(T1004) INVESTIGATING THE USE OF ALLELE-SPECIFIC GENOME EDITING IN A RARE AUTOSOMAL DOMINANT FORM OF BLINDNESS

Abstract: Best disease is an autosomal dominant inherited macular disease, caused by over 600 individual mutations in the BEST1 gene, leading to degeneration of the Retinal Pigment Epithelium (RPE) and central vision loss. Currently, there are no treatments for Best disease. We explore the therapeutic potential of targeted inactivation of the mutant BEST1 allele in patient-derived induced Pluripotent Stem Cells (iPSCs), using a mutation agnostic CRISPR/Cas9 dual-guide strategy targeting common Single Nucleotide Polymorphism (SNP)-induced Protospacer Adjacent Motif (PAM) sites (SiPAMs) in the dominant allele. A skin biopsy was taken from a patient with Best disease (BEST1, c.37C>T, p.R13C) reprogrammed into iPSCs using episomal vectors. SiPAMs were identified in the dominant BEST1 allele by PCR amplification and phase sequencing. Synthetic sgRNA guides targeting two SiPAMs and spCas9 were nucleofected into iPSCs as ribonucleoproteins. Editing of the dominant allele was confirmed by PCR and Sanger sequencing. RPE was generated via a directed differentiation protocol. An iPSC line carrying the BEST1 pR13C mutation was generated and pluripotency was confirmed. Immunostaining of BEST1R13C iPSC-derived RPE revealed mislocalisation of BEST1 and flattened cell morphology. Phase sequencing of BEST1 highlighted seven SNPs unique to the dominant allele, of which two generated SiPAM sites that flanked BEST1 start codon. Dual sgRNAs nucleofection of iPSC and clonal expansion revealed clones had the predicted excision of 1672bp in the dominant allele, while the wildtype allele remained unedited. Basolateral Best1 protein expression and polarised RPE cell morphology was restored in the edited iPSC-derived RPE, compared to BEST1R13C cells. We have demonstrated that targeting two SiPAMs specific to the dominant BEST1 allele excises the start codon, inactivating mutant BEST1 expression without affecting the wildtype allele. Our preliminary data shows the restoration of RPE morphology and protein localisation in patient cells, highlighting this approach as a potential therapy for Best disease. We will perform Next Generation Sequencing in our Best disease cohort to identify additional mutant allele-specific SiPAMs, enabling the development of a mutation-agnostic guide library to broaden therapy accessibility.

Funding Source: University College London, Moorfields Eye Charity

Clinical Trial ID number: