{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,6,19]],"date-time":"2026-06-19T21:32:56Z","timestamp":1781904776364,"version":"3.54.5"},"posted":{"date-parts":[[2026,5,15]]},"group-title":"Cancer Biology","reference-count":48,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,5,15]],"date-time":"2026-05-15T00:00:00Z","timestamp":1778803200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["F31 AI186545"],"award-info":[{"award-number":["F31 AI186545"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Kentucky IDeA Networks of Biomedical Research Excellence (KY INBRE) grant","award":["P20GM103436"],"award-info":[{"award-number":["P20GM103436"]}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health","doi-asserted-by":"crossref","award":["S10OD034432-01"],"award-info":[{"award-number":["S10OD034432-01"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,5,15]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>Lentiviral vectors are commonly used to introduce chimeric antigen receptor transgenes into T cells, but routine assays quantify vector copy number or integration sites without sequencing full-length integrated vectors. HIV-1 proviruses often acquire large deletions and cytidine deaminase-driven hypermutation; whether similar variation occurs in therapeutic lentiviral vectors is unclear. We adapted a novel long-read capture approach to enrich long fragments spanning vector DNA and adjacent human sequence, enabling simultaneous integration-site mapping and proviral integrity analysis with single-molecule resolution. In research-grade CAR T cells produced with an experimental, transient-transfection lentiviral vector workflow, 40% of integrated vectors carried recurrent deletions that removed the internal promoter or parts of the chimeric antigen receptor cassette. The dominant promoter deletion was present in the viral stock. In clinical chimeric antigen receptor T cell products, promoter deletions were less frequent, but detectable pre-infusion and post-infusion. Across datasets we observed widespread G-to-A substitutions consistent with restriction factor editing, including changes predicted to introduce premature stop codons within the transgene open reading frame. Our method reveals proviral variants invisible to standard quality-control assays and provides a framework to improve vector production and monitor transgene integrity in clinical products.<\/jats:p>","DOI":"10.64898\/2026.05.13.724601","type":"posted-content","created":{"date-parts":[[2026,5,16]],"date-time":"2026-05-16T02:20:15Z","timestamp":1778898015000},"source":"Crossref","is-referenced-by-count":1,"title":["LVV SMRTcap reveals extensive proviral variation in lentiviral vector-transduced CAR T cells"],"prefix":"10.64898","author":[{"given":"Catherine W.","family":"Kaiser","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, University of Louisville, Louisville, KY, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ghazal S.","family":"Mehs","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, University of Louisville, Louisville, KY, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Erin M.","family":"Elliott","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, University of Louisville, Louisville, KY, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joanna E.","family":"Mroczkowska","sequence":"additional","affiliation":[{"name":"Department of Pediatrics Division of Hematology, Oncology and Stem Cell Transplantation, University of Louisville School of Medicine, Louisville, KY, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ankita","family":"Jain","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA"},{"name":"Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA"},{"name":"Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA"},{"name":"Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael","family":"Ferguson","sequence":"additional","affiliation":[{"name":"Department of Pediatrics Division of Hematology, Oncology and Stem Cell Transplantation, University of Louisville School of Medicine, Louisville, KY, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alfred L.","family":"Garfall","sequence":"additional","affiliation":[{"name":"Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4740-4056","authenticated-orcid":false,"given":"Frederic D.","family":"Bushman","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joseph A.","family":"Fraietta","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA"},{"name":"Center for Cellular Immunotherapies, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA"},{"name":"Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA"},{"name":"Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3487-6572","authenticated-orcid":false,"given":"Eric C.","family":"Rouchka","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, University of Louisville, Louisville, KY, USA"},{"name":"Kentucky Biomedical Research Infrastructure Network Data Science Core, University of Louisville, Louisville, KY, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Melissa L.","family":"Smith","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Molecular Genetics, University of Louisville, Louisville, KY, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"issue":"8","key":"2026051910001643000_2026.05.13.724601v1.1","doi-asserted-by":"crossref","first-page":"1741","DOI":"10.1016\/j.ymthe.2017.06.005","article-title":"A fourth generation lentiviral vector: Simplifying genomic gymnastics","volume":"25","year":"2017","journal-title":"Mol Ther"},{"key":"2026051910001643000_2026.05.13.724601v1.2","doi-asserted-by":"publisher","DOI":"10.1038\/s41375-018-0106-0"},{"key":"2026051910001643000_2026.05.13.724601v1.3","doi-asserted-by":"publisher","DOI":"10.1002\/bit.28498"},{"key":"2026051910001643000_2026.05.13.724601v1.4","doi-asserted-by":"publisher","DOI":"10.1038\/s41571-023-00754-1"},{"key":"2026051910001643000_2026.05.13.724601v1.5","doi-asserted-by":"publisher","DOI":"10.1182\/blood-2014-12-580068"},{"key":"2026051910001643000_2026.05.13.724601v1.6","unstructured":"FDA Investigating Serious Risk of T-cell Malignancy Following BCMA-Directed or CD19-Directed Autologous Chimeric Antigen Receptor (CAR) T cell Immunotherapies [Article]. Vaccines, Blood & Biologics, Safety & Availability [updated 11\/28\/2023. Available from: https:\/\/www.fda.gov\/vaccines-blood-biologics\/safety-availability-biologics\/fda-investigating-serious-risk-t-cell-malignancy-following-bcma-directed-or-cd19-directed-autologous."},{"issue":"40","key":"2026051910001643000_2026.05.13.724601v1.7","doi-asserted-by":"crossref","first-page":"eadg9959","DOI":"10.1126\/sciadv.adg9959","article-title":"Integrome signatures of lentiviral gene therapy for SCID-X1 patients","volume":"9","year":"2023","journal-title":"Sci Adv"},{"key":"2026051910001643000_2026.05.13.724601v1.8","doi-asserted-by":"publisher","DOI":"10.1186\/s12967-022-03729-5"},{"key":"2026051910001643000_2026.05.13.724601v1.9","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.omto.2023.05.004","article-title":"Tagmentation-based analysis reveals the clonal behavior of CAR-T cells in association with lentivector integration sites","volume":"30","year":"2023","journal-title":"Mol Ther Oncolytics"},{"issue":"9","key":"2026051910001643000_2026.05.13.724601v1.10","doi-asserted-by":"crossref","first-page":"4459","DOI":"10.1016\/j.ymthe.2025.05.034","article-title":"Quantifying the mutational landscape of retroviral and lentiviral vectors in gene therapy patients","volume":"33","year":"2025","journal-title":"Mol Ther"},{"key":"2026051910001643000_2026.05.13.724601v1.11","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2013.09.020"},{"key":"2026051910001643000_2026.05.13.724601v1.12","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-019-0898-8"},{"key":"2026051910001643000_2026.05.13.724601v1.13","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1914183116"},{"key":"2026051910001643000_2026.05.13.724601v1.14","doi-asserted-by":"crossref","first-page":"62","DOI":"10.1016\/j.virol.2020.12.018","article-title":"Potential APOBEC-mediated RNA editing of the genomes of SARS-CoV-2 and other coronaviruses and its impact on their longer term evolution","volume":"556","year":"2021","journal-title":"Virology"},{"key":"2026051910001643000_2026.05.13.724601v1.15","doi-asserted-by":"publisher","DOI":"10.2217\/fvl.09.59"},{"key":"2026051910001643000_2026.05.13.724601v1.16","doi-asserted-by":"publisher","DOI":"10.1186\/s12929-022-00865-4"},{"key":"2026051910001643000_2026.05.13.724601v1.17","doi-asserted-by":"publisher","DOI":"10.1006\/mthe.2000.0094"},{"key":"2026051910001643000_2026.05.13.724601v1.18","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.72.11.8463-8471.1998"},{"issue":"2","key":"2026051910001643000_2026.05.13.724601v1.19","first-page":"673","article-title":"CD19-targeting CAR T cell immunotherapy outcomes correlate with genomic modification by vector integration","volume":"130","year":"2020","journal-title":"J Clin Invest"},{"key":"2026051910001643000_2026.05.13.724601v1.20","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.2331"},{"key":"2026051910001643000_2026.05.13.724601v1.21","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa2411507"},{"key":"2026051910001643000_2026.05.13.724601v1.22","doi-asserted-by":"publisher","DOI":"10.1038\/s41591-024-03478-6"},{"issue":"9","key":"2026051910001643000_2026.05.13.724601v1.23","doi-asserted-by":"crossref","first-page":"536","DOI":"10.1038\/s41434-022-00315-8","article-title":"Lentiviral standards to determine the sensitivity of assays that quantify lentiviral vector copy numbers and genomic insertion sites in cells","volume":"29","year":"2022","journal-title":"Gene Ther"},{"key":"2026051910001643000_2026.05.13.724601v1.24","doi-asserted-by":"crossref","first-page":"448","DOI":"10.1016\/j.omtm.2020.02.003","article-title":"Optimized assessment of qPCR-based vector copy numbers as a safety parameter for GMP-grade CAR T cells and monitoring of frequency in patients","volume":"17","year":"2020","journal-title":"Mol Ther Methods Clin Dev"},{"issue":"1","key":"2026051910001643000_2026.05.13.724601v1.25","doi-asserted-by":"crossref","first-page":"954","DOI":"10.1038\/s42003-023-05322-y","article-title":"Repetitive DNA sequence detection and its role in the human genome","volume":"6","year":"2023","journal-title":"Commun Biol"},{"issue":"1","key":"2026051910001643000_2026.05.13.724601v1.26","doi-asserted-by":"crossref","first-page":"e1013171","DOI":"10.1371\/journal.ppat.1013171","article-title":"Development and validation of HIV SMRTcap for the characterization of HIV-1 reservoirs across tissues and subtypes","volume":"22","year":"2026","journal-title":"PLoS Pathog"},{"key":"2026051910001643000_2026.05.13.724601v1.27","doi-asserted-by":"crossref","unstructured":"Serrao E , Cherepanov P , Engelman AN . Amplification, next-generation sequencing, and genomic DNA mapping of retroviral integration sites. J Vis Exp. 2016(109).","DOI":"10.3791\/53840"},{"key":"2026051910001643000_2026.05.13.724601v1.28","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(02)00864-4"},{"key":"2026051910001643000_2026.05.13.724601v1.29","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1105422108"},{"key":"2026051910001643000_2026.05.13.724601v1.30","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/bts004"},{"key":"2026051910001643000_2026.05.13.724601v1.31","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-018-0178-z"},{"key":"2026051910001643000_2026.05.13.724601v1.32","doi-asserted-by":"crossref","unstructured":"Uriu K , Kosugi Y , Ito J , Sato K . The battle between retroviruses and APOBEC3 genes: Its past and present. Viruses. 2021;13(1).","DOI":"10.3390\/v13010124"},{"issue":"4","key":"2026051910001643000_2026.05.13.724601v1.33","doi-asserted-by":"crossref","first-page":"200873","DOI":"10.1016\/j.omton.2024.200873","article-title":"APOBEC3B expression in 293T viral producer cells drives mutations in chimeric antigen receptors and reduces CAR T cell efficacy","volume":"32","year":"2024","journal-title":"Mol Ther Oncol"},{"key":"2026051910001643000_2026.05.13.724601v1.34","doi-asserted-by":"publisher","DOI":"10.1172\/JCI126397"},{"key":"2026051910001643000_2026.05.13.724601v1.35","doi-asserted-by":"publisher","DOI":"10.1101\/gr.229202. Article published online before March 2002"},{"key":"2026051910001643000_2026.05.13.724601v1.36","doi-asserted-by":"publisher","DOI":"10.1186\/s12864-015-1370-2"},{"key":"2026051910001643000_2026.05.13.724601v1.37","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/bty191"},{"key":"2026051910001643000_2026.05.13.724601v1.38","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btp352"},{"key":"2026051910001643000_2026.05.13.724601v1.39","unstructured":"Wickham H , Henry L. purrr: Functional Programming Tools. R package version 1.0.4. 2025."},{"key":"2026051910001643000_2026.05.13.724601v1.40","unstructured":"Wickham H , Girlich M. tidyr: Tidy Messy Data. 2022."},{"key":"2026051910001643000_2026.05.13.724601v1.41","unstructured":"Wickham H , Fran\u00e7ois R , Henry L , M\u00fcller K , Vaughan D. dplyr: A Grammar of Data Manipulation. R package version 1.1.4. 2023."},{"key":"2026051910001643000_2026.05.13.724601v1.42","unstructured":"Wickham H. stringr: Simple, Consistent Wrappers for Common String Operations. R package version 1.5.1. 2023."},{"key":"2026051910001643000_2026.05.13.724601v1.43","unstructured":"Team RC. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing. Vienna2023."},{"key":"2026051910001643000_2026.05.13.724601v1.44","doi-asserted-by":"crossref","unstructured":"Dohm JC , Peters P , Stralis-Pavese N , Himmelbauer H . Benchmarking of long-read correction methods. NAR Genomics and Bioinformatics. 2020;2(2).","DOI":"10.1093\/nargab\/lqaa037"},{"key":"2026051910001643000_2026.05.13.724601v1.45","doi-asserted-by":"publisher","DOI":"10.1101\/gr.148718.112"},{"key":"2026051910001643000_2026.05.13.724601v1.46","doi-asserted-by":"publisher","DOI":"10.1006\/jmbi.1990.9999"},{"key":"2026051910001643000_2026.05.13.724601v1.47","doi-asserted-by":"publisher","DOI":"10.1038\/nbt.1754"},{"key":"2026051910001643000_2026.05.13.724601v1.48","unstructured":"Scherer S. Guide to the human genome: Chromosomes and DNA. Cold Spring Harbor Laboratory Press, (n.d.) [Available from: https:\/\/www.cshlp.org\/ghg5_all\/section\/dna.shtml#:~:text=Gene%20density%20varies%20greatly%20among,x-axis%20of%20the%20figure."}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.05.13.724601","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,19]],"date-time":"2026-05-19T17:00:31Z","timestamp":1779210031000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.05.13.724601"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,15]]},"references-count":48,"URL":"https:\/\/doi.org\/10.64898\/2026.05.13.724601","relation":{},"subject":[],"published":{"date-parts":[[2026,5,15]]},"subtype":"preprint"}}