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Recessive Dystrophic Epidermolysis Bullosa: Advances in the Laboratory Leading to New Therapies

  • David T. Woodley
    Correspondence
    USC Norris Comprehensive Cancer Center, Topping #3405, 1441 Eastlake Avenue, Los Angeles, California 90033, USA
    Affiliations
    Department of Dermatology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA
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  • Mei Chen
    Affiliations
    Department of Dermatology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA
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      Recessive dystrophic epidermolysis bullosa (RDEB) is a rare, devastating, hereditary mechanobullous disease of the skin in which patients have marked skin fragility, widespread bullae, and erosions that characteristically heal with exuberant scarring and milia formation (
      • Fine J.D.
      • Bruckner-Tuderman L.
      • Eady R.A.
      Inherited epidermolysis bullosa: updated recommendations on diagnosis and classification.
      ). Our laboratory and several others have long aspired to develop an effective treatment—a goal now seemingly far closer than we had dared hope only a few years ago. Here we summarize relevant laboratory-based advances in understanding RDEB pathogenesis as well as recent steps toward translating this knowledge into targeted therapies.
      RDEB is attributable to mutations in the COL7A1 gene that encodes type VII collagen (C7). C7 forms large structures called anchoring fibrils (AFs) that localize to the dermal–epidermal junction (DEJ) and are required for epidermal–dermal adherence (
      • Fine J.D.
      • Bruckner-Tuderman L.
      • Eady R.A.
      Inherited epidermolysis bullosa: updated recommendations on diagnosis and classification.
      ). In human skin, both keratinocytes and fibroblasts synthesize C7 α1 chains that form homotrimeric C7 molecules. The cells then secrete C7 molecules into the high extracellular space of the papillary dermis. It is not clear how C7 in the papillary dermis actually gets to the DEJ to form AFs, but we demonstrated that C7 has specific binding domains for type IV collagen and laminin 332 in the DEJ (
      • Chen M.
      • Marinkovich M.P.
      • Veis A.
      Interactions of the amino-terminal noncollagenous (NC1) domain of type VII collagen with extracellular matrix components: a potential role in epidermal–dermal adherence in human skin.
      ,
      • Chen M.
      • Marinkovich M.P.
      • Jones J.J.
      NC1 domain of type VII collagen binds to the b3 chain of laminin 5 via a unique sub-domain within the fibronectin-like repeats.
      ). Therefore, C7 likely binds to components of the DEJ, condenses into antiparallel dimers, and then forms AFs. Using normal mouse wound healing models and mouse RDEB models, we and others have learned that all one needs to do is get C7 into the high papillary dermis: it will then self-assemble and form correctly localized AFs (
      • Chen M.
      • Keene D.R.
      • Chan L.S.
      Restoration of type VII collagen expression and function in dystrophic epidermolysis bullosa.
      ;
      • Ortiz-Urda S.
      • Lin Q.
      • Green C.L.
      Injection of genetically engineered fibroblasts corrects regenerated human epidermolysis bullosa skin.
      ;
      • Remington J.
      • Wang X.Y.
      • Hou Y.P.
      Injection of recombinant human type VII collagen corrects the disease phenotype in a murine model of dystrophic epidermolysis bullosa.
      ;
      • Wang X.Y.
      • Ghasri P.
      • Aimr M.
      Topical application of recombinant type VII collagen promotes wound closure and corrects recessive dystrophic epidermolysis bullosa.
      ;
      • Woodley D.T.
      • Krueger G.G.
      • Fairley J.A.
      Normal and gene-corrected dystrophic epidermolysis bullosa fibroblasts alone can produce type VII collagen at the basement membrane zone.
      ,
      • Woodley D.T.
      • Atha T.
      • Huang Y.
      Injection of recombinant human type VII collagen restores type VII collagen expression and function in dystrophic epidermolysis bullosa in vivo.
      ,
      • Woodley D.T.
      • Remington J.
      • Huang Y.
      Intravenously injected human fibroblasts home to skin wounds, deliver type VII collagen and promote wound healing.
      ,
      • Woodley D.T.
      • Wang X.
      • Amir M.
      Intravenously injected recombinant human type VII collagen homes to skin wounds and restores skin integrity of dystrophic epidermolysis bullosa.
      a).
      The ultimate treatment for RDEB may be gene therapy, provided the RDEB patient’s skin cells have a normal COL7A1 gene and express normal, functional C7. The COL7A1 gene is quite large, more than 9 kb, which exceeds the gene-packaging capability of most viral vectors. Nevertheless, we used a fourth-generation lentiviral vector engineered to express full-length C7 and demonstrated that RDEB keratinocytes and fibroblasts, when infected with this vector, were then able to synthesize and secrete full-length C7 (
      • Chen M.
      • Keene D.R.
      • Chan L.S.
      Restoration of type VII collagen expression and function in dystrophic epidermolysis bullosa.
      ;
      • Woodley D.T.
      • Atha T.
      • Huang Y.
      Injection of recombinant human type VII collagen restores type VII collagen expression and function in dystrophic epidermolysis bullosa in vivo.
      ). Therefore, one promising method of treating RDEB would be to take a biopsy from the patient, place the patient’s keratinocytes and/or fibroblasts into tissue culture in the laboratory, gene-correct the cultured RDEB cells such that they now express full-length C7, and then transplant the gene-corrected cells back onto the RDEB patient as a cultured autograft. Scientists in the Department of Dermatology at Stanford have taken this approach using gene-corrected cultured keratinocyte autografts and have achieved proof of principle for this approach in one RDEB patient. The Stanford team is now evaluating the safety and efficacy of this approach with other RDEB patients in a phase I clinical trial.
      Using RDEB murine models and murine wound models, we (
      • Woodley D.T.
      • Krueger G.G.
      • Fairley J.A.
      Normal and gene-corrected dystrophic epidermolysis bullosa fibroblasts alone can produce type VII collagen at the basement membrane zone.
      ) and Ortiz-Urda and colleagues (
      • Ortiz-Urda S.
      • Lin Q.
      • Green C.L.
      Injection of genetically engineered fibroblasts corrects regenerated human epidermolysis bullosa skin.
      ) demonstrated that cultured dermal fibroblasts (either from normal human subjects or from RDEB patients, engineered to express C7) can be injected into murine skin or transplanted RDEB skin equivalents and that the injected cells then secrete C7 into the papillary dermis. There, C7 incorporates into the DEJ, forms new AFs, and reverses the RDEB phenotype of poor epidermal–dermal adherence. Interestingly, we also showed that the cells could be administered intravenously (IV) and home to open wounds in the skin, promoting healing (
      • Woodley D.T.
      • Remington J.
      • Huang Y.
      Intravenously injected human fibroblasts home to skin wounds, deliver type VII collagen and promote wound healing.
      ). This suggests that such cells, injected IV into an RDEB patient, might localize within healing wounds and continually secrete C7 that can then incorporate into the DEJ and form new AFs that promote healing.
      Along the lines of cellular therapy for RDEB, two studies attempted to treat RDEB patients with intradermal injections of normal cultured dermal fibroblasts obtained from closely related relatives (
      • Wong T.
      • Gammon L.
      • Liu L.
      Potential of fibroblast cell therapy for recessive dystrophic epidermolysis bullosa.
      ;
      • Venugopal S.S.
      • Yan W.
      • Frew J.W.
      A phase II randomized vehicle-controlled trial of intradermal allogeneic fibroblasts for recessive dystrophic epidermolysis bullosa.
      ). These injected cells did not persist in the skin very long, but the treated RDEB patients had increased C7 expression at their DEJ and improved skin fragility and blistering. The increased C7, however, was not produced by the injected normal fibroblasts. Rather, the allogeneic fibroblasts or perhaps even the process of creating an injection wound itself (
      • Venugopal S.S.
      • Yan W.
      • Frew J.W.
      A phase II randomized vehicle-controlled trial of intradermal allogeneic fibroblasts for recessive dystrophic epidermolysis bullosa.
      ) stimulated the RDEB patients’ endogenous skin cells to synthesize increased amounts of mutated C7. Although the C7 was mutated, it was partially functional. Therefore, increased endogenous mutated C7 improved the epidermal–dermal adherence of these RDEB patients, leading to clinical improvement.
      Cell therapy for RDEB has also been demonstrated using another approach: delivery of allogeneic bone marrow/stem cells from normal relatives of RDEB patients who were closely HLA matched using a protocol similar to those used for the treatment of cancer and leukemia patients. This therapy requires immunosuppression and ablation of the patient’s normal bone marrow prior to the administration of the donor bone marrow/stem cells and has substantial inherent risks. This therapy has been shown to have remarkably positive effects in some RDEB patients by increasing C7 at the patient’s DEJ, with subsequent improvement in their skin disease and quality of life (
      • Wagner J.E.
      • Ishida-Yamamoto A.
      • McGrath J.A.
      Bone marrow transplantation for recessive dystrophic epidermolysis bullosa.
      ), although there were also some deaths and untoward side effects. Nevertheless, the success rate appears to be improving with this therapy as investigators optimize their protocols, reduce the intensity of immunosuppression, optimize the selection of candidates, and identify the best population of stem cells and more closely HLA-matched donors.
      Most collagens, if injected intravenously, activate platelets and the plasma clotting system and induce vascular apoplexy and death. C7 is different. C7 is soluble in neutral buffers and blood. Therefore, in addition to cellular therapy for RDEB, it might be possible to simply provide full-length functional C7 itself to patients, so-called “protein replacement therapy.” Our laboratory developed a method by which we could obtain milligram quantities of purified human recombinant C7 (rhC7) (
      • Chen M.
      • Keene D.R.
      • Chan L.S.
      Restoration of type VII collagen expression and function in dystrophic epidermolysis bullosa.
      ;
      • Woodley D.T.
      • Atha T.
      • Huang Y.
      Injection of recombinant human type VII collagen restores type VII collagen expression and function in dystrophic epidermolysis bullosa in vivo.
      ). Using RDEB mouse models, we have shown that one can administer rhC7 intradermally (
      • Woodley D.T.
      • Atha T.
      • Huang Y.
      Injection of recombinant human type VII collagen restores type VII collagen expression and function in dystrophic epidermolysis bullosa in vivo.
      ;
      • Remington J.
      • Wang X.Y.
      • Hou Y.P.
      Injection of recombinant human type VII collagen corrects the disease phenotype in a murine model of dystrophic epidermolysis bullosa.
      ), topically to wounds (
      • Wang X.Y.
      • Ghasri P.
      • Aimr M.
      Topical application of recombinant type VII collagen promotes wound closure and corrects recessive dystrophic epidermolysis bullosa.
      ), or even IV (
      • Woodley D.T.
      • Wang X.
      • Amir M.
      Intravenously injected recombinant human type VII collagen homes to skin wounds and restores skin integrity of dystrophic epidermolysis bullosa.
      a) to these animals, and it will incorporate into the DEJ and form new human AFs, improve epidermal–dermal adherence, and increase the survival of the animals. Importantly, the IV rhC7 only went to wounded skin and not to any internal organs (
      • Woodley D.T.
      • Wang X.
      • Amir M.
      Intravenously injected recombinant human type VII collagen homes to skin wounds and restores skin integrity of dystrophic epidermolysis bullosa.
      a). Because RDEB skin probably has widespread subclinical, microscopic wounds, conceptually, IV rhC7 could home to all of these skin sites and prevent frank skin blisters. We have completed toxicity studies in rats and minipigs with intradermal rhC7 and found no untoward side effects (unpublished data). IV administration is appealing because patients with RDEB characteristically have widespread skin lesions as well as lesions in areas that are less accessible, such as the upper third of the esophagus. Safe IV administration of rhC7 has been shown in several “preclinical” animal models (normal mice, RDEB mice, hypomorphic RDEB mice, and RDEB Golden Retriever dogs). Shire Pharmaceuticals (Dublin, Ireland) is currently exploring the possibility of developing IV rhC7 for use in RDEB patients. If IV rhC7 behaves in RDEB patients as it does in these preclinical animal models, it would home to the open or subclinical microscopic wounds, incorporate into the DEJ, generate new AFs, and improve, if not normalize, the epidermal–dermal adherence at that site. How often these patients would need to be treated with IV rhC7 to maintain improved epidermal–dermal adherence and prevent new blister formation remains unknown. Type I collagen injected into human skin for improvement in photoaging, however, persists for about 6 months. In RDEB mice and RDEB dogs, injected rhC7 persists for months, but the half-life of rhC7 in human skin may differ.
      RDEB patients heal their skin wounds with severe scarring that leads to esophageal strictures and fusion of the digits on their hands and feet (so-called “mitten” deformities). The conventional wisdom is that this occurs because the blister cleavage plane in RDEB is below the lamina densa zone of the DEJ. We recently found, however, that RDEB skin and cultured fibroblasts exhibit upregulation of profibrotic isoforms of transforming growth factor-β and its downstream signaling pathways, suggesting that the reason RDEB patients have such horrible scarring is because the absence of C7 produces a proscarring microenvironment. Further evidence that C7 may play a role in wound healing includes experiments in which exogenous rhC7 was administered to wounds topically or IV and, surprisingly, wound closure was dramatically accelerated by promoting reepithelialization (
      • Wang X.Y.
      • Ghasri P.
      • Aimr M.
      Topical application of recombinant type VII collagen promotes wound closure and corrects recessive dystrophic epidermolysis bullosa.
      ). Even more surprising, exogenously administered rhC7 to standardized murine skin wounds resulted in healed wounds with less scarring and an associated downregulation of fibrosis markers such as profibrotic forms of transforming growth factor-β, type I collagen, connective tissue growth factor, and α-smooth muscle actin–positive myofibroblasts (
      • Wang X.Y.
      • Ghasri P.
      • Aimr M.
      Topical application of recombinant type VII collagen promotes wound closure and corrects recessive dystrophic epidermolysis bullosa.
      ).
      Gentamicin, an aminoglycoside antibiotic, and its derivatives are old drugs traditionally used to resolve serious Gram-negative bacterial infections and are known to be ototoxic and nephrotoxic in some patients. Interestingly, this class of antibiotics has been shown in some cases to have the ability to “read-through” premature stop codons generated by nonsense mutations in certain types of gene defects. Approximately 10–25% of RDEB patients have nonsense mutations resulting in a truncated C7 or no C7 at all. We recently identified, genotyped, and characterized clinically, histologically, immunologically, and by electron microscopy 22 bona fide RDEB patients (
      • Woodley D.T.
      • Cogan J.
      • Wang X.Y.
      De novo anti-type VII collagen antibodies in patients with recessive dystrophic epidermolysis bullosa.
      b), of whom two had nonsense COL7A1 mutations. We cultured their keratinocytes and fibroblasts with varying doses of aminoglycoside antibiotics. Without aminoglycosides, these cells synthesized no C7. By contrast, in the presence of noncytotoxic doses of aminoglycosides, both the RDEB cultured keratinocytes and the fibroblasts synthesized and secreted full-length C7 at a level between 10 and 35% that of normal cells (
      • Cogan J.
      • Weinstein J.
      • Wang X.Y.
      Aminoglycosides restore full-length type VII collagen by overcoming premature termination codons: therapeutic implications for dystrophic epidermolysis bullosa.
      ). The C7 generated was structurally identical to normal C7 and incorporated correctly into the DEJ of a human skin equivalent in vitro. RDEB cells expressing C7 also exhibited a reversal of the abnormal cellular motility that is characteristic of RDEB cells. Finally, we generated 22 published RDEB nonsense mutations by site-directed mutagenesis and transfected these constructs into human 293 epithelial cells. Without aminoglycosides, these cells produced no C7. By contrast, treatment of the cells with aminoglycosides induced C7 expression between 10 and 80% of the level of C7 expressed in cells transfected with an expression vector for normal C7. These data suggest that aminoglycosides administered judiciously and cautiously may provide benefit for selected RDEB patients with nonsense mutations that create premature stop codons.
      In summary, recent federally funded laboratory research has generated reagents and concepts that are currently being translated into therapies for the devastating disease recessive dystrophic epidermolysis bullosa.

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