Abstract
Purpose :
One of the significant challenges in treating any blinding disease is to reduce or delay neuronal death. Overexpressing neuroprotective factors in a controlled manner is one strategy that can help preserve vision. The main objective of this project is to develop and validate artificial transcription factors (ATFs) for gene regulation-based therapies for neuroprotection. The generated ATFs will be utilized for in vivo neuroprotective therapies with the aim of multiplexed overexpression of neuroprotective genes in Muller glia (MG) at various doses.
Methods :
A stable cell line of MG was generated by transfection and sorting of MG with a dCas9-VP64-mCherry vector. The dCas9-VP64 line was further transfected with a vector that encodes gRNA and an eGFP reporter to develop a repertoire of ATFs that overexpress Fgf2, Lif, Cntf, and Manf. The transfected dCas9-VP64 MG was sorted, and the overexpression levels of each gene were assessed using the ddPCR method. Selected gRNAs that induce different doses of overexpression were further tested with a more potent activator using the dCas9-SPH stable MG, which was generated by immortalization of MG from the dCas9-SPH mouse line.
Results :
Gene expression analysis was conducted on two different MG, dCas9-VP64 and dCas9-SPH, containing different activators. We cloned several gRNAs targeting the promoter regions of Fgf2, Lif, Cntf, and Manf genes. Based on ddPCR results, we found that multiple gRNAs caused upregulation of gene expression at various dosages. These gRNAs were then cloned into Cre-recombinase and GFP vectors to assess their upregulation levels in the dCas9-SPH cell line. We showed that dCas9-SPH induced several-fold higher upregulation of Fgf2 and Cntf compared to dCas9-VP64. This suggests that activators are critical components for controlling the dosage of neuroprotective factors. These results also demonstrate that ATFs can be used to regulate the overexpression of neuroprotective factors in a dose-dependent manner.
Conclusions :
We previously anticipated that our ATFs would produce different levels of upregulation, which was essential for establishing dosages for neuroprotective factors. Our results for Fgf2, Lif, Cntf, and Manf are already encouraging, suggesting that dose modification is feasible. This will also enable us to test the neuroprotective effects of our ATFs in in vivo models.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.