Medical and health sciences
- Transcription and translation
- Stem cell biology
- Molecular medicine
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.