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EP2329017A2 - Nouvelle méthode pour générer des méganucléases ayant des caractéristiques modifiées - Google Patents

Nouvelle méthode pour générer des méganucléases ayant des caractéristiques modifiées

Info

Publication number
EP2329017A2
EP2329017A2 EP09785836A EP09785836A EP2329017A2 EP 2329017 A2 EP2329017 A2 EP 2329017A2 EP 09785836 A EP09785836 A EP 09785836A EP 09785836 A EP09785836 A EP 09785836A EP 2329017 A2 EP2329017 A2 EP 2329017A2
Authority
EP
European Patent Office
Prior art keywords
meganuclease
target
variants
altered
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP09785836A
Other languages
German (de)
English (en)
Inventor
Frédéric PAQUES
Sylvestre Grizot
Philippe Duchateau
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cellectis SA
Original Assignee
Cellectis SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/IB2008/002999 external-priority patent/WO2009019614A2/fr
Priority claimed from PCT/IB2008/003744 external-priority patent/WO2009074873A1/fr
Application filed by Cellectis SA filed Critical Cellectis SA
Publication of EP2329017A2 publication Critical patent/EP2329017A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1058—Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • LAGLIDADG proteins are central and form two packed ⁇ -helices where a 2-fold (pseudo-) symmetry axis separates two monomers or apparent domains.
  • residues 28 to 40 and 44 to 77 of I-Crel were shown to form two separable functional subdomains, able to bind distinct parts of a homing endonuclease half-site (Smith et al. Nucleic Acids Res., 2006, 34, el 49; International PCT Applications WO 2007/049095 and WO
  • This cleavage induces homologous recombination between the direct repeats, resulting in a functional reporter gene (LacZ, for example), whose expression can be monitored by an appropriate assay.
  • the specificity of the cleavage by the variant may be assessed by comparing the cleavage of the (non-palindromic) DNA target sequence with that of the two palindromic sequences cleaved by the parent homodimeric meganucleases or compared with wild type meganuclease.
  • target-site "target” , "site”; “site of interest”; "recognition site”, “recognition sequence”, “homing recognition site”, “horning site”, “cleavage site” is intended a 20 to 24 bp double-stranded palindromic, partially palindromic (pseudo-palindromic) or non-palindromic polynucleotide sequence that is recognized and cleaved by a LAGLIDADG homing endonuclease such as l-Crel, or a variant, or a single-chain chimeric meganuclease derived from I-Crel.
  • LAGLIDADG homing endonuclease
  • DNA target sequence from the beta-2-microglobulin gene it 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.
  • This new method has a number of advantages over prior art methods and in particular a major advantage of the method according to the present invention is that synergistic mutations can be found, these are mutations which do not generate the desired characteristic by themselves but instead act with other mutations to elicit the desired characteristic. Such synergistic mutations cannot be found using prior art methods, as the selection at different times of the two or more desired characteristics means that mutations which give rise to both characterisitics simultaneously are not selected for and hence cannot be found.
  • a further advantage of this method is that it can be used to generate meganuclease enzymes to targets for which previous attempts with prior art methods have failed. Examples of this are set out in the detailed description below.
  • this method is useful for generating and selected an altered meganuclease, starting with a parent meganuclease which is a member from the LAGLIDADG family.
  • this method involves the construction in steps a. and c. of a first and a second series of variants which differ from their respective parent meganuclease by at least one amino acid substitution in at least one of the functional domains or subdomains of said first and/or second series of variants.
  • the parent meganuclease is either a wildtype meganuclease or a functional variant of a wild type meganuclease.
  • DmoCre I-CreIII-DmoI hybrids
  • the present invention relates to a method to generate a meganuclease which has at least two altered characteristics in comparison to the parent meganuclease.
  • the present invention can also however be used to generate a meganuclease which comprises additional altered characteristics.
  • steps c. and d. of the method are repeated and the selection of meganuclease mutants showing the required combination of altered characteristics are made in each iteration of step d.
  • Steps c. and d. can be repeated a number of times 'n' so as to generate a meganuclease with a number of altered characteristics n (plus the original two altered characteristics).
  • the I-Dmol domain is modified in step a. and/or step c.
  • the present invention also relates to a polynucleotide, this polynucleotide being characterized in that it encodes a polypeptide according to the present invention.
  • IL2RG3 (SEQ ID NO: 86) is the DNA sequence located in the human IL2RG gene at position 1686.
  • IL2RG3.2 target (SEQ ID NO: 87) the TCTC sequence in the middle of the target is replaced with GTAC, the bases found in C1221.
  • the IL2RG3.6 target DNA sequence (SEQ ID NO: 7) differs from
  • IL2RG3.4 (SEQ ID NO: 6) only by the four central base pairs that are called 2NN 2NN.
  • IL2RG3.4 carries GTAC as the C1221 target (SEQ ID NO: 8) while
  • the SeqLib2 library that contains mutations at positions 28, 32 and 33 was built using the same method but with the use of the primer 10RG34Rev2 (5'- caagcttagctgatgtttaaacttmbnmbnctggtttggmbnaatctgagc-3'; SEQ ID NO: 14) instead of 10RG34Revl.
  • the MBN code in the oligonucleotide resulting in a NVK codon at positions 28, 32 and 33 allows the degeneracy at these positions among all the 20 possible amino acids but F, L, M, I and V.
  • the 1132V and E80K mutations were introduced on a DNA pool consisting of DNA molecules encoding Seq4, Seq5 and Seq7 I-Crel mutants from Table I below.
  • This further modification of the variants isolated in step d. of the method according to the present invention shows that further iterative steps can be used to introduce further altered characteristics into a meganuclease generated according to the method.
  • Site-directed mutagenesis libraries were created by PCR. For example, to introduce the Il 32V substitution into the coding sequences of the mutants, two separate overlapping PCR reactions were carried out that amplify the 5' end (residues 1-137) or the 3' end (residues 127-167) of the 1-OeI coding sequence.
  • the IL2RG3.3 and IL2RG3.4 mutants were coexpressed in yeast.
  • the co-expression lead to the formation of heterodimers, whose activity toward the IL2RG3 target was monitored.
  • PCR amplification is carried out using a primer specific to the pCLS0542 vector (GaIlOF 5'-GCAACTTTAGTGCTGACACATACAGG-S' (SEQ ID NO: 12)) and a primer specific to the I-Crel coding sequence for amino acids 25-32 (SeqlOBMRevl 5'-agactggtttggtttaatctgagc-3' (SEQ ID NO: 103)).
  • the NVK codon at positions 33 and 38 allows the degeneracy at these positions among all the 20 possible amino acids but F, L, M, I and V.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Ecology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

La présente invention concerne une méthode permettant de générer et de sélectionner une méganucléase ayant au moins deux caractéristiques modifiées par rapport à une méganucléase parente. Ladite méthode comporte les étapes consistant à : a. construire à partir d'une méganucléase parente une première série de variants qui diffèrent de ladite méganucléase parente par au moins une substitution d'acide aminé ; b. cribler les variants issus de ladite première série de l'étape a. et sélectionner ceux qui possèdent une première caractéristique modifiée ; c. construire à partir des variants sélectionnés de l'étape b. une seconde série de variants ayant au moins une autre substitution d'acide aminé ; d. cribler les variants issus de ladite série de l'étape b. et sélectionner ceux qui possèdent ladite première caractéristique modifiée et une seconde caractéristique modifiée. L'invention concerne également le polypeptide obtenu à partir de ladite méthode.
EP09785836A 2008-08-04 2009-02-09 Nouvelle méthode pour générer des méganucléases ayant des caractéristiques modifiées Withdrawn EP2329017A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
PCT/IB2008/002999 WO2009019614A2 (fr) 2007-08-03 2008-08-04 Variants de méganucléase clivant une séquence cible d'adn provenant du gène de la chaîne gamma du récepteur de l'interleukine-2 humain, et leurs utilisations
PCT/IB2008/003744 WO2009074873A1 (fr) 2007-12-13 2008-12-12 Enzymes améliorées de méganucléase chimère et leurs utilisations
PCT/IB2009/000486 WO2010015899A2 (fr) 2008-08-04 2009-02-09 Nouvelle méthode pour générer des méganucléases ayant des caractéristiques modifiées

Publications (1)

Publication Number Publication Date
EP2329017A2 true EP2329017A2 (fr) 2011-06-08

Family

ID=41566035

Family Applications (1)

Application Number Title Priority Date Filing Date
EP09785836A Withdrawn EP2329017A2 (fr) 2008-08-04 2009-02-09 Nouvelle méthode pour générer des méganucléases ayant des caractéristiques modifiées

Country Status (2)

Country Link
EP (1) EP2329017A2 (fr)
WO (1) WO2010015899A2 (fr)

Families Citing this family (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011104382A1 (fr) * 2010-02-26 2011-09-01 Cellectis Utilisation d'endonucléases pour insérer des transgènes dans des locus safe harbor
EP2702160B1 (fr) 2011-04-27 2020-05-27 Amyris, Inc. Procédés de modification génomique
EP2687605A1 (fr) 2012-07-19 2014-01-22 Biogemma Procédé pour effectuer une recombinaison homologue
CN106029886B (zh) 2013-12-19 2021-02-05 阿迈瑞斯公司 基因组整合的方法
WO2016073955A2 (fr) * 2014-11-06 2016-05-12 President And Fellows Of Harvard College Cellules ne présentant pas d'expression en surface de b2m et procédés pour l'administration allogène de ces cellules
CA2988854A1 (fr) 2015-05-08 2016-11-17 President And Fellows Of Harvard College Cellules souches de donneur universel et procedes associes
MX2018004146A (es) 2015-10-05 2018-11-09 Prec Biosciences Inc Células modificadas genéticamente que comprenden un gen mano modificado de la región constante alfa del receptor de células t.
EP3359660B1 (fr) 2015-10-05 2019-12-04 Precision Biosciences, Inc. Variantes de méganucléase clivant une séquence cible d'adn dans les domaines constants alpha de récepteur de lymphocyte t
JP6846429B2 (ja) * 2015-12-23 2021-03-24 プレシジョン バイオサイエンシズ，インク． ヒトβ−２ミクログロブリン遺伝子に見られる認識配列を有する操作されたメガヌクレアーゼ
EP3458595A2 (fr) 2016-05-18 2019-03-27 Amyris, Inc. Compositions et procédés d'intégration génomique d'acides nucléiques dans des plaques d'atterrissage exogènes
US11053484B2 (en) 2017-06-30 2021-07-06 Precision Biosciences, Inc. Genetically-modified T cells comprising a modified intron in the T cell receptor alpha gene
CA3095795A1 (fr) 2018-04-12 2019-10-17 Precision Biosciences, Inc. Nucleases modifiees optimisees ayant une specificite pour le gene de region constante du recepteur alpha des lymphocytes t humain

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050003420A1 (en) * 2003-07-01 2005-01-06 Xu Shuang-Yong Recycled mutagenesis of restriction endonuclease toward enhanced catalytic activity
WO2006097854A1 (fr) * 2005-03-15 2006-09-21 Cellectis Meganucleases heterodimeriques et utilisation de ces dernieres
WO2006097784A1 (fr) * 2005-03-15 2006-09-21 Cellectis Variants de meganuclease i-crei presentant une specificite modifiee, leur procede de preparation, et leurs utilisations
WO2007060495A1 (fr) * 2005-10-25 2007-05-31 Cellectis Variants de l'endonuclease homing i-crei a nouvelle specificite de clivage et leur utilisation
WO2007049095A1 (fr) * 2005-10-25 2007-05-03 Cellectis Variants d'endonuclease de liaison a laglidadg comprenant des mutations dans deux sous-domaines fonctionnels et leur utilisation

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2010015899A3 *

Also Published As

Publication number Publication date
WO2010015899A3 (fr) 2010-03-25
WO2010015899A2 (fr) 2010-02-11

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