In vivo engineering of murine T cells using the evolved adeno-associated virus variant Ark313

Abstract

Genetic engineering of T cells in mouse models is essential for investigating immune mechanisms. We aimed to develop an approach to manipulate T cells in vivo using an evolved adeno-associated virus (AAV) capsid named Ark313. Delivery of a transient transgene expression cassette was feasible using Ark313, and this serotype outperformed natural serotypes. A single intravenous injection of a Cre recombinase-expressing Ark313 in the Ai9 fluorescent reporter mouse model achieved permanent genetic modifications of T cells. Ark313 facilitated in vivo gene editing in both tissue-resident and splenic T cells and validation of immunotherapy targets in solid tumor models. Ark313 delivered large DNA donor templates to T cells in vivo and integrated transgenes in primary CD4⁺ and CD8⁺ T cells, including naive T cells. Ark313-mediated transgene delivery presents an efficient approach to target mouse T cells in vivo and a resource for the interrogation of T cell biology and for immunotherapy applications.