Multiplex Cripsr/Cas9 Genome Editing to Generate Potent Universal CART and PD1-Deficient Cells Against Leukemia (2015)

Abstract Engineered CAR T (CART) cell treatments of cancer patients have shown promising results. The majority of current CART clinical trials utilizes autologous T cells and might therefore be hampered by the poor quality and quantity of T cells as well as the time and expense of manufacturing autologous T cell products. These limitations would be circumvented by the use of allogeneic T cells. However, the endogenous TCR on allogeneic T cells may recognize the alloantigens of the recipient, leading to graft-versus-host disease (GVHD); furthermore, the expression of HLA on the surface of allogeneic T cells causes rapid rejection by the host immune system. Therefore, simple and efficient methods are needed for multiplex genomic editing of T cells. The CRISPR/Cas9 system has recently emerged as a potentially robust alternative for inducing targeted genetic alterations and as a process for multiplex genome engineering. In the present study, by using CRISPR/Cas9 system to simultaneously disrupt multiple genomic loci. we have generated CART cells deficient in the expression of endogenous TCR, HLA class I (HLA-I) and PD1 that can be used as allogeneic universal CART cells. We found that TCR α chain, TCR β chain, beta-2 microglobulin (B2M) and PD1 genes could be disrupted with high efficiency through the co-introduction of mRNA encoding the Cas9 with gRNAs targeting these genes by electroporation. We observed over 80% percent gene ablation at protein levels by single gene disruption and over 70% percent of TCR and HLA-I double ablation by multiplex gene disruption. Universal PD1 deficient CD19 CART cells could be generated by integrating this multiple gene editing technology into to the standard clinical CD19 CAR lentiviral transduction procedure and expanded to the clinical scale. The TCR/HLA-I double-negative CD19 CART showed robust in vitro anti-tumor activities, such as lytic capacity, cytokine secretion and proliferation, as potent as those of the wild-type CD19 CART cells. The T cells were tested in NSG mice bearing disseminated Nalm6 leukemia. Mice treated with CD19 CART cells with endogenous TCR disrupted or with a simultaneous disruption of TCR and HLA-I exhibited tumor regression similar to that of mice treated with wild-type CD19 CART cells, suggesting that the disruption of TCR alone or together with B2M does not affect CART cell anti-tumor activity. In addition, we found that simultaneous disruption of endogenous PD1 enhances the efficacy of the universal CD19 CART therapy in vivo in a Nalm6-PDL1 leukemia model. The universal T cells we generated showed reduced alloreactivity, demonstrated in a one-way MLR using an IFNg Elispot assay when incubated with irradiated allogeneic PBMCs. The GVHD effect of the engineered T cells was also tested in different NSG models. In a Nalm6 leukemia NSG model, the mice treated with the double or triple knock out CAR T cells did not develop any signs of GVHD. By contrast, 3 out of 4 mice from the wild-type CD19 CART group developed xenogeneic GVHD at day 65, which was confirmed by histological examination of different organs. In another experiment, T cells were infused intravenously into sub-lethally irradiated mice. GVHD was monitored 2 to 3 times per week, and 4 out 5 mice receiving wild type T cell developed GVHD, while PBS treated, TCR single and TCR/HLA-I double ablated T cell treated group did not show any signs of GVHD. In summary clinical scale universal CD19 CART cells, with potent anti-leukemia activity and reduced alloreactivity can be efficiently generated using multiplex CRISPR/Cas9 technology. This approach can be incorporated into current GMP-compliant manufacturing procedures and has a high potential for translation, and thus provides an alternative to autologous CART cells. Disclosures Ren: University of Pennsylevania: Patents & Royalties. Liu:University of Pennsylevania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight. June:University of Pennsylvania: Patents & Royalties: financial interests due to intellectual property and patents in the field of cell and gene therapy. Conflicts of interest are managed in accordance with University of Pennsylvania policy and oversight; Novartis: Research Funding. Zhao:Novartis: Patents & Royalties, Research Funding.