Non-viral DNA delivery and TALEN editing correct the sickle cell mutation in hematopoietic stem cells.

Sickle cell disease is a devastating blood disorder that originates from a single point mutation in the HBB gene coding for hemoglobin. Here, we develop a GMP-compatible TALEN-mediated gene editing process enabling efficient HBB correction via a DNA repair template while minimizing risks associated with HBB inactivation. Comparing viral versus non-viral DNA repair template delivery in hematopoietic stem and progenitor cells in vitro, both strategies achieve comparable HBB correction and result in over 50% expression of normal adult hemoglobin in red blood cells without inducing β-thalassemic phenotype.

Comprehensive analysis of the editing window of C-to-T TALE base editors.

One of the most recent advances in the genome editing field has been the addition of "TALE Base Editors", an innovative platform for cell therapy that relies on the deamination of cytidines within double strand DNA, leading to the formation of an uracil (U) intermediate. These molecular tools are fusions of transcription activator-like effector domains (TALE) for specific DNA sequence binding, split-DddA deaminase halves that will, upon catalytic domain reconstitution, initiate the conversion of a cytosine (C) to a thymine (T), and an uracil glycosylase inhibitor (UGI). We developed a high throughput screening strategy capable to probe key editing parameters in a precisely defined genomic context in cellulo, excluding or minimizing biases arising from different microenvironmental and/or epigenetic contexts.

Efficient multitool/multiplex gene engineering with TALE-BE.

TALE base editors are a recent addition to the genome editing toolbox. These molecular tools are fusions of a transcription activator-like effector domain (TALE), split-DddA deaminase halves, and an uracil glycosylase inhibitor (UGI) that have the distinct ability to directly edit double strand DNA, converting a cytosine (C) to a thymine (T). To dissect the editing rules of TALE-BE, we combined the screening of dozens of TALE-BE targeting nuclear genomic loci with a medium/high throughput strategy based on precise knock-in of TALE-BE target site collections into the cell genome.

Optimized two-step electroporation process to achieve efficient nonviral-mediated gene insertion into primary T cells.

The development of gene editing technologies over the past years has allowed the precise and efficient insertion of transgenes into the genome of various cell types. Knock-in approaches using homology-directed repair and designer nucleases often rely on viral vectors, which can considerably impact the manufacturing cost and timeline of gene-edited therapeutic products. An attractive alternative would be to use naked DNA as a repair template.

Preclinical Evaluation of a Novel TALEN Targeting CCR5 Confirms Efficacy and Safety in Conferring Resistance to HIV-1 Infection.

Therapies to treat patients infected with human immunodeficiency virus (HIV) aim at preventing viral replication but fail to eliminate the virus. Although transplantation of allogeneic CCR5Δ32 homozygous stem cell grafts provided a cure for a few patients, this approach is not considered a general therapeutic strategy because of potential side effects. Conversely, gene editing to disrupt the C-C chemokine receptor type 5 (CCR5) locus, which encodes the major HIV coreceptor, has shown to confer resistance to CCR5-tropic HIV strains.

Modulation of chimeric antigen receptor surface expression by a small molecule switch.

Background: Engineered therapeutic cells have attracted a great deal of interest due to their potential applications in treating a wide range of diseases, including cancer and autoimmunity. Chimeric antigen receptor (CAR) T-cells are designed to detect and kill tumor cells that present a specific, predefined antigen. The rapid expansion of targeted antigen beyond CD19, has highlighted new challenges, such as autoactivation and T-cell fratricide, that could impact the capacity to manufacture engineered CAR T-cells.

A Versatile Safeguard for Chimeric Antigen Receptor T-Cell Immunotherapies.

CAR T-cell therapies hold great promise for treating a range of malignancies but are however challenged by the complexity of their production and by the adverse events related to their activity. Here we report the development of the CubiCAR, a tri-functional CAR architecture that enables CAR T-cell detection, purification and on-demand depletion by the FDA-approved antibody Rituximab. This novel architecture has the potential to streamline the manufacturing of CAR T-cells, allow their tracking and improve their overall safety.

Fine and Predictable Tuning of TALEN Gene Editing Targeting for Improved T Cell Adoptive Immunotherapy.

Using a TALEN-mediated gene-editing approach, we have previously described a process for the large-scale manufacturing of "off-the-shelf" CAR T cells from third-party donor T cells by disrupting the gene encoding TCRα constant chain (TRAC). Taking advantage of a previously described strategy to control TALEN targeting based on the exclusion capacities of non-conventional RVDs, we have developed highly efficient and specific nucleases targeting a key T cell immune checkpoint, PD-1, to improve engineered CAR T cells' functionalities. Here, we demonstrate that this approach allows combined TRAC and PDCD1 TALEN processing at the desired locus while eliminating low-frequency off-site processing.

An oxygen sensitive self-decision making engineered CAR T-cell.

A key to the success of chimeric antigen receptor (CAR) T-cell based therapies greatly rely on the capacity to identify and target antigens with expression restrained to tumor cells. Here we present a strategy to generate CAR T-cells that are only effective locally (tumor tissue), potentially also increasing the choice of targetable antigens. By fusing an oxygen sensitive subdomain of HIF1α to a CAR scaffold, we generated CAR T-cells that are responsive to a hypoxic environment, a hallmark of certain tumors.

Design of chimeric antigen receptors with integrated controllable transient functions.

The ability to control T cells engineered to permanently express chimeric antigen receptors (CARs) is a key feature to improve safety. Here, we describe the development of a new CAR architecture with an integrated switch-on system that permits to control the CAR T-cell function. This system offers the advantage of a transient CAR T-cell for safety while letting open the possibility of multiple cytotoxicity cycles using a small molecule drug.

Multiplex Genome-Edited T-cell Manufacturing Platform for "Off-the-Shelf" Adoptive T-cell Immunotherapies.

Adoptive immunotherapy using autologous T cells endowed with chimeric antigen receptors (CAR) has emerged as a powerful means of treating cancer. However, a limitation of this approach is that autologous CAR T cells must be generated on a custom-made basis. Here we show that electroporation of transcription activator-like effector nuclease (TALEN) mRNA allows highly efficient multiplex gene editing in primary human T cells.

A Multidrug-resistant Engineered CAR T Cell for Allogeneic Combination Immunotherapy.

The adoptive transfer of chimeric antigen receptor (CAR) T cell represents a highly promising strategy to fight against multiple cancers. The clinical outcome of such therapies is intimately linked to the ability of effector cells to engraft, proliferate, and specifically kill tumor cells within patients. When allogeneic CAR T-cell infusion is considered, host versus graft and graft versus host reactions must be avoided to prevent rejection of adoptively transferred cells, host tissue damages and to elicit significant antitumoral outcome.

Optimized tuning of TALEN specificity using non-conventional RVDs.

A key feature when designing DNA targeting tools and especially nucleases is specificity. The ability to control and tune this important parameter represents an invaluable advance to the development of such molecular scissors. Here, we identified and characterized new non-conventional RVDs (ncRVDs) that possess novel intrinsic targeting specificity features.

Efficient strategies for TALEN-mediated genome editing in mammalian cell lines.

TALEN is one of the most widely used tools in the field of genome editing. It enables gene integration and gene inactivation in a highly efficient and specific fashion. Although very attractive, the apparent simplicity and high success rate of TALEN could be misleading for novices in the field of gene editing.

Exploring the transcription activator-like effectors scaffold versatility to expand the toolbox of designer nucleases.

Background: The past decade has seen the emergence of several molecular tools that render possible modification of cellular functions through accurate and easy addition, removal, or exchange of genomic DNA sequences. Among these technologies, transcription activator-like effectors (TALE) has turned out to be one of the most versatile and incredibly robust platform for generating targeted molecular tools as demonstrated by fusion to various domains such as transcription activator, repressor and nucleases.

Results: In this study, we generated a novel nuclease architecture based on the transcription activator-like effector scaffold.

Efficient design of meganucleases using a machine learning approach.

Background: Meganucleases are important tools for genome engineering, providing an efficient way to generate DNA double-strand breaks at specific loci of interest. Numerous experimental efforts, ranging from in vivo selection to in silico modeling, have been made to re-engineer meganucleases to target relevant DNA sequences.

Results: Here we present a novel in silico method for designing custom meganucleases that is based on the use of a machine learning approach.

Comprehensive analysis of the specificity of transcription activator-like effector nucleases.

A key issue when designing and using DNA-targeting nucleases is specificity. Ideally, an optimal DNA-targeting tool has only one recognition site within a genomic sequence. In practice, however, almost all designer nucleases available today can accommodate one to several mutations within their target site.

Gene correction of a duchenne muscular dystrophy mutation by meganuclease-enhanced exon knock-in.

Duchenne muscular dystrophy (DMD) is a severe inherited, muscle-wasting disorder caused by mutations in the DMD gene. Gene therapy development for DMD has concentrated on vector-based DMD minigene transfer, cell-based gene therapy using genetically modified adult muscle stem cells or healthy wild-type donor cells, and antisense oligonucleotide-induced exon-skipping therapy to restore the reading frame of the mutated DMD gene. This study is an investigation into DMD gene targeting-mediated correction of deletions in human patient myoblasts using a target-specific meganuclease (MN) and a homologous recombination repair matrix.

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

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 endonuclease-mediated 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.

Context dependence between subdomains in the DNA binding interface of the I-CreI homing endonuclease.

Homing endonucleases (HE) have emerged as precise tools for achieving gene targeting events. Redesigned HEs with tailored specificities can be used to cleave new sequences, thereby considerably expanding the number of targetable genes and loci. With HEs, as well as with other protein scaffolds, context dependence of DNA/protein interaction patterns remains one of the major limitations for rational engineering of new DNA binders.

Generation of redesigned homing endonucleases comprising DNA-binding domains derived from two different scaffolds.

Homing endonucleases have become valuable tools for genome engineering. Their sequence recognition repertoires can be expanded by modifying their specificities or by creating chimeric proteins through domain swapping between two subdomains of different homing endonucleases. Here, we show that these two approaches can be combined to create engineered meganucleases with new specificities.

Engineered I-CreI derivatives cleaving sequences from the human XPC gene can induce highly efficient gene correction in mammalian cells.

Meganucleases are sequence-specific endonucleases which recognize large (>12 bp) target sites in living cells and can stimulate homologous gene targeting by a 1000-fold factor at the cleaved locus. We have recently described a combinatorial approach to redesign the I-CreI meganuclease DNA-binding interface, in order to target chosen sequences. However, engineering was limited to the protein regions shown to directly interact with DNA in a base-specific manner.

A combinatorial approach to create artificial homing endonucleases cleaving chosen sequences.

Meganucleases, or homing endonucleases (HEs) are sequence-specific endonucleases with large (>14 bp) cleavage sites that can be used to induce efficient homologous gene targeting in cultured cells and plants. These findings have opened novel perspectives for genome engineering in a wide range of fields, including gene therapy. However, the number of identified HEs does not match the diversity of genomic sequences, and the probability of finding a homing site in a chosen gene is extremely low.

Large-scale gene discovery in the pea aphid Acyrthosiphon pisum (Hemiptera).

Aphids are the leading pests in agricultural crops. A large-scale sequencing of 40,904 ESTs from the pea aphid Acyrthosiphon pisum was carried out to define a catalog of 12,082 unique transcripts. A strong AT bias was found, indicating a compositional shift between Drosophila melanogaster and A.