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

#The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Aug. 13, 2010, is named 355503US.txt and is 13,703 bytes in size.

#The present invention relates to the use of meganucleases for inducing homologous recombination ex vivo and in toto in vertebrate somatic tissues and to its application for genome engineering and gene therapy.

#Homologous gene targeting has been widely used in the past to obtain site-specific and precise genome surgery (Thomas and Capecchi, 1986, Nature, 324, 34-8; Thomas et al., 1986, Cell, 44, 419-28; Thomas and Capecchi, 1986, Cold Spring Harb Symp Quant Biol, 51 Pt 2, 1101-13; Doetschman et al., 1988, PNAS, 85, 8583-7). Homologous gene targeting relies on the homologous recombination machinery, one of the endogenous maintenance systems of the cell. Since this system has been well conserved throughout evolution, gene targeting could be used in organisms as different as bacteria, yeast, filamentous fungi, mammals, insects, and plants.

#The present invention will be further illustrated by the additional description and drawings which follows, which refers to examples illustrating the use of meganucleases for inducing homologous recombination in somatic tissues according to the invention. It should be understood however that these examples are given only by way of illustration of the invention and do not constitute in anyway a limitation thereof

#FIG. 1 discloses the amino acid sequence of a single chain I-Cre I meganuclease and one polynucleotide encoding said single chain meganuclease. In the protein sequence, the two first N-terminal residues are methionine and alanine (MA), and the three C-terminal residues alanine, alanine and aspartic acid (AAD). These sequences allow having DNA coding sequences comprising the NcoI (CCATGG) and EagI (CGGCCG) restriction sites, which are used for cloning into various vectors.

#FIG. 2 discloses polynucleotide sequences. FIG. 2A discloses a polynucleotide called “Natural” encoding the I-Cre I homing endonuclease. FIG. 2B discloses a polynucleotide sequence called “Non homologous” encoding the I-Cre I homing endonuclease. FIG. 2C discloses a polynucleotide sequence called “Template” encoding the I-Cre I homing endonuclease comprising the mutation D75NJ. Each I-Cre I homing endonuclease has two additional amino acids (MA) at the N terminal end and three additional amino acids (AAD) at the C-terminal ends. FIG. 2D discloses the polynucleotide sequences of the primers, called UlibIfor, UlibIrev, UlibIIfor, and UlibIIrev, used for the generation of the libraries UlibI and UlibII.

#Some LAGLIDADG (SEQ ID NO: 24) homing endonucleases are active as homodimer. Each monomer mainly dimerizes through their dodecapeptide motifs. A single-chain meganuclease can be engineered by covalently binding two monomers modified such as to introduce a covalent link between the two sub-units of this enzyme. Preferably, the covalent link is introduced by creating a peptide bond between the two monomers. However, other convenient covalent links are also contemplated. The single-chain meganuclease preferably comprises two subunits from the same homing endonuclease such as single-chain I-Cre I and single-chain I-Ceu I. A single-chain meganuclease has multiple advantages. For example, a single-chain meganuclease is easier to manipulate. The single-chain meganuclease is thermodynamically favored, for example for the recognition of the target sequence, compared to a dimer formation. The single-chain meganuclease allows the control of the oligomerisation.

#A single chain version of I-CreI (scI-CreI) was modeled and engineered. scI-CreI cleaves its cognate DNA substrate in vitro and induces homologous recombination both in yeast and mammalian cells.

#Design of the Single Chain I-CreI Meganuclease