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.

Preclinical Evidence of an Allogeneic Dual CD20xCD22 CAR to Target a Broad Spectrum of Patients with B-cell Malignancies.

Despite the remarkable success of autologous chimeric antigen receptor (CAR) T cells, some patients relapse due to tumor antigen escape and low or uneven antigen expression, among other mechanisms. Therapeutic options after relapse are limited, emphasizing the need to optimize current approaches. In addition, there is a need to develop allogeneic "off-the-shelf" therapies from healthy donors that are readily available at the time of treatment decision and can overcome limitations of current autologous approaches.

Allogeneic TCRαβ deficient CAR T-cells targeting CD123 in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a disease with high incidence of relapse that is originated and maintained from leukemia stem cells (LSCs). Hematopoietic stem cells can be distinguished from LSCs by an array of cell surface antigens such as CD123, thus a candidate to eliminate LSCs using a variety of approaches, including CAR T cells. Here, we evaluate the potential of allogeneic gene-edited CAR T cells targeting CD123 to eliminate LSCs (UCART123).

Targeting CD123 in blastic plasmacytoid dendritic cell neoplasm using allogeneic anti-CD123 CAR T cells.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare hematologic malignancy with poor outcomes with conventional therapy. Nearly 100% of BPDCNs overexpress interleukin 3 receptor subunit alpha (CD123). Given that CD123 is differentially expressed on the surface of BPDCN cells, it has emerged as an attractive therapeutic target.

Gene editing of PKLR gene in human hematopoietic progenitors through 5' and 3' UTR modified TALEN mRNA.

Pyruvate Kinase Deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene, which encodes the erythroid specific Pyruvate Kinase enzyme. Erythrocytes from PKD patients show an energetic imbalance and are susceptible to hemolysis. Gene editing of hematopoietic stem cells (HSCs) would provide a therapeutic benefit and improve safety of gene therapy approaches to treat PKD patients.

TALEN-mediated genetic inactivation of the glucocorticoid receptor in cytomegalovirus-specific T cells.

Cytomegalovirus (CMV) infection is responsible for substantial morbidity and mortality after allogeneic hematopoietic stem cell transplant. T-cell immunity is critical for control of CMV infection, and correction of the immune deficiency induced by transplant is now clinically achievable by the adoptive transfer of donor-derived CMV-specific T cells. It is notable, however, that most clinical studies of adoptive T- cell therapy exclude patients with graft-versus-host disease (GVHD) from receiving systemic corticosteroid therapy, which impairs cellular immunity.

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.

Pre-TCRα supports CD3-dependent reactivation and expansion of TCRα-deficient primary human T-cells.

Chimeric antigen receptor technology offers a highly effective means for increasing the anti-tumor effects of autologous adoptive T-cell immunotherapy, and could be made widely available if adapted to the use of allogeneic T-cells. Although gene-editing technology can be used to remove the alloreactive potential of third party T-cells through destruction of either the α or β T-cell receptor (TCR) subunit genes, this approach results in the associated loss of surface expression of the CD3 complex. This is nonetheless problematic as it results in the lack of an important trophic signal normally mediated by the CD3 complex at the cell surface, potentially compromising T-cell survival in vivo, and eliminating the potential to expand TCR-knockout cells using stimulatory anti-CD3 antibodies.

megaTALs: a rare-cleaving nuclease architecture for therapeutic genome engineering.

Rare-cleaving endonucleases have emerged as important tools for making targeted genome modifications. While multiple platforms are now available to generate reagents for research applications, each existing platform has significant limitations in one or more of three key properties necessary for therapeutic application: efficiency of cleavage at the desired target site, specificity of cleavage (i.e.

Targeted gene therapy of xeroderma pigmentosum cells using meganuclease and TALEN™.

Xeroderma pigmentosum group C (XP-C) is a rare human syndrome characterized by hypersensitivity to UV light and a dramatic predisposition to skin neoplasms. XP-C cells are deficient in the nucleotide excision repair (NER) pathway, a complex process involved in the recognition and removal of DNA lesions. Several XPC mutations have been described, including a founder mutation in North African patients involving the deletion of a TG dinucleotide (ΔTG) located in the middle of exon 9.

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.

Coupling endonucleases with DNA end-processing enzymes to drive gene disruption.

Targeted DNA double-strand breaks introduced by rare-cleaving designer endonucleases can be harnessed for gene disruption applications by engaging mutagenic nonhomologous end-joining DNA repair pathways. However, endonuclease-mediated DNA breaks are often subject to precise repair, which limits the efficiency of targeted genome editing. To address this issue, we coupled designer endonucleases to DNA end-processing enzymes to drive mutagenic break resolution, achieving up to 25-fold enhancements in gene disruption rates.

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.

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.

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

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.

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

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.

Impaired gametogenesis in mice that overexpress the RNA-binding protein HuR.

A series of experiments, using cell culture models or in vitro assays, has shown that the RNA-binding protein HuR increases the half-life of some messenger RNAs that contain adenylate/uridylate-rich decay elements. However, its function in an integrated system has not yet been investigated. Here, using a mouse model, we report that misregulation of HuR, due to expression of an HuR transgene, prevents the production of fully functional gametes.