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Gene Therapy for Epidermolysis Bullosa

  • M. Peter Marinkovich
    Correspondence
    Correspondence: M. Peter Marinkovich, 269 Campus Drive, Room 2145a, Stanford, California 94305, USA.
    Affiliations
    Department of Dermatology, Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California, USA

    Department of Dermatology, Palo Alto Veterans Affairs Medical Center, Palo Alto, California, USA
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  • Jean Y. Tang
    Affiliations
    Department of Dermatology, Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California, USA
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Open ArchivePublished:May 05, 2019DOI:https://doi.org/10.1016/j.jid.2018.11.036
      Epidermolysis bullosa is a family of diseases characterized by blistering and fragility of the skin in response to mechanical trauma. Advances in our understanding of epidermolysis bullosa pathophysiology have provided the necessary foundation for the first clinical trials of gene therapy for junctional and dystrophic epidermolysis bullosa. These therapies show that gene therapy is both safe and effective, with the potential to correct the molecular and clinical phenotype of patients with epidermolysis bullosa. Improvements in gene delivery and in preventing immune reactions will be among the challenges that lie ahead during further therapeutic development.

      Abbreviations:

      C7 (type VII collagen), EB (epidermolysis bullosa), DEB (dystrophic epidermolysis bullosa), JEB (junctional epidermolysis bullosa), RDEB (recessive dystrophic epidermolysis bullosa)

      Background

      The epidermolysis bullosa (EB) family of inherited diseases is characterized by blistering in response to mechanical trauma. EB can produce painful wounds and erosions in skin, eyes, and mucosal tissues; can be mild or severe; and can heal with severe scarring or no scarring at all. EB subtypes display such marked variation in severity, prognosis, extracutaneous involvement, and inheritance that, after the clinical discovery of EB (

      Hebra FV. Arztlicher Bericht des K.K allegemeinen Krankenhauses zu Wien vom Jare 1870. Vienna, Austria; 1870.

      ), well over a century passed before a basic molecular understanding of EB was developed.
      Structural basement membrane alterations in EB subtypes seen by electron microscopy (
      • Palade G.E.
      • Farquar M.G.
      A special fibril of the dermis.
      ,
      • Pearson R.W.
      Studies on the pathogenesis of epidermolysis bullosa.
      ) provided initial mechanistic clues. Abnormal intermediate filaments seen in EB simplex eventually led to the discovery of EB simplex-associated mutations in genes coding for intermediate filament proteins keratin 5 and 14 (
      • Bonifas J.M.
      • Rothman A.L.
      • Epstein Jr., E.H.
      Epidermolysis bullosa simplex: evidence in two families for keratin gene abnormalities.
      ,
      • Coulombe P.A.
      • Hutton M.E.
      • Letai A.
      • Hebert A.
      • Paller A.
      • Fuchs E.
      Point mutations in human keratin 14 genes of epidermolysis bullosa simplex patients: genetic and functional analyses.
      ). Anchoring filament abnormalities seen by electron microscopy in junctional EB (JEB) skin, followed by the discovery of the anchoring filament protein laminin 332 (L332) (
      • Marinkovich M.P.
      • Lunstrum G.P.
      • Keene D.R.
      • Burgeson R.E.
      The dermal-epidermal junction of human skin contains a novel laminin variant.
      ,
      • Rousselle P.
      • Lunstrum G.P.
      • Keene D.R.
      • Burgeson R.E.
      Kalinin: an epithelium-specific basement membrane adhesion molecule that is a component of anchoring filaments.
      ) and its absence in JEB tissues (
      • Marinkovich M.P.
      • Verrando P.
      • Keene D.R.
      • Meneguzzi G.
      • Lunstrum G.P.
      • Ortonne J.P.
      • et al.
      Basement membrane proteins kalinin and nicein are structurally and immunologically identical.
      ,
      • Meneguzzi G.
      • Marinkovich M.P.
      • Aberdam D.
      • Pisani A.
      • Burgeson R.
      • Ortonne J.P.
      Kalinin is abnormally expressed in epithelial basement membranes of Herlitz’s junctional epidermolysis bullosa patients.
      ), set the stage for the discovery of JEB-associated mutations in L332 genes (
      • Uitto J.
      • McGrath J.A.
      • Pulkkinen L.
      • Christiano A.M.
      Molecular basis of the junctional forms of epidermolysis bullosa, a disorder of the cutaneous basement membrane zone.
      ). Identification of anchoring fibril deficiencies in dystrophic EB (DEB), the discovery of the anchoring fibril component type VII collagen (C7) (
      • Burgeson R.E.
      • Morris N.P.
      • Murray L.W.
      • Duncan K.G.
      • Keene D.R.
      • Sakai L.Y.
      The structure of type VII collagen.
      ,
      • Keene D.R.
      • Sakai L.Y.
      • Lunstrum G.P.
      • Morris N.P.
      • Burgeson R.E.
      Type VII collagen forms an extended network of anchoring fibrils.
      ), the discovery of C7’s absence in recessive DEB (RDEB) skin (
      • Bruckner-Tuderman L.
      • Mitsuhashi Y.
      • Schnyder U.W.
      • Bruckner P.
      Anchoring fibrils and type VII collagen are absent from skin in severe recessive dystrophic epidermolysis bullosa.
      ), and the molecular cloning of the C7 COL7A1 gene (
      • Parente M.G.
      • Chung L.C.
      • Ryynanen J.
      • Uitto J.
      Human type VII collagen: cDNA cloning and chromosomal mapping of the gene (COL7A1) on chromosome 3 to dominant dystrophic epidermolysis bullosa.
      ) led to the discovery of DEB-associated COL7A1 mutations (
      • Christiano A.M.
      • Greenspan D.S.
      • Hoffman G.G.
      • Zhang X.
      • Tamai Y.
      • Lin A.N.
      • et al.
      A missense mutation in type VII collagen in two affected siblings with recessive dystrophic epidermolysis bullosa.
      ,
      • Hilal L.
      • Rochat A.
      • Duquesnoy P.
      • Blanchet-Bardon C.
      • Wechsler J.
      • Martin N.
      • et al.
      A homozygous insertion-deletion in the type VII collagen gene (COL7A1) in Hallopeau-Siemens dystrophic epidermolysis bullosa.
      ). Distinct molecular defects were later found in other rare EB simplex and JEB subtypes (
      • Has C.
      • Nystrom A.
      • Saeidian A.H.
      • Bruckner-Tuderman L.
      • Uitto J.
      Epidermolysis bullosa: molecular pathology of connective tissue components in the cutaneous basement membrane zone.
      ,
      • McGrath J.A.
      Recently identified forms of epidermolysis bullosa.
      ). Through all of these efforts, the molecular basis of the vast majority of EB subtypes was established.
      Once the therapeutic EB gene targets were identified, cell-based delivery methods were needed. One of the first was a pioneering study of two JEB patients with chronic facial wounds in whom healing was observed after the transfer of autologous patient keratinocytes cultured atop collagen sponges (
      • Carter D.M.
      • Lin A.N.
      • Varghese M.C.
      • Caldwell D.
      • Pratt L.A.
      • Eisinger M.
      Treatment of junctional epidermolysis bullosa with epidermal autografts.
      ). Allogeneic tissue engineered with neonatal keratinocytes and fibroblasts (
      • Falabella A.F.
      • Valencia I.C.
      • Eaglstein W.H.
      • Schachner L.A.
      Tissue-engineered skin (Apligraf) in the healing of patients with epidermolysis bullosa wounds.
      ,
      • Fivenson D.P.
      • Scherschun L.
      • Choucair M.
      • KuKuruga D.
      • Young J.
      • Shwayder T.
      Graftskin therapy in epidermolysis bullosa.
      ,
      • Phillips J.
      • Rockwell W.B.
      Surgical treatment of recessive dystrophic epidermolysis bullosa in the hand: use of tissue-engineered skin (Apligraf).
      ), allogeneic fibroblasts (
      • Petrof G.
      • Martinez-Queipo M.
      • Mellerio J.E.
      • Kemp P.
      • McGrath J.A.
      Fibroblast cell therapy enhances initial healing in recessive dystrophic epidermolysis bullosa wounds: results of a randomized, vehicle-controlled trial.
      ,
      • Venugopal S.S.
      • Yan W.
      • Frew J.W.
      • Cohn H.I.
      • Rhodes L.M.
      • Tran K.
      • et al.
      A phase II randomized vehicle-controlled trial of intradermal allogeneic fibroblasts for recessive dystrophic epidermolysis bullosa.
      ), and allogeneic mesenchymal stem cells (
      • Conget P.
      • Rodriguez F.
      • Kramer S.
      • Allers C.
      • Simon V.
      • Palisson F.
      • 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.
      ,
      • El-Darouti M.
      • Fawzy M.
      • Amin I.
      • Abdel Hay R.
      • Hegazy R.
      • Gabr H.
      • et al.
      Treatment of dystrophic epidermolysis bullosa with bone marrow non-hematopoeitic stem cells: a randomized controlled trial.
      ,
      • Petrof G.
      • Lwin S.M.
      • Martinez-Queipo M.
      • Abdul-Wahab A.
      • Tso S.
      • Mellerio J.E.
      • et al.
      Potential of systemic allogeneic mesenchymal stromal cell therapy for children with recessive dystrophic epidermolysis bullosa.
      ) each showed favorable short-term effects on wound healing after transfer to EB patient tissues. Allogeneic cell therapies appear to exert their favorable effects on wound healing primarily through modulation of cytokines and growth factors in the skin (
      • Qi Y.
      • Jiang D.
      • Sindrilaru A.
      • Stegemann A.
      • Schatz S.
      • Treiber N.
      • et al.
      TSG-6 released from intradermally injected mesenchymal stem cells accelerates wound healing and reduces tissue fibrosis in murine full-thickness skin wounds.
      ,
      • Ren G.
      • Zhang L.
      • Zhao X.
      • Xu G.
      • Zhang Y.
      • Roberts A.I.
      • et al.
      Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide.
      ).
      Bone marrow transplantation prevents rejection and allows the potential for long-term persistence of allogeneic cells in recipient tissues. In this approach, RDEB patients are treated with a high dose immunoablative chemotherapy regimen before whole bone marrow transplantation from a tissue-matched donor (
      • Wagner J.E.
      • Ishida-Yamamoto A.
      • McGrath J.A.
      • Hordinsky M.
      • Keene D.R.
      • Riddle M.J.
      • et al.
      Bone marrow transplantation for recessive dystrophic epidermolysis bullosa.
      ). Bone marrow-derived cells of donor origin were detected in patient wounds, showing C7 expression detected by immunofluorescence microscopy in recipient skin. Clinical improvement was also seen; however, anchoring fibrils were not shown in recipient skin, and as a result, correlation between molecular correction and clinical improvement was incomplete.
      Further characterization of bone marrow-derived C7-expressing cells remains an interesting question, as is their presence in skin, which appears to involve a high mobility group box protein, HMGB-1 (
      • Tamai K.
      • Yamazaki T.
      • Chino T.
      • Ishii M.
      • Otsuru S.
      • Kikuchi Y.
      • et al.
      PDGFRα-positive cells in bone marrow are mobilized by high mobility group box 1 (HMGB1) to regenerate injured epithelia.
      ). Despite the positive findings, major risks were noted with this procedure. Of the seven patients who initially entered the trial, one died before receiving a transplant, likely due to cyclophosphamide cardiotoxicity, and another died after transplantation from infections associated with graft failure (
      • Wagner J.E.
      • Ishida-Yamamoto A.
      • McGrath J.A.
      • Hordinsky M.
      • Keene D.R.
      • Riddle M.J.
      • et al.
      Bone marrow transplantation for recessive dystrophic epidermolysis bullosa.
      ). Bone marrow replacement was unable to favorably affect two patients with severe generalized JEB, with each patient dying between 3 and 5 months after therapy (
      • Hammersen J.
      • Has C.
      • Naumann-Bartsch N.
      • Stachel D.
      • Kiritsi D.
      • Soder S.
      • et al.
      Genotype, clinical course, and therapeutic decision making in 76 infants with severe generalized junctional epidermolysis bullosa.
      ). Thus, the challenge remaining with this mode of therapy is to further reduce the serious risks it poses to patients.

      Gene Therapy for JEB

      Early preclinical JEB studies used an approach of retroviral ex vivo gene transfer to primary patient keratinocytes, which were then constructed into skin equivalents and xenografted to immunodeficient mice (
      • Robbins P.B.
      • Sheu S.M.
      • Goodnough J.B.
      • Khavari P.A.
      Impact of laminin 5 β3 gene versus protein replacement on gene expression patterns in junctional epidermolysis bullosa.
      ). Similar studies of in vivo correction of BP180/collagen XVII-deficient primary engineered JEB skin were performed after retroviral transfer of COL17 cDNA and xenografting (
      • Seitz C.S.
      • Giudice G.J.
      • Balding S.D.
      • Marinkovich M.P.
      • Khavari P.A.
      BP180 gene delivery in junctional epidermolysis bullosa.
      ). Viral-mediated gene transfer has the advantage of high durability because chromosomal insertion of the therapeutic gene. However, because insertion is random, it could modulate expression of a gene influencing neoplasia development. This was shown in patients with retroviral therapy for X-linked severe combined immunodeficiency disease (
      • Hacein-Bey-Abina S.
      • von Kalle C.
      • Schmidt M.
      • Le Deist F.
      • Wulffraat N.
      • McIntyre E.
      • et al.
      A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency.
      ,
      • Hacein-Bey-Abina S.
      • Von Kalle C.
      • Schmidt M.
      • McCormack M.P.
      • Wulffraat N.
      • Leboulch P.
      • et al.
      LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1.
      ) but not in patients with immunodeficiency associated with adenosine deaminase deficiency (
      • Aiuti A.
      • Cattaneo F.
      • Galimberti S.
      • Benninghoff U.
      • Cassani B.
      • Callegaro L.
      • et al.
      Gene therapy for immunodeficiency due to adenosine deaminase deficiency.
      ). These results stimulated exploration of nonviral gene transfer methods including transposon, integrase (
      • Ortiz-Urda S.
      • Lin Q.
      • Yant S.R.
      • Keene D.
      • Kay M.A.
      • Khavari P.A.
      Sustainable correction of junctional epidermolysis bullosa via transposon-mediated nonviral gene transfer.
      ,
      • Ortiz-Urda S.
      • Thyagarajan B.
      • Keene D.R.
      • Lin Q.
      • Calos M.P.
      • Khavari P.A.
      φC31 integrase-mediated nonviral genetic correction of junctional epidermolysis bullosa.
      ), and adeno-associated virus (
      • Melo S.P.
      • Lisowski L.
      • Bashkirova E.
      • Zhen H.H.
      • Chu K.
      • Keene D.R.
      • et al.
      Somatic correction of junctional epidermolysis bullosa by a highly recombinogenic AAV variant.
      ). Clustered regularly interspaced short palindromic repeats (i.e., CRISPR)/Cas9- and transcription activator-like effector nuclease (TALEN)-based gene correction methodologies have also become active areas of exploration in EB therapy (
      • Osborn M.J.
      • Starker C.G.
      • McElroy A.N.
      • Webber B.R.
      • Riddle M.J.
      • Xia L.
      • et al.
      TALEN-based gene correction for epidermolysis bullosa.
      ,
      • Osborn M.J.
      • Lees C.J.
      • McElroy A.N.
      • Merkel S.C.
      • Eide C.R.
      • Mathews W.
      • et al.
      CRISPR/Cas9-based cellular engineering for targeted gene overexpression.
      ,
      • Webber B.R.
      • Osborn M.J.
      • McElroy A.N.
      • Twaroski K.
      • Lonetree C.L.
      • DeFeo A.P.
      • et al.
      CRISPR/Cas9-based genetic correction for recessive dystrophic epidermolysis bullosa.
      ). Despite these new areas of study, ex vivo retroviral therapy has remained of high interest because of the efficiency of gene transfer to primary cells. Nevertheless, because the efficiency of gene transfer is high, and because insertional oncogenesis on the skin would be more easily detected early and excised surgically, compared with leukemia, ex vivo viral-based gene transfer in the skin proved to be the first method of gene therapy to be extended to clinical trials.
      The first clinical demonstration of ex vivo retroviral medicated genetic correction of JEB was performed on the upper right leg of an adult JEB patient with a LAMB3 mutation (
      • 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.
      ). The researchers adapted a method of autologous epidermal sheet transfer used to treat burn patients (
      • Boyce S.T.
      • Kagan R.J.
      • Greenhalgh D.G.
      • Warner P.
      • Yakuboff K.P.
      • Palmieri T.
      • et al.
      Cultured skin substitutes reduce requirements for harvesting of skin autograft for closure of excised, full-thickness burns.
      ) to graft the culture to patient skin, adding an ex vivo retroviral LAMB3 gene transfer step, delivering full-length, wild-type LAMB3 to patient keratinocytes. The grafted area showed both clinical improvement and restoration of laminin 332 expression. A later follow-up of the patient showed continued clinical benefits and laminin 332 expression of the transgene for more than 6.5 years (
      • 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.
      ).
      Successful gene therapy for JEB was later shown in a 49-year-old woman (
      • Bauer J.W.
      • Koller J.
      • Murauer E.M.
      • De Rosa L.
      • Enzo E.
      • Carulli S.
      • et al.
      Closure of a large chronic wound through transplantation of gene-corrected epidermal stem cells.
      ) and a 7-year-old boy (
      • Hirsch T.
      • Rothoeft T.
      • Teig N.
      • Bauer J.W.
      • Pellegrini G.
      • De Rosa L.
      • et al.
      Regeneration of the entire human epidermis using transgenic stem cells.
      ), both with LAMB3 mutations. In the latter instance, 80% of the total skin area was grafted with genetically engineered sheets overexpressing an ex vivo, retrovirally delivered, full-length LAMB3 transgene. The child had suffered a major loss of epidermis due to a severe infection, and replacement of skin was possibly lifesaving. Laminin 332 expression in the dermal-epidermal junction was maintained out to 21 months. Integration profile measurements of patient keratinocyte cultures taken from skin biopsy samples over an 8-month period showed a gradual trend toward reduction of transgene-containing meroclones and paraclones and an increase in transgene-containing holoclones, suggesting the successful targeting and maintenance of epidermal stem cell LAMB3 expression.
      The success of these two case studies can be attributed to several factors. First, the group monitored holoclone stem cells (
      • Barrandon Y.
      • Green H.
      Three clonal types of keratinocyte with different capacities for multiplication.
      ), giving an indirect indication of the efficiency of stem cell targeting, which can be used to gauge durability. A second reason for success lies in the key role that laminin 332 plays in mediating keratinocyte adhesion. Laminin 332 is the major epidermal adhesive ligand, and deprived of laminin 332, keratinocytes were rounded up and detached from the culture surface (
      • Rousselle P.
      • Lunstrum G.P.
      • Keene D.R.
      • Burgeson R.E.
      Kalinin: an epithelium-specific basement membrane adhesion molecule that is a component of anchoring filaments.
      ). Retroviral LAMB3 transduction thus provides laminin 332-expressing patient cells with a selective in vitro adhesive advantage over nontransduced patient cells during culture expansion during sheet production, which helps boost numbers of transduced cells and limit numbers of nontransduced cells in epidermal sheets before grafting. Similarly, LAMB3 transduction offers a selective in vivo advantage. Laminin 332 inhibitory antibodies dissociate epidermis from dermis (
      • Lazarova Z.
      • Yee C.
      • Darling T.
      • Briggaman R.A.
      • Yancey K.B.
      Passive transfer of anti-laminin 5 antibodies induces subepidermal blisters in neonatal mice.
      ,
      • Rousselle P.
      • Lunstrum G.P.
      • Keene D.R.
      • Burgeson R.E.
      Kalinin: an epithelium-specific basement membrane adhesion molecule that is a component of anchoring filaments.
      ). Thus, LAMB3-transduced patient keratinocytes may outcompete nontransduced patient cells during wound re-epithelialization in vivo. This may account for the high level of graft acceptance and clinical improvement seen in the second patient (
      • Hirsch T.
      • Rothoeft T.
      • Teig N.
      • Bauer J.W.
      • Pellegrini G.
      • De Rosa L.
      • et al.
      Regeneration of the entire human epidermis using transgenic stem cells.
      ).
      Another success of gene therapy studies with these two JEB patients was the lack of immune reactions to the therapeutic gene product. The laminin β3 chain is antigenic, exemplified by the fact that it is targeted by autoantibodies in patients with the autoimmune blistering disease anti-laminin cicatricial pemphigoid (
      • Domloge-Hultsch N.
      • Gammon W.R.
      • Briggaman R.A.
      • Gil S.G.
      • Carter W.G.
      • Yancey K.B.
      Epiligrin, the major human keratinocyte integrin ligand, is a target in both an acquired autoimmune and an inherited subepidermal blistering skin disease.
      ,
      • Hisamatsu Y.
      • Nishiyama T.
      • Amano S.
      • Matsui C.
      • Ghohestani R.
      • Hashimoto T.
      Usefulness of immunoblotting using purified laminin 5 in the diagnosis of anti-laminin 5 cicatricial pemphigoid.
      ). However, a potential reason for this lack of therapeutic immunoreactivity could lie in the patient selection. Each of the JEB patients selected for these gene therapy studies had missense rather than null LAMB3 mutations. In the first patient, only a single amino acid was altered by the underlying mutation, and the second patient showed altered splicing in exon 14. These mutations would have given each patient’s developing immune system the opportunity to develop self-tolerance to most of the laminin β3 chain. These single-amino acid or small-deletion mutations reduce the chance of immune reactions to the wild-type laminin β3 introduced after gene therapy. However, most cases of JEB are associated with null mutations and are lethal (
      • Laimer M.
      • Lanschuetzer C.M.
      • Diem A.
      • Bauer J.W.
      Herlitz junctional epidermolysis bullosa.
      ) (generalized severe JEB). The immune systems in these patients would be expected to have an increased chance of recognizing laminin β3 as non-self, compared with patients with less severe JEB with missense or deletion mutations. Therefore, because the majority of JEB patients have null mutations, they would likely carry a greater risk of immunoreactivity to gene therapy, compared with the two patients selected for this study.
      Another consequence of null mutations in severe generalized JEB—which, as mentioned, represents the majority of JEB cases–is the occurrence of severe internal complications, especially respiratory disease, believed to be the primary cause of death in these patients (
      • Laimer M.
      • Lanschuetzer C.M.
      • Diem A.
      • Bauer J.W.
      Herlitz junctional epidermolysis bullosa.
      ). Therefore, even gene therapy-mediated correction of a JEB patient’s entire skin would not affect the severe fatal internal disease. Thus, although LAMB3 gene therapy studies to date in missense/deletion patients are truly remarkable, additional measures are needed to address increased immune risk and fatal internal disease in patients with null mutations who display the generalized severe JEB phenotype, which represents the majority of JEB patients.

      Gene Therapy for RDEB

      One of the most significant obstacles in gene therapy development for RDEB has been the large size of COL7A1, the gene coding for type VII collagen, which contains an 8,833-nucleotide open reading frame (
      • Christiano A.M.
      • Greenspan D.S.
      • Lee S.
      • Uitto J.
      Cloning of human type VII collagen. Complete primary sequence of the α1(VII) chain and identification of intragenic polymorphisms.
      ). With this discovery, it became clear that it would be challenging to deliver COL7A1 to RDEB skin. The large cDNA size of COL7A1 has a negative impact on virus packaging, substantially reducing viral titer and limiting the efficiency of transduction. Despite these challenges, a variety of preclinical efforts took on the difficult problem of C7 skin delivery to RDEB skin, including direct intradermal injection of C7-expressing lentiviral vectors (
      • Woodley D.T.
      • Keene D.R.
      • Atha T.
      • Huang Y.
      • Ram R.
      • Kasahara N.
      • et al.
      Intradermal injection of lentiviral vectors corrects regenerated human dystrophic epidermolysis bullosa skin tissue in vivo.
      ) and C7 protein delivery (
      • Remington J.
      • Wang X.
      • Hou Y.
      • Zhou H.
      • Burnett J.
      • Muirhead T.
      • et al.
      Injection of recombinant human type VII collagen corrects the disease phenotype in a murine model of dystrophic epidermolysis bullosa.
      ,
      • Woodley D.T.
      • Keene D.R.
      • Atha T.
      • Huang Y.
      • Lipman K.
      • Li W.
      • et al.
      Injection of recombinant human type VII collagen restores collagen function in dystrophic epidermolysis bullosa.
      ). These studies made it clear that i) delivery of C7 to skin had the potential to safely reverse the RDEB phenotype and ii) the half-life of C7 appeared to be long, between 1 and 2 months (
      • Kuhl T.
      • Mezger M.
      • Hausser I.
      • Guey L.T.
      • Handgretinger R.
      • Bruckner-Tuderman L.
      • et al.
      Collagen VII half-life at the dermal-epidermal junction zone: implications for mechanisms and therapy of genodermatoses.
      ,
      • Remington J.
      • Wang X.
      • Hou Y.
      • Zhou H.
      • Burnett J.
      • Muirhead T.
      • et al.
      Injection of recombinant human type VII collagen corrects the disease phenotype in a murine model of dystrophic epidermolysis bullosa.
      ). A major breakthrough in the development of a clinical gene therapy for RDEB came with the cloning of COL7A1 into an extensively modified Moloney murine leukemia virus, pLZRS, in which many nonessential viral sequences were removed (
      • Siprashvili Z.
      • Nguyen N.T.
      • Bezchinsky M.Y.
      • Marinkovich M.P.
      • Lane A.T.
      • Khavari P.A.
      Long-term type VII collagen restoration to human epidermolysis bullosa skin tissue.
      ). After transduction of this modified COL7A1 LZRS vector into primary C7-null RDEB keratinocytes, and epidermal sheet grafting of C7-engineered keratinocytes to immunodeficient mice, C7 expression was noted for up to 1 year, suggesting targeting of patient stem cells.
      This advance set the stage for the first successful COL7A1 gene therapy trial in seven adult RDEB patients (
      • Siprashvili Z.
      • Nguyen N.T.
      • Gorell E.S.
      • Loutit K.
      • Khuu P.
      • Furukawa L.K.
      • et al.
      Phase I/IIa clinical trial for recessive dystrophic epidermolysis bullosa using genetically corrected autologous keratinocytes.
      ). An important initial part of the trial was focused on patient selection (
      • Gorell E.S.
      • Nguyen N.
      • Siprashvili Z.
      • Marinkovich M.P.
      • Lane A.T.
      Characterization of patients with dystrophic epidermolysis bullosa for collagen VII therapy.
      ). As touched on in the laminin 332 gene therapy discussion, it would be expected that patients with completely null mutation would have a greater chance of immune reaction to therapeutic C7 compared with patients who expressed some protein. The large NC1 domain is thought to be the most immunogenic portion of C7, as determined by epitope mapping studies of sera from patients with autoimmunity against C7, epidermolysis bullosa acquisita (
      • Lapiere J.C.
      • Woodley D.T.
      • Parente M.G.
      • Iwasaki T.
      • Wynn K.C.
      • Christiano A.M.
      • et al.
      Epitope mapping of type VII collagen. Identification of discrete peptide sequences recognized by sera from patients with acquired epidermolysis bullosa.
      ). Therefore, one important inclusion criterion was positive expression of the NC1 domain to reduce the potential of immune reactions against the therapy. To that end, low levels of the NC1 domain protein were detected in approximately 70% of primary RDEB patient cell cultures by Western blot (
      • Gorell E.S.
      • Nguyen N.
      • Siprashvili Z.
      • Marinkovich M.P.
      • Lane A.T.
      Characterization of patients with dystrophic epidermolysis bullosa for collagen VII therapy.
      ,
      • Ortiz-Urda S.
      • Garcia J.
      • Green C.L.
      • Chen L.
      • Lin Q.
      • Veitch D.P.
      • et al.
      Type VII collagen is required for Ras-driven human epidermal tumorigenesis.
      ). Another key patient selection criterion was choosing patients with severe generalized RDEB who showed a lack of full-length C7 expression, either by Western blot of keratinocyte medium or by indirect immunofluorescence microscopy with the monoclonal antibody LH24 (
      • Sinclair R.
      • Wojnarowska F.
      • Leigh I.
      • Dawber R.
      The basement membrane zone of the nail.
      ), which recognizes a portion of C7 near the NC2 domain (
      • Siprashvili Z.
      • Nguyen N.T.
      • Gorell E.S.
      • Loutit K.
      • Khuu P.
      • Furukawa L.K.
      • et al.
      Safety and wound outcomes following genetically corrected autologous epidermal grafts in patients with recessive dystrophic epidermolysis bullosa.
      ). Use of only LH24-negative patients provided the opportunity to use the positive staining of LH24 expression after C7 therapy, for indication of full-length C7 expression, as evidence of molecular correction in RDEB skin.
      Seven adult NC1+/LH24 patients have been enrolled in a single center phase 1/2A clinical trial at Stanford University, where wounds were engrafted with six C7-engineered autologous epidermal sheets 35 cm2 in size (
      • Siprashvili Z.
      • Nguyen N.T.
      • Gorell E.S.
      • Loutit K.
      • Khuu P.
      • Furukawa L.K.
      • et al.
      Phase I/IIa clinical trial for recessive dystrophic epidermolysis bullosa using genetically corrected autologous keratinocytes.
      ). Results of the first four patients have been published (
      • Siprashvili Z.
      • Nguyen N.T.
      • Gorell E.S.
      • Loutit K.
      • Khuu P.
      • Furukawa L.K.
      • et al.
      Safety and wound outcomes following genetically corrected autologous epidermal grafts in patients with recessive dystrophic epidermolysis bullosa.
      ) and showed that all grafts (n = 24) were well tolerated without serious adverse events; linear C7 expression was noted in 90% of biopsy samples at 3 months, 66% at 6 months, and 42% at 12 months. Immunoelectron microscopy showed the localization of therapeutic C7 into mature anchoring fibrils. Coincident with the molecular correction, grafted sites showed 75% or greater wound healing in 87% of sites at 3 months, 67% of sites at 6 months, and 50% of sites at 12 months compared with baseline wound sites. In total, these results showed a correlation of C7 basement membrane expression with clinical improvement over the year of the study. The ability to properly immobilize the grafts for the first several days after placement was a critical factor believed to contribute to some of the variability of the results. For example, grafts placed on the back were in general more difficult to immobilize than grafts on the extremities.
      One explanation for the gradual decline in results after 1 year of the RDEB trial compared with the JEB case studies might be due to limitations in the numbers of stem cells targeted in the therapy. Holoclone analysis on the RDEB trial was not performed, so as a consequence, stem cells were not quantified as in the JEB trials. Age-related decline in regenerative potential could also have been a factor, because the average age of patients in the RDEB trials was 23 years, in contrast to the second JEB study, in which the patient was 7 years old. Also, C7-overexpressing keratinocytes may not have the strong selective in vitro and in vivo adhesive advantages over their nontransduced counterparts that laminin 332-expressing keratinocytes display, as described. This is because keratinocytes do not directly bind C7 as an adhesive ligand but rather bind with C7 indirectly, through their interaction with laminin 332 (
      • Chen M.
      • Marinkovich M.P.
      • Jones J.C.
      • O’Toole E.A.
      • Li Y.Y.
      • Woodley D.T.
      NC1 domain of type VII collagen binds to the β3 chain of laminin 5 via a unique subdomain within the fibronectin-like repeats.
      ,
      • Rousselle P.
      • Keene D.R.
      • Ruggiero F.
      • Champliaud M.F.
      • Rest M.
      • Burgeson R.E.
      Laminin 5 binds the NC-1 domain of type VII collagen.
      ). These studies point to the need for new methods to increase stem cell delivery to the skin, such as the use of induced pluripotent stem cells (
      • Sebastiano V.
      • Zhen H.H.
      • Derafshi B.H.
      • Bashkirova E.
      • Melo S.P.
      • Wang P.
      • et al.
      Human COL7A1-corrected induced pluripotent stem cells for the treatment of recessive dystrophic epidermolysis bullosa.
      ,
      • Tolar J.
      • McGrath J.A.
      • Xia L.
      • Riddle M.J.
      • Lees C.J.
      • Eide C.
      • et al.
      Patient-specific naturally gene-reverted induced pluripotent stem cells in recessive dystrophic epidermolysis bullosa.
      ,
      • Wenzel D.
      • Bayerl J.
      • Nystrom A.
      • Bruckner-Tuderman L.
      • Meixner A.
      • Penninger J.M.
      Genetically corrected iPSCs as cell therapy for recessive dystrophic epidermolysis bullosa.
      ).
      Although no serious adverse events occurred, including recombination competent retrovirus or evidence of squamous cell carcinoma, C7 antibodies were detected in one patient in the RDEB trial (
      • Siprashvili Z.
      • Nguyen N.T.
      • Gorell E.S.
      • Loutit K.
      • Khuu P.
      • Furukawa L.K.
      • et al.
      Safety and wound outcomes following genetically corrected autologous epidermal grafts in patients with recessive dystrophic epidermolysis bullosa.
      ). Pretherapy screening of this individual by indirect immunofluorescence microscopy showed a lack of C7 reactivity; however, after detection of C7 antibodies by indirect immunofluorescence microscopy 1 month after grafting, serum was examined by a more sensitive Western blot test, and low levels of C7 antibodies were detected in pretherapy serum (patient 4). Therefore, it is possible that the therapy did not initiate a de novo antibody response but, rather, exacerbated a preexisting immune reaction. Epitope mapping of this patient’s sera showed reactivity to the NC2 domain rather than the more antigenic NC1 domain typically targeted by epidermolysis bullosa acquisita autoantibodies. Because the patient’s screening data indicated that his cells expressed a C7 molecule-containing NC1 domain but lacked NC2, it is possible that his reaction was not autoimmune against his own expressed protein but, rather, was an allo-reaction against the therapeutic gene product. Low levels of circulating C7 antibodies previously showed by ELISA in some RDEB patients (
      • Pendaries V.
      • Gasc G.
      • Titeux M.
      • Leroux C.
      • Vitezica Z.G.
      • Mejia J.E.
      • et al.
      Immune reactivity to type VII collagen: implications for gene therapy of recessive dystrophic epidermolysis bullosa.
      ,
      • Tampoia M.
      • Bonamonte D.
      • Filoni A.
      • Garofalo L.
      • Morgese M.G.
      • Brunetti L.
      • et al.
      Prevalence of specific anti-skin autoantibodies in a cohort of patients with inherited epidermolysis bullosa.
      ,
      • Woodley D.T.
      • Cogan J.
      • Wang X.
      • Hou Y.
      • Haghighian C.
      • Kudo G.
      • et al.
      De novo anti-type VII collagen antibodies in patients with recessive dystrophic epidermolysis bullosa.
      ) have been disregarded as nonpathogenic. However, the results of the RDEB gene therapy studies suggest that the existence of even low levels of C7 antibodies in RDEB patients should be treated with caution when considering such patients for C7 replacement therapies.
      Fibroblasts are also known to naturally contribute C7 into the basement membrane (
      • Marinkovich M.P.
      • Keene D.R.
      • Rimberg C.S.
      • Burgeson R.E.
      Cellular origin of the dermal-epidermal basement membrane.
      ). Intradermal injection of C7-expressing fibroblasts has been shown to reverse the RDEB phenotype in preclinical xenograft models (
      • Jackow J.
      • Titeux M.
      • Portier S.
      • Charbonnier S.
      • Ganier C.
      • Gaucher S.
      • et al.
      Gene-corrected fibroblast therapy for recessive dystrophic epidermolysis bullosa using a self-inactivating COL7A1 retroviral vector.
      ,
      • Ortiz-Urda S.
      • Lin Q.
      • Green C.L.
      • Keene D.R.
      • Marinkovich M.P.
      • Khavari P.A.
      Injection of genetically engineered fibroblasts corrects regenerated human epidermolysis bullosa skin tissue.
      ), and delivery of autologous C7-overexpressing fibroblasts represents another important focus in current ongoing RDEB gene therapy studies. Recently, a clinical trial studying genetically corrected, C7-expressing autologous human dermal fibroblasts injected into the skin of RDEB patients was started (ClinicalTrials.gov identifier: NCT02810951), and five patients have already been treated (
      • Marinkovich M.
      • Lane A.
      • Sridhar K.
      • Keene D.
      • Malyala A.
      • Maslowski J.
      A phase 1/2 study of genetically-corrected, collagen VII expressing autologous human dermal fibroblasts injected into the skin of patients with recessive dystrophic epidermolysis bullosa (RDEB).
      ).
      All of the gene delivery approaches described thus far represent ex vivo approaches, whereby the gene is transferred and the cells expanded are in good manufacturing practice (i.e., GMP) tissue culture facilities. Two new in vivo topical COL7A1 gene therapies for RDEB recently began early-phase clinical trials. These therapies do not require anesthesia or hospitalization, increasing convenience and accessibility for EB patients. One approach uses a topical preparation containing oligonucleotides, which mediate antisense-mediated exon skipping, removing premature termination codon mutations in COL7A1 exon 73, producing a slightly truncated C7 molecule (
      • Turczynski S.
      • Titeux M.
      • Tonasso L.
      • Decha A.
      • Ishida-Yamamoto A.
      • Hovnanian A.
      Targeted exon skipping restores type VII collagen expression and anchoring fibril formation in an in vivo RDEB model.
      ) (ClinicalTrials.gov identifier: NCT03605069). The second clinical trial uses a modified replication-deficient herpes simplex virus 1 virus to deliver therapeutic genes. Chronic administration of this vector was successfully found to be safe and effective in melanoma clinical trials (
      • Rehman H.
      • Silk A.W.
      • Kane M.P.
      • Kaufman H.L.
      Into the clinic: Talimogene laherparepvec (T-VEC), a first-in-class intratumoral oncolytic viral therapy.
      ), whereas the current clinical trial uses a modified herpes simplex virus 1 vector to topically deliver the wild-type COL7A1 gene to skin cells in patient wounds (ClinicalTrials.gov identifier: NCT03536143).

      Conclusions

      Advances in our understanding of the biology, biochemistry, and molecular biology of the dermal-epidermal basement membrane and its alterations in EB have provided the necessary foundation for the first clinical trials of gene therapy for JEB and RDEB. These have shown that gene therapy is both safe and effective, with the potential to make positive changes in the lives of patients with EB. Improvements in gene therapy, including refining ex vivo approaches, developing new in vivo approaches, and preventing immune reactions, will be among the challenges that lie ahead.

      Conflict of Interest

      MPM and JYT are investigators for Abeona Therapeutics and Fibrocell, and MPM is an investigator for ProQR and Krystal Biotech. These companies develop gene therapies for epidermolysis bullosa.

      Acknowledgments

      Funding from the Office of Research, Palo Alto Veterans Affairs Medical Center , the Epidermolysis Bullosa Medical Research Foundation , the Epidermolysis Bullosa Research Partnership , DEBRA International , and the National Institutes of Health ( R13-AR009431-52 ; principal investigator: Kulesz-Martin) is gratefully acknowledged.

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