A Destabilizing Domain Allows For Fast, Noninvasive, Conditional Control Of Protein Abundance In The Mouse Eye - Implications For Ocular Gene Therapy

PURPOSE. Temporal and reversible control of protein expression in vivo is a central goal for many gene therapies, especially for strategies involving proteins that are detrimental to physiology if constitutively expressed. Accordingly, we explored whether protein abundance in the mouse retina could be effectively controlled using a destabilizing Eschericbia coil dihydrofolatc reductasc (DHFR) domain whose stability is dependent on the small molecule, trimethoprim (TMP).METHODS. We intravitreally injected wild-type C57BL6/J mice with an adeno-associated vector (rAAV2/2[MAX]) constitutively expressing separate fluorescent reporters: MIK fused to yellow fluorescent protein (DHFR.YFP) and mCherry. TMP or vehicle was administered to mice via oral gavage, drinking water, or eye drops. Ocular TMP levels post treatment were quantified by LC-MS/MS. Protein abundance was measured by ftuidus fluorescence imaging and western blotting. Visual acuity, response to light stimulus, retinal structure, and gene expression were evaluated after long-term (3 months) TMP treatment.RESULTS. Without TMP, DIIIRNIP was efficiently degraded in the retina. 'IMP achieved ocular concentrations of 13.6 pM (oral gavage), 331 nM (drinking water), and 636 nM (eye drops). Oral gavage and TMP eye drops stabilized. DLIFRATP as quickly as 6 hours, whereas continuous 'IMP drinking water could stabilize DHIR.YFP for >3 months. Stabilization was completely and repeatedly reversible following removal/addition of TMP in all regimens. Long-term TMP treatment had no impact on retina fiinction/stnicture and had no effect on >99.9% of tested genes.CONCLUSIONS. This DHFR-based conditional system is a rapid, efficient, and reversible tool to effectively control protein expression in the retina.