Branched oligonucleotides
Provided herein are branched oligonucleotides exhibiting efficient and specific tissue distribution, cellular uptake, minimum immune response and off-target effects, without formulation.
1. A method of delivering an siRNA molecule to the central nervous system of a subject, the method comprising intrastriatally, intrathecally, or intracerebroventricularly administering to the subject a di-branched oligonucleotide compound comprising two siRNA molecules covalently bound to one another by way of a linker,
wherein:
(a) each siRNA molecule of the di-branched oligonucleotide compound comprises an antisense strand having complementarity to a target gene and a sense strand having complementarity to the antisense strand;
(b) from 80% to 100% of the nucleotides in each siRNA molecule of the di-branched oligonucleotide compound are chemically modified; and
(c) each antisense strand and each sense strand are, independently, from 15 to 30 nucleotides in length; and
(d) wherein the 5′ end of each antisense strand, independently, comprises a terminal group represented by a formula selected from (X1)-(X8):
2. The method of claim 1 , wherein each sense strand is, independently, from 15 to 20 nucleotides in length.
3. The method of claim 1 , wherein each antisense strand is, independently, from 18 to 30 nucleotides in length.
4. The method of claim 1 , wherein the nucleotides of each antisense strand and each sense strand consist of chemically modified nucleotides selected from 2′-methoxy-ribonucleotides and 2′-fluoro-ribonucleotides.
5. The method of claim 1 , wherein each antisense strand and each sense strand comprise alternating 2′-methoxy-ribonucleotides and 2′-fluoro-ribonucleotides.
6. The method of claim 2 , wherein each sense strand is, independently, 15, 16, or 17 nucleotides in length.
7. The method of claim 6 , wherein each sense strand is, independently, 16 nucleotides in length.
8. The method of claim 6 , wherein each antisense strand is, independently, from 18 to 30 nucleotides in length.
9. The method of claim 7 , wherein each antisense strand is, independently, from 18 to 30 nucleotides in length.
10. The method of claim 1 , wherein the di-branched oligonucleotide compound comprises 5-20 phosphorothioated bonds.
11. The method of claim 1 , wherein the nucleotides at positions 1-6 from the 3′ end of each antisense strand, independently, or positions 1-7 from the 3′ end of each antisense strand, independently, are connected to adjacent nucleotides via phosphorothioate linkages.
12. The method of claim 1 , wherein the nucleotides at positions 1 and 2 from the 5′ end of each sense and antisense strands are, independently, connected to adjacent nucleotides via phosphorothioate linkages.
13. The method of claim 1 , wherein each siRNA molecule comprises an unpaired overhang of at least 2 nucleotides.
14. The method of claim 13 , wherein the nucleotides of the overhang are connected via phosphorothioate linkages.
15. The method of claim 1 , wherein each antisense strand has, independently, complementarity to a target mRNA in a neuronal cell.
16. A method of delivering an siRNA molecule to the central nervous system of a subject, the method comprising intrastriatally, intrathecally, or intracerebroventricularly administering to the subject a di-branched oligonucleotide compound comprising two siRNA molecules covalently bound to one another by way of a linker,
wherein:
(a) each siRNA molecule of the di-branched oligonucleotide compound comprises an antisense strand having complementarity to a target gene and a sense strand having complementarity to the antisense strand;
(b) from 80% to 100% of the nucleotides in each siRNA molecule of the di-branched oligonucleotide compound are chemically modified; and
(c) each antisense strand and each sense strand are, independently, from 15 to 30 nucleotides in length; and
(d) wherein the 5′ end of each antisense strand comprises a terminal group represented by formula (X3):
17. The method of claim 16 , wherein each siRNA molecule comprises an unpaired overhang of at least 2 nucleotides.
18. The method of claim 17 , wherein the nucleotides of the overhang are connected via phosphorothioate linkages.
19. A method of delivering an siRNA molecule to the central nervous system of a subject, the method comprising intrastriatally, intrathecally, or intracerebroventricularly administering to the subject a di-branched oligonucleotide compound comprising two siRNA molecules covalently bound to one another by way of a linker,
wherein:
(a) each siRNA molecule of the di-branched oligonucleotide compound comprises an antisense strand having complementarity to a target gene and a sense strand having complementarity to the antisense strand;
(b) from 80% to 100% of the nucleotides in each siRNA molecule of the di-branched oligonucleotide compound are chemically modified; and
(c) each antisense strand and each sense strand are, independently, from 15 to 30 nucleotides in length; and
(d) wherein the 5′ end of each antisense strand comprises an (E)-vinylphosphonate terminal group.
20. The method of claim 19 , wherein each antisense strand and each sense strand comprise alternating 2′-methoxy-ribonucleotides and 2′-fluoro-ribonucleotides.