{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,26]],"date-time":"2026-06-26T22:52:47Z","timestamp":1782514367093,"version":"3.54.5"},"reference-count":27,"publisher":"MDPI AG","issue":"23","license":[{"start":{"date-parts":[[2025,11,24]],"date-time":"2025-11-24T00:00:00Z","timestamp":1763942400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000053","name":"US NIH award","doi-asserted-by":"publisher","award":["R41EY030807"],"award-info":[{"award-number":["R41EY030807"]}],"id":[{"id":"10.13039\/100000053","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Norrin\u2013Wnt signaling is essential for retinal vascular development and generation of the inner blood retinal barrier. Norrin itself is a potential therapeutic for retinal vascular repair. We explored the feasibility of producing a recombinant protein therapeutic based on human Norrin for intravitreal injection. NorrinK86P production was tested using MBP fusion and non-tagged versions. FZD4 binding was evaluated by an ELISA, and the activation of AXIN2 gene expression in primary human retinal microvascular endothelial cells was measured by qPCR. Intravitreal injection was tested in the rat eye, evaluated by fluoresceine angiography, OCT, and ERG. MBP-tagged Norrin was resistant to HRV3C protease cleavage unless linker polypeptides were also incorporated. MBP\u2013Norrin or cleaved MBP\u2013Norrin also required refolding with disulfide reshuffling to generate FZD4-binding activity and to affect AXIN-2 gene expression. A production strategy based upon untagged NorrinK86P refolded from bacterial inclusion bodies was selected. Intravitreal injection of NorrinK86P did not affect retinal thickness nor retinal function, the latter monitored by the ERG A-wave and B-wave amplitudes. We concluded that MBP\u2013Norrin, cleaved Norrin, and untagged Norrin from inclusion bodies display Norrin-like biological activity after refolding with disulfide reshuffling. The untagged, bacterial inclusion body process was selected for future large-scale bacterial fermentation. NorrinK86P could be produced with Norrin-like biochemical and biological activities and was tolerated after intravitreal injection into the rat eye.<\/jats:p>","DOI":"10.3390\/ijms262311340","type":"journal-article","created":{"date-parts":[[2025,11,24]],"date-time":"2025-11-24T11:47:22Z","timestamp":1763984842000},"page":"11340","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Development of Norrin-Based Protein Therapeutic for Activation of Norrin-Wnt Signaling in Human Retinal Endothelial Cells"],"prefix":"10.3390","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4751-7440","authenticated-orcid":false,"given":"Kenneth P.","family":"Mitton","sequence":"first","affiliation":[{"name":"Eye Research Institute, Oakland University, Rochester, MI 48309, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wendy A.","family":"Dailey","sequence":"additional","affiliation":[{"name":"RetiNova Therapeutics, San Diego, CA 94402, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Steven Q.","family":"Krikor","sequence":"additional","affiliation":[{"name":"Eye Research Institute, Oakland University, Rochester, MI 48309, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kimberly A.","family":"Drenser","sequence":"additional","affiliation":[{"name":"RetiNova Therapeutics, San Diego, CA 94402, USA"},{"name":"Associated Retinal Consultants P.C., Royal Oak, MI 48037, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,11,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"417","DOI":"10.1016\/j.molmed.2010.07.003","article-title":"The Norrin\/Frizzled4 Signaling Pathway in Retinal Vascular Development and Disease","volume":"16","author":"Ye","year":"2010","journal-title":"Trends Mol. 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