title: siRNA silencing of the mutant keratin 12 allele in corneal limbal epithelial cells grown from patients with Meesmann's epithelial corneal dystrophy.
abstract: OBJECTIVE
  The aim of this study is to further assess our previously reported keratin 12 (K12)-Leu132Pro specific siRNA in silencing the mutant allele in Meesmann's Epithelial Corneal Dystrophy (MECD) in experimental systems more akin to the in vivo situation through simultaneous expression of both wild-type and mutant alleles.
  
  
  METHODS
  Using KRT12 exogenous expression constructs transfected into cells, mutant allele specific knockdown was quantified using pyrosequencing and infrared Western blot analysis, while the silencing mechanism was assessed by a modified rapid amplification of cDNA ends (5'RACE) method. Corneal limbal biopsies taken from patients suffering from MECD were used to establish cultures of MECD corneal limbal epithelial stem cells and the ability of the siRNA to silence the endogenous mutant KRT12 allele was assessed by a combination of pyrosequencing, qPCR, ELISA, and quantitative-fluorescent immunohistochemistry (Q-FIHC).
  
  
  RESULTS
  The siRNA displayed a potent and specific knockdown of K12-Leu132Pro at both the mRNA and protein levels with exogenous expression constructs. Analysis by the 5'RACE method confirmed siRNA-mediated cleavage. In the MECD cells, an allele-specific knockdown of 63% of the endogenous mutant allele was observed without effect on wild-type allele expression.
  
  
  CONCLUSIONS
  Combined with an effective delivery vehicle this siRNA approach represents a viable treatment option for prevention of the MECD pathology observed in K12-Leu132Pro heterozygous individuals.
authors.affiliation: School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland, United Kingdom.
authors.forename: David G
authors.initials: DG
authors.lastname: Courtney
authors.affiliation: School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland, United Kingdom.
authors.forename: Sarah D
authors.initials: SD
authors.lastname: Atkinson
authors.affiliation: School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland, United Kingdom.
authors.forename: Edwin H A
authors.initials: EH
authors.lastname: Allen
authors.affiliation: School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland, United Kingdom.
authors.forename: Johnny E
authors.initials: JE
authors.lastname: Moore
authors.affiliation: School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland, United Kingdom.
authors.forename: Colum P
authors.initials: CP
authors.lastname: Walsh
authors.affiliation: Dermatology and Genetic Medicine, Colleges of Life Sciences and Medicine, Dentistry & Nursing, University of Dundee, Dundee, Scotland, United Kingdom.
authors.forename: Deena M Leslie
authors.initials: DM
authors.lastname: Pedrioli
authors.affiliation: Ophthalmology Department, University of Dundee, Ninewells Hospital, Dundee, Scotland, United Kingdom.
authors.forename: Caroline J
authors.initials: CJ
authors.lastname: MacEwen
authors.affiliation: Centre for Regenerative Medicine, Stefano Ferrari, University of Modena and Reggio Emilia, Modena, Italy.
authors.forename: Graziella
authors.initials: G
authors.lastname: Pellegrini
authors.affiliation: School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland, United Kingdom Centre for Regenerative Medicine, Stefano Ferrari, University of Modena and Reggio Emilia, Modena, Italy.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Maurizi
authors.affiliation: Centre for Regenerative Medicine, Stefano Ferrari, University of Modena and Reggio Emilia, Modena, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Serafini
authors.affiliation: Centre for Regenerative Medicine, Stefano Ferrari, University of Modena and Reggio Emilia, Modena, Italy.
authors.forename: Monica
authors.initials: M
authors.lastname: Fantacci
authors.affiliation: Dermatology and Genetic Medicine, Colleges of Life Sciences and Medicine, Dentistry & Nursing, University of Dundee, Dundee, Scotland, United Kingdom.
authors.forename: Haihui
authors.initials: H
authors.lastname: Liao
authors.affiliation: Institute of Molecular Medicine, Trinity College Dublin, Dublin, Ireland.
authors.forename: Alan D
authors.initials: AD
authors.lastname: Irvine
authors.affiliation: Dermatology and Genetic Medicine, Colleges of Life Sciences and Medicine, Dentistry & Nursing, University of Dundee, Dundee, Scotland, United Kingdom.
authors.forename: W H Irwin
authors.initials: WH
authors.lastname: McLean
authors.affiliation: School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland, United Kingdom Dermatology and Genetic Medicine, Colleges of Life Sciences and Medicine, Dentistry & Nursing, University of Dundee, Dundee, Scotland, United Kingdom Centre for Regenerative Medicine, Stefano Ferrari, University of Modena and Reggio Emilia, Modena, Italy.
authors.forename: C B Tara
authors.initials: CB
authors.lastname: Moore
mesh_terms: D000483:Alleles; D049109:Cell Proliferation; D002478:Cells, Cultured; D053559:Corneal Dystrophy, Juvenile Epithelial of Meesmann / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D004247:DNA / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D005091:Exons; D006579:Heterozygote; D006801:Humans; D007150:Immunohistochemistry; D053551:Keratin-12 / Q000235:genetics* / Q000378:metabolism; D016850:Limbus Corneae / Q000378:metabolism / Q000473:pathology*; D020125:Mutation, Missense; D010375:Pedigree; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D053551:Keratin-12; D034741:RNA, Small Interfering; D004247:DNA
keywords: MECD; allele discrimination; ex vivo; keratin 12; siRNA