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 IC(50) 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.
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 IC(50) 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.
Authors: Mark E Kleinman; Hiroki Kaneko; Won Gil Cho; Sami Dridi; Benjamin J Fowler; Alexander D Blandford; Romulo J C Albuquerque; Yoshio Hirano; Hiroko Terasaki; Mineo Kondo; Takashi Fujita; Balamurali K Ambati; Valeria Tarallo; Bradley D Gelfand; Sasha Bogdanovich; Judit Z Baffi; Jayakrishna Ambati Journal: Mol Ther Date: 2011-10-11 Impact factor: 11.454
Authors: Francis L Munier; Beatrice E Frueh; Philippe Othenin-Girard; Sylvie Uffer; Pascal Cousin; Ming X Wang; Elise Héon; Graeme C M Black; Maria A Blasi; Emilio Balestrazzi; Birgit Lorenz; Rafael Escoto; Rafael Barraquer; Maria Hoeltzenbein; Balder Gloor; Maurizio Fossarello; Arun D Singh; Yvan Arsenijevic; Léonidas Zografos; Daniel F Schorderet Journal: Invest Ophthalmol Vis Sci Date: 2002-04 Impact factor: 4.799
Authors: Jürgen Soutschek; Akin Akinc; Birgit Bramlage; Klaus Charisse; Rainer Constien; Mary Donoghue; Sayda Elbashir; Anke Geick; Philipp Hadwiger; Jens Harborth; Matthias John; Venkitasamy Kesavan; Gary Lavine; Rajendra K Pandey; Timothy Racie; Kallanthottathil G Rajeev; Ingo Röhl; Ivanka Toudjarska; Gang Wang; Silvio Wuschko; David Bumcrot; Victor Koteliansky; Stefan Limmer; Muthiah Manoharan; Hans-Peter Vornlocher Journal: Nature Date: 2004-11-11 Impact factor: 49.962
Authors: Sancy A Leachman; Robyn P Hickerson; Mary E Schwartz; Emily E Bullough; Stephen L Hutcherson; Kenneth M Boucher; C David Hansen; Mark J Eliason; G Susan Srivatsa; Douglas J Kornbrust; Frances Jd Smith; Wh Irwin McLean; Leonard M Milstone; Roger L Kaspar Journal: Mol Ther Date: 2009-11-24 Impact factor: 11.454
Authors: Robyn P Hickerson; Frances J D Smith; Robert E Reeves; Christopher H Contag; Devin Leake; Sancy A Leachman; Leonard M Milstone; W H Irwin McLean; Roger L Kaspar Journal: J Invest Dermatol Date: 2007-10-11 Impact factor: 8.551
Authors: Raymond Kwan; Lu Chen; Koksun Looi; Guo-Zhong Tao; Sujith V Weerasinghe; Natasha T Snider; Mary Anne Conti; Robert S Adelstein; Qing Xie; M Bishr Omary Journal: Hepatology Date: 2015-08-25 Impact factor: 17.425
Authors: Robyn P Hickerson; Tycho J Speaker; Maria Fernanda Lara; Emilio González-González; Manuel A Flores; Christopher H Contag; Roger L Kaspar Journal: Mol Imaging Biol Date: 2016-02 Impact factor: 3.488
Authors: Jingyuan Sun; Vincent E Groppi; Honglian Gui; Lu Chen; Qing Xie; Li Liu; M Bishr Omary Journal: Methods Enzymol Date: 2015-11-19 Impact factor: 1.600
Authors: Edwin H A Allen; David G Courtney; Sarah D Atkinson; Johnny E Moore; Laura Mairs; Ebbe Toftgaard Poulsen; Davide Schiroli; Eleonora Maurizi; Christian Cole; Robyn P Hickerson; John James; Helen Murgatroyd; Frances J D Smith; Carrie MacEwen; Jan J Enghild; M Andrew Nesbit; Deena M Leslie Pedrioli; W H Irwin McLean; C B Tara Moore Journal: Hum Mol Genet Date: 2016-01-11 Impact factor: 6.150
Authors: Judy L Chen; Benjamin R Lin; Katherine M Gee; Jessica A Gee; Duk-Won D Chung; Ricardo F Frausto; Sophie X Deng; Anthony J Aldave Journal: Mol Vis Date: 2015-12-31 Impact factor: 2.367
Authors: Kathleen A Christie; David G Courtney; Larry A DeDionisio; Connie Chao Shern; Shyamasree De Majumdar; Laura C Mairs; M Andrew Nesbit; C B Tara Moore Journal: Sci Rep Date: 2017-11-23 Impact factor: 4.379
Authors: Dun Jack Fu; Edwin H A Allen; Robyn P Hickerson; Deena M Leslie Pedrioli; W H Irwin McLean Journal: Transl Vis Sci Technol Date: 2020-12-29 Impact factor: 3.283
Authors: F Novelli; A M Lena; E Panatta; W Nasser; R Shalom-Feuerstein; E Candi; G Melino Journal: Cell Death Dis Date: 2016-05-19 Impact factor: 8.469
Authors: D G Courtney; J E Moore; S D Atkinson; E Maurizi; E H A Allen; D M L Pedrioli; W H I McLean; M A Nesbit; C B T Moore Journal: Gene Ther Date: 2015-08-20 Impact factor: 5.250