Adrian Westhaus, Elena Barba-Sarasua, Yuyan Chen, Kenneth Hsu, Suzanne Scott, Maddison Knight, Florencia Haase, Santiago Mesa Mora, Benjamin C Houghton, Ramon Roca-Pinilla, Predrag Kalajdzic, Geraldine O'Neill, Adrian J Thrasher, Giorgia Santilli, Leszek Lisowski
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Tailoring capsid directed evolution technology for improved AAV-mediated CAR-T generation.
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.
期刊介绍:
Molecular Therapy is the leading journal for research in gene transfer, vector development, stem cell manipulation, and therapeutic interventions. It covers a broad spectrum of topics including genetic and acquired disease correction, vaccine development, pre-clinical validation, safety/efficacy studies, and clinical trials. With a focus on advancing genetics, medicine, and biotechnology, Molecular Therapy publishes peer-reviewed research, reviews, and commentaries to showcase the latest advancements in the field. With an impressive impact factor of 12.4 in 2022, it continues to attract top-tier contributions.