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首页> 外文期刊>Investigative ophthalmology & visual science >SiRNA silencing of the mutant keratin 12 allele in corneal limbal epithelial cells grown from patients with Meesmann's epithelial corneal dystrophy
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SiRNA silencing of the mutant keratin 12 allele in corneal limbal epithelial cells grown from patients with Meesmann's epithelial corneal dystrophy

机译：Meesmann上皮角膜营养不良患者角膜缘上皮细胞中突变型角蛋白12等位基因的SiRNA沉默

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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.
机译：目的。这项研究的目的是进一步评估我们先前报道的角蛋白12（K12）-Leu132Pro特异的siRNA，该基因在实验系统中通过类似于梅斯曼上皮角膜营养不良症（MECD）的突变等位基因沉默，通过同时表达两种野生型来更类似于体内情况型和突变等位基因。方法。使用转染到细胞中的KRT12外源表达构建体，通过焦磷酸测序和红外Western印迹分析对突变体等位基因特异性敲低进行定量，同时通过改良的cDNA末端快速扩增（5'RACE）方法评估沉默机制。取自患有MECD的患者的角膜缘活检样本用于建立MECD角膜缘上皮干细胞的培养，并通过焦磷酸测序，qPCR，ELISA和定量荧光检测评估siRNA沉默内源突变KRT12等位基因的能力。免疫组化（Q-FIHC）。结果。 siRNA在外源表达构建体的mRNA和蛋白质水平上均显示出有力且特异性的K12-Leu132Pro敲低。通过5'RACE方法的分析证实了siRNA介导的切割。在MECD细胞中，观察到内源突变型等位基因的等位基因特异性敲低63％，而对野生型等位基因表达没有影响。结论。结合有效的递送载体，这种siRNA方法代表了一种预防在K12-Leu132Pro杂合子个体中观察到的MECD病理的可行治疗选择。

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