Abstract
Purpose :
Best disease is an autosomal dominant inherited maculopathy, caused by over 600 individual mutations in the BEST1 gene, which lead to degeneration of the Retinal Pigment Epithelium (RPE) and central vision loss. Currently there are no treatments for Best disease. We explore targeted inactivation of the mutant BEST1 allele as a therapy for Best disease in patient-derived induced Pluripotent Stem Cells (iPSCs) using a mutation agnostic CRISPR/Cas9 dual guided strategy that targets common Single Nucleotide Polymorphism (SNP)-induced Protospacer Adjacent Motif (PAM) sites (SiPAMs) in the dominant allele.
Methods :
A skin biopsy was taken from a patient with Best disease (BEST1, c.37C>T, p.R13C) and used to generate iPSCs using episomal vectors. SiPAMs were identified in the dominant BEST1 allele by PCR amplification and phase sequencing. Synthetic sgRNA guides targeting two SiPAMs were combined with spCas9 and 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.
Results :
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.
Conclusions :
We have demonstrated that simultaneously targeting two SiPAMs unique to the dominant BEST1 allele leads to the excision of the dominant allele, inactivating its expression, without editing the wildtype allele. Our preliminary data shows that this approach can restore normal RPE morphology and protein localisation in patient cells, highlighting this approach as a potential therapy for Best disease. Next, we will perform Next Generation Sequencing in our Best disease cohort to identify other common, mutant allele-specific SiPAMs. This will allow us to build a mutation-agnostic guide library that will make this therapy more widely accessible.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.