Abstract

Development and Assessment of Gene Therapies for Inherited Blinding Diseases

By

Kathleen Durgin Kolstad

Doctor of Philosophy in Molecular and Cell Biology

University of California, Berkeley

Professor John Flannery, Committee Chair

There are two therapeutic approaches for inherited retinal disease addressed in

this dissertation: we sought to slow retinal degeneration and reverse visual loss after

complete photoreceptor apoptosis. In the first approach, by viral gene transfer to the

support cells of the retina, Müller glia (RMCs), we achieved sustained secretion of

human glial derived neurotrophic factor (hGDNF) (Chapter 3). We hypothesized that

hGDNF production by retinal glia will enhance the protective affects of RMCs in the

diseased retina and help slow photoreceptor degeneration. Furthermore, this method avoids extra photoreceptor stress caused by direct hGDNF gene transfer to cells that are already stressed. We were able to optimize gene transfer to RMCs and observe the beginnings of functional rescue in an animal model of autosomal dominant retinitis pigmentosa with this technique. One major advantage of this therapeutic approach is that it is applicable to multiple retinal disease genotypes.

The second approach to ocular gene therapy presented in this dissertation was to

re-introduce photosensitivity to the retina after complete photoreceptor degeneration

(Chapter 4). To this end, we employed the engineered light activated glutamate receptor (LiGluR) to confer light sensitivity on retinal ganglion cells (RGCs) in the diseased retina. We first showed LiGluR mediated RGC photo-activation in in vitro retinal tissue preparation. We then characterized in vivo cell population responses (visually evoked potentials, VEPs) in V1 when retinal input was limited to LiGluR induced activity in the retina. VEPs driven by LiGluR are approximately 50% of the amplitude of full field light flash driven responses in the wild type animal. LiGluR driven cortical responses in blind animals suggest that it is a promising therapy for restoring visual function and processing in the late stages of retinal degeneration.

In the third part of this dissertation (Chapter 2a and 2b), the goal was to develop

and assess methods of making ocular gene therapies safer and more efficacious. Current gene therapies for retinal degenerative diseases rely on subretinal delivery of viral vectors carrying therapeutic DNA. However, this method of delivery limits the viral transduction profile to the region of injection and seriously compromises the retina during detachment. We have identified natural barriers to viral vector delivery to the outer retina from the vitreous. Furthermore, we have developed artificial methods and characterized disease states that allow these barriers to be overcome. The understanding of and the ability to manipulate barriers to vitreal delivery of viral vectors will help avoid the limitations, risks, and damage associated with subretinal injections.