Establishing a Flow Cytometry Protocol for Screening AAV Transduction in Photoreceptors after Intravitreal Injection

Abstract

Purpose : Current ocular gene therapy for targeting the outer retina uses subretinal injection (SR) as the most effective route for transduction. However, subretinal injection is considered invasive and can lead to potential surgical complications and so, there is a high medical need for treating the outer retina through intravitreal injection (IVT). But there are natural physiological barriers, such as the inner limiting membrane (ILM), that prevent viral vectors reaching the outer retina. There are new generations of adeno-associated viruses (AAVs) being created by directed evolution to overcome these barriers and to evaluate their transduction, many use immunohistochemistry (IHC) and quantitative polymerase chain reaction (qPCR). A quantitative method to specifically count the amount of transduced photoreceptor cells was needed. For this study, a flow cytometry protocol was established to measure the amount of transduced photoreceptor cells after IVT injection of AAVs.

Methods : To establish the flow cytometry protocol, CD73 was evaluated as a reliable photoreceptor marker in adult mice through single-cell sequencing data, immunohistochemistry, and qPCR. Wild-type AAV2 with the CAG promoter was injected either intravitreally or subretinally in C57BL/6J mice at a titer of about 1E12 to 2E12 vg/ml in 1 ul. Fundus autofluorescence (FAF) images and optical coherence tomography (OCT) images were taken 4-6 weeks post-injection. Eyes were collected 6-8 weeks post-injection for IHC and flow cytometry.

Results : For proof of concept, AAV2-CAG-GFP was injected intravitreally or subretinally. FAF images showed GFP expression at 8 weeks. Retinas were dissected, dissociated, and stained for flow cytometry. After applying the gating strategy, CD73+GFP+ cells accounted for about 4% of the intravitreally injected group and 38% in the subretinally injected group.

Conclusions : We have established a relatively straightforward method of quantifying the number of transduced photoreceptor cells after intravitreal injection in mice. This will support efforts to find an AAV capsid that can penetrate the inner retina to treat the outer retina with inherited retinal diseases. More optimization is needed for quantifying other retinal cell populations.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.