Project Summary The ability to spatially and temporally control cellular signaling is a fundamental requirement of organismal and tissue development/function. As an organ, the eye is not exempt from these requirements. In fact, arguably, the retina is even more sensitive than other tissues to environmental or genetic disturbances which can affect the transduction of light into electrochemical potential that is ultimately recognized by the brain. Thus, unregulated gene expression (also called phenotoxicity) during inappropriate times (temporally agnostic), or pan-retina activation/repression of cellular signaling using small molecules regardless of cell type (spatially agnostic), have the potential to compromise physiologic signaling with dire consequences. To circumvent these potential adverse consequences, and to develop an idealized gene-therapy platform, we have thoroughly validated and utilized a chemical biology approach involving destabilizing domains (DDs) in the mouse retina. These DD tools allow for conditional control of protein abundance through the addition of an exogenously-added small molecule stabilizer (typically trimethoprim, TMP, for the E. coli dihydrofolate reductase [DHFR] domain) and can achieve temporal control on near physiologically-relevant timescales within the retina. We envision that such approaches will provide idealized therapeutics that can target desired cell types affected in disease and according to defined timeframes. The overall goal of this R21 is to substantially expand the novelty, capability, and applicability of available small molecule probes that can be used in vivo for conditionally regulating retinal stress responsive signaling using DDs. This work is important because it will significantly enable the scientific community to interrogate retinal biologic phenomena with increasing spatio-temporal precision and flexibility, and it will test the ideological boundaries and therapeutic utility of conditional gene therapies.