David G Courtney1, Sarah D Atkinson1, Edwin H A Allen1, Johnny E Moore1, Colum P Walsh1, Deena M Leslie Pedrioli2, Caroline J MacEwen3, Graziella Pellegrini4, Eleonora Maurizi5, Chiara Serafini4, Monica Fantacci4, Haihui Liao2, Alan D Irvine6, W H Irwin McLean2, C B Tara Moore7. 1. School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland, United Kingdom. 2. Dermatology and Genetic Medicine, Colleges of Life Sciences and Medicine, Dentistry & Nursing, University of Dundee, Dundee, Scotland, United Kingdom. 3. Ophthalmology Department, University of Dundee, Ninewells Hospital, Dundee, Scotland, United Kingdom. 4. Centre for Regenerative Medicine, Stefano Ferrari, University of Modena and Reggio Emilia, Modena, Italy. 5. 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. 6. Institute of Molecular Medicine, Trinity College Dublin, Dublin, Ireland. 7. 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.
Abstract
PURPOSE: 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. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.
PURPOSE: 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. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.
Entities:
Keywords:
MECD; allele discrimination; ex vivo; keratin 12; siRNA
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