Control of Microbial Opsin Expression in Stem Cell Derived Cones for Improved Outcomes in Cell Therapy

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.

Red Light Optogenetics in Neuroscience

“…Organoids are organ-mimicking multicellular 3D structures. Retinal organoids have been utilized for optogenetic vision restoration in mice by Garita-Hernandez et al (2019 , 2021) . In their studies, human-induced pluripotent stem cells were differentiated into retinal organoids.…”

Outer Retinal Cell Replacement: Putting the Pieces Together

“…One intriguing approach to this conundrum is the use of optogenetically engineered hiPSC-PRP, which have recently been shown to generate modest responses to bright light in vitro and in vivo. 163 , 189 However, such a genetic modification presumes that hPSC-derived PRs cannot innately respond to light and also introduces aforementioned regulatory hurdles. In the absence of genetic modification of hPSC-PRP, other potency assays may prove useful for authenticating batches of transplantable hPSC-PRP, including examinations of cell polarity, synaptic marker expression, 190 PR marker expression, axon outgrowth, and membrane electrophysiology.…”

“…A number of recent xenograft studies have demonstrated proof-of-concept for PR survival and anatomic engraftment (see Supplementary Note S1 ) following transplantation of dissociated hPSC-derived cell suspensions or retinal sheets in rodents 146 , 149 , 161 – 163 , 179 , 189 and non-human primates (NHPs), 147 , 160 using controls for biomaterial transfer. Dissociated cell injections have the advantage of being relatively simple, cost-effective, rapid, and minimally invasive, although graft organization and cell survival is often suboptimal.…”

“… 134 However, no study to date has definitively shown that hPSC-PRPs can form new functional synapses after being isolated from retinal organoids. Evidence of functional post-transplant synaptogenesis currently includes modest light responses recorded with multi-electrode array (MEA) and micro-electroretinography (mERG), 146 , 147 , 161 , 189 , 205 and often does not conclusively distinguish light-induced donor cell responses from possible neuroprotective effects on residual host retinal circuitry. Reproducible, well-controlled approaches for assessing de novo synaptogenesis at the level of individual donor hPSC-PRPs (via calcium imaging or viral monosynaptic circuit tracing), particularly in the context of xenogeneic transplantation, 206 will be necessary to further clarify mechanisms of functional recovery.…”

“…Rodents have historically been the preferred model system for retinal cell replacement studies due to cost, ease of genetic manipulation, and widespread availability. Several reports have shown that transplanted hPSC-PRP can survive and be associated with varying degrees of light-evoked behavior and/or electrophysiologic activity in degenerating rodent retinas, 146 , 149 , 161 – 163 , 179 , 189 but there is not yet direct evidence of causation. The well-documented neuroprotection caused by virtually any subretinally transplanted material (including control vehicles 110 , 207 ) in the Royal College of Surgeons (RCS) rat makes it highly difficult to fully control for alternate mechanisms in this model.…”