title: Development of allele-specific therapeutic siRNA in Meesmann epithelial corneal dystrophy.
abstract: BACKGROUND
  Meesmann epithelial corneal dystrophy (MECD) is an inherited eye disorder caused by dominant-negative mutations in either keratins K3 or K12, leading to mechanical fragility of the anterior corneal epithelium, the outermost covering of the eye. Typically, patients suffer from lifelong irritation of the eye and/or photophobia but rarely lose visual acuity; however, some individuals are severely affected, with corneal scarring requiring transplant surgery. At present no treatment exists which addresses the underlying pathology of corneal dystrophy. The aim of this study was to design and assess the efficacy and potency of an allele-specific siRNA approach as a future treatment for MECD.
  
  
  RESULTS
  We studied a family with a consistently severe phenotype where all affected persons were shown to carry heterozygous missense mutation Leu132Pro in the KRT12 gene. Using a cell-culture assay of keratin filament formation, mutation Leu132Pro was shown to be significantly more disruptive than the most common mutation, Arg135Thr, which is associated with typical, mild MECD. A siRNA sequence walk identified a number of potent inhibitors for the mutant allele, which had no appreciable effect on wild-type K12. The most specific and potent inhibitors were shown to completely block mutant K12 protein expression with negligible effect on wild-type K12 or other closely related keratins. Cells transfected with wild-type K12-EGFP construct show a predominantly normal keratin filament formation with only 5% aggregate formation, while transfection with mutant K12-EGFP construct resulted in a significantly higher percentage of keratin aggregates (41.75%; p<0.001 with 95% confidence limits). The lead siRNA inhibitor significantly rescued the ability to form keratin filaments (74.75% of the cells contained normal keratin filaments; p<0.001 with 95% confidence limits).
  
  
  CONCLUSIONS
  This study demonstrates that it is feasible to design highly potent siRNA against mutant alleles with single-nucleotide specificity for future treatment of MECD.
authors.affiliation: Division of Molecular Medicine, Colleges of Life Sciences and Medicine, Dentistry & Nursing, University of Dundee, Dundee, Scotland.
authors.forename: Haihui
authors.initials: H
authors.lastname: Liao
authors.forename: Alan D
authors.initials: AD
authors.lastname: Irvine
authors.forename: Caroline J
authors.initials: CJ
authors.lastname: Macewen
authors.forename: Kathryn H
authors.initials: KH
authors.lastname: Weed
authors.forename: Louise
authors.initials: L
authors.lastname: Porter
authors.forename: Laura D
authors.initials: LD
authors.lastname: Corden
authors.forename: A Bethany
authors.initials: AB
authors.lastname: Gibson
authors.forename: Jonathan E
authors.initials: JE
authors.lastname: Moore
authors.forename: Frances J D
authors.initials: FJ
authors.lastname: Smith
authors.forename: W H Irwin
authors.initials: WH
authors.lastname: McLean
authors.forename: C B Tara
authors.initials: CB
authors.lastname: Moore
mesh_terms: D000483:Alleles; D001483:Base Sequence; D002478:Cells, Cultured; D053559:Corneal Dystrophy, Juvenile Epithelial of Meesmann / Q000235:genetics* / Q000628:therapy*; D020868:Gene Silencing; D018014:Gene Transfer Techniques; D006801:Humans; D007633:Keratins / Q000737:chemistry; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry; D020836:Protein Structure, Quaternary; D034741:RNA, Small Interfering / Q000235:genetics* / Q000627:therapeutic use*
chemical_list: D050505:Mutant Proteins; D034741:RNA, Small Interfering; D007633:Keratins