Single-nucleotide-specific siRNA targeting in a dominant-negative skin model
Single-nucleotide-specific siRNA targeting in a dominant-negative skin model
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DOI：
10.1038/sj.jid.5701060
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发表时间：
2008-03-01
影响因子：
6.5
通讯作者：
Kaspar, Roger L.
中科院分区：
文献类型：
--
作者：
Hickerson, Robyn P.;Smith, Frances J. D.;Kaspar, Roger L.
RNA干扰为开发治疗显性阴性遗传疾病的新方法提供了新的途径。抑制突变等位基因表达而不影响野生型基因表达的能力可能是一种强有力的新治疗选择。针对罕见的单基因皮肤疾病先天性厚甲癣（PC）的单核角蛋白6a (K6a) N171K突变，我们证明了小干扰rna （sirna）可以有效和选择性地阻断突变K6a的表达。为了测试在野生型和突变型存在的情况下，铅sirna是否能区分突变mRNA，建立了一个显性阴性的PC细胞培养模型。正如对显性阴性疾病的预测，同时表达野生型和突变型K6a导致角蛋白丝形成缺陷。添加突变体特异性sirna允许正常的丝形成，表明选择性抑制突变体K6a。我们的siRNA在皮肤中的有效性通过将萤火虫荧光素酶/突变K6a双电报告结构和突变特异性siRNA共同递送到小鼠脚掌来测试。利用Xenogen IVIS200体内成像系统证明了对荧光报告基因的有效抑制作用。此外，野生型特异性sirna敲低了人角质形成细胞中预先存在的内源性K6a的表达。这些结果表明，这些“设计sirna”的有效递送可能允许有效治疗包括PC在内的许多遗传疾病。
RNA interference offers a novel approach for developing therapeutics for dominant-negative genetic disorders. The ability to inhibit expression of the mutant allele without affecting wild-type gene expression could be a powerful new treatment option. Targeting the single-nucleoticle keratin 6a (K6a) N171K mutation responsible for the rare monogenic skin disorder pachyonychia congenita (PC), we demonstrate that small interfering RNAs (siRNAs) can potently and selectively block expression of mutant K6a. To test whether lead siRNAs could discriminate mutant mRNA in the presence of both wild-type and mutant forms, a dominant-negative PC cell culture model was developed. As predicted for a dominant-negative disease, simultaneous expression of both wild-type and mutant K6a resulted in defective keratin filament formation. Addition of mutant-specific siRNAs allowed normal filament formation, suggesting selective inhibition of mutant K6a. The effectiveness of our siRNA in skin was tested by co-delivering a firefly luciferase/mutant K6a bicistronic reporter construct and mutant-specific siRNAs to mouse footpads. Potent inhibition of the fluorescent reporter was demonstrated using the Xenogen IVIS200 in vivo imaging system. Additionally, wild type-specific siRNAs knocked down the expression of pre-existing endogenous K6a in human keratinocytes. These results suggest that efficient delivery of these "designer siRNAs" may allow effective treatment of numerous genetic disorders including PC.