Faculty Collaboration Database

A Destabilizing Domain Allows for Fast, Noninvasive, Conditional Control of Protein Abundance in the Mouse Eye - Implications for Ocular Gene Therapy. Invest Ophthalmol Vis Sci 2018 Oct 01;59(12):4909-4920

Date

10/23/2018

Pubmed ID

30347085

Pubmed Central ID

PMC6181441

DOI

10.1167/iovs.18-24987

Scopus ID

2-s2.0-85055194829 (requires institutional sign-in at Scopus site)   15 Citations

Abstract

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.

Author List

Datta S, Renwick M, Chau VQ, Zhang F, Nettesheim ER, Lipinski DM, Hulleman JD

Author

Daniel M. Lipinski PhD Associate Professor in the Ophthalmology and Visual Sciences department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Administration, Oral
Animals
Bacterial Proteins
Blotting, Western
Chromatography, Liquid
Dependovirus
Electroretinography
Escherichia coli
Folic Acid Antagonists
Genetic Therapy
Genetic Vectors
Intravitreal Injections
Luminescent Agents
Luminescent Proteins
Mice
Mice, Inbred C57BL
Parvovirinae
Plasmids
Real-Time Polymerase Chain Reaction
Retina
Tandem Mass Spectrometry
Tetrahydrofolate Dehydrogenase
Trimethoprim
Visual Acuity