{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,24]],"date-time":"2026-08-24T12:24:44Z","timestamp":1787574284879,"version":"build-2736575974"},"reference-count":44,"publisher":"Wiley","issue":"8","license":[{"start":{"date-parts":[[2026,8,11]],"date-time":"2026-08-11T00:00:00Z","timestamp":1786406400000},"content-version":"vor","delay-in-days":10,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,8,1]],"date-time":"2026-08-01T00:00:00Z","timestamp":1785542400000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"funder":[{"DOI":"10.13039\/100009619","name":"Japan Agency for Medical Research and Development","doi-asserted-by":"publisher","award":["JP21fk0108560"],"award-info":[{"award-number":["JP21fk0108560"]}],"id":[{"id":"10.13039\/100009619","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100009619","name":"Japan Agency for Medical Research and Development","doi-asserted-by":"publisher","award":["JP24fk0310523"],"award-info":[{"award-number":["JP24fk0310523"]}],"id":[{"id":"10.13039\/100009619","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["JP19K06476"],"award-info":[{"award-number":["JP19K06476"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["JP23K27095"],"award-info":[{"award-number":["JP23K27095"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100001691","name":"Japan Society for the Promotion of Science","doi-asserted-by":"publisher","award":["JP24K21938"],"award-info":[{"award-number":["JP24K21938"]}],"id":[{"id":"10.13039\/501100001691","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["The Journal of Gene Medicine"],"published-print":{"date-parts":[[2026,8]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>Adenovirus vectors (AdVs) are widely used and have an advantage of large insert capacity compared with adeno\u2010associated virus vectors. However, AdVs have scarcely been used in genome\u2010editing knock\u2010in strategies because of low efficiency.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      Novel AdVs possessing a very large, 3.7\u2009kb donor DNA fragment and six or eight multiplex gRNA expression units were developed for CRISPR\/Cas9\u2010mediated knock\u2010in to correct a phenylalanine hydroxylase (\n                      <jats:italic>Pah<\/jats:italic>\n                      ) gene in a\n                      <jats:italic>Pah<\/jats:italic>\n                      <jats:sup>enu2<\/jats:sup>\n                      phenylketonuria mouse model. These AdVs were co\u2010infected to Hepa1\u20136 cells or liver cells in\u00a0vivo together with an AdV expressing either native Cas9 or Cas9 nickase (Cas9n) for double\u2010nicking cleavage.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>In vitro knock\u2010in of the AdVs carrying 3.7\u2009kb donor DNA and six gRNA units targeting the cell genome was observed in both cases using Cas9 and Cas9n, though their efficiencies were low. Therefore, we generated AdVs carrying an additional two gRNA units that cleave the donor DNA terminus in the AdV genome via native Cas9 or Cas9 nickase. The knock\u2010in efficiency increased approximately twofold for both vectors and reached a maximum of 8% for native Cas9 without selection. Newborn phenylketonuria model mice were intravenously administered the knock\u2010in AdV together with the native\u2010Cas9 AdV. Although the knock\u2010in efficiency by homologous recombination occurred in only approximately 1% of hepatocytes, blood phenylalanine levels were reduced by up to 30%. Also, unintended fragments produced by nonhomologous end\u2010joining were observed between the cleavage site at the terminus of the donor DNA in the AdV genome and the target site in the cell genome.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusions<\/jats:title>\n                    <jats:p>The knock\u2010in efficiency of AdVs can be increased by cleaving the terminus of the donor DNA, although it would be desirable to avoid nonhomologous end\u2010joining between double\u2010strand break termini.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.1002\/jgm.70104","type":"journal-article","created":{"date-parts":[[2026,8,11]],"date-time":"2026-08-11T23:58:34Z","timestamp":1786492714000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Adenovirus Vector\u2010Mediated In\u00a0Vivo Knock\u2010in Treatment of Neonatal Phenylketonuria Mice Using Terminally Cleaved Donor DNA"],"prefix":"10.1002","volume":"28","author":[{"given":"Megumi","family":"Yamaji","sequence":"first","affiliation":[{"name":"Center for Biomedical Research Resources Juntendo University Graduate School of Medicine  Tokyo Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hirotaka","family":"Tabata","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Engineering, Faculty of Engineering Toyama Prefectural University  Toyama Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mariko","family":"Nakamura","sequence":"additional","affiliation":[{"name":"Center for Biomedical Research Resources Juntendo University Graduate School of Medicine  Tokyo Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ryoko","family":"Fuse","sequence":"additional","affiliation":[{"name":"Laboratory of Virology Institute of Microbial Chemistry (BIKAKEN), Microbial Chemistry Foundation  Tokyo 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