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Barbara Bogner, Sanford Boye, Seok-Hong Min, James Peterson, Qing Ruan, Vince Chiodo, Renee Ryals, Herbert Reitsamer, William Hauswirth, Shannon Boye; Transduction Profile of Anterior Chamber-injected Capsid Mutated Self-complementary Adeno-Associated Virus Type 2 in Rodents. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2735.
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© ARVO (1962-2015); The Authors (2016-present)
The surface properties of the adeno-associated virus (AAV) capsid are essential for cellular tropism and efficiency of transgene expression. The transduction properties of AAV2-based capsid mutants have not been investigated in the anterior chamber (AC). In this study we investigate transduction profiles of AAV2 mutants containing self-complementary AAV (scAAV) genomes coding for green fluorescent protein (GFP) by localizing GFP in AC-injected mice and rats. scAAV2 mutants contain single or combinations of triple and septuple tyrosine (Y) to phenylalanine (F) mutations in the highly conserved surface-exposed capsid tyrosine residues.
AC injections were performed in C57BL/6 mice (1 µl) and Wistar rats (2 µl). After general and topical anesthesia, the peripheral cornea was punctured (33G needle) anterior of the iridocorneal angle. scAAVs expressing GFP under the constitutive small CMV-chicken β-actin (smCBA) promoter and vehicle control (BSS+0.014% Tween 20) were infused slowly using a micropump. 4 weeks post-injection, animals were sacrificed and GFP-expression was evaluated by immunohistochemistry and confocal microscopy. In Table 1 experimental groups and titers for scAAVs are listed.
scAAV-injected eyes showed intense GFP-signals in the corneal endothelium, the ciliary non-pigmented epithelium (NPE), the chamber angle and the iris. Depending on the scAAV mutant, the expression pattern ranged from mosaic-like to homogenous. Both, vehicle control and control without injection revealed no immunopositivity. Table 1 summarizes the localization and semiquantitative evaluation of the GFP-signal in injected eyes.
scAAVs bypass rate-limiting second-strand DNA synthesis to obtain the transcriptionally active AAV genome. This results in earlier onset of transgene expression and facilitates the transduction of difficult-to-transduce cells (e. g. trabecular meshwork). This study shows that mutagenesis of surface-exposed tyrosine residues affects the tropism and transduction efficiency of scAAV2 in the AC. While single and triple mutants show enhanced transduction efficiency, the septuple mutant reveals no improvement compared to scAAV2 WT. These results are of interest for the development of transgene-based models to investigate pathological processes in the AC and for the development of gene-based therapies targeted to the trabecular meshwork.
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