Projects per year
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 | |
Publication status | Published - 2013 |
Keywords
- DISEASES
- EPIDERMOLYSIS-BULLOSA SIMPLEX
- CELL-LINE
- RNAI THERAPEUTICS
- GENE-THERAPY
- KNOCKING
- INCLUDING PACHYONYCHIA-CONGENITA
- DISORDERS
- INTERFERENCE
- DELIVERY
Fingerprint
Dive into the research topics of 'Allele-specific siRNA silencing for the common keratin 12 founder mutation in Meesmann epithelial corneal dystrophy'. Together they form a unique fingerprint.Projects
- 1 Finished
-
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