Enhancing ocular gene therapy using glycosidic enzymes

Gene therapy shows great promise in treating human blindness due to genetic diseases affecting the retina. Healthy genes, contained in modified viruses, are delivered by injection under the retina, a thin later of tissue that converts light into signals that are sent to our brain. However, this approach can potentially damage the retina and cannot deliver the genes across the entire retina. A potentially safer and more effective approach would be to inject the gene-carrying viruses into the vitreous, a gel-like structure in front of the retina. However, to date this approach has not worked very well with only a small number of retinal cells receiving the gene.

My research to date has shown that if we use enzymes in combination with the viruses we achieve much better gene delivery to the retina following injection into the vitreous. The aim of this project is to optimise the intravitreal combination of virus and enzymes to deliver genes right across the retina. I will then see if I can demonstrate recovery of retinal function in a mouse that, because of genetic alterations, has no vision. This may ultimately lead to exciting advancements in treating patients with a variety of retinal diseases.

The overall aim of this study is to: Investigate strategies to improve retinal transduction efficiency using combinations of intravitreally delivered AAV serotypes and glycosidic enzymes.



Specific objectives of this study are to:

1. Optimize combinations of glycosidic enzymes and AAV serotypes for the transduction of inner and outer retina using an intravitreal approach in a mouse model

2. Assess the safety of these treatment combinations in the mouse using functional studies

3. Confirm our ability to target cells in ganglion cell layer and photoreceptor layer in a therapeutically relevant manner using this technique



Methodology:

AAV containing a cDNA encoding enhanced GFP under the control of a chicken beta-actin promoter will be delivered intravitreally into anaesthetized adult mouse eyes in conjunction with enzymes. Various combinations and doses of glycosidic enzymes including hyaluronan lyase, chondroitin ABC lyase and heparinase III will be co-injected with the AAV2. In addition, I will investigate whether other AAV serotypes (AAV5, AAV8 and AAV9) can achieve retinal transduction following intravitreal injection when used in combination with these enzymes. Two weeks later retina flat mounts will be examined by confocal microscopy to analyze the amount and depth of penetration of the GFP fluorescence (using ImageJ software). Other retinas will be snap frozen, cryostat sectioned and co-stained with antibodies to GFP and to proteins expressed in specific types of retinal neurons to establish which particular cell types are transfected. At two weeks post treatment retinal function will be evaluated with dark- and light- adapted electroretinograms (ERG) and visually evoked potentials (VEPs), which will be compared to control eyes. Next, I will generate triple-knockout (Opn4-/-, Rho-/-, Cnga3-/-) mice lacking rod, cone and melanopsin transduction. I will then be able to use AAV containing cDNA for melanopsin (Opn4), rod opsin (Rho) or the cone cyclic GMP-gated channel A-subunit 3 (Cnga3) to restore phototransduction in melanopsin ganglion cells, rods or cones respectively. The readouts for these experiments will be circadian photo-entrainment (melanopsin photoreceptor activity), pupil reflex activity, ERGs and VEPs. The retinas will also be examined using immunohistochemistry.



Scientific and medical opportunities of the study:

If successful, this work will validate an intravitreal injection as a route for ocular gene therapy. As the current the approach of sub-retinal injection has many practical limitations, my work will meet an immediate clinical need, which is only likely to grow as the range of ocular disorders amenable to gene therapy increases.