Safe selection of genetically manipulated human primary keratinocytes with very high growth potential using CD24

Stable and safe corrective gene transfer in stem keratinocytes is necessary for ensuring success in cutaneous gene therapy. There have been numerous encouraging preclinical approaches to cutaneous gene therapy in the past decade, but it is only recently that a human volunteer suffering from junctional epidermolysis bullosa could be successfully grafted using his own non-selected, genetically corrected epidermal keratinocytes. However, ex vivo correction of cancer-prone genetic disorders necessitates a totally pure population of stably transduced stem keratinocytes for grafting. Antibiotic selection is not compatible with the need for full respect for natural cell fate potential and avoidance of immunogenic response in vivo. In order to surmount these problems, we developed a strategy for selecting genetically modified stem cell keratinocytes. Driving ectopic expression of CD24 (a marker of post-mitotic keratinocytes) at the surface of clonogenic keratinocytes permitted their full selection. Engineered keratinocytes expressing CD24 and the green fluorescent protein (GFP) tracer gene were shown to retain their original growth and differentiation potentials both in vitro and in vivo over 300 generations. Also, they did not exhibit signs of genetic instability. Using ectopic expression of CD24 as a selective marker of genetically modified human epidermal stem cells appears to be the first realistic approach to safe cutaneous gene therapy in cancer-prone disease conditions.We are indebted to Françoise Bernerd (L'Oréal Advanced Research, Clichy, France) and Mathilde Frechet (Centre National de la Recherche Scientifique (CNRS), FRE2939, Villejuif, France) for their expert help with organotypic skin cultures. We thank Yann Lecluse (Institut Gustave Roussy, Villejuif, France) for his expert help with flow cytometry. Françoise Viala (CNRS, Toulouse, France) is gratefully acknowledged for excellent artwork contribution. We thank Claire Marionnet (L'Oréal Advanced Research, Clichy, France) for kindly helping us with statistical analysis and Mandy Schwint for kindly editing the manuscript. Gim Meneguzzi (Institut National de la Santé et de la Recherche Médicale, U634, Nice, France) is acknowledged for the generous gift of the GB3 anti-laminin 5 antibody. James R. Rheinwald and Howard Green (Harvard, Women';s Hospital, Boston, MA) are gratefully acknowledged for the generous gift of 3T3-J2 cells. We thank the Production and Control department of Genethon which is supported by the Association Française contre les Myopathie, within the Gene Vector Production Network (http://www.gvpn.org). This work was supported by funds from CNRS and Centro de Investigación Biomedica en Red de Enfermedades Raras, Spain, and grants SAF-2004-07717 to M.D.R. and FIS OI051577 to F.L. T.M. gratefully acknowledges funding from the Association pour la Recherche sur le Cancer (No. 3590), the Fondation de l'Avenir, the Société Française de Dermatologie, and the Association Française contre les Myopathies

Preclinical corrective gene transfer in Xeroderma pigmentosum human skin stem cells

Xeroderma pigmentosum (XP) is a devastating disease associated with dramatic skin cancer proneness. XP cells are deficient in nucleotide excision repair (NER) of bulky DNA adducts including ultraviolet (UV)-induced mutagenic lesions. Approaches of corrective gene transfer in NER-deficient keratinocyte stem cells hold great hope for the long-term treatment of XP patients. To face this challenge, we developed a retrovirus-based strategy to safely transduce the wild-type XPC gene into clonogenic human primary XP-C keratinocytes. De novo expression of XPC was maintained in both mass population and derived independent candidate stem cells (holoclones) after more than 130 population doublings (PD) in culture upon serial propagation (> 10(40) cells). Analyses of retrovirus integration sequences in isolated keratinocyte stem cells suggested the absence of adverse effects such as oncogenic activation or clonal expansion. Furthermore, corrected XP-C keratinocytes exhibited full NER capacity as well as normal features of epidermal differentiation in both organotypic skin cultures and in a preclinical murine model of human skin regeneration in vivo. The achievement of a long-term genetic correction of XP-C epidermal stem cells constitutes the first preclinical model of ex vivo gene therapy for XP-C patients.F.L. was supported in part by grants PI081054 from ISCIII and PBIO-0306-2006 from Comunidad de Madrid (CAM). M.D.R. was supported by grant SAF2010-16976 from MICINN. The authors declared no conflict of interest

PTCH1+/− Dermal Fibroblasts Isolated from Healthy Skin of Gorlin Syndrome Patients Exhibit Features of Carcinoma Associated Fibroblasts

Gorlin's or nevoid basal cell carcinoma syndrome (NBCCS) causes predisposition to basal cell carcinoma (BCC), the commonest cancer in adult human. Mutations in the tumor suppressor gene PTCH1 are responsible for this autosomal dominant syndrome. In NBCCS patients, as in the general population, ultraviolet exposure is a major risk factor for BCC development. However these patients also develop BCCs in sun-protected areas of the skin, suggesting the existence of other mechanisms for BCC predisposition in NBCCS patients. As increasing evidence supports the idea that the stroma influences carcinoma development, we hypothesized that NBCCS fibroblasts could facilitate BCC occurence of the patients. WT (n = 3) and NBCCS fibroblasts bearing either nonsense (n = 3) or missense (n = 3) PTCH1 mutations were cultured in dermal equivalents made of a collagen matrix and their transcriptomes were compared by whole genome microarray analyses. Strikingly, NBCCS fibroblasts over-expressed mRNAs encoding pro-tumoral factors such as Matrix Metalloproteinases 1 and 3 and tenascin C. They also over-expressed mRNA of pro-proliferative diffusible factors such as fibroblast growth factor 7 and the stromal cell-derived factor 1 alpha, known for its expression in carcinoma associated fibroblasts. These data indicate that the PTCH1+/− genotype of healthy NBCCS fibroblasts results in phenotypic traits highly reminiscent of those of BCC associated fibroblasts, a clue to the yet mysterious proneness to non photo-exposed BCCs in NBCCS patients

Thérapie génique cutanée

International audienc

Long-term complementation of DNA repair deficient human primary fibroblasts by retroviral transduction of the XPD gene

International audienceDue to their limited life time in culture and their relative resistance to DNA transfection, primary fibroblasts derived from UV-hypersensitive patients could not be used for cloning DNA repair gene and studying stable complementation with wild-type DNA repair genes. Primary cells were only used for complementation analysis after transient expression through cell fusion. DNA microinjection and transfection. We report the retroviral-mediated highly efficient transfer and stable expression of XPD/ERCC2 gene in fibroblast strains from eight different patients using the LXPDSN retroviral vector. Cells derived from skin biopsies of xeroderma pigmentosum and trichothiodystrophy patients were incubated with vector-containing suspension and selected with the neomycin-analog G418. LXPDSN vector specifically complemented cells belonging to the XP-D group. Long-term reversion of repair-deficient phenotype, monitored by UV survival and UDS analysis, has been achieved in these diploid fibroblasts. We demonstrate this methodology is a powerful tool to study phenotypic reversion of nucleotide excision repair-deficient cells such as cellular DNA repair properties and we suggest that it may be used to study other cellular parameters (cell cycle regulation, p53 stability or immunosurveillance-controlling factors) involved in UV-induced skin cancers and which reliability requires the use of untransformed cells