Valérie Guyot

Bio: Valérie Guyot is an academic researcher from Royal Holloway, University of London. The author has contributed to research in topics: Genome engineering & Genome editing. The author has an hindex of 10, co-authored 11 publications receiving 719 citations.

Genome engineering empowers the diatom Phaeodactylum tricornutum for biotechnology

Abstract: Diatoms, a major group of photosynthetic microalgae, have a high biotechnological potential that has not been fully exploited because of the paucity of available genetic tools. Here we demonstrate targeted and stable modifications of the genome of the marine diatom Phaeodactylum tricornutum, using both meganucleases and TALE nucleases. When nuclease-encoding constructs are co-transformed with a selectable marker, high frequencies of genome modifications are readily attained with 56 and 27% of the colonies exhibiting targeted mutagenesis or targeted gene insertion, respectively. The generation of an enhanced lipid-producing strain (45-fold increase in triacylglycerol accumulation) through the disruption of the UDP-glucose pyrophosphorylase gene exemplifies the power of genome engineering to harness diatoms for biofuel production. Diatoms are photosynthetic microalgae with underutilized biotechnological potential. Here, the authors carry out targeted gene modifications of lipid metabolism genes in the diatom, Phaeodactylum tricornutum, resulting in a strain that exhibits a 45-fold increase in triacylglycerol accumulation.

Chromosomal context and epigenetic mechanisms control the efficacy of genome editing by rare-cutting designer endonucleases

Abstract: The ability to specifically engineer the genome of living cells at precise locations using rare-cutting designer endonucleases has broad implications for biotechnology and medicine, particularly for functional genomics, transgenics and gene therapy. However, the potential impact of chromosomal context and epigenetics on designer endonucleasemediated genome editing is poorly understood. To address this question, we conducted a comprehensive analysis on the efficacy of 37 endonucleases derived from the quintessential I-CreI meganuclease that were specifically designed to cleave 39 different genomic targets. The analysis revealed that the efficiency of targeted mutagenesis at a given chromosomal locus is predictive of that of homologous gene targeting. Consequently, a strong genome-wide correlation was apparent between the efficiency of targeted mutagenesis (� 0.1% to � 6%) with that of homologous gene targeting (� 0.1% to � 15%). In contrast, the efficiency of targeted mutagenesis or homologous gene targeting at a given chromosomal locus does not correlate with the activity of individual endonucleases on transiently transfected substrates. Finally, we demonstrate that chromatin accessibility modulates the efficacy of rare-cutting endonucleases, accounting for strong position effects. Thus, chromosomal context and epigenetic mechanisms may play a major role in the efficiency rare-cutting endonuclease-induced genome engineering.

Efficient in toto targeted recombination in mouse liver by meganuclease-induced double-strand break.

Abstract: Background Sequence-specific endonucleases with large recognition sites can cleave DNA in living cells, and, as a consequence, stimulate homologous recombination (HR) up to 10 000-fold. The recent development of artificial meganucleases with chosen specificities has provided the potential to target any chromosomal locus. Thus, they may represent a universal genome engineering tool and seem to be very promising for acute gene therapy. However, in toto applications depend on the ability to target somatic tissues as well as the proficiency of somatic cells to perform double-strand break (DSB)-induced HR. Methods In order to investigate DSB-induced HR in toto, we have designed transgenic mouse lines carrying a LagoZ gene interrupted by one I-SceI cleavage site surrounded by two direct repeats. The LagoZ gene can be rescued upon cleavage by I-SceI and HR between the two repeats in a process called single-strand annealing. β-Galactosidase activity is monitored in liver after tail vein injection of adenovirus expressing the meganuclease I-SceI. Results In toto staining revealed a strong dotted pattern in all animals injected with adenovirus expressing I-SceI. In contrast, no staining could be detected in the control. β-Galactosidase activity in liver extract, tissue section staining, and PCR analysis confirmed the presence of the recombined LagoZ gene. Conclusions We demonstrate for the first time that meganucleases can be successfully delivered in animal and induce targeted genomic recombination in mice liver in toto. These results are an essential step towards the use of designed meganucleases and show the high potential of this technology in the field of gene therapy. Copyright © 2006 John Wiley & Sons, Ltd.

Factors affecting double-strand break-induced homologous recombination in mammalian cells

Abstract: Double-strand break (DSB)-induced homologous recombination (HR) of direct repeats is a powerful means to achieve gene excision, a critical step in genome engineering. In this report we have used an extrachrmosomal reporter system to monitor the impact of different parameters on meganuclease-induced HR in CHO-K1 cells. We found that repeat homology length is critical. Virtually no HR could be detected with a 15-bp duplication, while, with repeats larger than 400 bp, recombination efficiency became less dependent on homology length. The presence of an intervening sequence between the duplications dramatically impairs HR, independent of the cleavage position; by 3 kb of insertion, HR is virtually undetectable. Efficient HR can be restored by positioning cleavage sites at both ends of the intervening sequence, allowing a constant level of excision with up to 10 kb of intervening sequences. Using similar constructs, 2.8-kb inserts could be efficiently removed from several chromosomal loci, illustrating the wide potential of this technology. These results fit current models of direct repeat recombination and identify DSB-induced HR as a powerful tool for gene excision.

Multiplex Genome Editing to Generate Universal CAR T Cells Resistant to PD1 Inhibition

Abstract: Purpose: Using gene-disrupted allogeneic T cells as universal effector cells provides an alternative and potentially improves current chimeric antigen receptor (CAR) T-cell therapy against cancers and infectious diseases.Experimental Design: The CRISPR/Cas9 system has recently emerged as a simple and efficient way for multiplex genome engineering. By combining lentiviral delivery of CAR and electro-transfer of Cas9 mRNA and gRNAs targeting endogenous TCR, β-2 microglobulin (B2M) and PD1 simultaneously, to generate gene-disrupted allogeneic CAR T cells deficient of TCR, HLA class I molecule and PD1.Results: The CRISPR gene-edited CAR T cells showed potent antitumor activities, both in vitro and in animal models and were as potent as non-gene-edited CAR T cells. In addition, the TCR and HLA class I double deficient T cells had reduced alloreactivity and did not cause graft-versus-host disease. Finally, simultaneous triple genome editing by adding the disruption of PD1 led to enhanced in vivo antitumor activity of the gene-disrupted CAR T cells.Conclusions: Gene-disrupted allogeneic CAR and TCR T cells could provide an alternative as a universal donor to autologous T cells, which carry difficulties and high production costs. Gene-disrupted CAR and TCR T cells with disabled checkpoint molecules may be potent effector cells against cancers and infectious diseases. Clin Cancer Res; 23(9); 2255-66. ©2016 AACR.

NK cells for cancer immunotherapy.

Abstract: Natural killer (NK) cells can swiftly kill multiple adjacent cells if these show surface markers associated with oncogenic transformation. This property, which is unique among immune cells, and their capacity to enhance antibody and T cell responses support a role for NK cells as anticancer agents. Although tumours may develop several mechanisms to resist attacks from endogenous NK cells, ex vivo activation, expansion and genetic modification of NK cells can greatly increase their antitumour activity and equip them to overcome resistance. Some of these methods have been translated into clinical-grade platforms and support clinical trials of NK cell infusions in patients with haematological malignancies or solid tumours, which have yielded encouraging results so far. The next generation of NK cell products will be engineered to enhance activating signals and proliferation, suppress inhibitory signals and promote their homing to tumours. These modifications promise to significantly increase their clinical activity. Finally, there is emerging evidence of increased NK cell-mediated tumour cell killing in the context of molecularly targeted therapies. These observations, in addition to the capacity of NK cells to magnify immune responses, suggest that NK cells are poised to become key components of multipronged therapeutic strategies for cancer. Natural killer (NK) cells have a primordial role in tumour immunosurveillance. Given their potent antitumour activity, therapeutic manipulation of NK cells provides an attractive strategy for cancer treatment. This Review discusses new approaches to activate NK cells, increase their proliferation in vivo and increase their capacity to recognize tumour cells.

Homing endonuclease structure and function.

Abstract: Homing endonucleases are encoded by open reading frames that are embedded within group I, group II and archael introns, as well as inteins (intervening sequences that are spliced and excised post-translationally). These enzymes initiate transfer of those elements (and themselves) by generating strand breaks in cognate alleles that lack the intervening sequence, as well as in additional ectopic sites that broaden the range of intron and intein mobility. Homing endonucleases can be divided into several unique families that are remarkable in several respects: they display extremely high DNA-binding specificities which arise from long DNA target sites (14-40 bp), they are tolerant of a variety of sequence variations in these sites, and they display disparate DNA cleavage mechanisms. A significant number of homing endonucleases also act as maturases (highly specific cofactors for the RNA splicing reactions of their cognate introns). Of the known homing group I endonuclease families, two (HNH and His-Cys box enzymes) appear to be diverged from a common ancestral nuclease. While crystal structures of several representatives of the LAGLIDADG endonuclease family have been determined, only structures of single members of the HNH (I-HmuI), His-Cys box (I-PpoI) and GIY-YIG (I-TevI) families have been elucidated. These studies provide an important source of information for structure-function relationships in those families, and are the centerpiece of this review. Finally, homing endonucleases are significant targets for redesign and selection experiments, in hopes of generating novel DNA binding and cutting reagents for a variety of genomic applications.

'Off-the-shelf' allogeneic CAR T cells: development and challenges.

Abstract: Autologous chimeric antigen receptor (CAR) T cells have changed the therapeutic landscape in haematological malignancies. Nevertheless, the use of allogeneic CAR T cells from donors has many potential advantages over autologous approaches, such as the immediate availability of cryopreserved batches for patient treatment, possible standardization of the CAR-T cell product, time for multiple cell modifications, redosing or combination of CAR T cells directed against different targets, and decreased cost using an industrialized process. However, allogeneic CAR T cells may cause life-threatening graft-versus-host disease and may be rapidly eliminated by the host immune system. The development of next-generation allogeneic CAR T cells to address these issues is an active area of research. In this Review, we analyse the different sources of T cells for optimal allogeneic CAR-T cell therapy and describe the different technological approaches, mainly based on gene editing, to produce allogeneic CAR T cells with limited potential for graft-versus-host disease. These improved allogeneic CAR-T cell products will pave the way for further breakthroughs in the treatment of cancer. The use of allogeneic chimeric antigen receptor T cells from donors has many potential advantages over autologous approaches, such as immediate availability, standardization and the possibility of redosing or combination. This Review analyses the different sources of T cells and technological approaches to produce optimal allogeneic chimeric antigen receptor T cells with limited potential for graft-versus-host disease and increased persistence.