Modelling the outer retina utilising ex vivo tissue and stem cells to optimise complement gene therapy

Abstract

Age-related macular degeneration is a leading cause of blindness worldwide. However, a gap remains due to no available model with a truly representative Bruch’s membrane (BrM), which is key to realistically model the disease and evaluate therapies. This study addressed this gap by attempting to recreate the human RPE/BrM/choroid complex by recellularising decellularised ex-vivo human retina. Firstly, an optimal decellularisation protocol for BrMchoroidal complex was selected, ensuring cellular removal while preserving ECM proteins. Second, the ability of iPSC-RPE to form a functional monolayer on decellularised BrM (dBrM) was assessed. Lastly, apical transduction capabilities of AAV2 vs AAV8 GFP and AAV2 sCR1 CCP1-11 Flag His tag (truncated sCR1) vectors were evaluated. Human donor eyes were dissected and decellularised using three protocols: Chirco K et al. (CP), and two shortened protocols (SP1 and SP2). iPSC-RPE cells were cultured on dBrM or Matrigel, and evaluated with proteomics, imaging, functional assays (VEGF and PEDF secretion) and phagocytosis of photoreceptor outer segments. Transduction of cells on Matrigel at day 3 vs 23 with AAV2 and AAV8 GFP was performed and quantified by flow cytometry for optimisation. Final transduction of day 23 cells on dBrM with AAV2 truncated sCR1 was followed by FLAG ELISA and fluid-phase co-factor assay. CP was the most effective decellularisation protocol with the least DNA persistence. Proteomic analysis confirmed nuclear protein removal and ECM retention, particularly laminin and collagen. From day 21, iPSC-RPE on dBrM formed a ZO1-positive monolayer with higher basal VEGF and apical PEDF. Imaging revealed representative RPE morphology, while proteomics identified known RPE cell markers. GFP transduction at day 23 showed AAV2 outperformed AAV8. Finally, 6-, 13- and 22-days post AAV2 truncated sCR1 transduction, iPSC-RPE cells released functional truncated sCR1. These results showed successful decellularisation of the human BrM/choroidal complex, recellularisation with iPSC-RPE and efficient AAV2 truncated sCR1 transduction secreting functional protein.