Use of meganucleases 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.

What is claimed: 
     
         1 . A method for inducing recombination at a targeted genomic locus in the liver of a vertebrate, comprising;
 intravenous administration to said vertebrate of (i) a vector comprising a sequence encoding a meganuclease under the control of transcriptional regulatory elements for expression, wherein said meganuclease is expressed by said transcriptional regulatory elements in liver cells, and can induce a genomic DNA double-stranded break at a genomic site of interest in the liver, the genomic site comprising a recognition site and a cleavage site of a binding site of the meganuclease;   (ii) a targeting DNA comprising a repair-DNA flanked by 5′ and 3′ DNA arms homologous to the genomic DNA surrounding said genomic site of interest, wherein targeting DNA is inserted into the vector comprising the sequence encoding the meganuclease or into a second vector wherein the vector(s) is/are selected from the group consisting of a plasmid vector, an adenoviral vector, a lentiviral vector and an adeno-associated viral vector, and wherein homologous recombination between said targeting DNA and said genomic DNA surrounding the genomic site of interest repairs said double-stranded break and introduces said repair-DNA into said genomic site of interest.   
     
     
         2 . The method of  claim 1 , wherein the vertebrate comprises a genetic liver disease and the method prevents, improves or cures the genetic liver disease. 
     
     
         3 . The method of  claim 1 , wherein said transcriptional regulatory elements consist of a promoter. 
     
     
         4 . The method of  claim 3 , wherein said promoter is a liver tissue specific or an inducible promoter. 
     
     
         5 . The method of  claim 1 , wherein said vector includes a nuclear localisation signal. 
     
     
         6 . The method of  claim 1 , wherein the targeting DNA is in the vector encoding said meganuclease. 
     
     
         7 . The method of  claim 1 , wherein the targeting DNA 5′ and 3′ DNA arms comprise at least 50 bp homology with said targeted genomic locus. 
     
     
         8 . The method of  claim 1 , wherein said genomic site of interest comprises a mutated gene sequence and said repair DNA comprises a non-mutated sequence of said gene. 
     
     
         9 . The method of  claim 2 , wherein the vertebrate comprises a genetic liver disease and the method prevents, improves, or cures the genetic liver disease. 
     
     
         10 . The method of  claim 9 , wherein said genetic liver disease is selected from the group consisting of apolipoprotein deficiencies (apoA-I or apoB genes), familial hypercholesterolemia (FH; LDL receptor gene), Wilson's disease (WND gene), Sickle cell anemia (hemoglobin beta gene (HBB)), alpha-1 antitrypsin deficiency (alpha-1 antitrypsin gene), Hereditary hemochromatosis (HFE gene), Hemophilia A or B (Factor IX or X genes), Aminoacidopathies (tyrosinemia (fumarylacetoacetate hydrolase gene (FAH)), and phenylketonurea (phenylalanine hydroxylase gene (PAH)). 
     
     
         11 . The method of  claim 9 , wherein the vertebrate comprises a genetic liver disease and the method prevents, improves or cures the genetic disease. 
     
     
         12 . The method of  claim 9 , wherein said genetic disease is caused by dominant or compound heterozygous mutations is selected from the group consisting of familial hypercholesterolaemia and familial hyperlipidaemia. 
     
     
         13 . The method of  claim 1 , wherein said vector comprising the polynucleotide construct encoding said meganuclease and said targeting DNA are administered in association with either at least an appropriate vehicle or carrier. 
     
     
         14 . The method of  claim 1 , wherein said meganuclease is selected from the group consisting of a homing endonuclease and a zinc-finger nuclease. 
     
     
         15 . The method of  claim 1 , wherein the targeting DNA 5′ and 3′ DNA arms comprise at least 100 bp homology with said targeted genomic locus. 
     
     
         16 . The method of  claim 1 , wherein the targeting DNA 5′ and 3′ DNA arms comprise at least 200 bp with said targeted genomic locus.