Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells

Abstract

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.

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Figure 1: Analysis of the skin of patient KEP25.
Figure 2: Correction of LAM5-β3 deficiency in epidermal cells of KEP25.
Figure 3: Regeneration of a genetically corrected, functional epidermis.
Figure 4: Absence of humoral or cytotoxic immune response to LAM5-β3.

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Acknowledgements

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).

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Contributions

F.M., G.P. and M.D.L. designed and directed the study. G.P carried out the clonal analysis, transduced the patient's cells and prepared the skin implants. S.F, F.D.N., E.D.I. and G.M. carried out the hystological and molecular follow-up, G.F. and F.M. constructed the retroviral vectors and the packaging cell line, E.P. and C.B. performed the immunological analysis, S.C., A.C., C.M. and A.G. carried out the transplantation and were responsible for the clinical management of the patient.

Corresponding authors

Correspondence to Fulvio Mavilio or Michele De Luca.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Regeneration of a genetically corrected epidermis. (PDF 605 kb)

Supplementary Fig. 2

Integration site analysis in cultured keratinocytes. (PDF 59 kb)

Supplementary Table 1

Complete list of retroviral integration sites in skin biopsies 1 and 4 months after transplantation (PDF 85 kb)

Supplementary Table 2

Analysis of retroviral integration sites in skin biopsies (PDF 20 kb)

Supplementary Methods (PDF 126 kb)

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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

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