Viral vector-free generation of orthogonal IL-2-responsive CAR T cells through gene editing of IL-2 and its receptor.

Building on the enhancing effects of the orthogonal interleukin-2 (oIL-2) system for T-cell immunotherapy, we explored a viral vector-free strategy for generating orthogonal chimeric antigen receptor (orthoCAR) T cells. We show that prime editing can generate orthogonal IL-2Rβ (oIL-2Rβ) mutations in T cells with an average efficiency of 72% (range, 54%-89%). The mutations are functional, enrich ex vivo when cultured with oIL-2 and enhance the engraftment, efficacy, and toxicity of CAR T cells in vivo comparable to orthoCAR T cells generated by cotransduction of the oIL-2Rβ and CAR. We further show that the insertion of a CD19-specific CAR with a chicken β-globin promoter into the IL-2 locus (IL-2-CAR19) can be achieved by a CRISPR/CRISPR-associated protein 9 (Cas9)-triggered homology-directed repair with an average efficiency of 46% (range, 30%-65%) in primary T cells. IL-2-CAR19 T cells show a >3-log reduction in IL-2 production consistent with a >96% IL-2 gene disruption. Despite the loss of autocrine IL-2 secretion, IL-2-CAR19 T cells rapidly and potently kill NALM-6 leukemic cells in vitro and in vivo without the need for exogenous IL-2 support. Combining the prime editing of oIL-2Rβ with IL-2-CAR19 T cells yields orthoCAR T cells that exhibit in vitro and in vivo function comparable to lentivirally generated orthoCAR T cells. The prime editing of oIL-2Rβ expands the potential application of the orthogonal IL-2 system to existing T-cell therapies. Moreover, the robust functional activity of IL-2-CAR19 cells highlights the dispensable nature of the IL-2 gene in CAR T cells and its availability for the target insertion of genetic payloads.