Mutations in the bestrophin 1 (BEST1) gene lead to a variety of bestrophinopathies. To identify the exact location and function of BEST1 is key to understanding the mechanisms that cause bestrophinopathies. Thus, it was decided to study autosomal-recessive bestrophinopathy (ARB), a distinct inherited bestrophinopathy caused by BEST1, a protein located in the retinal pigment epithelium (RPE), which is a monolayer of epithelial cells located at the back of the eye between the photosensitive retinal layer and the choroid. The RPE closely interacts with the photoreceptor layer. Hence, mutations in BEST1 cause the RPE to dysfunction, which in turn leads to photoreceptor degeneration and eventual blindness. The ARB mutation studied in this thesis is a pR200X mutation that is a premature stop mutation causing alteration in the RPE and subretinal deposits in the macular area. Currently, patients with bestrophinopaties, such as ARB, do not have any available treatments and loss of vision cannot be prevented. As mentioned earlier, resolving the exact location and function of the BEST1 in the RPE is a crucial step in identifying therapies for these patient groups and understanding the pathology underlying bestrophinopathies. Human induced pluripotent stem cells (hiPSCs) are a promising source of cells to model a patient-specific disease in vitro and identify potential therapies. It has been previously demonstrated that RPE could be produced from the iPSCs and human embryonic stem cells (hESCs). Thus, in order to understand the role of BEST1 in RPE cells iPSCs were created from pR200X patient fibroblasts by reprogramming with episomal vectors (C-MYC, KLF4, LIN28, OCT4 and SOX2). iPSC colonies were isolated and expanded. After optimising the stem cell culture methods for patient-specific iPSCs, the iPSCs were differentiated into RPE by a mainly spontaneous differentiation method with an initial burst of activin A. After approximately 6 weeks pigmented foci were purified by manual dissection and cells were seeded to encourage monolayer formation. Patient iPSCs and iPSC-derived RPE cells were assessed by standard molecular and cellular protocols, including immunocytochemistry, electron microscopy, western blots, PCR and teratoma assay, in comparison to the control iPSCs and iPSC-derived RPE cells. To assess any functional abnormalities in the pR200X iPSC-derived RPE cells transepithelial resistance, phagocytosis and patch-clamping experiments were performed on patient and control iPSC-derived RPE. Additionally, gene therapy was investigated as a potential therapeutic option for the bestrophinopathy patients with a premature stop mutation
