Allele-specific siRNA silencing for the common keratin 12 founder mutation in Meesmann epithelial corneal dystrophy

Edwin H. A. Allen; Sarah D. Atkinson; Haihui Liao; Jonathan E. Moore; Deena M. Leslie Pedrioli; Frances J. D. Smith; William H. Irwin McLean; C. B. Tara Moore

doi:10.1167/iovs.12-10528

Allele-specific siRNA silencing for the common keratin 12 founder mutation in Meesmann epithelial corneal dystrophy

Edwin H. A. Allen, Sarah D. Atkinson, Haihui Liao, Jonathan E. Moore, Deena M. Leslie Pedrioli, Frances J. D. Smith, William H. Irwin McLean, C. B. Tara Moore

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

31 Citations (Scopus)

Abstract

PURPOSE. To identify an allele-specific short interfering RNA (siRNA), against the common KRT12 mutation Arg135Thr in Meesmann epithelial corneal dystrophy (MECD) as a personalized approach to treatment.

METHODS. siRNAs against the K12 Arg135Thr mutation were evaluated using a dual luciferase reporter gene assay and the most potent and specific siRNAs were further screened by Western blot. Off-target effects on related keratins were assessed and immunological stimulation of TLR3 was evaluated by RT-PCR. A modified 5' rapid amplification of cDNA ends method was used to confirm siRNA-mediated mutant knockdown. Allele discrimination was confirmed by quantitative infrared immunoblotting.

RESULTS. The lead siRNA, with an IC50 of thirty picomolar, showed no keratin off-target effects or activation of TLR3 in the concentration ranges tested. We confirmed siRNA-mediated knockdown by the presence of K12 mRNA fragments cleaved at the predicted site. A dual tag infrared immunoblot showed knockdown to be allele-specific, with 70% to 80% silencing of the mutant protein.

CONCLUSIONS. A potent allele-specific siRNA against the K12 Arg135Thr mutation was identified. In combination with efficient eyedrop formulation delivery, this would represent a personalized medicine approach, aimed at preventing the pathology associated with MECD and other ocular surface pathologies with dominant-negative or gain-of-function pathomechanisms. (Invest Ophthalmol Vis Sci. 2013;54:494-502) DOI:10.1167/iovs.12-10528

Original language	English
Pages (from-to)	494-502
Number of pages	9
Journal	Investigative Ophthalmology & Visual Science
Volume	54
Issue number	1
DOIs	https://doi.org/10.1167/iovs.12-10528
Publication status	Published - 2013

Keywords

DISEASES
EPIDERMOLYSIS-BULLOSA SIMPLEX
CELL-LINE
RNAI THERAPEUTICS
GENE-THERAPY
KNOCKING
INCLUDING PACHYONYCHIA-CONGENITA
DISORDERS
INTERFERENCE
DELIVERY

Access to Document

10.1167/iovs.12-10528

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

Cite this

@article{654a4313216b4245bc674d21562d46cb,

title = "Allele-specific siRNA silencing for the common keratin 12 founder mutation in Meesmann epithelial corneal dystrophy",

abstract = "PURPOSE. To identify an allele-specific short interfering RNA (siRNA), against the common KRT12 mutation Arg135Thr in Meesmann epithelial corneal dystrophy (MECD) as a personalized approach to treatment.METHODS. siRNAs against the K12 Arg135Thr mutation were evaluated using a dual luciferase reporter gene assay and the most potent and specific siRNAs were further screened by Western blot. Off-target effects on related keratins were assessed and immunological stimulation of TLR3 was evaluated by RT-PCR. A modified 5' rapid amplification of cDNA ends method was used to confirm siRNA-mediated mutant knockdown. Allele discrimination was confirmed by quantitative infrared immunoblotting.RESULTS. The lead siRNA, with an IC50 of thirty picomolar, showed no keratin off-target effects or activation of TLR3 in the concentration ranges tested. We confirmed siRNA-mediated knockdown by the presence of K12 mRNA fragments cleaved at the predicted site. A dual tag infrared immunoblot showed knockdown to be allele-specific, with 70% to 80% silencing of the mutant protein.CONCLUSIONS. A potent allele-specific siRNA against the K12 Arg135Thr mutation was identified. In combination with efficient eyedrop formulation delivery, this would represent a personalized medicine approach, aimed at preventing the pathology associated with MECD and other ocular surface pathologies with dominant-negative or gain-of-function pathomechanisms. (Invest Ophthalmol Vis Sci. 2013;54:494-502) DOI:10.1167/iovs.12-10528",

keywords = "DISEASES, EPIDERMOLYSIS-BULLOSA SIMPLEX, CELL-LINE, RNAI THERAPEUTICS, GENE-THERAPY, KNOCKING, INCLUDING PACHYONYCHIA-CONGENITA, DISORDERS, INTERFERENCE, DELIVERY",

author = "Allen, {Edwin H. A.} and Atkinson, {Sarah D.} and Haihui Liao and Moore, {Jonathan E.} and Pedrioli, {Deena M. Leslie} and Smith, {Frances J. D.} and McLean, {William H. Irwin} and Moore, {C. B. Tara}",

year = "2013",

doi = "10.1167/iovs.12-10528",

language = "English",

volume = "54",

pages = "494--502",

journal = "Investigative Ophthalmology & Visual Science",

issn = "0146-0404",

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

number = "1",

}

TY - JOUR

T1 - Allele-specific siRNA silencing for the common keratin 12 founder mutation in Meesmann epithelial corneal dystrophy

AU - Allen, Edwin H. A.

AU - Atkinson, Sarah D.

AU - Liao, Haihui

AU - Moore, Jonathan E.

AU - Pedrioli, Deena M. Leslie

AU - Smith, Frances J. D.

AU - McLean, William H. Irwin

AU - Moore, C. B. Tara

PY - 2013

Y1 - 2013

N2 - PURPOSE. To identify an allele-specific short interfering RNA (siRNA), against the common KRT12 mutation Arg135Thr in Meesmann epithelial corneal dystrophy (MECD) as a personalized approach to treatment.METHODS. siRNAs against the K12 Arg135Thr mutation were evaluated using a dual luciferase reporter gene assay and the most potent and specific siRNAs were further screened by Western blot. Off-target effects on related keratins were assessed and immunological stimulation of TLR3 was evaluated by RT-PCR. A modified 5' rapid amplification of cDNA ends method was used to confirm siRNA-mediated mutant knockdown. Allele discrimination was confirmed by quantitative infrared immunoblotting.RESULTS. The lead siRNA, with an IC50 of thirty picomolar, showed no keratin off-target effects or activation of TLR3 in the concentration ranges tested. We confirmed siRNA-mediated knockdown by the presence of K12 mRNA fragments cleaved at the predicted site. A dual tag infrared immunoblot showed knockdown to be allele-specific, with 70% to 80% silencing of the mutant protein.CONCLUSIONS. A potent allele-specific siRNA against the K12 Arg135Thr mutation was identified. In combination with efficient eyedrop formulation delivery, this would represent a personalized medicine approach, aimed at preventing the pathology associated with MECD and other ocular surface pathologies with dominant-negative or gain-of-function pathomechanisms. (Invest Ophthalmol Vis Sci. 2013;54:494-502) DOI:10.1167/iovs.12-10528

AB - PURPOSE. To identify an allele-specific short interfering RNA (siRNA), against the common KRT12 mutation Arg135Thr in Meesmann epithelial corneal dystrophy (MECD) as a personalized approach to treatment.METHODS. siRNAs against the K12 Arg135Thr mutation were evaluated using a dual luciferase reporter gene assay and the most potent and specific siRNAs were further screened by Western blot. Off-target effects on related keratins were assessed and immunological stimulation of TLR3 was evaluated by RT-PCR. A modified 5' rapid amplification of cDNA ends method was used to confirm siRNA-mediated mutant knockdown. Allele discrimination was confirmed by quantitative infrared immunoblotting.RESULTS. The lead siRNA, with an IC50 of thirty picomolar, showed no keratin off-target effects or activation of TLR3 in the concentration ranges tested. We confirmed siRNA-mediated knockdown by the presence of K12 mRNA fragments cleaved at the predicted site. A dual tag infrared immunoblot showed knockdown to be allele-specific, with 70% to 80% silencing of the mutant protein.CONCLUSIONS. A potent allele-specific siRNA against the K12 Arg135Thr mutation was identified. In combination with efficient eyedrop formulation delivery, this would represent a personalized medicine approach, aimed at preventing the pathology associated with MECD and other ocular surface pathologies with dominant-negative or gain-of-function pathomechanisms. (Invest Ophthalmol Vis Sci. 2013;54:494-502) DOI:10.1167/iovs.12-10528

KW - DISEASES

KW - EPIDERMOLYSIS-BULLOSA SIMPLEX

KW - CELL-LINE

KW - RNAI THERAPEUTICS

KW - GENE-THERAPY

KW - KNOCKING

KW - INCLUDING PACHYONYCHIA-CONGENITA

KW - DISORDERS

KW - INTERFERENCE

KW - DELIVERY

U2 - 10.1167/iovs.12-10528

DO - 10.1167/iovs.12-10528

M3 - Article

C2 - 23233254

SN - 0146-0404

VL - 54

SP - 494

EP - 502

JO - Investigative Ophthalmology & Visual Science

JF - Investigative Ophthalmology & Visual Science

IS - 1

ER -

Allele-specific siRNA silencing for the common keratin 12 founder mutation in Meesmann epithelial corneal dystrophy

Abstract

Keywords

Access to Document

Fingerprint

Projects

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

Cite this