Engineered I-CreI Derivatives Cleaving Sequences from the Human XPC Gene can Induce Highly Efficient Gene Correction in Mammalian Cells

Arnould, Sylvain; Pérez, C.; Cabaniols, Jean-Pierre; Smith, Julianne; Gouble, Agnès; Grizot, Sylvestre; Epinat, Jean‐Charles; Duclert, Aymeric; Duchâteau, Philippe; Pâques, Frédéric

doi:10.1016/j.jmb.2007.04.079

Cited by 132 publications

(112 citation statements)

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“…The previously described I-CreI engineered derivatives are heterodimers. Therefore, they are obtained by coexpression of 2 different monomers in the target cell (10,30,33), resulting in the formation of 3 molecular species (the heterodimer and 2 homodimers). Studies with heterodimeric Zinc Finger Nucleases (34,35) showed that such homodimeric byproducts can be cytotoxic.…”

Section: Discussionmentioning

confidence: 99%

Crystal structure of I-DmoI in complex with its target DNA provides new insights into meganuclease engineering

Marcaida

Prieto

Redondo

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Homing endonucleases, also known as meganucleases, are sequencespecific enzymes with large DNA recognition sites. These enzymes can be used to induce efficient homologous gene targeting in cells and plants, opening perspectives for genome engineering with applications in a wide series of fields, ranging from biotechnology to gene therapy. Here, we report the crystal structures at 2.0 and 2.1 Å resolution of the I-DmoI meganuclease in complex with its substrate DNA before and after cleavage, providing snapshots of the catalytic process. Our study suggests that I-DmoI requires only 2 cations instead of 3 for DNA cleavage. The structure sheds light onto the basis of DNA binding, indicating key residues responsible for nonpalindromic target DNA recognition. In silico and in vivo analysis of the I-DmoI DNA cleavage specificity suggests that despite the relatively few protein-base contacts, I-DmoI is highly specific when compared with other meganucleases. Our data open the door toward the generation of custom endonucleases for targeted genome engineering using the monomeric I-DmoI scaffold.gene targeting ͉ genetics ͉ protein-DNA interactions ͉ X-ray crystallography M eganucleases are sequence-specific enzymes that recognize large (12-45 bp) DNA target sites. These enzymes are often encoded by introns or inteins behaving as mobile genetic elements. They produce double strand breaks (DSB) that get repaired by homologous recombination with an intron-or intein-containing gene, resulting in the insertion of the intron or intein in that particular site (1). Meganucleases are being used to stimulate homologous gene targeting in the vicinity of their target sequences with the aim of improving current genome engineering approaches, alleviating the risks due to the randomly inserted transgenes (2, 3).The use of meganuclease-induced recombination has long been limited by the repertoire of natural meganucleases. In nature, meganucleases are essentially represented by homing endonucleases, a family of enzymes encoded by mobile genetic elements whose function is to initiate DSB induced recombination events in a process referred to as homing (4). The probability of finding a homing endonuclease cleavage site in a chosen gene is extremely low. Thus, making artificial meganucleases with custom-made substrate specificity is an intense area of research.Sequence homology has been used to classify homing endonucleases into 5 families, the largest one having the conserved LAGLI-DADG sequence motif. Homing endonucleases containing one such motif function as homodimers. In contrast, homing endonucleases containing 2 motifs are single chain proteins (5, 6). Structural information for several members of the LAGLIDADG endonuclease family indicate that these proteins adopt a similar active conformation as homodimers or as monomers with 2 separate domains (7-9). The LAGLIDADG motifs form structurally conserved ␣-helices tightly packed at the center of the interdomain or intermonomer interface. The last acidic residue of this LAGLI-DADG motif part...

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Section: Discussionmentioning

confidence: 99%

Crystal structure of I-DmoI in complex with its target DNA provides new insights into meganuclease engineering

Marcaida

Prieto

Redondo

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…Site-specific meganucleases have challenging design criteria owing to the single-chained recognition and cleavage domains, although successful modification has been described for a number of applications [15–17]. In the context of gene therapy, meganucleases have for example been modeled for targeted recombination and correction of the RAG1 gene associated with severe combined immunodeficiency (SCID) [18] in hematopoietic stem cells (HSCs) and the XPC gene associated with Xeroderma Pigmentosum in skin cells [19]. Applications within T cell therapies include prevention of graft-versus-host disease (GvHD) via meganuclease-mediated TCR α-chain knockout under conditions for optimal T cell stimulation and meganuclease cleavage efficiency [15].…”

Section: Toolsmentioning

confidence: 99%

Genome-Edited T Cell Therapies

Delhove

Qasim

2017

Curr Stem Cell Rep

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Purpose of ReviewAlternative approaches to conventional drug-based cancer treatments have seen T cell therapies deployed more widely over the last decade. This is largely due to their ability to target and kill specific cell types based on receptor recognition. Introduction of recombinant T cell receptors (TCRs) using viral vectors and HLA-independent T cell therapies using chimeric antigen receptors (CARs) are discussed. This article reviews the tools used for genome editing, with particular emphasis on the applications of site-specific DNA nuclease mediated editing for T cell therapies.Recent FindingsGenetic engineering of T cells using TCRs and CARs with redirected antigen-targeting specificity has resulted in clinical success of several immunotherapies. In conjunction, the application of genome editing technologies has resulted in the generation of HLA-independent universal T cells for allogeneic transplantation, improved T cell sustainability through knockout of the checkpoint inhibitor, programmed cell death protein-1 (PD-1), and has shown efficacy as an antiviral therapy through direct targeting of viral genomic sequences and entry receptors.SummaryThe combined use of engineered antigen-targeting moieties and innovative genome editing technologies have recently shown success in a small number of clinical trials targeting HIV and hematological malignancies and are now being incorporated into existing strategies for other immunotherapies.

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“…33 Finally, two engineered MGNs cleaving two different human genes, XPC and RAG1, have been described in the literature. 21,[33][34][35] These recent developments provide the potential to target any chromosomal locus with engineered MGNs. Thus, these proteins represent a universal tool to mutate the genome at specific sequence target and are thus very promising for precise and efficient genome modifications.…”

Section: Introductionmentioning

confidence: 99%

Meganucleases can restore the reading frame of a mutated dystrophin

et al. 2010

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Mutations in Duchenne muscular dystrophy (DMD) are either inducing a nonsense codon or a frameshift. Meganucleases (MGNs) can be engineered to induce double-strand breaks (DSBs) at specific DNA sequences. These breaks are repaired by homologous recombination or by non-homologous end joining (NHEJ), which results in insertions or deletions (indels) of a few base pairs. To verify whether MGNs could be used to restore the normal reading frame of a dystrophin gene with a frameshift mutation, we inserted in a plasmid coding for the dog m-dystrophin sequences containing a MGN target. The number of base pairs in these inserted sequences changed the reading frame. One of these modified target m-dystrophin plasmids and an appropriate MGN were then transfected in 293FT cells. The MGN induced micro-deletion or micro-insertion in the m-dystrophin that restored dystrophin expression. MGNs also restored m-dystrophin expression in myoblasts in vitro and in muscle fibers in vivo. The mutation of the targeted m-dystrophin was confirmed by PCR amplification followed by digestion with the Surveyor enzyme and by cloning and sequencing of the amplicons. These experiments are thus a proof of principle that MGNs that are adequately engineered to target appropriate sequences in the human dystrophin gene should be able to restore the normal reading frame of that gene in DMD patients with an out-of-frame deletion. New MGNs engineered to target a sequence including or near nonsense mutation could also be used to delete it.

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Engineered I-CreI Derivatives Cleaving Sequences from the Human XPC Gene can Induce Highly Efficient Gene Correction in Mammalian Cells

Cited by 132 publications

References 38 publications

Crystal structure of I-DmoI in complex with its target DNA provides new insights into meganuclease engineering

Crystal structure of I-DmoI in complex with its target DNA provides new insights into meganuclease engineering

Genome-Edited T Cell Therapies

Meganucleases can restore the reading frame of a mutated dystrophin

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