Allele-specific silencing of EEC p63 mutant R304W restores p63 transcriptional activity.
Allele-specific silencing of EEC p63 mutant R304W restores p63 transcriptional activity.
复制标题
DOI：
10.1038/cddis.2016.118
复制
发表时间：
2016-05-19
影响因子：
9
通讯作者：
Melino G
中科院分区：
文献类型：
--
作者：
Novelli F;Lena AM;Panatta E;Nasser W;Shalom-Feuerstein R;Candi E;Melino G
EEC（ectrodactyly-ectodermal dysplasia and cleft lip/palate）综合征是一种罕见的常染色体显性遗传疾病。它是由TP 63基因杂合突变引起的外胚层发育不良疾病的一部分。EEC患者存在肢体畸形、口面裂、皮肤和皮肤附件缺陷、眼部异常。由TP 63编码的转录因子p63是用于外胚层衍生组织定型的主基因，其在顶端外胚层嵴中表达对于脊椎动物肢体形成以及在后期阶段对于皮肤和皮肤附属物发育至关重要。Δ Np 63 α亚型主要在上皮细胞中表达，对于保持成体干细胞的自我更新能力和参与特定的上皮分化程序是必不可少的。小干扰RNA（siRNA）通过选择性沉默突变等位基因为EEC患者提供了一种潜在的治疗方法。在这里，使用基于双荧光素酶报告基因测定的系统筛选，我们已经成功地鉴定了用于抑制引起EEC的p63突变体R304 W的特异性siRNA。在siRNA处理后，我们能够在源自EEC患者活检的诱导多能干细胞中恢复Δ Np 63-WT等位基因转录功能。这项研究表明，siRNA方法是有前途的，可能为治愈/延缓年轻EEC患者的主要症状，如角膜变性和皮肤糜烂铺平道路。
EEC (ectrodactily-ectodermal dysplasia and cleft lip/palate) syndrome is a rare genetic disease, autosomal dominant inherited. It is part of the ectodermal dysplasia disorders caused by heterozygous mutations in TP63 gene. EEC patients present limb malformations, orofacial clefting, skin and skin's appendages defects, ocular abnormalities. The transcription factor p63, encoded by TP63, is a master gene for the commitment of ectodermal-derived tissues, being expressed in the apical ectodermal ridge is critical for vertebrate limb formation and, at a later stage, for skin and skin's appendages development. The ΔNp63α isoform is predominantly expressed in epithelial cells and it is indispensable for preserving the self-renewal capacity of adult stem cells and to engage specific epithelial differentiation programs. Small interfering RNA (siRNA) offers a potential therapy approach for EEC patients by selectively silencing the mutant allele. Here, using a systemic screening based on a dual-luciferase reported gene assay, we have successfully identified specific siRNAs for repressing the EEC-causing p63 mutant, R304W. Upon siRNA treatment, we were able to restore ΔNp63-WT allele transcriptional function in induced pluripotent stem cells that were derived from EEC patient biopsy. This study demonstrates that siRNAs approach is promising and, may pave the way for curing/delaying major symptoms, such as cornea degeneration and skin erosions in young EEC patients.