Recent advances in viral vector engineering, as well as an increased understanding of the cellular and molecular mechanism of retinal diseases, have led to the development of novel gene therapy approaches. Furthermore, ease of accessibility and ocular immune privilege makes the retina an ideal target for gene therapies. In this study, the nuclear hormone receptor gene Nr2e3 was evaluated for efficacy as broad-spectrum therapy to attenuate early to intermediate stages of retinal degeneration in five unique mouse models of retinitis pigmentosa (RP). RP is a group of heterogenic inherited retinal diseases associated with over 150 gene mutations, affecting over 1.5 million individuals worldwide. RP varies in age of onset, severity, and rate of progression. In addition,~40% of RP patients cannot be genetically diagnosed, confounding the ability to develop personalized RP therapies. Remarkably, Nr2e3 administered therapy resulted in reduced retinal degeneration as observed by increase in photoreceptor cells, improved electroretinogram, and a dramatic molecular reset of key transcription factors and associated gene networks. These therapeutic effects improved retinal homeostasis in diseased tissue. Results of this study provide evidence that Nr2e3 can serve as a broad-spectrum therapy to treat multiple forms of RP.

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.

High-temperature requirement A1 (HTRA1) is a secreted serine protease reported to play a role in the development of several cancers and neurodegenerative diseases. Still, the mechanism underlying the disease processes largely remains undetermined. In age-related macular degeneration (AMD), a common cause of vision impairment and blindness in industrialized societies, two synonymous polymorphisms (rs1049331:C>T, and rs2293870:G>T) in exon 1 of the HTRA1 gene were associated with a high risk to develop disease. Here, we show that the two polymorphisms result in a protein with altered thermophoretic properties upon heat-induced unfolding, trypsin accessibility and secretion behavior, suggesting unique structural features of the AMD-risk-associated HTRA1 protein. Applying MicroScale Thermophoresis and protease digestion analysis, we demonstrate direct binding and proteolysis of transforming growth factor β1 (TGF-β1) by normal HTRA1 but not the AMD-risk-associated isoform. As a consequence, both HTRA1 isoforms strongly differed in their ability to control TGF-β mediated signaling, as revealed by reporter assays targeting the TGF-β1-induced serpin peptidase inhibitor (SERPINE1, alias PAI-1) promoter. In addition, structurally altered HTRA1 led to an impaired autocrine TGF-β signaling in microglia, as measured by a strong down-regulation of downstream effectors of the TGF-β cascade such as phosphorylated SMAD2 and PAI-1 expression. Taken together, our findings demonstrate the effects of two synonymous HTRA1 variants on protein structure and protein interaction with TGF-β1. As a consequence, this leads to an impairment of TGF-β signaling and microglial regulation. Functional implications of the altered properties on AMD pathogenesis remain to be clarified.