PROJECT SUMMARY 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 the human glaucomatous retina. We identified the secreted apoA-I binding protein (AIBP) 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 the 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 the 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 findings we proposed the development of AAV2-hAIBP-based therapy to reduce retinal neuroinflammation and provide effective neuroprotection to glaucoma patients receiving standard- of-care IOP-lowering treatment. This work is currently funded by the 1UG3NS129684-01A1 grant award. The overall goal of the UG3/UH3 project is to conduct preclinical studies of an AAV-AIBP for the treatment of glaucoma. The current lead construct encodes a secreted form of AIBP-His in which the C-terminal 6xHis augments the biological activity of AIBP. The goal of this supplemental proposal is to assess the relative risk of immunogenicity between the two test samples, an AIBP-His recombinant protein, and tagless AIBP, and in comparison, them with the clinically relevant controls with known immunogenicity. These results will provide necessary data for an assessment of the risk of clinical immunogenicity for AIBP-His and the selection of the final candidate for the development of the AAV-AIBP construct for neuroprotection in glaucoma. It would also provide the flexibility needed to undertake the high-impact 1UG3NS129684-01A1 project and its on-schedule progression to the UH3 phase.