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J. Cehajic, R. Lucas, P. N. Bishop; Making the Blind See Again - A Gene Therapy Approach to Converting Retinal Ganglion Cells Into Visual Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3007.
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Many patients are blind due to degeneration of the outer retina, but the retinal ganglion cells (RGCs) are still functional. Some RGCs have photosensitive properties, but are not concerned with vision. We explore the possibility of converting RGCs into directly visual photoreceptors (PRs) using gene therapy approach. However, in order to achieve useful vision there will need to be a high level of retinal transfection. So far, only low transfection rates have been achieved with gene delivery to the eye using adeno-associated viral (AAV) vectors in an experimental mouse model. We further hypothesize that, enzymes that breakdown barriers such as vitreous, inner limiting lamina (ILL) and retinal matrix can increase retinal transfection rates. Therefore, our overall aim is to convert RGCs into functional visual PRs in humans using gene therapy. Our specific aim is to improve retinal transfection rates with AAV2 vector using enzymes to breakdown vitreous, inner limiting lamina and retinal matrix.
In this study AAV2 vector carrying a gene encoding green fluorescent protein (GFP) was delivered into the vitreous of mice in conjunction with an enzyme. Collagenase and glycosidic enzymes including hyaluronidase, chondroitinase and heparinase were used in increasing concentrations. AAV2-GFP complex has to enter the cell, which needs to be viable to produce GFP. This expression of GFP was analyzed in retinal flat mounts at two weeks post injections using confocal microscopy.
Confocal microscopy images show GFP fluorescence in retinas in order of magnitude: heparinase (n=10) > chondroitinase (n=10) > hyaluronidase (n=10) > collagenase (n=10) > no enzyme (n=8). We also show that various cell types of the retina were capable of transfection and produced robust levels of GFP expression that were enzyme dose-dependant.
This study demonstrates that gene delivery to the retina can be achieved with AAV vectors. Retinal gene expression is significantly increased using enzymes that breakdown vitreous, ILL and retinal matrix. This treatment strategy provides a potential for future efficient delivery of various opsin genes in order to convert RGCs into visual PRs and restore vision after retinal degeneration.
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