Correction of Mutant p63 in EEC Syndrome Using siRNA Mediated Allele-Specific Silencing Restores Defective Stem Cell Function
Correction of Mutant p63 in EEC Syndrome Using siRNA Mediated Allele-Specific Silencing Restores Defective Stem Cell Function
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DOI：
10.1002/stem.2343
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发表时间：
2016-06-01
期刊：
影响因子：
5.2
通讯作者：
Di Iorio, Enzo
中科院分区：
文献类型：
--
作者：
Barbaro, Vanessa;Nasti, Annamaria A.;Di Iorio, Enzo
缺指-外胚层发育不良-裂（EEC）综合征是一种罕见的常染色体显性遗传疾病，由p63基因杂合突变引起，以肢体缺陷、口面裂、外胚层发育不良和眼部缺陷为特征。患者发展为进行性双侧角膜缘干细胞缺乏，最终导致角膜盲。内科和外科治疗无效，效果有限。口腔粘膜上皮干细胞（OMESCs）是一种能够再生角膜上皮的干细胞的替代来源，与基因治疗相结合，可以提供一种有吸引力的治疗途径。对来自携带最严重p63突变（p.R279H和p.R304Q）的EEC患者的OMESC进行表征，并使用等位基因特异性（AS）小干扰RNA（siRNA）沉默p.R279H的遗传缺陷。在（i）稳定的WT-Δ Np 63 α-RFP和R279 H-Delta Np 63 α-EGFP细胞系、（ii）瞬时双转染细胞系和（iii）p.R279 H OMESC中针对R279 H-p63等位基因的锁核酸（LNA）-siRNA的系统筛选，鉴定了许多针对突变等位基因的有效siRNA抑制剂，其对野生型p63没有影响。此外，与对照相比，siRNA处理导致突变干细胞的获得寿命更长，体外干细胞分化加速更少，增殖特性降低，以及纠正上皮发育不全的有效能力，从而产生全厚度分层和分化的上皮细胞。本研究证明了突变干细胞（OMESC）在EEC综合征的表型校正的siRNA介导的AS沉默与功能的恢复。siRNA单独或与基于细胞的疗法组合的应用为EEC综合征中的角膜盲提供了治疗策略。
Ectrodactyly-Ectodermal dysplasia-Clefting (EEC) syndrome is a rare autosomal dominant disease caused by heterozygous mutations in the p63 gene and characterized by limb defects, orofacial clefting, ectodermal dysplasia, and ocular defects. Patients develop progressive total bilateral limbal stem cell deficiency, which eventually results in corneal blindness. Medical and surgical treatments are ineffective and of limited benefit. Oral mucosa epithelial stem cells (OMESCs) represent an alternative source of stem cells capable of regenerating the corneal epithelium and, combined with gene therapy, could provide an attractive therapeutic avenue. OMESCs from EEC patients carrying the most severe p63 mutations (p.R279H and p.R304Q) were characterized and the genetic defect of p.R279H silenced using allele-specific (AS) small interfering RNAs (siRNAs). Systematic screening of locked nucleic acid (LNA)-siRNAs against R279H-p63 allele in (i) stable WT-Delta Np63 alpha-RFP and R279H-Delta Np63 alpha-EGFP cell lines, (ii) transient doubly transfected cell lines, and (iii) p.R279H OMESCs, identified a number of potent siRNA inhibitors for the mutant allele, which had no effect on wild-type p63. In addition, siRNA treatment led to longer acquired life span of mutated stem cells compared to controls, less accelerated stem cell differentiation in vitro, reduced proliferation properties, and effective ability in correcting the epithelial hypoplasia, thus giving rise to full thickness stratified and differentiated epithelia. This study demonstrates the phenotypic correction of mutant stem cells (OMESCs) in EEC syndrome by means of siRNA mediated AS silencing with restoration of function. The application of siRNA, alone or in combination with cell-based therapies, offers a therapeutic strategy for corneal blindness in EEC syndrome.