Abstract
Precise targeting of large transgenes to Tcells using homology-directed repair has been transformative for adoptive cell therapies and Tcell biology. Delivery of DNA templates via adeno-associated virus (AAV) has greatly improved knockin efficiencies, but the tropism of current AAV serotypes restricts their use to human Tcells employed in immunodeficient mouse models. To enable targeted knockins in murine Tcells, we evolved Ark313, a synthetic AAV that exhibits high transduction efficiency in murine Tcells. We performed a genome-wide knockout screen and identified QA2 as an essential factor for Ark313 infection. We demonstrate that Ark313 can be used for nucleofection-free DNA delivery, CRISPR-Cas9-mediated knockouts, and targeted integration of large transgenes. Ark313 enables preclinical modeling of Trac-targeted CAR-T and transgenic TCR-T cells in immunocompetent models. Efficient gene targeting in murine Tcells holds great potential for improved cell therapies and opens avenues in experimental Tcell immunology.
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