Correction of Mutant p63 in EEC Syndrome Using siRNA Mediated Allele-Specific Silencing Restores Defective Stem Cell Function

Vanessa Barbaro, Annamaria A. Nasti, Claudia Del Vecchio, Stefano Ferrari, Angelo Migliorati, Paolo Raffa, Vincenzo Lariccia, Patrizia Nespeca, Mariangela Biasolo, Colin Willoughby, Diego Ponzin, Giorgio Palù, Cristina Parolin, Enzo Di Iorio

    Research output: Contribution to journalArticle

    7 Citations (Scopus)

    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.
    LanguageEnglish
    Pages1588-1600
    JournalStem Cells
    Volume34
    Issue number6
    DOIs
    Publication statusAccepted/In press - 1 Jan 2016

    Fingerprint

    Small Interfering RNA
    Stem Cells
    Alleles
    Mouth Mucosa
    Epithelial Cells
    Ectodermal Dysplasia
    Blindness
    Ectrodactyly-cleft lip-palate syndrome
    Cell Line
    Corneal Epithelium
    Mutation
    Therapeutics
    Cell- and Tissue-Based Therapy
    Genetic Therapy
    Cell Differentiation
    Extremities
    Epithelium
    Genes

    Keywords

    • Ectrodactyly-ectodermal dysplasia-clefting syndrome
    • Epithelial stem cells
    • Gene therapy
    • p63
    • small interfering RNAs

    Cite this

    Barbaro, V., Nasti, A. A., Del Vecchio, C., Ferrari, S., Migliorati, A., Raffa, P., ... Di Iorio, E. (Accepted/In press). Correction of Mutant p63 in EEC Syndrome Using siRNA Mediated Allele-Specific Silencing Restores Defective Stem Cell Function. Stem Cells, 34(6), 1588-1600. https://doi.org/10.1002/stem.2343
    Barbaro, Vanessa ; Nasti, Annamaria A. ; Del Vecchio, Claudia ; Ferrari, Stefano ; Migliorati, Angelo ; Raffa, Paolo ; Lariccia, Vincenzo ; Nespeca, Patrizia ; Biasolo, Mariangela ; Willoughby, Colin ; Ponzin, Diego ; Palù, Giorgio ; Parolin, Cristina ; Di Iorio, Enzo. / Correction of Mutant p63 in EEC Syndrome Using siRNA Mediated Allele-Specific Silencing Restores Defective Stem Cell Function. In: Stem Cells. 2016 ; Vol. 34, No. 6. pp. 1588-1600.
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    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.",
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    author = "Vanessa Barbaro and Nasti, {Annamaria A.} and {Del Vecchio}, Claudia and Stefano Ferrari and Angelo Migliorati and Paolo Raffa and Vincenzo Lariccia and Patrizia Nespeca and Mariangela Biasolo and Colin Willoughby and Diego Ponzin and Giorgio Pal{\`u} and Cristina Parolin and {Di Iorio}, Enzo",
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    Barbaro, V, Nasti, AA, Del Vecchio, C, Ferrari, S, Migliorati, A, Raffa, P, Lariccia, V, Nespeca, P, Biasolo, M, Willoughby, C, Ponzin, D, Palù, G, Parolin, C & Di Iorio, E 2016, 'Correction of Mutant p63 in EEC Syndrome Using siRNA Mediated Allele-Specific Silencing Restores Defective Stem Cell Function', Stem Cells, vol. 34, no. 6, pp. 1588-1600. https://doi.org/10.1002/stem.2343

    Correction of Mutant p63 in EEC Syndrome Using siRNA Mediated Allele-Specific Silencing Restores Defective Stem Cell Function. / Barbaro, Vanessa; Nasti, Annamaria A.; Del Vecchio, Claudia; Ferrari, Stefano; Migliorati, Angelo; Raffa, Paolo; Lariccia, Vincenzo; Nespeca, Patrizia; Biasolo, Mariangela; Willoughby, Colin; Ponzin, Diego; Palù, Giorgio; Parolin, Cristina; Di Iorio, Enzo.

    In: Stem Cells, Vol. 34, No. 6, 01.01.2016, p. 1588-1600.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Correction of Mutant p63 in EEC Syndrome Using siRNA Mediated Allele-Specific Silencing Restores Defective Stem Cell Function

    AU - Barbaro, Vanessa

    AU - Nasti, Annamaria A.

    AU - Del Vecchio, Claudia

    AU - Ferrari, Stefano

    AU - Migliorati, Angelo

    AU - Raffa, Paolo

    AU - Lariccia, Vincenzo

    AU - Nespeca, Patrizia

    AU - Biasolo, Mariangela

    AU - Willoughby, Colin

    AU - Ponzin, Diego

    AU - Palù, Giorgio

    AU - Parolin, Cristina

    AU - Di Iorio, Enzo

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    KW - Epithelial stem cells

    KW - Gene therapy

    KW - p63

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