Abstract
Purpose :
To develop and produce a non-toxic protein therapeutic for the repair and regeneration of the retinal vasculature in eyes affected by ischemic retinopathies, by targeting and stimulating endogenous retinal endothelial cells. A strategy for producing Noregen protein in bacteria, based on the human norrin protein, was developed and tested for toxicity, in vitro activity and in vivo efficacy.
Methods :
A method was refined to produce Noregen ( human-derived growth factor) protein as inclusion bodies in E. Coli, to meet several milestones. 1) Culturing at least 1,000 mg per Liter of protein. 2) Purifying protein sufficiently for safe intravitreal injection in the rat eye as confirmed by ERG. 3) Refolding Noregen to produce active protein. Activity was measured by a receptor (Frizzled-4) binding assay, RT-PCR to determine alterations in Norrin-target gene expression (AXIN-2 & PLVAP) in primary human retinal endothelial cells and use of a mouse OIR model to evaluate accelerated vascular regrowth in vivo. OIR mice (n=11) were removed from 75% oxygen at post-natal day (P) 12 and received a single injection of either Noregen 40 ng (n=6) or Vehicle (n=5) in the left eye at P14. Eyes were evaluated by retina whole mount and lectin staining at P17.
Results :
Production of protein met and then exceeded the desired culture concentrations. Noregen protein was refolded into a biologically active state and achieved sufficient binding to Frizzled-4, matching the binding EC50 of Norrin (30-150 ng/mL). Purification was reached to minimize endotoxin and no toxicity was detected in the Long Evans rat eye based upon analysis with fluorescein angiography, Spectral Domain-Optical Coherence Tomography (SD-OCT), and full-field electroretinogram (ERG) up to 6 weeks following a single intravitreal injection of Noregen 250 ng. Significant vascular regrowth was seen in the Noregen treated eyes compared to vehicle treated eyes (p=0.027). Noregen treatment of primary HRMECs increased AXIN-2 gene expression (Wnt pathway activation) similar to human norrin, increased cell proliferation in a dose dependent fashion, and suppressed PLVAP (marker of transcytosis).
Conclusions :
Noregen, a novel protein therapeutic, was successfully produced in E. Coli at sufficient scale, purity and biological activity to enable the continued development of Noregen for future GMP grade manufacturing.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.