyl RNA and 2'-fluoro RNA. In some embodiments, 15, 16, 17, 18, 19, 20, 21, 22, or 23 modified nucleotides of the first nucleotide sequence are independently selected from 2'-O-methyl RNA and 2'-fluoro RNA.

[0127] In some embodiments, the sense strand may further comprise one or more internucleoside linkages independently selected from a phosphodiester (PO) internucleoside linkage, a phosphorothioate (PS) internucleoside linkage, a mesyl phosphoramidate internucleoside linkage (Ms), a phosphorodithioate internucleoside linkage, and a PS-mimetic internucleoside linkage. In some embodiments, the PS-mimetic internucleoside linkage is a sulfonucleoside linkage.

[0128] In some embodiments, the sense strand may further comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more phosphorothioate internucleoside linkages. In some embodiments, the sense strand comprises 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 or fewer phosphorothioate internucleoside linkages. In some embodiments, the sense strand comprises 2-10, 2-8, 2-6, 1-5, 1-4, 1-3, or 1-2 phosphorothioate internucleoside linkages. In some embodiments, the sense strand comprises 1-2 phosphorothioate internucleoside linkages. In some embodiments, the sense strand comprises 2-4 phosphorothioate internucleoside linkages. In some embodiments, at least one phosphorothioate internucleoside linkage is between nucleotides 1 and 2 from the 5' end of the first nucleotide sequence. In some embodiments, at least one phosphorothioate internucleoside linkage is between nucleotides 2 and 3 from the 5' end of the first nucleotide sequence. In some embodiments, the sense strand comprises two phosphorothioate internucleoside linkages between nucleotides 1 to 3 from the 5' end of the first nucleotide sequence.

[0129] In some embodiments, the sense strand may further comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more mesyl phosphoramidate internucleoside linkages. In some embodiments, the sense strand comprises 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 or fewer mesyl phosphoramidate internucleoside linkages. In some embodiments, the sense strand comprises 2-10, 2-8, 2-6, 1-5, 1-4, 1-3, or 1-2 mesyl phosphoramidate internucleoside linkages. In some embodiments, the sense strand comprises 1-2 mesyl phosphoramidate internucleoside linkages. In some embodiments, the sense strand contains 2-4 mesyl phosphoramidate internucleoside linkages.

[0130] In some embodiments, the sense strand may include any of the modified nucleotides disclosed below in the subsection entitled "Modified Nucleotides." In some embodiments, the sense strand may include a 5' stabilized endcap, which may be selected from those disclosed below in the subsection entitled "5' Stabilized Endcaps."

[0131] siNA antisense strand Any of the siNA molecules described herein can comprise an antisense strand. The antisense strand can comprise a second nucleotide sequence. The second nucleotide sequence can be 15-30, 15-25, 15-23, 17-23, 19-23, or 19-21 nucleotides in length. In some embodiments, the second nucleotide sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the second nucleotide sequence is at least 19 nucleotides in length. In some embodiments, the second nucleotide sequence is at least 21 nucleotides in length.

[0132] In some embodiments, the antisense strand is the same length as the second nucleotide sequence. In some embodiments, the antisense strand is longer than the second nucleotide sequence. In some embodiments, the antisense strand may comprise 1, 2, 3, 4, 5, or more nucleotides longer than the second nucleotide sequence. In some embodiments, the antisense strand is the same length as the sense strand. In some embodiments, the antisense strand is longer than the sense strand. In some embodiments, the antisense strand may comprise 1, 2, 3, 4, 5, or more nucleotides longer than the sense strand. In some embodiments, the antisense strand may further comprise deoxyribonucleic acid (DNA). In some embodiments, the DNA is thymine (T). In some embodiments, the antisense strand may further comprise a TT sequence. In some embodiments, the antisense strand may further comprise one or more modified nucleotides adjacent to the second nucleotide sequence. In some embodiments, the one or more modified nucleotides are independently selected from any of the modified nucleotides disclosed herein (e.g., 2'-fluoro nucleotides, 2'-O-methyl nucleotides, 2'-fluoro nucleotide mimics, 2'-O-methyl nucleotide mimics, 2'-ocp nucleotides, 2'-omcp nucleotides, 3'-ocp nucleotides, 2'-OMe-3'-xylonucleotides, 2'-F-3'-xylonucleotides, vmX nucleotides, ganciclovir nucleotides, and denavir nucleotides or nucleotides comprising modified nucleobases).

[0133] In some embodiments, the second nucleotide sequence comprises 15, 16, 17, 18, 19, 20, 21, 22, 23, or more modified nucleotides independently selected from 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-ocp nucleotides, and 2'-omcp nucleotides, hi some embodiments, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the nucleotides in the second nucleotide sequence are modified nucleotides independently selected from 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-ocp nucleotides, 2'-omcp nucleotides, 3'-ocp nucleotides, 2'-OMe-3'-xylonucleotides, 2'-F-3'-xylonucleotides, vmX nucleotides, ganciclovir nucleotides, and denavir nucleotides. In some embodiments, 100% of the nucleotides in the second nucleotide sequence are modified nucleotides independently selected from 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-ocp nucleotides, 2'-omcp nucleotides, 3'-ocp nucleotides, 2'-OMe-3'-xylonucleotides, 2'-F-3'-xylonucleotides, vmX nucleotides, ganciclovir nucleotides, and denavir nucleotides.

[0134] In some embodiments, about 15-30, 15-25, 15-24, 15-23, 15-22, 15-21, 17-30, 17-25, 17-24, 17-23, 17-22, 17-21, 18-30, 18-25, 18-24, 18-23, 18-22, 18-21, 19-30, 19-25, 19-24, 19-23, 19-22, 19-21, 20-25, 20-24, 20-23, 21-25, 21-24, or 21-23 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, not more than one of the modified nucleotides of the second nucleotide sequence is a 2'-O-methyl nucleotide. In some embodiments, about 2-20 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, about 5-25 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, about 10-25 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, about 12-25 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 12 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 13 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 14 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 15 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 16 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides.In some embodiments, at least about 17 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 18 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 19 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 21 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 20 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 19 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 18 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 17 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 16 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 15 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 14 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 13 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least one modified nucleotide of the second nucleotide sequence is a 2'-O-methyl pyrimidine. In some embodiments, at least 5, 6, 7, 8, 9, or 10 modified nucleotides of the second nucleotide sequence are 2'-O-methylpyrimidines.In some embodiments, at least one modified nucleotide of the second nucleotide sequence is a 2'-O-methyl purine. In some embodiments, at least 5, 6, 7, 8, 9, or 10 modified nucleotides of the second nucleotide sequence are 2'-O-methyl purines. In some embodiments, the 2'-O-methyl nucleotides are 2'-O-methyl nucleotide mimics.

[0135] In some embodiments, 2 to 15 modified nucleotides of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 2 to 10 modified nucleotides of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 2 to 6 modified nucleotides of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 1 to 6, 1 to 5, 1 to 4, or 1 to 3 modified nucleotides of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least 1, 2, 3, 4, 5, or 6 modified nucleotides of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least 1 modified nucleotide of the second nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, at least 2 modified nucleotides of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least 3 modified nucleotides of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least 4 modified nucleotides of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least 5 modified nucleotides in the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 10, 9, 8, 7, 6, 5, 4, 3, or fewer modified nucleotides in the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 10 or fewer modified nucleotides in the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 7 or fewer modified nucleotides in the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 6 or fewer modified nucleotides in the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 5 or fewer modified nucleotides in the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 4 or fewer modified nucleotides in the second nucleotide sequence are 2'-fluoro nucleotides.In some embodiments, no more than three modified nucleotides in the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, no more than two modified nucleotides in the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least one modified nucleotide in the second nucleotide sequence is a 2'-fluoro pyrimidine. In some embodiments, 1, 2, 3, 4, 5, or 6 modified nucleotides in the second nucleotide sequence are 2'-fluoro pyrimidines. In some embodiments, at least one modified nucleotide in the second nucleotide sequence is a 2'-fluoro purine. In some embodiments, 1, 2, 3, 4, 5, or 6 modified nucleotides in the second nucleotide sequence are 2'-fluoro purines. In some embodiments, the 2'-fluoro nucleotides are 2'-fluoro nucleotide mimics.

[0136] In some embodiments, the 2'-fluoro or 2'-O-methyl nucleotide is a 2'-fluoro or 2'-O-methyl nucleotide mimic. In some embodiments, the 2'-fluoro or 2'-O-methyl nucleotide mimic is a nucleotide mimic of formula (V): [ka] In the formula, R x are independently a nucleobase, aryl, heteroaryl, or H, and Q 1 and Q 2 are independently S or O, and R 5 are independently -OCD3, -F, or -OCH3, and R 6 and R 7 is independently H, D, or CD3. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0137] In some embodiments, the 2'-fluoro or 2'-O-methyl nucleotide mimic is a nucleotide mimic of Formula (16) through Formula (20): [ka] In the formula, R x is a nucleobase, aryl, heteroaryl, or H, and R 2 are independently F or -OCH3, TIFF2025535539000112.tif8161 represents a phosphodiester bond, a phosphorothioate bond, a mesyl phosphoramidate bond, or H. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0138] In some embodiments, the sense strand, the antisense strand, or both, each independently comprise: [ka] wherein B is a nucleobase, aryl, heteroaryl, or H; TIFF2025535539000114.tif8161 represents a phosphodiester bond, a phosphorothioate bond, a mesyl phosphoramidate bond, or H. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0139] In some embodiments, the antisense strand, the sense strand, or both, each independently comprise: [ka] and the modified nucleotides may comprise at least one, at least two, at least three, at least four, or at least five, or more modified nucleotides having the chemical structure: y is a nucleobase, TIFF2025535539000116.tif8161 represents a phosphodiester bond, a phosphorothioate bond, a mesyl phosphoramidate bond, or H. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0140] For purposes of this disclosure, modified nucleotides may be at any position in the antisense strand, hi some embodiments, modified nucleotides may be at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 of the antisense strand relative to the 5' end.

[0141] In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides at positions 2, 5, 6, 8, 10, 14, 16, 17, and / or 18 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotides at positions 2, 5, 6, 8, 10, 14, 16, 17, and / or 18 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least two nucleotides at positions 2, 5, 6, 8, 10, 14, 16, 17, and / or 18 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least three nucleotides at positions 2, 5, 6, 8, 10, 14, 16, 17, and / or 18 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least four nucleotides at positions 2, 5, 6, 8, 10, 14, 16, 17, and / or 18 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least five nucleotides at positions 2, 5, 6, 8, 10, 14, 16, 17, and / or 18 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotides at positions 2 and / or 14 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotides at positions 2, 6, and / or 16 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotides at positions 2, 6, 14, and / or 16 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotide at positions 2, 6, 10, 14, and / or 18 from the 5' end of the second nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at positions 2, 5, 8, 14, and / or 17 from the 5' end of the second nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 2 from the 5' end of the second nucleotide sequence is a 2'-fluoro nucleotide.In some embodiments, the nucleotide at position 5 from the 5' end of the second nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 6 from the 5' end of the second nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 8 from the 5' end of the second nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 10 from the 5' end of the second nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 14 from the 5' end of the second nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 16 from the 5' end of the second nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 17 from the 5' end of the second nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 18 from the 5' end of the second nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the 2'-fluoro nucleotide is a 2'-fluoro nucleotide mimic.

[0142] In some embodiments, the nucleotides in the second nucleotide sequence are arranged in an alternating 1:3 modification pattern, where one nucleotide is a 2'-fluoro nucleotide and three nucleotides are 2'-O-methyl nucleotides, and the alternating 1:3 modification pattern occurs at least twice. In some embodiments, the alternating 1:3 modification pattern occurs 2 to 5 times. In some embodiments, at least two of the alternating 1:3 modification patterns occur consecutively. In some embodiments, at least two of the alternating 1:3 modification patterns occur non-consecutively. In some embodiments, at least 1, 2, 3, 4, or 5 alternating 1:3 modification patterns begin 2, 6, 10, 14, and / or 18 nucleotide positions from the 5'-end of the antisense strand. In some embodiments, at least one alternating 1:3 modification pattern begins 2 nucleotide positions from the 5'-end of the antisense strand. In some embodiments, at least one alternating 1:3 modification pattern begins 6 nucleotide positions from the 5'-end of the antisense strand. In some embodiments, at least one alternating 1:3 modification pattern begins 10 nucleotide positions from the 5'-end of the antisense strand. In some embodiments, at least one alternating 1:3 modification pattern begins 14 nucleotide positions from the 5'-end of the antisense strand. In some embodiments, at least one alternating 1:3 modification pattern begins 18 nucleotide positions from the 5'-end of the antisense strand. In some embodiments, the 2'-fluoro nucleotide is a 2'-fluoro nucleotide mimic.

[0143] In some embodiments, the nucleotides in the second nucleotide sequence are arranged in an alternating 1:2 modification pattern, where one nucleotide is a 2'-fluoro nucleotide and two nucleotides are 2'-O-methyl nucleotides, and the alternating 1:2 modification pattern occurs at least twice. In some embodiments, the alternating 1:2 modification pattern occurs 2 to 5 times. In some embodiments, at least two of the alternating 1:2 modification patterns occur consecutively. In some embodiments, at least two of the alternating 1:2 modification patterns occur non-consecutively. In some embodiments, at least 1, 2, 3, 4, or 5 alternating 1:2 modification patterns begin 2, 5, 8, 14, and / or 17 nucleotide positions from the 5'-end of the antisense strand. In some embodiments, at least one alternating 1:2 modification pattern begins 2 nucleotide positions from the 5'-end of the antisense strand. In some embodiments, at least one alternating 1:2 modification pattern begins 5 nucleotide positions from the 5'-end of the antisense strand. In some embodiments, at least one alternating 1:2 modification pattern begins 8 nucleotide positions from the 5'-end of the antisense strand. In some embodiments, at least one alternating 1:2 modification pattern begins 14 nucleotide positions from the 5'-end of the antisense strand. In some embodiments, at least one alternating 1:2 modification pattern begins 17 nucleotide positions from the 5'-end of the antisense strand. In some embodiments, the 2'-fluoro nucleotide is a 2'-fluoro nucleotide mimic.

[0144] In some embodiments, the second nucleotide sequence comprises, consists of, or consists essentially of ribonucleic acid (RNA). In some embodiments, the second nucleotide sequence comprises, consists of, or consists essentially of modified RNA. In some embodiments, the modified RNA is selected from 2'-O-methyl RNA and 2'-fluoro RNA. In some embodiments, 15, 16, 17, 18, 19, 20, 21, 22, or 23 modified nucleotides of the second nucleotide sequence are independently selected from 2'-O-methyl RNA and 2'-fluoro RNA. In some embodiments, the 2'-fluoro nucleotide is a 2'-fluoro nucleotide mimic.

[0145] In some embodiments, the sense strand may further comprise one or more internucleoside linkages independently selected from a phosphodiester (PO) internucleoside linkage, a phosphorothioate (PS) internucleoside linkage, a phosphorodithioate internucleoside linkage, and a PS-mimetic internucleoside linkage. In some embodiments, the PS-mimetic internucleoside linkage is a sulfonucleoside linkage.

[0146] In some embodiments, the antisense strand may further comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more phosphorothioate internucleoside linkages. In some embodiments, the antisense strand comprises 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 or fewer phosphorothioate internucleoside linkages. In some embodiments, the antisense strand comprises 2-10, 2-8, 2-6, 1-5, 1-4, 1-3, or 1-2 phosphorothioate internucleoside linkages. In some embodiments, the antisense strand comprises 2-10, 2-8, 2-6, 1-5, 1-4, 1-3, or 1-2 phosphorothioate internucleoside linkages. In some embodiments, the antisense strand comprises 2 to 8 phosphorothioate internucleoside linkages. In some embodiments, the antisense strand comprises 3 to 8 phosphorothioate internucleoside linkages. In some embodiments, the antisense strand comprises 4 to 8 phosphorothioate internucleoside linkages. In some embodiments, at least one phosphorothioate internucleoside linkage is between nucleotides 1 and 2 from the 5' end of the second nucleotide sequence. In some embodiments, at least one phosphorothioate internucleoside linkage is between nucleotides 2 and 3 from the 5' end of the second nucleotide sequence. In some embodiments, at least one phosphorothioate internucleoside linkage is between nucleotides 1 and 2 from the 3' end of the second nucleotide sequence. In some embodiments, at least one phosphorothioate internucleoside linkage is between nucleotides 2 and 3 from the 3' end of the second nucleotide sequence. In some embodiments, the antisense strand comprises two phosphorothioate internucleoside linkages between nucleotides 1 to 3 from the 5' end of the first nucleotide sequence. In some embodiments, the antisense strand comprises two phosphorothioate internucleoside linkages between nucleotides 1 to 3 from the 3' end of the first nucleotide sequence.In some embodiments, the antisense strand comprises (a) two phosphorothioate internucleoside linkages between nucleotides 1 to 3 from the 5' end of the first nucleotide sequence, and (b) two phosphorothioate internucleoside linkages between nucleotides 1 to 3 from the 3' end of the first nucleotide sequence.

[0147] In some embodiments, the antisense strand may further comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more mesyl phosphoramidate internucleoside linkages. In some embodiments, the antisense strand comprises 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 or fewer mesyl phosphoramidate internucleoside linkages. In some embodiments, the antisense strand comprises 2-10, 2-8, 2-6, 1-5, 1-4, 1-3, or 1-2 mesyl phosphoramidate internucleoside linkages. In some embodiments, the antisense strand comprises 2 to 10, 2 to 8, 2 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 mesyl phosphoramidate internucleoside linkages. In some embodiments, the antisense strand comprises 2 to 8 mesyl phosphoramidate internucleoside linkages. In some embodiments, the antisense strand comprises 3 to 8 mesyl phosphoramidate internucleoside linkages. In some embodiments, the antisense strand comprises 4 to 8 mesyl phosphoramidate internucleoside linkages.

[0148] In some embodiments, at least one end of the ds-siNA is blunt-ended. In some embodiments, at least one end of the ds-siNA comprises an overhang, the overhang comprising at least one nucleotide. In some embodiments, both ends of the ds-siNA comprise an overhang, the overhang comprising at least one nucleotide. In some embodiments, the overhang comprises 1 to 5 nucleotides, 1 to 4 nucleotides, 1 to 3 nucleotides, or 1 to 2 nucleotides. In some embodiments, the overhang consists of 1 to 2 nucleotides.

[0149] In some embodiments, the sense strand may include any of the modified nucleotides disclosed below in the subsection entitled "Modified Nucleotides." In some embodiments, the sense strand may include a 5' stabilized endcap, which may be selected from those disclosed below in the subsection entitled "5' Stabilized Endcaps."

[0150] Modified Nucleotides The present disclosure provides oligonucleotides comprising one or more modified nucleotides disclosed herein. The oligonucleotides may be selected from short interfering nucleic acids (siNAs), antisense oligonucleotides (ASOs), steric blockers, short hairpin RNAs (shRNAs), and mRNAs.

[0151] The oligonucleotide may be a siNA, which may comprise a sense strand and an antisense strand. In some embodiments, the sense strand disclosed herein comprises one or more modified nucleotides. In some embodiments, any of the first nucleotide sequences disclosed herein comprises one or more modified nucleotides. In some embodiments, the antisense strand disclosed herein comprises one or more modified nucleotides. In some embodiments, any of the second nucleotide sequences disclosed herein comprises one or more modified nucleotides. In some embodiments, one or more modified nucleotides are adjacent to the first nucleotide sequence. In some embodiments, at least one modified nucleotide is adjacent to the 5' end of the first nucleotide sequence. In some embodiments, at least one modified nucleotide is adjacent to the 3' end of the first nucleotide sequence. In some embodiments, at least one modified nucleotide is adjacent to the 5' end of the first nucleotide sequence and at least one modified nucleotide is adjacent to the 3' end of the first nucleotide sequence. In some embodiments, one or more modified nucleotides are adjacent to the second nucleotide sequence. In some embodiments, at least one modified nucleotide is adjacent to the 5' end of the second nucleotide sequence. In some embodiments, at least one modified nucleotide is adjacent to the 3' end of the second nucleotide sequence. In some embodiments, at least one modified nucleotide is adjacent to the 5' end of the second nucleotide sequence and at least one modified nucleotide is adjacent to the 3' end of the second nucleotide sequence. In some embodiments, a 2'-O-methyl nucleotide in any of the sense strand or first nucleotide sequences disclosed herein is replaced with a modified nucleotide. In some embodiments, a 2'-O-methyl nucleotide in any of the antisense strand or second nucleotide sequences disclosed herein is replaced with a modified nucleotide.

[0152] In some embodiments, any of the siNA molecules, siNAs, sense strands, first nucleotide sequences, antisense strands, and second nucleotide sequences disclosed herein contain 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 or more modified nucleotides. In some embodiments, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the nucleotides in the siNA molecule, siNA, sense strand, first nucleotide sequence, antisense strand, or second nucleotide sequence are modified nucleotides.

[0153] In some embodiments, the modified nucleotide is selected from the group consisting of a 2'-fluoronucleotide, a 2'-O-methyl nucleotide, a 2'-fluoronucleotide mimic, a 2'-O-methyl nucleotide mimic, a 2'-ocp nucleotide, a 2'-omcp nucleotide, a 3'-ocp nucleotide, a 2'-OMe-3'-xylonucleotide, a 2'-F-3'-xylonucleotide, a vmX nucleotide, a ganciclovir nucleotide, or a denovir nucleotide, a locked nucleic acid, an unlocked nucleic acid, a nucleotide analog, and a nucleotide comprising a modified nucleobase. In some embodiments, the unlocked nucleic acid is a 2',3'-unlocked nucleic acid. In some embodiments, the unlocked nucleic acid is a 3',4'-unlocked nucleic acid in which the furanose ring lacks a bond between the 3' and 4' carbons (e.g., 3',4'-seco and mun34).

[0154] In some embodiments, the siNA of the present disclosure: [ka] [ka] or a combination thereof. In some embodiments, the siNA may contain at least two, at least three, at least four, or at least five or more of these modified nucleotides. In some embodiments, the sense strand comprises [ka] [ka] In some embodiments, the antisense strand may comprise at least one, at least two, at least three, at least four, or at least five or more of: [ka] [ka] or a combination thereof, wherein B is a nucleobase, aryl, heteroaryl, or H. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0155] In some embodiments, the siNA of the present disclosure optionally comprises: [ka] wherein Rx is a nucleobase, aryl, heteroaryl, or H; [ka] (In the formula, R y is a nucleobase), and [ka] (In the formula, Ry is a nucleobase), or combinations thereof. In some embodiments, the siNA may comprise at least two, three, four, or five or more of these modified nucleotides. In some embodiments, the sense strand optionally comprises [ka] wherein Rx is a nucleobase, aryl, heteroaryl, or H; [ka] (In the formula, R y is a nucleobase), and [ka] (In the formula, R y In some embodiments, the antisense strand may comprise at least one, two, three, four, or five or more of: [ka] wherein Rx is a nucleobase, aryl, heteroaryl, or H; [ka] (In the formula, R y is a nucleobase), and [ka] (In the formula, R y In some embodiments, both the sense strand and the antisense strand may each independently comprise at least one, two, three, four, or five or more of: [ka] (In the formula, R xis a nucleobase, aryl, heteroaryl, or H; [ka] (In the formula, R y are nucleobases), [ka] (In the formula, R y are nucleobases), or combinations thereof. In some embodiments, the nucleobases are selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof. For example, [ka] In some embodiments, the modified nucleotide is [ka] It may have the structure:

[0156] In some embodiments, any of the siNAs disclosed herein can optionally contain other modified nucleotides, such as 2'-fluoro or 2'-O-methyl nucleotide mimics. For example, the disclosed siNAs can contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more 2'-fluoro or 2'-O-methyl nucleotide mimics. In some embodiments, any of the sense strands disclosed herein contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more 2'-fluoro or 2'-O-methyl nucleotide mimics. In some embodiments, any of the first nucleotide sequences disclosed herein contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more 2'-fluoro or 2'-O-methyl nucleotide mimics. In some embodiments, any of the antisense strands disclosed herein comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more 2'-fluoro or 2'-O-methyl nucleotide mimics. In some embodiments, any of the second nucleotide sequences disclosed herein comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more 2'-fluoro or 2'-O-methyl nucleotide mimics. In some embodiments, the 2'-fluoro or 2'-O-methyl nucleotide mimics are nucleotide mimics of Formula (16) through Formula (20): [ka] In the formula, R x is a nucleobase, aryl, heteroaryl, or H, and R 2 is independently F or —OCH 3 . In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0157] In some embodiments, siNA molecules disclosed herein comprise at least one 2'-fluoro nucleotide, at least one 2'-O-methyl nucleotide, and at least one 2'-fluoro or 2'-O-methyl nucleotide mimic. In some embodiments, at least one 2'-fluoro or 2'-O-methyl nucleotide mimic is adjacent to a first nucleotide sequence. In some embodiments, at least one 2'-fluoro or 2'-O-methyl nucleotide mimic is adjacent to the 5' end of the first nucleotide sequence. In some embodiments, at least one 2'-fluoro or 2'-O-methyl nucleotide mimic is adjacent to the 3' end of the first nucleotide sequence. In some embodiments, at least one 2'-fluoro or 2'-O-methyl nucleotide mimic is adjacent to a second nucleotide sequence. In some embodiments, at least one 2'-fluoro or 2'-O-methyl nucleotide mimic is adjacent to the 5' end of the second nucleotide sequence. In some embodiments, at least one 2'-fluoro or 2'-O-methyl nucleotide mimic is adjacent to the 3' end of the second nucleotide sequence. In some embodiments, the first nucleotide sequence does not include a 2'-fluoro nucleotide mimic. In some embodiments, the first nucleotide sequence does not include a 2'-O-methyl nucleotide mimic. In some embodiments, the second nucleotide sequence does not include a 2'-fluoro nucleotide mimic. In some embodiments, the second nucleotide sequence does not include a 2'-O-methyl nucleotide mimic.

[0158] In some embodiments, any of the siNA, sense strand, first nucleotide sequence, antisense strand, or second nucleotide sequence disclosed herein is [ka] wherein Rx is a nucleobase, aryl, heteroaryl, or H; or [ka] (In the formula, R y is a nucleobase).

[0159] In some embodiments, any of the siNA, sense strand, first nucleotide sequence, antisense strand, or second nucleotide sequence disclosed herein optionally comprises: [ka] wherein B is a nucleobase, aryl, heteroaryl, or H; TIFF2025535539000141.tif8161 provides oligonucleotides exhibiting phosphodiester, phosphorothioate, or mesyl phosphoramidate linkages.

[0160] Phosphorylation Blockers Also disclosed herein are siNA molecules comprising phosphorylation blockers. In some embodiments, 2'-O-methyl nucleotides in any of the sense strands or first nucleotide sequences disclosed herein are replaced with nucleotides containing phosphorylation blockers. In some embodiments, 2'-O-methyl, 2'-ocp, or 2'-omcp nucleotides in any of the antisense strands or second nucleotide sequences disclosed herein are replaced with nucleotides containing phosphorylation blockers. In some embodiments, 2'-O-methyl, 2'-ocp, or 2'-omcp nucleotides in any of the sense strands or first nucleotide sequences disclosed herein are further modified to contain phosphorylation blockers. In some embodiments, 2'-O-methyl, 2'-ocp, or 2'-omcp nucleotides in any of the antisense strands or second nucleotide sequences disclosed herein are further modified to contain phosphorylation blockers.

[0161] In some embodiments, any of the siNA molecules disclosed herein has the formula (IV): [ka] wherein R y is the nucleobase and R 4 -OR 30 or -NR 31 R 32 and R 30 is a C1-C8 substituted or unsubstituted alkyl, and R 31 and R 32 together with the nitrogen to which they are attached form a substituted or unsubstituted heterocyclic ring. In some embodiments, the nucleobases are selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0162] In some embodiments, any of the siNA molecules disclosed herein has the formula (IV): [ka] phosphorylation blockers of formula (IV), wherein R y is the nucleobase and R 4 is —OCH 3 or —N(CH 2 CH 2 ) 2 O. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0163] In some embodiments, the siNA molecule comprises (a) a molecule of formula (IV): [ka] wherein R y is the nucleobase and R 4 -OR 30 or -NR 31 R 32 and R 30 is a C1-C8 substituted or unsubstituted alkyl, and R 31 and R 32and (b) a short interfering nucleic acid (siNA), wherein the phosphorylation blocker, together with the nitrogen to which they are attached, forms a substituted or unsubstituted heterocyclic ring, and the phosphorylation blocker is conjugated to the siNA. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0164] In some embodiments, the siNA molecule comprises (a) a molecule of formula (IV): [ka] A phosphorylation blocker of formula (IV), wherein R y is the nucleobase and R 4 comprises (a) a phosphorylation blocker that is -OCH3 or -N(CH2CH2)2O, and (b) a short interfering nucleic acid (siNA), wherein the phosphorylation blocker is bound to the siNA.

[0165] In some embodiments, the phosphorylation blocker is attached to the 3' end of the sense strand or the first nucleotide sequence. In some embodiments, the phosphorylation blocker is attached to the 3' end of the sense strand or the first nucleotide sequence via one, two, three, four, five, or more linkers. In some embodiments, the phosphorylation blocker is attached to the 5' end of the sense strand or the first nucleotide sequence. In some embodiments, the phosphorylation blocker is attached to the 5' end of the sense strand or the first nucleotide sequence via one, two, three, four, five, or more linkers. In some embodiments, the phosphorylation blocker is attached to the 3' end of the antisense strand or the second nucleotide sequence. In some embodiments, the phosphorylation blocker is attached to the 3' end of the antisense strand or the second nucleotide sequence via one, two, three, four, five, or more linkers. In some embodiments, the phosphorylation blocker is attached to the 5' end of the antisense strand or the second nucleotide sequence. In some embodiments, the phosphorylation blocker is attached to the 5' end of the antisense strand or the second nucleotide sequence via one, two, three, four, five, or more linkers, in some embodiments, the one or more linkers are independently selected from the group consisting of a phosphodiester linker, a phosphorothioate linker, a mesyl phosphoramidate linker, and a phosphorodithioate linker.

[0166] Conjugation Moiety Further disclosed herein are siNA molecules comprising a conjugation moiety. In some embodiments, the conjugation moiety is selected from galactosamine, peptide, protein, sterol, lipid, phospholipid, biotin, phenoxazine, active drug substance, cholesterol, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dye. In some embodiments, the conjugation moiety is attached to the 3' end of the sense strand or the first nucleotide sequence. In some embodiments, the conjugation moiety is attached to the 3' end of the sense strand or the first nucleotide sequence via one, two, three, four, five, or more linkers. In some embodiments, the conjugation moiety is attached to the 5' end of the sense strand or the first nucleotide sequence. In some embodiments, the conjugation moiety is attached to the 5' end of the sense strand or the first nucleotide sequence via one, two, three, four, five, or more linkers. In some embodiments, the conjugated moiety is attached to the 3'-end of the antisense strand or the second nucleotide sequence. In some embodiments, the conjugated moiety is attached to the 3'-end of the antisense strand or the second nucleotide sequence via one, two, three, four, five, or more linkers. In some embodiments, the conjugated moiety is attached to the 5'-end of the antisense strand or the second nucleotide sequence. In some embodiments, the conjugated moiety is attached to the 5'-end of the antisense strand or the second nucleotide sequence via one, two, three, four, five, or more linkers. In some embodiments, the one or more linkers are independently selected from the group consisting of a phosphodiester linker, a phosphorothioate linker, a phosphorodithioate linker, and a mesyl phosphoramidate linker.

[0167] In some embodiments, the conjugation moiety is galactosamine. In some embodiments, any of the siNAs disclosed herein are linked to a conjugation moiety that is galactosamine. In some embodiments, the galactosamine is N-acetylgalactosamine (GalNAc). In some embodiments, any of the siNA molecules disclosed herein comprise GalNAc. In some embodiments, GalNAc is represented by formula (VI): [ka] wherein m is 1, 2, 3, 4, or 5, each n is independently 1 or 2, p is 0 or 1, each R is independently H or a first protecting group, each Y is independently selected from -OP(=O)(SH)-, -OP(=O)(O)-, -OP(=O)(OH)-, -OP(S)S-, and -O-, Z is H or a second protecting group, either L is a linker, or L and Y together are a linker, and A is H, OH, a third protecting group, an activating group, or an oligonucleotide. In some embodiments, the first protecting group is acetyl. In some embodiments, the second protecting group is trimethoxytrityl (TMT). In some embodiments, the activating group is a phosphoramidite group. In some embodiments, the phosphoramidite group is a cyanoethoxy N,N-diisopropyl phosphoramidite group. In some embodiments, the linker is a C6-NH2 group. In some embodiments, A is a short interfering nucleic acid (siNA) or siNA molecule. In some embodiments, m is 3. In some embodiments, R is H, Z is H, and n is 1. In some embodiments, R is H, Z is H, and n is 2.

[0168] In some embodiments, GalNAc is of formula (VII): [ka] In the formula, R zis OH or SH, and each n is independently 1 or 2. In some embodiments, the targeting ligand can be GalNAc, and the targeting ligand can include 1, 2, 3, 4, 5, or 6 GalNAc units. In some embodiments, the targeting ligand can be GalNAc2, GalNAc3, GalNAc4 (GalNAc of Formula VII, where n=1 and R z ═OH), GalNAc5, and GalNAc6.

[0169] In some embodiments, GalNAc can be a GalNAc amidite (e.g., compound 40-9, see Example 22), GalNAc4 CPG (e.g., compound 40-8, see Examples 22 and 23), GalNAc phosphoramidite, or GalNAc4-ps-GalNAc4-ps-GalNAc4. These GalNAc moieties are shown below. [Table 1]

[0170] GalNAc3, GalNAc4, GalNAc5, and GalNAc6 may be conjugated to the siNAs disclosed herein during synthesis using moieties 1, 2, or 3. Additional GalNAc moieties, such as GalNAc1 and GalNAc2, can be used to form 5'- and 3'-GalNAc using post-synthesis conjugation. [Table 2]

[0171] In some embodiments, the galactosamine is attached to the 3' end of the sense strand or the first nucleotide sequence. In some embodiments, the galactosamine is attached to the 3' end of the sense strand or the first nucleotide sequence via one, two, three, four, five, or more linkers. In some embodiments, the galactosamine is attached to the 5' end of the sense strand or the first nucleotide sequence. In some embodiments, the galactosamine is attached to the 5' end of the sense strand or the first nucleotide sequence via one, two, three, four, five, or more linkers. In some embodiments, the galactosamine is attached to the 3' end of the antisense strand or the second nucleotide sequence. In some embodiments, the galactosamine is attached to the 3' end of the antisense strand or the second nucleotide sequence via one, two, three, four, five, or more linkers. In some embodiments, the galactosamine is attached to the 5' end of the antisense strand or the second nucleotide sequence. In some embodiments, the galactosamine is attached to the 5'-end of the antisense strand or the second nucleotide sequence via one, two, three, four, five, or more linkers. In some embodiments, one or more linkers are independently selected from the group consisting of a phosphodiester (p or po) linker, a phosphorothioate (ps) linker, a mesyl phosphoramidate linker (Ms), a phosphoramidite (HEG) linker, a triethylene glycol (TEG) linker, and / or a phosphorodithioate linker. In some embodiments, one or more linkers are independently selected from the group consisting of p-(PS)2, (PS)2-p-TEG-p, (PS)2-p-HEG-p, and (PS)2-p-(HEG-p)2.

[0172] In some embodiments, the conjugation moiety is a lipid moiety. In some embodiments, any of the siNAs disclosed herein is conjugated to a conjugation moiety that is a lipid moiety. Examples of lipid moieties include, but are not limited to, cholesterol moieties, thioethers such as hexyl-S-tritylthiol, thiocholesterol, aliphatic chains such as dodecanediol or undecyl residues, phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1-di-O-hexadecyl-rac-glycero-SH-phosphonate, polyamines or polyethylene glycol chains, adamantane acetic acid, palmityl moieties, or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties.

[0173] In some embodiments, the conjugation moiety is an active drug substance. In some embodiments, any of the siNAs disclosed herein is linked to a conjugation moiety that is an active drug substance. Examples of active drug substances include, but are not limited to, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (5)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folic acid, benzothiadiazide, chlorothiazide, diazepines, indomethicin, barbiturates, cephalosporins, sulfa drugs, antidiabetics, antibacterial agents, or antibiotics.

[0174] 5' stabilized end cap Further disclosed herein are oligonucleotides (e.g., siNAs) comprising a 5' stabilized endcap. The terms "5' stabilized endcap" and "5' endcap" are used interchangeably herein. In some embodiments, 2'-O-methyl, 2'-ocp, or 2'-omcp nucleotides in any of the sense strands or first nucleotide sequences disclosed herein are replaced with nucleotides containing a 5' stabilized endcap. In some embodiments, 2'-O-methyl, 2'-ocp, or 2'-omcp nucleotides in any of the antisense strands or second nucleotide sequences disclosed herein are replaced with nucleotides containing a 5' stabilized endcap. In some embodiments, 2'-O-methyl, 2'-ocp, or 2'-omcp nucleotides in any of the sense strands or first nucleotide sequences disclosed herein are further modified to contain a 5' stabilized endcap. In some embodiments, the 2'-O-methyl, 2'-ocp, or 2'-omcp nucleotides in any of the antisense strands or second nucleotide sequences disclosed herein are further modified to contain a 5' stabilized end cap.

[0175] In some embodiments, the 5'-stabilized endcap is a 5'-phosphate mimic. In some embodiments, the 5'-stabilized endcap is a modified 5'-phosphate mimic. In some embodiments, the modified 5'-phosphate is a chemically modified 5'-phosphate. In some embodiments, the 5'-stabilized endcap is a 5'-vinyl phosphonate. In some embodiments, the 5'-vinyl phosphonate is 5'-(E)-vinyl phosphonate or 5'-(Z)-vinyl phosphonate. In some embodiments, the 5'-vinyl phosphonate is a deuterated vinyl phosphonate. In some embodiments, the deuterated vinyl phosphonate is a mono-deuterated vinyl phosphonate. In some embodiments, the deuterated vinyl phosphonate is a di-deuterated vinyl phosphonate. In some embodiments, the 5'-stabilized endcap is a phosphate mimic. Examples of phosphate mimetics are disclosed in Parmar et al., J Med Chem, 201861(3):734-744, WO 2018 / 045317 and WO 2018 / 044350, and U.S. Pat. No. 10,087,210, each of which is incorporated by reference in its entirety.

[0176] In some aspects, the present disclosure provides short interfering nucleic acids (siNAs) comprising a sense strand and an antisense strand, wherein the antisense strand comprises: [ka] and a 5' vinyl phosphonate dimer moiety comprising the structure wherein each B is independently selected from a nucleobase, an aryl, a heteroaryl, and H; TIFF2025535539000151.tif8161 represents a phosphodiester bond, a phosphorothioate bond, or a mesyl phosphoramidate bond. In some embodiments, the chiral center is in the S configuration. In some embodiments, the chiral center is in the R configuration. In some embodiments, each B of the vinyl phosphonate dimer moiety may be the same nucleobase, while in some embodiments, each B may be a different nucleobase. The nucleobase may be selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof. In some embodiments, each B is independently thymine, cytosine, guanine, adenine, or uracil. For example, a vinyl phosphonate dimer of the present disclosure may be [ka] As shown in the structure of

[0177] In some aspects, the present disclosure provides a method for manufacturing a semiconductor device, comprising: [ka] wherein R y is the nucleobase and R 15 is F or CH3. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof. In some embodiments, the disclosed nucleotide phosphate mimics are [ka] [ka] [ka] [ka] [ka] These include, but are not limited to, the structure 15 is H or CH3.

[0178] In some aspects, the present disclosure provides a method for manufacturing a semiconductor device, comprising: [ka] wherein R 15 is H or CH. In some embodiments, one of these novel nucleotide phosphate mimetics (e.g., omeco-d3 nucleotide, 4h nucleotide, v-mun nucleotide, c2o-4h nucleotide, coc-4h nucleotide, omeco-munb nucleotide, or d2vm nucleotide) can be located at the 5'-end of the antisense strand. However, these novel nucleotide phosphate mimetics can also be incorporated at the 5'-end of the sense strand, the 3'-end of the antisense strand, or the 3'-end of the sense strand.

[0179] Additionally or alternatively, the siNA molecules disclosed herein may comprise a nucleotide sequence of formula (Ia) in the sense strand, the antisense strand, or both. [ka] wherein R x is H, a nucleobase, an aryl, or a heteroaryl; R 26 teeth, [ka] -CH=CD-Z, -CD=CH-Z, -CD=CD-Z, -(CR 21 R 22 ) n -Z, or -(C-C alkenylene)-Z, and R 20 is H or R 26 and R 20 Both are -(CR 21 R 22 ) nforming a 3- to 7-membered carbocyclic ring substituted with -Z or -(C2-C6 alkenylene)-Z, where n is 1, 2, 3, or 4, and Z is -ONR 23 R 24 , -OP(O)OH(CH2) m CO2R 23 , -OP(S)OH(CH2) m CO2R 23 , -P(O)(OH)2, -P(O)(OH)(OCH3), -P(O)(OH)(OCD3), -SO2(CH2) m P(O)(OH)2, -SO2NR 23 R 25 , -NR 23 R 24 , -NR 23 SO2R 24 and R 21 and R 22 are independently hydrogen or C1-C6 alkyl, or R 21 and R 22 together form an oxo group, and R 23 is hydrogen or C1-C6 alkyl, and R 24 is -SO2R 25 or -C(O)R 25 and R 23 and R 24 together with the nitrogen to which they are attached form a substituted or unsubstituted heterocyclic ring, and R 25 is hydrogen or C1-C6 alkyl, and m is 1, 2, 3, or 4. In some embodiments, R 1 is aryl. In some embodiments, aryl is phenyl.

[0180] Additionally or alternatively, the siNA molecules disclosed herein may comprise a nucleotide sequence of formula (Ib) in the sense strand, the antisense strand, or both. [ka] wherein R x is H, a nucleobase, an aryl, or a heteroaryl; R 26 teeth, [ka] -CH=CD-Z, -CD=CH-Z, -CD=CD-Z, -(CR 21 R 22 ) n -Z, or -(C-C alkenylene)-Z, and R 20 is H or R 26 and R 20 Both are -(CR 21 R 22 ) n forming a 3- to 7-membered carbocyclic ring substituted with -Z or -(C2-C6 alkenylene)-Z, where n is 1, 2, 3, or 4, and Z is -ONR 23 R 24 , -OP(O)OH(CH2) m CO2R 23 , -OP(S)OH(CH2) m CO2R 23 , -P(O)(OH)2, -P(O)(OH)(OCH3), -P(O)(OH)(OCD3), -SO2(CH2) m P(O)(OH)2, -SO2NR 23 R 25 , -NR 23 R 24 , -NR 23 SO2R 24 and R 21 and R 22 are independently hydrogen or C1-C6 alkyl, or R 21 and R 22 together form an oxo group, and R 23 is hydrogen or C1-C6 alkyl, and R 24 is -SO2R 25 or -C(O)R 25 and R 23 and R 24 together with the nitrogen to which they are attached form a substituted or unsubstituted heterocyclic ring, and R 25 is C1-C6 alkyl, and m is 1, 2, 3, or 4. In some embodiments, R 1 is aryl. In some embodiments, aryl is phenyl.

[0181] Additionally or alternatively, the siNA molecules disclosed herein may comprise a nucleotide sequence of formula (Ic) in the sense strand, the antisense strand, or both. [ka] wherein R x is a nucleobase, aryl, heteroaryl, or H; R 26 teeth, [ka] -CH=CD-Z, -CD=CH-Z, -CD=CD-Z, -(CR 21 R 22 ) n -Z, or -(C-C alkenylene)-Z, and R 20 is hydrogen, or R 26 and R 20 Both are -(CR 21 R 22 ) n forming a 3- to 7-membered carbocyclic ring substituted with -Z or -(C2-C6 alkenylene)-Z, where n is 1, 2, 3, or 4; Z is -ONR 23 R 24 , -OP(O)OH(CH2) m CO2R 23 , -OP(S)OH(CH2) m CO2R 23 , -P(O)(OH)2, -P(O)(OH)(OCH3), -P(O)(OH)(OCD3), -SO2(CH2) m P(O)(OH)2, -SO2NR 23 R 25 , -NR 23 R 24 , or -NR 23 SO2R 24 and R 21 and R 22 are independently hydrogen or C1-C6 alkyl, or R 21 and R 22 together form an oxo group, and R 23is hydrogen or C1-C6 alkyl, and R 24 is -SO2R 25 or -C(O)R 25 or R 23 and R 24 together with the nitrogen to which they are attached form a substituted or unsubstituted heterocyclic ring, and R 25 is C1-C6 alkyl, and m is 1, 2, 3, or 4. In some embodiments, R 1 is aryl. In some embodiments, aryl is phenyl.

[0182] Additionally or alternatively, the siNA molecules disclosed herein may comprise a nucleotide sequence of formula (IIa) in the sense strand, the antisense strand, or both. [ka] wherein R x is a nucleobase, aryl, heteroaryl, or H, and R 26 teeth, [ka] and R 9 is -SO2CH3 or -COCH3, TIFF2025535539000168.tif8161 is a double bond or a single bond, R 10 = -CH2PO3H or -NHCH3, and R 11 is -CH2- or -CO-, and R 12 is H and R 13 is CH3 or R 12 and R 13 together form -CHCHCH-. In some embodiments, R 1 is aryl. In some embodiments, aryl is phenyl.

[0183] Additionally or alternatively, the siNA molecules disclosed herein may comprise a nucleotide sequence of formula (IIb) in the sense strand, the antisense strand, or both. [ka] wherein R x is a nucleobase, aryl, heteroaryl, or H, and R 26 teeth, [ka] and R 9 is -SO2CH3 or -COCH3, TIFF2025535539000171.tif8161 is a double bond or a single bond, R 10 = -CH2PO3H or -NHCH3, and R 11 is -CH2- or -CO-, and R 12 is H and R 13 is CH3 or R 12 and R 13 together form -CHCHCH-. In some embodiments, R 1 is aryl. In some embodiments, aryl is phenyl.

[0184] Additionally or alternatively, the siNA molecules disclosed herein may comprise a nucleotide sequence of formula (III) in the sense strand, the antisense strand, or both. [ka] wherein R x is a nucleobase, aryl, heteroaryl, or H; L is -CH2-, -CH=CH-, -CO-, or -CH2CH2-; A is -ONHCOCH3, -ONHSO2CH 3、 -POH, -OP(SOH)CHCOH, -SOCHPOH, -SONHCH, -NHSOCH, or -N(SOCHCHCHCH). In some embodiments, R 1 is aryl. In some embodiments, aryl is phenyl.

[0185] Additionally or alternatively, the siNA molecules disclosed herein include those of Formulas (1) to (16), (9X) to (12X), (16X), (9Y) to (12Y), (16Y), (21) to (36), (36X), (41) to (56), (49X) to (52X), (49Y) to (52Y), (56X), (56Y), (61), (62), and (63): [ka] [ka] [ka] [ka] [ka] wherein R x is a nucleobase, aryl, heteroaryl, or H.

[0186] In some embodiments, any of the siNA molecules disclosed herein comprises a nucleotide sequence represented by Formula (50), Formula (50X), Formula (50Y), Formula (56), Formula (56X), Formula (56Y), Formula (61), Formula (62), and Formula (63): [ka] wherein R x is a nucleobase, aryl, heteroaryl, or H.

[0187] In some embodiments, any of the siNA molecules disclosed herein comprises a nucleotide sequence selected from the group consisting of Formulas (71)-(86), (79X)-(82X), (79Y)-(82Y), 86X, 86X', 86Y, and 86Y': [ka] [ka] wherein R x is a nucleobase, aryl, heteroaryl, or H.

[0188] In some embodiments, any of the siNA molecules disclosed herein comprises a nucleotide sequence selected from the group consisting of Formula (78), Formula (79), Formula (79X), Formula (79Y), Formula (86), Formula (86X), and Formula (86X'): [ka] wherein R x is a nucleobase, aryl, heteroaryl, or H.

[0189] In some embodiments, any of the siNA molecules disclosed herein comprises a 5' stabilizing end cap selected from the group consisting of formulas (1A) to (15A), formulas (1A-1) to (7A-1), formulas (1A-2) to (7A-2), formulas (1A-3) to (7A-3), formulas (1A-4) to (7A-4), formulas (9B) to (12B), formulas (9AX) to (12AX), formulas (9AY) to (12AY), formulas (9BX) to (12BX), and formulas (9BY) to (12BY). [ka] [ka] [ka] [ka]

[0190] In some embodiments, any of the siNA molecules disclosed herein comprises a 5' stabilizing end cap selected from the group consisting of formulas (21A) to (35A), formulas (29B) to (32B), formulas (29AX) to (32AX), formulas (29AY) to (32AY), formulas (29BX) to (32BX), and formulas (29BY) to (32BY). [ka] [ka]

[0191] In some embodiments, any of the siNA molecules disclosed herein comprises a 5' stabilizing endcap selected from the group consisting of formula (71A) to (86A), formula (79XA) to (82XA), formula (79YA) to (82YA), formula (86XA), formula (86X'A), formula (86Y), and formula (86Y'). [ka] [ka]

[0192] In some embodiments, any of the siNA molecules disclosed herein comprises a 5' stabilized endcap selected from the group consisting of formula (78A), formula (79A), formula (79XA), formula (79YA), formula (86A), formula (86XA), and formula (86X'A). [ka]

[0193] In some embodiments, a 5'-stabilized endcap is attached to the 5'-end of the antisense strand. In some embodiments, the 5'-stabilized endcap is attached to the 5'-end of the antisense strand via one, two, three, four, five, or more linkers. In some embodiments, one or more linkers are independently selected from the group consisting of a phosphodiester (p or po) linker, a phosphorothioate (ps) linker, a mesyl phosphoramidate (Ms) linker, a phosphoramidate (HEG) linker, a triethylene glycol (TEG) linker, and / or a phosphorodithioate linker. In some embodiments, one or more linkers are independently selected from the group consisting of p-(PS)2, (PS)2-p-TEG-p, (PS)2-p-HEG-p, and (PS)2-p-(HEG-p)2.

[0194] As mentioned above, the present disclosure provides compositions comprising any of the siNA molecules, sense strands, antisense strands, first nucleotide sequences, or second nucleotide sequences described herein. The siNAs and compositions thereof of the present disclosure can be used to treat various diseases and conditions (e.g., viral diseases, liver diseases, etc.).

[0195] Linker In some embodiments, any of the siNA, sense strand, first nucleotide sequence, antisense strand, and / or second nucleotide sequence disclosed herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or more internucleoside linkers. In some embodiments, the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more internucleoside linkers are independently selected from the group consisting of phosphodiester (p or po) linkers, phosphorothioate (ps) linkers, mesyl phosphoramidate (Ms) linkers, or phosphorodithioate linkers.

[0196] In some embodiments, any of the siNA, sense strand, first nucleotide sequence, antisense strand, and / or second nucleotide sequence disclosed herein further comprises one, two, three, four, or more linkers connecting the conjugation moiety, phosphorylation blocker, and / or 5' end cap to the siNA, sense strand, first nucleotide sequence, antisense strand, and / or second nucleotide sequence. In some embodiments, the one, two, three, four, or more linkers are independently selected from the group consisting of phosphodiester (p or po) linkers, phosphorothioate (ps) linkers, mesyl phosphoramidate (Ms) linkers, phosphoramidite (HEG) linkers, triethylene glycol (TEG) linkers, and / or phosphorodithioate linkers. In some embodiments, one or more linkers are independently selected from the group consisting of p-(PS)2, (PS)2-p-TEG-p, (PS)2-p-HEG-p, and (PS)2-p-(HEG-p)2.

[0197] Exemplary siNA As described above, the siNAs disclosed herein can contain modified nucleotides (i.e., N1 stabilizers) at one or two positions from the 3' end of the antisense strand. The N1 stabilizing nucleotides (e.g., moe, ln, cp, mun34, bl-m, tn, 3m, and those shown in bold in Table 1) comprise one or more of the disclosed N1 stabilizing nucleotides, and the one or more N1 stabilizing nucleotides can be present in the sense strand, the antisense strand, or both. Table 1 shows exemplary siNAs containing these N1 stabilizing nucleotides. [Table 3-1] [Table 3-2]

[0198] Additionally or alternatively, the disclosed siNAs can also incorporate novel nucleotides (e.g., 2'-ocp and 2'-omcp). Table 2 shows exemplary siNAs containing these nucleotides. siNAs containing the disclosed novel nucleotides (e.g., 2'-ocp and 2'-omcp, and those shown in bold in Table 2) can contain one or more of the disclosed novel nucleotides, and the one or more novel nucleotides can be present in the sense strand, the antisense strand, or both. [Table 4-1] [Table 4-2]

[0199] Additionally or alternatively, the disclosed siNAs may also incorporate a conjugation moiety. In some embodiments, the conjugation moiety is galactosamine. In some embodiments, any of the siNAs disclosed herein is linked to a conjugation moiety that is galactosamine. In some embodiments, the galactosamine is N-acetylgalactosamine (GalNAc4). Table 3 shows exemplary siNAs that incorporate novel nucleotides (e.g., 2'-ocp and 2'-omcp, and those shown in bold in the table) and contain these conjugation sites. In some embodiments, a siNA may include one or more of the disclosed conjugation moieties, and the one or more conjugation moieties may be present on the sense strand, the antisense strand, or both. [Table 5-1] [Table 5-2] [Table 5-3]

[0200] Additionally or alternatively, the disclosed siNAs can incorporate alternating 2'-ocp or 2'-omcp nucleotides. Table 4 shows these alternating 2'-ocp Illustrated are exemplary siNAs containing alternating 2'-ocp or 2'-omcp nucleotides. siNAs containing alternating 2'-ocp or 2'-omcp nucleotides (shown in bold in the table) can contain one or more alternating 2'-ocp or 2'-omcp nucleotides, and the one or more alternating 2'-ocp or 2'-omcp nucleotides can be present in the sense strand, the antisense strand, or both. [Table 6-1] [Table 6-2] [Table 6-3]

[0201] Additionally or alternatively, the disclosed siNAs can also incorporate 2'-ocp or 2'-omcp nucleotides at the 5' and 3' ends of the antisense strand, respectively. Table 5 shows exemplary siNAs containing these terminally modified duplexes (shown in bold in the table). Terminally modified siNAs can contain 2'-ocp nucleotides and / or 2'-omcp nucleotides instead of 2'-O-methyl nucleotides at positions 1, 2, 3, and / or 4 from the end of either the sense strand or the antisense strand, or both. [Table 7]

[0202] Additionally or alternatively, the disclosed siNAs can also incorporate 2'-ocp or 2'-omcp nucleotides in the 3' overhang of the antisense strand to replace one or more of the 2'O-methyl nucleotides. Table 6 shows exemplary siNAs containing these overhang-modified duplexes (shown in bold in the table). [Table 8]

[0203] Additionally or alternatively, the disclosed siNAs can also incorporate 2'-ocp nucleotides or 2'-omcp nucleotides to replace most or all of the 2'-O-methyl nucleotides. Table 5 shows exemplary siNAs containing these fully modified duplexes (shown in bold in the table). Fully modified siNAs can contain most or all of the 2'-ocp nucleotides and / or 2'-omcp nucleotides in place of 2'-O-methyl nucleotides in the sense strand, the antisense strand, or both. [Table 9]

[0204] Additionally or alternatively, the disclosed siNAs may also incorporate novel unlocked nucleotide monomers. These novel unlocked nucleotides include: [ka] (In the formula, R x is a nucleobase, aryl, heteroaryl, or H), or more specifically, [ka] (In the formula, R y These unlocked nucleotides may have the structure: [ka] ) These siNAs differ from unlocked nucleotides (UNAs) known in the art. Table 7 shows exemplary siNAs containing these unlocked nucleotides (shown in bold in the table). siNAs containing 3',4' UNAs (e.g., mun34) can contain one or more of the disclosed 3',4' UNAs, and the one or more 3',4' UNAs can be present in the sense strand, the antisense strand, or both. [Table 10]

[0205] Additionally or alternatively, the disclosed siNAs can also incorporate additional modifications to the nucleotide monomers. These modifications include 3m, 3oh, un, mun34, and alterations to the 2'-fluoro nucleotide pattern. Table 9 shows exemplary siNAs containing these additional modifications (shown in bold in the table). In some embodiments, a siNA can contain one or more of the disclosed modifications, and one or more of the disclosed modifications can be present in the sense strand, the antisense strand, or both. [Table 11]

[0206] Additionally or alternatively, the disclosed siNAs may also incorporate further modifications to the nucleotide monomers. In some embodiments, the modification may be a 5'-cyclopropyl modification. For example, the siNA may comprise 5 cpr^mA, 5 cps^mA, 5 mcpr^mA, or 5 mcps^mA. Table 10 shows exemplary siNAs comprising these additional modifications. In some embodiments, the siNA may comprise one or more of the disclosed modifications, and the one or more disclosed modifications may be present in the sense strand, the antisense strand, or both. [Table 12-1] [Table 12-2]

[0207] Additionally or alternatively, the disclosed siNAs can also incorporate additional modifications to the nucleotide monomers. In some embodiments, the modification can be a 2'-F'3'-xylo modification. For example, the siNA can include lfG, lfA, lfC, and / or lfU. Table 11 shows exemplary siNAs containing these additional modifications (in bold). In some embodiments, the siNA can include one or more of the disclosed modifications, and the one or more disclosed modifications can be present in the sense strand, the antisense strand, or both. [Table 13-1] [Table 13-2]

[0208] Additionally or alternatively, the disclosed siNAs can also incorporate additional modifications to the nucleotide monomers. In some embodiments, the modifications include additional 2'-F nucleotides at different positions along the antisense strand. For example, a siNA can include additional fG, fA, fC, and / or fU. Table 12 shows exemplary siNAs that include these additional modifications (in bold). In some embodiments, a siNA can include one or more of the disclosed modifications, and one or more of the disclosed modifications can be present in the sense strand, the antisense strand, or both. [Table 14]

[0209] Additionally or alternatively, the disclosed siNAs can also incorporate additional modifications to the nucleotide monomers. In some embodiments, the modifications include ganciclovir, denvir, and 3'-ocp nucleotides along the sense and / or antisense strands. For example, a siNA can include ganr^G, gans^G, denr^G, dens^G, and / or 3'-ocp. Table 13 shows exemplary siNAs containing these modifications (in bold). In some embodiments, a siNA can include one or more of the disclosed modifications, and one or more of the disclosed modifications can be present in the sense or antisense strand, or both. [Table 15-1] [Table 15-2]

[0210] Additionally or alternatively, the disclosed siNAs can also incorporate additional modifications to the nucleotide monomers. In some embodiments, the modifications include 2'-OMe-3'-xylonucleotides along the antisense strand. For example, a siNA can include 1mG, 1mG, 1mG, and / or 1mG. Table 14 shows exemplary siNAs containing these modifications (in bold). In some embodiments, a siNA can include one or more of the disclosed modifications, and one or more of the disclosed modifications can be present in the sense strand, the antisense strand, or both. [Table 16-1] [Table 16-2]

[0211] Additionally or alternatively, the disclosed siNAs can also incorporate additional modifications to the nucleotide monomers. In some embodiments, the modifications include 2'-ocp, 2'-omcp, and / or 5'-vinylphosphonate 2'-O-methyl nucleotides along the antisense strand. For example, a siNA can include 2ocpA, 2ocpC, 2ocpG, 2ocpU, 2omcpA, 2omcpC, 2omcpG, 2omcpU, and / or vmU. Table 15 shows exemplary siNAs containing these modifications (in bold). In some embodiments, a siNA can include one or more of the disclosed modifications, and one or more of the disclosed modifications can be present in the sense strand, the antisense strand, or both. [Table 17-1] [Table 17-2] [Table 17-3]

[0212] Additionally or alternatively, siNAs of the present disclosure may also incorporate additional modifications to the nucleotide monomers. In some embodiments, modifications include vinyl phosphonate 5' end caps, such as vmU, and / or G-analog nucleotides, such as dens^G and mun12G, along the antisense strand. Table 16 shows exemplary siNAs containing these modifications (bold). In some embodiments, siNAs may include one or more of the disclosed modifications, and one or more of the disclosed modifications may be present in the sense strand, the antisense strand, or both. [Table 18]

[0213] Additionally or alternatively, the disclosed siNAs can also incorporate further modifications to the nucleotide monomers. In some embodiments, the modifications include a 5' TNA modification, such as coc-4h, on the antisense strand. Table 17 shows exemplary siNAs containing these modifications (in bold). In some embodiments, the siNAs can include one or more of the disclosed modifications, and the one or more disclosed modifications can be present in the sense strand, the antisense strand, or both. [Table 19]

[0214] Additionally or alternatively, the disclosed siNAs can also incorporate further modifications at the nucleotide linkages. In some embodiments, the modifications include sterically defined PS linkages, such as psr and pss on the antisense strand. Table 18 shows exemplary siNAs containing these modifications (in bold). In some embodiments, the siNAs can include one or more of the disclosed modifications, and one or more of the disclosed modifications can be present in the sense strand, the antisense strand, or both. [Table 20]

[0215] Target gene Without wishing to be bound by theory, once inside a cell, any of the ds-siNA molecules disclosed herein can interact with proteins in the cell to form an RNA-induced silencing complex (RISC).When ds-siNA is part of RISC, ds-siNA can unwind to form single-stranded siNA (ss-siNA).Ss-siNA can contain the antisense strand of ds-siNA.The antisense strand can bind to complementary messenger RNA (mRNA), thereby silencing the gene encoding the mRNA.

[0216] The target gene may be any gene in a cell. In some embodiments, the target gene is a viral gene. In some embodiments, the viral gene is from a DNA virus. In some embodiments, the DNA virus is a double-stranded DNA (dsDNA) virus. In some embodiments, the dsDNA virus is a hepadnavirus. In some embodiments, the hepadnavirus is hepatitis B virus (HBV). In some embodiments, the HBV is selected from HBV genotypes A-J. In some embodiments, the viral disease is caused by an RNA virus. In some embodiments, the RNA virus is a single-stranded RNA virus (ssRNA virus). In some embodiments, the ssRNA virus is a positive-sense single-stranded RNA virus ((+)ssRNA virus). In some embodiments, the (+)ssRNA virus is a coronavirus. In some embodiments, the coronavirus is a β-coronavirus. In some embodiments, the β-coronavirus is selected from the group consisting of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (also known by the working name of 2019 novel coronavirus or 2019-nCoV), human coronavirus OC43 (hCoV-OC43), Middle East respiratory syndrome-related coronavirus (MERS-CoV, also known by the working name of 2012 novel coronavirus or 2012-nCoV), and severe acute respiratory syndrome-related coronavirus (SARS-CoV, also known as SARS-CoV-1). In some embodiments, the β-coronavirus is SARS-CoV-2, the causative agent of COVID-19. Some exemplary target genes are listed in Table 23 at the end of this specification.

[0217] In some embodiments, the target gene is selected from the S gene or the X gene of HBV. In some embodiments, the HBV has a genome sequence set forth in the nucleotide sequence of SEQ ID NO: 89, which corresponds to the nucleotide sequence of GenBank Accession No. U95551.1, which is incorporated by reference in its entirety.

[0218] An exemplary HBV genome sequence is set forth in SEQ ID NO:81, corresponding to Genbank Accession No. KC315400.1, which is incorporated by reference in its entirety. Nucleotides 2307-3215, 1-1623 of SEQ ID NO:94 correspond to the polymerase / RT gene sequence encoding the polymerase protein. Nucleotides 2848-3215, 1-835 of SEQ ID NO:94 correspond to the PreS1 / S2 / S gene sequence encoding the large S protein. Nucleotides 3205-3215, 1-835 of SEQ ID NO:94 correspond to the PreS2 / S gene sequence encoding the middle S protein. Nucleotides 155-835 of SEQ ID NO:94 correspond to the S gene sequence encoding the small S protein. Nucleotides 1374-1838 of SEQ ID NO:94 correspond to the X gene sequence encoding the X protein. Nucleotides 1814-2452 of SEQ ID NO:94 correspond to the PreC / C gene sequence encoding the precore / core protein. Nucleotides 1901 to 2452 of SEQ ID NO:94 correspond to the C gene sequence encoding the core protein. The HBV genome further comprises viral regulatory elements such as viral promoters (preS2, preS1, Core, and X) and enhancer elements (ENH1 and ENH2). Nucleotides 1624 to 1771 of SEQ ID NO:94 correspond to ENH2. Nucleotides 1742 to 1849 of SEQ ID NO:94 correspond to the core promoter. Nucleotides 1818 to 3215, 1 to 1930 of SEQ ID NO:94 correspond to the pregenomic RNA (pgRNA) encoding the core and polymerase proteins.

[0219] In some embodiments, the sense strand comprises a sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to or hybridizes to a viral target RNA sequence beginning in the X region of HBV or the S region of HBV. The target of interest may begin, for example, at the 5' end of the target site in acc.KC315400.1 (genotype B, "gt B") or in any one of the A, C, or D genotypes. Those skilled in the art will be familiar with HBV locations, for example, as described in Wing-Kin Sung, et al., Nature Genetics 44:765 (2012). In some embodiments, the S region is defined as from the start of the small S protein (genotype B KC315400.1 isolate, position 155) to before the start of the X protein (genotype B KC315400.1 isolate, position 1373). In some embodiments, the X region is defined as from the start of the X protein (genotype B KC315400.1 isolate, position 1374) to the end of the DR2 site (genotype B KC315400.1 isolate, position 1603).

[0220] In some embodiments, the second nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to 15-30, 15-25, 15-23, 15-22, 15-21, 17-25, 17-23, 17-22, 17-21, or 19-21 nucleotides within positions 200-720, or 1100-1700 of SEQ ID NO:89. In some embodiments, the second nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to 15-30, 15-25, 15-23, 15-22, 15-21, 17-25, 17-23, 17-22, 17-21, or 19-21 nucleotides within positions 200-280, 300-445, 460-510, 650-720, 1170-1220, 1250-1300, or 1550-1630 of SEQ ID NO:89. In some embodiments, the second nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to 15-30, 15-25, 15-23, 15-22, 15-21, 17-25, 17-23, 17-22, 17-21, or 19-21 nucleotides within positions 200-230, 250-280, 300-330, 370-400, 405-445, 460-500, 670-700, 1180-1210, 1260-1295, 1520-1550, or 1570-1610 of SEQ ID NO:89. In some embodiments, the second nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to 15-30, 15-25, 15-23, 15-22, 15-21, 17-25, 17-23, 17-22, 17-21, or 19-21 nucleotides starting at position 203, 206, 254, 305, 375, 409, 412, 415, 416, 419, 462, 466, 467, 674, 676, 1182, 1262, 1263, 1268, 1526, 1577, 1578, 1580, 1581, 1583, or 1584 of SEQ ID NO:89.

[0221] In some embodiments, the first nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a nucleotide region within SEQ ID NO: 89, except that thymine (Ts) in SEQ ID NO: 89 is replaced with uracil (U). In some embodiments, the first nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to 15-30, 15-25, 15-23, 15-22, 15-21, 17-25, 17-23, 17-22, 17-21, or 19-21 nucleotides within positions 200-720, or 1100-1700 of SEQ ID NO: 89. In some embodiments, the first nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to 15-30, 15-25, 15-23, 15-22, 15-21, 17-25, 17-23, 17-22, 17-21, or 19-21 nucleotides within positions 200-280, 300-445, 460-510, 650-720, 1170-1220, 1250-1300, or 1550-1630 of SEQ ID NO:89. In some embodiments, the first nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to 15-30, 15-25, 15-23, 15-22, 15-21, 17-25, 17-23, 17-22, 17-21, or 19-21 nucleotides within positions 200-230, 250-280, 300-330, 370-400, 405-445, 460-500, 670-700, 1180-1210, 1260-1295, 1520-1550, or 1570-1610 of SEQ ID NO:89.In some embodiments, the first nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to 15-30, 15-25, 15-23, 15-22, 15-21, 17-25, 17-23, 17-22, 17-21, or 19-21 nucleotides starting at position 203, 206, 254, 305, 375, 409, 412, 415, 416, 419, 462, 466, 467, 674, 676, 1182, 1262, 1263, 1268, 1526, 1577, 1578, 1580, 1581, 1583, or 1584 of SEQ ID NO:89.

[0222] Several disease-causing coronaviruses share a high degree of homology in the region of their genomes encoding nonstructural proteins (nsps), more specifically, the region encoding nsp8 through nsp15. Indeed, approximately 65% ​​identity exists across the approximately 7 kB sequence of the β-coronavirus, from approximately 12,900 nucleotides to approximately 19,900 nucleotides in 2019-nCoV, and some subsections of the nsp8 through nsp15 genome span may contain greater than 95% identity. All genes within this region encode nonstructural proteins involved in replication. Therefore, this segment of the genome is suitable for targeting with siNAs, which can provide broad-spectrum therapy against multiple different types of coronaviruses, including MERS-CoV, SARS-CoV-1, and SARS-CoV-2.

[0223] In some embodiments, the target gene is selected from the genome of SARS-CoV-2. In some embodiments, SARS-CoV-2 has a genome sequence set forth in the nucleotide sequence of SEQ ID NO: 97, which corresponds to the nucleotide sequence of GenBank Accession No. NC_045512.2, which is incorporated by reference in its entirety. In some embodiments, the target gene sequence within SEQ ID NO: 97 is 15-30, 15-25, 15-23, 17-23, 19-23, or 19-21 nucleotides in length, preferably 19 or 21 nucleotides in length. In some embodiments, the antisense strand sequence is selected from the group consisting of 190 to 216, 233 to 279, 288 to 324, 455 to 477, 626 to 651, 704 to 723, 3352 to 3378, 5384 to 5403, 6406 to 6483, 7532 to 7551, 9588 to 9606, 10484 to 10509, 11609 to 11630, 11834 to 11853, 12023 to 12045, 12212 to 12234, and the like of SEQ ID NO: 97. 12401~12420, 12839~12867, 12885~12924, 12966~12990, 13151~13176, 13363~13386, 13388~13416, 13458~13416, 13458~13520, 13762~13790, 14290~14312, 14404~14429, 14500~14531, 14623~14642, 14650~14687, 14698~14700 717, 14722~14748, 14750~14777, 14821~14846, 14854~14873, 14875~14903, 14962~14990, 14992~15020, 15055~15140, 15172~15200, 15310~15332, 15346~15367, 15496~15518, 15622~15644, 15838~15869, 15886~15905, 1598 5~16010, 16057~16079, 16186~16205, 16430~16448, 16822~16865, 16954~16976, 17008~17042, 17080~17111, 17137~17156, 17269~17289, 17530~17549, 17563~17582, 17680~17699, 17746~17765, 17857~17876, 17956~17975,18100~18122, 18196~18218, 19618~19639, 19783~19802, 19831~19850, 20107~20130, 20776~20795, 21502~21524, 24302~24325, 24446~24465, 24620~24651, 24662~24684, 25034~25057, 25104~25128, 25364~25365 87, 25502~25530, 26191~26227, 26232~26267, 26269~26330, 26332~26394, 26450~26481, 26574~26600, 27003~27064, 27093~27111, 27183~27212, 27382~27407, 27511~27533, 27771~27818, 28270~28296, 28397~2 8434, 28513~28546, 28673~28692, 28706~28726, 28744~28794, 28799~28827, 28946~28972, 28976~29034, 29144~29172, 29174~29196, 29228~29259, 29285~29305, 29342~29394, 29444~29463, 29543~29566, 2959 It is complementary to 15 to 30, 15 to 25, 15 to 23, 15 to 22, 15 to 21, 17 to 25, 17 to 23, 17 to 22, 17 to 21, or 19 to 21 nucleotides, preferably 19 to 21 nucleotides, more preferably 19 to 21 nucleotides, within positions 8 to 29630, 29652 to 29687, 29689 to 29731, 29733 to 29757, or 29770 to 29828. In some embodiments, the sense strand sequence is selected from the group consisting of 190 to 216, 233 to 279, 288 to 324, 455 to 477, 626 to 651, 704 to 723, 3352 to 3378, 5384 to 5403, 6406 to 6483, 7532 to 7551, 9588 to 9606, 10484 to 10509, 116 09~11630, 11834~11853, 12023~12045, 12212~12234, 12401~12420, 12839~12867, 12885~12924, 12966~12990, 13151~13176, 13363~13386, 13388~13416, 13458~13416,13458~13520、13762~13790、14290~14312、14404~14429、14500~14531、14623~14642、14650~14687、14698~14717、14722~14748、14750~14777、14821~14846、14854~14873、14875~14903、14962~14990、14992~15020、15055~15140、15172~15200、15310~15332、15346~15367、15496~15518、15622~15644、15838~15869、15886~15905、15985~16010、16057~16079、16186~16205、16430~16448、16822~16865、16954~16976、17008~17042、17080~17111、17137~17156、17269~17289、17530~17549、17563~17582、17680~17699、17746~17765、17857~17876、17956~17975、18100~18122、18196~18218、19618~19639、19783~19802、19831~19850、20107~20130、20776~20795、21502~21524、24302~24325、24446~24465、24620~24651、24662~24684、25034~25057、25104~25128、25364~25387、25502~25530、26191~26227、26232~26267、26269~26330、26332~26394、26450~26481、26574~26600、27003~27064、27093~27111、27183~27212、27382~27407、27511~27533、27771~27818、28270~28296、28397~28434、28513~28546、28673~28692、28706~28726、28744~28794、28799~28827、28946~28972、28976~29034、29144~29172、29174~29196、29228~29259、29285~29305、29342~29394、29444~29463、29543~29566、It is identical to 15 to 30, 15 to 25, 15 to 23, 15 to 22, 15 to 21, 17 to 25, 17 to 23, 17 to 22, 17 to 21, or 19 to 21 nucleotides, preferably 19 to 21 nucleotides, more preferably 19 to 21 nucleotides, within positions 29598 to 29630, 29652 to 29687, 29689 to 29731, 29733 to 29757, or 29770 to 29828.

[0224] In some embodiments, the target gene is selected from the genome of SARS-CoV, which has a genome corresponding to the nucleotide sequence of GenBank Accession No. NC_004718.3, which is incorporated by reference in its entirety.

[0225] In some embodiments, the target gene is selected from the genome of MERS-CoV, which has a genome corresponding to the nucleotide sequence of GenBank Accession No. NC_019843.3, which is incorporated by reference in its entirety.

[0226] In some embodiments, the target gene is selected from the genome of hCoV-OC43, which has a genome corresponding to the nucleotide sequence of GenBank Accession No. NC_006213.1, which is incorporated by reference in its entirety.

[0227] In some embodiments, the target gene is involved in liver metabolism. In some embodiments, the target gene is an inhibitor of the electron transport chain. In some embodiments, the target gene encodes an MCJ protein (MCJ / DnaJC15 or a methylation-regulated J protein). In some embodiments, the MCJ protein is encoded by the mRNA sequence of SEQ ID NO: 90, which corresponds to the nucleotide sequence of GenBank Accession No. NM_013238.3, which is incorporated by reference in its entirety.

[0228] In some embodiments, the target gene is TAZ. In some embodiments, TAZ comprises the nucleotide sequence of SEQ ID NO: 91, which corresponds to the nucleotide sequence of GenBank Accession No. NM_000116.5, the entire contents of which are incorporated by reference.

[0229] In some embodiments, the target gene is angiopoietin-like 3 (ANGPTL3). In some embodiments, ANGPTL3 has a genomic sequence set forth in the nucleotide sequence of SEQ ID NO: 92, which corresponds to the nucleotide sequence of GenBank Accession No. NM_014495.4, which is incorporated by reference in its entirety. In some embodiments, the target gene is diacylglycerol acyltransferase 2 (DGAT2). In some embodiments, DGAT2 has a genomic sequence set forth in the nucleotide sequence of SEQ ID NO: 93, which corresponds to the nucleotide sequence of GenBank Accession No. NM_001253891.1, which is incorporated by reference in its entirety.

[0230] composition As described above, the present disclosure provides compositions comprising any of the oligonucleotides, siNA molecules, sense strands, antisense strands, first nucleotide sequences, or second nucleotide sequences described herein. The compositions may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more siNA molecules described herein. The compositions may comprise a first nucleotide sequence comprising the nucleotide sequence of any one of SEQ ID NOs: 1, 11, 28, 30-56, 69, 73, 98-103, 106, 158-160, and 165. In some embodiments, the compositions comprise a second nucleotide sequence comprising the nucleotide sequence of any one of SEQ ID NOs: 2-10, 12-27, 29, 57-68, 70-72, 74-87, 104-157, and 161-164. In some embodiments, the composition comprises a sense strand comprising the nucleotide sequence of any one of SEQ ID NOs: 1, 11, 28, 30-56, 69, 73, 98-103, 106, 158-160, and 165. In some embodiments, the composition comprises an antisense strand comprising the nucleotide sequence of any one of SEQ ID NOs: 2-10, 12-27, 29, 57-68, 70-72, 74-87, 104-157, and 161-164.

[0231] Alternatively, the composition may comprise (a) a phosphorylation blocker and (b) a short interfering nucleic acid (siNA). In some embodiments, the phosphorylation blocker is any of the phosphorylation blockers disclosed herein. In some embodiments, the siNA is any of the siNAs disclosed herein. In some embodiments, the siNA comprises any of the sense strand, antisense strand, first nucleotide sequence, or second nucleotide sequence described herein. In some embodiments, the siNA comprises any of the sense strand, antisense strand, first nucleotide sequence, or second nucleotide sequence described herein. In some embodiments, the siNA comprises one or more modified nucleotides. In some embodiments, the one or more modified nucleotides are independently selected from 2'-fluoro nucleotides, 2'-O-methyl nucleotides, 2'-ocp nucleotides, 2'-omcp nucleotides, 3'-ocp nucleotides, 3'-omcp nucleotides, 2'-OMe-3'-xylonucleotides, 2'-F-3'-xylonucleotides, ganciclovir nucleotides, denvir nucleotides, 5'-vinylphosphonate 2'-O-methyl nucleotides, and nucleotide analogs. In some embodiments, the 2'-fluoro nucleotides or 2'-O-methyl nucleotides are independently selected from any of the 2'-fluoro or 2'-O-methyl nucleotide mimics disclosed herein. In some embodiments, the siNA comprises a nucleotide sequence comprising any of the modification patterns disclosed herein.

[0232] In some embodiments, the composition may comprise (a) a conjugated moiety and (b) a short interfering nucleic acid (siNA). In some embodiments, the conjugated moiety is any of the galactosamines disclosed herein. In some embodiments, the siNA is any of the siNAs disclosed herein. In some embodiments, the siNA comprises any of the sense strand, antisense strand, first nucleotide sequence, or second nucleotide sequence described herein. In some embodiments, the siNA comprises any of the sense strand, antisense strand, first nucleotide sequence, or second nucleotide sequence described herein. In some embodiments, the siNA comprises one or more modified nucleotides. In some embodiments, the one or more modified nucleotides are independently selected from 2'-fluoro nucleotides, 2'-O-methyl nucleotides, 2'-ocp nucleotides, 2'-omcp nucleotides, 3'-ocp nucleotides, 3'-omcp nucleotides, 2'-OMe-3'-xylonucleotides, 2'-F-3'-xylonucleotides, ganciclovir nucleotides, denvir nucleotides, 5'-vinylphosphonate 2'-O-methyl nucleotides, and nucleotide analogs. In some embodiments, the 2'-fluoro nucleotides or 2'-O-methyl nucleotides are independently selected from any of the 2'-fluoro or 2-O-methyl nucleotide mimics disclosed herein. In some embodiments, the siNA comprises a nucleotide sequence comprising any of the modification patterns disclosed herein.

[0233] In some embodiments, the composition may comprise (a) a 5'-stabilized endcap and (b) a short interfering nucleic acid (siNA). In some embodiments, the 5'-stabilized endcap is any of the 5'-stabilized endcaps disclosed herein. In some embodiments, the siNA is any of the siNAs disclosed herein. In some embodiments, the siNA comprises any of the sense strand, antisense strand, first nucleotide sequence, or second nucleotide sequence described herein. In some embodiments, the siNA comprises one or more modified nucleotides. In some embodiments, the one or more modified nucleotides are independently selected from 2'-fluoro nucleotides, 2'-O-methyl nucleotides, 2'-ocp nucleotides, 2'-omcp nucleotides, 3'-ocp nucleotides, 3'-omcp nucleotides, 2'-OMe-3'-xylonucleotides, 2'-F-3'-xylonucleotides, ganciclovir nucleotides, denvir nucleotides, 5'-vinylphosphonate 2'-O-methyl nucleotides, and nucleotide analogs. In some embodiments, the 2'-fluoro nucleotides or 2'-O-methyl nucleotides are independently selected from any of the 2'-fluoro or 2-O-methyl nucleotide mimics disclosed herein. In some embodiments, the siNA comprises a nucleotide sequence comprising any of the modification patterns disclosed herein.

[0234] In some embodiments, the composition may comprise (a) at least one phosphorylation blocker, conjugation moiety, or 5'-stabilized endcap; and (b) a short interfering nucleic acid (siNA). In some embodiments, the phosphorylation blocker is any of the phosphorylation blockers disclosed herein. In some embodiments, the conjugation moiety is any of the galactosamines disclosed herein. In some embodiments, the 5'-stabilized endcap is any of the 5'-stabilized endcaps disclosed herein. In some embodiments, the siNA is any of the siNAs disclosed herein. In some embodiments, the siNA comprises any of the sense strand, antisense strand, first nucleotide sequence, or second nucleotide sequence described herein. In some embodiments, the siNA comprises one or more modified nucleotides. In some embodiments, the one or more modified nucleotides are independently selected from 2'-fluoro nucleotides, 2'-O-methyl nucleotides, 2'-ocp nucleotides, 2'-omcp nucleotides, 3'-ocp nucleotides, 3'-omcp nucleotides, 2'-OMe-3'-xylonucleotides, 2'-F-3'-xylonucleotides, ganciclovir nucleotides, denvir nucleotides, 5'-vinylphosphonate 2'-O-methyl nucleotides, and nucleotide analogs. In some embodiments, the 2'-fluoro nucleotides or 2'-O-methyl nucleotides are independently selected from any of the 2'-fluoro or 2-O-methyl nucleotide mimics disclosed herein. In some embodiments, the siNA comprises a nucleotide sequence comprising any of the modification patterns disclosed herein.

[0235] The composition may be a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises an amount of one or more siNA molecules described herein formulated with one or more pharmaceutically acceptable carriers (excipients) and / or diluents. The pharmaceutical composition may be specifically formulated for administration in solid or liquid form, including those suitable for (1) oral administration, e.g., drench (aqueous or non-aqueous solution or suspension), tablet, e.g., intended for buccal, sublingual, and systemic absorption, bolus, powder, granule, or paste for application to the tongue; (2) parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as a sterile solution or suspension, or sustained-release formulation; (3) topical / transdermal administration, e.g., as a cream, ointment, or controlled-release patch or spray applied to the skin; (4) vaginal or rectal administration, e.g., as a pessary, cream, or foam; (5) sublingual; (6) ophthalmic; (7) transdermal; or (8) nasal administration.

[0236] As used herein, the phrase "therapeutically effective amount" means an amount of a compound, material, or composition, including a siNA of the present disclosure, effective to produce some desired therapeutic effect in at least a subpopulation of cells in an animal at a reasonable benefit / risk ratio applicable to any medical treatment.

[0237] The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0238] Wetting agents, emulsifying agents, and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring, and perfuming agents, preservatives, and antioxidants can also be present in the compositions.

[0239] Pharmaceutically acceptable antioxidants include, for example, (1) water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfite, sodium metabisulfite, and sodium sulfite; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), lecithin, propyl gallate, and α-tocopherol; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0240] Formulations of the present disclosure include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, and / or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which may be combined with a carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. The amount of active ingredient which may be combined with a carrier material to produce a single dosage form will generally be that amount of compound (e.g., siNA molecule) which produces a therapeutic effect. Generally, out of 100%, this amount will range from about 0.1% to about 99% of the active ingredient, preferably from about 5% to about 70%, and most preferably from about 10% to about 30%.

[0241] In certain embodiments, a formulation of the present disclosure comprises an excipient selected from the group consisting of cyclodextrins, celluloses, liposomes, micelle-forming agents such as bile acids, and polymeric carriers such as polyesters and polyanhydrides, and a compound of the present disclosure (e.g., a siNA molecule). In certain embodiments, the formulation renders the compound of the present disclosure (e.g., a siNA molecule) orally bioavailable.

[0242] Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present disclosure (e.g., siNA molecule) with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present disclosure (e.g., siNA molecule) with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0243] Formulations of the present disclosure suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), powder, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a pastille (using an inert base such as gelatin and glycerin, or sucrose and acacia), and / or as a mouthwash, each containing a predetermined amount of a compound of the present disclosure (e.g., siNA molecule) as an active ingredient. A compound of the present disclosure (e.g., siNA molecule) may also be administered as a bolus, electuary, or paste.

[0244] In solid dosage forms of the present disclosure for oral administration (such as capsules, tablets, pills, dragees, powders, granules, lozenges, and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarders, such as paraffin; (6) absorption enhancers, such as quaternary ammonium compounds; (7) surfactants such as poloxamer and sodium lauryl sulfate, (8) wetting agents such as cetyl alcohol, glycerol monostearate and nonionic surfactants, (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, zinc stearate, sodium stearate, stearic acid, and mixtures thereof, (10) coloring agents, and (11) controlled-release agents such as crospovidone or ethylcellulose.

[0245] In the case of capsules, tablets, and pills, the pharmaceutical compositions may also contain buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-shell gelatin capsules using such excipients as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like.

[0246] Tablets can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using binders (e.g., gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surface-active agents, or dispersing agents. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0247] Tablets and other solid dosage forms of the pharmaceutical compositions of the present disclosure, such as dragees, capsules, pills, and granules, can optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They can also be formulated to provide sustained or controlled release of the active ingredient therein, for example, using various proportions of hydroxypropylmethylcellulose, other polymer matrices, liposomes, and / or microspheres to provide the desired release profile. They can also be formulated for immediate release, e.g., lyophilized.

[0248] They may be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injectable medium immediately before use. These compositions may also optionally contain an opacifying agent, and may be of a composition that releases the active ingredient(s) only, or preferentially, in a certain part of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient may also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0249] Liquid dosage forms for oral administration of compounds (e.g., siNA molecules) of the present disclosure include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof.

[0250] Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.

[0251] Suspensions may contain, in addition to the active compound (e.g., siNA molecule), suspending agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, and mixtures thereof.

[0252] Formulations of pharmaceutical compositions of the present disclosure for rectal or vaginal administration may be presented as suppositories, which may be prepared by mixing one or more compounds of the present disclosure (e.g., siNA molecules) with one or more suitable non-irritating excipients or carriers including, for example, cocoa butter, polyethylene glycol, suppository waxes or salicylates, which are solid at room temperature but liquid at body temperature and therefore will melt in the rectum or vaginal cavity and release the active compound (e.g., siNA molecule).

[0253] Formulations of the present disclosure which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams, or spray formulations containing such carriers as are known in the art to be appropriate.

[0254] Dosage forms for topical or transdermal administration of a compound (e.g., siNA molecule) of the present disclosure include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound (e.g., siNA molecule) may be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0255] The ointments, pastes, creams and gels may contain, in addition to the active compounds of the present disclosure (e.g., siNA molecules), excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0256] Powders and sprays can contain, in addition to the compounds of the present disclosure (e.g., siNA molecules), excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain conventional propellants, such as chlorofluorohydrocarbons, and volatile unsubstituted hydrocarbons, such as butane and propane.

[0257] Transdermal patches have the additional advantage of providing controlled delivery of the disclosed compounds (e.g., siNA molecules) to the body. Such dosage forms can be made by dissolving or dispersing the compound (e.g., siNA molecules) in a suitable medium. Absorption enhancers can also be used to increase the flux of the compound (e.g., siNA molecules) through the skin. The rate of such flux can be controlled by either providing a rate-controlling membrane or dispersing the compound (e.g., siNA molecules) in a polymer matrix or gel.

[0258] Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of this disclosure.

[0259] Pharmaceutical compositions of the present disclosure suitable for parenteral administration comprise one or more compounds of the present disclosure (e.g., siNA molecules) in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions immediately before use, and may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0260] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0261] These compositions may contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms on the subject compounds can be ensured by including various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0262] In some cases, in order to prolong the effect of a drug, it is desirable to delay the absorption of the drug from subcutaneous or intramuscular injection.This can be achieved by using a liquid suspension of crystalline or amorphous material that is poorly water-soluble.The absorption rate of the drug then depends on its dissolution rate, which in turn depends on the crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drug form can be achieved by dissolving or suspending the drug in an oil vehicle.

[0263] Injectable depot forms are made by forming microencapsule matrices of the subject compounds (e.g., siNA molecules) in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Injectable depot forms are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0264] When compounds of the present disclosure (e.g., siNA molecules) are administered to humans and animals as pharmaceuticals, the compounds of the present disclosure can be administered per se or as a pharmaceutical composition containing, for example, 0.1 to 99% (more preferably, 10 to 30%) of the active ingredient in combination with a pharmaceutically acceptable carrier.

[0265] Treatment and Administration The siNA molecules of the present disclosure can be used to treat a disease in a subject in need thereof. In some embodiments, the method for treating a disease in a subject in need thereof comprises administering any of the siNA molecules disclosed herein to the subject. In some embodiments, the method for treating a disease in a subject in need thereof comprises administering any of the compositions disclosed herein to the subject.

[0266] Preparations (e.g., siNA molecules or compositions) of the present disclosure can be administered orally, parenterally, topically, or rectally. They will, of course, be administered in a form suitable for each administration route. For example, they may be administered in tablet or capsule form, by injection, infusion, or inhalation, topically in lotions or ointments, and rectally in suppositories. Oral administration is preferred.

[0267] As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal and intrapleural injection and infusion.

[0268] As used herein, the phrases "systemic administration," "systemically administered," "peripheral administration," and "peripherally administered" refer to administration of a compound, drug, or other substance other than by direct administration into the central nervous system so that it enters the patient's system and therefore undergoes metabolism and other similar processes, e.g., subcutaneous administration.

[0269] These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, e.g., by spray, rectally, vaginally, parenterally, intracisternally, and topically, e.g., by powder, ointment or drops, bucally and sublingually.

[0270] Regardless of the route of administration selected, the compounds (e.g., siNA molecules) and / or pharmaceutical compositions of the present disclosure, which may be used in a suitable hydrated form, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0271] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient.

[0272] The selected dosage level will depend on a variety of factors, including the activity of the particular compound of the present disclosure (e.g., siNA molecule) or ester, salt, or amide thereof used, the route of administration, the time of administration, the rate of excretion or metabolism, the rate and extent of absorption of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular compound used, the age, sex, weight, condition, general health, and medical history of the patient being treated, and similar factors well known in the medical arts.

[0273] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian can start dosages of the disclosed compounds (e.g., siNA molecules) used in the pharmaceutical composition at levels lower than needed to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0274] Generally, a suitable daily dose of a compound (e.g., a siNA molecule) of the present disclosure will be the lowest effective dose of the compound to produce a therapeutic effect. Such an effective dose will generally depend on the factors described above. Preferably, the compound is administered at about 0.01 mg / kg to about 200 mg / kg, more preferably about 0.1 mg / kg to about 100 mg / kg, and even more preferably about 0.5 mg / kg to about 50 mg / kg. In some embodiments, the compounds of the invention are administered at a dose of 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1 mg / kg or more. In some embodiments, the compound is administered at a dose of less than or equal to 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, or 15 mg / kg. In some embodiments, the total daily dose of the compound is 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 100 mg or more.

[0275] When a compound described herein (eg, a siNA molecule) is co-administered with another, the effective amount may be less than when that compound is used alone.

[0276] If desired, the effective daily dose of an active compound (e.g., siNA molecule) may be administered as two, three, four, five, six, or more subdoses administered at appropriate intervals throughout the day, optionally administered separately in unit dosage forms. Preferred dosing is once daily. In some embodiments, the compound is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 times per week. In some embodiments, the compound is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 times per month. In some embodiments, the compound is administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days. In some embodiments, the compound is administered once every 1, 2, 3, 4, 5, 6, 7, or 8 weeks.

[0277] disease The siNA molecules and compositions described herein can be administered to a subject to treat a disease. Also disclosed herein is the use of any of the siNA molecules or compositions disclosed herein in the manufacture of a medicament for treating a disease.

[0278] In some embodiments, the disease is a viral disease. In some embodiments, the viral disease is caused by a DNA virus. In some embodiments, the DNA virus is a double-stranded DNA (dsDNA) virus. In some embodiments, the dsDNA virus is a hepadnavirus. In some embodiments, the hepadnavirus is hepatitis B virus (HBV).

[0279] In some embodiments, the disease is a liver disease. In some embodiments, the liver disease is non-alcoholic fatty liver disease (NAFLD). In some embodiments, the NAFLD is non-alcoholic steatohepatitis (NASH). In some embodiments, the liver disease is hepatocellular carcinoma (HCC).

[0280] The siNA molecules of the present disclosure may be used to treat or prevent a disease in a subject in need thereof. In some embodiments, a method of treating or preventing a disease in a subject in need thereof comprises administering to the subject any of the siNA molecules disclosed herein. In some embodiments, a method of treating or preventing a disease in a subject in need thereof comprises administering to the subject any of the compositions disclosed herein.

[0281] In some embodiments of the methods and uses of the present disclosure, the disease is a respiratory disease. In some embodiments, the respiratory disease is a viral infection. In some embodiments, the respiratory disease is viral pneumonia. In some embodiments, the respiratory disease is an acute respiratory infection. In some embodiments, the respiratory disease is a cold. In some embodiments, the respiratory disease is severe acute respiratory syndrome (SARS). In some embodiments, the respiratory disease is Middle East respiratory syndrome (MERS). In some embodiments, the disease is coronavirus disease 2019 (e.g., COVID-19). In some embodiments, the respiratory disease can comprise one or more symptoms selected from cough, sore throat, runny nose, sneezing, headache, fever, shortness of breath, muscle pain, abdominal pain, fatigue, difficulty breathing, persistent chest pain or pressure, difficulty waking up, loss of smell and taste, muscle or joint pain, chills, nausea or vomiting, nasal congestion, diarrhea, hemoptysis, conjunctival congestion, sputum production, chest pressure, and palpitations. In some embodiments, the respiratory disorder can include a complication selected from sinusitis, otitis media, pneumonia, acute respiratory distress syndrome, disseminated intravascular coagulation, pericarditis, and renal failure, hi some embodiments, the respiratory disorder is idiopathic.

[0282] In some embodiments, the present disclosure provides a method for treating or preventing a coronavirus infection, comprising administering a therapeutically effective amount of one or more of the siNAs or pharmaceutical compositions disclosed herein to a subject in need thereof. In some embodiments, the coronavirus infection is selected from the group consisting of Middle East Respiratory Syndrome (MERS), Severe Acute Respiratory Syndrome (SARS), and COVID-19. In some embodiments, the subject is being treated with one or more additional coronavirus therapeutic agents. In some embodiments, the subject is being treated concurrently with one or more additional coronavirus therapeutic agents.

[0283] siNA administration Administration of any of the siNAs disclosed herein may be by methods known in the art. In some embodiments, siNAs are administered by subcutaneous (SC) or intravenous (IV) delivery. The preparations (e.g., siNAs or compositions) of the present disclosure may be given orally, parenterally, topically, or rectally. They are, of course, given in a form suitable for each administration route. For example, they may be administered in tablet or capsule form, by injection, infusion, or inhalation, topically in lotions or ointments, and rectally in suppositories. In some embodiments, subcutaneous administration is preferred.

[0284] As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intraspinal and intrapleural injection and infusion.

[0285] As used herein, the phrases "systemic administration," "systemically administered," "peripheral administration," and "peripherally administered" refer to administration of a compound, drug, or other substance other than by direct administration into the central nervous system so that it enters the patient's system and therefore undergoes metabolism and other similar processes, e.g., subcutaneous administration.

[0286] These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, e.g., by spray, rectally, vaginally, parenterally, intracisternally, and topically, e.g., by powder, ointment or drops, bucally and sublingually.

[0287] Regardless of the route of administration selected, the compounds of the present disclosure (e.g., siNA) and / or pharmaceutical compositions of the present disclosure, which may be used in a suitable hydrated form, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0288] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient.

[0289] The selected dosage level will depend on a variety of factors, including the activity of the particular compound of the present disclosure (e.g., siNA) or ester, salt, or amide thereof used, the route of administration, the time of administration, the rate of excretion or metabolism, the rate and extent of absorption of the particular compound used, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular compound used, the age, sex, weight, condition, general health, and medical history of the patient being treated, and similar factors well known in the medical arts.

[0290] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian can start the dosage of the compound of the present disclosure (e.g., siNA) used in the pharmaceutical composition at a level lower than needed to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0291] In general, a suitable daily dose of a compound of the present disclosure (e.g., siNA) will be the lowest effective dose of the compound to produce a therapeutic effect. Such an effective dose will generally depend on the factors described above. Preferably, the compound is administered at about 0.01 mg / kg to about 200 mg / kg, more preferably about 0.1 mg / kg to about 100 mg / kg, and even more preferably about 0.5 mg / kg to about 50 mg / kg. In some embodiments, the compound is administered at about 1 mg / kg to about 40 mg / kg, about 1 mg / kg to about 30 mg / kg, about 1 mg / kg to about 20 mg / kg, about 1 mg / kg to about 15 mg / kg, or about 1 mg / kg to about 10 mg / kg. In some embodiments, the compounds of the invention are administered at a dose of 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1 mg / kg or more. In some embodiments, the compound is administered at a dose of 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 mg / kg or more. In some embodiments, the compound is administered at a dose of 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, or 15 mg / kg or less. In some embodiments, the total daily dose of the compound is 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 100 mg or more.

[0292] If desired, an effective daily dose of an active compound (e.g., siNA) may be administered as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more doses or subdoses administered separately at appropriate intervals throughout the day, optionally in unit dosage forms. In some embodiments, the compound is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 times. Preferred dosing is once daily. In some embodiments, the compound is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 times per week. In some embodiments, the compound is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 times per month. In some embodiments, the compound is administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days. In some embodiments, the compound is administered every 3 days. In some embodiments, the compound is administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 weeks. In some embodiments, the compound is administered monthly. In some embodiments, the compound is administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 months.In some embodiments, the compound is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53 times. Administered over 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 days. In some embodiments, the compound comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 5 Three doses are administered over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53 weeks.In some embodiments, the compound comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53 and / or over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53 months. In some embodiments, the compound is administered at least once per week for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 weeks. In some embodiments, the compound is administered at least once per week for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 months.In some embodiments, the compound is administered at least twice a week for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 weeks. In some embodiments, the compound is administered at least twice a week for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 months. In some embodiments, the compound is administered at least once every two weeks for a period of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 weeks.In some embodiments, the compound is administered at least once every two weeks for a period of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 months. In some embodiments, the compound is administered once every four weeks for a period of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 weeks. In some embodiments, the compound is administered at least once every four weeks for a period of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 months.

[0293] In some embodiments, any one of the siNAs or compositions disclosed herein is administered in a particle or viral vector.In some embodiments, the viral vector is an adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpes simplex virus, lentivirus, measles virus, picornavirus, poxvirus, retrovirus, or rhabdovirus vector.In some embodiments, the viral vector is a recombinant viral vector.In some embodiments, the viral vector is selected from AAVrh.74, AAVrh.10, AAVrh.20, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13.

[0294] The subject of the described methods may be a mammal, including human and non-human mammals, hi some embodiments, the subject is a human, such as an adult human.

[0295] Some embodiments include a method for treating HBV in a subject infected with the virus, comprising administering a therapeutically effective amount of one or more siNAs of the present disclosure or compositions of the present disclosure to a subject in need thereof, thereby reducing the viral load of the virus in the subject and / or reducing the level of viral antigens in the subject. The siNA may be complementary to or hybridize to a portion of a target RNA in the virus, for example, the X region and / or S region of HBV.

[0296] Combination therapy Any of the methods disclosed herein may further comprise administering to the subject an additional HBV therapeutic agent. Any of the compositions disclosed herein may further comprise an additional HBV therapeutic agent. In some embodiments, the additional HBV therapeutic agent is selected from nucleotide analogs, nucleoside analogs, capsid assembly modulators (CAMs), recombinant interferon, entry inhibitors, small molecule immune modulators, and oligonucleotide therapy. In some embodiments, the additional HBV therapeutic agent is HBV STOPS™ ALG-010133, HBV CAMs, or other HBV therapeutic agents. ALG-000184, ASO1 (SEQ ID NO: 95), ASO2 (SEQ ID NO: 96), recombinant interferon alpha 2b, IFN-a, PEG-IFN-a-2a, lamivudine, telbivudine, adefovir dipivoxil, clevudine, entecavir, tenofovir alafenamide, tenofovir disoproxil, NVR3-778, BAY41-4109, JNJ-632, JNJ-3989 ( In some embodiments, the oligonucleotide therapy is selected from a nucleic acid polymer or an S-antigen transport inhibitory oligonucleotide polymer (NAP or STOPS), an siRNA, and an ASO. In some embodiments, the oligonucleotide therapy is an additional siNA. In some embodiments, the additional siNA is selected from any of ds-siNA-001 through ds-siNA-092. In some embodiments, the oligonucleotide therapy is an antisense oligonucleotide (ASO). In some embodiments, the ASO is ASO1 (SEQ ID NO: 95) or ASO2 (SEQ ID NO: 96). In some embodiments, any of the siNAs disclosed herein are co-administered with STOPS.Exemplary STOPS are described in International Publication No. 2020 / 097342 and U.S. Publication No. 2020 / 0147124, both of which are incorporated by reference in their entireties. In some embodiments, the STOPS is ALG-010133. In some embodiments, any of the siNAs disclosed herein are co-administered with tenofovir. In some embodiments, any of the siNAs disclosed herein are co-administered with CAM. For exemplary CAMs, see Berke et al., Antimicrob Agents Chemother, 2017, 61(8):e00560-17, Klumpp, et al., Gastroenterology, 2018, 154(3):652-662.e8, International Application Nos. PCT / US2020 / 017974, PCT / US2020 / 026116, and PCT / US2020 / 028349, and U.S. Application Nos. 16 / 789,298, 16 / 837,515, and 16 / 849,851. In some embodiments, the CAM is ALG-000184, ALG-001075, ALG-001024, JNJ-632, BAY41-4109, or NVR3-778. In some embodiments, the siNA and the HBV therapeutic agent are administered simultaneously. In some embodiments, the siNA and the HBV therapeutic agent are administered simultaneously. In some embodiments, the siNA and the HBV therapeutic agent are administered sequentially. In some embodiments, the siNA is administered before the HBV therapeutic agent is administered. In some embodiments, the siNA is administered after the HBV therapeutic agent is administered. In some embodiments, the siNA and the HBV therapeutic agent are in separate containers. In some embodiments, the siNA and the HBV therapeutic agent are in the same container.

[0297] Any of the methods disclosed herein may further comprise administering to the subject a drug for treating liver disease. Any of the compositions disclosed herein may further comprise a drug for treating liver disease. In some embodiments, the drug for treating liver disease is selected from a peroxisome proliferator-activated receptor (PPAR) agonist, a farnesoid X receptor (FXR) agonist, a lipid-altering agent, and an incretin-based therapy. In some embodiments, the PPAR agonist is selected from a PPARα agonist, a dual PPARα / δ agonist, a PPARγ agonist, and a dual PPARα / γ agonist. In some embodiments, the dual PPARα agonist is a fibrate. In some embodiments, the PPARα / δ agonist is elafibranor. In some embodiments, the PPARγ agonist is a thiazolidinedione (TZD). In some embodiments, the TZD is pioglitazone. In some embodiments, the dual PPARα / γ agonist is saroglitazar. In some embodiments, the FXR agonist is obeticholacis (OCA). In some embodiments, the lipid-altering agent is aramchol. In some embodiments, the incretin-based therapy is a glucagon-like peptide 1 (GLP-1) receptor agonist or a dipeptidyl peptidase 4 (DPP-4) inhibitor. In some embodiments, the GLP-1 receptor agonist is exenatide or liraglutide. In some embodiments, the DPP-4 inhibitor is sitagliptin or vildapriptin. In some embodiments, the siNA and the liver disease therapeutic agent are administered simultaneously. In some embodiments, the siNA and the liver disease therapeutic agent are administered sequentially. In some embodiments, the siNA is administered before the liver disease therapeutic agent. In some embodiments, the siNA is administered after the liver disease therapeutic agent. In some embodiments, the siNA and the liver disease therapeutic agent are in separate containers. In some embodiments, the siNA and the liver disease therapeutic agent are in the same container.

[0298] phosphoramidite In addition to the disclosed oligonucleotides containing novel Nusselt dimonomers, the present disclosure also provides [ka] [ka] wherein * is a chiral center, [ka] is.

[0299] Those skilled in the art will understand that the disclosed phosphoramidites may be used to synthesize the disclosed nucleotide monomers.

[0300] definition Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs. The following references provide those skilled in the art with general definitions of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al., (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless otherwise specified. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the disclosure.

[0301] As used herein, the words "a" and "an" mean "one or more" and include the plural forms unless the context is inappropriate.

[0302] As used herein, the terms "patient" and "subject" refer to an organism treated by the methods of the present disclosure. Such organisms are preferably mammals (e.g., marine organisms, apes, horses, cows, pigs, dogs, cats, etc.), and more preferably humans.

[0303] As used herein, the term "effective amount" refers to an amount of a compound (e.g., a siNA of the present disclosure) sufficient to produce a beneficial or desired result. An effective amount can be administered in one or more administrations, applications, or dosages, and is not intended to be limited to a particular formulation or route of administration.

[0304] As used herein, the term "treating" includes any effect, e.g., alleviating, reducing, modulating, ameliorating, or eliminating, that results in the improvement of a condition, disease, disorder, etc., or that ameliorates the symptoms thereof.

[0305] As used herein, the terms "alleviate" and "alleviating" refer to reducing the severity of a condition, such as reducing the severity by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

[0306] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent with an inert or active carrier that makes the composition particularly suitable for diagnostic or therapeutic use in vivo or ex vivo.

[0307] As used herein, the term "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, such as phosphate-buffered saline, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents. The composition may also contain stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA

[1975] .

[0308] As used herein, the term "about," when referring to measurable values ​​(e.g., weight, time, and dose), is meant to encompass variations such as ±10%, ±5%, ±1%, or ±0.1% of the specified value.

[0309] As used herein, the term "nucleobase" refers to nitrogen-containing biological compounds that form nucleosides, including, but not limited to, thymine, uracil, adenine, cytosine, guanine, and analogs or derivatives thereof.

[0310] Throughout this specification, when compositions are described as having, including, or comprising particular components, or processes and methods are described as having, including, or comprising particular steps, it is contemplated that there are additionally compositions of the disclosure that consist essentially of, or consist of, the recited components, and processes and methods according to the disclosure that consist essentially of, or consist of, the recited processing steps.

[0311] As a general matter, compositions specifying percentages are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, the variable's previous definition controls.

[0312] All publications and patents cited herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited, as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed. [Example]

[0313] Example 1: siNA synthesis This example describes an exemplary method for synthesizing ds-siNAs, for example, the siNAs disclosed in Tables 1-15 (as identified by ds-siNA ID).

[0314] The 2'-OMe phosphoramidites 5'-O-DMT-deoxyadenosine (NH-Bz), 3'-O-(2-cyanoethyl-N,N-diisopropyl phosphoramidite, 5'-O-DMT-deoxyguanosine (NH-ibu), 3'-O-(2-cyanoethyl-N,N-diisopropyl phosphoramidite, 5'-O-DMT-deoxycytosine (NH-Bz), 3'-O-(2-cyanoethyl-N,N-diisopropyl phosphoramidite, 5'-O-DMT-uridine 3'-O-(2-cyanoethyl-N,N-diisopropyl phosphoramidite), and solid supports were purchased from Chemgenes Corp., MA. [ka]

[0315] 2'-F-5'-O-DMT-(NH-Bz)adenosine-3'-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite, 2'-F-5'-O-DMT-(NH-ibu)-guanosine, 3'-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite, 5'-O-DMT-(NH-Bz)-cytosine, 2'-F-3'-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite, 5'-O-DMT-uridine, 2'-F-3'-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite), and solid supports were purchased from Milwaukee, WI, USA. [ka]

[0316] All monomers were dried in a vacuum oven using a desiccant (PO at room temperature for 24 hours). Nucleosides and universal support-bound solid supports (CPG) were obtained from LGC and Chemgenes. Chemicals and solvents for the post-synthesis workflow were purchased from commercially available sources such as VWR / Sigma and used without any purification or treatment. Solvents (acetonitrile) and solutions (amidites and activators) were stored over molecular sieves during synthesis.

[0317] Oligonucleotides were synthesized on a DNA / RNA synthesizer (Expedite 8909, ABI-394, or MM-48) using standard oligonucleotide phosphoramidite chemistry, starting from the 3' residue of an oligonucleotide preloaded on a CPG support. Modified oligonucleotides were obtained by extension coupling of a 0.1 M phosphoramidite solution in CHCN to the solid-bound oligonucleotide in the presence of 5-(ethylthio)-1H-tetrazole activator, followed by standard capping, oxidation, and deprotection. DDTT ((dimethylamino-methylidene)amino)-3H-1,2,4-dithiazaolin-3-thione) was used as the sulfur transfer agent for the synthesis of oligoribonucleotide phosphorothioates, with 0.1 M I, THF:pyridine:water (7:2:1) as the oxidizing agent. The stepwise coupling efficiency of all modified phosphoramidites was greater than 98%. TIFF2025535539000229.tif51166

[0318] Cleavage and deprotection: Deprotection and cleavage from the solid support was achieved with a mixture of ammonia methylamine (1:1, AMA) for 15 min at 65 °C. When a universal linker was used, deprotection was allowed to proceed at 65 °C for 90 min, or the solid support was heated in aqueous ammonia (28%) at 55 °C for 8–16 h to deprotect base-labile protecting groups.

[0319] Quantitative or raw material analysis of crude siNA Samples were dissolved in deionized water (1.0 mL) and quantified as follows: First, a Thermo Scientific™ Nanodrop UV spectrophotometer or a BioTek™ Epoch™ plate reader was used to blank with water only (2 μl), and then oligo sample readings were obtained at 260 nm. The crude product was dried and stored at −20° C.

[0320] Crude HPLC / LC-MS analysis For crude HPLC and LC-MS analysis, 0.1 OD of crude sample was analyzed. After confirming the crude LC-MS data, purification steps were performed as needed based on purity.

[0321] HPLC purification Unconjugated GalNac-modified oligonucleotides were purified by anion-exchange HPLC. The buffers were 20 mM sodium phosphate in 10% CHCN, pH 8.5 (Buffer A) and 20 mM sodium phosphate, 1.0 M NaBr, pH 8.5 in 10% CHCN (Buffer B). Fractions containing the full-length oligonucleotide were pooled.

[0322] Desalting of purified SiNA The purified, dried siNA was then desalted using Sephadex G-25M (Amersham Biosciences). The cartridge was conditioned three times with 10 mL of deionized water. Finally, the purified siNA, completely dissolved in 2.5 mL of RNAse-free water, was applied to the cartridge with very slow, dropwise elution. Salt-free siNA was eluted directly into a screw-cap vial with 3.5 mL of deionized water. Alternatively, some unconjugated siNA was desalted using a Pall AcroPrep™ 3K MWCO desalting plate.

[0323] IEX HPLC and electrospray LC / MS analysis Approximately 0.10 OD of siNA was dissolved in water and then pipetted into an HPLC autosampler vial for IEX-HPLC and LC / MS analysis. Analytical HPLC and ES LC-MS confirmed the identity and purity of the compound.

[0324] Preparation of double strands: The single-stranded oligonucleotides (sense and antisense strands) were annealed (1:1 molar equivalents at 90°C for 2 minutes, followed by slow cooling to room temperature) to obtain double-stranded ds-siNA. The final compounds were analyzed by size exclusion chromatography (SEC).

[0325] Example 2: ds-siNA activity This example investigates the activity of the ds-siNA synthesized in Example 1.

[0326] Homo sapiens HepG2.2.15 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (ATCC 30-2002) supplemented with 10% fetal calf serum (FCS). Cells were incubated at 37°C in a humidified incubator with a 5% CO2 atmosphere. For transfection of HepG2.2.15 cells with HBV-targeting siRNAs, cells were seeded at a density of 15,000 cells / well in 96-well standard tissue culture plates. Cell transfection was performed using RNAiMAX (Invitrogen / Life Technologies) according to the manufacturer's instructions. Dose-response experiments were performed at oligo concentrations of 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, and 0.07813 nM. For each HBV targeting siRNA treatment, four wells were transfected in parallel, and individual data points were collected from each well. After 24 hours of incubation with siRNA, the medium was removed, and cells were lysed and analyzed with a QuantiGene 2.0 branched DNA (bDNA) probe set specific for HBV genotype D (also known as hepatitis B virus subtype ayw, the 3182 base pair complete genome) present in the cell line HepG2.2.15.

[0327] For each well, HBV on-target mRNA levels were normalized to GAPDH mRNA levels. As shown in Tables 9-17, the activity of HBV targeting ds-siRNA was expressed as EC50 (50% reduction in normalized HBV RNA levels from the no-drug control). As shown in Tables 9-17, the cytotoxicity of HBV targeting ds-siRNA was expressed by CC50 for 50% reduction in GAPDH mRNA from the no-drug control.

[0328] Example 3: Use of ds-siNA to treat hepatitis B virus infection In this example, the ds-siNA synthesized in Example 1 is used to treat Hepatitis B virus infection in a subject. Generally, a composition comprising a ds-siNA (as identified by the ds-siNA ID) from Tables 1-8 and a pharmaceutically acceptable carrier is administered to a subject suffering from Hepatitis B virus. The ds-siNA from Tables 1-8 is conjugated to N-acetylgalactosamine. The ds-siNA is administered at a dose of 0.3-5 mg / kg by subcutaneous injection or intravenous infusion every three weeks.

[0329] Example 4: siNA activity assay This example provides an exemplary method for testing the activity of the siNAs disclosed herein.

[0330] In vitro assay: HepG2.2.15 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (ATCC 30-2002) supplemented with 10% fetal calf serum (FCS). Cells were incubated at 37°C in a humidified incubator with a 5% CO2 atmosphere. For transfection of HepG2.2.15 cells with HBV-targeting siRNA, cells were seeded at a density of 15,000 cells / well in 96-well standard tissue culture plates. Cell transfection was performed using RNAiMAX (Invitrogen / Life Technologies) according to the manufacturer's instructions. Dose-response experiments were performed at oligo concentrations of 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, and 0.07813 nM. For each HBV targeting siRNA (e.g., ds-siRNA identified by ds-siNA ID in Tables 9-17), four wells were transfected in parallel, and individual data points were collected from each well. After 24 hours of incubation with siRNA, the medium was removed, and cells were lysed and analyzed with a QuantiGene 2.0 branched DNA (bDNA) probe set specific for HBV genotype D (also known as hepatitis B virus subtype ayw, 3182 base pair complete genome) present in the cell line HepG2.2.15.

[0331] For each well, HBV on-target mRNA levels were normalized to GAPDH mRNA levels. As shown in Tables 19-33 and 36-40, the activity of HBV targeting ds-siRNA was expressed as EC50 (50% reduction in normalized HBV RNA levels from the no-drug control). As shown in Tables 19-33 and 36-40, the cytotoxicity of HBV targeting ds-siRNA was expressed by CC50 for 50% reduction in GAPDH mRNA from the no-drug control.

[0332] Off-target effects were also measured for certain siNAs. Table 34 shows the IC50s for variants containing ganocyclovir and danovir nucleotides, which improved off-target activity by over 1000-fold compared to the control. Table 35 shows the IC50s for variants containing G analogs and vinylphosphonate 5' end caps, which also improved off-target activity compared to the control. [Table 21] [Table 22] [Table 23] [Table 24] [Table 25] [Table 26] [Table 27] [Table 28] [Table 29] [Table 30] [Table 31] [Table 32] [Table 33] [Table 34] [Table 35] [Table 36] [Table 37] [Table 38] [Table 39] [Table 40] [Table 41] [Table 42]

[0333] Example 5 - In vivo activity of ds-siNA containing a 5' vinylphosphonate moiety and a modified unlocked nucleotide. Mice were infected with AAV-HBV on day 28 of the study. Test ds-siNAs, ds-siNA-159, ds-siNA-160, ds-siNA-077, ds-siNA-078, or ds-siNA-161, or a negative control ds-siNA, were administered subcutaneously at a single dose of 5 mg / kg on day 0. Serial blood samples were collected on days 0, 7, 14, 21, and 28. Serum HBsAg was measured by ELISA.

[0334] As shown in Figure 9, the presence of the 5' vinyl phosphonate moiety and modified unlocked nucleotide on the antisense strand of ds-siNA-077 and ds-siNA-078 reduced the activity compared to their counterpart ds-siNAs, ds-siNA-159 and ds-siNA-160, which do not have such modifications.

[0335] Example 6 - In vitro stability and in vivo activity of ds-siNA containing 5'-cyclopropyl nucleotides. The stability of ds-siNA-085, ds-siNA-086, ds-siNA-087, ds-siNA-088, ds-siNA-089, ds-siNA-090, ds-siNA-091, ds-siNA-092, and ds-siNA-159 was measured in mouse liver homogenate. Mouse livers were pulverized, and 50 mg of pulverized liver was transferred to individual microcentrifuge tubes placed on dry ice. Homogenization buffer (50 mM Tris·HCl, 150 mM KCl, pH 7.2) was added to a concentration of 200 mg / ml. The microcentrifuge tubes were placed in a heated multishaker and shaken at 40°C and 1,200 rpm for 1 hour. 40 μL of 100 mM MgCl2 and 40 μL of 100x concentrated antibiotic were added per mL of homogenate. Liver homogenates were stored at -20°C. On the day of the experiment, the required amount of liver homogenate was thawed and added to the oligos (the final concentration of oligos in the matrix was 5 μM). The microcentrifuge tubes were incubated at 37°C with gentle shaking (approximately 400 rpm) on a heated shaker. At the end of each time point (e.g., 48 hours), 20 μL of internal standard (2,000 ng / mL in nuclease-free water), 200 μL of 10% phosphoric acid, and 600 μL of Clarity lysis loading buffer were added to the microcentrifuge tube, with mixing by inversion after each addition. Solid-phase extraction was then performed.

[0336] As shown in Figure 10, ds-siNA-088 and ds-siNA-090 showed the highest stability after 48 hours. Therefore, ds-siNA-088 and ds-siNA-090 were selected for in vivo activity analysis. Mice were infected with AAV-HBV on day 28 of the study. Test ds-siNAs, ds-siNA-088 and ds-siNA-090, positive control ds-siNA-159, or vehicle were administered subcutaneously at a single dose of 5 mg / kg on day 0. Serial blood samples were collected on days 0, 7, 14, 21, and 28. Serum HBsAg was measured by ELISA.

[0337] As shown in Figure 11, the high durability and in vitro activity of ds-siNA-088 and ds-siNA-090 was not reflected in their in vivo performance, with HBsAg levels slightly higher than the positive control.

[0338] Example 7 - In vivo activity of ds-siNA containing 3'OH and unlocked modified nucleotides on the antisense strand. Mice were infected with AAV-HBV on study day 28. Test ds-siNAs, ds-siNA-080 and ds-siNA-081, negative control ds-siNA-083, positive control ds-siNA-084, or vehicle were administered subcutaneously at a single dose of 5 mg / kg on day 0. Serial blood samples were collected on days 0, 7, 14, 21, and 28. Serum HBsAg was measured by ELISA.

[0339] As shown in Figure 12, the presence of 3'OH and unlocked modified nucleotides on the antisense strand of ds-siNA-080 and ds-siNA-081 significantly reduced in vivo activity.

[0340] Example 8 - In vivo activity of ds-siNA containing a 5' end cap on the antisense strand. Mice were infected with AAV-HBV on study day 28. Test ds-siNA, ds-siNA-162, control ds-siNA-151, or vehicle was administered subcutaneously at a single dose of 5 mg / kg on day 0. Serial blood samples were taken on days 0, 7, 14, 21, and 28. Serum HBsAg was measured by ELISA.

[0341] As shown in Figure 13, the presence of a 5' endcap on the antisense strand of ds-siNA-162 provided similar in vivo activity compared to ds-siNA-151, which does not have a 5' endcap.

[0342] Example 9 - Comparison of in vivo activity of ds-siNA analogs. The stability of ds-siNA-159, a ds-siNA-159 analog with a single nucleotide modification in the 3' overhang of the antisense strand, and ds-siNA-009 was measured in mouse liver homogenate. Mouse livers were pulverized, and 50 mg of pulverized liver was transferred to individual microcentrifuge tubes placed on dry ice. Homogenization buffer (50 mM Tris·HCl, 150 mM KCl, pH 7.2) was added to a concentration of 200 mg / ml. The microcentrifuge tubes were placed in a heated multishaker and shaken at 40°C and 1,200 rpm for 1 hour. 40 μL of 100 mM MgCl2 and 40 μL of 100x concentrated antibiotic were added per mL of homogenate. The liver homogenates were stored at -20°C. On the day of the experiment, the required amount of liver homogenate was thawed and added to the oligos (the final concentration of oligos in the matrix was 5 μM). The microcentrifuge tubes were incubated at 37°C with gentle shaking (approximately 400 rpm) on a heated shaker. At the end of each time point (e.g., 48 hours), 20 μL of internal standard (2,000 ng / mL in nuclease-free water), 200 μL of 10% phosphoric acid, and 600 μL of Clarity Lysis Loading Buffer were added to the microcentrifuge tube, with mixing by inversion after each addition. Solid-phase extraction was then performed.

[0343] As shown in Figure 14A, ds-siNA-009 exhibited improved stability compared to the parental ds-siNA-159 after 48 hours. To determine whether the increased in vitro stability was correlated with improved in vivo activity, an in vivo activity assay was performed.

[0344] Mice were infected with AAV-HBV on study day 28. Test ds-siNA, ds-siNA-159, ds-siNA-009, or vehicle was administered subcutaneously at a single dose of 5 mg / kg on day 0. Serial blood samples were taken on days 0, 5, and then every 5 days until day 100. Serum HBsAg was measured by ELISA.

[0345] As shown in Figure 14B, ds-siNA-009 exhibited moderately reduced in vivo activity compared to the parental ds-siNA-159.

[0346] Example 10 - In vitro stability and in vivo activity of ds-siNA containing xylo-modified nucleotides. The stability of ds-siNA-131 was measured in mouse liver homogenate. Mouse liver was pulverized, and then 50 mg of pulverized liver was transferred to each microcentrifuge tube placed on dry ice. Homogenization buffer (50 mM Tris·HCl, 150 mM KCl, pH 7.2) was added to a concentration of 200 mg / ml. The microcentrifuge tubes were placed in a heated multishaker and shaken at 40°C and 1,200 rpm for 1 hour. 40 μL of 100 mM MgCl2 and 40 μL of 100x concentrated antibiotic were added per mL of homogenate. The liver homogenate was stored at -20°C. On the day of the experiment, the required amount of liver homogenate was thawed and added to the oligo (the final concentration of oligo in the matrix was 5 μM). The microcentrifuge tubes were incubated at 37°C with gentle shaking (approximately 400 rpm) on a heated multishaker. At the end of each time point (e.g., 48 hours), 20 μL of internal standard (2,000 ng / mL in nuclease-free water), 200 μL of 10% phosphoric acid, and 600 μL of Clarity Lysis Loading Buffer were added to the microcentrifuge tube, with mixing by shaking upside down after each addition, followed by solid-phase extraction.

[0347] As shown in Figure 15A, ds-siNA-131 showed improved stability after 48 hours compared with ds-siNA-159 and ds-siNA-009 in Figure 14A. Therefore, ds-siNA-131 was selected for in vivo activity analysis. Mice were infected with AAV-HBV on day 28 of the study. Test ds-siNAs, ds-siNA-131, ds-siNA-009, or vehicle were administered subcutaneously at a single dose of 5 mg / kg on day 0. Serial blood samples were collected on days 0, 7, 14, and 21. Serum HBsAg was measured by ELISA.

[0348] As shown in Figure 15B, the improved stability of ds-siNA-131 was correlated with in vivo activity comparable to that of ds-siNA-009.

[0349] Example 11 - In vivo activity and in vitro stability of ds-siNA containing 2'F modified nucleotides. Mice were infected with AAV-HBV on study day 28. Test ds-siNAs, including ds-siNA-103, ds-siNA-104, ds-siNA-105, ds-siNA-084, ds-siNA-106, ds-siNA-108, and ds-siNA-109, or vehicle were administered subcutaneously at a single dose of 5 mg / kg on day 0. Serial blood samples were taken on days 0, 5, and then every 5 days until day 25. Serum HBsAg was measured by ELISA.

[0350] As shown in Figure 16A, ds-siNA-084 exhibited the highest in vivo activity compared to the test ds-siNAs. Therefore, the in vitro stability of ds-siNA-084 was measured in mouse liver homogenate according to the previously described method. As can be seen in Figure 16B, ds-siNA-084 exhibited improved stability after 48 hours compared to ds-siNA-159 and ds-siNA-009 (Figure 15A).

[0351] The efficacy of ds-siNA-108, which showed the second highest in vivo activity in Figure 17A, was compared with that of Vir-2218. ds-siNA-108, the test ds-siNA, Vir-2218, or vehicle was administered subcutaneously at a single dose of 5 mg / kg on day 0 and every 14 days thereafter for 70 days. Serial blood samples were taken on day 0 and every 7 days thereafter for up to 164 days. Serum HBsAg, HBeAg, and alanine aminotransferase (ALT) levels were measured by ELISA.

[0352] As shown in Figure 17, ds-siNA-108 exhibited significantly increased activity compared to Vir-2218 (A) as shown with HBsAg. Serum ALT levels were comparable between the test ds-siNAs ds-siNA-108 and Vir-2218 (B).

[0353] Example 12 - Comparison of the in vivo activity of ds-siNA analogs with HBV treatment Vir-2218. Mice were infected with AAV-HBV on study day 28. Test ds-siNAs ds-siNA-159, ds-siNA-084, Vir-2218, or vehicle were administered subcutaneously at a single dose of 5 mg / kg on day 0 and every 14 days thereafter for 70 days. Serial blood samples were collected on day 0 and every 7 days thereafter for up to 168 days. Serum HBsAg, HBeAg, and alanine aminotransferase (ALT) levels were measured by ELISA.

[0354] As shown in Figure 18, ds-siNA-159 and ds-siNA-084 showed increased activity compared to Vir-2218, as indicated by (A) HBsAg and (B) HBeAg levels. Serum ALT levels were comparable between the test ds-siNAs ds-siNA-159 and Vir-2218, as shown in (C). ds-siNA-084 showed a sharp increase in ALT after the first administration on day 0, which then returned to normal by day 14. Further repeated administration did not result in a re-elevation of ALT.

[0355] Example 13 - Administration of 10-fold more effective dose of ds-siNA does not result in ALT signaling in uninfected mice. To measure ALT, a highly sensitive marker of drug-induced hepatotoxicity, uninfected mice were subcutaneously administered either 5 mg / kg, 15 mg / kg, or 50 mg / kg of the test ds-siNA, ds-siNA-084, or either 5 mg / kg or 15 mg / kg of the control siNA on day 0. Serial blood samples were taken on days 0, 7, and 14. Serum ALT levels were measured by ELISA.

[0356] As shown in Figure 19, ALT levels were not significantly affected by a 10-fold effective dose increase of either siNA-084 or Roche / Discerna. Comparing Figures 18C and 19, ds-siNA-084 only increased ALT in AAV-HBV-infected mice, suggesting that ALT is an immune-related factor that can occur when siNA-084 activates mouse CD8+ T cells to eliminate infected hepatocytes.

[0357] Example 14 - In vivo activity of ganciclovir and denavir modified ds-siNA. Mice were infected with AAV-HBV on day 28 of the study. Test ds-siNAs, ds-siNA-111, ds-siNA-112, ds-siNA-113, ds-siNA-116, control ds-siNA-084, or vehicle were administered subcutaneously at a single dose of 5 mg / kg on day 0. Serial blood samples were collected on days 0, 7, 14, and 21. Serum HBsAg and ALT levels were measured by ELISA.

[0358] As shown in Figure 20, ds-siNA-111, ds-siNA-112, ds-siNA-113, and ds-siNA-116 reduced ALT levels (A) but lost significant efficacy as shown by HBsAg levels (B).

[0359] Example 15 - In vitro stability and in vivo activity of ds-siNA containing xylo-modified nucleotides. The stability of parental ds-siNA-084 and xylo-modified ds-siNA-125 was measured in mouse liver homogenate. Mouse liver was pulverized, and then 50 mg of pulverized liver was transferred to each microcentrifuge tube placed on dry ice. Homogenization buffer (50 mM Tris·HCl, 150 mM KCl, pH 7.2) was added to a concentration of 200 mg / ml. The microcentrifuge tubes were placed in a heated multishaker and shaken at 40°C and 1,200 rpm for 1 hour. 40 μL of 100 mM MgCl2 and 40 μL of 100x concentrated antibiotic were added per mL of homogenate. The liver homogenate was stored at -20°C. On the day of the experiment, the required amount of liver homogenate was thawed and added to the oligos (the final concentration of oligos in the matrix was 5 μM). The microcentrifuge tubes were incubated at 37°C with gentle shaking (approximately 400 rpm) on a heated shaker. At the end of each time point (e.g., 48 hours), 20 μL of internal standard (2,000 ng / mL in nuclease-free water), 200 μL of 10% phosphoric acid, and 600 μL of Clarity Lysis Loading Buffer were added to the microcentrifuge tube, with mixing by inversion after each addition. Solid-phase extraction was then performed.

[0360] As shown in Figure 21, parental ds-siNA-084 (A) and xylo-modified ds-siNA-125 (B) showed similar stability after 48 hours. The in vivo activity of ds-siNA-084 and ds-siNA-125 was measured. Mice were infected with AAV-HBV on day 28 of the study. ds-siNA-084, ds-siNA-125, or vehicle was administered subcutaneously at a single dose of 5 mg / kg on day 0. Serial blood samples were taken on days 0, 7, 14, and 21. Serum HBsAg and ALT were measured by ELISA.

[0361] As shown in Figure 22A, ds-siNA-125 maintained its potency in reducing ALT compared to ds-siNA-084 (Figure 22B).

[0362] Example 16 - In vitro stability and in vivo activity of ds-siNA containing stereodefined PS linkages. The stability of parent ds-siNA-143, PS(S)-modified ds-siNA-157, and PS(R)-modified ds-siNA-156 was measured in mouse liver homogenate. Mouse liver was ground, and 50 mg of ground liver was transferred to each microcentrifuge tube placed on dry ice. Homogenization buffer (50 mM Tris·HCl, 150 mM KCl, pH 7.2) was added to a concentration of 200 mg / ml. The microcentrifuge tube was placed in a heated multishaker and shaken at 40°C and 1,200 rpm for 1 hour. 40 μL of 100 mM MgCl2 and 40 μL of 100x concentrated antibiotic were added per mL of homogenate. The liver homogenate was stored at -20°C. On the day of the experiment, the required amount of liver homogenate was thawed and added to the oligos (the final concentration of oligos in the matrix was 5 μM). The microcentrifuge tubes were incubated at 37°C with gentle shaking (approximately 400 rpm) on a heated shaker. At the end of each time point (e.g., 48 hours), 20 μL of internal standard (2,000 ng / mL in nuclease-free water), 200 μL of 10% phosphoric acid, and 600 μL of Clarity Lysis Loading Buffer were added to the microcentrifuge tube, with mixing by inversion after each addition. Solid-phase extraction was then performed.

[0363] Figure 23 shows the stability of the sense and antisense strands of (A) parent ds-siNA-143, (B) PS(S)-modified ds-siNA-157, and (C) PS(R)-modified ds-siNA-156. The stability of the antisense strands of parent ds-siNA-143 and PS(S)-modified ds-siNA-157 was comparable, whereas the stability of the antisense strand of PS(R)-modified ds-siNA-156 was low. The sense strands of all ds-siNAs included in the assay were degraded within 48 hours.

[0364] The in vivo activity of each ds-siNA was tested. Mice were infected with AAV-HBV on day 28 of the study. ds-siNA-143, ds-siNA-157, ds-siNA-156, or vehicle was administered subcutaneously at a single dose of 5 mg / kg on day 0. Serial blood samples were collected on days 0, 7, 14, and 21. Serum HBsAg was measured by ELISA. The results show that ds-siNA-156 modified with a PS(R) bond exhibited slightly improved activity compared with the parent chain and s-siNA-157 modified with a PS(S) bond (Figure 24).

[0365] Example 17 - In vivo activity of ds-siNA containing denavir (S) and mun12 modified nucleotides. Mice were infected with AAV-HBV on study day 28. Test ds-siNAs ds-siNA-147, ds-siNA-148, parental ds-siNA-149, or vehicle were administered subcutaneously at a single dose of 5 mg / kg on day 0. Serial blood samples were taken on days 0, 7, 14, and 21. Serum HBsAg was measured by ELISA.

[0366] As shown in Figure 25, ds-siNA-147, which was shown in Table 35 to improve the off-target profile, maintained potency, while ds-siNA-148 exhibited reduced potency.

[0367] Example 18: Preparation of Compound 40-9 (GalNAc4 Amidite) Compound 40-9 can be conjugated to any siNA disclosed herein as a targeting moiety. This compound, shown below, can be prepared according to the following brief description. [ka]

[0368] Building block compound 40-9 is useful for generating embodiments of modified phosphorothioated oligonucleotides. Compound 40-9 was prepared as follows.

[0369] Preparation of compound 40-2To a solution of commercially available glucosamine hydrochloride 40-1 (60 g, 278.25 mmol, 1 equiv) in DCM (300 mL) was added AcO (323.83 g, 3.17 mol, 297.09 mL, 11.4 equiv) dropwise at 0 °C, followed by the addition of pyridine (300 mL) and DMAP (3.40 g, 27.83 mmol, 0.1 equiv). The mixture was gradually warmed to 20 °C and stirred at 20 °C for 24 h. Upon completion as monitored by LCMS, the mixture was concentrated under reduced pressure, diluted with DCM (900 mL), and extracted with NaHCO (saturated, 300 mL aqueous solution). The combined organic layer was washed with brine (300 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give compound 40-2 (89.5 g, crude product) as a yellow solid. 1 H NMR(400MHz,CDCl3)δ=6.16(d,J=3.8Hz,1H),5.62(d,J=9.0Hz,1H),5.27-5.16(m,2H),4.54-4.43(m,1H),4.24(dd, LCMS(ESI):m / z C 16 H 23 NaNO 10 Calculated value for [M+Na] 412.34 + , actual measured value 412.0).

[0370] Preparation of compound 40-3 To a solution of compound 40-2 (40 g, 102.73 mmol, 1 equiv) in DCE (320 mL) at 25 °C, TMSOTf (23.98 g, 107.87 mmol, 19.49 mL, 1.05 equiv) was added dropwise, and the mixture was stirred at 60 °C for 4 h. Upon completion as monitored by LCMS, the mixture was quenched by adding TEA (60 mL) at 20 °C, stirred for 15 min, diluted with DCM (500 mL), and washed with NaHCO (saturated, 300 mL*2 aqueous solution). The organic layer was washed with brine (300 mL), dried over anhydrous NaSO, filtered, and concentrated under reduced pressure to give compound 40-3 (32.5 g, crude product) as a yellow oil. 1H NMR (400MHz, CDCl3) δ=5.96(d,J=7.3Hz,1H),5.25(t,J=2.4Hz,1H),4.95-4.88(m,1H),4.19-4.08(m,3H),3.59(m,1H),2.13-2.05(m,12H...

Claims

1. An oligonucleotide comprising: 【Chemical 1】 and a nucleotide having a structure selected from where B is a nucleobase, aryl, heteroaryl, or H; 【change】 represents a phosphodiester bond, a phosphorothioate bond, a mesyl phosphoramidate bond, or H in an oligonucleotide.

2. 2. The oligonucleotide of claim 1, wherein B is selected from adenine, guanine, cytosine, thymine, and uracil.

3. The nucleotide is 【Chemistry 2】 【Chemistry 3】 and where B is a nucleobase, aryl, heteroaryl, or H; 【change】 The oligonucleotide of claim 1 , wherein represents a phosphodiester bond, a phosphorothioate bond, a mesyl phosphoramidate bond, or H.

4. The oligonucleotide is 【Chemistry 4】 and at least two, at least three, at least four, or at least five nucleotides comprising a structure independently selected from: where B is a nucleobase, aryl, heteroaryl, or H; 【change】 The oligonucleotide according to any one of claims 1 to 3, wherein represents a phosphodiester bond, a phosphorothioate bond, a mesyl phosphoramidate bond, or H.

5. An oligonucleotide comprising: 【Chemistry 5】 and a nucleotide analogue comprising the structure where B is a nucleobase, aryl, heteroaryl, or H; 【change】 represents a phosphodiester bond, a phosphorothioate bond, or H, and * represents a chiral center in oligonucleotides.

6. 6. The nucleotide analogue of claim 5, wherein B is selected from adenine, guanine, cytosine, thymine, and uracil.

7. The oligonucleotide is 【Chemistry 6】 and at least two, at least three, at least four, or at least five nucleotide analogs comprising structures independently selected from: where B is a nucleobase, aryl, heteroaryl, or H; 【change】 The oligonucleotide according to claim 5 or 6, wherein represents a phosphodiester bond, a phosphorothioate bond, or H, and * represents a chiral center.

8. An oligonucleotide comprising: 【Chemistry 7】 and a nucleotide comprising a structure selected from where B is a nucleobase, aryl, heteroaryl, or H; 【change】 represents a phosphodiester bond, a phosphorothioate bond, or a mesyl phosphoramidate bond in an oligonucleotide.

9. An oligonucleotide comprising: 【Chemistry 8】 The structure of wherein each B is independently selected from a nucleobase, an aryl, a heteroaryl, and H; 【change】 represents a phosphodiester bond, a phosphorothioate bond, or a mesyl phosphoramidate bond in an oligonucleotide.

10. The oligonucleotide of any one of claims 1 to 9, wherein the oligonucleotide is selected from short interfering nucleic acids (siNA), antisense oligonucleotides (ASO), steric blockers, short hairpin RNAs (shRNA), and mRNAs.

11. A short interfering nucleic acid (siNA) comprising a sense strand and an antisense strand, The sense strand, the antisense strand, or both, 【Chemistry 9】 【Chemistry 10】 at least one, at least two, at least three, at least four, or at least five nucleotides independently selected from or 【Chemistry 11】 and at least one, at least two, at least three, at least four, or at least five nucleotide analogues independently selected from: where B is a nucleobase, aryl, heteroaryl, or H; 【change】 represents a phosphodiester bond, a phosphorothioate bond, or H, and * represents a chiral center. Short interfering nucleic acids (siNA).

11. a short interfering nucleic acid (siNA), (a) a sense strand comprising a first nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to an RNA corresponding to a target gene, wherein the first nucleotide sequence is (ix) is 15 to 30 nucleotides in length; and (x) a sense strand comprising 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least one modified nucleotide is a 2'-O-methyl nucleotide and the nucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and / or 19 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides, or at least one modified nucleotide is a 2'-O-methyl nucleotide and at least one modified nucleotide is a 2'-fluoro nucleotide; an antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to an RNA corresponding to the target gene, wherein the second nucleotide sequence comprises: (xi) is 15 to 30 nucleotides in length; and (xii) an antisense strand comprising 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least one modified nucleotide is a 2'-O-methyl nucleotide and at least one modified nucleotide is a 2'-fluoro nucleotide; or (b) a sense strand comprising a first nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to an RNA corresponding to the target gene, wherein the first nucleotide sequence is (i) is 15 to 30 nucleotides in length; and (ii) a sense strand comprising 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least one modified nucleotide is a 2'-O-methyl nucleotide and at least one modified nucleotide is a 2'-fluoro nucleotide; and an antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to an RNA corresponding to the target gene, wherein the second nucleotide sequence comprises: (iii) is 15 to 30 nucleotides in length; and (iv) an antisense strand comprising 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least one modified nucleotide is a 2'-O-methyl nucleotide and the nucleotides at positions 2, 5, 6, 7, 8, 10, 14, 16, 17, and / or 18 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides; The sense strand and / or the antisense strand are 【Chemistry 12】 at least one, at least two, at least three, at least four, or at least five nucleotides independently selected from or 【Chemistry 13】 and at least one, at least two, at least three, at least four, or at least five nucleotide analogues independently selected from: where B is a nucleobase, aryl, heteroaryl, or H; 【change】 represents a phosphodiester bond, a phosphorothioate bond, or H, and * represents a chiral center. Short interfering nucleic acids (siNA).

12. The antisense strand is 【Chemistry 14】 and a 5' stabilized end cap selected from Here, R ｙ is a nucleobase, and R １５ is H or CH ３ and 【change】 The siNA of claim 10 or 11, wherein represents a phosphodiester bond, a phosphorothioate bond, or a mesyl phosphoramidate bond.

13. The antisense strand comprises a 5'-stabilized end cap selected from the group consisting of formulas (1) to (16), formulas (9X) to (12X), formula (16X), formulas (9Y) to (12Y), formula (16Y), formulas (21) to (36), formula 36X, formulas (41) to (56), formulas (49X) to (52X), formulas (49Y) to (52Y), formulas 56X, formula 56Y, formula (61), formula (62), and formula (63); Here, R ｘ The siNA of claim 10 or 11, wherein is a nucleobase, aryl, heteroaryl, or H.

14. the antisense strand comprises a 5' stabilized end cap selected from the group consisting of formulas (71) to (86), formulas (79X) to (82X), formulas (79Y) to (82Y), formula 86X, formula 86X', formula 86Y, and formula 86Y'; Here, R ｘ The siNA of claim 10 or 11, wherein is a nucleobase, aryl, heteroaryl, or H.

15. The siNA of claim 10 or 11, wherein the antisense strand comprises a 5'-stabilized end cap selected from the group consisting of formulas (1A) to (15A), (1A-1) to (7A-1), (1A-2) to (7A-2), (1A-3) to (7A-3), (1A-4) to (7A-4), (9B) to (12B), (9AX) to (12AX), (9AY) to (12AY), (9BX) to (12BX), and (9BY) to (12BY).

16. The siNA of claim 10 or 11, wherein the antisense strand comprises a 5' stabilized end cap selected from the group consisting of formulas (21A) to (35A), (29B) to (32B), (29AX) to (32AX), (29AY) to (32AY), (29BX) to (32BX), and (29BY) to (32BY).

17. The siNA of claim 10 or 11, wherein the antisense strand comprises a 5' stabilized end cap selected from the group consisting of formula (71A) to (86A), formula (79XA) to (82XA), formula (79YA) to (82YA), formula (86XA), formula (86X'A), formula (86Y), and formula (86Y').

18. A short interfering nucleic acid (siNA) comprising a sense strand and an antisense strand, The antisense strand is 【Chemistry 15】 and a 5' vinyl phosphonate moiety comprising the structure wherein each B is independently selected from a nucleobase, an aryl, a heteroaryl, and H; 【change】 represents a phosphodiester, phosphorothioate, or mesyl phosphoramidate linkage; short interfering nucleic acid (siNA).

19. The structure is 【Chemistry 16】 The siNA of claim 18,

20. The structure is 【Chemistry 17】 The siNA of claim 18,

21. The sense strand, the antisense strand, or both, 【Chemistry 18】 【Chemistry 19】 at least one, at least two, at least three, at least four, or at least five nucleotides independently selected from or 【Chemistry 20】 and at least one, at least two, at least three, at least four, or at least five nucleotide analogs comprising a structure independently selected from: where B is a nucleobase, aryl, heteroaryl, or H; 【change】 The siNA of any one of claims 18 to 20, wherein represents a phosphodiester bond, a phosphorothioate bond, or H, and * represents a chiral center.

22. The siNA of any one of claims 10 to 21, wherein the sense strand, the antisense strand, or both each independently comprise one or more phosphorothioate internucleoside linkages.

23. The siNA of any one of claims 10 to 22, further comprising a phosphorylation blocker.

24. The siNA molecule of any one of claims 10-23, wherein the sense strand comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more phosphorothioate internucleoside linkages.

25. (i) at least one phosphorothioate internucleoside linkage of the sense strand is between the nucleotides at positions 1 and 2 from the 5' end of the sense strand; and / or 25. The siNA molecule of claim 24, wherein (ii) at least one phosphorothioate internucleoside linkage of the sense strand is between the nucleotides at positions 2 and 3 from the 5' end of the sense strand.

26. The siNA molecule of any one of claims 10-25, wherein the antisense strand further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more phosphorothioate internucleoside linkages.

27. (i) at least one phosphorothioate internucleoside linkage of the antisense strand is between the nucleotides at positions 1 and 2 from the 5′ end of the antisense strand; (ii) at least one phosphorothioate internucleoside linkage of the antisense strand is between the nucleotides at positions 2 and 3 from the 5' end of the antisense strand; (iii) at least one phosphorothioate internucleoside linkage of the antisense strand is between the nucleotides at positions 1 and 2 from the 3' end of the secondary sense strand; and / or (iv) the siNA molecule of claim 26, wherein at least one phosphorothioate internucleoside linkage is present between the nucleotides at positions 2 and 3 from the 3' end of the antisense strand.

28. The siNA molecule of any one of claims 10-27, wherein the sense strand comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mesyl phosphoramidate internucleoside linkages.

29. (i) at least one mesyl phosphoramidate internucleoside linkage of the sense strand is between the nucleotides at positions 1 and 2 from the 5' end of the sense strand; and / or 29. The siNA molecule of claim 28, wherein (ii) at least one mesyl phosphoramidate internucleoside linkage is located between the nucleotides at positions 2 and 3 from the 5' end of the sense strand.

30. The siNA molecule of any one of claims 10-29, wherein the antisense strand further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mesyl phosphoramidate internucleoside linkages.

31. (i) at least one mesyl phosphoramidate internucleoside linkage of the antisense strand is between the nucleotides at positions 1 and 2 from the 5′ end of the antisense strand; (ii) at least one mesyl phosphoramidate internucleoside linkage of the antisense strand is between the nucleotides at positions 2 and 3 from the 5′ end of the antisense strand; (iii) at least one mesyl phosphoramidate internucleoside linkage of the antisense strand is between the nucleotides at positions 1 and 2 from the 3' end of the antisense strand; and / or (iv) the siNA molecule of claim 30, wherein at least one mesyl phosphoramidate internucleoside linkage is located between the nucleotides at positions 2 and 3 from the 3' end of the antisense strand.

32. the sense strand, the antisense strand, or both, each independently comprise: 【Chemical 21】 (where R ｘ is a nucleobase, aryl, heteroaryl, H), 【Chemical 22】 (where R ｙ is a nucleobase), 【Chemical 23】 (where R ｙ The siNA molecule of any one of claims 10-31, comprising at least one, at least two, at least three, at least four, or at least five or more of the following:

33. The siNA of any one of claims 10 to 32, further comprising galactosamine.

34. The galactosamine is N-acetylgalactosamine (GalNAc) of formula (VI): 【Chemistry 24】 wherein m is 1, 2, 3, 4, or 5; each n is independently 1 or 2; p is 0 or 1; each R is independently H; each Y is independently selected from —O—P(═O)(SH)—, —O—P(═O)(O)—, —O—P(═O)(OH)—, and —O—P(S)S—; Z is H or a second protecting group; any L is a linker, or L and Y together are a linker; and The siNA of claim 33, wherein A is H, OH, a third protecting group, an activating group, or an oligonucleotide.

35. The galactosamine is N-acetylgalactosamine (GalNAc) of formula (VII): 【Chemistry 25】 In the formula, R ｚ The siNA of claim 33, wherein n is OH or SH, and each n is independently 1 or 2.

36. (i) at least one end of the siNA is blunt-ended; (ii) at least one end of the siNA comprises an overhang, the overhang comprising at least one nucleotide; or (iii) The siNA of any one of claims 10 to 35, wherein both ends of the siNA comprise an overhang, and the overhang comprises at least one nucleotide.

37. (i) the target gene is a viral gene; (ii) the target gene is a gene derived from a DNA virus; (iii) the target gene is a gene derived from a double-stranded DNA (dsDNA) virus; (iv) the target gene is a gene derived from a hepadnavirus; (v) the target gene is a gene derived from hepatitis B virus (HBV); (vi) the target gene is a gene derived from any one of HBV genotypes A to J; or (vii) The siNA of any one of claims 10 to 36, wherein the target gene is selected from the S gene or the X gene of HBV.

38. An siNA shown in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, or Table 10, Table 11, Table 12, Table 13, Table 14, Table 15, Table 16, Table 17, or Table 18.

39. A composition comprising the siNA of any one of claims 10 to 38 and a pharmaceutically acceptable excipient.

40. The composition of claim 39, further comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, or more siNAs of any one of claims 10 to 38.

41. 41. The composition of claim 39 or 40, further comprising an additional therapeutic agent.

42. 42. The composition of claim 41, wherein the additional therapeutic agent is selected from a nucleotide analog, a nucleoside analog, a capsid assembly modulator (CAM), a recombinant interferon, an entry inhibitor, a small molecule immune modulator, and an oligonucleotide therapy.

43. 43. The composition of claim 42, wherein the oligonucleotide therapy is an additional siNA, an antisense oligonucleotide (ASO), a NAP, or STOPS™.

44. 1. A method of treating a disease in a subject in need thereof, comprising: A method comprising administering to the subject a siNA of any one of claims 10 to 38 or a composition of any one of claims 39 to 43.

45. 45. The method of claim 44, wherein the disease is a viral disease, optionally caused by a DNA virus or a double-stranded DNA (dsDNA) virus.

46. 46. ​​The method of claim 45, wherein the dsDNA virus is a hepadnavirus.

47. 47. The method of claim 46, wherein the hepadnavirus is a hepatitis B virus (HBV), and optionally the HBV is selected from HBV genotypes A-J.

48. 48. The method of claim 47, further comprising administering an additional HBV therapeutic agent.

49. The method of claim 48, wherein the siNA or the composition and the additional HBV therapeutic agent are administered simultaneously or sequentially.

50. 50. The method of claim 48 or 49, wherein the additional HBV therapeutic agent is selected from a nucleotide analog, a nucleoside analog, a capsid assembly modulator (CAM), a recombinant interferon, an entry inhibitor, a small molecule immune modulator, and an oligonucleotide therapy.

51. 46. ​​The method of claim 45, wherein the viral disease is a disease caused by a coronavirus, optionally wherein the coronavirus is SARS-CoV-2.

52. 45. The method of claim 44, wherein the disease is a liver disease.

53. 53. The method of claim 52, wherein the liver disease is nonalcoholic fatty liver disease (NAFLD) or hepatocellular carcinoma (HCC).

54. 54. The method of claim 53, wherein the NAFLD is nonalcoholic steatohepatitis (NASH).

55. 55. The method of any one of claims 52 to 54, further comprising administering to the subject a drug for treating liver disease.

56. 56. The method of claim 55, wherein the liver disease therapeutic agent is selected from a peroxisome proliferator-activated receptor (PPAR) agonist, a farnesoid X receptor (FXR) agonist, a lipid-altering agent, and an incretin-based therapy.

57. 57. The method of claim 56, wherein (i) the PPAR agonist is selected from a PPARα agonist, a dual PPARα / δ agonist, a PPARγ agonist, and a dual PPARα / γ agonist; (ii) the lipid-altering agent is aramchol; or (iii) the incretin-based therapy is a glucagon-like peptide 1 (GLP-1) receptor agonist or a dipeptidyl peptidase 4 (DPP-4) inhibitor.

58. The method of any one of claims 55 to 57, wherein the siNA or the composition and the liver disease therapeutic agent are administered simultaneously or sequentially.

59. The method of any one of claims 44 to 58, wherein the siNA or composition is administered at a dose of at least 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, or 15 mg / kg.

60. The siNA or the composition may be administered in a range of 0.5 mg / kg to 50 mg / kg, 0.5 mg / kg to 40 mg / kg, 0.5 mg / kg to 30 mg / kg, 1 mg / kg to 50 mg / kg, 1 mg / kg to 40 mg / kg, 1 mg / kg to 30 mg / kg, 1 mg / kg to 20 mg / kg, 3 mg / kg to 50 mg / kg, 3 mg / kg to 40 mg / kg, 3 mg / kg to 30 mg / kg, 3 mg / kg to 20 mg / kg, 3 mg / kg to 15 mg / kg, 3 mg / kg to 10 mg / kg 59. The method of any one of claims 44-58, wherein the patient is administered at a dose of 4 mg / kg to 50 mg / kg, 4 mg / kg to 40 mg / kg, 4 mg / kg to 30 mg / kg, 4 mg / kg to 20 mg / kg, 4 mg / kg to 15 mg / kg, 4 mg / kg to 10 mg / kg, 5 mg / kg to 50 mg / kg, 5 mg / kg to 40 mg / kg, 5 mg / kg to 30 mg / kg, 5 mg / kg to 20 mg / kg, 5 mg / kg to 15 mg / kg, or 5 mg / kg to 10 mg / kg.

61. The method of any one of claims 44 to 60, wherein the siNA or the composition is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

62. The method of any one of claims 44 to 61, wherein the siNA or composition is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per day, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per week, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times per month.

63. The method of any one of claims 44 to 61, wherein the siNA or composition is administered at least once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days.

64. The method of any one of claims 44 to 61, wherein the siNA or composition is administered for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 51, 52, 53, 54, or 55 weeks.

65. The method of any one of claims 44 to 64, wherein the siNA or composition is administered at a single dose of 5 mg / kg or 10 mg / kg, three doses of 10 mg / kg once a week, three doses of 10 mg / kg once every three days, or five doses of 10 mg / kg once every three days.

66. The method of any one of claims 44 to 64, wherein the siNA or composition is administered in six doses of 1 mg / kg to 15 mg / kg, 1 mg / kg to 10 mg / kg, 2 mg / kg to 15 mg / kg, 2 mg / kg to 10 mg / kg, 3 mg / kg to 15 mg / kg, or 3 mg / kg to 10 mg / kg, wherein the first and second doses are optionally administered at least three days apart, the second and third doses are optionally administered at least four days apart, and the third and fourth doses, the fourth and fifth doses, or / and the fifth and sixth doses are optionally administered at least seven days apart.

67. The method of any one of claims 44 to 66, wherein the siNA or the composition is administered in a particle or viral vector, and the viral vector is optionally selected from adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpes simplex virus, lentivirus, measles virus, picornavirus, poxvirus, retrovirus, and rhabdovirus vectors.

68. 68. The method of claim 67, wherein the viral vector is a recombinant viral vector.

69. 69. The method of claim 67 or 68, wherein the viral vector is selected from AAVrh.74, AAVrh.10, AAVrh.20, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13.

70. The method of any one of claims 44 to 69, wherein the siNA or composition is administered systemically or locally.

71. The method of any one of claims 44 to 70, wherein the siNA or composition is administered intravenously, subcutaneously, or intramuscularly.

72. Use of a siNA according to any one of claims 10 to 38 or a composition according to any one of claims 39 to 43 for treating a disease in a subject.

73. 73. The use of claim 72, wherein the disease is a viral disease, optionally caused by a DNA virus or a double-stranded DNA (dsDNA) virus or disease.

74. 73. The use of claim 72, wherein the disease is a liver disease, optionally selected from non-alcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC).

75. 44. The siNA of any one of claims 10 to 38, or the composition of any one of claims 39 to 43, for use in treating a disease in a subject.

76. 76. The siNA or composition of claim 75, wherein the disease is a viral disease, and the viral disease is optionally caused by a DNA virus or a double-stranded DNA (dsDNA) virus or disease.

77. The siNA or composition of claim 75, wherein the disease is a liver disease, optionally selected from non-alcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC).

78. A short interfering nucleic acid (siNA) comprising a sense strand and an antisense strand, The antisense strand is 【Chemical 26】 wherein B is a nucleobase, aryl, heteroaryl, or H; 【change】 is a phosphodiester linkage, a phosphorothioate linkage, a mesyl phosphoramidate linkage, or H; a short interfering nucleic acid (siNA).

79. The modified nucleotide is 【Chemical 27】 The siNA of claim 78, selected from:

80. The siNA of claim 78 or 79, wherein the modified nucleotide is the last or penultimate nucleotide at the 3' end of the antisense strand.

81. The siNA of any one of claims 78 to 80, wherein the siNA is resistant to nuclease activity compared to a siNA of the same sequence that does not have the modified nucleotide in the 3' overhang.

82. 【Catalog 28】 【Chemical Formula 29】 【Chemistry 30】 【Chemical 31】 Phosphoramidites including the structure:

Images