{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"medRxiv"}],"indexed":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T17:09:48Z","timestamp":1789664988625,"version":"4.0.1"},"posted":{"date-parts":[[2026,9,14]]},"group-title":"Genetic and Genomic Medicine","reference-count":30,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T00:00:00Z","timestamp":1789344000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.medrxiv.org\/about\/FAQ#license"}],"funder":[{"id":[{"id":"https:\/\/ror.org\/00ax59295","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R01-AI128756"],"award-info":[{"award-number":["R01-AI128756"]}],"id":[{"id":"https:\/\/ror.org\/043z4tv69","id-type":"ROR","asserted-by":"publisher"}]},{"id":[{"id":"https:\/\/ror.org\/03rx10x31","id-type":"ROR","asserted-by":"publisher"}]},{"id":[{"id":"https:\/\/ror.org\/02r3e0967","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,14]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Sequencing of liver tissue from a patient treated with the rAAV gene therapy Zolgensma for spinal muscular atrophy recently revealed contaminating plasmid sequences derived from rAAV manufacturing within the patient\u2019s hepatocytes. In particular, REP\/CAP-derived sequences were remarkably abundant, corresponding to 0.5-1% of the therapeutic transgene. We hypothesized that these contaminants originated through Rep-mediated incorporation initiated at the AAV P5 promoter. Through reanalysis of the sequencing data, we inferred that an intact P5 promoter had been placed directly downstream of the rAAV capsid gene in the manufacturing plasmid.\n                  <jats:italic>De novo<\/jats:italic>\n                  assembly revealed a contiguous contaminant sequence spanning the rAAV REP and CAP genes and terminating within P5 at the Rep nicking site, immediately downstream of the Rep-binding element (RBE). This analysis also revealed a distinct vector-plasmid backbone contig consistent with reverse packaging. Among partially aligned REP\/CAP reads, nearly 17% were linked to rAAV ITR-derived sequence at heterogeneous junctions. Long-read data independently identified the P5 promoter as the most frequent recombination breakpoint region. Together, these findings identify a defined, avoidable mechanism by which REP\/CAP manufacturing contaminants arise. Positioning of the P5 promoter within the manufacturing plasmid is therefore a modifiable determinant of rAAV product purity and the transfer of rAAV DNA contaminants to recipient patients.\n                <\/jats:p>","DOI":"10.64898\/2026.09.10.26362787","type":"posted-content","created":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T14:30:20Z","timestamp":1789396220000},"source":"Crossref","is-referenced-by-count":0,"title":["P5 promoter-mediated incorporation explains REP\/CAP manufacturing contaminants in patient liver after rAAV gene therapy"],"prefix":"10.64898","author":[{"given":"Mark A.","family":"Brimble","sequence":"first","affiliation":[{"name":"Division of Allergy and Pulmonary Medicine, Dept of Pediatrics, Washington University School of Medicine, St. Louis, MO, USA"},{"name":"Department of Host-Microbe Interactions, St. Jude Children\u2019s Research Hospital, Memphis, TN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shaoyuan","family":"Tan","sequence":"additional","affiliation":[{"name":"Department of Host-Microbe Interactions, St. Jude Children\u2019s Research Hospital, Memphis, TN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sarah","family":"Buddle","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Virology and Infection Control, Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK"},{"name":"Infection, Immunity and Inflammation Department, Great Ormond Street Institute of Child Health, University College London, London, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Li-An K.","family":"Brown","sequence":"additional","affiliation":[{"name":"Infection, Immunity and Inflammation Department, Great Ormond Street Institute of Child Health, University College London, London, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Judith","family":"Breuer","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Virology and Infection Control, Great Ormond Street Hospital for Children NHS Foundation Trust, London, UK"},{"name":"Infection, Immunity and Inflammation Department, Great Ormond Street Institute of Child Health, University College London, London, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4096-6048","authenticated-orcid":false,"given":"Jeremy Chase","family":"Crawford","sequence":"additional","affiliation":[{"name":"Department of Host-Microbe Interactions, St. Jude Children\u2019s Research Hospital, Memphis, TN, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026091709451003000_2026.09.10.26362787v1.1","doi-asserted-by":"publisher","DOI":"10.3390\/pharmaceutics17111492"},{"key":"2026091709451003000_2026.09.10.26362787v1.2","unstructured":"Approved Cellular and Gene Therapy Products. https:\/\/www.fda.gov\/vaccines-blood-biologics\/cellular-gene-therapy-products\/approved-cellular-and-gene-therapy-products (2026)."},{"key":"2026091709451003000_2026.09.10.26362787v1.3","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymthe.2026.07.031"},{"key":"2026091709451003000_2026.09.10.26362787v1.4","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymthe.2026.05.016"},{"key":"2026091709451003000_2026.09.10.26362787v1.5","doi-asserted-by":"publisher","DOI":"10.1177\/10430342261423168"},{"key":"2026091709451003000_2026.09.10.26362787v1.6","unstructured":"Borrell, B. 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