A destabilizing domain allows for fast, noninvasive, conditional control of protein abundance in the mouse eye – Implications for ocular gene therapy

Shyamtanu Datta, Marian Renwick, Viet Q. Chau, Fang Zhang, Emily R. Nettesheim, Daniel M. Lipinski, John D Hulleman

Research output: Contribution to journalArticle

1 Citation (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)4909-4920
Number of pages12
JournalInvestigative Ophthalmology and Visual Science
Volume59
Issue number12
DOIs
StatePublished - Oct 1 2018

Fingerprint

Trimethoprim
Genetic Therapy
Tetrahydrofolate Dehydrogenase
Proteins
Retina
Ophthalmic Solutions
Drinking Water
Optical Imaging
Visual Acuity
Therapeutics
Western Blotting
Escherichia coli
Gene Expression
Light

Keywords

  • Chemical biology
  • DHFR
  • Gene therapy
  • Inducible
  • Trimethoprim

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Cite this

A destabilizing domain allows for fast, noninvasive, conditional control of protein abundance in the mouse eye – Implications for ocular gene therapy. / Datta, Shyamtanu; Renwick, Marian; Chau, Viet Q.; Zhang, Fang; Nettesheim, Emily R.; Lipinski, Daniel M.; Hulleman, John D.

In: Investigative Ophthalmology and Visual Science, Vol. 59, No. 12, 01.10.2018, p. 4909-4920.

Research output: Contribution to journalArticle

Datta, Shyamtanu ; Renwick, Marian ; Chau, Viet Q. ; Zhang, Fang ; Nettesheim, Emily R. ; Lipinski, Daniel M. ; Hulleman, John D. / A destabilizing domain allows for fast, noninvasive, conditional control of protein abundance in the mouse eye – Implications for ocular gene therapy. In: Investigative Ophthalmology and Visual Science. 2018 ; Vol. 59, No. 12. pp. 4909-4920.
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T1 - A destabilizing domain allows for fast, noninvasive, conditional control of protein abundance in the mouse eye – Implications for ocular gene therapy

AU - Datta, Shyamtanu

AU - Renwick, Marian

AU - Chau, Viet Q.

AU - Zhang, Fang

AU - Nettesheim, Emily R.

AU - Lipinski, Daniel M.

AU - Hulleman, John D

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AB - 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.

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