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

Abstract

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

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 241,935, filed September 8, 2021, the disclosure of which is incorporated herein by reference in its entirety.

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

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

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

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

[0006] In a first aspect, the present disclosure provides a compound having the following structure: [ka] The present invention provides nucleic acid sequences and siNAs comprising any one of the foregoing nucleotides or combinations of these nucleotides.

[0007] In a second aspect, the present disclosure provides a compound having the following structure:

[0008] [ka] wherein Rx is a nucleobase, aryl, heteroaryl, or H. For example, the nucleotide may have the following structure: [ka] wherein R y is a nucleobase.

[0009] In a third aspect, the present disclosure provides a compound having the following structure: [ka] wherein R y is a nucleobase, and the nucleic acid sequence and siNA comprise the above nucleotides. In some embodiments, the nucleotides can comprise the following structures: [ka]

[0010] In a fourth aspect, the present disclosure provides a compound having the following structure: [ka] wherein R y is the nucleobase, and R 15 is H or CH 3 It is.

[0011] The present disclosure provides short interfering nucleic acid (siNA) molecules according to the first, second or third aspect, comprising at least one, at least two, at least three, at least four or at least five nucleotides, which may optionally be located in the seed region of the siNA and / or may be capable of destabilizing it. In some embodiments, the antisense strand is [ka] wherein R y is the nucleobase, and R 15 is H or CH 3 It is.

[0012] The present disclosure provides short interfering nucleic acid (siNA) molecules comprising a sense strand and an antisense strand, the antisense strand comprising at its 5' end a nucleotide phosphate mimic conforming to the fourth embodiment.

[0013] The present disclosure provides a short interfering nucleic acid (siNA) molecule comprising: (a) a sense strand comprising a first nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to an RNA corresponding to a target gene, wherein the first nucleotide sequence is 15-30 nucleotides in length, a sense strand comprising 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least one modified nucleotide is a 2'-O-methyl nucleotide and nucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and / or 19 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides or at least one modified nucleotide is a 2'-O-methyl nucleotide and at least one modified nucleotide is a 2'-fluoro nucleotide; an antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to an RNA corresponding to a target gene, the second nucleotide sequence being 15-30 nucleotides in length, an antisense strand comprising 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least one modified nucleotide is a 2'-O-methyl nucleotide and at least one modified nucleotide is a 2'-fluoro nucleotide; or (b) a sense strand comprising a first nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to an RNA corresponding to the target gene, wherein the first nucleotide sequence is (i) a length of 15 to 30 nucleotides; (ii) a sense strand comprising 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least one modified nucleotide is a 2'-O-methyl nucleotide and at least one modified nucleotide is a 2'-fluoro nucleotide; and an antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to an RNA corresponding to the target gene, wherein the second nucleotide sequence is (iii) 15 to 30 nucleotides in length; (iv) an antisense strand that 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 positions 2, 5, 6, 8, 10, 14, 16, 17, and / or 18 from the 5' end of the second nucleotide sequence is a 2'-fluoro nucleotide; The present invention provides a siNA molecule, wherein the sense strand and / or the antisense strand comprises at least one, at least two, at least three, at least four, or at least five nucleotides according to the first, second, or third aspect. In some embodiments, the antisense strand comprises

[0014] [ka] wherein R y is the nucleobase, and R 15 is H or CH 3 It is.

[0015] The present disclosure provides a short interfering nucleic acid (siNA) molecule comprising: (a) a sense strand comprising a first nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to an RNA corresponding to a target gene, wherein the first nucleotide sequence is 15-30 nucleotides in length, a sense strand comprising 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least one modified nucleotide is a 2'-O-methyl nucleotide and nucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and / or 19 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides or at least one modified nucleotide is a 2'-O-methyl nucleotide and at least one modified nucleotide is a 2'-fluoro nucleotide; an antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to an RNA corresponding to a target gene, the second nucleotide sequence being 15-30 nucleotides in length, an antisense strand comprising 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least one modified nucleotide is a 2'-O-methyl nucleotide and at least one modified nucleotide is a 2'-fluoro nucleotide; or (b) a sense strand comprising a first nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to an RNA corresponding to the target gene, wherein the first nucleotide sequence is (i) a length of 15 to 30 nucleotides; (ii) a sense strand comprising 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least one modified nucleotide is a 2'-O-methyl nucleotide and at least one modified nucleotide is a 2'-fluoro nucleotide; and an antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to an RNA corresponding to the target gene, wherein the second nucleotide sequence is (iii) 15 to 30 nucleotides in length; (iv) an antisense strand that 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 positions 2, 5, 6, 8, 10, 14, 16, 17, and / or 18 from the 5' end of the second nucleotide sequence is a 2'-fluoro nucleotide; A siNA molecule is provided in which the antisense strand comprises a nucleotide phosphate mimic according to the fourth aspect at its 5' end.

[0016] In some embodiments of the disclosed siNA molecules, the sense strand and / or the antisense strand independently comprise one or more phosphorothioate internucleoside linkages.

[0017] In some embodiments of the disclosed siNA molecules, the sense strand and / or the antisense strand independently contain one or more mesyl phosphoramidate internucleoside linkages.

[0018] In some embodiments of the disclosed siNA molecules, the siNA further comprises a phosphorylation blocker, a galactosamine, and / or a 5' stabilizing endcap.

[0019] In some embodiments of the disclosed siNA molecules, 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. In some embodiments, (i) at least one phosphorothioate internucleoside linkage of the sense strand is between nucleotides 1 and 2 positions from the 5' end of the first nucleotide sequence, and (ii) at least one phosphorothioate internucleoside linkage is between nucleotides 2 and 3 positions from the 5' end of the first nucleotide sequence.

[0020] In some embodiments of the disclosed siNA molecules, 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. In some embodiments, (i) at least one phosphorothioate internucleoside linkage of the antisense strand is between nucleotides 1 and 2 positions from the 5' end of the second nucleotide sequence, (ii) at least one phosphorothioate internucleoside linkage of the antisense strand is between nucleotides 2 and 3 positions from the 5' end of the second nucleotide sequence, (iii) at least one phosphorothioate internucleoside linkage of the antisense strand is between nucleotides 1 and 2 positions from the 3' end of the second nucleotide sequence, and / or (iv) at least one phosphorothioate internucleoside linkage is between nucleotides 2 and 3 positions from the 3' end of the second nucleotide sequence.

[0021] In some embodiments of the disclosed siNA molecules, the sense strand comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mesyl phosphoramidate internucleoside linkages. In some embodiments, (i) at least one mesyl phosphoramidate internucleoside linkage of the sense strand is between nucleotides 1 and 2 positions from the 5' end of the first nucleotide sequence, and (ii) at least one mesyl phosphoramidate internucleoside linkage is between nucleotides 2 and 3 positions from the 5' end of the first nucleotide sequence.

[0022] In some embodiments of the disclosed siNA molecules, the antisense strand further comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mesyl phosphoramidate internucleoside linkages. In some embodiments, (i) at least one mesyl phosphoramidate internucleoside linkage of the antisense strand is between nucleotides 1 and 2 positions from the 5' end of the second nucleotide sequence, (ii) at least one mesyl phosphoramidate internucleoside linkage of the antisense strand is between nucleotides 2 and 3' from the 5' end of the second nucleotide sequence, (iii) at least one mesyl phosphoramidate internucleoside linkage of the antisense strand is between nucleotides 1 and 2' from the 3' end of the second nucleotide sequence, and / or (iv) at least one mesyl phosphoramidate internucleoside linkage is between nucleotides 2 and 3 positions from the 3' end of the second nucleotide sequence.

[0023] The present disclosure further provides short interfering nucleic acids (siNAs) comprising a sense strand and an antisense strand, wherein the sense strand and / or the antisense strand independently comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more mesyl phosphoramidate internucleoside linkages.

[0024] In some embodiments of the disclosed siNA molecules, the antisense strand is selected from the group consisting of Formulas (1)-(16), (9X)-(12X), (16X), (9Y)-(12Y), (16Y), (21)-(36), (36X), (41)-(56), (49X)-(52X), (49Y)-(52Y), (56X), (56Y), (61), (62), and (63): [ka] [ka] [ka] [ka] wherein R x is a nucleobase, aryl, heteroaryl, or H.

[0025] In some embodiments of the disclosed siNA molecules, the antisense strand is selected from the group consisting of Formulas (71)-(86), (79X)-(82X), (79Y)-(82Y), 86X, 86X', 86Y, and 86Y': [ka] [ka] wherein R x is a nucleobase, aryl, heteroaryl, or H.

[0026] In some embodiments of the disclosed siNA molecules, the antisense strand comprises a 5' stabilized endcap selected from the group consisting of Formulas (1A)-(15A), Formulas (1A-1)-(7A-1), Formulas (1A-2)-(7A-2), Formulas (1A-3)-(7A-3), Formulas (1A-4)-(7A-4), Formulas (9B)-(12B), Formulas (9AX)-(12AX), Formulas (9AY)-(12AY), Formulas (9BX)-(12BX), and Formulas (9BY)-(12BY). [ka] [ka] [ka] [ka]

[0027] In some embodiments of the disclosed siNA molecules, the antisense strand comprises a 5' stabilized endcap selected from the group consisting of formulas (21A)-(35A), (29B)-(32B), (29AX)-(32AX), (29AY)-(32AY), (29BX)-(32BX), and (29BY)-(32BY). [ka] [ka]

[0028] In some embodiments of the disclosed siNA molecules, the antisense strand comprises a 5' stabilized endcap 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'). [ka] [ka]

[0029] In some embodiments of the disclosed siNA molecules, the siNA further comprises a galactosamine. In some embodiments, the galactosamine has the formula (VI): [ka] N-acetylgalactosamine (GalNAc) of the formula: m is 1, 2, 3, 4, or 5; each n is independently 1 or 2; p is 0 or 1; each R is independently H; each Y is independently selected from -OP(=O)(SH)-, -OP(=O)(O)-, -OP(=O)(OH)-, and -OP(S)S-; Z is H or a second protecting group; any L is a linker or L and Y together are a linker; A is H, OH, a third protecting group, an activating group, or an oligonucleotide.

[0030] In some embodiments of the disclosed siNA molecules, the galactosamine has formula (VII): [ka] N-acetylgalactosamine (GalNAc) of the formula z is OH or SH, and each n is independently 1 or 2.

[0031] In some embodiments of the disclosed siNA molecules, (i) at least one end of the siNA is blunt-ended, (ii) at least one end of the siNA comprises an overhang, the overhang comprising at least one nucleotide, or (iii) both ends of the siNA comprise an overhang, the overhang comprising at least one nucleotide.

[0032] In some embodiments of the disclosed siNA molecules, (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.

[0033] The present disclosure provides siNAs as shown in Tables 1, 2, 3, 4, and 5.

[0034] The present disclosure provides a composition comprising a siNA disclosed herein and a pharma- ceutically acceptable excipient. In some embodiments, the composition may further comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, or more siNAs disclosed herein. In some embodiments, the composition may further comprise an additional therapeutic agent. For example, the additional therapeutic agent is selected from a nucleotide analog, a nucleoside analog, a capsid assembly modulator (CAM), a recombinant interferon, an entry inhibitor, a small molecule immunomodulator, and an oligonucleotide therapy such as an additional siNA, an antisense oligonucleotide (ASO), a NAP, or STOPS™.

[0035] 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 a siNA disclosed herein. The present disclosure further provides for the use of the disclosed siNAs and compositions for treating a disease in a subject. The present disclosure further provides for the siNAs and compositions for use in treating a disease in a subject.

[0036] In some embodiments of the methods and uses of the present disclosure, the disease is a viral disease, optionally caused by a DNA virus or a double-stranded DNA (dsDNA) virus. In some embodiments, the dsDNA virus is a hepadnavirus. In some embodiments, the hepadnavirus is a Hepatitis B virus (HBV), optionally, the HBV is selected from HBV genotypes A-J. In some embodiments, the methods and uses may further comprise administering an additional HBV therapeutic agent. In some embodiments, the siNA or composition and the additional HBV therapeutic agent are administered simultaneously or sequentially. In some embodiments, the additional HBV therapeutic agent is selected from a nucleotide analog, a nucleoside analog, a capsid assembly modulator (CAM), a recombinant interferon, an entry inhibitor, a small molecule immune modulator, and an oligonucleotide therapy. In some embodiments, the viral disease is a disease caused by a coronavirus, optionally, the coronavirus is SARS-CoV-2.

[0037] In some embodiments of the disclosed methods and uses, the disease is a liver disease. In some embodiments, the liver disease is non-alcoholic fatty liver disease (NAFLD) or hepatocellular carcinoma (HCC). In some embodiments, the NAFLD is non-alcoholic steatohepatitis (NASH). Some embodiments may further comprise administering a liver disease treatment drug to the subject. In some embodiments, the liver disease treatment drug is selected from a peroxisome proliferator-activated receptor (PPAR) agonist, a farnesoid X receptor (FXR) agonist, a lipid-modifying agent, and an incretin system therapy. In some embodiments, (i) the PPAR agonist is selected from a PPARα agonist, a dual PPARα / δ agonist, a PPARγ agonist, and a dual PPARα / γ agonist, (ii) the lipid modifying agent is aramchol, or (iii) the incretin system therapy is a glucagon-like peptide 1 (GLP-1) receptor agonist or a dipeptidyl peptidase 4 (DPP-4) inhibitor. In some embodiments, the siNA or composition and the liver disease therapeutic agent are administered simultaneously or sequentially.

[0038] In some embodiments of the methods and uses of the disclosure, the siNA or composition is administered at a dose of at least 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, or 15 mg / kg.

[0039] In some embodiments of the disclosed methods and uses, the siNA or composition may be administered in an amount of from 0.5 mg / kg to 50 mg / kg, from 0.5 mg / kg to 40 mg / kg, from 0.5 mg / kg to 30 mg / kg, from 1 mg / kg to 50 mg / kg, from 1 mg / kg to 40 mg / kg, from 1 mg / kg to 30 mg / kg, from 1 mg / kg to 20 mg / kg, from 3 mg / kg to 50 mg / kg, from 3 mg / kg to 40 mg / kg, from 3 mg / kg to 30 mg / kg, from 3 mg / kg to 20 mg / kg, from 3 mg / kg to It is administered at a dose of 15mg / kg, 3mg / kg to 10mg / kg, 4mg / kg to 50mg / kg, 4mg / kg to 40mg / kg, 4mg / kg to 30mg / kg, 4mg / kg to 20mg / kg, 4mg / kg to 15mg / kg, 4mg / kg to 10mg / kg, 5mg / kg to 50mg / kg, 5mg / kg to 40mg / kg, 5mg / kg to 30mg / kg, 5mg / kg to 20mg / kg, 5mg / kg to 15mg / kg, or 5mg / kg to 10mg / kg.

[0040] In some embodiments of the methods and uses of the disclosure, the siNA or composition is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0041] In some embodiments of the methods and uses of the disclosure, the siNA or composition is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times 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.

[0042] In some embodiments of the methods and uses of the disclosure, the siNA or composition is administered at least once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days.

[0043] In some embodiments of the disclosed methods and uses, the siNA or composition is administered for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days, or for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 51, 52, 53, 54, or 55 weeks.

[0044] In some embodiments of the methods and uses of the present disclosure, the siNA or composition is administered at a single dose of 5 mg / kg or 10 mg / kg, three doses of 10 mg / kg once a week, three doses of 10 mg / kg once every three days, or five doses of 10 mg / kg once every three days.

[0045] In some embodiments of the methods and uses of the disclosure, the siNA or 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, where the first and second doses are optionally administered at least 3 days apart, the second and third doses are optionally administered at least 4 days apart, and the third and fourth doses, the fourth and fifth doses, and / or the fifth and sixth doses are optionally administered at least 7 days apart.

[0046] In some embodiments of the methods and uses of the present disclosure, the siNA or composition is administered in a particle or a viral vector, and the viral vector is optionally selected from adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpes simplex virus, lentivirus, measles virus, picornavirus, poxvirus, retrovirus, and rhabdovirus vectors.In some embodiments, the viral vector is a recombinant viral vector.In some embodiments, the viral vector is selected from AAVrh.74, AAVrh.10, AAVrh.20, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13.

[0047] In some embodiments of the methods and uses of the present disclosure, the siNA or composition is administered systemically or locally.

[0048] In some embodiments of the methods and uses of the disclosure, the siNA or composition is administered intravenously, subcutaneously, or intramuscularly.

[0049] The foregoing summary and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the present disclosure as claimed. Other objects, advantages, and novel features will become readily apparent to those skilled in the art from the following brief description of the drawings and detailed description of the present disclosure. [Brief description of the drawings]

[0050] [Figure 1] An exemplary siNA molecule is shown. [Diagram 2] An exemplary siNA molecule is shown. [Figure 3A-3D] An exemplary double-stranded siNA molecule is shown. [Figure 3E-3H] An exemplary double-stranded siNA molecule is shown. [Figure 4]A graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01), control 2, ds-siNA-009, or ds-siNA-010 is shown. [Figure 5A] A graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01), control 2, ds-siNA-017 (with the addition of GalNAc), or ds-siNA-018 (with the addition of GalNAc) is shown. [Figure 5B] Graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01), control 2, control 7, or control 8 is shown. [Figure 6] A graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01), control 2, ds-siNA-011, ds-siNA-012, or ds-siNA-013 is shown. [Figure 7] A graph showing the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01), control 2, ds-siNA-026, ds-siNA-027, ds-siNA-028, ds-siNA-029, ds-siNA-030, ds-siNA-031, or ds-siNA-032 is shown. [Figure 8] A graph showing the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01), control 2, ds-siNA-046, ds-siNA-047, ds-siNA-048, or ds-siNA-049 is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] Disclosed herein are novel modified nucleobase monomers that may contain unique chemical moieties in place of a base, may lack a bond between the 3' and 4' carbons of the central furanose ring (i.e., unlocked nucleotides), and / or may have phosphate mimetic groups (such nucleotides may hereinafter be referred to as "nucleotide phosphate mimics"). Also disclosed herein are short interfering nucleic acid (siNA) molecules that contain modified nucleobases (i.e., nucleotides).

[0052] In general, the siNA molecules described herein may be double-stranded siNA (ds-siNA) molecules. The siNA molecules described herein may comprise modified nucleotides selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides. The siNA molecules described herein may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more phosphorothioate internucleoside linkages. The siNA molecules described herein may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more mesyl phosphoramidate internucleoside linkages. The siNA molecules described herein may comprise at least one phosphorylation blocker. The siNA molecules described herein may comprise a 5' stabilized end cap (including, but not limited to, a nucleotide phosphate mimic of the present disclosure). The siNA molecules described herein may comprise a galactosamine. The siNA molecules described herein may comprise one or more blunt ends. The siNA molecules described herein may comprise one or more overhangs.

[0053] For example, the disclosure provides modified nucleotides comprising the following structure: [ka] (In the formula, R y are nucleobases), and [ka] A modified nucleotide having the structure x is a nucleobase, aryl, heteroaryl, or H. In some embodiments, the modified nucleotide has the following structure: [ka] wherein R yis a nucleobase. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0054] The present disclosure also provides nucleotide phosphate mimetics that can function as a stabilized end cap at the 5' end of the antisense strand of any of the siNAs of the present disclosure. Nucleotide phosphate mimetics of the present disclosure include, but are not limited to, the following structures: [ka] In the formula, R y is the nucleobase, and R 15 is H or CH 3 In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof. In some embodiments, nucleotide phosphate mimetics of the present disclosure include, but are not limited to, the following structures: [ka] [ka] [ka] [ka] [ka] and R 15 is H or CH 3 It is.

[0055] The short interfering nucleic acid (siNA) molecules of the present disclosure may contain at least one, at least two, at least three, at least four, or at least five of the above-mentioned modified nucleotides and / or one of the above-mentioned nucleotide phosphate mimetics at the 5' end of the antisense strand. Indeed, the short interfering nucleic acid (siNA) molecules of the present disclosure: (a) a sense strand comprising a first nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to an RNA corresponding to a target gene, wherein the first nucleotide sequence is (i) a length of 15 to 30 nucleotides; (ii) a sense strand comprising 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least one modified nucleotide is a 2'-O-methyl nucleotide and nucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and / or 19 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides, or at least one modified nucleotide is a 2'-O-methyl nucleotide and at least one modified nucleotide is a 2'-fluoro nucleotide; an antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to an RNA corresponding to a target gene, the second nucleotide sequence being (iii) 15 to 30 nucleotides in length; (iv) an antisense strand that 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 the target gene, wherein the first nucleotide sequence is (i) a length of 15 to 30 nucleotides; (ii) a sense strand that 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 an RNA corresponding to a target gene, the second nucleotide sequence being (iii) 15 to 30 nucleotides in length; (iv) an antisense strand that 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, 8, 10, 14, 16, 17, and / or 18 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides; or (c) a sense strand comprising a first nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to an RNA corresponding to the target gene, wherein the first nucleotide sequence is (i) a length of 15 to 30 nucleotides; (ii) a sense strand comprising 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least one modified nucleotide is a 2'-O-methyl nucleotide and nucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and / or 19 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides, or at least one modified nucleotide is a 2'-O-methyl nucleotide and at least one modified nucleotide is a 2'-fluoro nucleotide; an antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to an RNA corresponding to a target gene, the second nucleotide sequence being (iii) 15 to 30 nucleotides in length; (iv) an antisense strand that 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 positions 2, 5, 6, 8, 10, 14, 16, 17, and / or 18 from the 5' end of the second nucleotide sequence is a 2'-fluoro nucleotide; The sense strand and / or the antisense strand are [ka] wherein Rx is a nucleobase, aryl, heteroaryl, or H; and / or the antisense strand comprises at least one, at least two, at least three, at least four, or at least five modified nucleotides selected from [ka] (In the formula, R 15 is H or CH 3 Insofar as the nucleotide phosphomimetic is selected from

[0056] Additionally, the siNAs of the present disclosure may each independently comprise a sense strand and / or an antisense strand comprising one or more phosphorothioate internucleoside linkages, one or more mesyl phosphoramidate internucleoside linkages, or a combination thereof. The siNAs may comprise a phosphorylation blocker, a galactosamine, and / or a 5' stabilized end cap (other than those described above). The siNAs may be conjugated to a targeting moiety, such as a galactosamine.

[0057] Further disclosed herein are compositions comprising two or more of the siNA molecules described herein.

[0058] Further disclosed herein are compositions comprising any of the described siNA molecules and a pharma- ceutically acceptable carrier or diluent. Such compositions may also include an additional therapeutic agent or may be administered in conjunction (simultaneously or sequentially) with an additional therapeutic agent.

[0059] Further disclosed herein are compositions comprising two or more of the siNA molecules described herein for use as a medicament.

[0060] Further disclosed herein is a composition comprising any of the described siNA molecules and a pharma- ceutically acceptable carrier or diluent for use as a medicament. Such a medicament may also include an additional therapeutic agent or may be administered in conjunction with (simultaneously or sequentially) an additional therapeutic agent.

[0061] Further disclosed herein is a method of treating a disease in a subject in need thereof, comprising administering to the subject any of the siNA molecules (or combinations thereof) or compositions / medicines described herein.

[0062] Further disclosed herein is the use of any of the siNA molecules described herein (or a combination thereof) in the manufacture of a medicament for treating a disease.

[0063] Short interfering nucleic acid (siNA) molecules As mentioned above, the present disclosure provides siNA molecules that include modified nucleotides. Any of the siNA molecules described herein may be double-stranded siNA (ds-siNA) molecules. The terms "siNA molecule" and "ds-siNA molecule" may be used interchangeably. In some embodiments, the ds-siNA molecule comprises a sense strand and an antisense strand.

[0064] For purposes of this disclosure, a siNA molecule disclosed herein may generally comprise (a) at least one phosphorylation blocker, conjugation moiety, and / or 5' stabilized endcap, and (b) a short interfering nucleic acid (siNA). In some embodiments, the phosphorylation blocker is a phosphorylation blocker disclosed herein. In some embodiments, the conjugation moiety is a galactosamine disclosed herein. In some embodiments, the 5' stabilized endcap is a 5' stabilized endcap disclosed herein.

[0065] The siNA may comprise any of the first nucleotide, second nucleotide, sense strand, or antisense strand sequences disclosed herein. The siNA may comprise 5-100, 5-90, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-30, 10-25, 15-100, 15-90, 15-80, 15-70, 15-60, 15-50, 15-30, or 15-25 nucleotides. The siNA may 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 no more than 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 siNA may be single stranded (ss-siNA). The siNA may be double stranded (ds-siNA).

[0066] The ds-siNA comprises (a) a sense strand containing 15-30, 15-25, 15-24, 15-23, 15-22, 15-21, 17-30, 17-25, 17-24, 17-23, 17-22, 17-21, 18-30, 18-25, 18-24, 18-23, 18-22, 18-21, 19-30, 19-25, 19-24, 19-23, 19-22, 19-21, 20-25, 20-24, 20-23, 21-25, 21-24, or 21-23 nucleotides; and (b) a sense strand containing 15-30, 15-25, 15-25, 15-24, 15-23, 15-22, 15-21, 17-30, 17-25, 17-24, 17-23, 17-22, 17-21, 18-30, 18-25, 18-24, 18-23, 18-22, 18-21, 19-30, 19-25, 19-24, 19-23, 19-22, 19-21, 20-25, 20-24, 20-23, 21-25, 21-24, or 21-23 nucleotides. ) 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.

[0067] Any of the siNA molecules disclosed herein may further comprise one or more linkers independently selected from a phosphodiester (PO) linker, a phosphorothioate (PS) linker, a phosphorodithioate linker, a mesyl phosphoramidate (Ms), and a PS-mimetic linker. In some embodiments, the PS-mimetic linker is a sulfur linker. In some embodiments, the linker is an internucleoside linker. Alternatively or additionally, the linker may connect a nucleotide of the siNA molecule to at least one phosphorylation blocker, a conjugation moiety, or a 5'-stabilized endcap. In some embodiments, the linker connects a conjugation moiety to a phosphorylation blocker or a 5'-stabilized endcap.

[0068] An exemplary siNA molecule of the present disclosure is shown in Figure 1. As shown in Figure 1, the exemplary siNA molecule includes a sense strand (101) and an antisense strand (102). The sense strand (101) may include a first oligonucleotide sequence (103). The first oligonucleotide sequence (103) may include one or more phosphorothioate internucleoside linkages (109). The phosphorothioate internucleoside linkages (109) may be between nucleotides at the 5' or 3' end of the first oligonucleotide sequence (103). The phosphorothioate internucleoside linkages (109) may be between the first three nucleotides from the 5' end of the first oligonucleotide sequence (103). The first oligonucleotide sequence (103) may include one or more 2'-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 linkages (109). The phosphorothioate internucleoside linkages (109) may be between nucleotides at the 5' or 3' end of the second oligonucleotide sequence (104). The phosphorothioate internucleoside linkage (109) may be between the first three nucleotides from the 5' end of the second oligonucleotide sequence (104). The phosphorothioate internucleoside linkage (109) may be between the first three nucleotides from the 3' end of the second oligonucleotide sequence (104). The second oligonucleotide sequence (104) may include one or more 2'-fluoro nucleotides (110).The second oligonucleotide sequence (104) may comprise one or more 2'-O-methyl nucleotides (111). The second oligonucleotide sequence (104) may comprise 15 or more modified nucleotides independently selected from 2'-fluoro nucleotides (110) and 2'-O-methyl nucleotides (111). The antisense strand (102) may further comprise a 5' stabilized end cap (107). The siNA may further comprise one or more blunt ends. Alternatively, or in addition, one end of the siNA may comprise an overhang (108). The overhang (108) may be part of the sense strand (101). The overhang (108) may be part of the antisense strand (102). The overhang (108) may be different from the first nucleotide sequence (103). The overhang (108) may be different from the second nucleotide sequence (104). The overhang (108) may be part of the first nucleotide sequence (103). The overhang (108) may be part of the second nucleotide sequence (104). The overhang (108) may comprise one or more nucleotides. The overhang (108) may comprise one or more deoxyribonucleotides. The overhang (108) may comprise one or more modified nucleotides. The overhang (108) may comprise one or more modified ribonucleotides. The sense strand (101) may be shorter than the antisense strand (102). The sense strand (101) may be the same length as the antisense strand (102). The sense strand (101) may be longer than the antisense strand (102).

[0069] An exemplary siNA molecule of the present disclosure is shown in Figure 2. As shown in Figure 2, the exemplary siNA molecule includes a sense strand (201) and an antisense strand (202). The sense strand (201) may include a first oligonucleotide sequence (203). The first oligonucleotide sequence (203) may include one or more phosphorothioate internucleoside linkages (209). The phosphorothioate internucleoside linkages (209) may be between nucleotides at the 5' or 3' end of the first oligonucleotide sequence (203). The phosphorothioate internucleoside linkages (209) may be between the first three nucleotides from the 5' end of the first oligonucleotide sequence (203). The first oligonucleotide sequence (203) may include one or more 2'-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 linkages (209). The phosphorothioate internucleoside linkages (209) may be between nucleotides at the 5' or 3' end of the second oligonucleotide sequence (204). The phosphorothioate internucleoside linkage (209) may be between the first three nucleotides from the 5' end of the second oligonucleotide sequence (204). The phosphorothioate internucleoside linkage (209) may be between the first three nucleotides from the 3' end of the second oligonucleotide sequence (204). The second oligonucleotide sequence (204) may include one or more 2'-fluoro nucleotides (210).The second oligonucleotide sequence (204) may comprise one or more 2'-O-methyl nucleotides (211). The second oligonucleotide sequence (204) may comprise 15 or more modified nucleotides independently selected from 2'-fluoro nucleotides (210) and 2'-O-methyl nucleotides (211). The antisense strand (202) may further comprise a 5' stabilized end cap (207). The siNA may further comprise one or more overhangs (208). The overhang (208) may be part of the sense strand (201). The overhang (208) may be part of the antisense strand (202). The overhang (208) may be different from the first nucleotide sequence (203). The overhang (208) may be different from the second nucleotide sequence (204). The overhang (208) may be part of the first nucleotide sequence (203). The overhang (208) may be part of the second nucleotide sequence (204). The overhang (208) may be adjacent to the 3' end of the first nucleotide sequence (203). The overhang (208) may be adjacent to the 5' end of the first nucleotide sequence (203). The overhang (208) may be adjacent to the 3' end of the second nucleotide sequence (204). The overhang (208) may be adjacent to the 5' end of the second nucleotide sequence (204). The overhang (208) may comprise one or more nucleotides. The overhang (208) may comprise one or more deoxyribonucleotides. The overhang (208) may comprise a TT sequence. The overhang (208) may comprise one or more modified nucleotides. The overhang (208) may comprise one or more modified nucleotides disclosed herein (e.g., 2'-fluoro nucleotides, 2'-O-methyl nucleotides, 2'-fluoro nucleotide mimics, 2'-O-methyl nucleotide mimics, or nucleotides containing modified nucleobases). The overhang (208) may comprise one or more modified ribonucleotides. The sense strand (201) may be shorter than the antisense strand (202).The sense strand (201) may be the same length as the antisense strand (202). The sense strand (201) may be longer than the antisense strand (202).

[0070] 3A-3H show exemplary ds-siNA modification patterns. As shown in FIG. 3A-3G, an exemplary ds-siNA molecule may have the following formula: 5'-A n 1 B n 2 A n 3 B n 4 A n 5 B n 6 A n 7 B n 8 A n 9 -3' 3'-C q 1 A q 2 B q 3 A q 4 B q 5 A q 6 B q 7 A q 8 B q 9 A q 10 B q 11 A q 12 -5' During the ceremony, the top strand is a sense strand comprising a first nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to an RNA corresponding to a target gene, the first nucleotide sequence comprising 15 to 30 nucleotides; the lower strand is an antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to an RNA corresponding to a target gene, the second nucleotide sequence comprising 15 to 30 nucleotides; each A is independently a 2'-O-methyl nucleotide, or a nucleotide containing a 5' stabilized end cap or phosphorylation blocker; B is a 2'-fluoro nucleotide; C represents an overhanging nucleotide and is a 2'-O-methyl nucleotide, a deoxynucleotide, or uracil; n 1 = 1 to 6 nucleotides in length, each n 2 , n 6 , n 8 , q 3 , q 5 , q 7 , q 9 , q 11 , and q 12 are independently 0 to 1 nucleotides in length, each n 3 and n 4 are independently 1 to 3 nucleotides in length; n 5 is 1 to 10 nucleotides in length, n 7 is 0 to 4 nucleotides in length, each n 9 , q 1 , and q 2 are independently 0 to 2 nucleotides in length, q 4 is 0 to 3 nucleotides in length, q 6 is 0 to 5 nucleotides in length, q 8 is 2 to 7 nucleotides in length, q 10 However, they are between 2 and 11 nucleotides in length. The ds-siNA may further comprise a conjugation moiety. The conjugation moiety may comprise any of the galactosamines disclosed herein. The ds-siNA may further comprise (i) a phosphorothioate internucleoside linkage between nucleotides at positions 1 and 2 and 2 and 3 from the 5' end of the sense strand, and (ii) a phosphorothioate internucleoside linkage between nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 from the 5' end of the antisense strand. The ds-siNA may further comprise a 5' stabilizing endcap. The 5' stabilizing endcap may be a vinyl phosphonate. The 5' stabilizing endcap may be attached to the 5' end of the antisense strand. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the sense strand is further modified to contain a 5' stabilizing endcap. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the antisense strand is further modified to contain a 5' stabilized 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. Exemplary ds-siNA molecules may have the formula: 5'-A 2-4 B 1 A 1-3 B 2-3 A 2-10 B 0-1 A 0-4 B 0-1 A 0-2 -3' 3'-C 2 A 0-2 B 0-1 A0-3 B 0-1 A 0-5 B 0-1 A 2-7 B 1 A 2-11 B 1 A 1 -5' During the ceremony, the top strand is a sense strand comprising a first nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to an RNA corresponding to a target gene, the first nucleotide sequence comprising 15 to 30 nucleotides; the lower strand is an antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to an RNA corresponding to a target gene, the second nucleotide sequence comprising 15 to 30 nucleotides; each A is independently a 2'-O-methyl nucleotide, or a nucleotide containing a 5' stabilized end cap or phosphorylation blocker; B is a 2'-fluoro nucleotide; C represents an overhanging nucleotide and is a 2'-O-methyl nucleotide, a deoxynucleotide, or uracil; The ds-siNA may further comprise a conjugation moiety. The conjugation moiety may comprise any of the galactosamines disclosed herein. The ds-siNA may further comprise (i) phosphorothioate internucleoside linkages between nucleotides at positions 1 and 2 and 2 and 3 from the 5' end of the sense strand, and (ii) phosphorothioate internucleoside linkages between nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 from the 5' end of the antisense strand. The ds-siNA may further comprise a 5' stabilizing end cap. The 5' stabilizing end cap may be a vinyl phosphonate. The vinyl phosphonate may be a deuterated vinyl phosphonate. The deuterated vinyl phosphonate may be a mono-deuterated vinyl phosphonate. The deuterated vinyl phosphonate may be a mono-di-deuterated vinyl phosphonate. The 5' stabilized end cap may be attached to the 5' end of the antisense strand. The 5' stabilized end cap may be attached to the 3' end of the antisense strand. The 5' stabilized end cap may be attached to the 5' end of the sense strand. The 5' stabilized end cap may be attached to the 3' end of the sense strand. In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 5' end of the sense strand is further modified to contain a 5' stabilized end cap. In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 5' end of the antisense strand is further modified to contain a 5' stabilized end cap. In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 5' end of the sense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 3' end of the sense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 5' end of the antisense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 3' end of the antisense strand is further modified to contain a phosphorylation blocker.

[0071] The exemplary ds-siNA shown in Figures 3A-3H includes (i) a sense strand including 19-21 nucleotides and (ii) an antisense strand including 21-23 nucleotides. The ds-siNA may optionally further include (iii) a conjugation moiety, where the conjugation moiety (e.g., GalNAc shown as G3 in Figures 3A-3G) is attached to the 3' or 5' end of the sense or antisense strand. The ds-siNA may include a two nucleotide overhang consisting of nucleotides 20 and 21 from the 5' end of the antisense strand. The ds-siNA may include a two nucleotide overhang consisting of nucleotides 22 and 23 from the 5' end of the antisense strand. The ds-siNA may further comprise 1, 2, 3, 4, 5, 6 or more phosphorothioate (ps) internucleoside linkages or mesyl phosphoramidate internucleoside linkages (Ms). At least one phosphorothioate internucleoside linkage or mesyl phosphoramidate internucleoside linkage (Ms) may be between nucleotides 1 and 2 positions or 2 and 3 positions from the 5' end of the sense strand. At least one phosphorothioate internucleoside linkage or mesyl phosphoramidate internucleoside linkage (Ms) may be between nucleotides 1 and 2 positions or 2 and 3 positions from the 5' end of the antisense strand. At least one phosphorothioate internucleoside linkage or mesyl phosphoramidate internucleoside linkage (Ms) may be between nucleotides at positions 19 and 20, 20 and 21, 21 and 22, or 22 and 23 from the 5' end of the antisense strand. As shown in Figures 3A-3H, 4-6 nucleotides in the sense strand may be 2'-fluoro nucleotides. As shown in Figures 3A-3H, 2-5 nucleotides in the antisense strand may be 2'-fluoro nucleotides. As shown in Figures 3A-3H, 13-15 nucleotides in the sense strand may be 2'-O-methyl nucleotides. As shown in Figures 3A-3H, 14-19 nucleotides in the antisense strand may be 2'-O-methyl nucleotides.As shown in Figures 3A-3H, ds-siNA does not contain a base pair between 2'-fluoro nucleotides on the sense strand and the antisense strand. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the sense strand is further modified to contain a 5' stabilized 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 contain a 5' stabilized 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 5' end of the antisense strand is further modified to contain a phosphorylation blocker.

[0072] As shown in Figure 3A, the ds-siNA may comprise (a) a sense strand of 19 nucleotides, where 2'-fluoro nucleotides are located at positions 3, 7-9, 12, and 17 from the 5' end of the sense strand, and 2'-O-methyl nucleotides are located at positions 1, 2, 4-6, 10, 11, 13-16, 18, and 19 from the 5' end of the sense strand, and (b) an antisense strand of 21 nucleotides, where the nucleotides at positions 2 and 14 from the 5' end of the antisense strand are 2'-fluoro nucleotides, and the nucleotides at positions 1, 3-13, and 15-21 from the 5' end of the antisense strand are 2'-O-methyl nucleotides. The ds-siNA may further comprise a conjugated moiety attached to the 3' end of the sense strand. The ds-siNA may further comprise (i) phosphorothioate internucleoside linkages between nucleotides at positions 1 and 2 and 2 and 3 from the 5' end of the sense strand, and (ii) phosphorothioate internucleoside linkages between nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 from the 5' end of the antisense strand. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the sense strand is further modified to contain a 5' stabilized 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 contain a 5' stabilized 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.In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 5' end of the sense strand is 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-mun nucleotide, a d2vm nucleotide, or a d2vmA nucleotide. In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 5' end of the antisense strand is 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-mun nucleotide, a d2vm nucleotide, or a d2vmA nucleotide. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 3' end of the sense strand is 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-mun nucleotide, a d2vm nucleotide, or a d2vmA nucleotide. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 3' end of the antisense strand is 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-mun 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 or antisense strand are 2'-fluoro nucleotide mimics. In some embodiments, at least 1, 2, 3, 4, or more of the 2'-fluoro nucleotides on the sense or antisense strand are fB, fN, f(4nh)Q, f4P, f2P, or fX nucleotides.In some embodiments, at least one, two, three, four, or more 2'-O-methyl nucleotides on the sense or antisense strand are 2'-O-methyl nucleotide mimics. In some embodiments, one or more nucleotides in the sense and / or antisense strand may be a 3',4' seco modified nucleotide in which the bond between the 3' and 4' positions of the furanose ring is broken (e.g., mun34).

[0073] As shown in Figure 3B, the ds-siNA may comprise (a) a sense strand of 19 nucleotides, where 2'-fluoro nucleotides are located at positions 3, 7, 8, and 17 from the 5' end of the sense strand, and 2'-O-methyl nucleotides are located at positions 1, 2, 4-6, 9-16, 18, and 19 from the 5' end of the sense strand, and (b) an antisense strand of 21 nucleotides, where nucleotides 2 and 14 from the 5' end of the antisense strand are 2'-fluoro nucleotides, and nucleotides 1, 3-13, and 15-21 from the 5' end of the antisense strand are 2'-O-methyl nucleotides. The ds-siNA may further comprise a conjugated moiety attached to the 3' end of the sense strand. The ds-siNA may further comprise (i) phosphorothioate internucleoside linkages between nucleotides at positions 1 and 2 and 2 and 3 from the 5' end of the sense strand, and (ii) phosphorothioate internucleoside linkages between nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 from the 5' end of the antisense strand. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the sense strand is further modified to contain a 5' stabilized 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 contain a 5' stabilized 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.In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 5' end of the sense strand is 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-mun nucleotide, a d2vm nucleotide, or a d2vmA nucleotide. In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 5' end of the antisense strand is 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-mun nucleotide, a d2vm nucleotide, or a d2vmA nucleotide. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 3' end of the sense strand is 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-mun nucleotide, a d2vm nucleotide, or a d2vmA nucleotide. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 3' end of the antisense strand is 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-mun 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 or antisense strand are 2'-fluoro nucleotide mimics. In some embodiments, at least 1, 2, 3, 4, or more of the 2'-fluoro nucleotides on the sense or antisense strand are fB, fN, f(4nh)Q, f4P, f2P, or fX nucleotides.In some embodiments, at least one, two, three, four, or more 2'-O-methyl nucleotides on the sense or antisense strand are 2'-O-methyl nucleotide mimics. In some embodiments, one or more nucleotides in the sense and / or antisense strand may be a 3',4' seco modified nucleotide in which the bond between the 3' and 4' positions of the furanose ring is broken (e.g., mun34).

[0074] As shown in Figure 3C, the ds-siNA may comprise (a) a sense strand of 19 nucleotides, where 2'-fluoro nucleotides are at positions 3, 7-9, 12, and 17 from the 5' end of the sense strand, and 2'-O-methyl nucleotides are at positions 1, 2, 4-6, 10, 11, 13-16, 18, and 19 from the 5' end of the sense strand, and (b) an antisense strand of 21 nucleotides, where the nucleotides in the antisense strand comprise an alternating 1:3 modification pattern, where one nucleotide is a 2'-fluoro nucleotide and three nucleotides are 2'-O-methyl nucleotides. The ds-siNA may further comprise a conjugation 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 and 2 and 3 from the 5' end of the sense strand, and (ii) phosphorothioate internucleoside linkages between nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 from the 5' end of the antisense strand. The ds-siNA may comprise 2-5 alternating 1:3 modification patterns on the antisense strand. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the sense strand is further modified to contain a 5' stabilizing endcap. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the antisense strand is further modified to contain a 5' stabilizing endcap. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the sense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at the 1 position 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 the 1 position from the 5' end of the antisense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 3' end of the antisense strand is further modified to contain a phosphorylation blocker.In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 5' end of the sense strand is 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-mun nucleotide, a d2vm nucleotide, or a d2vmA nucleotide. In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 5' end of the antisense strand is 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-mun nucleotide, a d2vm nucleotide, or a d2vmA nucleotide. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 3' end of the sense strand is 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-mun nucleotide, a d2vm nucleotide, or a d2vmA nucleotide. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 3' end of the antisense strand is 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-mun 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 or antisense strand are 2'-fluoro nucleotide mimics. In some embodiments, at least 1, 2, 3, 4, or more of the 2'-fluoro nucleotides on the sense strand are fB, fN, f(4nh)Q, f4P, f2P, or fX nucleotides.In some embodiments, at least one, two, three, four, or more 2'-fluoro nucleotides on the antisense strand are fB, fN, f(4nh)Q, f4P, f2P, or fX nucleotides. In some embodiments, at least one, two, three, four, or more 2'-O-methyl nucleotides on the sense or antisense strand are 2'-O-methyl nucleotide mimics. In some embodiments, one or more nucleotides in the sense and / or antisense strand may be a 3',4' seco modified nucleotide in which the bond between the 3' and 4' positions of the furanose ring is broken (e.g., mun34).

[0075] As shown in Figure 3D, the ds-siNA may comprise (a) a sense strand of 19 nucleotides, where 2'-fluoro nucleotides are at positions 5 and 7-9 from the 5' end of the sense strand, and 2'-O-methyl nucleotides are at positions 1-4, 6, and 10-19 from the 5' end of the sense strand, and (b) an antisense strand of 21 nucleotides, where the nucleotides in the antisense strand comprise an alternating 1:3 modification pattern, where one nucleotide is a 2'-fluoro nucleotide and three nucleotides are 2'-O-methyl nucleotides. The ds-siNA may further comprise a conjugation 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 and 2 and 3 from the 5' end of the sense strand, and (ii) phosphorothioate internucleoside linkages between nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 from the 5' end of the antisense strand. The ds-siNA may comprise 2-5 alternating 1:3 modification patterns on the antisense strand. The alternating 1:3 modification patterns may begin at any of nucleotide positions 2, 6, 10, 14, and / or 18 from the 5' end of the antisense strand. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the sense strand is further modified to contain a 5' stabilizing endcap. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the antisense strand is further modified to contain a 5' stabilizing endcap. In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 5' end of the sense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 3' end of the sense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 5' end of the antisense strand is further modified to contain a phosphorylation blocker.In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 3' end of the antisense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 5' end of the sense strand is 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-mun nucleotide, a d2vm nucleotide, or a d2vmA nucleotide. In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 5' end of the antisense strand is 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-mun nucleotide, a d2vm nucleotide, or a d2vmA nucleotide. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 3' end of the sense strand is 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-mun nucleotide, a d2vm nucleotide, or a d2vmA nucleotide. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 3' end of the antisense strand is 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-mun 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 or antisense strand are 2'-fluoro nucleotide mimics.In some embodiments, at least 1, 2, 3, 4, or more 2'-fluoro nucleotides on the sense strand are 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 fB, fN, f(4nh)Q, f4P, f2P, or fX nucleotides. In some embodiments, at least 1, 2, 3, 4, or more 2'-O-methyl nucleotides on the sense or antisense strand are 2'-O-methyl nucleotide mimics. In some embodiments, one or more nucleotides in the sense and / or antisense strand may be 3',4' seco modified nucleotides in which the bond between the 3' and 4' positions of the furanose ring is broken (e.g., mun34).

[0076] As shown in Figure 3E, the ds-siNA may comprise (a) a sense strand of 19 nucleotides, where 2'-fluoro nucleotides are at positions 5 and 7-9 from the 5' end of the sense strand, and 2'-O-methyl nucleotides are at positions 1-4, 6, and 10-19 from the 5' end of the sense strand, and (b) an antisense strand of 21 nucleotides, where the nucleotides in the antisense strand comprise an alternating 1:2 modification pattern, where one nucleotide is a 2'-fluoro nucleotide and two nucleotides are 2'-O-methyl nucleotides. The ds-siNA may further comprise a conjugation 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 and 2 and 3 from the 5' end of the sense strand, and (ii) phosphorothioate internucleoside linkages between nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 from the 5' end of the antisense strand. The ds-siNA may comprise 2-5 alternating 1:2 modification patterns on the antisense strand. The alternating 1:2 modification patterns may begin at any of nucleotide positions 2, 5, 8, 14, and / or 17 from the 5' end of the antisense strand. In some embodiments, the ds-siNA comprises (a) a sense strand of 19 nucleotides, where the 2'-fluoro nucleotides are at positions 5 and 7-9 from the 5' end of the sense strand, and the 2'-O-methyl nucleotides are at positions 1-4, 6, and 10-19 from the 5' end of the sense strand, and (b) an antisense strand of 21 nucleotides, where the 2'-fluoro nucleotides are at positions 2, 5, 8, 14, and 17 from the 5' end of the antisense strand, and the 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 sense strand. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the sense strand is further modified to contain a 5' stabilized end cap.In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 5' end of the antisense strand is further modified to contain a 5' stabilized end cap. In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 5' end of the sense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 3' end of the sense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 5' end of the antisense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 3' end of the antisense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 5' end of the sense strand is 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-mun nucleotide, a d2vm nucleotide, or a d2vmA nucleotide. In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 5' end of the antisense strand is 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-mun nucleotide, a d2vm nucleotide, or a d2vmA nucleotide. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 3' end of the sense strand is 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-mun nucleotide, a d2vm nucleotide, or a d2vmA nucleotide.In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 3' end of the antisense strand is 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-mun nucleotide, a d2vm nucleotide, or a d2vmA nucleotide. In some embodiments, at least one, two, three, four, or more 2'-fluoro nucleotides on the sense strand or the antisense strand are 2'-fluoro nucleotide mimics. In some embodiments, at least one, two, three, four, or more 2'-fluoro nucleotides on the sense strand are fB, fN, f(4nh)Q, f4P, f2P, or fX nucleotides. In some embodiments, at least one, two, three, four, or more 2'-fluoro nucleotides on the antisense strand are fB, fN, f(4nh)Q, f4P, f2P, or fX nucleotides. In some embodiments, at least one, two, three, four, or more 2'-O-methyl nucleotides on the sense or antisense strand are 2'-O-methyl nucleotide mimics. In some embodiments, one or more nucleotides in the sense and / or antisense strand may be a 3',4' seco modified nucleotide in which the bond between the 3' and 4' positions of the furanose ring is broken (e.g., mun34).

[0077] As shown in Figure 3F, the ds-siNA may comprise (a) a sense strand of 19 nucleotides, with 2'-fluoro nucleotides at positions 5 and 7-9 from the 5' end of the sense strand and 2'-O-methyl nucleotides at positions 1-4, 6, and 10-19 from the 5' end of the sense strand, and (b) an antisense strand of 21 nucleotides, with 2'-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand and 2'-O-methyl nucleotides at positions 1, 3-5, 7-13, 15, and 17-21 from the 5' end of the antisense strand. The ds-siNA may further comprise a conjugated moiety attached to the 3' end of the sense strand. The ds-siNA may further comprise (i) phosphorothioate internucleoside linkages between nucleotides at positions 1 and 2 and 2 and 3 from the 5' end of the sense strand, and (ii) phosphorothioate internucleoside linkages between nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 from the 5' end of the antisense strand. In some embodiments, at least 1, 2, 3, 4, or more 2'-fluoro nucleotides on the sense or antisense strand are 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 sense or antisense strand are f4P nucleotides. In some embodiments, at least 1, 2, 3, or 4 of the 2'-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand are f4P nucleotides. In some embodiments, at least one of the 2'-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand is an f4P nucleotide. In some embodiments, at least two of the 2'-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand are f4P nucleotides. In some embodiments, no more than three of the 2'-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand are f4P nucleotides.In some embodiments, no more than two of the 2'-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand are f4P nucleotides. In some embodiments, the 2'-fluoro nucleotide at position 2 from the 5' end of the antisense strand is an f4P nucleotide. In some embodiments, the 2'-fluoro nucleotide at position 6 from the 5' end of the antisense strand is an f4P nucleotide. In some embodiments, the 2'-fluoro nucleotide at position 14 from the 5' end of the antisense strand is an f4P nucleotide. In some embodiments, the 2'-fluoro nucleotide at position 16 from the 5' end of the antisense strand is an f4P nucleotide. In some embodiments, at least one, two, three, four, or more 2'-fluoro nucleotides on the sense or antisense strand are f2P nucleotides. In some embodiments, at least one, two, three, or four of the 2'-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand are f2P nucleotides. In some embodiments, at least one of the 2'-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand is an f2P nucleotide. In some embodiments, at least two of the 2'-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand are f2P nucleotides. In some embodiments, no more than three of the 2'-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand are f2P nucleotides. In some embodiments, no more than two of the 2'-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand are f2P nucleotides. In some embodiments, the 2'-fluoro nucleotide at position 2 from the 5' end of the antisense strand is an f2P nucleotide. In some embodiments, the 2'-fluoro nucleotide at position 6 from the 5' end of the antisense strand is an f2P nucleotide. In some embodiments, the 2'-fluoro nucleotide at position 14 from the 5' end of the antisense strand is an f2P nucleotide.In some embodiments, the 2'-fluoro nucleotide at position 16 from the 5' end of the antisense strand is an f2P nucleotide. In some embodiments, at least 1, 2, 3, 4, or more 2'-fluoro nucleotides on the sense strand or antisense strand are fX nucleotides. In some embodiments, at least 1, 2, 3, or 4 of the 2'-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand are fX nucleotides. In some embodiments, at least one of the 2'-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand is an fX nucleotide. In some embodiments, at least two of the 2'-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand are fX nucleotides. In some embodiments, no more than three of the 2'-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand are fX nucleotides. In some embodiments, no more than two of the 2'-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand are fX nucleotides. In some embodiments, the 2'-fluoro nucleotide at position 2 from the 5' end of the antisense strand is a fX nucleotide. In some embodiments, the 2'-fluoro nucleotide at position 6 from the 5' end of the antisense strand is a fX nucleotide. In some embodiments, the 2'-fluoro nucleotide at position 14 from the 5' end of the antisense strand is a fX nucleotide. In some embodiments, the 2'-fluoro nucleotide at position 16 from the 5' end of the antisense strand is a fX nucleotide. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the sense strand is further modified to contain a 5' stabilized 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 contain a 5' stabilized 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 the 1 position 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 the 1 position from the 5' end of the antisense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 3' end of the antisense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 5' end of the sense strand is 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-mun nucleotide, a d2vm nucleotide, or a d2vmA nucleotide. In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 5' end of the antisense strand is 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-mun nucleotide, a d2vm nucleotide, or a d2vmA nucleotide. In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 3' end of the sense strand is 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-mun nucleotide, a d2vm nucleotide, or a d2vmA nucleotide. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 3' end of the antisense strand is 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-mun 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 mimics. In some embodiments, at least 1, 2, 3, 4, or more 2'-fluoro nucleotides on the sense strand are 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 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 mimics. In some embodiments, one or more nucleotides in the sense strand and / or antisense strand may be 3', 4' seco modified nucleotides in which the bond between the 3' and 4' positions of the furanose ring is broken (e.g., mun34).

[0078] As shown in Figure 3G, the ds-siNA may comprise (a) a sense strand of 21 nucleotides, with 2'-fluoro nucleotides at positions 5, 9-11, 14, and 19 from the 5' end of the sense strand, and 2'-O-methyl nucleotides at positions 1-4, 6-8, 12, 13, 15-18, 20, and 21 from the 5' end of the sense strand, and (b) an antisense strand of 23 nucleotides, with 2'-fluoro nucleotides at positions 2 and 14 from the 5' end of the antisense strand, and 2'-O-methyl nucleotides at positions 1, 3-13, and 15-23 from the 5' end of the antisense strand. The ds-siNA may further comprise a conjugated moiety attached to the 3' end of the sense strand. The ds-siNA may further comprise (i) phosphorothioate internucleoside linkages between nucleotides at positions 1 and 2 and 2 and 3 from the 5' end of the sense strand, and (ii) phosphorothioate internucleoside linkages between nucleotides at positions 1 and 2, 2 and 3, 19 and 20, and 20 and 21 from the 5' end of the antisense strand. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the sense strand is further modified to contain a 5' stabilized 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 contain a 5' stabilized 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.In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 5' end of the sense strand is 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-mun nucleotide, a d2vm nucleotide, or a d2vmA nucleotide. In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 5' end of the antisense strand is 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-mun nucleotide, a d2vm nucleotide, or a d2vmA nucleotide. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 3' end of the sense strand is 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-mun nucleotide, a d2vm nucleotide, or a d2vmA nucleotide. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 3' end of the antisense strand is 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-mun 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 or antisense strand are 2'-fluoro nucleotide mimics. In some embodiments, at least 1, 2, 3, 4, or more of the 2'-fluoro nucleotides on the sense strand are fB, fN, f(4nh)Q, f4P, f2P, or fX nucleotides.In some embodiments, at least one, two, three, four, or more 2'-fluoro nucleotides on the antisense strand are fB, fN, f(4nh)Q, f4P, f2P, or fX nucleotides. In some embodiments, at least one, two, three, four, or more 2'-O-methyl nucleotides on the sense or antisense strand are 2'-O-methyl nucleotide mimics. In some embodiments, one or more nucleotides in the sense and / or antisense strand may be a 3',4' seco modified nucleotide in which the bond between the 3' and 4' positions of the furanose ring is broken (e.g., mun34).

[0079] As shown in Figure 3H, the ds-siNA may include (a) a sense strand of 21 nucleotides, with 2'-fluoro nucleotides at positions 7 and 9-11 from the 5' end of the sense strand, and 2'-O-methyl nucleotides at positions 1-6, 8, and 12-21 from the 5' end of the sense strand, and (b) an antisense strand of 23 nucleotides, with 2'-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand, and 2'-O-methyl nucleotides at positions 1, 3-5, 7-13, 15, and 17-23 from the 5' end of the antisense strand. Optionally, the nucleotides at positions 22 and 23 from the 5' end of the antisense strand may be unlocked nucleotides. Optionally, the ds-siNA may further include a conjugation moiety (not shown) attached to the 3' end of the sense strand. The ds-siNA may optionally include a vinyl phosphonate (shown) attached to the 5' end of the antisense strand, although in some embodiments, a 5' end cap as disclosed herein may be suitable as well. The ds-siNA may further include (i) phosphorothioate internucleoside linkages between nucleotides at positions 1 and 2, 2 and 3, and 20 and 21 from the 5' end of the sense strand, and (ii) phosphorothioate internucleoside linkages between nucleotides at positions 1 and 2, 2 and 3, 21 and 22, and 22 and 23 from the 5' end of the antisense strand. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 5' end of the sense strand is further modified to contain a 5' stabilized 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 contain a 5' stabilized 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 the 1 position from the 5' end of the antisense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 3' end of the antisense strand is further modified to contain a phosphorylation blocker. In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 5' end of the sense strand is 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-mun 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-mun nucleotide, or a d2vmA nucleotide. In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 5' end of the antisense strand is 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-mun nucleotide, a d2vm nucleotide, or a d2vmA nucleotide. In some embodiments, the 2'-O-methyl nucleotide at the 1 position from the 3' end of the sense strand is 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-mun nucleotide, a d2vm nucleotide, or a d2vmA nucleotide. In some embodiments, the 2'-O-methyl nucleotide at position 1 from the 3' end of the antisense strand is 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-mun 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 mimics. In some embodiments, at least 1, 2, 3, 4, or more 2'-fluoro nucleotides on the sense strand are 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 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 mimics. In some embodiments, one or more nucleotides in the sense strand and / or antisense strand may be 3', 4' seco modified nucleotides in which the bond between the 3' and 4' positions of the furanose ring is broken (e.g., mun34).

[0080] siNA sense strand Any of the siNA molecules described herein may comprise a sense strand. The sense strand may comprise a first nucleotide sequence. The first nucleotide sequence may be 15-30, 15-25, 15-23, 17-23, 19-23, or 19-21 nucleotides in length. In some embodiments, the first nucleotide sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the first nucleotide sequence is at least 19 nucleotides in length. In some embodiments, the first nucleotide sequence is at least 21 nucleotides in length.

[0081] In some embodiments, the sense strand is the same length as the first nucleotide sequence. In some embodiments, the sense strand is longer than the first nucleotide sequence. In some embodiments, the sense strand may further comprise 1, 2, 3, 4, or 5 or more nucleotides than the first nucleotide sequence. In some embodiments, the sense strand may further comprise deoxyribonucleic acid (DNA). In some embodiments, the DNA is thymine (T). In some embodiments, the sense strand may further comprise a TT sequence. In some embodiments, the sense strand may further comprise one or more modified nucleotides adjacent to the first nucleotide sequence. In some embodiments, the one or more modified nucleotides are independently selected from any of the modified nucleotides disclosed herein (e.g., 2'-fluoro nucleotides, 2'-O-methyl nucleotides, 2'-fluoro nucleotide mimics, 2'-O-methyl nucleotide mimics, or nucleotides comprising modified nucleobases).

[0082] 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 and 2'-fluoro nucleotides. In some embodiments, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the nucleotides in the first nucleotide sequence are modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides. In some embodiments, 100% of the nucleotides in the first nucleotide sequence are modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides. In some embodiments, the 2'-O-methyl nucleotides are 2'-O-methyl nucleotide mimics. In some embodiments, the 2'-fluoro nucleotides are 2'-fluoro nucleotide mimics.

[0083] In some embodiments, about 15-30, 15-25, 15-24, 15-23, 15-22, 15-21, 17-30, 17-25, 17-24, 17-23, 17-22, 17-21, 18-30, 18-25, 18-24, 18-23, 18-22, 18-21, 19-30, 19-25, 19-24, 19-23, 19-22, 19-21, 20-25, 20-24, 20-23, 21-25, 21-24, or 21-23 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, about 2-20 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, about 5-25 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, about 10-25 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, about 12-25 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 12 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 13 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 14 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 15 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 16 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 17 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides.In some embodiments, at least about 18 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 19 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 21 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 20 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 19 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 18 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 17 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 16 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 15 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 14 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 13 modified nucleotides of the first nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least one modified nucleotide of the first nucleotide sequence is a 2'-O-methyl pyrimidine. In some embodiments, at least 5, 6, 7, 8, 9, or 10 modified nucleotides of the first nucleotide sequence are 2'-O-methyl pyrimidines. In some embodiments, at least one modified nucleotide of the first nucleotide sequence is a 2'-O-methyl purine.In some embodiments, at least 5, 6, 7, 8, 9, or 10 modified nucleotides of the first nucleotide sequence are 2'-O-methyl purines. In some embodiments, the 2'-O-methyl nucleotides are 2'-O-methyl nucleotide mimics.

[0084] In some embodiments, 2 to 15 modified nucleotides of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 2 to 10 modified nucleotides of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 2 to 6 modified nucleotides of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 1 to 6, 1 to 5, 1 to 4, or 1 to 3 modified nucleotides of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least 1, 2, 3, 4, 5, or 6 modified nucleotides of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least 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, no more than four modified nucleotides of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, no more than three modified nucleotides of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, no more than two modified nucleotides of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least one modified nucleotide of the first nucleotide sequence is a 2'-fluoro pyrimidine. In some embodiments, 1, 2, 3, 4, 5, or 6 modified nucleotides of the first nucleotide sequence are 2'-fluoro pyrimidines. In some embodiments, at least one modified nucleotide of the first nucleotide sequence is a 2'-fluoro purine. In some embodiments, 1, 2, 3, 4, 5, or 6 modified nucleotides of the first nucleotide sequence are 2'-fluoro purines. In some embodiments, the 2'-fluoro nucleotides are 2'-fluoro nucleotide mimics.

[0085] In some embodiments, the nucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and / or 19 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least two nucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and / or 19 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least three nucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and / or 19 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least four nucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and / or 19 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least five nucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and / or 19 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and / or 19 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotide at position 3 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 7 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 8 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 9 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 12 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 17 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the 2'-fluoro nucleotide is a 2'-fluoro nucleotide mimic.

[0086] 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 2 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 3 nucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and / or 19 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotide at position 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and / or 19 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 3 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 5 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 7 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 8 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 9 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 10 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 11 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 12 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide.In some embodiments, the nucleotide at position 14 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 17 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 19 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at positions 3, 7, 8, 9, 12, and / or 17 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at positions 3, 7, 8, and / or 17 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at positions 3, 7, 8, 9, 12, and / or 17 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at positions 5, 7, 8, and / or 9 from the 5' end of the first nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotides at positions 5, 9, 10, 11, 12, and / or 19 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the 2'-fluoro nucleotide is a 2'-fluoro nucleotide mimic.

[0087] In some embodiments, the 2'-fluoro or 2'-O-methyl nucleotide mimic is a nucleotide mimic of formula (V): [ka] In the formula, R x is independently a nucleobase, aryl, heteroaryl, or H; Q 1 and Q 2 are independently S or O, R 5 Independently, -OCD 3 , -F, or -OCH 3 and R 6 and R 7 are independently H, D, or CD 3In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0088] In some embodiments, the 2'-fluoro or 2'-O-methyl nucleotide mimic is a nucleotide mimic of formula (16)-(20): [ka] In the formula, R x is independently a nucleobase, aryl, heteroaryl, or H; R 2 But F or -OCH 3 In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0089] In some embodiments, the sense strand, the antisense strand, or both, each independently have the following chemical structure: [ka] wherein R y is the nucleobase, and R x is a nucleobase, aryl, heteroaryl, or H. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0090] In some embodiments, the sense strand, the antisense strand, or both, each independently have the following chemical structure: [ka] wherein R yis a nucleobase. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0091] For purposes of this disclosure, the modified nucleotide may be at any position on the sense strand. In some embodiments, the modified nucleotide may 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 on the sense strand relative to the 5' end. For example, the modified nucleotide may be [ka] In some embodiments, the modified nucleotide is: [ka] If it is, it may be located at position 3, 16, 17, or 18 relative to the 5' end of the sense strand.

[0092] 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.

[0093] In some embodiments, the sense strand may further comprise one or more internucleoside linkages independently selected from a phosphodiester (PO) internucleoside linkage, a phosphorothioate (PS) internucleoside linkage, a mesyl phosphoramidate internucleoside linkage (Ms), a phosphorodithioate internucleoside linkage, and a PS-mimetic internucleoside linkage. In some embodiments, the PS-mimetic internucleoside linkage is a sulfonucleoside linkage.

[0094] In some embodiments, the sense strand may further comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more phosphorothioate internucleoside linkages. In some embodiments, the sense strand comprises 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 or fewer phosphorothioate internucleoside linkages. In some embodiments, the sense strand comprises 2-10, 2-8, 2-6, 1-5, 1-4, 1-3, or 1-2 phosphorothioate internucleoside linkages. In some embodiments, the sense strand comprises 1-2 phosphorothioate internucleoside linkages. In some embodiments, the sense strand comprises 2-4 phosphorothioate internucleoside linkages. In some embodiments, at least one phosphorothioate internucleoside linkage is between nucleotides 1 and 2 positions from the 5' end of the first nucleotide sequence. In some embodiments, at least one phosphorothioate internucleoside linkage is between nucleotides 2 and 3 positions from the 5' end of the first nucleotide sequence. In some embodiments, the sense strand comprises two phosphorothioate internucleoside linkages between nucleotides 1-3 positions from the 5' end of the first nucleotide sequence.

[0095] In some embodiments, the sense strand may further comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more mesyl phosphoramidate internucleoside linkages. In some embodiments, the sense strand comprises 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 or fewer mesyl phosphoramidate internucleoside linkages. In some embodiments, the sense strand comprises 2-10, 2-8, 2-6, 1-5, 1-4, 1-3, or 1-2 mesyl phosphoramidate internucleoside linkages. In some embodiments, the sense strand comprises 1-2 mesyl phosphoramidate internucleoside linkages. In some embodiments, the sense strand contains 2-4 mesyl phosphoramidate internucleoside linkages.

[0096] In some embodiments, the sense strand may include any of the modified nucleotides disclosed below in the subsection entitled "Modified Nucleotides." In some embodiments, the sense strand may include a 5' stabilized endcap, which may be selected from those disclosed below in the subsection entitled "5' stabilized endcaps."

[0097] siNA antisense strand Any of the siNA molecules described herein may comprise an antisense strand. The antisense strand may comprise a second nucleotide sequence. The second nucleotide sequence may be 15-30, 15-25, 15-23, 17-23, 19-23, or 19-21 nucleotides in length. In some embodiments, the second nucleotide sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the second nucleotide sequence is at least 19 nucleotides in length. In some embodiments, the second nucleotide sequence is at least 21 nucleotides in length.

[0098] In some embodiments, the antisense strand is the same length as the second nucleotide sequence. In some embodiments, the antisense strand is longer than the second nucleotide sequence. In some embodiments, the antisense strand may further comprise 1, 2, 3, 4, or 5 or more nucleotides than the second nucleotide sequence. In some embodiments, the antisense strand is the same length as the sense strand. In some embodiments, the antisense strand is longer than the sense strand. In some embodiments, the antisense strand may further comprise 1, 2, 3, 4, or 5 or more nucleotides than the sense strand. In some embodiments, the antisense strand may further comprise deoxyribonucleic acid (DNA). In some embodiments, the DNA is thymine (T). In some embodiments, the antisense strand may further comprise a TT sequence. In some embodiments, the antisense strand may further comprise one or more modified nucleotides adjacent to the second nucleotide sequence. In some embodiments, the one or more modified nucleotides are independently selected from any of the modified nucleotides disclosed herein (e.g., a 2'-fluoro nucleotide, a 2'-O-methyl nucleotide, a 2'-fluoro nucleotide mimic, a 2'-O-methyl nucleotide mimic, or a nucleotide comprising a modified nucleobase).

[0099] In some embodiments, the second nucleotide sequence comprises 15, 16, 17, 18, 19, 20, 21, 22, 23 or more modified nucleotides independently selected from 2'-O-methyl and 2'-fluoro 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 2'-O-methyl and 2'-fluoro nucleotides. In some embodiments, 100% of the nucleotides in the second nucleotide sequence are modified nucleotides independently selected from 2'-O-methyl and 2'-fluoro nucleotides.

[0100] In some embodiments, about 15-30, 15-25, 15-24, 15-23, 15-22, 15-21, 17-30, 17-25, 17-24, 17-23, 17-22, 17-21, 18-30, 18-25, 18-24, 18-23, 18-22, 18-21, 19-30, 19-25, 19-24, 19-23, 19-22, 19-21, 20-25, 20-24, 20-23, 21-25, 21-24, or 21-23 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, about 2-20 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, about 5-25 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, about 10-25 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, about 12-25 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 12 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 13 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 14 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 15 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 16 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 17 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides.In some embodiments, at least about 18 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least about 19 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 21 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 20 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 19 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 18 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 17 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 16 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 15 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 14 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, no more than 13 modified nucleotides of the second nucleotide sequence are 2'-O-methyl nucleotides. In some embodiments, at least one modified nucleotide of the second nucleotide sequence is a 2'-O-methyl pyrimidine. In some embodiments, at least 5, 6, 7, 8, 9, or 10 modified nucleotides of the second nucleotide sequence are 2'-O-methyl pyrimidines. In some embodiments, at least one modified nucleotide of the second nucleotide sequence is a 2'-O-methyl purine.In some embodiments, at least 5, 6, 7, 8, 9, or 10 modified nucleotides of the second nucleotide sequence are 2'-O-methyl purines. In some embodiments, the 2'-O-methyl nucleotides are 2'-O-methyl nucleotide mimics.

[0101] In some embodiments, 2 to 15 modified nucleotides of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 2 to 10 modified nucleotides of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 2 to 6 modified nucleotides of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, 1 to 6, 1 to 5, 1 to 4, or 1 to 3 modified nucleotides of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least 1, 2, 3, 4, 5, or 6 modified nucleotides of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least 1 modified nucleotide of the second nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, at least 2 modified nucleotides of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least 3 modified nucleotides of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least 4 modified nucleotides of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least 5 modified nucleotides 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, no more than three modified nucleotides of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, no more than two 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'-fluoro pyrimidine. In some embodiments, 1, 2, 3, 4, 5, or 6 modified nucleotides of the second nucleotide sequence are 2'-fluoro pyrimidines. In some embodiments, at least one modified nucleotide of the second nucleotide sequence is a 2'-fluoro purine. In some embodiments, 1, 2, 3, 4, 5, or 6 modified nucleotides of the second nucleotide sequence are 2'-fluoro purines. In some embodiments, the 2'-fluoro nucleotides are 2'-fluoro nucleotide mimics.

[0102] In some embodiments, the 2'-fluoro or 2'-O-methyl nucleotide is a 2'-fluoro or 2-O-methyl nucleotide mimic. In some embodiments, the 2'-fluoro or 2'-O-methyl nucleotide mimic is a nucleotide mimic of formula (V): [ka] In the formula, R x is independently a nucleobase, aryl, heteroaryl, or H; Q 1 and Q 2 are independently S or O, R 5 Independently, -OCD 3 , -F, or -OCH 3 and R 6 and R 7 are independently H, D, or CD 3 In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0103] In some embodiments, the 2'-fluoro or 2'-O-methyl nucleotide mimic is a nucleotide mimic of formula (16)-(20): [ka] In the formula, R x is a nucleobase, aryl, heteroaryl, or H; R 2 is independently F or -OCH 3 In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0104] In some embodiments, the antisense strand, the sense strand, or both, each independently have the following chemical structure: [ka] wherein R y is a nucleobase and Rx is a nucleobase, aryl, heteroaryl, or H. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0105] In some embodiments, the antisense strand, the sense strand, or both, each independently have the following chemical structure: [ka] wherein R y is a nucleobase. In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0106] For purposes of this disclosure, the modified nucleotide may be at any position in the antisense strand. In some embodiments, the modified nucleotide may be at the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 position in the antisense strand relative to the 5' end.

[0107] In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides at positions 2, 5, 6, 8, 10, 14, 16, 17, and / or 18 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotides at positions 2, 5, 6, 8, 10, 14, 16, 17, and / or 18 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least two nucleotides at positions 2, 5, 6, 8, 10, 14, 16, 17, and / or 18 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least three nucleotides at positions 2, 5, 6, 8, 10, 14, 16, 17, and / or 18 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least four nucleotides at positions 2, 5, 6, 8, 10, 14, 16, 17, and / or 18 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, at least five nucleotides at positions 2, 5, 6, 8, 10, 14, 16, 17, and / or 18 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotides at positions 2 and / or 14 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotides at positions 2, 6, and / or 16 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotides at positions 2, 6, 14, and / or 16 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides. In some embodiments, the nucleotide at position 2, 6, 10, 14, and / or 18 from the 5' end of the second nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 2, 5, 8, 14, and / or 17 from the 5' end of the second nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 2 from the 5' end of the second nucleotide sequence is a 2'-fluoro nucleotide.In some embodiments, the nucleotide at position 5 from the 5' end of the second nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 6 from the 5' end of the second nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 8 from the 5' end of the second nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 10 from the 5' end of the second nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 14 from the 5' end of the second nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 16 from the 5' end of the second nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 17 from the 5' end of the second nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the nucleotide at position 18 from the 5' end of the second nucleotide sequence is a 2'-fluoro nucleotide. In some embodiments, the 2'-fluoro nucleotide is a 2'-fluoro nucleotide mimic.

[0108] In some embodiments, the nucleotides in the second nucleotide sequence are arranged in an alternating 1:3 modification pattern, one nucleotide is a 2'-fluoro nucleotide and three nucleotides are 2'-O-methyl nucleotides, and the alternating 1:3 modification pattern occurs at least twice. In some embodiments, the alternating 1:3 modification pattern occurs 2-5 times. In some embodiments, at least two of the alternating 1:3 modification patterns occur consecutively. In some embodiments, at least two of the alternating 1:3 modification patterns occur non-consecutively. In some embodiments, at least one, two, three, four, or five alternating 1:3 modification patterns begin 2, 6, 10, 14, and / or 18 nucleotide positions from the 5' end of the antisense strand. In some embodiments, at least one alternating 1:3 modification pattern begins 2 nucleotide positions from the 5' end of the antisense strand. In some embodiments, at least one alternating 1:3 modification pattern begins 6 nucleotide positions from the 5' end of the antisense strand. In some embodiments, at least one alternating 1:3 modification pattern begins 10 nucleotide positions from the 5' end of the antisense strand. In some embodiments, at least one alternating 1:3 modification pattern begins 14 nucleotide positions from the 5' end of the antisense strand. In some embodiments, at least one alternating 1:3 modification pattern begins 18 nucleotide positions from the 5' end of the antisense strand. In some embodiments, the 2'-fluoro nucleotide is a 2'-fluoro nucleotide mimic.

[0109] In some embodiments, the nucleotides in the second nucleotide sequence are arranged in an alternating 1:2 modification pattern, one nucleotide is a 2'-fluoro nucleotide and two nucleotides are 2'-O-methyl nucleotides, and the alternating 1:2 modification pattern occurs at least twice. In some embodiments, the alternating 1:2 modification pattern occurs 2-5 times. In some embodiments, at least two of the alternating 1:2 modification patterns occur consecutively. In some embodiments, at least two of the alternating 1:2 modification patterns occur non-consecutively. In some embodiments, at least one, two, three, four, or five alternating 1:2 modification patterns begin 2, 5, 8, 14, and / or 17 nucleotide positions from the 5' end of the antisense strand. In some embodiments, at least one alternating 1:2 modification pattern begins 2 nucleotide positions from the 5' end of the antisense strand. In some embodiments, at least one alternating 1:2 modification pattern begins 5 nucleotide positions from the 5' end of the antisense strand. In some embodiments, at least one alternating 1:2 modification pattern begins 8 nucleotide positions from the 5' end of the antisense strand. In some embodiments, at least one alternating 1:2 modification pattern begins 14 nucleotide positions from the 5' end of the antisense strand. In some embodiments, at least one alternating 1:2 modification pattern begins 17 nucleotide positions from the 5' end of the antisense strand. In some embodiments, the 2'-fluoro nucleotide is a 2'-fluoro nucleotide mimic.

[0110] In some embodiments, the second nucleotide sequence comprises, consists of, or consists essentially of ribonucleic acid (RNA). In some embodiments, the second nucleotide sequence comprises, consists of, or consists essentially of modified RNA. In some embodiments, the modified RNA is selected from 2'-O-methyl RNA and 2'-fluoro RNA. In some embodiments, 15, 16, 17, 18, 19, 20, 21, 22, or 23 modified nucleotides of the second nucleotide sequence are independently selected from 2'-O-methyl RNA and 2'-fluoro RNA. In some embodiments, the 2'-fluoro nucleotide is a 2'-fluoro nucleotide mimic.

[0111] In some embodiments, the sense strand may further comprise one or more internucleoside linkages independently selected from a phosphodiester (PO) internucleoside linkage, a phosphorothioate (PS) internucleoside linkage, a phosphorodithioate internucleoside linkage, and a PS-mimetic internucleoside linkage. In some embodiments, the PS-mimetic internucleoside linkage is a sulfonucleoside linkage.

[0112] In some embodiments, the antisense strand may further comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more phosphorothioate internucleoside linkages. In some embodiments, the antisense strand comprises 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 or fewer phosphorothioate internucleoside linkages. In some embodiments, the antisense strand comprises 2-10, 2-8, 2-6, 1-5, 1-4, 1-3, or 1-2 phosphorothioate internucleoside linkages. In some embodiments, the antisense strand comprises 2-10, 2-8, 2-6, 1-5, 1-4, 1-3, or 1-2 phosphorothioate internucleoside linkages. In some embodiments, the antisense strand comprises 2-8 phosphorothioate internucleoside linkages. In some embodiments, the antisense strand comprises 3-8 phosphorothioate internucleoside linkages. In some embodiments, the antisense strand comprises 4-8 phosphorothioate internucleoside linkages. In some embodiments, at least one phosphorothioate internucleoside linkage is between nucleotides 1 and 2 positions from the 5' end of the second nucleotide sequence. In some embodiments, at least one phosphorothioate internucleoside linkage is between nucleotides 2 and 3 positions from the 5' end of the second nucleotide sequence. In some embodiments, at least one phosphorothioate internucleoside linkage is between nucleotides 1 and 2 positions from the 3' end of the second nucleotide sequence. In some embodiments, at least one phosphorothioate internucleoside linkage is between nucleotides 2 and 3 positions from the 3' end of the second nucleotide sequence. In some embodiments, the antisense strand comprises two phosphorothioate internucleoside linkages between nucleotides 1-3 from the 5' end of the first nucleotide sequence. In some embodiments, the antisense strand comprises two phosphorothioate internucleoside linkages between nucleotides 1-3 from the 3' end of the first nucleotide sequence.In some embodiments, the antisense strand comprises (a) two phosphorothioate internucleoside linkages between nucleotides 1 to 3 from the 5' end of the first nucleotide sequence and (b) two phosphorothioate internucleoside linkages between nucleotides 1 to 3 from the 3' end of the first nucleotide sequence.

[0113] In some embodiments, the antisense strand may further comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more mesyl phosphoramidate internucleoside linkages. In some embodiments, the antisense strand comprises 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 or fewer mesyl phosphoramidate internucleoside linkages. In some embodiments, the antisense strand comprises 2-10, 2-8, 2-6, 1-5, 1-4, 1-3, or 1-2 mesyl phosphoramidate internucleoside linkages. In some embodiments, the antisense strand comprises 2-10, 2-8, 2-6, 1-5, 1-4, 1-3, or 1-2 mesyl phosphoramidate internucleoside linkages. In some embodiments, the antisense strand comprises 2-8 mesyl phosphoramidate internucleoside linkages. In some embodiments, the antisense strand comprises 3-8 mesyl phosphoramidate internucleoside linkages. In some embodiments, the antisense strand comprises 4-8 mesyl phosphoramidate internucleoside linkages.

[0114] In some embodiments, at least one end of the ds-siNA is blunt-ended. In some embodiments, at least one end of the ds-siNA comprises an overhang, the overhang comprising at least one nucleotide. In some embodiments, both ends of the ds-siNA comprise an overhang, the overhang comprising at least one nucleotide. In some embodiments, the overhang comprises 1-5 nucleotides, 1-4 nucleotides, 1-3 nucleotides, or 1-2 nucleotides. In some embodiments, the overhang consists of 1-2 nucleotides.

[0115] In some embodiments, the sense strand may include any of the modified nucleotides disclosed below in the subsection entitled "Modified Nucleotides." In some embodiments, the sense strand may include a 5' stabilized endcap, which may be selected from those disclosed below in the subsection entitled "5' stabilized endcaps."

[0116] Modified Nucleotides The siNA molecules disclosed herein comprise one or more modified nucleotides. 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, 2'-O-methyl nucleotides in any of the sense strands or first nucleotide sequences disclosed herein are replaced with modified nucleotides. In some embodiments, 2'-O-methyl nucleotides in any of the antisense strands or second nucleotide sequences disclosed herein are replaced with modified nucleotides.

[0117] In some embodiments, any of the siNA molecules, siNAs, sense strands, first nucleotide sequences, antisense strands, and second nucleotide sequences disclosed herein contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more modified nucleotides. In some embodiments, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the nucleotides in the siNA molecule, siNA, sense strand, first nucleotide sequence, antisense strand, or second nucleotide sequence are modified nucleotides.

[0118] 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, locked nucleic acids, unlocked nucleic acids, 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 in which the furanose ring lacks a bond between the 3' and 4, carbons (e.g., mun34).

[0119] In some embodiments, the siNA of the present disclosure comprises: [ka] wherein Rx is a nucleobase, aryl, heteroaryl, or H; [ka] wherein Ry is a nucleobase; [ka] wherein Ry is a nucleobase, or a combination thereof. In some embodiments, the siNA may contain at least two, at least three, at least four, or at least five or more of these modified nucleotides. In some embodiments, the sense strand is [ka] wherein Rx is a nucleobase, aryl, heteroaryl, or H; [ka] (In the formula, R y are nucleobases), [ka] (In the formula, R y are nucleobases), or combinations thereof. In some embodiments, the antisense strand may comprise at least one, at least two, at least three, at least four, or at least five or more of: [ka] wherein Rx is a nucleobase, aryl, heteroaryl, or H; [ka] (In the formula, R y are nucleobases), [ka] (In the formula, R y are nucleobases), or combinations thereof. In some embodiments, both the sense strand and the antisense strand may each independently comprise at least one, at least two, at least three, at least four, or at least five or more of: [ka] (In the formula, R x is a nucleobase, aryl, heteroaryl, or H; [ka] (In the formula, R y are nucleobases), [ka] (In the formula, R y are nucleobases), or combinations thereof. In some embodiments, the nucleobases are selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof. For example, [ka] In some embodiments, the modified nucleotide is [ka] The structure may be:

[0120] In some embodiments, any of the siRNAs disclosed herein may optionally include other modified nucleotides, such as 2'-fluoro or 2-O-methyl nucleotide mimics. For example, the disclosed siNAs may include 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 include 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 include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more 2'-fluoro or 2'-O-methyl nucleotide mimics. In some embodiments, any of the antisense strands disclosed herein comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more 2'-fluoro or 2'-O-methyl nucleotide mimics. In some embodiments, any of the second nucleotide sequences disclosed herein comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more 2'-fluoro or 2'-O-methyl nucleotide mimics. In some embodiments, the 2'-fluoro or 2'-O-methyl nucleotide mimics are nucleotide mimics of formula (16)-(20): [ka] In the formula, R x is a nucleobase, aryl, heteroaryl, or H; R 2 is independently F or -OCH 3 In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0121] 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, the at least one 2'-fluoro or 2'-O-methyl nucleotide mimic is adjacent to a first nucleotide sequence. In some embodiments, the 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, the 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, the at least one 2'-fluoro or 2'-O-methyl nucleotide mimic is adjacent to a second nucleotide sequence. In some embodiments, the 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, the at least one 2'-fluoro or 2'-O-methyl nucleotide mimic is adjacent to the 3' end of the second nucleotide sequence. In some embodiments, the first nucleotide sequence does not include a 2'-fluoro nucleotide mimic. In some embodiments, the first nucleotide sequence does not include a 2'-O-methyl nucleotide mimic. In some embodiments, the second nucleotide sequence does not include a 2'-fluoro nucleotide mimic. In some embodiments, the second nucleotide sequence does not include a 2'-O-methyl nucleotide mimic.

[0122] In some embodiments, any of the siRNAs, sense strands, first nucleotide sequences, antisense strands, or second nucleotide sequences disclosed herein are [ka] wherein Rx is a nucleobase, aryl, heteroaryl, or H; or [ka] (In the formula, R y is a nucleobase).

[0123] Phosphorylation blockers Further disclosed herein are siNA molecules that contain phosphorylation blockers. In some embodiments, 2'-O-methyl nucleotides in any of the sense strands or first nucleotide sequences disclosed herein are replaced with nucleotides that contain phosphorylation blockers. In some embodiments, 2'-O-methyl nucleotides in any of the antisense strands or second nucleotide sequences disclosed herein are replaced with nucleotides that contain phosphorylation blockers. In some embodiments, 2'-O-methyl nucleotides in any of the sense strands or first nucleotide sequences disclosed herein are further modified to contain phosphorylation blockers. In some embodiments, 2'-O-methyl nucleotides in any of the antisense strands or second nucleotide sequences disclosed herein are further modified to contain phosphorylation blockers.

[0124] In some embodiments, any of the siNA molecules disclosed herein has formula (IV): [ka] Phosphorylation blocker of formula (wherein R y is the nucleobase, and R 4 But -OR 30 or -NR 31 R 32 and R 30 But, C 1 -C 8 is a substituted or unsubstituted alkyl; R 31 and R 32 together with the nitrogen to which they are attached form a substituted or unsubstituted heterocyclic ring. In some embodiments, the nucleobases are selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0125] In some embodiments, any of the siNA molecules disclosed herein comprises a phosphorylation blocker of formula (IV): [ka] Formula (IV), where R y is the nucleobase, and R 4 But, -OCH 3 or -N(CH 2 CH 2 ) 2 In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof.

[0126] In some embodiments, the siNA molecule has (a) formula (IV): [ka] Phosphorylation blocker of formula (wherein R y is the nucleobase, and R 4 But -OR 30 or -NR 31 R 32 and R 30 But, C 1 -C 8 is a substituted or unsubstituted alkyl; R 31 and R 32 together with the nitrogen to which they are attached form a substituted or unsubstituted heterocyclic ring; and (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.

[0127] In some embodiments, the siNA molecule comprises (a) a phosphorylation blocker of formula (IV): [ka] Formula (IV) (wherein, R y is the nucleobase, and R 4 But, -OCH3 or -N(CH 2 CH 2 ) 2 O), and (b) a short interfering nucleic acid (siNA), wherein the phosphorylation blocker is conjugated to the siNA.

[0128] In some embodiments, the phosphorylation blocker is attached to the 3' end of the sense strand or the first nucleotide sequence. In some embodiments, the phosphorylation blocker is attached to the 3' end of the sense strand or the first nucleotide sequence via one, two, three, four, or five or more linkers. In some embodiments, the phosphorylation blocker is attached to the 5' end of the sense strand or the first nucleotide sequence. In some embodiments, the phosphorylation blocker is attached to the 5' end of the sense strand or the first nucleotide sequence via one, two, three, four, or five or more linkers. In some embodiments, the phosphorylation blocker is attached to the 3' end of the antisense strand or the second nucleotide sequence. In some embodiments, the phosphorylation blocker is attached to the 3' end of the antisense strand or the second nucleotide sequence via one, two, three, four, or five or more linkers. In some embodiments, the phosphorylation blocker is attached to the 5' end of the antisense strand or the second nucleotide sequence. In some embodiments, the phosphorylation blocker is attached to the 5' end of the antisense strand or the second nucleotide sequence via one, two, three, four, or five or more linkers. In some embodiments, the one or more linkers are independently selected from the group consisting of a phosphodiester linker, a phosphorothioate linker, a mesyl phosphoramidate linker, and a phosphorodithioate linker.

[0129] Conjugation Moiety Further disclosed herein are siNA molecules comprising a conjugation moiety. In some embodiments, the conjugation moiety is selected from galactosamine, peptide, protein, sterol, lipid, phospholipid, biotin, phenoxazine, active drug substrate, cholesterol, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dye. In some embodiments, the conjugation moiety is attached to the 3' end of the sense strand or the first nucleotide sequence. In some embodiments, the conjugation moiety is attached to the 3' end of the sense strand or the first nucleotide sequence via one, two, three, four, or five or more linkers. In some embodiments, the conjugation moiety is attached to the 5' end of the sense strand or the first nucleotide sequence. In some embodiments, the conjugation moiety is attached to the 5' end of the sense strand or the first nucleotide sequence via one, two, three, four, or five or more linkers. In some embodiments, the conjugated moiety is attached to the 3' end of the antisense strand or the second nucleotide sequence. In some embodiments, the conjugated moiety is attached to the 3' end of the antisense strand or the second nucleotide sequence via one, two, three, four, or five or more linkers. In some embodiments, the conjugated moiety is attached to the 5' end of the antisense strand or the second nucleotide sequence. In some embodiments, the conjugated moiety is attached to the 5' end of the antisense strand or the second nucleotide sequence via one, two, three, four, or five or more linkers. In some embodiments, the one or more linkers are independently selected from the group consisting of a phosphodiester linker, a phosphorothioate linker, a phosphorodithioate linker, and a mesyl phosphoramidate linker.

[0130] In some embodiments, the conjugation moiety is galactosamine. In some embodiments, any of the siNAs disclosed herein are linked to a conjugation moiety that is galactosamine. In some embodiments, the galactosamine is N-acetylgalactosamine (GalNAc). In some embodiments, any of the siNA molecules disclosed herein comprise GalNAc. In some embodiments, the GalNAc has the formula (VI): [ka] wherein m is 1, 2, 3, 4, or 5, each n is independently 1 or 2, p is 0 or 1, each R is independently H or a first protecting group, each Y is independently selected from -OP(=O)(SH)-, -OP(=O)(O)-, -OP(=O)(OH)-, -OP(S)S-, and -O-, Z is H or a second protecting group, either L is a linker or L and Y 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-diisopropyl phosphoramidite group. In some embodiments, the linker is a C6-NH 2 In some embodiments, A is a group. In some embodiments, A is a short interfering nucleic acid (siNA) or a 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.

[0131] In some embodiments, GalNAc has formula (VII): [ka] where R zR is OH or SH, and each n is independently 1 or 2. In some embodiments, the targeting ligand can be GalNAc, and the targeting ligand can include 1, 2, 3, 4, 5, or 6 GalNAc units. In some embodiments, the targeting ligand can be GalNAc2, GalNAc3, GalNAc4 (GalNAc of formula VII, where n=1 and R z ═OH), GalNAc5, and GalNAc6.

[0132] In some embodiments, GalNAc can be a GalNAc amidite (i.e., compound 40-9, see Example 22), GalNAc4 CPG (i.e., compound 40-8, see Examples 22 and 23), GalNAc phosphoramidite, or GalNAc4-ps-GalNAc4-ps-GalNAc4. These GalNAc moieties are shown below. [Table 1]

[0133] GalNAc3, GalNAc4, GalNAc5 and GalNAc6 may be conjugated to the siNAs disclosed herein during synthesis using moieties 1, 2, or 3. Additional GalNAc moieties, such as GalNAc1 and GalNAc2, can be used to form 5' and 3'-GalNAc using post-synthetic conjugation.

[0134] GalNAc phosphoramidite [Table 2] In some embodiments, the galactosamine is attached to the 3' end of the sense strand or the first nucleotide sequence. In some embodiments, the galactosamine is attached to the 3' end of the sense strand or the first nucleotide sequence via one, two, three, four, or five or more linkers. In some embodiments, the galactosamine is attached to the 5' end of the sense strand or the first nucleotide sequence. In some embodiments, the galactosamine is attached to the 5' end of the sense strand or the first nucleotide sequence via one, two, three, four, or five or more linkers. In some embodiments, the galactosamine is attached to the 3' end of the antisense strand or the second nucleotide sequence. In some embodiments, the galactosamine is attached to the 3' end of the antisense strand or the second nucleotide sequence via one, two, three, four, or five or more linkers. In some embodiments, the galactosamine is attached to the 5' end of the antisense strand or the second nucleotide sequence. In some embodiments, the galactosamine is linked to the 5' end of the antisense strand or the second nucleotide sequence via one, two, three, four, or five or more linkers. In some embodiments, the one or more linkers are independently selected from the group consisting of a phosphodiester (p or po) linker, a phosphorothioate (ps) linker, a mesyl phosphoramidate linker (Ms), a phosphoramidite (HEG) linker, a triethylene glycol (TEG) linker, and / or a phosphorodithioate linker. In some embodiments, 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.

[0135] In some embodiments, the conjugation moiety is a lipid moiety. In some embodiments, any of the siNAs disclosed herein are linked to a conjugation moiety that is a lipid moiety. Examples of lipid moieties include, but are not limited to, cholesterol moieties, thioethers such as hexyl-S-tritylthiol, thiocholesterol, aliphatic chains such as dodecanediol or undecyl residues, phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1-di-O-hexadecyl-rac-glycero-SH-phosphonate, polyamines or polyethylene glycol chains, adamantane acetic acid, palmityl moieties, or octadecylamine or hexylamino-carbonyl-oxycholesterol moieties.

[0136] In some embodiments, the conjugated moiety is an active drug substrate. In some embodiments, any of the siNAs disclosed herein are linked to a conjugated moiety that is an active drug substrate. Examples of active drug substrates include, but are not limited to, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (5)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folic acid, benzothiadiazid, chlorothiazide, diazepines, indomethicine, barbiturates, cephalosporins, sulfa drugs, antidiabetic drugs, antibacterial agents, or antibiotics.

[0137] 5' stabilized end cap Further disclosed herein are siNA molecules comprising a 5' stabilized endcap. As used herein, the terms "5' stabilized endcap" and "5' endcap" are used interchangeably. In some embodiments, a 2'-O-methyl nucleotide in any of the sense strand or the first nucleotide sequence disclosed herein is replaced with a nucleotide containing a 5' stabilized endcap. In some embodiments, a 2'-O-methyl nucleotide in any of the antisense strand or the second nucleotide sequence disclosed herein is replaced with a nucleotide containing a 5' stabilized endcap. In some embodiments, a 2'-O-methyl nucleotide in any of the sense strand or the first nucleotide sequence disclosed herein is further modified to contain a 5' stabilized endcap. In some embodiments, a 2'-O-methyl nucleotide in any of the antisense strand or the second nucleotide sequence disclosed herein is further modified to contain a 5' stabilized endcap.

[0138] In some embodiments, the 5' stabilized end cap is a 5' phosphate mimic. In some embodiments, the 5' stabilized end cap is a modified 5' phosphate mimic. In some embodiments, the modified 5' phosphate is a chemically modified 5' phosphate. In some embodiments, the 5' stabilized 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 mono-deuterated vinyl phosphonate. In some embodiments, the deuterated vinyl phosphonate is a di-deuterated vinyl phosphonate. In some embodiments, the 5' stabilized end cap is a phosphate mimic. Examples of phosphate mimetics are disclosed in Parmar et al.,, J Med Chem, 201861(3):734-744, WO 2018 / 045317 and WO 2018 / 044350, and U.S. Pat. No. 10,087,210, each of which is incorporated by reference in its entirety.

[0139] In some aspects, the disclosure provides a siNA comprising a nucleotide phosphate mimic selected from: [ka] In the formula, R y is the nucleobase, and R 15 is H or CH 3 In some embodiments, the nucleobase is selected from thymine, cytosine, guanine, adenine, uracil, and analogs or derivatives thereof. In some embodiments, nucleotide phosphate mimetics of the present disclosure include, but are not limited to, the following structures: [ka] [ka] [ka] [ka] [ka] where R 15 is H or CH 3 It is.

[0140] In some aspects, the disclosure provides a siNA comprising a nucleotide phosphate mimic selected from: [ka] In the formula, R 15 is H or CH 3 In some embodiments, one of these novel nucleotide phosphate mimics (e.g., an omeco-d3 nucleotide, a 4h nucleotide, a v-mun nucleotide, a co2o-4h nucleotide, a coc-4h nucleotide, an omeco-mun nucleotide, a 4h-vp nucleotide, or a d2vm nucleotide) is located at the 5'-end of the antisense strand, although these novel nucleotide phosphate mimics may 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.

[0141] Additionally or alternatively, the siNA molecules disclosed herein can comprise a nucleotide sequence of formula (Ia) in the sense strand, the antisense strand, or both. [ka] wherein R x is H, a nucleobase, an aryl, or a heteroaryl; R 26 teeth, [ka] -CH=CD-Z, -CD=CH-Z, -CD=CD-Z, -(CR 21 R 22 ) n -Z or -(C 2 -C 6 alkenylene)-Z, and R 20 is H or R 26 and R 20 Together, -(CR 21 R 22 ) n -Z or -(C 2 -C 6 alkenylene)-Z, n is 1, 2, 3, or 4, and Z is -ONR 23 R 24 , -OP(O)OH(CH 2 ) m CO 2 R 23 , -OP(S)OH(CH 2 ) m CO 2 R 23 , -P(O)(OH) 2 , -P(O)(OH)(OCH 3 ), -P(O)(OH)(OCD 3 ), -SO 2 (CH 2 ) m P(O)(OH) 2 , -SO 2 NR 23 R 25 , -NR 23 R 24 , -NR 23 SO 2 R 24 and R 21 and R 22 any of the following is independently hydrogen or C 1 -C 6 alkyl or R 21 and R 22 together form an oxo group, and R 23 is hydrogen or C 1 -C 6 is alkyl, R 24 -SO 2 R 25 Or -C(O)R25 or R 23 and R 24 together with the nitrogen to which they are attached form a substituted or unsubstituted heterocyclic ring; R 25 is C 1 -C 6 alkyl, and m is 1, 2, 3, or 4. In some embodiments, R 1 is aryl. In some embodiments, aryl is phenyl.

[0142] Additionally or alternatively, the siNA molecules disclosed herein can comprise a nucleotide sequence of formula (Ib) in the sense strand, the antisense strand, or both. [ka] wherein R x is H, a nucleobase, an aryl, or a heteroaryl; R 26 teeth, [ka] -CH=CD-Z, -CD=CH-Z, -CD=CD-Z, -(CR 21 R 22 ) n -Z or -(C 2 -C 6 alkenylene)-Z, and R 20 is H or R 26 and R 20 Together, -(CR 21 R 22 ) n -Z or -(C 2 -C 6 alkenylene)-Z, n is 1, 2, 3, or 4, and Z is -ONR 23 R 24 , -OP(O)OH(CH 2 ) m CO 2 R 23 , -OP(S)OH(CH 2 ) m CO 2 R23 , -P(O)(OH) 2 , -P(O)(OH)(OCH 3 ), -P(O)(OH)(OCD 3 ), -SO 2 (CH 2 ) m P(O)(OH) 2 , -SO 2 NR 23 R 25 , -NR 23 R 24 , -NR 23 SO 2 R 24 and R 21 and R 22 any of the following is independently hydrogen or C 1 -C 6 alkyl or R 21 and R 22 together form an oxo group, and R 23 is hydrogen or C 1 -C 6 is alkyl, R 24 -SO 2 R 25 Or -C(O)R 25 or R 23 and R 24 together with the nitrogen to which they are attached form a substituted or unsubstituted heterocyclic ring; R 25 is C 1 -C 6 alkyl, and m is 1, 2, 3, or 4. In some embodiments, R 1 is aryl. In some embodiments, aryl is phenyl.

[0143] Additionally or alternatively, the siNA molecules disclosed herein can comprise a nucleotide sequence of formula (Ic) in the sense strand, the antisense strand, or both. [ka] wherein R x is a nucleobase, aryl, heteroaryl, or H; R 26but, [ka] -CH=CD-Z, -CD=CH-Z, -CD=CD-Z, -(CR 21 R 22 ) n -Z or -(C 2 -C 6 alkenylene)-Z, and R 20 is hydrogen or R 26 and R 20 But together, -(CR 21 R 22 ) n -Z or -(C 2 -C 6 alkenylene)-Z, forming a 3-7 membered carbocyclic ring substituted therewith, n is 1, 2, 3, or 4; Z is -ONR 23 R 24 , -OP(O)OH(CH 2 ) m CO 2 R 23 , -OP(S)OH(CH 2 ) m CO 2 R 23 , -P(O)(OH) 2 , -P(O)(OH)(OCH 3 ), -P(O)(OH)(OCD 3 ), -SO 2 (CH 2 ) m P(O)(OH) 2 , -SO 2 NR 23 R 25 , -NR 23 R 24 , or -NR 23 SO 2 R 24 and R 21 and R 22 are independently hydrogen or C 1 -C 6 alkyl or R 21 and R 22 together form an oxo group, and R 23 is hydrogen or C1 -C 6 is alkyl, R 24 But -SO 2 R 25 Or -C(O)R 25 or

[0144] R 23 and R 24 together with the nitrogen to which they are attached form a substituted or unsubstituted heterocyclic ring; R 25 But, C 1 ~C 6 alkyl, and m is 1, 2, 3, or 4. In some embodiments, R 1 is aryl. In some embodiments, aryl is phenyl.

[0145] Additionally or alternatively, the siNA molecules disclosed herein can comprise a nucleotide sequence of formula (IIa) in the sense strand, the antisense strand, or both. [ka] wherein R x is a nucleobase, aryl, heteroaryl, or H; R 26 but, [ka] -CH 2 SO 2 NHCH 3 or [ka] and R 9 But -SO 2 CH 3 or -COCH 3 and [ka] is a double bond or a single bond, and R 10 =-CH 2 PO 3 H or -NHCH 3and R 11 But -CH 2 - or -CO-, R 12 is H and R 13 But, CH 3 or R 12 and R 13 But together, -CH 2 CH 2 CH 2 In some embodiments, R 1 is aryl. In some embodiments, aryl is phenyl.

[0146] Additionally or alternatively, the siNA molecules disclosed herein can comprise a nucleotide sequence of formula (IIb) in the sense strand, the antisense strand, or both. [ka] wherein R x is a nucleobase, aryl, heteroaryl, or H; R 26 but, [ka] -CH 2 SO 2 NHCH 3 or [ka] and R 9 But -SO 2 CH 3 or -COCH 3 and [ka] is a double bond or a single bond, and R 10 =-CH 2 PO 3 H or -NHCH 3 and R 11 But -CH 2 - or -CO-, R 12 is H and R 13 But, CH3 or R 12 and R 13 But together, -CH 2 CH 2 CH 2 In some embodiments, R 1 is aryl. In some embodiments, aryl is phenyl.

[0147] Additionally or alternatively, the siNA molecules disclosed herein can comprise a nucleotide sequence of formula (III) in the sense strand, the antisense strand, or both. [ka] wherein R x is a nucleobase, aryl, heteroaryl, or H; L is -CH 2 -, -CH=CH-, -CO-, or -CH 2 CH 2 - and A is -ONHCOCH 3 , -ONHSO 2 CH 3、 -PO 3 H, -OP(SOH)CH 2 CO 2 -H, -SO 2 CH 2 PO 3 H, -SO 2 NHCH 3 , -NHSO 2 CH 3 , or -N(SO 2 CH 2 CH 2 CH 2 In some embodiments, R 1 is aryl. In some embodiments, aryl is phenyl.

[0148] Additionally or alternatively, the siNA molecules disclosed herein include those of Formulas (1)-(16), (9X)-(12X), (16X), (9Y)-(12Y), (16Y), (21)-(36), (36X), (41)-(56), (49X)-(52X), (49Y)-(52Y), (56X), (56Y), (61), and (62): [ka] [ka] [ka] [ka] wherein R x is a nucleobase, aryl, heteroaryl, or H.

[0149] In some embodiments, any of the siNA molecules disclosed herein comprises a nucleotide sequence represented by Formula (50), Formula (50X), Formula (50Y), Formula (56), Formula (56X), Formula (56Y), Formula (61), Formula (62), and Formula (63): [ka] wherein R x is a nucleobase, aryl, heteroaryl, or H.

[0150] In some embodiments, any of the siNA molecules disclosed herein comprises a nucleotide sequence selected from the group consisting of Formula (71)-(86), Formula (79X)-(82X), Formula (79Y)-(82Y), Formula 86X, Formula 86X', Formula 86Y, and Formula 86Y': [ka] [ka] wherein R x is a nucleobase, aryl, heteroaryl, or H.

[0151] In some embodiments, any of the siNA molecules disclosed herein comprises a nucleotide sequence represented by formula (78), formula (79), formula (79X), formula (79Y), formula (86), formula (86X), and formula (86X'): [ka] wherein R x is a nucleobase, aryl, heteroaryl, or H.

[0152] In some embodiments, any of the siNA molecules disclosed herein comprises a 5' stabilized endcap selected from the group consisting of formulas (1A)-(15A), (1A-1)-(7A-1), (1A-2)-(7A-2), (1A-3)-(7A-3), (1A-4)-(7A-4), (9B)-(12B), (9AX)-(12AX), (9AY)-(12AY), (9BX)-(12BX), and (9BY)-(12BY). [ka] [ka] [ka] [ka]

[0153] In some embodiments, any of the siNA molecules disclosed herein comprises a 5' stabilizing endcap selected from the group consisting of formulas (21A)-(35A), (29B)-(32B), (29AX)-(32AX), (29AY)-(32AY), (29BX)-(32BX), and (29BY)-(32BY). [ka] [ka]

[0154] In some embodiments, any of the siNA molecules disclosed herein comprises a 5' stabilized endcap selected from the group consisting of formulas (71A)-(86A), (79XA)-(82XA), (79YA)-(82YA), (86XA), (86X'A), (86Y), and (86Y'). [ka] [ka]

[0155] In some embodiments, any of the siNA molecules disclosed herein comprises a 5' stabilized endcap selected from the group consisting of formula (78A), formula (79A), formula (79XA), formula (79YA), formula (86A), formula (86XA), and formula (86X'A). [ka]

[0156] In some embodiments, the 5' stabilized end cap is attached to the 5' end of the antisense strand. In some embodiments, the 5' stabilized end cap is attached to the 5' end of the antisense strand via one, two, three, four, or five or more linkers. In some embodiments, the one or more linkers are independently selected from the group consisting of a phosphodiester (p or po) linker, a phosphorothioate (ps) linker, a mesyl phosphoramidate (Ms) linker, a 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.

[0157] As mentioned above, the present disclosure provides compositions comprising any of the siNA molecules, sense strands, antisense strands, first nucleotide sequences, or second nucleotide sequences described herein. The siNAs and compositions thereof of the present disclosure can be used to treat a variety of diseases and conditions (e.g., viral diseases, liver diseases, etc.).

[0158] Linker In some embodiments, any of the siRNAs, sense strands, first nucleotide sequences, antisense strands, and / or second nucleotide sequences disclosed herein comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or more internucleoside linkers. In some embodiments, the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more internucleoside linkers are independently selected from the group consisting of phosphodiester (p or po) linkers, phosphorothioate (ps) linkers, mesyl phosphoramidate (Ms) linkers, or phosphorodithioate linkers.

[0159] In some embodiments, any of the siRNAs, sense strands, first nucleotide sequences, antisense strands, and / or second nucleotide sequences disclosed herein further comprise one, two, three, four, or more linkers connecting the conjugation moiety, phosphorylation blocker, and / or 5' end cap to the siRNAs, sense strands, first nucleotide sequences, antisense strands, and / or second nucleotide sequences. In some embodiments, the one, two, three, four, or more linkers are independently selected from the group consisting of phosphodiester (p or po) linkers, phosphorothioate (ps) linkers, mesyl phosphoramidate (Ms) linkers, phosphoramidite (HEG) linkers, triethylene glycol (TEG) linkers, and / or phosphorodithioate linkers. In some embodiments, 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.

[0160] Exemplary siNA As described above, the siNA disclosed herein may contain modified nucleotides such as 2'-fluoro nucleotides fB, fN, or 4(4nh)Q. Other 2'-fluoro nucleotides such as f2P, f4P, and fX may also be incorporated into the siNA of the present disclosure. The siNAs containing the disclosed 2'-fluoro nucleotides (e.g., fB, fN, or 4(4nh)Q, which are bolded in the table) may contain one or more of the 2'-fluoro nucleotides of the present disclosure, and the one or more 2'-fluoro nucleotides may be present in the sense strand or antisense strand, or both. Table 1 shows exemplary siNAs containing these 2'-fluoro nucleotides. [Table 3-1] [Table 3-2]

[0161] Additionally or alternatively, siNAs of the present disclosure may also incorporate novel nucleotide phosphate mimetics (e.g., omeco-d3U, 4hU, v-mun, c2o-4h, omeco-mun, d2vmA, coc-4h, 4H-VP nucleotides). Table 2 shows exemplary siNAs that include these nucleotide phosphate mimetics. siNAs that include the disclosed novel phosphate mimetics (e.g., omeco-d3U, 4hU, v-mun, c2o-4h, omeco-mun, coc-4h, or d2vmA, shown in bold in the table) may include one or more of the novel phosphate mimetics of the present disclosure, and the one or more novel phosphate mimetics may be present in the sense strand or antisense strand, or both. [Table 4-1] [Table 4-2]

[0162] Additionally or alternatively, the siNAs of the present disclosure may also incorporate novel unlocked nucleotide monomers. These novel unlocked nucleotides include: [ka] (In the formula, R x is a nucleobase, aryl, heteroaryl, or H), or more specifically, [ka] (In the formula, R y These unlocked nucleotides can have the structure: [ka] ) are distinct from unlocked nucleic acids (UNAs) known in the art. Table 3 shows exemplary siNAs that contain these unlocked nucleotides. siNAs that contain a 3',4' UNA (e.g., mun34) can contain one or more of the disclosed 3',4' UNAs, and the one or more 3',4' UNAs can be present in the sense strand or antisense strand, or both. [Table 5-1] [Table 5-2]

[0163] Additionally or alternatively, the siNAs of the present disclosure may also incorporate one or more mesyl phosphoramidate internucleoside linkages. A mesyl phosphoramidate internucleoside linkage (also known as "yp") is [ka] Table 4 shows exemplary siNAs containing these mesyl phosphoramidate internucleoside linkages. siNAs containing mesyl phosphoramidate internucleoside linkages (shown in bold in the table as "yp") may contain one or more yp linkages, which may be present in the sense strand, the antisense strand, or both. [Table 6]

[0164] Additionally or alternatively, the siNA of the present disclosure may also comprise a nucleotide sequence in which Ry represents a nucleobase (e.g., U, A, G, T, C). [ka] In some embodiments, the apN may incorporate a novel monomer designated "apN" having the structure: [ka] Table 5 shows exemplary siNAs that contain these modified nucleotides. siNAs that contain apU nucleotides (designated as "aU" and shown in bold in the table) can contain one or more apU nucleotides, and the one or more apU nucleotides can be present in the sense strand or antisense strand, or both. [Table 7-1] [Table 7-2]

[0165] Target Gene Without wishing to be bound by theory, once inside a cell, any of the ds-siNA molecules disclosed herein can interact with proteins in the cell to form an RNA-induced silencing complex (RISC). When ds-siNA is part of RISC, ds-siNA can unwind to form single-stranded siNA (ss-siNA). ss-siNA can include the antisense strand of ds-siNA. The antisense strand can bind to complementary messenger RNA (mRNA), thereby silencing the gene that codes for the mRNA.

[0166] The target gene may be any gene in a cell. In some embodiments, the target gene is a viral gene. In some embodiments, the viral gene is from a DNA virus. In some embodiments, the DNA virus is a double-stranded DNA (dsDNA) virus. In some embodiments, the dsDNA virus is a hepadnavirus. In some embodiments, the hepadnavirus is Hepatitis B virus (HBV). In some embodiments, the HBV is selected from HBV genotypes A-J. In some embodiments, the viral disease is caused by an RNA virus. In some embodiments, the RNA virus is a single-stranded RNA virus (ssRNA virus). In some embodiments, the ssRNA virus is a positive-sense single-stranded RNA virus ((+)ssRNA virus). In some embodiments, the (+)ssRNA virus is a coronavirus. In some embodiments, the coronavirus is a β-coronavirus. In some embodiments, the β-coronavirus is selected from the group consisting of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (also known under the provisional name of 2019 novel coronavirus, or 2019-nCoV), human coronavirus OC43 (hCoV-OC43), Middle East respiratory syndrome-related coronavirus (MERS-CoV, also known under the provisional name of 2012 novel coronavirus, or 2012-nCoV), and severe acute respiratory syndrome-related coronavirus (SARS-CoV, also known as SARS-CoV-1). In some embodiments, the β-coronavirus is SARS-CoV-2, the causative agent of COVID-19. Some exemplary target genes are shown in Table 17 at the end of this specification.

[0167] In some embodiments, the target gene is selected from the S or X genes of HBV. In some embodiments, the HBV has a genomic sequence set forth in the nucleotide sequence of SEQ ID NO: 55, which corresponds to the nucleotide sequence of GenBank Accession No. U95551.1, which is incorporated by reference in its entirety.

[0168] An exemplary HBV genome sequence is set forth in SEQ ID NO:60, which corresponds to Genbank Accession No. KC315400.1, which is incorporated by reference in its entirety. Nucleotides 2307-3215, 1-1623 of SEQ ID NO:60 correspond to the polymerase / RT gene sequence encoding the polymerase protein. Nucleotides 2848-3215, 1-835 of SEQ ID NO:60 correspond to the PreS1 / S2 / S gene sequence encoding the large S protein. Nucleotides 3205-3215, 1-835 of SEQ ID NO:60 correspond to the PreS2 / S gene sequence encoding the middle S protein. Nucleotides 155-835 of SEQ ID NO:60 correspond to the S gene sequence encoding the small S protein. Nucleotides 1374-1838 of SEQ ID NO:60 correspond to the X gene sequence encoding the X protein. Nucleotides 1814-2452 of SEQ ID NO:60 correspond to the PreC / C gene sequence encoding the precore / core protein. Nucleotides 1901-2452 of SEQ ID NO:60 correspond to the C gene sequence encoding the Core protein. The HBV genome further comprises viral regulatory elements such as viral promoters (preS2, preS1, Core, and X) and enhancer elements (ENH1 and ENH2). Nucleotides 1624-1771 of SEQ ID NO:60 correspond to ENH2. Nucleotides 1742-1849 of SEQ ID NO:60 correspond to the Core promoter. Nucleotides 1818-3215, 1-1930 of SEQ ID NO:60 correspond to the pregenomic RNA (pgRNA) encoding the Core and polymerase proteins.

[0169] In some embodiments, the sense strand comprises a sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to or hybridizes to a viral target RNA sequence beginning with the X region of HBV or the S region of HBV. The viral target may begin, for example, at the 5' end of the target site in acc.KC315400.1 (genotype B, "gt B") or in any one of the A, C, or D genotypes. One of skill in the art will be familiar with HBV locations, for example, as described in Wing-Kin Sung, et al., Nature Genetics 44:765 (2012). In some embodiments, the S region is defined as from the start of the small S protein (genotype BKC315400.1 isolate, position #155) to before the start of the X protein (genotype BKC315400.1 isolate, position #1373). In some embodiments, the X region is defined as from the start of the X protein (genotype BKC315400.1 isolate, position #1374) to the end of the DR2 site (genotype BKC315400.1 isolate, position #1603).

[0170] In some embodiments, the second nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% complementary to 15-30, 15-25, 15-23, 15-22, 15-21, 17-25, 17-23, 17-22, 17-21, or 19-21 nucleotides within positions 200-720, or 1100-1700 of SEQ ID NO:55. In some embodiments, the second nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% complementary to 15-30, 15-25, 15-23, 15-22, 15-21, 17-25, 17-23, 17-22, 17-21, or 19-21 nucleotides within positions 200-280, 300-445, 460-510, 650-720, 1170-1220, 1250-1300, or 1550-1630 of SEQ ID NO:55. In some embodiments, the second nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to 15-30, 15-25, 15-23, 15-22, 15-21, 17-25, 17-23, 17-22, 17-21, or 19-21 nucleotides within positions 200-230, 250-280, 300-330, 370-400, 405-445, 460-500, 670-700, 1180-1210, 1260-1295, 1520-1550, or 1570-1610 of SEQ ID NO:55. In some embodiments, the second nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to 15-30, 15-25, 15-23, 15-22, 15-21, 17-25, 17-23, 17-22, 17-21, or 19-21 nucleotides beginning 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:55.

[0171] In some embodiments, the first nucleotide is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a nucleotide region in SEQ ID NO: 55, except that a thymine (Ts) in SEQ ID NO: 55 is replaced with a uracil (U). In some embodiments, the first nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to 15-30, 15-25, 15-23, 15-22, 15-21, 17-25, 17-23, 17-22, 17-21, or 19-21 nucleotides within positions 200-720, or 1100-1700 of SEQ ID NO: 55. In some embodiments, the first nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% identical to 15-30, 15-25, 15-23, 15-22, 15-21, 17-25, 17-23, 17-22, 17-21, or 19-21 nucleotides within positions 200-280, 300-445, 460-510, 650-720, 1170-1220, 1250-1300, or 1550-1630 of SEQ ID NO:55. In some embodiments, the first nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to 15-30, 15-25, 15-23, 15-22, 15-21, 17-25, 17-23, 17-22, 17-21, or 19-21 nucleotides within positions 200-230, 250-280, 300-330, 370-400, 405-445, 460-500, 670-700, 1180-1210, 1260-1295, 1520-1550, or 1570-1610 of SEQ ID NO:55.In some embodiments, the first nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to 15-30, 15-25, 15-23, 15-22, 15-21, 17-25, 17-23, 17-22, 17-21, or 19-21 nucleotides beginning 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:55.

[0172] Several disease-causing coronaviruses share a high degree of homology in the region of the genome that encodes the nonstructural proteins (nsps), more specifically, the region encoding nsp8 to nsp15. Indeed, there is approximately 65% ​​identity across the approximately 7 kB sequence of β-coronaviruses from approximately 12,900 nucleotides to approximately 19,900 nucleotides of 2019-nCoV, and some subsections of the genome span from nsp8 to nsp15 may contain 95% or more identity. All genes within this region encode nonstructural proteins associated with replication. Therefore, this segment of the genome is amenable to targeting with siNA, which can provide broad spectrum therapy against multiple different types of coronaviruses, such as MERS-CoV, SARS-CoV-1, and SARS-CoV-2.

[0173] In some embodiments, the target gene is selected from the genome of SARS-CoV-2. In some embodiments, SARS-CoV-2 has a genome sequence as set forth in the nucleotide sequence of SEQ ID NO: 74, which corresponds to the nucleotide sequence of GenBank Accession No. NC_045512.2, which is incorporated by reference in its entirety. In some embodiments, the target gene sequence in SEQ ID NO: 74 is 15-30, 15-25, 15-23, 17-23, 19-23, or 19-21 nucleotides in length, preferably 19 or 21 nucleotides in length. In some embodiments, the antisense strand sequence is selected from the group consisting of 190 to 216, 233 to 279, 288 to 324, 455 to 477, 626 to 651, 704 to 723, 3352 to 3378, 5384 to 5403, 6406 to 6483, 7532 to 7551, 9588 to 9606, 10484 to 10509, 11609 to 11630, 11834 to 11853, 12023 to 12045, 12212 to 12234, 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, 14698~14 717, 14722~14748, 14750~14777, 14821~14846, 14854~14873, 14875~14903, 14962~14990, 14992~15020, 15055~15140, 15172~15200, 15310~15332, 15346~15367, 15496~15518, 15622~15644, 15838~15869, 15886~15905, 1598 5~16010, 16057~16079, 16186~16205, 16430~16448, 16822~16865, 16954~16976, 17008~17042, 17080~17111, 17137~17156, 17269~17289, 17530~17549, 17563~17582, 17680~17699, 17746~17765, 17857~17876, 17956~17975,18100~18122, 18196~18218, 19618~19639, 19783~19802, 19831~19850, 20107~20130, 20776~20795, 21502~21524, 24302~24325, 24446~24465, 24620~24651, 24662~24684, 25034~25057, 25104~25128, 25364~253 87, 25502-25530, 26191-26227, 26232-26267, 26269-26330, 26332-26394, 26450-26481, 26574-26600, 27003-27064, 27093-27111, 27183-27212, 27382-27407, 27511-27533, 27771-27818, 28270-28296, 28397-2 8434, 28513~28546, 28673~28692, 28706~28726, 28744~28794, 28799~28827, 28946~28972, 28976~29034, 29144~29172, 29174~29196, 29228~29259, 29285~29305, 29342~29394, 29444~29463, 29543~29566, 2959 It is complementary to 15 to 30, 15 to 25, 15 to 23, 15 to 22, 15 to 21, 17 to 25, 17 to 23, 17 to 22, 17 to 21, or 19 to 21 nucleotides, preferably 19 to 21 nucleotides, and more preferably 19 to 21 nucleotides, within positions 8 to 29630, 29652 to 29687, 29689 to 29731, 29733 to 29757, or 29770 to 29828. In some embodiments, the sense strand sequence is selected from the group consisting of 190 to 216, 233 to 279, 288 to 324, 455 to 477, 626 to 651, 704 to 723, 3352 to 3378, 5384 to 5403, 6406 to 6483, 7532 to 7551, 9588 to 9606, 10484 to 10509, 116 09~11630, 11834~11853, 12023~12045, 12212~12234, 12401~12420, 12839~12867, 12885~12924, 12966~12990, 13151~13176, 13363~13386, 13388~13416, 13458~13416,13458~13520、13762~13790、14290~14312、14404~14429、14500~14531、14623~14642、14650~14687、14698~14717、14722~14748、14750~14777、14821~14846、14854~14873、14875~14903、14962~14990、14992~15020、15055~15140、15172~15200、15310~15332、15346~15367、15496~15518、15622~15644、15838~15869、15886~15905、15985~16010、16057~16079、16186~16205、16430~16448、16822~16865、16954~16976、17008~17042、17080~17111、17137~17156、17269~17289、17530~17549、17563~17582、17680~17699、17746~17765、17857~17876、17956~17975、18100~18122、18196~18218、19618~19639、19783~19802、19831~19850、20107~20130、20776~20795、21502~21524、24302~24325、24446~24465、24620~24651、24662~24684、25034~25057、25104~25128、25364~25387、25502~25530、26191~26227、26232~26267、26269~26330、26332~26394、26450~26481、26574~26600、27003~27064、27093~27111、27183~27212、27382~27407、27511~27533、27771~27818、28270~28296、28397~28434、28513~28546、28673~28692、28706~28726、28744~28794、28799~28827、28946~28972、28976~29034、29144~29172、29174~29196、29228~29259、29285~29305、29342~29394、29444~29463、29543~29566、It is identical to 15 to 30, 15 to 25, 15 to 23, 15 to 22, 15 to 21, 17 to 25, 17 to 23, 17 to 22, 17 to 21, or 19 to 21 nucleotides, preferably 19 to 21 nucleotides, more preferably 19 to 21 nucleotides, within positions 29598 to 29630, 29652 to 29687, 29689 to 29731, 29733 to 29757, or 29770 to 29828.

[0174] In some embodiments, the target gene is selected from the genome of SARS-CoV, which in some embodiments has a genome corresponding to the nucleotide sequence of GenBank Accession No. NC_004718.3, which is incorporated by reference in its entirety.

[0175] In some embodiments, the target gene is selected from the genome of MERS-CoV, which has a genome corresponding to the nucleotide sequence of GenBank Accession No. NC_019843.3, which is incorporated by reference in its entirety.

[0176] In some embodiments, the target gene is selected from the genome of hCoV-OC43, which has a genome corresponding to the nucleotide sequence of GenBank Accession No. NC_006213.1, the entirety of which is incorporated by reference.

[0177] In some embodiments, the target gene is involved in liver metabolism. In some embodiments, the target gene is an inhibitor of the electron transport chain. In some embodiments, the target gene encodes an MCJ protein (MCJ / DnaJC15 or a methylation-regulated J protein). In some embodiments, the MCJ protein is encoded by the mRNA sequence of SEQ ID NO: 56, which corresponds to the nucleotide sequence of GenBank Accession No. NM_013238.3, which is incorporated by reference in its entirety.

[0178] In some embodiments, the target gene is TAZ. In some embodiments, TAZ comprises the nucleotide sequence of SEQ ID NO: 57, which corresponds to the nucleotide sequence of GenBank Accession No. NM_000116.5, the entirety of which is incorporated by reference.

[0179] In some embodiments, the target gene is angiopoietin-like 3 (ANGPTL3). In some embodiments, ANGPTL3 comprises the nucleotide sequence of SEQ ID NO: 60, which corresponds to the nucleotide sequence of GenBank Accession No. NM_014495.4, the entirety of which is incorporated by reference. In some embodiments, the target gene is diacylglycerol acyltransferase 2 (DGAT2). In some embodiments, DGAT2 comprises the nucleotide sequence of SEQ ID NO: 59, which corresponds to the nucleotide sequence of GenBank Accession No. NM_001253891.1, which is incorporated by reference in its entirety.

[0180] composition As mentioned above, the present disclosure provides a composition comprising any of the siNA molecules, sense strands, antisense strands, first nucleotide sequences, or second nucleotide sequences described herein. The composition may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more siNA molecules described herein. The composition may comprise a first nucleotide sequence comprising the nucleotide sequence of any one of SEQ ID NOs: 1 and 2. In some embodiments, the composition comprises a second nucleotide sequence comprising the nucleotide sequence of any one of SEQ ID NOs: 51-74. In some embodiments, the composition comprises a sense strand comprising the nucleotide sequence of any one of SEQ ID NOs: 1 and 2. In some embodiments, the composition comprises an antisense strand comprising the nucleotide sequence of any one of SEQ ID NOs: 51-74.

[0181] Alternatively, the composition may comprise (a) a phosphorylation blocker and (b) a short interfering nucleic acid (siNA). In some embodiments, the phosphorylation blocker is any of the phosphorylation blockers disclosed herein. In some embodiments, the siNA is any of the siNAs disclosed herein. In some embodiments, the siNA comprises any of the sense strand, antisense strand, first nucleotide sequence, or second nucleotide sequence described herein. In some embodiments, the siNA comprises any of the sense strand, antisense strand, first nucleotide sequence, or second nucleotide sequence described herein. In some embodiments, the siNA comprises one or more modified nucleotides. In some embodiments, the one or more modified nucleotides are independently selected from 2'-fluoro nucleotides and 2'-O-methyl nucleotides. In some embodiments, the 2'-fluoro nucleotides or 2'-O-methyl nucleotides are independently selected from any of the 2'-fluoro or 2-O-methyl nucleotide mimics disclosed herein. In some embodiments, the siNA comprises a nucleotide sequence comprising any of the modification patterns disclosed herein.

[0182] In some embodiments, the composition comprises (a) a conjugated moiety and (b) a short interfering nucleic acid (siNA). In some embodiments, the conjugated moiety is any of the galactosamines disclosed herein. In some embodiments, the siNA is any of the siNAs disclosed herein. In some embodiments, the siNA comprises any of the sense strand, antisense strand, first nucleotide sequence, or second nucleotide sequence described herein. In some embodiments, the siNA comprises any of the sense strand, antisense strand, first nucleotide sequence, or second nucleotide sequence described herein. In some embodiments, the siNA comprises one or more modified nucleotides. In some embodiments, the one or more modified nucleotides are independently selected from 2'-fluoro nucleotides and 2'-O-methyl nucleotides. In some embodiments, the 2'-fluoro nucleotides or 2'-O-methyl nucleotides are independently selected from any of the 2'-fluoro or 2-O-methyl nucleotide mimics disclosed herein. In some embodiments, the siNA comprises a nucleotide sequence comprising any of the modification patterns disclosed herein.

[0183] In some embodiments, the composition comprises (a) a 5' stabilized endcap and (b) a short interfering nucleic acid (siNA). In some embodiments, the 5' stabilized endcap is any of the 5' stabilized endcaps disclosed herein. In some embodiments, the siNA is any of the siNAs disclosed herein. In some embodiments, the siNA comprises any of the sense strand, antisense strand, first nucleotide sequence, or second nucleotide sequence described herein. In some embodiments, the siNA comprises one or more modified nucleotides. In some embodiments, the one or more modified nucleotides are independently selected from 2'-fluoro nucleotides and 2'-O-methyl nucleotides. In some embodiments, the 2'-fluoro nucleotides or 2'-O-methyl nucleotides are independently selected from any of the 2'-fluoro or 2-O-methyl nucleotide mimics disclosed herein. In some embodiments, the siNA comprises a nucleotide sequence comprising any of the modification patterns disclosed herein.

[0184] In some embodiments, the composition comprises (a) at least one phosphorylation blocker, conjugation moiety, or 5' stabilized endcap, and (b) a short interfering nucleic acid (siNA). In some embodiments, the phosphorylation blocker is any of the phosphorylation blockers disclosed herein. In some embodiments, the conjugation moiety is any of the galactosamines disclosed herein. In some embodiments, the 5' stabilized endcap is any of the 5' stabilized endcaps disclosed herein. In some embodiments, the siNA is any of the siNAs disclosed herein. In some embodiments, the siNA comprises any of the sense strand, antisense strand, first nucleotide sequence, or second nucleotide sequence described herein. In some embodiments, the siNA comprises one or more modified nucleotides. In some embodiments, the one or more modified nucleotides are independently selected from 2'-fluoro nucleotides and 2'-O-methyl nucleotides. In some embodiments, the 2'-fluoro nucleotides or 2'-O-methyl nucleotides are independently selected from any of the 2'-fluoro or 2-O-methyl nucleotide mimics disclosed herein. In some embodiments, the siNA comprises a nucleotide sequence comprising any of the modification patterns disclosed herein.

[0185] The composition may be a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises an amount of one or more of the siNA molecules described herein, formulated with one or more pharma- ceutically acceptable carriers (additives) and / or diluents. The pharmaceutical composition may be specially formulated for administration in solid or liquid form, including those adapted for: (1) oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., buccal, sublingual, and targeted for systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., sterile solutions or suspensions, or sustained release formulations; (3) topical administration, e.g., creams, ointments, or controlled release patches or sprays applied to the skin; (4) vaginally or rectally, e.g., as a pessary, cream, or foam; (5) sublingually; (6) ophthalmic; (7) transdermal; or (8) nasal.

[0186] As used herein, the phrase "therapeutically effective amount" means an amount of a compound, material, or composition, including a siNA of the present disclosure, effective to produce some desired therapeutic effect in at least a subpopulation of cells in an animal at a reasonable benefit / risk ratio applicable to any medical treatment.

[0187] The phrase "pharmacologically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment, commensurate with a reasonable benefit / risk ratio.

[0188] Wetting agents, emulsifying agents, 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 composition.

[0189] Examples of pharma- ceutically 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 chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0190] The formulations of the present disclosure include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and / or parenteral administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any method well known in the art of pharmacy. 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, the particular 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 that amount of compound (e.g., siNA molecule) that produces a therapeutic effect. Generally, out of 100%, this amount will range from about 0.1% to about 99% of the active ingredient, preferably from about 5% to about 70%, and most preferably from about 10% to about 30%.

[0191] In certain embodiments, a formulation of the present disclosure comprises an excipient selected from the group consisting of cyclodextrins, celluloses, liposomes, micelle forming agents such as bile acids, and polymeric carriers such as polyesters and polyanhydrides, and a compound of the present disclosure (e.g., a siNA molecule). In certain embodiments, the formulation renders the compound of the present disclosure (e.g., a siNA molecule) orally bioavailable.

[0192] Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present disclosure (e.g., siNA molecule) with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present disclosure (e.g., siNA molecule) with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0193] Formulations of the present disclosure suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), 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 a pastille (using an inert base such as gelatin and glycerin, or sucrose and acacia), and / or as a mouthwash, each containing a predetermined amount of a compound of the present disclosure (e.g., siNA molecule) as an active ingredient. A compound of the present disclosure (e.g., siNA molecule) may also be administered as a bolus, electuary, or paste.

[0194] In 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 pharma- ceutically 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) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (5) solution retarding agents, such as paraffin, and (6) quaternary ammonium salts, such as poloxamer and sodium laurate. (7) absorption enhancers such as compounds and surfactants; (8) wetting agents such as cetyl alcohol, glycerol monostearate, and nonionic surfactants; (9) absorbents such as talc, calcium stearate, stearic acid, solid magnesium stearate, polyethylene glycol, sodium lauryl phosphate, sodium stearate, and mixtures thereof; (10) colorants; and (11) controlled release agents such as ketyl alcohol, and nonionic surfactants.

[0195] In the case of capsules, tablets, and pills, the pharmaceutical compositions may also contain buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard shelled gelatin capsules using such excipients as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like.

[0196] Tablets can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using binders (e.g., gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surface active agents or dispersing agents. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

[0197] Tablets and other solid dosage forms of the pharmaceutical compositions of the present disclosure, such as dragees, capsules, pills and granules, can be optionally scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. They can also be formulated to provide sustained or controlled release of the active ingredient therein, for example, using various proportions of hydroxypropylmethylcellulose, other polymer matrices, liposomes and / or microspheres to provide the desired release profile. They can also be formulated for immediate release, for example, lyophilized.

[0198] They may be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating a sterilizing agent in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injectable medium immediately before use. These compositions may also optionally contain an opacifying agent, and may be of a composition that releases the active ingredient(s) only, or preferentially, in a certain part of the gastrointestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes. The active ingredient may also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

[0199] Liquid dosage forms for oral administration of the compounds (e.g., siNA molecules) of the present disclosure include pharma- ceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.In addition to the active ingredient, the liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, and mixtures thereof.

[0200] 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.

[0201] Suspensions may contain, in addition to the active compound (e.g., siNA molecule), suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

[0202] Formulations of pharmaceutical compositions of the disclosure for rectal or vaginal administration may be presented as suppositories, which may be prepared by mixing one or more compounds of the disclosure (e.g., siNA molecules) with one or more suitable non-irritating excipients or carriers including, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, 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).

[0203] 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.

[0204] Dosage forms for topical or transdermal administration of the disclosed compounds (e.g., siNA molecules) include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound (e.g., siNA molecule) may be mixed under sterile conditions with a pharma- ceutically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0205] The ointments, pastes, creams and gels may contain, in addition to the active compounds of the present disclosure (e.g., siNA molecules), excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0206] Powders and sprays can contain, in addition to the compounds of the present disclosure (e.g., siNA molecules), excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these substances. Sprays can additionally contain conventional propellants, such as chlorofluorohydrocarbons, and volatile unsubstituted hydrocarbons, such as butane and propane.

[0207] Transdermal patches have the added advantage of providing controlled delivery of the disclosed compounds (e.g., siNA molecules) to the body. Such dosage forms can be made by dissolving or dispersing the compounds (e.g., siNA molecules) in a suitable medium. Absorption enhancers can also be used to increase the flux of the compounds (e.g., siNA molecules) through the skin. The rate of such flux can be controlled by either providing a rate-controlling membrane or dispersing the compounds (e.g., siNA molecules) in a polymer matrix or gel.

[0208] Ophthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope of the present disclosure.

[0209] Pharmaceutical compositions of the disclosure suitable for parenteral administration comprise one or more compounds of the disclosure (e.g., siNA molecules) in combination with one or more pharma- ceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted into sterile injectable solutions or dispersions immediately prior to use, and may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents.

[0210] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present disclosure include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0211] These compositions may contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of microbial action on the subject compounds may be ensured by including various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0212] In some cases, it is desirable to delay the absorption of drugs from subcutaneous or intramuscular injections in order to prolong the effect of drugs.This can be achieved by using liquid suspensions of crystalline or amorphous substances that are poorly water-soluble.The absorption rate of the drug then depends on its dissolution rate, which in turn depends on the crystal size and crystalline form.Alternatively, delayed absorption of parenterally administered drug forms can be achieved by dissolving or suspending the drug in an oil vehicle.

[0213] Injectable depot forms are made by forming microencapsule matrices of the subject compounds (e.g., siNA molecules) in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer and the nature of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Injectable depot forms are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0214] When the compounds of the present disclosure (e.g., siNA molecules) are administered to humans and animals as pharmaceuticals, the compounds of the present disclosure can be provided by themselves or as a pharmaceutical composition containing, for example, 0.1 to 99% (more preferably, 10 to 30%) of the active ingredient in combination with a pharma- ceutically acceptable carrier.

[0215] Treatment and Administration The siNA molecule of the present disclosure may be used to treat a disease in a subject in need of the treatment.In some embodiments, the method of treating a disease in a subject in need of the treatment comprises administering any of the siNA molecules disclosed herein to the subject.In some embodiments, the method of treating a disease in a subject in need of the treatment comprises administering any of the compositions disclosed herein to the subject.

[0216] The preparations (e.g., siNA molecules or compositions) of the present disclosure can be given orally, parenterally, topically, or rectally. They are, of course, given in a form suitable for each administration route. For example, they are given in tablet or capsule form, administered by injection, infusion or inhalation, topically by lotion or ointment, and rectally by suppository. Oral administration is preferred.

[0217] As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrapleural injection and infusion.

[0218] As used herein, the phrases "systemic administration," "systemically administered," "peripheral administration," and "administered peripherally" refer to administration of a compound, drug, or other substance other than directly into the central nervous system so that it enters the patient's system and thus undergoes metabolic and other like processes, e.g., subcutaneous administration.

[0219] These compounds may be administered to humans and other animals for treatment by any suitable route of administration, including orally, nasally, e.g., by spray, rectally, vaginally, parenterally, intracisternally, and topically, e.g., by powders, ointments or drops, buccally and sublingually.

[0220] Regardless of the route of administration selected, the compounds (e.g., siNA molecules) and / or pharmaceutical compositions of the present disclosure, which may be used in a suitable hydrated form, are formulated into pharma- ceutically acceptable dosage forms by conventional methods known to those of skill in the art.

[0221] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient.

[0222] The selected dosage level will depend on a variety of factors, including the activity of the particular compound of the present disclosure (e.g., a siNA molecule), or ester, salt, or amide thereof, being used, the route of administration, the time of administration, the rate of excretion or metabolism, the rate and extent of absorption of the particular compound being used, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular compound being used, the age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors well known in the medical arts.

[0223] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian can start the dosage of the disclosed compound (e.g., siNA molecule) used in the pharmaceutical composition at a lower level than is needed to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0224] In general, a suitable daily dose of a compound (e.g., siNA molecule) of the present disclosure is the amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend on the factors mentioned above. Preferably, the compound is administered at about 0.01 mg / kg to about 200 mg / kg, more preferably about 0.1 mg / kg to about 100 mg / kg, and even more preferably about 0.5 mg / kg to about 50 mg / kg. In some embodiments, a compound of the invention is administered at a dose of 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1 mg / kg or more. In some embodiments, the compound is administered at a dose of less than or equal to 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, or 15 mg / kg. In some embodiments, the total daily dose of the compound is greater than or equal to 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.

[0225] When a compound described herein (eg, a siNA molecule) is co-administered with another, the effective amount may be less than when that compound is used alone.

[0226] If desired, the effective daily dose of the active compound (e.g., siNA molecule) may be administered as 2, 3, 4, 5, 6, or more subdoses administered at appropriate intervals throughout the day, optionally in unit dosage form. Preferred dosing 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 per week. In some embodiments, the compound is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 times per month. In some embodiments, the compound is administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days. In some embodiments, the compound is administered once every 1, 2, 3, 4, 5, 6, 7, or 8 weeks.

[0227] disease The siNA molecules and compositions described herein may 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.

[0228] 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).

[0229] In some embodiments, the disease is a liver disease. In some embodiments, the liver disease is non-alcoholic fatty liver disease (NAFLD). In some embodiments, the NAFLD is non-alcoholic steatohepatitis (NASH). In some embodiments, the liver disease is hepatocellular carcinoma (HCC).

[0230] The siNA molecules of the present disclosure may be used to treat or prevent a disease in a subject in need thereof. In some embodiments, a method of treating or preventing a disease in a subject in need thereof comprises administering to the subject any of the siNA molecules disclosed herein. In some embodiments, a method of treating or preventing a disease in a subject in need thereof comprises administering to the subject any of the compositions disclosed herein.

[0231] In some embodiments of the methods and uses of the present disclosure, the disease is a respiratory disease. In some embodiments, the respiratory disease is a viral infection. In some embodiments, the respiratory disease is viral pneumonia. In some embodiments, the respiratory disease is an acute respiratory infection. In some embodiments, the respiratory disease is a cold. In some embodiments, the respiratory disease is Severe Acute Respiratory Syndrome (SARS). In some embodiments, the respiratory disease is Middle East Respiratory Syndrome (MERS). In some embodiments, the disease is a coronavirus disease 2019 (e.g., COVID-19). In some embodiments, the respiratory disease can include one or more symptoms selected from cough, sore throat, runny nose, sneezing, headache, fever, shortness of breath, muscle pain, abdominal pain, fatigue, difficulty breathing, persistent chest pain or pressure, difficulty waking up, loss of smell and taste, muscle or joint pain, chills, nausea or vomiting, stuffy nose, diarrhea, hemoptysis, conjunctival congestion, sputum production, chest pressure, and palpitations. In some embodiments, the respiratory disease can include a complication selected from sinusitis, otitis media, pneumonia, acute respiratory distress syndrome, disseminated intravascular coagulation, pericarditis, and renal failure, hi some embodiments, the respiratory disease is idiopathic.

[0232] In some embodiments, the disclosure provides a method for treating or preventing a coronavirus infection comprising administering a therapeutically effective amount of one or more of the siNAs or pharmaceutical compositions disclosed herein to a subject in need thereof. In some embodiments, the coronavirus infection is selected from the group consisting of Middle East Respiratory Syndrome (MERS), Severe Acute Respiratory Syndrome (SARS), and COVID-19. In some embodiments, the subject is being treated with one or more additional coronavirus therapeutic agents. In some embodiments, the subject is being treated concurrently with one or more additional coronavirus therapeutic agents.

[0233] Administration of siNA Administration of any of the siNAs disclosed herein may be by methods known in the art. In some embodiments, the siNAs are administered by subcutaneous (SC) or intravenous (IV) delivery. The preparations (e.g., siNAs or compositions) of the present disclosure may be given orally, parenterally, topically, or rectally. They are, of course, given in a form suitable for each administration route. For example, they 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.

[0234] As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrapleural injection and infusion.

[0235] As used herein, the phrases "systemic administration," "systemically administered," "peripheral administration," and "administered peripherally" refer to administration of a compound, drug, or other substance other than directly into the central nervous system so that it enters the patient's system and thus undergoes metabolic and other like processes, e.g., subcutaneous administration.

[0236] These compounds may be administered to humans and other animals for treatment by any suitable route of administration, including orally, nasally, e.g., by spray, rectally, vaginally, parenterally, intracisternally, and topically, e.g., by powders, ointments or drops, buccally and sublingually.

[0237] Regardless of the route of administration selected, the compounds of the present disclosure (e.g., siNA), which may be used in a suitable hydrated form, and / or pharmaceutical compositions of the present disclosure are formulated into pharma- ceutically acceptable dosage forms by conventional methods known to those of skill in the art.

[0238] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient.

[0239] The selected dosage level will depend on a variety of factors, including the activity of the particular compound of the disclosure (e.g., siNA), or ester, salt, or amide thereof, being used, the route of administration, the time of administration, the rate of excretion or metabolism, the rate and extent of absorption of the particular compound being used, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular compound being used, the age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors well known in the medical arts.

[0240] A physician or veterinarian having ordinary skill in the art can easily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian can start the dosage of the compound of the present disclosure (e.g., siNA) used in the pharmaceutical composition at a lower level than is needed to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0241] In general, a suitable daily dose of a compound of the present disclosure (e.g., siNA) is the amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend on the factors described above. Preferably, the compound is administered at about 0.01 mg / kg to about 200 mg / kg, more preferably about 0.1 mg / kg to about 100 mg / kg, and even more preferably about 0.5 mg / kg to about 50 mg / kg. In some embodiments, the compound is administered at about 1 mg / kg to about 40 mg / kg, about 1 mg / kg to about 30 mg / kg, about 1 mg / kg to about 20 mg / kg, about 1 mg / kg to about 15 mg / kg, or 1 mg / kg to about 10 mg / kg. In some embodiments, a compound of the invention is administered at a dose of 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1 mg / kg or more. In some embodiments, the compound is administered at a dose of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mg / kg or more. In some embodiments, the compound is administered at a dose of 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, or 15 mg / kg or less. In some embodiments, the total daily dose of the compound is greater than or equal to 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.

[0242] If desired, the effective daily dose of the active compound (e.g., siNA) may be administered as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses or subdoses, which are administered separately at appropriate intervals throughout the day, optionally in unit dosage forms. In some embodiments, the compound is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 times. The preferred dosage 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 per week. In some embodiments, the compound is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 times per month. In some embodiments, the compound is administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days. In some embodiments, the compound is administered every 3 days. In some embodiments, the compound is administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 weeks. In some embodiments, the compound is administered monthly. In some embodiments, the compound is administered once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 months.In some embodiments, the compound is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53 times. The vaccine is administered over a period of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 days. In some embodiments, the compound comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 5 Three doses are administered over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53 weeks.In some embodiments, the compound comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53 The therapeutic agent is administered once or twice a day 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, or 53 months. In some embodiments, the compound is administered at least once per week for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 weeks. In some embodiments, the compound is administered at least once per week for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 months.In some embodiments, the compound is administered at least twice per week for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 weeks. In some embodiments, the compound is administered at least twice a week for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 months. In some embodiments, the compound is administered at least once every two weeks for a period of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 weeks.In some embodiments, the compound is administered at least once every two weeks for a period of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 months. In some embodiments, the compound is administered once every four weeks for a period of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 weeks. In some embodiments, the compound is administered at least once every four weeks for a period of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 months.

[0243] In some embodiments, any one of the siNA or compositions disclosed herein is administered in a particle or viral vector.In some embodiments, the viral vector is 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, or a rhabdovirus vector.In some embodiments, the viral vector is a recombinant viral vector.In some embodiments, the viral vector is selected from AAVrh.74, AAVrh.10, AAVrh.20, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, and AAV-13.

[0244] The subject of the described methods may be a mammal, including humans and non-human mammals, hi some embodiments, the subject is a human, such as an adult human.

[0245] Some embodiments include a method for treating HBV virus in a subject infected with the virus, comprising administering a therapeutically effective amount of one or more siNAs of the present disclosure or compositions of the present disclosure to a subject in need thereof, thereby reducing the viral load of the virus in the subject and / or reducing the level of viral antigens in the subject. The siNA may be complementary to or hybridize to a portion of a target RNA in the virus, for example, the X and / or S region of HBV.

[0246] Combination therapy Any of the methods disclosed herein may further comprise administering to the subject an additional HBV therapeutic agent. Any of the compositions disclosed herein may further comprise an additional HBV therapeutic agent. In some embodiments, the additional HBV therapeutic agent is selected from nucleotide analogs, nucleoside analogs, capsid assembly modulators (CAMs), recombinant interferons, entry inhibitors, small molecule immunomodulators, and oligonucleotide therapy. In some embodiments, the additional HBV therapeutic agent is HBV STOPS™ ALG-010133, HBV CAMs, or combinations thereof. ALG-000184, ASO1 (SEQ ID NO: 61), ASO2 (SEQ ID NO: 62) recombinant interferon alpha 2b, IFN-a, PEG-IFN-a-2a, lamivudine, telbivudine, adefovir dipivoxil, clevudine, entecavir, tenofovir alafenamide, tenofovir disoproxil, NVR3-778, BAY41-4109, JNJ-632, JNJ-3989 ( 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 inhibitor oligonucleotide polymer (NAP or STOPS), an siRNA, and an ASO. In some embodiments, the oligonucleotide therapy is an additional siNA. In some embodiments, the additional siNA is selected from any of ds-siNA-001 to ds-siNA-025. In some embodiments, the oligonucleotide therapy is an antisense oligonucleotide (ASO). In some embodiments, the ASO is ASO1 (SEQ ID NO: 61) or ASO2 (SEQ ID NO: 62). In some embodiments, any of the siNAs disclosed herein are co-administered with STOPS.Exemplary STOPS are described in International Publication No. WO 2020 / 097342 and U.S. Publication No. 2020 / 0147124, both of which are incorporated by reference in their entireties. In some embodiments, the STOPS is ALG-010133. In some embodiments, any of the siNAs disclosed herein are co-administered with tenofovir. In some embodiments, any of the siNAs disclosed herein are co-administered with CAM. For exemplary CAMs, see Berke et al., Antimicrob Agents Chemother, 2017, 61(8):e00560-17, Klumpp, et al., Gastroenterology, 2018, 154(3):652-662.e8, International Application Nos. PCT / US2020 / 017974, PCT / US2020 / 026116, and PCT / US2020 / 028349, and U.S. Application Nos. 16 / 789,298, 16 / 837,515, and 16 / 849,851. In some embodiments, the CAM is ALG-000184, ALG-001075, ALG-001024, JNJ-632, BAY41-4109, or NVR3-778. In some embodiments, the siNA and the HBV therapeutic agent are administered simultaneously. In some embodiments, the siNA and the HBV therapeutic agent are administered simultaneously. In some embodiments, the siNA and the HBV therapeutic agent are administered sequentially. In some embodiments, the siNA is administered prior to administration of the HBV therapeutic agent. In some embodiments, the siNA is administered after administration of the HBV therapeutic agent. In some embodiments, the siNA and the HBV therapeutic agent are in separate containers. In some embodiments, the siNA and the HBV therapeutic agent are in the same container.

[0247] Any of the methods disclosed herein may further comprise administering to the subject a liver disease therapeutic drug. Any of the compositions disclosed herein may further comprise a liver disease therapeutic drug. In some embodiments, the liver disease therapeutic drug is selected from a peroxisome proliferator-activated receptor (PPAR) agonist, a farnesoid X receptor (FXR) agonist, a lipid-altering agent, and an incretin system therapy. In some embodiments, the PPAR agonist is selected from a PPARα agonist, a dual PPARα / δ agonist, a PPARγ agonist, and a dual PPARα / γ agonist. In some embodiments, the dual PPARα agonist is a fibrate. In some embodiments, the PPARα / δ agonist is elafibranor. In some embodiments, the PPARγ agonist is a thiazolidinedione (TZD). In some embodiments, the TZD is pioglitazone. In some embodiments, the dual PPARα / γ agonist is saroglitazar. In some embodiments, the FXR agonist is obetichol acys (OCA). In some embodiments, the lipid-altering agent is aramchol. In some embodiments, the incretin system therapy is a glucagon-like peptide 1 (GLP-1) receptor agonist or a dipeptidyl peptidase 4 (DPP-4) inhibitor. In some embodiments, the GLP-1 receptor agonist is exenatide or liraglutide. In some embodiments, the DPP-4 inhibitor is sitagliptin or vildapriptin. In some embodiments, the siNA and the liver disease therapeutic agent are administered simultaneously. In some embodiments, the siNA and the liver disease therapeutic agent are administered sequentially. In some embodiments, the siNA is administered prior to administering the liver disease therapeutic agent. In some embodiments, the siNA is administered after administering the liver disease therapeutic agent. In some embodiments, the siNA and the liver disease therapeutic agent are in separate containers. In some embodiments, the siNA and the liver disease therapeutic agent are in the same container.

[0248] definition Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this disclosure belongs. The following references provide those skilled in the art with general definitions of many of the terms used in the present invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed.1994), The Cambridge Dictionary of Science and Technology (Walker, ed., 1988), The Glossary of Genetics, 5th Ed., R. Rieger et al., (eds.), Springer Verlag (1991), and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless otherwise specified. The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the present disclosure.

[0249] As used herein, the words "a" and "an" mean "one or more" and include the plural forms unless the context is inappropriate.

[0250] As used herein, the terms "patient" and "subject" refer to an organism that is treated by the methods of the present disclosure. Such organisms are preferably mammals (e.g., marine organisms, apes, horses, cows, pigs, dogs, cats, etc.), and more preferably humans.

[0251] As used herein, the term "effective amount" refers to an amount of a compound (e.g., a siNA of the present disclosure) sufficient to produce a beneficial or desired result. An effective amount can be administered in one or more administrations, applications, or dosages, and is not intended to be limited to a particular formulation or route of administration.

[0252] As used herein, the term "treating" includes any effect, e.g., alleviating, reducing, modulating, ameliorating, or eliminating, that results in the improvement of a condition, disease, disorder, or the like, or that ameliorates the symptoms thereof.

[0253] As used herein, the terms "ameliorate" and "ameliorating" refer to reducing the severity of a condition, such as reducing the severity by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.

[0254] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent with an inert or active carrier that makes the composition particularly suitable for in vivo or ex vivo diagnostic or therapeutic uses.

[0255] As used herein, the term "pharmaceutical acceptable carrier" refers to any of the standard pharmaceutical carriers, such as phosphate buffered saline, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA

[1975] .

[0256] As used herein, the term "about" when referring to measurable values ​​(e.g., weight, time, and dose) is meant to encompass variations such as ±10%, ±5%, ±1%, or ±0.1% of the specified value.

[0257] As used herein, the term "nucleobase" refers to a nitrogen-containing biological compound that forms a nucleoside. Examples of nucleobases include, but are not limited to, thymine, uracil, adenine, cytosine, guanine, and analogs or derivatives thereof.

[0258] Throughout this specification, when compositions are described as having, including, or comprising certain components, or processes and methods are described as having, including, or comprising certain steps, it is contemplated that there are additionally compositions of the disclosure that consist essentially of or consist of the recited components, and processes and methods according to the disclosure that consist essentially of or consist of the recited processing steps.

[0259] As a general matter, compositions specifying percentages are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, the previous definition of the variable is controlling.

[0260] All publications and patents cited herein are incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference herein, and are incorporated by reference herein to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the publication dates provided may be different from the actual publication dates which may need to be independently confirmed. EXAMPLES

[0261] Example 1: siNA synthesis This example describes an exemplary method for synthesizing ds-siNAs, for example, siNAs disclosed in Tables 1-5 (as identified by ds-siNA ID).

[0262] 2'-O-Me phosphoramidites 5'-O-DMT-deoxyadenosine (NH-Bz), 3'-O-(2-cyanoethyl-N,N-diisopropyl phosphoramidite, 5'-O-DMT-deoxyguanosine (NH-ibu), 3'-O-(2-cyanoethyl-N,N-diisopropyl phosphoramidite, 5'-O-DMT-deoxycytosine (NH-Bz), 3'-O-(2-cyanoethyl-N,N-diisopropyl phosphoramidite, 5'-O-DMT-uridine 3'-O-(2-cyanoethyl-N,N-diisopropyl phosphoramidite, and solid supports were purchased from Chemgenes Corp. MA. [ka]

[0263] 2'-F-5'-O-DMT-(NH-Bz) adenosine-3'-O-(2-cyanoethyl-N,N-diisopropyl phosphoramidite, 2'-F-5'-O-DMT-(NH-ibu)-guanosine, 3'-O-(2-cyanoethyl-N,N-diisopropyl phosphoramidite, 5'-O-DMT-(NH-Bz)-cytosine, 2'-F-3'-O-(2-cyanoethyl-N,N-diisopropyl phosphoramidite, 5'-O-DMT-uridine, 2'-F-3'-O-(2-cyanoethyl-N,N-diisopropyl phosphoramidite, and solid supports were purchased from Milwaukee WI, USA. [ka]

[0264] All monomers were mixed with a desiccant (P 2 O 5The nucleosides and universal support-bound solid supports (CPG) were obtained from LGC and Chemgenes. Chemicals and solvents for the post-synthesis workflow were purchased from commercially available sources such as VWR / Sigma and used without any purification or treatment. Solvents (acetonitrile) and solutions (amidites and activators) were stored over molecular sieves during the synthesis.

[0265] Oligonucleotides were synthesized on a DNA / RNA synthesizer (Expedite 8909 or ABI-394 or MM-48) using standard oligonucleotide phosphoramidite chemistry starting from the 3′ residue of the oligonucleotide preloaded on the CPG support. CH 3 Extended coupling of a 0.1 M solution of phosphoramidite in CN to the solid-bound oligonucleotide, followed by standard capping, oxidation and deprotection, afforded the modified oligonucleotide. 2 DDTT ((dimethylamino-methylidene)amino)-3H-1,2,4-dithiazaolin-3-thione was used as the sulfur transfer agent for the synthesis of oligoribonucleotide phosphorothioates, with THF:pyridine:water-7:2:1 as the oxidizing agent. The stepwise coupling efficiency of all modified phosphoramidites was greater than 98%. [Table 8]

[0266] Cleavage and deprotection: Deprotection and cleavage from the solid support was achieved with a mixture of ammonia methylamine (1:1, AMA) at 65 °C for 15 min when the universal linker was used, leaving the deprotection at 65 °C for 90 min, or by heating the solid support with aqueous ammonia (28%) at 55 °C for 8–16 h to deprotect the base labile protecting groups.

[0267] Quantitative or raw material analysis of crude siNA Samples were dissolved in deionized water (1.0 mL) and quantified as follows: Blanking was first performed with water alone (2 μl) on a Thermo Scientific™ Nanodrop UV spectrophotometer or a BioTek™ Epoch™ plate reader before obtaining oligo sample readings at 260 nm. Crude material is dried and stored at -20°C.

[0268] Crude HPLC / LC-MS analysis For crude HPLC and LC-MS analysis, 0.1 OD of crude sample was analyzed. After reviewing the crude LC-MS data, purification steps were performed if necessary based on purity.

[0269] HPLC purification Unconjugated GalNac modified oligonucleotides were purified by anion exchange HPLC. The buffer was 10% CH 3 20 mM sodium phosphate, pH 8.5 (buffer A) in CN, and 10% CH 3 20 mM sodium phosphate, 1.0 M NaBr in CN, pH 8.5 (Buffer B). Fractions containing the full length oligonucleotide were pooled.

[0270] Desalting of purified SiNA The purified dried siNA was then desalted using Sephadex G-25M (Amersham Biosciences). The cartridge was conditioned three times with 10 mL of deionized water. Finally, the purified siNA, completely dissolved in 2.5 mL of RNAse-free water, was applied to the cartridge with very slow dropwise elution. The salt-free siNA was eluted directly into a screw-cap vial with 3.5 ml of deionized water. Alternatively, some unconjugated siNA was desalted using a Pall AcroPrep™ 3K MWCO desalting plate.

[0271] IEX HPLC and electrospray LC / MS analysis Approximately 0.10 OD of siNA was dissolved in water and then pipetted into an HPLC autosampler vial for IEX-HPLC and LC / MS analysis. Analytical HPLC and ES LC-MS confirmed compound identity and purity.

[0272] Preparation of double strands: The single-stranded oligonucleotides (sense and antisense strands) were annealed (1:1 by molar equivalent, 90°C for 2 min, followed by slow cooling at room temperature) to obtain double-stranded ds-siNA. The final compounds were analyzed by size exclusion chromatography (SEC).

[0273] Example 2 [ka]

[0274] Preparation of PH-ALIG-14-1-1 A 5000 mL 3-neck round bottom flask purged and maintained with an inert atmosphere of argon was charged with uridine (150.00 g, 614.24 mmol, 1.00 equiv), pyridine (2.2 L), TBDPSCl (177.27 g, 644.95 mmol, 1.05 equiv). The resulting solution was stirred at room temperature overnight. The resulting mixture was concentrated. The resulting solution was extracted with 3×1000 mL of dichloromethane and the organic layers were combined. The resulting mixture was diluted with 3×1 L of 0.5 N HCl (aq) and 2×500 mL of 0.5 N NaHCO 3 The resulting mixture was washed with 2×1 L of H 2 The mixture was washed with O. The mixture was dried over anhydrous sodium sulfate. The solids were filtered off. The filtrate was concentrated. This gave 262 g (crude) PH-ALIG-14-1-1. LC-MS (m / z) 483.00 [M+H] + ; 1 H NMR (400MHz, DMSO-d 6) δ 11.35(d,J=2.2Hz,1H),7.70(d,J=8.1Hz,1H),7.64(m,4H),7.52-7.40(m,6H),5.80(d,J=4.1Hz,1H),5.50(d,J=5.1Hz,1H) ,5.28(dd,J=8.0,2.2Hz,1H),5.17(d,J=5.3Hz,1H),4.15-4.05(m,2H),4.00-3.85(m,2H),3.85-3.73(m,1H),1.03(s,9H).

[0275] Preparation of PH-ALIG-14-1-2 A solution of PH-ALIG-14-1-1 (260.00 g, 538.7 mmol, 1.0 equiv) in MeOH (5000 mL) was placed in a 10 L, 3-neck round bottom flask purged and maintained with an inert atmosphere of argon. This was followed by H 2 NaIO in O (1600 mL) 4 (126.8 g, 592.6 mmol, 1.1 equiv) was added in several batches at 0° C. The resulting solution was stirred at room temperature for 1 h. Then, 3 L of Na 2 S 2 O 3 The reaction was quenched by the addition of (saturated) at 0° C. The resulting solution was extracted with 3×1 L of dichloromethane, and the organic layers were combined and dried over anhydrous sodium sulfate. The solids were filtered off. The filtrate was concentrated. This gave 290 g (crude) of PH-ALIG-14-1-2 as a white solid.

[0276] Preparation of PH-ALIG-14-1-3 Into a 5 L 3-neck round bottom flask purged and maintained with an inert atmosphere of argon was placed PH-ALIG-14-1-2 (290 g, 603.4 mmol, 1.0 equiv), EtOH (3 L). This was followed by NaBH 4(22.8 g, 603.4 mmol, 1.0 equiv) was added in portions at 0° C. The resulting solution was stirred at room temperature for 1 h. The reaction was then quenched by the addition of 2000 mL of water / ice. The resulting solution was extracted with 3×1000 mL of dichloromethane, the organic layers were combined and dried over anhydrous sodium sulfate. The solids were filtered off. The filtrate was concentrated. This gave 230 g (crude) of PH-ALIG-14-1-3 as a white solid. LC-MS: m / z 485.10 [M+H] + . 1 H NMR (400MHz, DMSO-d 6 )δ 11.28(d,J=2.2Hz,1H),7.63-7.37(m,11H),5.84(dd,J=6.4,4.9Hz,1H),5.44(dd,J=8.0,2.2Hz,1H),5.11(t,J=6.0 Hz,1H),4.78(t,J=5.2Hz,1H),3.65(dd,J=11.4,5.7Hz,1H),3.60-3.52(m,5H),3.18(d,J=5.2Hz,1H),0.96(s,9H).

[0277] Preparation of PH-ALIG-14-1-4 A 5000 mL 3-neck round bottom flask purged and maintained with an inert atmosphere of argon was charged with a solution of PH-ALG-14-1-3 (120 g, 1 equiv.) in DCM (1200 mL). This was followed by the addition of DIEA (95.03 g, 3 equiv.) at 0° C. To this was added methanesulfonic anhydride (129 g, 3 equiv.) in portions at 0° C. The resulting solution was stirred at room temperature for 1 h. The reaction was then quenched by the addition of 1000 mL of water / ice. The resulting solution was extracted with 3×500 mL of dichloromethane and the organic layers were combined and dried over anhydrous magnesium sulfate. The solids were filtered off. The filtrate was concentrated. This gave 160 g (crude) of PH-ALG-14-1-4 as a yellow solid, LC-MS (m / z) 641.05 [M+H]. +

[0278] Preparation of PH-ALIG-14-1-5 A 1 L round bottom flask was charged with a solution of PH-ALG-14-1-4 (160.00 g, 1.00 equiv.) in THF (1600 mL), DBU (108 g, 2.8 equiv.). The resulting solution was stirred at 30° C. for 1 h. The reaction was then quenched by the addition of 3000 mL of water / ice. The resulting solution was extracted with 3×500 mL of dichloromethane, the organic layers were combined and dried over anhydrous sodium sulfate. The solids were filtered off. The filtrate was concentrated. This gave 150 g (crude) of PH-ALG-14-1-5 as a brown oil, LC-MS: (ES, m / z): 567.25 [M+H] + 1 HNMR (400MHz, DMSO-d 6 )δ 7.83(d,J=7.4Hz,1H),7.67-7.55(m,4H),7.55-7.35(m,6H),6.05(dd,J=5.9,1.7Hz,1H),5.72(d,J=7.4Hz,1H),4.81(dd,J=10.4,5 .8Hz,1H),4.58-4.46(m,2H),4.42(p,J=5.2,4.6Hz,1H),4.33(dd,J=10.6,5.9Hz,1H),3.79-3.70(m,2H),3.23(s,3H),0.98(s,9H).

[0279] Preparation of PH-ALIG-14-1-6 A 3000 mL round bottom flask purged and maintained with an inert atmosphere of argon was charged with PH-ALIG-14-1-5 (150.00 g, 201.950 mmol, 1 equiv), DMF (1300.00 mL), potassium benzoate (44.00 g, 1.0 equiv). The resulting solution was stirred at 80° C. for 1.5 h. The reaction was then quenched by the addition of 500 mL of water / ice. The resulting solution was extracted with 3×500 mL of dichloromethane. The resulting mixture was diluted with 3×1000 mL of H 2 The mixture was concentrated. The residue was applied to a silica gel column with EA / PE (99:1). The collected fractions were combined and concentrated. This gave 40 g of PH-ALIG-14-1-6 as a yellow oil. LC-MS: m / z 571.20 [M+H] +;1HNMR: (400MHz, DMSO-d 6 )δ 7.97-7.91(m,2H),7.89(d,J=7.4Hz,1H),7.74-7.51(m,7H),7.51-7.31(m,6H),6.16(m,1H),5.76(d,J= 7.4Hz,1H),4.78(m,1H),4.61(m,1H),4.55-4.46(m,2H),4.38(m,1H),3.82(d,J=5.0Hz,2H),0.97(s,9H)

[0280] Preparation of PH-ALIG-14-1-7A A 2-L round-bottom flask was charged with PH-ALIG-14-1-6 (30.00 g, 1 equiv.), MeOH (1.20 L), and p-toluenesulfonic acid (4.50 g, 0.5 equiv.). The resulting solution was stirred at 70° C. for 2 h. Then, 3 L of NaHCO 3 The reaction was quenched by the addition of (saturated). The pH value of the solution was adjusted to 0.05 with NaHCO 3 The acid was adjusted to 7 with (saturated). The resulting solution was extracted with 3×1 L of ethyl acetate, and the organic layers were combined and dried over anhydrous sodium sulfate. The solids were filtered off. The filtrate was concentrated under vacuum. The crude product was purified by flash prep-HPLC using the following conditions (IntelFlash-1): column, silica gel; mobile phase, PE / EA=50 / 50 increased to PE / EA=25 / 75 within 30; detector, 254. This gave 11.5 g (3.1% yield for 7 steps) of PH-ALIG-14-1-7A as a white solid. LC-MS: m / z 625.15 [M+Na] + ; 1 HNMR: (400MHz, DMSO-d 6 )δ 11.37(d,J=2.3Hz,1H),7.99-7.93(m,2H),7.74-7.65(m,1H),7.63-7.50(m,7H),7.50-7.33(m,6H),6.08(t,J=6.0Hz,1H),5 .49(m,1H),4.60(m,1H),4.43(m,1H),4.03-3.96(m,1H),3.70(d,J=5.3Hz,2H),3.62-3.49(m,2H),3.21(s,3H),0.97(s,9H).

[0281] Preparation of PH-ALIG-14-1-7 In a 2 L round bottom flask, add PH-ALIG-14-1-7A (11.50 g). The above 7M NH 3 (g) was introduced at 30° C. The resulting solution was stirred at 30° C. overnight. The resulting mixture was concentrated under vacuum. The crude product was purified by flash using the following conditions (IntelFlash-1): column, silica gel; mobile phase, PE / EA=60 / 40 increased to PE / EA=1 / 99 within 60; detector, 254. This gave 8.1 g (97% yield) of PH-ALIG-14-1-7 as a white solid. LC-MS-: m / z 499.35 [M+H] + ; 1 HNMR: (300MHz, DMSO-d 6 )δ 11.31(s,1H),7.64-7.50(m,5H),7.48-7.35(m,6H),6.02(t,J=5.8Hz,1H),5.45 (d,J=8.0Hz,1H),4.80(t,J=5.1Hz,1H),3.58(m,7H),3.27(s,3H),0.96(s,9H).

[0282] Preparation of PH-ALIG-14-1-8 A 250 mL round bottom flask was charged with PH-ALIG-14-1-7 (8.10 g, 1 equiv.), pyridine (80.0 mL), and DMTr-Cl (7.10 g, 1.3 equiv.). The flask was evacuated and flushed with argon three times. The resulting solution was stirred at room temperature for 2 h. Then, 500 mL of NaHCO 3 The reaction was quenched by the addition of (saturated). The resulting solution was extracted with 2×500 mL of ethyl acetate, and the organic layers were combined and dried over anhydrous sodium sulfate. The solids were filtered off. The filtrate was concentrated under vacuum. The crude product was purified using the following conditions (IntelFlash-1): column, C18; mobile phase, ACN / H 2 O=5 / 95 within 30 ACN / H 2O = 95 / 5; purified by flash with detector, 254. This gave 11.5 g (88% yield) of PH-ALIG-14-1-8 as a white solid. LC-MS: m / z 823.40 [M+Na] + ;1HNMR: (300MHz, DMSO-d 6 )δ 11.37(s,1H),7.55-7.18(m,20H),6.92-6.83(m,4H),6.14(t,J=5.9Hz,1H) ,5.48(d,J=8.0Hz,1H),3.74(m,7H),3.57(m,4H),3.25(m,5H),0.84(s,9H).

[0283] Preparation of PH-ALIG-14-1-9 A 1000 mL round bottom flask was charged with PH-ALIG-14-1-8 (11.5 g, 1.00 equiv), THF (280.00 mL), TBAF (14.00 mL, 1.00 equiv). The resulting solution was stirred at room temperature for 3 hours. The reaction was then quenched by the addition of 1 L of water. The resulting solution was extracted with 3×500 mL of ethyl acetate, and the organic layers were combined and dried over anhydrous sodium sulfate. The solids were filtered off. The filtrate was concentrated under vacuum. The crude product was purified using the following conditions (IntelFlash-1): column, C18; mobile phase, ACN / H 2 O=5 / 95 within 30 ACN / H 2 O = 95 / 5; purified by flash with detector, 254. This gave 7.8 g (98% yield) of PH-ALIG-14-1-9 as a white solid. LC-MS: m / z 561.20 [M−H] - ; 1 HNMR: (300MHz, DMSO-d 6 )δ 11.32(s,1H),7.66(d,J=8.1Hz,1H),7.52-7.39(m,2H),7.39-7.20(m,7H),6.96-6.83(m,4H),6.17(t,J=5 .9Hz,1H),5.63(d,J=8.0Hz,1H),4.63(t,J=5.6Hz,1H),3.90-3.46(m,9H),3.26(s,5H),3.19-2.98(m,2H).

[0284] Preparation of PH-ALIG-14-1-10 In a 3-L round-bottom flask, add PH-ALIG-14-1-9 (7.80 g, 1.00 equiv.), DCM (300.00 mL), NaHCO 3 (3.50 g, 3 equiv.) was added. This was followed by the addition of Dess-Martin (7.06 g, 1.2 equiv.) with stirring at 0° C., and the resulting solution was stirred at 0° C. for 20 min. The resulting solution was stirred at room temperature for 5 h. The reaction mixture was cooled to 0° C. with a water / ice bath. Then, 500 mL of NaHCO 3 :Na 2 S 2 O 3 The reaction was quenched by the addition of 1:1 NaCl / NaCl. The resulting solution was extracted with 3×500 mL of ethyl acetate, and the organic layers were combined and dried over anhydrous sodium sulfate. The solids were filtered off. The filtrate was concentrated under vacuum. The crude product was purified using the following conditions (IntelFlash-1): column, C18; mobile phase, ACN / H 2 O=5 / 95 within 30 ACN / H 2 O = 95 / 5; purified by flash with detector, 254. This gave 5.8 g (75% yield) of PH-ALIG-14-1-1-110 as a white solid. LC-MS: m / z 558.80 [M−H] - ; 1 HNMR: (300MHz, DMSO-d 6 )δ 11.35-11.22(m,1H),9.43(s,1H),7.75(d,J=8.1Hz,1H),7.49-7.19(m,8H),6.90(m,5H),6.00(t ,J=5.9Hz,1H),5.66(m,1H),4.40(m,1H),3.75(s,7H),3.70-3.56(m,3H),3.29(d,J=3.7Hz,3H).

[0285] Preparation of PH-ALIG-14-1-11 A 250 mL 3-neck round bottom flask was charged with THF (150.00 mL), NaH (1.07 g, 60% w, 3.00 equiv). The flask was evacuated and flushed with argon three times and the reaction mixture was cooled to -78°C. This was followed by the dropwise addition of [[(bis[[(2,2-dimethylpropanoyl)oxy]methoxy]phosphoryl)methyl([(2,2-dimethylpropanoyl)oxy]methoxy)phosphoryl]oxy]methyl 2,2-dimethylpropanoate (14.60 g, 2.6 equiv in 60 mL THF) with stirring at -78°C in 10 min and the resulting solution was stirred at -78°C for 30 min. This was followed by the dropwise addition of PH-ALIG-14-1-10 (5.00 g, 1.00 equiv in 50 mL THF) with stirring at -78°C in 10 min. The resulting solution was stirred at room temperature for 4 h. Then, 400 mL of NH 4 The reaction was quenched by the addition of Cl(sat). The resulting solution was extracted with 3×400 mL of ethyl acetate, and the organic layers were combined and dried over anhydrous sodium sulfate. The solids were filtered off. The filtrate was concentrated under vacuum. The crude product was purified using the following conditions (IntelFlash-1): column, C18; mobile phase, ACN / H 2 O=5 / 95 within 30 ACN / H 2 O = 95 / 5; purified by flash with detector, 254. This gave 7.2 g (crude) of PH-ALIG-14-1-11a solid. LC-MS: m / z: 865.10 [M−H] -

[0286] Preparation of PH-ALIG-14-1-12 A 500 mL round bottom flask was charged with PH-ALIG-14-1-1-11. (6.00g), H 2 O (30.00 mL), AcOH (120.00 mL). The resulting solution was stirred at 50 °C for 1 h. The reaction mixture was cooled to 0 °C in a water / ice bath. Then, 2 L of NaHCO 3 The reaction was quenched by the addition of (saturated). The pH value of the solution was adjusted to 0.05 with NaHCO 3The pH was adjusted to 7 with (saturated). The resulting solution was extracted with 3×500 mL of ethyl acetate, and the organic layers were combined and dried over anhydrous sodium sulfate. The solids were filtered off. The filtrate was concentrated under vacuum. The crude product was purified using the following conditions (IntelFlash-1): column, C18; mobile phase, ACN / H 2 O=5 / 95 within 30 ACN / H 2 Purified by flash with detector, 254. This gave 2.6 g (44% over two steps) of PH-ALIG-14-1-12 as a yellow oil. LC-MS: m / z 587.25 [M+Na] + ; 1 HNMR: (300MHz, DMSO-d 6 )δ 11.31(s,1H),7.73(d,J=8.1Hz,1H),6.63(ddd,J=24.2,17.2,4.2Hz,1H),6.14-5.96(m,2H),5.65-5.48(m,5H) ),5.09(t,J=5.6Hz,1H),4.17(s,1H),3.65(d,J=6.1Hz,2H),3.52(m,2H),3.27(s,3H),1.15(d,J=3.7Hz,18H); 31 PNMR: (162MHz, DMSO-d 6 )δ 17.96.

[0287] Preparation of PH-ALIG-14-1-0 A 250 mL 3-neck round bottom flask was charged with DCM (60.00 mL), DCI (351.00 mg, 1.2 equiv), 3-[[bis(diisopropylamino)phosphanyl]oxy]propanenitrile (971.00 mg, 1.3 equiv), 4A MS. The flask was evacuated and flushed with argon three times and the reaction mixture was cooled to 0° C. Following this, PH-ALIG-14-1-12 (1.40 g, 1.00 equiv in 30 mL DCM) was added dropwise within 30 seconds with stirring at 0° C. The resulting solution was stirred at room temperature for 1 hour. The reaction was then quenched by the addition of 50 mL water. The resulting solution was extracted with 3×50 mL ethyl acetate and the organic layers were combined. The resulting mixture was washed with 3×50 ml NaCl (sat.). The mixture was dried over anhydrous magnesium sulfate. The solids were filtered off. The filtrate was concentrated under vacuum. The crude product was purified using the following conditions: Column: Ultimate Diol, 2*25cm, 5 [ka] Im, mobile phase A:CO 2 Purification by Prep-Archiral-SFC using HPLC, mobile phase A: ACN (0.2% TEA), flow rate: 50 mL / min, gradient: isocratic 30% B, column temperature (20 °C): 35, back pressure (bar): 100, wavelength: 254 nm, retention time 1 (min): 2.58, sample solvent: MeOH--HPLC, injection volume: 1 mL, number of runs: 4. This gave 1.31 g (65% yield) of PH-ALIG-14-1-0 as a yellow oil. LC-MS: m / z 763.40 [MH] - ;1HNMR-: (300MHz, acetonitrile-d 3 ) δ 9.05(s,1H), 7.51(d,J=8.1Hz,1H), 6.64(dddd,J=23.8,17.1,4.8,1.9Hz,1H), 6.23-5.92(m,2H), 5.70-5.51(m,5H), 4.38(d,J=4.9Hz,1H), 3.96-3.56(m,8H), 3.35(s,3H), 2.70(m,2H), 1.33-1.14(m,30H);31PNMR-:(acetonitrile-d 3 )δ 148.75,148.53,16.68.

[0288] Example 3 [ka]

[0289] Preparation of PH-ALIG-14-1-7B A solution of PH-ALIG-14-1-6 (23 g, 40.300 mmol, 1.00 equiv) and p-TsOH (9.02 g, 52.390 mmol, 1.3 equiv) in MeOH (1000 mL) was stirred at 40 °C overnight under argon atmosphere. The reaction was quenched with saturated sodium bicarbonate (aq) at 0 °C. The resulting mixture was extracted with EtOAc (2 × 500 mL). The combined organic layers were washed with water (2 × 500 mL) and diluted with anhydrous MgSO 4 The mixture was dried at 4°C for 30 min. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse flash chromatography under the following conditions: column, C18 silica gel, mobile phase, ACN in water, gradient 10% to 90% in 30 min, detector, UV 254 nm. This gave PH-ALIG-14-1-7B (5.3 g, 36%) as a colorless oil. LC-MS: (ES, m / z): 365 [M+H] + ; 1 H-NMR: (300MHz, DMSO-d 6 )δ 11.20(s,1H),8.09-7.78(m,2H),7.63-7.50(m,2H),7.51-7.35(m,2H) ,5.95(t,J=5.9Hz,1H),5.51(d,J=8.1Hz,1H),4.73(t,J=5.7Hz,1H),4. 41(dd,J=11.9,3.3Hz,1H),4.17(dd,J=11.9,6.3Hz,1H),3.69(dq,J=10.1,6.8,6.3Hz,1H),3.48-3.40(m,2H),3.39-3.29(m,2H),3.07(s,3H).

[0290] Preparation of PH-ALIG-14-3-1 In a 250 mL 3-neck round bottom flask, add PH-ALIG-14-1-7B (7.00 g, 19.212 mmol, 1.00 equiv), ACN (60.00 mL), H 2 0 (60.00 mL), TEMPO (0.72 g, 4.611 mmol, 0.24 equiv), BAIB (13.61 g, 42.267 mmol, 2.20 equiv). The resulting solution was stirred at 30° C. overnight. The reaction was then quenched by the addition of 200 mL of water / ice. The resulting solution was extracted with 2×200 mL of ethyl acetate and the resulting mixture was washed with 2×200 mL of water. The mixture was dried over anhydrous sodium sulfate and concentrated. The crude product was purified using the following conditions (IntelFlash-1): column, C18 silica gel; mobile phase, ACN / H 2 O=5 / 95 in 30 minutes or less ACN / H 2 The product was purified by flash preparative HPLC with UV 254 nm detector to give 5 g (68.8%) of PH-ALIG-14-3-1 as a solid. LC-MS: (ES, m / z): 379 [M+H] + ; 1 H NMR (300MHz, DMSO-d 6 )δ 13.24(s,1H),11.31(d,J=2.2Hz,1H),8.18-7.83(m,2H),7.81-7.63(m,2H),7.61-7.42(m,2H),6.0 1(t,J=6.0Hz,1H),5.61(dd,J=8.0,2.2Hz,1H),4.72-4.40(m,3H),3.73-3.55(m,2H),3.22(s,3H).

[0291] Preparation of PH-ALIG-14-3-2 In a 250 mL round bottom flask, add PH-ALIG-14-3-1 (4.5 g, 11.894 mmol, 1.00 equiv), DMF (90.00 mL, 1.00 eq.), Pb(OAc) 4(15.82 g, 35.679 mmol, 3.00 equiv). The resulting solution was stirred at 30° C. overnight. The reaction was then quenched by the addition of 200 mL of water / ice. The resulting solution was extracted with 2×200 mL of ethyl acetate and the resulting mixture was washed with 2×200 mL of water. The mixture was dried over anhydrous sodium sulfate and concentrated. The crude product was purified using the following conditions (IntelFlash-1): column, C18 silica gel; mobile phase, ACN / H 2 O=5 / 95 in 30 minutes or less ACN / H 2 The product was purified by flash with detector, UV 254 nm to give 4 g of PH-ALIG-14-3-2 as an oil. LC-MS: (ES, m / z): 415 [M+Na] + ; 1 H NMR (300MHz, DMSO-d 6 )δ 11.39(s,1H),7.93(dd,J=24.2,7.6Hz,2H),7.75-7.46(m,4H),6.35-6.03(m,2H),5.71-5.47 (m,1H),4.60-4.14(m,2H),3.88-3.54(m,2H),3.26(d,J=6.7Hz,3H),2.03(d,J=49.7Hz,3H).

[0292] Preparation of PH-ALIG-14-3-3 Into a 250 mL, 3-neck round bottom flask purged and maintained with an inert atmosphere of argon was added PH-ALIG-14-3-2 (4.00 g, 10.195 mmol, 1.00 equiv), DCM (80.00 mL), dimethylhydroxymethylphosphonate (22.85 g, 163.114 mmol, 16.00 equiv), BF 3 .Et 20 (28.94 g, 203.91 mmol, 20 equiv.) was charged. The resulting solution was stirred at room temperature overnight. The reaction was then quenched by the addition of 500 mL of water / ice. The resulting solution was extracted with 2×500 mL of ethyl acetate and the resulting mixture was washed with 2×500 mL of water. The mixture was dried over anhydrous sodium sulfate and concentrated. The residue was applied to a silica gel column with dichloromethane / methanol (20 / 1). This gave 2 g (41.5%) of PH-ALIG-14-3-3 as a solid. LC-MS: (ES, m / z): 490 [M+H 2 O]+; 1H-NMR (300 MHz, DMSO-d 6 )δ 11.39(d,J=5.4Hz,1H),7.96(dt,J=11.5,9.3Hz,2H),7.81-7.40(m,4H),6.29-5.98(m,1H),5.56(dd,J=12.2,8.1Hz ,1H),5.28-4.99(m,1H),4.29(dp,J=25.1,5.9Hz,2H),4.16-3.84(m,2H),3.75-3.53(m,7H),3.28(d,J=12.5Hz,2H).

[0293] Preparation of PH-ALIG-14-3-4 In a 100 mL round bottom flask, place PH-ALIG-14-3-3 (2.00 g, 4.234 mmol, 1.00 equiv) in 7 M NH in THF (20.00 mL). 3 (g) was added. The resulting solution was stirred at 25° C. overnight. The resulting mixture was concentrated under vacuum. The crude product was purified by HPLC using a preparative-sfc column: Lux5um i-cellulose-5, 3*25cm, 5μm, mobile phase A: CO 2 , Mobile phase B: MeOH (0.1% 2M NH 3-MEOH), flow rate: 70 mL / min, gradient: isocratic 50% B, column temperature (25 °C): 35, back pressure (bar): 100, wavelength: 220 nm, retention time 1 (min): 3.75, retention time 2 (min): 4.92, sample solvent: MeOH:DCM = 1:1, injection volume: 1 mL, run number: 15, which gave 330 mg (21.2%) of PH-ALIG-14-3-4 as a solid. 1H-NMR-: (300 MHz, DMSO-d 6 )δ 11.14(s,1H),7.63(d,J=8.1Hz,1H),6.06(t,J=5.9Hz,1H),5.64(d,J=8.0Hz,1H),4.89(s,1H),4.63(t,J= 5.3Hz,1H), 3.98(d,J=9.8Hz,2H),3.70(dd,J=10.7,1.2Hz,8H),3.63(dd,J=6.0,3.2Hz,1H),3.29(s,3H).

[0294] Preparation of PH-ALIG-14-3-0 To a stirred solution of 3-{[bis(diisopropylamino)phosphanyl]oxy}propanenitrile (324.10 mg, 1.075 mmol, 1.2 equiv) and 1H-imidazole-4,5-dicarbonitrile (126.99 mg, 1.075 mmol, 1.2 equiv) in DCM (10 mL) was added PH-ALIG-14-3-4 (330 mg, 0.9 mmol, 1.00 equiv) dropwise under argon atmosphere at 25 °C. The resulting mixture was stirred at 25 °C for 30 min. The reaction was quenched with water / ice. The resulting mixture was extracted with EtOAc (2 × 10 mL). The combined organic layers were washed with water (2 × 10 mL) and diluted with anhydrous MgSO 4 The mixture was dried at 37° C. After filtration, the filtrate was concentrated under reduced pressure. Column: Ultimate Diol, 2*25 cm, 5 μm, Mobile phase A: CO2, Mobile phase B: ACN, Flow rate: 50 mL / min, Gradient: Isocratic 30% B, Column temperature (25° C.): 35, Back pressure (bar): 100, Wavelength: 254 nm, Retention time 1 (min): 3.95, Sample solvent: ACN, Injection volume: 1 mL, Number of runs: 10, This gave PH-ALIG-14-3-0 (349 mg, 68.4%) as a pale yellow oil. LC-MS: (ES, m / z): 567.25 [M+H]+ ;1H-NMR: (300MHz, DMSO-d 6 )δ 11.38(s,1H),7.64(dd,J=8.0,1.3Hz,1H),6.09(dt,J=5.8,3.4Hz,1H),5.65(dd,J=8.0,3.2Hz,1H),4.83(q,J=5.5Hz,1H) ,4.03(dt,J=9.7,2.2Hz,2H),3.83-3.40(m,14H),3.30(s,3H),2.77(t,J=5.9Hz,2H),1.12(ddd,J=9.2,6.7,1.7Hz,12H); 31 PNMR (DMSO-d 6 ) δ 148.0, 147.6, 23.1

[0295] Example 4 [ka]

[0296] Preparation of PH-ALIG-14-3-40 In a 100 mL round bottom flask, place 2PH-ALIG-14-3-3 (2.00 g, 4.234 mmol, 1.00 equiv) in 7 M NH in THF (20.00 mL). 3 (g) was added. The resulting solution was stirred at 25° C. overnight. The resulting mixture was concentrated under vacuum. The crude product was purified by HPLC using a preparative-sfc column: Lux5um i-cellulose-5, 3*25cm, 5μm, mobile phase A: CO2, mobile phase B: MeOH (0.1% 2M NH 3 -MeOH), flow rate: 70 mL / min, gradient: isocratic 50% B, column temperature (°C): 35, back pressure (bar): 100, wavelength: 220 nm, retention time 1 (min): 3.75, retention time 2 (min): 4.92, sample solvent: MeOH:DCM=1:1, injection volume: 1 mL, number of runs: 15, which gave 320 mg (22.8%) of PH-ALIG-14-3-40 as a solid. 1 H-NMR--14-3-40: (300 MHz, DMSO-d 6) δ 11.11(s,1H),7.70(d,J=8.0Hz,1H),6.03(t,J=6.1Hz,1H),5.64(d,J=8.0Hz,1H),4.97(s,1H),4.76(t,J=5.3H) z,1H),4.07-3.85(m,1H),3.79(dd,J=13.9,9.3Hz,1H),3.73-3.55(m,9H),3.41(d,J=5.0Hz,2H),3.28(s,3H).

[0297] Preparation of PH-ALIG-14-3-100 To a stirred solution / mixture of 3-{[bis(diisopropylamino)phosphanyl]oxy}propanenitrile (517.58 mg, 1.717 mmol, 1.2 equiv) and 1H-imidazole-4,5-dicarbonitrile (202.79 mg, 1.717 mmol, 1.2 equiv) in DCM, PH-ALIG-14-3-40 (527 mg, 1.431 mmol, 1.00 equiv) was added dropwise at 25° C. under argon atmosphere. The resulting mixture was stirred at 25° C. for 30 min. The reaction was quenched with water / ice. The resulting mixture was extracted with EtOAc (2×10 mL). The combined organic layers were washed with water (2×10 mL) and dried over anhydrous MgSO4. After filtration, the filtrate was concentrated under reduced pressure. Column: Ultimate Diol, 2*25 cm, 5 μm, Mobile phase A: CO 2 , Mobile phase B: ACN (0.1% DEA)-HPLC-merk, Flow rate: 50 mL / min, Gradient: Isocratic 30% B, Column temperature (°C): 35, Back pressure (bar): 100, Wavelength: 254 nm, Retention time 1 (min): 4.57, Sample solvent: ACN, Injection volume: 1 mL, Number of runs: 10, PH-ALIG-14-3-100 (264.8 mg, 31.7%) was obtained as a pale yellow oil. LC-MS: (ES, m / z): 567.25 [MH] - ;:1H NMR (300MHz, DMSO-d 6)δ 13.24(s,1H),11.31(d,J=2.2Hz,1H),8.18-7.83(m,2H),7.81-7.63(m,2H),7.61-7.42(m,2H),6.0 1(t,J=6.0Hz,1H),5.61(dd,J=8.0,2.2Hz,1H),4.72-4.40(m,3H),3.73-3.55(m,2H),3.22(s,3H); 31 PNMR (DMSO-d 6 ) δ 148.01,147.67,22.8

[0298] Example 5 [ka]

[0299] Preparation of PH-ALIG-14-4-1 H 2 Ascorbic acid (100.00 g, 567.78 mmol, 1.00 equiv.) and CaCO in O (1.00 L) 3 (113.0 g, 1129.02 mmol, 2 equiv.) was added to a stirred mixture of H 2 O 2 (30%) (236.0 g, 6938.3 mmol, 12.22 equiv) was added dropwise at 0° C. The resulting mixture was stirred at room temperature overnight. The mixture was treated with charcoal and heated to 70° C. until no peroxide was detected. The resulting mixture was filtered and the filter cake was washed with hot water (3×300 mL). The filtrate was concentrated under reduced pressure. The solid was diluted with MeOH (200 mL) and the mixture was stirred for 5 h. The resulting mixture was filtered and the filter cake was washed with MeOH (3×80 mL). The filtrate was concentrated under reduced pressure to give L-threonate (86 g, 96.6%) as a crude white solid. 1H-NMR-:(300MHz, Deuterium Oxide) δ 4.02(dd,J=4.6,2.4Hz,1H),3.91(ddt,J=7.6,5.3,2.2Hz,1H),3.78-3.44(m,2H).

[0300] Preparation of PH-ALIG-14-4-2 In a 5 L round bottom flask, add L-threonate (70.00 g, 518.150 mmol, 1.00 equiv.) and H 2 O (2 L) was added at room temperature. The residue was acidified to pH=1 with Dowex 50wX8, H(+) form. The resulting mixture was stirred at 70° C. for 1 h. The resulting mixture was filtered and the filter cake was washed with water (2×1 L). The filtrate was concentrated under reduced pressure. The solid was co-evaporated with (2×2 L). The solid was then diluted with ACN (700.00 mL) and TsOH (5.35 g, 31.089 mmol, 0.06 equiv.) was added. The resulting mixture was stirred at 80° C. for 1 h under air atmosphere. The resulting mixture was filtered and the filter cake was washed with ACN (2×500 mL). The filtrate was concentrated under reduced pressure to give PH-ALIG-14-4-2 (70 g, crude) as a yellow oil.

[0301] Preparation of PH-ALIG-14-4-3 To a stirred solution of (PH-ALIG-14-4-2 (crude 70.0 g, 593.2 mmol, 1.00 equiv) in pyridine (280.00 mL) was added benzoyl chloride (207.62 g, 1.483 mol, 2.5 equiv) dropwise at 0 °C under an argon atmosphere. The resulting mixture was stirred at room temperature under an argon atmosphere for 1 h. The reaction was cooled to 0 °C with saturated NaHCO 3 The mixture was quenched by the addition of (aq) (500 mL). 2 Cl 2 (3×500 mL). The combined organic layers were washed with brine (2×300 mL) and anhydrous Na 2 SO 4 The mixture was dried at 40° C. for 1 hour. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with PE / EtOAc to give (PH-ALIG-14-4-3 (80 g, 41.4%) as an off-white solid. LC-MS: (ES, m / z): 327 [M+H] + ;1H-NMR: (300MHz, CDCl 3)δ 8.18-8.04(m,4H),7.68-7.61(m,2H),7.50(tt,J=7.1,1.4Hz,4H),5.96-5.57(m,2H),5.11-5.00(m,1H),4.45-4.35(m,1H).

[0302] Preparation of PH-ALIG-14-4-4 To a stirred solution of PH-ALIG-14-4-3 (125 g, 383.078 mmol, 1.00 equiv) in THF (1.50 L) was added DIBAL-H (1M) (600 mL, 2 equiv) dropwise at −78° C. under argon atmosphere. The resulting mixture was stirred at −78° C. for 1 h under argon atmosphere. The desired product was detected by LCMS. The reaction was quenched with MeOH at 0° C. The resulting mixture was diluted with EtOAc (600 mL). The resulting mixture was filtered and the filter cake was washed with EtOAc (3×800 mL). The filtrate was concentrated under reduced pressure. This gave PH-ALIG-14-4-4 (73 g, crude) as a colorless solid. LC-MS: (ES, m / z): 392 [M+Na+ACN]+; 1H-NMR-: (400 MHz, chloroform-d) δ 8.22-7.99(m,8H),7.62(dtd,J=7.4,4.4,2.2Hz,4H),7.48(td,J=7.8,2 .4Hz,8H),5.87(d,J=4.3Hz,1H),5.77(dt,J=6.6,3.6Hz,1H),5.56(d,J =4.9Hz,2H),5.50(t,J=4.3Hz,1H),4.73(s,1H),4.63(ddd,J=10.4,7.9,6.1Hz,2H),4.28(dd,J=10.3,3.8Hz,1H),3.99(dd,J=10.6,3.2Hz,1H).

[0303] Preparation of PH-ALIG-14-4-5 To a stirred solution of (PH-ALIG-14-4-4 (73.00 g, 222.344 mmol, 1.00 equiv.) and DMAP (271.63 mg, 2.223 mmol, 0.01 equiv.) and pyridine (365.00 mL) in DCM (365.00 mL) was added Ac 2O (24.97 g, 244.6 mmol, 1.1 equiv) was added dropwise at 0 °C under an argon atmosphere. The resulting mixture was stirred at room temperature under an argon atmosphere for 1 h. The reaction was diluted with saturated NaHCO 3 (aqueous) at 0 °C. The resulting mixture was diluted with CH 2 Cl 2 (3×500 mL). The combined organic layers were washed with saturated CuSO 4 (3×200 mL), and washed with anhydrous Na 2 SO 4 The mixture was dried at 4° C. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography, eluted with PE / EtOAc to give PH-ALIG-14-4-5 (60 g, 73%) as a colorless oil. LC-MS: (ES, m / z): 434 [M+Na+ACN] + ;1H-NMR:(400MHz,chloroform-d)δ 8.17-8.02(m,8H),7.63(tddd,J=7.9,6.6,3.2,1.6Hz,4H),7.57-7.44(m,8H),6.66(d,J=4.5Hz,1H),6.40(s,1H),5.83-5.53(m ,4H),4.67(ddd,J=23.4,10.5,6.2Hz,2H),4.24(dd,J=10.5,3.8Hz,1H),4.19-4.01(m,1H),2.18(s,3H),2.06(d,J=3.2Hz,3H).

[0304] Preparation of PH-ALIG-14-4-6 To a stirred mixture of PH-ALIG-14-4-5 (50.00 g, 135.005 mmol, 1.00 equiv.) and uracil (15.13 g, 135.005 mmol, 1 equiv.) in a can (500.00 mL), BSA (54.81 g, 270.010 mmol, 2 equiv.) was added portionwise at room temperature under air atmosphere. The resulting mixture was stirred at 60° C. for 1 h under argon atmosphere. TMSOTf (90.02 g, 405.0 mmol, 3 equiv.) was then added dropwise at 0° C. The resulting mixture was stirred at 60° C. for 2 h under argon atmosphere. The mixture was diluted with saturated NaHCO 3 (aqueous) at 0 °C to pH = 7. The resulting mixture was diluted with CH 2Cl 2 (3×400 mL). The combined organic layers were washed with brine (2×400 mL) and anhydrous Na 2 SO 4 The mixture was dried at 70° C. for 1 hour. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with PE / EtOAc (1:1) to give PH-ALIG-14-4-6 (43 g, 75.4%) as a white solid. LC-MS: (ES, m / z): [M+H] + ;423 464[M+H+ACN]+;1H-NMR-:(300MHz,chloroform-d)δ 9.08-8.89(m,1H),8.17-7.94(m,4H),7.70-7.43(m,7H),6.19(d,J=1.9Hz,1H),5.84-5 .71(m,2H),5.62(td,J=3.3,2.8,1.4Hz,1H),4.59-4.44(m,2H),4.14(q,J=7.2Hz,1H).

[0305] Preparation of PH-ALIG-14-4-7 A solution of PH-ALIG-14-4-6 (52.00 g, 123.108 mmol, 1 equiv.) was dissolved in 642 mL of MeOH / H 2 The residue was dissolved in EtOAc (600 mL) and the organic layer was extracted with water (5×800 mL). The aqueous layer was concentrated under vacuum to give PH-ALIG-14-4-7 (21 g, crude) as an off-white solid. The crude product was used directly in the next step without further purification. LC-MS-: (ES, m / z): 213 [MH]-; 1 H-NMR: (300 MHz, DMSO-d 6 ) δ 11.26(s,1H),7.68(d,J=8.1Hz,1H),5.75(s,1H),5.65(d,J=1.2Hz,1H),5.59(d,J=8.1Hz,1H),5.39(s,1H),4.10-3.97(m,4H).

[0306] Preparation of PH-ALIG-14-4-8 To a stirred mixture of PH-ALIG-14-4-7 (16.00 g, 74.705 mmol, 1.00 equiv) and DBU (22.75 g, 149.409 mmol, 2 equiv) in DCM (80.00 mL) and DMF (200.00 mL), DMTr-Cl (7.88 g, 25.680 mmol, 1.1 equiv) was added dropwise at room temperature under an argon atmosphere. The resulting mixture was stirred at room temperature under an argon atmosphere for 2 h. The reaction was cooled to 0 °C with saturated NaHCO 3 The mixture was quenched by the addition of (aq) (100 mL). The mixture was extracted with EtOAc (3×60 mL). The combined organic layers were washed with brine (2×50 mL) and anhydrous Na 2 SO 4 The mixture was dried at 4° C. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with PE (0.5% TEA) / EtOAc (2:3) to give PH-ALIG-14-4-8 (25 g, 64.8%) as an off-white solid. LC-MS: (ES, m / z): 515 [MH] - ; 1 H-NMR: (400MHz, DMSO-d 6 )δ 11.33(s,1H),7.57(d,J=8.1Hz,1H),7.45-7.13(m,9H),6.86(t,J=8.5Hz,4H),5.94(d,J=1.7Hz,1H),5.5 8(d,J=8.1Hz,1H),5.15(d,J=2.6Hz,1H),3.97-3.79(m,3H),3.73(d,J=2.3Hz,6H),3.33(d,J=2.5Hz,1H).

[0307] Preparation of PH-ALIG-14-4-9A To a stirred solution of PH-ALIG-14-4-8 (6.00 g, 11.616 mmol, 1.00 equiv) in THF (240.00 mL) was added NaH (60%) (1.40 g, 35.003 mmol, 3 equiv) dropwise at 0° C. under an argon atmosphere. The resulting mixture was stirred at 0° C. for 30 min under an argon atmosphere. Dimethyl ethenyl phosphonate (15.81 g, 116.2 mmol, 10.00 equiv) was then added and the resulting mixture was stirred at room temperature under an argon atmosphere overnight. The reaction was purified with saturated NH4 The mixture was quenched with Cl(aq) at room temperature. The resulting mixture was extracted with EtOAc (3×100 mL). The combined organic layers were washed with brine (3×80 mL) and anhydrous Na 2 SO 4 The mixture was dried at 4° C. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by reverse flash chromatography using the following conditions: column, C18 mobile phase, ACN in water, gradient 5% to 95% in 30 min, detector, UV 254 nm to give PH-ALIG-14-4-9A (3.65 g, 48.15%) as a white solid. LC-MS:(ES,m / z):675[M+Na]+;1H-NMR-:(300MHz, DMSO-d 6 )δ 11.39(s,1H),7.44-7.36(m,3H),7.34-7.21(m,7H),6.93-6.83(m,4H),6.08(d,J=2.0Hz,1H),5.55(d,J=8.1Hz,1H),4.08(d,J=11.0Hz,1H) ,3.92(d,J=2.0Hz,1H),3.82-3.71(m,7H),3.57(dd,J=10.9,3.6Hz,6H),3.30-3.23(m,1H),3.06-2.86(m,2H),1.96(dt,J=18.1,7.1Hz,2H).

[0308] Preparation of PH-ALIG-14-4-10A AcOH (12.00 mL) and H 2 A solution of PH-ALIG-14-4-9A (2.80 g, 4.3 mmol, 1.00 equiv) in 2H2O (3.00 mL) was stirred at room temperature under air overnight. The reaction was diluted with saturated NaHCO 3 (aq) was quenched at 0 °C. The resulting mixture was diluted with 3 × 20 mL of CH 2 Cl 2 The product in the aqueous layer was concentrated under reduced pressure. The product was purified under the following conditions (preparative SFC80-2): column, Green Sep basic, 3*15cm, mobile phase, CO 2 (70%) and IPA (0.5% 2M NH 3-MeOH) (30%), detector, UV 254 nm to give the product. This gave 870 mg (57.89%) of PH-ALIG-14-4-10A as a white solid. LC-MS: (ES, m / z): 351 [M+Na]+; 1H-NMR-: (300 MHz, DMSO-d 6 )δ 11.28(s,1H),7.56(d,J=8.1Hz,1H),5.86(d,J=4.4Hz,1H),5.65(d,J=1.6Hz,1H),5.56(d,J=8.1Hz,1H),4.17(d,J=10.1Hz,1H),4.10(d,J =4.3Hz,1H),4.00(dd,J=10.1,3.9Hz,1H),3.87(dt,J=4.1,1.3Hz,1H),3.72-3.49(m,8H),2.08(dd,J=7.1,2.8Hz,1H),2.05-1.96(m,1H).

[0309] Preparation of PH-ALIG-14-4-100 To a 250 mL three-neck round bottom flask, molecular sieves and ACN (30.00 mL) were added at room temperature. The resulting mixture was stirred at room temperature for 10 min under argon atmosphere. Then, 3-[[bis(diisopropylamino)phosphanyl]oxy]propanenitrile (1058.46 mg, 3.512 mmol, 1.5 equiv) and DCI (359.12 mg, 3.043 mmol, 1.30 equiv) were added to the stirred solution. Then, dimethyl PH-ALIG-14-4-10A (820.00 mg, 2.341 mmol, 1.00 equiv) in 30 mL of ACN was added dropwise at room temperature under argon atmosphere. The resulting mixture was stirred at room temperature for 1 h under argon atmosphere. The resulting mixture was diluted with CHCl (60 mL). The combined organic layer was washed with water (3×40 mL) after filtration and dried over anhydrous MgSO. After filtration, the filtrate was concentrated under reduced pressure. The residue was purified by preparative TLC (10% EtOH in 0.5% TEA / EtOAc 1:9 in PE) to give PH-ALIG-14-4-100 (800 mg, 62.1%) as a colorless oil. LC-MS: (ES, m / z): 549 [MH] - ;1H-NMR: (300MHz, DMSO-d 6)δ 11.34(s,1H),7.61(dd,J=8.1,1.7Hz,1H),5.80(dd,J=15.0,1.8Hz,1H),5.60(d,J=8.1Hz,1H),4.48-4.23(m,2H),4.17-3.98(m,2H), 3.88-3.73(m,2H),3.72-3.51(m,10H),2.79(q,J=5.9Hz,2H),2.07(dtt,J=17.9,7.1,3.2Hz,2H),1.15(ddd,J=6.3,3.8,2.1Hz,12H); 31 P NMR (DMSO-d 6 ) δ 149.71, 149.35, 30.85, 30.75

[0310] Example 6 [ka]

[0311] Preparation of 2: (J. Chem. Soc., Perkin Trans. 1, 1992, 1943-1952) To a solution of 1 (150.0 g, 1.0 mol) in DMF (2.0 L), 2,2-dimethoxypropane (312.0 g, 3.0 mol) and p-TsOH (1.7 g, 10.0 mmol) were added, then the reaction mixture was stirred at room temperature for 4 h, after the reaction, the solvent was concentrated to give the crude product, which was used directly in the next step.

[0312] Preparation of 3: (J. Chem. Soc., Perkin Trans. 1, 1992, 1943-1952) To a solution of 2 (190.0 g, 1.0 mol) in pyridine (2.0 L), BzCl (560.0 g, 4.0 mol) was added, then the reaction mixture was stirred at room temperature for 2 h, after the reaction, the reaction mixture was poured into ice water, extracted by adding EA, the organic phase was washed with brine, and Na 2 SO 4 The mixture was dried at 40° C. and concentrated to give the crude product, which was purified by silica gel column (EA:PE=1:5 to 1:1) to give 3 (350.0 g, yield 87.9%), ESI-LCMS: m / z=421.2 [M+Na] + .

[0313] Preparation of 4: (J. Chem. Soc., Perkin Trans. 1, 1992, 1943-1952) To a solution of 3 (240.0 g, 815.5 mmol) in MeCN (3.0 L), N-(2-oxo-1H-pyrimidin-4-yl)benzamide (193.0 g, 897.0 mmol) and BSA (496.6 g, 2.4 mol) were added, then the reaction mixture was stirred at 50° C. for 30 min, then the reaction mixture was cooled to 0° C., TMSOTf (271.5 g, 1.2 mol) was added to the mixture at 0° C., then the reaction mixture was stirred at 70° C. for 2 h, after the reaction, the solvent was concentrated to obtain an oil, and then NaHCO was added to keep the mixture. 3 Pour oil into a solution of slightly alkaline, add EA, wash the organic phase with brine and add Na 2 SO 4 The mixture was dried at 40° C. and concentrated to give a crude product, which was purified by silica gel column (EA:EP=1:3 to 1:1) to give 4 (180.0 g, 44.9% yield). ESI-LCMS: m / z=491.2 [M+H] + ; 1 H NMR (400MHz, DMSO-d 6 )δ 11.19(s,1H),8.20(d,J=7.6Hz,1H),8.01-7.84(m,4H),7.73-7.57(m,2H),7.50(dt,J=10.4,7.7Hz,4H),7.40(d,J=7.4Hz,1H),6.03(d,J=9. 4Hz,1H), 5.33(dd,J=9.4,7.3Hz,1H),4.66(dd,J=7.3,5.3Hz,1H),4.45-4.35(m,2H),4.22(dd,J=13.7,2.5Hz,1H),1.58(s,3H),1.34(s,3H).

[0314] Preparation of 5: A solution of 4 (78.0 g, 158.7 mmol) in pyridine (800.0 mL) was added with H 2 A solution of NaOH (6.3 g, 158.7 mmol) in a mixed solvent of O and MeOH (4:1, 2N) was added, and the reaction mixture was then stirred at 0° C. for 20 min, LC-MS and TLC showed that the starting material had disappeared, and the mixture was then diluted with NH 4The organic phase was poured into a solution of 0.5% NaCl, EA was added for extraction, and the organic phase was washed with brine and 2 SO 4 The mixture was dried at 40° C. and concentrated to give the crude product, which was purified by silica gel column (DCM:MeOH=30:1 to 10:1) to give 5 (56.0 g, 91.0% yield). ESI-LCMS: m / z=388.1 [M+H] + ; 1 H NMR (400MHz, DMSO-d 6 )δ 11.29(s,1H),8.16(d,J=7.6Hz,1H),8.08-7.99(m,2H),7.67-7.60(m,1H),7.53(t,J=7.6Hz,2H),7.35(d,J=7.6Hz,1H),5.63(d,J= 6.1Hz,1H),5.51(d,J=9.5Hz,1H),4.35-4.13(m,3H),3.78(dt,J=9.6,6.5Hz,1H),3.19(d,J=5.1Hz,1H),1.53(s,3H),1.32(s,3H).

[0315] Preparation of 6: To a solution of 5 (15.0 g, 38.7 mmol) in DCM (200.0 mL), 2 O (35.8 g, 154.8 mmol), CH 3 I (54.6 g, 387.2 mmol) and NaI (1.1 g, 7.7 mmol) were added, and then the reaction mixture was stirred at room temperature overnight. After the reaction, the filtrate was obtained by filtration, and the solvent was concentrated from the filtrate to obtain product 6 (13.0 g, 75.2% yield). ESI-LCMS: m / z=402.30 [M+H] + ; 1 H NMR (400MHz, DMSO-d 6 )δ 11.30(s,1H),8.22(s,1H),8.00(d,J=7.6Hz,2H),7.71-7.20(m,4H),5.56(d,J=9.3Hz,1H),4.33(t,J=6.1Hz,1H),4.26(dd,J=6.2,2 .1Hz,1H),4.20(d,J=13.5Hz,1H),3.98(dd,J=13.5,2.5Hz,1H),3.66(dd,J=9.3,6.6Hz,1H),3.34(s,3H),1.57(s,3H),1.32(s,3H).

[0316] Preparation of 7: A solution of 6 (12.0 g, 29.9 mmol) was added to CH 3 COOH (120.0 mL) was added, and the mixture was stirred at room temperature for 2 h. LC-MS and TLC showed that the raw material had disappeared, and the solvent was then concentrated to give crude product 7 (10.0 g, 83.3% yield). ESI-LCMS: m / z=362.1 [M+H] + .

[0317] Preparation of 8: Dioxane:H 2 A solution of 7 (10.0 g, 24.9 mmol) in 3:1 HO (120.0 mL) was added to NaIO 4 (8.8 g, 41.5 mmol) was added, and the reaction mixture was then stirred at room temperature for 2 h, LC-MS and TLC showed that the starting material had disappeared, and the reaction mixture was then cooled to 0° C. and NaBH 4 (2.4 g, 41.5 mmol) was added to the mixture and stirred at 0 °C for 0.5 h, LC-MS and TLC showed that the starting material had disappeared, and NH 4 The mixture was added with Cl to adjust the pH to slightly alkaline and concentrated to give the crude product, which was purified by silica gel column (PE:EA=5:1 to 1:1) to give 8 (8.0 g, 79.5% yield). ESI-LCMS: m / z=364.1 [M+H] + ; 1 H NMR (400MHz, DMSO-d 6 )δ 11.26(s,1H),8.14(d,J=7.5Hz,1H),8.07-7.94(m,2H),7.67-7.59(m,1H),7.52(t,J=7.6Hz,2H),7.37(s,1H),5.91(d,J=6 .0Hz,1H),4.77(t,J=5.6Hz,1H),4.70(t,J=5.1Hz,1H),3.70(ddd,J=11.5,5.0,2.5Hz,1H),3.57-3.39(m,6H),3.31(s,3H).

[0318] Preparation of 9: To a solution of 8 (4.0 g, 11.0 mmol) in pyridine (50.0 mL), DMTrCl (5.5 g, 16.5 mmol) was added, then the reaction mixture was stirred at room temperature for 2 h, LC-MS showed that the starting material was 20.0% and the product to by-product ratio was 3.5:1. The solvent was then concentrated to give a residue, which was purified on a silica gel column to give the purified product, the by-product was 5 g in total, and the product was then purified by SFC to give 9 (3.0 g, 40.9% yield). ESI-LCMS: m / z=666.2 [M+H] + ; 1 H NMR (400MHz, DMSO-d 6 )δ 11.33(s,1H),8.20(d,J=7.4Hz,1H),8.04(d,J=7.7Hz,2H),7.64(t,J=7.4Hz,1H),7. 53(t,J=7.6Hz,2H),7.40(d,J=7.8Hz,3H),7.36-7.18(m,7H),6.89(d,J=8.4Hz,4H), 5.96(d,J=5.7Hz,1H),4.79(t,J=5.7Hz,1H),3.73(s,6H),3.66-3.46(m,4H),3.37(s ,3H),3.16(ddd,J=10.1,7.1,3.0Hz,1H),3.04(dt,J=10.9,3.4Hz,1H),2.08(s,1H).

[0319] Preparation of 10: To a solution of 9 (2.8 g, 4.2 mmol) in DCM (30.0 mL), add CEP[N(iPr) 2 ] 2 (1.3 g, 4.2 mmol) and DCI (601.2 mg, 5.1 mmol) were added. The mixture was stirred at room temperature for 1 h. LC-MS showed that 9 was completely consumed. The solution was diluted with NaHCO 3 Wash twice with solution, then with brine, and finally with Na 2 SO 4 The mixture was then dried at 40° C. and concentrated to give a residue, which was then run under the following conditions (IntelFlash-1): column, C 18 Silica gel, mobile phase, CH 3 CN / H 2 O(0.5%NH 4 HCO 3) = 1 / 1 in 20.0 minutes or less 3 CN / H 2 O(0.5%NH 4 HCO 3 The mixture was purified by Flash-Prep-HPLC while increasing the concentration of 1:1 to 1:0. The eluted product was 3 CN / H 2 O(0.5%NH 4 HCO 3 ) = 90 / 10, detector, UV 254 nm. This gave 10 (2.8 g, 76.8% yield). ESI-LCMS: m / z = 866.2 [M+H] + ; 1 H NMR (400MHz, DMSO-d 6 )δ 11.34(s,1H),8.22(d,J=7.4Hz,1H),8.09-7.98(m,2H),7.64(t,J=7.4Hz,1H),7.53(t,J=7.6Hz,2H),7.45(d,J= 7.3Hz,1H),7.39(d,J=7.5Hz,2H),7.31(t,J=7.6Hz,2H),7.24(t,J=9.1Hz,5H),6.89(d,J=8.8Hz,4H),5.96(d,J= 6.1Hz,1H),4.02-3.86(m,1H),3.84-3.63(m,11H),3.56(dtq,J=13.3,6.6,3.5,3.1Hz,3H),3.37(s,2H),3.16(d dd,J=10.0,6.8,3.3Hz,1H),3.04(ddd,J=10.7,5.5,3.0Hz,1H),2.75(td,J=5.9,2.3Hz,2H),1.18-1.07(m,12H); 31 P NMR (DMSO-d 6 ) δ 148.02(d,J=12.0Hz).

[0320] Example 7 [ka]

[0321] Preparation of 10: A solution of 3 (200.0 g, 0.5 mol) in ACN (2000.0 mL) was 2 SnCl in DCM (1000.0 mL) at 0 °C4 A solution of 1000 ml of ethyl alcohol was added and the reaction mixture was subjected to nitrogen 2 The mixture was stirred at 0° C. under atmospheric pressure for 4 hours. The reaction solution was then poured into saturated sodium bicarbonate solution, and the resulting product was extracted with EA (3*500.0 mL). The combined organic layers were washed with water and brine, and then extracted with Na 2 SO 4 After 20 min, the mixture was dried at 40° C. and concentrated to give the crude material, which was purified by silica gel column (PE:EA=5:1 to 0:1) to give 10 (65.0 g, 31.4% yield) as a white solid. ESI-LCMS: m / z=412.0 [M+H] + ; 1 H NMR (400MHz, DMSO-d 6 )δ 8.27(s,1H),8.09(s,1H),7.74-7.60(m,2H),7.59-7.57(m,1H),7.44-7.40(m,2H),7.24(s,2H),5.90(d,J=9.6Hz ,1H),5.73(dd,J=7.4Hz,1H),4.63(t,1H),4.50-4.30(m,2H),4.21(dd,J=13.6Hz,1H),1.61(s,3H),1.35(s,3H).

[0322] Preparation of 11: To a solution of 10 (40.0 g, 97.3 mmol) in DCM (500.0 mL) was added Et 3 N (30.0 g, 297.0 mmol) and DMAP (1.2 g, 9.8 mmol) were added at room temperature. The reaction mixture was stirred at 4°C for 1 h. 2 The mixture was then replaced with 3 portions of NaHCO in ice water, and then MMTrCl (45.0 g, 146.1 mmol) was added to the mixture. The reaction mixture was stirred at room temperature overnight. TLC and LC-MS showed that 10 was consumed and the reaction mixture was dissolved in NaHCO in ice water. 3 The product was then extracted with EA, the organic phase was washed with brine, and the organic phase was added with Na 2 SO 4 Drying at rt and then concentration gave 11 (66.5 g, ) as crude material which was used directly in the next step.

[0323] Preparation of 12: A solution of 11 (66.5 g, 97.3 mmol) in pyridine (600.0 mL) was diluted with 2 N NaOH (H 2 O:MeOH=4:1) (200.0 mL) was added at room temperature. The reaction mixture was then stirred at 0° C. for 30 min, LC-MS and TLC showed that the starting material had disappeared, and the mixture was then diluted with NH 4 Cl solution, EA was added for extraction, the organic phase was washed with brine and Na 2 SO 4 The crude product was purified by silica gel column (EA:PE=1:5 to 1:1) to give 12 (50.0 g, 88.7% yield in two steps). ESI-LCMS: m / z=580.4 [M+H] + ; 1 H NMR (400MHz, DMSO-d 6 )δ 8.44(s,1H),7.92(s,1H),7.36-7.16(m,13H),6.89-6.80(m,2H),5.59(d,J=6.0Hz,1H),5.35(d,J=9.6Hz,1H),4 .32-4.12(m,4H),4.08-3.95(m,3H),3.72(s,3H),1.99(s,3H),1.54(s,3H),1.32(s,3H),1.17(t,J=7.1Hz,3H).

[0324] Preparation of 13:CH 3 To a solution of 12 (46.0 g, 79.4 mmol) in I (200.0 mL), 2 O (36.6 g, 158.4 mmol) and NaI (6.0 g, 42.5 mmol) were added and the reaction mixture was stirred at room temperature for 4 h, then the reaction mixture was filtered and the solvent was concentrated to give product 13 (46.0 g, 97.6% yield), which was used directly in the next step. ESI-LCMS: m / z=594.3 [M+H] + .

[0325] Preparation of 14: To a stirred solution of DCA (22.5 mL) in DCM (750.0 mL) was added 13 (46.0 g, 77.5 mmol) and Et 3Si (185.0 mL) was added at room temperature. The reaction mixture was stirred at room temperature for 12 h. The reaction solution was evaporated to dryness under reduced pressure to give a residue which was dissolved in NaHCO 3 (50.0 mL) to give 14 (19.0 g, 76% yield), which was used directly in the next step.

[0326] Preparation of 15: To a solution of 14 (16.0 g, 49.7 mmol) in pyridine (200.0 mL) was added BzCl (9.0 g, 64.7 mmol) at 0° C. The reaction mixture was then stirred at room temperature for 2 h. LC-MS showed complete consumption of 6, then the mixture was cooled to 0° C. and purified with MeOH and H 2 A solution of NaOH in O (2N, 50.0 mL) was added to the reaction mixture, the mixture was stirred at 0 °C for 1 h, and then the mixture was treated with NH 4 The product was extracted with EA (300.0 mL) and the organic layer was washed with brine and poured into a solution of Na 2 SO 4 The organic layer was then concentrated to give a residue, which was purified by slurry with PE:EA (8:1, 900.0 mL) to give 15 (20.0 g, 95.0% yield). ESI-LCMS: m / z=426.2 [M+H] + ;1H NMR(400MHz,DMSO-d6)δ 11.21(s,1H),8.77-8.69(m,2H),8.06(d,J=7.6Hz,2H),7.65(t,J=7.4Hz,1H),7.56(t,J=7.6Hz,2 H),7.34-7.23(m,4H),7.23-7.12(m,5H),6.89-6.80(m,4H),5.90(d,J=7.9Hz,1H),4.36-4.29(m,1 H),4.06(t,J=8.8Hz,1H),3.92(dd,J=25.0,6.9Hz,0H),3.72(d,J=1.0Hz,7H),3.59(dt,J=10.4,6 .6Hz,1H),3.24(s,3H),2.97(d,J=7.7Hz,1H),2.76(q,J=5.5Hz,2H),1.14(dd,J=9.2,5.7Hz,12H).

[0327] Preparation of 16: HCOOH (180.0 mL) and H 2 To a mixture solution of 2H2O (20.0 mL) was added 15 (19.0 g, 44.7 mmol). The reaction mixture was stirred at room temperature for 4 h. LC-MS showed that 15 was completely consumed. The reaction mixture was then concentrated to give a residue, which was purified by slurrying with MeOH (100.0 mL) to give 16 (16.0 g, 92.7% yield) as a white solid. ESI-LCMS: m / z=385.9 [M+H] + ;1H NMR (400MHz, DMSO-d 6 )δ 11.21(s,1H),8.77(d,J=1.2Hz,2H),8.09-8.02(m,2H),7.70-7.61(m,1H),7.56(t,J=7.6Hz,2H),5.56(d,J=9.2Hz,1H),5.2 1(d,J=6.1Hz,1H),4.94(d,J=4.5Hz,1H),4.18(t,J=9.1Hz,1H),4.09(q,J=5.2Hz,1H),3.88-3.71(m,4H),3.21-3.14(m,6H).

[0328] Preparation of 17: A solution of 16 (16.0 g, 41.4 mmol) in dioxane (200.0 mL) was added with H 2 O (32.0 mL), and NaIO 4 (9.7 g, 45.5 mmol) was added, then the reaction mixture was stirred at room temperature for 1 h, LC-MS and TLC showed that the starting material had disappeared, then the reaction mixture was cooled to 0 °C, NaBH4 (1.7 g, 45.5 mmol) was added to the mixture, stirred at 0 °C for 0.5 h, LC-MS and TLC showed that the intermediate had disappeared, then NH 4 The mixture was added with Cl to adjust the pH to slightly alkaline and concentrated at room temperature to give the crude product, which was purified by silica gel column (DCM:MeOH=20:1 to 8:1) to give 17 (16.0 g, 99.5% yield). ESI-LCMS: m / z=388.0 [M+H] + ; 1 H NMR (400MHz, DMSO-d 6)δ 11.18(s,1H),8.75(s,1H),8.67(s,1H),8.09-7.99(m,2H),7.65(t,J=7.4Hz ,1H),7.56(t,J=7.6Hz,2H),5.90(d,J=7.6Hz,1H),4.88(t,J=5.7Hz,1H),4.6 7(t,J=5.5Hz,1H),4.08-3.98(m,2H),3.78(ddd,J=12.1,5.2,3.1Hz,1H),3.6 8-3.39(m,4H),3.36(s,0H),3.20(s,3H),1.99(s,1H),1.17(t,J=7.1Hz,1H).

[0329] Preparation of 18: To a solution of 17 (12.0 g, 31.0 mmol) in pyridine (50.0 mL), DMTrCl (11.5 g, 34.1 mmol) was added, and the reaction mixture was then stirred at room temperature for 2 h, and LC-MS showed that 15.0% starting material remained and the product to by-product ratio was 3.5:1. The reaction solution was then poured into ice water, extracted with EA, washed with brine, and diluted with Na. 2 SO 4 The mixture was dried at 40° C., filtered and concentrated to give a residue which was purified by silica gel column to give a total of 13.0 g of purified product and by-products, and then 4.0 g of the crude material was purified by SFC to give 18 (3.3 g, 15.4% yield). ESI-LCMS: m / z=690.3 [M+H] + ; 1 H NMR (400MHz, DMSO-d 6)δ 11.21(s,1H),8.75(s,1H),8.69(s,1H),8.10-8.03(m,2H),7.70-7.61(m,1H),7.56(t,J=7.6Hz,2H),7.35-7.12(m,9H) ,6.90-6.80(m,4H),5.94(d,J=7.5Hz,1H),4.88(t,J=5.6Hz,1H),4.36(t,J=5.1Hz,1H),4.11(dt,J=7.4,3.6Hz,1H),3. 82(ddd,J=11.9,5.1,3.1Hz,1H),3.72(d,J=1.3Hz,7H),3.64(ddd,J=11.9,6.2,4.2Hz,1H),3.45(qd,J=7.0,4.9Hz,2H) ,3.24(s,3H),3.09(ddd,J=9.9,6.4,3.2Hz,1H),2.97(ddd,J=9.9,5.7,3.2Hz,1H),1.23(s,0H),1.06(t,J=7.0Hz,1H).

[0330] Preparation of 19: A suspension of 18 (3.3 g, 4.8 mmol) in DCM (40.0 mL) was treated with DCI (0.5 g, 4.0 mmol) and CEP[N(iPr) 2 ] 2 (1.6 g, 5.3 mmol) was added. The mixture was stirred at room temperature for 0.5 h. LC-MS showed that 10 was completely consumed. The solution was diluted with NaHCO 3 Wash twice with solution, then with brine, and finally with Na 2 SO 4 The mixture was then dried at 40° C. and concentrated to give a residue, which was then run under the following conditions (IntelFlash-1): column, C 18 Silica gel, mobile phase, CH 3 CN / H 2 O(0.5%NH 4 HCO 3 )=1 / 1 in 20 minutes or less 3 CN / H 2 O(0.5%NH 4 HCO 3 The mixture was purified by Flash-Prep-HPLC while increasing the concentration of 1:1 to 1:0. The eluted product was 3 CN / H 2 O(0.5%NH 4 HCO3 )=1 / 0, detector, UV 254 nm. This gave 19 (3.0 g, 3.9 mmol, 81.2% yield) as a white solid. ESI-LCMS: m / z=765.3 [M+H] + ; 1 H NMR (400MHz, DMSO-d 6 )δ 11.22(s,1H),8.80-8.71(m,2H),8.11-8.04(m,2H),7.65(t,J=7.3Hz,1H),7.56(t,J=7.5Hz,2H),7.36-7.24(m,4H) ,7.24-7.15(m,5H),6.89-6.82(m,4H),5.92(d,J=7.7Hz,1H),4.34(dt,J=7.5,3.5Hz,1H),4.08(ddd,J=10.7,7.3,2. 7Hz,1H),4.03-3.89(m,1H),3.80-3.72(m,10H),3.67-3.53(m,2H),3.47(dp,J=10.5,3.4Hz,1H),3.26(s,3H)3.11( ddd,J=10.3,6.2,3.5Hz,1H),3.00(q,J=6.6,5.2Hz,1H),2.77(q,J=5.6Hz,2H),2.08(s,1H),1.15(t,J=7.0Hz,12H). ; 31 P NMR (162MHz, DMSO-d 6 )δ 148.30,147.99.

[0331] Example 8 [ka]

[0332] Preparation of 19: A solution of 8 (8.0 g, 22.0 mmol) in EtOH (50.0 mL) was added to CH 3 NH 2 (50.0 mL) solution was added, then the reaction mixture was stirred at room temperature for 4 h, after the reaction, the solvent was concentrated to give the crude material, which was added to a mixed solvent of EA (20.0 mL) and PE (10.0 mL), then the mixture was stirred for 30 min, and filtered to give 19 (5.5 g, yield 96.5%), which was used directly in the next step.

[0333] Preparation of 20: (J. Chem. Soc., Perkin Trans. 1, 1992, 1943-1952) H 2 To a solution of 19 (5.0 g, 19.3 mmol) in 2H2O (50.0 mL) and AcOH (50.0 mL) was added NaNO 2 (65.0 g, 772.0 mmol) was added, and then the reaction mixture was stirred at room temperature for 2 hours. After the reaction, the reaction mixture was concentrated to obtain a crude product, which was purified by silica gel column (DCM:MeOH=20:1 to 6:1) and MPLC (ACN:H 2 O = 0:100 to 10:90) to give 20 (3.0 g, 59.6% yield). ESI-LCMS: m / z = 261.2 (M+H) + ; 1 H NMR (400MHz, DMSO-d 6 ) δ 11.29(s,1H),7.66(d,J=8.0Hz,1H),5.67(dd,J=17.5,7.6Hz,2H),4.74(d,J=36.0Hz,2H),3.86-3.63(m,1H),3.58-3.40(m,6H).

[0334] Preparation of 21: To a solution of 20 (3.0 g, 11.5 mmol) in pyridine (30.0 mL), DMTrCl (3.9 g, 11.5 mmol) was added, and the reaction mixture was stirred at room temperature for 2 h. LC-MS showed 20.0% starting material and a 3:1 product to by-product ratio. The mixture was then purified by precipitation with NaHCO 3 (100.0 mL), extracted with EA (100.0 mL), washed with brine and added Na 2 SO 4 After drying at 40° C., filtration and concentration gave a residue which was purified by silica gel column to give a total of 5.0 g of purified product and by-products, and the product was then purified by SFC to give 21 (1.8 g,). ESI-LCMS: m / z=561.2 [M+H] + ; 1 H NMR (400MHz, DMSO-d 6) δ 11.31(s,1H),7.69(d,J=8.1Hz,1H),7.45-7.15(m,8H),6.88(d,J=8.5Hz,4H),5.71(d,J=6.8Hz,1H),5.64(d,J=8.0Hz,1 H),4.79(t,J=5.5Hz,1H),3.74(s,6H),3.60(s,1H),3.51(d,J=5.5Hz,3H),3.11(d,J=6.7Hz,1H),3.02(d,J=7.0Hz,1H).

[0335] Preparation of 22: To a solution of 21 (1.8 g, 3.2 mmol) in DCM (20.0 mL), CEP[N(iPr) 2 ] 2 (1.0 g, 3.4 mmol) and DCI (321.0 mg, 2.7 mmol) were added. The mixture was stirred at room temperature for 1 h. LC-MS showed that 21 was completely consumed. The solution was diluted with NaHCO 3 Wash twice with solution, then with brine, and finally with Na 2 SO 4 The mixture was then dried at 40° C. and concentrated to give a residue, which was then run under the following conditions (IntelFlash-1): column, C 18 Silica gel, mobile phase, CH 3 CN / H 2 O(0.5%NH 4 HCO 3 )=1 / 1 in 20 minutes or less 3 CN / H 2 O(0.5%NH 4 HCO 3 The mixture was purified by Flash-Prep-HPLC while increasing the concentration of 1:1 to 1:0. The eluted product was 3 CN / H 2 O(0.5%NH 4 HCO 3 )=90 / 10, detector, UV 254 nm. This gave 22 (2.0 g, 82% yield). ESI-LCMS: m / z=761.2 [M+H] + ; 1 H NMR (400MHz, DMSO-d 6)δ 11.35(s,1H),7.73(dd,J=8.0,2.0Hz,1H),7.39(d,J=7.4Hz,2H),7.35-7.1 8(m,7H),6.94-6.82(m,4H),5.81-5.74(m,1H),5.67(d,J=8.0Hz,1H),4.11 -3.85(m,1H),3.82-3.67(m,11H),3.67-3.50(m,5H),3.17-3.09(m,1H),3. 09-3.01(m,1H),2.74(td,J=5.8,2.9Hz,2H),1.13(dd,J=9.2,6.7Hz,13H); 31 P NMR (DMSO-d 6 ) δ 148.09(d,J=41.8Hz).

[0336] Example 9 [ka]

[0337] Preparation of 2 (J. Chem. Soc., Perkin Trans. 1, 1992, 1943-1952): To a solution of 1 (150.0...

Claims

1. The following structure: 【Chemical 1】 wherein Rx is a nucleobase, aryl, heteroaryl, or H; 【Chemistry 2】 indicates a phosphodiester linkage.

2. The following structure: 【Chemistry 3】 wherein R ｙ is the nucleobase, 【Chemistry 4】 The nucleotide of claim 1, wherein: represents a phosphodiester linkage.

3. The following structure: 【Chemistry 5】 Including, 【Chemistry 6】 indicates a phosphodiester linkage.

4. A method for detecting a nucleotide sequence comprising a sense strand and an antisense strand, the method comprising: 【Chemistry 7】 wherein Rx is a nucleobase, aryl, heteroaryl, or H; 【Chemistry 8】 (In the formula, R ｙ A short interfering nucleic acid (siNA) molecule comprising at least one modified nucleotide selected from the group consisting of nucleotides 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, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,

5. A short interfering nucleic acid (siNA) molecule comprising: (a) a sense strand comprising a first nucleotide sequence that is at least about 90% identical to an RNA corresponding to a target gene, wherein the first nucleotide sequence is (i) is 15 to 30 nucleotides in length; (ii) a sense strand comprising 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least one modified nucleotide is a 2'-O-methyl nucleotide and the nucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and / or 19 from the 5' end of the first nucleotide sequence are 2'-fluoro nucleotides, or at least one modified nucleotide is a 2'-O-methyl nucleotide and at least one modified nucleotide is a 2'-fluoro nucleotide; an antisense strand comprising a second nucleotide sequence that is at least about 90% complementary to the RNA corresponding to the target gene, wherein the second nucleotide sequence is (iii) is 15 to 30 nucleotides in length; (iv) an antisense strand comprising 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least one modified nucleotide is a 2'-O-methyl nucleotide and at least one modified nucleotide is a 2'-fluoro nucleotide; or (b) a sense strand comprising a first nucleotide sequence that is at least about 60% identical to an RNA corresponding to the target gene, wherein the first nucleotide sequence is (i) is 15 to 30 nucleotides in length; (ii) a sense strand comprising 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least one modified nucleotide is a 2'-O-methyl nucleotide and at least one modified nucleotide is a 2'-fluoro nucleotide; and an antisense strand comprising a second nucleotide sequence that is at least about 60% complementary to the RNA corresponding to the target gene, wherein the second nucleotide sequence is (iii) is 15 to 30 nucleotides in length; (iv) an antisense strand comprising 15 or more modified nucleotides independently selected from 2'-O-methyl nucleotides and 2'-fluoro nucleotides, wherein at least one modified nucleotide is a 2'-O-methyl nucleotide and the nucleotides at positions 2, 5, 6, 8, 10, 14, 16, 17, and / or 18 from the 5' end of the second nucleotide sequence are 2'-fluoro nucleotides; An siNA molecule, wherein the sense strand and / or the antisense strand comprises at least one modified nucleotide according to claim 1, and wherein the modified nucleotide is not the 5' end cap of the sense strand or the antisense strand.

6. The antisense strand is 【Chemistry 9】 wherein R ｙ is a nucleobase, and R １５ is H or CH ３ The siNA of claim 4, wherein:

7. The siNA molecule of claim 4, wherein the antisense strand comprises a 5' stabilized 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 (56), Formula (49X) to Formula (52X), Formula (49Y) to (52Y), Formula 56X, Formula 56Y, Formula (61), Formula (62), Formula (63), Formula (71) to Formula (86), Formula (79X) to Formula (82X), Formula (79Y) to (82Y), Formula 86X, Formula 86X', Formula 86Y, and Formula 86Y'.

8. The antisense strand has formulas (1A) to (15A), formulas (1A-1) to (7A-1), formulas (1A-2) to (7A-2), formulas (1A-3) to (7A-3), formulas (1A-4) to (7A-4), formulas (9B) to (12B), formulas (9AX) to (12AX), and formulas (9AY) to (12). AY), formulas (9BX) to (12BX), formulas (9BY) to (12BY), formulas (21A) to (35A), formulas (29B) to (32B), formulas (29AX) to (32AX), formula (29AY) to (32AY), formula (29BX) to (32BX), and formula (29BY) to (32BY) The siNA of claim 4, comprising a 5'-stabilizing endcap selected from the group consisting of formulas (71A) to (86A), formulas (79XA) to (82XA), formulas (79YA) to (82YA), formula (86XA), formula (86X'A), formula (86Y), and formula (86Y').

9. The siNA of claim 4, wherein the sense strand and / or the antisense strand independently comprise one or more phosphorothioate internucleoside linkages, one or more mesyl phosphoramidate internucleoside linkages, or a combination thereof.

10. The siNA of claim 4, wherein the siNA further comprises a phosphorylation blocker.

11. (i) at least one phosphorothioate internucleoside linkage of the sense strand is between the nucleotides at positions 1 and 2 from the 5' end of the first nucleotide sequence; (ii) at least one phosphorothioate internucleoside linkage of the sense strand is between the nucleotides at positions 2 and 3 from the 5' end of the first nucleotide sequence; (iii) at least one phosphorothioate internucleoside linkage of the antisense strand is between the nucleotides at positions 1 and 2 from the 5' end of the second nucleotide sequence; (iv) at least one phosphorothioate internucleoside linkage of the antisense strand is between the nucleotides at positions 2 and 3 from the 5' end of the second nucleotide sequence; (v) at least one phosphorothioate internucleoside linkage of the antisense strand is between the nucleotides at positions 1 and 2 from the 3' end of the second nucleotide sequence; and / or (vi) at least one phosphorothioate internucleoside linkage of the antisense strand is between the nucleotides at positions 2 and 3 from the 3' end of the second nucleotide sequence; The siNA molecule of claim 9.

12. (i) at least one mesyl phosphoramidate internucleoside linkage of the sense strand is between the nucleotides at positions 1 and 2 from the 5' end of the first nucleotide sequence; (ii) at least one mesyl phosphoramidate internucleoside linkage of the sense strand is between the nucleotides at positions 2 and 3 from the 5' end of the first nucleotide sequence; (iii) at least one mesyl phosphoramidate internucleoside linkage of the antisense strand is between the nucleotides at positions 1 and 2 from the 5' end of the second nucleotide sequence; (iv) at least one mesyl phosphoramidate internucleoside linkage of the antisense strand is between the nucleotides at positions 2 and 3 from the 5' end of the second nucleotide sequence; (v) at least one mesyl phosphoramidate internucleoside linkage of the antisense strand is between the nucleotides at positions 1 and 2 from the 3' end of the second nucleotide sequence; and / or (vi) at least one mesyl phosphoramidate internucleoside linkage of the antisense strand is between the nucleotides at positions 2 and 3 from the 3' end of the second nucleotide sequence; The siNA molecule of claim 9.

13. the siNA further comprises galactosamine; The galactosamine has the formula (VI): 【Chemistry 10】 N-acetylgalactosamine (GalNAc) of the formula: m is 1, 2, 3, 4, or 5; each n is independently 1 or 2; p is 0 or 1; each R is independently H; each Y is independently selected from —O—P(═O)(SH)—, —O—P(═O)(O)—, —O—P(═O)(OH)—, and —O—P(S)S—; Z is H or a second protecting group; any L is a linker, or L and Y together are a linker; A is H, OH, a third protecting group, an activating group, or an oligonucleotide; or The galactosamine has the formula (VII): 【Chemistry 11】 5. The siNA of claim 4, wherein R z is OH or SH, and each n is independently 1 or 2.

14. The siNA described in claim 9, wherein at least one end of the siNA comprises an overhang, the overhang comprising at least one nucleotide.

15. The siNA described in claim 9, wherein the target gene is a viral gene.

16. An siNA comprising a nucleotide sequence of any of SEQ ID NOs: 36, 38, 40, 77, 79, 81, 83, 85, 87, 88, 90, 93, and 95 to 105.

17. A composition comprising the siNA of claim 4 and a pharmaceutically acceptable excipient.

18. A method for detecting a nucleotide sequence comprising: 【Chemistry 12】 wherein Rx is a nucleobase, aryl, heteroaryl, or H; 【Chemistry 13】 18. The composition of claim 17, further comprising two or more siNAs comprising at least one nucleotide selected from: wherein R y is a nucleobase, and any combination thereof.

19. The composition of claim 17, further comprising an additional therapeutic agent selected from a nucleotide analog, a nucleoside analog, a capsid assembly modulator (CAM), a recombinant interferon, an entry inhibitor, a small molecule immunomodulator, and an oligonucleotide therapy.

20. Use of a siNA described in any one of claims 4 to 16 or a composition described in any one of claims 17 to 19 in the manufacture of a medicament for the treatment of a disease in a subject in need thereof.

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