CR2-fH, A Targeted Inhibitor of Alternative Complement Pathway Reduces Dry AMD-Like Phenotype in AMD Patient...

To investigate whether the alternative pathway (AP) of complement inhibition restores the epithelial phenotype and ameliorates dry AMD biomarkers in AMD patient induced pluripotent stem cell-derived RPE (iPSC-RPE) monolayers.

iPSC-RPE monolayers were established from an AMD patient iPSC line; ARPE-19 monolayers were used for comparison. For AP inhibition, monolayers were treated with CR2-fH (fusion protein composed of the iC3b/C3d/C3dg-binding region of complement receptor type 2 [CR2] linked to the inhibitory domain of human factor H [fH]) either as a soluble protein or by adeno-associated virus transduction. Complement challenge used 5% complement competent human serum (CC-HS); 5% heat-inactivated serum served as a control. Membrane attack complex, apolipoprotein E, lipid deposits, and autophagy were analyzed by immunostaining. Morphology and integrity were evaluated in brightfield images, using zonula occludens-1 (ZO-1) immunostaining, followed by ImageJ analysis, and transepithelial electrical resistance measurements.

iPSC-RPE monolayers expressed the RPE-specific markers zonula occludens-1 and RPE65 and exhibited a honeycomb pattern, as confirmed by nearest-neighbor analysis. Exposure to CC-HS significantly reduced transepithelial electrical resistance; increased membrane attack complex, apolipoprotein E, and lipid deposits; and altered levels of autophagy-related proteins. Preincubation with soluble CR2-fH or CR2-fH expression reversed these features. Quantitatively, similar changes were observed in ARPE-19 cell monolayers.

The AP inhibitor CR2-fH delivered either as a soluble protein or via gene therapy is efficacious in preventing serum-induced complement activation that led to monolayer disruption and AMD-like pathology in iPSC-RPE cells. These results contribute to existing data highlighting the importance of the AP of complement in dry AMD pathology and its potential role in AMD treatment.