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Enzo Di Iorio, Stefano Ferrari, Diego Ponzin, Colin E. Willoughby, Elisabetta Bohm, Vanessa Barbaro; Gene Therapy Approach Through siRNA Against p63 To Correct Ocular Surface Disorders In Ectrodactyly–Ectodermal Dysplasia–Clefting (EEC) Syndrome. Invest. Ophthalmol. Vis. Sci. 2011;52(14):487.
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To describe the ocular phenotype in patients with EEC syndrome, to determine the pathogenic basis of visual morbidity and finally to investigate gene silencing approaches through allele specific siRNA, in order to assess new therapeutic approaches based on transplantation of genetically corrected autologous corneal limbal stem cells.
Genetic and ophthalmologic analyses of 19 families consisting of 25 patients with EEC syndrome were assessed. All patients with EEC syndrome resulting from p63 mutations had a complete ophthalmic examination and ocular surface assessment. Limbal stem cell deficiency was diagnosed clinically on the basis of corneal conjunctivalisation, anatomy of the limbal palisades of Vogt, and immunohistochemical analysis performed on both pannus explant and impression cytology specimens. Keratinocytes stem cells from EEC patients carrying the R279H and the R304Q mutations have been analysed to obtain informations about their proliferative potential. Gene silencing approaches have been investigated through siRNA against R279H–p63 mutant.
All EEC patients had ocular involvement and the major cause of visual morbidity was limbal stem cell deficiency (LSCD) which was seen in 13/25 (52%) of cases. Keratinocytes stem cells from EEC patients carrying the R279H and the R304Q mutations showed a rapid decrease of colony forming efficiency (CFE) and clonogenic cell number and an increased replicative senescence. In vitro organotypic cultures showed defects in EEC epithelial stem cell stratification. Allele-specific siRNA against R279H–p63 mutant provided interesting results in order to assess new therapeutic approaches based on transplantation of genetically corrected autologous corneal limbal stem cells.
Our study confirms that the major cause of visual morbidity in EEC syndrome is limbal stem cell failure, resulting in a vascularised corneal pannus, leading to visual loss. Mutations in p63 gene severly compromise the ability of stem cells to give rise to a full tickness stratified and differentiated corneal tissue. The only effective management for EEC patients would therefore be to restore the limbus by grafting cultured limbal stem cells following correction of the p63 gene defect.
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