Retinal Gene Transfer for AAV Serotypes 1, 2 and 5 Assayed in Retinal Explants Versus In Vivo Injection

Abstract

Abstract: : Purpose: To compare transduction efficiency and cell specificity among AAV vector serotypes 1, 2 and 5 in retinal explants and in injected mice. Methods: Retinal explants (Ogilvie et al., 1999) prepared from C57BL/6 mice on postnatal day (P)7 were exposed to AAV vectors containing the CBA (chicken beta-actin) promoter driving expression of GFP. 10 µl (approx. 5x10exp11 vector particles) of either AAV-CBA-GFP serotype 1, 2 or 5 was placed on the inner retinal surface (upper face of the explant). Alternatively, 10 µl of each vector was diluted with 1 ml of DMEM in the well under the explant to expose the outer retinal surface in contact with the supporting membrane. Explants were infected for 24 hours, rinsed and incubated in vector-free DMEM containing 10% FCS for another 5 days. They were then lightly fixed, photographed, and frozen sections made for fluorescent microscopy. The same vectors were injected intravitreally or subretinally in P3 mice and assayed similarly 5 weeks later.Results: When vector was placed on the retinal ganglion cell (RGC) side, serotype 2 treated explants showed the highest fraction of GFP-positive cells while serotype 1 showed the least. When the photoreceptor (PR) side of explant contacted the vector, serotype 5 was the most efficient. With serotype 2, primarily RGCs and a few Muller cells were transduced. Serotype 1 vector only infected an occasional Muller cell. In contrast, serotype 5 transduced primarily PR cells with an occasional central Muller cell. When serotype 2 vector was injected intravitreally, transduction was similar to that when vector was placed on the inner retinal surface of explant. When serotype 5 vector was injected subretinally, transduction mimicked the explant results when the outer retina was exposed to vector. Subretinal injection of serotype 1 vector gave mainly RPE cell transduction.Conclusions: AAV-CBA-GFP serotype 2 vector has the highest transduction efficiency in the inner retina (primarily RGCs) when assayed either by intravitreal injection or by exposing the RGCs surface of the retinal explant to vector. Type 5 vector has the highest transduction efficiency in PR cells either by subretinal injection or by exposing the outer retina to vector. This retinal explant vector assay method therefore faithfully reproduces in vivo results and shortens analysis from 5 weeks to one week, thus making accurate and rapid validation of a wide variety of gene-based approaches for retinal disease therapy possible.

Keywords: retinal culture • gene/expression • injection