Capsid-modified raav vectors and methods of use

Abstract

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 - 36 . (canceled) 
     
     
         37 . A modified adeno associated virus (AAV) capsid protein comprising a non-native amino acid at one or more positions that correspond to Tyr576 or Tyr700 of a wild-type AAV2 capsid protein. 
     
     
         38 . The modified AAV capsid protein according to  claim 37 , wherein the non-native amino acid is a phenylalanine. 
     
     
         39 . The modified AAV capsid protein according to  claim 37 , wherein the capsid protein is an AAV serotype 1 (AAV1), AAV2, AAV3, AAV4, AAV5 or AAV6 capsid protein. 
     
     
         40 . The modified AAV capsid protein according to  claim 37 , wherein the capsid protein is comprised within an AAV particle. 
     
     
         41 . A recombinant adeno-associated viral (rAAV) particle that comprises the modified AAV capsid protein according to  claim 37 . 
     
     
         42 . The rAAV particle of  claim 41 , wherein the rAAV particle is an AAV1, AAV2, AAV3, AAV4, AAV5 or AAV6 particle. 
     
     
         43 . The rAAV particle of  claim 41 , wherein the transduction efficiency of the rAAV particle comprising the modified AAV capsid protein is higher than that of an AAV particle comprising a corresponding, unmodified AAV capsid protein. 
     
     
         44 . The rAAV particle according to  claim 41 , further comprising a nucleic acid segment that encodes a therapeutic agent or a diagnostic agent operably linked to a promoter capable of expressing the nucleic acid segment in a host cell. 
     
     
         45 . The rAAV particle according to  claim 44 , further comprising an enhancer sequence operably linked to the nucleic acid segment. 
     
     
         46 . The rAAV particle according to  claim 45 , wherein the enhancer sequence is 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, or an islet cell-specific enhancer. 
     
     
         47 . The rAAV particle according to  claim 44 , wherein the promoter is a heterologous, tissue-specific, constitutive or inducible promoter. 
     
     
         48 . The rAAV particle according to  claim 47 , wherein the promoter is 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, or a VP16-LexA promoter. 
     
     
         49 . The rAAV particle according to  claim 44 , wherein the therapeutic agent is a polypeptide, a peptide, an antibody, an antigen binding fragment, a ribozyme, a peptide nucleic acid, an siRNA, an RNAi, an antisense oligonucleotide, or an antisense polynucleotide. 
     
     
         50 . A composition comprising the rAAV particle according to  claim 41 . 
     
     
         51 . A composition comprising the rAAV particle according to  claim 44 . 
     
     
         52 . A method for administering a therapeutic or a diagnostic agent to a mammal in need thereof, the method comprising: providing to a cell, tissue or organ of the mammal the composition according to  claim 51 , wherein the transduction efficiency of the rAAV particle comprised in the composition is higher than that of an AAV particle comprising a corresponding, unmodified, wild-type capsid protein. 
     
     
         53 . The method of  claim 52 , wherein the rAAV particle is an AAV1, AAV2, AAV3, AAV4, AAV5 or AAV6 particle. 
     
     
         54 . The method according to  claim 52 , wherein the transduction efficiency of the rAAV particle comprising the modified capsid protein is at least 4-fold higher than that of an AAV particle comprising the corresponding, unmodified capsid protein. 
     
     
         55 . The method according to  claim 52 , wherein the non-native amino acid is a phenylalanine. 
     
     
         56 . The method according to  claim 52 , wherein the rAAV particle is comprised within a mammalian host cell. 
     
     
         57 . The method according to  claim 56 , wherein the mammalian host cell is a human endothelial, epithelial, vascular, liver, lung, heart, pancreas, intestinal, kidney, muscle, bone, neural, blood, or brain cell. 
     
     
         58 . 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 41 ;   wherein the rAAV particle comprises a nucleic acid segment that encodes the therapeutic peptide, polypeptide, or RNA, and that is operably linked to at least one promoter that expresses the nucleic acid segment in one or more cells of the selected population.   
     
     
         59 . The method according to  claim 58 , wherein the rAAV particle is an AAV1, AAV2, AAV3, AAV4, AAV5 or AAV6 particle. 
     
     
         60 . The method according to  claim 58 , 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; and/or 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. 
     
     
         61 . The method according to  claim 58 , wherein the nucleic acid segment is further operably linked to at least one enhancer sequence. 
     
     
         62 . The method according to  claim 58 , 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 cells. 
     
     
         63 . The method according to  claim 58 , 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. 
     
     
         64 . The method according to  claim 58 , wherein the mammal has cancer, diabetes, autoimmune disease, kidney disease, cardiovascular disease, pancreatic disease, intestinal disease, liver disease, neurological disease, neuromuscular disease, Batten disease, Alzheimer's disease, Huntington disease, Parkinson's disease, pulmonary disease, an α-1 antitrypsin deficiency, a neurological disability, a neuromotor deficit, a neuroskeletal impairment, ischemia, stroke, or any combination thereof. 
     
     
         65 . A method of treating or ameliorating one or more symptoms of a disease, a disorder, a dysfunction, an injury, or trauma in a mammal, the method comprising administering to the mammal an effective amount of the recombinant adeno-associated viral (rAAV) particle according to  claim 41 ; wherein the particle comprises a nucleic acid segment that encodes a therapeutic peptide, polypeptide, or RNA, and that is operably linked to at least one promoter that expresses the nucleic acid segment in one or more cells of a selected population. 
     
     
         66 . The method of  claim 65 , wherein the rAAV particle is an AAV1, AAV2, AAV3, AAV4, AAV5 or AAV6 particle. 
     
     
         67 . The method according to  claim 65 , wherein the one or more cells are human endothelial, epithelial, vascular, liver, lung, heart, pancreas, intestinal, kidney, muscle, bone, neural, blood, or brain cells.