Affecting about two million people worldwide, inherited retinal diseases (IRDs) have a huge socio-economic impact. Causing impaired vision or even blindness, IRDs hamper quality of life of affected individuals, since daily activities become difficult or even impossible. IRDs also impact the social environment of affected individuals, as they need support and help of people such as caretakers, friends and family. Providing effective treatment to IRD patients has increased priority as illustrated by available IRD gene therapies and many ongoing studies. Luxturna is the first approved gene augmentation therapy to treat patients with confirmed (likely) pathogenic biallelic recessive RPE65 mutations. However, many RPE65 variants are considered variants of uncertain or unknown significance (VUS) and patients carrying these VUS are not eligible for Luxturna. The main aim of this thesis is to decipher the functional effect of 53 RPE65 VUS in order to reclassify these variants. Ultimately this may impact patient’s eligibility for RPE65 gene therapy. This project also focusses on the identification of a novel ‘Belgian’ dominant RPE65 variant, which can improve molecular diagnoses in unresolved IRD patients. Functional assessment of the variant can help unravel disease mechanisms, possibly leading to new treatment opportunities. Providing a molecular diagnosis is useful for family planning, which might include pre-implantation genetic testing (PGT) to prevent passing on this dominant variant to the next generation.