Development of gene therapy for the treatment of inherited retinal degeneration.

Retinitis pigmentosa (RP) is the name given to a group of inherited retinal diseases that cause atrophy of the retina and retinal pigment epithelium (RPE), and frequently lead to a progressive loss of vision. Molecular techniques have implicated a large number of gene defects as causes of RP. In general the genes in which these occur are expressed in the photoreceptor and RPE cells of the retina. Currently there is no available treatment that can alter the primary defect of these diseases. This thesis describes a programme of work that examines the potential of gene therapy in the treatment for RP. It centres on the use of recombinant adeno-associated virus (rAAV) for the transfer of a therapeutic gene to photoreceptor cells. Vector plasmids containing a photoreceptor cell-specific promoter and a reporter gene were cloned. These were used to produce high titre rAAV using a novel method involving replicating herpes simplex amplicons. In subsequent in vivo studies these viruses and others were injected sub-retinally into normal mouse eyes. The resulting expression of the reporter gene in photoreceptor cells demonstrated the potential of rAAV in the efficient transduction of this cell type. Expression of the reporter gene did not decrease before the final time point of 1 year. The information gained from these studies was then utilised in the treatment of the Prph2Rd2/Rd2 mouse, an animal model of RP that results from a null mutation in a photoreceptor cell-specific gene. A vector plasmid encoding a wild type copy of this gene was cloned and used to produce rAAV. Sub-retinal injection of this virus into mice resulted in partial rescue of the disease phenotype by re-establishing the structural integrity of the photoreceptor cell layer. Functional tests performed on treated animals showed this was accompanied by a partial correction of electrophysiological responses