Transduction of self-complementary AAV in the Trabecular Meshwork and Anterior Chamber in mouse

Abstract

Purpose : Adeno-Associated Virus (AAV)-based gene therapies have been developed for various anterior segment diseases involved cornea, trabecular meshwork and iris. Intracameral injection is the appropriate procedure to deliver AAV to transduce anterior segment. Self-complementary AAV (scAAV) can bypass the rate-limiting step of second-strand synthesis and has shown to mediate a rapid onset and higher level of transgene expression compared to the parent vector. However, the transduction profile of different scAAV serotypes following intracameral injection has not been established. This study aims to evaluate transduction of scAAV2, 3b, 6, 8 and 9 in anterior segment tissues.

Methods : EGFP gene was packaged in conventionally used AAV serotypes 2, 3b, 6, 8 and 9, with the chimeric CMV–chicken ß–actin (CBA) promoter. In 8-week old C57BL/6J mice, 1.0μl scAAV-CBA-EGFP solution for one serotype was injected into the anterior chamber of right eye for each mouse (n=6). The formulation buffer injected eyes serve as the negative control (n=6). Four weeks post injection, the intracameral injected eyes were enucleated and subjected to immunohistochemistry. The anterior segment including cornea, anterior chamber angle, ciliary body and iris was imaged and evaluated for the transduction efficiency and tropism using EGFP as a surrogate marker. EGFP-positive cells/structures in the anterior segment was analyzed for distribution and EGFP-signal was quantitated.

Results : The self-complementary AAVs resulted in the transduction of corneal endothelium, corneal keratocyte, trabecular meshwork and ciliary body but with varied distribution, transduction efficiency and cell specificity. The inflammatory cells infiltration was observed in different levels for serotypes studied.

Conclusions : Our results provides the dataset applicable to the development of AAV-based therapy, targeting the ocular diseases involved the anterior segment. In addition, this data generated from this study may also be applied to the appropriate AAV vector selection for the gene therapy for glaucoma and corneal diseases, which can be targeted by intracameral administration.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.