Systematic Identification and Mechanistic Characterization of Novel Oxidative Protective Genes to Prevent RPE Degeneration

Project Summary / Abstract Age-related macular degeneration (AMD) affects approximately 196 million people globally and is primarily caused by oxidative stress in the retinal pigment epithelium (RPE) cells. These crucial cells lie between the oxygen-rich choroid and the photo-oxidatively stressed retina, making them highly susceptible to damage from reactive oxygen species (ROS). Given the prevalence of AMD and the lack of highly effective treatments, there is a pressing need for novel interventions that enhance the oxidative defense mechanisms of RPE cells. In this project, I have initiated, to my knowledge, the first-ever genome-wide overexpression screen specifically in RPE cells, identifying ten novel gene candidates that enhance cell survival against oxidative stress, particularly NaIO3 which is known to cause mitochondrial dysfunction. Among these, the cardiac-specific transcription factor NKX2-5 emerged as a standout due to its potential to confer heart-like antioxidative properties to RPE cells. Preliminary data suggest that NKX2-5 can significantly ameliorate oxidative stress in these cells, thereby potentially mitigating AMD progression. The project is structured into three primary aims: Aim 1 focuses on elucidating the mechanistic pathways through which NKX2-5 confers oxidative protection, including direct ROS reduction, enhancement of mitochondrial biogenesis, and activation of anti-apoptotic pathways. This will be assessed using iPSC-derived RPE cells expressing NKX2-5, where cellular ROS levels, mitochondrial metrics, and genomic changes will be evaluated. Aim 2 tests the hypothesis that NKX2-5 can improve both the morphology and function of RPE cells in vivo. This will involve developing a new adeno-associated virus (AAV) construct for inducible NKX2-5 expression specifically in RPE cells, which will then be administered in both an acute model of RPE degeneration and in aged mice to assess improvements in RPE health and visual function. Aim 3 expands the investigation to include all identified genes from the screen, assessing their ability to protect against various oxidative stressors relevant to AMD, such as oxidized-LDL and cigarette smoke extract. The ultimate goal is to identify gene combinations that provide the broadest oxidative protection. These combinations will be tested in a comprehensive mouse model of AMD that incorporates genetic risk factors, a high-fat diet, and aging influences. Throughout this project, I aim to gain a deep understanding of cellular defense mechanisms against ROS, paving the way for novel gene therapy approaches to treat AMD and potentially other ocular diseases influenced by oxidative stress.