Development of a Corneal Bioluminescence Mouse for Real-Time In Vivo Evaluation of Gene Therapies

Dun Jack Fu; Edwin H. A. Allen; Robyn P. Hickerson; Deena M. Leslie Pedrioli; W. H. Irwin McLean

doi:10.1167/tvst.9.13.44

Development of a Corneal Bioluminescence Mouse for Real-Time In Vivo Evaluation of Gene Therapies

Dun Jack Fu, Edwin H. A. Allen, Robyn P. Hickerson (Lead / Corresponding author), Deena M. Leslie Pedrioli, W. H. Irwin McLean

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Abstract

Purpose: The purpose of this study was to develop and characterize a novel bioluminescence transgenic mouse model that facilitates rapid evaluation of genetic medicine delivery methods for inherited and acquired corneal diseases.

Methods: Corneal expression of the firefly luciferase transgene (luc2) was achieved via insertion into the Krt12 locus, a type I intermediate filament keratin that is exclusively expressed in the cornea, to generate the Krt12luc2 mouse. The transgene includes a multiple target cassette with human pathogenic mutations in K3 and K12.

Results: The Krt12luc2 mouse exclusively expresses luc2 in the corneal epithelium under control of the keratin K12 promoter. The luc2 protein is enzymatically active, can be readily visualized, and exhibits a symmetrically consistent readout. Moreover, structural integrity of the corneal epithelium is preserved in mice that are heterozygous for the luc2 transgene (Krt12+/luc2).

Conclusions: This novel Krt12luc2 mouse model represents a potentially ideal in vivo system for evaluating the efficacies of cornea-targeting gene therapies and for establishing and/or validating new delivery modalities. Importantly, the multiple targeting cassette that is included in the Luc2 transgene will greatly reduce mouse numbers required for in vivo therapy evaluation.

Original language	English
Article number	44
Number of pages	10
Journal	Translational Vision Science and Technology
Volume	9
Issue number	13
DOIs	https://doi.org/10.1167/tvst.9.13.44
Publication status	Published - 29 Dec 2020

Keywords

MECD
keratin 12
cornea knock-in-mouse

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1167/tvst.9.13.44Licence: CC BY-NC-ND

Final Published VersionFinal published version, 4.31 MBLicence: CC BY-NC-ND

SiTransporter - Identification of Molecular Transporters that Facilitate SiRNA Skin Delivery (People IIF)
Hickerson, R. (Investigator) & McLean, I. (Investigator)
COMMISSION OF THE EUROPEAN COMMUNITIES
1/04/14 → 31/03/16
Project: Research
Dermatology and Genetic Medicine (Strategic Grant) (Joint with Kings College London)
Barton, G. (Investigator), Campbell, P. (Investigator), Hickerson, R. (Investigator), Leigh, I. (Investigator), McLean, I. (Investigator) & Wyatt, P. (Investigator)
1/08/12 → 30/04/19
Project: Research
Aref#d: 21558. SIRNA Therapy in Dominant Skin and Eye Disorders (Programme Grant)
McLean, I. (Investigator) & Smith, F. (Investigator)
Medical Research Council
18/02/10 → 17/02/15
Project: Research

Cite this

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title = "Development of a Corneal Bioluminescence Mouse for Real-Time In Vivo Evaluation of Gene Therapies",

abstract = "Purpose: The purpose of this study was to develop and characterize a novel bioluminescence transgenic mouse model that facilitates rapid evaluation of genetic medicine delivery methods for inherited and acquired corneal diseases.Methods: Corneal expression of the firefly luciferase transgene (luc2) was achieved via insertion into the Krt12 locus, a type I intermediate filament keratin that is exclusively expressed in the cornea, to generate the Krt12luc2 mouse. The transgene includes a multiple target cassette with human pathogenic mutations in K3 and K12.Results: The Krt12luc2 mouse exclusively expresses luc2 in the corneal epithelium under control of the keratin K12 promoter. The luc2 protein is enzymatically active, can be readily visualized, and exhibits a symmetrically consistent readout. Moreover, structural integrity of the corneal epithelium is preserved in mice that are heterozygous for the luc2 transgene (Krt12+/luc2).Conclusions: This novel Krt12luc2 mouse model represents a potentially ideal in vivo system for evaluating the efficacies of cornea-targeting gene therapies and for establishing and/or validating new delivery modalities. Importantly, the multiple targeting cassette that is included in the Luc2 transgene will greatly reduce mouse numbers required for in vivo therapy evaluation.",

keywords = "MECD, keratin 12, cornea knock-in-mouse",

author = "Fu, {Dun Jack} and Allen, {Edwin H. A.} and Hickerson, {Robyn P.} and {Leslie Pedrioli}, {Deena M.} and McLean, {W. H. Irwin}",

note = "Supported by the United Kingdom Medical Research Council (MRC) Grants G0801742 and G0802780 (to W.H.I.M.), Fight for Sight (UK) (to W.H.I.M.), and Wellcome Trust Strategic Award 098439/Z/12/Z (to W.H.I.M.). R.P.H. was supported by Marie Curie IIF award 626530 (to W.H.I.M.). The research was funded by Fight for Sight and a Wellcome Trust Strategic Award supporting The Centre for Dermatology and Genetic Medicine. The sponsor and funding organization had no role in the design or conduct of this research. ",

year = "2020",

month = dec,

day = "29",

doi = "10.1167/tvst.9.13.44",

language = "English",

volume = "9",

journal = "Translational Vision Science and Technology",

issn = "2164-2591",

publisher = "Association for Research in Vision and Ophthalmology",

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T1 - Development of a Corneal Bioluminescence Mouse for Real-Time In Vivo Evaluation of Gene Therapies

AU - Fu, Dun Jack

AU - Allen, Edwin H. A.

AU - Hickerson, Robyn P.

AU - Leslie Pedrioli, Deena M.

AU - McLean, W. H. Irwin

N1 - Supported by the United Kingdom Medical Research Council (MRC) Grants G0801742 and G0802780 (to W.H.I.M.), Fight for Sight (UK) (to W.H.I.M.), and Wellcome Trust Strategic Award 098439/Z/12/Z (to W.H.I.M.). R.P.H. was supported by Marie Curie IIF award 626530 (to W.H.I.M.). The research was funded by Fight for Sight and a Wellcome Trust Strategic Award supporting The Centre for Dermatology and Genetic Medicine. The sponsor and funding organization had no role in the design or conduct of this research.

PY - 2020/12/29

Y1 - 2020/12/29

N2 - Purpose: The purpose of this study was to develop and characterize a novel bioluminescence transgenic mouse model that facilitates rapid evaluation of genetic medicine delivery methods for inherited and acquired corneal diseases.Methods: Corneal expression of the firefly luciferase transgene (luc2) was achieved via insertion into the Krt12 locus, a type I intermediate filament keratin that is exclusively expressed in the cornea, to generate the Krt12luc2 mouse. The transgene includes a multiple target cassette with human pathogenic mutations in K3 and K12.Results: The Krt12luc2 mouse exclusively expresses luc2 in the corneal epithelium under control of the keratin K12 promoter. The luc2 protein is enzymatically active, can be readily visualized, and exhibits a symmetrically consistent readout. Moreover, structural integrity of the corneal epithelium is preserved in mice that are heterozygous for the luc2 transgene (Krt12+/luc2).Conclusions: This novel Krt12luc2 mouse model represents a potentially ideal in vivo system for evaluating the efficacies of cornea-targeting gene therapies and for establishing and/or validating new delivery modalities. Importantly, the multiple targeting cassette that is included in the Luc2 transgene will greatly reduce mouse numbers required for in vivo therapy evaluation.

AB - Purpose: The purpose of this study was to develop and characterize a novel bioluminescence transgenic mouse model that facilitates rapid evaluation of genetic medicine delivery methods for inherited and acquired corneal diseases.Methods: Corneal expression of the firefly luciferase transgene (luc2) was achieved via insertion into the Krt12 locus, a type I intermediate filament keratin that is exclusively expressed in the cornea, to generate the Krt12luc2 mouse. The transgene includes a multiple target cassette with human pathogenic mutations in K3 and K12.Results: The Krt12luc2 mouse exclusively expresses luc2 in the corneal epithelium under control of the keratin K12 promoter. The luc2 protein is enzymatically active, can be readily visualized, and exhibits a symmetrically consistent readout. Moreover, structural integrity of the corneal epithelium is preserved in mice that are heterozygous for the luc2 transgene (Krt12+/luc2).Conclusions: This novel Krt12luc2 mouse model represents a potentially ideal in vivo system for evaluating the efficacies of cornea-targeting gene therapies and for establishing and/or validating new delivery modalities. Importantly, the multiple targeting cassette that is included in the Luc2 transgene will greatly reduce mouse numbers required for in vivo therapy evaluation.

KW - MECD

KW - keratin 12

KW - cornea knock-in-mouse

U2 - 10.1167/tvst.9.13.44

DO - 10.1167/tvst.9.13.44

M3 - Article

C2 - 33442498

SN - 2164-2591

VL - 9

JO - Translational Vision Science and Technology

JF - Translational Vision Science and Technology

IS - 13

M1 - 44

ER -

Development of a Corneal Bioluminescence Mouse for Real-Time In Vivo Evaluation of Gene Therapies

Abstract

Keywords

UN SDGs

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Projects

SiTransporter - Identification of Molecular Transporters that Facilitate SiRNA Skin Delivery (People IIF)

Dermatology and Genetic Medicine (Strategic Grant) (Joint with Kings College London)

Aref#d: 21558. SIRNA Therapy in Dominant Skin and Eye Disorders (Programme Grant)

Cite this