An integrated CRISPR/iPSC-based approach to elucidate uncertain variation in RPE65, a target for gene therapy

01 November 2022 → 31 October 2024
Research Foundation - Flanders (FWO)
Research disciplines
  • Medical and health sciences
    • Transcription and translation
    • Ophtalmology
    • Stem cell biology
    • Molecular medicine
    • Genetics
iPSC-derived retinal pigment epithelium (iPSC-RPE) CRISPR/Cas9 RPE65 VUS target for gene therapy
Project description

In 2017 and 2018, Luxturna received FDA and EMA approval as the first gene therapy product to treat patients with biallelic RPE65-mutations, causing severe inherited blindness. Eligibility for gene therapy requires a complete molecular diagnosis, which is often hampered by the identification of variants of uncertain significance (VUS). To this end, we first will set up a biochemical assay to assess the pathogenicity of coding RPE65 VUS, by simulating the visual cycle in HEK293-F cells via overexpression of relevant genes, incl. RPE65. RPE65 is a key enzyme in the visual cycle, uniquely expressed in the retinal pigment epithelium (RPE), which is characterized by tissue-specific splicing and expression patterns. Expanding on the first assay, a second, biologically relevant disease model will be developed using human iPSC-derived RPE and CRISPR-editing. In addition, we will investigate the regulatory network underlying RPE65 expression by interrogating 9 candidate cis-regulatory elements of RPE65 via genome editing in ARPE-19 cells. This integrated approach, using established and novel, biologically relevant cellular models and genome editing will elucidate the regulation and pathomechanisms of coding and non-coding variants in RPE65. The set-up will improve eligibility of patients with uncertain RPE65 variants for gene therapy (trials) but can ultimately serve as platform to assess all genomic variation in RPE-expressed genes and impact a substantially larger group of patients.