Chimeric antigen receptor (CAR) T-cell (CAR-T) therapies present options for patients diagnosed with certain leukemias. Recent advances of the technology included a method to integrate the CAR into the T-cell receptor alpha constant (TRAC) locus to take advantage of the endogenous promoter and regulatory elements for CAR expression. This method used adeno-associated viral (AAV) vectors based on AAV6 to deliver the donor template encoding the CAR construct. Since the original publication, improvements have been made to this targeted CAR integration technique, however, none of those techniques focused on improving the AAV vector used to deliver the therapeutic cargo. The herein presented study developed a novel AAV capsid directed evolution platform that allows to specifically select for novel AAV capsid variants that enable more efficient targeted gene editing-mediated CAR construct integration into the TRAC locus in primary T-cells. Using this new platform, we selected several novel AAVs that enable more efficient editing in T-cells than AAV6. Two novel capsids, AAV-T1 and AAV-T2, were able to mediate five-fold improvement for on-target knock-in, which resulted in five-fold reduction of the vector dose to produce highly cytolytic T-cells against a brain tumor cell line.
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