Duplexed Parvovirus Vectors

• US 20100310516A1
• Filed: 08/02/2010
• Published: 12/09/2010
• Est. Priority Date: 06/01/2000
• Status: Active Grant

• Current Assignee: University of North Carolina At Chapel Hill (University of North Carolina System)
• Original Assignee: University of North Carolina At Chapel Hill (University of North Carolina System)
• Inventors: Samulski, Richard Jude, McCarty, Douglas M.
• Granted Patent: US 8,361,457 B2
• Time in Patent Office: Days
• US Class Current: 424/93.2
• 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 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 33/00 Antiparasitic agents
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 15/864 Parvoviral vectors , e.g. parvovirus, densovirus
• CPC Class Codes: C12N 2750/14121 Viruses as such, e.g. new isolates, mutants or their genomic sequences
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00

Abstract

The present invention provides duplexed parvovirus vector genomes that are capable under appropriate conditions of forming a double-stranded molecule by intrastrand base-pairing. Also provided are duplexed parvovirus particles comprising the vector genome. Further disclosed are templates and methods for producing the duplexed vector genomes and duplexed parvovirus particles of the invention. Methods of administering these reagents to a cell or subject are also described. Preferably, the parvovirus capsid is an AAV capsid. It is further preferred that the vector genome comprises AAV terminal repeat sequences.

Claims

1. A parvovirus particle comprising:

a parvovirus capsid; and

a vector genome comprising in the 5′

to 3′

direction;

(i) a 5′

parvovirus terminal repeat;

(ii) a first heterologous nucleotide sequence;

(iii) a terminal repeat that is resolved by Rep protein to a lesser extent as compared with the 5′

parvovirus terminal repeat and a 3′

parvovirus terminal repeat;

(iv) a separate heterologous nucleotide sequence; and

(v) the 3′

parvovirus terminal repeat;

wherein the first and the separate heterologous nucleotide sequences are essentially self-complementary and may form a hairpin structure.

2. The parvovirus particle of claim 1, wherein the 5′

and 3′

parvovirus terminal repeats are 5′ and

3′

adeno-associated virus (AAV) terminal repeats.

3. The parvovirus particle of claim 2, wherein the 5′

and 3′

AAV terminal repeats are selected from the group consisting of 5′ and

3′

AAV1, AAV2, AAV3, AAV4, AAV5 and AAV6 terminal repeats.

4. The parvovirus particle of claim 1, wherein the vector genome is approximately the size of the wild-type adeno-associated (AAV) genome.

5. The parvovirus particle of claim 1, wherein a polypeptide is encoded by the first and the separate heterologous nucleotide sequences.

6. The parvovirus particle of claim 5, wherein the polypeptide is a therapeutic polypeptide.

7. The parvovirus particle of claim 5, wherein the polypeptide is an immunogenic polypeptide.

8. The parvovirus particle of claim 5, wherein the polypeptide is an endostatin, an angiostatin, a superoxide dismutase, an erythropoietin, a monoclonal antibody, a Factor IX, a Factor X, a lysosomal enzyme, a globin, a leptin, a catalase, a tyrosine hydroxylase, a cytokine, a peptide growth factor, a hormone, a receptor, a suicide gene product, a tumor suppressor gene product or an ornithine transcarbamylase.

9. The parvovirus particle of claim 5, wherein the polypeptide is a cancer or a tumor antigen.

10. The parvovirus particle of claim 5, wherein the polypeptide is a bacterial antigen, a viral antigen, a protozoan antigen, or a parasite antigen.

11. The parvovirus particle of claim 1, wherein the first and the separate heterologous nucleotide sequences encode an antisense nucleic acid, a ribozyme, an RNA that effects spliceosome-mediated trans-splicing, an interfering RNA (RNAi), or a guide RNA.

12. The parvovirus particle of claim 1, wherein said parvovirus capsid is an adeno-associated virus (AAV) capsid.

13. The parvovirus particle of claim 12, wherein said parvovirus capsid is selected from the group consisting of an AAV1, AAV2, AAV3, AAV4, AAV5 and AAV6 capsid.

14. The parvovirus particle of claim 1, wherein the 5′

and 3′

halves of the vector genome are essentially completely complementary to each other.

15. A pharmaceutical formulation comprising a plurality of the parvovirus particles of claim 1 in a pharmaceutically acceptable carrier.

26. A method of delivering the vector genome to a mammalian cell, comprising contacting the mammalian cell with a parvovirus particle according to claim 1 under conditions sufficient for the parvovirus particle to enter the mammalian cell.

27. The method of claim 26, wherein the cell is selected from the group consisting of a cancer cell, a tumor cell, a brain cell, a muscle cell, an airway epithelial cell, a liver cell, a dendritic cell, and an eye cell.

28. The method of claim 26, wherein a polypeptide is encoded by the first and separate heterologous nucleotide sequences.

29. The method of claim 28, wherein the polypeptide is a therapeutic polypeptide.

30. The method of claim 28, wherein the polypeptide is an immunogenic polypeptide.

31. The method of claim 28, wherein the polypeptide is an endostatin, an angiostatin, a superoxide dismutase, an erythropoietin, a monoclonal antibody, a Factor IX, a Factor X, a lysosomal enzyme, a globin, a leptin, a catalase, a tyrosine hydroxylase, a cytokine, a peptide growth factor, a hormone, a receptor, a suicide gene product, a tumor suppressor gene product or an ornithine transcarbamylase.

32. The method of claim 28, wherein the polypeptide is a cancer or a tumor antigen.

33. The method of claim 28, wherein the polypeptide is a bacterial antigen, a viral antigen, a protozoan antigen, or a parasite antigen.

34. The method of claim 26, wherein the vector genome encodes an antisense nucleic acid, a ribozyme, an RNA that effects spliceosome-mediated trans-splicing, an interfering RNA (RNAi), or a guide RNA.

35. The method of claim 26, wherein the parvovirus capsid is an adeno-associated virus (AAV) capsid.

36. The method of claim 26, wherein the 5′

and 3′

parvovirus terminal repeats are 5′ and

3′

adeno-associated virus (AAV) repeats.

38. A mammalian cell produced by the method of claim 26.

40. A method of administering the vector genome to a mammalian subject comprising administering a mammalian cell according to claim 38 to the mammalian subject.

42. A method of administering the vector genome to a mammalian subject comprising administering to the mammalian subject a parvovirus particle according to claim 1.

16. A parvovirus particle comprising:

an adeno-associated virus (AAV) capsid; and

a vector genome comprising in the 5′

to 3′

direction;

(a) a 5′

AAV terminal repeat;

(ii) a first heterologous nucleotide sequence;

(iii) a terminal repeat that is resolved by Rep protein to a lesser extent as compared to the 5′

AAV terminal repeat and a 3′

AAV terminal repeat;

(iv) a separate heterologous nucleotide sequence; and

(v) the 3′

AAV terminal repeat;

wherein the first and the separate heterologous nucleotide sequences are essentially self-complementary and may form a hairpin structure.

17. The parvovirus particle of claim 16, wherein the vector genome is approximately the size of the wild-type adeno-associated (AAV) genome.

18. The parvovirus particle of claim 16, wherein a polypeptide is encoded by the first and separate heterologous nucleotide sequences.

19. The parvovirus particle of claim 18, wherein the polypeptide is a therapeutic polypeptide.

20. The parvovirus particle of claim 18, wherein the polypeptide is an immunogenic polypeptide.

21. The parvovirus particle of claim 18, wherein the polypeptide is an endostatin, an angiostatin, a superoxide dismutase, an erythropoietin, a monoclonal antibody, a Factor IX, a Factor X, a lysosomal enzyme, globin, a leptin, a catalase, a tyrosine hydroxylase, a cytokine, a peptide growth factor, a hormone, a receptor, a suicide gene product, a tumor suppressor gene product or an ornithine transcarbamylase.

22. The parvovirus particle of claim 18, wherein the polypeptide is a cancer or a tumor antigen.

23. The parvovirus particle of claim 18, wherein the polypeptide is a bacterial antigen, a viral antigen, a protozoan antigen, or a parasite antigen.

24. The parvovirus particle of claim 16, wherein the first and separate heterologous nucleotide sequences encode an antisense nucleic acid, a ribozyme, an RNA that effects spliceosome-mediated trans-splicing, an interfering RNA (RNAi), or a guide RNA.

25. A pharmaceutical formulation comprising a plurality of the parvovirus particles of claim 16 in a pharmaceutically acceptable carrier.

37. A method of delivering the vector genome to a mammalian cell, comprising contacting the mammalian cell with a parvovirus particle according to claim 16 under conditions sufficient for the parvovirus particle to enter the mammalian cell.

39. A mammalian cell produced by the method of claim 37.

41. A method of administering the vector genome to a mammalian subject comprising administering a mammalian cell according to claim 39 to the mammalian subject.

43. A method of administering the vector genome to a mammalian subject comprising administering to the mammalian subject a parvovirus particle according to claim 16.