WO2008102199A1 - Variants de méganucléase clivant une séquence cible d'adn provenant du gène de la bêta-2-microglobuline et utilisations de ceux-ci - Google Patents

Variants de méganucléase clivant une séquence cible d'adn provenant du gène de la bêta-2-microglobuline et utilisations de ceux-ci Download PDF

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WO2008102199A1
WO2008102199A1 PCT/IB2007/001532 IB2007001532W WO2008102199A1 WO 2008102199 A1 WO2008102199 A1 WO 2008102199A1 IB 2007001532 W IB2007001532 W IB 2007001532W WO 2008102199 A1 WO2008102199 A1 WO 2008102199A1
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positions
beta
sequence
variant
crel
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PCT/IB2007/001532
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Sylvain Arnould
André CHOULIKA
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Cellectis
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Priority to PCT/IB2007/001532 priority Critical patent/WO2008102199A1/fr
Priority to AU2008218605A priority patent/AU2008218605A1/en
Priority to JP2009550342A priority patent/JP2010518832A/ja
Priority to PCT/IB2008/001334 priority patent/WO2008102274A2/fr
Priority to EP08751044A priority patent/EP2121036A2/fr
Priority to CA002678709A priority patent/CA2678709A1/fr
Priority to CN200880005471A priority patent/CN101678126A/zh
Publication of WO2008102199A1 publication Critical patent/WO2008102199A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • 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.
  • MHC Major Histocompatibility Complex
  • HLA HLA
  • 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 ⁇ 2-microglobulin, necessary for assembly of MHC I complexes at the cell surface (Zijlstra et al, Nature, 1990, 344, 742-746).
  • knocking-out the ⁇ 2-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).
  • MHC proteins are also major players in graft rejection.
  • disruption of MHC class I proteins would at least partly alleviate graft rejection.
  • studies in mice have provided a more mitigated picture. Whereas hematopoietic stem cells from ⁇ 2m -/- mice are quickly rejected (Bix et al., Nature, 1991, 349, 329-331 ; Liao et al., Science, 1991, 253, 199-202 ; Huang et al, J.
  • 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: 4811-4821). This includes Hemodialysis-Related Amyloidosis (HRA; Gorevic et al, 1986, Proc. Nat. Acad. Sci. 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.
  • beta-2 microglobuline 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.
  • 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 ⁇ 2m) gene (Roller 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 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 reasinstasinsta of alleles in meiosis (Roeder, Genes Dev., 1997, 11, 2600-2621), mating type interconversion in yeast (Haber, Annu. Rev.
  • 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.
  • 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.
  • 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 FoM (Catto et al, Nucleic Acids Res., 2006, 34, 1711-1720). Thus, specificity might be very degenerate, as illustrated by toxicity in mammalian cells and Drosophila (Bibikova et al, Genetics, 2002, 161, 1169-1175 ; Bibikova et al, Science, 2003, 300, 764-.).
  • HEs Homing Endonucleases
  • proteins families Cholier, B. S. and B. L. Stoddard, Nucleic Acids Res., 2001, 29, 3757-3774.
  • 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.
  • 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 monomeric and display two LAGLIDADG motifs, a few ones have only one motif, but dimerize to cleave palindromic or pseudo-palindromic target sequences.
  • 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 l-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, l-Dmol (Silva et al, J.
  • 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., Epub 27 November 2006).
  • 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., Epub 27 November 2006).
  • 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.
  • 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 Inventors have identified a series of DNA targets in the beta-2 microglobulin gene that could be cleaved by l-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.
  • the invention relates to an l-Crel 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 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, 3 I 5 2952-2962; CIiames et al, Nucleic Acids Res., 2005, 33, el78 and Arnould et al, J. MoI. Biol., 2006, 355, 443-458.
  • 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.
  • nucleosides 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.
  • 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 repre- sents 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
  • n represents g, a, t or c.
  • 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 monomeric enzyme comprising the two domains on a single polypeptide.
  • 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.
  • 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.
  • such variants have amino acid variation at positions contacting the DNA target sequence or interacting directly or indirectly with said DNA target.
  • “meganuclease variant with novel specificity” is intended a variant having a pattern of cleaved targets different from that of the parent meganuclease.
  • novel specificity refers 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 in the sequence listing.
  • - by “domain” or “core domain” is intended the "LAGLIDADG hominR endonuclease core domain” which is the characteristic ⁇ ! ⁇ ! ⁇ 2 ⁇ 2 ⁇ 3 ⁇ 4 ⁇ 3 fold of the homing endonucleases of the LAGLIDADG family, corresponding to a sequence of about one hundred amino acid residues.
  • Said domain comprises four beta-strands ( ⁇ i ⁇ 2 ⁇ 3 ⁇ 4 ) 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.
  • the LAGLIDADG homing endonuclease core domain corresponds to the residues 6 to 94.
  • 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.
  • 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.
  • Two different subdomains behave independently and the mutation in one subdomain does not alter the binding and cleavage properties of the other subdomain. Therefore, two subdomains bind 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 (( ⁇ i ⁇ 2 or, ⁇ 3 ⁇ 4) which are connected by a loop or a turn,
  • l-Crel 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 l-Crel homing site and the derived palindromic sequences such as the sequence 5'- L 1 2C-ua -1 oa-9a-8a -7 c-6g-5t-4C -3 g -2 t -1 a +1 c + 2g+3a+4C+ 5 g+6t+7t+8tf9t+i 0 g+iia +12 also called C 1221 (SEQ ID NO :2; figure 5).
  • 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.
  • 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 l-Cre I meganuclease variant occurs, corresponds to the cleavage site on the sense strand of the DNA target.
  • DNA target half-site 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.
  • chimeric DNA target or “hybrid DNA target” is intended the fusion of a different half of two parent meganuclease target sequences.
  • 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).
  • beta-2-microglobulin gene is intended the beta-2- microglobulin gene of a mammal.
  • the human beta-2-microglobulin gene (B2M, 6673bp) is situated from positions 42790977 to 42797649 of the sequence corresponding to accession number NC_000015.
  • 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).
  • DNA target sequence from the beta-2-microglobulin gene 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.
  • vector a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • 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.
  • mammals as well as other vertebrates (e.g., birds, fish and reptiles).
  • mammals e.g., birds, fish and reptiles.
  • 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.
  • said substitution(s) in the subdomain situated from positions 44 to 77 of I-Crel are in positions 44, 68, 70, 75 and/or 77.
  • said substitution ⁇ ) in the subdomain situated from positions 26 to 40 of l-Crel are in positions 26, 28, 30, 32, 33, 38 and/or 40.
  • said variant 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 etal, J. MoI. Biol., 2003, 329, 253-269).
  • additional substitutions may be introduced at positions contacting the phosphate backbone, for example in the final C-terminal loop (positions 137 to 143).
  • said residues are involved in binding and cleavage of said DNA cleavage site.
  • 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 l-Crel site.
  • 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.
  • 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.
  • said variant 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 l-Crel (positions 80 to 163).
  • the variant comprises one or more additional substitutions in positions: 2, 19, 31, 43, 49, 50, 53 ' , 54, 56, 57, 59, 60, 64, 66, 69, 72, 73, 80, 81, 82, 83, 85, 87, 92, 94, 96, 100, 103, 105, 107, 110, 111, 117, 120, 129, 132, 135, 140, 142, 147, 153, 154, 155, 156, 157, 158, 159, 161, 163.
  • the variant may also comprise one or two additional residues inserted at the C-terminus of the l-Crel sequence (positions 164 and 165).
  • 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 wil-type l-Crel
  • SEQ ID NO: 1 or an l-Crel scaffold protein, such as the scaffold of SEQ ID NO: 106 (167 amino acids) having the insertion of an alanine in position 2, the substitutions A42T, D75N, WI lOE, Rl I lQ and the insertion of AAD at the C- terminus (positions 164 to 166) of the l-Crel sequence.
  • variants of the invention may include one or more residues inserted at the NH 2 terminus and/or COOH terminus of the sequence.
  • a tag epipe or polyhistidine sequence
  • 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.
  • 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 l-Crel, 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.
  • said DNA target sequence is from the human beta-2-microglobulin gene (B2M gene).
  • B2M gene human beta-2-microglobulin gene
  • said DNA target sequence is selected from the group consisting of the sequences SEQ ID NO: 82 to 91 ( Figures 4 and 15). 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.
  • the monomers of the variant have at least the following substitutions, respectively for the first and the second monomer:
  • this variant cleaves the B2M20 target which is located at the intron 2- exon 3 junction (figures 4 and 15), and - K28T, Y33R, S40R, Q44T, R70S and D75Y (first monomer), and
  • said variant consist of a first monomer having any of the sequences SEQ ID NO: 24 to 28 and a second monomer having any of the sequences SEQ ID NO: 37 to 77; 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).
  • 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 prevent the formation of functional homodimers from each monomer and allow the formation of a functional heterodimer, able to cleave the genomic DNA target from the beta-2 microglobulin gene, as described in the International PCT Application from CELLECTIS, filed first february 2007.
  • 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.
  • 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
  • the first monomer may have the mutation D137R and the second monomer, the mutation R51D.
  • 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-Cre ⁇ ) or a combination of both.
  • 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.
  • 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, semisynthetic or synthetic nucleic acids.
  • Preferred vectors are those capable of autonomous replication (episomal vector) and/or expression of nucleic acids to which they are linked (expression vectors). Large numbers of suitable vectors are known to those of skill in the art and commercially available.
  • Viral vectors include retrovirus, adenovirus, parvovirus (e. g. adeno- associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e. g., influenza virus), rhabdovirus (e. g., rabies and vesicular stomatitis virus), paramyxovirus (e. g. measles and Sendai), positive strand RNA viruses such as 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.
  • orthomyxovirus e. g., influenza virus
  • rhabdovirus e. g., rabies and vesicular stomatitis virus
  • paramyxovirus e. g. measles and Sendai
  • viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus, for example.
  • retroviruses include: avian leukosis- sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retro viridae: 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.
  • 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 translationai control elements to permit production or synthesis of said variant.
  • 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.
  • 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-O- D-thiogalacto-pyranoside (IPTG) and eukaryotic heat shock promoter which is induced by increased temperature.
  • tissue specific promoters are skeletal muscle creatine kinase, prostate-specific antigen (PSA), ⁇ -antitrypsin protease, human surfactant (SP) A and B proteins, ⁇ -casein and acidic whey protein genes.
  • PSA prostate-specific antigen
  • SP human surfactant
  • a and B proteins ⁇ -casein and acidic whey protein genes.
  • said vector includes a targeting construct comprising sequences sharing homologies with the region surrounding the genomic DNA cleavage site as defined above.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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).
  • 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 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 W 2
  • 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).
  • a meganuclease cleaving B2M18 or B2M20 SEQ ID NO: 90, 91.
  • 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 the targets (SEQ ID NO: 82 to 91) presented in figures 4 and 15.
  • homologous sequences of at least 50 bp, preferably more than 100 bp and more preferably more than 200 bp are used.
  • shared DNA homologies are located in regions flanking upstream and downstream the site of the break and the DNA sequence to be introduced should be located between the two arms.
  • the 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.
  • 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.
  • 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. 2
  • said composition 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.
  • 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).
  • 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).
  • 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 frameshifit mutation.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • said variant, polynucleotide(s), vector are associated with a targeting DNA construct as defined above.
  • the meganuclease 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.
  • the meganuclease 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.
  • the meganuclease 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:
  • step (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,
  • step (c) developping the genomically modified animal precursor cell or embryo of step (b) into a chimeric animal
  • 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:
  • step (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,
  • step (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.
  • said targeting DNA construct is inserted in a vector.
  • 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:
  • step (b) developping the genomically modified animal precursor cell or embryo of step (a) into a chimeric animal
  • step (c) deriving a transgenic animal from a chimeric animal of step (b).
  • 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
  • step(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.
  • the cells are pluripotent precursor cells such as embryo- derived stem (ES) cells, which are well-kown in the art.
  • 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.
  • 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 ceils
  • 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.
  • the meganuclease (polypeptide) is associated with: - liposomes, polyethyleneimine (PEI); in such a case said association is administered and therefore introduced into somatic target cells.
  • PKI polyethyleneimine
  • 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.
  • a nuclear localization signal into the recombinant protein to be sure that it is expressed within the nucleus.
  • 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.
  • 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.
  • 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.
  • the meganuclease is substantially non-immunogenic, i.e., engender little or no adverse immunological response.
  • 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.
  • 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.
  • 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.
  • 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 l-Crel 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 l-Crel 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,
  • step (c) 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 -10 to -8 of the l-Crel 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,
  • step (d) 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 -5 to -3 of the l-Crel 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,
  • step (e) selecting and/or screening the variants from the first series of step (a) which are able to cleave a mutant I-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,
  • step (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 l-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,
  • step (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 1-OeI 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,
  • step (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
  • step (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.
  • 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.
  • 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, el78.
  • 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 1-OeI variant having mutations in positions 26 to 40 and/or 44 to 77 of l-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.
  • the invention encompasses the l-Crel variants as defined in step (c) to (f) of the method for engineering l-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.
  • 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.
  • FIG. 1 illustrates gene targeting strategies
  • 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.
  • 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 ⁇ 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.
  • FIG. 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 ORP is from position 61 (Exon 1) to positions 4856 (Exon 3).
  • Various meganuclease sites (B2Mn) are indicated.
  • B2M11.2 and B2M11.3 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 10GAA_P and 5TAG_P (B2M11.2) and the 1 OCTGJP and the 5TTT_P targets (B2M11.3) found to be cleaved by l-Crel 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.
  • the transcription termination sequences will result in a truncated transcript, and therefore, a truncated protein,
  • the restored cleavage site can be cleaved again by the meganuclease.
  • 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 l-Crel combinatorial mutants with the B2M11.2 target.
  • H12 is a positive control (C).
  • the sequence of positive mutant at position B 3 (circle) is KNAHQS/AYSYK (same nomenclature as for Table I).
  • the sequence of positive mutant at position F7 is KNGHQS/ A YS YK.
  • - figure 8 illustrates cleavage of the of B2M11.2 target by optimized mutants.
  • a series of 1-OeI 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.
  • FIG 9 illustrates cleavage of the B 2Ml 1.3 target by combinatorial mutants.
  • the figure displays an example of primary screening of l-Crel combinatorial mutants with the B2M11.3 target.
  • HlO, HI l and H12 are respectively negative (Cl) and two positive controls (C2 and C3) of different strength.
  • the sequence of positive mutant at position G5 (circle) is KQSGCS/QNSNR (same nomenclature as for Table III).
  • FIG. 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.
  • (1) and (2) are yeast strain with B2M11 target and mutant respectively
  • 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.
  • FIG. 12 represents the pCLS1055 vector map.
  • figure 13 represents teh 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.
  • 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.
  • l-Crel sequence SWISSPROT P05725 SEQ ID NO: 1 ; l-Crel 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.
  • Example 1 Strategy for engineering novel meganucleases cleaving the human B2M gene
  • the B2M11 sequence is partly a patchwork of the 10GAA_P, 10CTG_P, 5TAGJP and 5 TTTJP 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., Epub 27 november 2006.
  • B2M11 could be cleaved by meganucleases combining the mutations found in the l-Crel derivatives cleaving these four targets.
  • the 10GAA_P, 10CTG_P, 5TAG_P and 5TTTJP sequences are 24 bp derivatives of C 1221, a palindromic sequence cleaved by 1-OeI (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., Epub 27 november 2006).
  • B2M11 ( Figure 5). Since B2M11.2 and B2M11.3 are palindromic, they should be cleaved by homodimeric proteins. Thus, proteins able to cleave the B2M11.2 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
  • 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 Arnould et al, J. MoI.
  • 5TAG_P (caaaactaggt_P; SEQ ID NO: 94) were combined with the 28, 30, 32, 33, 38 and 40 mutations from proteins cleaving 10GAA_P (cgaaacgtcgt_P; SEQ ID NO: 92).
  • 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.
  • PCR amplification is carried out using GaIlOF (5'- gcaacrttagtgctgacacatacagg-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 l-Crel coding sequence for amino acids 39-43 (assF 5'-ctannnttgacctttt-3'(SEQ ID NO: 101 ) or assR 5'-aaaggtcaannntag- 3'(SEQ ID NO: 102)) where nnn code for residue 40.
  • GaIlOF 5'- gcaacrttagtgctgacacatacagg-3'; SEQ ID NO: 99
  • GaIlOR 5'- acaaccttgattggagacttgacc-3'; SEQ
  • 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.
  • filters were transferred to synthetic medium, lacking leucine and tryptophan, with galactose (2%) as a carbon source, and incubated for five days at 37 0 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 ⁇ -mercaptoethanol, 1% agarose, and incubated at 37 °C, to monitor ⁇ -galactosidase activity. Results were analyzed by scanning and quantification was performed using appropriate software. d) Sequencing of mutants
  • 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 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 I- OeI N75 or D75 scaffold, resulting in a library of a complexity of 2014. Examples of combinations are displayed on Table 1.
  • Random mutagenesis libraries were created on a pool of chosen mutants, by PCR using Mn 2+ 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'- gcataaattactatacttctatagacacgcaaacacaaatacacacagcggccttgccacc-3'; SEQ ID NO: 103) and ICrelpostRev (5'-ggctcgaggagctcgtctagaggatcgctcgagttatcagtcggccgG-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).
  • Example 4 Making of meganucleases cleaving B2M11.3
  • 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 5TTTJP target (caaaactttgt_P; SEQ ID NO: 95) were previously obtained by mutagenesis on l-Crel N75 at positions 44, 68, 70, 75 and 77, as described in Arnould et al, J. MoI.
  • I-Crel mutants cleaving 10CTG_P or 5TTT_P were identified as described previously in Smith et al, Nucleic Acids Res. Epub 27 november 2006, and Arnould et al, J. MoI. Biol., 2006, 355, 443-458; International PCT Applications WO 2006/097784, WO 2006/097853, respectively for the 10CTG_P and 5TTT JP targets.
  • 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 l-Crel coding sequence for amino acids 39-43 (assF 5'-ctannnttgacctttt-3'(SEQ ID NO: 101) or assR 5'-aaaggtcaannntag- 3'(SEQ ID NO: 102)) where nnn code for residue 40.
  • GaIlOF 5'-gcaactttagtgctgacacatacagg-3': SEQ ID NO: 99
  • GaIlOR 5'- acaaccttgattggagacttgacc-3': SEQ ID NO: 100
  • 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.
  • 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 (MAT ⁇ , trpl ⁇ 63, leu2 ⁇ l, his3 ⁇ 200) 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 l-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.
  • Example 5 Making of meganucleases cleaving B2M11 by coexpression of meganucleases cleaving B2M11.2 assembly with proteins cleaving B2M11.3 l-Crel 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 B2M11.2 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
  • yeast strain FYBL2-7B (MAT a, ura3 ⁇ 851, trpl ⁇ 63, leu2 ⁇ l, lys2 ⁇ 202) containing the B2M11 target into yeast reporter vector (pCLS1055, Figure 12) is transformed with optimised mutants cutting B2M11.2 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 B2M11.3, in kanamycin vector (pCLS1107).
  • 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/cm 2 ). 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 3O 0 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.
  • 1-OeI mutants able to cleave the palindromic B2M11 target were identified by co-expression of mutants cleaving the palindromic B2M11.2 and B2M11.3 targets (Example 5). However, efficiency and number of positive combinations able to cleave B2M11 were minimal.
  • the protein cleaving B2M11.3 was mutagenized and variants cleaving B2M11 with better efficiency, when combined to optimized mutants for B2M11.2, were screened.
  • the residues .used for the first combinatorial approach 28, 30, 32, 33, 38 and 40 vs 44, 68, 70, 75 and 77
  • the l-Crel protein Choevalier 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.
  • a random mutagenesis library was created on the chosen mutant, by PCR using Mn 2+ 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'- gcataaattactatacttctatagacacgcaaacacaaatacacacagcggccttgccaco-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).
  • 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.

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Abstract

L'invention porte sur un variant I-CreI présentant au moins deux substitutions, une dans chacun des deux sous-domaines fonctionnels du domaine central LAGLIDADG situé des positions 26 à 40 et 44 à 77 de I-CreI, ledit variant étant capable de cliver une séquence cible d'ADN provenant du gène de la bêta-2-microglobuline. L'invention porte sur l'utilisation dudit variant et de produits dérivés pour la prévention et le traitement d'un rejet de xénogreffe et de conditions pathologiques associées à une conformation fibrillaire de la bêta-2-microglobuline, ainsi que pour la synthèse par génie génétique d'animaux transgéniques et de lignées cellulaires recombinantes exprimant une protéine d'intérêt hétérologue.
PCT/IB2007/001532 2007-02-20 2007-02-20 Variants de méganucléase clivant une séquence cible d'adn provenant du gène de la bêta-2-microglobuline et utilisations de ceux-ci WO2008102199A1 (fr)

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AU2008218605A AU2008218605A1 (en) 2007-02-20 2008-02-20 Meganuclease variants cleaving a DNA target sequence from the beta-2-microglobulin gene and uses thereof
JP2009550342A JP2010518832A (ja) 2007-02-20 2008-02-20 ベータ−２−ミクログロブリン遺伝子からのｄｎａ標的配列を切断するメガヌクレアーゼ変異型及びその使用
PCT/IB2008/001334 WO2008102274A2 (fr) 2007-02-20 2008-02-20 Variants de méganucléase clivant une séquence d'adn cible du gène de la bêta-2-microglobuline et leurs utilisations
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CA002678709A CA2678709A1 (fr) 2007-02-20 2008-02-20 Variants de meganuclease clivant une sequence d'adn cible du gene de la beta-2-microglobuline et leurs utilisations
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CN101678126A (zh) 2010-03-24
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