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• W2000007632 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." @default.
• W2000007632 created "2016-06-24" @default.
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• W2000007632 date "2014-05-29" @default.
• W2000007632 modified "2023-10-17" @default.
• W2000007632 title "siRNA Silencing of the Mutant Keratin 12 Allele in Corneal Limbal Epithelial Cells Grown From Patients With Meesmann's Epithelial Corneal Dystrophy" @default.
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• W2000007632 doi "https://doi.org/10.1167/iovs.13-12957" @default.
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