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

Engineering Genetic Predisposition in Human Neuroepithelial Stem Cells Recapitulates Medulloblastoma Tumorigenesis.

Human neural stem cell cultures provide progenitor cells that are potential cells of origin for brain cancers. However, the extent to which genetic predisposition to tumor formation can be faithfully captured in stem cell lines is uncertain. Here, we evaluated neuroepithelial stem (NES) cells, representative of cerebellar progenitors. We transduced NES cells with MYCN, observing medulloblastoma upon orthotopic implantation in mice. Significantly, transcriptomes and patterns of DNA methylation from xenograft tumors were globally more representative of human medulloblastoma compared to a MYCN-driven genetically engineered mouse model. Orthotopic transplantation of NES cells generated from Gorlin syndrome patients, who are predisposed to medulloblastoma due to germline-mutated PTCH1, also generated medulloblastoma. We engineered candidate cooperating mutations in Gorlin NES cells, with mutation of DDX3X or loss of GSE1 both accelerating tumorigenesis. These findings demonstrate that human NES cells provide a potent experimental resource for dissecting genetic causation in medulloblastoma

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

Aspects moleculaires de la regulation de la differenciation epidermique par l'acide retinoique. Clonage et regulation de deux nouveaux marqueurs humains: la loricrine et la proargine

SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : T 82808 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Rôles de la voie SONIC HEDGEHOG/PATCHED et des interactions dermo-épidermiques dans la prédisposition aux carcinomes basocellulaires chez les patients atteints du syndrome de Gorlin

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

ALDH1A1 Deficiency in Gorlin Syndrome Suggests a Central Role for Retinoic Acid and ATM Deficits in Radiation Carcinogenesis

We hypothesize that aldehyde dehydrogenase 1A1 (ALDH1A1) deficiency will result in impaired ataxia-telangiectasia mutated (ATM) activation in a retinoic acid-sensitive fashion. Data supporting this hypothesis include (1) reduced ATM activation in irradiated primary dermal fibroblasts from ALDH1A1-deficient Gorlin syndrome patients (GDFs), relative to ALDH1A1-positive normal human dermal fibroblasts (NHDFs) and (2) increased ATM activation by X-radiation in GDFs pretreated with retinoic acid, however, the impact of donor variability on ATM activation in fibroblasts was not assessed and is a prudent consideration in future studies. Clonogenic survival of irradiated cells showed differential responses to retinoic acid as a function of treatment time. Long-term (5 Day) retinoic acid treatment functioned as a radiosensitizer and was associated with downregulation of ATM protein levels. Short-term (7 h) retinoic acid treatment showed a trend toward increased survival of irradiated cells and did not downregulate ATM protein levels. Using a newly developed IncubATR technology, which defines changes in bulk chemical bond patterns in live cells, we can discriminate between the NHDF and GDF phenotypes, but treatment of GDFs with retinoic acid does not induce reversion of bulk chemical bond patterns associated with GDFs toward the NHDF phenotype. Collectively, our preliminary investigation of the Gorlin phenotype has identified deficient ALDH1A1 expression associated with deficient ATM activation as a possible susceptibility factor that is consistent with the high incidence of spontaneous and radiation-induced carcinogenesis in these patients. The IncubATR technology exhibits sufficient sensitivity to detect phenotypic differences in live cells that may be relevant to radiation health effects