title: Development of allele-specific gene-silencing siRNAs for TGFBI Arg124Cys in lattice corneal dystrophy type I.
abstract: OBJECTIVE
  This study aimed to investigate the potency and specificity of short-interfering RNA (siRNA) treatment for TGFBI-Arg124Cys lattice corneal dystrophy type I (LCDI) using exogenous expression constructs in model systems and endogenous gene targeting in an ex vivo model using corneal epithelial cell cultures.
  
  
  METHODS
  A panel of 19 TGFBI-Arg124Cys-specific siRNAs were assessed by a dual-luciferase reporter assay. Further assessment using pyrosequencing and qPCR was used to identify the lead siRNA; suppression of mutant TGFBIp expression was confirmed by Western blot and Congo red aggregation assays. An ex vivo model of LCDI was established using limbal biopsies from corneal dystrophy patients harboring the Arg124Cys mutation. Treatment efficiency of the siRNA was assessed for the inhibition of the mutant allele in the primary patient's corneal epithelial cells using pyrosequencing, quantitative PCR (qPCR), and an ELISA.
  
  
  RESULTS
  A lead siRNA was identified, and demonstrated to be potent and specific in inhibiting the TGFBI-Arg124Cys mutant allele at the mRNA and protein levels. Besides high allele specificity, siRNA treatment achieved a 44% reduction of the endogenous Arg124Cys allele in an ex vivo model of LCDI.
  
  
  CONCLUSIONS
  We have identified a lead siRNA specific to the TGFBI-Arg124Cys mutant allele associated with LCDI. Silencing of exogenous TGFBI was observed at mRNA and protein levels, and in an ex vivo model of LCDI with an efficient suppression of the endogenous mutant allele. This result indicates the potential of siRNA treatment as a personalized medicine approach for the management of heritable TGFBI-associated corneal dystrophies.
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.forename: Sarah D
authors.initials: SD
authors.lastname: Atkinson
authors.forename: Johnny E
authors.initials: JE
authors.lastname: Moore
authors.forename: Eleonora
authors.initials: E
authors.lastname: Maurizi
authors.forename: Chiara
authors.initials: C
authors.lastname: Serafini
authors.forename: Graziella
authors.initials: G
authors.lastname: Pellegrini
authors.forename: Graeme C
authors.initials: GC
authors.lastname: Black
authors.forename: Forbes D
authors.initials: FD
authors.lastname: Manson
authors.forename: Gary H F
authors.initials: GH
authors.lastname: Yam
authors.forename: Caroline J
authors.initials: CJ
authors.lastname: Macewen
authors.forename: Edwin H A
authors.initials: EH
authors.lastname: Allen
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; D015153:Blotting, Western; D018929:Cell Culture Techniques; D003317:Corneal Dystrophies, Hereditary / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D016326:Extracellular Matrix Proteins / Q000235:genetics*; D020868:Gene Silencing; D006801:Humans; D017354:Point Mutation; D034741:RNA, Small Interfering / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000235:genetics*; D000099222:betaIG-H3 Protein
chemical_list: D016326:Extracellular Matrix Proteins; D034741:RNA, Small Interfering; D016212:Transforming Growth Factor beta; D000099222:betaIG-H3 Protein
keywords: RNA interference; TGFBI; allele discrimination; lattice corneal dystrophy; siRNA