Methods and compositions for nuclease-mediated targeted integration of transgenes into mammalian liver cells

Abstract

Disclosed herein are methods and compositions for targeted, nuclease-mediated insertion of transgene sequences into the genome of a cell.

1 . A method of integrating one or more transgenes into a genome of an isolated cell, the method comprising:
 introducing, into the cell,   (a) an adeno-associated type virus (AAV) donor vector comprising the one or more transgenes, wherein the one or more transgenes encode a protein selected from the group consisting of: Factor VII, Factor VIII, Factor IX, and any protein lacking in a lysosomal storage disease, hemoglobinopathy, or metabolic disorder;   (b) at least one fusion protein comprising a DNA binding domain and cleavage domain or cleavage half-domain that cleaves an endogenous gene,   such that the one or more transgenes are integrated into the cleaved endogenous gene.   
     
     
         2 . The method of  claim 1 , wherein the at least one fusion protein is introduced at least 4 hours before the AAV donor vector. 
     
     
         3 . (canceled) 
     
     
         4 . (canceled) 
     
     
         5 . (canceled) 
     
     
         6 . The method of  claim 5 , wherein the endogenous gene is a safe-harbor locus. 
     
     
         7 . A method of providing a functional protein lacking or deficient in a subject, the method comprising integrating one or more transgenes into an isolated cell according to the method of  claim 1  and administering the isolated cell to the subject. 
     
     
         8 . The method of  claim 7 , wherein the cell is a stem cell. 
     
     
         9 . The method of  claim 1 , wherein the AAV vector comprises an AAV2 vector, an AAV6 vector or chimeric AAV2/6. 
     
     
         10 . The method of  claim 6 , wherein the safe-harbor locus is selected from the group consisting of: CCR5, HRPT, AAVS1, Rosa26, and albumin. 
     
     
         11 . The method of  claim 1 , wherein the DNA binding domain is selected from the group consisting of a meganuclease DNA binding domain, a leucine zipper DNA binding domain, a transcription activator-like (TAL) DNA binding domain, a RNA-guided CRISPR-Cas, a recombinase, a zinc finger protein DNA binding domain, and chimeric combinations of any of the foregoing. 
     
     
         12 . The method of  claim 1 , wherein the cleavage domain or cleavage half-domain is selected from the group consisting of a cleavage half-domain from a type IIS restriction endonuclease, a cleavage half-domain from FokI endonuclease, a cleavage half-domain from StsI endonuclease, and a homing endonuclease. 
     
     
         13 . The method of  claim 1 , wherein the fusion protein is a zinc finger nuclease (ZFN). 
     
     
         14 . The method of  claim 13 , wherein the ZFN comprises first and second ZFNs. 
     
     
         15 . The method of  claim 14 , wherein the first and second ZFNs are administered as RNA. 
     
     
         16 . The method of  claim 15 , wherein the RNA is mRNA. 
     
     
         17 . The method of  claim 14 , wherein the first and second ZFNs are administered using one or more AAV vectors. 
     
     
         18 . The method of  claim 1 , wherein the ZFN is introduced before the donor vector. 
     
     
         19 . The method of  claim 14 , wherein the donor vector, first ZFN, and second ZFN are administered in a ratio of about 8:1:1. 
     
     
         20 . A method for treating a Factor VII (F.VII), Factor VIII (F.VIII), or Factor IX (F.IX) deficiency in a mammal, the method comprising:
 (i) intravenously administering one or more adeno-associated viral (AAV) vectors encoding a pair of zinc finger nucleases (ZFNs) or a pair of TAL-effector protein nucleases (TALENs) into a mammal with a F.VII, F.VIII, or F.IX deficiency such that a double-stranded break in an endogenous gene occurs in liver cells of the mammal, and   (ii) intravenously administering one or more AAV vectors comprising a donor sequences comprising a transgene encoding a functional protein into the mammal after administering the one or more AAV vectors encoding the pair of ZFNs or TALENs, wherein the transgene is flanked by nucleic acid sequences homologous to the endogenous gene, such that the transgene is integrated into the endogenous gene,   the functional F.VII or F.VIII protein is expressed in cells of the mammal.   
     
     
         21 . The method of  claim 20 , wherein the endogenous gene is a safe-harbor locus selected from the group consisting of: CCR5, HRPT, AAVS1, Rosa26, and albumin.