US2020208176A1PendingUtilityA1

Method of increasing the function of an aav vector

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Assignee: UNIV PENNSYLVANIAPriority: Apr 7, 2005Filed: Mar 13, 2020Published: Jul 2, 2020
Est. expiryApr 7, 2025(expired)· nominal 20-yr term from priority
Inventors:Luk H. VandenbergheGuangping GaoJames M. Wilson
C12N 2750/14142A61K 48/005C12N 15/86C12N 2750/14143A61P 31/12A61K 48/00C07K 14/005A61P 43/00A61K 48/0091C12N 2750/14152C12N 7/00A61P 37/00C12N 2750/14122
75
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Claims

Abstract

A method of correcting singletons in a selected AAV sequence in order to increasing the packaging yield, transduction efficiency, and/or gene transfer efficiency of the selected AAV is provided. This method involves altering one or more singletons in the parental AAV capsid to conform the singleton to the amino acid in the corresponding position(s) of the aligned functional AAV capsid sequences.

Claims

exact text as granted — not AI-modified
1 . An adeno-associated virus (AAV) comprising an AAV capsid and a minigene having AAV inverted terminal repeats and a heterologous gene operably linked to regulatory sequences which direct expression of the heterologous gene in a host cell, wherein the AAV capsid comprises AAV vp1 proteins, AAV vp2 proteins, and AAV vp3 proteins, wherein the AAV vp1 proteins have i) the sequence of amino acids 1 to 738 of SEQ ID NO: 4 (AAVrh46) with a D41N modification, or ii) a sequence with at least 95% identity to amino acids 1 to 738 of SEQ ID NO:4 wherein amino acid residue 41 is N (D41N). 
     
     
         2 . An adeno-associated virus (AAV) comprising an AAV capsid and a minigene having AAV inverted terminal repeats and a heterologous gene operably linked to regulatory sequences which direct expression of the heterologous gene in a host cell, wherein the AAV capsid comprises AAV vp1 proteins, AAV vp2 proteins, and AAV vp3 proteins, wherein the AAV vp1 proteins have i) the sequence of amino acids 1 to 738 of SEQ ID NO: 4 (AAVrh46) with a G135P modification, or ii) a sequence with at least 95% identity to amino acids 1 to 738 of SEQ ID NO:4 wherein amino acid residue 135 is P (G135P). 
     
     
         3 . An adeno-associated virus (AAV) comprising an AAV capsid and a minigene having AAV inverted terminal repeats and a heterologous gene operably linked to regulatory sequences which direct expression of the heterologous gene in a host cell, wherein the AAV capsid comprises AAV vp1 proteins, AAV vp2 proteins, and AAV vp3 proteins, wherein the AAV vp1 proteins have i) the sequence of amino acids 1 to 738 of SEQ ID NO: 4 (AAVrh46) with a A136V modification, or ii) a sequence with at least 95% identity to amino acids 1 to 738 of SEQ ID NO:4 wherein amino acid residue 136 is V (A136V). 
     
     
         4 . The AAV according to  claim 1 , wherein the AAV inverted terminal repeats are from a different AAV than the AAV supplying the capsid proteins. 
     
     
         5 . A composition comprising the AAV according to  claim 1  and a physiologically compatible carrier. 
     
     
         6 . The AAV according to  claim 1 , wherein the heterologous gene encodes an ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase, fumarylacetacetate hydrolase, carbamoyl phosphate synthetase I, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, cystathione beta-synthase, branched chain ketoacid decarboxylase, isovaleryl-coA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase (GCDH), betaglucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, P-glucuronidase (GUSB), or glycine decarboxylase, a low density lipoprotein (LDL) receptor, high density lipoprotein (HDL) receptor, very low density lipoprotein (VLDL) receptor, scavenger receptor, glucocorticoid receptor, estrogen receptor, Vitamin D receptor, nuclear receptor, cystic fibrosis transmembrane regulator (CFTR) sequence, Factor IX or variants thereof, Factor VIII or variants thereof, a dystrophin gene product, or an immunoglobulin. 
     
     
         7 . The AAV according to  claim 6 , wherein the dystrophin gene product is a mini-dystrophin or micro-dystrophin. 
     
     
         8 . The AAV according to  claim 6 , wherein the immunoglobulin comprises IgG, IgM, IgA, IgD, IgE, a chimeric immunoglobulin, a humanized antibody, and/or a single chain antibody. 
     
     
         9 . A recombinant nucleic acid molecule encoding an AAV vp1 protein according to  claim 1 . 
     
     
         10 . A cultured host cell containing a recombinant nucleic acid molecule according to  claim 9 . 
     
     
         11 . The cultured host cell according to  claim 10 , which further comprises a rep gene. 
     
     
         12 . The cultured host cell according to  claim 10 , wherein the recombinant nucleic acid molecule is a plasmid. 
     
     
         13 . The AAV according to  claim 2 , wherein the AAV inverted terminal repeats are from a different AAV than the AAV supplying the capsid proteins. 
     
     
         14 . A composition comprising the AAV according to  claim 2  and a physiologically compatible carrier. 
     
     
         15 . The AAV according to  claim 2 , wherein the heterologous gene encodes an ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase, fumarylacetacetate hydrolase, carbamoyl phosphate synthetase I, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, cystathione beta-synthase, branched chain ketoacid decarboxylase, isovaleryl-coA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase (GCDH), betaglucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, P-glucuronidase (GUSB), or glycine decarboxylase, a low density lipoprotein (LDL) receptor, high density lipoprotein (HDL) receptor, very low density lipoprotein (VLDL) receptor, scavenger receptor, glucocorticoid receptor, estrogen receptor, Vitamin D receptor, nuclear receptor, cystic fibrosis transmembrane regulator (CFTR) sequence, Factor IX or variants thereof, Factor VIII or variants thereof, a dystrophin gene product, or an immunoglobulin. 
     
     
         16 . The AAV according to  claim 15 , wherein the dystrophin gene product is a mini-dystrophin or micro-dystrophin. 
     
     
         17 . The AAV according to  claim 15 , wherein the immunoglobulin comprises IgG, IgM, IgA, IgD, IgE, a chimeric immunoglobulin, a humanized antibody, and/or a single chain antibody. 
     
     
         18 . A recombinant nucleic acid molecule encoding an AAV vp1 protein according to  claim 2 . 
     
     
         19 . A cultured host cell containing a recombinant nucleic acid molecule according to  claim 18 . 
     
     
         20 . The cultured host cell according to  claim 19 , which further comprises a rep gene. 
     
     
         21 . The cultured host cell according to  claim 19 , wherein the recombinant nucleic acid molecule is a plasmid. 
     
     
         22 . The AAV according to  claim 3 , wherein the AAV inverted terminal repeats are from a different AAV than the AAV supplying the capsid proteins. 
     
     
         23 . A composition comprising the AAV according to  claim 3  and a physiologically compatible carrier. 
     
     
         24 . The AAV according to  claim 3 , wherein the heterologous gene encodes an ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase, fumarylacetacetate hydrolase, carbamoyl phosphate synthetase I, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, cystathione beta-synthase, branched chain ketoacid decarboxylase, isovaleryl-coA dehydrogenase, propionyl-CoA carboxylase, methylmalonyl-CoA mutase, glutaryl-CoA dehydrogenase (GCDH), betaglucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, P-glucuronidase (GUSB), or glycine decarboxylase, a low density lipoprotein (LDL) receptor, high density lipoprotein (HDL) receptor, very low density lipoprotein (VLDL) receptor, scavenger receptor, glucocorticoid receptor, estrogen receptor, Vitamin D receptor, nuclear receptor, cystic fibrosis transmembrane regulator (CFTR) sequence, Factor IX or variants thereof, Factor VIII or variants thereof, a dystrophin gene product, or an immunoglobulin. 
     
     
         25 . The AAV according to  claim 24 , wherein the dystrophin gene product is a mini-dystrophin or micro-dystrophin. 
     
     
         26 . The AAV according to  claim 24 , wherein the immunoglobulin comprises IgG, IgM, IgA, IgD, IgE, a chimeric immunoglobulin, a humanized antibody, and/or a single chain antibody. 
     
     
         27 . A recombinant nucleic acid molecule encoding an AAV vp1 protein according to  claim 3 . 
     
     
         28 . A cultured host cell containing a recombinant nucleic acid molecule according to  claim 27 . 
     
     
         29 . The cultured host cell according to  claim 28 , which further comprises a rep gene. 
     
     
         30 . The cultured host cell according to  claim 28 , wherein the recombinant nucleic acid molecule is a plasmid.

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