Fast, efficient and virus-free generation of TRAC-replaced CAR T cells

Chimeric Antigen Receptor (CAR) redirected T cells are a potent treatment option for certain hematological malignancies. Recently, site-specific insertion of CARs into the T cell receptor (TCR) alpha constant (TRAC) locus using gene editing and adeno-associated viruses was shown to generate CAR T cells with improved functionality over their retrovirally transduced counterparts. However, the development of viruses for gene transfer is complex and associated with extensive costs at early clinical stages. Here, we provide an economical and virus-free method for efficient CAR insertion into the TRAC locus of primary human T cells via CRISPR-Cas mediated homology-directed repair (HDR). While the toxicity induced by transfected double-stranded template (donor) DNA was not fully prevented by pharmacological means, the combination of DNA-sensor inhibitors and HDR enhancers resulted in highly efficient gene editing with TCR-to-CAR replacement rates reaching up to 68%. The resulting TCR-deficient CAR T cells show antigen-specific cytotoxicity and cytokine production in vitro. Our GMP-compatible non-viral platform technology lays the foundation for clinical trials and fast-track generation of novel CAR T cells applicable for autologous or allogeneic off-the-shelf use

Pharmacological interventions enhance virus-free generation of TRAC-replaced CAR T cells

Chimeric Antigen Receptor (CAR) redirected T-cells are potent therapeutic options against hematological malignancies. The current dominant manufacturing approach for CAR T cells depends on retroviral transduction. With the advent of gene editing, insertion of a CD19-CAR into the T cell receptor (TCR) alpha constant (TRAC) locus using adeno-associated viruses for gene transfer was demonstrated, and these CD19-CAR T-cells showed improved functionality over their retrovirally transduced counterparts. However, clinical-grade production of viruses is complex and associated with extensive costs. Here, we optimized a virus-free genome editing method for efficient CAR insertion into the TRAC locus of primary human T-cells via nuclease-assisted homology-directed repair (HDR) using CRISPR-Cas and double-stranded template DNA (dsDNA). We evaluated DNA-sensor inhibition and HDR enhancement as two pharmacological interventions to improve cell viability and relative CAR knock-in rates, respectively. While the toxicity of transfected dsDNA was not fully prevented, the combination of both interventions significantly increased CAR knock-in rates and CAR T-cell yield. Resulting TRAC-replaced CD19-CAR T-cells showed antigen-specific cytotoxicity and cytokine production in vitro and slowed leukemia progression in a xenograft mouse model. Amplicon-sequencing did not reveal significant indel formation at potential off-target sites with or without exposure to DNA-repair modulating small molecules. With TRAC-integrated CAR+ T-cell frequencies exceeding 50%, this study opens new perspectives to exploit pharmacological interventions to improve non-viral gene editing in T-cells

On the mechanisms governing gas penetration into a tokamak plasma during a massive gas injection

A new 1D radial fluid code, IMAGINE, is used to simulate the penetration of gas into a tokamak plasma during a massive gas injection (MGI). The main result is that the gas is in general strongly braked as it reaches the plasma, due to mechanisms related to charge exchange and (to a smaller extent) recombination. As a result, only a fraction of the gas penetrates into the plasma. Also, a shock wave is created in the gas which propagates away from the plasma, braking and compressing the incoming gas. Simulation results are quantitatively consistent, at least in terms of orders of magnitude, with experimental data for a D 2 MGI into a JET Ohmic plasma. Simulations of MGI into the background plasma surrounding a runaway electron beam show that if the background electron density is too high, the gas may not penetrate, suggesting a possible explanation for the recent results of Reux et al in JET (2015 Nucl. Fusion 55 093013)

Gas jet disruption mitigation studies on Alcator C-Mod and DIII-D

High-pressure noble gas jet injection is a mitigation technique which potentially satisfies the requirements of fast response time and reliability, without degrading subsequent discharges. Previously reported gas jet experiments on DIII-D showed good success at reducing deleterious disruption effects. In this paper, results of recent gas jet disruption mitigation experiments on Alcator C-Mod and DIII-D are reported. Jointly, these experiments have greatly improved the understanding of gas jet dynamics and the processes involved in mitigating disruption effects. In both machines, the sequence of events following gas injection is observed to be quite similar: the jet neutrals stop near the plasma edge, the edge temperature collapses and large MHD modes are quickly destabilized, mixing the hot plasma core with the edge impurity ions and radiating away the plasma thermal energy. High radiated power fractions are achieved, thus reducing the conducted heat loads to the chamber walls and divertor. A significant (2 × or more) reduction in halo current is also observed. Runaway electron generation is small or absent. These similar results in two quite different tokamaks are encouraging for the applicability of this disruption mitigation technique to ITER.United States. Dept. of Energy (Coop. Agreements DE-FC02-99ER54512 and DE-FC02-04ER54698, Grants DE-FG02-04ER54758 and DE-FG02-04ER54762, and Contracts DE-AC05-00OR22725, W-7405-ENG-48, and W-7405-ENG-36