An evolved AAV variant enables efficient genetic engineering of murine T cells

Precise targeting of large transgenes to T cells using homology-directed repair has been transformative for adoptive cell therapies and T cell 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 T cells employed in immunodeficient mouse models. To enable targeted knockins in murine T cells, we evolved Ark313, a synthetic AAV that exhibits high transduction efficiency in murine T cells. We performed a genome-wide knockout screen and identified QA2 as an essential factor for Ark313 infection. We demon-strate 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 T cells holds great potential for improved cell therapies and opens avenues in experimental T cell immunology.

Automated summary

The precise targeting of large transgenes to T cells using homology-directed repair has been significant for adoptive cell therapies and T cell biology. The use of adeno-associated virus (AAV) for delivery of DNA templates has improved knockin efficiencies, but current AAV serotypes have limited tropism in human T cells used in immunodeficient mouse models. To overcome this, Ark313, a synthetic AAV that exhibits high transduction efficiency in murine T cells, was developed. Ark313 enables targeted knockins in murine T cells, allowing for preclinical modeling of Trac-targeted CAR-T and transgenic TCR-T cells in immunocompetent models. Efficient gene targeting in murine T cells holds great potential for improved cell therapies and advances in experimental T cell immunology.