Abstract
Purpose :
Retinal ganglion cells (RGCs) are often injured in retinal and optic nerve diseases, such as head trauma or glaucoma, and in these diseases, RGCs mostly die, and axons barely regrow. Unfortunately, there are no effective treatments to prevent RGC loss and promote efficient regeneration to restore vision loss in any of these conditions. Understanding how RGCs detect and respond to such insults could hold the key to developing novel protective and regenerative strategies. A specific cell state ultimately represents the readout of specific gene expression programs expressed at the time. When we and others investigated the transcriptional state of injured RGC, we found that they are unable to maintain gene programs needed for survival and axon regeneration, while sustaining gene programs for cell death. Thus, the cenral vision is to rewire the transcriptional state of the damaged RGCs, so as to improve their ability to survive and regenerate.
Methods :
We applied in vivo perturb-seq to screen key transcription factors (TFs) to rewire the non-permissive growth state of damaged RGCs. This approach couples CRISPR deletions in pools with single-cell transcriptome profiling, providing insight into the gene-regulatory effects across multiple TF perturbations at once.
Results :
We intravitreally delivered lentiviral vectors perturbing CTCF/SIN3A/REST/TCF3/TGIF1/EBF3 and LHX2/6, followed by optic nerve crush injury and purification of perturbed RGC at 3 days, and single-cell RNA-seq to investigate how these TFs function together to dictate the transcriptional output of damaged RGCs. We showed that those TFs act in distinct and shared pathways involving regeneration processes.
Conclusions :
Our results provide insight into potential TF combinations to rewire the transcriptional state of damaged RGCs. Future directions include incoporating key TF combinations into gene therapy for glaucoma in mice.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.