Evolving Adeno-Associated Virus for Editing T-Lymphocytes

Abstract

Adeno-Associated Virus (AAV) is a gene therapy vector with immense clinical importance. However, its use as a template for homology directed repair has come under greater examination particularly for the generation of site-specific recombined Chimeric antigen receptor or CAR T-cells. This is because traditional CAR T-cells generated from retro- or lentiviral vectors have risks for insertional oncogenesis or exhaustion from tonic signaling due to use of a constitutively active promoter, both problems which AAV directed knockins may overcome. In fact, the use of site-specific knockin CAR T-cells have now entered clinical trials for the treatment of CD19+ blood-borne cancers. While the use of these next-gen AAV generated CARs have excelled for liquid tumors, their use for the treatment of solid tumors has lagged. This is due to poor preclinical modeling for solid-tumor directed CARs which take place in immunocompromised mouse models that do not fully recapitulate the tumor microenvironment known to be problematic for infiltrating lymphocytes. Thus, there is a clear need to evaluate these therapeutics in immunocompetent hosts, however, there exist no known AAV serotype that can effectively target murine T-lymphocytes to generate these site-specific knockins. To ameliorate this problem, we employed a capsid evolution from the AAV6 background to generate a murine T-lymphocyte tropic AAV variant dubbed Ark313. Ark313 is vastly superior to the parent serotype in transducing, gene editing and site-specific knockins in murine T-cells. To characterize how this was happening, we employed a genome wide CRISPR knockout screen in murine primary T-cells to reveal the essential factor for Ark313 transduction to be Qa-2, a non-classical MHC-1b molecule. Due to the restricted tissue expression of the Qa-2 antigen, we injected mice systemically with Ark313 and saw it could transduce up to 25% of spleen resident T-cells including naïve/memory/effector subsets when using a self-complementary transgene. Additionally, Ark313 displayed a liver de-targeted tropism reducing potential off target tissue transduction when employing an ubiquitous promoter. Together we have generated a novel tool for the facile genetic manipulation of murine T-cells both ex and in vivo. We believe Ark313 will be a fundamental reagent to employ when interrogating T-lymphocyte immunotherapeutic questions and for investigating immune basic biology. This work lays the groundwork for the development of human lymphocyte targeting AAVs for generating CARs to combat liquid and solid tumors via systemic dosing.