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Robert W Nickells, Cassandra Schlamp, Heather M Schmitt; Characteristics of AAV2 mediated gene transfer in retinas after optic nerve injury. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4081.
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While a great deal of knowledge has been obtained on the characteristics of AAV2-mediated transduction of healthy rodent retinas, there have been no studies addressing if AAV2 gene therapy can successfully target and modify RGCs after optic nerve damage. This scenario, however, is most likely to be the condition of tissue being treated for optic neuropathies, such as glaucoma.
B6.Rosa26-(LoxP)-tdTomato reporter mice were crossed with B6.Bax-deficient animals. The presence of the Bax mutant allele provides a block of RGC apoptosis, while not interfering with early onset atrophic events, including chromatin condensation and gene silencing: conditions which may interfere with normal transgene expression. One eye of Bax-/- or Bax+/- mice was subjected to optic nerve crush (ONC). AAV2-Pgk-Cre (1e9 particles) was then injected into the vitreous of that eye at times between 2 and 30 days after ONC. Contralateral control and naïve eyes were also injected and examined. Retinas were evaluated for tdTomato expressing cells 30 days after injection.
Expression of the reporter indicates successful transduction of cells with the AAV2 virus, expression of the transgene, and modification of the cellular genome to enable it to express the tdTomato reporter. RGCs were successfully transduced at all time points after ONC based on reporter expression in the axons of the optic nerve, and co-localization with BRN3A. Contralateral eyes also demonstrated moderate transduction of a population of cells in the innermost layer of the inner nuclear layer (presumptive amacrine cells) and Müller cells. Transduction of these two populations was significantly elevated in damaged retinas however, increasing 2-fold for presumptive amacrines and nearly 4-fold for Müller cells (both P<0.001). In addition, AAV2-Pgk-Cre efficiently transduces non-pigmented epithelial cells of the ciliary process. Co-localization with cell-specific markers is ongoing to confirm identity of the non-RGC populations that are transduced.
Damaged RGCs can be efficiently transduced by AAV2 and their genome can be modified even after early formation of heterochromatin. Damage also significantly increases the transduction efficiency of at least 3 other cell-types in the eye. These findings may have ramifications in strategies to treat RGCs using AAV2-mediated gene therapy.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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