Adeno-associated virus vector variants for high efficiency genome editing and methods thereof

Abstract

Adeno-associated virus (AAV) Clade F vectors or AAV vector variants (relative to AAV9) for precise editing of the genome of a cell and methods and kits thereof are provided. Targeted genome editing using the AAV Clade F vectors or AAV vector variants provided herein occurred at frequencies that were shown to be 1,000 to 100,000 fold more efficient than has previously been reported. Also provided are methods of treating a disease or disorder in a subject by editing the genome of a cell of the subject via transducing the cell with an AAV Clade F vector or AAV vector variant as described herein and further transplanting the transduced cell into the subject to treat the disease or disorder of the subject. Also provided herein are methods of treating a disease or disorder in a subject by in vivo genome editing by directly administering the AAV Clade F vector or AAV vector variant as described herein to the subject.

1 - 146 . (canceled) 
     
     
         147 . A method for producing a replication-defective adeno-associated virus (AAV), the method comprising introducing into a mammalian cell:
 (a) a correction genome comprising a first nucleotide sequence comprising (i) an editing element comprising an internucleotide bond or a nucleotide sequence for integration into a target locus of a chromosome in a cell, (ii) a 5′ homologous arm nucleotide sequence 5′ of the editing element, having homology to a 5′ region of the chromosome relative to the target locus, and (iii) a 3′ homologous arm nucleotide sequence 3′ of the editing element, having homology to a 3′ region of the chromosome relative to the target locus; and   (b) a packaging system comprising (i) a second nucleotide sequence encoding one or more AAV Rep proteins, (ii) a third nucleotide sequence encoding one or more AAV capsid proteins, and (iii) a fourth nucleotide sequence comprising one or more herpes simplex virus (HSV) genes selected from the group consisting of UL5/8/52, ICPO, ICP4, ICP22 and UL30/UL42, under conditions whereby the correction genome is enclosed by AAV capsid proteins to form the replication-defective AAV.   
     
     
         148 . The method of  claim 147 , wherein the fourth nucleotide sequence encodes each one of the HSV genes UL5/8/52, ICPO, ICP4, ICP22 and UL30/UL42. 
     
     
         149 . The method of  claim 147 , wherein the first, second, third or fourth nucleotide sequences are contained within a virus. 
     
     
         150 . The method of  claim 149 , wherein the virus is a herpes simplex virus (HSV). 
     
     
         151 . The method of  claim 147 , wherein the packaging system comprises an HSV comprising the second and third nucleotide sequences. 
     
     
         152 . The method of  claim 147 , wherein the packaging system comprises an HSV comprising the first and fourth nucleotide sequences. 
     
     
         153 . The method of  claim 147 , wherein the packaging system comprises an HSV comprising the second, third and fourth nucleotide sequences. 
     
     
         154 . The method of  claim 147 , wherein the packaging system comprises a first HSV comprising the first nucleotide sequence, and a second HSV comprising the second, third and fourth nucleotide sequences. 
     
     
         155 . The method of  claim 147 , wherein the packaging system comprises a first HSV comprising the first and fourth nucleotide sequences, and a second HSV comprising the second and third nucleotide sequences. 
     
     
         156 . The method of  claim 147 , wherein the first, second, third or fourth nucleotide sequences are contained within a plasmid. 
     
     
         157 . The method of  claim 156 , wherein the packaging system comprises a plasmid comprising the second and third nucleotide sequences. 
     
     
         158 . The method of  claim 156 , wherein the packaging system comprises a first plasmid comprising the first nucleotide sequence and a second plasmid comprising the second and third nucleotide sequences. 
     
     
         159 . The method of  claim 156 , wherein the packaging system comprises: a first plasmid comprising the first nucleotide sequence; a second plasmid comprising the second and third nucleotide sequences; and an HSV comprising the fourth nucleotide sequence. 
     
     
         160 . The method of claim  1 , wherein one or more of the AAV capsid proteins is a Clade F capsid protein. 
     
     
         161 . The method of  claim 160 , wherein the Clade F capsid protein is a capsid protein of an AAV selected from the group consisting of AAV9, AAVHSC1, AAVHSC2, AAVHSC3, AAVHSC4, AAVHSC5, AAVHSC6, AAVHSC7, AAVHSC8, AAVHSC9, AAVHSC11, AAVHSC12, AAVHSC13, AAVHSC14, AAVHSC15, AAVHSC16, AAVHSC17, AAVHU31, and AAVHU32.