Project Summary/Abstract Thyroid eye disease (TED) is a debilitating orbital and ocular condition that affects up to 50% of patients with Grave’s hyperthyroidism. Tissue fibrosis and hyaluronan overproduction in active TED leads to proptosis, eye movement restriction, eyelid retraction, dry eye, and optic nerve compression. After 2-3 years, patients enter a chronic phase of TED where symptoms persist but do not acutely worsen. The molecular basis of acute and chronic TED is a mystery. Prior studies have found insulin like growth factor 1 receptor (IGF-1R) to be an important cell surface protein in effecting the fibrosis and hyaluronan production of active TED. IGF-1R is important in a variety of body processes from metabolism to cancer to TED and was found to be overexpressed in TED orbital tissue. Exactly how IGF-1R effects the phenotypic changes in TED, and how the IGF-1R gene is regulated is unknown. The utility of IGF-1R or other related targets in chronic TED is also not known. This study examines the cell type landscape of acute and chronic TED and how IGF-1R is expressed and regulated in the two phases of TED. Aim 1 will determine the cell type trophism of IGF-1R in acute and chronic TED compared to un-diseased orbit tissue using single nuclei multiome sequencing of orbit tissue isolated from patients undergoing surgery. Spatial transcriptomics will be used to distinguish microenvironments of isolated cell types and interacting cell surface receptors. Aim 2 will investigate the regulation of IGF-1R expression by cis-regulatory elements from single-nuclear Assay for Transposase-Accessible Chromatin (ATAC)-sequencing. Promising targets will be tested through an in-vitro CRISPR-screen on human orbital fibroblasts from acute and chronic TED patients to determine the effect on fibroblast proliferation of knocking down key regulatory regions. These studies will generate a greater understanding of the cell type landscape of TED and how IGF-1R and other key cell surface proteins are regulated in acute and chronic TED. The results could yield new therapeutic targets or better understanding of how to use existing treatments most effectively. The proposed research training plan features mentorship from the basic sciences and clinical sciences and access to state-of-the-art techniques and facilities. This plan also incorporates professional development and unites collaborative resources between the department of cellular and molecular medicine and the department of ophthalmology to maximize my training potential.