{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T01:53:01Z","timestamp":1792633981915,"version":"4.5.0"},"reference-count":42,"publisher":"Wiley","issue":"12","license":[{"start":{"date-parts":[[2022,11,14]],"date-time":"2022-11-14T00:00:00Z","timestamp":1668384000000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["faseb.onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["The FASEB Journal"],"published-print":{"date-parts":[[2022,12]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>\n                    Recombinant adeno\u2010associated viruses (rAAVs) are useful vectors for expressing genes of interest in vivo because of their low immunogenicity and long\u2010term gene expression. Various mutations have been introduced in recent years and have enabled high\u2010efficacy, stabilized, and organ\u2010oriented transduction. Our purpose for using rAAV is to express our target gene in the mouse lung to investigate pulmonary artery hypertension. We constructed a self\u2010complementary AAV having mutant capsids with the ESGHGYF insert, which directs the vectors to lung endothelial cells. However, when this mutant virus was purified from the producing cells by the conventional method using an ultracentrifuge, it resulted in a low yield. In addition, the purification method using an ultracentrifuge is tedious and labor\u2010intensive. Therefore, we aimed to develop a simple, high\u2010quality method for obtaining enough lung\u2010targeted rAAV. First, we modified amino acids (T491V and Y730F) of the capsid to stabilize the rAAV from degradation, and we optimized culture conditions. Next, we noticed that many rAAVs were released from the cells into the culture medium. We, therefore, improved our purification method by purifying from the culture medium without the ultracentrifugation step. Purification without ultracentrifugation had the problem that impurities were mixed in, causing inflammation. However, by performing PEG precipitation and chloroform extraction twice, we were able to purify rAAV that caused only as little inflammation as that obtained by the ultracentrifuge method. Sufficient rAAV was obtained and can now be administered to a rat as well as mice from a single dish: 1.50\u2009\u00d7\u200910\n                    <jats:sup>13<\/jats:sup>\n                    \u2009\u00b1\u20093.58\u2009\u00d7\u200910\n                    <jats:sup>12<\/jats:sup>\n                    vector genome from one \u03c6150\u2009mm dish (mean\u2009\u00b1\u2009SEM).\n                  <\/jats:p>","DOI":"10.1096\/fj.202200840rr","type":"journal-article","created":{"date-parts":[[2022,11,14]],"date-time":"2022-11-14T10:53:48Z","timestamp":1668423228000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Development of a high\u2010yield, high\u2010quality purification process for adeno\u2010associated virus vectors that can be used in vivo without ultracentrifugation: Application to a lung endothelial cell\u2010targeted adeno\u2010associated virus"],"prefix":"10.1096","volume":"36","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5901-4026","authenticated-orcid":false,"given":"Yasunaga","family":"Shiraishi","sequence":"first","affiliation":[{"name":"Division of Environmental Medicine National Defense Medical College Research Institute, National Defense Medical College  Saitama Japan"},{"name":"Division of Cardiovascular Medicine, Department of Internal Medicine National Defense Medical College  Saitama Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takeshi","family":"Adachi","sequence":"additional","affiliation":[{"name":"Division of Cardiovascular Medicine, Department of Internal Medicine National Defense Medical College  Saitama Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jose M.","family":"Cacicedo","sequence":"additional","affiliation":[{"name":"Department of Research and Development ALPCO Diagnostics  Salem New Hampshire USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yasuo","family":"Ido","sequence":"additional","affiliation":[{"name":"Division of Cardiovascular Medicine, Department of Internal Medicine National Defense Medical College  Saitama Japan"},{"name":"Department of Medicine Boston University School of Medicine  Boston Massachusetts USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2022,11,14]]},"reference":[{"key":"e_1_2_11_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/sj.gt.3301514"},{"key":"e_1_2_11_3_1","doi-asserted-by":"publisher","DOI":"10.1038\/mt.2008.171"},{"key":"e_1_2_11_4_1","doi-asserted-by":"publisher","DOI":"10.1038\/nbt856"},{"key":"e_1_2_11_5_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.omtm.2019.01.008"},{"key":"e_1_2_11_6_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0059142"},{"key":"e_1_2_11_7_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0802866105"},{"key":"e_1_2_11_8_1","doi-asserted-by":"publisher","DOI":"10.1038\/mt.2016.62"},{"key":"e_1_2_11_9_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598\u2010019\u201049624\u2010w"},{"key":"e_1_2_11_10_1","doi-asserted-by":"publisher","DOI":"10.1038\/gt.2009.157"},{"key":"e_1_2_11_11_1","doi-asserted-by":"publisher","DOI":"10.4161\/bioe.22293"},{"key":"e_1_2_11_12_1","doi-asserted-by":"publisher","DOI":"10.1038\/sj.gt.3300680"},{"key":"e_1_2_11_13_1","doi-asserted-by":"publisher","DOI":"10.1128\/JVI.72.3.2224-2232.1998"},{"key":"e_1_2_11_14_1","doi-asserted-by":"publisher","DOI":"10.1089\/hgtb.2011.034"},{"key":"e_1_2_11_15_1","doi-asserted-by":"publisher","DOI":"10.3390\/antiox10030471"},{"key":"e_1_2_11_16_1","doi-asserted-by":"publisher","DOI":"10.1167\/iovs.10\u20105784"},{"key":"e_1_2_11_17_1","doi-asserted-by":"publisher","DOI":"10.1128\/jvi.75.4.1824\u20101833.2001"},{"key":"e_1_2_11_18_1","doi-asserted-by":"publisher","DOI":"10.1172\/jci8317"},{"key":"e_1_2_11_19_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymthe.2005.12.002"},{"key":"e_1_2_11_20_1","doi-asserted-by":"publisher","DOI":"10.1038\/mt.2010.172"},{"key":"e_1_2_11_21_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jviromet.2012.04.004"},{"key":"e_1_2_11_22_1","unstructured":"AtkinsonEM TakeyaRK AranhaIL.Methods for generating high titer helper\u2010free preparations of released recombinant AAV vectors.2003."},{"key":"e_1_2_11_23_1","unstructured":"SakamotoS KawanoY DodoK et al.Method for manufacturing non\u2010enveloped virus.2018."},{"key":"e_1_2_11_24_1","doi-asserted-by":"publisher","DOI":"10.1101\/488585"},{"key":"e_1_2_11_25_1","doi-asserted-by":"publisher","DOI":"10.1089\/hum.2010.107"},{"key":"e_1_2_11_26_1","doi-asserted-by":"publisher","DOI":"10.1089\/hgtb.2015.117"},{"key":"e_1_2_11_27_1","doi-asserted-by":"publisher","DOI":"10.1038\/mtm.2016.15"},{"key":"e_1_2_11_28_1","doi-asserted-by":"publisher","DOI":"10.1182\/blood\u20102003\u201002\u20100495"},{"key":"e_1_2_11_29_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1538\u20107836.2006.02220.x"},{"key":"e_1_2_11_30_1","doi-asserted-by":"publisher","DOI":"10.1182\/blood\u20102013\u201001\u2010306647"},{"key":"e_1_2_11_31_1","doi-asserted-by":"publisher","DOI":"10.1056\/NEJMoa1108046"},{"key":"e_1_2_11_32_1","doi-asserted-by":"publisher","DOI":"10.1089\/humc.2013.087"},{"key":"e_1_2_11_33_1","doi-asserted-by":"publisher","DOI":"10.1016\/s0140\u20106736(17)31868\u20108"},{"key":"e_1_2_11_34_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jviromet.2004.07.001"},{"key":"e_1_2_11_35_1","doi-asserted-by":"publisher","DOI":"10.1089\/10430349950017563"},{"key":"e_1_2_11_36_1","doi-asserted-by":"publisher","DOI":"10.1038\/sj.gt.3300938"},{"key":"e_1_2_11_37_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598\u2010017\u201016882\u20105"},{"key":"e_1_2_11_38_1","doi-asserted-by":"publisher","DOI":"10.1002\/hep.21896"},{"key":"e_1_2_11_39_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymthe.2019.12.010"},{"key":"e_1_2_11_40_1","doi-asserted-by":"publisher","DOI":"10.1053\/j.gastro.2011.04.002"},{"key":"e_1_2_11_41_1","doi-asserted-by":"publisher","DOI":"10.1089\/ars.2021.0052"},{"key":"e_1_2_11_42_1","doi-asserted-by":"publisher","DOI":"10.1089\/hum.2010.143"},{"key":"e_1_2_11_43_1","doi-asserted-by":"publisher","DOI":"10.1046\/j.1523\u20101755.2002.0610s1009.x"}],"container-title":["The FASEB Journal"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1096\/fj.202200840RR","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/full-xml\/10.1096\/fj.202200840RR","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/faseb.onlinelibrary.wiley.com\/doi\/pdf\/10.1096\/fj.202200840RR","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,29]],"date-time":"2025-10-29T01:16:19Z","timestamp":1761700579000},"score":1,"resource":{"primary":{"URL":"https:\/\/faseb.onlinelibrary.wiley.com\/doi\/10.1096\/fj.202200840RR"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,11,14]]},"references-count":42,"journal-issue":{"issue":"12","published-print":{"date-parts":[[2022,12]]}},"alternative-id":["10.1096\/fj.202200840RR"],"URL":"https:\/\/doi.org\/10.1096\/fj.202200840rr","archive":["Portico"],"relation":{},"ISSN":["0892-6638","1530-6860"],"issn-type":[{"value":"0892-6638","type":"print"},{"value":"1530-6860","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,11,14]]},"assertion":[{"value":"2022-06-03","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2022-10-31","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2022-11-14","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}],"article-number":"e22653"}}