Development of Norrin-Based Protein Therapeutic for Activation of Norrin-Wnt Signaling in Human Retinal Endothelial Cells

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. Norrin^K86P production was tested using MBP fusion and non-tagged versions. FZD4 binding was evaluated by an ELISA, and the activation of <i>AXIN2</i> 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 <i>AXIN-2</i> gene expression. A production strategy based upon untagged Norrin^K86P refolded from bacterial inclusion bodies was selected. Intravitreal injection of Norrin^K86P did not affect retinal thickness nor retinal function, the latter 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. Norrin^K86P could be produced with Norrin-like biochemical and biological activities and was tolerated after intravitreal injection into the rat eye.