Abstract
Purpose :
Our aim is to investigate the role of photoreceptor-specific miRNAs in preventing cone photoreceptor degeneration and their potential to boost cone maturation in human stem cell-derived retinal organoids (RO). MiR-182, -183 and -124 are important for cone outer segment maintenance, survival, and function. We intend to harness their protective biological effects to preserve light perception by preventing cone photoreceptor degeneration. We also aim to accelerate cone photoreceptor outer segment maturation in growing RO.
Methods :
The miRNA precursor sequences for miR-182, miR-183, and miR-124 were flanked by an intronic gene sequence followed by the enhanced green fluorescent protein (EGFP) gene. MiRNAs were expressed using the mouse cone arrestin (mCAR) promotor. We opted for adeno-associated viral (AAV) vectors for gene delivery. To ensure viral transduction of photoreceptor cells we selected the AAV capsid serotype 2.NN. The AAV particles were injected into the subretinal space of 1-day old retinal degeneration mice 1 (rd1). We conducted vision-guided behavioral tests to analyze improvement of the animal’s visual performance 6-, 9-, and 13-weeks after injection. To track successful cone photoreceptor transduction, we analyzed the EGFP signal ex vivo. Retinas were isolated for live imaging, ex vivo retinal recordings and immunohistochemistry. Additionally, miR-183 was applied to human RO. The improvement in cone maturation was tracked using RT-qPCR and imaging methods at different timepoints.
Results :
Subretinal AAV injections in rd1 mice yielded robust EGFP expression in photoreceptors of retinal explants that were isolated at 6-, 9-, and 13-weeks post-injection. Ongoing investigations involve vision-guided behavioral testing, ex vivo retinal recordings using multi-electrode-arrays and imaging analyses. Organoids that expressed miR-183 through modification of stem cells were cultured. The miRNA expression was confirmed in 4-week-old RO based on the EGFP signal in live images. RT-qPCR and imaging analyses are currently in progress.
Conclusions :
To date, we have developed a method to overexpress photoreceptor-specific miRNAs in cone photoreceptors of rd1 retinas and in human stem cell-derived RO. Next, we will examine their protective nature in the rd1 mouse model, both functionally and histopathologically, and their potential to boost cone maturation in human retinal models.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.