Tailoring capsid-directed evolution technology for improved AAV-mediated CAR-T generation
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.
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BibTeX
@article{westhaus2026tailoring,
title = {{Tailoring capsid-directed evolution technology for improved AAV-mediated CAR-T generation}},
author = {Adrian Westhaus and Elena Barba-Sarasua and Yuyan Chen and Kenneth Hsu and Suzanne Scott and Maddison Knight and Florencia Haase and Santiago Mesa Mora and Benjamin Houghton and Ramon Roca-Pinilla and Predrag Kalajdzic and Geraldine O'Neill and Adrian Thrasher and Giorgia Santilli and Leszek Lisowski and Geraldine O’neill},
journal = {Molecular Therapy},
year = {2026},
month = sep,
doi = {10.1016/j.ymthe.2024.12.012},
url = {https://hal.science/hal-05772056v1},
note = {CC BY-NC-ND},
}
RIS
TY - JOUR
TI - Tailoring capsid-directed evolution technology for improved AAV-mediated CAR-T generation
AU - Adrian Westhaus
AU - Elena Barba-Sarasua
AU - Yuyan Chen
AU - Kenneth Hsu
AU - Suzanne Scott
AU - Maddison Knight
AU - Florencia Haase
AU - Santiago Mesa Mora
AU - Benjamin Houghton
AU - Ramon Roca-Pinilla
AU - Predrag Kalajdzic
AU - Geraldine O'Neill
AU - Adrian Thrasher
AU - Giorgia Santilli
AU - Leszek Lisowski
AU - Geraldine O’neill
PY - 2026
DA - 2026/09/30
JO - Molecular Therapy
DO - 10.1016/j.ymthe.2024.12.012
UR - https://hal.science/hal-05772056v1
AB - 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.
SN - 1525-0016
SN - 1525-0024
ER -