Modulation of mTOR signaling in human induced pluripotent stem cell-derived retinal ganglion cells through the genetic regulation of RHEB

Abstract

Purpose : Retinal ganglion cells (RGCs) serve as the sole connection between the eye and the brain, and damage to this pathway results in vision loss included in diseases such as glaucoma. Signaling through the mammalian target of rapamycin (mTOR) pathway is associated with the maintenance of RGC homeostasis and neuronal growth, and previous studies have demonstrated that a decrease in mTOR signaling is associated with RGC damage in optic neuropathies. We aimed to determine if manipulation of the mTOR signaling pathway could serve as a means of neuroprotection for RGCs, potentially providing a significant target for preventing loss of vision for patients.

Methods : CRISPR/Cas9 gene editing was used to insert the Ras Homolog Enriched in Brain (RHEB) gene, a known activator of the mTOR signaling pathway, downstream of the BRN3b locus to confer specificity to RGCs. Further, both RHEB-edited cells as well as isogenic controls were further edited to introduce the Thy1.2 antigen to allow for RGC selectability. Both iPSC lines were then differentiated toward a retinal lineage through the formation of retinal organoids, followed by the purification and further maturation of RGCs from these organoids. Activation of the mTOR signaling pathway was confirmed by Western blots, and the effects of RHEB overexpression upon RGCs were determined by measurements of neurite complexity.

Results : We have successfully generated a novel iPS cell line that drives the overexpression of RHEB at the RGC-specific BRN3b locus, as well as an isogenic control cell line. Analyses of off-target effects from the gene editing procedures revealed no observable off-target effects following Sanger sequencing of the top predicted off-target loci. Both cell lines were capable of robust differentiation into retinal organoids that served as a source of RGCs for experimental analyses. Ongoing studies are determining the effects of RHEB overexpression in RGCs through western blots and immunostaining, as well functional effects of RHEB overexpression based upon RGC morphological analyses.

Conclusions : The ability to induce the overexpression of RHEB in retinal ganglion cells provides a new tool to investigate the effects of mTOR expression upon RGC health, and may provide a novel opportunity to provide neuroprotection of RGCs following injury or disease through manipulation of this signaling pathway.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.