The development of gene therapy techniques and animal models for Retinitis Pigmentosa and towards the identification of a novel RP-causing gene in a family of Irish origin

Abstract

The work presented in this thesis pertains to several different stages in the preclinical development of therapeutics for inherited retinal disease. Chapter two addresses the difficult challenge of delivery of therapeutics to the retina. The technique of subretinal vector delivery is described and successfully illustrated. In addition, a comparison of two different types of viral vectors expressing reporter genes in the retina is described, including an evaluation of the enhancement of reporter gene expression by using immunesupressants. The chapter concludes with an analysis of the therapeutic effect of subretinal delivery, to the rhodopsin knockout mouse (Humphries et al., 1997), of an adeno-associated viral vector expressing rhodopsin. Chapter three describes the generation and characterisation of an animal model of human RP in the mouse. Rhodopsin knockout mice (Humphries et al., 1997) were bred against transgenic mice carrying a Pro-23-His mutant rhodopsin transgene (Olsson JE et al., 1992) producing a mouse expressing the mutant rhodopsin on a Rho-I- background. This new animal (Rho-/-, P23H) was subsequently bred to another transgenic mouse carrying a normal human rhodopsin gene on a rhodopsin knockout background (McNally N et al., 1999). The resulting transgenic mice express both mutant and wild type human rhodopsin at normal levels on a murine Rho-I- background. This mouse will be valuable for testing ribozyme based therapies targeting human rhodopsin sequences. The pathology observed in Rho-/- P23H NHR mice is described. Chapter four describes an extensive study to find the mutated gene in the last known large family of Irish origin segregating with autosomal dominant Retinitis Pigmentosa. A description of some interesting findings at previously described adRP loci in addition to the exclusion of all previously described loci for RP is presented. Also included are the exclusion results of an extensive genome-wide search for the disease causing locus and the sequencing of the PRPF31 gene previously implicated in the pathology of RP.