![]() 17 – 20 Furthermore, particular tet/dox systems have been found to be leaky in vivo 21 (although headway has been made in this regard 22), and high levels of tetracyclines have been suggested to disrupt mitochondrial homeostasis. Secondly, mouse in vivo tet/dox reversibility experiments typically use a 1- to 2-week induction “on” period for full activation, followed by a 1- to 3-week washout “off” period for full reversibility. Firstly, tet/dox systems regulate gene expression at the transcriptional level, requiring subsequent protein translation, which takes additional processing time. While this inducible system has been validated and utilized successfully in the mouse eye, 14 – 16 it is not an ideal approach for reversibly and repeatedly regulating protein abundance. The most commonly used conditional gene therapy approach relies on the tetracycline/doxycycline (tet/dox)-inducible system. Therefore, there is an urgent need to develop and validate a conditional and reversible therapeutic strategy that allows for experimental control of the timing and extent of production of a protein of interest (e.g., a stress-responsive transcription factor) in the eye. 8 – 13 However, the technical capability to regulate these signaling pathways in a physiological context (i.e., conditional sinusoidal expression) and under spatial and temporal control is lacking. 5 – 7 These risks are especially problematic when attempting to control cellular signaling pathways responsible for promoting protein homeostasis such as the unfolded protein response, oxidative stress response, inflammation and autophagy, all of which are incontrovertibility linked to incurable eye diseases. 4 While appropriate in certain contexts, such as for restoring genes that are incapacitated by loss-of-function mutations or premature stop codons, constitutive ectopic gene expression does bear the potential risk of unanticipated side effects due to sustained, elevated expression in a nonphysiologic (and potentially disadvantageous) manner. This DHFR-based conditional system is a rapid, efficient, and reversible tool to effectively control protein expression in the retina.Ĭonventional gene therapy approaches utilize constitutive gene expression to combat ocular diseases such as Leber's congenital amaurosis (LCA), 1 choroideremia, 2 MERTK-associated retinitis pigmentosa, 3 amongst others. Long-term TMP treatment had no impact on retina function/structure and had no effect on >99.9% of tested genes. Stabilization was completely and repeatedly reversible following removal/addition of TMP in all regimens. 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. TMP achieved ocular concentrations of ∼13.6 μM (oral gavage), ∼331 nM (drinking water), and ∼636 nM (eye drops). Without TMP, DHFR.YFP was efficiently degraded in the retina. Visual acuity, response to light stimulus, retinal structure, and gene expression were evaluated after long-term (3 months) TMP treatment. Protein abundance was measured by fundus fluorescence imaging and western blotting. Ocular TMP levels post treatment were quantified by LC-MS/MS. TMP or vehicle was administered to mice via oral gavage, drinking water, or eye drops. We intravitreally injected wild-type C57BL6/J mice with an adeno-associated vector (rAAV2/2) constitutively expressing separate fluorescent reporters: DHFR fused to yellow fluorescent protein (DHFR.YFP) and mCherry. 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). 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.
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