miRNA Mimic Screen to Identify Genetic Pathways Involved in Maintenance of RPE Phenotype

Abstract

 

Purpose

 

Functional defects in the Retinal Pigment Epithelium (RPE) are implicated in a number of different retinal diseases including Age Related Macular Degeneration (AMD), a leading cause of vision loss in the United States. Loss of RPE phenotype and cell death leads to a stage of AMD called Geographic Atrophy (GA), where the patient’s vision is severally compromised. RPE-specific signaling pathways and genes involved in GA are not well understood. This project seeks to identify genes and pathways essential to maintaining RPE phenotype and identify potential drugs for treating GA.

 

Methods

 

We developed a reporter induced pluripotent stem (iPS) cell line in which a RPE-specific enhancer of Tyrosinase drives GFP expression and a constitutively active promoter drives RFP expression. We have shown previously that expression of GFP is indicative of RPE fate. RFP expression is indicative of overall cell health. In collaboration with NCATS, we developed a miRNA mimic screen for quantifying upregulation or downregulation of GFP expression in RPE derived from the reporter iPS cell line. RPE cells are seeded in 384-well plates and transduced with 825 different miRNA mimics.

 

Results

 

We compiled our results of the high throughput screen in a list of the top 10% miRNA mimics that resulted in more than 250% upregulation or downregulation of GFP signal without affecting RFP signal. Using bioinformatics analysis, we identified target genes of these miRNAs. These target genes were analyzed for most impacted pathways important in the eye. Both established and novel pathways were found to be linked to upregulation and downregulation of TYROSINASE.

 

Conclusions

 

We plan to validate identified genes and pathways using molecular biology and physiology assays. In addition we will test identified compounds for their ability to rescue RPE cellular endophenotypes in wound healing response assays and in vitro AMD stress models. This work will provide data for reconstructing an entire set of genetic pathways and networks essential for maintaining RPE phenotype and survival and lead to the development of a therapeutic intervention for geographic atrophy.

 

Keywords: 701 retinal pigment epithelium • 412 age-related macular degeneration