부산대학교 도서관

Age-related macular degeneration (AMD) is the most common cause of visual loss amongst the elderly in developed countries. AMD is a complex disease with a highly variable phenotype, which makes generating reliable disease models difficult. In addition, lack of knowledge of the underlying pathological mechanisms makes development of an effective treatment difficult. To address the lack of knowledge of the molecular mechanisms affected in AMD a robust cell-based model system is needed. Late-onset retinal degeneration (L-ORD) is a rare autosomal dominant disorder caused by a p.S163R, p.P188T, or p.G216C missense mutation in the C1q and tumor necrosis factor-related protein 5 (C1QTNF5) gene. L-ORD has a very similar phenotype to AMD, including sub-RPE deposit formation, the hallmark of AMD. Studying L-ORD could therefore potentially reveal common molecular pathways affected in macular degenerations. Here, I used CRISPR/Cas9 gene editing to correct the p.S163R mutation in C1QTNF5 in patient-derived induced pluripotent stem cells (iPSCs) and generated C1QTNF5 null iPSCs. The iPSC lines were differentiated into retinal pigment epithelium (RPE), the cell type that highly expresses C1QTNF5 and is severely affected in macular degenerations. RNA sequencing of L-ORD and control iPSC-RPE revealed that the extracellular matrix, complement system, lipid and general cell metabolism, and oxidative stress pathways are affected in L-ORD. C1QTNF5S163R in L-ORD iPSC-RPE was found to form less high molecular weight multimeric structures compared to its isogenic gene-corrected control. In addition, L-ORD iPSC-RPE were also found to phagocytose and possibly adhere differently compared to their isogenic controls. Both oxidative stress and the complement system play an important role in the pathology of macular degenerations. RPE were found to undergo regulated necrosis in response to oxidative stress and L-ORD iPSC-RPE were found to be more sensitive to UV light-induced oxidative stress. In addition, the terminal complement system complex C5b-9 was found to bind more frequently to L-ORD iPSC-RPE and colocalises with APOE in sub-RPE drusen-like deposits after human serum exposure. Together, the results in this thesis reveal that L-ORD iPSC-RPE have a molecular phenotype that could explain part of the clinical presentation of this disease and possibly of AMD. Further study of the affected molecular mechanisms could potentially lead to therapies for both L-ORD and AMD.