Abstract
Purpose :
Our goal is to unravel the potential of photoreceptor-specific miRNAs to prevent cone photoreceptor degeneration. A subset of miRNAs have been identified as particularly important for maintenance, survival and function of cone outer segments. This includes the photoreceptor-specific miRNAs miR-182, 183 and 124. We aim to utilize the protective biological properties of photoreceptor-specific miRNAs to prevent cone photoreceptors from degeneration and thereby, preserve light perception.
Methods :
DNA-encoded miRNA precursor sequences of miR-182, 183 and 124 were placed within an intron followed by the enhanced green fluorescent protein (EGFP) gene. We included a scrambled control sequence that is structurally similar to the miRNA sequences, yet not having any target mRNAs. We confirmed miRNA and EGFP expression within human stem-cell derived neurons using imaging and RT-qPCR. For in vivo applications, miRNA expression was controlled by the mouse cone arrestin promoter. For gene transfer, adeno-associated-viruses (AAV) were utilized. We selected AAV capsid serotype 2.NN for optimal viral transduction of photoreceptors. AAV particles were injected into the subretinal space of 3-week-old retinal degeneration mice 1 (rd1). The right eye was injected while the contralateral eye served as an untreated internal control. In vivo and ex vivo EGFP expression enabled tracking of successful cone photoreceptor transduction. Retinal tissues were isolated for live fluorescent imaging, ex vivo electrophysiology and immunohistochemistry.
Results :
RT-qPCR indicated that all miRNA overexpression constructs were functional and resulted in significantly higher expression levels in human stem cell-derived neurons compared to control samples. We detected that the scrambled control miRNA sequence caused altered neuronal morphologies and cell death. Therefore, we omitted the scrambled control from in vivo applications. Subretinal AAV injections in rd1 mice resulted in strong EGFP expression in photoreceptors of retinal explants that were isolated 3- and 6 weeks after injection. Ex vivo retinal recordings using multi-electrode-arrays and imaging analyses are ongoing.
Conclusions :
So far, we have developed a method to overexpress photoreceptor-specific miRNAs in cone photoreceptors of rd1 retinas. Next, we will study the protective nature photoreceptor-specific miRNAs have in the rd1 mouse model at a functional and histopathological level.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.