US20220364096A1 - Modified Short Interfering Nucleic Acid (siNA) Molecules and Uses Thereof - Google Patents

Modified Short Interfering Nucleic Acid (siNA) Molecules and Uses Thereof Download PDF

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US20220364096A1
US20220364096A1 US17/194,079 US202117194079A US2022364096A1 US 20220364096 A1 US20220364096 A1 US 20220364096A1 US 202117194079 A US202117194079 A US 202117194079A US 2022364096 A1 US2022364096 A1 US 2022364096A1
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nucleotide
nucleotides
nucleotide sequence
sina
fluoro
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Leonid Beigelman
Vivek Kumar Rajwanshi
Markus Hossbach
Rajendra K. Pandey
Jin Hong
Laxman Eltepu
Saul MARTINEZ MONTERO
N. Tilani S. De Costa
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Aligos Therapeutics Inc
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Aligos Therapeutics Inc
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Priority to US17/194,079 priority Critical patent/US20220364096A1/en
Assigned to ALIGOS THERAPEUTICS, INC. reassignment ALIGOS THERAPEUTICS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: MONTERO, Saul MARTINEZ, BEIGELMAN, LEONID, HONG, JIN, HOSSBACH, MARKUS, RAJWANSHI, VIVEK KUMAR, DE COSTA, N. Tilani S., ELTEPU, LAXMAN, PANDEY, RAJENDRA K.
Priority to US17/672,268 priority patent/US11549110B2/en
Publication of US20220364096A1 publication Critical patent/US20220364096A1/en
Priority to US18/059,561 priority patent/US20230332153A1/en
Priority to US18/361,363 priority patent/US12129469B2/en
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Definitions

  • siNA short interfering nucleic acid
  • RNA interference 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.
  • the short interfering nucleic acids such as siRNA, have been developed for RNAi therapy to treat a variety of diseases.
  • RNAi therapy has been proposed for the treatment of metabolic diseases, neurodegenerative diseases, cancer, and pathogenic infections (See e.g., Rondindone, Biotechniques, 2018, 40(4S), doi.org/10.2144/000112163, Boudreau and Davidson, Curr Top Dev Biol, 2006, 75:73-92, Chalbatani et al., Int J Nanomedicine, 2019, 14:3111-3128, Arbuthnot, Drug News Perspect, 2010, 23(6):341-50, and Chernikov et. al., Front. Pharmacol., 2019, doi.org/10.3389/fphar.2019.00444, each of which are incorporated by reference in their entirety).
  • major limitations of RNAi therapy are the ability to effectively deliver siRNA to target cells and the degradation of the siRNA.
  • the present disclosure improves the delivery and stability of siNA molecules by providing siNA molecules comprising modified nucleotides.
  • the siNA molecules of the present disclosure provide optimized combinations and numbers of modified nucleotides, nucleotide lengths, design (e.g., blunt ends or overhangs, internucleoside linkages, conjugates), and modification patterns for improving the delivery and stability of siNA molecules.
  • 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: (i) is 15 to 30 nucleotides in length; and (ii) comprises 15 or more modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide, wherein at least one modified nucleotide is a 2′-O-methyl nucleotide and the nucleotide at position 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and/or 19 from the 5′ end of the first nucleotide sequence is a 2′-fluoro nucleotide; and (b) an antisense strand comprising a second nucleotide
  • 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: (i) is 15 to 30 nucleotides in length; and (ii) comprises 15 or more modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide, wherein at least one modified nucleotide is a 2′-O-methyl nucleotide and at least one modified nucleotide is a 2′-fluoro nucleotide; and (b) an antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
  • the first nucleotide sequence comprises 16, 17, 18, 19, 20, 21, 22, 23, or more modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide.
  • 70%, 75%, 80%, 85%, 90%, 95% or 100% of the nucleotides in the first nucleotide sequence are modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide.
  • between 2 to 15 modified nucleotides of the first nucleotide sequence are 2′-fluoro nucleotides.
  • between 2 to 10 modified nucleotides of the first nucleotide sequence are 2′-fluoro nucleotides. In some embodiments, between 2 to 6 modified nucleotides of the first nucleotide sequence are 2′-fluoro nucleotides. In some embodiments, at least 2, 3, 4, 5, or 6 modified nucleotides of the first nucleotide sequence are 2′-fluoro nucleotides. In some embodiments, less than or equal to 10, 9, 8, 7, 6, 5, 4, 3, or 2 modified nucleotides of the first nucleotide sequence are 2′-fluoro nucleotides.
  • between about 2 to 25 modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, between about 2 to 20 modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, between about 5 to 25 modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, between about 10 to 25 modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, between about 12 to 25 modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides.
  • At least 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, less than or equal to 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides.
  • the second nucleotide sequence comprises 16, 17, 18, 19, 20, 21, 22, 23, or more modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide.
  • 70%, 75%, 80%, 85%, 90%, 95% or 100% of the nucleotides in the second nucleotide sequence are modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide.
  • between 2 to 15 modified nucleotides of the second nucleotide sequence are 2′-fluoro nucleotides.
  • between 2 to 10 modified nucleotides of the second nucleotide sequence are 2′-fluoro nucleotides. In some embodiments, between 2 to 6 modified nucleotides of the second nucleotide sequence are 2′-fluoro nucleotides. In some embodiments, at least 2, 3, 4, 5, or 6 modified nucleotides of the second nucleotide sequence are 2′-fluoro nucleotides. In some embodiments, less than or equal to 10, 9, 8, 7, 6, 5, 4, 3, or 2 modified nucleotides of the second nucleotide sequence are 2′-fluoro nucleotides.
  • between about 2 to 25 modified nucleotides of the second nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, between about 2 to 20 modified nucleotides of the second nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, between about 5 to 25 modified nucleotides of the second nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, between about 10 to 25 modified nucleotides of the second nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, between about 12 to 25 modified nucleotides of the second nucleotide sequence are 2′-O-methyl nucleotides.
  • 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, less than or equal to 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 modified nucleotides of the second nucleotide sequence are 2′-O-methyl nucleotides.
  • 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: (i) is 15 to 30 nucleotides in length; (ii) comprises 15 or more modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide, wherein at least one modified nucleotide is a 2′-O-methyl nucleotide and at least one modified nucleotide is a 2′-fluoro nucleotide; and (iii) comprises 1 or more phosphorothioate internucleoside linkage; and (b) an antisense strand comprising a second nucleotide sequence that is
  • 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: (i) is 15 to 30 nucleotides in length; and (ii) comprises 15 or more modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide, wherein at least one modified nucleotide is a 2′-O-methyl nucleotide and at least one modified nucleotide is a 2′-fluoro nucleotide; and (b) an antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
  • At least 1, 2, 3, 4, 5, 6, or 7 nucleotides at position 3, 5, 7, 8, 9, 10, 11, 12, and/or 17 from the 5′ end of the first nucleotide sequence is a 2′-fluoro nucleotide.
  • the nucleotide at position 3 from the 5′ end of the first nucleotide sequence is a 2′-fluoro nucleotide.
  • the nucleotide at position 5 from the 5′ end of the first nucleotide sequence is a 2′-fluoro nucleotide.
  • the nucleotide at position 7 from the 5′ end of the first nucleotide sequence is a 2′-fluoro nucleotide.
  • 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.
  • 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.
  • At least 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides at position 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 nucleotide at position 2 from the 5′ end of the second nucleotide sequence is a 2′-fluoro nucleotide.
  • the nucleotide at position 5 from the 5′ end of the second nucleotide sequence is a 2′-fluoro nucleotide.
  • 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.
  • the nucleotides 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.
  • the nucleotides in the second nucleotide sequence are arranged in an alternating 1:3 modification pattern, and wherein 1 nucleotide is a 2′-fluoro nucleotide and 3 nucleotides are 2′-O-methyl nucleotides.
  • the alternating 1:3 modification pattern occurs 2-5 times.
  • at least two of the alternating 1:3 modification pattern occur consecutively.
  • at least two of the alternating 1:3 modification pattern occurs nonconsecutively.
  • at least 1, 2, 3, 4, or 5 alternating 1:3 modification pattern begins at nucleotide position 2, 6, 10, 14, and/or 18 from the 5′ end of the antisense strand.
  • At least one alternating 1:3 modification pattern begins at nucleotide position 2 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:3 modification pattern begins at nucleotide position 6 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:3 modification pattern begins at nucleotide position 10 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:3 modification pattern begins at nucleotide position 14 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:3 modification pattern begins at nucleotide position 18 from the 5′ end of the antisense strand.
  • the nucleotides in the second nucleotide sequence are arranged in an alternating 1:2 modification pattern, and wherein 1 nucleotide is a 2′-fluoro nucleotide and 2 nucleotides are 2′-O-methyl nucleotides.
  • the alternating 1:2 modification pattern occurs 2-5 times.
  • at least two of the alternating 1:2 modification pattern occurs consecutively.
  • at least two of the alternating 1:2 modification pattern occurs nonconsecutively.
  • at least 1, 2, 3, 4, or 5 alternating 1:2 modification pattern begins at nucleotide position 2, 5, 8, 14, and/or 17 from the 5′ end of the antisense strand.
  • At least one alternating 1:2 modification pattern begins at nucleotide position 2 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:2 modification pattern begins at nucleotide position 5 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:2 modification pattern begins at nucleotide position 8 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:2 modification pattern begins at nucleotide position 14 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:2 modification pattern begins at nucleotide position 17 from the 5′ end of the antisense strand.
  • siNA short interfering nucleic acid
  • 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, wherein the first nucleotide sequence comprises 15 to 30 nucleotides; the bottom 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 the RNA corresponding to the target gene, wherein the second nucleotide sequence comprises
  • siNA short interfering nucleic acid
  • 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, wherein the first nucleotide sequence comprises 15 to 30 nucleotides;
  • the bottom 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 the RNA corresponding to the target gene, wherein the second nucleotide sequence comprises 15 to 30 nucleotides; each A is independently a 2′-
  • a short interfering nucleic acid (siNA) molecule comprising (a) a sense strand comprising a first nucleotide sequence consisting of 17 to 23 nucleotides, wherein 2′-fluoro nucleotides are at positions 3, 7-9, 12, and 17 from the 5′ end of the first nucleotide sequence, and wherein 2′-O-methyl nucleotides are at positions 1, 2, 4-6, 10, 11, and 13-16 from the 5′ end of the first nucleotide sequence; and (b) an antisense strand comprising a second nucleotide sequence consisting of 17 to 23 nucleotides, wherein 2′-fluoro nucleotides are at positions 2 and 14 from the 5′ end of the second nucleotide sequence, and wherein 2′-O-methyl nucleotides are at positions 1, 3-13, and 15-17 from the 5′ end of the second nucleotide sequence.
  • siNA short interfering nucleic acid
  • the first nucleotide sequence consists of 19 nucleotides. In some embodiments, 2′-O-methyl nucleotides are at positions 18 and 19 from the 5′ end of the first nucleotide sequence. In some embodiments, the second nucleotide sequence consists of 21 nucleotides. In some embodiments, 2′-O-methyl nucleotides are at positions 18-21 from the 5′ end of the second nucleotide sequence.
  • a short interfering nucleic acid (siNA) molecule comprising (a) a sense strand comprising a first nucleotide sequence consisting of 17 to 23 nucleotides, wherein 2′-fluoro nucleotides are at positions 3, 7, 8, and 17 from the 5′ end of the first nucleotide sequence, and wherein 2′-O-methyl nucleotides are at positions 1, 2, 4-6, and 9-16 from the 5′ end of the first nucleotide sequence; and (b) an antisense strand comprising a second nucleotide sequence consisting of 17 to 23 nucleotides, wherein 2′-fluoro nucleotides are at positions 2 and 14 from the 5′ end of the first nucleotide sequence; and wherein 2′-O-methyl nucleotides are at positions 1, 3-13, and 15-17 from the 5′ end of the first nucleotide sequence.
  • siNA short interfering nucleic acid
  • the first nucleotide sequence consists of 19 nucleotides. In some embodiments, 2′-O-methyl nucleotides are at positions 18 and 19 from the 5′ end of the first nucleotide sequence. In some embodiments, the second nucleotide sequence consists of 21 nucleotides. In some embodiments, 2′-O-methyl nucleotides are at positions 18-21 from the 5′ end of the second nucleotide sequence.
  • a short interfering nucleic acid (siNA) molecule comprising (a) a sense strand comprising a first nucleotide sequence consisting of 17 to 23 nucleotides, wherein 2′-fluoro nucleotides are at positions 3, 7-9, 12 and 17 from the 5′ end of the first nucleotide sequence, and wherein 2′-O-methyl nucleotides are at positions 1, 2, 4-6, 10, 11, and 13-16 from the 5′ end of the first nucleotide sequence; and (b) an antisense strand comprising a second nucleotide sequence consisting of 17 to 23 nucleotides, wherein the nucleotides in the second nucleotide sequence are arranged in an alternating 1:3 modification pattern, and wherein 1 nucleotide is a 2′-fluoro nucleotide and 3 nucleotides are 2′-O-methyl nucleotides.
  • the first nucleotide sequence consists of 19 nucleotides. In some embodiments, 2′-O-methyl nucleotides are at positions 18 and 19 from the 5′ end of the first nucleotide sequence. In some embodiments, the second nucleotide sequence consists of 21 nucleotides. In some embodiments, 2′-O-methyl nucleotides are at positions 19-21 from the 5′ end of the second nucleotide sequence. 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 pattern occur consecutively. In some embodiments, at least two of the alternating 1:3 modification pattern occurs nonconsecutively.
  • At least 1, 2, 3, 4, or 5 alternating 1:3 modification pattern begins at nucleotide position 2, 6, 10, 14, and/or 18 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:3 modification pattern begins at nucleotide position 2 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:3 modification pattern begins at nucleotide position 6 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:3 modification pattern begins at nucleotide position 10 from the 5′ end of the antisense strand.
  • At least one alternating 1:3 modification pattern begins at nucleotide position 14 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:3 modification pattern begins at nucleotide position 18 from the 5′ end of the antisense strand.
  • a short interfering nucleic acid (siNA) molecule comprising (a) a sense strand comprising a first nucleotide sequence consisting of 17 to 23 nucleotides, wherein 2′-fluoro nucleotides are at positions 5 and 7-9 from the 5′ end of the first nucleotide sequence, and wherein 2′-O-methyl nucleotides are at positions 1-4, 6, and 10-17 from the 5′ end of the first nucleotide sequence; and (b) an antisense strand comprising a second nucleotide sequence consisting of 17 to 23 nucleotides, wherein the nucleotides in the second nucleotide sequence are arranged in an alternating 1:3 modification pattern, and wherein 1 nucleotide is a 2′-fluoro nucleotide and 3 nucleotides are 2′-O-methyl nucleotides.
  • the first nucleotide sequence consists of 19 nucleotides. In some embodiments, 2′-O-methyl nucleotides are at positions 18 and 19 from the 5′ end of the first nucleotide sequence. In some embodiments, the second nucleotide sequence consists of 21 nucleotides. In some embodiments, 2′-O-methyl nucleotides are at positions 19-21 from the 5′ end of the second nucleotide sequence. 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 pattern occur consecutively. In some embodiments, at least two of the alternating 1:3 modification pattern occurs nonconsecutively.
  • At least 1, 2, 3, 4, or 5 alternating 1:3 modification pattern begins at nucleotide position 2, 6, 10, 14, and/or 18 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:3 modification pattern begins at nucleotide position 2 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:3 modification pattern begins at nucleotide position 6 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:3 modification pattern begins at nucleotide position 10 from the 5′ end of the antisense strand.
  • At least one alternating 1:3 modification pattern begins at nucleotide position 14 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:3 modification pattern begins at nucleotide position 18 from the 5′ end of the antisense strand.
  • a short interfering nucleic acid (siNA) molecule comprising (a) a sense strand comprising a first nucleotide sequence consisting of 17 to 23 nucleotides, wherein 2′-fluoro nucleotides are at positions 5 and 7-9 from the 5′ end of the first nucleotide sequence, and wherein 2′-O-methyl nucleotides are at positions 1-4, 6, and 10-17 from the 5′ end of the first nucleotide sequence; and (b) an antisense strand comprising a second nucleotide sequence consisting of 17 to 23 nucleotides, wherein the nucleotides in the second nucleotide sequence are arranged in an alternating 1:2 modification pattern, and wherein 1 nucleotide is a 2′-fluoro nucleotide and 2 nucleotides are 2′-O-methyl nucleotides.
  • the first nucleotide sequence consists of 19 nucleotides. In some embodiments, 2′-O-methyl nucleotides are at positions 18 and 19 from the 5′ end of the first nucleotide sequence. In some embodiments, the second nucleotide sequence consists of 21 nucleotides. In some embodiments, 2′-O-methyl nucleotides are at positions 18-21 from the 5′ end of the second nucleotide sequence. 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 pattern occur consecutively. In some embodiments, at least two of the alternating 1:2 modification pattern occurs nonconsecutively.
  • At least 1, 2, 3, 4, or 5 alternating 1:2 modification pattern begins at nucleotide position 2, 5, 8, 14, and/or 17 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:2 modification pattern begins at nucleotide position 2 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:2 modification pattern begins at nucleotide position 5 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:2 modification pattern begins at nucleotide position 8 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:2 modification pattern begins at nucleotide position 14 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:2 modification pattern begins at nucleotide position 17 from the 5′ end of the antisense strand.
  • a short interfering nucleic acid (siNA) molecule comprising (a) a sense strand comprising a first nucleotide sequence consisting of 17 to 23 nucleotides, wherein 2′-fluoro nucleotides are at positions 5 and 7-9 from the 5′ end of the first nucleotide sequence, and wherein 2′-O-methyl nucleotides are at positions 1-4, 6, and 10-17 from the 5′ end of the first nucleotide sequence; and (b) an antisense strand comprising a second nucleotide sequence consisting of 17 to 23 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 6, 14, and 16 from the 5′ end of the second nucleotide sequence, and wherein 2′-O-methyl nucleotides are at positions 1, 3-5, 7-13, 15, and 17 from the 5′ end the second nucleotide sequence.
  • siNA short interfering nucleic acid
  • the first nucleotide sequence consists of 19 nucleotides. In some embodiments, 2′-O-methyl nucleotides are at positions 18 and 19 from the 5′ end of the first nucleotide sequence. In some embodiments, the second nucleotide sequence consists of 21 nucleotides. In some embodiments, 2′-O-methyl nucleotides are at positions 18-21 from the 5′ end of the second nucleotide sequence.
  • a short interfering nucleic acid (siNA) molecule comprising: (a) a sense strand comprising a first nucleotide sequence consisting of 17 to 23 nucleotides, wherein 2′-fluoro nucleotides are at positions 5, 9-11, and 14 from the 5′ end of the first nucleotide sequence, and wherein 2′-O-methyl nucleotides are at positions 1-4, 6-8, and 12-17 from the 5′ end of the first nucleotide sequence; and (b) an antisense strand comprising a second nucleotide sequence consisting of 17 to 23 nucleotides, wherein 2′-fluoro nucleotides are at positions 2 and 14 from the 5′ end of the second nucleotide sequence, and wherein 2′-O-methyl nucleotides are at positions 1, 3-13, and 15-17 from the 5′ end the second nucleotide sequence.
  • the first nucleotide sequence consists of 21 nucleotides. In some embodiments, 2′-O-methyl nucleotides are at positions 18-21 from the 5′ end of the first nucleotide sequence. In some embodiments, the second nucleotide sequence consists of 23 nucleotides. In some embodiments, 2′-O-methyl nucleotides are at positions 18-23 from the 5′ end of the second nucleotide sequence.
  • any of the sense strands disclosed herein further comprise a TT sequence adjacent to the first nucleotide sequence.
  • any of the sense strands disclosed herein further comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more phosphorothioate internucleoside linkages.
  • at least one phosphorothioate internucleoside linkage is between the nucleotides at positions 1 and 2 from the 5′ end of the first nucleotide sequence.
  • at least one phosphorothioate internucleoside linkage is between the nucleotides at positions 2 and 3 from the 5′ end of the first nucleotide sequence.
  • any of the antisense strands disclosed herein further comprise TT sequence adjacent to the second nucleotide sequence.
  • 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.
  • at least one phosphorothioate internucleoside linkage is between the nucleotides at positions 1 and 2 from the 5′ end of the second nucleotide sequence.
  • at least one phosphorothioate internucleoside linkage is between the nucleotides at positions 2 and 3 from the 5′ end of the second nucleotide sequence.
  • At least one phosphorothioate internucleoside linkage is between the nucleotides at positions 1 and 2 from the 3′ end of the second nucleotide sequence. In some embodiments, at least one phosphorothioate internucleoside linkage is between the nucleotides at positions 2 and 3 from the 3′ end of the second nucleotide sequence.
  • the first nucleotide from the 5′ end of any of the first nucleotide sequences disclosed herein comprises a 5′ stabilizing end cap.
  • the first nucleotide from the 5′ end of any of the second nucleotide sequences disclosed herein comprise a 5′ stabilizing end cap.
  • the first nucleotide from the 5′ end of any of the first nucleotide sequences disclosed herein comprises a phosphorylation blocker.
  • the first nucleotide from the 5′ end of any of the second nucleotide sequences disclosed herein comprises a phosphorylation blocker.
  • any of the first nucleotide sequences or second nucleotide sequences disclosed herein comprise at least one modified nucleotide selected from
  • R is H or alkyl (or AmNA(N-Me)) when R is alkyl);
  • B is a nucleobase
  • siNA short-interfering nucleic acid
  • R 1 is a nucleobase
  • R 4 is —O—R 30 or —NR 31 R 32
  • R 30 is C 1 -C 8 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
  • a short interfering nucleic acid siNA
  • the siNA is any of the siNAs disclosed herein.
  • the siNA comprises any of the sense strands, first nucleotide sequences, antisense strands, or second nucleotide sequences disclosed herein.
  • the siNA comprises any of the sense strands disclosed herein.
  • the siNA comprises any of the antisense strand disclosed herein.
  • the siNA comprises a first nucleotide sequence selected from any one of SEQ ID NOs: 1-56, 103-158, and 205-260.
  • the siNA comprises a second nucleotide sequence selected from any one of SEQ ID NOs: 57-102, 159-204, and 261-306.
  • the siNA comprises a sense sequence selected from any one of SEQ ID NOs: 307-362 and 415-444.
  • the siNA comprises an antisense sequence selected from any one of SEQ ID NOs: 363-409, 445-533, and 536-539.
  • the siNA comprises a ds-siNA sequence selected from any one of ds-siNA-001 to ds-siNA-0178. In some embodiments, the siNA further comprises any of the 5′ end caps disclosed herein. In some embodiments, the siNA further comprises any of the conjugated moieties disclosed herein. In some embodiments, the siNA further comprises any of the destabilizing nucleotides disclosed herein. In some embodiments, the siNA further comprises any of the modified nucleotides disclosed herein.
  • siNA short-interfering nucleic acid
  • R 1 is a nucleobase, aryl, heteroaryl, or H
  • R 20 is hydrogen; or R 2 and R 20 together form a 3- to 7-membered carbocyclic ring substituted with —(CR 21 R 22 ) n —Z or —(C 2 -C 6 alkenylene)-Z; 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
  • the siNA comprises any of the sense strands disclosed herein. In some embodiments, the siNA comprises any of the antisense strand disclosed herein. In some embodiments, the siNA comprises a first nucleotide sequence selected from any one of SEQ ID NOs: 1-56, 103-158, and 205-260. In some embodiments, the siNA comprises a second nucleotide sequence selected from any one of SEQ ID NOs: 57-102, 159-204, and 261-306. In some embodiments, the siNA comprises a sense sequence selected from any one of SEQ ID NOs: 307-362 and 415-444.
  • the siNA comprises an antisense sequence selected from any one of SEQ ID NOs: 363-409, 445-533, and 536-539. In some embodiments, the siNA comprises a ds-siNA sequence selected from any one of ds-siNA-001 to ds-siNA-0178. In some embodiments, the siNA further comprises any of the phosphorylation blockers disclosed herein. In some embodiments, the siNA further comprises any of the conjugated moieties disclosed herein. In some embodiments, the siNA further comprises any of the destabilizing nucleotides disclosed herein. In some embodiments, the siNA further comprises any of the modified nucleotides disclosed herein.
  • siNA short-interfering nucleic acid
  • R 1 is a nucleobase, aryl, heteroaryl, or H
  • R 20 is hydrogen; or R 2 and R 20 together form a 3- to 7-membered carbocyclic ring substituted with —(CR 21 R 22 ) n —Z or —(C 2 -C 6 alkenylene)-Z; 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
  • the siNA comprises any of the sense strands disclosed herein. In some embodiments, the siNA comprises any of the antisense strand disclosed herein. In some embodiments, the siNA comprises a first nucleotide sequence selected from any one of SEQ ID NOs: 1-56, 103-158, and 205-260. In some embodiments, the siNA comprises a second nucleotide sequence selected from any one of SEQ ID NOs: 57-102, 159-204, and 261-306. In some embodiments, the siNA comprises a sense sequence selected from any one of SEQ ID NOs: 307-362 and 415-444.
  • the siNA comprises an antisense sequence selected from any one of SEQ ID NOs: 363-409, 445-533, and 536-539. In some embodiments, the siNA comprises a ds-siNA sequence selected from any one of ds-siNA-001 to ds-siNA-0178. In some embodiments, the siNA further comprises any of the phosphorylation blockers disclosed herein. In some embodiments, the siNA further comprises any of the conjugated moieties disclosed herein. In some embodiments, the siNA further comprises any of the destabilizing nucleotides disclosed herein. In some embodiments, the siNA further comprises any of the modified nucleotides disclosed herein.
  • a short-interfering nucleic acid (siNA) molecule comprising: (a) a 5′-stabilized end cap selected from the group consisting of Formula (1) to Formula (15), Formula (9X) to Formula (12X), and Formula (9Y) to Formula (12Y):
  • R 1 is a nucleobase, aryl, heteroaryl, or H; and (b) a short interfering nucleic acid (siNA).
  • the siNA comprises any of the sense strands disclosed herein.
  • the siNA comprises any of the antisense strand disclosed herein.
  • the siNA comprises a first nucleotide sequence selected from any one of SEQ ID NOs: 1-56, 103-158, and 205-260.
  • the siNA comprises a second nucleotide sequence selected from any one of SEQ ID NOs: 57-102, 159-204, and 261-306.
  • the siNA comprises a sense sequence selected from any one of SEQ ID NOs: 307-362 and 415-444. In some embodiments, the siNA comprises an antisense sequence selected from any one of SEQ ID NOs: 363-409, 445-533, and 536-539. In some embodiments, the siNA comprises a ds-siNA sequence selected from any one of ds-siNA-001 to ds-siNA-0178. In some embodiments, the siNA further comprises any of the phosphorylation blockers disclosed herein. In some embodiments, the siNA further comprises any of the conjugated moieties disclosed herein. In some embodiments, the siNA further comprises any of the destabilizing nucleotides disclosed herein. In some embodiments, the siNA further comprises any of the modified nucleotides disclosed herein.
  • a short-interfering nucleic acid (siNA) molecule comprising: (a) a 5′-stabilized end cap selected from the group consisting of Formulas (1A)-(15A), Formulas (9B)-(12B), Formulas (9AX)-(12AX), Formulas (9AY)-(12AY), Formulas (9BX)-(12BX), and Formulas (9BY)-(12BY):
  • the siNA comprises any of the sense strands disclosed herein. In some embodiments, the siNA comprises any of the antisense strand disclosed herein. In some embodiments, the siNA comprises a first nucleotide sequence selected from any one of SEQ ID NOs: 1-56, 103-158, and 205-260. In some embodiments, the siNA comprises a second nucleotide sequence selected from any one of SEQ ID NOs: 57-102, 159-204, and 261-306. In some embodiments, the siNA comprises a sense sequence selected from any one of SEQ ID NOs: 307-362 and 415-444.
  • the siNA comprises an antisense sequence selected from any one of SEQ ID NOs: 363-409, 445-533, and 536-539. In some embodiments, the siNA comprises a ds-siNA sequence selected from any one of ds-siNA-001 to ds-siNA-0178. In some embodiments, the siNA further comprises any of the phosphorylation blockers disclosed herein. In some embodiments, the siNA further comprises any of the conjugated moieties disclosed herein. In some embodiments, the siNA further comprises any of the destabilizing nucleotides disclosed herein. In some embodiments, the siNA further comprises any of the modified nucleotides disclosed herein.
  • siNA short-interfering nucleic acid
  • R 1 is a nucleobase, aryl, heteroaryl, or H
  • R 20 is hydrogen; or R 2 and R 20 together form a 3- to 7-membered carbocyclic ring substituted with —(CR 21 R 22 ) n —Z or —(C 2 -C 6 alkenylene)-Z; 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
  • the siNA comprises any of the sense strands disclosed herein. In some embodiments, the siNA comprises any of the antisense strand disclosed herein. In some embodiments, the siNA comprises a first nucleotide sequence selected from any one of SEQ ID NOs: 1-56, 103-158, and 205-260. In some embodiments, the siNA comprises a second nucleotide sequence selected from any one of SEQ ID NOs: 57-102, 159-204, and 261-306. In some embodiments, the siNA comprises a sense sequence selected from any one of SEQ ID NOs: 307-362 and 415-444.
  • the siNA comprises an antisense sequence selected from any one of SEQ ID NOs: 363-409, 445-533, and 536-539. In some embodiments, the siNA comprises a ds-siNA sequence selected from any one of ds-siNA-001 to ds-siNA-0178. In some embodiments, the siNA further comprises any of the phosphorylation blockers disclosed herein. In some embodiments, the siNA further comprises any of the conjugated moieties disclosed herein. In some embodiments, the siNA further comprises any of the destabilizing nucleotides disclosed herein. In some embodiments, the siNA further comprises any of the modified nucleotides disclosed herein.
  • siNA short-interfering nucleic acid
  • R 1 is a nucleobase, aryl, heteroaryl, or H; and (b) a short interfering nucleic acid (siNA).
  • the siNA comprises any of the sense strands disclosed herein.
  • the siNA comprises any of the antisense strand disclosed herein.
  • the siNA comprises a first nucleotide sequence selected from any one of SEQ ID NOs: 1-56, 103-158, and 205-260.
  • the siNA comprises a second nucleotide sequence selected from any one of SEQ ID NOs: 57-102, 159-204, and 261-306.
  • the siNA comprises a sense sequence selected from any one of SEQ ID NOs: 307-362 and 415-444. In some embodiments, the siNA comprises an antisense sequence selected from any one of SEQ ID NOs: 363-409, 445-533, and 536-539. In some embodiments, the siNA comprises a ds-siNA sequence selected from any one of ds-siNA-001 to ds-siNA-0178. In some embodiments, the siNA further comprises any of the phosphorylation blockers disclosed herein. In some embodiments, the siNA further comprises any of the conjugated moieties disclosed herein. In some embodiments, the siNA further comprises any of the destabilizing nucleotides disclosed herein. In some embodiments, the siNA further comprises any of the modified nucleotides disclosed herein.
  • siNA short-interfering nucleic acid
  • a 5′-stabilized end cap selected from the group consisting of Formulas (21A)-(35A), Formulas (29B)-(32B), Formulas (29AX)-(32AX), Formulas (29AY)-(32AY), Formulas (29BX)-(32BX), and Formulas (29BY)-(32BY):
  • the siNA comprises any of the sense strands disclosed herein. In some embodiments, the siNA comprises any of the antisense strand disclosed herein. In some embodiments, the siNA comprises a first nucleotide sequence selected from any one of SEQ ID NOs: 1-56, 103-158, and 205-260. In some embodiments, the siNA comprises a second nucleotide sequence selected from any one of SEQ ID NOs: 57-102, 159-204, and 261-306. In some embodiments, the siNA comprises a sense sequence selected from any one of SEQ ID NOs: 307-362 and 415-444.
  • the siNA comprises an antisense sequence selected from any one of SEQ ID NOs: 363-409, 445-533, and 536-539. In some embodiments, the siNA comprises a ds-siNA sequence selected from any one of ds-siNA-001 to ds-siNA-0178. In some embodiments, the siNA further comprises any of the phosphorylation blockers disclosed herein. In some embodiments, the siNA further comprises any of the conjugated moieties disclosed herein. In some embodiments, the siNA further comprises any of the destabilizing nucleotides disclosed herein. In some embodiments, the siNA further comprises any of the modified nucleotides disclosed herein.
  • 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 comprises a nucleotide sequence of any one SEQ ID NOs: 1-56, 103-158, and 205-260; and (b) an antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the RNA corresponding to the target gene, wherein the second nucleotide sequence comprises a nucleotide sequence of any one of SEQ ID NOs: 57-102, 159-204, and 261-306.
  • the siNA further comprises any of the 5′ end caps disclosed herein. In some embodiments, the siNA further comprises any of the phosphorylation blockers disclosed herein. In some embodiments, the siNA further comprises any of the conjugated moieties disclosed herein. In some embodiments, the siNA further comprises any of the destabilizing nucleotides disclosed herein. In some embodiments, the siNA further comprises any of the modified nucleotides disclosed herein.
  • a interfering nucleic acid (siNA) molecule comprising: (a) a sense strand comprising a nucleotide sequence of any one of SEQ ID NOs: 307-362 and 415-444; and (b) an antisense strand comprising a nucleotide sequence of any one of SEQ ID NOs: 363-409, 445-533, and 536-539.
  • any of the siNA disclosed herein further comprise a phosphorylation blocker.
  • the phosphorylation blocker has the structure of Formula (IV):
  • R 1 is a nucleobase
  • R 4 is —O—R 30 or —NR 31 R 32
  • R 30 is C 1 -C 8 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.
  • R 4 is —OCH 3 or —N(CH 2 CH 2 ) 2 O.
  • the phosphorylation blocker is attached to the 5′ end of the sense strand.
  • the phosphorylation blocker is attached to the 5′ end of the sense strand via one or more linkers independently selected from a phosphodiester linker, phosphorothioate linker, and phosphorodithioate linker.
  • the phosphorylation blocker is attached to the 3′ end of the sense strand.
  • the phosphorylation blocker is attached to the 3′ end of the sense strand via one or more linkers independently selected from a phosphodiester linker, phosphorothioate linker, and phosphorodithioate linker.
  • the phosphorylation blocker is attached to the 5′ end of the antisense strand. In some embodiments, the phosphorylation blocker is attached to the 5′ end of the antisense strand via one or more linkers independently selected from a phosphodiester linker, phosphorothioate linker, and phosphorodithioate linker. In some embodiments, the phosphorylation blocker is attached to the 3′ end of the antisense strand. In some embodiments, the phosphorylation blocker is attached to the 3′ end of the antisense strand via one or more linkers independently selected from a phosphodiester linker, phosphorothioate linker, and phosphorodithioate linker.
  • any of the siNAs disclosed herein further comprise a conjugated moiety.
  • the conjugated moiety comprises a galactosamine.
  • the galactosamine is N-acetylgalactosamine (GalNAc) of Formula (VII):
  • the galactosamine is N-acetylgalactosamine (GaNAc) of Formula (VI):
  • 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; either L is a linker or L and Y in combination are a linker; and A is H, OH, a third protecting group, an activated group, or an oligonucleotide. In some embodiments, wherein A is an oligonucleotide.
  • A is 1-2 oligonucleotides.
  • the oligonucleotide is dTdT.
  • the galactosamine is attached to the 3′ end of the sense strand.
  • the galactosamine is attached to the 3′ end of the sense strand via one or more linkers independently selected from a phosphodiester linker, phosphorothioate linker, or phosphorodithioate linker.
  • the galactosamine is attached to the 5′ end of the sense strand.
  • the galactosamine is attached to the 5′ end of the sense strand via one or more linkers independently selected from a phosphodiester linker, phosphorothioate linker, or phosphorodithioate linker. In some embodiments, the galactosamine is attached to the 3′ end of the antisense strand. In some embodiments, the galactosamine is attached to the 3′ end of the atnisense strand via one or more linkers independently selected from a phosphodiester linker, phosphorothioate linker, or phosphorodithioate linker. In some embodiments, the galactosamine is attached to the 5′ end of the antisense strand.
  • the galactosamine is attached to the 5′ end of the atnisense strand via one or more linkers independently selected from a phosphodiester linker, phosphorothioate linker, or phosphorodithioate linker.
  • any of the siNAs disclosed herein further comprise a 5′-stabilized end cap.
  • the 5′-stabilized end cap is a 5′ vinyl phosphonate or deuterated 5′ vinyl phosphonate.
  • the 5′-stabilized end cap has the structure of Formula (Ia):
  • R 1 is a nucleobase, aryl, heteroaryl, or H
  • R 20 is hydrogen; or R 2 and R 20 together form a 3- to 7-membered carbocyclic ring substituted with —(CR 21 R 22 ) n —Z or —(C 2 -C 6 alkenylene)-Z; 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
  • R 1 is a nucleobase, aryl, heteroaryl, or H
  • R 20 is hydrogen; or R 2 and R 20 together form a 3- to 7-membered carbocyclic ring substituted with —(CR 21 R 22 ) n —Z or —(C 2 -C 6 alkenylene)-Z; 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
  • R 1 is a nucleobase, aryl, heteroaryl, or H
  • R 20 is hydrogen; or R 2 and R 20 together form a 3- to 7-membered carbocyclic ring substituted with —(CR 21 R 22 ) n —Z or —(C 2 -C 6 alkenylene)-Z; 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
  • R 1 is an aryl.
  • the aryl is a phenyl.
  • the 5′-stabilized end cap is selected from the group consisting of Formula (1) to Formula (15), Formula (9X) to Formula (12X), and Formula (9Y) to Formula (12Y):
  • the 5′-stabilized end cap is selected from the group consisting of Formulas (1A)-(15A), Formulas (9B)-(12B), Formulas (9AX)-(12AX), Formulas (9AY)-(12AY), Formulas (9BX)-(12BX), and Formulas (9BY)-(12BY):
  • the 5′-stabilized end cap is selected from the group consisting of Formula (21) to Formula (35):
  • R 1 is a nucleobase, aryl, heteroaryl, or H.
  • the 5′-stabilized end cap is selected from the group consisting of Formulas (21A)-(35A), Formulas (29B)-(32B), Formulas (29AX)-(32AX), Formulas (29AY)-(32AY), Formulas (29BX)-(32BX), and Formulas (29BY)-(32BY):
  • 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 or more linkers independently selected from a phosphodiester linker, phosphorothioate linker, or phosphorodithioate linker. In some embodiments, the 5′-stabilized end cap is attached to the 5′ end of the sense strand. In some embodiments, the 5′-stabilized end cap is attached to the 5′ end of the sense strand via one or more linkers independently selected from a phosphodiester linker, phosphorothioate linker, or phosphorodithioate linker.
  • any of the siNAs, sense strands, first nucleotide sequences, antisense strands, or second nucleotide sequences disclosed herein further comprise at least one thermally destabilizing nucleotides.
  • any of the antisense strands disclosed herein further comprise at least one thermally destabilizing nucleotide selected from:
  • any of the sense strands disclosed herein comprise at least one thermally destabilizing nucleotide selected from:
  • any of the first nucleotide sequences disclosed herein further comprise at least one thermally destabilizing nucleotide selected from:
  • any of the second nucleotide sequences disclosed herein further comprise at least one thermally destabilizing nucleotide selected from:
  • any of the modified nucleotides disclosed herein is a thermally destabilizing nucleotide.
  • any of the siNAs disclosed herein specifically downregulate or reduce expression of a target gene.
  • the target gene is a viral gene.
  • the viral gene is from a DNA virus.
  • the DNA virus is a double-stranded DNA (dsDNA) virus.
  • the dsDNA virus is a hepadnavirus.
  • the hepadnavirus is a hepatitis B virus (HBV).
  • HBV is selected from HBV genotypes A-J.
  • the target gene is selected from the S gene or X gene of the HBV.
  • the second nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to 15 to 30 nucleotides within positions 200-720 or 1100-1700 of SEQ ID NO: 410. In some embodiments, the second nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to 15 to 30 nucleotides within positions 200-280, 300-445, 460-510, 650-720, 1170-1220, 1250-1300, or 1550-1630 of SEQ ID NO: 410.
  • the second nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to 15 to 30 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: 410.
  • the second nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to 15 to 30 nucleotides starting at position 203, 206, 254, 305, 375, 409, 412, 415, 416, 419, 462, 466, 467, 674, 676, 1182, 1262, 1263, 1268, 1526, 1577, 1578, 1580, 1581, 1583, or 1584 of SEQ ID NO: 410.
  • the first nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to 15 to 30 nucleotides within positions 200-720 or 1100-1700 of SEQ ID NO: 410. In some embodiments, the first nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to 15 to 30 nucleotides within positions 200-280, 300-445, 460-510, 650-720, 1170-1220, 1250-1300, or 1550-1630 of SEQ ID NO: 410.
  • the first nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to 15 to 30 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: 410.
  • the first nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to 15 to 30 nucleotides starting at position 203, 206, 254, 305, 375, 409, 412, 415, 416, 419, 462, 466, 467, 674, 676, 1182, 1262, 1263, 1268, 1526, 1577, 1578, 1580, 1581, 1583, or 1584 of SEQ ID NO: 410.
  • the first nucleotide sequence comprises a nucleotide sequence of any one SEQ ID NOs: 1-56, 103-158, and 205-260.
  • the second nucleotide sequence comprises a nucleotide sequence of any one of SEQ ID NOs: 57-102, 159-204, and 261-306.
  • the sense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 307-362 and 415-444.
  • the antisense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 363-409, 445-533, and 536-539.
  • At least one end of the siNA is a blunt end.
  • At least one end of the siNA comprises an overhang, wherein the overhang comprises at least one nucleotide.
  • both ends of the siNA comprise an overhang, wherein the overhang comprises at least one nucleotide.
  • the siNA is selected from ds-siNA-001 to ds-siNA-0178.
  • At least one 2′-fluoro nucleotide or 2′-O-methyl nucleotide is a 2′-fluoro or 2-O-methyl nucleotide mimic of Formula (V):
  • R 1 is independently a nucleobase, aryl, heteroaryl, or H
  • Q 1 and Q 2 are independently S or O
  • R 5 is independently-OCD 3 , —F, or —OCH 3
  • R 6 and R 7 are independently H, D, or CD3.
  • the 2′-fluoro or 2′-O-methyl nucleotide mimic is a nucleotide mimic of Formula (16)-Formula (20):
  • R 1 is a nucleobase and R 2 is independently F or —OCH 3 .
  • compositions comprising any of the siNAs disclosed herein.
  • the siNA targets an S gene of HBV.
  • the siNA specifically downregulates or reduces expression of the S gene of HBV.
  • the siNA targets an X gene of HBV.
  • the siNA specifically downregulates or reduces expression of the X gene of HBV.
  • the siNA comprises a first nucleotide sequence.
  • the first nucleotide sequence comprises a nucleotide sequence of any one SEQ ID NOs: 1-56, 103-158, and 205-260.
  • the siNA comprises a second nucleotide sequence.
  • the second nucleotide sequence comprises a nucleotide sequence of any one of SEQ ID NOs: 57-102, 159-204, and 261-306.
  • the siNA comprises a sense strand.
  • the sense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 307-362 and 415-444.
  • the siNA comprises an antisense strand.
  • the antisense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 363-409, 445-533, and 536-539.
  • the siNA further comprises any of the 5′ end caps disclosed herein.
  • the siNA further comprises any of the phosphorylation blockers disclosed herein. In some embodiments, the siNA further comprises any of the conjugated moieties disclosed herein. In some embodiments, the siNA further comprises any of the destabilized nucleotides disclosed herein. In some embodiments, the siNA further comprises any of the modified nucleotides disclosed herein.
  • compositions comprising 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of any of the siNAs disclosed herein.
  • at least 1, 2, 3, 4, 5, or more siNAs target an S gene of HBV.
  • at least 1, 2, 3, 4, 5, or more siNAs specifically downregulate or reduce expression of the S gene of HBV.
  • at least 1, 2, 3, 4, 5, or more siNAs target an X gene of HBV.
  • at least 1, 2, 3, 4, 5, or more siNAs specifically downregulate or reduce expression of the X gene of HBV.
  • the siNA comprises a first nucleotide sequence.
  • the first nucleotide sequence comprises a nucleotide sequence of any one SEQ ID NOs: 1-56, 103-158, and 205-260.
  • the siNA comprises a second nucleotide sequence.
  • the second nucleotide sequence comprises a nucleotide sequence of any one of SEQ ID NOs: 57-102, 159-204, and 261-306.
  • the siNA comprises a sense strand.
  • the sense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 307-362 and 415-444.
  • the siNA comprises an antisense strand.
  • the antisense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 363-409, 445-533, and 536-539.
  • the siNA further comprises any of the 5′ end caps disclosed herein.
  • the siNA further comprises any of the phosphorylation blockers disclosed herein.
  • the siNA further comprises any of the conjugated moieties disclosed herein.
  • the siNA further comprises any of the destabilized nucleotides disclosed herein.
  • the siNA further comprises any of the modified nucleotides disclosed herein.
  • any of the compositions disclosed herein further comprise an additional HBV treatment agent.
  • the additional HBV treatment agent is selected from a nucleotide analog, nucleoside analog, a capsid assembly modulator (CAM), a recombinant interferon, an entry inhibitor, a small molecule immunomodulator and oligonucleotide therapy.
  • the oligonucleotide therapy is an additional siNA.
  • the additional siNA is selected from any of ds-siNA-001 to ds-siNA-0178.
  • the oligonucleotide therapy is an antisense oligonucleotide (ASO), NAPs, or STOPs.
  • the ASO is ASO 1 or ASO 2. In some embodiments, the ASO specifically targets the S gene of HBV. In some embodiments, the ASO specifically targets the X gene of HBV. In some embodiments, the additional HBV treatment agent is selected from HBV STOPS' ALG-010133, HBV CAM ALG-000184, ASO 1, 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
  • any of the compositions disclosed herein further comprise a liver disease treatment agent.
  • the liver disease treatment agent is selected from a peroxisome proliferator-activator receptor (PPAR) agonist, farnesoid X receptor (FXR) agonist, lipid-altering agent, and incretin-based therapy.
  • PPAR peroxisome proliferator-activator receptor
  • FXR farnesoid X receptor
  • the PPAR agonist is selected from a PPAR ⁇ agonist, dual PPAR ⁇ / ⁇ agonist, PPAR ⁇ agonist, and dual PPAR ⁇ / ⁇ agonist.
  • the dual PPAR ⁇ agonist is a fibrate.
  • the PPAR ⁇ / ⁇ agonist is elafibranor.
  • the PPAR ⁇ agonist is a thiazolidinedione (TZD). In some embodiments, TZD is pioglitazone. In some embodiments, the dual PPAR ⁇ / ⁇ agonist is saroglitazar. In some embodiments, the FXR agonist is obeticholic acis (OCA). In some embodiments, the lipid-altering agent is aramchol. In some embodiments, the incretin-based therapy is a glucagon-like peptide 1 (GLP-1) receptor agonist or 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 vildapliptin.
  • the siNA comprises a first nucleotide sequence.
  • the first nucleotide sequence comprises a nucleotide sequence of any one SEQ ID NOs: 1-56, 103-158, and 205-260.
  • the siNA comprises a second nucleotide sequence.
  • the second nucleotide sequence comprises a nucleotide sequence of any one of SEQ ID NOs: 57-102, 159-204, and 261-306.
  • the siNA comprises a sense strand.
  • the sense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 307-362 and 415-444.
  • the siNA comprises an antisense strand.
  • the antisense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 363-409, 445-533, and 536-539.
  • the siNA further comprises any of the 5′ end caps disclosed herein.
  • the siNA further comprises any of the phosphorylation blockers disclosed herein.
  • the siNA further comprises any of the conjugated moieties disclosed herein.
  • the siNA further comprises any of the destabilized nucleotides disclosed herein.
  • the siNA further comprises any of the modified nucleotides disclosed herein.
  • the composition comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of any of the siNAs disclosed herein.
  • the siNA comprises a first nucleotide sequence.
  • the first nucleotide sequence comprises a nucleotide sequence of any one SEQ ID NOs: 1-56, 103-158, and 205-260.
  • the siNA comprises a second nucleotide sequence.
  • the second nucleotide sequence comprises a nucleotide sequence of any one of SEQ ID NOs: 57-102, 159-204, and 261-306.
  • the siNA comprises a sense strand.
  • the sense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 307-362 and 415-444.
  • the siNA comprises an antisense strand.
  • the antisense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 363-409, 445-533, and 536-539.
  • the siNA further comprises any of the 5′ end caps disclosed herein.
  • the siNA further comprises any of the phosphorylation blockers disclosed herein. In some embodiments, the siNA further comprises any of the conjugated moieties disclosed herein. In some embodiments, the siNA further comprises any of the destabilized nucleotides disclosed herein. In some embodiments, the siNA further comprises any of the modified nucleotides disclosed herein. In some embodiments, the composition further comprises any of the additional HBV treatment agents disclosed herein.
  • 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.
  • the hepadnavirus is a hepatitis B virus (HBV).
  • HBV hepatitis B virus
  • the HBV is selected from HBV genotypes A-J.
  • the method further comprises administering an additional HBV treatment agent.
  • the siNA or the composition and the additional HBV treatment agent are administered concurrently.
  • the siNA or the composition and the additional HBV treatment agent are administered sequentially.
  • the siNA or the composition is administered prior to administering the additional HBV treatment agent.
  • the siNA or the composition is administered after administering the additional HBV treatment agent.
  • the additional HBV treatment agent is selected from a nucleotide analog, nucleoside analog, a capsid assembly modulator (CAM), a recombinant interferon, an entry inhibitor, a small molecule immunomodulator and oligonucleotide therapy.
  • the oligonucleotide therapy is an additional siNA.
  • the additional siNA is selected from any of ds-siNA-001 to ds-siNA-0178.
  • the oligonucleotide therapy is an antisense oligonucleotide (ASO), NAPs, or STOPs. In some embodiments, the ASO is ASO 1 or ASO 2.
  • the additional HBV treatment agent is selected from HBV STOPSTM ALG-010133, HBV CAM ALG-000184, ASO 1, 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 A
  • the disease is a liver disease.
  • the liver disease is a nonalcoholic fatty liver disease (NAFLD) or hepatocellular carcinoma (HCC).
  • NAFLD nonalcoholic steatohepatitis (NASH).
  • the method further comprises administering to the subject a liver disease treatment agent.
  • the liver disease treatment agent is selected from a peroxisome proliferator-activator receptor (PPAR) agonist, farnesoid X receptor (FXR) agonist, lipid-altering agent, and incretin-based therapy.
  • PPAR peroxisome proliferator-activator receptor
  • FXR farnesoid X receptor
  • the PPAR agonist is selected from a PPAR ⁇ agonist, dual PPAR ⁇ / ⁇ agonist, PPAR ⁇ agonist, and dual PPAR ⁇ / ⁇ agonist.
  • the dual PPAR ⁇ agonist is a fibrate.
  • the PPAR ⁇ / ⁇ agonist is elafibranor.
  • the PPAR ⁇ agonist is a thiazolidinedione (TZD).
  • TZD is pioglitazone.
  • the dual PPAR ⁇ / ⁇ agonist is saroglitazar.
  • the FXR agonist is obeticholic acis (OCA).
  • the lipid-altering agent is aramchol.
  • the incretin-based therapy is a glucagon-like peptide 1 (GLP-1) receptor agonist or dipeptidyl peptidase 4 (DPP-4) inhibitor.
  • GLP-1 receptor agonist is exenatide or liraglutide.
  • DPP-4 inhibitor is sitagliptin or vildapliptin.
  • the siNA or composition and the liver disease treatment agent are administered concurrently.
  • the siNA or composition and the liver disease treatment agent are administered sequentially.
  • the siNA or composition is administered prior to administering the liver disease treatment agent.
  • the siNA or composition is administered after administering the liver disease treatment agent.
  • the siNA or the composition is administered at a dose of at least 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg 14 mg/kg, or 15 mg/kg.
  • the siNA or the composition is administered at a dose of between 0.5 mg/kg to 50 mg/kg, 0.5 mg/kg to 40 mg/kg 0.5 mg/kg to 30 mg/kg, 1 mg/kg to 50 mg/kg, 1 mg/kg to 40 mg/kg, 1 mg/kg to 30 mg/kg, 1 mg/kg to 20 mg/kg, 3 mg/kg to 50 mg/kg, 3 mg/kg to 40 mg/kg, 3 mg/kg to 30 mg/kg, 3 mg/kg to 20 mg/kg, 3 mg/kg to 15 mg/kg, 3 mg/kg to 10 mg/kg, 4 mg/kg to 50 mg/kg, 4 mg/kg to 40 mg/kg, 4 mg/kg to 30 mg/kg, 4 mg/kg to 20 mg/kg, 4 mg/kg to 15 mg/kg, 4 mg/kg to 10 mg/kg, 5 mg/kg to 50 mg/kg, 5 mg/kg to 40 mg/kg, 5 mg/kg to 30 mg/kg, 5 mg/kg to 20 mg/kg, 5 mg/kg to 30 mg/
  • the siNA or the composition is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, the siNA or the composition is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times a day, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times a week, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times a month. In some embodiments, the siNA or the composition are 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.
  • the siNA or the composition is administered for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 51, 52, 53, 54, or 55 weeks.
  • the siNA or the composition is administered at a single dose of 5 mg/kg. In some embodiments, the siNA or the composition is administered at a single dose of 10 mg/kg. In some embodiments, the siNA or the composition is administered at three doses of 10 mg/kg once a week. In some embodiments, the siNA or the composition is administered at three doses of 10 mg/kg once every three days. In some embodiments, the siNA or the composition is administered at five doses of 10 mg/kg once every three days.
  • the siNA or the composition is administered at six doses of 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.
  • the first dose and second dose are administered at least 3 days apart.
  • the second dose and third dose are administered at least 4 days apart.
  • the third dose and fourth dose, fourth dose and fifth dose, or fifth dose and sixth dose are administered at least 7 days apart.
  • any of the siNAs or the compositions disclosed herein are formulated as a particle or viral vector.
  • the siNA or the composition are administered in a particle or viral vector.
  • the viral vector is a vector of adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpes simplex virus, lentivirus, measles virus, picornavirus, poxvirus, retrovirus, or rhabdovirus.
  • the viral vector is a recombinant viral vector.
  • 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.
  • the siNA or the composition is administered systemically. In some embodiments, the siNA or the composition is administered locally. In some embodiments, the siNA or the composition is administered intravenously, subcutaneously, or intramuscularly.
  • any of the siRNAs or compositions disclosed herein are used in the manufacture of a medicament for treating a disease.
  • the disease is a viral disease.
  • the viral disease is caused by a DNA virus.
  • the DNA virus is a double stranded DNA (dsDNA virus).
  • the dsDNA virus is a hepadnavirus.
  • the hepadnavirus is a hepatitis B virus (HBV).
  • HBV is selected from HBV genotypes A-J.
  • an additional HBV treatment agent is further used in the manufacture of the medicament.
  • the additional HBV treatment agent is selected from a nucleotide analog, nucleoside analog, a capsid assembly modulator (CAM), a recombinant interferon, an entry inhibitor, a small molecule immunomodulator and oligonucleotide therapy.
  • the oligonucleotide therapy is an additional siNA.
  • the additional siNA is selected from any of ds-siNA-001 to ds-siNA-0178.
  • the oligonucleotide therapy is an antisense oligonucleotide (ASO), NAPs, or STOPs. In some embodiments, the ASO is ASO 1 or ASO 2.
  • the additional HBV treatment agent is selected from HBV STOPSTM ALG-010133, HBV CAM ALG-000184, ASO 1, 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 A
  • any of the siRNAs or compositions disclosed herein are used in the manufacture of a medicament for treating a disease.
  • the disease is a liver disease.
  • the liver disease is a nonalcoholic fatty liver disease (NAFLD) or hepatocellular carcinoma (HCC).
  • the NAFLD is nonalcoholic steatohepatitis (NASH).
  • the siNA comprises a first nucleotide sequence.
  • the first nucleotide sequence comprises a nucleotide sequence of any one SEQ ID NOs: 1-56, 103-158, and 205-260.
  • the siNA comprises a second nucleotide sequence.
  • the second nucleotide sequence comprises a nucleotide sequence of any one of SEQ ID NOs: 57-102, 159-204, and 261-306.
  • the siNA comprises a sense strand.
  • the sense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 307-362 and 415-444.
  • the siNA comprises an antisense strand.
  • the antisense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 363-409, 445-533, and 536-539.
  • the siNA further comprises any of the 5′ end caps disclosed herein.
  • the siNA further comprises any of the phosphorylation blockers disclosed herein. In some embodiments, the siNA further comprises any of the conjugated moieties disclosed herein. In some embodiments, the siNA further comprises any of the destabilized nucleotides disclosed herein. In some embodiments, the siNA further comprises any of the modified nucleotides disclosed herein.
  • a liver disease treatment agent is further used in the manufacture of the medicament. In some embodiments, the liver disease treatment agent is selected from a peroxisome proliferator-activator receptor (PPAR) agonist, farnesoid X receptor (FXR) agonist, lipid-altering agent, and incretin-based therapy.
  • PPAR peroxisome proliferator-activator receptor
  • FXR farnesoid X receptor
  • the PPAR agonist is selected from a PPAR ⁇ agonist, dual PPAR ⁇ / ⁇ agonist, PPAR ⁇ agonist, and dual PPAR ⁇ / ⁇ agonist.
  • the dual PPAR ⁇ agonist is a fibrate.
  • the PPAR ⁇ / ⁇ agonist is elafibranor.
  • the PPAR ⁇ agonist is a thiazolidinedione (TZD).
  • TZD is pioglitazone.
  • the dual PPAR ⁇ / ⁇ agonist is saroglitazar.
  • the FXR agonist is obeticholic acis (OCA).
  • the lipid-altering agent is aramchol.
  • the incretin-based therapy is a glucagon-like peptide 1 (GLP-1) receptor agonist or dipeptidyl peptidase 4 (DPP-4) inhibitor.
  • GLP-1 receptor agonist is exenatide or liraglutide.
  • DPP-4 inhibitor is sitagliptin or vildapliptin.
  • any of the siNAs disclosed herein is used as a medicament.
  • the siNA comprises a first nucleotide sequence.
  • the first nucleotide sequence comprises a nucleotide sequence of any one SEQ ID NOs: 1-56, 103-158, and 205-260.
  • the siNA comprises a second nucleotide sequence.
  • the second nucleotide sequence comprises a nucleotide sequence of any one of SEQ ID NOs: 57-102, 159-204, and 261-306.
  • the siNA comprises a sense strand.
  • the sense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 307-362 and 415-444.
  • the siNA comprises an antisense strand.
  • the antisense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 363-409, 445-533, and 536-539.
  • the siNA further comprises any of the 5′ end caps disclosed herein.
  • the siNA further comprises any of the phosphorylation blockers disclosed herein.
  • the siNA further comprises any of the conjugated moieties disclosed herein.
  • the siNA further comprises any of the destabilized nucleotides disclosed herein.
  • the siNA further comprises any of the modified nucleotides disclosed herein.
  • any of the compositions disclosed herein are used as a medicament.
  • the composition comprises any of the siNAs disclosed herein.
  • the siNA comprises a first nucleotide sequence.
  • the first nucleotide sequence comprises a nucleotide sequence of any one SEQ ID NOs: 1-56, 103-158, and 205-260.
  • the siNA comprises a second nucleotide sequence.
  • the second nucleotide sequence comprises a nucleotide sequence of any one of SEQ ID NOs: 57-102, 159-204, and 261-306.
  • the siNA comprises a sense strand.
  • the sense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 307-362 and 415-444.
  • the siNA comprises an antisense strand.
  • the antisense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 363-409, 445-533, and 536-539.
  • the siNA further comprises any of the 5′ end caps disclosed herein.
  • the siNA further comprises any of the phosphorylation blockers disclosed herein.
  • the siNA further comprises any of the conjugated moieties disclosed herein.
  • the siNA further comprises any of the destabilized nucleotides disclosed herein.
  • the siNA further comprises any of the modified nucleotides disclosed herein.
  • any of the siNAs disclosed herein are used in the treatment of a disease.
  • the siNA comprises a first nucleotide sequence.
  • the first nucleotide sequence comprises a nucleotide sequence of any one SEQ ID NOs: 1-56, 103-158, and 205-260.
  • the siNA comprises a second nucleotide sequence.
  • the second nucleotide sequence comprises a nucleotide sequence of any one of SEQ ID NOs: 57-102, 159-204, and 261-306.
  • the siNA comprises a sense strand.
  • the sense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 307-362 and 415-444.
  • the siNA comprises an antisense strand.
  • the antisense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 363-409, 445-533, and 536-539.
  • the siNA further comprises any of the 5′ end caps disclosed herein.
  • the siNA further comprises any of the phosphorylation blockers disclosed herein.
  • the siNA further comprises any of the conjugated moieties disclosed herein.
  • the siNA further comprises any of the destabilized nucleotides disclosed herein.
  • the siNA further comprises any of the modified nucleotides disclosed herein.
  • the disease is a viral disease.
  • the viral disease is caused by a DNA virus.
  • the DNA virus is a double stranded DNA (dsDNA virus).
  • the dsDNA virus is a hepadnavirus.
  • the hepadnavirus is a hepatitis B virus (HBV).
  • HBV is selected from HBV genotypes A-J.
  • the disease is a liver disease.
  • the liver disease is a nonalcoholic fatty liver disease (NAFLD) or hepatocellular carcinoma (HCC).
  • the NAFLD is nonalcoholic steatohepatitis (NASH).
  • any of the compositions disclosed herein are used in the treatment of a disease.
  • the composition comprises any of the siNAs disclosed herein.
  • the siNA comprises a first nucleotide sequence.
  • the first nucleotide sequence comprises a nucleotide sequence of any one SEQ ID NOs: 1-56, 103-158, and 205-260.
  • the siNA comprises a second nucleotide sequence.
  • the second nucleotide sequence comprises a nucleotide sequence of any one of SEQ ID NOs: 57-102, 159-204, and 261-306.
  • the siNA comprises a sense strand.
  • the sense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 307-362 and 415-444.
  • the siNA comprises an antisense strand.
  • the antisense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 363-409, 445-533, and 536-539.
  • the siNA further comprises any of the 5′ end caps disclosed herein.
  • the siNA further comprises any of the phosphorylation blockers disclosed herein.
  • the siNA further comprises any of the conjugated moieties disclosed herein.
  • the siNA further comprises any of the destabilized nucleotides disclosed herein.
  • the siNA further comprises any of the modified nucleotides disclosed herein.
  • the disease is a viral disease.
  • the viral disease is caused by a DNA virus.
  • the DNA virus is a double stranded DNA (dsDNA virus).
  • the dsDNA virus is a hepadnavirus.
  • the hepadnavirus is a hepatitis B virus (HBV).
  • HBV is selected from HBV genotypes A-J.
  • the disease is a liver disease.
  • the liver disease is a nonalcoholic fatty liver disease (NAFLD) or hepatocellular carcinoma (HCC).
  • the NAFLD is nonalcoholic steatohepatitis (NASH).
  • FIG. 1 illustrates an exemplary siNA molecule.
  • FIG. 2 illustrates an exemplary siNA molecule.
  • FIGS. 3A-3G illustrate exemplary double-stranded siNA molecules.
  • FIG. 4 shows a graph of the change in serum HBsAg from AAV-HBV mice treated with ds-siNA-0160, ds-siNA-0165, ds-siNA-0163, or ds-siNA-0166.
  • FIG. 5A shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01) or ds-siNA-0160 (G03).
  • FIG. 5B shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01) or ds-siNA-0160 (G15).
  • FIG. 5C shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01) or ds-siNA-0160 (G03).
  • FIG. 5D shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01), ds-siNA-0160 (G03), or ds-siNA-0109 (G09).
  • FIGS. 5E-5F show a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01) or ds-siNA-0169 (G18).
  • FIG. 5G shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01) or ds-siNA-0169 (G04).
  • FIG. 5H shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01) or ds-siNA-0169 (G04).
  • FIG. 5I shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01), ds-siNA-0169 (G04), or ds-siNA-0147 (G08).
  • FIG. 5J shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01), ds-siNA-0166 (G06), or ds-siNA-0153 (G14).
  • FIG. 5K shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01), ds-siNA-0163 (G05), or ds-siNA-0119 (G13).
  • FIG. 6A shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01), ds-siNA-0160 (G15), or ds-siNA-080 (G14).
  • FIG. 6B shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01), ds-siNA-0169 (G16), or ds-siNA-081 (G13).
  • FIG. 7A shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01), ds-siNA-0165 (G18), or ds-siNA-0127 (G17).
  • FIG. 7B shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01), ds-siNA-0168 (G20), or ds-siNA-0150 (G19).
  • FIG. 8A shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01), ds-siNA-0160 (G06), ASO 1 (G18), or a combination of ds-siNA-0160 and ASO 1 (G20).
  • FIG. 8B shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01), ds-siNA-0160 (G06), ASO 1 (G18), or a combination of ds-siNA-0160 and ASO 1 (G20).
  • FIG. 8C shows a graph of a synergy analysis of a combination therapy with unconjugated forms of ds-siNA-0164 and ASO 2 (e.g., ds-siNA-0160 and ASO 1 without GalNac).
  • FIG. 9 shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01), ds-siNA-0166 (G03), ds-siNA-0155 (G08), or ds-siNA-0157.
  • FIG. 10 shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01), ds-siNA-0165 (G10), ds-siNA-0160 (G06), or a combination therapy with ds-siNA-0160 and ds-siNA-0165 (G14).
  • FIG. 11 shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01), ds-siNA-0165 (G05), or ds-siNA-0144 (G11).
  • FIG. 12 shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01), ds-siNA-0163 (G04), ds-siNA-0122 (G09), or ds-siNA-0123 (G13).
  • FIG. 13 shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G 15) or ds-siNA-0147 (G 19).
  • FIG. 14 shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G 15, square), ds-siNA-0109 (G 21, circle), or ds-siNA-0172 (G 27, diamond).
  • FIG. 15 shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G 01, circle), ds-siNA-0109 (G 07, square), ds-siNA-0119 (G 11, triangle), or ds-siNA-0153 (G 13, diamond).
  • FIG. 16 shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G 01, circle), ASO 1 (G 20, square), ds-siNA-0147 (G 24, diamond), or a combination of ASO 1 and ds-siNA-0147 (G 25, triangle).
  • FIG. 17 shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G 01, circle), ASO 1 (G 20, square), ds-siNA-0109 (G 26, diamond), or a combination of ASO 1 and ds-siNA-0109 (G 27, triangle).
  • siNA molecules comprising modified nucleotides.
  • 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 a phosphorylation blocker.
  • the siNA molecules described herein may comprise a 5′-stabilized end cap.
  • 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.
  • short interfering nucleic acid (siNA) molecules comprising (a) a phosphorylation blocker; and (b) a short interfering nucleic acid (siNA).
  • the siNA may comprise at least 5 nucleotides.
  • the nucleotides may be modified nucleotides, non-modified nucleotides, or any combination thereof.
  • the nucleotides may be ribonucleotides, deoxyribonucleotides, or any combination thereof.
  • the siNA may be single-stranded. Alternatively, the siNA is double-stranded.
  • the double-stranded siNA may comprise one or more blunt ends.
  • the double-stranded siNA may comprise one or more overhangs.
  • the double-stranded siNA may comprise a blunt end and an overhang.
  • short interfering nucleic acid (siNA) molecules comprising (a) a conjugated moiety; and (b) a short interfering nucleic acid (siNA).
  • the siNA may comprise at least 5 nucleotides.
  • the nucleotides may be modified nucleotides, non-modified nucleotides, or any combination thereof.
  • the nucleotides may be ribonucleotides, deoxyribonucleotides, or any combination thereof.
  • the siNA may be single-stranded. Alternatively, the siNA is double-stranded.
  • the double-stranded siNA may comprise one or more blunt ends.
  • the double-stranded siNA may comprise one or more overhangs.
  • the double-stranded siNA may comprise a blunt end and an overhang.
  • short interfering nucleic acid (siNA) molecules comprising (a) a 5′-stabilized end cap; and (b) a short interfering nucleic acid (siNA).
  • the siNA may comprise at least 5 nucleotides.
  • the nucleotides may be modified nucleotides, non-modified nucleotides, or any combination thereof.
  • the nucleotides may be ribonucleotides, deoxyribonucleotides, or any combination thereof.
  • the siNA may be single-stranded. Alternatively, the siNA is double-stranded.
  • the double-stranded siNA may comprise one or more blunt ends.
  • the double-stranded siNA may comprise one or more overhangs.
  • the double-stranded siNA may comprise a blunt end and an overhang.
  • short interfering nucleic acid (siNA) molecules comprising (a) at least one phosphorylation blocker, conjugated moiety, or 5′-stabilized end cap; and (b) a short interfering nucleic acid (siNA).
  • the siNA may comprise at least 5 nucleotides.
  • the nucleotides may be modified nucleotides, non-modified nucleotides, or any combination thereof.
  • the nucleotides may be ribonucleotides, deoxyribonucleotides, or any combination thereof.
  • the siNA may be single-stranded. Alternatively, the siNA is double-stranded.
  • the double-stranded siNA may comprise one or more blunt ends.
  • the double-stranded siNA may comprise one or more overhangs.
  • the double-stranded siNA may comprise a blunt end and an overhang.
  • an exemplary siNA molecule of the present disclosure is shown in FIG. 1 .
  • an exemplary siNA molecule comprises a sense strand ( 101 ) and an antisense strand ( 102 ).
  • the sense strand ( 101 ) may comprise a first oligonucleotide sequence ( 103 ).
  • the first oligonucleotide sequence ( 103 ) may comprise one or more phosphorothioate internucleoside linkages ( 109 ).
  • the phosphorothioate internucleoside linkage ( 109 ) may be between the nucleotides at the 5′ or 3′ terminal end of the first oligonucleotide sequence ( 103 ).
  • the phosphorothioate internucleoside linkage ( 109 ) may be between the first three nucleotides from the 5′ end of the first oligonucleotide sequence ( 103 ).
  • the first oligonucleotide sequence ( 103 ) may comprise one or more 2′-fluoro nucleotides ( 110 ).
  • the first oligonucleotide sequence ( 103 ) may comprise one or more 2′-O-methyl nucleotides ( 111 ).
  • the first oligonucleotide sequence ( 103 ) may comprise 15 or more modified nucleotides independently selected from 2′-fluoro nucleotides ( 110 ) and 2′-O-methyl nucleotides ( 111 ).
  • the sense strand ( 101 ) may further comprise a phosphorylation blocker ( 105 ).
  • the sense strand ( 101 ) may further comprise a galactosamine ( 106 ).
  • the antisense strand ( 102 ) may comprise a second oligonucleotide sequence ( 104 ).
  • the second oligonucleotide sequence ( 104 ) may comprise one or more phophorothioate internucleoside linkages ( 109 ).
  • the phosphorothioate internucleoside linkage ( 109 ) may be between the nucleotides at the 5′ or 3′ terminal 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 comprise one or more 2′-fluoro nucleotides ( 110 ).
  • the second oligonucleotide sequence ( 104 ) may comprise one or more 2′-O-methyl nucleotides ( 111 ).
  • the second oligonucleotide sequence ( 104 ) may comprise 15 or more modified nucleotides independently selected from 2′-fluoro nucleotides ( 110 ) and 2′-O-methyl nucleotides ( 111 ).
  • the antisense strand ( 102 ) may further comprise a 5′-stabilized end cap ( 107 ).
  • the siNA may further comprise one or more blunt ends.
  • 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 distinct from the first nucleotide sequence ( 103 ).
  • the overhang ( 108 ) may be distinct 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 1 or more nucleotides.
  • the overhang ( 108 ) may comprise 1 or more deoxyribonucleotides.
  • the overhang ( 108 ) may comprise 1 or more modified nucleotides.
  • the overhang ( 108 ) may comprise 1 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 ).
  • an exemplary siNA molecule of the present disclosure is shown in FIG. 2 .
  • an exemplary siNA molecule comprises a sense strand ( 201 ) and an antisense strand ( 202 ).
  • the sense strand ( 201 ) may comprise a first oligonucleotide sequence ( 203 ).
  • the first oligonucleotide sequence ( 203 ) may comprise one or more phophorothioate internucleoside linkages ( 209 ).
  • the phosphorothioate internucleoside linkage ( 209 ) may be between the nucleotides at the 5′ or 3′ terminal end of the first oligonucleotide sequence ( 203 ).
  • the phosphorothioate internucleoside linkage ( 209 ) may be between the first three nucleotides from the 5′ end of the first oligonucleotide sequence ( 203 ).
  • the first oligonucleotide sequence ( 203 ) may comprise one or more 2′-fluoro nucleotides ( 210 ).
  • the first oligonucleotide sequence ( 203 ) may comprise one or more 2′-O-methyl nucleotides ( 211 ).
  • the first oligonucleotide sequence ( 203 ) may comprise 15 or more modified nucleotides independently selected from 2′-fluoro nucleotides ( 210 ) and 2′-O-methyl nucleotides ( 211 ).
  • the sense strand ( 201 ) may further comprise a phosphorylation blocker ( 205 ).
  • the sense strand ( 201 ) may further comprise a galactosamine ( 206 ).
  • the antisense strand ( 202 ) may comprise a second oligonucleotide sequence ( 204 ).
  • the second oligonucleotide sequence ( 204 ) may comprise one or more phophorothioate internucleoside linkages ( 209 ).
  • the phosphorothioate internucleoside linkage ( 209 ) may be between the nucleotides at the 5′ or 3′ terminal 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 comprise one or more 2′-fluoro nucleotides ( 210 ).
  • the second oligonucleotide sequence ( 204 ) may comprise one or more 2′-O-methyl nucleotides ( 211 ).
  • the second oligonucleotide sequence ( 204 ) may comprise 15 or more modified nucleotides independently selected from 2′-fluoro nucleotides ( 210 ) and 2′-O-methyl nucleotides ( 211 ).
  • the antisense strand ( 202 ) may further comprise a 5′-stabilized end cap ( 207 ).
  • the siNA may further comprise one or more overhangs ( 208 ).
  • the overhang ( 208 ) may be part of the sense strand ( 201 ).
  • the overhang ( 208 ) may be part of the antisense strand. ( 202 ).
  • the overhang ( 208 ) may be distinct from the first nucleotide sequence ( 203 ).
  • the overhang ( 208 ) may be distinct 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 1 or more nucleotides.
  • the overhang ( 208 ) may comprise 1 or more deoxyribonucleotides.
  • the overhang ( 208 ) may comprise a TT sequence.
  • the overhang ( 208 ) may comprise 1 or more modified nucleotides.
  • the overhang ( 208 ) may comprise 1 or more modified nucleotides disclosed herein (e.g., 2-fluoro nucleotide, 2′-O-methyl nucleotide, 2′-fluoro nucleotide mimic, 2′-O-methyl nucleotide mimic, or a nucleotide comprising a modified nucleobase).
  • the overhang ( 208 ) may comprise 1 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 ).
  • FIGS. 3A-3G depict exemplary ds-siNA modification patterns.
  • an exemplary ds-siNA molecule may have the following formula:
  • 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, wherein the first nucleotide sequence comprises 15 to 30 nucleotides;
  • the bottom 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 the RNA corresponding to the target gene, wherein the second nucleotide sequence comprises 15 to 30 nucleotides;
  • 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, wherein the first nucleotide sequence comprises 15 to 30 nucleotides;
  • the bottom 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 the RNA corresponding to the target gene, wherein the second nucleotide sequence comprises 15 to 30 nucleotides;
  • the exemplary ds-siNA shown in FIGS. 3A-3G comprise (i) a sense strand comprising 19-21 nucleotides; and (ii) an antisense strand comprising 21-23 nucleotides.
  • the ds-siNA may further comprise (iii) a conjugated moiety, wherein the conjugated moiety is attached to the 3′ end of the antisense strand.
  • the ds-siNA may comprise a 2 nucleotide overhang consisting of nucleotides at positions 20 and 21 from the 5′ end of the antisense strand.
  • the ds-siNA may comprise a 2 nucleotide overhang consisting of nucleotides at positions 22 and 23 from the 5′ end of the antisense strand.
  • the ds-siNA may further comprise 1, 2, 3, 4, 5, 6 or more phosphorothioate (ps) internucleoside linkages. At least one phosphorothioate internucleoside linkage may be between the nucleotides at positions 1 and 2 or positions 2 and 3 from the 5′ end of the sense strand. At least one phosphorothioate internucleoside linkage may be between the nucleotides at positions 1 and 2 or positions 2 and 3 from the 5′ end of the antisense strand.
  • At least one phosphorothioate internucleoside linkage may be between the nucleotides at positions 19 and 20, positions 20 and 21, positions 21 and 22, or positions 22 and 23 from the 5′ end of the antisense strand.
  • 4-6 nucleotides in the sense strand may be 2′-fluoro nucleotides.
  • 2-5 nucleotides in the antisense strand may be 2′-fluoro nucleotides.
  • 13-15 nucleotides in the sense strand may be 2′-O-methyl nucleotides.
  • FIGS. 3A-3G 4-6 nucleotides in the sense strand may be 2′-fluoro nucleotides.
  • 2-5 nucleotides in the antisense strand may be 2′-fluoro nucleotides.
  • 13-15 nucleotides in the sense strand may be 2′-O-methyl nucleotides.
  • nucleotides in the antisense strand may be 2′-O-methyl nucleotides.
  • the ds-siNA does not contain a base pair between 2′-fluoro nucleotides on the sense and antisense strands.
  • the 2′-O-methyl nucleotide at position 1 from the 5′ end of the sense strand is further modified to contain a 5′ stabilizing end cap.
  • the 2′-O-methyl nucleotide at position 1 from the 5′ end of the antisense strand is further modified to contain a 5′ stabilizing end cap.
  • 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.
  • a ds-siNA may comprise (a) a sense strand consisting of 19 nucleotides, wherein 2′-fluoro nucleotides are at positions 3, 7-9, 12, and 17 from the 5′ end of the sense strand, and wherein 2′-O-methyl nucleotides are at positions 1, 2, 4-6, 10, 11, 13-16, 18, and 19 from the 5′ end of the sense strand; (b) an antisense strand consisting of 21 nucleotides, wherein nucleotides at positions 2 and 14 from the 5′ end of the antisense strand are 2′-fluoro nucleotides; and wherein nucleotides at positions 1, 3-13, and 15-21 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 the nucleotides at positions 1 and 2 and positions 2 and 3 from the 5′ end of the sense strand; and (ii) phosphorothioate internucleoside linkages between the nucleotides at positions 1 and 2; positions 2 and 3; positions 19 and 20; and positions 20 and 21 from the 5′ end of the antisense strand.
  • the 2′-O-methyl nucleotide at position 1 from the 5′ end of the sense strand is further modified to contain a 5′ stabilizing end cap.
  • the 2′-O-methyl nucleotide at position 1 from the 5′ end of the antisense strand is further modified to contain a 5′ stabilizing 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.
  • the 2′-O-methyl nucleotide at position 1 from the 3′ end of the antisense strand is further modified to contain a phosphorylation blocker.
  • the 2′-O-methyl nucleotide at position 1 from the 5′ end of the sense strand is a d2vd3 nucleotide.
  • the 2′-O-methyl nucleotide at position 1 from the 5′ end of the antisense strand is a d2vd3 nucleotide.
  • the 2′-O-methyl nucleotide at position 1 from the 3′ end of the sense strand is a d2vd3 nucleotide.
  • the 2′-O-methyl nucleotide at position 1 from the 3′ end of the antisense strand is a d2vd3 nucleotide.
  • at least 1, 2, 3, 4 or more 2′-fluoro nucleotides on the sense strand or antisense strand is a 2′-fluoro nucleotide mimic.
  • at least 1, 2, 3, 4 or more 2′-fluoro nucleotides on the sense strand or antisense strand is a f4P, f2P, or fX nucleotide.
  • at least 1, 2, 3, 4 or more 2′-O-methyl nucleotide on the sense or antisense strand is a 2′-O-methyl nucleotide mimic.
  • a ds-siNA may comprise (a) a sense strand consisting of 19 nucleotides, wherein 2′-fluoro nucleotides are at positions 3, 7, 8, and 17 from the 5′ end of the sense strand, and wherein 2′-O-methyl nucleotides are at positions 1, 2, 4-6, 9-16, 18, and 19 from the 5′ end of the sense strand; (b) an antisense strand consisting of 21 nucleotides, wherein nucleotides at positions 2 and 14 from the 5′ end of the antisense strand are 2′-fluoro nucleotides; and wherein nucleotides at positions 1, 3-13, and 15-21 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 the nucleotides at positions 1 and 2 and positions 2 and 3 from the 5′ end of the sense strand; and (ii) phosphorothioate internucleoside linkages between the nucleotides at positions 1 and 2; positions 2 and 3; positions 19 and 20; and positions 20 and 21 from the 5′ end of the antisense strand.
  • the 2′-O-methyl nucleotide at position 1 from the 5′ end of the sense strand is further modified to contain a 5′ stabilizing end cap.
  • the 2′-O-methyl nucleotide at position 1 from the 5′ end of the antisense strand is further modified to contain a 5′ stabilizing 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.
  • the 2′-O-methyl nucleotide at position 1 from the 3′ end of the antisense strand is further modified to contain a phosphorylation blocker.
  • the 2′-O-methyl nucleotide at position 1 from the 5′ end of the sense strand is a d2vd3 nucleotide.
  • the 2′-O-methyl nucleotide at position 1 from the 5′ end of the antisense strand is a d2vd3 nucleotide.
  • the 2′-O-methyl nucleotide at position 1 from the 3′ end of the sense strand is a d2vd3 nucleotide.
  • the 2′-O-methyl nucleotide at position 1 from the 3′ end of the antisense strand is a d2vd3 nucleotide.
  • at least 1, 2, 3, 4 or more 2′-fluoro nucleotides on the sense strand or antisense strand is a 2′-fluoro nucleotide mimic.
  • at least 1, 2, 3, 4 or more 2′-fluoro nucleotides on the sense strand or antisense strand is a f4P, f2P, or fX nucleotide.
  • at least 1, 2, 3, 4 or more 2′-O-methyl nucleotide on the sense or antisense strand is a 2′-O-methyl nucleotide mimic.
  • a ds-siNA may comprise (a) a sense strand consisting of 19 nucleotides, wherein 2′-fluoro nucleotides are at positions 3, 7-9, 12 and 17 from the 5′ end of the sense strand, and wherein 2′-O-methyl nucleotides are at positions 1, 2, 4-6, 10, 11, 13-16, 18, and 19 from the 5′ end of the sense strand; (b) an antisense strand consisting of 21 nucleotides, wherein the nucleotides in the antisense strand comprise an alternating 1:3 modification pattern, and wherein 1 nucleotide is a 2′-fluoro nucleotide and 3 nucleotides 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 the nucleotides at positions 1 and 2 and positions 2 and 3 from the 5′ end of the sense strand; and (ii) phosphorothioate internucleoside linkages between the nucleotides at positions 1 and 2; positions 2 and 3; positions 19 and 20; and positions 20 and 21 from the 5′ end of the antisense strand.
  • the ds-siNA may comprise 2-5 alternating 1:3 modification patterns on the antisense strand.
  • the 2′-O-methyl nucleotide at position 1 from the 5′ end of the sense strand is further modified to contain a 5′ stabilizing 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′ stabilizing 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.
  • 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 position 1 from the 5′ end of the sense strand is a d2vd3 nucleotide. In some embodiments, the 2′-O-methyl nucleotide at position 1 from the 5′ end of the antisense strand is a d2vd3 nucleotide.
  • the 2′-O-methyl nucleotide at position 1 from the 3′ end of the sense strand is a d2vd3 nucleotide. In some embodiments, the 2′-O-methyl nucleotide at position 1 from the 3′ end of the antisense strand is a d2vd3 nucleotide. In some embodiments, at least 1, 2, 3, 4 or more 2′-fluoro nucleotides on the sense strand or antisense strand is a 2′-fluoro nucleotide mimic.
  • At least 1, 2, 3, 4 or more 2′-fluoro nucleotides on the sense strand is a f4P, f2P, or fX nucleotide. In some embodiments, at least 1, 2, 3, 4 or more 2′-fluoro nucleotides on the antisense strand is a f4P, f2P, or fX nucleotide. In some embodiments, at least 1, 2, 3, 4 or more 2′-O-methyl nucleotide on the sense or antisense strand is a 2′-O-methyl nucleotide mimic.
  • a ds-siNA may comprise (a) a sense strand consisting of 19 nucleotides, wherein 2′-fluoro nucleotides are at positions 5 and 7-9 from the 5′ end of the sense strand, and wherein 2′-O-methyl nucleotides are at positions 1-4, 6, and 10-19 from the 5′ end of the sense strand; (b) an antisense strand consisting of 21 nucleotides, wherein the nucleotides in the antisense strand comprise an alternating 1:3 modification pattern, and wherein 1 nucleotide is a 2′-fluoro nucleotide and 3 nucleotides 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 the nucleotides at positions 1 and 2 and positions 2 and 3 from the 5′ end of the sense strand; and (ii) phosphorothioate internucleoside linkages between the nucleotides at positions 1 and 2; positions 2 and 3; positions 19 and 20; and positions 20 and 21 from the 5′ end of the antisense strand.
  • the ds-siNA may comprise 2-5 alternating 1:3 modification patterns on the antisense strand.
  • the alternating 1:3 modification pattern may start at the nucleotide at any of positions 2, 6, 10, 14, and/or 18 from the 5′ end of the antisense strand.
  • the 2′-O-methyl nucleotide at position 1 from the 5′ end of the sense strand is further modified to contain a 5′ stabilizing end cap.
  • the 2′-O-methyl nucleotide at position 1 from the 5′ end of the antisense strand is further modified to contain a 5′ stabilizing end cap.
  • the 2′-O-methyl nucleotide at position 1 from the 5′ end of the sense strand is further modified to contain a phosphorylation blocker.
  • 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 position 1 from the 5′ end of the sense strand is a d2vd3 nucleotide.
  • the 2′-O-methyl nucleotide at position 1 from the 5′ end of the antisense strand is a d2vd3 nucleotide. In some embodiments, the 2′-O-methyl nucleotide at position 1 from the 3′ end of the sense strand is a d2vd3 nucleotide. In some embodiments, the 2′-O-methyl nucleotide at position 1 from the 3′ end of the antisense strand is a d2vd3 nucleotide. In some embodiments, at least 1, 2, 3, 4 or more 2′-fluoro nucleotides on the sense strand or antisense strand is a 2′-fluoro nucleotide mimic.
  • At least 1, 2, 3, 4 or more 2′-fluoro nucleotides on the sense strand is a f4P, f2P, or fX nucleotide. In some embodiments, at least 1, 2, 3, 4 or more 2′-fluoro nucleotides on the antisense strand is a f4P, f2P, or fX nucleotide. In some embodiments, at least 1, 2, 3, 4 or more 2′-O-methyl nucleotide on the sense or antisense strand is a 2′-O-methyl nucleotide mimic.
  • a ds-siNA may comprise (a) a sense strand consisting of 19 nucleotides, wherein 2′-fluoro nucleotides are at positions 5 and 7-9 from the 5′ end of the sense strand, and wherein 2′-O-methyl nucleotides are at positions 1-4, 6, and 10-19 from the 5′ end of the sense strand; (b) an antisense strand consisting of 21 nucleotides, wherein the nucleotides in the antisense strand comprise an alternating 1:2 modification pattern, and wherein 1 nucleotide is a 2′-fluoro nucleotide and 2 nucleotides 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 the nucleotides at positions 1 and 2 and positions 2 and 3 from the 5′ end of the sense strand; and (ii) phosphorothioate internucleoside linkages between the nucleotides at positions 1 and 2; positions 2 and 3; positions 19 and 20; and positions 20 and 21 from the 5′ end of the antisense strand.
  • the ds-siNA may comprise 2-5 alternating 1:2 modification patterns on the antisense strand.
  • the alternating 1:2 modification pattern may start at the nucleotide at any of positions 2, 5, 8, 14, and/or 17 from the 5′ end of the antisense strand.
  • the ds-siNA comprises (a) a sense strand consisting of 19 nucleotides, wherein 2′-fluoro nucleotides are at positions 5 and 7-9 from the 5′ end of the sense strand, and wherein 2′-O-methyl nucleotides are at positions 1-4, 6, and 10-19 from the 5′ end of the sense strand; (b) an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 5, 8, 14, and 17 from the 5′ end of the antisense strand, and wherein 2′-O-methyl nucleotides are at positions 1, 3, 4, 6, 7, 9-13, 15, 16, and 18-21 from the 5′ end of the sense strand.
  • the 2′-O-methyl nucleotide at position 1 from the 5′ end of the sense strand is further modified to contain a 5′ stabilizing 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′ stabilizing 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.
  • 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 position 1 from the 5′ end of the sense strand is a d2vd3 nucleotide. In some embodiments, the 2′-O-methyl nucleotide at position 1 from the 5′ end of the antisense strand is a d2vd3 nucleotide.
  • the 2′-O-methyl nucleotide at position 1 from the 3′ end of the sense strand is a d2vd3 nucleotide. In some embodiments, the 2′-O-methyl nucleotide at position 1 from the 3′ end of the antisense strand is a d2vd3 nucleotide. In some embodiments, at least 1, 2, 3, 4 or more 2′-fluoro nucleotides on the sense strand or antisense strand is a 2′-fluoro nucleotide mimic.
  • At least 1, 2, 3, 4 or more 2′-fluoro nucleotides on the sense strand is a f4P, f2P, or fX nucleotide. In some embodiments, at least 1, 2, 3, 4 or more 2′-fluoro nucleotides on the antisense strand is a f4P, f2P, or fX nucleotide. In some embodiments, at least 1, 2, 3, 4 or more 2′-O-methyl nucleotide on the sense or antisense strand is a 2′-O-methyl nucleotide mimic.
  • a ds-siNA may comprise (a) a sense strand consisting of 19 nucleotides, wherein 2′-fluoro nucleotides are at positions 5 and 7-9 from the 5′ end of the sense strand, and wherein 2′-O-methyl nucleotides are at positions 1-4, 6, and 10-19 from the 5′ end of the sense strand; (b) an antisense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand, and wherein 2′-O-methyl nucleotides are at positions 1, 3-5, 7-13, 15, and 17-21 from the 5′ end 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 the nucleotides at positions 1 and 2 and positions 2 and 3 from the 5′ end of the sense strand; and (ii) phosphorothioate internucleoside linkages between the nucleotides at positions 1 and 2; positions 2 and 3; positions 19 and 20; and positions 20 and 21 from the 5′ end of the antisense strand.
  • At least 1, 2, 3, 4 or more 2′-fluoro nucleotides on the sense strand or antisense strand is a f4P, f2P, or fX nucleotide. In some embodiments, at least 1, 2, 3, 4 or more 2′-fluoro nucleotides on the sense strand or antisense strand is a f4P nucleotide. 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 is a f4P nucleotide.
  • At least one of the 2′-fluoro-nucleotides at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand is a 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 is a f4P nucleotide. In some embodiments, less than or equal to 3 of the 2′-fluoro-nucleotides at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand is a f4P nucleotide.
  • less than or equal to 2 of the 2′-fluoro-nucleotides at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand is a f4P nucleotide.
  • the 2′-fluoro-nucleotide at position 2 from the 5′ end of the antisense strand is a f4P nucleotide.
  • the 2′-fluoro-nucleotide at position 6 from the 5′ end of the antisense strand is a f4P nucleotide.
  • the 2′-fluoro-nucleotide at position 14 from the 5′ end of the antisense strand is a f4P nucleotide. In some embodiments, the 2′-fluoro-nucleotide at position 16 from the 5′ end of the antisense strand is a f4P nucleotide. In some embodiments, at least 1, 2, 3, 4 or more 2′-fluoro nucleotides on the sense strand or antisense strand is a f2P nucleotide.
  • 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 is a f2P nucleotide. 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 a 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 is a f2P nucleotide.
  • less than or equal to 3 of the 2′-fluoro-nucleotides at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand is a f2P nucleotide. In some embodiments, less than or equal to 2 of the 2′-fluoro-nucleotides at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand is a f2P nucleotide. In some embodiments, the 2′-fluoro-nucleotide at position 2 from the 5′ end of the antisense strand is a f2P nucleotide.
  • the 2′-fluoro-nucleotide at position 6 from the 5′ end of the antisense strand is a f2P nucleotide. In some embodiments, the 2′-fluoro-nucleotide at position 14 from the 5′ end of the antisense strand is a f2P nucleotide. In some embodiments, the 2′-fluoro-nucleotide at position 16 from the 5′ end of the antisense strand is a f2P nucleotide. In some embodiments, at least 1, 2, 3, 4 or more 2′-fluoro nucleotides on the sense strand or antisense strand is a fX nucleotide.
  • 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 is a fX nucleotide. 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 a 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 is a fX nucleotide.
  • less than or equal to 3 of the 2′-fluoro-nucleotides at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand is a fX nucleotide. In some embodiments, less than or equal to 2 of the 2′-fluoro-nucleotides at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand is a fX nucleotide. In some embodiments, the 2′-fluoro-nucleotide at position 2 from the 5′ end of the antisense strand is a fX nucleotide.
  • the 2′-fluoro-nucleotide at position 6 from the 5′ end of the antisense strand is a fX nucleotide.
  • the 2′-fluoro-nucleotide at position 14 from the 5′ end of the antisense strand is a fX nucleotide.
  • the 2′-fluoro-nucleotide at position 16 from the 5′ end of the antisense strand is a fX nucleotide.
  • the 2′-O-methyl nucleotide at position 1 from the 5′ end of the sense strand is further modified to contain a 5′ stabilizing end cap.
  • the 2′-O-methyl nucleotide at position 1 from the 5′ end of the antisense strand is further modified to contain a 5′ stabilizing 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.
  • the 2′-O-methyl nucleotide at position 1 from the 3′ end of the antisense strand is further modified to contain a phosphorylation blocker.
  • the 2′-O-methyl nucleotide at position 1 from the 5′ end of the sense strand is a d2vd3 nucleotide.
  • the 2′-O-methyl nucleotide at position 1 from the 5′ end of the antisense strand is a d2vd3 nucleotide.
  • the 2′-O-methyl nucleotide at position 1 from the 3′ end of the sense strand is a d2vd3 nucleotide.
  • the 2′-O-methyl nucleotide at position 1 from the 3′ end of the antisense strand is a d2vd3 nucleotide.
  • at least 1, 2, 3, 4 or more 2′-fluoro nucleotides on the sense strand or antisense strand is a 2′-fluoro nucleotide mimic.
  • at least 1, 2, 3, 4 or more 2′-fluoro nucleotides on the sense strand is a f4P, f2P, or fX nucleotide.
  • At least 1, 2, 3, 4 or more 2′-fluoro nucleotides on the antisense strand is a f4P, f2P, or fX nucleotide. In some embodiments, at least 1, 2, 3, 4 or more 2′-O-methyl nucleotide on the sense or antisense strand is a 2′-O-methyl nucleotide mimic.
  • a ds-siNA may comprise (a) a sense strand consisting of 21 nucleotides, wherein 2′-fluoro nucleotides are at positions 5, 9-11, 14, and 19 from the 5′ end of the sense strand, and wherein 2′-O-methyl nucleotides are at positions 1-4, 6-8, 12, 13, 15-18, 20, and 21 from the 5′ end of the sense strand; and (b) an antisense strand consisting of 23 nucleotides, wherein 2′-flouro nucleodies are at positions 2 and 14 from the 5′ end of the antisense strand, and wherein 2′-O-methyl nucleotides are at positions 1, 3-13, and 15-23 from the 5′ end of the antisense strand.
  • 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 the nucleotides at positions 1 and 2 and positions 2 and 3 from the 5′ end of the sense strand; and (ii) phosphorothioate internucleoside linkages between the nucleotides at positions 1 and 2; positions 2 and 3; positions 19 and 20; and positions 20 and 21 from the 5′ end of the antisense strand.
  • the 2′-O-methyl nucleotide at position 1 from the 5′ end of the sense strand is further modified to contain a 5′ stabilizing end cap.
  • the 2′-O-methyl nucleotide at position 1 from the 5′ end of the antisense strand is further modified to contain a 5′ stabilizing 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.
  • the 2′-O-methyl nucleotide at position 1 from the 3′ end of the antisense strand is further modified to contain a phosphorylation blocker.
  • the 2′-O-methyl nucleotide at position 1 from the 5′ end of the sense strand is a d2vd3 nucleotide.
  • the 2′-O-methyl nucleotide at position 1 from the 5′ end of the antisense strand is a d2vd3 nucleotide.
  • the 2′-O-methyl nucleotide at position 1 from the 3′ end of the sense strand is a d2vd3 nucleotide.
  • the 2′-O-methyl nucleotide at position 1 from the 3′ end of the antisense strand is a d2vd3 nucleotide.
  • at least 1, 2, 3, 4 or more 2′-fluoro nucleotides on the sense strand or antisense strand is a 2′-fluoro nucleotide mimic.
  • at least 1, 2, 3, 4 or more 2′-fluoro nucleotides on the sense strand is a f4P, f2P, or fX nucleotide.
  • At least 1, 2, 3, 4 or more 2′-fluoro nucleotides on the antisense strand is a f4P, f2P, or fX nucleotide. In some embodiments, at least 1, 2, 3, 4 or more 2′-O-methyl nucleotide on the sense or antisense strand is a 2′-O-methyl nucleotide mimic.
  • any of the siNAs disclosed herein may comprise a sense strand and an antisense strand.
  • the sense strand may comprise a first nucleotide sequence that is 15 to 30 nucleotides in length.
  • the antisense strand may comprise a second nucleotide sequence that is 15 to 30 nucleotides in length.
  • the double-stranded short interfering nucleic acid (ds-siNA) molecule comprises: (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: (i) is 15 to 30 nucleotides in length; and (ii) comprises 15 or more modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide, wherein at least one modified nucleotide is a 2′-O-methyl nucleotide and the nucleotide at position 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and/or 19 from the 5′ end of the first nucleotide sequence is a 2′-fluoro nucleotide; and (b) an antisense strand comprising a second
  • the double-stranded short interfering nucleic acid (ds-siNA) molecule comprises: (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: (i) is 15 to 30 nucleotides in length; and (ii) comprises 15 or more modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide, wherein at least one modified nucleotide is a 2′-O-methyl nucleotide and the nucleotide at position 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and/or 19 from the 5′ end of the first nucleotide sequence is a 2′-fluoro nucleotide; and (b) an antisense strand comprising a second
  • the double-stranded short interfering nucleic acid (ds-siNA) molecule comprises: (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: (i) is 15 to 30 nucleotides in length; and (ii) comprises 15 or more modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide, wherein at least one modified nucleotide is a 2′-O-methyl nucleotide and the nucleotide at position 7 from the 5′ end of the first nucleotide sequence is a 2′-fluoro nucleotide; and (b) an antisense strand comprising a second nucleotide sequence that is at least about 60%,
  • the double-stranded short interfering nucleic acid (ds-siNA) molecule comprises: (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: (i) is 15 to 30 nucleotides in length; and (ii) comprises 15 or more modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide, wherein at least one modified nucleotide is a 2′-O-methyl nucleotide and the nucleotide at position 7, 9, 10, and/or 11 from the 5′ end of the first nucleotide sequence is a 2′-fluoro nucleotide; and (b) an antisense strand comprising a second nucleotide sequence that
  • the double-stranded short interfering nucleic acid (ds-siNA) molecule comprises: (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: (i) is 15 to 30 nucleotides in length; and (ii) comprises 15 or more modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide, wherein at least one modified nucleotide is a 2′-O-methyl nucleotide and at least one modified nucleotide is a 2′-fluoro nucleotide; and (b) an antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%,
  • the double-stranded short interfering nucleic acid (ds-siNA) molecule comprises: (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: (i) is 15 to 30 nucleotides in length; and (ii) comprises 15 or more modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide, wherein at least one modified nucleotide is a 2′-O-methyl nucleotide and at least one modified nucleotide is a 2′-fluoro nucleotide; and (b) an antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%,
  • the double-stranded short interfering nucleic acid (ds-siNA) molecule comprises: (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: (i) is 15 to 30 nucleotides in length; (ii) comprises 15 or more modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide, wherein at least one modified nucleotide is a 2′-O-methyl nucleotide and at least one modified nucleotide is a 2′-fluoro nucleotide; and (iii) comprises 1 or more phosphorothioate internucleoside linkage; and (b) an antisense strand comprising a second nucleo
  • the double-stranded short interfering nucleic acid (ds-siNA) molecule comprises: (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: (i) is 15 to 30 nucleotides in length; and (ii) comprises 15 or more modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide, wherein at least one modified nucleotide is a 2′-O-methyl nucleotide and at least one modified nucleotide is a 2′-fluoro nucleotide; and (b) an antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%,
  • the double-stranded short interfering nucleic acid (ds-siNA) molecule comprises: (I) a sense strand comprising (A) a first nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to an RNA corresponding to a target gene, wherein the first nucleotide sequence: (i) is 15 to 30 nucleotides in length; and (ii) comprises 15 or more modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide, wherein at least one modified nucleotide is a 2′-O-methyl nucleotide and at least one modified nucleotide is a 2′-fluoro nucleotide; and (B) a phosphorylation blocker or a galactosamine; and (II) an antisense strand comprising a second nucle
  • the double-stranded short interfering nucleic acid (ds-siNA) molecule comprises: (I) 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: (a) is 15 to 30 nucleotides in length; and (b) comprises 15 or more modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide, wherein at least one modified nucleotide is a 2′-O-methyl nucleotide and at least one modified nucleotide is a 2′-fluoro nucleotide; and (II) an antisense strand comprising (A) a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%,
  • the double-stranded short interfering nucleic acid (ds-siNA) molecule comprises: (I) a sense strand comprising (A) a first nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to an RNA corresponding to a target gene, wherein the first nucleotide sequence: (i) is 15 to 30 nucleotides in length; and (ii) comprises 15 or more modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide, wherein at least one modified nucleotide is a 2′-O-methyl nucleotide and at least one modified nucleotide is a 2′-fluoro nucleotide; and (B) a phosphorylation blocker or a galactosamine; and (II) an antisense strand comprising (A) a sense strand compris
  • the double-stranded short interfering nucleic acid (ds-siNA) molecule comprises: (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 comprises a nucleotide sequence of any one SEQ ID NOs: 1-56, 103-158, and 205-260; and (b) an antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the RNA corresponding to the target gene, wherein the second nucleotide sequence comprises a nucleotide sequence of any one of SEQ ID NOs: 57-102, 159-204, and 261-306.
  • the double-stranded short interfering nucleic acid (ds-siNA) molecule comprises: (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 comprises a nucleotide sequence as shown in Tables 1-3; and (b) an antisense strand comprising a second nucleotide sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to the RNA corresponding to the target gene, wherein the second nucleotide sequence comprises a nucleotide sequence as shown in Tables 1-3.
  • the double-stranded short interfering nucleic acid (ds-siNA) molecule comprises: (a) a sense strand comprising a nucleotide sequence of any one of SEQ ID NOs: 307-362 and 415-444; and (b) an antisense strand comprising a nucleotide sequence of any one of SEQ ID NOs: 363-409, 445-533, and 536-539.
  • the ds-siNA molecule comprises a double-stranded molecule as identified by the duplex ID (e.g., ds-siNA-001 to ds-siNA-0178) shown in Tables 6 and 10.
  • compositions comprising two or more of the siNA molecules described herein.
  • compositions comprising any of the siNA molecule described and a pharmaceutically acceptable carrier or diluent.
  • compositions comprising two or more of the siNA molecules described herein for use as a medicament.
  • compositions comprising any of the siNA molecule described and a pharmaceutically acceptable carrier or diluent for use as a medicament.
  • siNA molecules comprising modified nucleotides.
  • Any of the siNA molecules described herein may be double-stranded siNA (ds-siNA) molecules.
  • ds-siNA double-stranded siNA
  • the terms “siNA molecules” and “ds-siNA molecules” may be used interchangeably.
  • the ds-siNA molecules comprise a sense strand and an antisense strand.
  • siNA molecules comprising (a) at least one phosphorylation blocker, conjugated moiety, or 5′-stabilized end cap; and (b) a short interfering nucleic acid (siNA).
  • the phosphorylation blocker is a phosphorylation blocker disclosed herein.
  • the conjugated moiety is a galactosamine disclosed herein.
  • the 5′-stabilized end cap is a 5′-stabilized end cap disclosed herein.
  • the siNA may comprise any of the first nucleotide, second nucleotide, sense strand, or antisense strand sequences disclosed herein.
  • the siNA may comprise 5 to 100, 5 to 90, 10 to 100, 10 to 90, 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 30, 10 to 25, 15 to 100, 15 to 90, 15 to 80, 15 to 70, 15 to 60, 15 to 50, 15 to 30, or 15 to 25 nucleotides.
  • the siNA may comprise at least 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides.
  • the siNA may comprise less than or equal to 50, 45, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, or 19 nucleotides.
  • the nucleotides may be modified nucleotides.
  • the siNA may be single stranded.
  • the siNA may be double stranded.
  • the siNA may comprise (a) a sense strand comprising 15 to 30, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 17 to 30, 17 to 25, 17 to 24, 17 to 23, 17 to 22, 17 to 21, 18 to 30, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 19 to 30, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 20 to 25, 20 to 24, 20 to 23, 21 to 25, 21 to 24, or 21 to 23 nucleotides; and (b) an antisense strand comprising 15 to 30, 15 to 25, 15 to 24, 15 to 23, 15 to 22, 15 to 21, 17 to 30, 17 to 25, 17 to 24, 17 to 23, 17 to 22, 17 to 21, 18 to 30, 18 to 25, 18 to 24, 18 to 23, 18 to 22, 18 to 21, 19 to 30, 19 to 25, 19 to 24, 19 to 23, 19 to 22, 19 to 21, 20 to 25, 20 to 24, 20 to 23, 21 to 25, 21 to 24,
  • the 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 siNA may comprise (a) a sense strand comprising about 19 nucleotides; and (b) an antisense strand comprising about 21 nucleotides.
  • the siNA may comprise (a) a sense strand comprising about 21 nucleotides; and (b) an antisense strand comprising about 23 nucleotides.
  • any of the siNA molecules disclosed herein further comprise one or more linkers independently selected from a phosphodiester (PO) linker, phosphorothioate (PS) linker, phosphorodithioate linker, and PS-mimic linker.
  • the PS-mimic linker is a sulfur linker.
  • the linkers are internucleoside linkers.
  • the linkers connect a nucleotide of the siNA molecule to at least one phosphorylation blocker, conjugated moiety, or 5′-stabilized end cap.
  • the linkers connect a conjugated moiety to a phosphorylation blocker or 5′-stabilized end cap.
  • the sense strand may comprise a first nucleotide sequence.
  • the first nucleotide sequence may be 15 to 30, 15 to 25, 15 to 23, 17 to 23, 19 to 23, or 19 to 21 nucleotides in length. In some embodiments, the 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.
  • 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 a 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 that are adjacent to the first nucleotide sequence.
  • the one or more modified nucleotides are independently selected from any of the modified nucleotides disclosed herein (e.g., 2′-fluoro nucleotide, 2′-O-methyl nucleotide, 2′-fluoro nucleotide mimic, 2′-O-methyl nucleotide mimic, or a nucleotide comprising a modified nucleobase).
  • the first nucleotide sequence comprises 15, 16, 17, 18, 19, 20, 21, 22, 23, or more modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide. 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 a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide.
  • 100% of the nucleotides in the first nucleotide sequence are modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide.
  • the 2′-O-methyl nucleotide is a 2′-O-methyl nucleotide mimic.
  • the 2′-fluoro nucleotide is a 2′-fluoro nucleotide mimic.
  • modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, between about 2 to 20 modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides.
  • between about 5 to 25 modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, between about 10 to 25 modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, between about 12 to 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.
  • 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.
  • At least about 17 modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, at least about 18 modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, at least about 19 modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, less than or equal to 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides.
  • less than or equal to 21 modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, less than or equal to 20 modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, less than or equal to 19 modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, less than or equal to 18 modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides.
  • less than or equal to 17 modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, less than or equal to 16 modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, less than or equal to 15 modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, less than or equal to 14 modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides.
  • modified nucleotides of the first nucleotide sequence are 2′-O-methyl nucleotides.
  • at least one modified nucleotide of the first nucleotide sequence is a 2′-O-methyl pyrimidine.
  • at least 5, 6, 7, 8, 9, or 10 modified nucleotides of the first nucleotide sequence are 2′-O-methyl pyrimidines.
  • at least one modified nucleotide of the first nucleotide sequence is a 2′-O-methyl purine.
  • at least 5, 6, 7, 8, 9, or 10 modified nucleotides of the first nucleotide sequence are 2′-O-methyl purines.
  • the 2′-O-methyl nucleotide is a 2′-O-methyl nucleotide mimic.
  • between 2 to 15 modified nucleotides of the first nucleotide sequence are 2′-fluoro nucleotides. In some embodiments, between 2 to 10 modified nucleotides of the first nucleotide sequence are 2′-fluoro nucleotides. In some embodiments, between 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.
  • 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.
  • 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.
  • 6 or fewer modified nucleotides of the first nucleotide sequence are 2′-fluoro nucleotides. In some embodiments, 5 or fewer modified nucleotides of the first nucleotide sequence are 2′-fluoro nucleotides. In some embodiments, 4 or fewer modified nucleotides of the first nucleotide sequence are 2′-fluoro nucleotides. In some embodiments, 3 or fewer modified nucleotides of the first nucleotide sequence are 2′-fluoro nucleotides. In some embodiments, 2 or fewer modified nucleotides of the first nucleotide sequence are 2′-fluoro nucleotides.
  • 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 nucleotide is a 2′-fluoro nucleotide mimic.
  • 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, 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.
  • 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.
  • 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.
  • 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.
  • At least 1, 2, 3, 4, 5, 6, or 7 nucleotides 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.
  • the nucleotide at positions 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.
  • 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.
  • At least three nucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and/or 19 from the 5′ end of the first nucleotide sequence are 2′-fluoro nucleotides. In some embodiments, the nucleotides at positions 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, and/or 19 from the 5′ end of the first nucleotide sequence are 2′-fluoro nucleotides. In some embodiments, the nucleotide at position 3 from the 5′ end of the first nucleotide sequence is a 2′-fluoro nucleotide.
  • 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.
  • 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.
  • the nucleotide at position 17 from the 5′ end of the first nucleotide sequence is a 2′-fluoro nucleotide. In some embodiments, the nucleotide at position 19 from the 5′ end of the first nucleotide sequence is a 2′-fluoro nucleotide. In some embodiments, the nucleotide at position 3, 7, 8, 9, 12, and/or 17 from the 5′ end of the first nucleotide sequence is a 2′-fluoro nucleotide. In some embodiments, the nucleotide at position 3, 7, 8, and/or 17 from the 5′ end of the first nucleotide sequence is a 2′-fluoro nucleotide.
  • the nucleotide at position 3, 7, 8, 9, 12, and/or 17 from the 5′ end of the first nucleotide sequence is a 2′-fluoro nucleotide. In some embodiments, the nucleotide at position 5, 7, 8, and/or 9 from the 5′ end of the first nucleotide sequence is a 2′-fluoro nucleotide. In some embodiments, the nucleotide at position 5, 9, 10, 11, 12, and/or 19 from the 5′ end of the first nucleotide sequence is a 2′-fluoro nucleotide. In some embodiments, the 2′-fluoro nucleotide is a 2′-fluoro nucleotide mimic.
  • the 2′-fluoro nucleotide 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):
  • R 1 is independently a nucleobase, aryl, heteroaryl, or H
  • Q 1 and Q 2 are independently S or O
  • R 5 is independently-OCD 3 , —F, or —OCH 3
  • R 6 and R 7 are independently H, D, or CD3.
  • the nucleobase is selected from cytosine, guanine, adenine, uracil, aryl, heteroaryl, and an analogue or derivative thereof.
  • the 2′-fluoro or 2′-O-methyl nucleotide mimic is a nucleotide mimic of Formula (16)-Formula (20):
  • R 1 is independently a nucleobase and R 2 is F or —OCH 3 .
  • the nucleobase is selected from cytosine, guanine, adenine, uracil, aryl, heteroaryl, and an analogue or derivative thereof.
  • the first nucleotide sequence comprises, consists of, or consists essentially of ribonucleic acids (RNAs). In some embodiments, the first nucleotide sequence comprises, consists of, or consists essentially of modified RNAs. In some embodiments, the modified RNAs are selected from a 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.
  • the sense strand may further comprise one or more internucleoside linkages independently selected from a phosphodiester (PO) internucleoside linkage, phosphorothioate (PS) internucleoside linkage, phosphorodithioate internucleoside linkage, and PS-mimic internucleoside linkage.
  • PO phosphodiester
  • PS phosphorothioate
  • phosphorodithioate internucleoside linkage phosphorodithioate internucleoside linkage
  • PS-mimic internucleoside linkage is a sulfo internucleoside linkage.
  • 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 to 10, 2 to 8, 2 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 phosphorothioate internucleoside linkages. In some embodiments, the sense strand comprises 1 to 2 phosphorothioate internucleoside linkages.
  • the sense strand comprises 2 to 4 phosphorothioate internucleoside linkages. In some embodiments, at least one phosphorothioate internucleoside linkage is between the nucleotides at positions 1 and 2 from the 5′ end of the first nucleotide sequence. In some embodiments, at least one phosphorothioate internucleoside linkage is between the nucleotides at positions 2 and 3 from the 5′ end of the first nucleotide sequence. In some embodiments, the sense strand comprises two phosphorothioate internucleoside linkages between the nucleotides at positions 1 to 3 from the 5′ end of the first nucleotide sequence.
  • any of the sense strands disclosed herein further comprise a monomer selected from Examples 21-32, 36, 37, 40-42, and 44-46 monomers. In some embodiments, any of the sense strands disclosed herein further comprise a 5′ end cap monomer. In some embodiments, the 5′ end cap monomer is selected from Examples 5-11, 33-35, 38, 39, 43, and 49-53 5′ end cap monomers.
  • any of the first nucleotide sequences disclosed herein further comprise a monomer selected from Examples 21-32, 36, 37, 40-42, and 44-46 monomers. In some embodiments, any of the first nucleotide sequences disclosed herein further comprise a 5′ end cap monomer. In some embodiments, the 5′ end cap monomer is selected from Examples 5-11, 33-35, 38, 39, 43, and 49-53 5′ end cap monomers.
  • the antisense strand may comprise a second nucleotide sequence.
  • the second nucleotide sequence may be 15 to 30, 15 to 25, 15 to 23, 17 to 23, 19 to 23, or 19 to 21 nucleotides in length. In some embodiments, the second nucleotide sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the second nucleotide sequence is at least 19 nucleotides in length. In some embodiments, the second nucleotide sequence is at least 21 nucleotides in length.
  • 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 a deoxyribonucleic acid (DNA).
  • DNA deoxyribonucleic acid
  • the DNA is thymine (T).
  • the antisense strand may further comprise a TT sequence.
  • the antisense strand may further comprise one or more modified nucleotides that are adjacent to the second nucleotide sequence.
  • the one or more modified nucleotides are independently selected from any of the modified nucleotides disclosed herein (e.g., 2′-fluoro nucleotide, 2′-O-methyl nucleotide, 2′-fluoro nucleotide mimic, 2′-O-methyl nucleotide mimic, or a nucleotide comprising a modified nucleobase).
  • the second nucleotide sequence comprises 15, 16, 17, 18, 19, 20, 21, 22, 23, or more modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide.
  • 70%, 75%, 80%, 85%, 90%, 95% or 100% of the nucleotides in the second nucleotide sequence are modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide.
  • 100% of the nucleotides in the second nucleotide sequence are modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide.
  • modified nucleotides of the second nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, between about 2 to 20 modified nucleotides of the second nucleotide sequence are 2′-O-methyl nucleotides.
  • between about 5 to 25 modified nucleotides of the second nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, between about 10 to 25 modified nucleotides of the second nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, between about 12 to 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.
  • 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.
  • 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, less than or equal to 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 modified nucleotides of the second nucleotide sequence are 2′-O-methyl nucleotides.
  • less than or equal to 21 modified nucleotides of the second nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, less than or equal to 20 modified nucleotides of the second nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, less than or equal to 19 modified nucleotides of the second nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, less than or equal to 18 modified nucleotides of the second nucleotide sequence are 2′-O-methyl nucleotides.
  • less than or equal to 17 modified nucleotides of the second nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, less than or equal to 16 modified nucleotides of the second nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, less than or equal to 15 modified nucleotides of the second nucleotide sequence are 2′-O-methyl nucleotides. In some embodiments, less than or equal to 14 modified nucleotides of the second nucleotide sequence are 2′-O-methyl nucleotides.
  • modified nucleotides of the second nucleotide sequence are 2′-O-methyl nucleotides.
  • at least one modified nucleotide of the second nucleotide sequence is a 2′-O-methyl pyrimidine.
  • at least 5, 6, 7, 8, 9, or 10 modified nucleotides of the second nucleotide sequence are 2′-O-methyl pyrimidines.
  • at least one modified nucleotide of the second nucleotide sequence is a 2′-O-methyl purine.
  • at least 5, 6, 7, 8, 9, or 10 modified nucleotides of the second nucleotide sequence are 2′-O-methyl purines.
  • the 2′-O-methyl nucleotide is a 2′-O-methyl nucleotide mimic.
  • between 2 to 15 modified nucleotides of the second nucleotide sequence are 2′-fluoro nucleotides. In some embodiments, between 2 to 10 modified nucleotides of the second nucleotide sequence are 2′-fluoro nucleotides. In some embodiments, between 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.
  • 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.
  • 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.
  • 5 or fewer modified nucleotides of the second nucleotide sequence are 2′-fluoro nucleotides. In some embodiments, 4 or fewer modified nucleotides of the second nucleotide sequence are 2′-fluoro nucleotides. In some embodiments, 3 or fewer modified nucleotides of the second nucleotide sequence are 2′-fluoro nucleotides. In some embodiments, 2 or fewer modified nucleotides of the second nucleotide sequence are 2′-fluoro nucleotides. In some embodiments, at least one modified nucleotide of the second nucleotide sequence is a 2′-fluoro pyrimidine.
  • 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 nucleotide is a 2′-fluoro nucleotide mimic.
  • the 2′-fluoro nucleotide 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):
  • R 1 is independently a nucleobase, aryl, heteroaryl, or H
  • Q 1 and Q 2 are independently S or O
  • R 5 is independently-OCD 3 , —F, or —OCH 3
  • R 6 and R 7 are independently H, D, or CD3.
  • the nucleobase is selected from cytosine, guanine, adenine, uracil, aryl, heteroaryl, and an analogue or derivative thereof.
  • the 2′-fluoro or 2′-O-methyl nucleotide mimic is a nucleotide mimic of Formula (16)-Formula (20):
  • R 1 is a nucleobase and R 2 is independently F or —OCH 3 .
  • the nucleobase is selected from cytosine, guanine, adenine, uracil, aryl, heteroaryl, and an analogue or derivative thereof.
  • At least 1, 2, 3, 4, 5, 6, 7, 8, or 9 nucleotides at position 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 nucleotide at position 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.
  • 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.
  • 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.
  • the nucleotides at positions 2 and/or 14 from the 5′ end of the second nucleotide sequence are 2′-fluoro nucleotides. In some embodiments, the nucleotides at positions 2, 6, and/or 16 from the 5′ end of the second nucleotide sequence are 2′-fluoro nucleotides. In some embodiments, the nucleotides at positions 2, 6, 14, and/or 16 from the 5′ end of the second nucleotide sequence are 2′-fluoro nucleotides. In some embodiments, the nucleotides at positions 2, 6, 10, 14, and/or 18 from the 5′ end of the second nucleotide sequence are 2′-fluoro nucleotides.
  • the nucleotides at positions 2, 5, 8, 14, and/or 17 from the 5′ end of the second nucleotide sequence are 2′-fluoro nucleotides. In some embodiments, the 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.
  • 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.
  • 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.
  • the nucleotides in the second nucleotide sequence are arranged in an alternating 1:3 modification pattern, wherein 1 nucleotide is a 2′-fluoro nucleotide and 3 nucleotides are 2′-O-methyl nucleotides, and wherein the alternating 1:3 modification pattern occurs at least 2 times. In some embodiments, the alternating 1:3 modification pattern occurs 2-5 times. In some embodiments, at least two of the alternating 1:3 modification pattern occur consecutively. In some embodiments, at least two of the alternating 1:3 modification pattern occurs nonconsecutively.
  • At least 1, 2, 3, 4, or 5 alternating 1:3 modification pattern begins at nucleotide position 2, 6, 10, 14, and/or 18 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:3 modification pattern begins at nucleotide position 2 from the 5′ end of the antisense strand. In some embodiments, wherein at least one alternating 1:3 modification pattern begins at nucleotide position 6 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:3 modification pattern begins at nucleotide position 10 from the 5′ end of the antisense strand.
  • At least one alternating 1:3 modification pattern begins at nucleotide position 14 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:3 modification pattern begins at nucleotide position 18 from the 5′ end of the antisense strand.
  • the 2′-fluoro nucleotide is a 2′-fluoro nucleotide mimic.
  • the nucleotides in the second nucleotide sequence are arranged in an alternating 1:2 modification pattern, wherein 1 nucleotide is a 2′-fluoro nucleotide and 2 nucleotides are 2′-O-methyl nucleotides, and wherein the alternating 1:2 modification pattern occurs at least 2 times. In some embodiments, the alternating 1:2 modification pattern occurs 2-5 times. In some embodiments, at least two of the alternating 1:2 modification pattern occurs consecutively. In some embodiments, at least two of the alternating 1:2 modification pattern occurs nonconsecutively. In some embodiments, at least 1, 2, 3, 4, or 5 alternating 1:2 modification pattern begins at nucleotide position 2, 5, 8, 14, and/or 17 from the 5′ end of the antisense strand.
  • At least one alternating 1:2 modification pattern begins at nucleotide position 2 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:2 modification pattern begins at nucleotide position 5 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:2 modification pattern begins at nucleotide position 8 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:2 modification pattern begins at nucleotide position 14 from the 5′ end of the antisense strand. In some embodiments, at least one alternating 1:2 modification pattern begins at nucleotide position 17 from the 5′ end of the antisense strand. In some embodiments, the 2′-fluoro nucleotide is a 2′-fluoro nucleotide mimic.
  • the second nucleotide sequence comprises, consists of, or consists essentially of ribonucleic acids (RNAs). In some embodiments, the second nucleotide sequence comprises, consists of, or consists essentially of modified RNAs. In some embodiments, the modified RNAs are selected from a 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.
  • the sense strand may further comprise one or more internucleoside linkages independently selected from a phosphodiester (PO) internucleoside linkage, phosphorothioate (PS) internucleoside linkage, phosphorodithioate internucleoside linkage, and PS-mimic internucleoside linkage.
  • PO phosphodiester
  • PS phosphorothioate
  • phosphorodithioate internucleoside linkage phosphorodithioate internucleoside linkage
  • PS-mimic internucleoside linkage is a sulfo internucleoside linkage.
  • 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 to 10, 2 to 8, 2 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 phosphorothioate internucleoside linkages.
  • the antisense strand comprises 2 to 10, 2 to 8, 2 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 phosphorothioate internucleoside linkages. In some embodiments, the antisense strand comprises 2 to 8 phosphorothioate internucleoside linkages. In some embodiments, the antisense strand comprises 3 to 8 phosphorothioate internucleoside linkages. In some embodiments, the antisense strand comprises 4 to 8 phosphorothioate internucleoside linkages. In some embodiments, at least one phosphorothioate internucleoside linkage is between the nucleotides at positions 1 and 2 from the 5′ end of the second nucleotide sequence.
  • At least one phosphorothioate internucleoside linkage is between the nucleotides at positions 2 and 3 from the 5′ end of the second nucleotide sequence. In some embodiments, at least one phosphorothioate internucleoside linkage is between the nucleotides at positions 1 and 2 from the 3′ end of the second nucleotide sequence. In some embodiments, at least one phosphorothioate internucleoside linkage is between the nucleotides at positions 2 and 3 from the 3′ end of the second nucleotide sequence.
  • the antisense strand comprises two phosphorothioate internucleoside linkages between the nucleotides at positions 1 to 3 from the 5′ end of the first nucleotide sequence. In some embodiments, the antisense strand comprises two phosphorothioate internucleoside linkages between the nucleotides at positions 1 to 3 from the 3′ end of the first nucleotide sequence.
  • the antisense strand comprises (a) two phosphorothioate internucleoside linkages between the nucleotides at positions 1 to 3 from the 5′ end of the first nucleotide sequence; and (b) two phosphorothioate internucleoside linkages between the nucleotides at positions 1 to 3 from the 3′ end of the first nucleotide sequence.
  • At least one end of the ds-siNA is a blunt end. In some embodiments, at least one end of the ds-siNA comprises an overhang, wherein the overhang comprises at least one nucleotide. In some embodiments, both ends of the ds-siNA comprise an overhang, wherein the overhang comprises at least one nucleotide. In some embodiments, the overhang comprises 1 to 5 nucleotides, 1 to 4 nucleotides, 1 to 3 nucleotides, or 1 to 2 nucleotides. In some embodiments, the overhang consists of 1 to 2 nucleotides.
  • the first nucleotide sequence comprises a nucleotide sequence of any one SEQ ID NOs: 1-56, 103-158, and 205-260.
  • the second nucleotide sequence comprises a nucleotide sequence of any one of SEQ ID NOs: 57-102, 159-204, and 261-306.
  • the sense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 307-362 and 415-444.
  • the antisense strand comprises a nucleotide sequence of any one of SEQ ID NOs: 363-409, 445-533, and 536-539.
  • any of the antisense strands disclosed herein further comprise a monomer selected from Examples 21-32, 36, 37, 40-42, and 44-46 monomers. In some embodiments, any of the antisense strands disclosed herein further comprise a 5′ end cap monomer. In some embodiments, the 5′ end cap monomer is selected from Examples 5-11, 33-35, 38, 39, 43, and 49-53 5′ end cap monomers.
  • any of the second nucleotide sequences disclosed herein further comprise a monomer selected from Examples 21-32, 36, 37, 40-42, and 44-46 monomers. In some embodiments, any of the second nucleotide sequences disclosed herein further comprise a 5′ end cap monomer. In some embodiments, the 5′ end cap monomer is selected from Examples 5-11, 33-35, 38, 39, 43, and 49-53 5′ end cap monomers.
  • siNA molecules comprising one or more modified nucleotides.
  • any of the siNAs disclosed herein comprise one or more modified nucleotides.
  • any of the sense strands disclosed herein comprise one or more modified nucleotides.
  • any of the first nucleotide sequences disclosed herein comprise one or more modified nucleotides.
  • any of the antisense strands disclosed herein comprise one or more modified nucleotides.
  • any of the second nucleotide sequences disclosed herein comprise one or more modified nucleotides.
  • the one or more modified nucleotides is adjacent to the first nucleotide sequence.
  • 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 is 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.
  • At least one modified nucleotide is adjacent to the 3′ end of the second nucleotide sequence. In some embodiments, at least one modified nucleotide is adjacent to the 5′ end of the second nucleotide sequence and at least one modified nucleotide is adjacent to the 3′ end of the second nucleotide sequence. In some embodiments, a 2′-O-methyl nucleotide in any of sense strands or first nucleotide sequences disclosed herein is replaced with a modified nucleotide. In some embodiments, a 2′-O-methyl nucleotide in any of antisense strands or second nucleotide sequences disclosed herein is replaced with a modified nucleotide.
  • any of the siNA molecules, siNAs, sense strands, first nucleotide sequences, antisense strands, and 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, 24, 25, 26, 27, 28, 29, or 30 or more modified nucleotides.
  • a modified nucleotide is selected from the group consisting of 2′-fluoro nucleotide, 2′-O-methyl nucleotide, 2′-fluoro nucleotide mimic, 2′-O-methyl nucleotide mimic, a locked nucleic acid, and a nucleotide comprising a modified nucleobase.
  • any of the siRNAs 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.
  • any of the sense 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.
  • any of the first nucleotide sequences disclosed herein comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more 2′-fluoro or 2′-O-methyl nucleotide mimics.
  • any of the antisense strand 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.
  • 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.
  • the 2′-fluoro or 2′-O-methyl nucleotide mimic is a nucleotide mimic of Formula (16)-Formula (20):
  • R 1 is a nucleobase and R 2 is independently F or —OCH 3 .
  • the nucleobase is selected from cytosine, guanine, adenine, uracil, aryl, heteroaryl, and an analogue or derivative thereof.
  • 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 the first nucleotide sequence.
  • the at least one 2′-fluoro or 2′-O-methyl nucleotide mimic is adjacent to the 5′ end of 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 first nucleotide sequence. In some embodiments, the at least one 2′-fluoro or 2′-O-methyl nucleotide mimic is adjacent to the 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 second nucleotide sequence.
  • the at least one 2′-fluoro or 2′-O-methyl nucleotide mimic is adjacent to the 3′ end of second nucleotide sequence.
  • the first nucleotide sequence does not comprise a 2′-fluoro nucleotide mimic. In some embodiments, the first nucleotide sequence does not comprise a 2′-O-methyl nucleotide mimic.
  • the second nucleotide sequence does not comprise a 2′-fluoro nucleotide mimic. In some embodiments, the second nucleotide sequence does not comprise a 2′-O-methyl nucleotide mimic.
  • any of the siRNAs disclosed herein comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more locked nucleic acids.
  • any of the sense strands disclosed herein comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more locked nucleic acids.
  • any of the first nucleotide sequences disclosed herein comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more locked nucleic acids.
  • any of the antisense strand disclosed herein comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more locked nucleic acids.
  • any of the second nucleotide sequences disclosed herein comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more locked nucleic acids.
  • the locked nucleic acid is selected from
  • R is H or alkyl (or AmNA(N-Me)) when R is alkyl);
  • any of the siRNAs, sense strands, first nucleotide sequences, antisense strands, or second nucleotide sequences disclosed herein comprise at least modified nucleotide that is
  • any of the siRNAs, sense strands, first nucleotide sequences, antisense strands, or second nucleotide sequences disclosed herein comprise at least modified nucleotide that is
  • any of the siRNAs, sense strands, first nucleotide sequences, antisense strands, or second nucleotide sequences disclosed herein comprise at least modified nucleotide that is
  • any of the siRNAs, sense strands, first nucleotide sequences, antisense strands, or second nucleotide sequences disclosed herein comprise at least modified nucleotide that is
  • any of the siRNAs, sense strands, first nucleotide sequences, antisense strands, or second nucleotide sequences disclosed herein comprise at least modified nucleotide that is
  • B is a nucleobase
  • siNA molecules comprising a phosphorylation blocker.
  • a 2′-O-methyl nucleotide in any of sense strands or first nucleotide sequences disclosed herein is replaced with a nucleotide containing a phosphorylation blocker.
  • a 2′-O-methyl nucleotide in any of antisense strands or second nucleotide sequences disclosed herein is replaced with a nucleotide containing a phosphorylation blocker.
  • a 2′-O-methyl nucleotide in any of sense strands or first nucleotide sequences disclosed herein is further modified to contain a phosphorylation blocker.
  • a 2′-O-methyl nucleotide in any of antisense strands or second nucleotide sequences disclosed herein is further modified to contain a phosphorylation blocker.
  • any of the siNA molecules disclosed herein comprise a phosphorylation blocker of Formula (IV):
  • R 1 is a nucleobase
  • R 4 is —O—R 30 or —NR 31 R 32
  • R 30 is C 1 -C 8 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.
  • any of the siNA molecules disclosed herein comprise a phosphorylation blocker of Formula (IV):
  • R 1 is a nucleobase
  • R 4 is —OCH 3 or —N(CH 2 CH 2 ) 2 O.
  • a siNA molecule comprises (a) a phosphorylation blocker of Formula (IV):
  • R 1 is a nucleobase
  • R 4 is —O—R 30 or —NR 31 R 32
  • R 30 is C 1 -C 8 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
  • siNA short interfering nucleic acid
  • a siNA molecule comprises (a) a phosphorylation blocker of Formula (IV):
  • R 1 is a nucleobase, and R 4 is —OCH 3 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.
  • siNA short interfering nucleic acid
  • the phosphorylation blocker is attached to the 3′ end of the sense strand or first nucleotide sequence. In some embodiments, the phosphorylation blocker is attached to the 3′ end of the sense strand or first nucleotide sequence via 1, 2, 3, 4, or 5 or more linkers. In some embodiments, the phosphorylation blocker is attached to the 5′ end of the sense strand or first nucleotide sequence. In some embodiments, the phosphorylation blocker is attached to the 5′ end of the sense strand or first nucleotide sequence via 1, 2, 3, 4, or 5 or more linkers. In some embodiments, the phosphorylation blocker is attached to the 3′ end of the antisense strand or second nucleotide sequence.
  • the phosphorylation blocker is attached to the 3′ end of the antisense strand or second nucleotide sequence via 1, 2, 3, 4, or 5 or more linkers. In some embodiments, the phosphorylation blocker is attached to the 5′ end of the antisense strand or second nucleotide sequence. In some embodiments, the phosphorylation blocker is attached to the 5′ end of the antisense strand or second nucleotide sequence via 1, 2, 3, 4, or 5 or more linkers. In some embodiments, the one or more linkers are independently selected from the group consisting of a phosphodiester linker, phosphorothioate linker, and phosphorodithioate linker.
  • siNA molecules comprising a conjugated moiety.
  • the conjugated moiety is selected from galactosamine, peptides, proteins, sterols, lipids, phosphohipids, biotin, phenoxazines, active drug substance, cholesterols, phenanthridine, anthraquinone, acridine, fluoresceins, rhodarnines, coumarins, and dyes.
  • the conjugated moiety is attached to the 3′ end of the sense strand or first nucleotide sequence.
  • the conjugated moiety is attached to the 3′ end of the sense strand or first nucleotide sequence via 1, 2, 3, 4, or 5 or more linkers. In some embodiments, the conjugated moiety is attached to the 5′ end of the sense strand or first nucleotide sequence. In some embodiments, the conjugated moiety is attached to the 5′ end of the sense strand or first nucleotide sequence via 1, 2, 3, 4, or 5 or more linkers. In some embodiments, the conjugated moiety is attached to the 3′ end of the antisense strand or second nucleotide sequence. In some embodiments, the conjugated moiety is attached to the 3′ end of the antisense strand or second nucleotide sequence via 1, 2, 3, 4, or 5 or more linkers.
  • the conjugated moiety is attached to the 5′ end of the antisense strand or second nucleotide sequence. In some embodiments, the conjugated moiety is attached to the 5′ end of the antisense strand or second nucleotide sequence via 1, 2, 3, 4, or 5 or more linkers. In some embodiments, the one or more linkers are independently selected from the group consisting of a phosphodiester linker, phosphorothioate linker, and phosphorodithioate linker.
  • the conjugated moiety is galactosamine.
  • any of the siNAs disclosed herein are attached to a conjugated moiety that is galactosamine.
  • the galactosamine is N-acetylgalactosamine (GalNAc).
  • any of the siNA molecules disclosed herein comprise GalNAc.
  • the GalNAc is of Formula (VI):
  • 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 —O—P( ⁇ O)(SH)—, —O—P( ⁇ O)(O)—, —O—P( ⁇ O)(OH)—, —O—P(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 activated group, or an oligonucleotide.
  • the first protecting group is acetyl.
  • the second protecting group is trimethoxytrityl (TMT).
  • the activated group is a phosphoramidite group.
  • the phosphoramidite group is a cyanoethoxy N,N-diisopropylphosphoramidite group.
  • the linker is a C6-NH 2 group.
  • A is a short interfering nucleic acid (siNA) or siNA molecule.
  • m is 3.
  • R is H, Z is H, and n is 1.
  • R is H, Z is H, and n is 2.
  • the GalNAc is of Formula (VII):
  • n is independently 1 or 2.
  • the galactosamine is attached to the 3′ end of the sense strand or first nucleotide sequence. In some embodiments, the galactosamine is attached to the 3′ end of the sense strand or first nucleotide sequence via 1, 2, 3, 4, or 5 or more linkers. In some embodiments, the galactosamine is attached to the 5′ end of the sense strand or first nucleotide sequence. In some embodiments, the galactosamine is attached to the 5′ end of the sense strand or first nucleotide sequence via 1, 2, 3, 4, or 5 or more linkers. In some embodiments, the galactosamine is attached to the 3′ end of the antisense strand or second nucleotide sequence.
  • the galactosamine is attached to the 3′ end of the antisense strand or second nucleotide sequence via 1, 2, 3, 4, or 5 or more linkers. In some embodiments, the galactosamine is attached to the 5′ end of the antisense strand or second nucleotide sequence. In some embodiments, the galactosamine is attached to the 5′ end of the antisense strand or second nucleotide sequence via 1, 2, 3, 4, or 5 or more linkers.
  • the one or more linkers are independently selected from the group consisting of a phosphodiester (p or po) linker, phosphorothioate (ps) linker, phosphoramidite (HEG) linker, triethylene glycol (TEG) linker, and/or phosphorodithioate linker.
  • 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 .
  • the conjugated moiety is a lipid moiety.
  • any of the siNAs disclosed herein are attached to a conjugated moiety that is a lipid moiety.
  • lipid moieties include, but are not limited to, a cholesterol moiety, a thioether, e.g., hexyl-S-tritylthiol a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylanmrmoniumr 1-di-O-hexadecyl-rac-glycero-S—H-phosphonate, a polyamine or a polyethylene glycol chain, adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino
  • the conjugated moiety is an active drug substance.
  • any of the siNAs disclosed herein are attached to a conjugated moiety that is an active drug substance.
  • active drug substances include, but are not limited to, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (5)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporir, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic.
  • siNA molecules comprising a 5′-stabilized end cap.
  • 5′-stabilized end cap As used herein the terms “5′-stabilized end cap” and “5′ end cap” are used interchangeably.
  • a 2′-O-methyl nucleotide in any of sense strands or first nucleotide sequences disclosed herein is replaced with a nucleotide containing a 5′-stabilized end cap.
  • a 2′-O-methyl nucleotide in any of antisense strands or second nucleotide sequences disclosed herein is replaced with a nucleotide containing a 5′-stabilized end cap.
  • a 2′-O-methyl nucleotide in any of sense strands or first nucleotide sequences disclosed herein is further modified to contain a 5′-stabilized end cap.
  • a 2′-O-methyl nucleotide in any of antisense strands or second nucleotide sequences disclosed herein is further modified to contain a 5′-stabilized end cap.
  • 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 5′-(Z)-vinyl phosphonate. In some embodiments, the 5′-vinyl phosphonate is a deuterated vinyl phosphonate.
  • 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 mimics are disclosed in Parmar et al., 2018 , J Med Chem, 61(3):734-744, International Publication Nos. WO2018/045317 and WO2018/044350, and U.S. Pat. No. 10,087,210, each of which is incorporated by reference in its entirety.
  • any of the siNA molecules, sense strands, first nucleotide sequences, antisense strands, or second nucleotide sequences disclosed herein comprise a 5′-stabilized end cap of Formula (Ia):
  • R 1 is H, a nucleobase, aryl, or heteroaryl; R 2 is
  • n 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 , —
  • any of the siNA molecules, sense strands, first nucleotide sequences, antisense strands, or second nucleotide sequences disclosed herein comprise a 5′-stabilized end cap of Formula (Ib):
  • R 1 is H, a nucleobase, aryl, or heteroaryl; R 2 is
  • n 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 , —
  • any of the siNA molecules, sense strands, first nucleotide sequences, antisense strands, or second nucleotide sequences disclosed herein comprise a 5′-stabilized end cap of Formula (Ic):
  • R 1 is a nucleobase, aryl, heteroaryl, or H
  • R 20 is hydrogen; or R 2 and R 20 together form a 3- to 7-membered carbocyclic ring substituted with —(CR 21 R 22 ) n —Z or —(C 2 -C 6 alkenylene)-Z; 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
  • any of the siNA molecules, sense strands, first nucleotide sequences, antisense strands, or second nucleotide sequences disclosed herein comprise a 5′-stabilized end cap of Formula (IIa):
  • R 1 is a nucleobase, aryl, heteroaryl, or H
  • R 2 is
  • R 9 is —SO 2 CH 3 or —COCH 3 , is a double or single bond
  • R 10 —CH 2 PO 3 H or —NHCH 3
  • R 11 is —CH 2 — or —CO—
  • R 12 is H and R 13 is CH 3 or R 12 and R 13 together form —CH 2 CH 2 CH 2 —.
  • R 1 is an aryl.
  • the aryl is a phenyl.
  • any of the siNA molecules, sense strands, first nucleotide sequences, antisense strands, or second nucleotide sequences disclosed herein comprise a 5′-stabilized end cap of Formula (IIb):
  • R 1 is a nucleobase, aryl, heteroaryl, or H
  • R 2 is
  • R 9 is —SO 2 CH 3 or —COCH 3 , is a double or single bond
  • R 10 —CH 2 PO 3 H or —NHCH 3
  • R 11 is —CH 2 — or —CO—
  • R 12 is H and R 15 is CH 3 or R 12 and R 13 together form —CH 2 CH 2 CH 2 —.
  • R 1 is an aryl.
  • the aryl is a phenyl.
  • any of the siNA molecules, sense strands, first nucleotide sequences, antisense strands, or second nucleotide sequences disclosed herein comprise a 5′-stabilized end cap of Formula (III):
  • R 1 is a nucleobase, aryl, heteroaryl, or H
  • L is —CH 2 —, —CH ⁇ CH—, —CO—, or —CH 2 CH 2 —
  • 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 ).
  • R 1 is an aryl.
  • the aryl is a phenyl.
  • any of the siNA molecules, sense strands, first nucleotide sequences, antisense strands, or second nucleotide sequences disclosed herein comprise a 5′-stabilized end cap selected from Examples 5-11, 33-35, 38, 39, 43, and 49-53 5′ end cap monomers.
  • siNA molecules comprising (a) a 5′-stabilized end cap of Formula (Ia):
  • R 1 is a nucleobase, aryl, heteroaryl, or H;
  • R 20 is H; or R 2 and R 20 together form a 3- to 7-membered carbocyclic ring substituted with —(CR 21 R 22 ) n —Z or —(C 2 -C 6 alkenylene)-Z; 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
  • siNA molecules comprising (a) a 5′-stabilized end cap of Formula (Ib):
  • R 1 is a nucleobase, aryl, heteroaryl, or H;
  • R 2 is
  • R 20 is H; or R 2 and R 20 together form a 3- to 7-membered carbocyclic ring substituted with —(CR 21 R 22 ) n —Z or —(C 2 -C 6 alkenylene)-Z; 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
  • siNA molecules comprising (a) a 5′-stabilized end cap of Formula (Ic):
  • R 1 is a nucleobase, aryl, heteroaryl, or H
  • R 2 is
  • R 20 is hydrogen; or R 2 and R 20 together form a 3- to 7-membered carbocyclic ring substituted with —(CR 21 R 22 ) n —Z or —(C 2 -C 6 alkenylene)-Z; 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
  • a siNA molecule comprises (a) a 5′-stabilized end cap of Formula (IIa):
  • R 1 is a nucleobase, aryl, heteroaryl, or H
  • R 2 is
  • R 1 is an aryl. In some embodiments, the aryl is a phenyl.
  • a siNA molecule comprises (a) a 5′-stabilized end cap of Formula (IIb):
  • R 1 is a nucleobase, aryl, heteroaryl, or H
  • R 2 is
  • R 1 is an aryl. In some embodiments, the aryl is a phenyl.
  • a siNA molecule comprises (a) a 5′-stabilized end cap of Formula (III):
  • R 1 is a nucleobase, aryl, heteroaryl, or H
  • L is —CH 2 —, —CH ⁇ CH—, —CO—, or —CH 2 CH 2 —
  • 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 ); and (b) a short interfering nucleic acid (siNA), wherein the 5′-stabilized end cap is conjugated to the siNA.
  • R 1 is an aryl.
  • the aryl is phenyl.
  • any of the siNA molecules disclosed herein comprise a 5′-stabilized end cap selected from the group consisting of Formula (1) to Formula (15), Formula (9X) to Formula (12X), and Formula (9Y) to Formula (12Y):
  • R 1 is a nucleobase, aryl, heteroaryl, or H.
  • R 1 is an aryl.
  • the aryl is a phenyl.
  • any of the siNA molecules disclosed herein comprise a 5′-stabilized end cap selected from the group consisting of Formulas (1A)-(15A), Formulas (9B)-(12B), Formulas (9AX)-(12AX), Formulas (9AY)-(12AY), Formulas (9BX)-(12BX), and Formulas (9BY)-(12BY):
  • any of the siNA molecules disclosed herein comprise a 5′-stabilized end cap selected from the group consisting of Formula (21) to Formula (35):
  • R 1 is a nucleobase, aryl, heteroaryl, or H.
  • R 1 is an aryl.
  • the aryl is a phenyl.
  • any of the siNA molecules disclosed herein comprise a 5′-stabilized end cap selected from the group consisting of Formulas (21A)-(35A), Formulas (29B)-(32B), Formulas (29AX)-(32AX), Formulas (29AY)-(32AY), Formulas (29BX)-(32BX), and Formulas (29BY)-(32BY):
  • 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 1, 2, 3, 4, or 5 or more linkers. In some embodiments, the one or more linkers are independently selected from the group consisting of a phosphodiester (p or po) linker, phosphorothioate (ps) linker (ps), phosphoramidite (HEG) linker, triethylene glycol (TEG) linker, and/or phosphorodithioate linker.
  • 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 .
  • 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.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more internucleoside linkers are independently selected from the group consisting of a phosphodiester (p or po) linker, phosphorothioate (ps) linker, or phosphorodithioate linker.
  • any of the siRNAs, sense strands, first nucleotide sequences, antisense strands, and/or second nucleotide sequences disclosed herein further comprise 1, 2, 3, 4 or more linkers that attach a conjugated moiety, phosphorylation blocker, and/or 5′ end cap to the siRNA, sense strand, first nucleotide sequence, antisense strand, and/or second nucleotide sequences.
  • the 1, 2, 3, 4 or more linkers are independently selected from the group consisting of a phosphodiester (p or po) linker, phosphorothioate (ps) linker, phosphoramidite (HEG) linker, triethylene glycol (TEG) linker, and/or phosphorodithioate linker.
  • 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 .
  • any of the ds-siNA molecules disclosed herein may interact with proteins in the cell to form a RNA-Induced Silencing Complex (RISC).
  • RISC RNA-Induced Silencing Complex
  • the ds-siNA may be unwound to form a single-stranded siNA (ss-siNA).
  • the ss-siNA may comprise the antisense strand of the ds-siNA.
  • the antisense strand may bind to a complementary messenger RNA (mRNA), which results in silencing of the gene that encodes the mRNA.
  • mRNA complementary messenger RNA
  • the target gene may be any gene in a cell.
  • the target gene is a viral gene.
  • the viral gene is from a DNA virus.
  • the DNA virus is a double-stranded DNA (dsDNA) virus.
  • the dsDNA virus is a hepadnavirus.
  • the hepadnavirus is a hepatitis B virus (HBV).
  • HBV is selected from HBV genotypes A-J.
  • the target gene is selected from the S gene or X gene of the HBV.
  • the HBV has a genome sequence shown in the nucleotide sequence of SEQ ID NO: 410, which corresponds to the nucleotide sequence of GenBank Accession No. U95551.1, which is incorporated by reference in its entirety.
  • SEQ ID NO: 415 An exemplary HBV genome sequence is shown in SEQ ID NO: 415, corresponding to Genbank Accession No. KC315400.1, which is incorporated by reference in its entirety.
  • Nucleotides 2307 . . . 3215, 1 . . . 1623 of SEQ ID NO: 415 correspond to the polymerase/RT gene sequence, which encodes for the polymerase protein.
  • Nucleotides 2848 . . . 3215, 1 . . . 835 of SEQ ID NO: 415 correspond to the PreS1/S2/S gene sequence, which encodes for the large S protein.
  • SEQ ID NO: 415 corresponds to the PreS2/S gene sequence, which encodes for the middle S protein.
  • Nucleotides 155 . . . 835 of SEQ ID NO: 415 correspond to the S gene sequence, which encodes the small S protein.
  • Nucleotides 1374 . . . 1838 of SEQ ID NO: 415 correspond to the X gene sequence, which encodes the X protein.
  • Nucleotides 1814 . . . 2452 of SEQ ID NO: 415 correspond to the PreC/C gene sequence, which encodes the precore/core protein.
  • Nucleotides 1901.2452 of SEQ ID NO: 415 correspond to the C gene sequence, which encodes the core protein.
  • the HBV genome further comprises viral regulatory elements, such as viral promoters (preS2, preS1, Core, and X) and enhancer elements (ENH1 and ENH2).
  • Nucleotides 1624 . . . 1771 of SEQ ID NO: 415 correspond to ENH2.
  • Nucleotides 1742 . . . 1849 of SEQ ID NO: 415 correspond to the Core promoter.
  • Nucleotides 1818 . . . 3215, 1 . . . 1930 of SEQ ID NO: 415 correspond to the pregenomic RNA (pgRNA), which encodes the core and polymerase proteins.
  • pgRNA pregenomic RNA
  • the ASO is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary or hybridizes to a viral target RNA sequence that begins in an X region of HBV or in an S region of HBV.
  • the viral target may, e.g., begin at the 5′-end of target-site in acc. KC315400.1 (genotype B, “gt B”), or in any one of genotypes A, C, or D.
  • gt B genotypes A, C, or D.
  • the S region is defined as from the beginning of small S protein (in genotype B KC315400.1 isolate, position #155) to before beginning of X protein (in genotype B KC315400.1 isolate, position #1373).
  • the X region is defined as from the beginning X protein (in genotype B KC315400.1 isolate, position #1374) to end of DR2 site (in genotype B KC315400.1 isolate, position #1603).
  • the second nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to 15 to 30, 15 to 25, 15 to 23, 15 to 22, 15 to 21, 17 to 25, 17 to 23, 17 to 22, 17 to 21, or 19 to 21 nucleotides within positions 200-720 or 1100-1700 of SEQ ID NO: 410.
  • the second nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to 15 to 30, 15 to 25, 15 to 23, 15 to 22, 15 to 21, 17 to 25, 17 to 23, 17 to 22, 17 to 21, or 19 to 21 nucleotides within positions 200-280, 300-445, 460-510, 650-720, 1170-1220, 1250-1300, or 1550-1630 of SEQ ID NO: 410.
  • the second nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to 15 to 30, 15 to 25, 15 to 23, 15 to 22, 15 to 21, 17 to 25, 17 to 23, 17 to 22, 17 to 21, or 19 to 21 nucleotides within positions 200-230, 250-280, 300-330, 370-400, 405-445, 460-500, 670-700, 1180-1210, 1260-1295, 1520-1550, or 1570-1610 of SEQ ID NO: 410.
  • the second nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary to 15 to 30, 15 to 25, 15 to 23, 15 to 22, 15 to 21, 17 to 25, 17 to 23, 17 to 22, 17 to 21,or 19 to 21 nucleotides starting at position 203, 206, 254, 305, 375, 409, 412, 415, 416, 419, 462, 466, 467, 674, 676, 1182, 1262, 1263, 1268, 1526, 1577, 1578, 1580, 1581, 1583, or 1584 of SEQ ID NO: 410.
  • the first nucleotide is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to a nucleotide region within SEQ ID NO: 410, with the exception that the thymines (Ts) in SEQ ID NO: 410 are replaced with uracil (U).
  • the first nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to 15 to 30, 15 to 25, 15 to 23, 15 to 22, 15 to 21, 17 to 25, 17 to 23, 17 to 22, 17 to 21, or 19 to 21 nucleotides within positions 200-720 or 1100-1700 of SEQ ID NO: 410.
  • the first nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to 15 to 30, 15 to 25, 15 to 23, 15 to 22, 15 to 21, 17 to 25, 17 to 23, 17 to 22, 17 to 21,or 19 to 21 nucleotides within positions 200-280, 300-445, 460-510, 650-720, 1170-1220, 1250-1300, or 1550-1630 of SEQ ID NO: 410.
  • the first nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to 15 to 30, 15 to 25, 15 to 23, 15 to 22, 15 to 21, 17 to 25, 17 to 23, 17 to 22, 17 to 21,or 19 to 21 nucleotides within positions 200-230, 250-280, 300-330, 370-400, 405-445, 460-500, 670-700, 1180-1210, 1260-1295, 1520-1550, or 1570-1610 of SEQ ID NO: 410.
  • the first nucleotide sequence is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to 15 to 30, 15 to 25, 15 to 23, 15 to 22, 15 to 21, 17 to 25, 17 to 23, 17 to 22, 17 to 21, or 19 to 21 nucleotides starting at position 203, 206, 254, 305, 375, 409, 412, 415, 416, 419, 462, 466, 467, 674, 676, 1182, 1262, 1263, 1268, 1526, 1577, 1578, 1580, 1581, 1583, or 1584 of SEQ ID NO: 410.
  • 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 the MCJ protein (MCJ/DnaJC15 or Methylation-Controlled J protein). In some embodiments, the MCJ protein is encoded by the mRNA sequence of SEQ ID NO: 411, which corresponds to the nucleotide sequence of GenBank Accession No. NM_013238.3, which is incorporated by reference in its entirety.
  • the target gene is TAZ.
  • TAZ comprises the nucleotide sequence of SEQ ID NO: 412, which corresponds to the nucleotide sequence of GenBank Accession No. NM_000116.5, which is incorporated by reference in its entirety.
  • the target gene is angiopoietin like 3 (ANGPTL3).
  • ANGPTL3 comprises the nucleotide sequence of SEQ ID NO: 413, which corresponds to the nucleotide sequence of GenBank Accession No. NM_014495.4, which is incorporated by reference in its entirety.
  • the target gene is diacylglycerol acyltransferase 2 (DGAT2).
  • DGAT2 comprises the nucleotide sequence of SEQ ID NO: 414, which corresponds to the nucleotide sequence of GenBank Accession No. NM_001253891.1, which is incorporated by reference in its entirety.
  • compositions comprising any of the siNA molecules, sense strands, antisense strands, first nucleotide sequences, or second nucleotide sequences described herein.
  • the compositions may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more siNA molecules described herein.
  • the compositions may comprise a first nucleotide sequence comprising a nucleotide sequence of any one SEQ ID NOs: 1-56, 103-158, and 205-260.
  • the composition comprises a second nucleotide sequence comprising a nucleotide sequence of any one of SEQ ID NOs: 57-102, 159-204, and 261-306.
  • the composition comprises a sense strand comprising a nucleotide sequence of any one of SEQ ID NOs: 307-362 and 415-444. In some embodiments, the composition comprises an antisense strand comprising a nucleotide sequence of any one of SEQ ID NOs: 363-409, 445-533, and 536-539.
  • compositions may comprise (a) a phosphorylation blocker; and (b) a short interfering nucleic acid (siNA).
  • the phosphorylation blocker is any of the phosphorylation blockers disclosed herein.
  • the siNA is any of the siNAs disclosed herein.
  • the siNA comprises any of the sense strands, antisense strands, first nucleotide sequences, or second nucleotide sequences described herein.
  • the siNA comprises any of the sense strands, antisense strands, first nucleotide sequences, or second nucleotide sequences described herein.
  • the siNA comprises one or more modified nucleotides.
  • the one or more modified nucleotides are independently selected from a 2′-fluoro nucleotide and a 2′-O-methyl nucleotide.
  • the 2′-fluoro nucleotide or the 2′-O-methyl nucleotide is independently selected from any of the 2′-fluoro or 2′-O-methyl nucleotide mimics disclosed herein.
  • the siNA comprises a nucleotide sequence comprising any of the modification patterns disclosed herein.
  • the composition comprises (a) a conjugated moiety; and (b) a short interfering nucleic acid (siNA).
  • the conjugated moiety is any of the galactosamines disclosed herein.
  • the siNA is any of the siNAs disclosed herein.
  • the siNA comprises any of the sense strands, antisense strands, first nucleotide sequences, or second nucleotide sequences described herein.
  • the siNA comprises any of the sense strands, antisense strands, first nucleotide sequences, or second nucleotide sequences described herein.
  • the siNA comprises one or more modified nucleotides.
  • the one or more modified nucleotides are independently selected from a 2′-fluoro nucleotide and a 2′-O-methyl nucleotide.
  • the 2′-fluoro nucleotide or the 2′-O-methyl nucleotide is independently selected from any of the 2′-fluoro or 2′-O-methyl nucleotide mimics disclosed herein.
  • the siNA comprises a nucleotide sequence comprising any of the modification patterns disclosed herein.
  • the composition comprises (a) a 5′-stabilized end cap; and (b) a short interfering nucleic acid (siNA).
  • the 5′-stabilized end cap is any of the 5-stabilized end caps disclosed herein.
  • the siNA is any of the siNAs disclosed herein.
  • the siNA comprises any of the sense strands, antisense strands, first nucleotide sequences, or second nucleotide sequences described herein.
  • the siNA comprises one or more modified nucleotides.
  • the one or more modified nucleotides are independently selected from a 2′-fluoro nucleotide and a 2′-O-methyl nucleotide.
  • the 2′-fluoro nucleotide or the 2′-O-methyl nucleotide is independently selected from any of the 2′-fluoro or 2′-O-methyl nucleotide mimics disclosed herein.
  • the siNA comprises a nucleotide sequence comprising any of the modification patterns disclosed herein.
  • the composition comprises (a) at least one phosphorylation blocker, conjugated moiety, or 5′-stabilized end cap; and (b) a short interfering nucleic acid (siNA).
  • the phosphorylation blocker is any of the phosphorylation blockers disclosed herein.
  • the conjugated moiety is any of the galactosamines disclosed herein.
  • the 5′-stabilized end cap is any of the 5-stabilized end caps disclosed herein.
  • the siNA is any of the siNAs disclosed herein.
  • the siNA comprises any of the sense strands, antisense strands, first nucleotide sequences, or second nucleotide sequences described herein.
  • the siNA comprises one or more modified nucleotides.
  • the one or more modified nucleotides are independently selected from a 2′-fluoro nucleotide and a 2′-O-methyl nucleotide.
  • the 2′-fluoro nucleotide or the 2′-O-methyl nucleotide is independently selected from any of the 2′-fluoro or 2′-O-methyl nucleotide mimics disclosed herein.
  • the siNA comprises a nucleotide sequence comprising any of the modification patterns disclosed herein.
  • the composition may be a pharmaceutical composition.
  • the pharmaceutical composition comprises an amount of one or more of the siNA molecules described herein formulated with one or more pharmaceutically acceptable carriers (additives) and/or diluents.
  • the pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or
  • terapéuticaally-effective amount means that amount of a compound, material, or composition comprising a siNA of the present disclosure which is effective for producing some desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit/risk ratio applicable to any medical treatment.
  • phrases “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • antioxidants examples include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), le
  • Formulations of the present disclosure include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound (e.g., siNA molecule) which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.
  • a formulation of the present disclosure comprises an excipient selected from the group consisting of cyclodextrins, celluloses, liposomes, micelle forming agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides; and a compound (e.g., siNA molecule) of the present disclosure.
  • an aforementioned formulation renders orally bioavailable a compound (e.g., siNA molecule) of the present disclosure.
  • Methods of preparing these formulations or compositions include the step of bringing into association a compound (e.g., siNA molecule) of the present disclosure with the carrier and, optionally, one or more accessory ingredients.
  • a compound e.g., siNA molecule
  • the formulations are prepared by uniformly and intimately bringing into association a compound (e.g., siNA molecule) of the present disclosure with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • Formulations of the disclosure suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound (e.g., siNA molecule) of the present disclosure as an active ingredient.
  • a compound (e.g., siNA molecule) of the present disclosure may also be administered as a bolus, electuary or paste.
  • the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, 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; (6) absorption accelerators, such as quaternary ammonium compounds and surfactants, such as poloxa
  • pharmaceutically-acceptable carriers such as sodium citrate or dicalcium phosphate
  • compositions may also comprise 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 sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets, and other solid dosage forms of the pharmaceutical compositions of the present disclosure may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be formulated for rapid release, e.g., freeze-dried.
  • compositions may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • embedding compositions which can be used include polymeric substances and waxes.
  • the active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
  • Liquid dosage forms for oral administration of the compounds (e.g., siNA molecules) of the disclosure include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (I particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art, such as, for example, water or
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions in addition to the active compounds (e.g., siNA molecules), may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • Formulations of the pharmaceutical compositions of the disclosure for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds (e.g., siNA molecules) of the disclosure with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is 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).
  • a suppository which may be prepared by mixing one or more compounds (e.g., siNA molecules) of the disclosure with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is 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.
  • 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.
  • Dosage forms for the topical or transdermal administration of a compound (e.g., siNA molecule) of this disclosure include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active compound (e.g., siNA molecule) may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
  • the ointments, pastes, creams and gels may contain, in addition to an active compound (e.g., siNA molecule) of this disclosure, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to a compound (e.g., siNA molecule) of this disclosure, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • Transdermal patches have the added advantage of providing controlled delivery of a compound (e.g., siNA molecule) of the present disclosure to the body.
  • dosage forms can be made by dissolving or dispersing the compound (e.g., siNA molecule) in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the compound (e.g., siNA molecule) across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound (e.g., siNA molecule) in a polymer matrix or gel.
  • Ophthalmic formulations are also contemplated as being within the scope of this invention.
  • compositions of this disclosure suitable for parenteral administration comprise one or more compounds (e.g., siNA molecules) of the disclosure in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • compounds e.g., siNA molecules
  • sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • 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.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms upon the subject compounds may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • the absorption of the drug in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
  • Injectable depot forms are made by forming microencapsule matrices of the subject compounds (e.g., siNA molecules) in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.
  • biodegradable polymers such as polylactide-polyglycolide.
  • Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue.
  • the compounds (e.g., siNA molecules) of the present disclosure are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99% (more preferably, 10 to 30%) of active ingredient in combination with a pharmaceutically acceptable carrier.
  • the siNA molecules of the present disclosure may be used to treat a disease in a subject in need thereof.
  • a method of treating a disease in a subject in need thereof comprises administering to the subject any of the siNA molecules disclosed herein.
  • a method of treating a disease in a subject in need thereof comprises administering to the subject any of the compositions disclosed herein.
  • the preparations (e.g., siNA molecules or compositions) of the present disclosure may be given orally, parenterally, topically, or rectally. They are of course given in forms suitable for each administration route. For example, they are administered in tablets or capsule form, administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral administrations are preferred.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • systemic administration means the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually.
  • the compounds (e.g., siNA molecules) of the present disclosure which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present disclosure, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of this disclosure may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular compound (e.g., siNA molecule) of the present disclosure employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the rate and extent of absorption, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • the particular compound e.g., siNA molecule
  • the route of administration e.g., the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the rate and extent of absorption, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start doses of the compounds (e.g., siNA molecules) of the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable daily dose of a compound (e.g., siNA molecule) of the disclosure is the amount of the compound that is the lowest dose effective to produce a therapeutic effect.
  • Such an effective dose generally depends upon the factors described above.
  • the compounds are administered at about 0.01 mg/kg to about 200 mg/kg, more preferably at about 0.1 mg/kg to about 100 mg/kg, even more preferably at about 0.5 mg/kg to about 50 mg/kg.
  • the compound is administered at a dose equal to or greater than 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1 mg/kg.
  • the compound is administered at a dose equal to or less than 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, or 15 mg/kg.
  • the total daily dose of the compound is equal to or greater than 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 100 mg.
  • the effective amount may be less than when the compound is used alone.
  • the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • Preferred dosing is one administration per day.
  • the compound is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 times a week.
  • the compound is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 times a month.
  • 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.
  • the compound is administered once every 1, 2, 3, 4, 5, 6, 7, or 8 weeks.
  • siNA molecules and compositions described herein may be administered to a subject to treat a disease. Further disclosed herein are uses of any of the siNA molecules or compositions disclosed herein in the manufacture of a medicament for treating a disease.
  • the disease is a viral disease.
  • the viral disease is caused by a DNA virus.
  • the DNA virus is a double stranded DNA (dsDNA virus).
  • the dsDNA virus is a hepadnavirus.
  • the hepadnavirus is a hepatitis B virus (HBV).
  • the disease is a liver disease.
  • the liver disease is nonalcoholic fatty liver disease (NAFLD).
  • the NAFLD is nonalcoholic steatohepatitis (NASH).
  • the liver disease is hepatocellular carcinoma (HCC).
  • the siNA is administered by subcutaneous (SC) or intravenous (IV) delivery.
  • SC subcutaneous
  • IV intravenous
  • 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 forms suitable for each administration route. For example, they are administered in tablets or capsule form, administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories.
  • subcutaneous administration is preferred.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • systemic administration means the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • These compounds may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spray, rectally, intravaginally, parenterally, intracisternally and topically, as by powders, ointments or drops, including buccally and sublingually.
  • the compounds (e.g., siNAs) of the present disclosure which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present disclosure, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of this disclosure may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular compound (e.g., siNA) of the present disclosure employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the rate and extent of absorption, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • the particular compound e.g., siNA
  • the route of administration e.g., the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the rate and extent of absorption, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start doses of the compounds (e.g., siNAs) of the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable daily dose of a compound (e.g., siNA) of the disclosure is the amount of the compound that is the lowest dose effective to produce a therapeutic effect.
  • Such an effective dose generally depends upon the factors described above.
  • the compounds are administered at about 0.01 mg/kg to about 200 mg/kg, more preferably at about 0.1 mg/kg to about 100 mg/kg, even more preferably at about 0.5 mg/kg to about 50 mg/kg.
  • 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.
  • the compound is administered at a dose equal to or greater than 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, or 1 mg/kg.
  • the compound is administered at a dose equal to or greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mg/kg. In some embodiments, the compound is administered at a dose equal to or less than 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, or 15 mg/kg.
  • the total daily dose of the compound is equal to or greater than 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 100 mg.
  • the effective daily dose of the active compound may be administered as two, three, four, five, six, seven, eight, nine, ten or more doses or sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • the compound is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 times.
  • Preferred dosing is one administration per day.
  • the compound is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 times a week.
  • the compound is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 times a month.
  • 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 every month. 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.
  • 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 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, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 days.
  • 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 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.
  • 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 over a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53 months.
  • the compound is administered at least once a week for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 weeks.
  • the compound is administered at least once a week for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 months.
  • the compound is administered at least twice a week for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 weeks.
  • 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.
  • 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.
  • 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.
  • the compound is administered at least once every four weeks for a period of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 weeks.
  • 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.
  • any one of the siNAs or compositions disclosed herein is administered in a particle or viral vector.
  • the viral vector is a vector of adenovirus, adeno-associated virus (AAV), alphavirus, flavivirus, herpes simplex virus, lentivirus, measles virus, picornavirus, poxvirus, retrovirus, or rhabdovirus.
  • the viral vector is a recombinant viral vector.
  • 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.
  • the subject of the described methods may be a mammal, and it includes humans and non-human mammals.
  • the subject is a human, such as an adult human.
  • Some embodiments include a method for treating an HBV virus in a subject infected with the virus comprising administering a therapeutically effective amount of one or more siNA of the present disclosure or a composition of the present disclosure to the subject in need thereof thereby reducing the viral load of the virus in the subject and/or reducing a level of a virus antigen in the subject.
  • the siNA may be complementary or hybridize to a portion of the target RNA in the virus, e.g., an X region and/or an S region of HBV.
  • any of the methods disclosed herein may further comprise administering to the subject an additional HBV treatment agent.
  • Any of the compositions disclosed herein may further comprise an additional HBV treatment agent.
  • the additional HBV treatment agent is selected from a nucleotide analog, nucleoside analog, a capsid assembly modulator (CAM), a recombinant interferon, an entry inhibitor, a small molecule immunomodulator and oligonucleotide therapy.
  • the additional HBV treatment agent is selected from HBV STOPSTM ALG-010133, HBV CAM ALG-000184, ASO 1, 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 A
  • the oligonucleotide therapy is selected from Nucleic Acid Polymers or S-Antigen Transport-inhibiting Oligonucleotide Polymers (NAPs or STOPS), siRNA, and ASO.
  • the oligonucleotide therapy is an additional siNA.
  • the additional siNA is selected from any of ds-siNA-001 to ds-siNA-0178.
  • the oligonucleotide therapy is an antisense oligonucleotide (ASO).
  • the ASO is ASO 1.
  • any of the siNAs disclosed herein are co-administered with STOPS.
  • any of the siNAs disclosed herein are co-administered with tenofovir. In some embodiments, any of the siNAs disclosed herein are co-administered with a CAM.
  • Exemplary CAMs are described in Berke et al., Antimicrob Agents Chemother, 2017, 61(8):e00560-17, Klumpp, et al., Gastroenterology, 2018, 154(3):652-662.e8, International Application Nos.
  • the CAM is ALG-000184, ALG-001075, ALG-001024, JNJ-632, BAY41-4109, or NVR3-778.
  • the siNA and the HBV treatment agent are administered simultaneously. In some embodiments, the siNA and the HBV treatment agent are administered concurrently. In some embodiments, the siNA and the HBV treatment agent are administered sequentially.
  • the siNA is administered prior to administering the HBV treatment agent. In some embodiments, the siNA is administered after administering the HBV treatment agent. In some embodiments, the siNA and the HBV treatment agent are in separate containers. In some embodiments, the siNA and the HBV treatment agent are in the same container.
  • any of the methods disclosed herein may further comprise administering to the subject a liver disease treatment agent.
  • Any of the compositions disclosed herein may further comprise a liver disease treatment agent.
  • the liver disease treatment agent is selected from a peroxisome proliferator-activator receptor (PPAR) agonist, farnesoid X receptor (FXR) agonist, lipid-altering agent, and incretin-based therapy.
  • PPAR peroxisome proliferator-activator receptor
  • FXR farnesoid X receptor
  • the PPAR agonist is selected from a PPAR ⁇ agonist, dual PPAR ⁇ / ⁇ agonist, PPAR ⁇ agonist, and dual PPAR ⁇ / ⁇ agonist.
  • the dual PPAR ⁇ agonist is a fibrate.
  • the PPAR ⁇ / ⁇ agonist is elafibranor. In some embodiments, the PPAR ⁇ agonist is a thiazolidinedione (TZD). In some embodiments, TZD is pioglitazone. In some embodiments, the dual PPAR ⁇ / ⁇ agonist is saroglitazar. In some embodiments, the FXR agonist is obeticholic acis (OCA). In some embodiments, the lipid-altering agent is aramchol. In some embodiments, the incretin-based therapy is a glucagon-like peptide 1 (GLP-1) receptor agonist or dipeptidyl peptidase 4 (DPP-4) inhibitor.
  • GLP-1 glucagon-like peptide 1
  • DPP-4 dipeptidyl peptidase 4
  • the GLP-1 receptor agonist is exenatide or liraglutide.
  • the DPP-4 inhibitor is sitagliptin or vildapliptin.
  • the siNA and the liver disease treatment agent are administered concurrently. In some embodiments, the siNA and the liver disease treatment agent are administered sequentially. In some embodiments, the siNA is administered prior to administering the liver disease treatment agent. In some embodiments, the siNA is administered after administering the liver disease treatment agent. In some embodiments, the siNA and the liver disease treatment agent are in separate containers. In some embodiments, the siNA and the liver disease treatment agent are in the same container.
  • the terms “patient” and “subject” refer to organisms to be treated by the methods of the present disclosure. Such organisms are preferably mammals (e.g., marines, simians, equines, bovines, porcinis, canines, felines, and the like), and more preferably humans.
  • mammals e.g., marines, simians, equines, bovines, porcinis, canines, felines, and the like
  • humans preferably humans.
  • an effective amount refers to the amount of a compound (e.g., a siNA of the present disclosure) sufficient to effect beneficial or desired results.
  • An effective amount can be administered in one or more administrations, applications, or dosages and is not intended to be limited to a particular formulation or administration route.
  • treating includes any effect, e.g., lessening, reducing, modulating, ameliorating or eliminating, that results in the improvement of the condition, disease, disorder, and the like, or ameliorating a symptom thereof.
  • the terms “alleviate” and “alleviating” refer to reducing the severity of the condition, such as reducing the severity by, for example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%.
  • composition refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo.
  • the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil/water or water/oil emulsions), and various types of wetting agents.
  • the compositions also can include stabilizers and preservatives.
  • stabilizers and adjuvants see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, Pa. [1975].
  • nucleobase refers to a nitrogen-containing biological compound that forms a nucleoside.
  • nucleobases include, but are not limited to, thymine, uracil, adenine, cytosine, guanine, aryl, heteroaryl, and an analogue or derivative thereof.
  • compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited processing steps.
  • compositions specifying a percentage are by weight unless otherwise specified. Further, if a variable is not accompanied by a definition, then the previous definition of the variable controls.
  • This example describes an exemplary method for synthesizing ds-siNAs, such as the siNAs disclosed in Table 6 (as identified by the ds-siNA ID).
  • oligonucleotides were synthesized on a DNA/RNA Synthesizers (Expedite 8909 or ABI-394) using standard oligonucleotide phosphoramidite chemistry starting from the 3′ residue of the oligonucleotide preloaded on CPG support.
  • the 0.1M I 2 , THF:Pyridine; Water-7:2:1 was used as oxidizing agent while DDTT ((dimethylamino-methylidene) amino)-3H-1,2,4-dithiazaoline-3-thione was used as the sulfur-transfer agent for the synthesis of oligoribonucleotide phosphorothioates.
  • DDTT (dimethylamino-methylidene) amino)-3H-1,2,4-dithiazaoline-3-thione was used as the sulfur-transfer agent for the synthesis of oligoribonucleotide phosphorothioates.
  • the stepwise coupling efficiency of all modified phosphoramidites was more than 98%.
  • the unconjugated and GalNac modified oligonucleotides were purified by anion-exchange HPLC.
  • the buffers were 20 mM sodium phosphate in 10% CH 3 CN, pH 8.5 (buffer A) and 20 mM sodium phosphate in 10% CH 3 CN, 1.0 M NaBr, pH 8.5 (buffer B). Fractions containing full-length oligonucleotides were pooled.
  • the purified dry siNA was then desalted using Sephadex G-25 M (Amersham Biosciences).
  • the cartridge was conditioned with 10 mL of deionized water thrice.
  • the purified siNA dissolved thoroughly in 2.5 mL RNAse free water was applied to the cartridge with very slow drop wise elution.
  • the salt free siNA was eluted with 3.5 ml deionized water directly into a screw cap vial.
  • siNA Approximately 0.10 OD of siNA is dissolved in water and then pipetted in special vials for IEX-HPLC and LC/MS analysis. Analytical HPLC and ES LC-MS established the integrity of the compounds.
  • Single strand oligonucleotides (Sense and Antisense strands) were annealed (1:1 by molar equivalents, heat 90° C. for 3 min followed by room temperature, 20 min) to give the duplex ds-siNA.
  • the final compounds were analyzed on size exclusion chromatography (SEC).
  • HepG2.2.15 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (ATCC 30-2002) supplemented to also contain 10% fetal calf serum (FCS). Cells were incubated at 37° C. in an atmosphere with 5% CO 2 in a humidified incubator. For transfection of HepG2.2.15 cells with HBV targeting siRNAs, cells were seeded at a density of 15000 cells/well in 96-well regular tissue culture plates. Transfection of cells was carried out using RNAiMAX (Invitrogen/Life Technologies) according to the manufacturer's instructions.
  • DMEM Dulbecco's Modified Eagle's Medium
  • FCS fetal calf serum
  • Dose-response experiments were done with oligo concentrations of 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625 and 0.07813 nM.
  • HBV targeting siRNA treatment e.g., ds-siRNA, as identified by the ds-siNA ID in Table 6
  • four wells were transfected in parallel, and individual data points were collected from each well.
  • bDNA QuantiGene2.0 branched DNA (bDNA) probe set specific for HBV genotype D (also called Hepatitis B virus subtype ayw, complete genome of 3182 base-pairs) as present in cell line HepG2.2.15.
  • bDNA QuantiGene2.0 branched DNA
  • the HBV on-target mRNA levels were normalized to the GAPDH mRNA level.
  • the activity of the HBV targeting ds-siRNAs was expressed as EC50, 50% reduction of normalized HBV RNA level from no drug control.
  • the cytotoxicity of the HBV targeting ds-siRNAs was expressed by CC50 of 50% reduction of GAPDH mRNA from no drug control.
  • the ds-siNAs synthesized in Example 1 are used to treat a hepatitis B virus infection in a subject.
  • a composition comprising a ds-siNA from Table 6 (as identified by the ds-siNA ID) and a pharmaceutically acceptable carrier is administered to the subject suffering from hepatitis B virus.
  • the ds-siNA from Table 6 is conjugated to N-acetylgalactosamine.
  • the ds-siNA is administered at a dose of 0.3 to 5 mg/kg every three weeks by subcutaneous injection or intravenous infusion.
  • the ds-siNAs from Tables 6A and 6B (as identified by the ds-siNA ID) will be evaluated for safety and efficacy in healthy volunteers and chronic hepatitis B patients.
  • ds-siNAs are being developed for the treatment of chronic hepatitis B (CHB) in adults.
  • CHB chronic hepatitis B
  • the study will be conducted in 3 parts, a single ascending-dose (SAD) phase in healthy volunteers (Group A), a single-dose (SD) phase in patients with CHB (Group B), and a multiple ascending-dose (MAD) phase in patients with CHB (Group C).
  • SAD single ascending-dose
  • SD single-dose
  • MAD multiple ascending-dose
  • Study Design Study Type Interventional (Clinical Trial) Estimated 50 participants Enrollment: Allocation: Randomized Intervention Sequential Assignment Model: Intervention Progression from the SAD phase to the first cohort in Model the MAD phase is contingent upon the Safety Review Description: Committee (SRC) review of a minimum of 14 days post- dose safety and tolerability data from all HV in at least the first 2 SAD cohorts.
  • the SRC will select one (or more) well-tolerated dose(s) from the SAD phase for administration in the SD and MAD phases. In all study phases, dosing will be staggered with the use of sentinel participants to allow time for the assessment of safety before additional subjects are exposed to study drug.
  • Masking Triple (Participant, Care Provider, Investigator) Masking This is a double-blind placebo-controlled study in Description: which the study site team, the Sponsor, and the participants will be blinded to treatment assignment. The unblinded pharmacist will cover each syringe, prior to transport to the bedside, to ensure blinding. Participants will be centrally assigned to randomized study intervention using an Interactive Voice/Web Response System (IVRS/IWRS). Primary Treatment Purpose:
  • ds-siNA ds-siNA ds-siNA is a synthetic ribonucleic Single dose, Subcutaneous acid interference (RNAi) drug that injection of 0.1 mg/kg of consists of double-stranded ds-siNA (HV) oligonucleotides conjugated to an N-acetyl-D-galactosamine (GalNAc) ligand.
  • RNAi Subcutaneous acid interference
  • HV double-strandeds-siNA
  • GalNAc N-acetyl-D-galactosamine
  • Placebo Comparator Cohort A1 Drug: Placebo for ds-siNA Placebo Sterile 9% saline for injection. Single dose, Subcutaneous Other Name: Placebo injection of 0.1 mg/kg of Placebo for ds-siNA (HV) Experimental: Cohort A2 Drug: ds-siNA ds-siNA ds-siNA is a synthetic ribonucleic Single dose, Subcutaneous acid interference (RNAi) drug that injection of 1.5 mg/kg of consists of double-stranded ds-siNA (HV) oligonucleotides conjugated to an N-acetyl-D-galactosamine (GalNAc) ligand.
  • RNAi Subcutaneous acid interference
  • ds-siNA sterile solution of the ds-siNA at a concentration of 185 mg/mL in water for injection (WFI).
  • Placebo Comparator Cohort A2 Drug: Placebo for ds-siNA Placebo Sterile 9% saline for injection.
  • ds-siNA ds-siNA ds-siNA is a synthetic ribonucleic Single dose, Subcutaneous acid interference (RNAi) drug that injection of 3 mg/kg of consists of double-stranded ds-siNA (HV) oligonucleotides conjugated to an N-acetyl-D-galactosamine (GalNAc) ligand.
  • RNAi Subcutaneous acid interference
  • HV double-strandeds-siNA
  • GalNAc N-acetyl-D-galactosamine
  • Placebo Comparator Cohort A3 Drug: Placebo for ds-siNA Placebo Sterile 9% saline for injection. Single dose, Subcutaneous Other Name: Placebo injection of 3 mg/kg of Placebo for ds-siNA (HV) Experimental: Cohort A4 Drug: ds-siNA ds-siNA ds-siNA is a synthetic ribonucleic Single dose, Subcutaneous acid interference (RNAi) drug that injection of 6 mg/kg of consists of double-stranded ds-siNA (HV) oligonucleotides conjugated to an N-acetyl-D-galactosamine (GalNAc) ligand.
  • RNAi Subcutaneous acid interference
  • ds-siNA sterile solution of the ds-siNA at a concentration of 185 mg/mL in water for injection (WFI).
  • Placebo Comparator Cohort A4 Drug: Placebo for ds-siNA Placebo Sterile 9% saline for injection.
  • ds-siNA ds-siNA ds-siNA is a synthetic ribonucleic Single dose, Subcutaneous acid interference (RNAi) drug that injection of 12 mg/kg of consists of double-stranded ds-siNA (HV) oligonucleotides conjugated to an N-acetyl-D-galactosamine (GalNAc) ligand.
  • RNAi Subcutaneous acid interference
  • HV double-strandeds-siNA
  • GalNAc N-acetyl-D-galactosamine
  • Placebo Comparator Cohort A5 Drug: Placebo for ds-siNA Placebo Sterile 9% saline for injection. Single dose, Subcutaneous Other Name: Placebo injection of 12 mg/kg of Placebo for ds-siNA (HV) Experimental: Cohort B Drug: ds-siNA ds-siNA ds-siNA is a synthetic ribonucleic Single dose, Subcutaneous acid interference (RNAi) drug that injection of 3 mg/kg of consists of double-stranded for ds-siNA (NUC na ⁇ ve, oligonucleotides conjugated to an CHB) N-acetyl-D-galactosamine (GalNAc) ligand.
  • RNAi Subcutaneous acid interference
  • Placebo Comparator Cohort B Drug: Placebo for ds-siNA Placebo Sterile 9% saline for injection.
  • Subcutaneous ds-siNA is a synthetic ribonucleic injection of 1.5 mg/kg of acid interference (RNAi) drug that ds-siNA administered every consists of double-stranded 28 days (NUC experienced, oligonucleotides conjugated to an CHB) N-acetyl-D-galactosamine (GalNAc) ligand.
  • RNAi acid interference
  • GalNAc N-acetyl-D-galactosamine
  • ds-siNA sterile solution of the ds-siNA at a concentration of 185 mg/mL in water for injection (WFI).
  • Placebo Comparator Cohort C1 Drug: Placebo for ds-siNA Placebo Sterile 9% saline for injection.
  • ds-siNA ds-siNA 4 doses- ds-siNA is a synthetic ribonucleic Subcutaneous injection acid interference (RNAi) drug that of 3 mg/kg of ds-siNA consists of double-stranded administered every 28 days oligonucleotides conjugated to an (NUC experienced, CHB) N-acetyl-D-galactosamine (GalNAc) ligand.
  • RNAi N-acetyl-D-galactosamine
  • ds-siNA sterile solution of the ds-siNA at a concentration of 185 mg/mL in water for injection (WFI).
  • Placebo Comparator Cohort C2 Drug: Placebo for ds-siNA Placebo 4 doses- Sterile 9% saline for injection.
  • ds-siNA ds-siNA 4 doses- ds-siNA is a synthetic ribonucleic
  • RNAi N-acetyl-D-galactosamine
  • Placebo Comparator Cohort C3 Drug: Placebo for ds-siNA Placebo 4 doses- Sterile 9% saline for injection. Subcutaneous injection Other Name: Placebo of 6 mg/kg of Placebo for ds-siNA administered every 28 days (NUC experienced, CHB)
  • ECG electrocardiogram
  • Negative screen for alcohol or drugs of abuse is a negative screen for alcohol or drugs of abuse.
  • Non-smokers for at least 3 months with a negative urinary cotinine concentration at screening are provided.
  • Chronic hepatitis B infection (Group B and C only).
  • NUC nucleotides
  • Antiviral therapy (other than entecavir or tenofovir) within 3 months of screening or treatment with interferon in the last 3 years (Group B and C only).
  • Example 7 monomer (1.2 g, 38.2% yield) as a white solid.
  • Example 8 monomer (3.54 g, 43.36% yield) as a yellow solid.
  • Example 9 monomer (5.75 g, 55.37% yield, 99.4% purity) as a white solid.
  • Example 10 monomer To a solution of 6 (8.4 g, 12.5 mmol) in MeCN (80 mL) was added P-1 (4.9 g, 16.26 mmol, 5.16 mL) at 0° C., followed by addition of DCI (1.624 g, 13.76 mmol) in one portion at 0° C. under Ar. The mixture was stirred at 25° C. for 2 h. Upon completion as monitored by LCMS, the reaction mixture was quenched with saturated aq.NaHCO 3 (20 mL) and extracted with DCM (50 mL*2). The combined organic layers were dried over anhydrous Na 2 SO 4 , filtered and concentrated under reduce pressure to give a residue.
  • P-1 4.9 g, 16.26 mmol, 5.16 mL
  • DCI 1.624 g, 13.76 mmol
  • Example 10 monomer (3.4 g, 72.1% yield) as a white foam.
  • reaction mixture was then diluted with DCM (100 mL) and washed with water (70 mL) and brine (70 mL), dried over Na 2 SO 4 , filtered and evaporated to give a residue.
  • the residue was purified by flash silica gel chromatography (ISCO®; 80 g SepaFlash® Silica Flash Column, Eluent of 0 ⁇ 100% Ethyl acetate/Petroleum ether gradient @ 60 mL/min) followed by reverse-phase HPLC (0.1% NH 3 .H 2 O condition, eluent at 74%) to give 4 (2.88 g, 25% yield) as a white solid.
  • This example provides an exemplary method for synthesizing the siNAs comprising a 5′-stabilized end caps disclosed herein.
  • the 5′-stabilized end cap and/or deuterated phosphoramidites were dissolved in anhydrous acetonitrile and oligonucleotide synthesis was performed on a Expedite 8909 Synthesizer using standard phosphoramidite chemistry.
  • An extended coupling (12 minutes) of 0.12 M solution of phosphoramidite in anhydrous CH 3 CN in the presence of Benzyl-thio-tetrazole (BTT) activator to a solid bound oligonucleotide followed by standard capping, oxidation and sulfurization produced modified oligonucleotides.
  • BTT Benzyl-thio-tetrazole
  • the crude oligonucleotides were precipitated with isopropanol and centrifuged (Eppendorf 5810R, 3000 g, 4° C., 15 min) to obtain a pellet.
  • the crude product was then purified using ion exchange chromatography (TSK gel column, 20 mM NaH 2 PO 4 , 10% CH 3 CN, 1 M NaBr, gradient 20-60% B over 20 column volumes) and fractions were analyzed by ion change chromatography on an HPLC. Pure fractions were pooled and desalted by Sephadex G-25 column and evaporated to dryness. The purity and molecular weight were determined by HPLC analysis and ESI-MS analysis. Single strand RNA oligonucleotides (sense and antisense strand) were annealed (1:1 by molar equivalents) at 90° C. for 3 min followed by RT 40 min) to produce the duplexes.
  • This example provides exemplary methods for testing the activity of the siNAs disclosed herein.
  • HepG2.2.15 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (ATCC 30-2002) supplemented to also contain 10% fetal calf serum (FCS). Cells were incubated at 37° C. in an atmosphere with 5% CO 2 in a humidified incubator. For transfection of HepG2.2.15 cells with HBV targeting siRNAs, cells were seeded at a density of 15000 cells/well in 96-well regular tissue culture plates. Transfection of cells was carried out using RNAiMAX (Invitrogen/Life Technologies) according to the manufacturer's instructions.
  • DMEM Dulbecco's Modified Eagle's Medium
  • FCS fetal calf serum
  • Dose-response experiments were done with oligo concentrations of 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625 and 0.07813 nM.
  • HBV targeting siRNA treatment e.g., ds-siRNA, as identified by the ds-siNA ID in Table 6
  • four wells were transfected in parallel, and individual data points were collected from each well.
  • bDNA QuantiGene2.0 branched DNA (bDNA) probe set specific for HBV genotype D (also called Hepatitis B virus subtype ayw, complete genome of 3182 base-pairs) as present in cell line HepG2.2.15.
  • bDNA QuantiGene2.0 branched DNA
  • the HBV on-target mRNA levels were normalized to the GAPDH mRNA level.
  • the activity of the HBV targeting ds-siRNAs was expressed as EC50, 50% reduction of normalized HBV RNA level from no drug control.
  • the cytotoxicity of the HBV targeting ds-siRNAs was expressed by CC50 of 50% reduction of GAPDH mRNA from no drug control.
  • Unconjugated siRNA 1) with or without a phosphorylation blocker; and 2) with or without end caps (e.g., 5′-stabilized end cap) are transfected into in vitro disease models or in vitro toxicity models. After transfection, target reduction and/or cell viability is measured and compared after a period of incubation.
  • exemplary disease cell models include, but are not limited to, HepG2.2.15, HepG2.117 or live HBV infected HepG2-NTCP or Primary Human Hepatocytes.
  • GalNAc conjugated siRNA 1) with or without phosphorylation blocker; and 2) with or without 5′-end caps are dosed subcutaneously or intravenously in animal disease models.
  • the target knockdown magnitude and duration is measured from serum or liver samples and compared to each other and/or control animals (e.g., non-treated diseased animals).
  • the toxicity of the siRNAs is compared through routine Clinpath or Histopath assays.
  • exemplary animal efficacy models include, but are not limited to, AAV-HBV mouse model, HBV transgenic mouse model, PXB or FRG mouse models.
  • ds-siNAs efficacy of ds-siNAs in treating HBV in an adeno-associated virus (AAV)-HBV mouse model was evaluated.
  • AAV-HBV mice were subcutaneously injected with a single dose of (a) 5 mL/kg of vehicle; or (b) 5 mg/kg a ds-siNA at day 0.
  • the sequences of the ds-siNA tested in this example are shown in Table 7.
  • FIG. 4 shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01), ds-siNA-0160 (G03), ds-siNA-0165 (G04), ds-siNA-0163 (G05), or ds-siNA-0166 (G06).
  • the ds-siNAs comprise a sense and antisense strand comprising a mixture of 2′-fluoro and 2′-O-methyl nucleotides.
  • the total number of 2′-fluoro nucleotides in the ds-siNAs are between 6-8.
  • the 2′-fluoro nucleotides may be at specific positions, such as nucleotide position 3, 5, 7, 8, 9, 10, 11, 12, and/or 17 from the 5′ end of the sense strand or 2, 5, 6, 8, 10, 14, 16, 17, and/or 18.
  • the 2′-fluoro nucleotides and 2′-O-methyl nucleotides might occur at specific patterns on the antisense strand, such as an alternating 1:2 or 1:3 pattern, wherein 1 nucleotide is a 2′-fluoro nucleotide and 2 or 3 nucleotides are 2-O-methyl nucleotides.
  • HepG2.2.15 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) (ATCC 30-2002) supplemented to also contain 10% fetal calf serum (FCS). Cells were incubated at 37° C. in an atmosphere with 5% CO 2 in a humidified incubator. For transfection of HepG2.2.15 cells with HBV targeting siRNAs, cells were seeded at a density of 15000 cells/well in 96-well regular tissue culture plates. Transfection of cells was carried out using RNAiMAX (Invitrogen/Life Technologies) according to the manufacturer's instructions.
  • DMEM Dulbecco's Modified Eagle's Medium
  • FCS fetal calf serum
  • Dose-response experiments were done with oligo concentrations of 40, 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625 and 0.07813 nM.
  • HBV targeting siRNA treatment e.g., ds-siRNA, as identified by the ds-siNA ID in Table 8
  • four wells were transfected in parallel, and individual data points were collected from each well.
  • bDNA QuantiGene2.0 branched DNA (bDNA) probe set specific for HBV genotype D (also called Hepatitis B virus subtype ayw, complete genome of 3182 base-pairs) as present in cell line HepG2.2.15.
  • bDNA QuantiGene2.0 branched DNA
  • AAV/HBV is a recombinant AAV carrying replicable HBV genome. Taking advantage of the highly hepatotropic feature of genotype 8 AAV, the HBV genome can be efficiently delivered to the mouse liver cells. Infection of immune competent mouse with AAV/HBV can result in long term HBV viremia, which mimics chronic HBV infection in patients.
  • the AAV/HBV model can be used to evaluate the in vivo activity of various types of anti-HBV agents. Mice were infected with AAV-HBV on day ⁇ 28 of the study.
  • the test articles or negative control (PBS) were dosed subcutaneously (unless specified otherwise) as single dose on days 0 at 5 mg/kg. Serial blood collections were usually taken every 5 days on day 0, 5, 10 and 15 etc. until the termination of studies. Serum HBV S antigen (HBsAg) was assayed through ELISA.
  • GalNAc conjugated ds-siNAs were further tested at a single dose of 5 mg/kg at day 0 in the adeno-associated virus (AAV)-HBV mouse model.
  • the resulting nadir log 10 reduction in serum HBsAg is presented in Table 8, where X ⁇ 1 log 10 reduction in HBsAg, Y is 0.5-1 log 10 reduction in HBsAg, and Z is ⁇ 0.5 log 10 reduction in HBsAg.
  • FIG. 5A shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01) or ds-siNA-0160 (G03).
  • AAV-HBV mice were subcutaneously injected with a single dose of 5 mL/kg of vehicle or 5 mg/kg of ds-siNA-0160 on day 0.
  • FIG. 5B shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01) or ds-siNA-0160 (G15).
  • AAV-HBV mice were subcutaneously injected with a single dose of 5 mL/kg of vehicle or 5 mg/kg of ds-siNA-0160 on day 0.
  • FIG. 5C shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01) or ds-siNA-0160 (G03).
  • AAV-HBV mice were subcutaneously injected with a single dose of 5 mL/kg of vehicle or 5 mg/kg of each ds-siNA on day 0.
  • FIG. 5D shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01), ds-siNA-0160 (G03), or ds-siNA-0109 (G09).
  • AAV-HBV mice were subcutaneously injected with a single dose of 5 mL/kg of vehicle or 5 mg/kg of each ds-siNA on day 0.
  • FIGS. 5E-5F show a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01) or ds-siNA-0169 (G18).
  • AAV-HBV mice were subcutaneously injected with a single dose of 5 mL/kg of vehicle or 5 mg/kg of ds-siNA-0169 on day 0.
  • FIG. 5G shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01) or ds-siNA-0169 (G04).
  • AAV-HBV mice were subcutaneously injected with a single dose of 5 mL/kg of vehicle or 5 mg/kg of ds-siNA-0169 on day 0.
  • FIG. 5H shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01) or ds-siNA-0169 (G04).
  • AAV-HBV mice were subcutaneously injected with a single dose of 5 mL/kg of vehicle or 5 mg/kg of each ds-siNA on day 0.
  • FIG. 5I shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01), ds-siNA-0169 (G04) or ds-siNA-0147 (G08).
  • AAV-HBV mice were subcutaneously injected with a single dose of 5 mL/kg of vehicle or 5 mg/kg of each ds-siNA on day 0.
  • FIG. 5J shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01), ds-siNA-0166 (G06), or ds-siNA-0153 (G14).
  • AAV-HBV mice were subcutaneously injected with a single dose of 5 mL/kg of vehicle or 5 mg/kg of each ds-siNA on day 0.
  • FIG. 5K shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01), ds-siNA-0163 (G05), or ds-siNA-0119 (G13).
  • AAV-HBV mice were subcutaneously injected with a single dose of 5 mL/kg of vehicle or 5 mg/kg of each ds-siNA on day 0.
  • ds-siNAs comprising combination of 2′-fluoro nucleotides and 2′-O-methyl nucleotides can be used to target HBV X and S gene sequences, which resulted in successful treatment of HBV.
  • ds-siNAs comprising (a) a sense strand comprising 19 nucleotides, wherein 6 nucleotides are 2′-fluoro nucleotides and 13 nucleotides are 2′-O-methyl nucleotides; (b) an antisense strand comprising 21 nucleotides, wherein 2 nucleotides are 2′-fluoro nucleotides and 19 nucleotides are 2′-O-methyl nucleotides; and (c) a conjugated moiety, wherein the conjugated moiety is attached to the 3′ end of the sense strand, resulted in successful treatment of HBV as evidenced by HBsAg reduction in serum.
  • ds-siNA-0160 and ds-siNA-0165 the 2′-fluoro nucleotides were located at positions 3, 7-9, 12, and 17 from the 5′ end of the sense strand and at positions 2 and 14 from the 5′ end of the antisense strand.
  • ds-siNAs comprising (a) a sense strand comprising 19 nucleotides, wherein 4 nucleotides are 2′-fluoro nucleotides and 15 nucleotides are 2′-O-methyl nucleotides; (b) an antisense strand comprising 21 nucleotides, wherein 2 nucleotides are 2′-fluoro nucleotides and 19 nucleotides are 2′-O-methyl nucleotides; and (c) a conjugated moiety, wherein the conjugated moiety is attached to the 3′ end of the sense strand, resulted in successful treatment of HBV as evidenced by HBsAg reduction in serum.
  • the 2′-fluoro nucleotides were located at positions 3, 7, 8, and 17 from the 5′ end of the sense strand and at positions 2 and 14 from the 5′ end of the antisense strand.
  • ds-siNAs comprising (a) a sense strand comprising 19 nucleotides; (b) an antisense strand comprising 21 nucleotides, wherein the nucleotides in the antisense strand comprise at least two alternating 1:3 modification pattern, and wherein approximate 1 nucleotide is a 2′-fluoro nucleotide and 3 nucleotides are 2′-O-methyl nucleotides in repeat pattern; and (c) a conjugated moiety, wherein the conjugated moiety is attached to the 3′ end of the sense strand, resulted in successful treatment of HBV as evidenced by HBsAg reduction in serum. See FIG.
  • the sense strand comprises 6 2′-fluoro nucleotides at positions 3, 7-9, 12, and 17 from the 5′ end of the sense strand.
  • the antisense strand comprises 5 repeats of the 1:3 modification pattern starting at position 2 from the 5′ end of the antisense strand.
  • ds-siNAs comprising (a) a sense strand comprising 19 nucleotides wherein 4 nucleotides are 2′-fluoro nucleotides and 15 nucleotides are 2′-O-methyl nucleotides; (b) an antisense strand comprising 21 nucleotides, wherein 4 nucleotides are 2′-fluoro nucleotides and 17 nucleotides are 2′-O-methyl nucleotides; and (c) a conjugated moiety, wherein the conjugated moiety is attached to the 3′ end of the sense strand, resulted in successful treatment of HBV as evidenced by HBsAg reduction in serum.
  • the sense strand comprises 4 2′-fluoro nucleotides at positions 5 and 7-9 from the 5′ end of the sense strand.
  • the antisense strand comprises 5 repeats of the 1:2 modification pattern starting at positions 2, 5, 8, 14, and 17 from the 5′ end of the antisense strand.
  • ds-siNAs comprising (a) a sense strand comprising 19 nucleotides; (b) an antisense strand comprising 21 nucleotides, wherein the nucleotides in the antisense strand comprise at least two alternating 1:2 modification pattern, and wherein approximate 1 nucleotide is a 2′-fluoro nucleotide and 2 nucleotides are 2′-O-methyl nucleotides in repeat pattern; and (c) a conjugated moiety, wherein the conjugated moiety is attached to the 3′ end of the sense strand, resulted in successful treatment of HBV as evidenced by HBsAg reduction in serum. See FIG.
  • the 2′-fluoro nucleotides were located at positions 5 and 7-9 from the 5′ end of the sense strand and at positions 2, 6, 14, and 16 from the 5′ end of the antisense strand.
  • FIG. 6A shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01), ds-siNA-0160 (G15) (ds-siNA without a 5′-stabilized end cap, e.g., vinyl phosphonate), or ds-siNA-080 (G14) (ds-siNA with a 5′-stabilized end cap, e.g., vinyl phosphonate).
  • vehicle G01
  • ds-siNA-0160 G15
  • ds-siNA-siNA-080 ds-siNA with a 5′-stabilized end cap, e.g., vinyl phosphonate
  • the resulting nadir log 10 reduction in serum HBsAg is presented in Table 9, where X ⁇ 1 log 10 reduction in HBsAg, Y is 0.5-1 log 10 reduction in HBsAg, and Z is ⁇ 0.5 log 10 reduction in HBsAg.
  • FIG. 6B shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01), ds-siNA-0169 (G16) (ds-siNA without a 5′-stabilized end cap, e.g., vinyl phosphonate), or ds-siNA-081 (G13) (ds-siNA with a 5′-stabilized end cap, e.g., vinyl phosphonate).
  • AAV-HBV mice were subcutaneously injected with a single dose of 5 mL/kg of vehicle or 5 mg/kg of each ds-siNA on day 0.
  • the resulting nadir log 10 reduction in serum HBsAg is presented in Table 9, where X ⁇ 1 log 10 reduction in HBsAg, Y is 0.5-1 log 10 reduction in HBsAg, and Z is ⁇ 0.5 log 10 reduction in HBsAg.
  • FIG. 7A shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01), ds-siNA-0165 (G18) (ds-siNA without a 5′-stabilized end cap, e.g., vinyl phosphonate), or ds-siNA-0127 (G17) (ds-siNA with a 5′-stabilized end cap, e.g., vinyl phosphonate).
  • AAV-HBV mice were subcutaneously injected with a single dose of 5 mL/kg of vehicle or 5 mg/kg of each ds-siNA on day 0.
  • the resulting nadir log 10 reduction in serum HBsAg is presented in Table 9, where X ⁇ 1 log 10 reduction in HBsAg, Y is 0.5-1 log 10 reduction in HBsAg, and Z is ⁇ 0.5 log 10 reduction in HBsAg.
  • FIG. 7B shows a graph of the change in serum HBsAg from AAV-HBV mice treated with vehicle (G01), ds-siNA-0168 (G20) (ds-siNA without a 5′-stabilized end cap, e.g., vinyl phosphonate), or ds-siNA-0150 (G19) (ds-siNA with a 5′-stabilized end cap, e.g., vinyl phosphonate).
  • AAV-HBV mice were subcutaneously injected with a single dose of 5 mL/kg of vehicle or 5 mg/kg of each ds-siNA on day 0.

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