Method for treating fibrosis using siRNA and a retinoid-lipid drug carrier

1. A method for treating a fibrotic disease in a subject in need thereof, the method comprising administering an effective amount of a pharmaceutical composition to the subject, wherein the pharmaceutical composition comprises a drug carrier and a double-stranded nucleic acid molecule, wherein the drug carrier comprises a lipid and a retinoid, wherein the lipid comprises a permanent cationic lipid or an ionizable cationic lipid, and wherein the double-stranded nucleic acid molecule comprises the structure (A2):

(A2) 5′ N1-(N)x - Z 3′ (antisense strand)

     3′ Z′-N2-(N′)y -z″ 5′ (sense strand)

wherein each of N 2 , N and N′ is an unmodified or modified ribonucleotide, or an unconventional moiety;

wherein each of (N) x and (N′) y is an oligonucleotide in which each consecutive N or N′ is joined to the adjacent N or N′ by a covalent bond;

wherein each of x and y is independently an integer between 17 and 39;

wherein the sequence of (N′) y has complementarity to the sequence of (N) x and (N) x has complementarity to a consecutive sequence in a target RNA;

wherein N 1 is covalently bound to (N) x and is mismatched to the target RNA or is a complementary DNA moiety to the target RNA;

wherein N 1 is a natural or modified uridine, deoxyribouridine, ribothymidine, deoxyribothymidine, adenosine or deoxyadenosine;

wherein z″ may be present or absent, but if present is a capping moiety covalently attached at the 5′-terminus of N 2 —(N′) y ;

wherein each of Z and Z′ is independently present or absent, but if present is independently 1-5 consecutive nucleotides, consecutive non-nucleotide moieties or a combination thereof covalently attached at the 3′-terminus of the strand in which it is present; and

wherein (N) x comprises an antisense sequence to the mRNA coding sequence for human hsp47 exemplified by SEQ ID NO:1.

2. The method according to claim 1 , wherein the pharmaceutical composition is parenterally administered.

3. The method of claim 1 , wherein the fibrotic disease is liver fibrosis.

4. The method of claim 3 , wherein liver fibrosis consists of non-alcoholic steatohepatitis; hepatitis; hepatic fibrosis; chronic hepatitis C virus infection; hepatic cirrhosis; chronic hepatic damage; or liver cancer.

5. The method of claim 1 , wherein the fibrotic disease is pulmonary fibrosis.

6. The method of claim 5 , wherein the pulmonary fibrosis consists of pulmonary fibrosis including idiopathic pulmonary fibrosis, interstitial lung disease, radiation pneumonitis leading to pulmonary fibrosis; or fibrotic lung disease.

7. The method of claim 1 , wherein the fibrotic disease is kidney fibrosis.

8. The method of claim 7 , wherein the kidney fibrosis is chronic renal failure or diabetic nephropathy.

9. The method of claim 1 , wherein the fibrotic disease is peritoneal fibrosis.

10. The method of claim 9 , wherein the peritoneal fibrosis is peritoneal sclerosis associated with continual ambulatory peritoneal dialysis.

11. The method of claim 1 , wherein the fibrotic disease is pancreatic fibrosis.

12. The method of claim 11 , wherein the pancreatic fibrosis consists of pancreatitis, pancreatic fibrosis, or pancreatic cancer.

13. The method of claim 1 , wherein the fibrotic disease is cardiac fibrosis.

14. The method of claim 13 , wherein the cardiac fibrosis consists of myocardial fibrosiscardiomyopathy, atherosclerosis, endomyocardial fibrosis, atrial fibrillation, or scarring post-myocardial infarction.

15. The method of claim 1 , wherein the fibrotic disease is intestinal fibrosis.

16. The method of claim 1 , wherein the fibrotic disease is fibrosis of the eye.

17. The method of claim 16 , wherein the fibrosis of the eye consists of ocular scarring including proliferative vitreoretinopathy (PVR) or scarring resulting from surgery to treat cataract or macular degeneration, glaucoma, Grave's ophthalmopathy; ocular cicatricial pemphigoid, or drug induced ergotism.

18. The method of claim 1 , wherein the fibrotic disease is skin fibrosis.

19. The method of claim 18 , wherein the skin fibrosis consists scleroderma, keloids, hypertrophic scars, psoriasis, or Kaposi's sarcoma.

20. The method of claim 1 , wherein the fibrotic disease is fibrosis of bone.

21. The method of claim 20 , wherein the fibrosis of bone consists of myelofibrosis; myeloid leukemia; acute myelogenous leukemia; myelodysplastic syndrome; lymphangioleiomyomatosis (LAM), chronic graft vs. host disease, polycythemia vera, essential thrombocythemia, or myeloproferative syndrome.

22. The method of claim 1 , wherein the fibrotic disease is inflammatory bowel disease of variable etiology.

23. The method of claim 1 , wherein the fibrotic disease is glioblastoma in Li-Fraumeni syndrome or sporadic glioblastoma.

24. The method of claim 1 , wherein the fibrotic disease is gynecological cancer.

25. The method of claim 1 , wherein the fibrotic disease is Hansen's disease.

26. The method of claim 1 , wherein the fibrotic disease is collagenous colitis.

27. The method of claim 1 , wherein the fibrotic disease is fibrillogenesis.

28. The method of claim 1 , wherein the fibrotic disease is fibrosis of vocal cords.

29. The method of claim 28 , wherein the fibrosis of vocal cords consists of vocal cord scarring, vocal cord mucosal fibrosis, or laryngeal fibrosis.

30. The method of claim 1 , wherein the retinoid is at least partially exposed on the exterior of the drug carrier before the drug carrier reaches a stellate cell.

31. The method of claim 1 , wherein the drug carrier comprises a retinoid in the form of a retinoid-PEG conjugate.

32. The method of claim 1 , wherein the drug carrier comprises a polymer micelle, a liposome, an emulsion, a microsphere, or a nanosphere.

33. The method of claim 1 , wherein the drug carrier comprises a lipid vesicle comprising a bilayer of lipid molecules.

34. The method of claim 33 , wherein the retinoid is 0.2 wt % to 20 wt % of the lipid molecules.

35. The method of claim 33 , wherein the double-stranded nucleic acid molecule is exposed on an exterior surface of the lipid vesicle.

36. The method of claim 33 , wherein the double-stranded nucleic acid molecule is encapsulated by the lipid vesicle.

37. The method of claim 36 , wherein the double-stranded nucleic acid molecule is resistant to nucleases.

38. The method of claim 1 , wherein one strand of the double-stranded nucleic acid molecule comprises SEQ ID NO:127 and the second strand comprises SEQ ID NO:60.

39. The method of claim 38 , wherein the antisense strand comprises SEQ ID NO:127, and the sense strand comprises SEQ ID NO:60.

40. The method of claim 39 , wherein the antisense strand further comprises a 2′-O-methyl modified ribonucleotide; a 2′-5′-ribonucleotide in at least one of positions 1, 5, 6, or 7; and a non-nucleotide moiety covalently attached to the 3′-terminus; and wherein the sense strand further comprises at least one 2′-5′-ribonucleotide or 2′-O-methyl modified ribonucleotide; a non-nucleotide moiety covalently attached at the 3′-terminus; and a cap moiety covalently attached at the 5′-terminus.

41. The method of claim 39 , wherein the antisense strand further comprises 2-′O-methyl modified ribonucleotides at positions 3, 5, 9, 11, 13, 15, 17, and 19; a 2′-5′-ribonucleotide in position 7; and a non-nucleotide moiety covalently attached at the 3′-terminus; and wherein the sense strand further comprises five consecutive 2′-5′-ribonucleotides in the 3′-terminal positions 15, 16, 17, 18, and 19; a non-nucleotide moiety covalently attached at the 3′-terminus; and an inverted abasic moiety covalently attached at the 5′-terminus.

42. The method of claim 39 , wherein the antisense strand further comprises 2′-O-methyl modified ribonucleotides at positions 1, 3, 5, 9, 11, 13, 15, 17, and 19; and a C3 or C3-C3 non-nucleotide moiety covalently attached at the 3′-terminus; and wherein the sense strand further comprises a C3Pi non-nucleotide moiety covalently attached at the 3′-terminus; and an inverted abasic moiety covalently attached at the 5′-terminus.

43. The method of claim 39 , wherein the antisense strand further comprises 2′-O-methyl modified ribonucleotides at positions 1, 3, 5, 9, 11, 13, 15, 17, and 19; a 2′-5′-ribonucleotide in position 7; and a C3Pi-C3OH non-nucleotide moiety covalently attached at the 3′-terminus; and wherein the sense strand further comprises five consecutive 2′-5′-ribonucleotides in the 3′-terminal positions 15, 16, 17, 18, and 19; a C3Pi non-nucleotide moiety covalently attached at the 3′-terminus; and an inverted abasic moiety covalently attached at the 5′-terminus.

44. The method of claim 39 , wherein the antisense strand further comprises 2-′O-methyl modified ribonucleotides in positions 1, 3, 5, 9, 11, 13, 15, 17, and 19; a 2′-5′-ribonucleotide in position 7; and a C3Pi-C3OH non-nucleotide moiety covalently attached at the 3′-terminus; and wherein the sense strand further comprises 2′-O-methyl modified ribonucleotides in positions 7, 13, 16 and 18; a 2′-5′-ribonucleotide at position 9; a C3 non-nucleotide moiety covalently attached at the 3′-terminus; and an inverted abasic moiety covalently attached at the 5′-terminus.

45. The method of claim 1 , wherein one strand of the double-stranded nucleic acid molecule comprises SEQ ID NO:98 and the second strand comprises SEQ ID NO:165.

46. The method of claim 45 , wherein the sense strand comprises SEQ ID NO:98 and the antisense strand comprises SEQ ID NO:165.

47. The method of claim 46 , wherein the sense strand further comprises 2′-5′-ribonucleotides in positions at or near the 3′-terminus; a non-nucleotide moiety covalently attached at the 3′-terminus; and a cap moiety covalently attached at the 5′-terminus; and the antisense strand further comprises a 2′-O-methyl modified ribonucleotide; a 2′-5′-ribonucleotide in at least one of positions 5, 6 or 7; and a non-nucleotide moiety covalently attached at the 3′-terminus.

48. The method of claim 46 , wherein the sense strand further comprises 2′-5′-ribonucleotides in positions 15, 16, 17, 18, and 19; a C3-OH 3′ moiety covalently attached at the 3′-terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′-terminus; and the antisense strand further comprises 2′-O-methyl modified ribonucleotides in positions 2, 4, 6, 8, 11, 13, 15, 17, and 19; a 2′-5′-ribonucleotide in position 7; and a C3Pi-C3OH moiety covalently attached at the 3′-terminus.

49. The method of claim 1 , wherein one strand of the double-stranded nucleic acid molecule comprises SEQ ID NO:101 and the second strand comprises SEQ ID NO:168.

50. The method of claim 49 , wherein the sense strand comprises SEQ ID NO:101 and the antisense strand comprises SEQ ID NO:168.

51. The method of claim 50 , wherein the sense strand further comprises 2′-O-methyl modified pyrimidine ribonucleotides; an optional 2′-5′-ribonucleotide in one of position 9 or 10; a non-nucleotide moiety covalently attached at the 3′-terminus; and a cap moiety covalently attached at the 5′-terminus; and the wherein antisense strand further comprises a 2′-O-methyl modified ribonucleotide; a 2′-5′-ribonucleotide in at least one of positions 5, 6, or 7; and a non-nucleotide moiety covalently attached at the 3′-terminus.

52. The method of claim 50 , wherein the sense strand further comprises 2′-O-methyl modified ribonucleotides in positions 4, 11, 13, and 17; a 2′-5′-ribonucleotide in position 9; a C3OH non-nucleotide moiety covalently attached at the 3′-terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′-terminus; and wherein the antisense strand further comprises 2′-O-methyl modified ribonucleotides in positions 1, 4, 8, 11 and 15; a 2′-5′-ribonucleotide in position 6; and a C3Pi-C3OH moiety covalently attached at the 3′-terminus.

53. The method of claim 50 , wherein sense strand further comprises 2′-O-methyl modified ribonucleotides in positions 4, 11, 13, and 17; a C3OH nonnucleotide moiety covalently attached at the 3′-terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′-terminus; and wherein the antisense strand further comprises 2′-O-methyl modified ribonucleotides in positions 1, 4, 8, 13 and 15; a 2′-5′-ribonucleotide in position 6; and a C3Pi-C3OH moiety covalently attached at the 3′-terminus.

54. The method of claim 50 , wherein the sense strand further comprises 2′-O-methyl modified ribonucleotides in positions 2, 4, 11, 13, and 17; a C3OH nonnucleotide moiety covalently attached at the 3′-terminus; and an inverted abasic deoxyribonucleotide moiety covalently attached at the 5′-terminus; and wherein the antisense strand further comprises 2′-O-methyl modified ribonucleotides in positions 1, 4, 8, 11 and 15; a 2′-5′-ribonucleotide in position 6; and a C3Pi-C3OH non-nucleotide moiety covalently attached at the 3′-terminus.

55. The method of claim 1 , wherein the double-stranded nucleic acid molecule reduces hsp47 expression in a stellate cell.

56. The method of claim 1 , wherein the drug carrier comprises a permanent cationic lipid and an ionizable cationic lipid.

57. The method of claim 1 , wherein the drug carrier comprises HEDC

58. The method of claim 1 , wherein the drug carrier comprises S104