A Destabilizing Domain Allows for Fast, Noninvasive, Conditional Control of Protein Abundance in the Mouse Eye – Implications for Ocular Gene Therapy

Abstract

PurposeTemporal 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 Escherichia coli dihydrofolate reductase (DHFR) domain whose stability is dependent on the small molecule, trimethoprim (TMP).MethodsWe intravitreally injected wild-type C57BL6/J mice with an adeno-associated vector (rAAV2/2[MAX]) constitutively expressing separate fluorescent reporters: DHFR 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 fundus 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.ResultsWithout TMP, DHFR.YFP was efficiently degraded in the retina. TMP achieved ocular concentrations of ∼13.6 μM (oral gavage), ∼331 nM (drinking water), and ∼636 nM (eye drops). Oral gavage and TMP eye drops stabilized DHFR.YFP as quickly as 6 hours, whereas continuous TMP drinking water could stabilize DHFR.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 function/structure and had no effect on >99.9% of tested genes.ConclusionsThis DHFR-based conditional system is a rapid, efficient, and reversible tool to effectively control protein expression in the retina.

A Conditional Protein Degradation System To Study Essential Gene Function in Cryptosporidium parvum

“…To optimize the DDD system for C. parvum , we administered doses of TMP that have been found to work efficiently in other in vivo studies. In rodent models, a range of 0.25 to 2.0 mg/ml TMP in drinking water has been shown to regulate temporal and dose-dependent expression of DDD-tagged fusion proteins in the brain, neuronal tissues, and eye ( 42 , 50 , 51 ). Daily change of water containing TMP or change of water every 3 days or weekly, with prolonged treatment for 3 to 6 weeks for DDD-mediated protein regulation in vivo has been reported ( 42 , 50 , 52 , 53 ).…”

Faculty Opinions recommendation of A Conditional Protein Degradation System To Study Essential Gene Function in Cryptosporidium parvum.

“…To optimize the DDD system for C. parvum, we administered doses of TMP that have been found to work efficiently in other in vivo studies. In rodent models, a range of 0.25 to 2.0 mg/ml TMP in drinking water has been shown to regulate temporal and dosedependent expression of DDD-tagged fusion proteins in the brain, neuronal tissues, and eye (42,50,51). Daily change of water containing TMP or change of water every 3 days or weekly, with prolonged treatment for 3 to 6 weeks for DDD-mediated protein regulation in vivo has been reported (42,50,52,53).…”

Protocol for Designing Small-Molecule-Regulated Destabilizing Domains for In Vitro Use

“…Note: TMP and its derivatives for the most part have limited water solubility (~0.2 mg/mL for TMP [~690 μM]). Nonetheless, this amount of soluble TMP is certainly sufficient for basic in vitro studies as well as for in vivo regulation as long as sufficient amounts of this solution is used/imbibed ( Datta et al, 2018 ). Additional solubility can be achieved through pH reduction and generation of salt forms of 2,4-diaminopyrimidine-containing compounds or TMP.…”

“…While these are the most characterized DDs, in theory, one could generate a DD from a destabilized form of any protein that retains an ability to bind a stabilizing ligand, thereby promoting protein abundance. Nonetheless, due to its extensive use in the literature both in vitro ( Shoulders et al, 2013a , Shoulders et al, 2013b ) and in vivo ( Datta et al, 2018 , Peng et al, 2019 , Ramadurgum et al, 2020 ), we have focused on optimization and use of the ecDHFR-DD.…”