{"doi":"10.1016/j.bict.2025.100019","title":"Viral vector-free generation of orthogonal IL-2–responsive CAR T cells through gene editing of IL-2 and its receptor","abstract":"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.","journal":"Blood Immunology & Cellular Therapy","year":2025,"id":580980,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9477,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1492019,"name":"Yihao Wu","orcid":"0000-0003-0163-8009","position":1,"is_corresponding":false},{"id":1492020,"name":"Jee‐Eun Yang","orcid":"0009-0000-0580-775X","position":2,"is_corresponding":false},{"id":860600,"name":"Kevin N. Zhou","orcid":"0000-0003-1567-3606","position":3,"is_corresponding":false},{"id":1492379,"name":"Eric Nigel Ebenezer Anand Manoharan","orcid":null,"position":4,"is_corresponding":false},{"id":1119284,"name":"Tyra Wentz","orcid":"0000-0002-1774-6349","position":5,"is_corresponding":false},{"id":625421,"name":"Leon Su","orcid":"0000-0001-7654-9997","position":6,"is_corresponding":false},{"id":230148,"name":"Ai‐Ming Yu","orcid":"0000-0003-1441-4012","position":7,"is_corresponding":false},{"id":63575,"name":"Hongyi Wang","orcid":"0000-0003-0125-6430","position":8,"is_corresponding":false},{"id":1492021,"name":"Laurakay Bruhn","orcid":"0000-0002-0708-4898","position":9,"is_corresponding":false},{"id":56499,"name":"Israel Steinfeld","orcid":null,"position":10,"is_corresponding":false},{"id":25156,"name":"Nicolas Gonzalez","orcid":null,"position":11,"is_corresponding":false},{"id":674388,"name":"Edward Z. Song","orcid":"0000-0002-4022-9753","position":12,"is_corresponding":false},{"id":1032171,"name":"Yunlin Zhang","orcid":"0000-0002-5608-0615","position":13,"is_corresponding":false},{"id":628612,"name":"Morgan Hresko","orcid":"0000-0003-1322-3791","position":14,"is_corresponding":false},{"id":1492380,"name":"Daniel E. Ryan","orcid":null,"position":15,"is_corresponding":false},{"id":660710,"name":"Nils W. Engel","orcid":"0000-0002-3514-5686","position":16,"is_corresponding":false},{"id":257344,"name":"Selene Nuñez-Cruz","orcid":null,"position":17,"is_corresponding":false},{"id":227870,"name":"Carl H. June","orcid":"0000-0003-0241-3557","position":18,"is_corresponding":false},{"id":218189,"name":"K. Christopher García","orcid":"0000-0001-9273-0278","position":19,"is_corresponding":false},{"id":242813,"name":"Michael C. 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