Compounds and methods for modulating expression of PCSK9
The present disclosure describes short antisense compounds, including such compounds comprising chemically-modified high-affinity monomers 8-16 monomers in length. Certain such short antisense compound are useful for the reduction of target nucleic acids and/or proteins in cells, tissues, and animals with increased potency and improved therapeutic index. Thus, provided herein are short antisense compounds comprising high-affinity nucleotide modifications useful for reducing a target RNA in vivo. Such short antisense compounds are effective at lower doses than previously described antisense compounds, allowing for a reduction in toxicity and cost of treatment. In addition, the described short antisense compounds have greater potential for oral dosing.
1. A short antisense compound 8 to 14 monomers in length, comprising a 2′-deoxyribonucleotide gap region flanked on each side by at least one wing, wherein each wing independently comprises 1 to 3 high-affinity modified monomers and wherein the short antisense compound is targeted to a nucleotide encoding PCSK9.
2. The short antisense compound of claim 1 , wherein said high-affinity modified monomers are sugar-modified nucleotides.
3. The short antisense compound of claim 2 , wherein at least one of the sugar-modified nucleotides comprises a bridge between the 4′ and the 2′ position of the sugar.
4. The short antisense compound of claim 2 , wherein each of said high-affinity modified nucleotides confers a ΔT m of 1 to 4 degrees per nucleotide.
5. The short antisense compound of claim 2 , wherein each of said sugar-modified nucleotides comprises a 2′-substituent group that is other than H or OH.
6. The short antisense compound of claim 5 , wherein at least one of said sugar-modified nucleotides is a 4′ to 2′ bridged bicyclic nucleotide.
7. The short antisense compound of claim 5 , wherein each of the 2′-substituent groups is, independently, alkoxy, substituted alkoxy, or halogen.
8. The short antisense compound of claim 7 , wherein each of the 2′-substituent groups is OCH 2 CH 2 OCH 3 .
9. The short antisense compound claim 3 , wherein the conformation of each of said sugar-modified nucleotides is, independently, β-D or α-L.
10. The short antisense compound claim 3 , wherein each of said bridges independently comprises 1 or from 2 to 4 linked groups independently selected from —[C(R 1 )(R 2 )] n —, —C(R 1 )═C(R 2 )—, —C(R 1 )═N—, —C(═NR 1 )—, —C(═O)—, —C(═S)—, —O—, —Si(R 1 ) 2 —, —S(═O) x — and —N(Ri)—;
wherein
x is 0, 1, or 2;
n is 1, 2, 3, or 4;
each R 1 and R 2 is, independently, H, a protecting group, hydroxyl, C 1 -C 12 alkyl, substituted C 1 -C 12 alkyl, C 2 -C 12 alkenyl, substituted C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, substituted C 2 -C 12 alkynyl, C 5 -C 20 aryl, substituted C 5 -C 20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C 5 -C 7 alicyclic radical, substituted C 5 -C 7 alicyclic radical, halogen, OJ 1 , NJ 1 J 2 , SJ 1 , N 3 , COOJ I , acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O) 2 -J 1 ), or sulfoxyl (S(═O)-J 1 ); and
each J 1 and J 2 is, independently, H, C 1 -C 12 alkyl, substituted C 1 -C 12 alkyl, C 2 -C 12 alkenyl, substituted C 2 -C 12 alkenyl, C 2 -C 12 alkynyl, substituted C 2 -C 12 alkynyl, C 5 -C 20 aryl, substituted C 5 -C 20 aryl, acyl (C(═O)—H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C 1 -C 12 aminoalkyl, substituted C 1 -C 12 aminoalkyl or a protecting group.
11. The short antisense compound of claim 10 , wherein each of said bridges is, independently, 4′-CH 2 -2′, 4′-(CH 2 ) 2 -2′, 4′-CH 2 —O-2′, 4′-(CH 2 ) 2 —O-2′, 4′-CH 2 —O—N(R 1 )-2′ and 4′-CH 2 —N(R 1 )—O-2′- wherein each R 1 is, independently, H, a protecting group or C 1 -C 12 alkyl.
12. The short antisense compound of claim 1 , wherein each of the high-affinity modified monomer is independently selected from bicyclic nucleotides or other 2′-modified nucleotides.
13. The short antisense compound of claim 12 , wherein the 2′-modified nucleotides are selected from halogen, allyl, amino, azido, thio, O-allyl, O—C 1 -C 10 alkyl, —OCF 3 , O—(CH 2 ) 2 —O—CH 3 , 2′-O(CH 2 ) 2 SCH 3 , O—(CH 2 ) 2 —O—N(R m )(R n ) or O—CH 2 —C(═O)—N(R m )(R n ), where each R m and R n is, independently, H or substituted or unsubstituted C 1 -C 10 alkyl.
14. The short antisense compound of claim 13 , wherein the 2′-modified nucleotide is a 2′-OCH 2 CH 2 OCH 3 nucleotide.
15. The short antisense compound of claim 1 , wherein at least one monomeric linkage is a modified monomeric linkage.
16. The antisense compound of claim 15 , wherein the modified monomeric linkage is a phosphorothioate linkage.
17. The short antisense compound of claim 1 , wherein each monomeric linkage is a phosphorothioate internucleoside linkage.
18. The short antisense compound of claim 1 , that is 8-13 monomers in length.
19. The short antisense compound of claim 18 that is 8-10 monomers in length.
20. The short antisense compound of claim 18 that is 11-13 monomers in length.
21. The short antisense compound of claim 1 that is 11-12 monomers in length.
22. The short antisense compound of claim 18 that is 9-10 monomers in length.
23. The short antisense compound of claim 18 that is 9-13 monomers in length.
24. The short antisense compound of claim 18 that is 10-13 monomers in length.
25. The short antisense compound of claim 18 that is 9-12 monomers in length.
26. The short antisense compound of claim 18 that is 10-12 monomers in length.
27. The short antisense compound of claim 18 that is 9-11 monomers in length.
28. The short antisense compound of claim 18 that is 10-11 monomers in length.
29. The short antisense compound of claim 18 that is 8 monomers in length.
30. The short antisense compound of claim 18 that is 9 monomers in length.
31. The short antisense compound of claim 18 that is 10 monomers in length.
32. The short antisense compound of claim 18 that is 11 monomers in length.
33. The short antisense compound of claim 18 that is 12 monomers in length.
34. The short antisense compound of claim 18 that is 13 monomers in length.
35. The short antisense compound of claim 1 that is 14 monomers in length.
36. The short antisense compound of claim 1 , having a motif selected from 1-12-1; 2-10-2; 1-10-1; 1-10-2; 1-9-1; 1-9-2; 2-9-1; 2-8-1; 2-7-1; 2-7-2; 2-6-3; 2-7-2; 2-6-2; 3-8-3; 2-8-2; 1-8-1; 3-6-3; and 1-6-1 wherein, the first number represents the number of monomers in the 5′-wing, the second number represents the number of monomers in the gap, and the third number represents the number of monomers in the 3′ wing.
37. The short antisense compound of claim 36 wherein the motif is selected from 1-10-1; 2-10-2; and 1-9-2.
38. The short antisense compound of claim 1 having a motif selected from 1-1-10-2, 1-1-8-2, 1-1-6-3, and 1-2-8-2, wherein the first number represents the number of monomers in a first 5′ wing, the second number represents the number of monomers in a second 5′ wing, the third number represents the number of monomers in the gap, and the fourth number represents the number of monomers in the 3′ wing.
39. The short antisense compound of claim 1 having a motif selected from 2-10-1-1, 2-8-1-1, 3-6-1-1, and 2-8-2-1, wherein the first number represents the number of monomers in the 5′ wing, the second number represents the number of monomers in the gap, the third number represents the number of monomers in a first 3′ wing, and the fourth number represents the number of monomers in a second 3′ wing.
40. The short antisense compound of claim 1 having a motif selected from 1-1-8-1-1; 2-1-6-1-1; and 1-2-8-2-1, wherein the first number represents the number of monomers in a first 5′ wing, the second number represents the number of monomers in a second 5′ wing, the third number represents the number of monomers in the gap, the fourth number represents the number of monomers in a first 3′ wing and the fifth number represents the number of monomers in a second 3′ wing.
41. A method of inhibiting PCSK9 expression comprising contacting a nucleic acid encoding PCSK9 with the short antisense compound of claim 1 .
42. The method of claim 41 wherein the PCSK9 nucleic acid is in a cell.
43. The method of claim 42 , wherein the PCSK9 nucleic acid is in an animal, and wherein the method further comprises administering the short antisense compound of claim 1 to the animal.
44. The method of claim 43 , wherein the animal is a human.
45. The method of claim 43 , wherein said contacting decreases total serum cholesterol, serum LDL, serum VLDL, serum HDL, serum triglycerides, serum apolipoprotein(s) and/or free fatty acids in the animal.
46. The method of claim 43 , wherein inhibiting PCSK9 expression treats a cardiovascular disorder in the animal.