Activities per year
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.
- allele discrimination
- lattice corneal dystrophy
- RNA interference
Development of allele specific gene silencing siRNAs for TGFBI Arg124Cys in Lattice Corneal Dystrophy Type I
Tara Moore (Speaker)
Activity: Talk or presentation › Oral presentation
Development of allele specific gene silencing siRNAs for TGFBI Arg124Cys in Lattice Corneal Dystrophy Type 1
Tara Moore (Invited speaker)
Activity: Talk or presentation › Invited talk
Research Output per year
Development of allele specific gene silencing siRNAs for TGFBI Arg124Cys in Lattice Corneal Dystrophy Type I.Courtney, D. G., Atkinson, S. D., Moore, J. E., Maurizi, E., Pellegrini, G., Black, G., Manson, F., Hin-Fai Yam, G., McLean, W. H. I. & Moore, C. B. T., 2014, Unknown Host Publication. 2 ed. Association for Research in Vision and Ophthalmology, Vol. 55. p. 977-985 8 p.
Research output: Chapter in Book/Report/Conference proceeding › Conference contribution