Simple Autofluorescence-Restrictive Sorting of eGFP+ RPE Cells Allows Reliable Assessment of Targeted Retinal Gene Therapy

Abstract

Gene therapy is a promising therapeutic modality for ocular diseases arising in and affecting the retina and choroid. In this context, delivering gene therapy to the multifunctional retinal pigment epithelium (RPE) cells situated between the retina and choroid is desired. Efficacy assessment of any gene therapy strategy, whether it is gene augmentation, inhibition, or editing is initially tested in vitro in cell models, where delivery is simple and efficient. However, efficacy assessment in vivo in animal models is far more complex and several factors can influence the result significantly. Here we report a simple fluorescence activated cell sorting (FACS)-based enrichment method for direct assessment of efficacy and potential off-target effects of gene therapy co-delivered with an eGFP reporter to murine RPE cells using subretinal administration. Isolation of true eGFP+ RPE cells by FACS is notoriously difficult due to their intrinsic autofluorescence resulting in decreased sensitivity and false positives. Combining retinal dissection and harvest of RPE cells with a FACS-gating strategy utilizing the GFP filter and a neighboring filter, to separate the eGFP signal from autofluorescence, allows a significant enrichment of gene therapy-targeted eGFP+ RPE cells. In our hands the method may provide quantitative and qualitative advances in terms of up to 7-fold enrichment of true eGFP+ RPE cells compared to a standard protocol. The isolated cells can subsequently be utilized for reliable assessment of changes in DNA, RNA, or protein. This method allows proof-of-principle analysis of early gene therapy development and investigation of new delivery strategies or therapeutic approaches targeting RPE cells in vivo.

Subretinal AAV delivery of RNAi-therapeutics targeting VEGFA reduces choroidal neovascularization in a large animal model

“…This enables the detection of changes in Vegfa mRNA levels specifically in transduced RPE cells ( Figure 2 C). 26 , 34 Vegfa mRNA levels were reduced by approximately 88% in GFP-positive RPE cells from mice injected with ssAAV2.8/13-12 compared with GFP-positive RPE cells from mice injected with ssAAV2.8/S1-S2 (p = 0.002) ( Figure 2 C). Representative examples of fundus imaging (FI) and fundus fluorescence imaging (FFI) from injected eyes and gating strategy for FACS are shown in Figure S1 .…”

“…RPE cells were gathered from ssAAV2.8/13-12-injected eyes (n = 15), ssAAV2.8/S1-S2-injected eyes (n = 16), and PBS-injected eyes (n = 4) as described previously ( Figures S1 A and S1B). 26 , 34 The eyes injected with AAV were further divided into groups of five or six eyes to obtain three pools of RPE cells for each vector construct. PBS injected eyes were pooled and used as negative control to set the gates for FACS sorting.…”

“…To avoid autofluorescent RPE cells, we identified the GFP-positive cell population based on fluorescence measured in the GFP and the neighboring PE detector ( Figure S1 C), as described previously. 34 Data were analyzed in FlowJo v10.8.1.…”