Methods of gene delivery using capsid-modified raav expression systems

Abstract

Disclosed are methods of gene delivery using capsid-modified recombinant adeno-associated viral (rAAV) vectors. Exemplary methods are provided employing vectors that have altered affinity for heparin or heparin sulfate, as well as vectors, expression systems, and rAAV virions that lack functional VP2 protein expression, but are nevertheless, fully virulent. Also provided by the invention are methods employing the rAAV vector-based compositions, virus particles, host cells, and pharmaceutical formulations in the expression of selected therapeutic proteins, polypeptides, peptides, antisense oligonucleotides and/or ribozymes in selected mammals, including organs, tissues, and human host cells.

What is claimed is: 
     
         1 . A method for targeting a selected therapeutic agent to a mammalian cell, the method comprising providing to the cell an effective amount of a composition comprising:
 (a) an rAAV vector system that comprises or that encodes a selected therapeutic agent, and that further comprises: (i) a first expression vector comprising a coding sequence of a first AAV capsid protein under control of an expression control sequence; and (ii) a second expression vector comprising coding sequences for a second AAV capsid protein under control of an expression control sequence and a third AAV capsid protein under control of an expression control sequence; wherein the first and the second expression vectors are not on the same nucleic acid segment, and wherein the first, the second, and the third capsid proteins are distinct capsid proteins selected from the group consisting of an AAV2 Vp1 protein, an AAV2 Vp2 protein and an AAV2 Vp3 protein, and further wherein the composition is modified 1) by a mutation in the coding sequence for the Vp2 protein such that the coding sequence for Vp2 does not express a functional AAV Vp2 capsid protein and/or 2) by at least one mutation such that binding to HPSG is altered, impaired, or prevented, wherein the at least one mutation is an arginine-to-alanine mutation, or an arginine-to-lysine mutation at an amino acid residue corresponding to R487, R585, or R588 of an AAV2 capsid protein; or   (b) an infectious rAAV virion that comprises or that encodes a selected therapeutic agent, and that further comprises: (i) a first expression vector comprising a coding sequence of a first AAV capsid protein under control of an expression control sequence; and (ii) a second expression vector comprising coding sequences for a second AAV capsid protein under control of an expression control sequence, and a third AAV capsid protein under control of an expression control sequence; wherein the first and the second expression vectors are not on the same nucleic acid segment, and wherein the first, the second, and the third capsid proteins are distinct capsid proteins selected from the group consisting of an AAV2 Vp1 protein, an AAV2 Vp2 protein and an AAV2 Vp3 protein, and further wherein the composition is modified 1) by a mutation in the coding sequence for the Vp2 protein such that the coding sequence for Vp2 does not express a functional AAV Vp2 capsid protein and/or 2) by at least one mutation such that binding to HPSG is altered, impaired, or prevented, wherein the at least one mutation is an arginine-to-alanine mutation, or an arginine-to-lysine mutation at an amino acid residue corresponding to R487, R585, or R588 of an AAV2 capsid protein.   
     
     
         2 . The method of  claim 1 , wherein the selected therapeutic agent comprises a ribozyme, an antisense molecule, a peptide, a polypeptide, a protein, an antibody or an antigen binding fragment thereof, or any combination thereof. 
     
     
         3 . The method of  claim 2 , wherein the selected therapeutic agent is a protein or a 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 kinase 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. 
     
     
         4 . The method of  claim 3 , wherein the protein or polypeptide is 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(187A), viral IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, and IL-18. 
     
     
         5 . The method of  claim 1 , wherein the mammalian cell is a human, a primate, a murine, a feline, a canine, a porcine, an ovine, a bovine, an equine, an epine, a caprine, or a lupine cell. 
     
     
         6 . The method of  claim 5 , wherein the mammalian cell is a human endothelial cell, a human vascular cell, a human epithelial cell, a human liver cell, a human lung cell, a human cardiac cell, a human pancreatic cell, a human renal cell, a human muscle cell, a human bone cell, a human neural cell, a human blood cell, or a human brain cell. 
     
     
         7 . The method of  claim 1 , wherein the composition further comprises a liposome, a lipid, a lipid complex, a microsphere, a microparticle, a nanosphere, a nanoparticle, or any combination thereof. 
     
     
         8 . A method for treating or ameliorating one or more symptoms of a disease, a dysfunction, or a deficiency in a mammal, the method comprising: administering to a mammal in need thereof, an effective amount of a composition comprising:
 (a) an rAAV vector system that comprises or that encodes a selected therapeutic agent, and that further comprises: (i) a first expression vector comprising a coding sequence of a first AAV capsid protein under control of an expression control sequence; and (ii) a second expression vector comprising coding sequences for a second AAV capsid protein under control of an expression control sequence and a third AAV capsid protein under control of an expression control sequence; wherein the first and the second expression vectors are not on the same nucleic acid segment, and wherein the first, the second, and the third capsid proteins are distinct capsid proteins selected from the group consisting of an AAV2 Vp1 protein, an AAV2 Vp2 protein and an AAV2 Vp3 protein, and further wherein the composition is modified 1) by a mutation in the coding sequence for the Vp2 protein such that the coding sequence for Vp2 does not express a functional AAV Vp2 capsid protein and/or 2) by at least one mutation such that binding to HPSG is altered, impaired, or prevented, wherein the at least one mutation is an arginine-to-alanine mutation, or an arginine-to-lysine mutation at an amino acid residue corresponding to R487, R585, or R588 of an AAV2 capsid protein; or   (b) an infectious rAAV virion that comprises or that encodes a selected therapeutic agent, and that further comprises: (i) a first expression vector comprising a coding sequence of a first AAV capsid protein under control of an expression control sequence; and (ii) a second expression vector comprising coding sequences for a second AAV capsid protein under control of an expression control sequence, and a third AAV capsid protein under control of an expression control sequence; wherein the first and the second expression vectors are not on the same nucleic acid segment, and wherein the first, the second, and the third capsid proteins are distinct capsid proteins selected from the group consisting of an AAV2 Vp1 protein, an AAV2 Vp2 protein and an AAV2 Vp3 protein, and further wherein the composition is modified 1) by a mutation in the coding sequence for the Vp2 protein such that the coding sequence for Vp2 does not express a functional AAV Vp2 capsid protein and/or 2) by at least one mutation such that binding to HPSG is altered, impaired, or prevented, wherein the at least one mutation is an arginine-to-alanine mutation, or an arginine-to-lysine mutation at an amino acid residue corresponding to R487, R585, or R588 of an AAV2 capsid protein;   
       in an amount and for a time sufficient to treat or to ameliorate the one or more symptoms of the disease, the dysfunction, or the deficiency in the mammal. 
     
     
         9 . The method of  claim 8 , wherein the composition is administered to the mammal intra-muscularly, intravenously, subcutaneously, intrathecally, intraperitoneally, or by direct injection into one or more organs or tissues of the mammal. 
     
     
         10 . The method of  claim 9 , wherein the one or more organs or tissues are selected from the group consisting of pancreas, liver, heart, lung, brain, kidney, joint, and muscle. 
     
     
         11 . The method of  claim 8 , wherein the rAAV vector system or the infectious rAAV virion further comprises a third distinct expression vector that comprises an expression cassette flanked by AAV2 terminal repeat sequences. 
     
     
         12 . The method of  claim 11 , wherein the expression cassette comprises a first polynucleotide that comprises a first nucleic acid segment that encodes a selected therapeutic agent. 
     
     
         13 . The method of  claim 12 , wherein the selected therapeutic agent is a peptide, a polypeptide, a protein, a catalytic RNA molecule, a ribozyme, an antisense oligonucleotide, or an antisense polynucleotide. 
     
     
         14 . The method of  claim 12 , wherein the first polynucleotide further comprises a promoter operably linked to the first nucleic acid segment, wherein the promoter controls expression of the selected therapeutic agent in a mammalian cell. 
     
     
         15 . The method of  claim 14 , wherein the promoter is a heterologous promoter, a tissue-specific promoter, a constitutive promoter, or an inducible promoter. 
     
     
         16 . The method of  claim 12 , wherein the first polynucleotide further comprises an enhancer sequence operably linked to the first nucleic acid segment. 
     
     
         17 . The method of  claim 16 , wherein the enhancer sequence comprises a CMV enhancer, a synthetic enhancer, a liver-specific enhancer, an 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. 
     
     
         18 . The method of  claim 12 , wherein the first polynucleotide further comprises a post-transcriptional regulatory sequence or a polyadenylation signal operably linked to the first nucleic acid segment. 
     
     
         19 . The method of  claim 18 , wherein the post-transcriptional regulatory sequence is obtained from a woodchuck hepatitis virus post-transcription regulatory element, or wherein the polyadenylation signal is obtained from a bovine growth hormone gene. 
     
     
         20 . A method for targeting an AAV virion or viral particle to a mammalian cell that comprises a cell-surface receptor, the method comprising: providing to a population of mammalian cells a recombinant adeno-associated viral expression system comprising:
 (i) a first expression vector comprising a coding sequence of a first AAV capsid protein under control of an expression control sequence; and   (ii) a second expression vector comprising coding sequences for a second AAV capsid protein under control of an expression control sequence and a third AAV capsid protein under control of an expression control sequence; wherein the first and the second expression vectors are not on the same nucleic acid segment, and wherein the first, the second, and the third capsid proteins are distinct capsid proteins selected from the group consisting of an AAV2 Vp1 protein, an AAV2 Vp2 protein and an AAV2 Vp3 protein, and further wherein the composition is modified 1) by a mutation in the coding sequence for the Vp2 protein such that the coding sequence for Vp2 does not express a functional AAV Vp2 capsid protein and/or 2) by at least one mutation such that binding to HPSG is altered, impaired, or prevented, wherein the at least one mutation is an arginine-to-alanine mutation, or an arginine-to-lysine mutation at an amino acid residue corresponding to R487, R585, or R588 of an AAV2 capsid protein;   in an amount and for a time effective to target the virion or the viral particle to one or more cells of the population that express the cell-surface receptor.   
     
     
         21 . The method of  claim 20 , wherein the rAAV vector system further comprises a third distinct expression vector that comprises an expression cassette flanked by AAV2 terminal repeat sequences, wherein the expression cassette comprises a first polynucleotide that comprises a first nucleic acid segment that encodes a selected therapeutic agent operably linked to a promoter that controls expression of the selected therapeutic agent in a mammalian cell. 
     
     
         22 . The method of  claim 21 , wherein the selected therapeutic agent is a peptide, a polypeptide, a protein, a catalytic RNA molecule, a ribozyme, an antisense oligonucleotide, or an antisense polynucleotide.