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

doi:10.1167/iovs.18-24987

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 journal › Article › peer-review

18 Scopus 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.

Original language	English (US)
Pages (from-to)	4909-4920
Number of pages	12
Journal	Investigative Ophthalmology and Visual Science
Volume	59
Issue number	12
DOIs	https://doi.org/10.1167/iovs.18-24987
State	Published - Oct 2018
Externally published	Yes

Bibliographical note

Funding Information:
Supported by funding from the UT Southwestern Summer Medical Student Research Program (VQC). Supported through a Foundation Fighting Blindness Individual Investigator Award and receives institutional support through the Medical College of Wisconsin NIH/NEI Visual Science Core Grant (P30 EY001931-37) and Training Grant (T32 EY014537-11) (DML). Supported by an endowment from the Roger and Dorothy Hirl Research Fund, a vision research grant from the Karl Kirchgessner Foundation, a National Eye Institute R21 Grant (EY028261), and a Career Development Award from Research to Prevent Blindness (JDH). Additional support was provided by a National Eye Institute Visual Science Core Grant (P30 EY020799) and an unrestricted grant from RPB (both to the UT Southwestern Department of Ophthalmology).

Funding Information:
The authors thank J. Cameron Millar (University of North Texas Health Science Center) for his training on intravitreal injections, Noelle Williams and Jessica Kilgore (UT Southwestern Preclinical Pharmacology Core) for their work on the quantification of TMP in mouse tissue, and Matt Petroll (UT Southwestern Department of Ophthalmology) for his assistance with fundus image quantification. Supported by funding from the UT Southwestern Summer Medical Student Research Program (VQC). Supported through a Foundation Fighting Blindness Individual Investigator Award and receives institutional support through the Medical College of Wisconsin NIH/NEI Visual Science Core Grant (P30 EY001931-37) and Training Grant (T32 EY014537-11) (DML). Supported by an endowment from the Roger and Dorothy Hirl Research Fund, a vision research grant from the Karl Kirchgessner Foundation, a National Eye Institute R21 Grant (EY028261), and a Career Development Award from Research to Prevent Blindness (JDH). Additional support was provided by a National Eye Institute Visual Science Core Grant (P30 EY020799) and an unrestricted grant from RPB (both to the UT Southwestern Department of Ophthalmology).

Publisher Copyright:
© 2018 The Authors.

Keywords

Chemical biology
DHFR
Gene therapy
Inducible
Trimethoprim

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10.1167/iovs.18-24987

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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. / Datta, Shyamtanu; Renwick, Marian; Chau, Viet Q. et al.
In: Investigative Ophthalmology and Visual Science, Vol. 59, No. 12, 10.2018, p. 4909-4920.

Research output: Contribution to journal › Article › peer-review

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

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 \textasciitilde{}13.6 μM (oral gavage), \textasciitilde{}331 nM (drinking water), and \textasciitilde{}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.",

keywords = "Chemical biology, DHFR, Gene therapy, Inducible, Trimethoprim",

author = "Shyamtanu Datta and Marian Renwick and Chau, \{Viet Q.\} and Fang Zhang and Nettesheim, \{Emily R.\} and Lipinski, \{Daniel M.\} and Hulleman, \{John D.\}",

note = "Funding Information: Supported by funding from the UT Southwestern Summer Medical Student Research Program (VQC). Supported through a Foundation Fighting Blindness Individual Investigator Award and receives institutional support through the Medical College of Wisconsin NIH/NEI Visual Science Core Grant (P30 EY001931-37) and Training Grant (T32 EY014537-11) (DML). Supported by an endowment from the Roger and Dorothy Hirl Research Fund, a vision research grant from the Karl Kirchgessner Foundation, a National Eye Institute R21 Grant (EY028261), and a Career Development Award from Research to Prevent Blindness (JDH). Additional support was provided by a National Eye Institute Visual Science Core Grant (P30 EY020799) and an unrestricted grant from RPB (both to the UT Southwestern Department of Ophthalmology). Funding Information: The authors thank J. Cameron Millar (University of North Texas Health Science Center) for his training on intravitreal injections, Noelle Williams and Jessica Kilgore (UT Southwestern Preclinical Pharmacology Core) for their work on the quantification of TMP in mouse tissue, and Matt Petroll (UT Southwestern Department of Ophthalmology) for his assistance with fundus image quantification. Supported by funding from the UT Southwestern Summer Medical Student Research Program (VQC). Supported through a Foundation Fighting Blindness Individual Investigator Award and receives institutional support through the Medical College of Wisconsin NIH/NEI Visual Science Core Grant (P30 EY001931-37) and Training Grant (T32 EY014537-11) (DML). Supported by an endowment from the Roger and Dorothy Hirl Research Fund, a vision research grant from the Karl Kirchgessner Foundation, a National Eye Institute R21 Grant (EY028261), and a Career Development Award from Research to Prevent Blindness (JDH). Additional support was provided by a National Eye Institute Visual Science Core Grant (P30 EY020799) and an unrestricted grant from RPB (both to the UT Southwestern Department of Ophthalmology). Publisher Copyright: {\textcopyright} 2018 The Authors.",

year = "2018",

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doi = "10.1167/iovs.18-24987",

language = "English (US)",

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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.

N1 - Funding Information: Supported by funding from the UT Southwestern Summer Medical Student Research Program (VQC). Supported through a Foundation Fighting Blindness Individual Investigator Award and receives institutional support through the Medical College of Wisconsin NIH/NEI Visual Science Core Grant (P30 EY001931-37) and Training Grant (T32 EY014537-11) (DML). Supported by an endowment from the Roger and Dorothy Hirl Research Fund, a vision research grant from the Karl Kirchgessner Foundation, a National Eye Institute R21 Grant (EY028261), and a Career Development Award from Research to Prevent Blindness (JDH). Additional support was provided by a National Eye Institute Visual Science Core Grant (P30 EY020799) and an unrestricted grant from RPB (both to the UT Southwestern Department of Ophthalmology). Funding Information: The authors thank J. Cameron Millar (University of North Texas Health Science Center) for his training on intravitreal injections, Noelle Williams and Jessica Kilgore (UT Southwestern Preclinical Pharmacology Core) for their work on the quantification of TMP in mouse tissue, and Matt Petroll (UT Southwestern Department of Ophthalmology) for his assistance with fundus image quantification. Supported by funding from the UT Southwestern Summer Medical Student Research Program (VQC). Supported through a Foundation Fighting Blindness Individual Investigator Award and receives institutional support through the Medical College of Wisconsin NIH/NEI Visual Science Core Grant (P30 EY001931-37) and Training Grant (T32 EY014537-11) (DML). Supported by an endowment from the Roger and Dorothy Hirl Research Fund, a vision research grant from the Karl Kirchgessner Foundation, a National Eye Institute R21 Grant (EY028261), and a Career Development Award from Research to Prevent Blindness (JDH). Additional support was provided by a National Eye Institute Visual Science Core Grant (P30 EY020799) and an unrestricted grant from RPB (both to the UT Southwestern Department of Ophthalmology). Publisher Copyright: © 2018 The Authors.

PY - 2018/10

Y1 - 2018/10

N2 - 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.

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.

KW - Chemical biology

KW - DHFR

KW - Gene therapy

KW - Inducible

KW - Trimethoprim

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DO - 10.1167/iovs.18-24987

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C2 - 30347085

AN - SCOPUS:85055194829

SN - 0146-0404

VL - 59

SP - 4909

EP - 4920

JO - Investigative Ophthalmology and Visual Science

JF - Investigative Ophthalmology and Visual Science

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ER -

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

Abstract

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