{"doi":"10.3791/68150","title":"Non-Viral Engineering of Primary Human T Cells &lt;em&gt;via&lt;/em&gt; Homology-Mediated End-Joining Targeted Integration of Large DNA Templates","abstract":"Many current adoptive cellular therapies rely on lenti- or retroviral vectors to engineer T cells for the expression of a chimeric antigen receptor (CAR) or exogenous T cell receptor (TCR) to target a specific tumor-associated antigen. Reliance on viral vectors for the production of therapeutic T cells significantly increases the timeline, cost, and complexity of manufacturing while limiting the translation of new therapies, particularly in the academic setting. A process is presented for efficient non-viral engineering of T cells using CRISPR/Cas9 and homology-mediated end joining to achieve targeted integration of large, multicistronic DNA cargo. This approach has achieved integration frequencies comparable to those of viral vectors while yielding highly functional T cells capable of potent anti-tumor efficacy both in vitro and in vivo. Notably, this method is rapidly adaptable to current good manufacturing practices (cGMP) and clinical scale-up, providing a near-term option for the manufacturing of therapeutic T cells for use in clinical trials.","journal":"Journal of Visualized Experiments","year":2025,"id":565591,"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":0.9435,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"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":1362756,"name":"Anthony P. DeFeo","orcid":null,"position":1,"is_corresponding":false},{"id":417672,"name":"Nicholas J. Slipek","orcid":"0000-0003-2664-6507","position":2,"is_corresponding":false},{"id":1226935,"name":"Timothy D. Folsom","orcid":null,"position":3,"is_corresponding":false},{"id":556683,"name":"Tom Henley","orcid":null,"position":4,"is_corresponding":false},{"id":556686,"name":"Modassir Choudhry","orcid":null,"position":5,"is_corresponding":false},{"id":254328,"name":"Beau R. Webber","orcid":"0000-0002-3950-8747","position":6,"is_corresponding":false},{"id":254329,"name":"Branden S. Moriarity","orcid":"0000-0002-3031-3767","position":7,"is_corresponding":false},{"id":6709,"name":"Matthew J. Johnson","orcid":"0000-0002-1313-1742","position":0,"is_corresponding":true}],"reference_count":0,"raw_metadata":null,"created_at":"2026-07-19T02:56:32.546082Z","pmid":"40418657","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}