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

Abstract

The present disclosure describes short interfering nucleic acid (siNA) molecules comprising modified nucleotides, compositions and methods and uses thereof.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority under 35 U.S.C. §119 to provisional application serial number 63 / 421,946, filed on November 2, 2022, and provisional application serial number 63 / 591,984, filed on October 20, 2023, the disclosures of which are incorporated herein by reference in their entireties. Technical Field

[0003] The present disclosure describes short interfering nucleic acid (siNA) molecules comprising modified nucleotides, compositions, and methods, and uses thereof. Background Art

[0004] RNA interference (RNAi) is a biological response to double-stranded RNA that mediates resistance to both endogenous parasite nucleic acids and exogenous pathogenic nucleic acids and regulates the expression of protein-coding genes. Short interfering nucleic acids (siNA) such as siRNA have been developed for RNAi therapy to treat a variety of diseases. For example, RNAi therapeutics have been proposed for the treatment of metabolic diseases, neurodegenerative diseases, cancer, and pathogenic infections (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 herein by reference in its entirety). However, a major limitation of RNAi therapeutics is the ability to efficiently deliver siRNA to target cells and to degrade the siRNA.

[0005] The present disclosure improves the delivery and stability of siNA molecules by providing siNA molecules comprising modified nucleobases. The siNA molecules of the present disclosure provide optimized combinations and quantities of modified nucleotides, nucleotide lengths, designs (e.g., blunt ends or overhangs, internucleoside bonds, conjugates), and modification patterns for improving the delivery and stability of siNA molecules. Summary of the Invention

[0006] Described herein are short interfering nucleic acid (siNA) molecules comprising 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.

[0007] In a first aspect, the present disclosure provides an oligonucleotide comprising a nucleotide comprising a structure selected from the group consisting of:

[0008]

[0009] wherein B is a nucleobase selected from the group consisting of adenine, guanine, cytosine, thymine and uracil, aryl, heteroaryl or H, and wherein represents a phosphodiester bond, a phosphorothioate bond, a methylsulfonylphosphoramidate bond, or H. For example, an oligonucleotide comprises a nucleotide comprising a structure selected from the group consisting of:

[0010]

[0011]

[0012] wherein B is a nucleobase, an aryl group, a heteroaryl group, or H, and wherein represents a phosphodiester bond, a phosphorothioate bond, a methylsulfonylphosphoramidate bond, or H.

[0013] In some embodiments, the oligonucleotide comprises at least 2, at least 3, at least 4, or at least 5 nucleotides comprising structures independently selected from:

[0014] wherein B is a nucleobase, an aryl group, a heteroaryl group, or H, and wherein represents a phosphodiester bond, a phosphorothioate bond, a methylsulfonylphosphoramidate bond, or H.

[0015] In a second aspect, the present disclosure provides an oligonucleotide comprising a nucleotide analog comprising the following structure:

[0016] wherein B is a nucleobase, an aryl group, a heteroaryl group, or H; wherein represents a phosphodiester bond, a phosphorothioate bond, or H; and wherein * represents a chiral center (e.g., an R or S isomer). For example, the oligonucleotide comprises at least 2, at least 3, at least 4, or at least 5 nucleotide analogs comprising structures independently selected from the group consisting of:

[0017] wherein B is a nucleobase, an aryl group, a heteroaryl group, or H; wherein represents a phosphodiester bond, a phosphorothioate bond, or H; and wherein * represents a chiral center (eg, R or S isomer).

[0018] In a third aspect, the present disclosure provides an oligonucleotide selected from the following structures:

[0019] wherein B is a nucleobase, an aryl group, a heteroaryl group, or H; wherein represents a phosphodiester bond, a phosphorothioate bond, or a methylsulfonylphosphoramidate bond.

[0020] In a fourth aspect, the present disclosure provides an oligonucleotide comprising the following structure:

[0021]

[0022]

[0023] wherein each B is independently selected from a nucleobase, an aryl group, a heteroaryl group, and H; wherein represents a phosphodiester bond, a phosphorothioate bond, or a methylsulfonylphosphoramidate bond.

[0024] In some embodiments, the oligonucleotide is selected from the group consisting of a short interfering nucleic acid (siNA), an antisense oligonucleotide (ASO), a steric blocker, a short hairpin RNA (shRNA), and an mRNA.

[0025] 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 at least 1, at least 2, at least 3, at least 4, or at least 5 nucleotides independently selected from:

[0026]

[0027] or at least 1, at least 2, at least 3, at least 4 or at least 5 nucleotide analogs independently selected from:

[0028] wherein B is a nucleobase, an aryl group, a heteroaryl group, or H; wherein represents a phosphodiester bond, a phosphorothioate bond, or H; and wherein * represents a chiral center (eg, R or S isomer).

[0029] In a sixth aspect, the present disclosure provides a short interfering nucleic acid (siNA) comprising:

[0030] (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:

[0031] (i) about 15 to 30 nucleotides in length; and

[0032] (ii) comprises 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 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, or wherein the at least one modified nucleotide is a 2′-O-methyl nucleotide and the at least one modified nucleotide is a 2′-fluoro nucleotide; and

[0033] 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 the RNA corresponding to the target gene, wherein the second nucleotide sequence:

[0034] (iii) about 15 to 30 nucleotides in length; and

[0035] (iv) comprises 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

[0036] (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 a target gene, wherein the first nucleotide sequence:

[0037] (i) 15 to 30 nucleotides in length; and

[0038] (ii) comprises 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

[0039] 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 the RNA corresponding to the target gene, wherein the second nucleotide sequence:

[0040] (iii) 15 to 30 nucleotides in length; and

[0041] (iv) comprises 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 nucleotide at position 2, 5, 6, 7, 8, 10, 14, 16, 17 and / or 18 from the 5′ end of the second nucleotide sequence is a 2′-fluoro nucleotide;

[0042] The sense strand and / or the antisense strand comprises at least 1, at least 2, at least 3, at least 4 or at least 5 nucleotides selected from:

[0043] or at least 1, at least 2, at least 3, at least 4 or at least 5 nucleotide analogs independently selected from:

[0044] wherein B is a nucleobase, an aryl group, a heteroaryl group, or H; wherein represents a phosphodiester bond, a phosphorothioate bond, or H; and wherein * represents a chiral center (eg, R or S isomer).

[0045] In some embodiments, the antisense strand of the siRNA comprises a 5' stable endcap selected from the group consisting of:

[0046] (omeco-d3 nucleotide), (4h nucleotide), (v-mun nucleotide), (C2O-4H nucleotides), where R y is a nucleobase, and R 15 is H or CH3, and wherein represents a phosphodiester bond, a phosphorothioate bond, or a methylsulfonylphosphoramidate bond.

[0047] In some embodiments, the antisense strand of the siRNA comprises a 5′ stable endcap 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 Formula (56), Formula (49X) to Formula (52X), Formula (49Y) to Formula (52Y), Formula 56X, Formula 56Y, Formula (61), Formula (62), and Formula (63), wherein R x is a nucleobase, an aryl group, a heteroaryl group or H.

[0048] In some embodiments, the antisense strand of the siRNA comprises a 5′ stable end cap selected from the group consisting of: Formula (71) to Formula (86), Formula (79X) to Formula (82X), Formula (79Y) to Formula (82Y), Formula 86X, Formula 86X′, Formula 86Y, and Formula 86Y′, wherein R x is a nucleobase, an aryl group, a heteroaryl group or H.

[0049] In some embodiments, the antisense strand of the siRNA comprises a 5′ stable end cap selected from the group consisting of: formula (1A)-(15A), formula (1A-1)-(7A-1), formula (1A-2)-(7A-2), formula (1A-3)-(7A-3), formula (1A-4)-(7A-4), formula (9B)-(12B), formula (9AX)-(12AX), formula (9AY)-(12AY), formula (9BX)-(12BX), and formula (9BY)-(12BY).

[0050] In some embodiments, the antisense strand of the siRNA comprises a 5′ stabilized endcap selected from the group consisting of: formula (21A)-(35A), formula (29B)-(32B), formula (29AX)-(32AX), formula (29AY)-(32AY), formula (29BX)-(32BX), and formula (29BY)-(32BY).

[0051] In some embodiments, the antisense strand of the siRNA comprises a 5′ stabilized end cap selected from the group consisting of: Formula (71A)-(86A), Formula (79XA)-(82XA), Formula (79YA)-(82YA); Formula (86XA), Formula (86X′A), Formula (86Y), and Formula (86Y′).

[0052] 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 a 5′ vinyl phosphonate moiety comprising the following structure:

[0053]

[0054] wherein each B is independently selected from a nucleobase, an aryl group, a heteroaryl group, and H; wherein represents a phosphodiester bond, a phosphorothioate bond, or a methylsulfonylphosphoramidate bond.

[0055] In some embodiments, the sense strand of the siNA, the antisense strand of the siNA, or both comprises at least 1, at least 2, at least 3, at least 4, or at least 5 nucleotides comprising structures independently selected from:

[0056] or at least 1, at least 2, at least 3, at least 4, or at least 5 nucleotide analogs comprising a structure independently selected from:

[0057] wherein B is a nucleobase, an aryl group, a heteroaryl group, or H; wherein represents a phosphodiester bond, a phosphorothioate bond, or H; and wherein * represents a chiral center (e.g., R or S isomer). In some embodiments, the sense strand, the antisense strand, or both independently comprise one or more phosphorothioate internucleoside bonds. In some embodiments, the siNA further comprises a phosphorylation blocker.

[0058] 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 at positions 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 at positions 2 and 3 from the 5' end of the sense strand.

[0059] 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 the nucleotides at positions 1 and 2 from the 5' end of the antisense strand, at least one phosphorothioate internucleoside linkage in the antisense strand is between the nucleotides at positions 2 and 3 from the 5' end of the antisense strand, at least one phosphorothioate internucleoside linkage in the antisense strand is between the nucleotides at positions 1 and 2 from the 3' end of the antisense strand, and / or at least one phosphorothioate internucleoside linkage is between the nucleotides at positions 2 and 3 from the 3' end of the antisense strand.

[0060] 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 methylsulfonylphosphoramidate internucleoside linkages. In some embodiments, at least one methylsulfonylphosphoramidate internucleoside linkage in the sense strand is between the nucleotides at positions 1 and 2 from the 5' end of the sense strand; and / or at least one methylsulfonylphosphoramidate internucleoside linkage is between the nucleotides at positions 2 and 3 from the 5' end of the sense strand.

[0061] 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 methylsulfonylphosphoramidate internucleoside linkages. In some embodiments, at least one methylsulfonylphosphoramidate internucleoside linkage in the antisense strand is between the nucleotides at positions 1 and 2 from the 5' end of the antisense strand, at least one methylsulfonylphosphoramidate internucleoside linkage in the antisense strand is between the nucleotides at positions 2 and 3 from the 5' end of the antisense strand, at least one methylsulfonylphosphoramidate internucleoside linkage in the antisense strand is between the nucleotides at positions 1 and 2 from the 3' end of the antisense strand, and / or at least one methylsulfonylphosphoramidate internucleoside linkage is between the nucleotides at positions 2 and 3 from the 3' end of the antisense strand.

[0062] In some embodiments, the sense strand of the siNA, the antisense strand of the siNA, or both independently comprise at least 1, at least 2, at least 3, at least 4, or at least 5 or more where R x is a nucleobase, an aryl group, a heteroaryl group or H, where R y It is a nucleobase, where R y is a nucleobase, or a combination thereof.

[0063] In some embodiments, the siNA further comprises galactosamine. In some embodiments, the galactosamine is N-acetylgalactosamine (GalNAc) of formula (VI): in

[0064] m is 1, 2, 3, 4 or 5;

[0065] Each n is independently 1 or 2;

[0066] p is 0 or 1;

[0067] Each R is independently H;

[0068] each Y is independently selected from -OP(=O)(SH)-, -OP(=O)(O)-, -OP(=O)(OH)-, and -OP(S)S-;

[0069] Z is H or a second protecting group;

[0070] L is a linker or L and Y in combination are a linker; and

[0071] A is H, OH, a third protecting group, an activating group or an oligonucleotide. In some embodiments, galactosamine is N-acetylgalactosamine (GalNAc) of formula (VII):

[0072]

[0073] where R z is OH or SH; and each n is independently 1 or 2.

[0074] In some embodiments, at least one end of the siNA is blunt-ended, at least one end of the siNA comprises an overhang, wherein the overhang comprises at least one nucleotide, or both ends of the siNA comprise overhangs, wherein the overhang comprises at least one nucleotide.

[0075] In some embodiments, the target gene of the siNA is a viral gene, a gene from a DNA virus, a gene from a double-stranded DNA (dsDNA) virus, a gene from a hepadnavirus, a gene from hepatitis B virus (HBV), a gene from HBV of any one of genotypes AJ, or the target gene is selected from the S gene or the X gene of HBV.

[0076] In a seventh aspect, the present disclosure provides a siNA as 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.

[0077] The present disclosure provides a composition comprising siNA according to any one of the siNAs disclosed herein and a pharmaceutically acceptable excipient. In some embodiments, the composition comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of any one 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 nucleotide analogs, nucleoside analogs, capsid assembly regulators (CAMs), recombinant interferons, entry inhibitors, small molecule immunomodulators, and oligonucleotide therapies. In some embodiments, the oligonucleotide therapy is an additional siNA, antisense oligonucleotide (ASO), NAP, or STOPS. TM .

[0078] The present disclosure provides a method of treating a disease in a subject in need thereof, comprising administering to the subject a siNA disclosed herein or a composition comprising siNA disclosed herein. The present disclosure further provides uses of the disclosed siNA and compositions for treating a disease in a subject. The present disclosure further provides siNA and compositions for treating a disease in a subject.

[0079] In some embodiments of the disclosed methods and uses, the viral disease is 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), and optionally wherein the HBV is selected from HBV genotypes AJ. In some embodiments, the methods and uses further comprise administering an additional HBV therapeutic agent. In some embodiments, the siNA or the composition and the additional HBV therapeutic agent are administered simultaneously or sequentially. In some embodiments, the additional HBV therapeutic agent is selected from nucleotide analogs, nucleoside analogs, capsid assembly regulators (CAMs), recombinant interferons, entry inhibitors, small molecule immunomodulators, and oligonucleotide therapies. In some embodiments, the viral disease is a disease caused by a coronavirus, and optionally wherein the coronavirus is SARS-CoV-2.

[0080] In some embodiments of the disclosed methods and uses, the disease is liver disease. In some embodiments, the liver disease is non-alcoholic fatty liver disease (NAFLD) or hepatocellular carcinoma (HCC). In some embodiments, NAFLD is non-alcoholic steatohepatitis (NASH). Some embodiments may further include administering a liver disease therapeutic agent to the subject. In some embodiments, the liver disease therapeutic agent is selected from peroxisome proliferator-activated receptor (PPAR) agonists, farnesoid X receptor (FXR) agonists, lipid-regulating agents, and incretin-based therapies. In some embodiments, (i) the PPAR agonist is selected from PPARα agonists, PPARα / δ dual agonists, PPARγ agonists, and PPARα / γ dual agonists; (ii) the lipid-regulating 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 the composition and the agent treating liver disease are administered simultaneously or sequentially.

[0081] In some embodiments, the siNA or the 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.

[0082] In some embodiments, the siNA or the composition is administered at a dose of between 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, 4 mg / kg to 50 mg / kg. The invention also provides a method for the administration of a dose of between 5 mg / kg, 20 mg / kg, 4 ...

[0083] In some embodiments of the disclosed methods and uses, the siNA or the composition is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0084] In some embodiments of the disclosed methods and uses, the siNA or the 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.

[0085] In some embodiments of the disclosed methods and uses, the siNA or the 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.

[0086] In some embodiments of the disclosed methods and uses, the siNA or the composition is 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, 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.

[0087] In some embodiments of the disclosed methods and uses, the siNA or the composition is administered as 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.

[0088] In some embodiments of the disclosed methods and uses, the siNA or the composition is administered in six doses ranging from 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 dose and the second dose are optionally administered at least 3 days apart; wherein the second dose and the third dose are optionally administered at least 4 days apart; and wherein the third dose and the fourth dose, the fourth dose and the fifth dose, and or the fifth dose and the sixth dose are optionally administered at least 7 days apart.

[0089] In some embodiments of the disclosed methods and uses, the siNA or the composition is administered in the form of a particle or viral vector, wherein the viral vector is optionally selected from the following vectors: adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpes simplex virus, lentivirus, measles virus, picornavirus, poxvirus, retrovirus and rhabdomyovirus. 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.

[0090] In some embodiments of the disclosed methods and uses, the siNA or the composition is administered systemically or locally.

[0091] In some embodiments of the disclosed methods and uses, the siNA or the composition is administered intravenously, subcutaneously, or intramuscularly.

[0092] In an eighth aspect, the present disclosure provides a siNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises a 3' overhang, the 3' overhang comprising at least one modified nucleotide, the at least one modified nucleotide being selected from the following structures:

[0093] wherein B is a nucleobase, an aryl group, a heteroaryl group, or H, and wherein represents a phosphodiester bond, a phosphorothioate bond, a methylsulfonylphosphoramidate bond, or H. For example, the nucleotide comprises a structure selected from the group consisting of: In some embodiments, the modified nucleotide is the last nucleotide or the penultimate nucleotide at the 3' end of the antisense strand. In some embodiments, the siNA is resistant to nuclease activity relative to a siNA of the same sequence without the modified nucleotide in the 3' overhang.

[0094] In a seventh aspect, the present disclosure provides a phosphoramidite comprising the following structure:

[0095]

[0096]

[0097]

[0098] The above 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 be apparent to those skilled in the art from the following description of the drawings and the detailed description of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] Figure 1 Exemplary siNA molecules are shown.

[0100] Figure 2 Exemplary siNA molecules are shown.

[0101] Figures 3A-3J An exemplary double-stranded siNA molecule is shown. G3 represents the GalNAc-conjugated portion.

[0102] Figures 4A-4AB An exemplary double-stranded siNA molecule is shown. G3 represents the GalNAc-conjugated portion.

[0103] Figures 5A-5F An exemplary double-stranded siNA molecule is shown. G3 represents the GalNAc-conjugated portion.

[0104] Figures 6A-6K An exemplary double-stranded siNA molecule is shown. G3 represents the GalNAc-conjugated portion.

[0105] Figures 7A-7D An exemplary double-stranded siNA molecule is shown. G3 represents the GalNAc-conjugated portion.

[0106] Figures 8A-8B An exemplary double-stranded siNA molecule is shown. G3 represents the GalNAc-conjugated portion.

[0107] Figure 9 The in vivo activity of ds-siNA containing a 5′ vinylphosphonate moiety and a modified unlocked nucleotide on the antisense strand is demonstrated. Activity was determined by measuring serum HBsAg levels as determined by ELISA. Error bars represent standard error of the mean.

[0108] Figure 10 The effect of 5'-cyclopropyl nucleotides on the stability of siNA in mouse liver homogenate is demonstrated.

[0109] Figure 11 In vivo activity of ds-siNA containing 5′-cyclopropyl nucleotides on the antisense strand is demonstrated. Activity was determined by measuring serum HBsAg levels as determined by ELISA. Error bars represent standard error of the mean.

[0110] Figure 12 The bioavailability of ds-siNA containing 3OH and unlocked modified nucleotides on the antisense strand is shown. Activity was determined by measuring serum HBsAg levels determined by ELISA. Error bars represent standard error of the mean.

[0111] Figure 13 The bioavailability of ds-siNA containing a 5′-end cap on the antisense strand is shown. Activity was determined by measuring serum HBsAg levels by ELISA. Error bars represent standard error of the mean.

[0112] Figures 14A-14B show (Figure 14A) in vitro stability measured in mouse liver homogenate, and ( Figure 14B ) In vivo activity of ds-siNA analogs determined by measuring serum HBsAg levels by ELISA. Error bars represent standard error of the mean.

[0113] Figures 15A-15B show (Figure 15A) the effect of xylo modification on the stability of siNA in mouse liver homogenate, and ( Figure 15B ) Energetic activity of xylo-modified ds-siNAs determined by measuring serum HBsAg levels by ELISA. Error bars represent standard error of the mean.

[0114] Figures 16A-16B show (Figure 16A) the bioavailability of ds-siNA containing a 2'F modification along the antisense strand. Activity was determined by measuring serum HBsAg levels as determined by ELISA. Error bars represent standard error of the mean. ( Figure 16B ) Effect of 2′F modification on the stability of siNA in mouse liver homogenate.

[0115] Figures 17A-17B show a comparison of the in vivo activity of ds-siNA containing a 2'F modification along the antisense strand and the HBV treatment Vir-2218. The results were obtained by measuring serum HBsAg, ( Figure 17B ) Alanine aminotransferase (ALT) levels were used to determine activity. Error bars represent standard error of the mean.

[0116] Figures 18A-18C show the in vivo activity of ds-siNA analogs and the HBV treatment Vir-2218. The results were obtained by measuring serum HBsAg (Figure 18A), HBeAg (Figure 18B), and HBV antigen levels (Figure 18C). Figure 18C ) Alanine aminotransferase (ALT) levels were used to determine activity. Error bars represent standard error of the mean.

[0117] Figure 19 The physical activity of ds-siNA and Roch / Discerna administered at various concentrations to uninfected mice is shown. Activity was determined by measuring serum ALT levels determined by ELISA. Error bars represent standard error of the mean.

[0118] Figures 20A-20B show the bioavailability of ganciclovir, denvir and 3ocp modified ds-siNA. The results were obtained by measuring serum ALT and ( Figure 20B ) Serum HBsAg levels were used to determine activity. Error bars represent standard error of the mean.

[0119] 21A-21B demonstrate the effect of xylo modification on the stability of siNA in mouse liver homogenate.

[0120] Figures 22A-22B show the bioavailability of xylo-modified ds-siNA. The bioavailability of xylo-modified ds-siNA was determined by measuring the expression of serum HBsAg and ( Figure 22B) Serum ALT levels were used to determine activity. Error bars represent standard error of the mean.

[0121] 23A-23B show the effect of a well-defined PS bond on the stability of siNA in mouse liver homogenate.

[0122] Figure 24 The bioavailability of ds-siNA containing a well-defined PS bond is demonstrated. Activity was determined by measuring serum HBsAg levels by ELISA. Error bars represent standard error of the mean.

[0123] Figure 25 In vivo activity of ds-siNA containing denavir(S)-modified nucleotides on the antisense strand is shown. Activity was determined by measuring serum HBsAg levels by ELISA. Error bars represent standard error of the mean. DETAILED DESCRIPTION

[0124] Disclosed herein are oligonucleotide molecules (including short interfering nucleic acids or "siNA") comprising novel, modified nucleotide monomers and dimers comprising unique chemical moieties and / or other modifications. Also disclosed herein are methods of using the disclosed oligonucleotides and siNA molecules to treat various diseases and conditions.

[0125] Typically, the siNA molecules described herein can be double-stranded siNA (ds-siNA) molecules. The siNA molecules described herein can comprise modified nucleotides selected from 2′-O-methyl nucleotides and 2′-fluoro nucleotides. The siNA molecules described herein can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more phosphorothioate internucleoside linkages. The siNA molecules described herein can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more methylsulfonylphosphoramidate internucleoside linkages. The siNA molecules described herein can comprise at least one phosphorylation blocker. The siNA molecules described herein can comprise a 5′-stabilized end cap. The siNA molecules described herein can comprise galactosamine. The siNA molecules described herein can comprise one or more blunt ends. The siNA molecules described herein can comprise one or more overhangs.

[0126] For example, the present disclosure provides a modified nucleotide comprising the following structure:

[0127] wherein B is a nucleobase, an aryl group, a heteroaryl group, or H, and wherein represents a phosphodiester bond, a phosphorothioate bond, a methylsulfonylphosphoramidate 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 may comprise the following structure: In some embodiments, the modified nucleotide may comprise the following structure:

[0128] The present disclosure also provides a modified nucleotide comprising the following structure:

[0129] wherein B is a nucleobase, an aryl group, a heteroaryl group, or H, and wherein and represents a phosphodiester bond, a phosphorothioate bond, a methylsulfonylphosphoramidate 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 may comprise the following structure: In some embodiments, the modified nucleotide may comprise the following structure:

[0130] The present disclosure also provides a modified nucleotide comprising the following structure: wherein B is a nucleobase, an aryl group, a heteroaryl group, or H, and wherein and represents a phosphodiester bond, a phosphorothioate bond, a methylsulfonylphosphoramidate 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 may comprise the following structure: wherein A is adenine and G is guanine.

[0131] The present disclosure also provides a modified nucleotide comprising the following structure: wherein B is a nucleobase, an aryl group, a heteroaryl group, or H, and wherein represents a phosphodiester bond, a phosphorothioate bond, a methylsulfonylphosphoramidate bond, or H. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0132] The present disclosure also provides a modified nucleotide comprising the following structure: wherein B is a nucleobase, an aryl group, a heteroaryl group, or H, and wherein represents a phosphodiester bond, a phosphorothioate bond, a methylsulfonylphosphoramidate bond, or H. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0133] The present disclosure also provides a modified nucleotide comprising the following structure: wherein B is a nucleobase, an aryl group, a heteroaryl group, or H, and wherein represents a phosphodiester bond, a phosphorothioate bond, a methylsulfonylphosphoramidate 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 may comprise the following structure:

[0134]

[0135] The present disclosure also provides a modified nucleotide comprising the following structure: wherein B is a nucleobase, an aryl group, a heteroaryl group, or H, and wherein represents a phosphodiester bond, a phosphorothioate bond, a methylsulfonylphosphoramidate 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 may comprise the following structure:

[0136]

[0137] The present disclosure also provides a modified nucleotide comprising the following structure: wherein B is a nucleobase, an aryl group, a heteroaryl group, or H, and wherein represents a phosphodiester bond, a phosphorothioate bond, a methylsulfonylphosphoramidate 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 may comprise the following structure:

[0138]

[0139] The present disclosure also provides a modified nucleotide comprising the following structure: wherein B is a nucleobase, an aryl group, a heteroaryl group, or H, and wherein represents a phosphodiester bond, a phosphorothioate bond, a methylsulfonylphosphoramidate 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 may comprise the following structure:

[0140]

[0141] The present disclosure also provides a modified nucleotide comprising the following structure: wherein B is a nucleobase, an aryl group, a heteroaryl group, or H, and wherein represents a phosphodiester bond, a phosphorothioate bond, a methylsulfonylphosphoramidate 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 may comprise the following structure:

[0142]

[0143] The present disclosure also provides a modified nucleotide comprising the following structure: wherein B is a nucleobase, an aryl group, a heteroaryl group, or H, and wherein represents a phosphodiester bond, a phosphorothioate bond, a methylsulfonylphosphoramidate 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 may comprise the following structure:

[0144]

[0145] The present disclosure also provides nucleotide analogs comprising the following structure: wherein B is a nucleobase, an aryl group, a heteroaryl group, or H; wherein represents a phosphodiester bond, a phosphorothioate bond, or H; and wherein * represents a chiral center (e.g., an R or S isomer). In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0146] The present disclosure further provides a modified nucleotide comprising the following structure:

[0147] In a third aspect, the present disclosure provides an oligonucleotide selected from the following structures:

[0148] wherein B is a nucleobase, an aryl group, a heteroaryl group or H; wherein and represent a phosphodiester bond, a phosphorothioate bond or a methylsulfonylphosphoramidate bond.

[0149] The present disclosure further provides a modified nucleotide comprising the following structure:

[0150] and a modified nucleotide comprising the following structure: where R x is a nucleobase, an aryl group, a heteroaryl group, or H. In some embodiments, the modified nucleotide may comprise the following structure: where R y In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0151] The present disclosure also provides oligonucleotides comprising structures that can serve as stable endcaps at the 5′ end of the antisense strand of any disclosed siNA. The disclosed 5′-stable endcaps can include, but are not limited to, structures: wherein each B is independently selected from a nucleobase, an aryl group, a heteroaryl group, and H; wherein represents a phosphodiester bond, a phosphorothioate bond, or a methylsulfonylphosphoramidate bond. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0152] The present disclosure also provides for the use of 5-mercaptoethanol in the antisense strand of any disclosed siNA. ′ The disclosed 5′ stable end cap may include but is not limited to the structure:

[0153] (omeco-d3 nucleotide), (4h nucleotide), (v-mun nucleotide), (C2O-4H nucleotides), (coc-4h 4h-vp nucleotides)) and (4h-vp nucleotide); where R y is a nucleobase, and R 15 is H or CH3, and wherein represents a phosphodiester bond, a phosphorothioate bond, or a methylsulfonylphosphoramidate bond. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof. In some embodiments, the 5′-stabilizing end cap may be selected from, but not limited to, the structures: where R 15 is H or CH3, and wherein represents a phosphodiester bond, a phosphorothioate bond, or a methylsulfonylphosphoramidate bond.

[0154] The disclosed short interfering nucleic acid (siNA) molecules may comprise 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'-stable end caps at the 5' end of the antisense strand. In fact, the disclosed short interfering nucleic acid (siNA) molecules may comprise:

[0155] (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:

[0156] (i) about 15 to 30 nucleotides in length; and

[0157] (ii) comprises 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 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, or wherein the at least one modified nucleotide is a 2′-O-methyl nucleotide and the at least one modified nucleotide is a 2′-fluoro nucleotide; and

[0158] 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 the RNA corresponding to the target gene, wherein the second nucleotide sequence:

[0159] (iii) about 15 to 30 nucleotides in length; and

[0160] (iv) comprises 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

[0161] (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 a target gene, wherein the first nucleotide sequence:

[0162] (i) 15 to 30 nucleotides in length; and

[0163] (ii) comprises 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

[0164] 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 the RNA corresponding to the target gene, wherein the second nucleotide sequence:

[0165] (iii) 15 to 30 nucleotides in length; and

[0166] (iv) comprises 15 or more modified nucleotides and / or nucleotide analogues 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 nucleotide at position 2, 5, 6, 7, 8, 10, 14, 16, 17 and / or 18 from the 5′ end of the second nucleotide sequence is a 2′-fluoro nucleotide; or

[0167] (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 a target gene, wherein the first nucleotide sequence:

[0168] (v) about 15 to 30 nucleotides in length; and

[0169] (vi) comprises 15 or more modified nucleotides and / or nucleotide analogues independently selected from 2′-O-methyl nucleotides and 2′-fluoro nucleotides, wherein at least one modified nucleotide and / or nucleotide analogue is a 2′-O-methyl nucleotide and 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, or wherein at least one modified nucleotide is a 2′.O-methyl nucleotide and at least one modified nucleotide is a 2′-fluoro nucleotide; and

[0170] 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 the RNA corresponding to the target gene, wherein the second nucleotide sequence:

[0171] (vii) about 15 to 30 nucleotides in length; and

[0172] (viii) comprises 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 nucleotide at position 2, 5, 6, 7, 8, 10, 14, 16, 17 and / or 18 from the 5′ end of the second nucleotide sequence is a 2′-fluoro nucleotide;

[0173] As long as the sense strand and / or antisense strand comprises at least one, at least two, at least three, at least four or at least five modified nucleotides and / or nucleotide analogs, the modified nucleotides and / or nucleotide analogs are selected from: as well as or at least 1, at least 2, at least 3, at least 4 or at least 5 nucleotide analogs independently selected from: wherein B is a nucleobase, an aryl group, a heteroaryl group, or H; wherein represents a phosphodiester bond, a phosphorothioate bond, or H; and wherein * represents a chiral center (eg, R or S isomer).

[0174] In addition, the siNA of the present disclosure can comprise a sense strand and / or an antisense strand, each independently comprising one or more phosphorothioate internucleoside bonds, one or more methylsulfonylphosphoramidate internucleoside bonds, or a combination thereof. siNA can comprise a phosphorylation blocker, galactosamine, and / or a 5'-stabilizing end cap (in addition to those described above). siNA can be conjugated to a targeting moiety such as galactosamine.

[0175] The present disclosure also provides a siNA comprising a sense strand and an antisense strand, wherein the antisense strand comprises a 3′ overhang, the 3′ overhang comprising at least one modified nucleotide, the at least one modified nucleotide being selected from:

[0176] Wherein B is a nucleobase, an aryl group, a heteroaryl group or H, and wherein, represents a phosphodiester bond, a phosphorothioate bond, a methylsulfonylphosphoramidate 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 nucleotides include but are not limited to the structure: In some embodiments, the modified nucleotide is the last nucleotide or the penultimate nucleotide at the 3' end of the antisense strand. In some embodiments, the siNA is resistant to nuclease activity relative to a siNA of the same sequence without the modified nucleotide in the 3' overhang.

[0177] Further disclosed herein are compositions comprising two or more siNA molecules described herein.

[0178] Further disclosed herein are compositions comprising any of the described siNA molecules and a pharmaceutically acceptable carrier or diluent. Such compositions may also comprise additional therapeutic agents, or may be administered in combination with additional therapeutic agents (simultaneously or sequentially).

[0179] Further disclosed herein are compositions comprising two or more siNA molecules described herein for use as medicaments.

[0180] Further disclosed herein are compositions comprising any of the described siNA molecules for use as a medicament and a pharmaceutically acceptable carrier or diluent. Such medicaments may also comprise additional therapeutic agents or may be administered in combination with additional therapeutic agents (simultaneously or sequentially).

[0181] Further disclosed herein are methods of treating a disease in a subject in need thereof, the method comprising administering to the subject any siNA molecule (or combination thereof) or composition / medicament described herein.

[0182] Further disclosed herein is the use of any of the siNA molecules described herein (or combinations thereof) in the preparation of a medicament for treating a disease.

[0183] Short interfering nucleic acid (siNA) molecules

[0184] As described above, the present disclosure provides siNA molecules comprising modified nucleotides. Any siNA molecule described herein can be a double-stranded siNA (ds-siNA) molecule. The terms "siNA molecule" and "ds-siNA molecule" can be used interchangeably. In some embodiments, the ds-siNA molecule comprises a sense strand and an antisense strand.

[0185] For the purposes of this disclosure, siNA molecules disclosed herein generally comprise (a) at least one phosphorylation blocker, a conjugated moiety, and / or a 5′-stable end cap; and (b) a short interfering nucleic acid (siNA). In some embodiments, the phosphorylation blocker is a phosphorylation blocker disclosed herein. In some embodiments, the conjugated moiety is a galactosamine disclosed herein. In some embodiments, the 5′-stable end cap is a 5′-stable end cap disclosed herein.

[0186] siNA can comprise any of the first nucleotide, second nucleotide, sense strand or antisense strand sequences disclosed herein. siNA can 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. siNA can comprise 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 comprise less than or equal to 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. 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).

[0187] The ds-siNA may comprise (a) a sense 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; and (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.

[0188] Any siNA molecule disclosed herein may further comprise one or more linkers independently selected from the group consisting of a phosphodiester (PO) linker, a phosphorothioate (PS) linker, a phosphorodithioate linker, a methylsulfonylphosphoramidate (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 additionally, the linker can connect the nucleotides of the siNA molecule to at least one phosphorylation blocker, a conjugate moiety, or a 5'-stable end cap. In some embodiments, the linker connects the conjugate moiety to the phosphorylation blocker or the 5'-stable end cap.

[0189] Figure 1 An exemplary siNA molecule of the present disclosure is shown in FIG. Figure 1As shown, an exemplary siNA molecule comprises a sense strand (101) and an antisense strand (102). The sense strand (101) may comprise a first oligonucleotide sequence (103). The first oligonucleotide sequence (103) may comprise one or more phosphorothioate internucleoside linkages (109). The phosphorothioate internucleoside linkage (109) may be located between nucleotides at the 5′ or 3′ end of the first oligonucleotide sequence (103). The phosphorothioate internucleoside linkage (109) may be between the first three nucleotides from the 5′ end of the first oligonucleotide sequence (103). The first oligonucleotide sequence (103) may comprise one or more 2′-fluoro nucleotides (110). The first oligonucleotide sequence (103) may comprise one or more 2′-O-methyl nucleotides (111). The first oligonucleotide sequence (103) may comprise 15 or more modified nucleotides independently selected from 2′-fluoro nucleotides (110) and 2′-O-methyl nucleotides (111). The sense strand (101) may further comprise a phosphorylation blocker (105). The sense strand (101) may further comprise a galactosamine (106). The antisense strand (102) may comprise a second oligonucleotide sequence (104). The second oligonucleotide sequence (104) may comprise one or more phosphorothioate internucleoside bonds (109). The phosphorothioate internucleoside bonds (109) may be located between nucleotides at the 5′ or 3′ end of the second oligonucleotide sequence (104). The phosphorothioate internucleoside bonds (109) may be between the first three nucleotides from the 5′ end of the second oligonucleotide sequence (104). The phosphorothioate internucleoside bonds (109) may be between the first three nucleotides from the 3′ end of the second oligonucleotide sequence (104). The second oligonucleotide sequence (104) may comprise 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 additionally, 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 length of the sense strand (101) may be the same as the length of the antisense strand (102). The sense strand (101) may be longer than the antisense strand (102).

[0190] Figure 2 An exemplary siNA molecule of the present disclosure is shown in FIG. Figure 2As shown, an exemplary siNA molecule comprises a sense strand (201) and an antisense strand (202). The sense strand (201) may comprise a first oligonucleotide sequence (203). The first oligonucleotide sequence (203) may comprise one or more phosphorothioate internucleoside bonds (209). The phosphorothioate internucleoside bond (209) may be located between nucleotides at the 5′ or 3′ end of the first oligonucleotide sequence (203). The phosphorothioate internucleoside bond (209) may be between the first three nucleotides from the 5′ end of the first oligonucleotide sequence (203). The first oligonucleotide sequence (203) may comprise one or more 2′-fluoro nucleotides (210). The first oligonucleotide sequence (203) may comprise one or more 2′-O-methyl nucleotides (211). The first oligonucleotide sequence (203) may comprise 15 or more modified nucleotides independently selected from 2′-fluoro nucleotides (210) and 2′-O-methyl nucleotides (211). The sense strand (201) may further comprise a phosphorylation blocker (205). The sense strand (201) may further comprise a galactosamine (206). The antisense strand (202) may comprise a second oligonucleotide sequence (204). The second oligonucleotide sequence (204) may comprise one or more phosphorothioate internucleoside bonds (209). The phosphorothioate internucleoside bond (209) may be located between nucleotides at the 5′ or 3′ end of the second oligonucleotide sequence (204). The phosphorothioate internucleoside bond (209) may be between the first three nucleotides from the 5′ end of the second oligonucleotide sequence (204). The phosphorothioate internucleoside bond (209) may be between the first three nucleotides from the 3′ end of the second oligonucleotide sequence (204). The second oligonucleotide sequence (204) may comprise 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 comprising a modified nucleobase). The overhang (208) may comprise one or more modified ribonucleotides. The sense strand (201) may be shorter than the antisense strand (202). The length of the sense strand (201) may be the same as the length of the antisense strand (202). The sense strand (201) may be longer than the antisense strand (202).

[0191] Figures 3A-3J , 4A-4AB, 5A-F, 6A-K, 7A-D, and 8A-B depict exemplary ds-siNA modification patterns. Figures 3A-3J As shown, an exemplary ds-siNA molecule can have the following formula:

[0192] 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′

[0193] 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 9A q 10 B q 11 A q 12 -5′

[0194] in:

[0195] The top strand is the sense strand, the 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 comprises 15 to 30 nucleotides;

[0196] the bottom strand is the antisense strand, the antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the RNA corresponding to the target gene, wherein the second nucleotide sequence comprises 15 to 30 nucleotides;

[0197] Each A is independently a 2′-O-methyl nucleotide or a nucleotide comprising a 5′ stabilizing endcap or a phosphorylation blocker;

[0198] B is a 2′-fluoro nucleotide;

[0199] C represents a protruding nucleotide and is a 2′-O-methyl nucleotide, a deoxynucleotide, or uracil;

[0200] n 1 = 1-6 nucleotides in length;

[0201] Each n 2 、n 6 、n 8 ,q 3 ,q 5 ,q 7 ,q 9 ,q 11 and q 12 are independently 0-1 nucleotides in length;

[0202] Each n 3 and n 4 are independently 1-3 nucleotides in length;

[0203] n 5 The length is 1-10 nucleotides;

[0204] n 7 The length is 0-4 nucleotides;

[0205] Each n 9 ,q 1 and q 2are independently 0-2 nucleotides in length;

[0206] q 4 The length is 0-3 nucleotides;

[0207] q 6 The length is 0-5 nucleotides;

[0208] q 8 is 2-7 nucleotides in length; and

[0209] q 10 The length of the nucleotide sequence is approximately 2-11 nucleotides.

[0210] ds-siNA may further comprise a conjugate moiety. The conjugate moiety may comprise any galactamide disclosed herein. ds-siNA may further comprise (i) phosphorothioate internucleoside bonds between nucleotides at positions 1 and 2 and positions 2 and 3 from the 5′ end of the sense strand; and (ii) ds-siNA may further comprise a 5′-stable endcap. The 5′-stable endcap may be a vinylphosphonate. The 5′-stable 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 comprise a 5′-stable endcap. In some embodiments, the 2′-O-methyl nucleotide at position 1 from the 5′ end of the antisense strand is further modified to comprise a 5′-stable endcap. In some embodiments, the 2′-O-methyl nucleotide at position 1 from the 5′ end of the sense strand is further modified to comprise 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 comprise 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 can have the following formula:

[0211] 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′

[0212] 3′-C2A 0-2 B 0-1 A 0-3 B 0-1 A 0-5 B 0-1 A2-7 B1A 2-11 B1A1-5′

[0213] in:

[0214] The top strand is the sense strand, the 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 comprises 15 to 30 nucleotides;

[0215] the bottom strand is the antisense strand, the antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the RNA corresponding to the target gene, wherein the second nucleotide sequence comprises 15 to 30 nucleotides;

[0216] Each A is independently a 2′-O-methyl nucleotide or a nucleotide comprising a 5′ stabilizing endcap or a phosphorylation blocker;

[0217] B is a 2′-fluoro nucleotide;

[0218] C represents a protruding nucleotide and is a 2'-O-methyl nucleotide, a deoxynucleotide, or uracil.

[0219] The ds-siNA may further comprise a conjugation moiety. The conjugation moiety may comprise any galactamide disclosed herein. The ds-siNA may further comprise (i) phosphorothioate internucleoside linkages between the 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′-stable endcap. The 5′-stable endcap may be a vinylphosphonate. The vinylphosphonate may be a deuterated vinylphosphonate. The deuterated vinylphosphonate may be a monodeuterated vinylphosphonate. The deuterated vinylphosphonate may be a mono-di-deuterated vinylphosphonate. The 5′-stable endcap may be attached to the 5′ end of the antisense strand. The 5′-stable endcap may be attached to the 3′ end of the antisense strand. The 5′-stable endcap may be attached to the 5′ end of the sense strand. The 5′-stable 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 include a 5' stable end cap. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the antisense strand is further modified to include a 5' stable end cap. 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.

[0220] Figures 3A-3JThe exemplary ds-siNA shown in Figures 4A-4AB, 5A-F, 6A-K, 7A-D, and 8A-B comprises (i) a sense strand comprising 19-21 nucleotides; and (ii) an antisense strand comprising 21-23 nucleotides. The ds-siNA may optionally further comprise (iii) a conjugate moiety, wherein the conjugate moiety (e.g., GalNAc, designated as G3 in the Figures) is attached to the 3' end or the 5' end of the sense or antisense strand. The ds-siNA may comprise a two-nucleotide overhang consisting of nucleotides at positions 20 and 21 from the 5' end of the antisense strand. The ds-siNA may comprise a two-nucleotide overhang consisting of nucleotides at positions 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 methylsulfonyl phosphoramidate internucleoside linkages (Ms). At least one phosphorothioate internucleoside linkage or methylsulfonyl phosphoramidate internucleoside linkage (Ms) may be between nucleotides at positions 1 and 2 or positions 2 and 3 from the 5' end of the sense strand. At least one phosphorothioate internucleoside linkage or methylsulfonyl phosphoramidate internucleoside linkage (Ms) may be between nucleotides at positions 1 and 2 or positions 2 and 3 from the 5' end of the antisense strand. At least one phosphorothioate internucleoside linkage or methylsulfonyl phosphoramidate internucleoside linkage (Ms) may be between nucleotides at positions 19 and 20, positions 20 and 21, positions 21 and 22, or positions 22 and 23 from the 5' end of the antisense strand. Figures 3A-3J As shown in , 4A-4AB, 5A-F, 6A-K, 7A-D and 8A-B, 4-6 nucleotides in the sense strand may be 2'-fluoro nucleotides. Figures 3A-3J As shown in , 4A-4AB, 5A-F, 6A-K, 7A-D and 8A-B, 2-5 nucleotides in the antisense strand may be 2'-fluoro nucleotides. Figures 3A-3J , 4A-4D, 4P, 4R-4AB, 5A-F, 6A-K, 7A-D and 8A-B, 13-15 nucleotides in the sense strand may be 2'-O-methyl nucleotides. Figures 3A-3J As shown in , 4E, 4F, 4O, 4R-X, 5A-F, 6A-K, 7A-D and 8A-B, 14-19 nucleotides in the antisense strand may be 2'-O-methyl nucleotides. Figure 4E and 4G As shown in -4J, up to 8 nucleotides (i.e., 1, 2, 3, 4, 5, 6, 7, 8) in the sense strand can be 2'-O-cyclopropane (2'-ocp). Figure 4A 、 4BAs shown in 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, 11) in the antisense strand can be 2'ocp. Figure 4F 、 4K As shown in -4N and 4Q, 9-15 nucleotides in the sense strand can be 2'-OMe-cyclopropane (2'-omcp). Figure 4J 、 4M , 4N, 4P, 4Q, 4T, 4U, 4W, 4Z, 4AB and 5C, 1-15 nucleotides in the sense strand can be 2'omcp. Figure 5A As shown in -C, E and F, position 1 from the 5' end of the antisense strand can be vmX. Figures 6A-6G As shown, one or both nucleotides in the antisense strand may be xylo nucleotides, i.e., 2′-OMe-3′-xylo or 2′-F-3′-xylo nucleotides. Figure 7A As shown in -D, one nucleotide in the antisense strand can be modified with Ganciclovir or Denavir. Figure 8A As shown in -B, one nucleotide in the antisense strand can be 3′-ocp. Figures 3A-3J , 4A-4AB, 5A-F, 6A-K, 7A-D, and 8A-B, the ds-siNA does not contain a base pair between the 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' stable 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' stable 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.

[0221] The ds-siNA may comprise: a sense strand consisting of 19 nucleotides wherein 2′-fluoro nucleotides are at positions 3, 7-9, 12, and 17 from the 5′ end of the sense strand, and wherein 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 ( Figure 3A ); a sense strand consisting of 19 nucleotides, wherein 2′-fluoro nucleotides are at positions 3, 7, 8, and 17 from the 5′ end of the sense strand, and wherein 2′-O-methyl nucleotides are at positions 1, 2, 4-6, 9-16, 18, and 19 from the 5′ end of the sense strand ( Figure 3B ); a sense strand consisting of 19 nucleotides, wherein 2′-fluoro nucleotides are at positions 3, 7-9, 12, and 17 from the 5′ end of the sense strand, and wherein 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 ( Figure 3C ); a sense strand consisting of 19 nucleotides, wherein 2′-fluoro nucleotides are at positions 5 and 7-9 from the 5′ end of the sense strand, and wherein 2′-O-methyl nucleotides are at positions 1-4, 6, and 10-19 from the 5′ end of the sense strand ( Figure 3D -F); a sense strand consisting of 21 nucleotides wherein 2′-fluoro nucleotides are at positions 5, 9-11, 14, and 19 from the 5′ end of the sense strand, and wherein 2′-O-methyl nucleotides are at positions 1-4, 6-8, 12, 13, 15-18, 20, and 21 from the 5′ end of the sense strand ( Figure 3G ); a sense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 7 and 9-11 from the 5′ end of the sense strand, and wherein 2′-O-methyl nucleotides are at positions 1-6, 8, and 12-21 from the 5′ end of the sense strand ( Figure 3H ); a sense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 3, 7-9, 12, and 17 from the 5′ end of the sense strand, and wherein 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 ( Figure 3I ); and a sense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 3, 7-9, 12, and 17 from the 5′ end of the sense strand, and wherein 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 ( Figure 3J ).

[0222] The ds-siNA may comprise an antisense strand consisting of 21 nucleotides, wherein the nucleotides at positions 2 and 14 from the 5' end of the antisense strand are 2'-fluoro nucleotides; and wherein the nucleotides at positions 1, 3-13, and 15-21 are 2'-O-methyl nucleotides ( Figure 3A and B); an antisense strand consisting of 21 nucleotides, wherein the nucleotides in the antisense strand contain an alternating 1:3 modification pattern, wherein one nucleotide is a 2′-fluoro nucleotide and three nucleotides are 2′-O-methyl nucleotides (Figures C and D); an antisense strand consisting of 21 nucleotides, wherein the nucleotides in the antisense strand contain an alternating 1:2 modification pattern, wherein one nucleotide is a 2′-fluoro nucleotide and two nucleotides are 2′-O-methyl nucleotides ( Figure 3E ); an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand, and wherein 2′-O-methyl nucleotides are at positions 1, 3-5, 7-13, 15, and 17-21 from the 5′ end ( Figure 3F ); an antisense strand consisting of 23 nucleotides, wherein 2′-fluoro nucleotides are at positions 2 and 14 from the 5′ end of the antisense strand, and wherein 2′-O-methyl nucleotides are at positions 1, 3-13, and 15-23 from the 5′ end of the antisense strand ( Figure 3G ); an antisense strand consisting of 23 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand, and wherein 2′-O-methyl nucleotides are at positions 1, 3-5, 7-13, 15, and 17-23 from the 5′ end of the antisense strand ( Figure 3H ); an antisense strand consisting of 23 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 7, and 14 from the 5′ end of the antisense strand, and wherein 2′-O-methyl nucleotides are at positions 1, 3-6, 8-13, and 15-23 from the 5′ end of the antisense strand ( Figure 3I ); and an antisense strand consisting of 23 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 14, and 16 from the 5′ end of the antisense strand, and wherein 2′-O-methyl nucleotides are at positions 1, 3-13, 15, and 17-23 from the 5′ end of the antisense strand ( Figure 3J ).

[0223] like Figure 3A As shown in -G, I and J, ds-siNA may further comprise a conjugate moiety attached to the 3' end of the sense strand. Figure 3AAs shown in Figures AF, I, and J, the ds-siNA may further comprise phosphorothioate internucleoside linkages between the nucleotides at positions 1 and 2 and positions 2 and 3 from the 5′ end of the sense strand. As shown in Figures AF, I, and J, the ds-siNA may further comprise phosphorothioate internucleoside linkages between the nucleotides at the following positions from the 5′ end of the antisense strand: positions 1 and 2; positions 2 and 3; positions 19 and 20; and positions 20 and 21. As shown in Figures G and H, the ds-siNA may further comprise phosphorothioate internucleoside linkages between the nucleotides at the following positions from the 5′ end of the antisense strand: positions 1 and 2; positions 2 and 3; positions 21 and 22; and positions 22 and 23.

[0224] Optionally, at a distance Figure 3G The nucleotides at positions 22 and 23 of the 5′ end of the antisense strand of the ds-siNA may be unlocked nucleotides. The ds-siNA may optionally contain a vinylphosphonate ( Figure 3H ), but in some embodiments, the 5' end caps disclosed herein may also be suitable.

[0225] In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the sense strand is further modified to include a 5' stable end cap. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the antisense strand is further modified to include a 5' stable end cap. 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.

[0226] The ds-siNA may comprise: a sense strand consisting of 19 nucleotides, wherein 2′-fluoro nucleotides are at positions 5 and 7-9 from the 5′ end of the sense strand, and wherein 2′-O-methyl nucleotides are at positions 1-4, 6, and 10-19 from the 5′ end of the sense strand ( Figure 4A -D, P, and R-AB); a sense strand composed of 19 nucleotides with 2′-fluoro nucleotides at positions 5 and 7-9 from the 5′ end of the sense strand, 2′-O-methyl nucleotides at positions 2, 4, 11, 13, 15, 17, and 19 from the 5′ end of the sense strand, and with 2′-ocp nucleotides at positions 1, 3, 6, 10, 12, 14, 16, and 18 from the 5′ end of the sense strand ( Figure 4E, GJ); a sense strand consisting of 19 nucleotides, wherein 2′-fluoro nucleotides are at positions 5 and 7-9 from the 5′ end of the sense strand, 2′-O-methyl nucleotides are at positions 2, 4, 11, 13, 15, and 17 from the 5′ end of the sense strand, and wherein 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 KN); or a sense strand consisting of 19 nucleotides, wherein 2′-fluoro nucleotides are at positions 5 and 7-9 from the 5′ end of the sense strand, and wherein 2′-omcp nucleotides are at positions 1-4, 6, and 10-19 from the 5′ end of the sense strand ( Figure 4O and Q).

[0227] The ds-siNA may comprise an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 5, 8, 14, and 17 from the 5′ end of the antisense strand, 2′-O-methyl nucleotides are at positions 3, 4, 6, 9, 11, 13, 16, 19, and 21 from the 5′ end of the antisense strand, and wherein 2′-ocp nucleotides are at positions 1, 7, 10, 12, 15, 18, and 20 from the 5′ end of the antisense strand ( Figure 4A , G, and K); an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 5, 8, 14, and 17 from the 5′ end of the antisense strand, 2′-O-methyl nucleotides are at positions 1, 3, 4, 6, 7, 9, 11, 13, 16, 19, and 21 from the 5′ end of the antisense strand, and wherein 2′-ocp nucleotides are at positions 10, 12, 15, 18, and 20 from the 5′ end of the antisense strand ( Figure 4B , H, and L); an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 5, 8, 14, and 17 from the 5′ end of the antisense strand, 2′-O-methyl nucleotides are at positions 3, 6, 9, 11, 13, 16, 19, and 21 from the 5′ end of the antisense strand, and wherein 2′-omcp nucleotides are at positions 1, 4, 7, 10, 12, 15, 18, and 20 from the 5′ end of the antisense strand ( Figure 4C , I and M); an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 5, 8, 14 and 17 from the 5′ end of the antisense strand, 2′-O-methyl nucleotides are at positions 1, 3, 4, 6, 7, 11, 13, 16, 19 and 21 from the 5′ end of the antisense strand, and wherein 2′-omcp nucleotides are at positions 9, 10, 12, 15, 18 and 20 from the 5′ end of the antisense strand ( Figure 4Dand J); an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 5, 8, 14, and 17 from the 5′ end of the antisense strand, and wherein 2′-O-methyl nucleotides are 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, wherein 2′-fluoro nucleotides are at positions 2, 5, 8, 14, and 17 from the 5′ end of the antisense strand, 2′-O-methyl nucleotides are at positions 1, 3, 4, 6, 7, 11, 13, 16, 19, and 21 from the 5′ end of the antisense strand, and wherein 2′-omcp nucleotides are at positions 9, 10, 12, 15, 18, and 20 from the 5′ end of the antisense strand ( Figure 4N ); an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 5, 8, 14, and 17 from the 5′ end of the antisense strand, and wherein 2′-omcp nucleotides are at positions 1, 3, 4, 6, 7, 9-12, 15, 16, and 18-21 from the 5′ end of the antisense strand ( Figure 4P and Q); an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 1, 3-5, 7-13, 15, and 17-20 from the 5′ end of the antisense strand, and wherein a 2′-ocp nucleotide is at position 21 from the 5′ end of the antisense strand ( Figure 4R ); an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 3-5, 7-13, 15, and 17-21 from the 5′ end of the antisense strand, and wherein a 2′-ocp nucleotide is at position 1 from the 5′ end of the antisense strand ( Figure 4S ); an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 1, 3-5, 7-13, 15, and 17-20 from the 5′ end of the antisense strand, and wherein a 2′-omcp nucleotide is at position 21 from the 5′ end of the antisense strand ( Figure 4T ); an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 3-5, 7-13, 15, and 17-21 from the 5′ end of the antisense strand, and wherein a 2′-omcp nucleotide is at position 1 from the 5′ end of the antisense strand ( Figure 4U); an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 3-5, 7-13, 15, and 17-20 from the 5′ end of the antisense strand, and wherein 2′-ocp nucleotides are at positions 1 and 21 from the 5′ end of the antisense strand ( Figure 4V ); an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 3-5, 7-13, 15, and 17-20 from the 5′ end of the antisense strand, and wherein 2′-omcp nucleotides are at positions 1 and 21 from the 5′ end of the antisense strand ( Figure 4W ); an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 3-5, 7-13, 15, and 17-19 from the 5′ end of the antisense strand, and wherein 2′-ocp nucleotides are at positions 1, 20, and 21 from the 5′ end of the antisense strand ( Figure 4X ); an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 1, 3-5, 7, 8, 10, 12, 18, and 20 from the 5′ end of the antisense strand, and wherein 2′-ocp nucleotides are at positions 9, 11, 13, 15, 17, 19, and 21 from the 5′ end of the antisense strand ( Figure 4Y ); an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 1, 3-5, 7, 8, 10, 12, 18, and 20 from the 5′ end of the antisense strand, and wherein 2′-omcp nucleotides are at positions 9, 11, 13, 15, 17, 19, and 21 from the 5′ end of the antisense strand ( Figure 4Z ); an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 4, 8, 10, 12, 18, and 20 from the 5′ end of the antisense strand, and wherein 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 ( Figure 4AA); or an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 4, 8, 10, 12, 18, and 20 from the 5′ end of the antisense strand, and wherein 2′-omcp nucleotides are at positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, and 21 from the 5′ end of the antisense strand ( Figure 4AB ).

[0228] Optionally, the ds-siNA may further comprise a conjugate 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, positions 2 and 3, and positions 20 and 21 from the 5′ end of the sense strand; and (ii) phosphorothioate internucleoside linkages between nucleotides at the following positions from the 5′ end of the antisense strand: 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. In some embodiments, the 2′-O-methyl nucleotide at position 1 from the 5′ end of the sense strand is further modified to comprise 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 comprise 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'-ocp 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.

[0229] The ds-siNA may comprise: a sense strand consisting of 19 nucleotides, wherein 2′-fluoro nucleotides are at positions 5 and 7-9 from the 5′ end of the sense strand, and wherein 2′-O-methyl nucleotides are at positions 1-4, 6, and 10-19 from the 5′ end of the sense strand ( Figure 5A -C, E, and F); or 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 with 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 ( Figure 5D ).

[0230] The ds-siNA may comprise: an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 5, 8, 14, and 17 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 3, 4, 6, 7, 9-13, 15, 16, and 18-21 from the 5′ end of the antisense strand, and wherein a vmX nucleotide is at position 1 ( Figure 5A , E, and F); an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 5, 8, 14, and 17 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 3, 4, 6, 7, 9-13, 15, 16, and 18-20 from the 5′ end of the antisense strand, wherein a 2′-ocp nucleotide is at position 21 from the 5′ end of the antisense strand, and wherein a vmX nucleotide is at position 1 from the 5′ end of the antisense strand ( 5b); an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 5, 8, 14, and 17 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 3, 4, 6, 7, 9-13, 15, 16, and 18-20 from the 5′ end of the antisense strand, wherein a 2′-omcp nucleotide is at position 21 from the 5′ end of the antisense strand, and wherein a vmX nucleotide is at position 1 from the 5′ end of the antisense strand ( Figure 5C ); or an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 7, and 14 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 1, 3-6, 8-13, and 15-20 from the 5′ end of the antisense strand, and wherein a 2′-ocp nucleotide is at position 21 from the 5′ end of the antisense strand ( Figure 5D ).

[0231] Optionally, the ds-siNA may further comprise a conjugate 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, positions 2 and 3, and positions 20 and 21 from the 5' end of the sense strand; and (ii) phosphorothioate internucleoside linkages between nucleotides at the following positions from the 5' end of the antisense strand: positions 1 and 2; positions 2 and 3; positions 19 and 20; and positions 20 and 21. In some embodiments, the phosphorothioate internucleoside linkage may be the S enantiomer. In some embodiments, the phosphorothioate internucleoside linkage may be the R enantiomer.

[0232] In some embodiments, the 2′-O-methyl nucleotide at position 1 from the 5′ end of the sense strand is further modified to include a 5′ stable end cap. In some embodiments, the vmX nucleotide at position 1 from the 5′ end of the antisense strand is further modified to include a 5′ stable end cap. 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.

[0233] The ds-siNA may comprise: a sense strand consisting of 19 nucleotides wherein 2′-fluoro nucleotides are at positions 3, 7-9, 12, and 17 from the 5′ end of the sense strand, and wherein 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 ( Figure 6A -G); or a sense strand consisting of 19 nucleotides, wherein 2'-fluoro nucleotides are at positions 5 and 7-9, 12 from the 5' end of the sense strand, and wherein 2'-O-methyl nucleotides are at positions 1-4, 6, and 10-19 from the 5' end of the sense strand ( Figure 6H -K).

[0234] The ds-siNA may comprise: an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 7, and 14 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 1, 3, 4, 6, 8-13, and 15-21 from the 5′ end of the antisense strand, and wherein a xylo nucleotide is at position 5 ( Figure 6A ); an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 7, and 14 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 1, 3-5, 8-13, and 15-21 from the 5′ end of the antisense strand, and wherein a xylo nucleotide is at position 6 from the 5′ end of the antisense strand ( Figure 6B ); an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2 and 14 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 1, 3-6, 8-13, and 15-21 from the 5′ end of the antisense strand, and wherein a xylo nucleotide is at position 7 from the 5′ end of the antisense strand ( Figure 6C); an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 7, and 14 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 1, 3-6, 9-13, and 15-21 from the 5′ end of the antisense strand, and wherein a xylo nucleotide is at position 8 from the 5′ end of the antisense strand ( Figure 6D ); an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 7, and 14 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 1, 3-6, 8-13, 15-19, and 21 from the 5′ end of the antisense strand, and wherein a xylo nucleotide is at position 20 from the 5′ end of the antisense strand ( Figure 6E ); an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 7, and 14 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 1, 3-5, 8-13, 15-19, and 21 from the 5′ end of the antisense strand, and wherein a xylo nucleotide is at position 6 from the 5′ end of the antisense strand ( Figure 6F ); an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 7, and 14 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 1, 3-6, 9-13, 15-19, and 21 from the 5′ end of the antisense strand, and wherein xylo nucleotides are at positions 8 and 20 from the 5′ end of the antisense strand ( Figure 6G ); an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 1, 3-5, 7-13, 15, and 17-20 from the 5′ end of the antisense strand, and wherein a xylo nucleotide is at position 21 from the 5′ end of the antisense strand ( Figure 6H ); an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 3-5, 7-13, 15, and 17-21 from the 5′ end of the antisense strand, and wherein a xylo nucleotide is at position 1 from the 5′ end of the antisense strand ( Figure 6I ); an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 3-5, 7-13, 15, and 17-20 from the 5′ end of the antisense strand, and wherein xylo nucleotides are at positions 1 and 21 from the 5′ end of the antisense strand ( Figure 6J); or an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 1, 3-5, 7-13, 15, 17-19, and 21 from the 5′ end of the antisense strand, and wherein a xylo nucleotide is at position 20 from the 5′ end of the antisense strand ( Figure 6K ).

[0235] In some embodiments, the xylo nucleotides may be 2′-OMe-3′-xylo nucleotides. In some embodiments, the xylo nucleotides may be 2′-F-3′-xylo nucleotides. Optionally, the ds-siNA may further comprise a conjugate moiety attached to the 3′ end of the sense strand. The ds-siNA may further comprise (i) phosphorothioate internucleoside linkages between the nucleotides at positions 1 and 2, positions 2 and 3, and positions 20 and 21 from the 5′ end of the sense strand; and (ii) phosphorothioate internucleoside linkages between the nucleotides at the following positions from the 5′ end of the antisense strand: 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. In some embodiments, the 2′-O-methyl nucleotide at position 1 from the 5′ end of the sense strand is further modified to comprise a 5′ stable endcap. In some embodiments, the 2′-O-methyl nucleotide at position 1 from the 5′ end of the antisense strand is further modified to comprise a 5′ stable 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.

[0236] The ds-siNA may comprise: a sense strand consisting of 19 nucleotides wherein 2′-fluoro nucleotides are at positions 3, 7-9, 12, and 17 from the 5′ end of the sense strand, and wherein 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 ( Figure 7A -D).

[0237] The ds-siNA may comprise an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 7, and 14 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 1, 3-5, 8-13, and 15-21 from the 5′ end of the antisense strand, and an acyclic ganciclovir nucleotide analog is at position 6 ( Figure 7A ); an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 7, and 14 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 1, 3-5, 8-13, and 15-21 from the 5′ end of the antisense strand, and wherein a denavir nucleotide analog is at position 6 from the 5′ end of the antisense strand ( Figure 7B ); an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 7, and 14 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 1, 3-6, 9-13, and 15-21 from the 5′ end of the antisense strand, and wherein a ganciclovir nucleotide is at position 8 from the 5′ end of the antisense strand ( Figure 7C ); or an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 7, and 14 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 1, 3-6, 9-13, and 15-21 from the 5′ end of the antisense strand, and wherein a denavir nucleotide is at position 8 from the 5′ end of the antisense strand ( Figure 7D ).

[0238] In some embodiments, the acyclic ganciclovir nucleotide analog is the S enantiomer. In some embodiments, the acyclic ganciclovir nucleotide analog is the R enantiomer. In some embodiments, the denavir nucleotide is the S enantiomer. In some embodiments, the denavir nucleotide is the R enantiomer.

[0239] Optionally, the ds-siNA may further comprise a conjugate 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, positions 2 and 3, and positions 20 and 21 from the 5′ end of the sense strand; and (ii) phosphorothioate internucleoside linkages between nucleotides at the following positions from the 5′ end of the antisense strand: 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. In some embodiments, the 2′-O-methyl nucleotide at position 1 from the 5′ end of the sense strand is further modified to include a 5′ stable endcap. In some embodiments, the 2′-O-methyl nucleotide at position 1 from the 5′ end of the antisense strand is further modified to include a 5′ stable 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.

[0240] The ds-siNA may comprise: a sense strand consisting of 19 nucleotides wherein 2′-fluoro nucleotides are at positions 3, 7-9, 12, and 17 from the 5′ end of the sense strand, and wherein 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 ( Figure 8A and B).

[0241] The ds-siNA may comprise: an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 7, and 14 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 1, 3-5, 8-13, and 15-21 from the 5′ end of the antisense strand, and wherein a denavir 3′-ocp nucleotide is at position 6 from the 5′ end of the antisense strand. Figure 8A ); or an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 7, and 14 from the 5′ end of the antisense strand, wherein 2′-O-methyl nucleotides are at positions 1, 3-6, 9-13, and 15-21 from the 5′ end of the antisense strand, and wherein a 3′-ocp nucleotide is at position 8 from the 5′ end of the antisense strand ( Figure 8B ).

[0242] Optionally, the ds-siNA may further comprise a conjugate 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, positions 2 and 3, and positions 20 and 21 from the 5′ end of the sense strand; and (ii) phosphorothioate internucleoside linkages between nucleotides at the following positions from the 5′ end of the antisense strand: 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. In some embodiments, the 2′-O-methyl nucleotide at position 1 from the 5′ end of the sense strand is further modified to include a 5′ stable endcap. In some embodiments, the 2′-O-methyl nucleotide at position 1 from the 5′ end of the antisense strand is further modified to include a 5′ stable 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.

[0243] 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 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 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 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., 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 1, 2, 3, 4, or more 2'-fluoro nucleotides on the sense strand or antisense strand are 2'-fluoro nucleotide mimetics. In some embodiments, at least 1, 2, 3, 4, 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 on 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 strand or antisense strand are 2'-O-methyl nucleotide mimetics. In some embodiments, at least 1, 2, 3, 4 or more nucleotides on the sense strand 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, wherein the bond between the 3' position and the 4' position 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 comprise one or more nucleotide analogs (e.g., An1 and An2).

[0244] siNA sense strand

[0245] Any of the siNA molecules described herein can comprise a sense strand. The sense strand can comprise a first nucleotide sequence. The length of the first nucleotide sequence can be 15 to 30, 15 to 25, 15 to 23, 17 to 23, 19 to 23, or 19 to 21 nucleotides. In some embodiments, the length of the first nucleotide sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the length of the first nucleotide sequence is at least 19 nucleotides. In some embodiments, the length of the first nucleotide sequence is at least 21 nucleotides.

[0246] In some embodiments, the length of the sense strand is the same as the length of the first nucleotide sequence. In some embodiments, the sense strand is longer than the first nucleotide sequence. In some embodiments, compared with the first nucleotide sequence, the sense strand may further include 1, 2, 3, 4 or 5 or more nucleotides. In some embodiments, the sense strand may further include deoxyribonucleic acid (DNA). In some embodiments, DNA is thymine (T). In some embodiments, the sense strand may further include a TT sequence. In some embodiments, the sense strand may further include 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 one 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 core bases).

[0247] 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′-xylo nucleotides, 2′-F-3′-xylo nucleotides, 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 the group consisting of 2′-O-methyl nucleotides, 2′-fluoro nucleotides, 2′-ocp nucleotides, 2′-omcp nucleotides, 3′-ocp nucleotides, 2′-OMe-3′-xylo nucleotides, 2′-F-3′-xylo nucleotides, 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 the group consisting of 2′-O-methyl nucleotides, 2′-fluoro nucleotides, 2′-ocp nucleotides, 2′-omcp nucleotides, 3′-ocp nucleotides, 2′-OMe-3′-xylo nucleotides, 2′-F-3′-xylo nucleotides, vmX nucleotides, ganciclovir nucleotides, and denavir nucleotides. In some embodiments, the 2′-O-methyl nucleotides are 2′-O-methyl nucleotide mimetics. In some embodiments, the 2'-fluoro nucleotide is a 2'-fluoro nucleotide mimetic.

[0248] In some embodiments, between 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 of the modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, one or no modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, between about 2 and 20 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, between about 5 and 25 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, between about 10 and 25 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, between about 12 and 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, less than or equal to 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, less than or equal to 21 modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, less than or equal to 20 modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, less than or equal to 19 modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, less than or equal to 18 modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, less than or equal to 17 modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, less than or equal to 16 modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, less than or equal to 15 modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, less than or equal to 14 modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, less than or equal to 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-methylpyrimidine. 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-methylpurine. In some embodiments, at least 5, 6, 7, 8, 9, or 10 modified nucleotides of the first nucleotide sequence are 2'-O-methylpurines. In some embodiments, the 2'-O-methyl nucleotides are 2'-O-methyl nucleotide mimics.

[0249] In some embodiments, between 2 and 15 modified nucleotides of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, between 2 and 10 modified nucleotides of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, between 2 and 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'-O-fluoro nucleotides. In some embodiments, at least 1 modified nucleotide of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, at least 2 modified nucleotides of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least 3 modified nucleotides of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least 4 modified nucleotides of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least 5 modified nucleotides of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least 6 modified nucleotides of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 10, 9, 8, 7, 6, 5, 4, 3 or fewer modified nucleotides of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 10 or fewer modified nucleotides of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 7 or fewer modified nucleotides of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 6 or fewer modified nucleotides of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 5 or fewer modified nucleotides of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 4 or fewer modified nucleotides of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 3 or fewer modified nucleotides of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 2 or fewer 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'-fluoropyrimidine. In some embodiments, one, two, three, four, five, or six 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'-fluoropurine. In some embodiments, one, two, three, four, five, or six modified nucleotides of the first nucleotide sequence are 2'-O-methylpurines. In some embodiments, the 2'-fluoro nucleotide is a 2'-fluoro nucleotide mimetic.

[0250] 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 mimetic.

[0251] 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, 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, 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 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 position 3 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotides at position 5 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotides at position 7 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotides at position 8 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotides at position 9 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotides at position 10 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotides at position 11 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotides at position 12 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides. 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 position 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 position 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 position 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 position 5, 7, 8 and / or 9 from the 5 ' end of the first nucleotide sequence is a 2'-fluoro nucleotide.In some embodiments, the nucleotide at position 5, 9, 10, 11, 12, and / or 19 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 mimetic.

[0252] In some embodiments, the 2'-fluoro or 2'-O-methyl nucleoside mimetic is a nucleoside mimetic of Formula (V): where R x are independently nucleobase, aryl, heteroaryl or H, Q 1 and Q 2 independently S or O, 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.

[0253] In some embodiments, the 2'-fluoro or 2'-O-methyl nucleotide mimetic is a nucleotide mimetic of formula (16) to formula (20):

[0254] where R x are independently nucleobase, arylheteroaryl or H, and R 2 is F or -OCH3. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0255] In some embodiments, the sense strand, the antisense strand, or both may each independently comprise at least 1, at least 2, at least 3, at least 4, or at least 5 or more modified nucleotides and / or nucleotide analogs having the following chemical structure: (An1; where * indicates a chiral center), (An2; where * indicates a chiral center), wherein B is a nucleobase, an aryl group, a heteroaryl group, or H, and wherein represents a phosphodiester bond, a phosphorothioate bond, a methylsulfonylphosphoramidate bond, or H. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0256] In some embodiments, the sense strand, the antisense strand, or both may each further independently comprise at least 1, at least 2, at least 3, at least 4, or at least 5 or more modified nucleotides having the following chemical structure: where R x is a nucleobase, an aryl group, a heteroaryl group, or H, and R y is a nucleobase, and wherein represents a phosphodiester bond, a phosphorothioate bond, a methylsulfonylphosphoramidate bond, or H. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0257] In some embodiments, the sense strand, the antisense strand, or both may each further independently comprise at least 1, at least 2, at least 3, at least 4, or at least 5 or more modified nucleotides having the following chemical structure: where R y is a nucleobase, and wherein represents a phosphodiester bond, a phosphorothioate bond, a methylsulfonylphosphoramidate bond, or H. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0258] For the purposes of this disclosure, the modified nucleotide or nucleotide analog can be at any position of the sense strand. In some embodiments, the modified nucleotide or nucleotide analog can be at position 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.

[0259] 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.

[0260] In some embodiments, the sense strand may further comprise one or more internucleoside bonds independently selected from the group consisting of phosphodiester (PO) internucleoside bonds, phosphorothioate (PS) internucleoside bonds, methylsulfonylphosphoramidate internucleoside bonds (Ms), phosphorodithioate internucleoside bonds, and PS-mimetic internucleoside bonds. In some embodiments, the PS-mimetic internucleoside bond is a sulfonyl internucleoside bond.

[0261] In certain 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 bonds. In certain 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 less phosphorothioate internucleoside bonds. In certain embodiments, the sense strand comprises 2 to 10, 2 to 8, 2 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 to 2 phosphorothioate internucleoside bonds. In certain embodiments, the sense strand comprises 1 to 2 phosphorothioate internucleoside bonds. In certain embodiments, the sense strand comprises 2 to 4 phosphorothioate internucleoside bonds. In certain embodiments, at least one phosphorothioate internucleoside bond is between the nucleotides at positions 1 and 2 of the 5' end of the first nucleotide sequence. In some embodiments, at least one phosphorothioate internucleoside linkage is between the nucleotides at positions 2 and 3 from the 5' end of the first nucleotide sequence. In some embodiments, the sense strand comprises two phosphorothioate internucleoside linkages between the nucleotides at positions 1 to 3 from the 5' end of the first nucleotide sequence.

[0262] In certain embodiments, sense strand can further include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 or more methylsulfonylphosphoramidate nucleoside interkey.In certain embodiments, sense strand includes 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 or less methylsulfonylphosphoramidate nucleoside interkey.In certain embodiments, sense strand includes 2 to 10, 2 to 8, 2 to 6, 1 to 5, 1 to 4, 1 to 3 or 1 to 2 methylsulfonylphosphoramidate nucleoside interkey.In certain embodiments, sense strand includes 1 to 2 methylsulfonylphosphoramidate nucleoside interkey.In certain embodiments, sense strand includes 2 to 4 methylsulfonylphosphoramidate nucleoside interkey.

[0263] In some embodiments, the sense strand can comprise any of the modified nucleotides disclosed below in the subsection entitled "Modified Nucleotides." In some embodiments, the sense strand can comprise a 5'-stable endcap, and the 5'-stable endcap can be selected from the endcaps disclosed below in the subsection entitled "5'-stable endcap."

[0264] siNA antisense strand

[0265] 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 to 30, 15 to 25, 15 to 23, 17 to 23, 19 to 23, or 19 to 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.

[0266] In certain embodiments, the length of the antisense strand is the same as the length of the second nucleotide sequence. In certain embodiments, the antisense strand is longer than the second nucleotide sequence. In certain embodiments, compared with the second nucleotide sequence, the antisense strand may further include 1, 2, 3, 4 or 5 or more nucleotides. In certain embodiments, the length of the antisense strand is the same as the length of the sense strand. In certain embodiments, the antisense strand is longer than the sense strand. In certain embodiments, the antisense strand may further include 1, 2, 3, 4 or 5 or more nucleotides compared with the sense strand. In certain embodiments, the antisense strand may further include deoxyribonucleic acid (DNA). In certain embodiments, DNA is thymine (T). In certain embodiments, the antisense strand may further include a TT sequence. In certain embodiments, the antisense strand may further include 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 one of the modified nucleotides disclosed herein (e.g., 2′-fluoro nucleotides, 2′-O-methyl nucleotides, 2′-fluoro nucleotide mimetics, 2′-O-methyl nucleotide mimetics, 2′-ocp nucleotides, 2′-omcp nucleotides, 3′-ocp nucleotides, 2′-OMe-3′-xylo nucleotides, 2′-F-3′-xylo nucleotides, vmX nucleotides, ganciclovir nucleotides, and denavir nucleotides, or a nucleotide comprising a modified nucleobase).

[0267] In some embodiments, the second nucleotide sequence comprises 15, 16, 17, 18, 19, 20, 21, 22, 23 or more modified nucleotides independently selected from the group consisting of 2′-O-methyl nucleotides, 2′-fluoro nucleotides, 2′-ocp nucleotides, and 2′-omcp nucleotides. In some embodiments, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the nucleotides in the second nucleotide sequence are modified nucleotides independently selected from the group consisting of 2′-O-methyl nucleotides, 2′-fluoro nucleotides, 2′-ocp nucleotides, 2′-omcp nucleotides, 3′-ocp nucleotides, 2′-OMe-3′-xylo nucleotides, 2′-F-3′-xylo nucleotides, 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 the following: 2′-O-methyl nucleotides, 2′-fluoro nucleotides, 2′-ocp nucleotides, 2′-omcp nucleotides, 3′-ocp nucleotides, 2′-OMe-3′-xylo nucleotides, 2′-F-3′-xylo nucleotides, vmX nucleotides, ganciclovir nucleotides, and denavir nucleotides.

[0268] In some embodiments, between 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 of the modified nucleotides of the second nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, one or no modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, between about 2 and 20 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, between about 5 and 25 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, between about 10 and 25 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, between about 12 and 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 of the modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 15 of the modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 16 of the modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 17 of the modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 18 of the modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 19 of the modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, less than or equal to 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 of the modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides.In some embodiments, less than or equal to 21 modified nucleotides of the second nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, less than or equal to 20 modified nucleotides of the second nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, less than or equal to 19 modified nucleotides of the second nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, less than or equal to 18 modified nucleotides of the second nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, less than or equal to 17 modified nucleotides of the second nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, less than or equal to 16 modified nucleotides of the second nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, less than or equal to 15 modified nucleotides of the second nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, less than or equal to 14 modified nucleotides of the second nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, less than or equal to 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-methylpyrimidine. In some embodiments, at least 5, 6, 7, 8, 9, or 10 of the 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-methylpurine. In some embodiments, at least 5, 6, 7, 8, 9, or 10 of the modified nucleotides of the second nucleotide sequence are 2'-O-methylpurines. In some embodiments, the 2'-O-methyl nucleotides are 2'-O-methyl nucleotide mimics.

[0269] In some embodiments, between 2 and 15 modified nucleotides of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, between 2 and 10 modified nucleotides of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, between 2 and 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'-O-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 of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 10, 9, 8, 7, 6, 5, 4, 3 or fewer modified nucleotides of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 10 or fewer modified nucleotides of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 7 or fewer modified nucleotides of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 6 or fewer modified nucleotides of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 5 or fewer modified nucleotides of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 4 or fewer modified nucleotides of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 3 or fewer modified nucleotides of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 2 or fewer modified nucleotides of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least one modified nucleotide of the second nucleotide sequence is a 2'-fluoropyrimidine. In some embodiments, 1, 2, 3, 4, 5 or 6 modified nucleotides of the second nucleotide sequence are 2'-O-methylpyrimidine. In some embodiments, at least one modified nucleotide of the second nucleotide sequence is a 2'-fluoropurine. In some embodiments, 1, 2, 3, 4, 5, or 6 modified nucleotides of the second nucleotide sequence are 2'-O-methylpurine. In some embodiments, the 2'-fluoro nucleotide is a 2'-fluoro nucleotide mimetic.

[0270] In some embodiments, the 2'-fluoro nucleotide or 2'-O-methyl nucleotide is a 2'-fluoro or 2'-O-methyl nucleotide mimetic. In some embodiments, the 2'-fluoro or 2'-O-methyl nucleotide mimetic is a nucleotide mimetic of formula (V): where R x are independently nucleobase, aryl, heteroaryl or H, Q 1 and Q 2 independently S or O, 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.

[0271] In some embodiments, the 2'-fluoro or 2'-O-methyl nucleotide mimetic is a nucleotide mimetic of formula (16) to formula (20):

[0272] where R x is a nucleobase, an aryl group, a heteroaryl group, or H, and R 2 are independently F or -OCH3, and wherein represents a phosphodiester bond, a phosphorothioate bond, a methylsulfonylphosphoramidate bond, or H. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0273] In some embodiments, the sense strand, the antisense strand, or both can each independently comprise at least 1, at least 2, at least 3, at least 4, or at least 5 or more modified nucleotides having the following chemical structure: wherein B is a nucleobase, an aryl group, a heteroaryl group, or H, and wherein represents a phosphodiester bond, a phosphorothioate bond, a methylsulfonylphosphoramidate bond, or H. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0274] In some embodiments, the antisense strand, the sense strand, or both may each further independently comprise at least 1, at least 2, at least 3, at least 4, or at least 5 or more modified nucleotides having the following chemical structure: where R y is a nucleobase, and wherein represents a phosphodiester bond, a phosphorothioate bond, a methylsulfonylphosphoramidate bond, or H. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0275] For the purposes of this disclosure, the modified nucleotide can be at any position of the antisense strand. In some embodiments, the modified nucleotide can be at position 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.

[0276] 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, 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, 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 nucleotides at positions 2, 6, 10, 14 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, 8, 14 and / or 17 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotides at position 2 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotides at position 5 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotides at position 6 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotides at position 8 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotides at position 10 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotides at position 14 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotides at position 16 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotides at position 17 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides.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 mimetic.

[0277] In some embodiments, the nucleotides in the second nucleotide sequence are arranged in an alternating 1:3 modification pattern, wherein one nucleotide is a 2′-fluoro nucleotide and three nucleotides are 2′-O-methyl nucleotides, and wherein the alternating 1:3 modification pattern occurs at least 2 times. In some embodiments, the alternating 1:3 modification pattern occurs 2-5 times. In some embodiments, at least two alternating 1:3 modification patterns occur consecutively. In some embodiments, at least two alternating 1:3 modification patterns occur non-consecutively. In some embodiments, at least 1, 2, 3, 4, or 5 of the alternating 1:3 modification patterns begin at nucleotide position 2, 6, 10, 14, and / or 18 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:3 modification pattern begins at nucleotide position 2 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:3 modification pattern begins at nucleotide position 6 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:3 modification pattern begins at nucleotide position 10 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:3 modification pattern begins at nucleotide position 14 from the 5' end of the antisense strand. In some embodiments, at least one alternating 1:3 modification pattern begins at nucleotide position 18 from the 5' end of the antisense strand. In some embodiments, the 2'-fluoro nucleotide is a 2'-fluoro nucleotide mimetic.

[0278] In some embodiments, the nucleotides in the second nucleotide sequence are arranged in an alternating 1:2 modification pattern, wherein one nucleotide is a 2′-fluoro nucleotide and two nucleotides are 2′-O-methyl nucleotides, and wherein the alternating 1:2 modification pattern occurs at least 2 times. In some embodiments, the alternating 1:2 modification pattern occurs 2-5 times. In some embodiments, at least two alternating 1:2 modification patterns occur consecutively. In some embodiments, at least two alternating 1:2 modification patterns occur non-consecutively. In some embodiments, at least 1, 2, 3, 4, or 5 of the alternating 1:2 modification patterns begin at nucleotide position 2, 5, 8, 14, and / or 17 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:2 modification pattern begins at nucleotide position 2 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:2 modification pattern begins at nucleotide position 5 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:2 modification pattern begins at nucleotide position 8 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:2 modification pattern begins at nucleotide position 14 from the 5' end of the antisense strand. In some embodiments, at least one alternating 1:2 modification pattern begins at nucleotide position 17 from the 5' end of the antisense strand. In some embodiments, the 2'-fluoro nucleotide is a 2'-fluoro nucleotide mimetic.

[0279] 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 mimetic.

[0280] In some embodiments, the sense strand may further comprise one or more internucleoside bonds independently selected from the group consisting of phosphodiester (PO) internucleoside bonds, phosphorothioate (PS) internucleoside bonds, phosphorodithioate internucleoside bonds, and PS-mimetic internucleoside bonds. In some embodiments, the PS-mimetic internucleoside bond is a sulfonucleoside bond.

[0281] In certain 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 thiophosphate nucleoside bonds. In certain 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 less thiophosphate nucleoside bonds. In certain 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 thiophosphate nucleoside bonds. In certain 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 thiophosphate nucleoside bonds. In certain embodiments, the antisense strand comprises 2 to 8 thiophosphate nucleoside bonds. In some embodiments, the antisense strand comprises 3 to 8 phosphorothioate nucleoside bonds. In some embodiments, the antisense strand comprises 4 to 8 phosphorothioate nucleoside bonds. In some embodiments, at least one phosphorothioate nucleoside bond is between the nucleotides at positions 1 and 2 from the 5' end of the second nucleotide sequence. In some embodiments, at least one phosphorothioate nucleoside bond is between the nucleotides at positions 2 and 3 from the 5' end of the second nucleotide sequence. In some embodiments, at least one phosphorothioate nucleoside bond is between the nucleotides at positions 1 and 2 from the 3' end of the second nucleotide sequence. In some embodiments, at least one phosphorothioate nucleoside bond is between the nucleotides at positions 2 and 3 from the 3' end of the second nucleotide sequence. In some embodiments, the antisense strand is included in two phosphorothioate nucleoside bonds between the nucleotides at positions 1 to 3 from the 5' end of the first nucleotide sequence. In some embodiments, the antisense strand is included in two phosphorothioate nucleoside bonds between the nucleotides at positions 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 at positions 1 to 3 from the 5′ end of the first nucleotide sequence; and (b) two phosphorothioate internucleoside linkages between nucleotides at positions 1 to 3 from the 3′ end of the first nucleotide sequence.

[0282] In certain 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 methanesulfonylphosphoramidate nucleoside interkeys. In certain 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 less methanesulfonylphosphoramidate nucleoside interkeys. In certain 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 methanesulfonylphosphoramidate nucleoside interkeys. In certain 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 methanesulfonylphosphoramidate nucleoside interkeys. In certain embodiments, the antisense strand comprises 2 to 8 methanesulfonylphosphoramidate nucleoside interkeys. In some embodiments, the antisense strand comprises 3 to 8 methylsulfonylphosphoramidate internucleoside linkages. In some embodiments, the antisense strand comprises 4 to 8 methylsulfonylphosphoramidate internucleoside linkages.

[0283] 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, wherein the overhang comprises at least one nucleotide. In some embodiments, both ends of the ds-siNA comprise an overhang, wherein the overhang comprises 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.

[0284] In some embodiments, the sense strand can comprise any of the modified nucleotides disclosed below in the subsection entitled "Modified Nucleotides." In some embodiments, the sense strand can comprise a 5'-stable endcap, and the 5'-stable endcap can be selected from the endcaps disclosed below in the subsection entitled "5'-stable endcap."

[0285] Modified nucleotides

[0286] The present disclosure provides oligonucleotides comprising one or more modified nucleotides disclosed herein. The oligonucleotides can be selected from short interfering nucleic acids (siNA), antisense oligonucleotides (ASO), steric blockers, short hairpin RNA (shRNA) and mRNA.

[0287] 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, the 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, the 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, the 2'-O-methyl nucleotides in any of the sense strands or first nucleotide sequences disclosed herein are replaced by modified nucleotides. In some embodiments, the 2'-O-methyl nucleotides in any of the antisense strands or second nucleotide sequences disclosed herein are replaced by modified nucleotides.

[0288] In some embodiments, any of the siNA molecules, siNA, sense strand, first nucleotide sequence, antisense strand, and 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, 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.

[0289] In some embodiments, the modified nucleotide is selected from the group consisting of 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'-xylo nucleotides, 2'-F-3'-xylo nucleotides, vmX nucleotides, ganciclovir nucleotides, or denovir nucleotides, locked nucleic acids, unlocked nucleic acids, nucleotide analogs, and nucleotides comprising modified nucleobases. 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 (e.g., 3', 4'-seco and mun34), wherein the furanose ring lacks a bond between the 3' and 4 carbons.

[0290] In certain aspects, the siNA of the present disclosure will comprise at least one modified nucleotide selected from: or a combination thereof. In some embodiments, the siNA may comprise at least 2, at least 3, at least 4, or at least 5 or more of these modified nucleotides. In some embodiments, the sense strand may comprise at least 1, at least 2, at least 3, at least 4, or at least 5 or more of the following: 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 the following: or a combination thereof; wherein B is a nucleobase, an aryl group, a heteroaryl group, or H. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0291] In some aspects, the siNA of the present disclosure will optionally further comprise one or more modified nucleotides selected from: (wherein Rx is a nucleobase, an aryl group, a heteroaryl group or H), (where R y are nucleobases) and as well as (where R yis a nucleobase) or a combination thereof. In some embodiments, the siNA may comprise 2, 3, 4, or 5 or more of these modified nucleotides. In some embodiments, the sense strand may optionally further comprise one or more modified nucleotides comprising 1, 2, 3, 4, or 5 or more of the following: (wherein RRx is nucleobase, aryl, heteroaryl or H), (where R y are nucleobases) and as well as (where R y In some embodiments, the antisense strand may comprise 1, 2, 3, 4, or 5 or more of the following: (wherein Rx is a nucleobase, an aryl group, a heteroaryl group or H), (where R y are nucleobases) and as well as (apN)(where R y In some embodiments, the sense strand and the antisense strand may each independently comprise 1, 2, 3, 4, 5 or more of the following: (where R x is a nucleobase, an aryl group, a heteroaryl group or H), (where Ry is a nucleobase) and as well as (where R y is a nucleobase) or a combination thereof. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof. For example, in In some embodiments, the modified nucleotide may have the following structure:

[0292] In some embodiments, any of the siNAs disclosed herein may additionally comprise other modified nucleotides, such as 2'-fluoro or 2'-O-methyl nucleotide mimics. For example, the disclosed siNAs may 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 sense strands disclosed herein may 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 first 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, 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 comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more 2′-fluoro or 2′-O-methyl nucleotide mimetics. In some embodiments, the 2′-fluoro or 2′-O-methyl nucleotide mimetics are nucleotide mimetics of formula (16) to formula (20):

[0293] where R x is a nucleobase, an aryl group, a heteroaryl group, or H, and R 2 is independently F or -OCH3. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0294] In some embodiments, the 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 comprise a 2′-fluoro nucleotide mimic. In some embodiments, the first nucleotide sequence does not comprise a 2′-O-methyl nucleotide mimic. In some embodiments, the second nucleotide sequence does not comprise a 2′-fluoro nucleotide mimic. In some embodiments, the second nucleotide sequence does not comprise a 2'-O-methyl nucleotide mimetic.

[0295] In some embodiments, any of the siNA, sense strand, first nucleotide sequence, antisense strand, or second nucleotide sequence disclosed herein can optionally comprise at least one modified nucleotide that is wherein Rx is a nucleobase, an aryl group, a heteroaryl group, or H; or where R y It is a nucleobase.

[0296] In some embodiments, any of the siNA, sense strand, first nucleotide sequence, antisense strand, or second nucleotide sequence disclosed herein can optionally comprise at least one modified nucleotide that is wherein B is a nucleobase, an aryl group, a heteroaryl group, or H; wherein represents a phosphodiester bond, a phosphorothioate bond, or a methylsulfonylphosphoramidate bond.

[0297] Phosphorylation blockers

[0298] Further disclosed herein are siNA molecules comprising phosphorylation blockers. In some embodiments, the 2'-O-methyl nucleotides in any one of the sense strand or the first nucleotide sequence disclosed herein are replaced by nucleotides containing a phosphorylation blocker. In some embodiments, the 2'-O-methyl, 2'-ocp, or 2'-omcp nucleotides in any one of the antisense strand or the second nucleotide sequence disclosed herein are replaced by nucleotides containing a phosphorylation blocker. In some embodiments, the 2'-O-methyl, 2'-ocp, or 2'-omcp nucleotides in any one of the sense strand or the first nucleotide sequence disclosed herein are further modified to include a phosphorylation blocker. In some embodiments, the 2'-O-methyl, 2'-ocp, or 2'-omcp nucleotides in any one of the antisense strand or the second nucleotide sequence disclosed herein are further modified to include a phosphorylation blocker.

[0299] In some embodiments, any of the siNA molecules disclosed herein comprises a phosphorylation blocker of Formula (IV): where R y is a nucleobase, R 4 Yes-OR 30 or -NR 31 R 32 , R 30 is a C1-C8 substituted or unsubstituted alkyl group; and R 31 and R 32 Together with the nitrogen to which it is attached, it forms a substituted or unsubstituted heterocyclic ring. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0300] In some embodiments, any of the siNA molecules disclosed herein comprises a phosphorylation blocker of Formula (IV): where R y is a nucleobase, and R 4 is -OCH3 or -N(CH2CH2)2O. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0301] In some embodiments, the siNA molecule comprises (a) a phosphorylation blocker of formula (IV): where R y is a nucleobase, R 4 Yes-OR 30 or -NR 31 R 32 , R 30 is a C1-C8 substituted or unsubstituted alkyl group; and R 31 and R 32and (b) a short interfering nucleic acid (siNA) wherein 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.

[0302] In some embodiments, the siNA molecule comprises (a) a phosphorylation blocker of formula (IV): where R y is a nucleobase, and R 4 is -OCH3 or -N(CH2CH2)2O; and (b) a short interfering nucleic acid (siNA), wherein a phosphorylation blocker is conjugated to the siNA.

[0303] 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 1, 2, 3, 4 or 5 or more joints. 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 1, 2, 3, 4 or 5 or more joints. 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 1, 2, 3, 4 or 5 or more joints. 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 1, 2, 3, 4 or 5 or more joints. In some embodiments, the one or more linkers are independently selected from the group consisting of a phosphodiester linker, a phosphorothioate linker, a methylsulfonylphosphoramidate linker, and a phosphorodithioate linker.

[0304] Conjugated moiety

[0305] Further disclosed herein are siNA molecules comprising a conjugate portion. In some embodiments, the conjugate portion is selected from galactosamine, peptides, proteins, sterols, lipids, phospholipids, biotin, phenoxazine, active pharmaceutical ingredients, cholesterol, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes. In some embodiments, the conjugate portion is attached to the 3' end of the sense strand or the first nucleotide sequence. In some embodiments, the conjugate portion is attached to the 3' end of the sense strand or the first nucleotide sequence via 1, 2, 3, 4, or 5 or more linkers. In some embodiments, the conjugate portion is attached to the 5' end of the sense strand or the first nucleotide sequence. In some embodiments, the conjugate portion is attached to the 5' end of the sense strand or the first nucleotide sequence via 1, 2, 3, 4, or 5 or more linkers. In some embodiments, the conjugate portion is attached to the 3' end of the antisense strand or the second nucleotide sequence. In some embodiments, the conjugate portion is attached to the 3' end of the antisense strand or the second nucleotide sequence via 1, 2, 3, 4, or 5 or more linkers. In some embodiments, the conjugate portion is attached to the 5' end of the antisense strand or the second nucleotide sequence. In some embodiments, the conjugate moiety is attached to the 5' end of the antisense strand or the second nucleotide sequence via 1, 2, 3, 4, or 5 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 methylsulfonylphosphoramidate linker.

[0306] In some embodiments, the conjugate moiety is galactosamine. In some embodiments, any of the siNAs disclosed herein are attached to a conjugate 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, the GalNAc has formula (VI): 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; L is a linker or L and Y in combination 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-diisopropylphosphoramidite 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.

[0307] In some embodiments, GalNAc is of formula (VII):

[0308]

[0309] where R z is OH or SH; and each n is independently 1 or 2. In some embodiments, the targeting ligand can be a GalNAc targeting ligand, which can comprise 1, 2, 3, 4, 5, or 6 GalNAc units. In some embodiments, the targeting ligand can be a GalNAc selected from the following: GalNAc2, GalNAc3, GalNAc4 (GalNAc of Formula VII, wherein n=1 and R z =OH), GalNAc5 and GalNAc6.

[0310] In some embodiments, GalNAc can be GalNAc amidite (ie, compound 40-9, see Example 22), GalNAc 4 CPG (ie, compound 40-8, see Examples 22 and 23), GalNAc phosphoramidite, or GalNAc4-ps-GalNAc4-ps-GalNAc4. These GalNAc moieties are shown below:

[0311]

[0312] GalNAc3, GalNAc4, GalNAc5, and GalNAc6 can be conjugated to the siNA disclosed herein using 1, 2, or 3 moieties during synthesis. Other GalNAc moieties, such as GalNAc1 and GalNAc2, can be used to form 5' and 3'-GalNAc using post-synthesis conjugation.

[0313] GalNAc phosphoramidite

[0314]

[0315]

[0316]

[0317] In some embodiments, galactosamine is attached to the 3' end of the sense strand or the first nucleotide sequence. In some embodiments, galactosamine is attached to the 3' end of the sense strand or the first nucleotide sequence via 1, 2, 3, 4 or 5 or more joints. In some embodiments, galactosamine is attached to the 5' end of the sense strand or the first nucleotide sequence. In some embodiments, galactosamine is attached to the 5' end of the sense strand or the first nucleotide sequence via 1, 2, 3, 4 or 5 or more joints. In some embodiments, galactosamine is attached to the 3' end of the antisense strand or the second nucleotide sequence. In some embodiments, galactosamine is attached to the 3' end of the antisense strand or the second nucleotide sequence via 1, 2, 3, 4 or 5 or more joints. In some embodiments, galactosamine is attached to the 5' end of the antisense strand or the second nucleotide sequence. In some embodiments, galactosamine is attached to the 5' end of the antisense strand or the second nucleotide sequence via 1, 2, 3, 4 or 5 or more joints. In some embodiments, the one or more linkers are independently selected from the group consisting of: phosphodiester (p or po) linkers, phosphorothioate (ps) linkers, mesylphosphoamide linkers (Ms), phosphoramidite (HEG) linkers, triethylene glycol (TEG) linkers and / or phosphorodithioate linkers. In some embodiments, the 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.

[0318] In some embodiments, the conjugate moiety is a lipid moiety. In some embodiments, any of the siNAs disclosed herein are attached to a conjugate moiety that is a lipid moiety. Examples of lipid moieties include, but are not limited to, a cholesterol moiety, a thioether (e.g., hexyl-S-tritylthiol), a thiocholesterol, an aliphatic chain (e.g., dodecyl glycol or undecyl residues), a phospholipid (e.g., di-hexadecyl-rac-glycerol or 1-di-O-hexadecyl-rac-tripropyl-SH-phosphonic acid triethylammonium), a polyamine or polyethylene glycol chain, adamantaneacetic acid, a palmityl moiety or octadecylamine or a hexylamino-carbonyl-hydroxycholesterol moiety.

[0319] In some embodiments, the conjugate moiety is an active drug substance. In some embodiments, any of the siNAs disclosed herein are attached to a conjugate 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, folinic acid, benzothiadiazine, chlorothiazide, diazepine, indomethacin, barbiturate, cephalosporin, sulfonamide, antidiabetic, antibacterial, or antibiotic.

[0320] 5′-Stable end cap

[0321] Further disclosed herein are oligonucleotides (e.g., siNA) comprising 5'-stable end caps. As used herein, the terms "5'-stable end caps" and "5' end caps" are used interchangeably. In some embodiments, the 2'-O-methyl, 2'-ocp, or 2'-omcp nucleotides in any one of the sense strand or the first nucleotide sequence disclosed herein are replaced by nucleotides containing 5'-stable end caps. In some embodiments, the 2'-O-methyl, 2'-ocp, or 2'-omcp nucleotides in any one of the antisense strand or the second nucleotide sequence disclosed herein are replaced by nucleotides containing 5'-stable end caps. In some embodiments, the 2'-O-methyl, 2'-ocp, or 2'-omcp nucleotides in any one of the sense strand or the first nucleotide sequence disclosed herein are further modified to include 5'-stable end caps. In some embodiments, the 2'-O-methyl, 2'-ocp, or 2'-omcp nucleotides in any one of the antisense strand or the second nucleotide sequence disclosed herein are further modified to include 5'-stable end caps.

[0322] In some embodiments, the 5′-stable end cap is a 5′ phosphate mimetic. In some embodiments, the 5′-stable end cap is a modified 5′ phosphate mimetic. In some embodiments, the modified 5′ phosphate is a chemically modified 5′ phosphate. In some embodiments, the 5′-stable end cap is a 5′-vinyl phosphonate. In some embodiments, the 5′-vinyl phosphonate is a 5′-(E)-vinyl phosphonate or a 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 monodeuterated vinyl phosphonate. In some embodiments, the deuterated vinyl phosphonate is a dideuterated vinyl phosphonate. In some embodiments, the 5′-stable end cap is a phosphate mimetic. Examples of phosphate mimetics are described in Parmar et al., J Med Chem, 2018 61(3):734-744, International Publication Nos. WO2018 / 045317 and WO2018 / 044350, and U.S. Patent No. 10,087,210, each of which is incorporated by reference in its entirety.

[0323] In some aspects, the present disclosure provides a short interfering nucleic acid (siNA) comprising a sense strand and an antisense strand, wherein the antisense strand comprises a 5′ vinyl phosphonate moiety comprising the following structure:

[0324]

[0325] wherein each B is independently selected from a nucleobase, an aryl group, a heteroaryl group, and H; wherein Represents a phosphodiester bond, a phosphorothioate bond, or a methylsulfonylphosphamidate 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 vinylphosphonate dimer portion can be the same nucleobase, while in some embodiments, each B can be a different nucleobase. The nucleobase can 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, the vinylphosphonate dimer of the present disclosure can contain two different nucleobases, as shown in the following structure:

[0326]

[0327] In certain aspects, the disclosure provides a siNA, optionally comprising a nucleotide phosphate mimetic selected from the group consisting of: (omeco-d3 nucleotide), (4h nucleotide), (v-mun nucleotide), (C2O-4H nucleotides), where R y is a nucleobase, and R 15 is H 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 mimetics include but are not limited to the structure: R 15 It is H or -CH3.

[0328] In some aspects, the disclosure provides a siNA, optionally comprising a nucleotide phosphate mimetic selected from the group consisting of:

[0329] (omeco-munbU, when R 15 is CH3); where R 15 is H or CH3. In some embodiments, any of these novel nucleotide phosphate mimetics (e.g., omeco-d3 nucleotides, 4H nucleotides, v-mun nucleotides, c2o-4h nucleotides, coc-4h nucleotides, omeco-munb nucleotides, or d2vm nucleotides) 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.

[0330] Additionally or alternatively, the siNA molecules disclosed herein can comprise a 5'-stabilized endcap of Formula (Ia) in the sense strand, the antisense strand, or both: where R x is H, nucleobase, aryl or heteroaryl; R 26 yes -CH=CD-Z, -CD=CH-Z, -CD=CD-Z, -(CR 21 R 22 ) n -Z or -(C2-C6 alkenylene)-Z, and R 20 It is H; or R 26 and R 20 Together they form the quilt-(CR 21 R 22 ) n -Z or -(C2-C6 alkenylene)-Z substituted 3- to 7-membered carbocyclic ring; 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 、-NR 23 SO2R 24 ; R 21 and R 22 Any one of which is independently hydrogen or C1-C6 alkyl, or R 21 and R 22 Together they form an oxygen group; R 23 is hydrogen or C1-C6 alkyl; R 24 Yes-SO2R 25 or -C(O)R 25 ; or R 23 and R 24 Together with the nitrogen to which it is attached, R forms a substituted or unsubstituted heterocyclic ring; 25 is C1-C6 alkyl; and m is 1, 2, 3 or 4. In some embodiments, R 1 In some embodiments, the aryl group is phenyl.

[0331] Additionally or alternatively, the siNA molecules disclosed herein can comprise a 5'-stabilized endcap of formula (Ib) in the sense strand, the antisense strand, or both: where R x is H, nucleobase, aryl or heteroaryl; R 26 yes -CH=CD-Z, -CD=CH-Z, -CD=CD-Z, -(CR 21 R 22 ) n -Z or -(C2-C6 alkenylene)-Z, and R 20 It is H; or R 26 and R 20 Together they form the quilt-(CR 21 R 22 ) n -Z or -(C2-C6 alkenylene)-Z substituted 3- to 7-membered carbocyclic ring; n is 1, 2, 3 or 4; Z is -ONR 23 R 24 、-OP(O)OH(CH2) m CO2R23 、-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 ; R 21 and R 22 Any one of which is independently hydrogen or C1-C6 alkyl, or R 21 and R 22 Together they form an oxygen group; R 23 is hydrogen or C1-C6 alkyl; R 24 Yes-SO2R 25 or -C(O)R 25 ; or R 23 and R 24 Together with the nitrogen to which it is attached, R forms a substituted or unsubstituted heterocyclic ring; 25 is C1-C6 alkyl; and m is 1, 2, 3 or 4. In some embodiments, R 1 In some embodiments, the aryl group is phenyl.

[0332] Additionally or alternatively, the siNA molecules disclosed herein can comprise a 5'-stabilized endcap of formula (Ic) in the sense strand, the antisense strand, or both: where R x is a nucleobase, an aryl group, a heteroaryl group, or H;

[0333] R 26 yes

[0334]

[0335] -CH=CD-Z, -CD=CH-Z, -CD=CD-Z, -(CR 21 R 22 ) n -Z or -(C2-C6 alkenylene)-Z, and R 20 is hydrogen; or R 26 and R 20 Together they form the quilt-(CR 21 R 22 ) n -Z or -(C2-C6 alkenylene)-Z substituted 3- to 7-membered carbocyclic ring; n is 1, 2, 3 or 4;

[0336] Z is for 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 ; R 21 and R 22 Any one of is independently hydrogen or C 1- C6 alkyl, or R 21 and R 22 Together they form an oxygen group; R 23 is hydrogen or C1-C6 alkyl; R 24 Yes-SO2R 25 or -C(O)R 25 ;or

[0337] R 23 and R 24 Together with the nitrogen to which it is attached, R forms a substituted or unsubstituted heterocyclic ring; 25 is C1-C6 alkyl; and m is 1, 2, 3 or 4. In some embodiments, R 1 In some embodiments, the aryl group is phenyl.

[0338] Additionally or alternatively, the siNA molecules disclosed herein can comprise a 5'-stabilized endcap of Formula (IIa) in the sense strand, the antisense strand, or both: where R x is a nucleobase, an aryl group, a heteroaryl group, or H, R 26 yes R 9 is -SO2CH3 or -COCH3, is a double or single bond, R 10 =-CH2PO3H or -NHCH3, R 11 is -CH2- or -CO-, and R 12 is H, and R 13 It is CH3, or R 12 and R 13 Together they form -CH2CH2CH2-. In some embodiments, R1 In some embodiments, the aryl group is phenyl.

[0339] Additionally or alternatively, the siNA molecules disclosed herein can comprise a 5'-stabilized endcap of formula (lib) in the sense strand, the antisense strand, or both: where R x is a nucleobase, an aryl group, a heteroaryl group, or H, R 26 yes R 9 is -SO2CH3 or -COCH3, is a double or single bond, R 10 =-CH2PO3H or -NHCH3, R 11 is -CH2- or -CO-, and R 12 is H, and R 13 It is CH3, or R 12 and R 13 Together they form -CH2CH2CH2-. In some embodiments, R 1 In some embodiments, the aryl group is phenyl.

[0340] Additionally or alternatively, the siNA molecules disclosed herein can comprise a 5'-stabilized end cap of (III) on the sense strand, the antisense strand, or both: where R x In some embodiments, R 1 In some embodiments, the aryl group is phenyl.

[0341] Additionally or alternatively, the siNA molecules disclosed herein can comprise a 5′ stabilizing 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 Formula (56), Formula (49X) to (52X), Formula (49Y) to (52Y), Formula 56X, Formula 56Y, Formula (61), Formula (62), and Formula (63):

[0342]

[0343]

[0344]

[0345] where R x is a nucleobase, an aryl group, a heteroaryl group or H.

[0346] In some embodiments, any of the siNA molecules disclosed herein comprises a 5′-stabilized end cap selected from the group consisting of: Formula (50), Formula (50X), Formula (50Y), Formula (56), Formula (56X), Formula (56Y), Formula (61), Formula (62), and Formula (63):

[0347] where R x is a nucleobase, an aryl group, a heteroaryl group or H.

[0348] In some embodiments, any of the siNA molecules disclosed herein comprises a 5′ stabilized end cap selected from the group consisting of: Formula (71) to Formula (86), Formula (79X) to Formula (82X), Formula (79Y) to Formula (82Y), Formula 86X, Formula 86X′, Formula 86Y, and Formula 86Y′:

[0349]

[0350] where R x is a nucleobase, an aryl group, a heteroaryl group or H.

[0351] In some embodiments, any of the siNA molecules disclosed herein comprises a 5′-stabilized end cap selected from the group consisting of: Formula (78), Formula (79), Formula (79X), Formula (79Y), Formula (86), Formula (86X), and Formula (86X′):

[0352] where R x is a nucleobase, an aryl group, a heteroaryl group or H.

[0353] In some embodiments, any of the siNA molecules disclosed herein comprises a 5′-stable end cap selected from the group consisting of: Formula (1A)-(15A), Formula (1A-1)-(7A-1), Formula (1A-2)-(7A-2), Formula (1A-3)-(7A-3), Formula (1A-4)-(7A-4), Formula (9B)-(12B), Formula (9AX)-(12AX), Formula (9AY)-(12AY), Formula (9BX)-(12BX), and Formula (9BY)-(12BY):

[0354]

[0355]

[0356]

[0357]

[0358] In some embodiments, any of the siNA molecules disclosed herein comprises a 5′-stable end cap selected from the group consisting of: Formula (21A)-(35A), Formula (29B)-(32B), Formula (29AX)-(32AX), Formula (29AY)-(32AY), Formula (29BX)-(32BX), and Formula (29BY)-(32BY):

[0359]

[0360]

[0361] In some embodiments, any of the siNA molecules disclosed herein comprises a 5′ stabilized end cap selected from the group consisting of: Formula (71A)-(86A), Formula (79XA)-(82XA), Formula (79YA)-(82YA); Formula (86XA), Formula (86X′A), Formula (86Y), and Formula (86Y′):

[0362]

[0363]

[0364] In some embodiments, any of the siNA molecules disclosed herein comprises a 5′-stabilized end cap selected from the group consisting of: Formula (78A), Formula (79A), Formula (79XA), Formula (79YA), Formula (86A), Formula (86XA), and Formula (86X′A):

[0365]

[0366] In some embodiments, the 5'-stable end cap is attached to the 5' end of the antisense strand. In some embodiments, the 5'-stable end cap is attached to the 5' end of the antisense strand via 1, 2, 3, 4 or 5 or more joints. In some embodiments, the one or more joints are independently selected from the group consisting of: phosphodiester (p or po) joints, phosphorothioate (ps) joints, mesylphosphoamide (Ms) joints, phosphoramidite (HEG) joints, triethylene glycol (TEG) joints and / or phosphorodithioate joints. In some embodiments, the one or more joints 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.

[0367] As described 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 disclosed siNA and compositions thereof can be used to treat various diseases and conditions (e.g., viral diseases, liver diseases, etc.).

[0368] connector

[0369] 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, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more internucleoside linkers are independently selected from the group consisting of a phosphodiester (p or po) linker, a phosphorothioate (ps) linker, a methylsulfonylphosphoramidate (Ms) linker, or a phosphorodithioate linker.

[0370] In some embodiments, any of the siNA, sense strand, first nucleotide sequence, antisense strand, and / or second nucleotide sequence disclosed herein further comprises 1, 2, 3, 4, or more linkers that attach a conjugated moiety, a phosphorylation blocker, and / or a 5' end cap to the siNA, sense strand, first nucleotide sequence, antisense strand, and / or second nucleotide sequence. In some embodiments, the 1, 2, 3, 4, or more linkers are independently selected from the group consisting of: a phosphodiester (p or po) linker, a phosphorothioate (ps) linker, a methylsulfonylphosphoamide (Ms), a phosphoramidite (HEG) linker, a triethylene glycol (TEG) linker, and / or a phosphorodithioate linker. In some embodiments, the 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.

[0371] Exemplary siNA

[0372] As described above, the siNA disclosed herein can include a modified nucleotide (i.e., N1-stabilizer) at position 1 or 2 from the 3' end of the antisense strand. The N1-stabilized nucleotides (e.g., moe, ln, cp, mun34, bl-m, tn, 3m, and those in bold in Table 1) can include one or more of the disclosed N1-stabilized nucleotides, and one or more N1-stabilized nucleotides can be present in the sense strand, the antisense strand, or both. Table 1 shows exemplary siNAs comprising these N1-stabilized nucleotides.

[0373] Table 1 - siNA containing N1 stabilized nucleotides

[0374]

[0375]

[0376]

[0377] Additionally or alternatively, the disclosed siNAs can also incorporate novel nucleotides (e.g., 2'-ocp and 2'-omcp). Table 2 shows exemplary siNAs comprising these nucleotides. siNAs comprising the disclosed novel nucleotides (e.g., 2'-ocp and 2'-omcp and those in bold in Table 2) can comprise 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.

[0378] Table 2 - siNA containing 2'-ocp and 2'-omcp nucleotides

[0379]

[0380]

[0381]

[0382]

[0383] Additionally or alternatively, the disclosed siNA may also incorporate a conjugate moiety. In some embodiments, the conjugate moiety is galactosamine. In some embodiments, any of the siNA disclosed herein is attached to a conjugate moiety that is galactosamine. In some embodiments, the galactosamine is N-acetylgalactosamine (GalNAc4). Table 3 shows exemplary siNAs that include these conjugate moieties in addition to novel nucleotides (e.g., 2'-ocp and 2'-omcp, and bold in the table). In some embodiments, siNA may comprise one or more of the disclosed conjugate moieties, and one or more conjugate moieties may be present in the sense strand or the antisense strand or both.

[0384] Table 3 - siNAs containing 2'-ocp and 2'-omcp nucleotides and conjugated moieties

[0385]

[0386]

[0387]

[0388]

[0389]

[0390] Additionally or alternatively, the disclosed siNAs can also incorporate alternative 2'-ocp or 2'-omcp nucleotides. Table 4 shows exemplary siNAs comprising these alternative 2'-ocp or 2'-omcp nucleotides. siNAs comprising alternative 2'-ocp or 2'-omcp nucleotides (in bold in the table) can comprise one or more alternative 2'-ocp or 2'-omcp nucleotides, and the one or more alternative 2'-ocp or 2'-omcp nucleotides can be present in the sense strand or the antisense strand or both.

[0391] Table 4 - Duplex siNA containing alternative 2'-ocp or 2'-omcp nucleotides

[0392]

[0393]

[0394]

[0395]

[0396] Additionally or alternatively, the disclosed siNAs may also incorporate 2'-ocp or 2'-omcp nucleotides at each of the 5' and 3' ends of the antisense strand. Table 5 shows exemplary siNAs containing these end-modified duplexes (in bold in the table). The end-modified siNAs may contain 2'-ocp and / or 2'-omcp nucleotides in place of 2'-O-methyl nucleotides in the first, second, third, and / or fourth position from either end of the sense strand, the antisense strand, or both.

[0397] Table 5 - siNAs containing 2'-ocp or 2'-omcp nucleotides at the 3' and 5' ends of the antisense strand

[0398]

[0399]

[0400]

[0401] Additionally or alternatively, the disclosed siNAs may also incorporate 2'-ocp or 2'-omcp nucleotides in place of one or more 2'O-methyl nucleotides in the 3'-overhang of the antisense strand. Table 6 shows exemplary siNAs containing duplexes (bold in the table) with these overhang modifications.

[0402] Table 6 - siNAs containing 2'-ocp or 2'-omcp nucleotides in the 3'-overhang of the antisense strand

[0403]

[0404]

[0405] Additionally or alternatively, the disclosed siNAs may also incorporate 2'-ocp or 2'-omcp nucleotides in place of most or all 2'-O-methyl nucleotides. Table 5 shows exemplary siNAs containing these fully modified duplexes (bold in the table). Fully modified siNAs may contain most or all 2'-ocp and / or 2'-omcp nucleotides in place of 2'-O-methyl nucleotides in either the sense strand or the antisense strand or both.

[0406] Table 7 - Modified duplex siNA containing high 2'-omcp nucleotide content

[0407]

[0408]

[0409] Additionally or alternatively, the disclosed siNA can also incorporate novel unlocked nucleotide monomers. These novel unlocked nucleotides can have the following structures: (where R X is a nucleobase, an aryl, a heteroaryl or H), or more specifically, where R y These unlocked nucleotides are different from unlocked nucleic acids (UNAs) known in the art that lack a 2' to 3' bond (e.g., Table 7 shows exemplary siNAs containing these unlocked nucleotides (bold in the table). siNAs containing 3', 4' UNAs (e.g., mun34) can contain one or more of the disclosed 3', 4' UNAs, and one or more 3', 4' UNAs can be present in the sense strand, the antisense strand, or both.

[0410] Table 8 - siNAs containing modified unlocked nucleotides and 5' end caps on the antisense strand

[0411]

[0412]

[0413] Additionally or alternatively, the disclosed siNAs may further incorporate modifications to nucleotide monomers. These modifications include 3m, 3oh, un, mun34, and changes to the 2'-fluoro nucleotide pattern. Table 9 shows exemplary siNAs containing these additional modifications (in bold in the table). 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.

[0414] Table 9 - siNA containing alternative 2'-fluoro nucleotide patterns

[0415]

[0416]

[0417] Additionally or alternatively, the disclosed siNAs can further incorporate modifications to the nucleomonomers. In some embodiments, the modification can be a 5'-cyclopropyl modification. For example, the siNA can comprise 5cpr2mA, 5cps2mA, 5mcpr2mA, or 5mcps2mA. Table 10 shows exemplary siNAs comprising these additional modifications. In some embodiments, the siNA can comprise 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.

[0418] Table 10 - siNA containing 5'-cyclopropyl nucleotides on the antisense strand

[0419]

[0420]

[0421] Additionally or alternatively, the disclosed siNAs may further incorporate modifications to the nucleotide monomers. In some embodiments, the modification may be a 2'-F'3'-xylo modification. For example, the siNA may comprise 1fG, 1fA, 1fC, and / or 1fU. Table 11 shows exemplary siNAs comprising these additional modifications (in bold). In some embodiments, the siNA may comprise 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.

[0422] Table 11 - siNA containing 2'-F-3'-xylo modified nucleotides on the sense or antisense strand

[0423]

[0424]

[0425]

[0426] Additionally or alternatively, the disclosed siNAs may further incorporate modifications to nucleotide monomers. In some embodiments, the modifications comprise additional 2'-F nucleotides at various positions along the antisense strand. For example, siNAs may comprise additional fG, fA, fC, and / or fU. Table 12 shows exemplary siNAs comprising these additional modifications (in bold). In some embodiments, siNAs may comprise 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.

[0427] Table 12 - siNAs containing a 2'F nucleotide "shift" on the antisense strand

[0428]

[0429]

[0430] Additionally or alternatively, the disclosed siNA may further incorporate modifications to nucleotide monomers. In some embodiments, the modifications include ganciclovir, Denvir, and 3′-ocp nucleotides along the sense strand and / or antisense strand. For example, siNA may include ganr^G, gans^G, denr^G, dens^G, and / or 3ocp. Table 13 shows exemplary siNAs (in bold) comprising these modifications. In some embodiments, siNA may include one or more of the disclosed modifications, and one or more disclosed modifications may be present in the sense strand or the antisense strand or both.

[0431] Table 13 - siNA containing ganciclovir, Denvir and 3'-ocp nucleotides on the sense or antisense strand

[0432]

[0433]

[0434]

[0435] Additionally or alternatively, the disclosed siNA can further incorporate modifications to nucleotide monomers. In some embodiments, the modifications comprise 2'-OMe-3'-Xylo nucleotides along the antisense strand. For example, the siNA can comprise 1mG, 1mG, 1mG, and / or 1mG. Table 14 shows exemplary siNAs comprising these modifications (in bold). In some embodiments, the siNA can comprise 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.

[0436] Table 14 - siNAs containing 2'-OMe-3'-xylo modified nucleotides on the antisense strand

[0437]

[0438]

[0439]

[0440] Additionally or alternatively, the disclosed siNA may further incorporate modifications to nucleotide monomers. In some embodiments, the modifications comprise 2'-ocp, 2'-omcp, and / or 5'-vinylphosphonate 2'-O-methyl nucleotides along the antisense strand. For example, siNA may comprise 2ocpA, 2ocpC, 2ocpG, 2ocpU, 2omcpA, 2omcpC, 2omcpG, 2omcpU, and / or vmU. Table 15 shows exemplary siNAs comprising these modifications (in bold). In some embodiments, siNA may comprise one or more of the disclosed modifications, and one or more of the disclosed modifications may be present in the sense strand or the antisense strand or both.

[0441] Table 15 - siNA containing 2'-ocp, 2'-omcp and vmX nucleotides

[0442]

[0443]

[0444]

[0445]

[0446] Additionally or alternatively, the disclosed siNAs may further incorporate modifications to nucleotide monomers. In some embodiments, the modifications include vinyl phosphate 5' end caps along the antisense strand, such as vmU and / or G mimetic nucleotides, such as dens^G and mun12G. Table 16 shows exemplary siNAs containing these modifications (in bold). In some embodiments, the siNA 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.

[0447] Table 16 - siRNAs containing G analogs and vinyl phosphate 5' end caps

[0448]

[0449]

[0450] Additionally or alternatively, the disclosed siNAs may further incorporate modifications to nucleotide monomers. In some embodiments, the modifications comprise a 5' TNA modification, such as coc-4h on the antisense strand. Table 17 shows exemplary siNAs comprising these modifications (in bold). In some embodiments, the siNA may comprise 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.

[0451] Table 17 - siRNAs with TNA phosphonate chemistry

[0452]

[0453] Additionally or alternatively, the disclosed siNAs can further incorporate modifications to nucleotide bonds. In some embodiments, the modifications include well-defined PS bonds, 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.

[0454] Table 18 - siRNAs containing well-defined PS bonds

[0455]

[0456]

[0457] target gene

[0458] Without wishing to be bound by theory, upon entry into a cell, any of the ds-siNA molecules disclosed herein may interact with proteins in the cell to form an RNA-induced silencing complex (RISC). Once the ds-siNA is part of the RISC, the ds-siNA may unwind to form a single-stranded siNA (ss-siNA). The ss-siNA may comprise the antisense strand of the ds-siNA. The antisense strand may bind to a complementary messenger RNA (mRNA), which results in silencing of the gene encoding the mRNA.

[0459] The target gene can be any gene in the 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, HBV is selected from HBV genotypes AJ. 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 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, beta-coronavirus is selected from the group consisting of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (also referred to as the temporary name 2019 novel coronavirus or 2019-nCoV), human coronavirus OC43 (hCoV-OC43), Middle East respiratory syndrome-related coronavirus (MERS-CoV, also referred to as the temporary name 2012 novel coronavirus or 2012-nCoV) and severe acute respiratory syndrome-related coronavirus (SARS-CoV, also referred to as SARS-CoV-1). In some embodiments, beta-coronavirus is SARS-CoV-2, the pathogen of COVID-19. Some exemplary target genes are shown in Table 23 at the end of the specification.

[0460] In some embodiments, the target gene is selected from the S gene or X gene of HBV. In some embodiments, the HBV has a genomic 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 herein by reference in its entirety.

[0461] An exemplary HBV genomic sequence is set forth in SEQ ID NO:81, corresponding to Genbank Accession No. KC315400.1, which is incorporated herein by reference in its entirety. Nucleotides 2307 to 3215, and 1 to 1623 of SEQ ID NO:94 correspond to the polymerase / RT gene sequence encoding the polymerase protein. Nucleotides 2848 to 3215 and 1 to 835 of SEQ ID NO:94 correspond to the PreS1 / S2 / S gene sequence encoding the large S protein. Nucleotides 3205 to 3215 and 1 to 835 of SEQ ID NO:94 correspond to the PreS2 / S gene sequence encoding the intermediate S protein. Nucleotides 155 to 835 of SEQ ID NO:94 correspond to the S gene sequence encoding the small S protein. Nucleotides 1374 to 1838 of SEQ ID NO:94 correspond to the X gene sequence encoding the X protein. Nucleotides 1814 to 2452 of SEQ ID NO:94 correspond to the PreC / C gene sequence, which encodes the precore / core protein. Nucleotides 1901 to 2452 of SEQ ID NO:94 correspond to the C gene sequence, which encodes 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), which encodes the core and polymerase proteins.

[0462] In some embodiments, the positive strand comprises a sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary or hybridizing to a viral target RNA sequence starting in the X region of HBV or the S region of HBV. For example, the viral target can begin at the 5′ end of the target site in aCC.KC315400.1 (genotype B, "gt B"), or any of genotypes A, C, or D. The skilled artisan will understand the HBV positions, e.g., as described in Wing-Kin Sung et al., Nature Genetics 44:765 (2012). In some embodiments, the S region is defined as extending from the beginning of the small S protein (position #155 in genotype B KC315400.1 isolates) to before the beginning of the X protein (position #1373 in genotype B KC315400.1 isolates). In some embodiments, the X region is defined as from the beginning of the X protein (in genotype B KC315400.1 isolate, position #1374) to the end of the DR2 locus (in genotype B KC315400.1 isolate, position #1603).

[0463] In some embodiments, the second nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% 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 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 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 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 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 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 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 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.

[0464] In some embodiments, the first nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a region of nucleotides within SEQ ID NO: 89, except that thymine (T) is replaced with uracil (U) in 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 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 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 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 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 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 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 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 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.

[0465] Several disease-causing coronaviruses have a high degree of homology in the region of the genome encoding non-structural proteins (nsp), and more specifically in the region encoding nsp8-nsp15. In fact, the approximately 7kB sequence of the beta coronavirus from about nucleotide 12900 to about nucleotide 19900 of 2019-nCoV has about 65% identity, and some sections of the genome span of nsp8 to nsp15 may contain 95% or more identity. All genes in this region encode non-structural proteins associated with replication. Therefore, this section of the genome is suitable for targeting with siNA, which can provide a wide range of treatments for a variety of different types of coronaviruses (such as MERS-CoV, SARS-CoV-1, and SARS-CoV-2).

[0466] In some embodiments, the target gene is selected from the genome of SARS-CoV-2. In some embodiments, SARS-CoV-2 has the genomic sequence shown in the nucleotide sequence of SEQ ID NO: 97, which corresponds to the nucleotide sequence of GenBank accession number NC_045512.2, which is incorporated herein by reference in its entirety. In some embodiments, the target gene a sequence within SEQ ID NO: 97 is 15 to 30, 15 to 25, 15 to 23, 17 to 23, 19 to 23, or 19 to 21 nucleotides in length, and is preferably 19 or 21 nucleotides in length. In some embodiments, the antisense strand sequence is identical to the sequence of SEQ ID NO: NO:97 position 190-216、233-279、288-324、455-477、626-651、704-723、3352-3378、5384-5403、6406-6483、7532-7551、9588-9606、10484-10509、11609-11630、11834-11853、12023-12045、12212-12234、12401-12420、12839-12867、1 2885-12924, 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, 1 4821-14846、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, 2 8513-28546, 28673-28692, 28706-28726, 28744-28794, 28799-28827, 28946-28972, 28976-29034, 29144-29172, 29174-29196, 29228-29259, 29285-29305, 29342-29394, 29444-29463, 29543-2 9566, 29598-29630, 29652-29687, 29689-29731, 29733-29757, or 29770-29828, and preferably 19 to 21 nucleotides, and more preferably 19 or 21 nucleotides. In some embodiments, the sense strand sequence is complementary to SEQ ID NO: 1. NO: 97's position 190-216, 233-279, 288-324, 455-477, 626-651, 704-723, 3352-3378, 5384-5403, 6406-6483, 7532-7551, 9588-9606, 10484-10509, 11609-11630, 11834-11853, 12023-12045, 12212-1 2234, 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、29598-29630、29652-29687、29689-29731、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, and preferably 19 to 21 nucleotides, and more preferably 19 or 21 nucleotides, within 29733-29757 or 29770-29828 are identical.

[0467] In some embodiments, the target gene is selected from the genome of SARS-CoV. In some embodiments, the SARS-CoV has a genome having a nucleotide sequence corresponding to GenBank Accession No. NC_004718.3, which is incorporated herein by reference in its entirety.

[0468] In some embodiments, the target gene is selected from the genome of MERS-CoV. In some embodiments, MERS-CoV has a genome corresponding to the nucleotide sequence of GenBank Accession No. NC_019843.3, which is incorporated herein by reference in its entirety.

[0469] In some embodiments, the target gene is selected from the genome of hCoV-OC43. In some embodiments, hCoV-OC43 has a genome corresponding to the nucleotide sequence of GenBank Accession No. NC_006213.1, which is incorporated herein by reference in its entirety.

[0470] 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 methylation-controlled 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 herein by reference in its entirety.

[0471] 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, which is herein incorporated by reference in its entirety.

[0472] In some embodiments, the target gene is angiopoietin-like 3 (ANGPTL3). In some embodiments, ANGPTL3 comprises the nucleotide sequence of SEQ ID NO: 92, which corresponds to the nucleotide sequence of GenBank Accession No. NM_014495.4, which is incorporated herein by reference in its entirety. In some embodiments, the target gene is diacylglycerol acyltransferase 2 (DGAT2). In some embodiments, DGAT2 comprises the nucleotide sequence of SEQ ID NO: 93, which corresponds to the nucleotide sequence of GenBank Accession No. NM_001253891.1, which is incorporated herein by reference in its entirety.

[0473] Composition

[0474] 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.

[0475] 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 phosphorylation blocker disclosed herein. In some embodiments, siNA is any siNA disclosed herein. In some embodiments, siNA comprises any of the sense strand, antisense strand, first nucleotide sequence, or second nucleotide sequence described herein. In some embodiments, siNA comprises any of the sense strand, antisense strand, first nucleotide sequence, or second nucleotide sequence described herein. In some embodiments, 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′-Xylo nucleotides, 2′-F-3′-xylo nucleotides, 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 mimetics disclosed herein. In some embodiments, the siNA comprises a nucleotide sequence comprising any modification pattern disclosed herein.

[0476] In some embodiments, the composition comprises (a) a conjugated portion; and (b) a short interfering nucleic acid (siNA). In some embodiments, the conjugated portion is any galactosamine disclosed herein. In some embodiments, siNA is any siNA disclosed herein. In some embodiments, siNA comprises any of the sense strand, antisense strand, first nucleotide sequence, or second nucleotide sequence described herein. In some embodiments, siNA comprises any of the sense strand, antisense strand, first nucleotide sequence, or second nucleotide sequence described herein. In some embodiments, 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′-Xylo nucleotides, 2′-F-3′-xylo nucleotides, 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 mimetics disclosed herein. In some embodiments, the siNA comprises a nucleotide sequence comprising any modification pattern disclosed herein.

[0477] In some embodiments, the composition comprises (a) a 5′-stable end cap; and (b) a short interfering nucleic acid (siNA). In some embodiments, the 5′-stable end cap is any 5′-stable end cap disclosed herein. In some embodiments, siNA is any siNA disclosed herein. In some embodiments, siNA comprises any of the sense strand, antisense strand, first nucleotide sequence, or second nucleotide sequence described herein. In some embodiments, 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′-Xylo nucleotides, 2′-F-3′-xylo nucleotides, ganciclovir nucleotides, Denvir nucleotides, 5′-vinylphosphonate 2′-O-methyl nucleotides, and nucleotide analogs. In some embodiments, 2′-fluoro nucleotides or 2′-O-methyl nucleotides are independently selected from any of the 2′-fluoro or 2′-O-methyl nucleotide mimetics disclosed herein. In some embodiments, the siNA comprises a nucleotide sequence comprising any of the modification patterns disclosed herein.

[0478] In some embodiments, the composition comprises (a) at least one phosphorylation blocker, conjugate moiety, or 5′-stable end cap; and (b) short interfering nucleic acid (siNA). In some embodiments, the phosphorylation blocker is any phosphorylation blocker disclosed herein. In some embodiments, the conjugate moiety is any galactosamine disclosed herein. In some embodiments, the 5′-stable end cap is any 5′-stable end cap disclosed herein. In some embodiments, siNA is any siNA disclosed herein. In some embodiments, siNA comprises any of the sense strand, antisense strand, first nucleotide sequence, or second nucleotide sequence described herein. In some embodiments, 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′-Xylo nucleotides, 2′-F-3′-xylo nucleotides, 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 mimetics disclosed herein. In some embodiments, the siNA comprises a nucleotide sequence comprising any modification pattern disclosed herein.

[0479] The composition can be a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises an amount of one or more siNA molecules described herein formulated together with one or more pharmaceutically acceptable carriers (additives) and / or diluents. The pharmaceutical composition can be specifically formulated for administration in solid or liquid form, including those suitable for: (1) oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets (e.g., those for oral, sublingual, and systemic absorption, for application to the tongue as pills, powders, granules, pastes); (2) parenteral administration, such as by subcutaneous, intramuscular, intravenous, or epidural injection, for example, as a sterile solution or suspension or sustained-release formulation; (3) topical administration, such as, for example, application to the skin as a cream, ointment, sustained-release patch, or spray; (4) intravaginally or intrarectally, such as, for example, as a pessary, cream, or foam; (5) sublingually; (6) ophthalmically; (7) transdermally; or (8) nasally.

[0480] As used herein, the phrase "therapeutically effective amount" refers to an amount of a compound, material, or composition comprising a siNA of the present disclosure effective to produce the desired therapeutic effect in at least a subpopulation of cells in an animal, at a reasonable benefit / risk ratio applicable to any medical treatment.

[0481] As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0482] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0483] Examples of pharmaceutically acceptable antioxidants include: (1) water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0484] The formulations of the present disclosure include formulations suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and / or parenteral administration. The formulations can be conveniently presented in unit dosage form and can be prepared by any method well known in the pharmaceutical art. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending on the host being treated and the specific mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be the amount of the compound (e.g., siNA molecule) that produces a therapeutic effect. Typically, out of 100%, this amount will be in the range of about 0.1% to about 99%, preferably about 5% to about 70%, and most preferably about 10% to about 30% of the active ingredient.

[0485] In certain embodiments, the formulations of the present disclosure comprise an excipient selected from the group consisting of cyclodextrins, celluloses, liposomes, micelle formers (e.g., bile acids), and polymeric carriers (e.g., polyesters and polyanhydrides); and a compound of the present disclosure (e.g., siNA molecules). In certain embodiments, the formulations described above render the compound of the present disclosure (e.g., siNA molecules) orally bioavailable.

[0486] Methods for preparing these formulations or compositions include the step of associating a compound of the present disclosure (e.g., siNA molecule) with a carrier and, optionally, one or more auxiliary ingredients. Typically, the formulations are prepared by uniformly and intimately associating a compound of the present disclosure (e.g., siNA molecule) with a liquid carrier or a finely divided solid carrier, or both, and then, if desired, shaping the product.

[0487] Formulations of the present disclosure suitable for oral administration can be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored base, typically sucrose and acacia or tragacanth), powders, 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 pastilles (using an inert base such as gelatin and glycerin or sucrose and acacia); and / or as a mouthwash, etc., each containing a predetermined amount of a compound of the present disclosure (e.g., siNA molecule) as the active ingredient. Compounds of the present disclosure (e.g., siNA molecules) can also be administered as a bolus, electuary, or paste.

[0488] In the solid dosage forms of the present disclosure for oral administration (capsules, tablets, pills, dragees, powders, granules, lozenges, etc.), 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, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants, such as glycerol; (4) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicic acids, or the like. salt and sodium carbonate; (5) solution retarders, such as paraffin; (6) absorption promoters, such as quaternary ammonium compounds, and surfactants, such as poloxamer and sodium lauryl sulfate; (7) wetting agents, such as, for example, cetyl alcohol and glyceryl monostearate, and nonionic surfactants; (8) absorbents, such as kaolin and bentonite clays; (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 cross-linked polyvinylpyrrolidone or ethylcellulose.

[0489] In the case of capsules, tablets and pills, the pharmaceutical compositions may also include buffering agents. Solid compositions of a similar type may also serve as fillers in soft and hard shell gelatin capsules using excipients such as lactose or milk sugar and high molecular weight polyethylene glycols.

[0490] Tablets can be prepared by optionally compressing or molding together with one or more auxiliary components. Compressed tablets can be prepared using a binding agent (e.g., gelatin or hydroxypropyl methylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium carboxymethyl starch or cross-linked sodium carboxymethyl cellulose), a surfactant or a dispersant. Molded tablets can be manufactured by molding a mixture of a powdered compound moistened with an inert liquid diluent in a suitable machine.

[0491] Tablets and other solid dosage forms of pharmaceutical compositions of the present disclosure, such as dragees, capsules, pills, and granules, can optionally be coated or prepared with coatings and shells (such as enteric coatings and other coatings well-known in the field of pharmaceutical formulation). The dosage forms can also be formulated to use, for example, different proportions of hydroxypropyl methylcellulose to provide a sustained or controlled release of the active ingredient therein to provide a desired release profile, other polymer matrices, liposomes, and / or microspheres. They can be formulated for rapid release, such as freeze drying.

[0492] They can for example be filtered by a bacteria-retaining filter before being about to use or sterilized by being incorporated into a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injectable media. These compositions can also optionally contain an emulsifier and the composition of the composition can make it only or preferably release one or more active components in a certain part of the gastrointestinal tract in a delayed manner. The example of operable embedding composition comprises polymeric substances and wax. Active component can also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0493] Liquid dosage forms for oral administration of compounds of the present disclosure (e.g., siNA molecules) include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage form may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers, and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (specifically, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuranol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof.

[0494] 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.

[0495] In addition to the active compound (e.g., siNA molecule), the suspension can contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, mixtures thereof, and the like.

[0496] Formulations of the pharmaceutical compositions of the present disclosure for rectal or vaginal administration can be presented as suppositories, which can 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 wax, 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).

[0497] 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.

[0498] Dosage forms for topical or transdermal administration of compounds of the present disclosure (e.g., siNA molecules) include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound (e.g., siNA molecule) can be mixed under sterile conditions with a pharmaceutically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0499] Ointments, pastes, creams, and gels can contain, in addition to the active compounds of the disclosure (e.g., siNA molecules), excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicones, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0500] In addition to the compounds of the present disclosure (e.g., siNA molecules), powders and sprays can also contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide powder, or mixtures of these substances. Sprays can additionally contain conventional propellants such as chlorofluorocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

[0501] Transdermal patches have the additional advantage of controlled delivery of the compounds of the present disclosure (e.g., siNA molecules) into the body. Such dosage forms can be prepared by dissolving or dispersing the compound (e.g., siNA molecule) in an appropriate medium. Absorption enhancers can also be used to increase the flux of the compound (e.g., siNA molecule) through the skin. The rate of such flux can be controlled by providing a rate-controlling membrane or dispersing the compound (e.g., siNA molecule) in a polymer matrix or gel.

[0502] Ophthalmic formulations, eye ointments, powders, solutions, etc. are also contemplated as being within the scope of this disclosure.

[0503] 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 just prior to use (which 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).

[0504] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (such as 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 using coating materials such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants.

[0505] These compositions can also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. Can be by comprising various antibacterials and antifungals, for example parabens, chlorobutanol, phenol, sorbic acid etc. to ensure that the effect of microorganisms on the subject compound is prevented. It is also possible to expect that isotonic agents, such as sugar, sodium chloride etc. are included in these compositions. In addition, the absorption of the extension of injectable drug form can be realized by comprising the medicament (such as aluminum monostearate and gelatin) that delays absorption.

[0506] In some cases, in order to prolong the effect of the drug, it is necessary to slow down the absorption of the drug from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of a crystalline or amorphous material with poor water solubility. The absorption rate of the drug then depends on its dissolution rate, and then may depend on the crystal size and crystalline form. Alternatively, delayed absorption of a parenteral drug form is achieved by dissolving or suspending the drug in an oily vehicle.

[0507] Injectable depot formulations are prepared by forming a microencapsulated matrix of the subject compound (e.g., siNA molecules) in a biodegradable polymer such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the properties of the specific polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0508] When the compounds of the present disclosure (e.g., siNA molecules) are administered as drugs to humans and animals, they can be administered per se or as a pharmaceutical composition containing, for example, 0.1% to 99% (more preferably 10% to 30%) of active ingredient in combination with a pharmaceutically acceptable carrier.

[0509] Treatment and administration

[0510] The siNA molecules disclosed herein can be used to treat a disease in a subject in need thereof. In some embodiments, a method of treating 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 a disease in a subject in need thereof comprises administering to the subject any of the compositions disclosed herein.

[0511] The formulations of the present disclosure (e.g., siNA molecules or compositions) can be administered orally, parenterally, topically, or rectally. Of course, they are administered in a form suitable for each route of administration. For example, they are administered in tablet or capsule form, by injection, infusion, or inhalation; topically by lotion or ointment; and rectally by suppository. Oral administration is preferred.

[0512] As used herein, the phrases "parenteral administration" and "administered parenterally" mean modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0513] As used herein, the phrases "systemic administration," "administered systemically," "peripheral administration," and "administered peripherally" refer to the administration of a compound, drug, or other material other than directly into the central nervous system so that it enters the patient's system and is therefore subject to metabolic and other similar processes, e.g., subcutaneous administration.

[0514] The compounds can be administered to humans and other animals for therapy by any suitable route of administration, including oral, nasal (e.g., by spray), rectal, vaginal, parenteral, intracisternal, and topical (e.g., by powders, ointments, or drops), including buccal and sublingual.

[0515] Regardless of the route of administration chosen, the compounds of the disclosure (e.g., siNA molecules) and / or pharmaceutical compositions of the disclosure, which can be used in a suitable hydrated form, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0516] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied so as 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 causing toxicity to the patient.

[0517] The selected dosage level will depend upon a variety of factors, including the activity of the particular compound of the disclosure (e.g., siNA molecule) employed, or its ester, salt, or amide, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound employed, the rate and extent of absorption, the duration of the treatment, other drugs, compounds, and / or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0518] 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, a physician or veterinarian can start a dose of a compound of the disclosure (e.g., siNA molecule) used in a pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved.

[0519] In general, a suitable daily dose of a compound of the present disclosure (e.g., siNA molecule) is the amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose generally depends on the factors described above. Preferably, the compound is administered at about 0.01 mg / kg to about 200 mg / kg, more preferably at about 0.1 mg / kg to about 100 mg / kg, and even more preferably at about 0.5 mg / kg to about 50 mg / kg. In some embodiments, the compound is administered at a dose equal to or greater than 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. In some embodiments, the compound is administered at a dose of equal to or less than 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 equal to or greater than 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.

[0520] When a compound described herein (e.g., a siNA molecule) is co-administered with another compound, the effective amount may be lower than when the compound is used alone.

[0521] If desired, the effective daily dose of the active compound (e.g., siNA molecule) can be administered as two, three, four, five, six or more sub-doses at appropriate intervals throughout the year, optionally in unit dosage form. The preferred dose is once a day. 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 a 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 a 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.

[0522] disease

[0523] The siNA molecules and compositions described herein can be administered to a subject to treat a disease. Further 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.

[0524] 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).

[0525] 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, NAFLD is non-alcoholic steatohepatitis (NASH). In some embodiments, the liver disease is hepatocellular carcinoma (HCC).

[0526] The siNA molecules disclosed herein can be used to treat or prevent a disease in a subject in need thereof. In some embodiments, the 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, the method of treating or preventing a disease in a subject in need thereof comprises administering to the subject any of the compositions disclosed herein.

[0527] In some embodiments of the disclosed methods and uses, 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 may include one or more symptoms selected from the following: cough, sore throat, runny nose, sneezing, headache, fever, shortness of breath, myalgia, abdominal pain, fatigue, dyspnea, 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, chest tightness and palpitations. In some embodiments, the respiratory disease may include complications selected from sinusitis, otitis media, pneumonia, acute respiratory distress syndrome, disseminated intravascular coagulation, pericarditis and renal failure. In some embodiments, the respiratory disease is idiopathic.

[0528] In some embodiments, the present disclosure provides a method for treating or preventing a coronavirus infection, comprising administering to a subject in need thereof a therapeutically effective amount of one or more siNA or pharmaceutical compositions disclosed herein. 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 has been treated with one or more additional coronavirus therapeutic agents. In some embodiments, the subject is simultaneously treated with one or more additional coronavirus therapeutic agents.

[0529] Administration of siNA

[0530] Administration of any of the siNAs disclosed herein can be performed by methods known in the art. In some embodiments, siNA is administered by subcutaneous (SC) or intravenous (IV) delivery. The formulations of the present disclosure (e.g., siNA or compositions) can be administered orally, parenterally, topically, or rectally. Of course, they are administered in a form suitable for each route of administration. For example, they are administered in tablet or capsule form, administered by injection, infusion, or inhalation; topically by lotion or ointment; and rectally by suppository. In some embodiments, subcutaneous administration is preferred.

[0531] As used herein, the phrases "parenteral administration" and "administered parenterally" mean modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, and intrasternal injection and infusion.

[0532] As used herein, the phrases "systemic administration," "administered systemically," "peripheral administration," and "administered peripherally" refer to the administration of a compound, drug, or other material other than directly into the central nervous system so that it enters the patient's system and is therefore subject to metabolic and other similar processes, e.g., subcutaneous administration.

[0533] The compounds can be administered to humans and other animals for therapy by any suitable route of administration, including oral, nasal (e.g., by spray), rectal, vaginal, parenteral, intracisternal, and topical (e.g., by powders, ointments, or drops), including buccal and sublingual.

[0534] Regardless of the route of administration selected, the compounds of the disclosure (e.g., siNA) and / or pharmaceutical compositions of the disclosure, which can be used in a suitable hydrated form, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

[0535] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied so as 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 causing toxicity to the patient.

[0536] The selected dosage level will depend upon a variety of factors, including the activity of the particular compound of the disclosure (e.g., siNA) employed, or its ester, salt, or amide, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound employed, the rate and extent of absorption, the duration of the treatment, other drugs, compounds, and / or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0537] 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, a physician or veterinarian can start a dose of a compound of the present disclosure (e.g., siNA) used in a pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved.

[0538] In general, a suitable daily dose of a compound of the present disclosure (e.g., siNA) is the amount of the compound at the lowest dose effective to produce a therapeutic effect. Such an effective dose generally depends on the factors described above. Preferably, the compound is administered at about 0.01 mg / kg to about 200 mg / kg, more preferably at about 0.1 mg / kg to about 100 mg / kg, and even more preferably at 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 1 mg / kg to about 10 mg / kg. In some embodiments, the compound is administered at a dose equal to or greater than 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. 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. 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. In some embodiments, the total daily dose of the compound is equal to or greater than 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.

[0539] If desired, the effective daily dose of the active compound (e.g., siNA) can be administered as two, three, four, five, six, seven, eight, nine, ten or more doses or sub-doses at appropriate intervals throughout the day, optionally in unit dosage form. 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. The preferred dose is once a day. 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 a 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 a 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, 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 47, 48, 49, 50, 51, 52, or 53 administrations of a compound within a 70, 80, 90, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100, 100In some embodiments, 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 45, 46, 47, 48, 49, 50, 51, 52, or 53 times within a time period. In some embodiments, the patient is treated for 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. 47, 48, 49, 50, 51, 52, or 53 administrations of the compound over a period of time. In some embodiments, the compound is administered at least once 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 once 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 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 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 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.

[0540] In some embodiments, any of the siNA or compositions disclosed herein is administered in a particle or viral vector. In some embodiments, the viral vector is a vector of an adenovirus, an adeno-associated virus (AAV), an alpha virus, a flavivirus, a herpes simplex virus, a lentivirus, a measles virus, a picornavirus, a poxvirus, a retrovirus, or a rhabdovirus. 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.

[0541] The subject of the method can be a mammal, and includes humans and non-human mammals. In some embodiments, the subject is a human, such as an adult.

[0542] Some embodiments include methods for treating HBV in a subject infected with the virus, the methods comprising administering to a subject in need thereof a therapeutically effective amount of one or more siNAs of the present disclosure or a composition of the present disclosure, thereby reducing the viral load in the subject and / or reducing the level of viral antigens in the subject. The siNA can be complementary to or hybridize with a portion of the target RNA in the virus, such as the X region and / or the S region of HBV.

[0543] Combination therapy

[0544] Any method disclosed herein may further comprise administering to the subject an additional HBV therapeutic agent. Any composition 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 regulators (CAMs), recombinant interferon, entry inhibitors, small molecule immunomodulators, and oligonucleotide therapies. In some embodiments, the additional HBV therapeutic agent is selected from HBV STOPS TMALG-010133, HBVCAM ALG-000184, ASO 1 (SEQ ID NO: 95), ASO 2 (SEQ ID NO: 96), recombinant interferon α 2b, IFN-a, PEG-IFN-a-2a, lamivudine, telbivudine, adefovir dipivoxil, clevudine, entecavir, tenofovir alafenamide, tenofovir disoproxil fumarate, disoproxil), NVR3-778, BAY41-4109, JNJ-632, JNJ-3989 (ARO-HBV), RG6004, GSK3228836, REP-2139, REP-2165, AB-729, VIR-2218, RG6346 (DCR-HBVS), JNJ-6379, GLS4, ABI-HO731, JNJ-440, NZ-4, RG7907, EDP-514, AB-423, AB-506, ABI-H03733 and ABI-H2158. In some embodiments, the oligonucleotide therapy is selected from a nucleic acid polymer or an S antigen transport inhibitory oligonucleotide polymer (NAP or STOPS), siRNA and ASO. In some embodiments, the oligonucleotide therapy is additional siNA. In some embodiments, the additional siNA is selected from any one of ds-siNA-001 to ds-siNA-092. In some embodiments, the oligonucleotide therapy is an antisense oligonucleotide (ASO). In some embodiments, the ASO is ASO 1 (SEQ ID NO: 95) or ASO 2 (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. WO2020 / 097342 and U.S. Publication No. 2020 / 0147124, both of which are incorporated herein by reference in their entirety. In some embodiments, 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.Exemplary CAMs are described in 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, each of which is incorporated herein by reference in its entirety. In some embodiments, the CAM is ALG-000184, ALG-001075, ALG-001024, JNJ-632, BAY41-4109, or NVR3-778. In some embodiments, siNA and the HBV therapeutic agent are administered simultaneously. In some embodiments, siNA and the HBV therapeutic agent are administered concurrently. In some embodiments, siNA and the HBV therapeutic agent are administered sequentially. In some embodiments, siNA is administered before the HBV therapeutic agent is administered. In some embodiments, siNA is administered after the HBV therapeutic agent is administered. In some embodiments, siNA and the HBV therapeutic agent are in separate containers. In some embodiments, siNA and the HBV therapeutic agent are in the same container.

[0545] Any method disclosed herein may further include administering a liver disease therapeutic agent to the subject. Any composition disclosed herein may further include a liver disease therapeutic agent. In some embodiments, the liver disease therapeutic agent is selected from peroxisome proliferator-activated receptor (PPAR) agonists, farnesoid X receptor (FXR) agonists, lipid-regulating agents, and incretin-based therapies. In some embodiments, the PPAR agonist is selected from PPARα agonists, PPARα / δ dual agonists, PPARγ agonists, and PPARα / γ dual agonists. In some embodiments, the PPARα dual agonist is a fibrate. In some embodiments, the PPARα / δ agonist is elarenolide. In some embodiments, the PPARγ agonist is a thiazolidinedione (TZD). In some embodiments, the TZD is pioglitazone. In some embodiments, the PPARα / γ dual agonist is saroglitazar. In some embodiments, the FXR agonist is obeticholic acid (OCA). In some embodiments, the lipid-regulating agent is aramhol. 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 vildagliptin. In some embodiments, siNA and the liver disease therapeutic agent are administered simultaneously. In some embodiments, siNA and the liver disease therapeutic agent are administered sequentially. In some embodiments, siNA is administered before the liver disease therapeutic agent is administered. In some embodiments, siNA is administered after the liver disease therapeutic agent is administered. In some embodiments, siNA and the liver disease therapeutic agent are in separate containers. In some embodiments, siNA and the liver disease therapeutic agent are in the same container.

[0546] Phosphoramidites

[0547] In addition to the disclosed oligonucleotides comprising novel oligonucleotide monomers, the present disclosure also provides phosphoramidites selected from the group consisting of: ( Where * is a chiral center),

[0548]

[0549] Those skilled in the art will appreciate that the disclosed phosphoramidites can be used to synthesize the disclosed nucleotide monomers.

[0550] definition

[0551] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present disclosure belongs. The following references provide general definitions of many of the terms used in the present invention for those skilled in the art: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd edition 1994); The Cambridge Dictionary of Science and Technology (Walker edited, 1988); The Glossary of Genetics, 5th edition, R. Rieger et al., (ed.), Springer Verlag (1991); and Hale & Marham, The HarperCollins Dictionary of Biology (1991). Unless otherwise stated, the following terms as used herein have the following meanings assigned to them. The terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the present disclosure.

[0552] As used herein, the terms "a" and "an" mean "one or more" and include the plural unless the context is inappropriate.

[0553] As used herein, the terms "patient" and "subject" refer to an organism to be treated by the methods of the present disclosure. Such an organism is preferably a mammal (e.g., marine, ape, equine, bovine, porcine, canine, feline, etc.), and more preferably a human.

[0554] As used herein, the term "effective amount" refers to an amount of a compound (e.g., siNA of the present disclosure) sufficient to achieve beneficial or desired results. 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.

[0555] As used herein, the term "treating" includes any effect that results in improvement of a condition, disease, disorder, or the like, or ameliorates the symptoms thereof, such as alleviating, reducing, modulating, improving, or eliminating.

[0556] As used herein, the terms "alleviate" and "alleviating" refer to reducing the severity of a condition, such as reducing the severity by, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%.

[0557] As used herein, the term "pharmaceutical composition" refers to the combination of an active agent and an inert or active carrier, making the composition particularly suitable for in vivo or ex vivo diagnostic or therapeutic use.

[0558] As used herein, the term "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, such as phosphate-buffered saline solution, water, emulsions (e.g., such as 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, e.g., Martin, Remington's Pharmaceutical Sciences, 15th Edition, Mack Publ. Co., Easton, PA

[1975] .

[0559] As used herein, the term "about" when referring to a measurable value (eg, weight, time, and dosage) is meant to encompass variations such as ±10%, ±5%, ±1%, or ±0.1% of the specified value.

[0560] As used herein, the term "nucleobase" refers to nitrogen-containing biological compounds that form nucleosides. Examples of nucleobases include, but are not limited to, thymine, uracil, adenine, cytosine, guanine, and analogs or derivatives thereof.

[0561] Throughout this specification, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that the compositions of the present disclosure also consist essentially of, or consist of, the recited components, and that processes and methods according to the present disclosure also consist essentially of, or consist of, the recited process steps.

[0562] Generally, compositions specified in percentage are by weight unless otherwise indicated. Additionally, if a variable is not accompanied by a definition, the preceding definition of the variable will prevail.

[0563] All publications and patents cited in this specification are incorporated herein by reference as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated herein by reference to disclose and describe the methods and / or materials associated with the cited publications. Citation of any publication is by virtue of 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.

[0564] Examples

[0565] Example 1: siNA synthesis

[0566] This example describes exemplary methods for synthesizing ds-siNAs, such as the siNAs disclosed in Tables 1-15 (as identified by ds-siNA IDs).

[0567] 2′-OMe phosphoramidite 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 phase support were purchased from Chemgenes Corp., MA.

[0568]

[0569] 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 Thermo Fischer, Milwaukee, WI, USA.

[0570]

[0571] All monomers are dried (P in the vacuum desiccator with desiccant O , room temperature 24h).The solid support (CPG) and the universal support that are attached to nucleosides are available from LGC and Chemgenes. Chemical and solvent for the synthesis of rear workflow are purchased from commercial sources such as VWR / Sigma, and can be used without any purification or processing. During the synthesis, solvent (acetonitrile) and solution (amides (amidite) and activator) are stored on molecular sieves.

[0572] Oligonucleotides were synthesized using standard oligonucleotide phosphoramidite chemistry starting from the 3′ residue of oligonucleotides preloaded on CPG supports on a DNA / RNA synthesizer (Expedite 8909 or ABI-394 or MM-48). Extended coupling of the solid-bound oligonucleotides with a 0.1 M solution of phosphoramidite in CH3CN in the presence of 5-(ethylthio)-1H-tetrazole activator, followed by standard capping, oxidation, and deprotection, yielded the modified oligonucleotides. 0.1 M I2, THF:pyridine:water (7:2:1) was used as the oxidant, while DDTT ((dimethylamino-methylene)amino)-3H-1,2,4-dithiazoline-3-thione) was used as the sulfur transfer agent for the synthesis of oligoribonucleotide phosphorothioates. Stepwise coupling efficiencies of greater than 98% were achieved for all modified phosphoramidites.

[0573]

[0574] Cleavage and deprotection:

[0575] Deprotection and cleavage from the solid support were achieved using a mixture of aminomethylamine (1:1, AMA) at 65°C for 15 min. When a universal linker was used, deprotection was performed at 65°C for 90 min, or the solid support was heated with ammonia (28%) solution at 55°C for 8-16 h to deprotect base-labile protecting groups.

[0576] Quantification or raw analysis of crude siNA

[0577] The samples were dissolved in deionized water (1.0 mL) and quantified as follows: First, a Thermo Scientific TM Nanodrop UV spectrophotometer or BioTek TM Epoch TM A blank was run on the plate reader with water only (2 ul) and then the oligonucleotide sample readings were obtained at 260 nm. The crude material was dried and stored at -20°C.

[0578] Crude HPLC / LC-MS analysis

[0579] Analyze 0.1 OD of the crude sample for crude HPLC and LC-MS analysis. After confirming the crude LC-MS data, then proceed with purification steps if necessary depending on the purity.

[0580] HPLC purification

[0581] Unconjugated and GalNac-modified oligonucleotides were purified by anion exchange HPLC. The buffers were 20 mM sodium phosphate in 10% CH3CN, pH 8.5 (buffer A) and 20 mM sodium phosphate in 10% CH3CN, 1.0 M NaBr, pH 8.5 (buffer B). Fractions containing full-length oligonucleotides were pooled.

[0582] Desalting of purified SiNA

[0583] 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, thoroughly dissolved in 2.5 mL of RNase-free water, was applied to the cartridge in a very slow, dropwise elution. The salt-free siNA was eluted directly into a screw-cap vial with 3.5 mL of deionized water. Alternatively, a Pall AcroPrep TM Some unconjugated siNA was desalted using a 3K MWCO desalting plate.

[0584] IEX HPLC and electrospray LC / MS analysis

[0585] Approximately 0.10 OD of siNA was dissolved in water and then transferred 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.

[0586] Duplex preparation:

[0587] Single-stranded oligonucleotides (sense and antisense strands) were annealed (at a molar ratio of 1:1, heated at 90°C for 2 minutes, and then gradually cooled at room temperature) to produce duplex ds-siNA. The final compound was analyzed by size exclusion chromatography (SEC).

[0588] Example 2: ds-siNA activity

[0589] This example studies the activity of the ds-siNA synthesized in Example 1.

[0590] Homo sapiens HepG2.2.15 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (ATCC 30-2002) and supplemented with 10% fetal calf serum (FCS). The cells were incubated at 37°C in 5% CO2 in a humidified incubator at atmospheric pressure. In order to transfect HepG2.2.15 cells with siRNA targeting HBV, the cells were seeded in 96-well conventional tissue culture plates at a density of 15,000 cells / well. Cells were transfected using RNAiMAX (Invitrogen / Life Technologies) according to the manufacturer's instructions. Dose-response experiments were performed with oligonucleotide concentrations of 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, and 0.07813 nM. For each siRNA treatment targeting HBV, four wells were transfected in parallel, and individual data points were collected from each well. After 24 hours of incubation with siRNA, the culture medium was removed and the cells were lysed and analyzed using a QuantiGene 2.0 branched DNA (bDNA) probe set specific for HBV genotype D (also known as hepatitis B virus subtype ayw, a complete genome of 3182 base pairs) as found in the cell line HepG2.2.15.

[0591] For each well, HBV target mRNA levels were normalized to GAPDH mRNA levels. As shown in Tables 9-17, the activity of HBV-targeting ds-siRNAs was expressed as EC50, which is a 50% reduction in the normalized HBV RNA level of the no-drug control. As shown in Tables 9-17, the cytotoxicity of HBV-targeting ds-siRNAs was expressed as CC50, which is a 50% reduction in GAPDH mRNA level of the no-drug control.

[0592] Example 3: Treatment of Hepatitis B Virus Infection with ds-siNA

[0593] 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 from Tables 1-8 (as identified by ds-siNA ID) and a pharmaceutically acceptable carrier is administered to a subject having hepatitis B virus. The ds-siNA from Tables 1-8 can be conjugated to N-acetylgalactosamine. The ds-siNA is administered by subcutaneous injection or intravenous infusion at a dose of 0.3 to 5 mg / kg every three weeks.

[0594] Example 4: siNA activity assay

[0595] This example provides exemplary methods for testing the activity of the siNAs disclosed herein.

[0596] In vitro assays:

[0597] HepG2.2.15 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (ATCC 30-2002) and supplemented with 10% fetal calf serum (FCS). The cells were incubated at 37°C in 5% CO2 in a humidified incubator at atmospheric pressure. In order to transfect HepG2.2.15 cells with siRNA targeting HBV, the cells were seeded in 96-well conventional tissue culture plates at a density of 15,000 cells / well. Cells were transfected using RNAiMAX (Invitrogen / Life Technologies) according to the manufacturer's instructions. Dose-response experiments were performed with oligonucleotide concentrations of 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, and 0.07813 nM. For each siRNA treatment targeting HBV (e.g., ds-siRNA, as identified by the ds-siNA ID in Tables 9-17), four wells were transfected in parallel, and a separate data point was collected from each well. After 24 hours of incubation with siRNA, the culture medium was removed and the cells were lysed and analyzed using a QuantiGene 2.0 branched DNA (bDNA) probe set specific for HBV genotype D (also known as hepatitis B virus subtype ayw, a complete genome of 3182 base pairs) as present in the cell line HepG2.2.15.

[0598] For each well, HBV target mRNA levels were normalized to GAPDH mRNA levels. As shown in Tables 19-33 and 36-40, the activity of HBV-targeting ds-siRNAs was expressed as EC50, which is a 50% reduction in the normalized HBV RNA level of the no-drug control. As shown in Tables 19-33 and 36-40, the cytotoxicity of HBV-targeting ds-siRNAs was expressed as CC50, which is a 50% reduction in GAPDH mRNA level of the no-drug control.

[0599] The off-target effects of certain siNAs were also measured. Table 34 shows that the IC50s of variants containing ganociclovir and danovir nucleotides increased off-target activity by more than 1000x compared to the control. Table 35 shows that the IC50s of variants containing a G analog and a vinyl phosphate 5' end cap further increased off-target activity compared to the control.

[0600] Table 19 - siNA containing N1 stabilized nucleotides

[0601]

[0602] Table 20 - siNA-1 containing 2'-ocp and 2'-omcp nucleotides

[0603]

[0604]

[0605] Table 21 - siNA-2 containing 2'-ocp and 2'-omcp nucleotides

[0606]

[0607] Table 22 - siNA-3 containing 2'-ocp and 2'-omcp nucleotides

[0608]

[0609] Table 23 - siNAs containing 2'-ocp and 2'-omcp nucleotides and conjugated moieties

[0610]

[0611]

[0612]

[0613] Table 24 - siNAs containing alternative 2'-ocp or 2'-omcp nucleotides

[0614]

[0615] Table 25 - siNAs containing 2'-2ocp or 2'-omcp nucleotides at the 3' and 5' ends of the antisense strand

[0616]

[0617] Table 26 - siNAs containing 2'-ocp or 2'-omcp nucleotides in the 3'-overhang of the antisense strand

[0618]

[0619] Table 27 - Modified duplex siNA containing high 2'-2ocp or 2'-omcp nucleotide content

[0620]

[0621] Table 28 - siNAs containing modified unlocked nucleotides on the antisense strand

[0622]

[0623] Table 29 - siNAs containing alternative 2'-fluoro nucleotide patterns

[0624]

[0625] Table 30 - siNA containing 5'-cyclopropyl nucleotides on the antisense strand

[0626]

[0627]

[0628] Table 31 - siNA containing 2'-F-3'-Xylo modified nucleotides on the sense or antisense strand

[0629]

[0630] Table 32 - siNA containing 2'F nucleotides on the sense or antisense strand

[0631]

[0632]

[0633] Table 33 - siNA containing ganciclovir, Denvir and 3'-ocp nucleotides on the sense or antisense strand

[0634]

[0635]

[0636] Table 34 - siNA Ganciclovir and Denovir modified nucleotides on the antisense strand

[0637]

[0638] Table 35 - siRNA G analogs with vinyl phosphate 5' end caps

[0639]

[0640] Table 36 - siNAs containing 2'-OMe-3'-Xylo modified nucleotides on the antisense strand

[0641]

[0642]

[0643] Table 37 - Additional si-NAs containing 2'-OMe-3'-Xylo modified nucleotides in the antisense

[0644]

[0645] Table 38 - siNA containing 2'-ocp, 2'-omcp and vmX nucleotides

[0646]

[0647]

[0648] Table 39 - siRNAs with TNA phosphonate chemistry

[0649]

[0650] Table 40 - siRNAs containing well-defined PS bonds

[0651]

[0652] Example 5 - In vivo activity of ds-siNA comprising a 5' vinylphosphonate moiety and a modified unlocked nucleotide.

[0653] On day -28 of the study, mice were infected with AAV-HBV. On day 0, the test ds-siNAs, ds-siNA-159, ds-siNA-160, ds-siNA-077, ds-siNA-078, or the negative control ds-siNA, ds-siNA-161, were administered subcutaneously as a single dose at 5 mg / kg. Serial blood samples were collected on days 0, 7, 14, 21, and 28. Serum HBsAg was measured by ELISA.

[0654] like Figure 9 As shown, the presence of a 5' vinylphosphonate moiety and modified unlocked nucleotide on the antisense strand of ds-siNA-077, ds-siNA-078 reduced activity compared to their counterparts lacking such modifications, ds-siNA-159, ds-siNA-160.

[0655] Example 6 - In vitro stability and in vivo activity of ds-siNA containing 5'-cyclopropyl nucleotides.

[0656] The stability of ds-siNA-085, ds-siNA-086, ds-siNA-087, ds-siNA-088, dssiNA-089, ds-siNA-090, ds-siNA-091, ds-siNA-092, and ds-siNA-159 was measured in mouse liver homogenate. The mouse liver was ground, and then 50 mg of the ground liver was transferred to each microcentrifuge tube set in 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 multi-oscillator and shaken at 40°C and 1,200 rpm for one hour. For each mL of homogenate, 40 μL of 100 mM MgCl2 and 40 μL of 100x antibiotics were added. The liver homogenate was stored at -20°C. On the day of the experiment, the required volume of liver homogenate was thawed and added to the oligonucleotides (the final oligonucleotide concentration in the matrix was 5 μM). A heated oscillator was used to incubate the microcentrifuge tubes at 37°C with gentle shaking (~400 rpm). 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 tubes, vortexed to mix after each addition. Solid phase extraction was then performed.

[0657] like Figure 10 As shown, 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. On day -28 of the study, mice were infected with AAV-HBV. On day 0, test ds-siNA ds-siNA-088 and ds-siNA-090, positive control ds-siNA-159, or vehicle were administered subcutaneously as a single dose at 5 mg / kg. Serial blood collections were performed on days 0, 7, 14, 21, and 28. Serum HBsAg was measured by ELISA.

[0658] like Figure 11 As shown, the higher durability and in vitro activity of ds-siNA-088 and ds-siNA-090 did not translate into in vivo performance, with HBsAg levels modestly higher than the positive control.

[0659] Example 7 - In vivo activity of ds-siNA containing a 3'OH and unlocked modified nucleotides on the antisense strand.

[0660] On day -28 of the study, mice were infected with AAV-HBV. On day 0, the test ds-siNAs ds-siNA-080 and ds-siNA-081, the negative control ds-siNA-083, the positive control ds-siNA-084, or the vehicle were administered subcutaneously as a single dose at 5 mg / kg. Serial blood samples were collected on days 0, 7, 14, 21, and 28. Serum HBsAg was measured by ELISA.

[0661] like Figure 12 As shown, 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.

[0662] Example 8 - In vivo activity of ds-siNA containing a 5′-end cap on the antisense strand

[0663] Mice were infected with AAV-HBV for the first 28 days of the study. On day 0, test ds-siNAds-siNA-162, control ds-siNA-151, or vehicle was administered subcutaneously as a single dose at 5 mg / kg. Serial blood samples were collected on days 0, 7, 14, 21, and 28. Serum HBsAg was measured by ELISA.

[0664] like Figure 13 As shown, the presence of a 5' end cap on the antisense strand of ds-siNA-162 provided similar in vivo activity compared to ds-siNA-151, which did not contain a 5' end cap.

[0665] Example 9 - Comparison of in vivo activity of ds-siNA analogs.

[0666] The stability of ds-siNA-159 and ds-siNA-159 analogs, which have a single nucleotide modification in the 3′ overhang of the antisense strand ds-siNA-009, was measured in mouse liver homogenate. The mouse liver was ground, and then 50 mg of the ground liver was transferred to each microcentrifuge tube set in 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 multi-oscillator and shaken at 40°C and 1,200 rpm for one hour. For each mL of homogenate, 40 μL of 100 mM MgCl2 and 40 μL of 100x antibiotics were added. The liver homogenate was stored at -20°C. On the day of the experiment, the required volume of liver homogenate was thawed and added to the oligonucleotides (the final oligonucleotide concentration in the matrix was 5 μM). The microcentrifuge tubes were incubated at 37°C with gentle shaking (~400 rpm) using 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 tubes, vortexing to mix after each addition. Solid phase extraction was then performed.

[0667] As shown in Figure 14A, ds-siNA-009 exhibited improved stability after 48 hours compared to the parental ds-siNA- 159. To determine whether the increased in vitro stability translated into improved in vivo activity, in vivo activity assays were performed.

[0668] On day -28 of the study, mice were infected with AAV-HBV. On day 0, test ds-siNAs, ds-siNA-159, ds-siNA-009, or vehicle were administered subcutaneously as a single dose at 5 mg / kg. Serial blood collections were performed on days 0, 5, and every 5 days thereafter until day 100. Serum HBsAg was measured by ELISA.

[0669] like Figure 14B As shown, ds-siNA-009 exhibited moderately reduced in vivo activity compared to the parental ds-siNA-159.

[0670] Example 10 - In vitro stability and in vivo activity of ds-siNA containing xylo-modified nucleotides.

[0671] The stability of ds-siNA-131 was measured in mouse liver homogenate. The mouse liver was ground, and then 50 mg of the ground liver was transferred to each microcentrifuge tube set in 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 multi-oscillator and shaken at 40 ° C and 1,200 rpm for one hour. For each mL of homogenate, 40 μL 100 mM MgCl2 and 40 μL 100x antibiotics were added. The liver homogenate was stored at -20 ° C. On the day of the experiment, the required volume of liver homogenate was thawed and added to the oligonucleotides (the final oligonucleotide concentration in the matrix was 5 μM). The microcentrifuge tubes were incubated at 37 ° C under slight shaking (~400 rpm) using a heated oscillator. 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 a microcentrifuge tube, vortexing to mix after each addition. Solid phase extraction was then performed.

[0672] As shown in Figure 15A, ds-siNA-131 exhibited increased stability after 48 hours compared to 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 for the first 28 days of the study. On day 0, the test ds-siNA ds-siNA-131, ds-siNA-009, or vehicle was administered subcutaneously as a single dose at 5 mg / kg. Serial blood samples were collected on days 0, 7, 14, and 21. Serum HBsAg was measured by ELISA.

[0673] like Figure 15B As shown, the increased stability of ds-siNA-131 translated into in vivo activity comparable to that of ds-siNA-009.

[0674] Example 11 - In vivo activity and in vitro stability of ds-siNA containing 2'F modified nucleotides.

[0675] On day -28 of the study, mice were infected with AAV-HBV. On day 0, test ds-siNAs ds-siNA-103, ds-siNA-104, ds-siNA-105, ds-siNA-084, ds-siNA-106, ds-siNA-108, ds-siNA-109, or vehicle were administered subcutaneously as a single dose at 5 mg / kg. Serial blood collections were performed on days 0, 5, and every 5 days thereafter until day 25. Serum HBsAg was measured by ELISA.

[0676] As shown in Figure 16A, ds-siNA-084 exhibited the best in vivo activity compared to the test ds-siNAs. Therefore, the in vitro stability of ds-siNA-084 was determined in mouse liver homogenate according to the previously described method. Figure 16B As can be seen, ds-siNA-084 exhibited increased stability after 48 hours compared to ds-siNA-159 and ds-siNA-009 in FIG. 15A .

[0677] The efficacy of ds-siNA-108 (which exhibited suboptimal physical activity in Figure 17A) was measured compared to Vir-2218. Test ds-siNAs ds-siNA-108, Vir-2218, or vehicle were administered as a single dose subcutaneously at 5 mg / kg on day 0 and every 14 days thereafter for 70 days. Serial blood collections were performed on day 0 and every 7 days thereafter until day 164. Serum HBsAg, HBeAg, and alanine aminotransferase (ALT) levels were determined by ELISA.

[0678] As shown in Figure 17. As shown in (A) HBsAg, ds-siNA-108 exhibited significantly increased activity compared to Vir-2218. As shown in (B), serum ALT levels were comparable between the tested ds-siNAs ds-siNA-108 and Vir-2218.

[0679] Example 12 - Comparison of the in vivo activity of ds-siNA analogs and HBV therapy Vir-2218.

[0680] On day -28 of the study, mice were infected with AAV-HBV. Test ds-siNAs ds-siNA-159, ds-siNA-084, Vir-2218, or vehicle were administered as a single dose subcutaneously at 5 mg / kg on day 0 and every 14 days thereafter for 70 days. Serial blood collections were performed on day 0 and every 7 days thereafter until day 168. Serum HBsAg, HBeAg, and alanine aminotransferase (ALT) levels were determined by ELISA.

[0681] As shown in Figure 18 , ds-siNA-159 and ds-siNA-084 exhibited significantly increased activity compared to Vir-2218, as shown in (A) HBsAg and (B) HBeAg levels. As shown in (C), serum ALT levels were comparable between the tested ds-siNAs ds-siNA-159 and Vir-2218. ds-siNA-084 showed a sudden increase in ALT after the first dose on day 0, which then returned to normal by day 14. Even repeated doses did not cause further ALT elevations.

[0682] Example 13 - 10X effective dose of ds-siNA does not produce ALT signal in uninfected mice.

[0683] To measure ALT, a very sensitive marker of drug hepatotoxicity, uninfected mice were subcutaneously administered 5 mg / kg, 15 mg / kg, or 50 mg / kg of the test ds-siNAds-siNA-084, or 5 mg / kg or 15 mg / kg of the control on day 0. Serial blood was collected on days 0, 7, and 14. Serum ALT levels were determined by ELISA.

[0684] like Figure 19 As shown, at 10X the effective dose, increasing doses of siNA-084 or Roche / Discerna did not significantly affect ALT levels. Figure 18C and 19 ds-siNA-084 showed ALT only in AAV-HBV-infected mice, suggesting that ALT may be immune-related, which may occur when siNA-084-activated mouse CD8+ T cells clear infected hepatocytes.

[0685] Example 14 - In vivo activity of ganciclovir and denavir modified ds-siNA.

[0686] On day -28 of the study, mice were infected with AAV-HBV. On day 0, test ds-siNAs ds-siNA-111, ds-siNA-112, ds-siNA-113, ds-siNA-116, control ds-siNA-084, or vehicle were administered subcutaneously as a single dose at 5 mg / kg. Serial blood samples were collected on days 0, 7, 14, and 21. Serum HBsAg and ALT levels were determined by ELISA.

[0687] As shown in FIG20 , 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).

[0688] Example 15 - In vitro stability and potency of ds-siNA comprising xylo-modified nucleotides.

[0689] The stability of parent ds-siNA-084 and xylo-modified ds-siNA-125 was measured in mouse liver homogenate. Mouse livers were ground, and 50 mg of ground liver was then transferred to each microcentrifuge tube set in 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 multi-oscillator and shaken at 40°C and 1,200 rpm for one hour. For each mL of homogenate, 40 μL of 100 mM MgCl2 and 40 μL of 100x antibiotics were added. The liver homogenate was stored at -20°C. On the day of the experiment, the required volume of liver homogenate was thawed and added to the oligonucleotides (the final oligonucleotide concentration in the matrix was 5 μM). The microcentrifuge tubes were incubated at 37°C with gentle shaking (~400 rpm) using a heated oscillator. 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 a microcentrifuge tube, vortexing to mix after each addition. Solid phase extraction was then performed.

[0690] As shown in Figure 21, the parental ds-siNA-084 (A) and the xylo-modified ds-siNA-125 (B) showed similar stability after 48 hours. The physical activity of ds-siNA-084 and ds-siNA-125 was analyzed. On day -28 of the study, mice were infected with AAV-HBV. On day 0, ds-siNA-084, ds-siNA-125, or vehicle was administered subcutaneously as a single dose at 5 mg / kg. Serial blood samples were collected on days 0, 7, 14, and 21. Serum HBsAg and ALT were measured by ELISA.

[0691] As shown in FIG22A , ds-siNA-125 maintained efficacy while reducing ALT ( Figure 22B ).

[0692] Example 16 - In vitro stability and bioactivity of ds-siNA containing a stereologically well-defined PS bond.

[0693] The stability of parental ds-siNA-143, ds-siNA-157, and ds-siNA-156 modified with PS(R) bonds was measured in mouse liver homogenate. Mouse livers were ground, and 50 mg of the ground liver was then transferred to each microcentrifuge tube set in 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 multi-oscillator and shaken at 40°C and 1,200 rpm for one hour. For each mL of homogenate, 40 μL of 100 mM MgCl2 and 40 μL of 100x antibiotic were added. The liver homogenate was stored at -20°C. On the day of the experiment, the required volume of liver homogenate was thawed and added to the oligonucleotides (the final oligonucleotide concentration in the matrix was 5 μM). The microcentrifuge tubes were incubated at 37°C with gentle shaking (~400 rpm) using 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 tubes, vortexing to mix after each addition. Solid phase extraction was then performed.

[0694] Figure 23 shows the stability of the sense and antisense strands of (A) parental ds-siNA-143, (B) ds-siNA-157 modified with PS(S) bonds, and (C) ds-siNA-156 modified with PS(R) bonds. The stability of the antisense strands of parental ds-siNA-143 and ds-siNA-157 modified with PS(S) bonds was comparable, while the stability of ds-siNA-156 modified with PS(R) bonds was less stable. The sense strands of all ds-siNAs included in the assay degraded within 48 hours.

[0695] Each ds-siNA was tested for in vivo activity. Mice were infected with AAV-HBV on day -28 of the study. On day 0, ds-siNA-143, ds-siNA-157, ds-siNA-156, or vehicle was administered subcutaneously as a single dose at 5 mg / kg. Serial blood collections were performed on days 0, 7, 14, and 21. Serum HBsAg was measured by ELISA. The results showed that ds-siNA-156 modified with PS(R) bonds showed slightly improved activity compared to the parent and s-siNA-157 modified with PS(S) bonds ( Figure 24 ).

[0696] Example 17 - In vivo activity of ds-siNA comprising denavir(S) and mun12 modified nucleotides.

[0697] On day -28 of the study, mice were infected with AAV-HBV. On day 0, test ds-siNAs ds-siNA-147, ds-siNA-148, parental ds-siNA-149, or vehicle were administered subcutaneously as a single dose at 5 mg / kg. Serial blood samples were collected on days 0, 7, 14, and 21. Serum HBsAg was measured by ELISA.

[0698] like Figure 25 As shown, ds-siNA-147 showed improved off-target profiles in Table 35, maintaining efficacy, whereas ds-siNA-148 had decreased efficacy.

[0699] Example 18: Preparation of Compound 40-9 (GalNAc4 amidite)

[0700] Compound 40-9 can be conjugated to any siNA as a targeting moiety disclosed herein. As shown in the figure below, the compound can be prepared according to the following short description.

[0701]

[0702] Building block compound 40-9 can be used to make examples of modified phosphorothioate oligonucleotides. Compound 40-9 was prepared as follows:

[0703] Preparation of compound 40-2: Ac2O (323.83 g, 3.17 mol, 297.09 mL, 11.4 eq) was added dropwise to a solution of commercially available glucosamine hydrochloride 40-1 (60 g, 278.25 mmol, 1 eq) in DCM (300 mL) at 0 ° C., followed by pyridine (300 mL) and DMAP (3.40 g, 27.83 mmol, 0.1 eq). The mixture was gradually warmed to 20 ° C. and stirred at 20 ° C. for 24 hours. After completion as monitored by LCMS, the mixture was concentrated under reduced pressure, diluted with DCM (900 mL), and extracted with NaHCO3 (saturated aqueous solution 300 mL * 3). The combined organic layer was washed with brine (300 mL), dried over Na2SO4, filtered and concentrated under reduced pressure to obtain compound 40-2 (89.5 g, crude product) as a yellow solid. 1H 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, J=4.0, 12.5Hz, 1H), 4.10-3.94 (m, 2H), 2.18 (s, 3H), 2.08 (s, 3H), 2.04 (d, J=4.0Hz, 6H), 1.93 (s, 3H; LCMS (ESI): m / z C 16 H 23 NaNO 10 of[M+Na] + Calculated value, 412.34, experimental value, 412.0).

[0704] Preparation of compound 40-3: TMSOTf (23.98 g, 107.87 mmol, 19.49 mL, 1.05 eq) was added dropwise to a solution of compound 40-2 (40 g, 102.73 mmol, 1 eq) in DCE (320 mL) at 25 ° C, and the mixture was stirred at 60 ° C for 4 hours. After 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 aqueous solution 300 mL * 2). The organic layer was washed with brine (300 mL), dried over Na SO, filtered and concentrated under reduced pressure to give compound 40-3 (32.5 g, crude product) as a yellow oil. 1 H 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).

[0705] Preparation of compound 40-4: To a mixture of compound 40-3 (32.5 g, 98.69 mmol, 1 eq) in DCM (250 mL) was added hex-5-ene-1-ol (11.86 g, 118.43 mmol, 13.96 mL, 1.2 eq) and 4AMS (32.5 g). The mixture was stirred at 30 ° C for 0.5 h, and then TMSOTf (13.16 g, 59.22 mmol, 10.70 mL, 0.6 eq) was added dropwise. The mixture was stirred at 30 ° C for...

Claims

1. An oligonucleotide comprising a nucleotide comprising a structure selected from the group consisting of: wherein B is a nucleobase, an aryl group, a heteroaryl group or H, and wherein and represent a phosphodiester bond, a phosphorothioate bond, a methylsulfonylphosphoramidate bond or H.

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

3. The oligonucleotide of claim 1 , wherein the nucleotide comprises a structure selected from the group consisting of: Know wherein B is a nucleobase, an aryl group, a heteroaryl group, or H, and wherein represents a phosphodiester bond, a phosphorothioate bond, a methylsulfonylphosphoramidate bond, or H.

4. The oligonucleotide according to any one of claims 1 to 3, wherein the oligonucleotide comprises at least 2, at least 3, at least 4 or at least 5 nucleotides comprising a structure independently selected from: wherein B is a nucleobase, an aryl group, a heteroaryl group or H, and wherein and represent a phosphodiester bond, a phosphorothioate bond, a methylsulfonylphosphoramidate bond or H.

5. An oligonucleotide comprising a nucleotide analogue, wherein the nucleotide analogue comprises the following structure: wherein B is a nucleobase, an aryl group, a heteroaryl group, or H; wherein represents a phosphodiester bond, a phosphorothioate bond, or H; and Where * indicates a chiral center. The nucleotide analogue according to claim 5 , wherein B is selected from adenine, guanine, cytosine, thymine and uracil.

7. The oligonucleotide of claim 5 or 6, wherein the oligonucleotide comprises at least 2, at least 3, at least 4 or at least 5 nucleotide analogues comprising structures independently selected from: wherein B is a nucleobase, an aryl group, a heteroaryl group, or H; wherein represents a phosphodiester bond, a phosphorothioate bond, or H; and wherein * represents a chiral center.

8. An oligonucleotide comprising a nucleotide comprising a structure selected from the group consisting of: wherein B is a nucleobase, an aryl group, a heteroaryl group, or H; wherein represents a phosphodiester bond, a phosphorothioate bond, or a methylsulfonylphosphoramidate bond.

9. An oligonucleotide comprising the following structure: wherein each B is independently selected from a nucleobase, an aryl group, a heteroaryl group, and H; wherein represents a phosphodiester bond, a phosphorothioate bond, or a methylsulfonylphosphoramidate bond.

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

11. A short interfering nucleic acid (siNA) comprising a sense strand and an antisense strand, wherein the sense strand, the antisense strand, or both comprise at least 1, at least 2, at least 3, at least 4, or at least 5 nucleotides independently selected from: or at least 1, at least 2, at least 3, at least 4 or at least 5 nucleotide analogs independently selected from: wherein B is a nucleobase, an aryl group, a heteroaryl group, or H; wherein represents a phosphodiester bond, a phosphorothioate bond, or H; and wherein * represents a chiral center.

12. A short interfering nucleic acid (siNA) 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: (ix) about 15 to 30 nucleotides in length; and (x) comprises 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 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, or 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 the RNA corresponding to the target gene, wherein the second nucleotide sequence: (xi) about 15 to 30 nucleotides in length; and (xii) comprises 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 a target gene, wherein the first nucleotide sequence: (i) 15 to 30 nucleotides in length; and (ii) comprises 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 the RNA corresponding to the target gene, wherein the second nucleotide sequence: (iii) 15 to 30 nucleotides in length; and (iv) comprises 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; wherein the sense strand and / or the antisense strand comprises at least 1, at least 2, at least 3, at least 4 or at least 5 nucleotides selected from: or at least 1, at least 2, at least 3, at least 4 or at least 5 nucleotide analogs selected from: wherein B is a nucleobase, an aryl group, a heteroaryl group, or H; wherein represents a phosphodiester bond, a phosphorothioate bond, or H; and wherein * represents a chiral center.

13. The siNA of claim 10 or 11, wherein the antisense strand comprises a 5' stabilizing end cap selected from the group consisting of: where R y is a nucleobase, and R 15 is H or CH3, and wherein represents a phosphodiester bond, a phosphorothioate bond, or a methylsulfonylphosphoramidate bond.

14. The siNA of claim 10 or 11, wherein the antisense strand 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 Formula (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, an aryl group, a heteroaryl group 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: Formula (71) to Formula (86), Formula (79X) to Formula (82X), Formula (79Y) to Formula (82Y), Formula 86X, Formula 86X′, Formula 86Y, and Formula 86Y′, wherein R x is a nucleobase, an aryl group, a heteroaryl group or H.

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

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 (21A)-(35A), formula (29B)-(32B), formula (29AX)-(32AX), formula (29AY)-(32AY), formula (29BX)-(32BX), and formula (29BY)-(32BY).

18. 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)-(86A), Formula (79XA)-(82XA), Formula (79YA)-(82YA); Formula (86XA), Formula (86X′A), Formula (86Y), and Formula (86Y′).

19. A short interfering nucleic acid (siNA) comprising a sense strand and an antisense strand, wherein the antisense strand comprises a 5' vinyl phosphonate moiety comprising the following structure: wherein each B is independently selected from a nucleobase, an aryl group, a heteroaryl group and H; wherein, represents a phosphodiester bond, a phosphorothioate bond or a methylsulfonylphosphoramidate bond.

20. The siNA of claim 18, wherein the structure is:

21. The siNA of claim 18, wherein the structure is:

22. The siNA of any one of claims 18 to 20, wherein the sense strand, the antisense strand, or both comprise at least 1, at least 2, at least 3, at least 4, or at least 5 nucleotides comprising a structure independently selected from: or at least 1, at least 2, at least 3, at least 4, or at least 5 nucleotide analogs comprising a structure independently selected from: wherein B is a nucleobase, an aryl group, a heteroaryl group, or H; wherein represents a phosphodiester bond, a phosphorothioate bond, or H; and wherein * represents a chiral center.

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

24. The siNA of any one of claims 10 to 22, wherein the siNA further comprises a phosphorylation blocker.

25. The siNA molecule of any one of claims 10 to 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.

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

27. The siNA molecule of any one of claims 10 to 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.

28. The siNA molecule of claim 26, wherein: (i) at least one phosphorothioate internucleoside linkage in 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 in 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 in the antisense strand is between the nucleotides at positions 1 and 2 from the 3′ end of the antisense strand; and / or (iv) at least one phosphorothioate internucleoside linkage is between the nucleotides at positions 2 and 3 from the 3′ end of the antisense strand.

29. The siNA molecule of any one of claims 10 to 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 methylsulfonylphosphoramidate internucleoside linkages.

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

31. The siNA molecule of any one of claims 10 to 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 methylsulfonylphosphoramidate internucleoside linkages.

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

33. The siNA molecule of any one of claims 10 to 31, wherein the sense strand, the antisense strand, or both independently comprise at least 1, at least 2, at least 3, at least 4, or at least 5 or more where R x is a nucleobase, an aryl group, a heteroaryl group or H), where R y is a nucleobase, where R y is a nucleobase, or a combination thereof.

34. The siNA of any one of claims 10 to 32, wherein the siNA further comprises galactosamine.

35. The siNA of claim 33, wherein the galactosamine is N-acetylgalactosamine (GalNAc) of formula (VI): in 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; L is a linker or L and Y in combination are a linker; and A is H, OH, a third protecting group, an activating group or an oligonucleotide.

36. The siNA of claim 33, wherein the galactosamine is N-acetylgalactosamine (GalNAc) of formula (VII): where R z is OH or SH; and each n is independently 1 or 2.

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

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

39. A siNA as 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.

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

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

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

43. The composition of claim 41, wherein the additional therapeutic agent is selected from the group consisting of nucleotide analogs, nucleoside analogs, capsid assembly modulators (CAMs), recombinant interferons, entry inhibitors, small molecule immunomodulators, and oligonucleotide therapies.

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

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

46. 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.

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

48. The method of claim 46, wherein the hepadnavirus is hepatitis B virus (HBV), and optionally wherein the HBV is selected from HBV genotypes AJ.

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

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

51. The method of claim 48 or 49, wherein the additional HBV therapeutic agent is selected from nucleotide analogs, nucleoside analogs, capsid assembly modulators (CAMs), recombinant interferons, entry inhibitors, small molecule immunomodulators, and oligonucleotide therapies.

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

53. The method of claim 44, wherein the disease is liver disease.

54. The method of claim 52, wherein the liver disease is non-alcoholic fatty liver disease (NAFLD) or hepatocellular carcinoma (HCC).

55. The method of claim 53, wherein the NAFLD is non-alcoholic steatohepatitis (NASH).

56. The method of any one of claims 52 to 54, further comprising administering to the subject a liver disease therapeutic agent.

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

58. The method of claim 56, wherein (i) the PPAR agonist is selected from a PPARα agonist, a PPARα / δ dual agonist, a PPARγ agonist, and a PPARα / γ dual agonist; (ii) the lipid-regulating 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.

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

60. The method of any one of claims 44 to 58, wherein the siNA or the 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.

61. The method of any one of claims 44 to 58, wherein the siNA or the composition is administered at a dose of between 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 The invention also provides a method for the administration of a dose of between 1 mg / kg and 15 mg / kg, 3 mg / kg and 10 mg / kg, 4 mg / kg and 50 mg / kg, 4 mg / kg and 40 mg / kg, 4 mg / kg and 30 mg / kg, 4 mg / kg and 20 mg / kg, 4 mg / kg and 15 mg / kg, 4 mg / kg and 10 mg / kg, 5 mg / kg and 50 mg / kg, 5 mg / kg and 40 mg / kg, 5 mg / kg and 30 mg / kg, 5 mg / kg and 20 mg / kg, 5 mg / kg and 15 mg / kg, or 5 mg / kg and 10 mg / kg.

62. 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.

63. The method of any one of claims 44 to 61, wherein the siNA or the 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.

64. The method of any one of claims 44 to 61, wherein the siNA or the 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.

65. The method of any one of claims 44 to 61, wherein said siNA or said composition is 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, 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.

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

67. The method of any one of claims 44 to 64, wherein the siNA or the composition is administered in six doses ranging from 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 3 days apart; wherein the second and third doses are optionally administered at least 4 days apart; and wherein the third and fourth doses, the fourth and fifth doses, and or the fifth and sixth doses are optionally administered at least 7 days apart.

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

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

70. The method of claim 67 or 68, wherein the viral vector is selected from the group consisting of 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.

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

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

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

74. Use according to claim 72, wherein the disease is a viral disease, optionally caused by a DNA virus or a double-stranded DNA (dsDNA) virus, or said disease.

75. Use according to claim 72, wherein the disease is a liver disease, optionally selected from non-alcoholic fatty liver disease (NAFLD) and hepatocellular carcinoma (HCC).

76. 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.

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

78. 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).

79. A short interfering nucleic acid (siNA) comprising a sense strand and an antisense strand, wherein the antisense strand comprises a 3' overhang, the 3' overhang comprising at least one modified nucleotide selected from wherein B is a nucleobase, an aryl group, a heteroaryl group or H, and wherein and represent a phosphodiester bond, a phosphorothioate bond, a methylsulfonylphosphoramidate bond or H.

80. The siNA of claim 78, wherein the modified nucleotides are selected from the group consisting of:

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

82. The siNA of any one of claims 78 to 80, wherein the siNA is resistant to nuclease activity relative to a siNA having the same sequence without the modified nucleotides in the 3' overhang.

83. A phosphoramidite comprising the structure:

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