Advertisement

Molecular Therapeutics for Heritable Skin Diseases

  • Jouni Uitto
    Affiliations
    Department of Dermatology and Cutaneous Biology, and Biochemistry and Molecular Biology, Jefferson Medical College, and Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania, USA
    Search for articles by this author

      Introduction

      Over the past two decades, there has been tremendous progress in molecular genetics of heritable skin diseases, and as many as 500 distinct human genes are now known to harbor mutations in these diseases (
      • Feramisco J.D.
      • Sadreyev R.I.
      • Murray M.L.
      • et al.
      Phenotypic and genotypic analyses of genetic skin disease through the Online Mendelian Inheritance in Man (OMIM) database.
      ). The clinical implications of this progress are evident in the diagnosis and management of these diseases. For example, (a) identification of the specific mutations can be used for confirmation of diagnosis with prognostic implications; (b) identification of mutations has facilitated assessment of the precise mode of inheritance, particularly in cases with no previous family history of the disease; and (c) identification of candidate genes and mutations has formed the basis for DNA-based prenatal testing and preimplantation genetic diagnosis in families at risk for recurrence (
      • Uitto J.
      Progress in heritable skin diseases: translational implications of mutation analysis and prospects of molecular therapies.
      ). However, in spite of significant progress in identification of the molecular bases of heritable skin diseases, there has been relatively little progress until very recently in developing effective and specific treatments. This synopsis will highlight some of the milestones in progress towards treatment and cure of heritable skin diseases, primarily focusing on epidermolysis bullosa (EB) as a paradigm of such conditions, with emphasis on development of gene-, protein-, and cell-based molecular strategies just entering the clinical arena (
      • Uitto J.
      • Christiano A.M.
      • McLean W.H.
      • et al.
      Novel molecular therapies for heritable skin disorders.
      ).

      Preclinical Model Systems

      EB is a heterogeneous group of heritable blistering disorders due to fragility of the cutaneous basement membrane zone (
      • Bruckner-Tuderman L.
      • Has C.
      Molecular heterogeneity of blistering disorders: the paradigm of epidermolysis bullosa.
      ,
      • Fine J.D.
      • Eady R.A.
      • Bauer E.A.
      • et al.
      The classification of inherited epidermolysis bullosa (EB): report of the Third International Consensus Meeting on Diagnosis and Classification of EB.
      ,
      • Intong L.R.
      • Murrell D.F.
      Inherited epidermolysis bullosa: new diagnostic criteria and classification.
      ). The development of molecular therapies for skin diseases has been largely predicated upon development of animal model systems, particularly transgenic mice that recapitulate their clinical, histopathological, and ultrastructural features (
      • Bruckner-Tuderman L.
      • McGrath J.A.
      • Robinson E.C.
      • et al.
      Animal models of epidermolysis bullosa: update 2010.
      ,
      • Natsuga K.
      • Shinkuma S.
      • Nishie W.
      • et al.
      Animal models of epidermolysis bullosa.
      ). In the case of EB, these include knockout mice with targeted ablation of the corresponding gene, such as those encoding type VII collagen, type XVII collagen, or the subunit polypeptides of laminin 332. Also, identification of naturally occurring spontaneous mutations in mice and other vertebrates has been helpful for development of preclinical approaches. In addition to transgenic animals, human xenograft transplants onto immunocompromised mice have provided useful model systems (
      • Siprashvili Z.
      • Nguyen N.T.
      • Bezchinsky M.Y.
      • et al.
      Long-term type VII collagen restoration to human epidermolysis bullosa skin tissue.
      ). Finally, novel vertebrate model systems, such as zebrafish, have been explored as potential models for heritable skin diseases (
      • Li Q.
      • Frank M.
      • Thisse C.I.
      • et al.
      Zebrafish: a model system to study heritable skin diseases.
      ). Information gleaned from the experiments utilizing preclinical animal models has been critical for the development of gene therapy approaches for EB.

      Prospects of Gene Therapy

      The first Milestone for applying gene therapy for EB took place in 2005, when cultured keratinocyte stem cells with holoclone potential were cultured from the skin of a patient with a junctional form of EB (
      • Mavilio F.
      • Pellegrini G.
      • Ferrari S.
      • et al.
      Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells.
      ). This patient was shown to have mutations in the LAMB3 gene, which encodes the (β3-subunit polypeptide of laminin 332. The patient, a 36-year-old man, had a form of non-Herlitz junctional EB caused by compound heterozygosity for a LAMB3 null mutation and a missense mutation (E210K) that, in addition to the amino acid substitution, disrupts intron splicing. Holoclone-forming cells were not present in the blistering areas of skin, most likely because the continuous proliferative stimulus associated with the chronic wound-healing process had depleted the stem cell pool. However, biopsy from the palm area, which blisters less, if at all, allowed a sufficient number of holoclones to be cultured. The cultured cells were transduced with a Maloney leukemia virus-derived retroviral vector expressing the LAMB3 complementary DNA, and these cells were then used to prepare genetically corrected epidermal grafts in culture (Figure 1). The grafts were transplanted onto selected areas of the patient’s legs, which were prepared for acceptance of the graft by laser ablation of the deficient epidermis. Engraftment was shown to result in synthesis and proper assembly of normal levels of functional laminin 332, which clinically resulted in development of adherent epidermis that remained stable and did not demonstrate blistering (Mavilio et al.,2006). A published 3.5 year follow-up noted that on clinical examination, no blisters were observed in the transplanted area of skin, and the regenerated skin was stable, normal looking, and functionally resilient to mechanical trauma (
      • De Luca M.
      • Pellegrini G.
      • Mavilio F.
      Gene therapy of inherited skin adhesion disorders: a critical overview.
      ). There was no evidence of inflammation, and specific tests carried out 3 and 6 months after the transplantation procedure indicated the absence of both humoral and T cell–mediated cytotoxic immune responses against the transgene product. It should be noted that one of the mutant alleles contained a single point mutation (E210K), which allowed residual expression of some laminin 332. Thus, the introduction of the complementary DNA-derived laminin b3 polypeptide was not recognized as a new protein by the patient’s immune system, and no evidence of rejection has been noted.
      Figure thumbnail gr1
      Figure 1Strategy for anex vivopatient-specific keratinocyte gene therapy. sKeratinocytes cultured from the patient’s skin biopsy are transduced with a vector expressing the transgene, and the transgenic cells are selected and grown into epithelial sheets that can be grafted back to the patient
      (adapted from
      • Tamai K.
      • Kaneda Y.
      • Uitto J.
      Molecular therapies for heritable blistering diseases.
      , with permission from Elsevier).
      Although this study clearly provided proof-of-principle of ex vivo keratinocyte therapy for EB, there have been significant concerns regarding this approach. One of them revolves around the safety of the retroviral vectors used to integrate the transgene into the genome, and specifically, the concern highlights the possibility that random insertion of the transgene results in activation of proto-oncogenes or inactivation of tumor suppressive genes (
      • De Luca M.
      • Pellegrini G.
      • Mavilio F.
      Gene therapy of inherited skin adhesion disorders: a critical overview.
      ). These concerns were raised in response to clinical trials in which introduction of the curative transgene into hematopoietic progenitor cells of patients with X-linked severe combined immunodeficiency caused activation of a T cell proto-oncogene, resulting in leukemia (
      • Hacein-Bey-Abina S.
      • Von Kalle C.
      • Schmidt M.
      • et al.
      LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1.
      ). Although it has been suggested that, on statistical grounds, the probability of such an event to occur is extremely low, and that the retroviral integration into the genome may not be random, these concerns have resulted in the conclusion that the Maloney leukemia virus long-terminal repeat-based vectors are not acceptable for genetic modification of stem cells, and such clinical trials are now banned by the regulatory agencies in many countries in Europe (see
      • De Luca M.
      • Pellegrini G.
      • Mavilio F.
      Gene therapy of inherited skin adhesion disorders: a critical overview.
      ). Nevertheless, there have been significant improvements in vector design, and many of them have potentially a more favorable safety profile due to targeted integration sites and self-inactivating properties (
      • Titeux M.
      • Pendaries V.
      • Zanta-Boussif M.A.
      • et al.
      SIN retroviral vectors expressing COL7A1 under human promoters for ex vivo gene therapy of recessive dystrophic epidermolysis bullosa.
      ). On the basis of these considerations, clinical gene therapy trials focusing on the recessive dystrophic forms of EB have been recently initiated (Dr. Alfred Lane, Stanford University, personal communication).
      In the context of ex vivo keratinocyte gene therapy, the phenomenon known as revertant mosaicism is of considerable interest (
      • Lai-Cheong J.E.
      • McGrath J.A.
      • Uitto J.
      Revertant mosaicism in skin: natural gene therapy.
      ,
      • Pasmooij A.M.
      • Jonkman M.F.
      First symposium on natural gene therapy of the skin.
      ). Revertant mosaicism in skin diseases was originally noted in a patient with a junctional form of EB, where areas of skin were shown to reacquire the wild-type phenotype through naturally occurring mitotic gene conversion (
      • Jonkman M.F.
      • Scheffer H.
      • Stulp R.
      • et al.
      Revertant mosaicism in epidermolysis bullosa caused by mitotic gene conversion.
      ). Thus, revertant mosaicism reflects “natural gene therapy,” which could provide corrected, patient-specific cells for grafting purposes (
      • Gostynski A.
      • Deviaene F.C.
      • Pasmooij A.M.
      • et al.
      Adhesive stripping to remove epidermis in junctional epidermolysis bullosa for revertant cell therapy.
      ).

      RNA Interference Technology

      Another gene therapy approach for treatment of inherited skin diseases utilizes silencing of the mutant RNA by short-interfering RNA (siRNA; Figure 2). Specifically, the safety and efficacy of a siRNA targeting mutant keratin 6a (N171K) has been recently reported in a patient with pachyonychia congenita (PC). The Milestone paper by
      • Leachman S.A.
      • Hickerson R.P.
      • Schwartz M.E.
      • et al.
      First-in-human mutation-targeted siRNA phase Ib trial of an inherited skin disorder.
      describes a patient with PC, a disabling keratinization disorder affecting the skin, nails, oral mucosa, hair, and teeth. This disease is inherited in an autosomal dominant fashion and often leads to painful lesions in the feet characterized by blistering and callus formation on the soles. PC is caused by mutations in either keratin 6, 16, or 17 genes, and the mutant polypeptides cause the clinical manifestation by dominant-negative interference (
      • McLean W.H.
      • Hansen C.D.
      • Eliason M.J.
      • et al.
      The phenotypic and molecular genetic features of pachyonychia congenita.
      ). The approach of this study was to elicit a selective depletion of the mutant keratin by injection of siRNA using a siRNA molecule carefully selected to be mutant-specific and not interfere with the wild-type K6a mRNA.
      Figure thumbnail gr2
      Figure 2Small interfering RNA (siRNA) strategies for autosomal dominant keratin 6a disorders by targeting either mutant or both mutant and wild-type alleles. (a) In normal keratinocytes, synthesis of K6a (blue), K6b (red), and K6c (green) occurs; (b) in pachyonychia congenita keratinocytes with a heterozygous missense mutation in KRT6A, there is a dominant-negative interference between the wild-type and mutant K6a protein that perturbs the entire keratin network and compromises cell integrity, leading to skin blistering as a result of minor trauma; (c) one siRNA approach is to target the mutant KRT6A allele to leave only residual wild-type KRT6A allele expression; (d) an alternative siRNA strategy is to silence all KRT6A, both mutant and wild-type—blistering does not occur in the absence of K6a because of functional redundancy with K6b and K6c, allowing normal intermediate filament network integrity
      (reproduced from
      • Uitto J.
      • Christiano A.M.
      • McLean W.H.
      • et al.
      Novel molecular therapies for heritable skin disorders.
      ).
      This double-blind study consisted of injection of the siRNA to one foot while the other foot received a vehicle–control solution (
      • Leachman S.A.
      • Hickerson R.P.
      • Schwartz M.E.
      • et al.
      First-in-human mutation-targeted siRNA phase Ib trial of an inherited skin disorder.
      ). The treatment consisted of 17 weeks of two times weekly injections and was followed by a 3-month washout. Careful assessment of the clinical signs and symptoms revealed definitive improvement, and the size of the callus in siRNA-injected sites was getting smaller. Thus, this first-in-human double-blinded phase 1b clinical trial suggested the proof-of-principle of RNA interference technology for patients with keratinization disorders.
      This study also highlighted some of the drawbacks of this approach. First, the siRNA was delivered by direct injection to the lesional skin, resulting in intense pain (
      • Leachman S.A.
      • Hickerson R.P.
      • Schwartz M.E.
      • et al.
      First-in-human mutation-targeted siRNA phase Ib trial of an inherited skin disorder.
      ). Consequently, significant efforts are now focusing on improved delivery methods, such as pharmaceutical formulations for topical, non-invasive delivery for PC, and other keratin disorders (
      • Kaspar R.L.
      • McLean W.H.
      • Schwartz M.E.
      Achieving successful delivery of nucleic acids to skin: 6th Annual Meeting of the International Pachyonychia Congenita Consortium.
      ;
      • McLean W.H.
      • Moore C.B.
      Keratin disorders: from gene to therapy.
      ). Another concern is that although there was a dramatic and specific response in the treated area of skin, the improvement appeared to be temporary, and upon discontinuation of the treatment, the lesions returned to their original size. Thus, continuous treatment by improved topical delivery system might be required for this approach (
      • Smith F.J.D.
      • Hickerson R.P.
      • Sayers J.M.
      • et al.
      Development of therapeutic siRNAs for pachyonychia congenita.
      ). Finally, it has been pointed out that one of the disadvantages of the allele–specific gene silencing approach is that the US Food and Drug Administration and corresponding international agencies may consider each mutation-specific siRNA as a separate entity requiring individual toxicity studies and regulatory approval (see
      • Uitto J.
      • Christiano A.M.
      • McLean W.H.
      • et al.
      Novel molecular therapies for heritable skin disorders.
      ). Thus, an alternative approach to the mutant allele-specific ablation, for example, in case of PC, would be total silencing of all KRT6A alleles, both mutant and wild type (Figure 2). It should be noted that the blistering phenotype does not occur in the complete absence of K6a because of functional redundancy with K6b and K6c, allowing normal intermediate filament network integrity. Clinical trials are now being contemplated using these approaches for PC and other keratinization disorders (Dr Irwin McLean, University of Dundee, personal communication).

      Allogeneic Fibroblast Therapy

      The Milestone article by
      • Wong T.
      • Gammon L.
      • Liu L.
      • et al.
      Potential of fibroblast cell therapy for recessive dystrophic epidermolysis bullosa.
      reported on a clinical trial in which the potential for intradermal injections of allogeneic fibroblasts were assessed in individuals with recessive dystrophic EB (RDEB). These studies were based on the premise that human skin fibroblasts, in addition to keratinocytes, express type VII collagen (
      • Stanley J.R.
      • Rubinstein N.
      • Klaus-Kovtun V.
      Epidermolysis bullosa acquisita antigen is synthesized by both human keratinocytes and human dermal fibroblasts.
      ,
      • Chen Y.Q.
      • Mauviel A.
      • Ryynanen J.
      • et al.
      Type VII collagen gene expression by human skin fibroblasts and keratinocytes in culture: influence of donor age and cytokine responses.
      ). Furthermore, previous animal studies have suggested that COL7A1 gene-corrected human RDEB fibroblasts overexpressing type VII collagen, when injected intradermally into immuno-deficient mouse skin or into a transplanted human RDEB skin xenograft, allowed sustained human type VII collagen deposition at the dermal epidermal junction, accompanied by anchoring fibril formation (
      • Woodley D.T.
      • Krueger G.G.
      • Jorgensen C.M.
      • et al.
      Normal and gene-corrected dystrophic epidermolysis bullosa fibroblasts alone can produce type VII collagen at the basement membrane zone.
      ). These observations lead the authors to postulate that injection of a sufficient number of normal fibroblasts could lead to amelioration of the skin phenotype in patients with RDEB.
      Five subjects were injected with cultured autologous fibroblasts into the edge of unblistered skin, and the expression of type VII collagen was evaluated by immunofluorescence in biopsies at 2 weeks and 3 months after a single injection (
      • Wong T.
      • Gammon L.
      • Liu L.
      • et al.
      Potential of fibroblast cell therapy for recessive dystrophic epidermolysis bullosa.
      ). Significant, up to ~2-fold increase in type VII collagen immunostaining was noted at sites injected with parental fibroblasts or from those of unrelated donors. The increased type VII collagen at the dermal–epidermal junction was accompanied with an increase in anchoring fibrils, although these were not fully developed. Nevertheless, this and complementary studies demonstrated the potential of allogeneic fibroblast therapy for treatment of RDEB (
      • Petrova A.
      • Ilic D.
      • McGrath J.A.
      Stem cell therapies for recessive dystrophic epidermolysis bullosa.
      ,
      • Yan W.F.
      • Murrell D.F.
      Fibroblast-based cell therapy strategy for recessive dystrophic epidermolysis bullosa.
      ).
      One of the observations in this study was that the allogeneic cells, as determined by Y chromosome positivity in female patients injected with male fibroblasts, were not detectable 2 weeks after the initial injection, yet type VII collagen deposition continued up to 3 months (
      • Wong T.
      • Gammon L.
      • Liu L.
      • et al.
      Potential of fibroblast cell therapy for recessive dystrophic epidermolysis bullosa.
      ). Although initial observations of the injection sites did not reveal evidence of significant inflammation, subsequent studies suggested a subclinical immunological mechanism by which the allogeneic fibroblasts elicit increased type VII collagen expression in the basal keratinocytes of the recipient’s skin (
      • Nagy N.
      • Almaani N.
      • Tanaka A.
      • et al.
      HBEGF induces COL7A1 expression in keratinocytes and fibroblasts: possible mechanism underlying allogeneic fibroblast therapy in recessive dystrophic epidermolysis bullosa.
      ) (Figure 3). This conclusion was supported by the observation that increased type VII collagen deposition at the dermal–epidermal junction was particularly pronounced in those patients who expressed some type VII collagen at the baseline, and who, therefore, had an enhanced capacity to increase synthesis of their own mutant type VII collagen. These studies identified heparin-binding epidermal growth factor–like growth factor as a novel putative mediator induced in the recipient cells by allogeneic fibroblast injection (Figure 3).
      Figure thumbnail gr3
      Figure 3Postulated mechanism by which fibroblast therapy may ameliorate the blistering tendency in recessive dystrophic epidermolysis bullosa (RDEB). (a) In normal skin, keratinocytes synthesize type VII collagen molecules (red), which assemble into anchoring fibrils. These fibrils entrap the interstitial collagen fibers in the dermis, securing the stable association at the dermal–epidermal junction. (b) In some patients with RDEB, there are only a few rudimentary anchoring fibrils, allowing formation of blisters below the lamina densa as a result of minor trauma. (c) Allogeneic fibroblasts injected directly into dermis elicit a subclinical immune reaction that leads to synthesis of heparin-binding epidermal growth factor–like growth factor (HB–EGF), which upregulates the synthesis and assembly of patient’s own mutated type VII collagen. The increase in the rudimentary anchoring fibrils, which are partially functional, stabilizes the association of epidermis to the underlying dermis and ameliorates the blistering tendency.
      (adapted from
      • Uitto J.
      Cell-based therapy for RDEB: how does it work?.
      )
      Although these initial studies utilized a single injection of allogeneic fibroblasts to the skin, the increase in type VII collagen persisted for several months. This can be explained, in part, by the observation that type VII collagen, once deposited to the cutaneous basement membrane zone, has a relatively long half-life as judged from mouse studies (
      • Kern J.S.
      • Loeckermann S.
      • Fritsch A.
      • et al.
      Mechanisms of fibroblast cell therapy for dystrophic epidermolysis bullosa: high stability of collagen VII favors long-term skin integrity.
      ). Clinical trials utilizing multiple injections at regular intervals are now being contemplated to counteract the development of chronic wounds in patients with RDEB (Dr John McGrath, St John’s Institute of Dermatology, personal communication).

      Bone Marrow Stem Cell Therapy

      Cell-based therapy for EB and potentially other heritable skin diseases has recently been extended to include bone marrow–derived adult stem cells. Although these cells are known to have a critical role in skin homeostasis, it has also become clear that the plasticity of these cells enables their differentiation into cell types responsible for skin regeneration after injury (
      • Badiavas E.V.
      • Abedi M.
      • Butmarc J.
      • et al.
      Participation of bone marrow derived cells in cutaneous wound healing.
      ,
      • Tamai K.
      • Kaneda Y.
      • Uitto J.
      Molecular therapies for heritable blistering diseases.
      ). The Milestone article describing the first clinical trial of allogeneic bone marrow transplantation for EB was reported by
      • Wagner J.E.
      • Ishida-Yamamoto A.
      • McGrath J.A.
      • et al.
      Bone marrow transplantation for recessive dystrophic epidermolysis bullosa.
      . In this study seven children with severe RDEB were enrolled to be recipients of bone marrow transplantation, using standard myeloablative approach (
      • Wagner J.E.
      • Ishida-Yamamoto A.
      • McGrath J.A.
      • et al.
      Bone marrow transplantation for recessive dystrophic epidermolysis bullosa.
      ). This study was predicated on previous animal studies, which have been conducted to evaluate the potential of bone marrow–derived stem cells to treat EB. For example, bone marrow transfer into the fetal circulation of mice that are deficient in type VII collagen resulted in deposition of type VII collagen in the skin, associated with reduction in the severity of the blistering in neonatal animals (
      • Chino T.
      • Tamai K.
      • Yamazaki T.
      • et al.
      Bone marrow cell transfer into fetal circulation can ameliorate genetic skin diseases by providing fibroblasts to the skin and inducing immune tolerance.
      ). Also, hematopoietic and non-hematopoetic cell populations were infused into type VII collagen knockout mice at birth, and this treatment was shown to extend the survival of the recipient mice by several weeks or months (
      • Tolar J.
      • Ishida-Yamamoto A.
      • Riddle M.
      • et al.
      Amelioration of epidermolysis bullosa by transfer of wild-type bone marrow cells.
      ). The EB phenotype was also rescued in a type XVII collagen knockout model by bone marrow transplantation (
      • Fujita Y.
      • Abe R.
      • Inokuma D.
      • et al.
      Bone marrow transplantation restores epidermal basement membrane protein expression and rescues epidermolysis bullosa model mice.
      ). Although type VII collagen is synthesized both by keratinocytes and fibroblasts, type XVII collagen, a component of hemidesmosomes, is synthesized exclusively by keratinocytes. Collectively, these studies demonstrated that different bone marrow–derived stem cells, including mesenchymal stem cells, can ameliorate the clinical symptoms and increase the survival rate of EB mice, thus paving a way to clinical trials in patients with different forms of EB.
      Bone marrow transplantation in the children with RDEB was noted to result in synthesis of new type VII collagen and clinical improvement that was sustained for at least 1 year after the transplantation (
      • Wagner J.E.
      • Ishida-Yamamoto A.
      • McGrath J.A.
      • et al.
      Bone marrow transplantation for recessive dystrophic epidermolysis bullosa.
      ;
      • Tolar J.
      • Blazar B.R.
      • Wagner J.E.
      Concise review: transplantation of human hematopoietic cells for extracellular matrix protein deficiency in epidermolysis bullosa.
      ). Although these preliminary studies were promising and generated cautious optimism, it should be noted that two of the seven children died as a result of complications of the bone marrow transplant procedure, which utilized traditional chemoablative preconditioning of the recipients. A second clinical bone marrow transplantation trial has been initiated with the approach to use reduced-intensity chemotherapy before transplantation, perhaps having lesser side effects with reduced morbidity and mortality (
      • Kiuru M.
      • Itoh M.
      • Cairo M.S.
      • et al.
      Bone marrow stem cell therapy for recessive dystrophic epidermolysis bullosa.
      ).
      In addition to bone marrow transplantation, a pilot study on two patients with severe RDEB has tested the efficacy of intradermal injection of allogeneic mesenchymal stem cells into chronic ulcerative sites in these patients (
      • Conget P.
      • Rodriguez F.
      • Kramer S.
      • et al.
      Replenishment of type VII collagen and re-epithelialization of chronically ulcerated skin after intradermal administration of allogeneic mesenchymal stromal cells in two patients with recessive dystrophic epidermolysis bullosa.
      ). Improved wound healing lasting up to 4 months was attributed to replenishment of type VII collagen, which was undetectable before the procedure. Similar studies are now being developed to examine whether intradermal or intravenous injection of bone marrow–derived mesenchymal stem cells can improve the clinical outcome. Collectively, these early observations support the usefulness of bone marrow stem cell populations in the treatment of heritable skin diseases, such as RDEB.

      Novel Therapeutic Approaches

      A number of new technologies are currently being developed for potential treatment of EB, but these have not reached the clinical trial stage as yet. One of such approaches focuses on induced pluripotent stem cells (iPSCs), which allow patient-specific cells to be corrected for the gene defect, followed by introduction of differentiated fibroblast and/or keratinocytes to the skin (Uitto et al.,2012) (Figure 4). This approach would circumvent the difficulties in obtaining sufficient numbers of patient-specific cells and avoid the problem of immune rejection. Recently, patient-specific iPSCs have been generated from several human diseases to investigate the disease mechanisms, test potential drugs, and develop cell-based therapies. In the case of heritable skin diseases, patient-specific iPSCs have been generated from patients with dyskeratosis congenita as well as RDEB (
      • Agarwal S.
      • Loh Y.H.
      • McLoughlin E.M.
      • et al.
      Telomere elongation in induced pluripotent stem cells from dyskeratosis congenita patients.
      ,
      • Itoh M.
      • Kiuru M.
      • Cairo M.S.
      • et al.
      Generation of keratinocytes from normal and recessive dystrophic epidermolysis bullosa-induced pluripotent stem cells.
      ;
      • Tolar J.
      • Xia L.
      • Riddle M.J.
      • et al.
      Induced pluripotent stem cells from individuals with recessive dystrophic epidermolysis bullosa.
      ). Although a number of technological issues still need to be resolved before iPSC-based therapy can be moved to the clinic, there is rapid technological progress in this area, and the first report of gene correction utilizing patient-specific iPSCs has already been published in a case with gyrate atrophy (
      • Howden S.E.
      • Gore A.
      • Li Z.
      • et al.
      Genetic correction and analysis of induced pluripotent stem cells from a patient with gyrate atrophy.
      ), providing a proof-of-concept for this technology.
      Figure thumbnail gr4
      Figure 4Schematic steps of reprogramming somatic cells, such as fibroblasts, to induced pluripotent stem (iPS) cells, and their differentiation into epidermal keratinocytes capable of forming skin-like structures. The reprogramming process is initiated by introduction of transcription factors (cMYC, SOX2, OCT4, and KLF4) into the somatic cells by transduction of expression vectors, synthetic mRNA, or recombinant protein. The iPS cells have characteristic features that allow their identification and enrichment. The iPS cells can then be differentiated into keratinocytes under specific culture conditions, e.g., medium supplemented with retinoic acid (RA) and bone morphogenic protein-4 (BMP4). BMZ, basement membrane zone.
      (adapted from
      • Uitto J.
      Regenerative medicine for skin diseases: iPS cells to the rescue.
      )
      Another approach to counteract the clinical manifestations of EB, which has not reached the clinical trial stage yet, is potential protein-replacement therapy. This concept is again predicated on the use of Col7a1 knockout mice as a platform, which demonstrated that infusion of a purified type VII collagen results in significantly reduced blistering and extends the lifespan of these mice (
      • Remington J.
      • Wang X.
      • Hou Y.
      • et al.
      Injection of recombinant human type VII collagen corrects the disease phenotype in a murine model of dystrophic epidermolysis bullosa.
      ). Again, once the type VII collagen has been properly deposited on the cutaneous basement membrane zone, the protein remains stably assembled for several months. Clinical trials utilizing GMP-purified type VII collagen are currently contemplated for patients with severe RDEB (Dr David Woodley, University of Southern California, personal communication).
      Collectively, regenerative medicine for heritable skin diseases is moving very rapidly, and with novel technological innovations the field is making progress towards treatment and cure of these, currently intractable disorders.

      Conflict of Interest

      The author states no conflict of interest.

      Acknowledgments

      The author thanks Angela Christiano, Irwin McLean, and John McGrath for helpful discussions. This study was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases.

      References

        • Agarwal S.
        • Loh Y.H.
        • McLoughlin E.M.
        • et al.
        Telomere elongation in induced pluripotent stem cells from dyskeratosis congenita patients.
        Nature. 2010; 464: 292-296
        • Badiavas E.V.
        • Abedi M.
        • Butmarc J.
        • et al.
        Participation of bone marrow derived cells in cutaneous wound healing.
        J Cell Physiol. 2003; 196: 245-250
        • Bruckner-Tuderman L.
        • Has C.
        Molecular heterogeneity of blistering disorders: the paradigm of epidermolysis bullosa.
        J Invest Dermatol. 2012; 132: E2-E5
        • Bruckner-Tuderman L.
        • McGrath J.A.
        • Robinson E.C.
        • et al.
        Animal models of epidermolysis bullosa: update 2010.
        J Invest Dermatol. 2010; 130: 1485-1488
        • Chen Y.Q.
        • Mauviel A.
        • Ryynanen J.
        • et al.
        Type VII collagen gene expression by human skin fibroblasts and keratinocytes in culture: influence of donor age and cytokine responses.
        J Invest Dermatol. 1994; 102: 205-209
        • Chino T.
        • Tamai K.
        • Yamazaki T.
        • et al.
        Bone marrow cell transfer into fetal circulation can ameliorate genetic skin diseases by providing fibroblasts to the skin and inducing immune tolerance.
        Am J Pathol. 2008; 173: 803-814
        • Conget P.
        • Rodriguez F.
        • Kramer S.
        • et al.
        Replenishment of type VII collagen and re-epithelialization of chronically ulcerated skin after intradermal administration of allogeneic mesenchymal stromal cells in two patients with recessive dystrophic epidermolysis bullosa.
        Cytotherapy. 2010; 12: 429-431
        • De Luca M.
        • Pellegrini G.
        • Mavilio F.
        Gene therapy of inherited skin adhesion disorders: a critical overview.
        Br J Dermatol. 2009; 161: 19-24
        • Feramisco J.D.
        • Sadreyev R.I.
        • Murray M.L.
        • et al.
        Phenotypic and genotypic analyses of genetic skin disease through the Online Mendelian Inheritance in Man (OMIM) database.
        J Invest Dermatol. 2009; 129: 2628-2636
        • Fine J.D.
        • Eady R.A.
        • Bauer E.A.
        • et al.
        The classification of inherited epidermolysis bullosa (EB): report of the Third International Consensus Meeting on Diagnosis and Classification of EB.
        J Am Acad Dermatol. 2008; 58: 931-950
        • Fujita Y.
        • Abe R.
        • Inokuma D.
        • et al.
        Bone marrow transplantation restores epidermal basement membrane protein expression and rescues epidermolysis bullosa model mice.
        Proc Natl Acad Sci USA. 2010; 107: 14345-14350
        • Gostynski A.
        • Deviaene F.C.
        • Pasmooij A.M.
        • et al.
        Adhesive stripping to remove epidermis in junctional epidermolysis bullosa for revertant cell therapy.
        Br J Dermatol. 2009; 161: 444-447
        • Hacein-Bey-Abina S.
        • Von Kalle C.
        • Schmidt M.
        • et al.
        LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1.
        Science. 2003; 302: 415-419
        • Howden S.E.
        • Gore A.
        • Li Z.
        • et al.
        Genetic correction and analysis of induced pluripotent stem cells from a patient with gyrate atrophy.
        Proc Natl Acad Sci USA. 2011; 108: 6537-6542
        • Intong L.R.
        • Murrell D.F.
        Inherited epidermolysis bullosa: new diagnostic criteria and classification.
        Clin Dermatol. 2012; 30: 70-77
        • Itoh M.
        • Kiuru M.
        • Cairo M.S.
        • et al.
        Generation of keratinocytes from normal and recessive dystrophic epidermolysis bullosa-induced pluripotent stem cells.
        Proc Natl Acad Sci USA. 2011; 108: 8797-8802
        • Jonkman M.F.
        • Scheffer H.
        • Stulp R.
        • et al.
        Revertant mosaicism in epidermolysis bullosa caused by mitotic gene conversion.
        Cell. 1997; 88: 543-551
        • Kaspar R.L.
        • McLean W.H.
        • Schwartz M.E.
        Achieving successful delivery of nucleic acids to skin: 6th Annual Meeting of the International Pachyonychia Congenita Consortium.
        J Invest Dermatol. 2009; 129: 2085-2087
        • Kern J.S.
        • Loeckermann S.
        • Fritsch A.
        • et al.
        Mechanisms of fibroblast cell therapy for dystrophic epidermolysis bullosa: high stability of collagen VII favors long-term skin integrity.
        Mol Ther. 2009; 17: 1605-1615
        • Kiuru M.
        • Itoh M.
        • Cairo M.S.
        • et al.
        Bone marrow stem cell therapy for recessive dystrophic epidermolysis bullosa.
        Dermatol Clin. 2010; 28: 371-382
        • Lai-Cheong J.E.
        • McGrath J.A.
        • Uitto J.
        Revertant mosaicism in skin: natural gene therapy.
        Trends Mol Med. 2011; 7: 140-148
        • Leachman S.A.
        • Hickerson R.P.
        • Schwartz M.E.
        • et al.
        First-in-human mutation-targeted siRNA phase Ib trial of an inherited skin disorder.
        Mol Ther. 2010; 18: 442-446
        • Li Q.
        • Frank M.
        • Thisse C.I.
        • et al.
        Zebrafish: a model system to study heritable skin diseases.
        J Invest Dermatol. 2011; 131: 565-571
        • Mavilio F.
        • Pellegrini G.
        • Ferrari S.
        • et al.
        Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells.
        Nat Med. 2006; 12: 1397-1402
        • McLean W.H.
        • Hansen C.D.
        • Eliason M.J.
        • et al.
        The phenotypic and molecular genetic features of pachyonychia congenita.
        J Invest Dermatol. 2011; 131: 1015-1017
        • McLean W.H.
        • Moore C.B.
        Keratin disorders: from gene to therapy.
        Hum Mol Genet. 2011; 20: R189-R197
        • Nagy N.
        • Almaani N.
        • Tanaka A.
        • et al.
        HBEGF induces COL7A1 expression in keratinocytes and fibroblasts: possible mechanism underlying allogeneic fibroblast therapy in recessive dystrophic epidermolysis bullosa.
        J Invest Dermatol. 2011; 131: 1771-1774
        • Natsuga K.
        • Shinkuma S.
        • Nishie W.
        • et al.
        Animal models of epidermolysis bullosa.
        Dermatol Clin. 2010; 28: 137-142
        • Pasmooij A.M.
        • Jonkman M.F.
        First symposium on natural gene therapy of the skin.
        Exp Dermatol. 2012; 21: 236-239
        • Petrova A.
        • Ilic D.
        • McGrath J.A.
        Stem cell therapies for recessive dystrophic epidermolysis bullosa.
        Br J Dermatol. 2010; 163: 1149-1156
        • Remington J.
        • Wang X.
        • Hou Y.
        • et al.
        Injection of recombinant human type VII collagen corrects the disease phenotype in a murine model of dystrophic epidermolysis bullosa.
        Mol Ther. 2009; 17: 26-33
        • Siprashvili Z.
        • Nguyen N.T.
        • Bezchinsky M.Y.
        • et al.
        Long-term type VII collagen restoration to human epidermolysis bullosa skin tissue.
        Hum Gene Ther. 2010; 21: 1299-1310
        • Smith F.J.D.
        • Hickerson R.P.
        • Sayers J.M.
        • et al.
        Development of therapeutic siRNAs for pachyonychia congenita.
        J Invest Dermatol. 2008; 128: 50-58
        • Stanley J.R.
        • Rubinstein N.
        • Klaus-Kovtun V.
        Epidermolysis bullosa acquisita antigen is synthesized by both human keratinocytes and human dermal fibroblasts.
        J Invest Dermatol. 1985; 85: 542-545
        • Tamai K.
        • Kaneda Y.
        • Uitto J.
        Molecular therapies for heritable blistering diseases.
        Trends Mol Med. 2009; 15: 285-292
        • Titeux M.
        • Pendaries V.
        • Zanta-Boussif M.A.
        • et al.
        SIN retroviral vectors expressing COL7A1 under human promoters for ex vivo gene therapy of recessive dystrophic epidermolysis bullosa.
        Mol Ther. 2010; 18: 1509-1518
        • Tolar J.
        • Blazar B.R.
        • Wagner J.E.
        Concise review: transplantation of human hematopoietic cells for extracellular matrix protein deficiency in epidermolysis bullosa.
        Stem Cells. 2011; 29: 900-906
        • Tolar J.
        • Ishida-Yamamoto A.
        • Riddle M.
        • et al.
        Amelioration of epidermolysis bullosa by transfer of wild-type bone marrow cells.
        Blood. 2009; 113: 1167-1174
        • Tolar J.
        • Xia L.
        • Riddle M.J.
        • et al.
        Induced pluripotent stem cells from individuals with recessive dystrophic epidermolysis bullosa.
        J Invest Dermatol. 2011; 131: 848-856
        • Uitto J.
        Progress in heritable skin diseases: translational implications of mutation analysis and prospects of molecular therapies.
        Acta Derm Venereol. 2009; 89: 228-235
        • Uitto J.
        Cell-based therapy for RDEB: how does it work?.
        J Invest Dermatol. 2011; 131: 1597-1599
        • Uitto J.
        Regenerative medicine for skin diseases: iPS cells to the rescue.
        J Invest Dermatol. 2011; 131: 812-814
        • Uitto J.
        • Christiano A.M.
        • McLean W.H.
        • et al.
        Novel molecular therapies for heritable skin disorders.
        J Invest Dermatol. 2012; 132: 820-828
        • Wagner J.E.
        • Ishida-Yamamoto A.
        • McGrath J.A.
        • et al.
        Bone marrow transplantation for recessive dystrophic epidermolysis bullosa.
        N Engl J Med. 2010; 363: 629-639
        • Wong T.
        • Gammon L.
        • Liu L.
        • et al.
        Potential of fibroblast cell therapy for recessive dystrophic epidermolysis bullosa.
        J Invest Dermatol. 2008; 128: 2179-2189
        • Woodley D.T.
        • Krueger G.G.
        • Jorgensen C.M.
        • et al.
        Normal and gene-corrected dystrophic epidermolysis bullosa fibroblasts alone can produce type VII collagen at the basement membrane zone.
        J Invest Dermatol. 2003; 121: 1021-1028
        • Yan W.F.
        • Murrell D.F.
        Fibroblast-based cell therapy strategy for recessive dystrophic epidermolysis bullosa.
        Dermatol Clin. 2010; 28: 367-370