title: Correction of Mutant p63 in EEC Syndrome Using siRNA Mediated Allele-Specific Silencing Restores Defective Stem Cell Function.
abstract: 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-ΔNp63α-RFP and R279H-ΔNp63α-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. Stem Cells 2016;34:1588-1600.
authors.affiliation: Research Centre, Fondazione Banca degli Occhi del Veneto, 30174, Venice, Italy.
authors.forename: Vanessa
authors.initials: V
authors.lastname: Barbaro
authors.affiliation: Department of Molecular Medicine, University of Padua, 35121, Padua, Italy.
authors.forename: Annamaria A
authors.initials: AA
authors.lastname: Nasti
authors.affiliation: Department of Molecular Medicine, University of Padua, 35121, Padua, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Del Vecchio
authors.affiliation: Research Centre, Fondazione Banca degli Occhi del Veneto, 30174, Venice, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Ferrari
authors.affiliation: Department of Molecular Medicine, University of Padua, 35121, Padua, Italy.
authors.forename: Angelo
authors.initials: A
authors.lastname: Migliorati
authors.affiliation: Department of Molecular Medicine, University of Padua, 35121, Padua, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Raffa
authors.affiliation: Department of Biomedical Sciences and Public Health, School of Medicine, University "Politecnica delle Marche", 60120, Ancona, Italy.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Lariccia
authors.affiliation: Department of Molecular Medicine, University of Padua, 35121, Padua, Italy.
authors.forename: Patrizia
authors.initials: P
authors.lastname: Nespeca
authors.affiliation: Department of Molecular Medicine, University of Padua, 35121, Padua, Italy.
authors.forename: Mariangela
authors.initials: M
authors.lastname: Biasolo
authors.affiliation: Department of Eye and Vision Science, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, United Kingdom.
authors.forename: Colin E
authors.initials: CE
authors.lastname: Willoughby
authors.affiliation: Research Centre, Fondazione Banca degli Occhi del Veneto, 30174, Venice, Italy.
authors.forename: Diego
authors.initials: D
authors.lastname: Ponzin
authors.affiliation: Department of Molecular Medicine, University of Padua, 35121, Padua, Italy.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Palù
authors.affiliation: Department of Molecular Medicine, University of Padua, 35121, Padua, Italy.
authors.forename: Cristina
authors.initials: C
authors.lastname: Parolin
authors.affiliation: Research Centre, Fondazione Banca degli Occhi del Veneto, 30174, Venice, Italy.|Department of Molecular Medicine, University of Padua, 35121, Padua, Italy.
authors.forename: Enzo
authors.initials: E
authors.lastname: Di Iorio
mesh_terms: D000293:Adolescent; D000375:Aging / Q000473:pathology; D000483:Alleles; D059447:Cell Cycle Checkpoints; D002454:Cell Differentiation; D049109:Cell Proliferation; D000066673:Cell Self Renewal; D002971:Cleft Lip / Q000235:genetics*; D002972:Cleft Palate / Q000235:genetics*; D002999:Clone Cells; D004476:Ectodermal Dysplasia / Q000235:genetics*; D004847:Epithelial Cells / Q000473:pathology; D020868:Gene Silencing; D057809:HEK293 Cells; D006801:Humans; D016850:Limbus Corneae / Q000473:pathology; D008954:Models, Biological; D009061:Mouth Mucosa / Q000473:pathology; D009154:Mutation / Q000235:genetics*; D009841:Oligonucleotides / Q000378:metabolism; D010641:Phenotype; D034741:RNA, Small Interfering / Q000378:metabolism*; D013234:Stem Cells / Q000378:metabolism*; D014019:Tissue Donors; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000235:genetics* / Q000378:metabolism; D055815:Young Adult
chemical_list: D009841:Oligonucleotides; D034741:RNA, Small Interfering; C115051:TP63 protein, human; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C477371:locked nucleic acid
keywords: Ectrodactyly-ectodermal dysplasia-clefting syndrome; Epithelial stem cells; Gene therapy; p63; small interfering RNAs