Abstract
Sub-retinal injection of the common AAV2 pseudotypes frequently results in strong transduction of the retinal pigment epithelium (RPE) as well as the retina itself. This has been of benefit to date in human clinical trials using AAV, where the disease target is in the RPE. However, many mutations predisposing to retinal disease are located in the photoreceptor cells, present in the neural retina and not the RPE; in this case the sub-retinal injection route may cause an effective “loss” of therapeutic AAV to the RPE. The αvβ5 integrin receptor is highly expressed on the apical surface of the RPE, and is essential to the daily phagocytosis of the outer segment tips of photoreceptor cells. The transduction efficiency of AAV was tested in the retinas of β5−/− mice lacking this receptor and showing defects in photoreceptor outer segment phagocytosis. Following sub-retinal injection of AAV2/5-eGFP, fluorescence was found to be stronger and more widespread in the neural retina of β5−/− mice compared to wild-types with greatly reduced fluorescence in the RPE. Increased levels of the phagocytic signalling protein MFG-E8, the ligand for the αvβ5 integrin receptor, is found to have a moderate inhibitory effect on AAV transduction of the retina. However the opposite effect is found when only the integrin-binding domain of MFG-E8, the RGD (Arginine-Glycine-Aspartic acid) domain, was increased. In this case RGD enhanced AAV-mediated retinal transduction relative to RPE transduction. These results are presented for their relevance for the design of AAV-based retinal gene therapy strategies strategies targeting retinal/photoreceptor cells.
Highlights
To date, the clinical success of retinal gene therapy has involved delivery of genes to the RetinalPigment Epithelium (RPE) [1,2] which, though essential to the function and the maintenance of the retina is not part of the neural retina itself
There are some problems with this: efforts to fine-tune Associated Virus (AAV)-mediated gene expression for photoreceptors have been complicated by the significant “loss” of viral titre to the juxtaposed retinal pigment epithelium (RPE) cells (Figure 1)
We propose two potential reasons for this transduction bias: (1) The reduced level of retinal adhesion and impaired phagocytosis in the β5−/− retina may lead to the retention of the AAV particle in the photoreceptor cell; (2) the loss of a potential coreceptor in, the αvβ5 integrin, normally abundant on the RPE surface, may increase the utilisation of alternative coreceptors by AAV, these other coreceptors, may be potentially more prominent on the retinal side; (3) the decreased level of adhesion between the neural retina and RPE of β5−/− mice may allow for greater retinal detachment upon subretinal injection and greater AAV
Summary
The clinical success of retinal gene therapy has involved delivery of genes to the Retinal. (2) the loss of a potential coreceptor in, the αvβ integrin, normally abundant on the RPE surface, may increase the utilisation of alternative coreceptors by AAV, these other coreceptors, may be potentially more prominent on the retinal side; (3) the decreased level of adhesion between the neural retina and RPE of β5−/− mice may allow for greater retinal detachment upon subretinal injection and greater AAV uptake. This may be due to the reduced level of rhythmic phagocytosis that occurs in this model leading to the retention of the AAV particle in the photoreceptor cell. We investigate these two possibilities further in wild-type (WT) mice by using the endogenous signalling ligand molecule for the αvβ receptor and consider its implications for the clinical use of AAV
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