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Establishing a Human Cell-Based Model System for Macular Degeneration Almar Neiteler PhD The University of Edinburgh 2019 Declaration I declare that this thesis was composed by myself and that all experiments in this thesis were performed by myself unless otherwise indicated in the text. This work has not been submitted for any other degree or professional qualification. Almar Neiteler 2019 ii Acknowledgements I would like to thank Prof James Ross, Prof Baljean Dhillon, and Prof Siddharthan Chandran for giving me the opportunity to carry out this work. I would like to thank all the members of Prof Ross’ Tissue Injury and Repair Group, and in particular Kathryn Sangster and James Black for their technical support, Dr Anwar Palakkan for his technical advice, especially regarding flow cytometry and qPCR, and his help with the stem cell cultures, and Kevin Gallagher for his advice on the cell viability assays. I would also like to thank all the members of Prof Chandran’s group, and in particular Dr Bhuvaneish Thangaraj Selvaraj for all his technical advice and especially regarding gene editing, Dr Karen Burr for her help with the stem cell cultures and her advice on differentiation protocols, James Cooper for his help with the gene editing, Dr James Longden for his help with systems biology analysis of the RNA-seq data, and David Story for his technical support. I am grateful for all the technical advice provided by Dr Shyamanga Borooah (University of California, San Diego), especially regarding L-ORD and the complement system. Furthermore, I would like to thank Prof David Hay and Prof Sarah Howie (The University of Edinburgh) for their advice and support; I would like to thank Dr Owen Dando and Prof Giles Hardingham (The University of Edinburgh) for their help with the RNA-seq data analysis and all their advice regarding the RNA-seq; I would like to thank Graham Anderson and Dr Pierre Bagnaninchi (The University of Edinburgh) for their collaboration on the UV and ECIS experiments; and I would like to thank Dr Widad Dantoft (Cardiff University) and Callum Idle for proofreading this thesis. Finally, I would like to thank my family, who have supported me in every way possible throughout this whole experience. iii Abstract 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. iv Lay Summary 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 clinical presentation which inevitably leads to difficulty when studying this disease. AMD always leads to the degeneration of the macula, the most light-sensitive part of the retina and is responsible for sharp central vision. Lack of knowledge of this disease is one of the main reasons why there is currently no effective treatment. Related diseases with similar characteristics to AMD, are caused by one mutation. Unlike AMD, these are much easier to study and could potentially provide more information about shared macular degeneration- causing mechanisms. One of these diseases is late-onset retinal degeneration (L-ORD), which is a rare disorder caused by a mutation in the gene C1QTNF5. L-ORD has a noticeably similar clinical presentation to AMD, and therefore the mutation in C1QTNF5 is likely to affect the same molecular mechanisms involved in causing AMD. Here, I used gene editing to correct the C1QTNF5 mutation in patient-derived stem cells. These stem cells were then converted into retinal pigment epithelial (RPE) cells, the type of eye cells most affected in macular degenerations. Comparison of gene-corrected RPE and uncorrected RPE from patients revealed some key differences. The protein encoded by the C1QTNF5 gene was found to have a different shape caused by the mutation, which likely affects its function. Moreover, RPE from patients had greater sensitivity to light-induced stress and increased accumulation of immune system proteins compared to the gene-corrected cells. Together, the results in this thesis reveal some of the molecular mechanisms affected in L-ORD that could explain part of the clinical presentation of this disease and possibly also of AMD. Further study of the affected molecular mechanisms could potentially lead to a therapy for both L-ORD and AMD. v Table of Contents Abstract . iv Lay Summary . v Table of Contents . vi List of Figures . x List of Tables . xiii List of Abbreviations . xiv Chapter 1 Introduction . 1 1.1 The retina . 1 1.2 Retinal pigment epithelium . 4 1.3 Age-related macular degeneration . 7 1.4 Late-onset retinal degeneration . 9 1.5 Malattia Leventinese / Doyne honeycomb retinal dystrophy and Sorsby's fundus dystrophy . 10 1.6 Aims of this study . 12 Chapter 2 Gene Editing C1QTNF5 . 14 2.1 Introduction . 14 2.1.1 Induced pluripotent stem cells . 14 2.1.2 CRISPR/Cas9 gene editing . 15 2.1.3 Aims and objectives . 17 2.2 Materials and methods . 18 2.2.1 Cell culture . 18 2.2.2 Pluripotency staining . 18 2.2.3 Karyotyping . 18 2.2.4 Designing CRISPR/Cas9(n) constructs and cloning . 19 2.2.5 Transfection . 20 2.2.6 PCR . 21 2.2.7 Restriction fragment length polymorphism screening and Sanger sequencing 22 2.2.8 Off-target screening . 22 2.2.9 C1QTNF5 variant effect prediction . 23 2.3 Results . 24 vi 2.3.1 iPSC lines have normal stem cell characteristics . 24 2.3.2 Design and construction of C1QTNF5S163R gene correction plasmids . 27 2.3.3 Gene correction gRNA specificity test transfection of HEK 293T cells . 29 2.3.4 Gene correction gRNA specificity test transfection of L-ORD iPSCs . 30 2.3.5 Gene correction of LORD4 using the RNP method . 32 2.3.6 Gene correction of LORD4 using the plasmid method . 34 2.3.7 Gene correction of LORD3 using the plasmid method . 35 2.3.8 Checking gene-corrected LORD3 and LORD4 clones for off-target activity . 36 2.3.9 Strategy for generating C1QTNF5 KO lines . 36 2.3.10 KO gRNAs test transfection of HEK 293T cells . 37 2.3.11 Generation of C1QTNF5 KO iPSC line . 38 2.3.12 Checking C1QTNF5 KO clones for off-target activity . 41 2.4 Discussion . 42 Chapter 3 Characteristics of L-ORD iPSC-RPE and C1QTNF5 . 45 3.1 Introduction . 45 3.1.1 Differentiation of iPSC to RPE . 45 3.1.2 C1QTNF5 . 47 3.1.3 Aims and objectives . 51 3.2 Materials and methods . 52 3.2.1 Cell culture .