398. Site-Specific Introduction of Chimeric Antigen Receptors to Primary Human T Cells

In current clinical trials, delivery of an anti-cancer chimeric antigen receptor (CAR) to patient T cells is accomplished using randomly integrating retroviruses. Designer nucleases and AAV donor templates allow an alternative delivery method that introduces the CAR gene cassette at a target locus via homologous recombination, with concomitant disruption of the endogenous gene. Here we show high-efficiency introduction of anti-CD19 and anti-BCMA CAR expression cassettes in primary human T cells at two clinically relevant loci: CCR5 and TCRa. These gene-targeted CAR+ cells (tCARs) exhibit equivalent activity in vitro against CD19+ and BCMA+ cell lines compared to lentiviral-delivered CARs, and effectively clear tumor in a murine xenograft model. Advantages over lentiviral delivery include utilization of a defined integration site for the CAR construct linked with endogenous gene disruption. The use of specific nucleases and AAV for CAR delivery expands the possibilities for a precisely engineered cell therapy product in the clinic. For example, a CCR5-tCAR may be clinically useful in HIV+ patients, where traditional delivery methods leave therapeutic cells vulnerable to HIV infection and elimination. Moreover, a TCRa-tCAR could allow for off-the-shelf CAR therapy, potentially removing limitations to current approaches. Finally, the approach described here is broadly applicable for targeted delivery of alternative therapeutic cassettes at translationally relevant sites across the human genome.