Integrated Molecular Profiling of Diabetic Retinopathy - PROJECT SUMMARY Diabetic retinopathy (DR) progression varies greatly among patients, with those who develop diabetic macular edema (DME) often showing inconsistent responses to standard treatments, such as anti-VEGF therapy. Defining the molecular and cellular basis of DR in living patients remains a critical unmet need for optimizing precision health. This knowledge gap reflects the difficulty of interrogating specific cell types and proteome- wide profiling in vivo within highly complex, dynamic DR patient tissues, which hinders the development of novel and effective therapeutics. To obviate this challenge, we developed a method named TEMPO (Tracing Expression of Multiple Protein Origins; Cell (2023), PMID 37863056) a powerful tool to examine disease mechanisms at the cell level in vivo by integrating aptamer-based microvolume liquid biopsy proteomics, single-cell transcriptomics, and artificial intelligence (AI). In preliminary studies, TEMPO revealed surprising, new features of disease mechanisms by tracing the cellular origins of 5,953 proteins detected in the aqueous humor (AH): we identified immune and vascular cells as key contributors to distinct stages of DR and observed that diabetes accelerates molecular aging in the eye, even before retinopathy becomes clinically detectable. Additionally, systemic proteins, particularly liver-derived inflammatory markers, were found to play a role in DR progression, highlighting the influence of both local ocular and systemic factors. Our long-term goal is to create sensitive and patient-specific molecular diagnostics, prognostics, and treatments for retinal disease. This proposal’s objective is to apply the TEMPO proteomic platform using human liquid biopsy samples to identify protein biomarker signatures and cells driving DR stages and DME in living patients, and using our proteomic clock, determine the role of accelerated molecular aging in DR. Our central hypothesis is that the high- resolution proteomic profiling of AH will show that the diverse retinal phenotypes observed in DR and treatment-responses are driven by distinct molecular pathways and cell types, and reveal that DR-specific protein signatures accelerate cellular aging. Our specific aims will utilize three different approaches focusing on each element of DR molecular-pathology: (1) determine the differential expression of DR protein networks in living humans; (2) determine the cellular drivers of DR in living humans using TEMPO; and (3) determine the role of DR-specific protein signatures in accelerated cellular aging using AI-based proteomic models for proteomic pathways and eye clocks. Impact. This translational study will allow us to understand the mechanisms, cellular drivers, and molecular aging underlying DR pathology and DME treatment response in living patients, which will further aid in the development of therapeutics, diagnostics, prognostics, and clinical trial design.
