Modified Blood Clotting Factors And Methods Of Use

• US 20110126303A1
• Filed: 06/04/2010
• Published: 05/26/2011
• Est. Priority Date: 03/22/2000
• Status: Abandoned Application

• Current Assignee: Children's Hospital of Philadelphia (Children's Hospital of Philadelphia Foundation)
• Original Assignee: Children's Hospital of Philadelphia (Children's Hospital of Philadelphia Foundation)
• Inventors: Margaritis, Paris, Camire, Rodney, High, Katherine A.
• Application Number:
  US12/794,215
• Publication Number:
  US 20110126303A1
• Time in Patent Office: Days
• US Class Current: 800/13
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 7/04 Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: C07K 14/745 Blood coagulation or fibrinolysis factors
• CPC Class Codes: C07K 14/755 Factors VIII , e.g. factor VIII C (AHF), factor VIII Ag (VWF)

Abstract

The invention provides compositions including modified blood clotting factors that have a non-native proteolytic cleavage site engineered into them allowing intracellular cleavage and secretion of an active form. The compositions are useful in the methods for treating a bleeding or clotting disorder.

Claims

1. A composition comprising a recombinant polynucleotide that encodes a modified blood clotting factor, said modified blood clotting factor comprising a proteolytic cleavage site not normally present in the blood clotting factor, wherein said cleavage site is located at a position that allows secretion of active blood clotting factor upon cleavage.

2. The composition of claim 1, wherein the blood clotting factor is a functional variant or a functional subsequence of a naturally occurring blood clotting factor.

3. The composition of claim 1, wherein the blood clotting factor is a vitamin K-dependent procoagulent or anticoagulant protein.

4. The composition of claim 3, wherein the vitamin K-dependent procoagulent protein comprises Factor VII, Factor IX or Factor X.

5. The composition of claim 3, wherein the vitamin K-dependent anticoagulant protein comprises protein C.

6. The composition of claim 1, wherein the proteolytic cleavage site is a mammalian amino acid sequence.

7. The composition of claim 1, wherein the proteolytic cleavage site comprises a PACE/furin amino acid sequence.

8. The composition of claim 1, wherein the proteolytic cleavage site comprises a plurality of basic amino acid sequences.

9. The composition of claim 1, wherein the proteolytic cleavage site comprises the amino acid sequence of SEQ ID NO:

1, SEQ ID NO;

2, or SEQ ID NO;

3.

10. The composition of claim 1, wherein the proteolytic cleavage site comprises a viral amino acid sequence cleavage site.

11. The composition of claim 10, wherein the viral cleavage site comprises a retroviral protein amino acid sequence.

12. The composition of claim 11, wherein the retroviral protein cleavage site is an envelope polypeptide cleavage site.

13. The composition of claim 4, wherein the proteolytic cleavage site is introduced between amino acids 152 and 153 of Factor VII.

14. The composition of claim 4, wherein the proteolytic cleavage site is introduced between arginine 152 and isoleucine 153 of Factor VII.

15. The composition of claim 2, wherein the functional variant has one or more conservative amino acid substitutions of wild type blood clotting factor.

16. The composition of claim 2, wherein the functional variant comprises a Factor VII having increased activity relative to wild type Factor VII.

17. The composition of claim 2, wherein the functional variant comprises a Factor VII having increased stability in vivo relative to wild type Factor VII.

18. The composition of claim 2, wherein the functional variant comprises a Factor VII having decreased immunogenicity relative to wild type Factor VII.

19. The composition of claim 1, wherein the blood clotting factor is mammalian.

20. The composition of claim 19, wherein the blood clotting factor is primate, canine, feline, porcine, equine or bovine.

21. The composition of claim 20, wherein the primate is human.

22. The composition of claim 1, wherein the recombinant polynucleotide encoding the modified blood clotting factor is operatively linked to a regulatable or tissue specific expression control element.

23. The composition of claim 22, wherein the regulatable or tissue specific expression control element comprises a promoter.

24. The composition of claim 22, wherein the promoter comprises a skeletal muscle actin promoter or a muscle creatine kinase promoter.

25. The composition of claim 22, wherein the tissue-specific expression control element confers expression of the modified blood clotting factor in muscle, liver, kidney or blood vessel endothelium.

26. The composition of claim 22, wherein the regulatable expression control element comprises elongation factor la promoter.

34. A kit comprising a composition of claim 1 or a polypeptide of claim 33.

35. A kit comprising a composition of claim 1 further including instructions for expressing the modified blood clotting factor in vitro, ex vivo or in vivo.

41. The composition of claim 1, further comprising a pharmaceutically acceptable carrier.

42. A method for treating a bleeding or clotting disorder of a subject having or at risk of having a bleeding or clotting disorder comprising administering to the subject an amount of the composition of claim 1 sufficient to ameliorate one or more symptoms of the disorder.

43. The method of claim 42, wherein the disorder is amenable to treatment with Factor VII, Factor VIII or Factor IX.

44. The method of claim 42, wherein the disorder is caused by insufficient activity or expression of a vitamin-K dependent procoagulent.

45. The method of claim 42, wherein the disorder is caused by insufficient platelet aggregation.

46. The method of claim 42, wherein the disorder comprises hemophilia or Factor VII deficiency.

47. The method of claim 46, wherein the hemophilia comprises hemophilia A or hemophilia B.

48. The method of claim 42, wherein the disorder comprises Glanzmann'"'"'s thrombasthenia.

49. The method of claim 42, wherein the disorder comprises Bemard-Soulier'"'"'s thrombasthenia.

50. The method of claim 42, wherein the subject produces inhibitory antibodies that bind to a clotting factor.

51. The method of claim 50, wherein the inhibitory antibodies bind Factor VIII or Factor IX.

52. The method of claim 42, wherein the subject is a mammal.

53. The method of claim 42, wherein the mammal is human.

54. The method of claim 42, wherein the composition is administered by injection or infusion.

55. The method of claim 42, wherein the composition is administered into the portal vein or spleen.

56. A method of decreasing clotting time in a subject in need of decreased clotting time comprising administering to the subject an amount of the composition of claim 1 sufficient to decrease clotting time in the subject.

57. The method of claim 56, wherein the modified blood clotting factor comprises Factor VII, Factor VIII or Factor IX.

58. The method of claim 56, wherein the subject is a mammal.

59. The method of claim 58, wherein the mammal is human.

60. A method of reducing the frequency or severity of bleeding in a subject in need of reduced frequency or severity of bleeding comprising administering to the subject an amount of the composition of claim 1 sufficient to reduce the incidence or severity of a bleeding in the subject.

61. The method of claim 60, wherein the composition comprises Factor VII, Factor VIII or Factor IX.

62. The method of claim 60, wherein the subject is a mammal.

63. The method of claim 62, wherein the mammal is a human.

27. A composition comprising a recombinant polynucleotide that encodes a modified Factor VII, said modified Factor VII comprising a proteolytic cleavage site, said proteolytic cleavage site comprising a PACE/furin proteolytic cleavage recognition site at a location that allows secretion of active Factor VII upon cleavage, wherein said proteolytic cleavage site is not normally present in Factor VII.

28. A composition comprising a recombinant polynucleotide that encodes a modified Factor IX, said modified Factor IX comprising a proteolytic cleavage site at a location that allows secretion of active Factor IX upon cleavage, said proteolytic cleavage site comprising a PACE/furin proteolytic cleavage recognition site, wherein said proteolytic cleavage site is not normally present in Factor IX.

29. A composition comprising a vector comprising a recombinant polynucleotide that encodes a modified blood clotting factor, said modified blood clotting factor comprising a proteolytic cleavage site not normally present in the blood clotting factor, wherein said cleavage site is located at a position that allows secretion of active blood clotting factor upon cleavage.

30. The composition of claim 29, wherein the vector comprises a vector suitable for introduction into a cell in vivo.

31. The composition of claim 30, wherein the vector comprises an adeno-associated virus (AAV), adenovirus, retrovirus, parvovirus, papilloma virus, reovirus, rotavirus or a herpes virus.

32. The composition of claim 30, wherein the vector comprises a plasmid vector.

33. A polypeptide encoded by a recombinant polynucleotide that encodes a modified blood clotting factor, said modified blood clotting factor comprising a proteolytic cleavage site not normally present in the blood clotting factor, wherein said cleavage site is located at a position that allows secretion of active blood clotting factor upon cleavage.

36. A cell comprising a recombinant polynucleotide that encodes a modified blood clotting factor, said modified blood clotting factor comprising a proteolytic cleavage site not normally present in the blood clotting factor, wherein said cleavage site is located at a position that allows secretion of active blood clotting factor upon cleavage.

37. The cell of claim 36, wherein the cell is a muscle, liver, kidney or blood vessel cell.

38. The cell of claim 36, wherein the cell is present in a subject.

39. The cell of claim 38, wherein the subject is a non-human transgenic animal.

40. The cell of claim 38, wherein the subject is human.