Validation of IRBP as a retinal treatment target and screening for human photoreceptor-specific nanoparticles for CRISPRa gene therapy

Abstract

My research focuses on a protein called interphotoreceptor retinoid-binding protein (IRBP), which is important for eye health. Problems with this protein can lead to retinal degeneration, a serious eye condition. I investigated two new methods to increase IRBP levels, which could potentially treat this condition.

The first method involves a molecule called PD98059, which upregulates IRBP by inhibiting the phosphorylation of ERK1/2 in Müller cells. This inhibition prevents the activation of the JAK/STAT3 pathway, which, in turn, downregulates IRBP in photoreceptors. I tested this approach in human retinal explants and the retinas of mice exposed to photo-oxidative stress. The results were promising: it reduced cell damage in Müller cells and improved IRBP levels, leading to healthier photoreceptors.

The second method I explored uses a gene-editing technology called CRISPR activation (CRISPRa). I established the method to synthesise functional mRNA in vitro and delivered mRNA to the retina with the help of different LNP formulations. I also confirmed that the upregulation of IRBP can protect photoreceptors from stress. I further studied the transfection efficiency of different LNP formulations with an ex vivo human retinal platform and single-cell RNA sequencing. We observed the differential efficacy of LNPs in various retinal cell types across macular and peripheral regions, suggesting potential for targeted gene delivery.

In conclusion, my research has identified IRBP as a potential therapeutic target for photoreceptor degeneration. The development of a cutting-edge LNP drug carrier screening platform based on human retinal explants and single-cell RNA sequencing has enabled the discovery of highly efficient LNP formulations capable of delivering RNA therapeutics targeting IRBP to photoreceptors. This work opens new doors for using RNA therapy in treating retinal diseases, with implications far beyond just IRBP-related conditions.