表皮松解性掌跖角化症个性化靶向治疗siRNA分子的系统性筛选及高效穿皮给药方式的探讨

Epidermolytic palmoplantar keratoderma (EPPK) is a relative common autosomal dominant inherited birth defect that often includes disabling diffuse hyperkeratosis of the palms and soles for which no satisfactory treatment is currently available. Onset is at birth or soon after. EPPK is caused by mutations in keratin 9 (KRT9) gene that act by a dominant-negative interference to cause the disease symptoms. To date, a handful of potentially therapeutic short interfering RNAs (siRNAs) have been identified for several keratinizing disorders, demonstrated that RNAi-based therapeutics are well suited for these disorders. This, along with the fact that keratin 9 is expressed in a very specific manner limited to terminally differentiated epidermal keratinocytes of palmoplantar skin, makes it an ideal model skin disease to develop gene-specific, as well as mutation-specific, siRNA therapies. Based on our previous researches both on molecular and mouse model, we are planning to develop therapeutic siRNA targeting the related pathogenic mutations in EPPK. Allele-specific inhibitors will be systematically identified in vitro by a luciferase reporter gene assay and validated by a dual-Flag/Strep-TagII quantitative immunoblot assay. Delivery in vivo is the major limitation in applying siRNA technology to skin diseases. Thus, transdermal delivery of siRNA-based spherical nucleic acid nanoparticle conjugates and a loadable, dissolvable protrusion array device (PAD)-mediated delivery of Accell-modified-siRNA will be investigated both in vitro and in vivo (including our established krt9-mutant knock-in mouse model).

表皮松解性掌跖角化症（EPPK）是相对常见的致残性常染色体显性遗传性皮肤病。患者出生后不久即有表征，持续终生。该出生缺陷尚无有效的治疗手段。已知表达于人皮肤表皮角质形成细胞的角蛋白9基因（KRT9）是EPPK的主要致病基因。本项目用荧光素报告基因检测和双标签定量免疫印迹实验等，对已知的相关KRT9突变类型开展高效特异性抑制突变等位基因的siRNA分子的系统性筛选及验证，并通过带Accell修饰联合可降解微针阵列皮下注射和纳米金颗粒附着联合乳膏局部涂抹给药途径，对靶向siRNA治疗进行药效评估及毒理分析。此外，拟在已自主建立的小鼠krt9基因突变敲入模型上，开展靶向siRNA治疗小鼠EPPK表型的疗效分析。本项目有望初步找到一种高效可行的EPPK个性化基因治疗方案，为后续深入研究和后续临床用药治疗等研究奠定基础。若获得成功，也必将为其他单基因遗传性皮肤病的基因治疗研究提供思路和借鉴。

表皮松解性掌跖角化症（EPPK）是相对常见的致残性常染色体显性遗传性皮肤病。患者出生后不久即有表征，持续终生。该出生缺陷尚无有效的治疗手段。已知表达于人皮肤表皮角质形成细胞的角蛋白9基因（KRT9）是EPPK的主要致病基因。本项目通过构建与EPPK患者KRT9突变c.500delAinsGGCT同源性的小鼠Krt9基因c.434delAinsGGCT突变载体，利用同源重组原理基因打靶小鼠胚胎干细胞-囊胚注射后，移植到假孕鼠子宫获得嵌合鼠的出生。通过嵌合鼠与野生型母鼠的配繁，筛选得到了稳定遗传的Krt9基因突变敲入小鼠。表型分析、组织学检测、蛋白印迹实验和免疫组化分析等均已证实了EPPK小鼠的表型。我们以p.Y167delinsWL小插缺突变为靶向治疗siRNA递送方式的研究对象，设计了16条siRNA分子，意在筛选出一个功能性和特异性都极高的siRNA分子，只knockdown突变的K9分子，而对正常K9无影响或影响极小。我们选取了pCDH质粒，构入luciferase之后，再分别连接入小鼠K9-wt（野生型）、K9-434 mutation（突变型）进行慢病毒包裹，形成稳定的表达细胞系。在阴性对照组中，加入NSF4后，两组细胞系的荧光表达无明显差异；而在阳性对照组中，加入抑制luciferase的siRNA后，两组细胞系的荧光表达均出现了下降。在对转染后除K9以外的其他角蛋白分子进行Western杂交检测后发现，K10、K6、K5、K2e和K1在siRNA8、siRNA9、siRNA14转染细胞系中的表达差异均无统计学差异，表明这3条siRNA的功能性和特异性相对最好。这为后续进一步的小鼠透皮给药研究奠定了基础。

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