Proof of concept for AAV-mediated gene transfer in iPSc-derived retinal pigment epithelium

Abstract

Purpose: Inherited retinal dystrophies (IRDs) are a large group of genetically and clinically heterogeneous diseases leading to progressive vision loss, for which a paucity of disease-mimicking animal models can render pre-clinical studies difficult. We sought to develop pertinent human cellular IRD models, beginning with choroideremia, caused by mutations in CHM encoding Rab escort protein 1 (REP1). Choroideremia represents 2% of IRDs and is characterised by night blindness in childhood leading to blindness by 50 y of age. Mouse and zebrafish REP1-deficient models are lethal.

Methods: To generate a pertinent human cellular model, we reprogrammed REP1-deficient fibroblasts from a CHM-/y patient into induced pluripotent stem cells (iPSc). We then differentiated these patient-specific iPSc into REP1-deficient retinal pigment epithelium (RPE).

Results: We demonstrated that the iPSc-derived RPE is a polarised monolayer with a classical morphology, expresses characteristic markers, is functional for fluid transport and phagocytosis, and mimics the biochemical phenotype of patients. We assayed a panel of AAV vector serotypes in this human RPE and identified the serotype with the highest transduction efficiency (up to 60% transduction). This high and unmatched transduction efficiency is likely due to the phagocytic capacity of the iPSc-derived RPE, and therefore this model mimics the scenario an AAV vector encounters in vivo in the subretinal space. Furthermore, we demonstrated that AAV-mediated CHM gene transfer normalises the biochemical phenotype.

Conclusions: We identify the superior AAV serotype in functional human RPE, and, for the first time, show the potential of patient iPSc-derived RPE to provide a proof-of-concept for gene replacement in the absence of an appropriate animal model. This data is particularly pertinent in light of ongoing gene transfer studies for choroideremia, in particular, and IRDs, in general.

Keywords: 538 gene transfer/gene therapy • 701 retinal pigment epithelium • 696 retinal degenerations: hereditary