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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...

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Autores principales: Datta, Shyamtanu, Renwick, Marian, Chau, Viet Q., Zhang, Fang, Nettesheim, Emily R., Lipinski, Daniel M., Hulleman, John D.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Association for Research in Vision and Ophthalmology 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6181441/
https://www.ncbi.nlm.nih.gov/pubmed/30347085
http://dx.doi.org/10.1167/iovs.18-24987
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author Datta, Shyamtanu
Renwick, Marian
Chau, Viet Q.
Zhang, Fang
Nettesheim, Emily R.
Lipinski, Daniel M.
Hulleman, John D.
author_facet Datta, Shyamtanu
Renwick, Marian
Chau, Viet Q.
Zhang, Fang
Nettesheim, Emily R.
Lipinski, Daniel M.
Hulleman, John D.
author_sort Datta, Shyamtanu
collection PubMed
description 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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spelling pubmed-61814412018-10-15 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. Invest Ophthalmol Vis Sci Retina 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. The Association for Research in Vision and Ophthalmology 2018-10 /pmc/articles/PMC6181441/ /pubmed/30347085 http://dx.doi.org/10.1167/iovs.18-24987 Text en Copyright 2018 The Authors http://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
spellingShingle Retina
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
title A Destabilizing Domain Allows for Fast, Noninvasive, Conditional Control of Protein Abundance in the Mouse Eye – Implications for Ocular Gene Therapy
title_full A Destabilizing Domain Allows for Fast, Noninvasive, Conditional Control of Protein Abundance in the Mouse Eye – Implications for Ocular Gene Therapy
title_fullStr A Destabilizing Domain Allows for Fast, Noninvasive, Conditional Control of Protein Abundance in the Mouse Eye – Implications for Ocular Gene Therapy
title_full_unstemmed A Destabilizing Domain Allows for Fast, Noninvasive, Conditional Control of Protein Abundance in the Mouse Eye – Implications for Ocular Gene Therapy
title_short A Destabilizing Domain Allows for Fast, Noninvasive, Conditional Control of Protein Abundance in the Mouse Eye – Implications for Ocular Gene Therapy
title_sort destabilizing domain allows for fast, noninvasive, conditional control of protein abundance in the mouse eye – implications for ocular gene therapy
topic Retina
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6181441/
https://www.ncbi.nlm.nih.gov/pubmed/30347085
http://dx.doi.org/10.1167/iovs.18-24987
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