Gene delivery to the eye using adeno-associated viral vectors
Introduction
The potential for gene delivery to the eye using adeno-associated virus (AAV) vectors has received much recent attention. Gene transfer experiments in animal models allow vision researchers to study the mechanisms of retinal degenerative diseases and to explore possible new treatments for ocular diseases using gene therapy techniques. Recombinant AAV vectors have a number of important advantages over other vectors which make them suitable for such studies, in particular a relative lack of pathogenicity and their ability to induce long-term transgene expression in the eye [1], [2], [3]. AAV vectors can be used to transfect a variety of ocular cell types including photoreceptors [4], [5], [6], retinal pigment epithelial cells [4], [7], [8], Muller cells [9], retinal ganglion cells (RGCs) [2], [10], trabecular meshwork cells, and corneal endothelial cells [11].
There are two main approaches by which therapeutic AAV-mediated gene transfer might be useful in the context of ocular disease. First, AAV-mediated gene therapy has the potential to correct the specific gene defect in conditions where the defect is well understood. Correction of an ocular genetic defect requires gene delivery directly to the defective cells and has been successfully used to slow photoreceptor loss in several rodent models of primary photoreceptor disease [6], [12], [13], [14]. As an example, AAV-mediated transgenes have recently been shown to restore photoreceptor structure and function in retinal degeneration slow (rds) mice [15]. rds mice have a mutation in the Prph2 gene, coding for a photoreceptor-specific membrane glycoprotein called peripherin-2, which causes development of photoreceptor disks and causes the outer segments to fail. Mutations in Prph2 have also been demonstrated in retinitis pigmentosa, a human disease which causes progressive visual loss. rds mice injected subretinally with AAV–Prph2 developed new outer segment structures containing rhodopsin and looking ultrastructurally similar to normal rod photoreceptor outer segments. Electroretinography of treated animals showed dramatic improvements in physiological measures of photoreceptor function in the short term [15], although follow-up studies showed that photoreceptor death continued despite transient restoration of function [14].
AAV-mediated gene replacement has also been used recently to restore visual function in a dog model of Leber’s congenital amaurosis (LCA), a retinal degeneration that can cause severe childhood visual loss [16]. The gene defect in the naturally occurring dog model, a mutation in the RPE65 gene which codes for a retinal pigment epithelium (RPE) cell membrane-associated protein involved in retinoid metabolism, also occurs in human LCA. An AAV carrying wild-type RPE65 was able to restore vision as assessed by electroretinography, pupillometry, and psychophysical and behavioral tests.
For many ocular conditions, however, no specific genetic defect has been characterized. It is likely that many of these diseases will turn out to involve pathology more complex than a well-characterized mutation in a single gene. Glaucoma, the second leading cause of blindness in the world [17], is an example of an ocular disease which is likely to involve the interaction of multiple genetic and environmental factors and as such is unlikely to be “cured” by the replacement of a single gene. Yet, in such circumstances, a second strategy for gene therapy may be useful. This involves not replacing a defective gene, but using gene transfer to reduce loss of function by ameliorating the effect of the primary defect(s). As examples of this approach, AAV-mediated transfection of retinal cells with the gene for basic fibroblast growth factor (FGF-2), glial cell line-derived neurotrophic factor, and ciliary neurotrophic factor have been demonstrated to slow photoreceptor loss in rat models of retinitis pigmentosa [9], [12], [13].
Recent successes with AAV vectors in animal models mean that human clinical trials of AAV-mediated gene therapy for some severe photoreceptor degenerative diseases are already being planned. However, success with disease models involving cells other than photoreceptors and RPE cells has been much more limited. Optimization of techniques to target appropriate genes to appropriate retinal cells therefore remains an important goal for the future development of ocular gene transfer technology. Here, we describe the selection of an appropriate AAV vector for ocular gene transfer studies and discuss the techniques used to deliver the virus to the eye and to assess ocular transfection. We emphasize the techniques for successful gene transfer to RGC, which have often proven challenging to transfect with high efficiency.
Section snippets
Factors influencing ocular transfection by AAV
The efficiency of transfection of particular cell types in the eye is determined by a number of variables including the site of injection, the AAV serotype and titer, the amount of passenger DNA, and the specific gene promoters and enhancing elements used.
Intravitreal injection technique
In our studies, we have used intravitreal injections of AAV vectors to transfect rat RGCs. All animals are treated in accordance with the ARVO Statement for Use of Animals in Ophthalmic and Vision Research using protocols approved and monitored by the Animal Care Committee of the Johns Hopkins University School of Medicine. Adult Wistar rats (375–425 g) are anesthetized with intraperitoneal ketamine (50 mg/kg) and xylazine (5 mg/kg) and topical 1% proparacaine eyedrops. Pupillary dilatation is
Suggestions for troubleshooting
When the transfection efficiency for the retinal cell type of interest appears poor, a number of explanations should be considered.
Concluding remarks
AAV-mediated gene transfer is a powerful technology with which to explore the pathology of ocular diseases and to investigate potential new therapeutic approaches. The eye is an excellent candidate for gene therapy, given its small size, its relative anatomical isolation, its numerous well-characterized genetic defects, and the ease with which vectors can be delivered to the immediate vicinity of cells involved in a particular disease. Successful gene transfer to specific retinal cell
Acknowledgements
This study was supported in part by PHS Research Grants EY 02120 (Dr. Quigley) and EY 01765 (Core Facilities Grant, Wilmer Institute), The TFC Frost Trust, UK (Dr. Martin), and University College, Oxford, UK (Dr. Martin), and the Alzheimer’s Association (Dr. Klein).
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