Smith, Joseph James; (2024) The use of CRISPR for allele specific gene editing in autosomal dominant Bestrophinopathies. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Bestrophinopathies are a group of clinically distinct diseases which affect the macular region of the retina, resulting in central vision loss. These diseases result from mutations in Bestrophin1 (BEST1), a calcium activated chloride channel expressed on the basolateral membrane of the retinal pigment epithelium. In this thesis, a potential CRISPR therapeutic editing approach for autosomal dominant bestrophinopathies has been investigated. Using the gnomAD v4.0.0, Leiden Open Variation Database 3 and Moorfields Eye Hospital OpenEyes Electronic Medical Record databases a list of disease-causing BEST1 variants have been compiled. With over 515 unique variants identified, therapeutic approaches using CRISPR Cas9-based strategies were assessed. An allele-specific approach was chosen to target the mutated allele. A model system to assess dysfunction and test the hypothesised editing approach was created using cellular reprogramming with episomal plasmids. A patient with autosomal dominant Best disease (NG_009033.1(NP_004174.1):p.(Arg13Cys)) was recruited through Moorfields Eye Hospital (London) with their fibroblast cells utilised for this project obtained under informed consent. Induced pluripotent stem cells were reprogrammed from these patient-derived fibroblast cells, characterised to ensure pluripotency and differentiated into RPE. During RPE characterisation, significant mislocalisation of BEST1 was observed in the patient IPSC-derived RPE cells. To identify potential CRISPR Cas9 targeting sites on the patients’ mutated allele, large amplicons of the BEST1 gene were PCR amplified, cloned into bacterial cells and phase sequenced. Phase maps of the dominant and wild-type allele were created and assessed for CRISPR spCas9 PAM sites (NGG) unique to the dominant allele. CRISPR guides were designed to target two single nucleotide polymorphism induced PAM sites (rs972353 and rs1800007) identified on the dominant allele only. Transfection of the guides into patient IPSC removed a 1675bp region of the BEST1 gene including the ATG start codon, with no off-target sites identified. This editing was specific for the dominant allele, suggesting this approach could be used to specifically knock out the mutated allele. In conclusion, this thesis has examined the landscape of BEST1 mutations and identifies a novel approach, targeting common SNPs on BEST1 allele, which could be developed in the future as an allele-specific approach to treating dominant bestrophinopathies. Additionally, the generation of a novel IPSC-derived RPE model of Best disease is described and utilised to test the novel approach developed for allele-specific gene editing and to investigate the consequences of the identified mutation.

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