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

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.

Lentiviral protein delivery of meganucleases in human cells mediates gene targeting and alleviates toxicity.

Site-specific endonucleases can be engineered for custom recognition of any genetic locus and used for gene targeting. Yet, the prolonged expression and accumulation of these nucleases in cells lead to toxic effect. Here we describe an efficient and quantitative method for introducing nucleases into cells as proteins packaged within lentiviral vector particles. I-CreI-derived meganucleases, which can be engineered as single-chain proteins, were incorporated into lentiviral vector particles either without modification or as fusions with cyclophilin A. The small amount of nuclease delivered by the viral particles is sufficient to induce efficient targeted mutagenesis in human HEK293H and primary T cells. When a repair template sequence was packaged in the lentiviral vector, high levels of homologous gene targeting were obtained and toxicity was markedly reduced.

Variable correction of Artemis deficiency by I-Sce1-meganuclease-assisted homologous recombination in murine hematopoietic stem cells.

The correction of genetic mutations by homologous recombination is an attractive approach to gene therapy. We used the DNA double-strand breaks introduced by the site-specific endonuclease I-Sce1 as a means of increasing homologous recombination of an exogenous DNA template in murine hematopoietic stem cells (mHSCs). To develop this approach, we chose an Artemis knockout (Art(-/-)) mouse in which exon 12 of the Artemis gene had been replaced by an I-Sce1 recognition site. The I-Sce1 enzyme and the Artemis correction template were each delivered by a self-inactivating (SIN)-integrase-defective lentiviral vector (SIN-IDLV-CMV-ISce1 and SIN-IDLV-Art, respectively). Transduction of Art(-/-) mHSCs with the two vectors successfully reverted the Art(-/-) phenotype in 2 of our 10 experiments. Even though the potential for genotoxicity has yet to be evaluated, this new approach to gene editing appears to be promising. Improving the efficacy of this approach will require further technical work.

Different modes of pathogenesis in T-cell-dependent autoimmunity: clues from two TCR transgenic systems.

T lymphocytes constantly flirt with reactivity to self peptides, a price they pay for their ability to recognize foreign peptides presented by self-MHC molecules, and autoreactivity in the T compartment occasionally gives rise to autoimmune disease. Pathology from T-cell autoimmunity can manifest itself through radically different strategies, as we have observed recently in two transgenic models. In the BDC2.5 diabetes model, T cells express a transgene-encoded T-cell receptor (TCR) with reactivity against a pancreatic antigen. This leads to a massive, if often controlled, infiltration of the pancreatic islets. Target cell destruction then results from the local consequences of this local immune/inflammatory process. On the other hand, the arthritic manifestations of the KRN transgenic model are indirect: the transgenic TCR confers a broad autoreactivity, through which T cells stimulate B cells to produce arthritogenic immunoglobulins. These molecules are then sufficient to produce the disease, even in the complete absence of any lymphocytes. Although important questions subsist in this model--how the KRN T cells interfere with B-cell tolerance, what the target of arthritogenic IgG is--its implication is that an isolated T-cell dysregulation may manifest itself through an Ig-mediated disease.