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(19) TZZ ¥_T (11) EP 2 522 723 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: C12N 9/22 (2006.01) C12N 15/55 (2006.01) 03.12.2014 Bulletin 2014/49 C12N 15/10 (2006.01) A61K 48/00 (2006.01) A01K 67/027 (2006.01) A01H 1/00 (2006.01) (21) Application number: 12176308.0 (22) Date of filing: 28.01.2004 (54) Custom-made meganuclease and use thereof Maßgeschneiderte Meganuklease und Verwendung davon Méganucléase sur mesure et son utilisation (84) Designated Contracting States: • Paques, Frédéric AT BE BG CH CY CZ DE DK EE ES FI FR GB GR 92340 Bourg-La-Reine (FR) HU IE IT LI LU MC NL PT RO SE SI SK TR •Perez-Michaut, Christophe 75016 Paris (FR) (30) Priority: 28.01.2003 US 442911 P • Smith, Julianne 01.08.2003 US 491535 P 92350 Le Plessis Robinson (FR) • Sourdive, David (43) Date of publication of application: 92300 Levallois Perret (FR) 14.11.2012 Bulletin 2012/46 (74) Representative: Leblois-Préhaud, Hélène Marthe (62) Document number(s) of the earlier application(s) in Georgette et al accordance with Art. 76 EPC: Cabinet Orès 04705873.0 / 1 590 453 36, rue de St Pétersbourg 75008 Paris (FR) (73) Proprietor: Cellectis 75013 Paris (FR) (56) References cited: • LUCAS PATRICK ET AL: "Rapid evolution of the (72) Inventors: DNA-binding site in LAGLIDADG homing • Arnould, Sylvain endonucleases", NUCLEIC ACIDS RESEARCH, 93200 Saint-Denis (FR) OXFORD UNIVERSITY PRESS, SURREY, GB, vol. • Bruneau, Sylvia 29, no. 4, 15 February 2001 (2001-02-15), pages 75015 Paris (FR) 960-969, XP002516751, ISSN: 0305-1048, DOI: • Cabaniols, Jean-Pierre 10.1093/NAR/29.4.960 95320 Saint Leu La Foret (FR) • CHEVALIER B S ET AL: "Design, activity, and • Chames, Patrick structure of a highly specific artificial 13012 Marseille (FR) endonuclease", MOLECULAR CELL, CELL • Choulika, André PRESS, CAMBRIDGE, MA, US, vol. 10, no. 4, 20 75015 Paris (FR) October 2002 (2002-10-20), pages 895-905, • Duchateau, Philippe XP002248750, ISSN: 1097-2765 93190 Livry Gargan (FR) • SELIGMAN LENNY M ET AL: "Mutations altering • Epinat, Jean-Charles the cleavage specificity of a homing 75020 Paris (FR) endonuclease.", NUCLEIC ACIDS RESEARCH. • Gouble, Agnès ENGLAND 1 SEP 2002, vol. 30, no. 17, 1 75017 Paris (FR) September 2002 (2002-09-01), pages 3870-3879, • Lacroix, Emmanuel XP002282592, ISSN: 1362-4962 1380 Lasne (BE) Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 522 723 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 2 522 723 B1 • EPINAT J-C ET AL: "A novel engineered • MORRISON H A ET AL: "Homing endonuclease I- meganuclease induces homologous CreI mutants with substitutions at residues 30, recombination in yeast and mammalian cells", 32 and 38 include altered specificity derivatives", NUCLEIC ACIDS RESEARCH, OXFORD ABSTRACTS OF THE GENERAL MEETING OF UNIVERSITY PRESS, SURREY, GB, vol. 31, no. THE AMERICAN SOCIETY FOR MICROBIOLOGY, 11, 1 June 2003 (2003-06-01), pages 2952-2962, THE SOCIETY, WASHINGTON, DC, US, vol. 104, XP002248751, ISSN: 0305-1048 1 January 2004 (2004-01-01), page 313, • TURMEL M ET AL: "Evolutionarily conserved and XP009150733, ISSN: 1060-2011 functionally important residues in the I-Ceul homing endonuclease", NUCLEIC ACIDS RESEARCH 1997 UNITED KINGDOM, vol. 25, no. 13, 1997, pages 2610-2619, XP002282594, ISSN: 0305-1048 • SELIGMAN ET AL: "Genetic analysis of the Chlamydomonas reinhardtii I-CreI mobile intron homing system in Escherichia coli.", GENETICS, vol. 147, no. 4, 1 December 1997 (1997-12-01), pages 1653-1664, XP55039708, ISSN: 0016-6731 2 EP 2 522 723 B1 Description [0001] The present invention relates to new rare-cutting endonucleases, also called custom-made meganucleases, which recognize and cleave a specific nucleotide sequence, to polynucleotide sequences encoding for said new rare- 5 cutting endonucleases, to a vector comprising one of said polynucleotide sequences, to a cell, an animal, or a plant comprising one of said polynucleotide sequences or said rare-cutting endonucleases, to a process for producing one of said rare-cutting endonucleases and any use of the disclosed products and methods. More particularly, this invention contemplates any use such rare-cutting endonuclease for genetic engineering, antiviral therapy, genome therapy and gene therapy. 10 [0002] Homing endonucleases constitute a family of very rare-cutting endonucleases. It was first characterised at beginning of the Nineties by the use ( in vivo) of the protein I-Sce I (Omega nuclease encoded by a mitochondrial group I intron of the yeast Saccharomyces cerevisiae). Homing endonucleases encoded by introns ORF, independent genes or intervening sequences (inteins) present striking structural and functional properties that distinguish them from "clas- sical" restriction enzymes (generally from bacterial system R/MII). They have recognition sequences that span 12-40 15 bp of DNA, whereas "classical" restriction enzymes recognise much shorter stretches of DNA, in the 3-8 bp range (up to 12 bp for rare-cutter). Therefore, the homing endonucleases present a very low frequency of cleavage, even in the human genome. [0003] Furthermore, general asymmetry of homing endonuclease target sequences contrasts with the characteristic dyad symmetry of most restriction enzyme recognition sites. Several homing endonucleases encoded by introns ORF 20 or inteins have been shown to promote the homing of their respective genetic elements into allelic intronless or inteinless sites. By making a site-specific double-strand break in the intronless or inteinless alleles, these nucleases create re- combinogenic ends, which engage in a gene conversion process that duplicates the coding sequence and leads to the insertion of an intron or an intervening sequence at the DNA level. [0004] Homing endonucleases fall into 4 separated families on the basis of pretty well conserved amino acids motifs. 25 For review, see Chevalier and Stoddard (2001, Nucleic Acids Research, 29, 3757-3774). One of them is the dodecapep- tide family (dodecamer, DOD, D1-D2, LAGLI-DADG, P1-P2). This is the largest family of proteins clustered by their most general conserved sequence motif: one or two copies (vast majority) of a twelve-residue sequence: the di-dodecapeptide. Homing endonucleases with one dodecapetide (D) are around 20 kDa in molecular mass and act as homodimer. Those with two copies (DD) range from 25 kDa (230 AA) to 50 kDa (HO, 545 AA) with 70 to 150 residues between each motif 30 and act as monomer. Cleavage is inside the recognition site, leaving 4 nt staggered cut with 3’OH overhangs. I-Ceu I and I-Cre I illustrate the homing endonucleases with one Dodecapeptide (mono dodecapeptide). I-Dmo I, I-Sce I, PI- Pfu I and PI-Sce I illustrate homing endonucleases with two Dodecapeptide motifs. Structural models using X-ray crys- tallography have been generated for I-Cre I, I-Dmo I, PI-Sce I, PI-Pfu I. structures of I-Cre I bound to its DNA site have also been elucidated leading to a number of predictions about specific protein-DNA contacts. Seligman et al (Nucleic 35 Acids Research, 2002, 30, 3870-3879) tests these predictions by analysing a set of endonuclease mutants and a complementary set of homing site mutants. Nevertheless, these endonuclease mutants have single nucleotide contacting amino acid mutations only. In parallel, Gruen et al (Nucleic Acids Research, 2002, 30, e29) developed an in vivo selection system to identify DNA target site variants that are still recognized by wild-type homing endonucleases. [0005] Endonucleases are requisite enzymes for today’s advanced genetic engineering techniques, notably for cloning 40 and analyzing genes. Homing endonucleases are very interesting as rare-cutter endonucleases because they have a very low recognition and cleavage frequency in large genome due to the size of their recognition site. Therefore, the homing endonucleases are used for molecular biology and for genetic engineering. [0006] More particularly, homologous recombination provides a method for genetically modifying chromosomal DNA sequences in a precise way. In addition to the possibility of introducing small precise mutations in order to alter the 45 activity of the chromosomal DNA sequences, such a methodology makes it possible to correct the genetic defects in genes which cause disease. Unfortunately, current methods for achieving homologous recombination are inherently inefficient, in that homologous recombination-mediated gene repair can usually be achieved in only a small proportion of cells that have taken up the relevant "targeting or correcting’ DNA. For example, in cultured mammalian cells, such recombinational events usually occur in only one in ten thousand transfected cells. 50 [0007] It has been shown that induction of double stranded DNA cleavage at a specific site in chromosomal DMA induces a cellular repair mechanism which leads to highly efficient recombinational events at that locus. Therefore, the introduction of the double strand break is accompanied by the introduction of a targeting segment of DNA homologous to the region surrounding the cleavage site, which results in the efficient introduction of the targeting sequences into the locus (either to repair a genetic lesion or to alter the chromosomal DNA in some specific way). Alternatively the induction 55 of a double stranded break at a site of interest is employed to obtain correction of a genetic lesion via a gene conversion event in which the homologous chromosomal DNA sequences from an other copy of the gene donates sequences to the sequences where the double stranded break was induced.