608. In Silico Reconstructed Ancestral Adeno-Associated Viruses Transduce Mouse Anterior Segment

Abstract

Adeno-associated virus (AAV) has been widely used as a gene delivery vehicle in ocular gene therapy, especially for the treatment of inherited retinal diseases. It has been proven in animal models and humans that many AAVs can efficiently transduce different cell types in the retina. However, there are very few reports showing that single strand AAVs can successfully transduce the anterior segment of the eye. Some self-complementary AAVs, such as self- complementary AAV2 (scAAV2), have been shown to have robust transduction in the anterior segment cells of mouse, rat, and monkey. Unfortunately, the maximum package genome size of the self-complementary AAV is approximately 2.2kbp, which is less than half the package capacity of single strand AAV vectors. In previous studies, we have shown that an ancestral AAV, Anc80, which was developed in our lab by in silico ancestral sequence reconstruction, can achieve fast onset and long-term stable expression in retinal cells. In the present study, we have explored the tropism and transduction efficiency of the ancestral AAV vector (Anc80) in the anterior segment of mice eyes and compared it with some other contemporary AAVs. Three different AAV vectors (single strand AAV2-ssAAV2, scAAV2 and Anc80) carrying eGFP reporter gene driven by CMV promoter. The viral vectors were injected into wild type C57Bl/6 mouse eyes either via anterior chamber injection at a dose of 1×109 particles, or via intravitreal injection at a dose of 2×109 particles. At day 28 post- injection, mice eyeballs were collected and sectioned to examine GFP expression in the anterior segment by using confocal microscopy. Our data shows all three AAV vectors can transduce cornea, trabecular meshwork, and ciliary body by anterior chamber injection, as determined by GFP expression level and expression area in the cornea, Anc80 > ssAAV2 > scAAV2, and in the trabecular meshwork and ciliary body, Anc80 > scAAV2 > ssAAV2. Via intravitreal injection, GFP expression was detected in cornea and ciliary body in the Anc80 group, while no GFP expression in cornea or ciliary body was found in the ssAAV2 group. In Conclusion, anterior chamber or intravitreal injections of Anc80 efficiently transduce all components of the mouse anterior segment. These results will inform future studies that aim to deliver therapeutics in such diseases as glaucoma, which affect the cornea, iris, or ciliary body.