Mutant adeno-associated virus virions and methods of use thereof

Abstract

The present invention provides mutant adeno-associated virus (AAV) that exhibit altered capsid properties, e.g., reduced binding to neutralizing antibodies in serum and/or altered heparin binding and/or altered infectivity of particular cell types. The present invention further provides libraries of mutant AAV comprising one or more mutations in a capsid gene. The present invention further provides methods of generating the mutant AAV and mutant AAV libraries, and compositions comprising the mutant AAV. The present invention further provides recombinant AAV (rAAV) virions that comprise a mutant capsid protein. The present invention further provides nucleic acids comprising nucleotide sequences that encode mutant capsid proteins, and host cells comprising the nucleic acids. The present invention further provides methods of delivering a gene product to an individual, the methods generally involving administering an effective amount of a subject rAAV virion to an individual in need thereof.

What is claimed is: 
     
         1 . An isolated nucleic acid comprising a nucleotide sequence that encodes a mutant adeno-associated virus (AAV) capsid protein, wherein the mutant AAV capsid protein comprises at least one amino acid substitution relative to a wild-type AAV capsid protein. 
     
     
         2 . The isolated nucleic acid of  claim 1 , wherein the encoded mutant AAV capsid protein exhibits increased heparin binding relative to a wild-type AAV capsid protein. 
     
     
         3 . The isolated nucleic acid of  claim 1 , wherein the encoded mutant AAV capsid protein exhibits decreased heparin binding relative to a wild-type AAV capsid protein. 
     
     
         4 . The isolated nucleic acid of  claim 1 , wherein the encoded mutant AAV capsid protein exhibits reduced binding to neutralizing antibody relative to a wild-type AAV capsid protein. 
     
     
         5 . An isolated host cell comprising the nucleic acid of  claim 1 . 
     
     
         6 . The isolated host cell of  claim 5 , wherein the host cell is stably transfected with the nucleic acid. 
     
     
         7 . The isolated host cell of  claim 5 , further comprising a nucleic acid comprising a nucleotide sequence encoding an adeno-associated virus (AAV) rep protein. 
     
     
         8 . The isolated host cell of  claim 7 , further comprising a recombinant AAV vector. 
     
     
         9 . An adeno-associated virus (AAV) library, comprising a plurality of nucleic acids comprising a nucleotide sequence that encodes a mutant AAV capsid protein, wherein the mutant AAV capsid protein comprises at least one amino acid substitution relative to a wild-type AAV capsid protein. 
     
     
         10 . A library of mutant adeno-associated virus (AAV) particles, comprising a plurality of AAV particles comprising an AAV capsid protein comprising at least one amino acid substitution relative to a wild-type AAV capsid protein. 
     
     
         11 . A recombinant adeno-associated virus (rAAV) virion comprising an AAV capsid protein comprising at least one amino acid substitution relative to a wild-type AAV capsid protein; and a heterologous nucleic acid. 
     
     
         12 . The rAAV virion of  claim 11 , wherein the rAAV virion exhibits reduced heparin binding relative to wild-type AAV. 
     
     
         13 . The rAAV virion of  claim 11 , wherein the rAAV virion exhibits increased heparin binding relative to wild-type AAV. 
     
     
         14 . The rAAV virion of  claim 11 , wherein the rAAV virion exhibits reduced inhibition of infection by neutralizing antibodies relative to the inhibition of infection by neutralizing antibodies of wild-type AAV virion. 
     
     
         15 . The rAAV virion of  claim 11 , wherein the rAAV virion exhibits increased capacity to cross an endothelial cell layer relative to wild-type AAV virion. 
     
     
         16 . The rAAV virion of  claim 11 , wherein the rAAV virion exhibits increased infectivity of a non-permissive cell relative to wild-type AAV virion. 
     
     
         17 . A method of generating a mutant adeno-associated virus (AAV) particle comprising an AAV capsid protein comprising at least one amino acid substitution relative to a wild-type AAV capsid protein, wherein the mutant AAV particle exhibits an altered capsid property, the method comprising:
 a) generating a library of AAV mutants comprising a mutation in an AAV cap gene;   b) packaging the AAV library into AAV particles, generating AAV library particles; and   c) selecting the AAV library particles for an altered capsid property.   
     
     
         18 . The method of  claim 17 , wherein generation of the library of AAV mutants comprises polymerase chain reaction mutagenesis. 
     
     
         19 . The method of  claim 18 , where the mutagenesis comprises a staggered extension process. 
     
     
         20 . The method of  claim 17 , wherein generation of the library of AAV mutants comprises oligonucleotide-directed mutagenesis. 
     
     
         21 . The method of  claim 17 , wherein the selecting step comprises selecting for reduced binding to heparin. 
     
     
         22 . The method of  claim 17 , wherein the selecting step comprises selecting for increased binding to heparin. 
     
     
         23 . The method of  claim 17 , wherein the selecting step comprises selecting for reduced binding to neutralizing antibodies. 
     
     
         24 . The method of  claim 17 , wherein the selecting step comprises selecting for increased infectivity of a non-permissive cell. 
     
     
         25 . The method of  claim 17 , wherein the selecting step comprises selecting for increased ability to cross an endothelial cell layer. 
     
     
         26 . A method of delivering a gene product to an individual in need thereof, the method comprising administering to the individual a recombinant adeno-associated virus (rAAV) virion according to  claim 11 . 
     
     
         27 . The method of  claim 26 , wherein the gene product is a protein. 
     
     
         28 . The method of  claim 26 , wherein the gene product is a short interfering RNA.