CRISPR/Cas9 gene-edited RPE cells to model early stages of AMD

Abstract

Purpose : Complement system plays a key role in the pathobiology of early AMD, but the underlying mechanisms are unknown, which makes it difficult to design effective therapies. The inherited EFEMP1-associated macular degeneration is clinically similar to AMD, and we have previously used this model to show that the formation of sub-RPE deposits is due to impaired extracellular matrix (ECM) and can be modulated in vitro via C3. We hypothesize that complement is activated by hydrolysis of C3 via tick-over, which forms a stable C3(H2O)-convertase by deposition on abnormal ECM or aged Bruch’s membrane (BrM). This causes chronic local complement activation and further formation of sub-RPE deposits. Our aim is to unravel the mechanisms by which complement activation causes deposit formation in early stages of AMD.

Methods : We engineered ARPE-19 cells to be homozygous for the R345W mutation in EFEMP1 using CRISPR/Cas9 – mediated genome editing. RPE-EFEMP1^R345W/R345W cells were grown on transwells for 4 weeks and decellularized to culture human fetal RPE (hfRPE) cells on the exposed ECM. Further formation of basal deposits and complement activation were characterized by scanning electron microscopy (SEM), immunofluorescence, and ELISA. Matrix metalloproteinase (MMP) activity was measured by zymography.

Results : SEM showed that RPE-EFEMP1^R345W/R345W cells make abnormal ECM, with structure similar to aged BrM. The abnormal ECM contained increased C3b/C3(H2O) compared to ECM produced by control cells. hfRPE cells grown on the abnormal ECM produced by RPE-EFEMP1^R345W/R345W cells make thick basal deposits comprised of EFEMP1, collagen IV, collagen VI, fibronectin, and laminin, which are main components of basal laminar deposits in AMD patients. hfRPE cells grown on ECM from normal RPE cells did not make these deposits. Increased CFH, C3a and MMP-2 activity were detected in basal conditioned media of the hfRPE cells grown on abnormal ECM.

Conclusions : RPE-EFEMP1^R345W/R345W make abnormal ECM, which anchors C3b/C3(H2O) and causes local complement activation by hfRPE cells, which make thick deposits comparable to basal laminar deposits in patients. This model provides a valuable tool to study the pathogenesis of the RPE/BrM in early stages of AMD. Also, this system can be used to test complement-modulating drugs to prevent deposit formation in AMD.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.