Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells Fulvio Mavilio 1 , Graziella Pellegrini 1,2 , Stefano Ferrari 2 , Francesca Di Nunzio 1 , Enzo Di Iorio 2 , Alessandra Recchia 1 , Giulietta Maruggi 1 , Giuliana Ferrari 3 , Elena Provasi 4 , Chiara Bonini 4 , Sergio Capurro 5 , Andrea Conti 6 , Cristina Magnoni 6 , Alberto Giannetti 6 & Michele De Luca 1,2 The continuous renewal of human epidermis is sustained by stem cells contained in the epidermal basal layer and in hair follicles 1,2 . Cultured keratinocyte stem cells, known as holoclones 3–6 , generate sheets of epithelium used to restore severe skin, mucosal and corneal defects 7–9 . Mutations in genes encoding the basement membrane component laminin 5 (LAM5) cause junctional epidermolysis bullosa (JEB), a devastating and often fatal skin adhesion disorder 10 . Epidermal stem cells from an adult patient affected by LAM5-b3–deficient JEB were transduced with a retroviral vector expressing LAMB3 cDNA (encoding LAM5-b3), and used to prepare genetically corrected cultured epidermal grafts. Nine grafts were transplanted onto surgically prepared regions of the patient’s legs. Engraftment was complete after 8 d. Synthesis and proper assembly of normal levels of functional LAM5 were observed, together with the development of a firmly adherent epidermis that remained stable for the duration of the follow-up (1 year) in the absence of blisters, infections, inflammation or immune response. Retroviral integration site analysis indicated that the regenerated epidermis is maintained by a defined repertoire of transduced stem cells. These data show that ex vivo gene therapy of JEB is feasible and leads to full functional correction of the disease. The patient enrolled in this phase I/II clinical trial was a 36-year-old male (referred to as KEP25) affected by nonlethal JEB. He was a double-heterozygous carrier of a null allele and a single point muta- tion (E210K) in the LAMB3 gene (encoding LAM5-b3), impairing the normal assembly of LAM5 (ref. 11). Since his birth, he had suffered from blistering of the skin that occurred either spontaneously or after minimal injury and culminated in infected lesions (Fig. 1a). Most of his body was covered by large, hard-to-heal blisters or infected crusts, with few moderately affected areas (Fig. 1a,b). To select a donor site suitable for epidermal stem cell correction, we took skin biopsies from different areas of his body for clonal analyses of keratinocytes. We were unable to obtain clonogenic and holoclone-forming cells from most of the patient’s skin (Fig. 1b), most probably because of the continuous proliferative stimulus associated with the wound-healing process. Only his palms contained a sufficient number of holoclones. Primary KEP25 keratinocytes, obtained from two palm biopsies (1.5 cm 2 ), were transduced by a retroviral vector expressing the full- length LAMB3 cDNA under the control of the Moloney leukemia virus (MLV) long terminal repeat (LTR) (Fig. 2a). Clonogenic cells were transduced at virtually 100% efficiency, as indicated by immuno- fluorescence analysis of cytoplasmic LAM5-b3(Fig. 2b). Southern blot analysis of genomic DNA indicated the presence of an average of two intact vector copies per genome, with no sign of rearranged proviruses (Fig. 2c). Northern analysis of total RNA showed abundant accumulation of a single vector-derived mRNA of the expected size (Fig. 2d). A LAM5-b3–specific antibody immunoprecipitated (from total lysates of transduced keratinocytes) an amount of heterotrimeric LAM5 virtually indistinguishable from that of normal keratinocytes (Fig. 2e). Untransduced KEP25 palm keratinocytes contained barely detectable amounts of LAM5-b3 protein and LAMB3 mRNA (Fig. 2b,d,e). Transgene expression persisted at constant levels throughout the lifespan of the culture (4120 cell doublings, data not shown). The anterior upper regions of the patient’s legs, which were covered by a very fragile epidermis and contained several infected nonhealing lesions, were selected for transplantation. We removed LAM5-b3– deficient epidermal remnants by Timedsurgery 12 under local anesthe- sia (Supplementary Fig. 1 online). We transplanted four and five genetically modified grafts, each 55 cm 2 (a total of B500 cm 2 ), on the right and left legs, respectively. We observed complete epidermal regeneration on both legs at day 8, and a normal-looking epidermis was maintained throughout the 1-year follow-up (Fig. 3a, © 2006 Nature Publishing Group http://www.nature.com/naturemedicine Received 8 June; accepted 11 October; published online 19 November 2006; doi:10.1038/nm1504 1 Department of Biomedical Sciences, University of Modena and Reggio Emilia, Via Campi 287, 41100 Modena, Italy. 2 Epithelial Stem Cell Research Center, Veneto Eye Bank Foundation, H. SS Giovanni and Paolo, Castello 6777, 30100 Venice, Italy. 3 Istituto Scientifico H. San Raffaele-Telethon Institute for Gene Therapy (HSR- TIGET) and Vita-Salute University, and 4 Cancer Immunotherapy and Gene Therapy Program, Istituto Scientifico H. San Raffaele, Via Olgettina 58, 20132 Milano, Italy. 5 Division of Plastic Surgery, H. San Martino, Largo Rosanna Benzi 10, 16132 Genova, Italy. 6 Department of Internal Medicine, University of Modena and Reggio Emilia, Via del Pozzo 71, 41100 Modena, Italy. Correspondence should be addressed to M.D.L. (michele.deluca@unimore.it) or F.M. (fulvio.mavilio@unimore.it). NATURE MEDICINE ADVANCE ONLINE PUBLICATION 1 LETTERS