Abstract
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.
Highlights
The transcription factor p63, encoded by TP63 gene, part of the p53 gene family, is a master gene for the commitment of ectodermal-derived tissues
For screening of small interfering RNAs (siRNAs) molecules blocking ΔNp63-R304W expression with little or no effect on ΔNp63-WT, we used two kinds of artificial report constructs[23] harboring Renilla luciferase or Photinus luciferase and 50-bp siRNA-targeted sequences with/without C4T substitution in their 3’-UTRs (Figures 1a-c)
Each siRNA molecule was investigated for its ability to suppress luciferase activities of ΔNp63-R304W mutated constructs significantly compared with those of ΔNp63-WT ones by transient transfection in HEK-293E epithelial cells
Summary
The transcription factor p63, encoded by TP63 gene, part of the p53 gene family, is a master gene for the commitment of ectodermal-derived tissues. The five missense mutations affect arginine residues (R204, R227, R279, R280 and R304) and represent hotspot mutations for EEC.[1] These mutants, to other p63 mutants, are translated into proteins with enhanced stability,[22] maintain the ability to bind DNA but, being transcriptionally deficient themselves and by inhibiting transactivation by the residual p63 wild-type allele, have a strong dominant-negative effect.[1,9,22] The lack of therapies and the presence of hotspot mutants in EEC patients, lead us to explore the possibility that using.
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