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Kenton T. Woodard, Takeshi Iwase, Luk H. Vandenberghe, Ping Jie Xiao, Josh C. Grieger, Albert M. Maguire, Katharine J. Liang, Jean Bennett, Peter A. Campochiaro, R. Jude Samulski; Trafficking of an Adeno-Associated Virus Variant in the Retina. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1223.
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We recently engineered a variant of AAV2, called AAV2.5, which demonstrated enhanced gene delivery in muscle, and tested the retinal tropism of this virus. Although both AAV2 and its variant have the same heparin binding profiles, AAV2.5 showed enhanced transduction of multiple retinal layers when delivered intravitreally (IVit) into normal rodent and pig eyes. The focus of this study is to better understand the molecular mechanism of AAV vector trafficking in the retina after IVit delivery.
We integrated single-particle fluorescence imaging with 3D de-convolution, isosurface rendering and quantification as well as Fundus, histology and DNA in situ hybridization to examine viral distribution and dynamics in retina of mice, pigs, and primates at the single particle level.
In both rodent and pig models, AAV2.5 transduced multiple retinal layers, distinguishing this capsid from AAV2. Fundus showed global expression of AAV2.5. However, in studies with primates, no transduction was observed, suggesting a striking difference in retinal barriers between these models. Further analysis using DNA in situ demonstrated a lack of detectable genomes in primate retinas. Infectious virions were still present in primate vitreous at 1 month post-administration. Vitreous from primates showed no inhibition of AAV transduction in an in vitro neutralizing assay.
Recently, we uncovered a rate-limiting step during cytosol-to-nuclear transport of virions in the first 8hrs post-infection. Further, we also observed that following intramuscular injection, AAV spread progressively across muscle tissues through endomysium between myofibers instead of traversing through target cells. We were interested in the diffusion kinetics of IVit delivered AAV2.5. Kinetic studies in the pig eyes revealed AAV2.5 does not penetrate the retina until after 6.5hrs post-administration. Taken together, we anticipate that ongoing studies will help elucidate the mechanism(s) underlying the lack of diffusion or penetration in the primate retina. Our results highlight important cross-species differences in the diffusional properties of AAV capsids within the vitreous.
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