Chimeric Vectors

• US 20080269149A1
• Filed: 12/15/2005
• Published: 10/30/2008
• Est. Priority Date: 12/15/2004
• Status: Active Grant

• Current Assignee: University of Florida Research Foundation, Incorporated (State University System of Florida), University of North Carolina At Chapel Hill (University of North Carolina System)
• Original Assignee: University of Florida Research Foundation, Incorporated (State University System of Florida)
• Inventors: Bowles, Dawn E., Samulski, Richard Jude, Grieger, Josh, Agbandje-McKenna, Mavis, Rabinowitz, Joseph E., Li, Chengwen
• Granted Patent: US 7,892,809 B2
• Time in Patent Office: Days
• US Class Current: 514/44
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/02 of urine or of the urinary tract, e.g. urine acidifiers
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 19/04 for non-specific disorders of the connective tissue
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/08 Antiepileptics; Anticonvulsants
• CPC Class Codes: A61P 25/14 for treating abnormal movements, e.g. chorea, dyskinesia
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/04 Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/04 : Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• CPC Class Codes: A61P 9/10 : for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C12N 15/86 : Viral vectors
• CPC Class Codes: C12N 2750/14122 : New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 2750/14143 : viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2750/14144 : Chimeric viral vector comprising heterologous viral elements for production of another viral vector

Abstract

The present invention is based, in part, on the discovery that parvovirus (including AAV) capsids can be engineered to incorporate small, selective regions from other parvoviruses that confer desirable properties. The inventors have discovered that in some cases as little as a single amino acid insertion or substitution from a first parvovirus (e.g., an AAV) into the capsid structure of another parvovirus (e.g., an AAV) to create a chimeric parvovirus is sufficient to confer one or more of the desirable properties of the first parvovirus to the resulting chimeric parvovirus and/or to confer a property that is not exhibited by the first parvovirus or is present to a lesser extent.

Claims

1. A chimeric virus vector comprising:

(a) a chimeric adeno-associated virus (AAV) capsid comprising a selective amino acid insertion following amino acid position 264 in an AAV2 capsid subunit or a corresponding change in a capsid subunit from other AAV; and

(b) a nucleic acid comprising an AAV terminal repeat sequence and a heterologous nucleic acid sequence;

wherein the nucleic acid is packaged within the chimeric AAV capsid.

2. The chimeric virus vector of claim 1, wherein said chimeric virus vector has enhanced transduction of a target cell as compared with the recipient parent AAV vector.

3. The chimeric virus vector of claim 2, wherein said chimeric virus vector has enhanced transduction of skeletal muscle.

4. The chimeric virus vector of claim 2, wherein said chimeric virus vector has enhanced transduction of cardiac muscle.

5. The chimeric virus vector of claim 2, wherein said chimeric virus vector has enhanced transduction of astrocytes and/or glial cells.

6. The chimeric virus vector of claim 1, wherein the insertion is an insertion of threonine into the capsid subunit.

7. The chimeric virus vector of claim 1, wherein the chimeric virus vector comprises a chimeric AAV2 capsid comprising an amino acid insertion following amino acid position 264 in an AAV2 capsid subunit.

8. The chimeric virus vector of claim 7, wherein the insertion is an insertion of threonine.

9. The chimeric virus vector of claim 1, wherein the chimeric virus vector comprises a chimeric AAV3b capsid comprising an amino acid insertion following amino acid position 264 in an AAV3b capsid subunit.

10. The chimeric virus vector of claim 9, wherein the insertion is an insertion of threonine.

11. The chimeric virus vector of claim 1, wherein the chimeric virus vector further comprises a selective amino acid substitution selected from the group consisting of:

(a) an amino acid substitution at amino acid position 263 in the AAV2 capsid subunit or a corresponding change in the capsid subunit from other AAV;

(b) an amino acid substitution at amino acid position 450 in the AAV2 capsid subunit or a corresponding change in the capsid subunit from other AAV;

(c) an amino substitution at amino acid position 457 in the AAV2 capsid subunit or a corresponding change in the capsid subunit from other AAV; and

(d) any combination of (a) to (c) above.

12. The chimeric virus vector of claim 11, wherein the chimeric virus vector comprises a chimeric AAV2 capsid comprising an amino acid substitution at amino acid position 263 in the AAV2 capsid subunit.

13. The chimeric virus vector of claim 12, wherein the substitution is a substitution of alanine for glutamine.

14. The chimeric virus vector of claim 11, wherein the chimeric virus vector comprises a chimeric AAV3b capsid comprising an amino acid substitution at amino acid position 263 in the AAV3b capsid subunit.

15. The chimeric virus vector of claim 14, wherein the substitution is a substitution of alanine for glutamine.

16. The chimeric virus vector of claim 11, wherein the chimeric virus vector comprises a chimeric AAV2 capsid comprising an amino acid substitution at amino acid position 450 in the AAV2 capsid subunit.

17. The chimeric virus vector of claim 16, wherein the substitution is a substitution of asparagine for threonine.

18. The chimeric virus vector of claim 11, wherein the chimeric virus vector comprises a chimeric AAV2 capsid comprising an amino acid substitution at amino acid position 457 in the AAV2 capsid subunit.

19. The chimeric virus vector of claim 18, wherein the substitution is a substitution of asparagine for glutamine.

29. The chimeric virus vector of claim 1, wherein the insertion following amino acid position 264 or corresponding change is present in all subunits of the AAV capsid.

30. The chimeric virus vector of claim 1, wherein the heterologous nucleic acid sequence encodes a polypeptide.

31. The chimeric virus vector of claim 30, wherein the polypeptide is a therapeutic polypeptide.

32. The chimeric virus vector of claim 31, wherein the therapeutic polypeptide is selected from the group consisting of:

dystrophin, mini-dystrophin, utrophin, a clotting factor including Factor VIII or Factor IX, a growth factor including insulin-like growth factor I, insulin-like growth factor II, platelet-derived growth factor, epidermal derived growth factor, fibroblast-derived growth factor, nerve-derived growth factor, glial-derived growth factor, transforming growth factor-α

or transforming growth factor-β

, a neurotrophic factor, an anti-inflammatory factor including transforming growth factor-a soluble receptor or IRAP, α

1-antitrypsin, lysosomal acid-α

glucosidase, β

-glucocerebrosidase, α

-galactosidase A, a cytokine, an interferon including β

-interferon, TRAIL, FAS-ligand, endostatin, angiostatin, cystic fibrosis transmembrane regulator protein, erythropoietin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, ,β

-globin, α

-globin, spectrin, adenosine deaminase, hypoxanthine guanine phosphoribosyl transferase, sphingomyelinase, lysosomal hexosaminidase, branched-chain keto acid dehydrogenase, a bone morphogenic protein including VEGF and RANKL, protein phosphatase inhibitor I, phospholamban, serca2a, catalase, tyrosine hydroxylase, superoxide dismutase, leptin, RP65 protein, galanin, α

-L-iduronidase, a hormone including insulin or somatotropin, galactocerebrosidase, phenylalanine hydroxylase, LDL receptor, soluble CD4, anti-apoptotic gene products, glutamate receptor, a lymphokine, barkct, β

2-adrenergic receptor, calsarcin, enos, inos, a sarcoglycan, Fc receptor, T cell receptor, ApoE, ApoC, a suicide gene product, a tumor suppressor gene product, and any combination thereof.

33. The chimeric virus vector of claim 1, wherein the heterologous nucleic acid sequence encodes an untranslated RNA.

34. The chimeric virus vector of claim 33, wherein the untranslated RNA is an antisense RNA or an interfering RNA (RNAi).

35. The chimeric virus vector of claim 33, wherein the untranslated RNA is directed to VEGF, the multiple drug resistance gene product, myostatin, or repeats in the Huntington Disease gene product.

36. A pharmaceutical formulation comprising the chimeric virus vector according to claim 1 in a pharmaceutically acceptable carrier.

37. A method of administering a nucleic acid to a cell comprising contacting the cell with the chimeric virus vector of claim 1.

38. A method of delivering a nucleic acid to a subject comprising administering to the subject the chimeric virus vector of claim 1.

39. The method of claim 38, wherein the subject is a human subject.

40. The method of claim 38, wherein the subject has or is at risk for a disorder selected from the group consisting of a muscular dystrophy including Duchenne or Becker muscular dystrophy, hemophilia A, hemophilia B, multiple sclerosis, diabetes mellitus, Gaucher disease, Fabry disease, Pompe disease, cancer, arthritis, muscle wasting, heart disease including congenital heart failure or peripheral artery disease, intimal hyperplasia, a neurological disorder including epilepsy, Huntington'"'"'s disease, Parkinson'"'"'s disease or Alzheimer'"'"'s disease, an autoimmune disease, cystic fibrosis, thalassemia, Hurler'"'"'s disease, Krabbe'"'"'s disease, phenylketonuria, Batten'"'"'s disease, spinal cerebral ataxia, LDL receptor deficiency, hyperammonemia, anemia, arthritis, a retinal degenerative disorder including macular degeneration, adenosine deaminase deficiency, and cancer including tumor-forming cancers.

41. The method of claim 38, wherein the chimeric virus vector or pharmaceutical formulation is administered to skeletal muscle.

42. The method of claim 41, wherein the subject has or is at risk for muscular dystrophy.

43. The method of claim 41, wherein the chimeric virus vector comprises a chimeric AAV capsid comprising a selective amino acid insertion following amino acid position 264 in an AAV2 capsid subunit.

44. The method of claim 41, wherein the chimeric virus vector comprises a chimeric AAV capsid comprising a selective amino acid insertion following amino acid position 264 in an AAV3b capsid subunit.

45. The method of claim 38, wherein the chimeric virus vector or pharmaceutical formulation is administered to cardiac muscle.

46. The method of claim 45, wherein the subject has or is at risk for heart disease.

47. The method of claim 46, wherein the subject has or is at risk for congestive heart failure or peripheral artery disease.

48. The method of claim 46, wherein the chimeric virus vector comprises a chimeric AAV capsid comprising a selective amino acid insertion following amino acid position 264 in an AAV3b capsid subunit.

49. The method of claim 38, wherein the chimeric virus vector or pharmaceutical formulation is administered to glial cells and/or astrocytes.

50. The method of claim 49, wherein the subject has or is at risk for Parkinson'"'"'s Disease, astrocytomas or glioblastoma.

51. The method of claim 49, wherein the chimeric virus vector comprises a chimeric AAV capsid comprising a selective amino acid insertion following amino acid position 264 in an AAV2 capsid subunit.

20. A chimeric virus vector comprising:

(a) a chimeric adeno-associated virus (AAV) capsid comprising;

(i) a selective amino acid substitution of an alanine for glutamine at amino acid position 263 in an AAV2 capsid subunit;

(ii) a selective amino acid insertion of a threonine following amino acid position 264 in the AAV2 capsid subunit;

(b) a nucleic acid comprising an AAV terminal repeat sequence and a heterologous nucleic acid sequence;

wherein the nucleic acid is packaged within the chimeric AAV capsid.

21. A chimeric virus vector comprising:

(a) a chimeric adeno-associated virus (AAV) capsid comprising;

(i) a selective amino acid substitution of an alanine for glutamine at amino acid position 263 in an AAV3b capsid subunit;

(ii) a selective amino acid insertion of a threonine following amino acid position 264 in the AAV3b capsid subunit;

(b) a nucleic acid comprising an AAV terminal repeat sequence and a heterologous nucleic acid sequence;

wherein the nucleic acid is packaged within the chimeric AAV capsid.

22. A chimeric virus vector comprising:

(a) a chimeric adeno-associated virus (AAV) capsid comprising a selective amino acid substitution at amino acid position 450 in an AAV2 capsid subunit or a corresponding change in a capsid subunit from other AAV;

(b) a nucleic acid comprising an AAV terminal repeat sequence and a heterologous nucleic acid sequence;

wherein the nucleic acid is packaged within the chimeric AAV capsid.

23. The chimeric virus vector of claim 22, wherein the substitution is a substitution of asparagine for threonine.

24. A chimeric virus vector comprising:

(a) a chimeric adeno-associated virus (AAV) capsid comprising a selective amino acid substitution at amino acid position 457 in an AAV2 capsid subunit or a corresponding change in a capsid subunit from other AAV;

(b) a nucleic acid comprising an AAV terminal repeat sequence and a heterologous nucleic acid sequence;

wherein the nucleic acid is packaged within the chimeric AAV capsid.

25. The chimeric virus vector of claim 24, wherein the substitution is a substitution of asparagine for glutamine.

26. The chimeric virus vector of claim 24, wherein the chimeric virus vector further comprises a selective amino acid substitution at amino acid position 450 in the AAV2 capsid subunit or a corresponding change in the capsid subunit from other AAV.

27. A chimeric virus vector comprising:

(a) a chimeric adeno-associated virus (AAV) capsid comprising;

(i) a selective amino acid insertion following amino acid position 264 in an AAV2 capsid subunit or a corresponding change in a capsid subunit from other AAV;

(ii) a selective amino acid substitution at amino acid position 263 in the AAV2 capsid subunit or a corresponding change in the capsid subunit from other AAV;

(iii) a selective amino acid substitution at amino acid position 705 in the AAV2 capsid subunit or a corresponding change in the capsid subunit from other AAV;

(iv) a selective amino substitution at amino acid position 708 in the AAV2 capsid subunit or a corresponding change in the capsid subunit from other AAV; and

(v) a selective amino substitution at amino acid position 716 in the AAV2 capsid subunit or a corresponding change in the capsid subunit from other AAV; and

(b) a nucleic acid comprising an AAV terminal repeat sequence and a heterologous nucleic acid sequence;

wherein the nucleic acid is packaged within the chimeric AAV capsid.

28. The chimeric virus vector of claim 27, wherein the chimeric AAV capsid comprises:

(a) a threonine insertion following amino acid position 264 in an AAV2 capsid subunit(s);

(b) an alanine for glutamine substitution at amino acid position 263 in the AAV2 capsid subunit(s);

(c) an alanine for asparagine substitution at amino acid position 705 in the AAV2 capsid subunit(s);

(d) an alanine for valine substitution at amino acid position 708 in the AAV2 capsid subunit(s); and

(e) an asparagine for threonine substitution at amino acid position 716 in the AAV2 capsid subunit(s).