Utility of the DHFR-based destabilizing domain across mouse models of retinal degeneration and aging.

The Escherichia coli dihydrofolate reductase (DHFR) destabilizing domain (DD) serves as a promising approach to conditionally regulate protein abundance in a variety of tissues. To test whether this approach could be effectively applied to a wide variety of aged and disease-related ocular mouse models, we evaluated the DHFR DD system in the eyes of aged mice (up to 24 months), a light-induced retinal degeneration (LIRD) model, and two genetic models of retinal degeneration (rd2 and Abca4 -/- mice). The DHFR DD was effectively degraded in all model systems, including rd2 mice, which showed significant defects in chymotrypsin proteasomal activity. Moreover, trimethoprim (TMP) administration stabilized the DHFR DD in all mouse models. Thus, the DHFR DD-based approach allows for control of protein abundance in a variety of mouse models, laying the foundation to use this strategy for the conditional control of gene therapies to potentially treat multiple eye diseases.

Nr2e3 is a genetic modifier that rescues retinal degeneration and promotes homeostasis in multiple models of retinitis pigmentosa.

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.

Fibulin-3 knockout mice demonstrate corneal dysfunction but maintain normal retinal integrity.

Fibulin-3 (F3) is an extracellular matrix glycoprotein found in basement membranes across the body. An autosomal dominant R345W mutation in F3 causes a macular dystrophy resembling dry age-related macular degeneration (AMD), whereas genetic removal of wild-type (WT) F3 protects mice from sub-retinal pigment epithelium (RPE) deposit formation. These observations suggest that F3 is a protein which can regulate pathogenic sub-RPE deposit formation in the eye. Yet the precise role of WT F3 within the eye is still largely unknown. We found that F3 is expressed throughout the mouse eye (cornea, trabecular meshwork (TM) ring, neural retina, RPE/choroid, and optic nerve). We next performed a thorough structural and functional characterization of each of these tissues in WT and homozygous (F3-/-) knockout mice. The corneal stroma in F3-/- mice progressively thins beginning at 2 months, and the development of corneal opacity and vascularization starts at 9 months, which worsens with age. However, in all other tissues (TM, neural retina, RPE, and optic nerve), gross structural anatomy and functionality were similar across WT and F3-/- mice when evaluated using SD-OCT, histological analyses, electron microscopy, scotopic electroretinogram, optokinetic response, and axonal anterograde transport. The lack of noticeable retinal abnormalities in F3-/- mice was confirmed in a human patient with biallelic loss-of-function mutations in F3. These data suggest that (i) F3 is important for maintaining the structural integrity of the cornea, (ii) absence of F3 does not affect the structure or function of any other ocular tissue in which it is expressed, and (iii) targeted silencing of F3 in the retina and/or RPE will likely be well-tolerated, serving as a safe therapeutic strategy for reducing sub-RPE deposit formation in disease. KEY MESSAGES: • Fibulins are expressed throughout the body at varying levels. • Fibulin-3 has a tissue-specific pattern of expression within the eye. • Lack of fibulin-3 leads to structural deformities in the cornea. • The retina and RPE remain structurally and functionally healthy in the absence of fibulin-3 in both mice and humans.

Small Molecule-Based Inducible Gene Therapies for Retinal Degeneration.

The eye is an excellent target organ for gene therapy. It is physically isolated, easily accessible, immune-privileged, and postmitotic. Furthermore, potential gene therapies introduced into the eye can be evaluated by noninvasive methods such as fundoscopy, electroretinography, and optical coherence tomography. In the last two decades, great advances have been made in understanding the molecular underpinnings of retinal degenerative diseases. Building upon the development of modern techniques for gene delivery, many gene-based therapies have been effectively used to treat loss-of-function retinal diseases in mice and men. Significant effort has been invested into making gene delivery vehicles more efficient, less toxic, and non-immunogenic. However, one challenge for the treatment of more complex gain-of-function diseases, many of which might be benefited by the regulation of cellular stress-responsive signaling pathways, is the ability to control the strategy in a physiological (conditional) manner. This review is focused on promising retinal gene therapy strategies that rely on small molecule-based conditional regulation and the inherent limitations and challenges of these strategies that need to be addressed prior to their extensive use.

Non-antibiotic Small-Molecule Regulation of DHFR-Based Destabilizing Domains In Vivo.

The E. coli dihydrofolate reductase (DHFR) destabilizing domain (DD), which shows promise as a biologic tool and potential gene therapy approach, can be utilized to achieve spatial and temporal control of protein abundance in vivo simply by administration of its stabilizing ligand, the routinely prescribed antibiotic trimethoprim (TMP). However, chronic TMP use drives development of antibiotic resistance (increasing likelihood of subsequent infections) and disrupts the gut microbiota (linked to autoimmune and neurodegenerative diseases), tempering translational excitement of this approach in model systems and for treating human diseases. Herein, we identified a TMP-based, non-antibiotic small molecule, termed 14a (MCC8529), and tested its ability to control multiple DHFR-based reporters and signaling proteins. We found that 14a is non-toxic and can effectively stabilize DHFR DDs expressed in mammalian cells. Furthermore, 14a crosses the blood-retinal barrier and stabilizes DHFR DDs expressed in the mouse eye with kinetics comparable to that of TMP (≤6 h). Surprisingly, 14a stabilized a DHFR DD in the liver significantly better than TMP did, while having no effect on the mouse gut microbiota. Our results suggest that alternative small-molecule DHFR DD stabilizers (such as 14a) may be ideal substitutes for TMP in instances when conditional, non-antibiotic control of protein abundance is desired in the eye and beyond.

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 Escherichia coli dihydrofolate reductase (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: 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. Results: Without 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. Conclusions: This DHFR-based conditional system is a rapid, efficient, and reversible tool to effectively control protein expression in the retina.

Synonymous variants in HTRA1 implicated in AMD susceptibility impair its capacity to regulate TGF-ß signaling.

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 ß1 (TGF-ß1) by normal HTRA1 but not the AMD-risk-associated isoform. As a consequence, both HTRA1 isoforms strongly differed in their ability to control TGF-ß mediated signaling, as revealed by reporter assays targeting the TGF-ß1-induced serpin peptidase inhibitor (SERPINE1, alias PAI-1) promoter. In addition, structurally altered HTRA1 led to an impaired autocrine TGF-ß signaling in microglia, as measured by a strong down-regulation of downstream effectors of the TGF-ß 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-ß1. As a consequence, this leads to an impairment of TGF-ß signaling and microglial regulation. Functional implications of the altered properties on AMD pathogenesis remain to be clarified.