Allele-specific suppression of pathogenic Bestrophin-1 transcripts by targeted CRISPR/Cas9-mediated genome editing

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In recent years, major advances in our understanding of the molecular underpinnings of orphan diseases have made it feasible to advise precise therapies, often individually tailored to a specific type of genetic defect. Most notably, gene replacement therapy has found wide attention for the autosomal recessive disorders where complete or partial loss of protein expression is rescued by safe and efficient adeno-associated viral gene transfer. In contrast, autosomal dominant mutations, specifically those leading to gain of function or acting in a dominant negative fashion, are still challenging to address as the mutated gene product may interact with the normal gene copy and thus needs to be removed first before a replacement treatment can be considered.With the present proposal we want to explore a novel therapeutic option to treat dominant-negative disease by specifically eliminating mutant transcripts via the CRISPR/Cas9 genome editing technology. To demonstrate feasibility of such an approach, we focus on a dominant-negative bestrophin-1 (BEST1) mutation (Y227N) known to cause Best disease, a sight-threatening maculopathy of early adulthood. To this end, we have established patient-derived cells and have subsequently differentiated these cells into retinal pigment epithelium (RPE), a post-mitotic tissue known to harbor the primary pathology of BD. In this in vivo cell culture system, we want to demonstrate feasibility of restoring (increasing) normal BEST1 channel activity after ablating dominant-negative gene expression. In a second line of experiments, we focus on translational aspects to explore delivery routes of the required genome editing tools to the RPE and efficacy of treatment in a living mouse eye. Together, our in vitro and in vivo work may serve as proof-of-concept to further develop CRISPR/Cas-driven approaches in precision medicine and to pave the way for treatment of patients with dominant-negative gene mutations.