Oligonucleotide compounds for targeting huntingtin mRNA
This disclosure relates to novel huntingtin targets. Novel oligonucleotides for the treatment of Huntington's disease are also provided.
1. A vector for inhibiting the expression of HTT gene in a cell, said vector comprising a regulatory sequence operably linked to a nucleotide sequence that encodes a dsRNA molecule substantially complementary to 5′ CAGUAAAGAGAUUAA 3′ (SEQ ID NO:1), 5′ AUAUCAGUAAAGAGA 3′ (SEQ ID NO:2) or 5′ CUCAGGAUUUAAAAU 3′ (SEQ ID NO:3), wherein said dsRNA molecule is between 10 and 35 bases in length.
2. A cell comprising the vector of claim 1 .
3. A dsRNA molecule that is between 15 and 35 base pairs in length, comprising a region of complementarity which is substantially complementary to 5′ CAGUAAAGAGAUUAA 3′ (SEQ ID NO:1), 5′ AUAUCAGUAAAGAGA 3′ (SEQ ID NO:2) or 5′ CUCAGGAUUUAAAAU 3′ (SEQ ID NO:3), wherein the RNA molecule targets an HTT mRNA and comprises at least one modified nucleotide.
4. A pharmaceutical composition for inhibiting the expression of the HTT gene in an organism, comprising the dsRNA of claim 3 and a pharmaceutically acceptable carrier.
5. The dsRNA molecule of claim 3 , wherein the dsRNA comprises a sense strand and an antisense strand, wherein the antisense strand comprises the region of complementarity which is substantially complementary to 5′ CAGUAAAGAGAUUAA 3′ (SEQ ID NO:1).
6. The dsRNA molecule of claim 3 , wherein the dsRNA comprises a sense strand and an antisense strand, wherein the antisense strand comprises the region of complementarity which is substantially complementary to 5′ AUAUCAGUAAAGAGA 3′ (SEQ ID NO:2).
7. The dsRNA molecule of claim 3 , wherein the dsRNA comprises a sense strand and an antisense strand, wherein the antisense strand comprises the region of complementarity which is substantially complementary to 5′ CUCAGGAUUUAAAAU 3′ (SEQ ID NO:3).
8. The dsRNA molecule of claim 3 , wherein the dsRNA comprises a sense strand and an antisense strand, wherein the antisense strand is complementary to at least 10, 11, 12 or 13 contiguous nucleotides of SEQ ID NO:1, 2 or 3.
9. The dsRNA molecule of claim 3 , wherein the dsRNA comprises a sense strand and an antisense strand, wherein the antisense strand contains no more than 3 mismatches with SEQ ID NO:1, 2 or 3.
10. The dsRNA molecule of claim 3 , wherein the dsRNA comprises a sense strand and an antisense strand, wherein the antisense strand is fully complementary to SEQ ID NO:1, 2 or 3.
11. The dsRNA molecule of claim 3 , which is between 30 and 35 base pairs in length.
12. The dsRNA molecule of claim 3 , which is blunt-ended.
13. The dsRNA molecule of claim 3 , which comprises at least one single stranded nucleotide overhang.
14. The dsRNA molecule of claim 3 , wherein the at least one modified nucleotide is selected from the group consisting of a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or dodecanoic acid bisdecylamide group.
15. The dsRNA molecule of claim 3 , wherein said modified nucleotide is selected from the group consisting of a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, at least one 2′-O-methyl modified nucleotide and at least one nucleotide comprising a 5′phosphorothioate group.
16. The dsRNA molecule of claim 3 , wherein the dsRNA molecule comprises alternating 2′-methoxy-ribonucleotides and 2′-fluoro-ribonucleotides.
17. The dsRNA molecule of claim 3 , wherein the nucleotides at positions 2 and 14 from the 5′ end are not 2′-methoxy-ribonucleotides.
18. The dsRNA molecule of claim 3 , wherein the nucleotides are connected via phosphodiester or phosphorothioate linkages.
19. The dsRNA molecule of claim 3 , wherein the nucleotides at positions 1-6 from the 3′ end, or positions 1-7 from the 3′ end, are connected to adjacent nucleotides via phosphorothioate linkages.
20. A di-branched RNA compound comprising a dsRNA of claim 3 connected to another dsRNA by one or more moieties independently selected from a linker, a spacer and a branching point.
21. A method for inhibiting expression of HTT gene in a cell, the method comprising:
(a) introducing into the cell a double-stranded ribonucleic acid (dsRNA) of claim 3 ; and
(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the HTT gene, thereby inhibiting expression of the HTT gene in the cell.
22. A method of treating or managing Huntington's disease comprising administering to a patient in need of such treatment or management a therapeutically effective amount of said dsRNA of claim 3 .
23. The method of claim 21 , wherein said dsRNA is administered to the brain of the patient.
24. The method of claim 21 , wherein said dsRNA is administered by intrastriatal infusion.
25. The method of claim 21 , wherein the dsRNA causes a decrease in HTT gene mRNA in the striatum.
26. The method of claim 21 , where the dsRNA causes a decrease in HTT gene mRNA in the cortex.
27. The vector of claim 1 , wherein the dsRNA comprises a sense strand and an antisense strand, wherein the antisense strand comprises the region of complementarity which is substantially complementary to 5′ CAGUAAAGAGAUUAA 3′ (SEQ ID NO:1).
28. The vector of claim 1 , wherein the dsRNA comprises a sense strand and an antisense strand, wherein the antisense strand comprises the region of complementarity which is substantially complementary to 5′ AUAUCAGUAAAGAGA 3′ (SEQ ID NO:2).
29. The vector of claim 1 , wherein the dsRNA comprises a sense strand and an antisense strand, wherein the antisense strand comprises the region of complementarity which is substantially complementary to 5′ CUCAGGAUUUAAAAU 3′ (SEQ ID NO:3).
30. The vector of claim 1 , wherein the dsRNA comprises a sense strand and an antisense strand, wherein the antisense strand is complementary to at least 10, 11, 12 or 13 contiguous nucleotides of SEQ ID NO:1, 2 or 3.
31. The vector of claim 1 , wherein the dsRNA comprises a sense strand and an antisense strand, wherein the antisense strand contains no more than 3 mismatches with SEQ ID NO:1, 2 or 3.
32. The vector of claim 1 , wherein the dsRNA comprises a sense strand and an antisense strand, wherein the antisense strand is fully complementary to SEQ ID NO:1, 2 or 3.
33. The vector of claim 1 , wherein the dsRNA is between 30 and 35 base pairs in length.
34. The vector of claim 1 , wherein the dsRNA blunt-ended.
35. The vector of claim 1 , wherein the dsRNA comprises at least one single stranded nucleotide overhang.
36. A method for inhibiting expression of HTT gene in a cell, the method comprising:
(a) introducing into the cell the vector of claim 1 ; and
(b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of the HTT gene, thereby inhibiting expression of the HTT gene in the cell.
37. A method of treating or managing Huntington's disease comprising administering to a patient in need of such treatment or management a therapeutically effective amount of the vector of claim 1 .
38. The method of claim 37 , wherein said vector is administered to the brain of the patient.
39. The method of claim 37 , wherein said vector is administered by intrastriatal infusion.
40. The method of claim 37 , wherein the dsRNA causes a decrease in HTT gene mRNA in the striatum.
41. The method of claim 37 , where the dsRNA causes a decrease in HTT gene mRNA in the cortex.