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

Introduction

La molteplicità delle cause nel pensiero greco dopo Aristotele

High resolution simulation of recent Arctic and Antarctic stratospheric chemical ozone loss compared to observations

Simulations of polar ozone losses were performed using the three-dimensional high-resolution (1° × 1°) chemical transport model MIMOSA-CHIM. Three Arctic winters 1999–2000, 2001–2002, 2002–2003 and three Antarctic winters 2001, 2002, and 2003 were considered for the study. The cumulative ozone loss in the Arctic winter 2002–2003 reached around 35% at 475K inside the vortex, as compared to more than 60% in 1999–2000. During 1999–2000, denitrification induces a maximum of about 23% extra ozone loss at 475K as compared to 17% in 2002–2003. Unlike these two colder Arctic winters, the 2001–2002 Arctic was warmer and did not experience much ozone loss. Sensitivity tests showed that the chosen resolution of 1° ×1° provides a better evaluation of ozone loss at the edge of the polar vortex in high solar zenith angle conditions. The simulation results for ozone, ClO, HNO3, N2O, and NOy for winters 1999–2000 and 2002–2003 were compared with measurements on board ER-2 and Geophysica aircraft respectively. Sensitivity tests showed that increasing heating rates calculated by the model by 50% and doubling the PSC (Polar Stratospheric Clouds) particle density (from 5 × 10-3 to 10-2 cm-3) refines the agreement with in situ ozone, N2O and NOy levels. In this configuration, simulated ClO levels are increased and are in better agreement with observations in January but are overestimated by about 20% in March. The use of the Burkholder et al. (1990) Cl2O2 absorption cross-sections slightly increases further ClO levels especially in high solar zenith angle conditions. Comparisons of the modelled ozone values with ozonesonde measurement in the Antarctic winter 2003 and with Polar Ozone and Aerosol Measurement III (POAM III) measurements in the Antarctic winters 2001 and 2002, shows that the simulations underestimate the ozone loss rate at the end of the ozone destruction period. A slightly better agreement is obtained with the use of Burkholder et al. (1990) Cl2O2 absorption cross-sections

Uncertainties in modeling heterogeneous chemistry and Arctic ozone depletion in the winter 2009/2010

Stratospheric chemistry and denitrification are simulated for the Arctic winter 2009/2010 with the Lagrangian Chemistry and Transport Model ATLAS. A number of sensitivity runs is used to explore the impact of uncertainties in chlorine activation and denitrification on the model results. In particular, the efficiency of chlorine activation on different types of liquid aerosol versus activation on nitric acid trihydrate clouds is examined. Additionally, the impact of changes in reaction rate coefficients, in the particle number density of polar stratospheric clouds, in supersaturation, temperature or the extent of denitrification is investigated. Results are compared to satellite measurements of MLS and ACE-FTS and to in-situ measurements onboard the Geophysica aircraft during the RECONCILE measurement campaign. It is shown that even large changes in the underlying assumptions have only a small impact on the modelled ozone loss, even though they can cause considerable differences in chemical evolution of other species and in denitrification. Differences in column ozone between the sensitivity runs stay below 10% at the end of the winter. Chlorine activation on liquid aerosols alone is able to explain the observed magnitude and morphology of the mixing ratios of active chlorine, reservoir gases and ozone. This is even true for binary aerosols (no uptake of HNO3 from the gas-phase allowed in the model). Differences in chlorine activation between sensitivity runs are within 30%. Current estimates of nitric acid trihydrate (NAT) number density and supersaturation imply that, at least for this winter, NAT clouds play a relatively small role compared to liquid clouds in chlorine activation. The change between different reaction rate coefficients for liquid or solid clouds has only a minor impact on ozone loss and chlorine activation in our sensitivity runs

Murine pre-eclampsia induced by unspecific activation of the immune system correlates with alterations in the eNOS and AT1 receptor expression in the kidneys and placenta

It remains arguable if an animal model can be of use in pre-eclampsia (PE) studies, as it is clearly a human disease not observed spontaneously in other species. The aim of this study was to investigate whether PE-like signs in mice inoculated with activated Th1 cells were accompanied by abnormal expression of molecules related to the regulation of blood pressure, viz. nitric oxide synthase enzymes (eNOS and iNOS) and angiotensin (Ang) II receptors (AT1R and AT2R), in order to analyse the relevance of this model for human disease. In this model, C57/BL6-mated BALB/c females received lymphocytes crosslined with anti-CD3 and cultured with interleukin (IL)-2 and IL-12 to mimic PE pathology. Control mice received PBS. eNOS, iNOS and AT1R but not AT2R expression was augmented in the kidneys of PE-mice compared with control pregnant mice. The expression of eNOS but not of iNOS was augmented at the fetal-maternal interface of PE-mice as compared with the controls. NOSs regulate the synthesis of NO, a blood pressure and parturition mediator. As its expression is increased in PE patients, our data suggest that the Th1 cells-induced signs in this model are due to similar mechanisms as in humans. AT1R and AT2R mediate the effect of Ang II, and particularly the AT1R appears to be involved in the pathogenesis of human PE. The increased AT1R expression in the kidneys of PE-mice reinforces the theory that Th1 cells elicit a pathological situation closely resembling the human PE. All together, our data support the use of this animal model to study mechanisms underlying clinically overt PE

Autophagy and intracellular product degradation genes identified by systems biology analysis reduce aggregation of bispecific antibody in CHO cells

Aggregation of therapeutic bispecific antibodies negatively affects the yield, shelf-life, efficacy and safety of these products. Pairs of stable Chinese hamster ovary (CHO) cell lines produced two difficult-to-express bispecific antibodies with different levels of aggregated product (10–75% aggregate) in a miniaturised bioreactor system. Here, transcriptome analysis was used to interpret the biological causes for the aggregation and to identify strategies to improve product yield and quality. Differential expression- and gene set analysis revealed upregulated proteasomal degradation, unfolded protein response and autophagy processes to be correlated with reduced protein aggregation. Fourteen candidate genes with the potential to reduce aggregation were co-expressed in the stable clones for validation. Of these, HSP90B1, DDIT3, AKT1S1, and ATG16L1, were found to significantly lower aggregation in the stable producers and two (HSP90B1 and DNAJC3) increased titres of the anti-HER2 monoclonal antibody trastuzumab by 50% during transient expression. It is suggested that this approach could be of general use for defining aggregation bottlenecks in CHO cells