{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,27]],"date-time":"2026-02-27T05:12:26Z","timestamp":1772169146501,"version":"3.50.1"},"reference-count":26,"publisher":"F1000 Research Ltd","license":[{"start":{"date-parts":[[2025,7,30]],"date-time":"2025-07-30T00:00:00Z","timestamp":1753833600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100010269","name":"Wellcome Trust","doi-asserted-by":"publisher","award":["226804"],"award-info":[{"award-number":["226804"]}],"id":[{"id":"10.13039\/100010269","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100010269","name":"Wellcome Trust","doi-asserted-by":"publisher","award":["227417"],"award-info":[{"award-number":["227417"]}],"id":[{"id":"10.13039\/100010269","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["wellcomeopenresearch.org"],"crossmark-restriction":false},"short-container-title":["Wellcome Open Res"],"abstract":"<ns3:p>\n                    Background Alport syndrome is a rare genetic disorder characterized by progressive kidney disease, hearing loss, and eye abnormalities. It is caused by variants in the\n                    <ns3:italic>COL4A3<\/ns3:italic>\n                    ,\n                    <ns3:italic>COL4A4<\/ns3:italic>\n                    or\n                    <ns3:italic>COL4A5<\/ns3:italic>\n                    genes, which disrupt the synthesis, secretion and assembly of the alpha-3, -4 and -5 chains of type IV collagen. The defective collagen IV network results in the formation of an abnormal glomerular basement membrane (GBM). Adeno-associated virus (AAV)-mediated gene therapy is a promising approach for treating Alport syndrome but has not yet been realized due to technical challenges, including effective transduction of target cells. Methods In this study, we evaluated the transduction efficacy of Adeno-Associated Virus-9 with a green fluorescent protein cargo (AAV9-GFP) as a gene delivery vehicle in healthy and Alport (\n                    <ns3:italic>Col4a5<\/ns3:italic>\n                    knockout) podocytes. We established a quantitative testing platform using podocytes in culture,\n                    <ns3:italic>ex vivo<\/ns3:italic>\n                    glomeruli, and a mouse model of Alport syndrome (male C57BL\/6\n                    <ns3:sup>tm1b<\/ns3:sup>\n                    mice). Results First we compared transduction levels of AAV9-GFP between healthy and Alport podocytes\n                    <ns3:italic>in vitro<\/ns3:italic>\n                    . Both immortalized human podocytes and isolated mouse primary podocytes exhibited similar transduction efficiency in culture. We then incubated\n                    <ns3:italic>ex vivo<\/ns3:italic>\n                    glomeruli with AAV9-GFP and found increased podocyte uptake in the Alport glomeruli compared to wild type controls. Finally in mice we found an increase in transduction of AAV9-GFP in Alport podocytes following a peripheral intravenous injection. The level of transduction was dose-dependent and increased with disease progression suggesting that the pathological environment may facilitate higher penetration of the vector. Conclusion These findings underscore the potential of AAV9 for effective gene delivery in the context of Alport syndrome and show that the stage of disease at the time of injection plays a role in determining the efficiency of AAV transduction. Overall, this study provides a foundation for future therapeutic strategies aimed at correcting the underlying genetic defects.\n                  <\/ns3:p>","DOI":"10.12688\/wellcomeopenres.24212.1","type":"journal-article","created":{"date-parts":[[2025,7,30]],"date-time":"2025-07-30T10:10:06Z","timestamp":1753870206000},"page":"386","update-policy":"https:\/\/doi.org\/10.12688\/wellcomeopenres.crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Increased in vivo transduction of AAV-9 cargo in Alport podocytes"],"prefix":"10.12688","volume":"10","author":[{"given":"Emily","family":"Williams","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9530-6392","authenticated-orcid":false,"given":"Maryline","family":"Fresquet","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gema","family":"Bolas","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shota","family":"Kaseda","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kevin A.","family":"Goncalves","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Andrew","family":"Steinsapir","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5408-0013","authenticated-orcid":false,"given":"Antony","family":"Adamson","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David R.","family":"Sherwood","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6400-0227","authenticated-orcid":false,"given":"Rachel","family":"Lennon","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"2560","published-online":{"date-parts":[[2025,7,30]]},"reference":[{"key":"ref-1","doi-asserted-by":"publisher","first-page":"112-27","DOI":"10.1038\/s41581-020-0329-y","article-title":"Complexities of the glomerular basement membrane.","volume":"17","author":"R Naylor","year":"2021","journal-title":"Nat Rev Nephrol."},{"key":"ref-2","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbc.2021.100590","article-title":"Collagen IV\u03b1345 dysfunction in glomerular basement membrane diseases. I. Discovery of a COL4A3 variant in familial Goodpasture's and Alport diseases.","volume":"296","author":"E Pokidysheva","year":"2021","journal-title":"J Biol Chem."},{"key":"ref-3","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbc.2021.100592","article-title":"Collagen IV\u03b1345 dysfunction in glomerular basement membrane diseases. III. A functional framework for \u03b1345 hexamer assembly.","volume":"296","author":"V Pedchenko","year":"2021","journal-title":"J Biol Chem."},{"key":"ref-4","doi-asserted-by":"publisher","DOI":"10.1016\/j.jbc.2021.100591","article-title":"Collagen IV\u03b1345 dysfunction in glomerular basement membrane diseases. II. Crystal structure of the \u03b1345 hexamer.","volume":"296","author":"S Boudko","year":"2021","journal-title":"J Biol Chem."},{"key":"ref-5","doi-asserted-by":"publisher","DOI":"10.1016\/j.omtm.2024.101333","article-title":"Gene therapy and kidney diseases.","volume":"32","author":"N Tavakolidakhrabadi","year":"2024","journal-title":"Mol Ther Methods Clin Dev."},{"key":"ref-6","doi-asserted-by":"publisher","first-page":"1471-9","DOI":"10.1681\/ASN.2008101086","article-title":"Cellular origins of type IV collagen networks in developing glomeruli.","volume":"20","author":"D Abrahamson","year":"2009","journal-title":"J Am Soc Nephrol."},{"key":"ref-7","doi-asserted-by":"publisher","first-page":"129-33","DOI":"10.2478\/jtim-2024-0005","article-title":"Progress in therapeutic targets on podocyte for Alport Syndrome.","volume":"12","author":"Q Zheng","year":"2024","journal-title":"J Transl Int Med."},{"key":"ref-8","doi-asserted-by":"publisher","DOI":"10.1186\/s12951-023-02044-5","article-title":"Emerging non-viral vectors for gene delivery.","volume":"21","author":"C Wang","year":"2023","journal-title":"J Nanobiotechnology."},{"key":"ref-9","doi-asserted-by":"publisher","DOI":"10.1038\/s41392-021-00487-6","article-title":"Viral vector platforms within the gene therapy landscape.","volume":"6","author":"J Bulcha","year":"2021","journal-title":"Signal Transduct Target Ther."},{"key":"ref-10","doi-asserted-by":"publisher","first-page":"317-34","DOI":"10.1007\/s40259-017-0234-5","article-title":"Adeno-Associated Virus (AAV) as a vector for gene therapy.","volume":"31","author":"M Naso","year":"2017","journal-title":"BioDrugs."},{"key":"ref-11","doi-asserted-by":"publisher","first-page":"1189-99","DOI":"10.1038\/mt.2008.103","article-title":"Recombinant Adeno-Associated Virus transduction and integration.","volume":"16","author":"B Schultz","year":"2008","journal-title":"Mol Ther."},{"key":"ref-12","doi-asserted-by":"publisher","first-page":"451-62","DOI":"10.1038\/s41581-023-00702-3","article-title":"Cell and gene therapy for kidney disease.","volume":"19","author":"J Peek","year":"2023","journal-title":"Nat Rev Nephrol."},{"key":"ref-13","doi-asserted-by":"publisher","first-page":"785","DOI":"10.3390\/cells12050785","article-title":"Various AAV serotypes and their applications in gene therapy: an overview.","volume":"12","author":"S Issa","year":"2023","journal-title":"Cells."},{"key":"ref-14","doi-asserted-by":"publisher","first-page":"2287-97","DOI":"10.1681\/ASN.2018040426","article-title":"Efficient gene transfer to kidney mesenchymal cells using a synthetic Adeno-Associated Viral vector.","volume":"29","author":"Y Ikeda","year":"2018","journal-title":"J Am Soc Nephrol."},{"key":"ref-15","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-019-11321-7","article-title":"Standard screening methods underreport AAV-mediated transduction and gene editing.","volume":"10","author":"J Lang","year":"2019","journal-title":"Nat Commun."},{"key":"ref-16","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-024-54475-9","article-title":"Enhancing gene transfer to renal tubules and podocytes by context-dependent selection of AAV capsids.","volume":"15","author":"T Furusho","year":"2024","journal-title":"Nat Commun."},{"key":"ref-17","doi-asserted-by":"publisher","first-page":"508-14","DOI":"10.1038\/nature19356","article-title":"High-throughput discovery of novel developmental phenotypes.","volume":"537","author":"M Dickinson","year":"2016","journal-title":"Nature."},{"key":"ref-18","doi-asserted-by":"publisher","first-page":"1231-8","DOI":"10.1038\/ng.3901","article-title":"Disease model discovery from 3,328 gene knockouts by the International Mouse Phenotyping Consortium.","volume":"49","author":"T Meehan","year":"2017","journal-title":"Nat Genet."},{"key":"ref-19","doi-asserted-by":"publisher","first-page":"1713-32","DOI":"10.1681\/ASN.2020101442","article-title":"Identification of an altered matrix signature in kidney aging and disease.","volume":"32","author":"M Randles","year":"2021","journal-title":"J Am Soc Nephrol."},{"key":"ref-20","doi-asserted-by":"publisher","first-page":"2175-93","DOI":"10.1681\/ASN.2020091346","article-title":"Tripartite separation of glomerular cell types and proteomes from reporter-free mice.","volume":"32","author":"F Hatje","year":"2021","journal-title":"J Am Soc Nephrol."},{"key":"ref-21","doi-asserted-by":"publisher","DOI":"10.1126\/scitranslmed.abc8226","article-title":"Adeno-Associated Virus gene therapy prevents progression of kidney disease in genetic models of nephrotic syndrome.","volume":"15","author":"W Ding","year":"2023","journal-title":"Sci Transl Med."},{"key":"ref-22","doi-asserted-by":"publisher","DOI":"10.1038\/s41392-024-01780-w","article-title":"Adeno-Associated Virus as a delivery vector for gene therapy of human diseases.","volume":"9","author":"J Wang","year":"2024","journal-title":"Signal Transduct Target Ther."},{"key":"ref-23","doi-asserted-by":"publisher","first-page":"618-28","DOI":"10.1038\/gt.2014.35","article-title":"rAAV9 combined with renal vein injection is optimal for kidney-targeted gene delivery: conclusion of a comparative study.","volume":"21","author":"C Rocca","year":"2014","journal-title":"Gene Ther."},{"key":"ref-24","doi-asserted-by":"publisher","first-page":"172-84","DOI":"10.1016\/j.molmed.2020.09.010","article-title":"Journey to the center of the cell: tracing the path of AAV transduction.","volume":"27","author":"B Dhungel","year":"2021","journal-title":"Trends Mol Med."},{"key":"ref-25","doi-asserted-by":"publisher","first-page":"80-6","DOI":"10.1038\/mt.2009.255","article-title":"Effect of genome size on AAV vector packaging.","volume":"18","author":"Z Wu","year":"2010","journal-title":"Mol Ther."},{"key":"ref-26","article-title":"Raw data - Increased in vivo transduction of AAV-9 cargo in Alport podocytes","author":"M Fresquet","year":"2025"}],"container-title":["Wellcome Open Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-386\/v1\/xml","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-386\/v1\/pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-386\/v1\/iparadigms","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,11]],"date-time":"2025-12-11T04:49:07Z","timestamp":1765428547000},"score":1,"resource":{"primary":{"URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-386\/v1"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,7,30]]},"references-count":26,"URL":"https:\/\/doi.org\/10.12688\/wellcomeopenres.24212.1","relation":{"has-review":[{"id-type":"doi","id":"10.21956\/wellcomeopenres.26707.r139469","asserted-by":"subject"},{"id-type":"doi","id":"10.21956\/wellcomeopenres.26707.r134220","asserted-by":"subject"},{"id-type":"doi","id":"10.21956\/wellcomeopenres.26707.r139468","asserted-by":"subject"},{"id-type":"doi","id":"10.21956\/wellcomeopenres.26707.r134220","asserted-by":"object"},{"id-type":"doi","id":"10.21956\/wellcomeopenres.26707.r139466","asserted-by":"object"},{"id-type":"doi","id":"10.21956\/wellcomeopenres.26707.r139468","asserted-by":"object"},{"id-type":"doi","id":"10.21956\/wellcomeopenres.26707.r139469","asserted-by":"object"}]},"ISSN":["2398-502X"],"issn-type":[{"value":"2398-502X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,7,30]]},"assertion":[{"value":"Indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-386\/v1#article-reports","order":0,"name":"referee-status","label":"Referee status","group":{"name":"current-referee-status","label":"Current Referee Status"}},{"value":"10.21956\/wellcomeopenres.26707.r134220, Cheng Xue, nephrology, shanghai changzheng hospital, Naval Medical University (Second Military Medical University), Shanghai, 07 Oct 2025, version 1, indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-386\/v1#referee-response-134220","order":0,"name":"referee-response-134220","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.21956\/wellcomeopenres.26707.r139468, Emine Bilge Caparali, Internal Medicine PSTP Resident at UT Southwestern, Dallas, 25 Nov 2025, version 1, indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-386\/v1#referee-response-139468","order":1,"name":"referee-response-139468","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.21956\/wellcomeopenres.26707.r139469, Hassan Saei, Laboratory of Hereditary Kidney Diseases, Institut Imagine Institut des Maladies Genetiques, Paris, \u00cele-de-France, 11 Dec 2025, version 1, indexed","URL":"https:\/\/wellcomeopenresearch.org\/articles\/10-386\/v1#referee-response-139469","order":2,"name":"referee-response-139469","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"This work was supported by Wellcome [226804; to R.L.] [227417; to R.L., D.R.S] [301803 to R.L.]; Kidney Research UK to R.L.; The Stoneygate Trust [Alport Research Hub to R.L.]; and the National Institute for Health Research [NIHR-Manchester BRC to R.L.]; NIH [R35GM118049; to D.R.S.]; Japan Agency for Medical Research and Development (AMED) fellowship to S.K. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.","order":3,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.","order":0,"name":"copyright-info","label":"Copyright"}]}}