Modified short interfering nucleic acid (siNA) molecules and uses thereof
Disclosed herein are short interfering nucleic acid (siNA) molecules comprising modified nucleotides and uses thereof. The siNA molecules may be double stranded and comprise modified nucleotides selected from 2′-O-methyl nucleotides and 2′-fluoro nucleotides. Further disclosed herein are siNA molecules comprising (a) a phosphorylation blocker, conjugated moiety, or 5′-stabilized end cap; and (b) a short interfering nucleic acid (siNA).
1. A double stranded short interfering nucleic acid (siNA) comprising:
(a) a sense strand comprising 19-21 nucleotides in a nucleic acid sequence that is at least 80% identical to SEQ ID NO: 40, wherein 15 or more of the nucleotides are modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide, and wherein at least 11 of the modified nucleotides are a 2′-O-methyl nucleotide and at least 4 of the modified nucleotides are a 2′-fluoro nucleotide; and
(b) an antisense strand comprising 19-21 nucleotides in a nucleic acid sequence that is at least 80% complementary to SEQ ID NO: 40, wherein 15 or more of the nucleotides are modified nucleotides independently selected from a 2′-O-methyl nucleotide and a 2′-fluoro nucleotide, and wherein at least 11 of the modified nucleotides are a 2′-O-methyl nucleotide and 4 to 6 of the modified nucleotides are a 2′-fluoro nucleotide.
2. The double stranded siNA of claim 1 , wherein:
(a) the nucleotide(s) at position 3, 5, 7, 8, 9, 10, 11, 12, 14, 17, or 19 from the 5′ end of the sense strand is a 2′-fluoro nucleotide; and
(b) the nucleotide(s) at position 2, 5, 6, 8, 10, 14, 16, 17, or 18 from the 5′ end of the antisense strand is a 2′-fluoro nucleotide.
3. The double stranded siNA of claim 1 , wherein the sense strand comprises SEQ ID NO: 438 or SEQ ID NO: 435, and the antisense strand comprises any one of SEQ ID NOs: 501-519, SEQ ID NO: 537, SEQ ID NO: 538, or SEQ ID NO: 539.
4. A double stranded short interfering nucleic acid (siNA) comprising:
(a) a sense strand comprising 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; and
(b) an antisense strand comprising 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.
5. The double stranded siNA of claim 4 further comprising (i) the nucleotides at positions 1 and 2 and positions 2 and 3 from the 5′ end of the sense strand are connected by phosphorothioate internucleoside linkages; and (ii) 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 are connected by phosphorothioate internucleoside linkages.
6. The double stranded siNA of claim 4 further comprising a conjugated moiety attached to the sense strand.
7. The double stranded siNA of claim 6 , wherein the conjugated moiety is a galactosamine.
8. The double stranded siNA of claim 6 , wherein the conjugated moiety is N-acetylgalactosamine (GalNAc).
9. The double stranded siNA of claim 4 , wherein the sense strand comprises SEQ ID NO: 438, and the antisense strand comprises any one of SEQ ID NO: 501, SEQ ID NO: 537, SEQ ID NO: 538, or SEQ ID NO: 539.
10. The double stranded siNA of claim 4 , wherein the sense strand is at least 80% identical to SEQ ID NO: 40 and the antisense strand is at least 80% complementary to SEQ ID NO: 40.
11. The double stranded siNA of claim 4 , wherein the sense strand or the antisense strand comprises at least one overhang consisting of 1 or 2 nucleotides.
12. The double stranded siNA of claim 4 , wherein both the sense strand and the antisense strand each independently comprise an overhang consisting of 1 or 2 nucleotides, wherein the overhang on the sense strand and the overhang on the antisense strand are on opposite ends of the double stranded siNA.
13. A short interfering nucleic acid (siNA) molecule comprising:
(a) a sense strand comprising a nucleic acid sequence consisting of SEQ ID NO: 438 or SEQ ID NO: 435, and
(b) an antisense strand comprising a nucleic acid sequence consisting of any one of SEQ ID NO: 501, SEQ ID NO: 505, SEQ ID NO: 506, SEQ ID NO: 537, SEQ ID NO: 538, and SEQ ID NO: 539.
14. The double stranded siNA of claim 9 , wherein the senses strand comprises SEQ ID NO: 438 and the antisense comprises SEQ ID NO: 501.
15. The double stranded siNA of claim 9 , wherein the senses strand comprises SEQ ID NO: 438 and the antisense comprises any one of SEQ ID NO: 537-539.
16. A double stranded short interfering nucleic acid (siNA) comprising:
(a) a sense strand of
(SEQ ID NO: 438)
5′-mGpsmUpsmGmGfUmGfGfAfCmUmUmCmUmCmUmCmAmAmU-p-
ps2-GalNAc-3′
and an antisense strand of
(SEQ ID NO: 501)
5′-mApsfUpsmUmGmAfGmAmGmAmAmGmUmCfCmAfCmCmAmCpsm
GpsmA-3′;
(b) a sense strand of
(SEQ ID NO: 438)
5′-mGpsmUpsmGmGfUmGfGfAfCmUmUmCmUmCmUmCmAmAmU-p-
ps2-GalNAc-3′
and an antisense strand of
(SEQ ID NO: 521)
5′-mApsfUpsmUmGmAfGmAfGfAmAmGmUmCfCmAfCmCmAmCpsm
GpsmA-3′;
(c) a sense strand of
(SEQ ID NO: 438)
5′-mGpsmUpsmGmGfUmGfGfAfCmUmUmCmUmCmUmCmAmAmU-p-
ps2-GalNAc-3′
and an antisense strand of
(SEQ ID NO: 522)
5′-mApsfUpsmUmGfAmGmAfGmAmAmGmUmCfCmAmCfCmAmCpsm
GpsmA-3′;
(d) a sense strand of
(SEQ ID NO: 438)
5′-mGpsmUpsmGmGfUmGfGfAfCmUmUmCmUmCmUmCmAmAmU-
p-ps2-GalNAc-3′
and an antisense strand of
(SEQ ID NO: 537)
5′-mApsf4PpsmUmGmAfGmAmGmAmAmGmUmCfCmAfCmCmAmCp
smGpsmA-3′;
(e) a sense strand of
(SEQ ID NO: 438)
5′-mGpsmUpsmGmGfUmGfGfAfCmUmUmCmUmCmUmCmAmAmU-
p-ps2-GalNAc-3′
and an antisense strand of
(SEQ ID NO: 538)
5′-mApsfUpsmUmGmAfGmAmGmAmAmGmUmCf2PmAfCmCmAmCp
smGpsmA-3′;
or
(f) a sense strand of
(SEQ ID NO: 438)
5′-mGpsmUpsmGmGfUmGfGfAfCmUmUmCmUmCmUmCmAmAmU-p-
ps2-GalNAc-3′
and an antisense strand of
(SEQ ID NO: 539)
5′-mApsfUpsmUmGmAfGmAmGmAmAmGmUmCfCmAfXmCmAmCpsm
GpsmA-3′,
wherein fX is
wherein GalNAc comprises a structure of Formula (VII):
wherein n is 1, and R is OH.
17. The double stranded siNA of claim 16 , comprising a sense strand of 5′-mGpsmUpsmGmGfUmGfGfAfCmUmUmCmUmCmUmCmAmAmU-p-ps2-GalNAc-3′ (SEQ ID NO: 438) and an antisense strand of 5′-mApsfUpsmUmGmAfGmAmGmAmAmGmUmCfCmAfCmCmAmCpsmGpsmA-3′ (SEQ ID NO: 501).
18. The double stranded siNA of claim 16 , comprising a sense strand of 5′-mGpsmUpsmGmGfUmGfGfAfCmUmUmCmUmCmUmCmAmAmU-p-ps2-GalNAc-3′ (SEQ ID NO: 438) and an antisense strand of 5′-mApsfUpsmUmGmAfGmAfGfAmAmGmUmCfCmAfCmCmAmCpsmGpsmA-3′ (SEQ ID NO: 521).
19. The double stranded siNA of claim 16 , comprising a sense strand of 5′-mGpsmUpsmGmGfUmGfGfAfCmUmUmCmUmCmUmCmAmAmU-p-ps2-GalNAc-3′ (SEQ ID NO: 438) and an antisense strand of 5′-mApsfUpsmUmGfAmGmAfGmAmAmGmUmCfCmAmCfCmAmCpsmGpsmA-3′ (SEQ ID NO: 522).
20. The double stranded siNA of claim 16 , comprising a sense strand of 5′-mGpsmUpsmGmGfUmGfGfAfCmUmUmCmUmCmUmCmAmAmU-p-ps2-GalNAc-3′ (SEQ ID NO: 438) and an antisense strand of 5′-mApsf4PpsmUmGmAfGmAmGmAmAmGmUmCfCmAfCmCmAmCpsmGpsmA-3′ (SEQ ID NO: 537).
21. The double stranded siNA of claim 16 , comprising a sense strand of 5′-mGpsmUpsmGmGfUmGfGfAfCmUmUmCmUmCmUmCmAmAmU-p-ps2-GalNAc-3′ (SEQ ID NO: 438) and an antisense strand of 5′-mApsfUpsmUmGmAfGmAmGmAmAmGmUmCf2PmAfCmCmAmCpsmGpsmA-3′ (SEQ ID NO: 538).
22. The double stranded siNA of claim 16 , comprising a sense strand of 5′-mGpsmUpsmGmGfUmGfGfAfCmUmUmCmUmCmUmCmAmAmU-p-ps2-GalNAc-3′ (SEQ ID NO: 438) and an antisense strand of 5′-mApsfUpsmUmGmAfGmAmGmAmAmGmUmCfCmAfXmCmAmCpsmGpsmA-3′ (SEQ ID NO: 539), wherein fX is
23. The double stranded siNA of claim 9 further comprising a galactosamine attached to the sense strand.
24. The double stranded siNA of claim 9 further comprising a N-acetylgalactosamine (GalNAc) attached to the 3′ of the sense strand.
25. The double stranded siNA of claim 24 , wherein the GalNAc comprises a structure of Formula (VII):
wherein n is 1, and R is OH.
26. The siNA molecule of claim 13 further comprising a galactosamine attached to the sense strand.
27. The siNA molecule of claim 13 further comprising a N-acetylgalactosamine (GalNAc) attached to the 3′ of the sense strand.
28. The siNA molecule of claim 27 , wherein the GalNAc comprises a structure of Formula (VII):
wherein n is 1, and R is OH.
29. The double stranded siNA of claim 14 further comprising a N-acetylgalactosamine (GalNAc) attached to the 3′ of the sense strand.
30. The double stranded siNA of claim 29 , wherein the GalNAc comprises a structure of Formula (VII):
wherein n is 1, and R is OH.