The continuous renewal of human epidermis is sustained by stem cells contained in the epidermal basal layer and in hair follicles1,2. Cultured keratinocyte stem cells, known as holoclones3,4,5,6, generate sheets of epithelium used to restore severe skin, mucosal and corneal defects7,8,9. Mutations in genes encoding the basement membrane component laminin 5 (LAM5) cause junctional epidermolysis bullosa (JEB), a devastating and often fatal skin adhesion disorder10. Epidermal stem cells from an adult patient affected by LAM5-β3–deficient JEB were transduced with a retroviral vector expressing LAMB3 cDNA (encoding LAM5-β3), 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.
Subscribe to Journal
Get full journal access for 1 year
only $18.75 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Watt, F.M. Stem cell fate and patterning in mammalian epidermis. Curr. Opin. Genet. Dev. 11, 410–417 (2001).
Alonso, L. & Fuchs, E. Stem cells of the skin epithelium. Proc. Natl. Acad. Sci. USA 100 (suppl. 1), 11830–11835 (2003).
Barrandon, Y. & Green, H. Three clonal types of keratinocyte with different capacities for multiplication. Proc. Natl. Acad. Sci. USA 84, 2302–2306 (1987).
Rochat, A., Kobayashi, K. & Barrandon, Y. Location of stem cells of human hair follicles by clonal analysis. Cell 76, 1063–1073 (1994).
Pellegrini, G. et al. Location and clonal analysis of stem cells and their differentiated progeny in the human ocular surface. J. Cell Biol. 145, 769–782 (1999).
Blanpain, C., Lowry, W.E., Geoghegan, A., Polak, L. & Fuchs, E. Self-renewal, multipotency, and the existence of two cell populations within an epithelial stem cell niche. Cell 118, 635–648 (2004).
Gallico, G.G., III, O'Connor, N.E., Compton, C.C., Kehinde, O. & Green, H. Permanent coverage of large burn wounds with autologous cultured human epithelium. N. Engl. J. Med. 311, 448–451 (1984).
Romagnoli, G. et al. Treatment of posterior hypospadias by the autologous graft of cultured urethral epithelium. N. Engl. J. Med. 323, 527–530 (1990).
Pellegrini, G. et al. Long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. Lancet 349, 990–993 (1997).
Christiano, A.M. & Uitto, J. Molecular complexity of the cutaneous basement membrane zone. Revelations from the paradigms of epidermolysis bullosa. Exp. Dermatol. 5, 1–11 (1996).
Posteraro, P. et al. Compound heterozygosity for an out-of-frame deletion and a splice site mutation in the LAMB3 gene causes nonlethal junctional epidermolysis bullosa. Biochem. Biophys. Res. Commun. 243, 758–764 (1998).
Guerra, L. et al. Treatment of “stable” vitiligo by Timedsurgery and transplantation of cultured epidermal autografts. Arch. Dermatol. 136, 1380–1389 (2000).
Mills, A.A. et al. p63 is a p53 homologue required for limb and epidermal morphogenesis. Nature 398, 708–713 (1999).
Yang, A. et al. p63 is essential for regenerative proliferation in limb, craniofacial and epithelial development. Nature 398, 714–718 (1999).
Parsa, R., Yang, A., McKeon, F. & Green, H. Association of p63 with proliferative potential in normal and neoplastic human keratinocytes. J. Invest. Dermatol. 113, 1099–1105 (1999).
Pellegrini, G. et al. p63 identifies keratinocyte stem cells. Proc. Natl. Acad. Sci. USA 98, 3156–3161 (2001).
Di Iorio, E. et al. Isoforms of DeltaNp63 and the migration of ocular limbal cells in human corneal regeneration. Proc. Natl. Acad. Sci. USA 102, 9523–9528 (2005).
Parker, K.C., Bednarek, M.A. & Coligan, J.E. Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains. J. Immunol. 152, 163–175 (1994).
Manici, S. et al. Melanoma cells present a MAGE-3 epitope to CD4+ cytotoxic T cells in association with histocompatibility leukocyte antigen DR11. J. Exp. Med. 189, 871–876 (1999).
Pellegrini, G. et al. The control of epidermal stem cells (holoclones) in the treatment of massive full-thickness burns with autologous keratinocytes cultured on fibrin. Transplantation 68, 868–879 (1999).
Bushman, F. et al. Genome-wide analysis of retroviral DNA integration. Nat. Rev. Microbiol. 3, 848–858 (2005).
De Luca, M., Pellegrini, G. & Green, H. Regeneration of squamous epithelia from stem cells of cultured grafts. Regenerative Med. 1, 45–57 (2006).
Hacein-Bey-Abina, S. et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302, 415–419 (2003).
Gaspar, H.B. et al. Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vector. Lancet 364, 2181–2187 (2004).
Aiuti, A. et al. Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Science 296, 2410–2413 (2002).
Recchia, A. et al. Retroviral vector integration deregulates gene expression but has no consequence on the biology and function of transplanted T cells. Proc. Natl. Acad. Sci. USA 103, 1457–1462 (2006).
Ott, M.G. et al. Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1–EVI1, PRDM16 or SETBP1. Nat. Med. 12, 401–409 (2006).
Dellambra, E. et al. Corrective transduction of human epidermal stem cells in laminin-5-dependent junctional epidermolysis bullosa. Hum. Gene Ther. 9, 1359–1370 (1998).
Mathor, M.B. et al. Clonal analysis of stably transduced human epidermal stem cells in culture. Proc. Natl. Acad. Sci. USA 93, 10371–10376 (1996).
Krall, W.J. et al. Increased levels of spliced RNA account for augmented expression from the MFG retroviral vector in hematopoietic cells. Gene Ther. 3, 37–48 (1996).
We thank S. Bondanza for the clonal analysis of KEP25 keratinocytes shown in Figure 1b, K. Fleishauer for the HLA genotyping, and C. Rossi and D. Sartori for technical assistance. We also thank H. Green (Harvard Medical School) for providing the 3T3-J2 cells and G. Meneguzzi (Institut National de la Santé et de la Recherche Médicale (INSERM) U 634) for providing the K140 antibody to LAM5-β3 and performing the immunofluorescence shown in Figure 3d. This work was supported by grants from Telethon, AFM-Telethon and the European Commission (VI Framework Program, SKINTHERAPY).
The authors declare no competing financial interests.
Regeneration of a genetically corrected epidermis. (PDF 605 kb)
Integration site analysis in cultured keratinocytes. (PDF 59 kb)
Complete list of retroviral integration sites in skin biopsies 1 and 4 months after transplantation (PDF 85 kb)
Analysis of retroviral integration sites in skin biopsies (PDF 20 kb)
About this article
Cite this article
Mavilio, F., Pellegrini, G., Ferrari, S. et al. Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells. Nat Med 12, 1397–1402 (2006). https://doi.org/10.1038/nm1504
Expert Opinion on Biological Therapy (2020)
Journal of Investigative Dermatology (2020)
SLAS TECHNOLOGY: Translating Life Sciences Innovation (2020)
Long-term outcomes of cultivated cell sheet transplantation for treating total limbal stem cell deficiency
The Ocular Surface (2020)