Retinitis pigmentosa GTPase regulator gene replacement - a potential treatment for x-linked retinitis pigmentosa

X-linked retinitis pigmentosa (XLRP) is the most severe form of retinitis pigmentosa, a neurodegenerative, blinding disorder of the retina. 70% of XLRP cases are due to mutations in the retina-specific isoform of the gene encoding retinitis pigmentosa GTPase regulator (RPGRORF15). Because of the relatively large prevalence and severe clinical phenotype, the responsible gene locus, RPGRORF15, is ideally suited for a tailored gene therapeutic approach. The overall objective of this work therefore was to develop an optimised gene replacement therapy for human XLRP disease. The human RPGRORF15 coding sequence (cds) was codon optimised and both wildtype (wt) and codon optimised (co) sequences were spliced into a specific vector backbone designed for production of adeno-associated virus (AAV). In vitro studies in HEK293T, SHSY-5Y and 661W cell lines showed that the co construct features greater sequence stability and expression levels when compared to the wt construct. After packaging the superior co construct into AAV8 capsids, subretinal injections were performed in a pilot study on relevant animal models with targeted disruption (Rpgr-/y) or natural mutation (C57BL/6JRd9/Boc) in the murine homologue Rpgr. Transgene expression and correct localisation to the physiological subcellular compartment could be demonstrated. A masked, randomised and controlled study in C57BL6/J wild type mice was able to show lack of toxic effects while application in Rpgr-/y and C57BL/6JRd9/Boc mice led to significant rescue as determined by electroretinography. Overall the work in this thesis provides the basis for a clinical trial development to treat patients with XLRP due to RPGR mutations. A patent application to protect the intellectual property of this work has been filed on behalf of the University of Oxford.