Glaucoma is a leading cause of blindness worldwide. Emerging evidence suggests that glia-neuroinflammation is a critical element driving retinal ganglion cell (RGC) death and optic nerve degeneration in the pathogenesis of glaucoma. Gene polymorphisms in toll-like receptor-4 (TLR4, inflammatory receptor) and ATP-binding cassette transporter A1 (ABCA1, cellular cholesterol transporter) are linked to the risk of primary open-angle glaucoma. Our studies demonstrated increased TLR4 and reduced ABCA1 expression in RGCs and glia in human glaucomatous retina. We identified the secreted apoA-I binding protein (AIBP; gene APOA1BP) as a key regulator of cellular cholesterol metabolism, which controls TLR4 activation via ABCA1-dependent cholesterol depletion from TLR4 occupied lipid rafts in inflammatory and activated cells. Similar to ABCA1, AIBP expression was reduced, while cholesterol levels and inflammatory markers increased in human and mouse glaucomatous retinas. Apoa1bp-/- mice had compromised visual acuity and compared to wild type, had increased retinal TLR4 and IL-1β expression, augmented microglial activation, and increased RGC death in response to elevated intraocular pressure (IOP). These findings support the rationale for restoration of AIBP expression in the retina to provide sustained neuroprotection in patients with glaucoma. In preliminary studies, adeno-associated virus (AAV)-AIBP protected RGCs and ameliorated visual dysfunction in experimental mouse models of glaucoma. In addition, recombinant AIBP protein promoted mitochondrial function as well as inhibited inflammatory responses in cultured Müller glia and microglia in response to elevated hydrostatic pressure. Based on our previous and these findings, we propose development of AAV2-hAIBP based therapy to reduce retinal neuroinflammation and provide effective neuroprotection to glaucoma patients receiving standard-of-care IOP lowering treatment. We propose the following milestone-driven Specific Aims: (1) Optimize AAV2-hAIBP and define AIBP distribution and target engagement in retina; (2) Characterize the in vivo efficacy and non-GLP toxicology of AAV2-hAIBP; and (3) Conduct IND-enabling studies and obtain FDA approval for first-in-human trial. The project will combine expertise and resources from University of California, San Diego, RAFT Pharmaceuticals, and the NIH Blueprint Neurotherapeutics Network – Biologic. Our proposed studies will develop novel gene therapy to reduce retinal neuroinflammation and mitochondrial dysfunction and provide effective neuroprotection to glaucoma patients.