Abstract
Human-induced pluripotent stem cell (hiPSC) derived organoids have become increasingly used systems allowing 3D-modeling of human organ development, and disease. They are also a reliable source of cells for transplantation in cell therapy and an excellent model to validate gene therapies. To make full use of these systems, a toolkit of genetic modification techniques is necessary to control their activity in line with the downstream application. We have previously described adeno-associated viruse (AAV) vectors for efficient targeting of cells within human retinal organoids. Here, we describe biological restriction and enhanced gene expression in cone cells of such organoids thanks to the use of a 1.7-kb L-opsin promoter. We illustrate the usefulness of implementing such a promoter to enhance the expression of the red-shifted opsin Jaws in fusion with a fluorescent reporter gene, enabling cell sorting to enrich the desired cell population. Increased Jaws expression after transplantation improved light responses promising better therapeutic outcomes in a cell therapy setting. Our results point to the importance of promoter activity in restricting, improving, and controlling the kinetics of transgene expression during the maturation of hiPSC retinal derivatives. Differentiation requires mechanisms to initiate specific transcriptional changes and to reinforce those changes when mature cell states are reached. By employing a cell-type-specific promoter we put transgene expression under the new transcriptional program of mature cells.
Highlights
The generation of human retinal organoids has opened up new ways to study the brain and retinal development and evolution, as well as to model and treat neurodegenerative disorders (Ahmad et al, 2019; Gagliardi et al, 2019)
To favor differentiation towards cone lineage DAPT was added at the time point corresponding to the onset of cone arrestin (CAR) positive cells within the organoids (Garita-Hernandez et al, 2019). 3D retinal organoids were optogenetically-transformed with the recombinant AAV2-7m8 capsid variant selected for its improved performance in retinal organoids when compared to other capsids known for their tropism towards cones (Garita-Hernandez et al, 2020)
The retinal organoids were transduced with associated viruse (AAV) on day 44 of differentiation corresponding to the onset of cone-specific gene expression (Garita-Hernandez et al, 2019)
Summary
The generation of human retinal organoids has opened up new ways to study the brain and retinal development and evolution, as well as to model and treat neurodegenerative disorders (Ahmad et al, 2019; Gagliardi et al, 2019). Retinal organoids are multicellular 3D structures that mimic certain aspects of the cytoarchitecture and cell-type composition of the human retina These structures are generated by the differentiation of Human-induced pluripotent stem cells (hiPSCs) or embryonic stem cells (ESCs; Llonch et al, 2018; Ahmad et al, 2019; Lidgerwood et al, 2019). Genetic modification is a powerful tool that allows for the introduction of alterations ranging from small changes in the genome to the removal or integration of entire genes to the expression of exogenous genes to control cellular function (Garita-Hernandez et al, 2016). This enables researchers to investigate individual genes relating to cellular functions, and interactions. The delivered genetic cargo is maintained even in the absence of chromosomal integration and the transgene expression is dependent on the recruitment of transcription factors by the promoter sequence preceding the transgene
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
