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Chimeric antigen receptors (CARs) are synthetic receptors that redirect and reprogram T cells to mediate tumor rejection. CD19 targeted CAR T cells have showed remarkable effacy for chemorefractory/relapsed B cell malignancies. Recombinant retroviral vectors mediated- CAR gene transfer is currently the standard method to generate cGMP grade CAR T cells. However, CAR gene expression is highly variable, owing to position effects and vector incorporation variations. CAR T cells engineered in this manner are capable of tumor eradication, but are prone to tonic signaling and accelerated exhaustion. We have developed a novel genetic engineering strategy to insert CAR genes into a precise genomic location in human peripheral blood T cells. T cells are electroporated with Cas9 mRNA or Cas9 protein and a guide RNA, followed by transduction with a recombinant Adeno-Associated Virus encoding the CAR sequence to facilitate the insertion of the CAR gene upstream of the constant region of TCR alpha chain. This results in the endogenous TCR promoter (TRAC) controlled CAR expression and abrogation of TCR surface expression. This strategy not only allows uniform CAR expression, but also delays T-cell differentiation and exhaustion, leading to enhanced T cell function and anti-tumor efficacy. The edited cells vastly outperformes retrovirally modified CAR T cells in a pre-B ALL NALM6 mouse model. The targeting of CARs to the TCR locus also provides a safer therapeutic T cell by minimizing the risks of insertional oncogenesis, and TCR-induced autoimmunity and alloreactivity (thus spanning both autologous and allogeneic T cell applications). In addition, we have incorporated in the CAR a mutant CD3z chain encoding a single immunoreceptor tyrosine-based activation motif that improves the balance between effector and memory T cells composition. We are in the process of translating this novel approach into the clinical setting by establishing cGMP conditions and protocols for the manufacturing of TRAC-CAR T cells. Using the 4D-LV electroporator, we have demonstrated that we can knockout the TCR at large scale (100 E06 CD3+ T cells) with high efficiency (70-80%) as that obtained at small scale. We have evaluated AAV6 to deliver the CAR transgene in the TCR locus. Data will be presented on optimizing the manufacturing of the TRAC-CAR T cells and on evaluating their anti-tumor efficacy in vivo. [ABSTRACT FROM AUTHOR]