Publications
Molecular therapy : the journal of the American Society of Gene TherapyDec 2024 DOI:
10.1016/j.ymthe.2024.12.012

Tailoring capsid-directed evolution technology for improved AAV-mediated CAR-T generation

Westhaus, Adrian; Barba-Sarasua, Elena; Chen, Yuyan; Hsu, Kenneth; Scott, Suzanne; Knight, Maddison; Haase, Florencia; Mesa Mora, Santiago; Houghton, Benjamin C; Roca-Pinilla, Ramon; Kalajdzic, Predrag; O'Neill, Geraldine; Thrasher, Adrian J; Santilli, Giorgia; Lisowski, Leszek
Product Used
Genes
Abstract
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 for specifically selecting 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 5-fold improvement for on-target knockin, which resulted in 5-fold reduction of the vector dose to produce highly cytolytic T cells against a brain tumor cell line.
Product Used
Genes

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