WO2008102274A2

WO2008102274A2 - Meganuclease variants cleaving a dna target sequence from the beta-2-microglobulin gene and uses thereof

Info

Publication number: WO2008102274A2
Application number: PCT/IB2008/001334
Authority: WO
Inventors: Sylvain Arnould; André CHOULIKA
Original assignee: Cellectis
Priority date: 2007-02-20
Filing date: 2008-02-20
Publication date: 2008-08-28
Also published as: EP2121036A2; CA2678709A1; WO2008102199A1; CN101678126A; JP2010518832A; WO2008102274A3; AU2008218605A1

Abstract

An I-CreI variant which has at least two substitutions, one in each of the two functional subdomains of the LAGLIDADG core domain situated from positions 26 to 40 and 44 to 77 of I-Crel, said variant being able to cleave a DNA target sequence from the beta-2 microglobulin gene. Use of said variant and derived products for the prevention and the treatment of xenograft rejection and pathological conditions associated with a fibrillar conformation of the beta-2 microglobulin,, as well as for the engineering of transgenic animals and recombinant cell lines expressing an heterologous protein of interest.

Description

MEGANUCLEASE VARIANTS CLEAVING A DNA TARGET SEQUENCE FROM THE BETA-2-MICROGLOBULIN GENE AND USES THEREOF

The invention relates to a meganuclease variant cleaving a DNA target sequence from the beta-2-microglobulin gene, to a vector encoding said variant, to a cell, an animal or a plant modified by said vector and to the use of said meganuclease variant and derived products for genome therapy ex vivo (gene cell therapy), and genome engineering.

Proteins from the Major Histocompatibility Complex (MHC, also called HLA for humans) have a major role in allorecognition. These proteins present antigenic peptides to T cells. They can be classified in two different complexes, MHC class I and MHC class II. MHC class I complexes are ligands or specific T cells and NK cells immunoglobulin-like receptors. They involve highly polymorphic proteins and a small polypeptide, the β2-microglobulin, necessary for assembly of MHC I complexes at the cell surface (Zijlstra et al, Nature, 1990, 344, 742-746). Since the other components of the complex are encoded by multigenic families, knocking-out the β2-microglobulin gene (B2M in human) is the simplest way to suppress MHC I complexes (Koller et al, Proc. Nat. Acad. Sci. U. S. A., 1989, 86, 8932-8935 ; Zijlstra et al, Nature, 1990, 344, 742-746).

Because of their pivotal role in allorecognition, MHC proteins are also major players in graft rejection. One could hypothesize that disruption of MHC class I proteins would at least partly alleviate graft rejection. However, studies in mice have provided a more mitigated picture. Whereas hematopoietic stem cells from β2m -/- mice are quickly rejected (Bix et al., Nature, 1991, 349, 329-331 ; Liao et al., Science, 1991, 253, 199-202 ; Huang et al, J. Immunol., 2005, 175, 3753-3761; Ruggeri et al, Immunol Rev, 2006, 214, 202-218), apparently as a consequence of NK cells activation, renal and pancreatic islets allograft from such KO animals are better tolerated (Markmann et al, Transplantation, 1992, 54, 1085-1089 ; Coffinan et al, J. Immunol., 1993, 151, 425-435). Thus, at least for certain tissues, the inactivation of the human B2M gene could provide a solution for one of the most recurrent problems in transplantation. This could be used in many applications in cell therapy, for pancreatic, renal, and muscular tissues, including heart: precursor cells could be cultivated, treated ex vivo, and retransplanted into the patient. In adition, like immunoglobulins, prealbumin, and the beta protein (APP) found in the amyloid of Alzheimer disease, beta-2-microglobulin has a predominantly beta-pleated sheet structure that may adopt the fibrillar conformation of amyloid in certain pathologic states (Cunningham et al, Biochemistry, 1973, 12: 481 1-4821). This includes Hemodialysis-Related Amyloidosis (HRA; Gorevic et al, 1986, Proc. Nat. Acad. ScL 83: 7908-7912). Zingraff et al. (New Eng. J. Med., 1990, 323: 1070-1071) described a patient with severe renal insufficiency who had beta-2- microglobulin amyloidosis despite the fact that dialysis had never been performed. The authors suggested also that some B2M variants are more amyloidogenic than others. Thus, the inactivation of the human B2M gene could also provide a solution for treating pathologies associated with a fibrillar conformation of beta-2 microglobulin such as HRA.

Furthermore, the beta-2 microglobulin is highly expressed in the majority of cells; insertion of an exogenous gene of interest at the beta-2 microglobulin locus has the advantage of reproducible expression levels of the recombinant protein. Thus, gene targeting at the beta-2 microglobulin locus allows the engineering of transgenic animals or recombinant cell lines producing high level of a protein of interest.

Homologous gene targeting strategies have been used to knock out endogenous genes (Capecchi, M.R., Science, 1989, 244, 1288-1292) including the mouse B2M (or β2m) gene (Koller et al, Proc. Natl. Acad. Sci. U. S. A., 1989, 86, 8932-8935), or knock-in exogenous sequences in the chromosome. Basically, a DNA sharing homology with the targeted sequence was introduced into the cell's nucleus, and the endogenous homologous recombination machinery provides for the next steps (figure Ia). Homologous recombination (HR), is a very conserved DNA maintenance pathway involved in the repair of DNA double-strand breaks (DSBs) and other DNA lesions (Rothstein, Methods Enzymol., 1983, 101, 202-211; Paques et al, Microbiol MoI Biol Rev, 1999, 63, 349-404; Sung et al, Nat. Rev. MoI. Cell. Biol., 2006, 7, 739-750) but it also underlies many biological phenomenon, such as the meiotic reassortiment of alleles in meiosis (Roeder, Genes Dev., 1997, 11, 2600-2621), mating type interconversion in yeast (Haber, Annu. Rev. Genet., 1998, 32, 561-599), and the "homing" of class I introns and inteins to novel alleles. HR usually promotes the exchange of genetic information between endogenous sequences, but in gene targeting experiments, it is used to promote exchange between an endogenous chromosomal sequence and an exogenous DNA construct. However, the process has a low efficiency (10^~6 to 10^"9 of transfected cells). This efficiency can be enhanced by a DNA double-strand break

(DSB) in the targeted locus. Such DSBs can be created by meganucleases, which are by definition sequence-specific endonucleases recognizing large sequences (Thierry, A. and B. Dujon, Nucleic Acids Res., 1992, 20, 5625-5631). These proteins can cleave unique sites in living cells, thereby enhancing gene targeting by 1000-fold or more in the vicinity of the cleavage site (Puchta et al, Nucleic Acids Res., 1993, 21, 5034-5040 ; Rouet et al, MoI. Cell. Biol, 1994, 14, 8096-8106 ; Choulika et al, MoI. Cell. Biol., 1995, 15, 1968-1973; Puchta et al, Proc. Natl. Acad. Sci. U.S.A., 1996, 93, 5055-5060 ; Sargent et al, MoI. Cell. Biol., 1997, 17, 261-211; Cohen-Tannoudji et al, MoI. Cell. Biol., 1998, 18, 1444-1448 ; Donoho, et al, MoI. Cell. Biol., 1998, 18, 4070-4078; Elliott et al, MoI. Cell. Biol., 1998, 18, 93-101), (figure Ib). However, although several hundreds of natural meganucleases, also referred to as "homing endonucleases" have been identified (Chevalier, B. S. and B. L. Stoddard, Nucleic Acids Res., 2001, 29, 3757-3774), the repertoire of cleavable sequences is too limited to address the complexity of the genomes, and there is usually no cleavable site in a chosen gene. Theoretically, the making of artificial sequence specific endonucleases with chosen specificities could alleviate this limit. Therefore, the making of meganucleases with tailored specificities is under intense investigation.

Recently, fusion of Zinc-Finger Proteins with the catalytic domain of the Fokl, a class IIS restriction endonuclease, were used to make functional sequence-specific endonucleases (Smith et al, Nucleic Acids Res., 1999, 27, 674-681; Bibikova et al, MoI. Cell. Biol., 2001, 21, 289-297 ; Bibikova et al, Genetics, 2002, 161, 1169-1175 ; Bibikova et al, Science, 2003, 300, 764 ; Porteus, M.H. and D. Baltimore, Science, 2003, 300, 763- ; Alwin et al, MoI. Ther., 2005, 12, 610-617; Urnov et al, Nature, 2005, 435, 646-651; Porteus, M.H., MoI. Ther., 2006, 13, 438- 446). Such nucleases could recently be used for the engineering of the ILR2G gene in human cells from the lymphoid lineage (Urnov et al, Nature, 2005, 435, 646-651). The binding specificity of Cys2-His2 type Zinc-Finger Proteins (ZFP), is easy to manipulate, probably because they represent a simple (specificity driven by essentially four residues per finger), and modular system (Pabo et al, Annu. Rev. Biochem., 2001, 70, 313-340 ; Jamieson et al, Nat. Rev. Drug Discov., 2003, 2, 361-368. Studies from the Pabo (Rebar, E.J. and CO. Pabo, Science, 1994, 263, 671- 673 ; Kim, J.S. and CO. Pabo, Proc. Natl. Acad. Sci. U S A, 1998, 95, 2812-2817), Klug (Choo, Y. and A. Klug, Proc. Natl. Acad. Sci. USA, 1994, 91, 11163-11 167 ; Isalan M. and A. Klug, Nat. Biotechnol., 2001, 19, 656-660) and Barbas (Choo, Y. and A. Klug, Proc. Natl. Acad. Sci. USA, 1994, 91, 11163-11167 ; Isalan M. and A. Klug, Nat. Biotechnol., 2001, 19, 656-660) laboratories resulted in a large repertoire of novel artificial ZFPs, able to bind most G/ ANNG/ ANNG/ ANN sequences.

Nevertheless, ZFPs might have their limitations, especially for applications requiring a very high level of specificity, such as therapeutic applications. It was recently shown that Fokl nuclease activity in fusion acts with either one recognition site or with two sites separated by varied distances via a DNA loop including in the presence of some DNA-binding defective mutants of Fokl (Catto et al., Nucleic Acids Res., 2006, 34, 171 1-1720). Thus, specificity might be very degenerate, as illustrated by toxicity in mammalian cells and Drosophila (Bibikova et al, Genetics, 2002, 161, 1169-1 175 ; Bibikova et al, Science, 2003, 300, 764-.). In the wild, meganucleases are essentially represented by homing endonucleases. Homing Endonucleases (HEs) 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 scaffold to derive novel, highly specific endonucleases. HEs belong to four 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 monomelic and display two LAGLIDADG motifs, a few ones have only one motif, but 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 (figure 2). The catalytic core is flanked by two DNA-binding domains with a perfect two-fold symmetry for homodimers such as 1-OeI (Chevalier, et al, Nat. Struct. Biol., 2001, 8, 312-316) and I-Msol (Chevalier et al, J. MoI. Biol., 2003, 329, 253-269) and with a pseudo symmetry fo monomers such as l-Scel (Moure et al, J. MoI. Biol., 2003, 334, 685-69, 1-Dmol (Silva et al, J. MoI. Biol., 1999, 286, 1123-1136) or l-Anil (Bolduc et al, Genes Dev., 2003, 17, 2875-2888). Both monomers, or both domains (for monomelic proteins) contribute to the catalytic core, organized around divalent cations. Just above the catalytic core, the two LAGLIDADG peptides play also an essential role in the dimerization interface. DNA binding depends on two typical saddle-shaped ββαββ 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. MoI. Biol., 2000, 300, 889-901) and PI-SceI (Moure et al, Nat. Struct. Biol., 2002, 9, 764-770), which protein splicing domain is also involved in DNA binding.

The making of functional chimeric meganucleases, by fusing the N- terminal 1-Dmol domain with an 1-CVeI monomer (Chevalier et al, MoI. Cell., 2002, 10, 895-905 ; Epinat et al, Nucleic Acids Res, 2003, 31, 2952-62; International PCT Applications WO 03/078619 and WO 2004/031346) have demonstrasted the plasticity of LAGLIDADG proteins.

Besides, different groups have have used a rational approach to locally alter the specificity of the 1-Crel (Seligman et al, Genetics, 1997, 147, 1653- 1664; Sussman et al, J. MoI. Biol., 2004, 342, 31-41; International PCT Applications WO 2006/097784 and WO 2006/097853; Arnould et al, J. MoI. Biol., 2006, 355, 443-458; Rosen et al, Nucleic Acids Res., 2006, 34, 4791-4800 ; Smith et al, Nucleic Acids Res., 2006, 34, el 49), l-Scel (Doyon et al, J. Am. Chem. Soc, 2006, 128, 2477-2484), Pl-Scel (Gimble et al, J. MoI. Biol., 2003, 334, 993-1008 ) and 1-Msol (Ashworth et al, Nature, 2006, 441, 656-659). In addition, hundreds of 1-OeI 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 1-OeI were mutagenized and a collection of variants with altered specificity in 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; Arnould et al, J. MoI. Biol., 2006, 355, 443-458; Smith et al, Nucleic Acids Res., 2006, 34, el49). - Residues K28, N30 and Q38, N30, Y33 and Q38 or K28, Y33, Q38 and S40 of l-Crel were mutagenized and a collection of variants with altered specificity in positions ± 8 to 10 of the DNA target (lONNN DNA target) were identified by screening (Smith et al, Nucleic Acids Res., 2006, 34, el 49).

Residues 28 to 40 and 44 to 77 of l-Crel were shown to form two separable functional subdomains, able to bind distinct parts of a homing endonuclease half-site (Smith et al. Nucleic Acids Res., 2006, 34, el49).

The combination of mutations from the two subdomains of l-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 in positions ± 3 to 5 and ± 8 to 10 which are bound by each subdomain (Smith et al, Nucleic Acids Res., 2006, 34, el 49).

Two different variants were combined and assembled in a functional heterodimeric endonuclease able to cleave a chimeric target resulting from the fusion of a different half of each variant DNA target sequence (Arnould et al., precited; International PCT Application WO 2006/097854). Interestingly, the novel proteins had kept proper folding and stability, high activity, and a narrow specificity

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, as illustrated on figure 3. 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-meganuclease" can result in an heterodimeric species cleaving the target of interest. The assembly of four set of mutations into heterodimeric endonucleases cleaving a model target sequence or a sequence from the human RAGl gene has been described in Smith et al. (Nucleic Acids Res., 2006, 34, el49).

These variants can be used to cleave genuine chromosomal sequences and have paved the way for novel perspectives in several fields, including gene therapy.

The Inventors have identified a series of DNA targets in the beta-2 microglobulin gene that could be cleaved by I-Crel variants (figure 4). The combinatorial approach described in figure 3 was used to entirely redesign the DNA binding domain of the l-Crel protein and thereby engineer novel meganucleases with fully engineered specificity, to cleave DNA targets from the human B2M gene. The I- OeI variants which are able to cleave a genomic DNA target from the human B2M gene can be used for inactivating the human B2M gene (figure 6) ex vivo, for the purpose of preventing xenograft rejection in human. Other potential applications include genome therapy of pathologies associated with a fibrillar conformation of beta-2 microglobulin and genome engineering at the beta-2 microglobulin locus (knock-out and knock in). The invention relates to an 1-Creϊ variant wherein at least one of the two l-Crel monomers has at least two substitutions, one in each of the two functional subdomains of the LAGLIDADG core domain situated from positions 26 to 40 and 44 to 77 of l-Crel, and is able to cleave a DNA target sequence from the beta-2 microglobulin gene. The cleavage activity of the variant according to the invention may be measured by any well-known, in vitro or in vivo cleavage assay, such as those described in the International PCT Application WO 2004/067736; Epinat et al, Nucleic Acids Res., 2003, 31, 2952-2962; Chames et al, Nucleic Acids Res., 2005, 33, el78 and Amould et al, J. MoI. Biol., 2006, 355, 443-458. For example, the cleavage activity of the variant of the invention may be measured by a direct repeat recombination assay, in yeast or mammalian cells, using a reporter vector. The reporter vector comprises two truncated, non-functional copies of a reporter gene (direct repeats) and the genomic DNA target sequence within the intervening sequence, cloned in a yeast or a mammalian expression vector. Expression of the variant results in a functional endonuclease which is able to cleave the genomic DNA target sequence. This cleavage induces homologous recombination between the direct repeats, resulting in a functional reporter gene, whose expression can be monitored by appropriate assay.

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.

- 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.

- by "meganuclease", is intended an endonuclease having a double- stranded DNA target sequence of 12 to 45 bp. Said meganuclease is either a dimeric enzyme, wherein each domain is on a monomer or a monomelic enzyme comprising the two domains on a single polypeptide.

- by "meganuclease domain" is intended 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.

- by "meganuclease variant" or "variant" is intented a meganuclease obtained by replacement of at least one residue in the amino acid sequence of the wild-type meganuclease (natural meganuclease) with a different amino acid.

- by "functional variant" is intended 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.

- by "meganuclease variant with novel specificity" is intended 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.

- by "1-OeI" is intended the wild-type 1-OeI having the sequence SWISSPROT P05725 or pdb accession code Ig9y, corresponding to the sequence

SEQ ID NO: 1 or SEQ ID NO: 107 in the sequence listing.

- by "domain" or "core domain" is intended the "LAGLIDADG homing endonuclease core domain" which is the characteristic

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 (βiβ2β3β4) folded in an antiparallel 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 1-OeI (163 amino acids), the LAGLIDADG homing endonuclease core domain corresponds to the residues 6 to 94.

- by "single-chain meganuclease" is intended 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.

- by "subdomain" is intended the region of a LAGLIDADG homing endonuclease core domain which interacts with a distinct part of a homing endonuclease DNA target half-site. - by "beta-hairpin" is intended two consecutive beta-strands of the antiparallel beta-sheet of a LAGLIDADG homing endonuclease core domain ((βiβ₂ or,β₃β₄) which are connected by a loop or a turn, - by "1-OeI site" is intended a 22 to 24 bp double-stranded DNA sequence which is cleaved by l-Crel. l-Crel sites include the wild-type (natural) non- palindromic 1-OeI homing site and the derived palindromic sequences such as the sequence 5'- t-i₂c-i₁a_-1oa_-9a_-8a_-7c_-6g_-5t_-4c_-3g_-2t_-Ia₊₁c₊₂g₊₃a₊₄c₊₅g₊₆t₊₇t₊₈t₊₉t₊.i_Og₊iia₊₁₂ also called C 1221 (SEQ ID NO :2; figure 5).

- by "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" is intended a 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 l-Crel, or a variant, or a single-chain chimeric meganuclease derived from l-Crel. 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 1221. Cleavage of the DNA target occurs at the nucleotides in positions +2 and -2, respectively for the sense and the antisense strand. Unless otherwiwe indicated, the position at which cleavage of the DNA target by an 1-Cre I meganuclease variant occurs, corresponds to the cleavage site on the sense strand of the DNA target. - by "DNA target half-site", "half cleavage site" or half-site" is intended the portion of the DNA target which is bound by each LAGLIDADG homing endonuclease core domain.

- by "chimeric DNA target" or "hybrid DNA target" is intended 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).

- by "beta-2-microglobulin gene" is intended the beta-2- microglobulin gene of a mammal. For example, the human beta-2-microglobulin gene (B2M, 6673bp) is situated from positions 42790977 to 42797649 of the sequence corresponding to accession number NC OOOOl 5. The B2M gene comprises four exons (Exon 1 : positions 1-127; Exon 2: positions 3937 to 4215; Exon 3: 4843 to 4870; Exon 4: positions 6121 to 6673). The ORF which is from position 61 (Exon 1) to positions 4856 (Exon 3), is flanked by a short and a long untranslated region, respectively at its 5' and 3' ends (Figure 4).

- by "DNA target sequence from the beta-2-micro globulin gene", "genomic DNA target sequence", " genomic DNA cleavage site", "genomic DNA target" or "genomic target" is intended a 20 to 24 bp sequence of the beta-2- microglobulin gene of a mammal which is recognized and cleaved by a meganuclease variant.

- by "vector" is intended a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. - by "homologous" is intended a sequence with enough identity to another one to lead to a homologous recombination between sequences, more particularly having at least 95 % identity, preferably 97 % identity and more preferably 99 %.

- "identity" refers to sequence 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 settings.

- "individual" includes mammals, as well as other vertebrates (e.g., birds, fish and reptiles). The terms "mammal" and "mammalian", as used herein, refer to any vertebrate animal, including monotremes, marsupials and placental, that suckle their young and either give birth to living young (eutharian or placental mammals) or are egg-laying (metatharian or nonplacental mammals). Examples of mammalian species include humans and other primates (e.g., monkeys, chimpanzees), rodents (e.g., rats, mice, guinea pigs) and others such as for example: cows, pigs and horses.

- by mutation is intended the substitution, deletion, insertion of one or more nucleotides/amino acids in a polynucleotide (cDNA, gene) or a polypeptide 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.

The variant according to the invention may be an homodimer or an heterodimer. Preferably, it is an heterodimer wherein both monomers are mutated in positions 26 to 40 and/or 44 to 77. More preferably, both monomers have different substitutions both in positions 26 to 40 and 44 to 77 of I-Crel.

In a preferred embodiment of said variant, said substitution(s) in the subdomain situated from positions 44 to 77 of 1-OeI are in positions 44, 68, 70, 75 and/or 77.

In another preferred embodiment of said variant, said substitution(s) in the subdomain situated from positions 26 to 40 of 1-OeI are in positions 26, 28, 30, 32, 33, 38 and/or 40.

In another preferred embodiment of said variant, it comprises one or more mutations at positions of other amino acid residues which contact the DNA target sequence or interact with the DNA backbone or with the nucleotide bases, directly or via a water molecule; these residues are well-known in the art (Jurica et al., Molecular Cell., 1998, 2, 469-476; Chevalier et al, J. MoI. Biol., 2003, 329, 253-269). In particular, additional substitutions may be introduced at positions contacting the phosphate backbone, for example in the final C-terminal loop (positions 137 to 143). Preferably said residues are involved in binding and cleavage of said DNA cleavage site. More preferably, said residues are in positions 138, 139, 142 or 143 of l-Crel. Two residues may be mutated in one variant provided that each mutation is in a different pair of residues chosen from the pair of residues in positions 138 and 139 and the pair of residues in positions 142 and 143. The mutations which are introduced modify the interaction(s) of said amino acid(s) of the final C-terminal loop with the phosphate backbone of the 1-OeI site. Preferably, the residue in position 138 or 139 is substituted by an hydrophobic amino acid to avoid the formation of hydrogen bonds with the phosphate backbone of the DNA cleavage site. For example, the residue in position 138 is substituted by an alanine or the residue in position 139 is substituted by a methionine. The residue in position 142 or 143 is advantageously substituted by a small amino acid, for example a glycine, to decrease the size of the side chains of these amino acid residues. More, preferably, said substitution in the final C-terminal loop modify the specificity of the variant towards the nucleotide in positions ± 1 to 2, ± 6 to 7 and/or ± 11 to 12 of the 1-OeI site.

In another preferred embodiment of said variant, it comprises one or more additional mutations that improve the binding and/or the cleavage properties of the variant towards the DNA target sequence from the beta-2 microglobulin gene.

The additional residues which are mutated may be on the entire I- OeI sequence, and in particular in the C-terminal half of 1-OeI (positions 80 to 163). For example, the variant comprises one or more additional substitutions in positions: 2, 4, 19, 24, 31, 43, 49, 50, 53, 54, 56, 57, 59, 60, 64, 66, 69, 72, 73, 80, 81, 82, 83, 85, 87, 89, 92, 94, 96, 100, 103, 105, 107, 110, 111, 1 17, 120, 128, 129, 132, 135, 140, 142, 147, 153, 154, 155, 156, 157, 158, 159, 161, 163. Preferably said substitutions are selected from the group consisting of: N2I, N2Y, K4Q, G19S, I24F, I24V, Q31L, F43L, T49A, Q50R, W53R, F54L, D56E, D56G, K57N, V59A, D60E, D60G, D60N, V64A, V64D, Y66C, D69G, D69E, S72F, S72P, S72T, V73I, E80G, 18 IT, 18 IV, K82E, K82R, P83Q, P83A, H85R, F87L, T89A, T89I, Q92L, Q92R, F94L, F94Y, K96R, KlOOR, KlOOQ, N103T, N103S, V105A, K107R, EI lOD, EI lOG, Ql I lL, E117G, D120G, W128R, V129A, I132V, L135P, T140M, K142R, T147A, T147N, D153G, D153V, S154G, L155Q, S156N, S156R, E157V, K158N, K159Q, K159R, S161P, S161F, S162F, P163L and P163Q. More preferably, said mutations are selected from the group consisting of: G19S, 124 V, F54L, F87L and I132V.

The variant may also comprise one or two additional residues inserted at the C-terminus of the 1-OeI sequence (positions 164 and 165); for example a G can be inserted at position 164, a T or a P at position 165

In another preferred embodiment of said variant, said substitutions are replacement of the initial amino acids with amino acids selected from the group consisting of: A, D, E, G, H, K, N, P, Q, R, S, T, Y, C, V, L and W.

The variant of the invention may be derived from the wild-type I- OeI (SEQ ID NO: 1) or an 1-OeI scaffold protein, such as the scaffold of SEQ ID NO: 106 (167 amino acids) having the insertion of an alanine in position 2, the substitution D75N, and the insertion of AAD at the C-terminus (positions 164 to 166) of the 1-Crel sequence.

In addition, the variants of the invention may include one or more residues inserted at the NH₂ terminus and/or COOH terminus of the sequence. For example, a tag (epitope or polyhistidine sequence) is introduced at the NH₂ terminus and/or COOH terminus; said tag is useful for the detection and/or the purification of said variant.

The variant according to the present invention may be an homodimer which is able to cleave a palindromic or pseudo-palindromic DNA target sequence.

Alternatively, said variant is an heterodimer, resulting from the association of a first and a second monomer having different substitutions in positions 26 to 40 and 44 to 77 of 1-OeI, said heterodimer being able to cleave a non- palindromic DNA target sequence from the beta-2 -microglobulin gene. The DNA target sequence which is cleaved by said variant may be in an exon or in an intron of the beta-2 -microglobulin gene.

In another preferred embodiment of said variant, said DNA target sequence is from the human beta-2-microglobulin gene (B2M gene). Preferably said

DNA target sequence is selected from the group consisting of the sequences SEQ ID NO: 82 to 91 (24 bp; Figures 4 and 15) and the 20 to 22 bp derived sequences lacking one or two of the terminal base pairs from one or both ends of said 24 bp sequence.

Since coding exons represent only a small fraction of the gene (Figure 4), most potential target sites are found in intronic sequences or untranslated exonic sequences.

However, targets such as B2M18 and B2M20 (SEQ ID NO: 89 and 90) are found in the B2M open reading frame.

More preferably, the monomers of the variant have at least the following substitutions, respectively for the first and the second monomer:

- Y33R, Q38A, Q44D, R68A, R70S, D75K and I77R (first monomer), and K28R, Y33A, Q38Y, S40Q, Q44A, R68Y, R70S, D75Y and I77K (second monomer); this variant cleaves the B2M4 target which is located in the first intron (figures 4 and 15), - S32T, Y33T, Q44T, R68Y, R70S, D75Y and I77V (first monomer), and Y33R, Q38A, Q44N, R68Q, R70S, D75S and I77V (second monomer); this variant cleaves the B2M10 target which is located in the first intron (figures 4 and 15),

-S32G, Y33H, Q44A, R68Y, R70S, D75Y and I77K or S32A, Y33H, Q44A, R68Y, R70S, D75Y and I77K (first monomer) and N30Q, Y33G, Q38C, R68N, R70S, D75N and I77R (second monomer); this variants cleave the B2M11 target which is located in the first intron (figures 4 and 15),

- S32G, Y33H, Q44A, R68Y, R70S, D75Y and I77K (first monomer), and S32T, Q38S, Q44K, R70S and I77A (second monomer); this variant cleaves the B2M13 target which is located in the first intron (figures 4 and 15),

- S32T, Y33T, Q44K, R68E, R70S and I77R (first monomer), and N30A, Y33T, Q44N, R68K, R70S, D75H and I77F (second monomer); this variant cleaves the B2M14 target which is located in the first intron (figures 4 and 15),

- S32R, Y33D, Q44A, R70S, D75E and I77R (first monomer), and N30D, Y33R, Q44K, R68Y, R70S, D75N and I77Q (second monomer); this variant cleaves the B2M16 target which is located in the first intron (figures 4 and 15),

- S32T, Q38W, Q44A, R70S, D75R and 177 Y (first monomer), and Y33H, S40Q, Q44N, R70S, D75R and I77Y (second monomer); this variant cleaves the B2M17 target which is located in the first intron (figures 4 and 15), - Y33H, Q38G, Q44N, R68Y, R70S, D75R and I77V (first monomer), and a second monomer selected from the group consisiting of: N30A, Y33T, Q44N, R68Y, R70S, D75R and I77V; N30H, Y33C, R68Y, R70S, D75R and I77Q; S32G, Y33C, R68Y, R70S, D75R and I77Q; S32R, Y33T, R68Y, R70S, D75R and I77Q; S32G, Y33C, Q44N, R68Y, R70S, D75R and I77Q; S32A, Y33C, Q44N, R68Y, R70S, D75R and I77Q; S32G, Y33S, Q44N, R68Y, R70S, D75R and I77Q; N30H, Y33C, Q44N, R68Y, R70S, D75R and I77V; S32G, Y33C, Q44N, R68Y, R70S, D75R and I77V; Y33C, S40Q, Q44N, R68Y, R70S, D75R and I77V; S32G, Y33S, Q44N, R68Y, R70S, D75R and I77V; S32G, Y33C, Q44T, R68Y, R70S, D75R and I77Q; S32A, Y33C, Q44T, R68Y, R70S, D75R and I77Q ; S32G, Y33C, R68Y, R70S, D75R and I77V; S32G, Y33C, R68Y, R70S and D75R; S32G, Y33C, Q44N, R68Y, R70S, D75R and I77Y; S32A, Y33C, Q44N, R68Y, R70S, D75R and I77Y; S32R, Y33T, R68Y, R70S, D75R, I77Q and D153G; N30H, Y33C, Q44R, R68Y, R70S, D75R and I77Q; these variants cleave the B2M18 target which is located in exon 2 (figures 4 and 15, Table VII),

- Y33T, S40N, Q44T, R68Y, R70S, D75R and I77V (first monomer), and a second monomer selected from the group consisiting of: K28R, Y33A, Q38Y, S40Q, Q44A, R68S, R70S, D75S and I77R; K28A, Y33S, S40R, R70S and D75N; K28A, Y33T, S40R, R70S and D75N; K28R, Y33A, Q38Y, S40Q, R70S and D75N; K28R, Y33N, Q38R, S40Q, R70S and D75N; K28R, Y33R, Q38Y, S40Q, R70S and D75N; K28R, Y33S, Q38R, S40Q, R70S and D75N; K28R, Y33S, Q38Y, S40Q, R70S and D75N; K28T, Y33T, S40D, R70S and D75; K28T, Y33T, S40R, R70S and D75N; S32G, Y33C and D75N; Y33C and D75N; Y33S, S40R and D75N; Y33S and S40R; N30A and Y33G; N30A and Y33T; N30C and Y33A; N30G and Y33C; N30S, Y33S and Q38T; N30A and S32W; N30H and Y33K; N30R and Y33P; N30K and Y33T; N30P and Y33W; S32G and Y33P; S32T and Y33A; Y33T and S40E; these variants cleave the B2M20 target which is located at the intron 2- exon 3 junction (figures 4 and 15; Table VIII), and

- K28T, Y33R, S40R, Q44T, R70S and D75Y (first monomer), and N30D, Y33R, Q44N, R68Y, R70S, D75Y and I77Q (second monomer); this variant cleaves the B2M33 target which is located in exon 4 (figures 4 and 15).

More preferably, said variant consist of a first monomer having any of the sequences SEQ ID NO: 24 to 28, 126 to 134 and a second monomer having any of the sequences SEQ ID NO: 37 to 77 and 135; this variant which derives from the monomers cleaving the B2M11 target, as defined above, has additional substitutions that increase the cleavage of the B2M11 target (figures 4 and 15; Table IV, V and VI).

The heterodimeric variant is advantageously an obligate heterodimer variant having at least one pair of mutations interesting corresponding residues of the first and the second monomers which make an intermolecular interaction between the two l-Crel monomers, wherein the first mutation of said pair(s) is in the first monomer and the second mutation of said pair(s) is in the second monomer and said pair(s) of mutations impairs the formation of functional homodimers from each monomer without preventing the formation of a functional heterodimer, able to cleave the genomic DNA target from the beta-2 microglobulin gene. The monomers have advantageously at least one of the following pairs of mutations, respectively for the first and the second monomer: a) the substitution of the glutamic acid in position 8 with a basic amino acid, preferably an arginine (first monomer) and the substitution of the lysine in position 7 with an acidic amino acid, preferably a glutamic acid (second monomer ) ; the first monomer may further comprise the substitution of at least one of the lysine residues in positions 7 and 96, by an arginine. b) the substitution of the glutamic acid in position 61 with a basic amino acid, preferably an arginine (first monomer) and the substitution of the lysine in position 96 with an acidic amino acid, preferably a glutamic acid (second monomer) ; the first monomer may further comprise the substitution of at least one of the lysine residues in positions 7 and 96, by an arginine c) the substitution of the leucine in position 97 with an aromatic amino acid, preferably a phenylalanine (first monomer) and the substitution of the phenylalanine in position 54 with a small amino acid, preferably a glycine (second monomer) ; the first monomer may further comprise the substitution of the phenylalanine in position 54 by a tryptophane and the second monomer may further comprise the substitution of the leucine in position 58 or lysine in position 57, by a methionine, and d) the substitution of the aspartic acid in position 137 with a basic amino acid, preferably an arginine (first monomer) and the substitution of the arginine in position 51 with an acidic amino acid, preferably a glutamic acid (second monomer).

For example, the first monomer may have the mutation D137R and the second monomer, the mutation R5 ID.

The obligate heterodimer meganuclease comprises advantageously, at least two pairs of mutations as defined in a), b) c) or d), above; one of the pairs of mutation is advantageously as defined in c) or d). Preferably, one monomer comprises the substitution of the lysine residues at positions 7 and 96 by an acidic amino acid and the other monomer comprises the substitution of the glutamic acid residues at positions 8 and 61 by a basic amino acid. More preferably, the obligate heterodimer meganuclease, comprises three pairs of mutations as defined in a), b) and c), above. The obligate heterodimer meganuclease consists advantageously of a first monomer (A) having at least the mutations selected from: (i) E8R, E8K or E8H, E61R, E61K or E61H and L97F, L97W or L97Y; (ii) K7R, E8R, E61R, K96R and L97F, or (iii) K7R, E8R, F54W, E61R, K96R and L97F and a second monomer (B) having at least the mutations (iv) K7E or K7D, F54G or F54A and K96D or K96E; (v) K7E, F54G, L58M and K96E, or (vi) K7E, F54G, K57M and K96E. For example, the first monomer may have the mutations K7R, E8R, E61R, K96R and L97F or K7R, E8R, F54W, E61R, K96R and L97F and the second monomer, the mutations K7E, F54G, L58M and K96E or K7E, F54G, K57M and K96E. The subject-matter of the present invention is also a single-chain chimeric meganuclease (fusion protein) derived from an l-Crel variant as defined above. The single-chain meganuclease may comprise two 1-OeI monomers, two I- Crel core domains (positions 6 to 94 of l-Creϊ) or a combination of both. Preferably, the two monomers /core domains or the combination of both, are connected by a peptidic linker.

The subject-matter of the present invention is also a polynucleotide fragment encoding a variant or a single-chain chimeric meganuclease as defined above; said polynucleotide may encode one monomer of an homodimeric or heterodimeric variant, or two domains/monomers of a single-chain chimeric meganuclease.

The subject-matter of the present invention is also a recombinant vector for the expression of a variant or a single-chain meganuclease according to the invention. The recombinant vector comprises at least one polynucleotide fragment encoding a variant or a single-chain meganuclease, as defined above. In a preferred embodiment, said vector comprises two different polynucleotide fragments, each encoding one of the monomers of an heterodimeric variant.

A vector which can be used 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, semi- synthetic 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. adeno- associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e. g., influenza virus), rhabdo virus (e. g., rabies and vesicular stomatitis virus), paramyxovirus (e. g. measles and Sendai), positive strand RNA viruses such as picor- navirus 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).

Preferred vectors include lentiviral vectors, and particularly self inactivacting lentiviral vectors.

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; TRPl for S. cerevisiae; tetracycline, rifampicin or ampicillin resistance in E. coli.

Preferably said vectors are expression vectors, wherein the sequence(s) encoding the variant/single-chain meganuclease of the invention is placed under control of appropriate transcriptional and translational control elements to permit production or synthesis of said variant. 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, an RNA-splicing site (when genomic DNA is used), a polyadenylation site and a transcription termination site. It also can comprise an enhancer. Selection of the promoter will depend upon the cell in which the poly- peptide is expressed. Preferably, when said variant is an heterodimer, the two polynucleotides encoding each of the monomers are included in one vector which is able to drive the expression of both polynucleotides, simultaneously. 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-D- 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), α-antitrypsin protease, human surfactant (SP) A and B proteins, β-casein and acidic whey protein genes.

According to another advantageous embodiment of said vector, it includes a targeting construct comprising sequences sharing homologies with the region surrounding the genomic DNA cleavage site as defined above.

Alternatively, the vector coding for an l-Crel variant/single-chain meganuclease and the vector comprising the targeting construct are different vectors.

More preferably, the targeting DNA construct comprises: a) sequences sharing homologies with the region surrounding the genomic DNA cleavage site as defined above, and b) a sequence to be introduced flanked by sequences as in a). For genome therapy or the making of knock-out animals/cells, the sequence to be introduced is a sequence which inactivates the beta-2 microglobulin gene. Both homologous chromosomes have to be targeted in order to totally inactivate the function of the gene. In addition, said sequence may also delete the b2- microglobulin gene or part thereof, and eventually introduce an exogenous gene or part thereof (knock-in/gene replacement). For making knock-in animals/cells the DNA which repairs the site of interest comprises the sequence of an exogenous gene of interest, and eventually a selection marker, such as the HPRT gene. Alternatively, the sequence to be introduced can be any other sequence used to alter the chromosomal DNA in some specific way including a sequence used to modify a specific sequence, to attenuate or activate the endogenous gene of interest or to introduce a mutation into a site of interest. Such chromosomal DNA alterations may be used for genome engineering (animal models and recombinant cell lines including human cell lines). Inactivation of the beta-2 microglobulin gene may occur by insertion of a transcription termination signal that will interrupt the transcription, and result in a truncated protein (Figure 6a). In this case, the sequence to be introduced comprises, in the 5' to 3' orientation: at least a transcription termination sequence (polyAl), preferably said sequence further comprises a marker cassette including a promoter and the marker open reading frame (ORF) and a second transcription termination sequence (polyA2; figure 6a). This strategy can be used with any meganuclease cleaving a target downstream of the B2M promoter and upsteam of the B2M stop codon, such as any of the targets B2M4, B2M10, B2M11, B2M13, B2M14, B2M16, B2M17, B2M18 and B2M20 (SEQ ID NO: 82 to 90; figures 4 and 15).

Inactivation of the beta-2 microglobulin gene may also occur by insertion of a marker gene within the B2M open reading frame (ORF), that would disrupt the coding sequence (figure 6b). The insertion can in addition be associated with deletions of ORF sequences flanking the cleavage site and eventually, the insertion of an exogeneous gene of interest (gene replacement). This strategy can be used with a meganuclease cleaving an exonic sequence, such as for example, a meganuclease cleaving B2M18 or B2M20 (SEQ ID NO: 90, 91).

In addition, inactivation of the beta-2 microglobulin gene may also occur by insertion of a sequence that would destabilize the transcript. This strategy can be used with any meganuclease cleaving a target downstream of the B2M promoter such as any of the targets (SEQ ID NO: 82 to 91) presented in figures 4 and 15.

Preferably, homologous sequences of at least 50 bp, preferably more than 100 bp and more preferably more than 200 bp are used. 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.

Therefore, the targeting construct is preferably from 200 pb to 6000 pb, more preferably from 1000 pb to 2000 pb; it comprises: a beta-2 microglobulin gene fragment which has at least 200 bp of homologous sequence flanking the target site for repairing the cleavage, and the sequence for inactivating the beta-2 microglobulin gene and eventually the sequence of an exogeneous gene of interest for replacing the beta-2 microglobulin gene, as defined above.

For the insertion of a sequence, DNA homologies are generally located in regions directly upstream and downstream to the site of the break (sequences immediately adjacent to the break; minimal repair matrix). However, when the insertion is associated with a deletion of ORF sequences flanking the cleavage site, shared DNA homologies are located in regions upstream and downstream the region of the deletion.

For example, the B2M target which is cleaved by each of the variant as defined above and the minimal matrix for repairing the cleavage with each variant are indicated in figure 15.

The subject-matter of the present invention is also a composition characterized in that it comprises at least one meganuclease as defined above (variant or single-chain derived chimeric meganuclease) and/or at least one expression vector encoding said meganuclease, as defined above.

In a preferred embodiment of said composition, it comprises a targeting DNA construct comprising a sequence which inactivates the beta-2 microglobulin gene, flanked by sequences sharing homologies with the genomic DNA cleavage site of said variant, as defined above. Preferably, said targeting DNA construct is either included in a recombinant vector or it is included in an expression vector comprising the polynucleotide(s) encoding the meganuclease according to the invention.

The subject-matter of the present invention is also the use of at least one meganuclease and/or one expression vector, as defined above, for the preparation of a medicament for preventing, improving or curing xenograft rejection during transplantation of cells from a donor into an individual (recipient) in need thereof.

The subject-matter of the present invention is also the use of at least one meganuclease and/or one expression vector, as defined above, for the preparation of a medicament for preventing, improving or curing a pathological condition associated with a fibrillar conformation of beta-2 microglobulin in an individual in need thereof. The use of the meganuclease may comprise at least the step of (a) inducing in somatic tissue(s) of the donor/ individual a double stranded cleavage at a site of interest of the beta-2 microglobulin gene comprising at least one recognition and cleavage site of said meganuclease by contacting said cleavage site with said meganuclease, and (b) introducing into said somatic tissue(s) a targeting DNA, wherein said targeting DNA comprises (1) DNA sharing homologies to the region surrounding the cleavage site and (2) DNA which inactivates the beta-2 microglobulin gene upon recombination between the targeting DNA and the chromosomal DNA, as defined above. The targeting DNA is introduced into the somatic tissues(s) under conditions appropriate for introduction of the targeting DNA into the site of interest. The targeting construct may comprise sequences for deleting the beta-2 microglobulin gene and eventually the sequence of an exogenous gene of interest (gene replacement).

Alternatively, the beta-2 microglobulin gene may be inactivated by the mutagenesis of the open reading frame, by repair of the double-strands break by non-homologous end joining (Figure 6c). In the absence of a repair matrix, the DNA double-strand break in an exon will be repaired essentially by the error-prone Non Homologous End Joining pathway NHEJ, resulting in small deletions (a few nucleotides), that will inactivate the cleavage site, and result in frameshift mutation. In this case the use of the meganuclease comprises at least the step of : inducing in somatic tissue(s) of the donor/individual a double stranded cleavage at a site of interest of the beta-2 microglobulin gene comprising at least one recognition and cleavage site of said meganuclease by contacting said cleavage site with said meganuclease, and thereby induce mutagenesis of the beta-2 microglobulin gene open reading frame by repair of the double-strands break by non-homologous end joining.

According to the present invention, said double-stranded cleavage may be induced, ex vivo by introduction of said meganuclease into somatic cells (pancreas, kidney, heart, muscle) from the donor/individual and then transplantation of the modified cells into the recipient (xenotransplantation) or back into the diseased individual (pathology associated with a fibrillar conformation of beta-2 microglobulin) . The subject-matter of the present invention is also a method for preventing, improving or curing xenograft rejection during transplantation, in an individual in need thereof, said method comprising at least the step of administering to said individual a composition as defined above, by any means. The subject-matter of the present invention is also a method for preventing, improving or curing a pathological condition associated with a fibrillar conformation of beta-2 microglobulin, in an individual in need thereof, said method comprising at least the step of administering to said individual a composition as defined above, by any means. The subject-matter of the present invention is further the use of a meganuclease as defined above, one or two polynucleotide(s), preferably included in expression vector(s), for genome engineering at the beta-2 microglobulin gene locus (animal models and recombinant cells generation: knock-in or knock-out), for non- therapeutic purposes. According to an advantageous embodiment of said use, it is for inducing a double-strand break in a site of interest of the beta-2 microglobulin gene comprising a genomic DNA target sequence, thereby inducing a DNA recombination event, a DNA loss or cell death.

According to the invention, said double-strand break is for: repairing a specific sequence in the beta-2 microglobulin gene, modifying a specific sequence in the beta-2 microglobulin gene, restoring a functional beta-2 microglobulin gene in place of a mutated one, attenuating or activating the endogenous beta-2 microglobulin gene, introducing a mutation into a site of interest of the beta-2 microglobulin gene, introducing an exogenous gene or a part thereof, inactivating or deleting the endogenous beta-2 microglobulin gene or a part thereof, translocating a chromosomal arm, or leaving the DNA unrepaired and degraded.

According to another advantageous embodiment of said use, said variant, polynucleotide(s), vector, are associated with a targeting DNA construct as defined above. In a first embodiment of the use of the meganuclease according to the present invention, it comprises at least the following steps: 1) introducing a double-strand break at a site of interest of the beta-2 microglobulin gene comprising at least one recognition and cleavage site of said meganuclease, by contacting said cleavage site with said meganuclease ; 2) providing a targeting DNA construct comprising the sequence to be introduced flanked by sequences sharing homologies to the targeted locus. Said meganuclease can be provided directly to the cell or through an expression vector comprising the polynucleotide sequence encoding said meganuclease and suitable for its expression in the used cell. This strategy is used to introduce a DNA sequence at the target site, for example to generate knock-in or knock-out animal models or cell lines that can be used for drug testing.

In a second embodiment of the use of the meganuclease according to the present invention, it comprises at least the following steps: 1) introducing a double-strand break at a site of interest of the beta-2 microglobulin gene comprising at least one recognition and cleavage site of said meganuclease, by contacting said cleavage site with said meganuclease ; 2) maintaining said broken genomic locus under conditions appropriate for homologous recombination with chromosomal DNA sharing homologies to regions surrounding the cleavage site.

In a third embodiment of the use of the meganuclease according to the present invention, it comprises at least the following steps: 1) introducing a double-strand break at a site of interest of the beta-2 microglobulin gene comprising at least one recognition and cleavage site of said meganuclease, by contacting said cleavage site with said meganuclease ; 2) maintaining said broken genomic locus under conditions appropriate for repair of the double-strands break by nonhomologous end joining.

The subject-matter of the present invention is also a method for making a beta-2 microglobulin knock-in or knock-out animal, comprising at least the step of:

(a) introducing into a pluripotent precursor cell or an embryo of an animal, a meganuclease, as defined above, so as to into induce a double stranded cleavage at a site of interest of the beta-2 microglobulin gene comprising a DNA recognition and cleavage site of said meganuclease; simultaneously or consecutively, (b) introducing into the animal precursor cell or embryo of step (a) a targeting DNA, wherein said targeting DNA comprises (1) DNA sharing homologies to the region surrounding the cleavage site and (2) DNA which repairs the site of interest upon recombination between the targeting DNA and the chromosomal DNA, so as to generate a genomically modified animal precursor cell or embryo having repaired the site of interest by homologous recombination,

(c) developping the genomically modified animal precursor cell or embryo of step (b) into a chimeric animal, and

(d) deriving a transgenic animal from the chimeric animal of step

(C).

Preferably, step (c) comprises the introduction of the genomically modified precursor cell generated in step (b) into blastocysts so as to generate chimeric animals.

The subject-matter of the present invention is also a method for making a beta-2 microglobulin knock-in or knock-out cell, comprising at least the step of:

(a) introducing into a cell, a meganuclease, as defined above, so as to into induce a double stranded cleavage at a site of interest of the beta-2 microglobulin gene comprising a DNA recognition and cleavage site for said meganuclease, simultaneously or consecutively,

(b) introducing into the cell of step (a), a targeting DNA, wherein said targeting DNA comprises (1) DNA sharing homologies to the region surrounding the cleavage site and (2) DNA which repairs the site of interest upon recombination between the targeting DNA and the chromosomal DNA, so as to generate a recombinant cell having repaired the site of interest by homologous recombination,

(c) isolating the recombinant cell of step (b), by any appropriate mean. The targeting DNA is introduced into the cell under conditions appropriate for introduction of the targeting DNA into the site of interest. hi a preferred embodiment, said targeting DNA construct is inserted in a vector.

Alternatively, the beta-2 microglobulin gene may be inactivated by repair of the double-strands break by non-homologous end joining (Figure 6c).

The subject-matter of the present invention is also a method for making a beta-2 microglobulin knock-out animal, comprising at least the step of: (a) introducing into a pluripotent precursor cell or an embryo of an animal, a meganuclease, as defined above, so as to induce a double stranded cleavage at a site of interest of the beta-2 microglobulin gene comprising a DNA recognition and cleavage site of said meganuclease, and thereby generate genomically modified precursor cell or embryo having repaired the double-strands break by non-homologous end joining,

(b) developping the genomically modified animal precursor cell or embryo of step (a) into a chimeric animal, and

(c) deriving a transgenic animal from a chimeric animal of step (b). Preferably, step (b) comprises the introduction of the genomically modified precursor cell obtained in step (a), into blastocysts, so as to generate chimeric animals.

The subject-matter of the present invention is also a method for making a beta-2 microglobulin-deficient cell, comprising at least the step of: (a) introducing into a cell, a meganuclease, as defined above, so as to induce a double stranded cleavage at a site of interest of the beta-2 microglobulin gene comprising a DNA recognition and cleavage site of said meganuclease, and thereby generate genomically modified HPRT deficient cell having repaired the double-strands break, by non-homologous end joining, and (b) isolating the genomically modified HPRT deficient cell of step(a), by any appropriate mean.

The cell which is modified may be any cell of interest. For making transgenic/knock-out animals, the cells are pluripotent precursor cells such as embryo- derived stem (ES) cells, which are well-kown in the art. For making recombinant cell lines, the cells may advantageously be human cells, for example PerC6 (Fallaux et al., Hum. Gene Ther. 9, 1909-1917, 1998) or HEK293 (ATCC # CRL- 1573) cells. Said meganuclease can be provided directly to the cell or through an expression vector comprising the polynucleotide sequence encoding said meganuclease and suitable for its expression in the used cell. For making transgenic animals/recombinant cell lines, including human cell lines expressing an heterologous protein of interest, the targeting DNA comprises the sequence of the exogenous gene encoding the protein of interest, and eventually a marker gene, flanked by sequences upstream and downsteam the beta-2 microglobulin gene, as defined above, so as to generate genomically modified cells (animal precursor cell or embryo/animal or human cell) having replaced the beta-2 microglobulin gene by the exogenous gene of interest, by homologous recombination. The exogenous gene and the marker gene are inserted in an appropriate expression cassette, as defined above, in order to allow expression of the heterologous protein/marker in the transgenic animal/recombinant cell line.

The meganuclease can be used either as a polypeptide or as a polynucleotide construct encoding said polypeptide. It is introduced into somatic cells of an individual, by any convenient means well-known to those in the art, which are appropriate for the particular cell type, alone or in association with either at least an appropriate vehicle or carrier and/or with the targeting DNA.

According to an advantageous embodiment of the uses according to the invention, the meganuclease (polypeptide) is associated with: - liposomes, polyethyleneimine (PEI); in such a case said association is administered and therefore introduced into somatic target cells.

- membrane translocating peptides (Bonetta, The Scientist, 2002, 16, 38; Ford et al., Gene Ther., 2001, 8, 1-4 ; Wadia and Dowdy, Curr. Opin. Biotechnol., 2002, 13, 52-56); in such a case, the sequence of the variant/single-chain meganuclease is fused with the sequence of a membrane translocating peptide (fusion protein).

According to another advantageous embodiment of the uses according to the invention, the meganuclease (polynucleotide encoding said meganuclease) and/or the targeting DNA is inserted in a vector. Vectors comprising targeting DNA and/or nucleic acid encoding a meganuclease can be introduced into a cell by a variety of methods (e.g., injection, direct uptake, projectile bombardment, liposomes, electroporation). Meganucleases can be stably or transiently expressed into cells using expression vectors. Techniques of expression in eukaryotic cells are well known to those in the art. (See Current Protocols in Human Genetics: Chapter 12 "Vectors For Gene Therapy" & Chapter 13 "Delivery Systems for Gene Therapy"). Optionally, it may be preferable to incorporate a nuclear localization signal into the recombinant protein to be sure that it is expressed within the nucleus. Once in a cell, the meganuclease and if present, the vector comprising targeting DNA and/or nucleic acid encoding a meganuclease are imported or translocated by the cell from the cytoplasm to the site of action in the nucleus.

For purposes of therapy, the meganucleases and a pharmaceutically acceptable excipient are administered in a therapeutically effective amount. Such a combination is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of the recipient. In the present context, an agent is physiologically significant if its presence results in a decrease in the severity of one or more symptoms of the targeted disease and in a genome correction of the lesion or abnormality.

In one embodiment of the uses according to the present invention, the meganuclease is substantially non-immunogenic, i.e., engender little or no adverse immunological response. A variety of methods for ameliorating or eliminating delete- rious immunological reactions of this sort can be used in accordance with the invention. In a preferred embodiment, the meganuclease is substantially free of N-formyl methionine. Another way to avoid unwanted immunological reactions is to conjugate meganucleases to polyethylene glycol ("PEG") or polypropylene glycol ("PPG") (preferably of 500 to 20,000 daltons average molecular weight (MW)). Conjugation with PEG or PPG, as described by Davis et al. (US 4,179,337) for example, can provide non-immunogenic, physiologically active, water soluble endonuclease conjugates with anti-viral activity. Similar methods also using a polyethylene— polypropylene glycol copolymer are described in Saifer et al. (US 5,006,333).

The invention also concerns a prokaryotic or eukaryotic host cell which is modified by a polynucleotide or a vector as defined above, preferably an expression vector.

The invention also concerns a non-human transgenic animal or a transgenic plant, characterized in that all or part of their cells are modified by a polynucleotide or a vector as defined above. As used herein, a cell refers to a prokaryotic cell, such as a bacterial cell, or an eukaryotic cell, such as an animal, plant or yeast cell. The subject-matter of the present invention is also the use of at least one meganuclease variant, as defined above, as a scaffold for making other meganucleases. For example a third round of mutagenesis and selection/screening can be performed on said variants, for the purpose of making novel, third generation meganucleases.

The different uses of the meganuclease and the methods of using said meganuclease according to the present invention include the use of the l-Crel variant, the single-chain chimeric meganuclease derived from said variant, the polynucleotide^), vector, cell, transgenic plant or non-human transgenic mammal encoding said variant or single-chain chimeric meganuclease, as defined above.

The 1-OeI variant according to the invention may be obtained by a method for engineering l-Crel variants able to cleave a genomic DNA target sequence from the beta-2 microglobulin gene, comprising at least the steps of:

(a) constructing a first series of 1-OeI variants having at least one substitution in a first functional subdomain of the LAGLIDADG core domain situated from positions 26 to 40 of l-Crel,

(b) constructing a second series of 1-OeI variants having at least one substitution in a second functional subdomain of the LAGLIDADG core domain situated from positions 44 to 77 of l-Crel, (c) selecting and/or screening the variants from the first series of step (a) which are able to cleave a mutant 1-OeI site wherein (i) the nucleotide triplet in positions -10 to -8 of the 1-OeI site has been replaced with the nucleotide triplet which is present in positions -10 to -8 of said genomic target and (ii) the nucleotide triplet in positions +8 to +10 has been replaced with the reverse complementary sequence of the nucleotide triplet which is present in positions -10 to -8 of said genomic target,

(d) selecting and/or screening the variants from the second series of step (b) which are able to cleave a mutant I-Crel site wherein (i) the nucleotide triplet in positions -5 to -3 of the 1-OeI site has been replaced with the nucleotide triplet which is present in positions -5 to -3 of said genomic target and (ii) the nucleotide triplet in positions +3 to +5 has been replaced with the reverse complementary sequence of the nucleotide triplet which is present in positions -5 to -3 of said genomic target,

(e) selecting and/or screening the variants from the first series of step (a) which are able to cleave a mutant l-Crel site wherein (i) the nucleotide triplet in positions +8 to +10 of the l-Crel site has been replaced with the nucleotide triplet which is present in positions +8 to +10 of said genomic target and (ii) the nucleotide triplet in positions -10 to -8 has been replaced with the reverse complementary sequence of the nucleotide triplet which is present in positions +8 to +10 of said genomic target, (f) selecting and/or screening the variants from the second series of step (b) which are able to cleave a mutant 1-OeI site wherein (i) the nucleotide triplet in positions +3 to +5 of the I-Crel site has been replaced with the nucleotide triplet which is present in positions +3 to +5 of said genomic target and (ii) the nucleotide triplet in positions -5 to -3 has been replaced with the reverse complementary sequence of the nucleotide triplet which is present in positions +3 to +5 of said genomic target,

(g) combining in a single variant, the mutation(s) in positions 26 to 40 and 44 to 77 of two variants from step (c) and step (d), to obtain a novel homodimeric I-Crel variant which cleaves a sequence wherein (i) the nucleotide triplet in positions -10 to -8 is identical to the nucleotide triplet which is present in positions -10 to -8 of said genomic target, (ii) the nucleotide triplet in positions +8 to +10 is identical to the reverse complementary sequence of the nucleotide triplet which is present in positions -10 to -8 of said genomic target, (iii) the nucleotide triplet in positions -5 to -3 is identical to the nucleotide triplet which is present in positions -5 to -3 of said genomic target and (iv) the nucleotide triplet in positions +3 to +5 is identical to the reverse complementary sequence of the nucleotide triplet which is present in positions -5 to -3 of said genomic target, and/or

(h) combining in a single variant, the mutation(s) in positions 26 to 40 and 44 to 77 of two variants from step (e) and step (f), to obtain a novel homodimeric l-Crel variant which cleaves a sequence wherein (i) the nucleotide triplet in positions +3 to +5 is identical to the nucleotide triplet which is present in positions +3 to +5 of said genomic target, (ii) the nucleotide triplet in positions -5 to - 3 is identical to the reverse complementary sequence of the nucleotide triplet which is present in positions +3 to +5 of said genomic target, (iii) the nucleotide triplet in positions +8 to +10 of the l-Crel site has been replaced with the nucleotide triplet which is present in positions +8 to +10 of said genomic target and (iv) the nucleotide triplet in positions -10 to -8 is identical to the reverse complementary sequence of the nucleotide triplet in positions +8 to +10 of said genomic target,

(i) combining the variants obtained in steps (g) and (h) to form heterodimers, and

(j) selecting and/or screening the heterodimers from step (i) which are able to cleave said genomic DNA target from the beta-2 microglobulin gene.

Steps (a), (b), (g), and (h) may comprise the introduction of additional mutations in order to improve the binding and/or cleavage properties of the mutants, particularly at other positions contacting the DNA target sequence or interacting directly or indirectly with said DNA target. This may be performed by generating a combinatorial library as described in the International PCT Application WO 2004/067736.

In addition, step (g) and/or (h) may further comprise the introduction of random mutations on the whole variant or in a part of the variant, in particular the C-terminal half of the variant (positions 80 to 163). This may be performed by generating random mutagenesis libraries on a pool of variants, according to standard mutagenesis methods which are well-known in the art and commercially available.

The (intramolecular) combination of mutations in steps (g) and (h) may be performed by amplifying overlapping fragments comprising each of the two subdomains, according to well-known overlapping PCR techniques. The (intermolecular) combination of the variants in step (i) is performed by co-expressing one variant from step (g) with one variant from step (h), so as to allow the formation of heterodimers. For example, host cells may be modified by one or two recombinant expression vector(s) encoding said variant(s). The cells are then cultured under conditions allowing the expression of the variant(s), so that heterodimers are formed in the host cells, as described previously in the International PCT Application WO 2006/097854 and Arnould et al., J. MoI. Biol., 2006, 355, 443- 458. The selection and/or screening in steps (c), (d), (e), (f) and/or (j) may be performed by using a cleavage assay in vitro or in vivo, as described in the

International PCT Application WO 2004/067736, Arnould et al, J. MoI. Biol., 2006,

355, 443-458, Epinat et al., Nucleic Acids Res., 2003, 31, 2952-2962 and Chames et al., Nucleic Acids Res., 2005, 33, el 78.

According to another advantageous embodiment of said method, steps (c), (d), (e), (f) and/or (j) are performed in vivo, under conditions where the double-strand break in the mutated DNA target sequence which is generated by said variant leads to the activation of a positive selection marker or a reporter gene, or the inactivation of a negative selection marker or a reporter gene, by recombination- mediated repair of said DNA double-strand break.

The subject matter of the present invention is also an Ϊ-Crel variant having mutations in positions 26 to 40 and/or 44 to 77 of I-Crel that is useful for engineering the variants able to cleave a DNA target from the beta-2 microglobulin gene, according to the present invention. In particular, the invention encompasses the l-Crel variants as defined in step (c) to (f) of the method for engineering I-Crel variants, as defined above, including the variants of the sequence SEQ ID NO: 78, 79, 80, 81 and 105. The invention encompasses also the l-Crel variants as defined in step (g) and (h) of the method for engineering I-Crel variants, as defined above, including the variants of the sequence SEQ ID NO: 29 to 36.

Single-chain chimeric meganucleases able to cleave a DNA target from the gene of interest are derived from the variants according to the invention by methods well-known in the art (Epinat et al., Nucleic Acids Res., 2003, 31, 2952-62; Chevalier et al, MoI. Cell., 2002, 10, 895-905; Steuer et al, Chembiochem., 2004, 5, 206-13; International PCT Applications WO 03/078619 and WO 2004/031346). Any of such methods, may be applied for constructing single-chain chimeric meganucleases derived from the variants as defined in the present invention.

The polynucleotide sequence(s) encoding the variant as defined in the present invention may be prepared by any method known by the man skilled in the art. For example, they are amplified from a cDNA template, by polymerase chain reaction with specific primers. Preferably the codons of said cDNA are chosen to favour the expression of said protein in the desired expression system. The recombinant vector comprising said polynucleotides may be obtained and introduced in a host cell by the well-known recombinant DNA and genetic engineering techniques.

The l-Crel variant or single-chain derivative as defined in the present the invention are produced by expressing the polypeptide(s) as defined above; preferably said polypeptide(s) are expressed or co-expressed (in the case of the variant only) in a host cell or a transgenic animal/plant modified by one expression vector or two expression vectors (in the case of the variant only), under conditions suitable for the expression or co-expression of the polypeptide(s), and the variant or single-chain derivative is recovered from the host cell culture or from the transgenic animal/plant.

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, VoIs.154 and 155 (Wu et al. eds.) and Vol. 185, "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 hi 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). In addition to the preceding features, the invention further comprises other features which will emerge from the description which follows, which refers to examples illustrating the I- OeI meganuclease variants and their uses according to the invention, as well as to the appended drawings in which: - figure 1 illustrates gene targeting strategies (a) A linear sequence containing a marker surrounded by sequences homologous to the targeted locus can be introduced into the nucleus, and recombine with the homologous targeted locus. This experimental design is today the most widespread one for gene knock-in and gene knock-out. Note that the insertion of the marker can be associated with a deletion within the targeted locus, resulting in gene replacement, (b) meganuclease-induced gene targeting. In this case, the targeting sequence is often part of a circular plasmid.

- figure 2 represents the tridimensional structure of the l-Crel homing endonuclease bound to its DNA target. The catalytic core is surrounded by two αββαββα folds forming a saddle-shaped interaction interface above the DNA major groove.

- figure 3 illustrates the combinatorial approach for the making of redesigned Homing Endonucleases. A large collection of l-Crel derivatives with locally altered specificity is generated. Then, a two step combinatorial approach is used to assemble these mutants into homodimeric proteins (by combinations of mutations within a same monomer), and then into heterodimers, resulting in meganucleases with fully redesigned specificity.

- figure 4 represents the human B2M gene (accession number NC_000015 ; 6673 pb). The Exons are boxed (Exon 1 : positions 1-127; Exon 2: positions 3937 to 4215; Exon 3: 4843 to 4870; Exon 4: positions 6121 to 6673). The ORF is from position 61 (Exon 1) to positions 4856 (Exon 3). Various meganuclease sites (B2Mn) are indicated.

- figure 5 represents the B2M series of target. B2M11.2 and B2M1 1.3 are two palindromic sequences derived from the B2M target by mirror duplication of one half of the target. These two targets can in turn be considered as combinations of the IOGAA P and 5TAG P (B2M11.2) and the IOCTG P and the 5TTT P targets (B2M1 1.3) found to be cleaved by 1-OeI targets, if nucleotides at positions ±11, ±7 and ±6 in the B2M11.2 and B2M11.3 targets are considered as having no impact on cleavage. All targets are aligned with the C 1221 target, a palindromic sequence cleaved by l-Crel.

- figure 6 represent three strategies for inactivation of a gene by meganuclease-induced recombination. Note that in all three cases, both homologous chromosomes have to be targeted in order to totally inactivate the function of the gene, (a) inactivation by cleavage in the intron: a transcription termination sequence (polyAl) and a marker cassette including a promoter, the marker ORF, and a second transcription termination sequence (polyA2). The transcription termination sequences will result in a truncated transcript, and therefore, a truncated protein, (b) Inactivation by cleavage in an exon, and knock-in of a marker cassette. Marker knock-in can be associated with deletion of exonic sequences (see Figure 1). (c) Inactivation by cleavage in an exon, and repair by the error-prone Non Homologous End Joining pathway. In the absence of a repair matrix, the DNA double-strand break will be repaired essentially by NHEJ, resulting most of the time in perfect rejoining of the 3' cohesive ends resulting from meganuclease cleavage. In this case, the restored cleavage site can be cleaved again by the meganuclease. However, this error prone repair pathway can also result in small deletions (a few nucleotides), that will inactivate the cleavage site, and result in frameshift mutation.

- figure 7 illustrates the cleavage of the B2M11.2 target by combinatorial mutants. The figure displays an example of primary screening of 1-OeI combinatorial mutants with the B2M11.2 target. Hl 2 is a positive control (C). In the first top filter, the sequence of positive mutant at position B3 (circle) is KNAHQS/AYSYK (same nomenclature as for Table I). In the second filter, bottom one's, the sequence of positive mutant at position F7 is KNGHQS/AYSYK. - figure 8 illustrates cleavage of the of B2M11.2 target by optimized mutants. A series of l-Crel N75 optimized mutants cutting B2M11.2 are obtained from random mutagenesis of the mutants KNAHQS/AYSYK and KNGHQS/AYSYK. Cleavage is tested with the B2M11.2 target. Mutants cleaving B2M11.2 are circled. For example, the sequence of positive mutant at position B3 is corresponding to 28K30N32A33H38Q40S44A68Y70S75Y77K/2Y53R66C (same nomenclature as for Table II). H12 is a positive control. - figure 9 illustrates cleavage of the B2M1 1.3 target by combinatorial mutants. The figure displays an example of primary screening of l-Crel combinatorial mutants with the B2M1 1.3 target. HlO, HI l and H12 are respectively negative (Cl) and two positive controls (C2 and C3) of different strength. In the filter, the sequence of positive mutant at position G5 (circle) is KQSGCS/QNSNR (same nomenclature as for Table III).

- figure 10 illustrates cleavage of B2M11 target by heterodimeric combinatorial mutants. The figure displays primary screening of combinations of I- OeI mutants with the B2M11 target. A column of positive heterodimeric combinatorial mutants are circled. In the example filter, (1) and (2) are yeast strain with B2M11 target and mutant respectively

28K30N32G33H38Q40S44A68Y70S75Y77K/2I96R105A (1) and

28K30N32A33H38Q40S44A68Y70S75Y77K/132V (2), matted with the yeast strain with 28K30Q32S33G38C40S44Q68N70S75N77R (Ml) (same nomenclature as for Table IV).

- figure 11 illustrates cleavage of B2M11 target by optimized heterodimeric combinatorial mutants. A series of l-Crel N75 optimized mutants cutting B2M11.3 are coexpressed with mutants cutting B2M11.2. Cleavage is tested with the B2M11 target. Mutants cleaving B2M11 are circled (as example G9, corresponding to an heterodimer of 28K30Q32S33G38C40S44Q68N70S75N77R vs 28K30N32A33H38Q40S44A68Y70S75Y77K/2Y53R66C. In the examples filter, the yeast strain with B2M11 target and mutant

28K30N32A33H38Q40S44A68Y70S75Y77K/2Y53R66C is matted with the yeast strain with 28K30Q32S33G38C40S44Q68N70S75N77R (MI) (same nomenclature as for Table V), or controls (Cl to C3) in diagonal. HlO, HI l and H12 are also respectively negative (Cl) and two positive controls (C2 and C3) of different strength.

- figure 12 represents the pCLS1055 vector map.

- figure 13 represents the pCLS0542 vector map.

- figure 14 represents the pCLSl 107 vector map. - figure 15 represents meganuclease target sequences found in the human B2M gene and the corresponding l-Crel variant which is able to cleave said DNA target. The exons closest to the target sequences, and the exons junctions are indicated (columns 2 and 3), the sequence of the DNA target is presented (column 4), with its position (column 5). The minimum repair matrix for repairing the cleavage at the target site is indicated by its first nucleotide (start, column 8) and last nucleotide (end, column 9). The sequence of each variant is defined by the mutated residues at the indicated positions. For example, the first heterodimeric variant of figure 15 consists of a first monomer having K, N, S, R, A, S, D, A, S, K, R in positions 28, 30, 32, 33, 38, 40, 44, 68, 70, 75 and 77, respectively and a second monomer having R, N, S, A, Y, Q, A, Y, S, Y and K in positions 28, 30, 32, 33, 38, 40, 44, 68, 70, 75 and 77, respectively. The positions are indicated by reference to I- OeI sequence SWISSPROT P05725 (SEQ ID NO: 1) ; 1-OeI has K, N, S, Y, Q, S, Q, R, R, D and I, in positions 28, 30, 32, 33, 38, 40, 44, 68, 70, 75 and 77 respectively.

- figure 16 represents the pCLS1069 vector map.

- figure 17 represents the pCLS1058 vector map.

- figure 18 represents the B2M18 series of target. B2M18.3 and B2M18.4 are two palindromic sequences derived from the B2M18 target by mirror duplication of one half of the target. These two targets can in turn be considered as combinations of 1 ONNN and 5NNN targets found to be cleaved by 1-OeI targets, if one considers that nucleotides at positions ±11, ±7 and ±6 in the B2M18.3 and B2M18.4 targets have no impact on cleavage. All targets are aligned with the C 1221 target, a palindromic sequence cleaved by I-Crel.

- figure 19 illustrates cleavage of the B2M18.4 target by combinatorial mutants. The figure displays an example of primary screening of I-Crel combinatorial mutants with the B2M18.4 target. Right double dots (C) on each quarter are alternatively respectively negative (Cl) and two positive controls (C2 and C3) of different strength. The left double dots (M) in the filter are combinatorial clone tested. The sequence of positive mutant at position BlO (circle) is KNGCQS/QYSRQ (same nomenclature as for Table VII).

- figure 20 represents the B2M20 series of target. B2M20.3 and B2M20.4 are two palindromic sequences derived from the B2M20 target by mirror duplication of one half of the target. These two targets can in turn be considered as combinations of 1 ONNN and 5NNN targets found to be cleaved by I-Crel targets, if one considers that nucleotides at positions ±11, ±7 and ±6 in the B2M20.3 and B2M20.4 targets have no impact on cleavage. All targets are aligned with the C 1221 target, a palindromic sequence cleaved by l-Crel.

- figure 21 illustrates cleavage of the B2M20.4 target by combinatorial mutants. The figure displays an example of primary screening of l-Crel combinatorial mutants with the B2M20.4 target. Right double dots (C) on each quarter are alternatively respectively negative (Cl) and two positive controls (C2 and C3) of different strength. The left double dots (M) in the filter are combinatorial clone tested. The sequence of positive mutant at position F6 (circle) is KNSTQE/QRRDI (same nomenclature as for Table VIII). Example 1 : Strategy for engineering novel meganucleases cleaving the human B2M gene

The combinatorial approach described in Smith et al, Nucleic Acids Res., 2006 and illustrated in figure 3, was used to engineer the DNA binding domain of l-Crel, and cleave one of the B2M targets, B2M11 (figures 5 and 15), a 24 bp (non- palindromic) target (figure 5) located at positions 2892 to 2915 of the human B2M gene (accession number NCJ)OOOl 5.8, positions 42790977 to 42797649). The meganucleases cleaving B2M11 can be used to inactivate the B2M gene by insertion of a transcription termination signal that will interrupt the transcription, and result in a truncated protein (Figure 6a). The B2M11 sequence is partly a patchwork of the 10GAA P, lOCTG P, 5TAG_P and 5TTT P targets (Figure 5), which are cleaved by previously identified meganucleases, obtained as described in International PCT Applications WO 2006/097784, WO 2006/097853; Arnould et al, J. MoI. Biol., 2006, 355, 443- 458 and Smith et al, Nucleic Acids Res., 2006, 34, el49. Thus B2M11 could be cleaved by meganucleases combining the mutations found in the 1-OeI derivatives cleaving these four targets.

The 10GAA_P, 10CTG_P, 5TAG_P and 5TTT P sequences are 24 bp derivatives of C 1221, a palindromic sequence cleaved by I-Oel (International PCT Applications WO 2006/097784, WO 2006/097853; Arnould et al, J. MoI. Biol., 2006, 355, 443-458 and Smith et al, Nucleic Acids Res., 2006, 34, el49).

However, the structure of l-Crel bound to its DNA target suggests that the two external base pairs of these targets (positions -12 and 12) have no impact on binding and cleavage (Chevalier et al., Nat. Struct. Biol., 2001, 8, 312-316; Chevalier B.S. and Stoddard B.L., Nucleic Acids Res., 2001, 29, 3757-3754; Chevalier et al., J. MoI. Biol., 2003, 329, 253-269), and in this study, only positions - 11 to 11 were considered. Consequently, the B2M1 1 series of targets were defined as 22 bp sequences instead of 24 bp.

Two palindromic targets, B2M11.2 and B2M11.3 were derived from B2M11 (Figure 5). Since B2M1 1.2 and B2M1 1.3 are palindromic, they should be cleaved by homodimeric proteins. Thus, proteins able to cleave the B2M1 1.2 and B2M11.3 sequences as homodimers, were first designed (examples 2 and 3), followed by an optimization of homodimers able to cleaved more efficiently B2M11.2 target (example 4), and then coexpression to obtain heterodimers cleaving B2M11.1 (example 5). Chosen mutant cleaving B2M11.3 were then refined; the chosen mutants were randomly mutagenized, and used to form novel heterodimers that were screened against the B2M11 target (example 6). Example 2: Making of meganucleases cleaving B2M11.2

This example shows that l-Crel mutants can cut the B2M11.2 DNA target sequence derived from the left part of the B2M11 target in a palindromic form (figure 5).

Target sequences described in this example are 22 bp palindromic sequences. Therefore, they will be described only by the first 11 nucleotides, followed by the suffix _P. For example, target B2M11.2 will be noted also tgaaattaggt P; SEQ ID NO: 96)).

B2M11.2 is similar to 5TAG_P in positions ±1, ±2, ±3, ±4, ±5 and ±7 and to 10GAA_P in positions ±1, ±2, ±7, ±8, ±9 and ±10. It was hypothesized that positions ±6 and ±11 would have little effect on the binding and cleavage activity. Mutants able to cleave 5TAG P target (caaaactaggt P; SEQ ID NO: 94) were previously obtained by mutagenesis on l-Crel N75 at positions 44, 68, 70, 75 and 77, as described in Araould et al, J. MoI. Biol., 2006, 355, 443-458 and International PCT Applications WO 2006/097784, WO 2006/097853. Mutants able to cleave the IOGAA P target (cgaaacgtcgt P; SEQ ID NO:92 ) were obtained by mutagenesis on l-Crel N75 and D75 at positions 28, 30, 32, 33, 38, 40, as described previously in Smith et al, Nucleic Acids Res., 2006, 34, el 49). Thus combining such pairs of mutants would allow for the cleavage of the B2M11.2 target.

Therefore, to check whether combined mutants could cleave the

B2M11.2 target, mutations at positions 44, 68, 70, 75 and 77 from proteins cleaving 5TAG P (caaaactaggt P; SEQ ID NO: 94) were combined with the 28, 30, 32, 33, 38 and 40 mutations from proteins cleaving IOGAA P (cgaaacgtcgt P; SEQ ID NO: 92).

1) Material and Methods

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 al, Nucleic Acids Res., 2005, 33, el 78, and Arnould et al, J. MoI. Biol., 2006, 355, 443-458. These assays result in a functional LacZ reporter gene which can be monitored by standard methods. a) Construction of target vector

The target was cloned as follow: oligonucleotide corresponding to the target sequence flanked by gateway cloning sequence was ordered from PROLIGO: 5' tggcatacaagttttgttctcaggtacctgagaacaacaatcgtctgtca 3' (SEQ ID NO: 98). Double- stranded target DNA, generated by PCR amplification of the single stranded oligonucleotide, was cloned using the Gateway protocol (INVITROGEN) into yeast reporter vector (pCLS1055, Figure 12). Yeast reporter vector was transformed into S. cerevisiae strain FYBL2-7B (MATa, ura3Δ851, trplΔ63, leu2Δl, lys2Δ202). b) Construction of combinatorial mutants

1-OeI mutants cleaving IOGAA P or 5TAG P were identified as described previously in Smith et al, Nucleic Acids Res., 2006, 34, el 49, and Arnould et al, J. MoI. Biol., 2006, 355, 443-458; International PCT Applications WO 2006/097784, WO 2006/097853, respectively for the 10GAA_P or 5TAG_P targets. In order to generate l-Crel derived coding sequence containing mutations from both series, separate overlapping PCR reactions were carried out that amplify the 5' end (aa positions 1-43) or the 3' end (positions 39-167) of the l-Crel coding sequence. For both the 5' and 3' end, PCR amplification is carried out using GaIlOF (5'- gcaactttagtgctgacacatacagg-3'; SEQ ID NO: 99) or GaIlOR (5'- acaaccttgattggagacttgacc-3'; SEQ ID NO: 100) primers specific to the vector (pCLS0542, Figure 13) and primers specific to the 1-OeI coding sequence for amino acids 39-43 (assF 5'-ctannnttgaccttt-3'(SEQ ID NO: 101 ) or assR 5'-aaaggtcaannntag- 3'(SEQ ID NO: 102)) where nnn code for residue 40. The PCR fragments resulting from the amplification reaction realized with the same primers and with the same coding sequence for residue 40 were pooled. Then, each pool of PCR fragments resulting from the reaction with primers GaIlOF and assR or assF and GaIlOR was mixed in an equimolar ratio. Finally, approximately 25 ng of each final pool of the two overlapping PCR fragments and 75 ng of vector DNA (pCLS0542) linearized by digestion with Ncol and Eagl were used to transform the yeast Saccharomyces cerevisiae strain strain FYC2-6A (M ATa, trplΔ63, leuΔl, his3Δ200) using a high efficiency LiAc transformation protocol (Gietz and Woods, Methods Enzymol., 2002, 350, 87-96). An intact coding sequence containing both groups of mutations is generated by in vivo homologous recombination in yeast. c) Mating of meganuclease expressing clones and screening in yeast:

Screening was performed as described previously (Arnould et ai, J. MoI. Biol., 2006, 355, 443-458). Mating was performed using a colony gridder (QpixII, Genetix). Mutants were gridded on nylon filters covering YPD plates, using a low gridding density (about 4 spots/cm²). A second gridding process was performed on the same filters to spot a second layer consisting of different reporter-harboring yeast strains for each target. Membranes were placed on solid agar YPD rich medium, and incubated at 30 °C for one night, to allow mating. Next, filters were transferred to synthetic medium, lacking leucine and tryptophan, with galactose (2 %) as a carbon source, and incubated for five days at 37 °C, to select for diploids carrying the expression and target vectors. After 5 days, filters were placed on solid agarose medium with 0.02 % X-GaI in 0.5 M sodium phosphate buffer, pH 7.0, 0.1 % SDS, 6 % dimethyl formamide (DMF), 7mM β-mercaptoethanol, 1% agarose, and incubated at 37 °C, to monitor β-galactosidase activity. Results were analyzed by scanning and quantification was performed using appropriate software. d) Sequencing of mutants

To recover the mutant expressing plasmids, yeast DNA was extracted using standard protocols and used to transform E. coli. Sequence of mutant ORF were then performed on the plasmids by MILLEGEN SA. Alternatively, ORFs were amplified from yeast DNA by PCR (Akada et al, Biotechniques, 2000, 28, 668-670), and sequence was performed directly on PCR product by MILLEGEN SA. 2) Results I-Crel combinatorial mutants were constructed by associating mutations at positions 44, 68, 70, 75 and 77 with the 28, 30, 33, 38 and 40 mutations on the l-Crel N75 or D75 scaffold, resulting in a library of a complexity of 2014. Examples of combinations are displayed on Table 1. These libraries were transformed into yeast and 4464 clones (2.2 times the diversity) were screened for cleavage against B2M11.2 DNA target (tgaaattaggt P; SEQ ID NO: 96). 2 positives clones were found with a very low level of activity, which after sequencing and validation by secondary screening turned out to correspond to 2 different novel endonucleases (see Table I). Positives are shown in Figure 7.

Throughout the text and figures, combinatorial mutants sequences are named with an eleven letter code, after residues at positions 28, 30, 32, 33, 38, 40, 44, 68 and 70, 75 and 77. For example, KNGHQS/AYSYK stands for I-Oel K28, N30, G32, H33, Q38, S40, A44, Y68, S70, Y75 and K77 (l-Crel 28K30N32G33H38Q40S44A68Y70S75Y77K). Parental mutants are named with a six letter code, after residues at positions 28, 30, 32, 33, 38 and 40 or a five letter code, after residues at positions 44, 68, 70, 75 and 77. For example, KNGHQS stands for I- OeI K28, N30, G32, H33, Q38 and S40, and AYSYK stands for 1-OeI A44, Y68, S70, Y75 and K77.

*Only 176 out of the 2014 combinations are displayed

+ indicates cleavage of the B2M11.2 target by the combinatorial variant

Example 3; Making of meganucleases cleaving B2M11.2 with higher efficacy by random mutagenesis of meganucleases cleaving B2M11.2.

1-OeI mutants able to cleave the palindromic B2M11.2 target have been identified by assembly of mutants cleaving the palindromic 10GAA_P and 5TAG_P target (example 2). However, only 2 of these combinations were able to cleave B2M11.2 and with a minimal efficiency.

Therefore the two protein combinations cleaving B2M11.2 were mutagenized and variants cleaving B2M1 1.2 with better efficiency were screened. According to the structure of the I- OeI protein bound to its target, there is no contact between the residues used for the first combinatorial approach (28, 30, 32, 33, 38 and 40 vs 44, 68, 70, 75 and 77) in the I-Crel protein (Chevalier et al., Nat. Struct. Biol., 2001, 8, 312-316; Chevalier B.S. and Stoddard B.L., Nucleic Acids Res., 2001, 29, 3757-3754; Chevalier et al, J. MoI. Biol., 2003, 329, 253-269). Thus, it is difficult to rationally choose a set of positions to mutagenize, and mutagenesis was done on the C-terminal part of the protein (83 last amino acids) or on the whole protein. 1) Material and Methods

Random mutagenesis libraries were created on a pool of chosen mutants, by PCR using Mn²⁺ or derivatives of dNTPs as 8-oxo-dGTP and dPTP in two-step PCR process, as described in the protocol from JENA BIOSCIENCE GmbH in JBS dNTP-Mutagenis kit. Primers used are preATGCreFor (5'- gcataaattactatacttctatagacacgcaaacacaaatacacagcggccttgccacc-3'; SEQ ID NO: 103) and ICrelpostRev (5'-ggctcgaggagctcgtctagaggatcgctcgagttatcagtcggccgc-3'; SEQ ID NO: 104). The new libraries were cloned in vivo in the yeast in the linearized kanamycin vector harbouring a galactose inducible promoter, a KanR as selectable marker and a 2 micron origin of replication. Positives resulting clones were verified by sequencing (MILLEGEN).

Pools of mutants were amplified by PCR reaction using these primers common for leucine vector (pCLS0542, Figure 13) and kanamycin vector (pCLSl 107, Figure 14). Approximately 75ng of PCR fragment and 75ng of vector DNA (pCLS0542) linearized by digestion with Ncol and Eagl are used to transform the yeast Saccharomyces cerevisiae strain FYC2-6A (MATα, trplΔ63, leu2Δl, his3Δ200) using a high efficiency LiAc transformation protocol. A library of intact coding sequence for the 1-OeI mutant is generated by in vivo homologous recombination in yeast.

Mating assays were done as described in example 2. 2) Results:

Two mutants cleaving B2M11.2, 1-OeI

28K30N32G33H38Q40S44A68Y70S75Y77K and 1-OeI

28K30N32A3338Q40SH44A68Y70S75Y77K, also called KNGHQS/AYSYK and KNAHQS/AYSYK according to nomenclature of Table I) were pooled, randomly mutagenized and transformed into yeast (Figure 13). 4464 transformed clones were then mated with a yeast strain that contains the B2M1 1.2 target in a reporter plasmid. Thirty-two clones were found to trigger cleavage of the B2M1 1.2 target when mated with such yeast strain, corresponding at least to 13 different novel endonucleases (see Table II). Example of positives is shown on Figure 8.

Table II: Optimized mutants towards the B2M11.2 target

Optimized Mutant B2M11.2^* Target (SEQ ID NO: 24 to 36) B2M11.2

I-Crel 28K30N32A33H38Q40S44A68Y70S75Y77K/2Y53R + I-Crel 28K30N32A33H38Q40S44A68Y70S75Y77K/2Y53R66C I-Crel 28K30N32A33H 38Q40S44A68Y70S75Y77K/132V + I-Crel 28K30N32G33H38Q40S44A68Y70S75Y77K/2I96R105A + I-Crel 28K30N32G33H38Q40S44A68Y70S75Y77K/120G + I-Crel 28K30N32A33H38Q40S44A68Y70S75Y77K/43L105A159R + I-Crel 28K30N32G33H38Q40S44A68Y70S75Y77K/50R I-Crel 28K30N32G33H38Q40S44A68Y70S75Y77K/49A50R + I-Crel 28K30N32G33H38Q40S44A68Y70S75Y77K/81V129A154G + I-Crel 28K30N32G33H38Q40S44A68Y70S75Y77K/129A161 P + I-Crel 28K30N32G33H38Q40S44A68Y70S75Y77K/117G + I-Crel 28K30N32G33H38Q40S44A68Y70S75Y77K/81T + I-Crel 28K30N32G33H38Q40S44A68Y70S75Y77K/103T

+ B2M11.2 target cleavage * optimized mutations are in bold

Example 4: Making of meganucleases cleaving B2M11.3

This example, shows that 1-OeI mutants can cleave the B2M11.3 DNA target sequence derived from the right part of the B2M11.1 target in a palindromic form (Figure 5). All target sequences described in this example are 22 bp palindromic sequences. Therefore, they will be described only by the first 11 nucleotides, followed by the suffix _P, solely to indicate that (for example, B2M11.3 will be called tctgactttgt P; SEQ ID NO: 97).

B2M11.3 is similar to 5TTT P in positions ±1, ±2, ±3, ±4, ±5, ±6 and ±7 and to 10CTG_P in positions ±1, ±2, ±6, ±7, ±8, ±9 and ±10. It was hypothesized that position ±11 would have little effect on the binding and cleavage activity. Mutants able to cleave 5TTT P target (caaaactttgt P; SEQ ID NO: 95) were previously obtained by mutagenesis on 1-OeI N75 at positions 44, 68, 70, 75 and 77, as described in Arnould et al, J. MoI. Biol., 2006, 355, 443-458 and International PCT Applications WO 2006/097784, WO 2006/097853. Mutants able to cleave the IOCTG P target (cctgacgtcgt_P; SEQ ID NO: 93) were obtained by mutagenesis on I- OeI N75 and D75 at positions 28, 30, 32, 33, 38, 40 and 70, as described in Smith et al, Nucleic Acids Res., 2006, 34, el 49). Thus combining such pairs of mutants would allow for the cleavage of the B2M11.3 target.

Both sets of proteins are mutated at position 70. However, it was previously demonstrated that two separable functional domains exist in I- OeI (Smith et ah, Nucleic Acids Res., 2006, 34, el49). That implies that this position has little impact on the specificity in base 10 to 8 of the target. Therefore, to check whether combined mutants could cleave the B2M11.3 target, mutations at positions 44, 68, 70, 75 and 77 from proteins cleaving 5TTT P (caaaactttgt P; SEQ ID NO: 95) were combined with the 28, 30, 32, 33, 38, 40 mutations from proteins cleaving 10CTG_P (cctgacgtcgt P; SEQ ID NO: 93). 1) Material and Methods l-Crel mutants cleaving IOCTG P or 5TTT P were identified as described previously in Smith et al, Nucleic Acids Res., 2006, 34, el49, and Arnould et ah, J. MoI. Biol., 2006, 355, 443-458; International PCT Applications WO 2006/097784, WO 2006/097853, respectively for the 10CTG_P and 5TTT P targets.

In order to generate l-Crel derived coding sequence containing mutations from both series, separate overlapping PCR reactions were carried out that amplify the 5' end (aa positions 1-43) or the 3' end (positions 39-167) of the l-Crel coding sequence. For both the 5' and 3' end, PCR amplification is carried out using GaIlOF (5'-gcaactttagtgctgacacatacagg-3': SEQ ID NO: 99) or GaIlOR (5'- acaaccttgattggagacttgacc-3': SEQ ID NO: 100) primers specific to the vector (pCLS0542, Figure 13) and primers specific to the 1-OeI coding sequence for amino acids 39-43 (assF 5'-ctannnttgaccttt-3'(SEQ ID NO: 101) or assR 5'-aaaggtcaannntag- 3'(SEQ ID NO: 102)) where nnn code for residue 40. The PCR fragments resulting from the amplification reaction realized with the same primers and with the same coding sequence for residue 40 were pooled. Then, each pool of PCR fragments resulting from the reaction with primers GaIlOF and assR or assF and GaIlOR was mixed in an equimolar ratio. Finally, approximately 25ng of each final pool of the two overlapping PCR fragments and 75ng of vector DNA, a kanamycin resistant yeast expression vector (pCLS1107, Figure 14), linearized by digestion with DraIII and NgoMIV were used to transform the yeast Saccharomyces cerevisiae strain strain FYC2-6A (MATα, trplΔ63, leu2Δl, his3Δ200) using a high efficiency LiAc transformation protocol (Gietz and Woods, methods Enzymol., 2002, 350, 87-96). An intact coding sequence containing both groups of mutations is generated by in vivo homologous recombination in yeast. 2) Results

1-OeI combinatorial mutants were constructed by associating mutations at positions 44, 68, 70, 75 and 77 with the 28, 30, 33, 38 and 40 mutations on the I-Crel N75 or D75 scaffold, resulting in a library of complexity 1600. Examples of combinatorial mutants are displayed on Table III. This library was transformed into yeast and 3348 clones (2.1 times the diversity) were screened for cleavage against B2M11.3 DNA target (tctgactttgt P; SEQ ID NO: 97). One positive clone was found, which after sequencing and validation by secondary screening turned out to be correspond to a novel endonuclease (see Table III). Positive is shown in Figure 9. Table III: Cleavage of the B2M11.3 target by the combinatorial mutants*

* Only 240 out of the 1600 combinations are displayed).

+ indicates cleavage of the B2M11.3 target by the combinatorial mutant.

Example 5: Making of meganucleases cleaving B2M11 by coexpression of meganucleases cleaving B2M11.2 assembly with proteins cleaving B2M11.3

1-OeI mutants able to cleave each of the palindromic B2M11 derived targets (B2M11.2 and B2M11.3) were identified in examples 2, 3 and 4. Pairs of such mutants (one cutting B2M1 1.2 and one cutting B2M11.3) were co-expressed in yeast Upon coexpression, there should be three active molecular species, two homodimers, and one heterodimer. It was assayed whether the heterodimers that should be formed cut the B2Ml l target. 1) Material and Methods a) Cloning of optimized mutants in leucine vector, in B2M11 target yeast

The yeast strain FYBL2-7B (MAT a, ura3Δ851, trplΔ63, leu2Δl, lys2Δ202) containing the B2M11 target into yeast reporter vector (pCLS1055, Figure 12) is transformed with optimised mutants cutting B2M1 1.2 target that were cloned in leucine vector (pCLS0542, Figure 13), using a high efficiency LiAc transformation protocol. Mutant-target yeasts are used as target for mating assays as described in examples 2 and 4, against the mutant cutting B2M1 1.3, in kanamycin vector (pCLS1107). b) Mating of meganucleases coexpressing clones and screening in yeast Mating was performed using a colony gridder (QpixII, Genetix).

Mutants were gridded on nylon filters covering YPD plates, using a low gridding density (about 4 spots/cm²). A second gridding process was performed on the same filters to spot a second layer consisting of different reporter-harbouring yeast strains for each mutant-target. Membranes were placed on solid agar YPD rich medium, and incubated at 30°C for one night, to allow mating. Next, filters were transferred to synthetic medium, lacking leucine and tryptophan, adding G418, with galactose (1 %) as a carbon source, and incubated for five days at 37 °C, to select for diploids carrying the expression and target vectors. After 5 days, filters were placed on solid agarose medium with 0.02 % X-GaI in 0.5 M sodium phosphate buffer, pH 7.0, 0.1 % SDS, 6 % dimethyl formamide (DMF), 7mM β-mercaptoethanol, 1% agarose, and incubated at 37 °C, to monitor β-galactosidase activity. Results were analyzed by scanning and quantification was performed using appropriate software 2) Results:

Coexpression of mutants cleaving the B2M1 1.2 and B2M11.3 resulted in the cleavage of the B2M11 target in most cases (Figure 10). Functional combinations are summarized in Table IV. Table IV: Combinations that resulted in cleavage of B2M11 target.

+ indicates that the heterodimeric mutant is cleaving the B2M 11 target

Example 6: Making of meganucleases cleaving B2M11 with higher efficacy by random mutagenesis of meganuclease cleaving B2M11.3 and co-expression with proteins cleaving B2M11.2

1-OeI mutants able to cleave the palindromic B2M11 target were identified by co-expression of mutants cleaving the palindromic B2M11.2 and B2M1 1.3 targets (Example 5). However, efficiency and number of positive combinations able to cleave B2M11 were minimal. Therefore, the protein cleaving B2M11.3 was mutagenized and variants cleaving B2M11 with better efficiency, when combined to optimized mutants for B2M11.2, were screened. According to the structure of the l-Crel protein bound to its target, there is no contact between the residues used for the first combinatorial approach (28, 30, 32, 33, 38 and 40 vs 44, 68, 70, 75 and 77) in the l-Crel protein (Chevalier et al., Nat. Struct. Biol., 2001, 8, 312-316; Chevalier B.S. and Stoddard B.L., Nucleic Acids Res., 2001, 29, 3757-3754; Chevalier et al, J. MoI. Biol., 2003, 329, 253-269). Thus, it is difficult to rationally choose a set of positions to mutagenize, and mutagenesis was done on the C-terminal part of the protein (83 last amino acids) or on the whole protein. 1) Material and Methods

A random mutagenesis library was created on the chosen mutant, by PCR using Mn²⁺ or derivatives of dNTPs as 8-oxo-dGTP and dPTP in two-step PCR process as described in the protocol from JENA BIOSCIENCE GmbH in JBS dNTP- Mutageneis kit. Primers used are: preATGCreFor (5'- gcataaattactatacttctatagacacgcaaacacaaatacacagcggccttgccacc-3'; SEQ ID NO: 103) and ICrelpostRev (5'-ggctcgaggagctcgtctagaggatcgctcgagttatcagtcggccgc-3'; SEQ ID NO: 104). The new libraries were cloned in vivo in the yeast in the linearized kanamycin vector harbouring a galactose inducible promoter, a KanR as selectable marker and a 2 micron origin of replication. Positives resulting clones were verified by sequencing (MILLEGEN).

Pools of mutants were amplified by PCR reaction using these primers common for leucine vector (pCLS0542, Figure 13) and kanamycin vector (pCLSl 107, Figure 14). Approximately 75ng of PCR fragment and 75ng of vector DNA (pCLS 1107) linearized by digestion with DraIII and NgoMIV are used to transform the yeast Saccharomyces cerevisiae strain FYC2-6A (M ATa, trplΔ63, leu2Δl, his3Δ200) using a high efficiency LiAc transformation protocol. A library of intact coding sequence for the l-Crel mutant is generated by in vivo homologous recombination in yeast.

Mutant-target yeasts are prepared and used as target for mating assays as described in example 5. 2) Results:

The mutants cleaving B2M11.3 (1-OeI

28K30Q32S33G38C40S44Q68N70S75N77R also called KQSGCS/QNSNR according to nomenclature of Table III) was randomly mutagenized and transformed into yeast. 6696 transformed clones were then mated with a yeast strain that (i) contains the B2M11 target in a reporter plasmid (ii) expresses a optimized variant cleaving the B2M11.2 target, chosen among those described in example 5. Four such strains were used, expressing either the l-Crel

28K30N32A33H38Q40S44A68Y70S75Y77K/132V mutant, the 1-OeI 28K30N32G33H38Q40S44A68Y70S75Y77K/2I96R105A mutant, the

28K30N32G33H38Q40S44A68Y70S75Y77K/120G mutant, or the

28K30N32A33H38Q40S44A68Y70S75Y77K/2Y53R66C mutant (see Table V). One hundred and one clones were found to trigger cleavage of the B2M11 target when mated with such yeast strain. In a control experiment, none of these clones was found to trigger cleavage of B2M11 without coexpression of the KQSGCS/QNSNR protein. It was concluded that 101 positives were containing proteins able to cleave B2M11 when forming heterodimers with KQSGCS/QNSNR. Examples of such heterodimeric mutants are listed in Table V. Examples of positives are shown on Figure 11. Table V : Combinations that resulted in cleavage of B2M11 target

Optimized Mutant Optimized Mutant B2M11.3* B2M11

B2M11.2* target (SEQ ID NO: 37 to 77) (SEQ ID NO: 25 to 28) cleavage

I-Crel 28K30Q33G38C40S44Q68N70S77R/19S72F +

I-Crel 28K30Q33G38C40S44Q68N70S77R/31 L83Q87L +

I-Crel 28K30Q33G38C40S44Q68N70S77R/43L117G +

I-Crel 28K30Q33G38C40S44Q68N70S77R/49A +

I-Crel 28K30Q33G38C40S44Q68N70S77R/50R107R +

I-Crel 28K30Q33G38C40S44Q68N70S77R/54L +

I-Crel 28K30Q33G38C40S44Q68N70S77R/56E +

I-Crel 28K30Q33G38C40S44Q68N70S77R/57N +

I-Crel

I-Crel 28K30Q33G38C40S44Q68N70S77R/59A60E163L + 32G33H44A68Y70S75Y77

I-Crel 28K30Q33G38C40S44Q68N70S77R/60G100R155Q165T +

K

I-Crel 28K30Q33G38C40S44Q68N70S77R/60N + /2I96R105A

I-Crel 28K30Q33G38C40S44Q68N70S77R/64A +

I-Crel 28K30Q33G38C40S44Q68N70S77R/64D69G +

Or

I-Crel 28K30Q33G38C40S44Q68N70S77R/69E82E +

I-Crel 28K30Q33G38C40S44Q68N70S77R/69G +

I-Crel

I-Crel 28K30Q33G38C40S44Q68N70S77R/72P154G + 32A33H44A68Y70S75Y77

I-Crel 28K30Q33G38C40S44Q68N70S77R/73I +

K

I-Crel 28K30Q33G38C40S44Q68N70S77R/73I156N + /132V

I-Crel 28K30Q33G38C40S44Q68N70S77R/103S + K30Q33G38C40S44Q68N70S77R/103S147N +

Or I-Crel 28

I-Crel 28K30Q33G38C40S44Q68N70S77R/105A +

I-Crel 28K30Q33G38C40S44Q68N70S77R/110D +

I-Crel

I-Crel 28K30Q33G38C40S44Q68N70S77R/110G153V + 32A33H44A68Y70S75Y77

I-Crel 28K30Q33G38C40S44Q68N70S77R/111 L +

K

I-Crel 28K30Q33G38C40S44Q68N70S77R/142R161 P + /2Y53R66C

I-Crel 28K30Q33G38C40S44Q68N70S77R/153G +

I-Crel 28K30Q33G38C40S44Q68N70S77R/153V +

Or

I-Crel 28K30Q33G38C40S44Q68N70S77R/156N +

I-Crel 28K30Q33G38C40S44Q68N70S77R/156R +

I-Crel

I-Crel 28K30Q33G38C40S44Q68N70S77R/157V + 32G33H44A68Y70S75Y77

I-Crel 28K30Q33G38C40S44Q68N70S77R/158N +

K

I-Crel 28K30Q33G38C40S44Q68N70S77R/80G94Y + /120G

I-Crel 28K30Q33G38C40S44Q68N70S77R/81 T83 A117G +

I-Crel 28K30Q33G38C40S44Q68N70S77R/81 V159Q +

I-Crel 28K30Q33G38C40S44Q68N70S77R/82E107R +

I-Crel 28K30Q33G38C40S44Q68N70S77R/85R +

I-Crel 28K30Q33G38C40S44Q68N70S77R/87L +

I-Crel 28K30Q33G38C40S44Q68N70S77R/92L135P142R164G165P +

I-Crel 28K30Q33G38C40S44Q68N70S77R/96R +

I-Crel 28K30Q33G38C40S44Q 68Y70S77R/72T140M +

I-Crel 28K30Q33G38S40S44Q68N70S77R +

. +: indicates that the heterodimeric variant is cleaving the B2M11 target, Optimized mutations are in bold

Example 7: Making of meganucleases cleaving B2M11.2 in an extrachromosomic model in CHO cells with high efficacy by random mutagenesis of meganucleases cleaving B2M11.3 and co-expression with proteins cleaving B2M11.

1-OeI mutants able to cleave the palindromic B2M11 target with a better efficiency have been identified in yeast by co-expression of mutants- optimized or not- cleaving palindromic B2M11.2 and B2M11.3 targets (Example 5). However, functional heterodimer in CHO cell are interesting and efficiency and number of positive combinations able to cleave B2M1 1 in mammalian cell cells using an extrachromosomal assay could be different than in yeast cell. Therefore the best proteins cleaving B2M11.2 were mutagenized as in example 5, and variants cleaving with good efficiency B2M11 when combined to optimized mutants for B2M11.3 were screened. According to the structure of the I- OeI protein bound to its target, there is no contact between the residues used for the first combinatorial approach (28, 30, 32, 33, 38 and 40 vs 44, 68, 70, 75 and 77) in the I-Crel protein (Chevalier et al, Nat. Struct. Biol., 2001, 8, 312-316; Chevalier B.S. and Stoddard B.L., Nucleic Acids Res., 2001, 29, 3757-3754; Chevalier et al, J. MoI. Biol., 2003, 329, 253-269). Thus, it is difficult to rationally choose a set of positions to mutagenize, and mutagenesis was done on the C-terminal part of the protein (83 last amino acids) or on the whole protein. 1) Material and Methods a) Construction of libraries by random mutagenesis

Random mutagenesis libraries on a pool of chosen mutants were created by PCR using Mn²⁺ or derivatives of dNTPs as 8-oxo-dGTP and dPTP in two- step PCR process as described in the protocol from JENA BIOSCIENCE GmbH in JBS dNTP-Mutageneis kit. Primers used are attBl-ICreIFor (5'- ggggacaagtttgtacaaaaaagcaggcttcgaaggagatagaaccatggccaataccaaatataacaaagagttcc-3¹: (SEQ ID NO: 120) and attB2-ICreIRev (5'- ggggaccactttgtacaagaaagctgggtttagtcggccgccggggaggatttcttcttctcgc-3': SEQ ID NO: 121). PCR products obtained were cloned in vitro in CHO Gateway expression vector pCDNA6.2 from INVITROGEN (pCLS1069, Figure 16). In parallel, chosen mutants used for libraries were cloned in the same way in this vector. Cloned mutants and positives resulting clones of libraries were verified by sequencing (MILLEGEN). b) Construction of B2M11 target in a vector for screening in CHO cells From yeast target vector (as example 1) the B2M11 target was amplified by two steps PCR using primers MIs (5'-aaaaagcaggctgattggcatacaagtt-3': SEQ ID NO: 122) and M2s (S'-agaaagctgggtgattgacagacgattg-S': SEQ ID NO: 123) followed by attBladapbis (5'-ggggacaagtttgtacaaaaaagca-3': SEQ ID NO: 124) and attB2adapbis (5'- ggggaccactttgtacaagaaagct-3': SEQ ID NO: 125). Primers are from PROLIGO. Final PCR was cloned using the Gateway protocol (INVITROGEN) into CHO reporter vector (pCLS1058, Figure 17). Cloned target was verified by sequencing (MILLEGEN). c) Extrachromosomal assay in mammalian cells

CHO cells were transfected with Polyfect transfection reagent according to the supplier's protocol (QIAGEN). 72 hours after transfection, culture medium was removed and 150 μl of lysis/revelation buffer added for β-galactosidase liquid assay (typically, 1 liter of buffer contained 100 ml of lysis buffer (Tris-HCl 10 mM pH7.5, NaCl 150 mM, Triton XlOO 0.1%, BSA 0.1 mg/ml, protease inhibitors), 10 ml of Mg IOOX buffer (MgCl₂ 100 mM, β-mercaptoethanol 35 %), 110ml ONPG 8 mg/ml and 780 ml of sodium phosphate 0.1 M pH7.5). After incubation at 37 °C, optical density (OD) was measured at 420 nm. The entire process was performed on an automated Velocity 1 1 BioCel platform. Per assay, 150 ng of target vector was cotransfected with 12.5 ng of each one of both mutants (12.5 ng of mutant cleaving palindromic B2M11.2 target and 12.5 ng of mutant cleaving palindromic B2M11.3 target) 2) Results The optimized mutants cleaving B2M11.2 (I-Oel

32G33H44A68Y70S75Y77K/120G, 32A33H44A68Y70S75Y77K/2Y53R66C,

32G33H44A68Y70S75Y77K/2I96R105A and 32A33H44A68Y70S75Y77K/132V as described into Table V) were randomly mutagenized and transformed into Gateway vector (Figure 16). DNA plasmid of 1920 transformed clones were purified and then cotransfected with the CHO B2M11 target vector and an optimized variant cleaving the B2M1 1.3 target, chosen among those described in example 6. Cotransfection of the transformed clones with the CHO B2M11 target vector and the initial mutant (30Q33G38C68N70S77R) cleaving B2M11.3 was included for comparison. Sixty clones were found to trigger cleavage of the B2M11 target when co-transfected with an optimized variant cleaving B2M11.3 target.

In a control experiment, none of these clones was found to trigger cleavage of B2M11 without cotransfection of a (optimized or not) variant cleaving the B2M1 1.3 target. It was thus concluded that 60 positives were containing proteins able to cleave B2M1 1 when forming heterodimers with optimized variant cleaving the B2M11.3 target. Examples of such heterodimeric mutants derived from two optimized variants cleaving the B2M11.3 target (30Q33G38C68N70S77R/43L115T117G and 30Q33G38C68N70S77R/110D) are listed in Table VI. Table VI: Combinations that resulted in cleavage* of the B2M11 target in CHO cells.

*(-):< 0.25. (±): 0.25 ≤ < 0,5. (+) : 0,5 ≤ <1,2. (-H-): ≥ 1,2. Values (absorbance unit) correspond to average of experimental results of the extrachromosomal assay in CHO cells Example 8: Making of meganucleases cleaving B2M18.

Two news series of palindromic targets, B2M18.3 and B2M18.4 were derived from B2M18 and B2M20.3 and B2M20.4 from B2M20 (Figures 18 and 21). Since B2M18.3, B2M18.4, B2M20.3 and B2M20.4 are palindromic, they should be cleaved by homodimeric proteins. First, proteins able to cleave the B2M18.4 sequence as homodimers were designed (example 8), and then proteins able to cleave the B2M20.4 sequences as homodimers were designed (example 9).

This example shows that l-Crel variants can cleave the B2M18.4 DNA target sequence derived from the right part of the B2M18 target in a palindromic form (Figure 18). All target sequences described in this example are 22 bp palindromic sequences. Therefore, they will be described only by the first 11 nucleotides, followed by the suffix P. For example, B2M18.4 will be called TTAACTATCGT P (SEQ ID NO: 113).

B2M18.4 is similar to 5ATC_P in positions ±1, ±2, ±3, ±4, ±5, ±8 and ±9 and to 10TAA_P in positions ±1, ±2, ±3, ±4, ±8, ±9 and ±10. It was hypothesized that positions ±6, ±7 and ±11 would have little effect on the binding and cleavage activity. Mutants able to cleave 5ATC_P target (CAAAACATCGT_P) were previously obtained by mutagenesis on I-Crel N75 at positions 44, 68, 70, 75 and 77, as described in Arnould et al, J. MoI. Biol., 2006, 355, 443-458 and International PCT Applications WO 2006/097784, WO 2006/097853. Mutants able to cleave the 5ATC_P target (CTAAACGTCGT P) were obtained by mutagenesis on 1-OeI N75 and D75 at positions 28, 30, 32, 33, 38, 40 and 70 as described in Smith et al., Nucleic Acids Res., 2006, 34, el 49 . Thus combining such pairs of mutants would allow for the cleavage of the B2M18.4 target. Both sets of proteins are mutated at position 70. However, the existence of two separable functional subdomains was hypothesized. That implies that this position has little impact on the specificity in base 10 to 8 of the target. Therefore, mutations at positions 44, 68, 70, 75 and 77 from proteins cleaving 5ATC P (CAAAACATCGT_P) were combined with the 28, 30, 32, 33, 38, 40 mutations from proteins cleaving IOTAA P (CTAAACGTCGT_P) to check whether combined mutants could cleave the B2M18.4 target. 1) Material and Methods

The experimental procedure is as described in example 2.

2) Results l-Crel combinatorial mutants were constructed by associating mutations at positions 44, 68, 70, 75 and 77 with the 28, 30, 33, 38 and 40 mutations on the 1-OeI N75 or D75 scaffold, resulting in a library of complexity 1600. Examples of combinatorial mutants are displayed on Table VII. This library was transformed into yeast and 3348 clones (2.1 times the diversity) were screened for cleavage against B2M18.4 DNA target (TTAACTATCGT P). 59 positives clones were found, which after sequencing and validation by secondary screening turned out to be correspond to 18 novel endonucleases (SEQ ID NO: 136 to 152 and 179; see Table VII). Positives are shown in Figure 19. One positive clone presents an additional mutation in position 153. The sequence of this clone is KNRTQS/ QYSRQ - 153G.

Table VII : Cleavage of the B2M18.4 target by the combinatorial mutants*

* Only 138 out of the 1600 combinations theoretically present in the combinatorial library are displayed. + indicates that the combinatorial mutant was found among the identified positives. Example 9: Making of meganucleases cleaving B2M20

This example shows that 1-OeI variants can cleave the B2M20.4 DNA target sequence derived from the right part of the B2M20.1 target in a palindromic form (Figure 19). All target sequences described in this example are 22 bp palindromic sequences. Therefore, they will be described only by the first 11 nucleotides, followed by the suffix P. For example, B2M20.4 will be called TTACATGTCGT P: SEQ ID NO: 119). B2M18.4 is similar to 5GTC_P in positions ±1, ±2, ±3, ±4, ±5 and ±7 and to 10TAC_P in positions ±1, ±2, ±3, ±4, ±5, ±7,±8, ±9 and ±10. It was hypothesized that positions ±6 and ±11 would have little effect on the binding and cleavage activity. Mutants able to cleave 5GTCJP target (CAAAACGTCGT_P) were previously obtained by directed mutagenesis on l-Crel at positions 70 and 75 as described in Arnould et al., J. MoI. Biol., 2006, 355, 443-458 and International PCT Applications WO 2006/097784, WO 2006/097853. Mutants able to cleave the IOTAC P target (CTACACGTCGTJP) were obtained by mutagenesis on 1-OeI N75 and D75 at positions 28, 30, 32, 33, 38, 40 and 70, as described in Smith et al., Nucleic Acids Res., 2006, 34, el49. Thus combining such pairs of mutants would allow for the cleavage of the B2M20.4 target.

Both sets of proteins are mutated at position 70. However, the existence of two separable functional subdomains was hypothesized. That implies that this position has little impact on the specificity in base 10 to 8 of the target. Therefore, mutations at positions 70 and 75 from proteins cleaving 5GTC P (CAAAACGTCGTJP) were combined with the 28, 30, 32, 33, 38, 40 mutations from proteins cleaving IOTAC P (CTACACGTCGT P) to check whether combined mutants could cleave the B2M20.4 target. 1) Material and Methods

The experimental procedure is as described in example 2. 2) Results

1-Crel combinatorial mutants were constructed by associating mutations at positions 70 and 75 with the 28, 30, 33, 38 and 40 mutations on the I- OeI N75 or D75 scaffold, resulting in a library of complexity 1536. Examples of combinatorial mutants are displayed on Table VIII. This library was transformed into yeast and 6696 clones (4.4 times the diversity) were screened for cleavage against B2M20.4 DNA target (TTACATGTCGT P). 196 positives clones were found, which after sequencing and validation by secondary screening turned out to be correspond to 164 novel endonucleases (see Table VIII showing SEQ ID NO: 153 to 178). Positive is shown in Figure 21. 12 clones present additional mutations (56G, 82R, 147 A or 161F), as in example 8. Table VIII : Cleavage of the B2M20.4 target by the combinatorial mutants*

* Only 230 out of the 1536 combinations theoretically present in the combinatorial library are displayed. + indicates that the combinatorial mutant was found among the identified positives.

Claims

1 °) An l-Crel variant, characterized in that at least one of the two I- Crel monomers has at least two substitutions, one in each of the two functional subdomains of the LAGLIDADG core domain situated from positions 26 to 40 and 44 to 77 of l-Crel, said variant being able to cleave a DNA target sequence from the beta- 2 microglobulin gene, and being obtainable by a method comprising at least the steps of:

(b) constructing a second series of l-Crέl variants having at least one substitution in a second functional subdomain of the LAGLIDADG core domain situated from positions 44 to 77 of l-Crel,

(c) selecting and/or screening the variants from the first series of step (a) which are able to cleave a mutant 1-OeI site wherein (i) the nucleotide triplet in positions -10 to -8 of the l-Crel site has been replaced with the nucleotide triplet which is present in position -10 to -8 of said DNA target sequence from the beta-2 microglobulin gene and (ii) the nucleotide triplet in positions +8 to +10 has been replaced with the reverse complementary sequence of the nucleotide triplet which is present in position -10 to -8 of said DNA target sequence from the beta-2 microglobulin gene,

(d) selecting and/or screening the variants from the second series of step (b) which are able to cleave a mutant 1-OeI site wherein (i) the nucleotide triplet in positions -5 to -3 of the 1-OeI site has been replaced with the nucleotide triplet which is present in position -5 to -3 of said DNA target sequence from the beta-2 microglobulin gene and (ii) the nucleotide triplet in positions +3 to +5 has been replaced with the reverse complementary sequence of the nucleotide triplet which is present in position -5 to -3 of said DNA target sequence from the beta-2 microglobulin gene, (e) selecting and/or screening the variants from the first series of step (a) which are able to cleave a mutant l-Crel site wherein (i) the nucleotide triplet in positions +8 to +10 of the I-Oel site has been replaced with the nucleotide triplet which is present in positions +8 to +10 of said DNA target sequence from the beta-2 microglobulin gene and (ii) the nucleotide triplet in positions -10 to -8 has been replaced with the reverse complementary sequence of the nucleotide triplet which is present in position +8 to +10 of said DNA target sequence from the beta-2 microglobulin gene,

(f) selecting and/or screening the variants from the second series of step (b) which are able to cleave a mutant l-Crel site wherein (i) the nucleotide triplet in positions +3 to +5 of the I-Crel site has been replaced with the nucleotide triplet which is present in positions +3 to +5 of said DNA target sequence from the beta-2 microglobulin gene and (ii) the nucleotide triplet in positions -5 to -3 has been replaced with the reverse complementary sequence of the nucleotide triplet which is present in position +3 to +5 of said DNA target sequence from the beta-2 microglobulin gene,

(g) combining in a single variant, the mutation(s) in positions 26 to 40 and 44 to 77 of two variants from step (c) and step (d), to obtain a novel homodimeric l-Crel variant which cleaves a sequence wherein (i) the nucleotide triplet in positions -10 to -8 is identical to the nucleotide triplet which is present in positions -10 to -8 of said DNA target sequence from the beta-2 microglobulin gene, (ii) the nucleotide triplet in positions +8 to +10 is identical to the reverse complementary sequence of the nucleotide triplet which is present in positions -10 to - 8 of said DNA target sequence from the beta-2 microglobulin gene, (iii) the nucleotide triplet in positions -5 to -3 is identical to the nucleotide triplet which is present in positions -5 to -3 of said DNA target sequence from the beta-2 microglobulin gene and (iv) the nucleotide triplet in positions +3 to +5 is identical to the reverse complementary sequence of the nucleotide triplet which is present in positions -5 to -3 of said DNA target sequence from the beta-2 microglobulin gene, and/or

(h) combining in a single variant, the mutation(s) in positions 26 to 40 and 44 to 77 of two variants from step (e) and step (f), to obtain a novel homodimeric 1-OeI variant which cleaves a sequence wherein (i) the nucleotide triplet in positions +3 to +5 is identical to the nucleotide triplet which is present in positions +3 to +5 of said DNA target sequence from the beta-2 microglobulin gene, (ii) the nucleotide triplet in positions -5 to -3 is identical to the reverse complementary sequence of the nucleotide triplet which is present in positions +3 to +5 of said DNA target sequence from the beta-2 microglobulin gene, (iii) the nucleotide triplet in positions +8 to +10 of the 1-OeI site has been replaced with the nucleotide triplet which is present in positions +8 to +10 of said DNA target sequence from the beta-2 microglobulin gene and (iv) the nucleotide triplet in positions -10 to -8 is identical to the reverse complementary sequence of the nucleotide triplet in positions +8 to +10 of said DNA target sequence from the beta-2 microglobulin gene,

(i) combining the variants obtained in steps (g) and (h) to form heterodimers, and (j) selecting and/or screening the heterodimers from step (i) which are able to cleave said DNA target sequence from the beta-2 microglobulin gene.

2°) The variant of claim 1, wherein said substitution(s) in the subdomain situated from positions 44 to 77 of l-Crel are in positions 44, 68, 70, 75 and/or 77. 3°) The variant of claim 1, wherein said substitution(s) in the subdomain situated from positions 26 to 40 of I-Crel are in positions 26, 28, 30, 32, 33, 38 and/or 40.

4°) The variant of anyone of claims 1 to 3, which comprises one or more substitutions in positions 137 to 143 of 1-OeI that modify the specificity of the variant towards the nucleotide in positions ± 1 to 2, ± 6 to 7 and/or ± 11 to 12 of the I- OeI site.

5°) The variant of anyone of claims 1 to 4, which comprises one or more substitutions on the entire 1-OeI sequence that improve the binding and/or the cleavage properties of the variant towards said DNA target sequence from the beta-2 microglobulin gene.

6°) The variant of anyone of claims 1 to 5, wherein said substitutions are replacement of the initial amino acids with amino acids selected in the group consisting of A, D, E, G, H, K, N, P, Q, R, S, T , Y, C, W, L and V.

7°) The variant of anyone of claims 1 to 6, which is an heterodimer, resulting from the association of a first and a second monomer having different mutations in positions 26 to 40 and 44 to 77 of I- OeI, said heterodimer being able to cleave a non-palindromic DNA target sequence from the beta-2 microglobulin gene. 8°) The variant of claim 7, wherein the first and the second monomer, respectively, have at least the following substitutions:

- Y33R, Q38A, Q44D, R68A, R70S, D75K, I77R and K28R, Y33A, Q38Y, S40Q, Q44A, R68Y, R70S, D75Y, I77K, - S32T, Y33T, Q44T, R68Y, R70S, D75Y, I77V and Y33R, Q38A,

Q44N, R68Q, R70S, D75S, I77V,

-S32G, Y33H, Q44A, R68Y, R70S, D75Y, I77K or S32A, Y33H, Q44A, R68Y, R70S, D75Y, I77K and N30Q, Y33G, Q38C, R68N, R70S, D75N, I77R, - S32G, Y33H, Q44A, R68Y, R70S, D75Y, I77K and S32T, Q38S,

Q44K, R70S, I77A,

- S32T, Y33T, Q44K, R68E, R70S, I77R and N30A, Y33T, Q44N, R68K, R70S, D75H, I77F,

- S32R, Y33D, Q44A, R70S, D75E, I77R and N30D, Y33R, Q44K, R68Y, R70S, D75N, I77Q,

- S32T, Q38W, Q44A, R70S, D75R, I77Y and Y33H, S40Q, Q44N, R70S, D75R, I77Y,

- Y33H, Q38G, Q44N, R68Y, R70S, D75R, I77V and N30A, Y33T, Q44N, R68Y, R70S, D75R, I77V, - Y33T, S40N, Q44T, R68Y, R70S, D75R, I77V and K28R, Y33A,

Q38Y, S40Q, Q44A, R68S, R70S, D75S, I77R, and

- K28T, Y33R, S40R, Q44T, R70S, D75Y and N30D, Y33R, Q44N, R68Y, R70S, D75Y, I77Q.

9°) The variant of claim 8, wherein the first monomer is of any of the sequences SEQ ID NO: 24 to 28, 126 to 134 and the second monomer is of any of the sequences SEQ ID NO: 37 to 77, 135 to 179.

10°) The variant of anyone of claims 1 to 9, wherein said DNA target sequence is from the human beta-2 microglobulin gene.

11°) The variant of claim 10 wherein said DNA target is selected from the group consisting of the sequences SEQ ID NO: 82 to 91. 12°) The variant of anyone of claims 7 to 11, which is an obligate heterodimer, wherein the first and the second monomer, respectively, further comprises the D137R mutation and the R51D mutation.

13°) The variant of anyone of claims 7 to 12, which is an obligate heterodimer, wherein the first monomer further comprises the K7R, E8R, E61R,

K96R and L97F or K7R, E8R, F54W, E61R, K96R and L97F mutations and the second monomer further comprises the K7E, F54G, L58M and K96E or K7E, F54G,

K57M and K96E mutations.

14°) A single-chain chimeric meganuclease comprising two monomers or core domains of one or two variant(s) of anyone of claims 1 to 13, or a combination of both

15°) The single-chain meganuclease of claim 14 which comprises the first and the second monomer as defined in anyone of claims 7, 8, 12 and 13, connected by a peptidic linker. 16°) A polynucleotide fragment encoding the variant of anyone of claims 1 to 13 or the single-chain chimeric meganuclease of claim 14 or claim 15.

17°) An expression vector comprising at least one polynucleotide fragment of claim 16.

18°) The expression of claim 17, which comprises two different polynucleotide fragments, each encoding one of the monomers of an heterodimeric variant of anyone of claims 7, 8, 12 and 13.

19°) A vector, which includes a targeting construct comprising a sequence to be introduced flanked by sequences sharing homologies with the regions surrounding the genomic DNA cleavage site of the beta-2 microglobulin gene, as defined in anyone of claims 1, 10 and 11.

20°) The vector of claim 17 or claim 18, which includes a targeting construct comprising a sequence to be introduced flanked by sequences sharing homologies with the regions surrounding the genomic DNA cleavage site present in the beta-2 microglobulin gene, as defined in anyone of claims 1, 10 and 11. 21°) The vector of claim 19 or claim 20, wherein said sequence to be introduced is a sequence which inactivates the beta-2 microglobulin gene. 22°) The vector of claim 21, wherein the sequence which inactivates the beta-2 microglobulin gene comprises in the 5' to 3' orientation : a first transcription termination sequence and a marker cassette including a promoter, the marker open reading frame and a second transcription termination sequence, and said sequence interrupts the transcription of the coding sequence.

23°) The vector of claim 22, wherein said genomic DNA cleavage site is SEQ ID NO: 82 to 90.

24°) The vector of claim 21 , wherein the sequence which inactivates the beta-2 microglobulin gene comprises a marker gene, so as to allow the disruption of the beta-2 microglobulin coding sequence.

25°) The vector of claim 24, wherein said sequence which inactivates the beta-2 microglobulin gene comprises an exogenous gene of interest, so as to allow the replacement of the beta-2 microglobulin gene by the exogenous gene.

26°) The vector of claim 24 or claim 25, wherein said genomic DNA cleavage site is SEQ ID NO: 89 or 90.

27°) The vector of anyone of claims 19 to 26, wherein said sequence sharing homologies with the regions surrounding the genomic DNA cleavage site present in the beta-2 microglobulin gene is a fragment of the human beta-2 microglobulin gene comprising the sequence selected from positions: 1164 to 1363, 2795 to 2994, 2803 to 3002, 3074 to 3273, 3275 to 3474, 3284 to 3483, 3387 to 3586, 4099 to 4298, 4765 to 4944 and 6451 to 6650.

28°) The vector of anyone of claims 24 to 26, wherein said sequence sharing homologies with the regions surrounding the genomic DNA cleavage site present in the beta-2 microglobulin gene is a fragment of the beta-2 microglobulin gene comprising sequences upstream and downstream of the cleavage site, so as to allow the deletion of coding sequences flanking immediately the cleavage site.

29°) A composition comprising at least one variant of anyone of claims 1 to 13, one single-chain chimeric meganuclease of claim 14 or claim 15, and/or at least one expression vector of anyone of claims 17 to 28. 30°) The composition of claim 29, which comprises a targeting

DNA construct comprising a sequence which inactivates the beta-2 microglobulin gene, flanked by sequences sharing homologies with the region surrounding the genomic DNA target cleavage site of said variant, as defined in anyone of claims 21 to 28.

31°) The use of at least one variant of anyone of claims 1 to 13, one single-chain chimeric meganuclease of claim 14 or claim 15, and/or one expression vector of anyone of claims 17 to 28, for the preparation of a medicament for preventing, improving or curing xenograft rejection during cell transplantation from a donor individual into a recipient individual in need thereof.

32°) The use of claim 31, wherein said transplantation is kidney, pancreas, muscle or heart cell transplantation. 33°) The use of at least one variant of anyone of claims 1 to 13, one single-chain chimeric meganuclease of claim 14 or claim 15, and/or one expression vector of anyone of claims 17 to 28, for the preparation of a medicament for preventing, improving or curing a pathological condition associated with a fibrillar conformation of the beta-2 microglobulin in an individual in need thereof. 34°) A host cell which is modified by a polynucleotide of claim 16 or a vector of anyone of claims 17 to 28.

35°) A non-human transgenic animal comprising one or two polynucleotide fragments as defined in claim 16 or claim 18.

36°) A transgenic plant comprising one or two polynucleotide fragments as defined in claim 16 or claim 18.

37°) Use of at least one variant of anyone of claims 1 to 13, one single-chain chimeric meganuclease of claim 14 or claim 15, one vector according to anyone of claims 17 to 28, for genome engineering, for non-therapeutic purposes.

38°) The use of claim 36, wherein said variant, single-chain chimeric meganuclease, or vector is associated with a targeting DNA construct as defined in anyone of claims 19 to 28.

39°) The use of claim 38, wherein said targeting DNA construct comprises an exogenous gene of interest, and eventually a marker gene, flanked by sequences uptstream an downstream the beta-2 microglobumin locus, to allow replacement of the beta-2 microglobulin gene by the exogenous gene of interest.

40°) The use of anyone of claims 37 to 39, for making a transgenic animal or a recombinant cell line expressing an heterologous protein of interest. 41°) The use of claims 40, wherein said recombinant cell line is a human recombinant cell line.