Cellectis S.a.

Cellectis

Emmanuel Lacroix

Jean-Charles Epinat

Sylvain Arnould

Patrick Chames

Frederic Paques

Philippe Duchateau

Andre Choulika

Phillippe Duchateau

Julianne Smith

Agnes Gouble

André Choulika

Jean-Pierre Cabaniols

David Sourdive

Christophe Perez-Michaut

Frédéric Paques

Sylvia Bruneau

Agnès Gouble

Use of meganucleases for cleaving DNA in a non-human or an isolated human cell and, in some instances, inducing homologous recombination in said cells and to its application for genome engineering and gene therapy.

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

A monomer of an I-CreI meganuclease variant wherein said monomer comprises mutations in the amino acid sequence of SEQ ID NO: 34, wherein said mutations include, (i) at least one and up to five amino acid substitutions from residue Q44 to residue R70, said substitutions selected from the group consisting of substitutions at positions Q44, T46, Y66, R68 and R7; and (ii) at least one and up to six amino acid substitutions from residue Q26 to residue Q38 said substitutions selected from the group consisting of substitutions at positions Q26, K28, N30, S32, Y33 and Q38, and wherein said monomer when in dimeric form binds and cleaves a DNA target sequence. Said dimeric forms include homodimeric, heterodimeric and single-chain I-CreI meganuclease variants.

The present invention relates to a polynucleotide encoding a monomer of an I-CreI variant said monomer comprising mutations in the amino acid sequence of SEQ ID NO: 34, wherein said mutations include: (i) at least one and up to eleven amino acid substitutions from residue S22 to Q44 said substitutions selected from the group consisting of substitutions at positions I24, Q26, K28, N30, S32, Y33, Q38, S40 and T42; and (ii) at least one and up to six amino acid substitutions from residue Y66 to I77 said substitutions selected from the group consisting of substitutions at positions Y66, R68, R70, V73 and I77; and wherein said monomer when in dimeric form binds and cleaves DNA.

I-DmoI derivatives with enhanced cleavage activity at 37&#xb0; C., said mutant comprising a sequence of a mutant of a I-DmoI endonuclease or a chimeric 5 derivative thereof including at least the first I-DmoIdomain, said sequence comprising the sub-situation of at least: (i) one of the residues in positions 4, 20, 49, 52, 92, 94 and/or 95 of said first I-DmoIdomain, and/or (ii) one of the residues in positions 101, 102, and/or 109 of the linker or the beginning of the second domain of I-DmoI, if present. 10 Polynucleotide encoding said derivatives, cell, animal or plant comprising said polynucleotide and use thereof for isolating meganucleases with new DNA target specificity.

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This patent application relates to hybrid and/or single-chain rare-cutting endonucleases, called meganucleases, which recognize and cleave a specific nucleotide sequence, to polynucleotide sequences encoding for said rare-cutting endonucleases, to a vector comprising one of said polynucleotide sequences, to a cell or animal 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 of such rare-cutting endonuclease for genetic engineering and gene therapy.

Hybrid and single chain meganucleases and use thereof

A single chain homing endonuclease, comprising a first variant of I-CreI having the amino acid sequence of accession number pdb 1g9y (SEQ ID NO: 23 is residues 1-153 of pdb Ig9y) and a second variant of I-CreI variant having the amino acid sequence of accession number pdb 1g9y (SEQ ID NO: 23 is residues 1-153 of pdb Ig9y) in a single polypeptide.

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Paris, France

Emmanuel Lacroix