Advertisement

Closure of a Large Chronic Wound through Transplantation of Gene-Corrected Epidermal Stem Cells

Open AccessPublished:November 10, 2016DOI:https://doi.org/10.1016/j.jid.2016.10.038

      Abbreviations:

      JEB (junctional epidermolysis bullosa), MLV (Moloney leukemia virus), RV (retroviral vector)
      To the Editor
      Generalized junctional epidermolysis bullosa (JEB) is caused by mutations in LAMA3, LAMB3, or LAMC2, which together encode laminin-332, a heterotrimeric protein consisting of α3, β3, and γ2 chains (
      • Fine J.D.
      • Bruckner-Tuderman L.
      • Eady R.A.
      • Bauer E.A.
      • Bauer J.W.
      • Has C.
      • et al.
      Inherited epidermolysis bullosa: updated recommendations on diagnosis and classification.
      ). In nonlethal generalized intermediate JEB, laminin-332 is highly reduced, and hemidesmosomes are rudimentary or completely absent, leading to blister formation within the lamina lucida of the basement membrane upon minor trauma. The resulting chronic skin wounds invariably develop recurrent infections and scarring, which greatly impair patients’ quality of life (
      • Fine J.D.
      • Bruckner-Tuderman L.
      • Eady R.A.
      • Bauer E.A.
      • Bauer J.W.
      • Has C.
      • et al.
      Inherited epidermolysis bullosa: updated recommendations on diagnosis and classification.
      ,
      • Laimer M.
      • Lanschuetzer C.M.
      • Diem A.
      • Bauer J.W.
      Herlitz junctional epidermolysis bullosa.
      ,
      • Nakano A.
      • Chao S.C.
      • Pulkkinen L.
      • Murrell D.
      • Bruckner-Tuderman L.
      • Pfendner E.
      • et al.
      Laminin 5 mutations in junctional epidermolysis bullosa: molecular basis of Herlitz vs. non-Herlitz phenotypes.
      ).
      There is no cure for JEB; treatments are symptomatic and aimed at relieving the devastating clinical manifestations (
      • Carulli S.
      • Contin R.
      • De Rosa L.
      • Pellegrini G.
      • De Luca M.
      The long and winding road that leads to a cure for epidermolysis bullosa.
      ). The only published evidence for the possibility of a permanent local treatment of JEB was provided by a phase I/II trial showing that autologous epidermal cultures containing genetically modified epidermal stem cells were able to restore a normal epidermis on a JEB patient (
      • De Rosa L.
      • Carulli S.
      • Cocchiarella F.
      • Quaglino D.
      • Enzo E.
      • Franchini E.
      • et al.
      Long-term stability and safety of transgenic cultured epidermal stem cells in gene therapy of junctional epidermolysis bullosa.
      ,
      • Mavilio F.
      • Pellegrini G.
      • Ferrari S.
      • Di Nunzio F.
      • Di Iorio E.
      • Recchia A.
      • et al.
      Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells.
      ). However, the transgenic epidermis was applied in areas still covered by a diseased but apparently functional epidermis, which was surgically removed before grafting (
      • Mavilio F.
      • Pellegrini G.
      • Ferrari S.
      • Di Nunzio F.
      • Di Iorio E.
      • Recchia A.
      • et al.
      Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells.
      ). Although it is clear that the ideal clinical application of transgenic epidermis would aim at preventing the development of devastating chronic lesions, many patients suffer from therapy-resistant chronic ulcerations that are highly predisposed to cancer development and need timely closure (
      • Goldberg G.I.
      • Eisen A.Z.
      • Bauer E.A.
      Tissue stress and tumor promotion. Possible relevance to epidermolysis bullosa.
      ,
      • Hoste E.
      • Arwert E.N.
      • Lal R.
      • South A.P.
      • Salas-Alanis J.C.
      • Murrell D.F.
      • et al.
      Innate sensing of microbial products promotes wound-induced skin cancer.
      ). We report on a patient in whom gene-corrected epidermal sheets were transplanted onto a large nonhealing epidermal ulceration following a good manufacturing practice protocol. This single-case study was authorized by the Austrian Ministry of Health, and all experiments were approved by the University Hospital of the Paracelsus Medical University, Salzburg. Written informed consent was given by the patient, who also consented on the publication of photographs and medical information.
      A 49-year-old woman with generalized intermediate, laminin-332-β3–dependent JEB presented with a large (approximately 80-cm2) wound on her lower right leg (Figure 1a) (
      • Buchroithner B.
      • Klausegger A.
      • Ebschner U.
      • Anton-Lamprecht I.
      • Pohla-Gubo G.
      • Lanschuetzer C.M.
      • et al.
      Analysis of the LAMB3 gene in a junctional epidermolysis bullosa patient reveals exonic splicing and allele-specific nonsense-mediated mRNA decay.
      ). Whereas recurrent blisters covering her body have been treated with conventional therapies with varying success, the large lesion on her right leg persisted for over 10 years, and recurrent infections led at one point to post-streptococcal glomerulonephritis. In July 2014 her ulceration was treated with a combined ex vivo gene- and autologous cell-therapy approach. Primary patient keratinocytes, obtained from a biopsy of the palm, were transduced by the same retroviral vector (RV) expressing the full-length LAMB3 cDNA under the control of the Moloney leukemia virus (MLV) long-terminal repeat, as used in the previous study (
      • Mavilio F.
      • Pellegrini G.
      • Ferrari S.
      • Di Nunzio F.
      • Di Iorio E.
      • Recchia A.
      • et al.
      Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells.
      ), at an efficiency of 99.4% and an average of two proviral copies per cell (see Supplementary Figure S1 online). Whereas untransduced patient keratinocytes contained barely detectable amounts of laminin-332-β3 (Figure 1b, lanes 2 and 4), genetically modified cells contained an amount of laminin-332-β3 (Figure 1b, lane 3) even higher than normal keratinocytes (Figure 1b, lane 1). Transgene expression persisted at constant levels throughout the lifespan of the culture (approximately 100 cell doublings, data not shown).
      Figure 1
      Figure 1Regeneration of a transgenic functional epidermis on the skin wound of the JEB patient. (a) The long-standing ulceration on the lower right leg of the patient 2 days before transplantation. (b) Western blot analysis of cell lysates (20 μg protein, 30 seconds exposure time) from (lane 1) normal control and patient keratinocyte cultures (lane 2) before and (lane 3) after gene correction, probed with a monoclonal antibody against laminin 332-β3. (lane 4) Western blot analysis of a higher amount of loaded protein (65 μg, 5 seconds exposure time) of uncorrected patient keratinocytes, and (lane 5) normal keratinocyte cultures using the same laminin-332-β3 antibody. The 75-kD band in lane 4 is consistent with the truncated laminin-332-β3 generated by the c.1903C>T; p.R635X mutation. (c) Transplantation of cultured transgenic epidermal sheets (asterisks) on the prepared wound bed. Grafts are overlaid with petrolatum gauze. (d) Initial epidermal regeneration at 14 days. (e) Complete epidermal regeneration at 3.5 months. (f) Stable epidermal regeneration at 16 months. Note crusting and erosions outside of the grafted area. JEB, junctional epidermolysis bullosa.
      Gene-corrected clonogenic cells (∼4 × 105) were expanded and used to grow two cohesive epidermal sheets of approximately 80 cm2 to be transplanted onto the leg ulceration after wound bed preparation (Figure 1c). Complete engraftment of the transgenic epidermis was observed after 14 days (Figure 1d). The skin was apparently normal, with some hyperkeratosis originating from the initial biopsy site, the lateral aspect of the left palm. The graft remained mechanically stable throughout the entire follow-up period (16 months) and without blister formation, even upon shear force by repeated rubbing of the skin (Figure 1e and f).
      On histological analysis of skin biopsy samples taken at 1-year follow-up, we observed a normal and fully differentiated epidermis and a normal dermal-epidermal junction (Figure 2b). Immunofluorescence analysis showed that the transgenic epidermis expressed a normal amount of laminin-332 (Figure 2c), comparable to that observed in a normal control (Figure 2d), that is properly located at the dermal-epidermal junction. Transmission electron microscopy showed appropriate morphology of the basement membrane zone in the transplanted area (Figure 2e), unlike the patient’s lesional skin before grafting (Figure 2f). In situ hybridization performed using vector-specific LAMB3 probes showed homogenous expression of LAMB3 mRNA in all epidermal layers (Figure 2g), confirming that the regenerated epidermis consists only of transgenic keratinocytes. Because human epidermis is renewed monthly, the patient’s transgenic epidermis underwent at least 16 complete renewal cycles during the 16 months of follow-up. Thus, the long-term maintenance of the regenerated epidermis must be due to the engraftment of self-renewing transduced epidermal stem cells. This assumption was confirmed by genome-wide analysis of RV integration sites performed on DNA extracted from a 3-mm2 punch biopsy sample of the transgenic epidermis at 1-year follow-up. Libraries of vector-genome junctions, generated by ligation-mediated nested PCR and sequenced to saturation, retrieved three independent integrations into genes unambiguously mapped on the human genome (see Supplementary Table S1 online). Quantitative real-time reverse transcriptase–PCR analysis performed on untransduced and transduced primary cultures showed no change in the expression of the three genes, indicating that they were not dysregulated through the proviral integration (data not shown). Given an average integration per cell of 1.5–2.0, as determined by Southern blot analysis and previously shown (
      • Mavilio F.
      • Pellegrini G.
      • Ferrari S.
      • Di Nunzio F.
      • Di Iorio E.
      • Recchia A.
      • et al.
      Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells.
      ), these results indicate that the ∼150 clonogenic cells present in the small area of the taken biopsy arise from at least two single stem cell clones. These data confirm the notion that the entire regenerated epidermis is sustained only by the engrafted stem cells (
      • De Rosa L.
      • Carulli S.
      • Cocchiarella F.
      • Quaglino D.
      • Enzo E.
      • Franchini E.
      • et al.
      Long-term stability and safety of transgenic cultured epidermal stem cells in gene therapy of junctional epidermolysis bullosa.
      ).
      Figure 2
      Figure 2Expression of laminin-332 protein and LAMB3 mRNA in the regenerated transgenic epidermis and restoration of a normal dermal-epidermal junction. (a) Sections (7 μm thick) from the patient’s skin before transplantation were immunostained using a polyclonal antibody against laminin-332. (b) Hematoxylin and eosin staining of a skin section from a biopsy sample at 1-year follow-up. (c) Immunofluorescence staining of laminin-332 in corrected skin after 1 year and (d) normal control skin sections. Scale bars = 10 μm. (e) Transmission electron microscopy performed on skin biopsy samples from the transplanted area of the patient’s leg and (f) from the patient’s skin before transplantation. Arrowheads indicate basement membrane; arrows show hemidesmosomes. Scale bars = 0.5 μm. In situ hybridization of LAMB3 mRNA using vector-specific LAMB3 probes was performed (g) in skin sections taken from the transplanted area of the patient’s leg at the 1-year follow-up and (h) in sections of normal control skin. Cell nuclei were counterstained with DAPI (blue). Dotted line shows dermal-epidermal junction. Scale bars = 20 μm.
      To ensure the safety of our approach, we further demonstrated the complete absence of pathogenic antibodies against the newly synthesized laminin-332-β3 protein by indirect immunofluorescence analysis using the patient’s plasma collected 12 months after transplantation (see Supplementary Figure S2 online).
      Despite the occurrence of severe adverse events in patients with genetic immunodeficiency treated with MLV-based RV-transduced hematopoietic stem cells (
      • Carulli S.
      • Contin R.
      • De Rosa L.
      • Pellegrini G.
      • De Luca M.
      The long and winding road that leads to a cure for epidermolysis bullosa.
      ), these vectors cannot per se be considered oncogenic, because other clinical trials using the same vector backbone in the same cell type have not been accompanied by any serious adverse effects (
      • Aiuti A.
      • Cattaneo F.
      • Galimberti S.
      • Benninghoff U.
      • Cassani B.
      • Callegaro L.
      • et al.
      Gene therapy for immunodeficiency due to adenosine deaminase deficiency.
      ). The safety of using an MLV-based RV for skin gene therapy approaches is underscored by the fact that during the 10 years’ follow-up of the previously treated JEB patient, the vector did not cause any adverse events (
      • De Rosa L.
      • Carulli S.
      • Cocchiarella F.
      • Quaglino D.
      • Enzo E.
      • Franchini E.
      • et al.
      Long-term stability and safety of transgenic cultured epidermal stem cells in gene therapy of junctional epidermolysis bullosa.
      ). This notion indicates that the risk of vector-induced mutagenesis might require other factors possibly related to cell type, genetic background, disease, and the clinical protocol.
      Taken together, our data show that a functional epidermis has been regenerated on a previously infected nonhealing skin ulceration by a discrete number of gene-corrected epidermal stem cells, which might also reduce the risk of cancer development in such treated wounds. This underscores the high therapeutic potential of ex vivo gene therapy in the skin. On the basis of these results, further affected skin areas of the patient in this study can be treated, and the technology can be transferred to treat other JEB and dystrophic epidermolysis bullosa patients.

      ORCID

      Conflict of Interest

      GP and MDL are members of the Board of Holostem Terapie Avanzate S.r.l.

      Acknowledgments

      This work was supported by DEBRA Austria, University Hospital Salzburg, and POR FESR 2014-2020 Asse 1 Regione Emilia-Romagna.

      Supplementary Material

      References

        • Aiuti A.
        • Cattaneo F.
        • Galimberti S.
        • Benninghoff U.
        • Cassani B.
        • Callegaro L.
        • et al.
        Gene therapy for immunodeficiency due to adenosine deaminase deficiency.
        N Engl J Med. 2009; 360: 447-458
        • Buchroithner B.
        • Klausegger A.
        • Ebschner U.
        • Anton-Lamprecht I.
        • Pohla-Gubo G.
        • Lanschuetzer C.M.
        • et al.
        Analysis of the LAMB3 gene in a junctional epidermolysis bullosa patient reveals exonic splicing and allele-specific nonsense-mediated mRNA decay.
        Lab Invest. 2004; 84: 1279-1288
        • Carulli S.
        • Contin R.
        • De Rosa L.
        • Pellegrini G.
        • De Luca M.
        The long and winding road that leads to a cure for epidermolysis bullosa.
        Regen Med. 2013; 8: 467-481
        • De Rosa L.
        • Carulli S.
        • Cocchiarella F.
        • Quaglino D.
        • Enzo E.
        • Franchini E.
        • et al.
        Long-term stability and safety of transgenic cultured epidermal stem cells in gene therapy of junctional epidermolysis bullosa.
        Stem Cell Reports. 2014; 2: 1-8
        • Fine J.D.
        • Bruckner-Tuderman L.
        • Eady R.A.
        • Bauer E.A.
        • Bauer J.W.
        • Has C.
        • et al.
        Inherited epidermolysis bullosa: updated recommendations on diagnosis and classification.
        J Am Acad Dermatol. 2014; 70: 1103-1126
        • Goldberg G.I.
        • Eisen A.Z.
        • Bauer E.A.
        Tissue stress and tumor promotion. Possible relevance to epidermolysis bullosa.
        Arch Dermatol. 1988; 124: 737-741
        • Hoste E.
        • Arwert E.N.
        • Lal R.
        • South A.P.
        • Salas-Alanis J.C.
        • Murrell D.F.
        • et al.
        Innate sensing of microbial products promotes wound-induced skin cancer.
        Nat Commun. 2015; 6: 5932
        • Laimer M.
        • Lanschuetzer C.M.
        • Diem A.
        • Bauer J.W.
        Herlitz junctional epidermolysis bullosa.
        Dermatol Clin. 2010; 28: 55-60
        • Mavilio F.
        • Pellegrini G.
        • Ferrari S.
        • Di Nunzio F.
        • Di Iorio E.
        • Recchia A.
        • et al.
        Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells.
        Nat Med. 2006; 12: 1397-1402
        • Nakano A.
        • Chao S.C.
        • Pulkkinen L.
        • Murrell D.
        • Bruckner-Tuderman L.
        • Pfendner E.
        • et al.
        Laminin 5 mutations in junctional epidermolysis bullosa: molecular basis of Herlitz vs. non-Herlitz phenotypes.
        Hum Genet. 2002; 110: 41-51