Project Abstract The goal of this proposal is to define the mechanisms of retinal vasculopathy with cerebral leukoencephalopathy (RVCL), an adult-onset vascular dementia associated with brain atrophy, blindness, and premature death, usually within 5-10 years of disease onset. RVCL is an autosomal dominant disease that is refractory to treatment with immunosuppression despite the fact that RVCL patients develop autoantibodies. RVCL-causing mutations in the TREX1 gene cause disease with 100% penetrance. Currently, there is no effective treatment for this devastating disease, and the mechanisms of disease pathogenesis have not yet been defined. Dr. Jonathan Miner (PI) directs the RVCL Research Center at the University of Pennsylvania and coordinates care of RVCL patients from around the United States and the world. Dr. Miner also leads a team of RVCL- focused clinicians and investigators. Preliminarily the Miner laboratory has generated numerous mouse and human cellular models of RVCL and discovered novel molecular mechanisms that may be driving endotheliopathy, cellular senescence, and the resulting vascular dementia. The co-investigator is Dr. Nouri Neamati, a medicinal chemist and biochemist at the University of Michigan. Dr. Neamati is developing TREX1 inhibitors and protein degraders. This partnership has led to novel animal models, single-cell RNA-seq studies, and first-in-class inhibitors of TREX1, as well as our discovery that RVCL-causing TREX1 mutations lead to failure of replicative senescence and dysregulation of endogenous retroelements. Here, we propose to use cellular and animal models to define the role of TREX1 in regulating DNA damage, retroelements, and pathology (Aim 1), to use animal models to define functions of TREX1 mutants and a DNA damage repair pathway in humoral immunity (Aim 2), and to determine the role of TREX1 DNase activity using an enzyme-dead TREX1 mutant mice and first-in-class TREX1 inhibitors and protein degraders (Aim 3). Our mechanistic studies will broadly elucidate functions of TREX1 and the role of TREX1-mediated DNA damage in a mouse model expressing TREX1 mutants known to cause small vessel disease and vascular dementia in humans. Insights gained from our work may clarify the role of TREX1 and endogenous retroviruses in cellular senescence, eventually leading to new therapies for this monogenic vascular dementia.