AAV-Mediated Gene Delivery to 3D Retinal Organoids Derived from Human Induced Pluripotent Stem Cells.

Marcela Garita-Hernandez,Jose-Alain Sahel,Fiona Routet,Laure Guibbal,Hanen Khabou,Jens Duebel,Sacha Reichman,Deniz Dalkara,Luisa Riancho,Lyes Toualbi,Olivier Goureau

doi:10.3390/ijms21030994

Abstract

Human induced pluripotent stem cells (hiPSCs) promise a great number of future applications to investigate retinal development, pathophysiology and cell therapies for retinal degenerative diseases. Specific approaches to genetically modulate hiPSC would be valuable for all of these applications. Vectors based on adeno-associated virus (AAV) have shown the ability for gene delivery to retinal organoids derived from hiPSCs. Thus far, little work has been carried out to investigate mechanisms of AAV-mediated gene delivery and the potential advantages of engineered AAVs to genetically modify retinal organoids. In this study, we compared the early transduction efficiency of several recombinant and engineered AAVs in hiPSC-derived RPE cells and retinal organoids in relation to the availability of their cell-surface receptors and as a function of time. The genetic variant AAV2-7m8 had a superior transduction efficiency when applied at day 44 of differentiation on retinal organoids and provided long-lasting expressions for at least 4 weeks after infection without compromising cell viability. All of the capsids we tested transduced the hiPSC-RPE cells, with the AAV2-7m8 variant being the most efficient. Transduction efficiency was correlated with the presence of primary cell-surface receptors on the hiPS-derived organoids. Our study explores some of the mechanisms of cell attachment of AAVs and reports long-term gene expression resulting from gene delivery in retinal organoids.

Highlights

HiPSCs recapitulate different aspects of retinal development contributing to our understanding of normal retinogenesis and providing opportunities for in vitro disease modelling
RAAV tropism in non-human primates is expected to be close to the outcome on human retinal cells, less work has been invested in testing Recombinant AAVs (rAAVs) tropism on human retinal cells derived from Human induced pluripotent stem cells (hiPSCs) [18,19,20,21]
We investigated the mechanisms by which different rAAV variants transduce retinal organoids and retinal pigment epithelium (RPE) derived from hiPSCs

Summary

Introduction

HiPSCs recapitulate different aspects of retinal development contributing to our understanding of normal retinogenesis and providing opportunities for in vitro disease modelling. Recombinant AAVs (rAAVs) are the first choice for gene delivery to retinal cells for gene therapy [8] They are non-pathogenic, non-integrative, and highly efficient as they can infect post-mitotic cells and provide long-term expression of a given transgene [9]. In our hiPSC-derived organoids [12,13], laminated neural retina obtained after several weeks of differentiation displays photoreceptor precursors on the outside and ganglion cell precursors in the center of the structures. In these organoids, the medium containing the rAAVs is first and foremost in contact with the photoreceptor precursors, mimicking subretinal injections. RAAV tropism in non-human primates is expected to be close to the outcome on human retinal cells, less work has been invested in testing rAAV tropism on human retinal cells derived from hiPSCs [18,19,20,21]

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Discussion

Conclusion