Noregen Promotes Growth to Barrier Formation of Retinal Endothelial Cells and Activates FZD4 Variants.

Abstract

Purpose : Noregen^TM is a recombinantly produced protein therapeutic based on the human Norrin protein. To establish cell-based models for investigation and functional analysis of Noregen, primary human retinal microvascular endothelial cells were adopted for functional barrier analysis with ECIS, and co-transfection of HEK293 cells was employed for dose-response analysis with normal and disease-causing variants of FZD4.

Methods : Noregen was supply-run material provided by Caeregen Therapeutics. Primary Human Retinal Microvascular Endothelial Cells (HRMECs) were cultured for real-time barrier modeling using Electric Cell-substrate Impedance Sensing (ECIS). HRMECs were seeded at low concentrations in ECIS 96-well plates and treated with Noregen. Impedance data was fit to a monolayer model with ECIS software (Applied Biophysics). HEK293 cells were co-transfected with expression plasmids for LRP5, FZD4, or FZD4^M105V, and a beta-Catenin reporter-plasmid expressing Firefly Luciferase. At 24-hrs post-transfection, cells were treated with multiple doses of Noregen, and dose-response activation data were fit to a 4-parameter log-logistic dose-response model using the DRC package of R.

Results : ECIS-based analysis showed that Norgen caused a dose-dependent acceleration of growth to barrier formation with primary HRMECs. ECIS modeling also demonstrated barrier formation following proliferation to monolayer. Dose-response analysis of beta-Catenin activation demonstrated a sigmoidal dose-response curve from activation by Noregen. The EC50 values were greater for the FEVR-related variant FZD4^(M105V) , 115 ng/mL Noregen, compared to FZD4, 16 ng/mL Noregen. Increased activation of FZD4^(M105V) was possible to a maximum level of about 32% of the maximum activation with FZD4.

Conclusions : Noregen affected the growth of primary Human Retinal Microvascular Endothelial Cells similar to that expected from past results with research grade Norrin protein. The retinal endothelial cell type is known to be responsive to Norrin Wnt-signaling. HEK293 cell-based co-transfection analysis revealed that Noregen causes a sigmoidal dose-response for beta-Catenin mediated activation of gene expression. The EC50 values for activation were congruent with the EC50 values for binding to FZD4 in vitro. Dose response data will be used to inform dose selection for future clinical testing.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.