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L. K. Buie, T. Borras; New Self-Complementary Adeno-Associated Virus (scAAV) Serotypes Result in Specific Gene Delivery to the Trabecular Meshwork in Living Rats. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1610.
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© ARVO (1962-2015); The Authors (2016-present)
Recombinant scAAV vectors can be generated using the genome and capsid from the same viral serotype, or by including the genome from serotype 2 and the capsid from an alternative AAV serotype. These heterologous vectors are known to confer specific cellular tropism as well as intensity of expression. Our goal is to develop a scAAV vector targeting specifically the trabecular meshwork cells. Our purpose was to evaluate scAAV2/1, scAAV2/2 and scAAV2/5 vectors in the anterior segment of the living rat’s eye.
Heterologous vectors carrying the EGFP transgene were prepared at the UNC vector core facility. Wistar rats received single dose intracamerally injection [6-8X10(9) viral particles, VP] of scAAV or vehicle. Intraocular pressures (IOP) (calibrated Tonometer) were taken at baseline and once a week thereafter. Animals were bled at pre- and post-injection and euthanized at 3 and 10.5 weeks post injection. Their anterior segments were processed for fluorescence histochemistry and immunohistochemistry. Immune response to the viral capsid in the rat’s serum was performed by ELISA using 1X10(9) VP of the corresponding serotype bound to the microtiter plate.
Anterior segment cryosections from rats injected with all serotypes were positive for GFP fluorescence. Eyes injected with scAAV2/2 showed intense GFP transduction in the posterior face of the iris, some corneal endothelium and good trabecular meshwork. Eyes injected with scAAV2/1 showed weak iris transduction, intense corneal endothelium and moderate trabecular meshwork. Eyes injected with scAAV2/5 showed intense trabecular meshwork transduction and negligible fluorescence in iris and cornea endothelium. Delta-IOP values of eyes injected with all serotypes were not significantly different from baseline or from eyes injected with vehicle. At 2 months post-injection, immune response to the AAV2 viral capsid in the serum was not significantly different than the one obtained in pre-injected serum. Comparison of the extent of expression of the heterologous serotypes and T cell response (CD8 immunohistochemistry) are in progress and would be presented.
Our current data supports that a scAAV2 vector containing the AAV5 capsid is the most specific and efficient serotype for trabecular meshwork gene transfer in living rats. The identification of new heterologous scAAV serotypes will considerably advance potential in vivo gene therapy of glaucoma.
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