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Christine N. Kay, Sanford L. Boye, Renee C. Ryals, Jingfen Sun, Andy W. Neeley, William W. Hauswirth, Shannon E. Boye; Enhanced Retinal Transduction Via The Vitreous With Aav8 Capsid Mutants. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1886.
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Several ongoing clinical trials for RPE65-associated Leber congenital amaurosis (LCA2) have demonstrated the ability to subretinally target therapeutic transgene to the retinal pigment epithelium (RPE) thereby restoring vision to these patients. However, because most retinal degenerations are caused by mutations in photoreceptor-specific rather than RPE-specific genes, development of photoreceptor-targeted gene therapies would be of significant benefit. Of equal importance is the need to develop a less invasive injection procedure, particularly for diseased retinas that are more prone to injury during the subretinal injection procedure. The purpose of this study was to evaluate the ability of adeno-associated virus (AAV) capsid mutants to transduce photoreceptors following intravitreal delivery to the mouse retina. These AAV capsid mutants originated from a serotype (AAV8) with native tropism for photoreceptors.
HEK293 and 661W cells were infected with self-complimentary (sc) AAV8-smCBA-mCherry capsid mutant vectors containing single or multiple tyrosine mutations on their capsid surface at and MOI of 10,000. Fluorescence-activated cell sorting (FACS) analysis was used to measure relative mCherry fluorescence 3 days post-infection. Subsequently, scAAV8 capsid mutants containing GFP cDNA were intravitreally injected into P60 C57Bl/6J wildtype (WT) mice at a concentration ~9 x 10e9 vg/ul. Mice were sacrificed at 1 month post-injection and eyes were enucleated, cryoprotected and sectioned. Frozen sections were immunostained with an antibody raised against GFP and imaged with confocal microscopy.
AAV-mediated mCherry expression in HEK293 and 661W cells varied depending on how many capsid mutations were present, with most exhibiting higher efficiency relative to standard AAV8. Injection of AAV8 capsid mutants resulted in robust expression in the outer retina of WT mice following delivery to the vitreous.
The authors demonstrate that AAV capsid mutant vectors based on a serotype with native tropism for photoreceptors, AAV8, has the ability to transduce outer retina following intravitreal delivery. These results suggest that AAV8 capsid mutant vectors could be used to deliver therapeutic transgene to photoreceptors, thereby conferring therapy to various models of retinal degeneration associated with photoreceptor defects.
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