title: Long-term engraftment of single genetically modified human epidermal holoclones enables safety pre-assessment of cutaneous gene therapy.
abstract: Predicting the risks of permanent gene therapy approaches involving the use of integrative gene-targeting vectors has become a critical issue after the unfortunate episode of a clinical trial in children with X-linked severe combined immunodeficiency (X-SCID). Safety pre-assessment of single isolated gene-targeted stem cells or their derivative clones able to regenerate their tissue of origin would be a major asset in addressing untoward gene therapy effects in advance. Human epidermal stem cells, which have extensive proliferative potential in vitro, theoretically offer such a possibility as a method of assessment. By means of optimized organotypic culture and grafting methods, we demonstrate the long-term in vivo regenerative capacity of single gene-targeted human epidermal stem cell clones (holoclones). Both histopathological analysis of holoclone-derived grafts in immunodeficient mice and retroviral insertion site mapping performed in the holoclone in vitro and after grafting provide proof of the feasibility of pre-assessing genotoxicity risks in isolated stem cells before transplantation into patients. Our results provide an experimental basis for previously untested assumptions about the in vivo behavior of epidermal stem cells prospectively isolated in vitro and pave the way for a safer approach to cutaneous gene therapy.
authors.affiliation: Epithelial Biomedicine Division, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas, Centro de Investigación Biomédica en Red de Enfermedades, Raras, Madrid, Spain. fernando.larcher@ciemat.es
authors.forename: Fernando
authors.initials: F
authors.lastname: Larcher
authors.forename: Elena
authors.initials: E
authors.lastname: Dellambra
authors.forename: Laura
authors.initials: L
authors.lastname: Rico
authors.forename: Sergio
authors.initials: S
authors.lastname: Bondanza
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Murillas
authors.forename: Claudia
authors.initials: C
authors.lastname: Cattoglio
authors.forename: Fulvio
authors.initials: F
authors.lastname: Mavilio
authors.forename: José L
authors.initials: JL
authors.lastname: Jorcano
authors.forename: Giovanna
authors.initials: G
authors.lastname: Zambruno
authors.forename: Marcela
authors.initials: M
authors.lastname: Del Rio
mesh_terms: D000818:Animals; D043171:Chromosomal Instability / Q000235:genetics; D004817:Epidermis / Q000378:metabolism*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000009:adverse effects / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007621:Karyotyping; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism; D051379:Mice; D016513:Mice, SCID; D012038:Regeneration; D012867:Skin / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D016038:Skin Transplantation / Q000379:methods; D033581:Stem Cell Transplantation; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D014183:Transplantation, Heterologous
chemical_list: D049452:Green Fluorescent Proteins