Highly Efficient Subretinal Gene Transfer in the Mouse by AAV Vectors with Capsid Tyrosine Mutations

Abstract

Purpose: : To evaluate the transduction efficiency and the pattern of transgene expression after subretinal delivery of AAV vectors with capsid tyrosine-to-phenylalanine (Y-F) mutations in wild-type mice.

Methods: : Several self-complementary (sc) AAV vectors containing mutations in surface-exposed capsid tyrosine residues in serotypes 2, 8 and 9, were used to deliver an enhanced green fluorescent protein (EGFP) transgene under the control of a small CBA promoter to the retina of adult mice by subretinal injection. Fundus photographs were taken at different time-points after injection to monitor EGFP fluorescence in the treated eyes of live animals. Confocal microscopy was used to examine the pattern of EGFP expression in frozen retinal sections, and the identity of specific cells transduced by these vectors was confirmed by immunohistochemistry in co-localization experiments. Western blot analysis was performed to estimate the total amount of EGFP protein expressed in treated eyes.

Results: : Several mutant AAV vectors significantly enhanced transgene expression relative to equivalent doses of each wild-type vector counterpart, as revealed by fundoscopic examination of treated eyes and analysis of retinal sections. Intense EGFP fluorescence was found in photoreceptors and RPE. In addition, a serotype 8 mutant vector strongly transduced Muller cells throughout the retina after subretinal delivery, while a serotype 2 mutant led to widespread EGFP expression in ganglion cells.

Conclusions: : AAV vectors with appropriate tyrosine mutations can be used to enhance vector transduction efficiency over their wild-type counterparts, and could become novel gene delivery tools for a variety of therapeutic proteins to the retina.

Keywords: gene transfer/gene therapy • retina • Muller cells