WO2012018726A1 - Method for increasing double-strand break-induced gene targeting - Google Patents

Abstract

A method for increasing double-strand break-induced gene targeting by homologous recombination and particularly for increasing double-strand break-induced gene targeting frequency in primary or stem cells, thereby giving new tools for genome engineering. The present invention also relates to specific matrixes, vectors, compositions and kits used to implement this method.

Description

TITLE OF THE INVENTION

METHOD FOR INCREASING DOUBLE-STRAND BREAK-INDUCED GENE TARGETING CROSS-REFERENCE TO RELATED APPLICATIONS

The present PCT International patent application claims priority to U.S. provisional patent application 61/370,010, filed on August 2, 2010, the contents of which are hereby incorporated by reference in their entirety. BACKGROUND OF THE INVENTION Field of the Invention

The present invention concerns a method for increasing double-strand break-induced gene targeting by homologous recombination and particularly for increasing double-strand break-induced gene targeting frequency in primary or stem cells, thereby providing new tools for genome engineering. The present invention also relates to specific matrixes, vectors, compositions, and kits useful for in conjunction with this method.

Description of the Related Art

Stem cells are characterized by the ability to undergo mitosis and to differentiate into a diverse range of specialized cell types. Therefore, they can be used for several purposes, including basic research, screening, but also for cell therapy. Embryonic Stem Cells (ES), Hematopoietic Stem Cells (HSCs), Mesenchymal Stem Cells (MSCs) and Epidermal Stem Cells (ESCs) are as many examples of cells having retained multipotency, i.e. the ability to differentiate into different cell types. Even differentiated cells can be reprogrammed into Stem Cells, by nuclear transfer into embryonic cells or fusion with embryonic stem cells [for review, see (Yamanaka, 2008)]. In 2006, induced pluripotent stem cells (iPSCs) were created by expression of the Oct3/4, Sox2, c-Myc and lf4 genes in mice fibroblasts. iPSCs have the essential characteristics of ES cells, in terms of growth, morphology, but also of pluripotency (Takahashi and Yamanaka, 2006). Similar results were obtained by several groups using a variety of mouse and human differentiated cells (Hanna et al., 2007; Meissner et al., 2008; Nakagawa et al., 2008; Park et al., 2008; Takahashi et al., 2009; Takahashi et al., 2007; Wernig et al., 2007; Yu et al., 2007). iPSCs can provide an alternative to ES cells and other stem cells, for basic research and for therapeutic purposes. Engineered stem cells are at the basis of many gene and cell therapy approaches. For example, engineered HSCs have been used to treat diseases such as Severe Combined Immune Deficiency (Aiuti et al., 2002; Cavazzana-Calvo et al., 2000; Cavazzana-Calvo et al., 2005; Gaspar et al., 2004; Hacein-Bey-Abina et al., 2003), Sickle Cell Anemia (Bank et al., 2005), and Adrenoleukodystrophy (Cartier et al., 2009), by ex vivo treatment of autologous cells and transplantation.

One of the major limitations of the preclinical studies that aim at exploring the efficacy of gene transfer in humans with immunodeficiency is the limited availability of patient- derived target cells. However, having an unlimited supply of stem cells derived from somatic tissues could alleviate several technical and ethical issues related to stem cells.

In a recent series of studies, it has been shown that mature fibroblasts can be reprogrammed in vitro into pluripotent stem cell-like cells (named "induced pluripotent stem cells," or iPSCs) through retroviral transfection of a combination of transcription factors (Park et al., 2008 ( 1 ); Maherali et al., 2007; Takahashi et al, 2007; Takahashi et al., 2006; Yu et al., 2007; Park et al., 2008). These cells were shown to have the potential for hematopoietic differentiation and the ability to serve as an ideal targets for correction of genetic disorders in mice (Hanna et al., 2008).

Engineering of stem cells can be performed by transduction of integrative viral vectors. However, several other approaches have been envisioned. For example, the Piggy Back transposon has been used to engineer iPSCs in the absence of any viral vector ( aji et al., 2009; Woltjen et al., 2009). Homologous gene targeting (HGT) is another way to engineer cells. HGT allow for precise, targeted modification, using homologies between a targeting vector and the locus one want to modify (Paques and Duchateau, 2007). HGT has been extensively used to modify mouse ES cells. However, its efficiency remains low, in the range of 10^"6 to 1 0^"9 (Capecchi, 1990; Capecchi, 1989; Doetschman et al., 1987; oller and Smithies, 1989; ansour et al., 1988; Smithies et al., 1985; Thomas and Capecchi, 1987), and human ES cells proved also difficult to engineer (Irion et al., 2007; Urbach et al., 2004; Zwaka and Thomson, 2003). HGT was also used to correct the HbS allele in iPSC derived from fibroblasts from a mouse humanized model for Sickle Cell Anemia (Hanna et al., 2007). The corrected iPS could then be differentiated into hematopoietic progenitors that could engraft into mice and restore their hematopoietic systems (Hanna et al., 2007).

Several techniques have been developed to enhance the efficacy of HGT (for review, see (Paques and Duchateau, 2007). The most popular one today is the use of rare-cutting endonucleases, such as meganucleases and zinc-finger nucleases, to induce a DNA double- strand break in the targeted locus, thereby enhancing the efficiency of HGT by a 100- or 1000-fold factor (Paques and Duchateau, 2007). Zinc-finger nucleases (ZFN) are generated by fusing Zinc-finger-based DNA binding domains to an independent catalytic domain via a flexible linker (Kim et al., 1996; Smith et al., 1999; Smith et al., 2000). The archetypal ZFNs are based on the catalytic domain of the Type IIS restriction enzyme Fokl and have been successfully used to induce gene correction, gene insertion, and gene deletion (Carroll, 2004; Carroll, 2008; Cathomen and Joung, 2008; Durai et al., 2005; Porteus and Carroll, 2005).

Meganucleases are natural endonucleases whose function is to induce homologous recombination events. Meganucleases, also called homing endonucleases, can be divided into five families based on sequence and structure motifs: LAGLIDADG, GIY-YIG, H H, His-Cys box and PD-(D/E)XK (Orlowski et al., 2007; Zhao et al., 2007). The most well studied family is that of the LAGLIDADG proteins, which have been found in all kingdoms of life and which are generally encoded within introns or inteins although freestanding members also exist. I-Scel, a meganuclease from the LAGLIDADG family, has been used to induce genome engineering events in a variety of cell types and organisms (Paques and Duchateau, 2007). In addition, tailored meganucleases derived from the I-Crel LAGLIDADG protein can be engineered, to recognize chosen sequences. An engineered meganuclease targeting the human Ragl gene has been designed and tested in cells (Grizot et al., 2009; Smith et al., 2006). Up to 6% of targeted modifications could be observed with a meganuclease targeting the Ragl gene, in transfection experiments in 293 cells (Grizot et al., 2009). Today, nuclease-based approaches can achieve today HGT frequencies in the range of 10% and even more in immortalized cells. However, these figures can strongly decrease in stem cells. Although many other cell lines are difficult to transfect, primary cells, for example stem cells, particularly fall into this category. Lombardo et al. used a ZFN targeting the CCR5 human gene to induce targeted insertion in different cell types (Lombardo et al., 2007). Using non-integrative lentiviral vectors instead of transfection, the authors observed up to 50% of targeted events in K562 and Jurkat cells, about 5% in ES cells, and close to 0. 1 % in CD34+ cord blood progenitor cells (Lombardo et al., 2007). With ZFNs targeting a reporter GFP gene, Zou and colleagues reported frequencies in the range of 0.1 -0.2% in both hES and iPSC (Zou et al., 2009). However, the same authors reported lower rates with a ZFN targeting the PIG-A gene, HGT efficiencies being about 2-4x 10^"4 in ES cells, and 10^"5 in iPSC. Customized ZFNs targeting the OCT4, AAVS 1 and PITX3 endogenous loci were shown to strongly stimulate HGT in both hES and iPSC loci (Dekelver et al., 2010; Hockemeyer et al., 2009), but targeted cells were identified after a selection process, and it is difficult to compare these studies with earlier ones. Nevertheless, the use of a selection procedure suggests that the absolute rate of gene targeting was low.

Therefore, in view of the obstacles described above there is still a need for methods that would allow for efficient gene targeting in iPS cells, with frequencies higher than 10^"3 in endogenous genes, which could thus give targeted events at frequencies more compatible with most applications and/or allow for the identification for targeted events without selection.

BRIEF SUMMARY OF THE INVENTION The present invention concerns a method for increasing double-stranded break-induced homologous recombination and particularly for increasing double-stranded break-induced gene targeting in primary or stem cells, thereby giving new tools for genome engineering, particularly to increase the integration efficiency of a transgene into a genome at a predetermined location, including therapeutic applications and cell line engineering. The present invention also relates to specific matrixes, vectors, compositions and kits used to implement this method as well as other applications of these products. The above objects highlight certain aspects of the invention. Additional objects, aspects and embodiments of the invention are found in the following detailed description of the invention. In addition to the preceding features, the invention further comprises other features which will emerge from the description which follows, as well as to the appended drawings. A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following Figures in conjunction with the detailed description below.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : /L4 (//-specific Homing Endonuclease (RHE)-mediated targeting of the endogenous RA G J locus in patient-derived fibroblasts.

A. Representation of the endogenous RA G I allele (upper scheme) and of the targeted allele (lower scheme) upon RHE-mediated homologous recombination with the repair plasmid. The RHE cleavage site and the coding region (exon 2) of the RAG1 gene are shown.

B. Amplification by PCR with forward (F) and reverse (R) primers yields a 4kb and a 5.8 kb products for the untargeted and the targeted alleles, respectively. Panel B PCR products detected after amplification of DNA from fibroblasts transfected with the RHE-coding plasmid and the repair plasmid (A), the repair plasmid only

(B) or with a plasmid carrying the NeoR gene only (C).

Figure 2: Schematic representation of lentiviral vectors used.

A lentiviral vector that codes for Ds-Red under the human EF l promoter followed by an IRES sequence and either the RHE sequence (A) or ZsGreen (B) were used for transfection of human iPSC. LTR: Long Terminal Repeats of HIV; HIVgag and ENV: HIV genes; WPRE: woodchuck hepatitis post-transcriptional regulatory element.

Figure 3: Production and characterization of OS-iPSCs (iPSCs derived from patients fibroblasts carrying mutations associated in vivo with an Omenn syndrome)

A. OS-iPSc have morphology similar to hES cells when grown in coculture with irradiated mouse embryonic feeder fibroblasts (iMEFs) and express pluripotency markers including Tra- 1-81 , NANOG, OCT4, Tra- 1 -60, SSEA3, and SSEA4 as shown demonstrated by immunohistochemistry. 4,6-Diamidino-2-phenylindole (DAPI) staining is shown at right and indicates the total cell content per image.

B. PCR amplification followed by DNA sequencing of genomic DNA derived from the PID-specific IPSC and their parental fibroblasts was performed using specific primers corresponding to the gene causing mutations in each of the lines as described under methods. The OS-IPSc were shown to carry the same disease causing mutations as their parental fibroblasts that were not detected in normal control genomic DNA.

C. OS-iPSc were analyzed for chromosomal integrity by karyotyping and G-banding

Cytogenic analysis was performed on twenty G-banded metaphase cells.

D. OS-iPSc were differentiated to embryoid bodies (EB) by culture in a bFGF free hES media and without coculture with feeder cells. Robust formation of tight and well formed cell clusters was detected by day 7 becoming cystic by day 10.

Figure 4: Transfection of OS-iPSCs with Red-RHE and Red-Green vectors.

20 to 25 OS-iPSc colonies were plated in a 6 well plate on a feeder iMEF layer. Either Red-RHE or Red-Green Lentiviral vector were added to the cell culture medium at an MOI of 5 in the presence of 1 μg/ul protamine sulfate. Cells were incubated with the virus for 24 hours and then washed 3 times with PBS, and the medium was then replaced with fresh iPSC culture media. Ds-Red expression was evaluated 48 hours following the initial transfection.

Figure 5: Targeting of the RAGl locus in OS-iPSC

A. Representation of the endogenous RAGl allele (upper scheme) and of the targeted allele (lower scheme) upon RHE-mediated homologous recombination with the repair plasmid. The RHE cleavage site and the coding region (exon 2) of the RAGl gene are shown.

B. Amplification by PCR using a forward primer upstream of the 5' end of the left homology arm, and a reverse primer in exon2 of RAGl yields a 2.6kb and a 4.4kb product for the untargeted and the targeted alleles, respectively. PCR amplification using a forward primer in the Neo cassette and a reverse primer downstream to the 3 ' homology arm yields a 3.3kb amplification product for the targeted allele only while the untargeted allele will not be amplified using this combination of primers.

Figure 6: RA G1 mRNA expression in targeted iPSC.

Total RNA was extracted from the different samples using TRIsol reagent. Reverse transcription was preformed on l ug of total RNA form each sample using Quanta qScript kit.

A. Semiquantitative PCR for RAG 1 (Forward primer- GCAAGAGGCAAAGCGATC SEQ ID NO: 4; Reverse Primer ATGATGATCGCCATACTG SEQ ID NO: 5) and Actin B was performed on 100 ng cDNA from the different samples using AmpliTaq Gold (Roche). PCR conditions were 95°C for 2 min, then 35 cycles of

95°C for 30 sec, 57°C for 30 sec, 72°C for 60 sec, and finally 7 min at 72°C.

B. Real time PCR for RAG 1 (Forward primer- GCAAGAGGCAAAGCGATC SEQ ID NO: 4; Reverse Primer TCACAGGATGGTGTGTGGG SEQ ID NO: 6) and Actin B was performed on 100 ng cDNA from the different samples using Power Syber Green PCR master mix (Applied Biosystems) acording to the manufecour protocol in a AB7500 Real-Time PCR system.

Figure 7: Vector map of pCLS2222 encoding RAG-1 (or Ragl) -specific homing endonuclease mentioned as SCOH RAG for Single Chain RAG-1 -specific endonuclease. Figure 8: Vector map of pCLS1866

Figure 9 : Vector map of pCLS0002

DETAILED DESCRIPTION OF THE INVENTION Unless specifically defined herein below, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled artisan in the fields of gene therapy, biochemistry, genetics, and molecular biology. All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. However, in case of conflict, the present specification, including definitions, will control. Further, the materials, methods, and examples are illustrative only and are not intended to be limiting, unless otherwise specified. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001 , Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683, 195; Nucleic Acid Hybridization (B. D. Harries & S. J. Higgins eds. 1984); Transcription And Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning ( 1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds. -in-chief, Academic Press, Inc., New York), specifically, Vols.154 and 155 (Wu et al. eds.) and Vol. 1 85, "Gene Expression Technology" (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986); and Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

One aspect of the present invention is a method for increasing double-strand break-induced Gene Targeting frequency in a eukaryotic cell, comprising the steps of :

i. introducing into a eukaryotic cell at least one double-strand break creating agent, using at least one first delivery vector, wherein said at least one double-strand break creating agent targets a sequence into a genomic locus of interest;

ii. expressing into said eukaryotic cell said at least one double-strand break creating agent; iii. then, using at least one second delivery vector to introduce into previously-modified eukaryotic cell of step (i), at least one donor nucleic acid sequence flanked by sequences homologous to genomic nucleic acid portions surrounding said double-strand break creating agent nucleic acid target, said second delivery vector being identical to said first delivery vector or different; thereby obtaining a eukaryotic cell in which double-strand break-induced Gene Targeting frequency is increased.

In a preferred embodiment, said double-strand break-induced Gene Targeting frequency is higher than 10^"5, preferably higher than 10^"4, more preferably higher than 10^"3. In a more preferred embodiment said double-strand break-induced Gene Targeting frequency is higher than 10^"5, preferably higher than 10^"4, more preferably higher than 10^"3 , at endogenous genomic locus of interest.

In a preferred embodiment, said at least one first delivery vector is a viral vector. In a more preferred embodiment, said at least one first delivery vector is an integrative viral vector. More preferably, this integrative viral vector is an integrative lentiviral vector (or LV). In another embodiment, said at least one first delivery vector is a non-integrative viral vector. More preferably, this non-integrative viral vector is a non-integrative lentiviral vector (or NILV). In a preferred embodiment, said at least one first delivery vector is a non viral vector. In a more preferred embodiment, said at least one first delivery vector is an electroporation method.

In another preferred embodiment, said at least one double-strand break creating agent targets a sequence into an endogenous genomic locus of interest. In another preferred embodiment, said at least one double-strand break creating agent targets a sequence into an exogenous gene introduced at a genomic locus of interest of an eukaryotic cell, such as a reporter gene or a tag fused or not to an endogenous gene.

In these preferred embodiments, said at least one double-strand break creating agent can be expressed into target cell for an appropriate time, more preferably at least 24 hours. In a more preferred embodiment, said double-strand break creating agent can be expressed into target cell between 24 and 48 hours. In another more preferred embodiment, said double- strand break creating agent can be expressed into target cell between 48 and 72 hours. In a more preferred embodiment, said double-strand break creating agent can be expressed into target cell between 72 and 96 hours. In another preferred embodiment, said double-strand break creating agent can be continuously expressed into target cell for the previously mentioned times. Alternatively, said double-strand break creating agent can be expressed discontinuously, i.e. by several pulses of appropriate times. Each pulse duration can be of 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10 hours. More preferably, these expression pulses can be of 10 hours. More preferably, each pulse duration can be of more than 10 hours. More than on expression pulse can be performed. Successive pulses can be of a different duration. As non limiting examples, 2, 3, 4, 5, 6, 7, 8, 9 successive pulses can be performed. As an illustrative example, a first pulse of 24 hours can be performed, followed by 4 pulses of 10 hours, times between each pulses being of 12 hours. Time between each expression pulses can be of several hours, identical or different. In these preferred embodiment, said double- strand break creating agent expression is inducible. By inducible, it is mean that said double-strand break creating agent only becomes active in response to an external stimulus. In a preferred embodiment, the sequence encoding said double-strand break creating agent is under the control of an inducible promoter. As non limiting examples, inducible promoter may be induced by a stress or a chemical agent.

In another preferred embodiment, said at least one double-strand break creating agent is an endonuclease. In a more preferred embodiment of this aspect said at least one double- strand break creating agent is a meganuclease.

In another preferred embodiment, said eukaryotic cell is a primary cell. In a more preferred embodiment, said eukaryotic cell is a stem cell. In another more preferred embodiment, said eukaryotic cell is an induced Pluripotent Stem (iPS) cell. In another preferred embodiment said method of the present invention is used for treating an individual by gene therapy. In another preferred embodiment said method of the present invention is used for inserting a transgene into the genome of a cell, tissue or non-human animal. In a more preferred embodiment said method of the present invention is used for inserting a transgene into the genome of a vegetal cell.

In another preferred embodiment said method of the present invention is used for producing a recombinant protein of interest from said eukaryotic cell.

In another preferred embodiment said method of the present invention is used for inactivating a gene of interest

In a second aspect of the present invention is an isolated eukaryotic cell, in which double- strand break-induced homologous recombination is increased by the method previously described. In a more preferred embodiment of this aspect of the present invention is an isolated eukaryotic cell, in which double-strand break-induced gene targeting frequency is increased by the method previously described.

In another preferred embodiment, said cell in this aspect of the present invention is modified with at least one delivery vector comprising at least one double-strand break creating agent wherein said at least one double-strand break creating agent targets a sequence into a genomic locus of interest.

In another preferred embodiment, said cell is a primary cell. In a more preferred embodiment, said cell is a stem cell. In another preferred embodiment, said cell is an iPS cell.

In a third aspect of the present invention is a composition for increasing double-strand break-induced gene targeting frequency in a eukaryotic cell. Said composition comprises at least one of: i. one first delivery vector as defined in previously described method; ii. one second delivery vector as defined in previously described method; iii. an isolated eukaryotic cell as previously described, and a carrier.

In a fourth aspect of the present invention is a composition for increasing double-strand break-induced gene targeting frequency in a eukaryotic cell. Said composition comprises at least one of: i. one first delivery vector as defined in previously described method; ii. one second delivery vector as defined in previously described method;

iii. an isolated eukaryotic cell as previously described, and instructions for use in increasing gene targeting efficiency.

Other aspects of the invention involve design of a targeting construct to knock out, knock- in, replace, correct, substitute, mutate or otherwise alter a polynucleotide or gene sequences. Such methods may employ homologous recombination that usually promotes the exchange of genetic information between endogenous sequences. In such gene targeting experiments, the exchange of genetic information can be promoted between an endogenous chromosomal sequence and an exogenous DNA construct. Depending on the design of the targeting construct, genes could be knocked out, knocked-in, replaced, corrected or mutated.

Definitions

Amino acid residues in a polypeptide sequence are designated herein according to the one- letter code, in which, for example, Q means Gin or Glutamine residue, R means Arg or Arginine residue and D means Asp or Aspartic acid residue.

Amino acid substitution means the replacement of one amino acid residue with another, for instance the replacement of an Arginine residue with a Glutamine residue in a peptide sequence is an amino acid substitution.

Nucleotides are designated as follows: one-letter code is used for designating the base of a nucleoside: a is adenine, t is thymine, c is cytosine, and g is guanine. For the degenerated nucleotides, r represents g or a (purine nucleotides), k represents g or t, s represents g or c, w represents a or t, m represents a or c, y represents t or c (pyrimidine nucleotides), d represents g, a or t, v represents g, a or c, b represents g, t or c, h represents a, t or c, and n represents g, a, t or c.

Altered/enhanced/increased/improved cleavage activity, refers to an increase in the detected level of meganuclease cleavage activity, see below, against a target DNA sequence by a second meganuclease in comparison to the activity of a first meganuclease against the target DNA sequence. Normally the second meganuclease is a variant of the first and comprises one or more substituted amino acid residues in comparison to the first meganuclease.

"iPS'Or "iPSC" refer to induced Pluripotent Stem Cells. A "reprogrammation process" is intended the process of dedifferentiation of a somatic cell toward iPS cells.

A "meganuclease" is an endonuclease having a double-stranded DNA target sequence of ranging from 12 to 45 bp or any intermediate value therebetween. Said meganuclease is either a dimeric enzyme, wherein each domain is on a monomer or a monomeric enzyme comprising the two domains on a single polypeptide.

A "meganuclease domain" comprises or consists of the region which interacts with one half of the DNA target of a meganuclease and is able to associate with the other domain of the same meganuclease which interacts with the other half of the DNA target to form a functional meganuclease able to cleave said DNA target.

A "meganuclease variant" or "variant" describes a meganuclease obtained by insertion, deletion, substitution or replacement of at least one, two, three, four, five, six, seven, eight, nine, ten or more residue(s) in the amino acid sequence of the parent meganuclease with a different amino acid. A variant endonuclease may be encoded by a polynucleotide that has at least 90%, 95%, 97.5%, 98%, 99% or more sequence identity with a polynucleotide encoding a non-variant endonuclease, such as a parent endonuclease or one that exists in nature. A polynucleotide sequence that encodes a variant endonuclase may hybridize to the sequence of a polynucleotide encoding the corresponding non-variant endonuclease under moderate, moderately high, or high stringency conditions, such as those characterized by washing at a temperature of 42°C in 0.2x SSC and 0.1 SDS, or of 68°C in O. l x SSC and 0.1 SDS, or those conditions identifying polynucleotides having 90%, 95%, 97.5%, 98%, 99% or more polynucleotide sequence mismatch with a polynucleotide encoding a non-variant endonuclease described herein. Suitable hybridization procedures and conditions are described by and incorporated by reference to Current Protocols in Molecular Biology, vol. 1 , unit 2.10 last referenced August, 2, 2010.

A "peptide linker" refers to peptide sequence of at least 10, 1 1 , 12, 13, 14, 15, 16 and preferably at least 17 amino acids which links the C-terminal amino acid residue of the first monomer to the N-terminal residue of the second monomer and which allows the two variant monomers to adopt the correct conformation for activity and which does not alter the specificity of either of the monomers for their targets.

A "tag" or "tags" describes epitope tags well known in the art including as nonlimiting examples FLAG, His, myc, Tap, HA or any detectable amino acid sequence.

A "subdomain" encompasses a region of a LAGLIDADG homing endonuclease core domain which interacts with a distinct part of a homing endonuclease DNA target half-site. The phrase "targeting DNA construct/minimal repair matrix/repair matrix" means a DNA construct comprising a first and second portions which are homologous to regions 5 ' and 3 ' of the DNA target in situ. The DNA construct also comprises a third portion positioned between the first and second portions which comprise some homology with the corresponding DNA sequence in situ or alternatively comprise no homology with the regions 5 ' and 3 ' of the DNA target in situ. Following double-strand break or cleavage of the DNA target, a homologous recombination event is stimulated between the genome containing the targeted gene comprised in the locus of interest and the repair matrix, wherein the genomic sequence containing the DNA target is replaced by the third portion of the repair matrix and a variable part of the first and second portions of the repair matrix. The expressions "donor sequence" or "donor nucleic acid sequence" also refer to the third portion mentioned above and positioned between the first and second portions which can comprise some homology with said double-strand break creating agent nucleic acid target of the present invention; these first and second portions are also mentioned as "sequences homologous to genomic nucleic portions surrounding said double-strand break creating agent nucleic acid target" of the present invention. The repair matrix can also be endogenous such as a chromosomal sequence of interest. The chromosomal sequence of interest can be either located on the same chromosome as the genomic locus of interest, or on a different chromosome.

Preferably, homologous sequences of at least 50 bp, preferably more than 100 bp and more preferably more than 200 bp are used. Therefore, the targeting DNA construct is preferably from 200 bp to 6000 bp, more preferably from 1000 bp to 2000 bp. Indeed, shared DNA homologies are located in regions flanking upstream and downstream the site of the break and the DNA sequence to be introduced should be located between the two arms. The sequence to be introduced may be any sequence used to alter the chromosomal DNA in some specific way including a sequence used to repair a mutation in the targeted gene, restore a functional targeted gene in place of a mutated one, modify a specific sequence in the targeted gene, to attenuate or activate the targeted gene, to inactivate or delete the targeted gene or part thereof, to introduce a mutation into a site of interest or to introduce an exogenous gene or part thereof. Such chromosomal DNA alterations are used for genome engineering (animal models/recombinant cell lines) or genome therapy (gene correction or recovery of a functional gene). The targeting construct can comprise advantageously a positive selection marker between the two homology arms and eventually a negative selection marker upstream of the first homology arm or downstream of the second homology arm. The marker(s) can allow the selection of cells having inserted the sequence of interest by homologous recombination at the target site.

A "functional variant" describes a variant which is able to cleave a DNA target sequence, preferably said target is a new target which is not cleaved by the parent meganuclease. For example, such variants have amino acid variation at positions contacting the DNA target sequence or interacting directly or indirectly with said DNA target.

The terms "selection or selecting" mean the isolation of one or more meganuclease variants based upon an observed specified phenotype, for instance altered cleavage activity. This selection can be of the variant in a peptide form upon which the observation is made or alternatively the selection can be of a nucleotide coding for selected meganuclease variant.

The word "screening" describes the sequential or simultaneous selection of one or more meganuclease variant (s) which exhibits a specified phenotype such as altered cleavage activity.

The term "derived from" describes a meganuclease variant which is created from a parent meganuclease and hence the peptide sequence of the meganuclease variant is related to (primary sequence level) but derived from (mutations) the sequence peptide sequence of the parent meganuclease.

"I-Oel" is intended to refer to the wild-type I-Cn?I having the sequence of pdb accession code l g9y, corresponding to the sequence SEQ ID NO: 1 in the sequence listing.

An "I-CVel variant with novel specificity" describes a variant having a pattern of cleaved targets different from that of the parent meganuclease.

The terms "novel specificity", "modified specificity", "novel cleavage specificity", "novel substrate specificity" which are equivalent and used indifferently, refer to the specificity of the variant towards the nucleotides of the DNA target sequence. In the present Patent Application all the I-Cre\ variants described comprise an additional Alanine after the first Methionine of the wild type I-Crel sequence (SEQ ID NO: 3). These variants also comprise two additional Alanine residues and an Aspartic Acid residue after the final Proline of the wild type I-Cre\ sequence. These additional residues do not affect the properties of the enzyme and to avoid confusion these additional residues do not affect the numeration of the residues in I-Cre\ or a variant referred in the present Patent Application, as these references exclusively refer to residues of the wild type 1-Cre\ enzyme (SEQ ID NO: 1 ) as present in the variant, so for instance residue 2 of I-Crel is in fact residue 3 of a variant which comprises an additional Alanine after the first Methionine.

"I-Oel site" refers to a 22 to 24 bp double-stranded DNA sequence which is cleaved by I-Oel. I-CVel sites include the wild-type non-palindromic \-Crel homing site and the derived palindromic sequences such as the sequence 5'- t-^C-i i a-ioa^a-ga-yaeg t^c^g^t. i a₊ iC₊2g+3a+4C₊5g₊6t₊₇t₊8t₊9t₊iog₊i ia₊i₂ (SEQ ID NO: 2), also called C I 221 .

A "domain" or "core domain" describes a "LAGLIDADG homing endonuclease core domain" which is the characteristic ι β ι β2 ₂β3β4 ₃ fold of the homing endonucleases of the LAGLIDADG family, corresponding to a sequence of about one hundred amino acid residues. Said domain comprises four beta-strands ((β ι β₂β3β4) folded in an anti-parallel beta-sheet which interacts with one half of the DNA target. This domain is able to associate with another LAGLIDADG homing endonuclease core domain which interacts with the other half of the DNA target to form a functional endonuclease able to cleave said DNA target. For example, in the case of the dimeric homing endonuclease \-Cre\ ( 163 amino acids), the LAGLIDADG homing endonuclease core domain corresponds to the residues 6 to 94.

A "subdomain" refers the region of a LAGLIDADG homing endonuclease core domain which interacts with a distinct part of a homing endonuclease DNA target half-site.

The term "chimeric DNA target" or "hybrid DNA target" describes the fusion of a different half of two parent meganuclease target sequences. In addition at least one half of said target may comprise the combination of nucleotides which are bound by at least two separate subdomains (combined DNA target).

A "beta-hairpin" is intended to describe two consecutive beta-strands of the antiparallel beta-sheet of a LAGLIDADG homing endonuclease core domain (β ι β₂ 0Γ,β₃β₄) which are connected by a loop or a turn,

A "single-chain meganuclease", "single-chain chimeric meganuclease", "single-chain meganuclease derivative", "single-chain chimeric meganuclease derivative" or "single- chain derivative" describes a meganuclease comprising two LAGLIDADG homing endonuclease domains or core domains linked by a peptidic spacer. The single-chain meganuclease is able to cleave a chimeric DNA target sequence comprising one different half of each parent meganuclease target sequence. A "DNA target", "DNA target sequence", "target sequence" , "target-site", "target" , "site", "site of interest", "recognition site", "recognition sequence", "homing recognition site", "homing site", "cleavage site" describes 20 to 24 bp double-stranded palindromic, partially palindromic (pseudo-palindromic) or non-palindromic polynucleotide sequence that is recognized and cleaved by a LAGLIDADG homing endonuclease such as \-Cre\, or a variant, or a single-chain chimeric meganuclease derived from \-Cre\. These terms refer to a distinct DNA location, preferably a genomic location, at which a double stranded break (cleavage) is to be induced by the meganuclease. The DNA target is defined by the 5 ' to 3 ' sequence of one strand of the double-stranded polynucleotide, as indicate above for C I 221 . Cleavage of the DNA target occurs at the nucleotides at positions +2 and -2, respectively for the sense and the antisense strand. Unless otherwise indicated, the position at which cleavage of the DNA target by an \-Cre I meganuclease variant occurs, corresponds to the cleavage site on the sense strand of the DNA target.

The terms "DNA target half-site", "half cleavage site" or half-site" are intended to describe the portion of the DNA target which is bound by each LAGLIDADG homing endonuclease core domain.

The term "chimeric DNA target" or "hybrid DNA target" is intended to describe the fusion of different halves of two parent meganuclease target sequences. In addition at least one half of said target may comprise the combination of nucleotides which are bound by at least two separate subdomains (combined DNA target).

The term "endonuclease" refers to any wild-type or variant enzyme capable of catalyzing the hydrolysis (cleavage) of bonds between nucleic acids within of a DNA or RNA molecule, preferably a DNA molecule. Endonucleases do not cleave the DNA or RNA molecule irrespective of its sequence, but recognize and cleave the DNA or RNA molecule at specific polynucleotide sequences, further referred to as "target sequences" or "target sites" and significantly increased HR by specific meganuclease-induced DNA double- strand break (DSB) at a defined locus (Rouet et al, 1994; Choulika et al, 1995). Endonucleases can for example be a homing endonuclease (Paques et al. Curr Gen Ther. 2007 7:49-66), a chimeric Zinc-Finger nuclease (ZFN) resulting from the fusion of engineered zinc-finger domains with the catalytic domain of a restriction enzyme such as Fokl (Porteus et al. Nat Biotechnol. 2005 23 :967-973) or a chemical endonuclease (Arimondo et al. Mol Cell Biol. 2006 26:324-333; Simon et al. NAR 2008 36:3531 -3538; Eisenschmidt et al. NAR 2005 33 :7039-7047; Cannata et al. PNAS 2008 105 :9576-9581 ). In chemical endonucleases, a chemical or peptidic cleaver is conjugated either to a polymer of nucleic acids or to another DNA recognizing a specific target sequence, thereby targeting the cleavage activity to a specific sequence. Chemical endonucleases also encompass synthetic nucleases like conjugates of orthophenanthroline, a DNA cleaving molecule, and triplex-forming oligonucleotides (TFOs), known to bind specific DNA sequences ( alish and Glazer Ann NY Acad Sci 2005 1058: 151 -61 ). Such chemical endonucleases are comprised in the term "endonuclease" according to the present invention. In the scope of the present invention is also intended any fusion between molecules able to bind DNA specific sequences and agent/reagent/chemical able to cleave DNA or interfere with cellular proteins implicated in the DSB repair (Majumdar et al. J. Biol. Chem 2008 283, 1 7: 1 1244-1 1252; Liu et al. NAR 2009 37:6378-6388); as a non limiting example such a fusion can be constituted by a specific DNA-sequence binding domain linked to a chemical inhibitor known to inhibate religation activity of a topoisomerase after DSB cleavage.

An endonuclease can be a homing endonuclease, also known under the name of meganuclease. Such homing endonucleases are well-known to the art (see e.g. Stoddard, Quarterly Reviews of Biophysics, 2006, 38:49-95). Homing endonucleases recognize a DNA target sequence and generate a single- or double-strand break. Homing endonucleases are highly specific, recognizing DNA target sites ranging from 12 to 45 base pairs (bp) in length, usually ranging from 14 to 40 bp in length. The homing endonuclease according to the invention may for example correspond to a LAGLIDADG endonuclease, to a HNH endonuclease, or to a GIY-YIG endonuclease.

In the wild, meganucleases are essentially represented by homing endonucleases (HEs). Homing Endonucleases are a widespread family of natural meganucleases including hundreds of proteins families (Chevalier, B.S. and B.L. Stoddard, Nucleic Acids Res., 2001 , 29, 3757-3774). These proteins are encoded by mobile genetic elements which propagate by a process called "homing": the endonuclease cleaves a cognate allele from which the mobile element is absent, thereby stimulating a homologous recombination event that duplicates the mobile DNA into the recipient locus. Given their exceptional cleavage properties in terms of efficacy and specificity, they could represent ideal scaffolds to derive novel, highly specific endonucleases.

HEs belong to five major families. The LAGLIDADG family, named after a conserved peptidic motif involved in the catalytic center, is the most widespread and the best characterized group. Seven structures are now available. Whereas most proteins from this family are monomeric and display two LAGLIDADG motifs, a few have only one motif, and thus dimerize to cleave palindromic or pseudo-palindromic target sequences. Although the LAGLIDADG peptide is the only conserved region among members of the family, these proteins share a very similar architecture. The catalytic core is flanked by two DNA-binding domains with a perfect two-fold symmetry for homodimers such as I-Oel (Chevalier, et al , Nat. Struct. Biol., 2001 , 8, 3 12-3 16) , I-Msol (Chevalier et al, J. Mol. Biol., 2003, 329, 253-269) and \-Ceul (Spiegel et al., Structure, 2006, 14, 869-880) and with a pseudo symmetry for monomers such as \-Scel (Moure et al , J. Mol. Biol., 2003, 334, 685-69, \-Dmo\ (Silva et al , J. Mol. Biol., 1999, 286, 1 123- 1 136) or \-Ani\ (Bolduc et al , Genes Dev., 2003, 17, 2875-2888). Both monomers and both domains (for monomeric proteins) contribute to the catalytic core, organized around divalent cations. Just above the catalytic core, the two LAGLIDADG peptides also play an essential role in the dimerization interface. DNA binding depends on two typical saddle-shaped αββαββα folds, sitting on the DNA major groove. Other domains can be found, for example in inteins such as PI-P/wI (Ichiyanagi et al , J. Mol. Biol., 2000, 300, 889-901 ) and PI-Scel (Moure et al , Nat. Struct. Biol., 2002, 9, 764-770), whose protein splicing domain is also involved in DNA binding.

The making of functional chimeric meganucleases, by fusing the N-terminal \-Dmo\ domain with an l-Crel monomer (Chevalier et al , Mol. Cell., 2002, 10, 895-905 ; Epinat et al , Nucleic Acids Res, 2003, 3 1 , 2952-62; International PCT Application WO 03/07861 9 (Cellectis) and WO 2004/031346 (Fred Hutchinson Cancer Research Center, Stoddard et al)) have demonstrated the plasticity of LAGLIDADG proteins.

Different groups have also used a semi-rational approach to locally alter the specificity of the I-Oel (Seligman et al , Genetics, 1997, 147, 1653- 1664; Sussman et al , J. Mol. Biol., 2004, 342, 3 1 -41 ; International PCT Applications WO 2006/097784, WO 2006/097853, WO 2007/060495 and WO 2007/049156 (Cellectis); Arnould et al. , J. Mol. Biol., 2006, 355, 443-458; Rosen et al , Nucleic Acids Res., 2006, 34, 4791 -4800 ; Smith et al , Nucleic Acids Res., 2006, 34, e l 49), l-Scel (Doyon et al., J. Am. Chem. Soc, 2006, 128, 2477-2484), P\-Scel (Gimble et al. , J. Mol. Biol., 2003, 334, 993- 1008 ) and \-Mso\ (Ashworth et al , Nature, 2006, 441 , 656-659).

In addition, hundreds of \-Cre\ derivatives with locally altered specificity were engineered by combining the semi-rational approach and High Throughput Screening:

- Residues Q44, R68 and R70 or Q44, R68, D75 and 177 of \-Crel were mutagenized and a collection of variants with altered specificity at positions ± 3 to 5 of the DNA target (5NNN DNA target) were identified by screening (International PCT Applications WO 2006/097784 and WO 2006/097853 (Cellectis); Arnould et al., J. Mol. Biol., 2006, 355, 443-458; Smith et al , Nucleic Acids Res., 2006, 34, e l 49).

- Residues 28, N30 and Q38 or N30, Y33 and Q38 or 28, Y33, Q38 and S40 of

I-Od were mutagenized and a collection of variants with altered specificity at positions ± 8 to 10 of the DNA target ( 10NNN DNA target) were identified by screening (Smith et al , Nucleic Acids Res., 2006, 34, e l 49; International PCT Applications WO 2007/060495 and WO 2007/049156).

Two different variants were combined and assembled in a functional heterodimeric endonuclease able to cleave a chimeric target resulting from the fusion of two different halves of each variant DNA target sequence (Arnould et al , precited; International PCT Applications WO 2006/097854 and WO 2007/034262).

Furthermore, residues 28 to 40 and 44 to 77 of \-Cre\ were shown to form two separable functional subdomains, able to bind distinct parts of a homing endonuclease target half-site (Smith et al. Nucleic Acids Res., 2006, 34, e l 49; International PCT Applications WO 2007/049095 and WO 2007/057781 (Cellectis)).

The combination of mutations from the two subdomains of \-Crel within the same monomer allowed the design of novel chimeric molecules (homodimers) able to cleave a palindromic combined DNA target sequence comprising the nucleotides at positions ± 3 to 5 and ± 8 to 10 which are bound by each subdomain (Smith et ai , Nucleic Acids Res., 2006, 34, el49; International PCT Applications WO 2007/049095 and WO 2007/057781 (Cellectis)). The method for producing meganuclease variants and the assays based on cleavage- induced recombination in mammal or yeast cells, which are used for screening variants with altered specificity are described in the International PCT Application WO 2004/067736; Epinat et al , Nucleic Acids Res., 2003, 31 , 2952-2962; Chames et ai , Nucleic Acids Res., 2005, 33, e l 78, and Arnould et ai , J. Mol. Biol., 2006, 355, 443-458. These assays result in a functional LacZ reporter gene which can be monitored by standard methods.

The combination of the two former steps allows a larger combinatorial approach, involving four different subdomains. The different subdomains can be modified separately and combined to obtain an entirely redesigned meganuclease variant (heterodimer or single- chain molecule) with chosen specificity. In a first step, couples of novel meganucleases are combined in new molecules ("half-meganucleases") cleaving palindromic targets derived from the target one wants to cleave. Then, the combination of such "half-meganucleases" can result in a heterodimeric species cleaving the target of interest. The assembly of four sets of mutations into heterodimeric endonucleases cleaving a model target sequence or a sequence from different genes has been described in the following Cellectis International patent applications: XPC gene (WO2007/093918), RAG gene (WO2008/010093), HPRT gene (WO2008/059382), beta-2 microglobulin gene (WO2008/102274), Rosa26 gene (WO2008/152523), Human hemoglobin beta gene (WO2009/13622) and Human interleukin-2 receptor gamma chain gene (WO2009019614).

These variants can be used to cleave genuine chromosomal sequences and have paved the way for novel perspectives in several fields, including gene therapy. Examples of such endonuclease include I-Sce I, I-Chu I, I-Cre I, I-Csm I, Pl-Sce I, PI-Tli I, PI-Mtu I, I-Ceu I, I-Sce II, I-Sce III, HO, Pi-Civ I, PI-Ctr I, PI-Aae I, PI-Bsu I, PI-Dha I, PI-Dra I, PI-Mav I, PI-Mch I, PI-Mfu I, PI-Mfl I, PI-Mga I, PI-Mgo I, PI-Min I, PI-Mka I, PI-Mle I, PI-Mma I PI-Msh I, PI-Msm I, PI-Mth I, PI-Mtu I, PI-Mxe I, PI-Npu I, PI-Pfu PI-Rma /, Pl-Spb I, PI-Ssp I, PI-Fac I, PI-Mja I, PI-Pho I Pi-Tag I, PI-Thy I, PI-Tko I, PI- Tsp I, I-Msol.

A homing endonuclease can be a LAGL1DADG endonuclease such as \-Scel l-Crel, 1- Ceul, \-MsoI, and \-DmoI.

Said LAGLIDADG endonuclease can be I-Sce I, a member of the family that contains two LAGLIDADG motifs and functions as a monomer, its molecular mass being approximately twice the mass of other family members like I-Crel which contains only one LAGLIDADG motif and functions as homodimers. Endonucleases mentioned in the present application encompass both wild-type (naturally-occurring) and variant endonucleases. Endonucleases according to the invention can be a "variant" endonuclease, i.e. an endonuclease that does not naturally exist in nature and that is obtained by genetic engineering or by random mutagenesis. This variant endonuclease can for example be obtained by substitution of at least one residue in the amino acid sequence of a wild-type, naturally-occurring, endonuclease with a different amino acid. Said substitution(s) can for example be introduced by site-directed mutagenesis and/or by random mutagenesis. In the frame of the present invention, such variant endonucleases remain functional, i.e. they retain the capacity of recognizing and specifically cleaving a target sequence to initiate gene targeting process.

The variant endonuclease according to the invention cleaves a target sequence that is different from the target sequence of the corresponding wild-type endonuclease. Methods for obtaining such variant endonucleases with novel specificities are well-known in the art.

Endonucleases variants may be homodimers (meganuclease comprising two identical monomers) or heterodimers (meganuclease comprising two non-identical monomers). Endonucleases with novel specificities can be used in the method according to the present invention for gene targeting and thereby integrating a transgene of interest into a genome at a predetermined location. Endonucleases according to the invention can be mentioned or defined as one "double- strand break creating agent" amongst other double-strand break creating agents well- known in the art. "Double-strand break creating agent" means any agent or chemical or molecule able to create DNA (or double-stranded nucleic acids) double-strand breaks (DSBs). As previously mentioned, endonucleases can be considered as double-strand break creating agent targeting specific DNA sequences. Other agents or chemicals or molecules are double-strand break creating agents whom DNA sequence targets are non-specific or non- predictable such as, in a non limiting list, alkylating agents (Methyl Methane Sulfonate or dimethane sulfonates family and analogs), zeocyn, enzyme inhibitors such as topoisomerase inhibitors (types I and II such as non limiting examples quinolones, fluoroquinolones, ciprofloxacin, irinotecan, lamellarin D, doxorubicin, etoposide) and ionizing radiations (x-rays, UltraViolet, gamma-rays).

A "parent meganuclease" means a wild type meganuclease or a variant of such a wild type meganuclease with identical properties or alternatively a meganuclease with some altered characteristic in comparison to a wild type version of the same meganuclease. In the present invention the parent meganuclease can refer to the initial meganuclease from which a first series of variants are derived or the meganuclease from which a second series of variants are derived, or the meganuclease from which a third series of variants are derived.

By "homologous" is intended a sequence with enough identity to another one to lead to homologous recombination between sequences, more particularly having at least 95% identity, preferably 97% identity and more preferably 99% sequence identity.

"Homologous recombination (HR)" refers to the very conserved DNA maintenance pathway involved in the repair of DSBs and other DNA lesions (Paques and Haber, 1999; Sung and Klein, 2006), that promotes the exchange of genetic information between endogenous sequences. In gene targeting experiments, the exchange of genetic information is promoted between an endogenous chromosomal sequence and an exogenous DNA construct. Depending of the design of the targeted construct, genes could be knocked out, knocked in, replaced, corrected or mutated, in a rational, precise and efficient manner. The process requires essentially a few hundred base pairs of homology between the targeting construct and the targeted locus (Hinnen et al, 1978) and is significantly stimulated by free DNA ends in the construct (Orr- Weaver et al, 1981 ; Orr- Weaver et al, 1983 ; Szostak et al, 1983). These free DNA ends label the construct as a substrate for the HR machinery.

By ''gene targeting" is intended a process whereby a targeted nucleic acid sequence modification is facilitated at a genetic locus of interest by an exogenous DNA construct, such as a delivery vector containing a donor sequence as mentioned herein. Typically, the targeted nucleic acid sequence at the genetic locus of interest is modified, removed, replaced or duplicated by the exogenous DNA construct. Such modifications include at least one insertion, deletion or substitution of one or more nucleotides at the targeted nucleic sequence of interest.

By "gene targeting frequency" is intended the quantification of gene targeting events. Gene targeting can be performed without selection if there is a sensitive method for identifying recombinants, for example if the targeted gene modification can be easily detected by PCR analysis, or if it results in a certain phenotype. Gene targeting frequencies can be given by the ratio between the number of identified recombinant events on the total number of measured events. Gene targeting frequencies can also be expressed as a percentage of recombinant events amongst a given total number of events. Identification of gene targeting events can be facilitated by the use of markers such as positive or negative selectable markers or visual markers. Selectable markers include genes carrying resistance to an antibiotic such as ampicillin, hygromycin, streptomycin, kanamycin, gentamycin, zeocyn as non limiting examples and other such genes known in the art. Amongst visual markers are Green Fluorescent Protein and derivatives, other fluorescent protein, reporter enzymes such as β-galactosidase, alkaline phosphatase, β-glucuronidase, luciferase and other known in the art.

The homologous recombination according to the invention can be an "endonuclease- induced homologous recombination", i.e. an homologous recombination event taking place after a double-strand break, wherein said double-strand break is due to cleavage by an endonuclease. The term "reporter gene", as used herein, refers to a nucleic acid sequence whose product can be easily assayed, for example, colorimetrically as an enzymatic reaction product, such as the lacZ gene which encodes for β-galactosidase. Examples of widely-used reporter molecules include enzymes such as β-galactosidase, β-glucoronidase, β-glucosidase; luminescent molecules such as green fluorescent protein and firefly luciferase; and auxotrophic markers such as His3p and Ura3p. (See, e. g., Chapter 9 in Ausubel, F. M., et al. Current Protocols in Molecular Biology, John Wiley & Sons, Inc. ( 1998)).

The term "sequence identity" refers to identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base, then the molecules are identical at that position. A degree of similarity or identity between nucleic acid or amino acid sequences is a function of the number of identical or matching nucleotides at positions shared by the nucleic acid sequences. Various alignment algorithms and/or programs may be used to calculate the identity between two sequences, including FASTA, or BLAST which are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default setting. Two amino-acid sequences or nucleotide sequences are said to be "identical" if the sequence of amino-acids or nucleotidic residues, in the two sequences is the same when aligned for maximum correspondence as described below. Sequence comparisons between two (or more) peptides or polynucleotides are typically performed by comparing sequences of two optimally aligned sequences over a segment or "comparison window" to identify and compare local regions of sequence similarity. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Ad. App. Math 2: 482 (1981 ), by the homology alignment algorithm of Neddleman and Wunsch, J. Mol. Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444 (1988), by computerized implementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by visual inspection.

"Percentage of sequence identity" (or degree or identity) is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the peptide or polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino-acid residue or nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Sequence identity by itself does not necessarily require any algorithm, but algorithms may be helpful to determine the optimal alignment of two sequences. BLAST and COBALT sequence alignment programs available via the U.S. National Institutes of Health (NIH) at the web site and may be downloaded at:

ftp://ftp.ncbi. nlm.nih.gov/blast/executables/blast+/LATEST/ and http://www.ncbi.nlm.nih.gov/tools/cobalt/cobalt.cgi

(last accessed August 2, 2010) are available and usable to those of skill in the art for comparing and determining the identity between two sequences.

By "mutation" is intended the substitution, deletion, insertion of one or more nucleotides/amino acids in a polynucleotide (cDNA, gene) or a polypeptide sequence, for example, at 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or 50 or more positions in an amino acid or polynucleotide sequence. Said mutation can affect the coding sequence of a gene or its regulatory sequence. It may also affect the structure of the genomic sequence or the structure/stability of the encoded mRNA.

By "gene" is meant the basic unit of heredity, consisting of a segment of DNA arranged in a linear manner along a chromosome, which codes for a specific protein or segment of protein. A gene typically includes a promoter, a 5' untranslated region, one or more coding sequences (exons), optionally introns, a 3' untranslated region. The gene may further comprise a terminator, enhancers and/or silencers. As used herein, the term "transgene" refers to a sequence encoding a polypeptide.

Preferably, the polypeptide encoded by the transgene is either not expressed, or

expressed but not biologically active, in the cell, tissue or individual in which the transgene is inserted. Most preferably, the transgene encodes a therapeutic polypeptide useful for the treatment of an individual.

The term "gene of interest" or "GOI" refers to any nucleotide sequence encoding a known or putative gene product. As used herein, the term "locus" is the specific physical location of a DNA sequence (e.g. of a gene) on a chromosome. The term "locus" usually refers to the specific physical location of an endonuclease's target sequence on a chromosome. Such a locus, which comprises a target sequence that is recognized and cleaved by an endonuclease according to the invention, is referred to as "locus according to the invention". Also, the expression "genomic locus of interest" is used to qualify a nucleic acid sequence in a genome that can be a putative target for a double-strand break creating agent according to the invention. By "endogenous genomic locus of interest" is intended a native nucleic acid sequence in a genome, i.e. a sequence or allelic variations of this sequence that is naturally present at this genomic locus.

By " delivery vector" or " delivery vectors" is intended any delivery vector which can be used in the present invention to put into cell contact or deliver inside cells or subcellular compartments agents/chemicals and molecules (proteins or nucleic acids) needed in the present invention. It includes, but is not limited to liposomal delivery vectors, viral delivery vectors, drug delivery vectors, chemical carriers, polymeric carriers, lipoplexes, polyplexes, dendrimers, microbubbles (ultrasound contrast agents), nanoparticles, emulsions or other appropriate transfer vectors. These delivery vectors allow delivery of molecules, chemicals, macromolecules (genes, proteins), or other vectors such as plasmids, peptides developed by Diatos. In these cases, delivery vectors are molecule carriers. By " delivery vector" or "delivery vectors" is also intended delivery methods to perform transfection

The terms "vector" or "vectors" refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. A "vector" in the present invention includes, but is not limited to, a viral vector, a plasmid, a RNA vector or a linear or circular DNA or RNA molecule which may consists of a chromosomal, non chromosomal, semisynthetic or synthetic nucleic acids. Preferred vectors are those capable of autonomous replication (episomal vector) and/or expression of nucleic acids to which they are linked (expression vectors). Large numbers of suitable vectors are known to those of skill in the art and commercially available.

Viral vectors include retrovirus, adenovirus, parvovirus (e. g. adenoassociated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e. g., influenza virus), rhabdovirus (e. g., rabies and vesicular stomatitis virus), paramyxovirus (e. g. measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double- stranded DNA viruses including adenovirus, herpesvirus (e. g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e. g., vaccinia, fowlpox and canarypox). Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example. Examples of retroviruses include: avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields, et al, Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

By "lentiviral vector" is meant HIV-Based lentiviral vectors that are very promising for gene delivery because of their relatively large packaging capacity, reduced immunogenicity and their ability to stably transduce with high efficiency a large range of different cell types. Lentiviral vectors are usually generated following transient transfection of three (packaging, envelope and transfer) or more plasmids into producer cells. Like HIV, lentiviral vectors enter the target cell through the interaction of viral surface glycoproteins with receptors on the cell surface. On entry, the viral RNA undergoes reverse transcription, which is mediated by the viral reverse transcriptase complex. The product of reverse transcription is a double-stranded linear viral DNA, which is the substrate for viral integration in the DNA of infected cells.

By "integrative lentiviral vectors (or LV)", is meant such vectors as non limiting example, that are able to integrate the genome of a target cell.

At the opposite by "non integrative lentiviral vectors (or NILV)" is meant efficient gene delivery vectors that do not integrate the genome of a target cell through the action of the virus integrase. One type of preferred vector is an episome, i.e., a nucleic acid capable of extra- chromosomal replication. Preferred vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors. A vector according to the present invention comprises, but is not limited to, a YAC (yeast artificial chromosome), a BAC (bacterial artificial), a baculovirus vector, a phage, a phagemid, a cosmid, a viral vector, a plasmid, a RNA vector or a linear or circular DNA or RNA molecule which may consist of chromosomal, non chromosomal, semi-synthetic or synthetic DNA. In general, expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer generally to circular double stranded DNA loops which, in their vector form are not bound to the chromosome. Large numbers of suitable vectors are known to those of skill in the art. Vectors can comprise selectable markers, for example: neomycin phosphotransferase, histidinol dehydrogenase, dihydrofolate reductase, hygromycin phosphotransferase, herpes simplex virus thymidine kinase, adenosine deaminase, glutamine synthetase, and hypoxanthine- guanine phosphoribosyl transferase for eukaryotic cell culture; TRP 1 for S. cerevisiae; tetracyclin, rifampicin or ampicillin resistance in E. coli. Preferably said vectors are expression vectors, wherein a sequence encoding a polypeptide of interest is placed under control of appropriate transcriptional and translational control elements to permit production or synthesis of said polypeptide. Therefore, said polynucleotide is comprised in an expression cassette. More particularly, the vector comprises a replication origin, a promoter operatively linked to said encoding polynucleotide, a ribosome binding site, a RNA-splicing site (when genomic DNA is used), a polyadenylation site and a transcription termination site. It also can comprise an enhancer or silencer elements. Selection of the promoter will depend upon the cell in which the polypeptide is expressed. Suitable promoters include tissue specific and/or inducible promoters. Examples of inducible promoters are: eukaryotic metallothionine promoter which is induced by increased levels of heavy metals, prokaryotic lacZ promoter which is induced in response to isopropyl-P-D- thiogalacto-pyranoside (IPTG) and eukaryotic heat shock promoter which is induced by increased temperature. Examples of tissue specific promoters are skeletal muscle creatine kinase, prostate-specific antigen (PSA), ot-antitrypsin protease, human surfactant (SP) A and B proteins, β-casein and acidic whey protein genes. Inducible promoters may be induced by pathogens or stress, more preferably by stress like cold, heat, UV light, or high ionic concentrations (reviewed in Potenza C et al. 2004, In vitro Cell Dev Biol 40: 1 -22). Inducible promoter may be induced by chemicals (reviewed in Moore et al 2006, Plant J. 45 :651 -83 ; Padidam et al 2003, Curr Opin Plant Biol. 6(2): 169-77; Wang et al 2003 Transgenic Res 12 (5):529-40; Zuo and Chua 2000, Curr Opin Biotechnol 1 1 (2): 146-51 .)

Delivery vectors and vectors can be associated or combined with any cellular permeabilization techniques such as sonoporation or electroporation or derivatives of these techniques.

By cell or cells is intended any prokaryotic or eukaryotic living cells, cell lines derived from these organisms for in vitro cultures, primary cells from animal or plant origin.

By "primary cell" or "primary cells" are intended cells taken directly from living tissue (i.e. biopsy material) and established for growth in vitro, that have undergone very few population doublings and are therefore more representative of the main functional components and characteristics of tissues from which they are derived from, in comparison to continuous tumorigenic or artificially immortalized cell lines. These cells thus represent a more valuable model to the in vivo state they refer to.

In the frame of the present invention, "eukaryotic cells" refer to a fungal, plant or animal cell or a cell line derived from the organisms listed below and established for in vitro culture. More preferably, the fungus is of the genus Aspergillus, Penicillium, Acremonium, Trichoderma, Chrysoporium, Mortierella, Kluyveromyces or Pichia; More preferably, the fungus is of the species Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae, Aspergillus terreus, Penicillium chrysogenum, Penicillium citrinum, Acremonium Chrysogenum, Trichoderma reesei, Mortierella alpine, Chrysosporium lucknowense, Kluyveromyces lactis, Pichia pastoris or Pichia ciferrii.

In those applications involving plants, a plant is advantageously of the genus Arabidospis, Nicotiana, Solanum, lactuca, Brassica, Oryza, Asparagus, Pisum, Medicago, Zea, Hordeum, Secale, Triticum, Capsicum, Cucumis, Cucurbita, Citrullis, Citrus, Sorghum; More preferably, the plant is of the species Arabidospis thaliana, Nicotiana tabaccum, Solanum lycopersicum, Solanum tuberosum, Solanum melongena, Solanum esculentum, Lactuca saliva, Brassica napus, Brassica oleracea, Brassica rapa, Oryza glaberrima, Oryza sativa, Asparagus officinalis, Pisum sativum, Medicago sativa, zea mays, Hordeum vulgare, Secale cereal, Triticum aestivum, Triticum durum, Capsicum sativus, Cucurbita pepo, Citrullus lanatus, Cucumis melo, Citrus aurantifolia, Citrus maxima, Citrus medica, Citrus reticulata. More preferably the animal cell is of the genus Homo, Rattus, Mus, Sus, Bos, Danio, Canis, Felis, Equus, Salmo, Oncorhynchus, Gallus, Meleagris, Drosophila, Caenorhabditis; more preferably, the animal cell is of the species Homo sapiens, Rattus norvegicus, Mus musculus, Sus scrofa, Bos taurus, Danio rerio, Canis lupus, Felis catus, Equus caballus, Salmo salar, Oncorhynchus mykiss, Gallus gallus, Meleagris gallopavo, Drosophila melanogaster, Caenorhabditis elegans.

By "target organism" or "target cell", is intended an organism or a cell which comprises at least one target polynucleotide to be modified. The expression "polynucleotide derivatives" refers to polynucleotide sequences that can be deduced and constructed from the respective sequence or a part of the respective sequence of identified-effector genes according to the present invention. These derivatives can refer to mRNAs, siRNAs, dsRNAs, miRNAs, cDNAs. These derivatives can be used directly or as part of a delivery vector or vector/plasmid/construct, by introducing them in an eukaryotic cell to increase gene targeting efficiency and/or endonuclease-induced homologous recombination.

"Transfection" is a generic term used to refer to "introduction" into a live cell, either in vitro or in vivo, of certain nucleic acid construct, preferably into a desired cellular location of a cell, said nucleic acid construct being functional once in the transfected cell. Such presence of the introduced nucleic acid may be stable or transient. Successful transfection will have an intended effect on the transfected cell, such as silencing or enhancing a gene target, or triggering target physiological event, like enhancing the frequency of HR. They are various methods of introducing foreign DNA into a eukaryotic cell and many materials have been used as carriers for transfection, which can be divided into three kinds: (cationic) polymers, liposomes and nanoparticles. Other methods of transfection include nucleotransfection, electroporation, proprietary techniques such as techniques developed by Amaxa, Maxcyte..., heat shock, magnetofection and proprietary transfection reagents such as Lipofectamine, Dojindo Hilymax, Fugene, JetPEI, Effectene, Dreamfect, Polyfect, Nucleofector, Lyovec, Attractene, Transfact, Optifect. Viral vectors as delivery vectors are also considered as transfection means. The above written description of the invention provides a manner and process of making and using it such that any person skilled in this art is enabled to make and use the same, this enablement being provided in particular for the subject matter of the appended claims, which make up a part of the original description. As used above, the phrases "selected from the group consisting of," "chosen from," and the like include mixtures of the specified materials.

Where a numerical limit or range is stated herein, the endpoints are included. Also, all values and subranges within a numerical limit or range are specifically included as if explicitly written out.

The above description is presented to enable a person skilled in the art to make and use the invention, and is provided in the context of a particular application and its requirements. Various modifications to the preferred embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the invention. Thus, this invention is not intended to be limited to the embodiments shown, but is to be accorded the widest scope consistent with the principles and features disclosed herein.

Having generally described this invention, a further understanding can be obtained by reference to certain specific examples, which are provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified. EXAMPLES

Severe combined immune deficiency (SCID) comprises a group of heterogeneous genetic disorders that are inevitably fatal, unless immune reconstitution is achieved. Hematopoietic cell transplantation (HCT) represents the mainstay of treatment for SCID. The outcome of HCT for SCID is influenced both by donor/recipient HLA matching and by the immunological phenotype, and is significantly worse for patients without T and B cells (T- B- SCID) (Antoine et al., 2003). The RAG 1 and RAG2 proteins form a complex that initiates V(D)J recombination, an essential step in the development of T and B lymphocytes (Dudley et al., 2005). Null mutations in either RAGl or RAG2 are the most common cause of T^" B^" NK⁺ SCID in humans (Schwarz et al., 1996). In contrast, hypomorphic mutations in the RAG genes that are permissive for some RAG protein expression and function, often result in leaky SCID or in Omenn syndrome (OS), a condition characterized by immunodeficiency and severe tissue damage due to infiltrating and oligoclonal T cells (Notarangelo et al., 2004; Signorini et al., 1999; Villa et al l 998). Furthermore, unpublished observations indicate that hypomorphic RAG mutations result also in significant B-cell-mediated autoimmunity, due to defects in receptor editing and altered peripheral B cell homeostasis. These abnormalities in T- and B-lymphocyte development and function may contribute to the significant immunopathology of Omenn syndrome and leaky SCID, and may also help explain why the results of HCT for leaky SCID and OS are even more problematic than for T- B- SCID, especially if recipient lymphocytes persist after transplant (Antoine et al., 2003 ; Aleman et al., 2001 ; Mazzolari et al., 2005). Current data indicate that survival after HCT for T- B- SCID and Omenn syndrome is approximately 50%. Overall, these observations indicate the need to explore novel and more effective forms of treatment for RAG-deficient patients.

Studies of virus-mediated gene transfer into hematopoietic CD34⁺ progenitor cells of SCID patients have offered proof-of-principal of the efficacy of gene therapy in both X-linked SCID (SCIDX1 ) and adenosine deaminase deficiency, and this approach has been successfully used in patients who lacked an HLA-identical family donor (Aiuti et al. 2002; Cavazzana-Calvo et al. 2000; Gaspar et al. 2004; Hacein-Bey-Abina et al. 2002). However, clonal proliferation due to insertional mutagenesis has been observed following gene therapy for SCIDX1 (Hacein-Bey-Abina et al. 2008; Hacein-Bey-Abina et al. 2003; Howe et al. 2008). Furthermore, attempts to rescue ragl deficiency in mice using a similar strategy have also led to deleterious oncoretroviral integration and malignant proliferative disease (Lagresle-Peyrou et al. 2006). Moreover, preliminary unpublished observations indicate that lentiviral-mediated gene transfer leads to modest immune reconstitution when applied to a mouse model of leaky SCID due to hypomorphic ragl mutation. Therefore, development of novel and safer approaches, based on gene correction, is of utmost importance for patients with RAG deficiency.

Preliminary data show that engineered meganuclease targeting the human Ragl gene (Smith, Grizot et al. 2006; Grizot, Smith et al. 2009) efficiently recognizes and cleaves the Rag l locus. Using a LacZ reporter assay, preliminary data also demonstrate the ability of the RHE to correct the mutation in both plasmid and chromosomal context in CHO cells.

Example 1 : RHE-mediated targeting of the endogenous RAGl locus in patient- derived fibroblasts.

To assess the ability of the RAGl-speciiic homing endonuclease (RHE) to target the endogenous human RAG1 locus, primary fibroblasts from a patient carrying the RAG1 c.256-257del and c.2164G>A mutations were nucleofected by AMAXA with the RHE- coding plasmid and a RAG1 repair plasmid that includes a Neo-resi stance (NeoR) gene. In parallel, cells were nucleofected nucleofected by AMAXA NHDF protocol program U-23. Cells were nucleofected with l ^g of the RHE-co&mg plasmid and the RAG l repair plasmid that includes a Neo resistance gene. In parallel, cells were nucleofected with \ .5μ% of the RAGl repair plasmid only or with l ^g plasmid vector coding for the NeoR gene. After selection for 6 days with G418, followed by 10 days of expansion in non selective medium, DNA from the surviving fibroblasts was extracted and amplified by PCR. In order to amplify only sequences within the RAGl locus and not within the repair plasmid, we used a forward primer (primer F in Fig. 1 ) upstream to the 5 ' end of the left homology arm and to the RHE-restriction site, and a reverse primer (primer R in Fig. 1 ) in the coding exon2 of RAGl, within the right homology arm. As shown in Figure 1 , a band of 4kb, corresponding to the endogenous locus, was detected in cells that had been nucleofected with the repair plasmid only or with the NeoR plasmid. In contrast, fibroblasts that had been nucleofected with both the RHE-coding plasmid and with the repair plasmid gave two products: a 4kb band (corresponding to the untargeted allele) and a 5.8kb band that corresponds to the targeted RAG1 locus that includes the SV40-NeoR-IRES-Myc sequence.

Example 2: Use of lentiviral vectors to improve the delivery of RHE in the cells a) Constructions

In order to improve the delivery of the RHE into the cells, the RHE coding sequence was cloned into a lentiviral vector that codes for Ds-Red under the human EF l promoter followed by an IRES sequence and the RHE sequence (Figure 2). This Red-RHE lentiviral vector efficiently infected 293T cells and resulted in robust expression of the Ds-Red and the RHE (Figure 2). As a control vector, an identical vector coding for Ds-Red and ZsGreen instead of the RHE was used. b) Production and characterization of OS-iPSCs

Next, we have targeted patient-specific induced pluripotent stem cells (iPSCs) that were derived by reprogramming primary fibroblasts from patients carrying the RAG1 c.256- 257del and c.2164G>A mutations. Compound heterozygosity for these mutations was associated in vivo with an Omenn syndrome (OS) phenotype, hence the iPSCs derived from the fibroblasts were named OS-iPSCs. To derive OS-iPSCs, patient fibroblasts were transduced with a single polycistronic lentiviral vector that included four reprogramming factors, OCT4, SOX2, KLF4, and c-MYC. The OS- iPSCs have a typical morphological appearance of iPSc and show a sternness and pluripotency profile that is comparable to that observed in human embryonic stem cells (Figure 3A). It has also been demonstrated the patient-specific origin of each iPSC lines (Fig. 3B) and the maintenance of karyotypic integrity (Fig. 4C) following the reprogramming process. In vitro differentiation of the OS- iPSC lines into embryoid bodies (Figure 3D) that had the potential to develop along specific lineages as confirmed by expression of markers of all three embryonic germ layers (data not shown) was induced. c) Transfection of OS-iPScs with Red-RHE and Red-Green vectors To test the ability of the RHE to induce correction of RAG1 gene mutations in the OS- iPSc, 20 of OS-iPSCs were transduced with the Red-RHE or the Red-Green lentiviral vector. All of the iPSC colonies showed robust Ds-Red staining both when incubated with the Red-RHE vector or with the Red-only control vector (Figure 4). d) Targeting of the RAG1 locus in iPScs

To achieve single-cell iPSCs suspensions, the transduced iPSC colonies were washed with PBS and incubated with 0.05% EDTA-Trypsin for 3 minutes, followed by manual release from the plate using a pipette tip. The supernatant was passed several times through a 1000 μΐ pipette and the cells were collected by centrifugation at 200g for 3 minutes.

The collected cells were then re-suspended in 100 ul AMAXA NHDF neucleofection media. 2μg of repair matrix PCLS-1866 (Figure 8) or control plasmid (PCLS-0003 ; Figure 9) were added. Three combinations were used, as detailed in Table 1 .

Table 1 : Different combinations of lentiviral vectors and repair matrix used for each treatment.

The cells were transferred to a cuvette and nucleofected with an AMAXA nucleofector using neucleofection protocol A23. 500 μΐ of iPS media were added and the cells were immediately plated onto Irradiated Mouse embryonic fibroblasts (iMEFs) in the presence of 10μΜ Y27632 in a 6-well plate and propagated according to standard iPSCs protocols without selection. iPSc colonies as well as differentiated cells started to appear after 14-21 days of culture. Cells were further expanded until they reached confluence and then both DNA and RNA were extracted. To evaluate correction of the c.256-257del mutation, we performed PCR amplification of genomic DNA extracted from iPSCs, using specific primers. Two different combinations of primers were used; a) a forward primer upstream of the 5 ' end of the left homology arm, and a reverse primer in exon2 of RAGl ; and, b) a forward primer in the Neo cassette and a reverse primer downstream to the 3 ' homology arm (Figure 5).

The cells that had been transduced with the RHE and later nucleofected with the RAG1 repair matrix demonstrated amplification of both the targeted allele as well as of the untargeted allele. In contrast, only the untargeted mutated allele could be amplified from genomic DNA extracted from the cells that had been transduced with the Red-only lentiviral vector, followed by nucleofection with the RAG1 repair matrix or with the control plasmid. With the first combination of primers, the non recombined endogenous locus as well as the recombined locus can be amplified (Fig. 5A). Thus, amplification of the non recombined locus provides an internal control that can be used, in semiquantitative or quantitative methods, to evaluate the frequencies of recombined loci. According to the band intensities on Figure 5B, a significant proportion (above 1 %) of the endogenous loci have been targeted. e) RAG1 mRNA expression in targeted iPScs

When controlled by its endogenous promoter, RAG J is expressed only at very specific stages of T and B lymphocyte differentiation. However, following targeting of the RAGl locus with the repair matrix, an SV40 promoter is introduced in the locus that is expected to drive RAGl expression in the targeted cells.

To evaluate RAGl mRNA expression in the targeted OS-iPSC cells, cDNA was transcribed and semi-quantitative PCR was performed using RAGl specific primers. Only the OS-iPSCs that had been transduced with the RHE lentiviral vector, and then nucleofected with the RAGl repair matrix, showed detectable levels of RAGl mRNA, as detected by RT-PCR (Fig. 6A). In contrast, RAGl mRNA was not detected in OS-iPSCs that had been transduced with the Red-only lentiviral vector, followed by nucleofection with the RAGl repair matrix or with the control plasmid (Figure 6A). Similar results were observed when real-time PCR was used to evaluate RAGl expression (Fig. 6B). These data indicate that the RHE can mediate targeting of the endogenous RAG J locus in patient-derived fibroblasts and iPSc. iPSCs represent a desirable target to achieve homologous recombination. In spite of advances in the treatment of SCID and related disorders, the outcome after HCT remains unsatisfactory, especially for patients with hypomorphic mutations that allow for residual T cell immunity. Retrovirus-mediated gene transfer has offered proof-of-principle of the potential benefits of gene therapy for SCID, but risks associated with random integration have indicated the need for novel approaches based on homologous recombination or targeted insertion of the transgene. The HE technology has been shown to efficiently and specifically target and correct various gene mutations in different cell types. This approach may be of particular interest for the correction of hypomorphic mutations that result in the expression of a mutant protein that might interfere with the wild-type protein, if a conventional gene therapy approach was used. As the first step toward a clinical application of HE to correct human genetic disorders, and using the highly innovative approach represented by iPS the ability of the newly developed 7L4G/-specific HE to correct a common RAG1 mutation and to restore RAG l expression in vitro has been shown with a frequency higher than with classical gene targeting standards.

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Claims

1 . A method for increasing double-strand break-induced gene targeting in a eukaryotic cell, comprising:

i. introducing into a eukaryotic cell at least one double-strand break creating agent, using at least one first delivery vector, wherein said at least one double-strand break creating agent targets a sequence into a genomic locus of interest

ii. expressing into said eukaryotic cell said at least one double-strand break creating agent;

iii. then, using at least one second delivery vector to introduce into previously- modified eukaryotic cell of step (i), at least one donor nucleic acid sequence flanked by sequences homologous to genomic nucleic acid portions surrounding said double-strand break creating agent nucleic acid target, said second delivery vector being identical to said first delivery vector or different;

thereby obtaining a eukaryotic cell in which double-strand break-induced gene targeting is increased.

2. The method according to claim 1 , wherein double-strand break-induced gene targeting frequency is higher than 10^"3.

3. The method according to claim 1 , wherein said at least one first delivery vector comprising at least one double-strand break creating agent is a viral vector.

4. The method according to claim 1 , wherein said at least one first delivery vector comprising at least one double-strand break creating agent is an integrative viral vector.

5. The method according to claim 1 , wherein said at least one double-strand break creating agent targets a sequence into a native endogenous genomic locus of interest.

6. The method according to claim 1 , wherein said at least one double-strand break creating agent is an endonuclease.

7. The method according to claim 1 , wherein said at least one double-strand break creating agent is a meganuclease.

8. The method according to claim 1 , wherein said eukaryotic cell is a primary cell.

9. The method according to claim 1 , wherein said eukaryotic cell is a stem cell.

10. The method according to claim 1 , wherein said eukaryotic cell is an induced Pluripotent Stem (iPS) cell.

1 1 . The method of claim 1 , wherein said eukaryotic cell is that of an individual being treated by gene therapy.

12. The method of claim 1 , which inserts a transgene into the genome of a cell, tissue or non-human animal.

13. The method of claim 1 , which inserts a transgene into the genome of a vegetal cell.

14. The method of claim 1 , further comprising producing a recombinant protein of interest from said eukaryotic cell.

1 5. The method of claim 1 , further comprising inactivating a gene of interest.

16. An isolated eukaryotic cell that has been produced or modified with at least one delivery vector comprising at least one double-strand break creating agent according to claim 1 .

17. The isolated eukaryotic cell according to claim 16, wherein said cell is a primary cell.

1 8. The isolated eukaryotic cell according to claim 16, wherein said cell is a stem cell.

19. The isolated eukaryotic cell according to claim 16, wherein said cell is an iPS cell.

20. An isolated eukaryotic cell in which double-strand break-induced gene targeting is increased by the method of claim 1 .

21. An isolated eukaryotic cell in which double-strand break-induced gene targeting frequency has been increased by the method of claim 1.

22. A composition for increasing double-strand break-induced gene targeting frequency in a eukaryotic cell comprising at least one of:

iv. one first delivery vector as defined in claim 1 ;

v. one second delivery vector as defined in claim 1 ;

vi. a carrier and an isolated eukaryotic cell that has been produced or modified with at least one delivery vector comprising at least one double-strand break creating agent according to claim 1 or in which double-strand break-induced gene targeting frequency is increased by the method of claim 1 .

23. A kit for increasing double-strand break-induced gene targeting frequency in a eukaryotic cell, wherein said kit comprises at least one of:

i. one first delivery vector as defined in claim 1 ;

ii. one second delivery vector as defined in claim 1 ;

iii. an isolated eukaryotic cell an isolated eukaryotic cell that has been produced or modified with at least one delivery vector comprising at least one double- strand break creating agent according to claim 1 or in which double-strand break-induced gene targeting frequency is increased by the method of claim 1 , and instructions for use in increasing gene targeting efficiency.

24. A method for inducing or enhancing homologous recombination in a target polynucleotide, gene, genomic locus or genome of a eukaryotic cell comprising: introducing into a eukaryotic cell a first vector that expresses at least one double- strand break creating agent, wherein said at least one double-strand break creating agent targets a sequence into a polynucleotide, gene, genomic locus, or genome of interest,

introducing into said eukaryotic cell a second vector containing at least one donor nucleic acid sequence flanked by sequences homologous to genomic nucleic acid portions surrounding said double-strand break creating agent nucleic acid target in the polynucleotide, gene, genomic locus or genome of said eukaryotic cell, and

isolating a eukaryotic cell in which homologous recombination has occurred.

25. The method of claim 24, wherein said double-strand break creating agent is an endonuclease.

26. The method of claim 24, wherein said double-strand break creating agent is a meganuclease.

27. A eukaryotic cell transformed with a polynucleotide that expresses at least one double-strand break creating agent,

wherein said at least one double-strand break creating agent induces a double- strand break at a specific target site in a polynucleotide, gene, genomic locus or genome of interest, and

wherein said double-stranded break creating agent increases the amount of homologous recombination at or around the target site compared to an otherwise identical eukaryotic cell not transformed with said polynucleotide expressing the double-strand break creating agent.

28. A kit comprising the eukaryotic cell of claim 27 and an isolated vector comprising at least one donor nucleic acid sequence flanked by sequences homologous to genomic nucleic acid portions surrounding a target polynucleotide sequence recognized by the double-strand break creating agent expressed by said eukaryotic cell.

29. A kit comprising:

a first polynucleotide that expresses at least one double-strand break creating agent that recognizes and cleaves a target polynucleotide sequence in a eukaryotic cell, and a second polynucleotide comprising at least one donor nucleic acid sequence flanked by sequences homologous to polynucleotide sequences surrounding said target polynucleotide sequence recognized by the double-strand break creating agent;

wherein said first and second polynucleotide sequences are optionally in forms suitable for transfection into a eukaryotic cell containing the target polynucleotide sequence.