Abstract
Purpose :
In this study, we evaluated role of OCU410 (human RORA) in controlling inflammation, a causative factor for dry age-related macular degeneration (dry-AMD) pathogenesis, in an in vitro assay. While there is no approved therapy for dry-AMD, most therapeutics in clinical trials target the complement system (cell death pathway), inflammation, or oxidative stress. However, these therapies target only one pathway. OCU410 is an orphan nuclear receptor, a transcription factor, that may target multiple pathways, particularly in suppressing inflammatory cytokine gene expression, which can potentially limit damage to the retina due to dry-AMD.
Methods :
To assess the effects of OCU410, we created an inflammatory model system using LPS in human immortalized retinal epithelial cells (hTERT-RPE1). Inflammatory cytokine genes for inflammatory cytokines (e.g., IL1b, IL6, CXCL8, etc.) were measured by quantitative reverse transcriptase real time polymerase chain reaction (qRT-PCR). LPS-induced expression of Gluc luciferase was compared in the presence and absence of OCU410 using a robust CXCL8 promoter driven secretory Gaussia Luciferase (GLuc). Secretory Alkaline Phosphatase (SEAP) driven by constitutive strong CMV promoter was used as the internal control for signal normalization. This promoter reporter luciferase system was used to access the direct role of RORA on CXCL8 promoter. In silico analysis also shows that human CXCL8 has a consensus RORA binding site within 1.5kb of the CXCL8 start site.
Results :
LPS stimulated the expression of inflammatory cytokines IL1b, CXCL8, and IL6 in a dose dependent manner in hTERT-RPE1 cells treated with OCU410. The expression of inflammatory cytokines in response to LPS was inversely related to the expression of RORA. Furthermore, the GLuc/SEAP luciferase assay on CXCL8 promoter reporter assay demonstrates that the suppression of luciferase activity is mediated through RORA binding on the CXCL8 promoter.
Conclusions :
Dry-AMD is characterized by expression of inflammatory cytokines. OCU410 transduction leads to the expression of nuclear hormone receptor RORA upon viral transduction which suppresses inflammatory cytokine gene expression, including CXCL8, in an LPS-induced inflammation model in hTERT-RPE1 cells. Thus, OCU410’s ability to inhibit multiple inflammatory cytokines strengthens its potential as a gene therapy molecule for dry-AMD.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.