Development of Norrin-Based Protein Therapeutic for Activation of Norrin-Wnt Signaling in Human Retinal Endothelial Cells
Year: 2025
Type: article
Abstract: Norrin-Wnt 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 testing using MBP-fusion and non-tagged versions. FZD4 binding was evaluated by ELISA and the activation of AXIN-2 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-Norrin or cleaved MBP-Norrin 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 later monitored by the ERG A-wave and B-wave amplitudes. We concluded that MBP-Norrin, 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.
Institutions: Oakland University, Associated Retinal Consultants
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $3,499
Funder: National Eye Institute
OpenAlex ID: W4416597141