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

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 -