{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,6,6]],"date-time":"2026-06-06T20:00:38Z","timestamp":1780776038114,"version":"3.54.1"},"posted":{"date-parts":[[2026,6,3]]},"group-title":"Microbiology","reference-count":97,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,6,3]],"date-time":"2026-06-03T00:00:00Z","timestamp":1780444800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,6,3]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Adeno-Associated Viruses (AAVs) are powerful platforms for delivering therapeutic transgenes via recombinant AAV (rAAV) vectors. However, a limited understanding of the regulation of AAV gene expression has narrowed the ability to efficiently express therapeutic transgenes from rAAV vectors. Since rAAVs retain only the wtAAV inverted terminal repeats (ITR), we hypothesized that regulatory elements outside the ITR that govern wild-type AAV (wtAAV) gene expression can be used to modify rAAV genomes to enhance vector performance. Through in silico analysis, biochemical pulldowns, and high-throughput sequencing, we have identified that the host architectural protein CCCTC-binding Factor (CTCF) associates with the wtAAV type 2 (wtAAV2) genome but is absent from rAAV vectors. Global knockdown and site-specific deletion revealed that the CTCF binding element (CBE) on the wtAAV2 genome, located upstream of the viral P5 promoter, regulates expression of the viral\n                  <jats:italic>Rep68\/78<\/jats:italic>\n                  genes. We have re-engineered new rAAV vectors expressing a GFP reporter transgene to contain the wtAAV2-CBE upstream of the vector promoter. Our results show that CTCF binding dramatically increased rAAV transduction efficiency and GFP expression by up to four-fold across multiple cell types. This enhancement was independent of the AAV capsid serotype used for packaging rAAV vectors. CUT&amp;RUN analysis revealed that this CBE was necessary and sufficient to regulate the chromatin landscape of wtAAV2 and rAAV2. Finally, we observed that CTCF-mediated chromatin remodeling of rAAV2 led to increased production of nascent RNA transcripts from the vector genome. Based on our findings, we propose that CTCF supports wtAAV2\/rAAV gene expression by shaping the local chromatin landscape.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>SIMPLE ABSTRACT<\/jats:title>\n                  <jats:p>\n                    Recombinant Adeno-Associated Viruses (rAAV) gene therapy vectors have been engineered from wild-type AAV (wtAAV) by inserting the viral telomeres (that serve as replication and packaging signals) on either side of therapeutic transgenes. However, efficient expression of transgenes using current rAAV technologies require high doses, which can lead to sporadic toxic side effects. We hypothesized that uncharacterized regulatory elements in the wtAAV2 genome drive efficient viral gene expression and are absent from the current generation of rAAV vectors. Using in-silico analysis combined with biochemical pulldowns, high-throughput sequencing, and mutant viral systems, we have identified a novel\n                    <jats:italic>cis<\/jats:italic>\n                    -acting element bound by the cellular architectural protein CCCTC-binding Factor (CTCF). This CTCF binding element is necessary for wtAAV2 gene expression and is sufficient to enhance the rAAV vector\u2019s ability to express reporter transgenes. This CTCF-binding element regulates the chromatin landscape of the virus and its vectors. Our discovery that adding the 19-bp AAV2 CTCF-binding element enhances transgene expression without affecting vector production efficiency presents a promising new rAAV gene therapy platform that is likely to reduce clinical doses and minimize toxicity in therapeutic applications.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.64898\/2026.06.03.729793","type":"posted-content","created":{"date-parts":[[2026,6,4]],"date-time":"2026-06-04T04:45:19Z","timestamp":1780548319000},"source":"Crossref","is-referenced-by-count":0,"title":["CTCF regulates wild-type and recombinant AAV gene expression by shaping viral chromatin"],"prefix":"10.64898","author":[{"given":"Clairine I.S.","family":"Larsen","sequence":"first","affiliation":[{"name":"Institute for Molecular Virology, University of Wisconsin-Madison, Madison, WI, 53706"},{"name":"McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, WI, 53706"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rhiannon R.","family":"Abrahams","sequence":"additional","affiliation":[{"name":"Institute for Molecular Virology, University of Wisconsin-Madison, Madison, WI, 53706"},{"name":"McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, WI, 53706"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rea","family":"Guertler","sequence":"additional","affiliation":[{"name":"Ultragenyx Pharmaceuticals, Somerville, MA, 02143"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elliott M.","family":"Wilion","sequence":"additional","affiliation":[{"name":"Ultragenyx Pharmaceuticals, Somerville, MA, 02143"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eda","family":"Erata","sequence":"additional","affiliation":[{"name":"Ultragenyx Pharmaceuticals, Somerville, MA, 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Wisconsin-Madison"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Richa","family":"Rai","sequence":"additional","affiliation":[{"name":"McPherson Eye Research Institute, University of Wisconsin-Madison"},{"name":"Waisman Center, University of Wisconsin-Madison"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elizabeth K.","family":"Hofer","sequence":"additional","affiliation":[{"name":"Institute for Molecular Virology, University of Wisconsin-Madison, Madison, WI, 53706"},{"name":"McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, WI, 53706"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eleanor","family":"Hart","sequence":"additional","affiliation":[{"name":"Institute for Molecular Virology, University of Wisconsin-Madison, Madison, WI, 53706"},{"name":"McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, WI, 53706"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David M.","family":"Gamm","sequence":"additional","affiliation":[{"name":"McPherson Eye Research Institute, University of Wisconsin-Madison"},{"name":"Waisman Center, University of Wisconsin-Madison"},{"name":"Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4704-572X","authenticated-orcid":false,"given":"Matthew S.","family":"Fuller","sequence":"additional","affiliation":[{"name":"Ultragenyx Pharmaceuticals, Somerville, MA, 02143"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1898-8251","authenticated-orcid":false,"given":"Kinjal","family":"Majumder","sequence":"additional","affiliation":[{"name":"Institute for Molecular Virology, University of Wisconsin-Madison, Madison, WI, 53706"},{"name":"McArdle Laboratory for Cancer Research, 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