Capsid modified rAAV vectors and methods of use
Disclosed are tyrosine-modified rAAV vectors, as well as infectious virions, compositions, and pharmaceutical formulations that comprise them. Also disclosed are methods of preparing and methods for using the disclosed tyrosine-phosphorylated capsid protein mutant rAAV vectors in a variety of diagnostic and therapeutic applications including in vivo and ex vivo gene therapy, and large-scale production of rAAV vectors.
1. A modified adeno associated virus (AAV) capsid protein comprising a non-tyrosine amino acid at one or more positions that correspond to Tyr252, Tyr272, Tyr275, Tyr281, Tyr444, Tyr500, Tyr508, Tyr612, Tyr704, Tyr720, Tyr730, or Tyr673 in a wild-type AAV2 capsid protein.
2. The modified AAV capsid protein according to claim 1 , wherein the non-tyrosine amino acid is a phenylalanine.
3. The modified AAV capsid protein according to claim 1 , wherein the capsid protein is of a serotype selected from the group consisting of an AAV serotype 1, an AAV serotype 2, an AAV serotype 3, an AAV serotype 4, an AAV serotype 5 and an AAV serotype 6 .
4. The modified AAV capsid protein according to claim 1 , wherein the capsid protein is comprised within an AAV particle.
5. A recombinant adeno-associated viral (rAAV) particle that comprises the modified AAV capsid protein according to claim 1 .
6. The rAAV particle of claim 5 , that is of a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5 and AAV6.
7. The rAAV particle according to claim 5 , further comprising at least a first nucleic acid segment that encodes a therapeutic agent operably linked to a promoter capable of expressing said segment in a host cell.
8. The rAAV particle according to claim 7 , further comprising an enhancer sequence operably linked to said nucleic acid segment.
9. The rAAV particle according to claim 8 , wherein the enhancer sequence is selected from the group consisting of a CMV enhancer, a synthetic enhancer, a liver-specific enhancer, a vascular-specific enhancer, a brain-specific enhancer, a neural cell-specific enhancer, a lung-specific enhancer, a muscle-specific enhancer, a kidney-specific enhancer, a pancreas-specific enhancer, and an islet cell-specific enhancer.
10. The rAAV particle according to claim 7 , wherein said promoter is a heterologous, tissue-specific, constitutive or inducible promoter.
11. The rAAV particle according to claim 10 , wherein said promoter is selected from the group consisting of a CMV promoter, a β-actin promoter, an insulin promoter, an enolase promoter, a BDNF promoter, an NGF promoter, an EGF promoter, a growth factor promoter, an axon-specific promoter, a dendrite-specific promoter, a brain-specific promoter, a hippocampal-specific promoter, a kidney-specific promoter, an elafin promoter, a cytokine promoter, an interferon promoter, a growth factor promoter, an alpha- 1 antitrypsin promoter, a brain-specific promoter, a neural cell-specific promoter, a central nervous system cell-specific promoter, a peripheral nervous system cell-specific promoter, an interleukin promoter, a serpin promoter, a hybrid CMV promoter, a hybrid β-actin promoter, an EF1 promoter, a U1a promoter, a U1b promoter, a Tet-inducible promoter and a VP16-LexA promoter.
12. The rAAV particle according to claim 7 , wherein the therapeutic agent is selected from the group consisting of a polypeptide, a peptide, an antibody, an antigen binding fragment, a ribozyme, a peptide nucleic acid, an siRNA, an RNAi, an antisense oligonucleotide and an antisense polynucleotide.
13. A composition comprising the rAAV particle according to claim 5 .
14. A method for administering a therapeutic or a diagnostic agent to a mammal in need thereof, the method comprising: providing to at least a first cell, tissue or organ of the mammal the composition according to claim 13 , wherein the transduction efficiency of the rAAV particle comprised in the composition is higher than that of a particle comprising a corresponding, unmodified, wild-type capsid protein.
15. The method of claim 14 , wherein the rAAV particle is a serotype selected from AAV1, AAV2, AAV3, AAV4, AAVS and AAV6.
16. The method according to claim 14 , wherein the transduction efficiency of the rAAV particle comprising the modified capsid protein is at least 4-fold higher than that of a rAAV particle comprising the corresponding, unmodified capsid protein.
17. The method according to claim 14 , wherein the non-tyrosine amino acid is a phenylalanine.
18. The method according to claim 14 , wherein the rAAV particle further comprises a polynucleotide that encodes a therapeutic agent operably linked to a promoter.
19. The method according to claim 14 , wherein the rAAV particle is comprised within a mammalian host cell.
20. The method according to claim 19 , wherein the mammalian host cell is a human endothelial, epithelial, vascular, liver, lung, heart, pancreas, intestinal, kidney, muscle, bone, neural, blood, or brain cell.
21. A method of providing a therapeutic peptide, polypeptide, or RNA to a mammal in need thereof, the method comprising:
introducing into a selected population of cells of the mammal an effective amount of a recombinant adeno-associated viral (rAAV) particle according to claim 5 ;
wherein the rAAV particle comprises a nucleic acid segment that encodes the therapeutic peptide, polypeptide, or RNA, and is operably linked to at least one promoter that expresses the nucleic acid segment in one or more cells of the selected population.
22. The method of claim 21 , wherein the rAAV particle is a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5 and AAV6.
23. The method according to claim 21 , wherein the therapeutic peptide or polypeptide is an antibody or an antigen-binding fragment thereof; or wherein the therapeutic RNA is a ribozyme, a peptide-nucleic acid, an siRNA, an RNAi, or an antisense oligonucleotide.
24. The method according to claim 21 , wherein the nucleic acid segment is further operably linked to at least one enhancer sequence.
25. The method according to claim 21 , wherein the promoter is a heterologous promoter, a tissue-specific promoter, a constitutive promoter, a cell-specific promoter, an inducible promoter, or any combination thereof.
26. The method of claim 21 , wherein the selected population of cells is a population of human endothelial, epithelial, vascular, liver, lung, heart, pancreas, intestinal, kidney, muscle, bone, neural, blood, or brain cell.
27. The method according to claim 21 , wherein the therapeutic peptide, polypeptide, or RNA, is expressed in one or more cells of the selected population in an amount, and for a time sufficient to treat or ameliorate at least one symptom of a disease, a disorder, a dysfunction, an injury, or trauma in a human patient.
28. The method according to claim 21 , wherein the mammal has cancer, diabetes, autoimmune disease, kidney disease, cardiovascular disease, pancreatic disease, intestinal disease, liver disease, neurological disease, neuromuscular disease, Bratten's disease, Alzheimer's disease, Huntington disease, Parkinson's disease, pulmonary disease, an a-1 antitrypsin deficiency, neurological disability, neuromotor deficit, neuroskeletal impairment, ischemia, stroke, or any combination thereof.
29. A method of treating or ameliorating one or more symptoms of a disease, a disorder, a dysfunction, an injury, or trauma in a mammal, comprising administering to the mammal an effective amount of the recombinant adeno-associated viral (rAAV) particle according to claim 5 ; wherein the particle comprises a nucleic acid segment that encodes a therapeutic peptide, polypeptide, or RNA, operably linked to at least one promoter that expresses the nucleic acid segment in one or more cells of the selected population.
30. The method of claim 29 , wherein the rAAV particle is a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5 and AAV6.
31. The method according to claim 29 , wherein the one or more cells are human endothelial, epithelial, vascular, liver, lung, heart, pancreas, intestinal, kidney, muscle, bone, neural, blood, or brain cells.