In vivo 2-photon imaging of retinal biochemistry before and after retinal organoid transplantation

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Project Summary Candidate and Career Development Plan: Dr. Browne is an assistant professor practicing vitreoretinal surgery and functioning as an engineer and physician-scientist at the University of California, Irvine. Dr. Browne established his engineering and basic biological sciences skills as a PhD student. He has balanced clinical duties with his laboratory research activities and. His long-term career goals are to engineer functional imaging tools that will advance the understanding of early and advanced eye diseases and facilitate the therapies needed to treat humans. His training thus far has been using in vitro imaging alone. To achieve his career goals, he is requesting support for training to develop functional imaging tools in vivo and the molecular tools to correctly interpret Two-photon imaging (2PI) observations from animal models. This K08 award will enable Dr. Browne to develop his scientific and professional skills in advanced imaging, fluorescence microscopy, and retinal cellular biology applied in vivo. Dr. Browne and his co-mentors, Drs. Palczewski, Seiler and Kuppermann, have developed a hands-on strategy to fulfill the training requirements through relevant course work, didactics, laboratory techniques, and collaborations at UC Irvine. The training program will prepare Dr. Browne to submit R01-level proposals to independently investigate and optimize functional retinal imaging as a tool for therapeutic discovery. Research Plan: Many blinding retinal conditions, like age-related macular degeneration, are the consequence of biochemical dysfunction and the secondary effects of inflammation and cell death. Conventional clinical tools provide valuable structural information about the retina but are unable to visualize retinal function. Non-linear optical imaging can now reveal subcellular biochemistry in vitro and has emerged as a reliable tool to study retinal disease. 2PI in mouse models demonstrates subcellular changes in energy and light cycle metabolism. Cell replacement therapy has emerged as a therapeutic candidate for advanced vision loss, and preclinical trials in rats have demonstrated visual function restoration. To understand metabolic function in retinal organoids (RtOg) before and after transplantation into blind rats, we will employ functional 2PI in vitro and in vivo as outlined in the following specific aims. 1) Investigate RtOg maturation with 2PI and correlate functional imaging data with cell-specific reporters in vitro and molecular signatures post vivo, 2) Identify functional imaging biomarkers for in vivo metabolism using 2PI of normal rats and rat with retinal degeneration, 3) Study in vivo functional 2PI of transplanted RtOgs in rats to identify alterations in functional imaging biomarkers and correlate imaging findings with visual function testing. Completion of these aims will yield time-resolved metabolic detail for specific cell populations in developing RtOgs, avail novel in vivo information about retinal biochemistry in healthy retina, diseased retina, and diseased retinas treated with retinal sheet transplantation. These discoveries will guide future translation of both functional imaging and tissue replacement therapy in humans.