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 recombinant adeno-associated viral (rAAV) particle comprising a mutated capsid protein, wherein the mutated capsid protein comprises a non-tyrosine amino acid at a position that corresponds to Tyr444 in a wild-type AAV2 capsid protein.
2. The rAAV particle according to claim 1 , wherein said particle further comprises 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.
3. The rAAV particle according to claim 2 , further comprising an enhancer sequence operably linked to said nucleic acid segment.
4. The rAAV particle according to claim 3 , wherein said particle further comprises an enhancer sequence 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.
5. The rAAV particle according to claim 2 , wherein said promoter is a heterologous, tissue-specific, constitutive or inducible promoter.
6. The rAAV particle according to claim 5 , 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.
7. The rAAV particle according to claim 5 , wherein said promoter is a mammalian or avian β-actin promoter.
8. The rAAV particle according to claim 2 , wherein said first nucleic acid segment further comprises a post-transcriptional regulatory sequence or a polyadenylation signal.
9. The rAAV particle according to claim 2 , wherein said first nucleic acid segment further comprises a woodchuck hepatitis virus post-transcription regulatory element.
10. The rAAV particle according to claim 2 , wherein said 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.
11. The rAAV particle according to claim 2 , wherein said therapeutic agent is a protein or polypeptide selected from the group consisting of an adrenergic agonist, an anti-apoptosis factor, an apoptosis inhibitor, a cytokine receptor, a cytokine, a cytotoxin, an erythropoietic agent, a glutamic acid decarboxylase, a glycoprotein, a growth factor, a growth factor receptor, a hormone, a hormone receptor, an interferon, an interleukin, an interleukin receptor, a kinase, a kinase inhibitor, a nerve growth factor, a netrin, a neuroactive peptide, a neuroactive peptide receptor, a neurogenic factor, a neurogenic factor receptor, a neuropilin, a neurotrophic factor, a neurotrophin, a neurotrophin receptor, an N-methyl-D-aspartate antagonist, a plexin, a protease, a protease inhibitor, a protein decarboxylase, a protein kinase, a protein kinsase inhibitor, a proteolytic protein, a proteolytic protein inhibitor, a semaphorin, a semaphorin receptor, a serotonin transport protein, a serotonin uptake inhibitor, a serotonin receptor, a serpin, a serpin receptor, and a tumor suppressor.
12. The rAAV particle according to claim 2 , wherein said therapeutic agent is a polypeptide selected from the group consisting of BDNF, CNTF, CSF, EGF, FGF, G-SCF, GM-CSF, gonadotropin, IFN, IFG-1, M-CSF, NGF, PDGF, PEDF, TGF, TGF-B2, TNF, VEGF, prolactin, somatotropin, XIAP1, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-10(I87A), viral IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, and IL-18.
13. The rAAV particle according to claim 2 , wherein said first nucleic acid segment encodes a peptide or polypeptide of human, murine, avian, porcine, bovine, ovine, feline, canine, equine, epine, caprine, lupine or primate origin.
14. The rAAV particle according to claim 2 , wherein the non-tyrosine amino acid is a phenylalanine.
15. The rAAV particle according to claim 2 , wherein the capsid protein is 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 capsid protein.
16. A composition comprising the rAAV particle in accordance with claim 2 .
17. A method for providing a mammal in need thereof with a therapeutically-effective amount of a therapeutic agent, said method comprising providing to a cell, tissue or organ of said mammal, an amount of a composition according to claim 16 ; for a time effective to provide said mammal with a therapeutically-effective amount of said therapeutic agent.
18. The method of claim 17 , wherein said 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.
19. The rAAV particle according to claim 2 , wherein the capsid protein is an AAV serotype 2 capsid protein.
20. A recombinant adeno-associated viral (rAAV) particle comprising a mutated capsid protein, wherein the mutated capsid protein comprises a non-tyrosine amino acid at a position that corresponds to Tyr730 in a wild-type AAV2 capsid protein.
21. The rAAV particle according to claim 20 , wherein said particle further comprises 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.
22. A recombinant adeno-associated viral (rAAV) particle comprising a mutated capsid protein, wherein the mutated capsid protein comprises a non-tyrosine amino acid at a position that corresponds to Tyr500 in a wild-type AAV2 capsid protein.
23. The rAAV particle according to claim 22 , wherein said particle further comprises 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.