Use of meganucelases for inducing homologous recombination ex vivo and in toto in vertebrate somatic tissues and application thereof

Abstract

A single chain homing endonuclease, comprising a first variant of I-CreI having the amino acid sequence of accession number pdb 1g9y and a second variant of I-CreI variant having the amino acid sequence of accession number pdb 1g9y in a single polypeptide.

1 - 16 . (canceled) 
     
     
         17 . A method of cleaving a targeted double stranded DNA in a liver cell of a mammal in toto, comprising steps of:
 (a) injecting intravenously a vector comprising a nucleic acid encoding a custom-made meganuclease specifically recognizing and cleaving a targeted double stranded DNA, comprising a recognition site 12 to 60 pb in length, and   (b) inducing a double strand break in the targeted double stranded DNA by expressing the custom-made meganuclease in the liver cell.   
     
     
         18 . The method of  claim 17 , wherein the vector is injected by hydrodynamic injection. 
     
     
         19 . The method of  claim 17 , wherein the double stranded DNA is, or is part of, an infectious agent. 
     
     
         20 . The method of  claim 19 , wherein the infectious agent is a virus. 
     
     
         21 . The method of  claim 20 , wherein the virus is selected from the groups consisting of adenoviruse, herpesviruse, hepadnaviruse, papovaviruse and poxviruse. 
     
     
         22 . The method of  claim 20 , wherein the virus is in a latent form. 
     
     
         23 . The method of  claim 20 , wherein the virus is in an episomal form. 
     
     
         24 . The method of  claim 20 , wherein the virus is integrated into the mammal's host genome. 
     
     
         25 . The method of  claim 20 , wherein the recognition and cleavage site is located in an ori gene. 
     
     
         26 . The method of  claim 21 , wherein the hepadnavirus is HBV. 
     
     
         27 . The method of  claim 26 , wherein the recognition and cleavage site is located in the gene X of HBV. 
     
     
         28 . The method of  claim 17 , wherein the custom-made meganuclease is a very rare-cutting endonuclease with a DNA recognition and cleavage site of at least 15 by in length, and preferably at least 18 by in length. 
     
     
         29 . The method of  claim 17 , wherein the custom-made meganuclease is derived from a homing endonuclease. 
     
     
         30 . The method of  claim 17 , wherein the custom-made meganuclease is a heterodimer of two fusion proteins. 
     
     
         31 . The method of  claim 30 , wherein the heterodimer comprises a Fokl catalytic domain. 
     
     
         32 . The method of  claim 31 , wherein the custom-made meganuclease comprises a zinc finger DNA-binding domain. 
     
     
         33 . The method of  claim 17 , wherein more than 1010 units of the vector are injected per mammal. 
     
     
         34 . The method of  claim 17 , wherein the expression of the meganuclease leads to partial or complete deletion of the viral sequence. 
     
     
         35 . The method of  claim 17 , wherein the vector is a recombinant virus. 
     
     
         36 . The method of  claim 17 , wherein the vector is introduced into the liver cells using liposomes. 
     
     
         37 . The method of  claim 17 , wherein the vector is conjugated with PEG, PPG or polyethylene-propylene glycol copolymer. 
     
     
         38 . The method of  claim 17 , wherein the meganuclease is expressed in fusion with a translocating peptide or a nuclear localization signal. 
     
     
         39 . The method of  claim 17 , wherein the mammal is a human. 
     
     
         40 . A therapeutic composition comprising a meganuclease and a pharmaceutically acceptable carrier or excipient for intravenous injection. 
     
     
         41 . The therapeutic composition of  claim 40 , wherein the pharmaceutically acceptable carrier is saline, sterile water, Ringers solution, or isotonic sodium chloride solution, or a mixture of one or more thereof. 
     
     
         42 . The therapeutic composition of  claim 40 , wherein the pharmaceutically acceptable excipient is sterile phosphate buffer saline. 
     
     
         43 . The therapeutic composition of  claim 40  for the treatment of hepatitis or liver cancer.