DOI: 10.1111/exd.12666
ADF Perspectives
www.wileyonlinelibrary.com/journal/EXD
Therapies for inherited skin fragility disorders Cristina Has and Dimitra Kiritsi Department of Dermatology, Medical Center – University of Freiburg, Freiburg, Germany Correspondence: Prof. Dr. Cristina Has, Department of Dermatology, Medical Center – University of Freiburg, Hauptstrasse 7, 79104 Freiburg, Germany, Tel.: +49 761 27069920, Fax: +49 761 27067200, e-mail: [email protected] Abstract: Inherited skin fragility comprises disorders characterized by mechanical induced blistering and erosions within the skin and mucosal membranes as a consequence of mutations in genes encoding proteins involved in intra-epidermal or dermal– epidermal adhesion. As the molecular pathology is largely known, it is a prototype group of disorders for which numerous experimental treatments have been developed. However, it became clear that single therapeutic strategies will not be able to address all molecular and clinical aspects. Significant progress has been achieved in gene, cell and protein therapies. Although the way towards clinical application seems obvious, major challenges must be addressed before these therapies become largely accessible. Until curative treatments will become available, alternative
strategies which aim at increasing protein stability, amending apoptosis, inflammation and scarring may alleviate skin fragility and prevent or delay the onset of complications.
Introduction
decades (Table 1). Regenerative medicine is moving very rapidly, and several approaches have recently entered the clinical arena. Although the pathway towards clinical application seems obvious, major investigative and regulatory hurdles must be addressed before it becomes largely accessible and affordable to patients and their physicians (5).
Inherited skin fragility comprises a spectrum of disorders, mainly designated as epidermolysis bullosa (EB), which are characterized by mechanical induced blistering and erosions within the skin and mucosal membranes as a consequence of mutations in genes encoding proteins involved in intra-epidermal or dermal–epidermal adhesion (1–3) (Table 1). The affected proteins have various functions, structure and cell compartment localization, such as keratin intermediate filaments, intra-cellular anchor, transmembrane or extracellular proteins (Fig. 1a). Altogether, mutations in 18 genes contribute to the molecular pathology of this group of disorders, with a large variety of mutation constellations accounting for the phenotypic diversity of more than 30 subtypes (4). In the most severe subtypes of inherited skin fragility, extensive erosions or blisters lead either to early demise or to chronic disease associated with complications such as non-healing wounds, infections, scars and epithelial skin cancers (Fig. 1b). In addition, the involvement of mucous membranes or of other organs results in multi-organ dysfunction. Besides, the clinical spectrum covers subtypes with intermediate severity evolving mainly with cutaneous features and a normal lifespan. In recent years, our knowledge extended to recognize mild forms of inherited skin fragility. These can begin later in life, or be unravelled and aggravated by additional factors to become clinically relevant. Such phenotypes are often underdiagnosed and inadequately treated, thus resulting in a significant disease burden. For all subtypes, precise molecular genetic analysis is prerequisite for the correct diagnosis and eventually for a targeted therapy. While currently only symptomatic management of fragile skin is available, the patients have high unmet therapeutic needs. As the molecular pathology is largely known, inherited skin fragility is a prototype group of disorders for which numerous studies on experimental therapies have been published in the past two
ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 325–331
Abbreviations: EB, epidermolysis bullosa; ESC, embryonic stem cells; iPSCs, induced pluripotent stem cells; MSC, mesenchymal stem/stromal cells; TALENs, transcription activator-like effector nucleases; USSCs, unrestricted somatic stem cells. Key words: adhesion – collagen VII – epidermolysis bullosa – keratin – laminin
Accepted for publication 10 February 2015
Therapeutic strategies Ideally, deficiency of gene expression may be overcome either by addition of the correct gene, gene product or by cells which express the respective protein. There are challenges and technical limits of these methods and many questions are still open: (i) whether sufficient protein can be generated for disease modification, (ii) which is the appropriate dosage and frequency of applications/administrations, (iii) how long do the effects last, (iv) how to reliably measure the therapeutic efficacy, (v) which are the consequences of repeated applications and of long-term therapy and (vi) specific safety concerns for each approach. Like for other rare disorders, the development of clinical trials is complicated by the lack of systematic studies of the natural history, of rigorous criteria for clinical evaluation or scores for disease activity, and by the relatively small number of patients. Proof of principle studies has been performed in vitro or in mouse models, which recapitulate only in part the human disorders. They have generated valuable knowledge regarding the potential benefits of strategies such as protein replacement or cell therapies. The study of genotype–phenotype correlations (6–8) and mouse models (9–11) revealed that expression of about 10% of the normal protein level makes the difference between severe and moderate disease. While these findings suggest that such amounts significantly alleviate the manifestations of the disorder, the long-term achievement of these thresholds in local or systemic therapy is far from being trivial.
325
Has and Kiritsi
Table 1. Summary of targeted therapies in skin fragility disorders
Type/subtype
Molecular pathology, inheritance
Clinical spectrum
Targeted therapeutic strategies, selected references
EBS suprabasal: basal:
Genetic heterogeneity Affected proteins: desmoplakin,
Severe generalized
No specific therapies
plakoglobin, plakophilin-1, transglutaminase 5 Autosomal recessive
Moderate to mild skin fragility
Genetic heterogeneity Affected proteins: keratin 5,
Blistering, inflammation,
keratin 14, plectin, BPAG1e, exophilin 5 Mostly autosomal dominant, rare autosomal recessive subtypes JEB
DEB
plantar keratoses, hyperhidrosis Acral blistering
Genetic heterogeneity Affected proteins: laminin 332,
Lethal subtypes Chronic wounds, mucosal
collagen XVII, integrin a6b4, integrin a3 subunit Autosomal recessive
Involvement of other organs
High allelic heterogeneity Affected protein: collagen VII Autosomal recessive and dominant subtypes
KS
erosions, early lethal outcome
Affected protein: kindlin-1 Autosomal recessive
–
developed so far
Gene therapy DNA editing (92) Trans-splicing (34, 35) siRNA (34, 39) Small molecules anti-IL1 (89) chaperones (90)
Gene therapy (18, 33, 80) Protein therapy (79) Revertant mosaicism (73–75)
preclinical preclinical preclinical clinical preclinical
clinical preclinical clinical
involvement
Blistering, scarring, squamous cell carcinomas Mild forms with localized skin fragility and nail dystrophy
Skin fragility, photosensitivity, poikiloderma, squamous cell carcinomas
Further, in patients with mutations leading to absence of protein, an immune response against the therapeutic protein could represent a potential complication, mimicking an autoimmune blistering disorder or neutralizing the therapeutic effect. Two recent back-to-back reports were published describing the presence of antibodies against collagen VII in patients with dystrophic EB; in both, the auto-antibodies were not found to be pathogenic (12,13). Similarly, an immune response was noted in a dystrophic EB mouse model after injection of collagen VII, but it did not prevent the incorporation of collagen VII into the basement membrane and the therapeutic effect (14). So far, most therapeutic studies have been concentrated on recessive dystrophic EB, one of the most severe types of skin fragility which affects a significant number of patients. Collagen VII, which is defective, is an extracellular protein which undergoes homotrimerization, processing and assembly in supramolecular structures, and builds the anchoring fibrils. Gene therapy was the initial focus of the researchers, while the interest in cell therapies and protein therapy grew progressively in recent years. In the meantime, new alternative therapeutic avenues emerged as it
326
Stage of development
Gene therapy COL7A1 correction in keratinocytes or fibroblasts [reviewed in (16)] Antisense oligonucleotides (93) siRNA (40) Trans-splicing (94) TALENs (32) Cell therapies Fibroblast therapy (52, 53, 56) Mesenchymal stem cells (54) Bone marrow transplantation (51) Revertant mosaicism Protein therapy (14, 77, 78, 95) No specific therapies developed so far
preclinical, clinical
preclinical preclinical preclinical preclinical preclinical clinical clinical clinical preclinical, clinical envisaged
became evident that the dysfunction of a single protein results in highly complex disease mechanisms.
Gene therapy The idea that the treatment of chronic wounds in patients with inherited skin fragility may benefit from ex vivo gene therapy (15) resulted in numerous studies which were recently reviewed (16,17). Although the concept of gene transfer is relatively straightforward and initial attempts showed promising results (18,19), gene therapeutic trials were tempered by the development of T-cell leukaemia in patients with severe combined immunodeficiency and Wiskott–Aldrich syndrome (20–22). This made researchers focus on challenges related to insertional mutagenesis, immunogenicity and vector stability in the host (23). Clear evidence of clinical benefits leads to increased gene therapy clinical studies, resulting in 1800 gene transfer trials around the world (16), but the challenges are still extremely high (24). The propensity of any type of retroviral vector to interfere with regulation of gene transcription is still raising safety concerns, regardless of the strategy used to correct the genetic defect, because in any case, the use of self-renewing stem cells to maintain stable skin grafts
ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 325–331
Therapies for inherited skin fragility disorders
Figure 1. Molecular and clinical spectrum of inherited skin fragility. a. Schematic representation of the epidermis and dermal–epidermal junction zone. On the left side, the levels of skin cleavage in EB are shown. The marked areas, representing cell-matrix adhesions, are schematically depicted in the right panel. The proteins involved in inherited skin fragility disorders are underlined. EBS, EB simplex; JEB, junctional EB; DEB, dystrophic EB; KS, Kindler syndrome. b. In the left panel, a young man with severe generalized dystrophic EB caused by COL7A1 loss-offunction mutations. Note chronic wounds on sites of mechanical pressure. In the right panel, a young woman with junctional EB and LAMB3 mutations, allowing residual expression of laminin 332. Note extensive scarring and non-healing wounds on sites exposed to mechanical stress.
in both cases recovering normal transcript and protein and correcting the enzyme deficiency (30). Gene editing is likely to become the next generation of gene therapy, by overcoming the potentially harmful effects of current gene replacement technology (25). The spectrum of targeted genome editing tools has enormous capabilities and applications, but each approach has distinct drawbacks and limitations (31); in particular, somatic cells have a low propensity to utilize homologous recombination to repair DNA (25). In the field of inherited skin fragility disorders, such strategies have been developed in preclinical studies (Table 1). For example, engineered transcription activator-like effector nucleases (TALENs) were able to induce site-specific doublestranded DNA breaks leading to homology-directed repair from an exogenous donor template. This process resulted in correction of the COL7A1 gene mutation in patient’s fibroblasts that were subsequently reprogrammed into inducible pluripotent stem cells and showed normal protein expression and deposition in a teratoma-based skin model in vivo (32). In another study, correction of LAMA3 mutations in primary keratinocytes derived from a patient affected by severe junctional EB was achieved using recombinant adeno-associated virus-mediated homologous recombination (33). The RNA transsplicing technology which aims at skipping or replacing exons harbouring mutations has been optimized to correct mutations in the genes for keratin 14, collagen XVII or plectin, in in vitro systems (34– 36). Finally, siRNA to knockdown the dominant negative allele was developed for the treatment of painful plantar keratoses in patients with pachyonychia congenita (37,38) and in preclinical settings for EB simplex and dominant dystrophic EB (39,40). Delivery of therapeutic siRNA is a hurdle to overcome. Manufacturing dissolvable microneedles to deliver siRNAs while minimizing pain has been accomplished for pachyonychia congenita, and a mouse study has been completed with no signs of toxicity (41).
requires permanent vector integration (25). Currently, clinical trials were initiated intending to develop safe and efficient ex vivo gene therapy approaches for a permanent treatment for recessive dystrophic EB [(26), http://clinicaltrials.gov/ct2/show/record/ NCT01263379?term=epidermolysis+bullosa&recr=Open&no_unk=Y&rank=1]. These approaches use autologous skin equivalents genetically corrected with safer retroviral or other vectors expressing type VII collagen (26,27) (28). The efficacy of antisense therapy for suppression of normal, pathogenic, or cryptic splice sites and skip exons bearing mutations has been demonstrated in cellular and animal models and has already reached the clinical trials phase for Duchenne muscular dystrophy. Preliminary data from early-phase clinical trials regarding an antisense oligonucleotide drug designed to correct the out-of-frame genetic defect via skipping of exon 51 of the dystrophin gene in patients with Duchenne muscular dystrophy had appeared encouraging. A failure in large-scale clinical trials was a clear setback for the field and should provide important lessons for future trials. The lack of therapeutic response seems to be due to two critical factors: (i) the amount of functional dystrophin protein generated, which was probably marginal for clinical efficacy and (ii) the primary outcome measure itself which was not appropriate (29). In different inherited metabolic diseases, spliceswitching oligonucleotides have been used with success in patients’ cells to force pseudoexon skipping or to block cryptic splice sites,
Cell-based therapies became highly popular among researchers in the EB field in the past few years, and the progress was recently reviewed (42–44). Early studies in mouse models served as a proof of principle and hold promise (9,45–50). Local injections of fibroblasts or mesenchymal stem/stromal cells (MSC) and bone marrow transplantation demonstrated a certain efficacy in patients with recessive dystrophic EB (51–54). It was suggested that injection of fibroblasts results in elevation of the heparin-binding EGFlike growth factor which may increase COL7A1 gene expression (55). The results of two clinical trials comparing allogeneic fibroblasts versus vehicle are contradictory (52,56), and the utility of this procedure remains to be defined. Injection of bone marrowderived MSC led to improvement of wound healing in two individuals with recessive dystrophic EB (54). Based on this and on studies in mice which showed that a specific MSC bone marrow subpopulation is mobilized to regenerate injured epithelia (50), systemic application of MSC is envisaged. Several clinical trials are ongoing and will hopefully shed light on the long-term outcome and on the precise mechanisms of the therapeutic effects. Beside the first seven patients with dystrophic EB treated with bone marrow transplantation in 2010 (51), the experience on overall 20 patients was recently reported (44). Five died due to complications of the transplantation or disease progression, whereas all others had at least some benefit from the procedure. Although these studies show efficacy of bone marrow transplanta-
(a)
(b)
ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 325–331
Cell therapies
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Has and Kiritsi
Table 2. Strengths and weaknesses of targeted therapies in skin fragility disorders Strategy Gene therapy
Gene correction
Pro
Contra
Effective for topical
treatment of chronic wounds
Cell therapies
Topical application Safety concerns Complex technology Expensive
Antisense oligonucleotides
Clinical trials for other genetic disorders
Limited stability in the skin High dosage required to achieve clinical benefit Toxicity
DNA editing Trans-splicing siRNA
Simple administration
Experimental approaches Low topical absorption in the skin Only topical application
Bone marrow transplantation Fibroblasts MSC
Established administration Systemic application Safe
Short survival of cells in the skin Low concentration of cells in unwounded skin High amounts of cells required to achieve therapeutic protein concentrations
Immune reaction after repeated administrations probable
IPSCs
Unlimited amount of autologous cells
Highly complex technology Safety concerns Expensive
Revertant mosaicism
Autologous cells which can be
Only topical application Revertant mosaicism must be present in the patient
used as grafts or epidermal sheets Simple administration Safe Inexpensive Protein therapy
Limited durability of the protein in the skin High amounts of protein required
Simple administration Potential systemic application Safe
to achieve therapeutic dosage
Immune reaction after repeated administrations probable
Not suited for intra-cellular structural proteins Drugs
Established for other disorders Simple administration Target pathogenic mechanisms Safe Inexpensive compared to experimental therapies
Unclear long-term effects Do not address the disease cause
BMT, bone marrow transplantation; MSC, mesenchymal stem cells; iPSCs, induced pluripotent stem cells.
tion in EB with some degree of amelioration, none of the patients had a complete cure of the disease. Recently, human umbilical cord blood-derived unrestricted somatic stem cells (USSCs) were induced to express genes that hallmark keratinocyte differentiation including collagen VII. USSCs have previously been demonstrated to have a broad differentiation potential and regenerative beneficial effects when administered in animal models of multiple degenerative diseases. In an initial study, USSCs promoted wound healing, suggesting that they could have a therapeutic potential for patients with recessive dystrophic EB (57).
328
Although skin biopsies are a regular source of keratinocytes and epidermal stem cells, keratinocytes were recently generated from embryonic stem cells (ESCs) (58) and from induced pluripotent stem cells (iPSCs) (59–61). The clinical use of ESCs has significant ethical and biological obstacles related to their derivation from embryos and potential for immunological rejection, respectively. These disadvantages can be circumvented by the alternative use of iPSCs, which are derived from an individual’s (autologous) somatic cells by exogenous expression of defined transcription factors and have biological characteristics similar to ESCs. Patient-specific
ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 325–331
Therapies for inherited skin fragility disorders
(a)
(b)
Figure 2. Revertant mosaicism in skin fragility. a. Areas of revertant mosaicism on the dorsal aspect of the hand of a young man with Kindler syndrome are outlined in green. The inset shows a magnification of the marked area, to better demonstrate the contrast between the affected, atrophic and the revertant skin with normal skin texture. b. Keratinocytes were isolated from the skin of a healthy control and from the revertant epidermis of a patient with Kindler syndrome. Kindlin-1 stains positive the control cells mainly at focal adhesions. Note that in the photographed field, only one cell derived from the revertant skin of the patient expresses kindlin-1, while the others stain negative, as expected by the mutation constellation.
mutant and corrected iPSCs were generated from individuals with recessive dystrophic EB (59,60,62,63) and junctional EB (64,65) and differentiated into epidermal keratinocytes which were able to build three-dimensional skin equivalents, suggesting their full functionality (66,67). Such models can be used to study disease mechanisms or for testing pharmacological agents. Safety issues and optimization of protocols must be addressed before application of iPSCs in clinical practice, but the field is changing very rapidly (Table 2). The translation of cell-based therapies for tissue repair remains limited by several factors, including poor cell survival and limited target site retention (Table 2). Advances in nanotechnology enable the development of specifically designed delivery matrices to address these limitations and thereby improve the efficacy of cellbased therapies (68).
Revertant mosaicism Spontaneous gene repair, also called revertant mosaicism or natural gene therapy, has been documented in all main types of EB (69). The interest in this topic has strongly increased in the past 5 years because of the therapeutic potential of these patients’ own, spontaneously corrected cells. It is conceivable that revertant mosaicism is more common than initially assumed (70–72) and that patients acquire revertant cells at some point, and the frequency, diversity and functionality of the revertant cells depend on various factors, including the strength of selection (2). Fundamental scientific questions regarding the mechanisms which drive revertant mutational events remain to be addressed. Clinically, revertant skin areas demonstrate a ‘normal’ or improved texture and mechanical resistance, compared to the surrounding affected skin (Fig. 2a). The difference can be confirmed through immunostaining with antibodies specific to the mutant protein. The elucidation of the reversion mechanisms on genomic level is more labour intensive and requires laser-dissection microscopy or deep sequencing. From a pragmatic perspective, patients benefit from identification and expansion of revertant tissue which provides material for cell therapies. In an initial revertant cell therapy on a patient with mosaic collagen XVII -deficient junctional EB, functional repair was not
ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 325–331
achieved due to the low percentage of revertant cells in the graft (73). Indeed, revertant epidermis is a mosaic, which only contains a certain percentage of revertant cells that can be lost in in vitro culture conditions (Fig. 2b). This behaviour seems to be dependent on the protein which is affected in each specific EB subtype. For example, in the case of the reversion of collagen XVII mutations, stainings showed 40% revertant cells after first passage, 25% revertant cells after the second passage and a drop of revertant cells to 15%, 1% and
ADF Perspectives
www.wileyonlinelibrary.com/journal/EXD
Therapies for inherited skin fragility disorders Cristina Has and Dimitra Kiritsi Department of Dermatology, Medical Center – University of Freiburg, Freiburg, Germany Correspondence: Prof. Dr. Cristina Has, Department of Dermatology, Medical Center – University of Freiburg, Hauptstrasse 7, 79104 Freiburg, Germany, Tel.: +49 761 27069920, Fax: +49 761 27067200, e-mail: [email protected] Abstract: Inherited skin fragility comprises disorders characterized by mechanical induced blistering and erosions within the skin and mucosal membranes as a consequence of mutations in genes encoding proteins involved in intra-epidermal or dermal– epidermal adhesion. As the molecular pathology is largely known, it is a prototype group of disorders for which numerous experimental treatments have been developed. However, it became clear that single therapeutic strategies will not be able to address all molecular and clinical aspects. Significant progress has been achieved in gene, cell and protein therapies. Although the way towards clinical application seems obvious, major challenges must be addressed before these therapies become largely accessible. Until curative treatments will become available, alternative
strategies which aim at increasing protein stability, amending apoptosis, inflammation and scarring may alleviate skin fragility and prevent or delay the onset of complications.
Introduction
decades (Table 1). Regenerative medicine is moving very rapidly, and several approaches have recently entered the clinical arena. Although the pathway towards clinical application seems obvious, major investigative and regulatory hurdles must be addressed before it becomes largely accessible and affordable to patients and their physicians (5).
Inherited skin fragility comprises a spectrum of disorders, mainly designated as epidermolysis bullosa (EB), which are characterized by mechanical induced blistering and erosions within the skin and mucosal membranes as a consequence of mutations in genes encoding proteins involved in intra-epidermal or dermal–epidermal adhesion (1–3) (Table 1). The affected proteins have various functions, structure and cell compartment localization, such as keratin intermediate filaments, intra-cellular anchor, transmembrane or extracellular proteins (Fig. 1a). Altogether, mutations in 18 genes contribute to the molecular pathology of this group of disorders, with a large variety of mutation constellations accounting for the phenotypic diversity of more than 30 subtypes (4). In the most severe subtypes of inherited skin fragility, extensive erosions or blisters lead either to early demise or to chronic disease associated with complications such as non-healing wounds, infections, scars and epithelial skin cancers (Fig. 1b). In addition, the involvement of mucous membranes or of other organs results in multi-organ dysfunction. Besides, the clinical spectrum covers subtypes with intermediate severity evolving mainly with cutaneous features and a normal lifespan. In recent years, our knowledge extended to recognize mild forms of inherited skin fragility. These can begin later in life, or be unravelled and aggravated by additional factors to become clinically relevant. Such phenotypes are often underdiagnosed and inadequately treated, thus resulting in a significant disease burden. For all subtypes, precise molecular genetic analysis is prerequisite for the correct diagnosis and eventually for a targeted therapy. While currently only symptomatic management of fragile skin is available, the patients have high unmet therapeutic needs. As the molecular pathology is largely known, inherited skin fragility is a prototype group of disorders for which numerous studies on experimental therapies have been published in the past two
ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 325–331
Abbreviations: EB, epidermolysis bullosa; ESC, embryonic stem cells; iPSCs, induced pluripotent stem cells; MSC, mesenchymal stem/stromal cells; TALENs, transcription activator-like effector nucleases; USSCs, unrestricted somatic stem cells. Key words: adhesion – collagen VII – epidermolysis bullosa – keratin – laminin
Accepted for publication 10 February 2015
Therapeutic strategies Ideally, deficiency of gene expression may be overcome either by addition of the correct gene, gene product or by cells which express the respective protein. There are challenges and technical limits of these methods and many questions are still open: (i) whether sufficient protein can be generated for disease modification, (ii) which is the appropriate dosage and frequency of applications/administrations, (iii) how long do the effects last, (iv) how to reliably measure the therapeutic efficacy, (v) which are the consequences of repeated applications and of long-term therapy and (vi) specific safety concerns for each approach. Like for other rare disorders, the development of clinical trials is complicated by the lack of systematic studies of the natural history, of rigorous criteria for clinical evaluation or scores for disease activity, and by the relatively small number of patients. Proof of principle studies has been performed in vitro or in mouse models, which recapitulate only in part the human disorders. They have generated valuable knowledge regarding the potential benefits of strategies such as protein replacement or cell therapies. The study of genotype–phenotype correlations (6–8) and mouse models (9–11) revealed that expression of about 10% of the normal protein level makes the difference between severe and moderate disease. While these findings suggest that such amounts significantly alleviate the manifestations of the disorder, the long-term achievement of these thresholds in local or systemic therapy is far from being trivial.
325
Has and Kiritsi
Table 1. Summary of targeted therapies in skin fragility disorders
Type/subtype
Molecular pathology, inheritance
Clinical spectrum
Targeted therapeutic strategies, selected references
EBS suprabasal: basal:
Genetic heterogeneity Affected proteins: desmoplakin,
Severe generalized
No specific therapies
plakoglobin, plakophilin-1, transglutaminase 5 Autosomal recessive
Moderate to mild skin fragility
Genetic heterogeneity Affected proteins: keratin 5,
Blistering, inflammation,
keratin 14, plectin, BPAG1e, exophilin 5 Mostly autosomal dominant, rare autosomal recessive subtypes JEB
DEB
plantar keratoses, hyperhidrosis Acral blistering
Genetic heterogeneity Affected proteins: laminin 332,
Lethal subtypes Chronic wounds, mucosal
collagen XVII, integrin a6b4, integrin a3 subunit Autosomal recessive
Involvement of other organs
High allelic heterogeneity Affected protein: collagen VII Autosomal recessive and dominant subtypes
KS
erosions, early lethal outcome
Affected protein: kindlin-1 Autosomal recessive
–
developed so far
Gene therapy DNA editing (92) Trans-splicing (34, 35) siRNA (34, 39) Small molecules anti-IL1 (89) chaperones (90)
Gene therapy (18, 33, 80) Protein therapy (79) Revertant mosaicism (73–75)
preclinical preclinical preclinical clinical preclinical
clinical preclinical clinical
involvement
Blistering, scarring, squamous cell carcinomas Mild forms with localized skin fragility and nail dystrophy
Skin fragility, photosensitivity, poikiloderma, squamous cell carcinomas
Further, in patients with mutations leading to absence of protein, an immune response against the therapeutic protein could represent a potential complication, mimicking an autoimmune blistering disorder or neutralizing the therapeutic effect. Two recent back-to-back reports were published describing the presence of antibodies against collagen VII in patients with dystrophic EB; in both, the auto-antibodies were not found to be pathogenic (12,13). Similarly, an immune response was noted in a dystrophic EB mouse model after injection of collagen VII, but it did not prevent the incorporation of collagen VII into the basement membrane and the therapeutic effect (14). So far, most therapeutic studies have been concentrated on recessive dystrophic EB, one of the most severe types of skin fragility which affects a significant number of patients. Collagen VII, which is defective, is an extracellular protein which undergoes homotrimerization, processing and assembly in supramolecular structures, and builds the anchoring fibrils. Gene therapy was the initial focus of the researchers, while the interest in cell therapies and protein therapy grew progressively in recent years. In the meantime, new alternative therapeutic avenues emerged as it
326
Stage of development
Gene therapy COL7A1 correction in keratinocytes or fibroblasts [reviewed in (16)] Antisense oligonucleotides (93) siRNA (40) Trans-splicing (94) TALENs (32) Cell therapies Fibroblast therapy (52, 53, 56) Mesenchymal stem cells (54) Bone marrow transplantation (51) Revertant mosaicism Protein therapy (14, 77, 78, 95) No specific therapies developed so far
preclinical, clinical
preclinical preclinical preclinical preclinical preclinical clinical clinical clinical preclinical, clinical envisaged
became evident that the dysfunction of a single protein results in highly complex disease mechanisms.
Gene therapy The idea that the treatment of chronic wounds in patients with inherited skin fragility may benefit from ex vivo gene therapy (15) resulted in numerous studies which were recently reviewed (16,17). Although the concept of gene transfer is relatively straightforward and initial attempts showed promising results (18,19), gene therapeutic trials were tempered by the development of T-cell leukaemia in patients with severe combined immunodeficiency and Wiskott–Aldrich syndrome (20–22). This made researchers focus on challenges related to insertional mutagenesis, immunogenicity and vector stability in the host (23). Clear evidence of clinical benefits leads to increased gene therapy clinical studies, resulting in 1800 gene transfer trials around the world (16), but the challenges are still extremely high (24). The propensity of any type of retroviral vector to interfere with regulation of gene transcription is still raising safety concerns, regardless of the strategy used to correct the genetic defect, because in any case, the use of self-renewing stem cells to maintain stable skin grafts
ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 325–331
Therapies for inherited skin fragility disorders
Figure 1. Molecular and clinical spectrum of inherited skin fragility. a. Schematic representation of the epidermis and dermal–epidermal junction zone. On the left side, the levels of skin cleavage in EB are shown. The marked areas, representing cell-matrix adhesions, are schematically depicted in the right panel. The proteins involved in inherited skin fragility disorders are underlined. EBS, EB simplex; JEB, junctional EB; DEB, dystrophic EB; KS, Kindler syndrome. b. In the left panel, a young man with severe generalized dystrophic EB caused by COL7A1 loss-offunction mutations. Note chronic wounds on sites of mechanical pressure. In the right panel, a young woman with junctional EB and LAMB3 mutations, allowing residual expression of laminin 332. Note extensive scarring and non-healing wounds on sites exposed to mechanical stress.
in both cases recovering normal transcript and protein and correcting the enzyme deficiency (30). Gene editing is likely to become the next generation of gene therapy, by overcoming the potentially harmful effects of current gene replacement technology (25). The spectrum of targeted genome editing tools has enormous capabilities and applications, but each approach has distinct drawbacks and limitations (31); in particular, somatic cells have a low propensity to utilize homologous recombination to repair DNA (25). In the field of inherited skin fragility disorders, such strategies have been developed in preclinical studies (Table 1). For example, engineered transcription activator-like effector nucleases (TALENs) were able to induce site-specific doublestranded DNA breaks leading to homology-directed repair from an exogenous donor template. This process resulted in correction of the COL7A1 gene mutation in patient’s fibroblasts that were subsequently reprogrammed into inducible pluripotent stem cells and showed normal protein expression and deposition in a teratoma-based skin model in vivo (32). In another study, correction of LAMA3 mutations in primary keratinocytes derived from a patient affected by severe junctional EB was achieved using recombinant adeno-associated virus-mediated homologous recombination (33). The RNA transsplicing technology which aims at skipping or replacing exons harbouring mutations has been optimized to correct mutations in the genes for keratin 14, collagen XVII or plectin, in in vitro systems (34– 36). Finally, siRNA to knockdown the dominant negative allele was developed for the treatment of painful plantar keratoses in patients with pachyonychia congenita (37,38) and in preclinical settings for EB simplex and dominant dystrophic EB (39,40). Delivery of therapeutic siRNA is a hurdle to overcome. Manufacturing dissolvable microneedles to deliver siRNAs while minimizing pain has been accomplished for pachyonychia congenita, and a mouse study has been completed with no signs of toxicity (41).
requires permanent vector integration (25). Currently, clinical trials were initiated intending to develop safe and efficient ex vivo gene therapy approaches for a permanent treatment for recessive dystrophic EB [(26), http://clinicaltrials.gov/ct2/show/record/ NCT01263379?term=epidermolysis+bullosa&recr=Open&no_unk=Y&rank=1]. These approaches use autologous skin equivalents genetically corrected with safer retroviral or other vectors expressing type VII collagen (26,27) (28). The efficacy of antisense therapy for suppression of normal, pathogenic, or cryptic splice sites and skip exons bearing mutations has been demonstrated in cellular and animal models and has already reached the clinical trials phase for Duchenne muscular dystrophy. Preliminary data from early-phase clinical trials regarding an antisense oligonucleotide drug designed to correct the out-of-frame genetic defect via skipping of exon 51 of the dystrophin gene in patients with Duchenne muscular dystrophy had appeared encouraging. A failure in large-scale clinical trials was a clear setback for the field and should provide important lessons for future trials. The lack of therapeutic response seems to be due to two critical factors: (i) the amount of functional dystrophin protein generated, which was probably marginal for clinical efficacy and (ii) the primary outcome measure itself which was not appropriate (29). In different inherited metabolic diseases, spliceswitching oligonucleotides have been used with success in patients’ cells to force pseudoexon skipping or to block cryptic splice sites,
Cell-based therapies became highly popular among researchers in the EB field in the past few years, and the progress was recently reviewed (42–44). Early studies in mouse models served as a proof of principle and hold promise (9,45–50). Local injections of fibroblasts or mesenchymal stem/stromal cells (MSC) and bone marrow transplantation demonstrated a certain efficacy in patients with recessive dystrophic EB (51–54). It was suggested that injection of fibroblasts results in elevation of the heparin-binding EGFlike growth factor which may increase COL7A1 gene expression (55). The results of two clinical trials comparing allogeneic fibroblasts versus vehicle are contradictory (52,56), and the utility of this procedure remains to be defined. Injection of bone marrowderived MSC led to improvement of wound healing in two individuals with recessive dystrophic EB (54). Based on this and on studies in mice which showed that a specific MSC bone marrow subpopulation is mobilized to regenerate injured epithelia (50), systemic application of MSC is envisaged. Several clinical trials are ongoing and will hopefully shed light on the long-term outcome and on the precise mechanisms of the therapeutic effects. Beside the first seven patients with dystrophic EB treated with bone marrow transplantation in 2010 (51), the experience on overall 20 patients was recently reported (44). Five died due to complications of the transplantation or disease progression, whereas all others had at least some benefit from the procedure. Although these studies show efficacy of bone marrow transplanta-
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ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 325–331
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Has and Kiritsi
Table 2. Strengths and weaknesses of targeted therapies in skin fragility disorders Strategy Gene therapy
Gene correction
Pro
Contra
Effective for topical
treatment of chronic wounds
Cell therapies
Topical application Safety concerns Complex technology Expensive
Antisense oligonucleotides
Clinical trials for other genetic disorders
Limited stability in the skin High dosage required to achieve clinical benefit Toxicity
DNA editing Trans-splicing siRNA
Simple administration
Experimental approaches Low topical absorption in the skin Only topical application
Bone marrow transplantation Fibroblasts MSC
Established administration Systemic application Safe
Short survival of cells in the skin Low concentration of cells in unwounded skin High amounts of cells required to achieve therapeutic protein concentrations
Immune reaction after repeated administrations probable
IPSCs
Unlimited amount of autologous cells
Highly complex technology Safety concerns Expensive
Revertant mosaicism
Autologous cells which can be
Only topical application Revertant mosaicism must be present in the patient
used as grafts or epidermal sheets Simple administration Safe Inexpensive Protein therapy
Limited durability of the protein in the skin High amounts of protein required
Simple administration Potential systemic application Safe
to achieve therapeutic dosage
Immune reaction after repeated administrations probable
Not suited for intra-cellular structural proteins Drugs
Established for other disorders Simple administration Target pathogenic mechanisms Safe Inexpensive compared to experimental therapies
Unclear long-term effects Do not address the disease cause
BMT, bone marrow transplantation; MSC, mesenchymal stem cells; iPSCs, induced pluripotent stem cells.
tion in EB with some degree of amelioration, none of the patients had a complete cure of the disease. Recently, human umbilical cord blood-derived unrestricted somatic stem cells (USSCs) were induced to express genes that hallmark keratinocyte differentiation including collagen VII. USSCs have previously been demonstrated to have a broad differentiation potential and regenerative beneficial effects when administered in animal models of multiple degenerative diseases. In an initial study, USSCs promoted wound healing, suggesting that they could have a therapeutic potential for patients with recessive dystrophic EB (57).
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Although skin biopsies are a regular source of keratinocytes and epidermal stem cells, keratinocytes were recently generated from embryonic stem cells (ESCs) (58) and from induced pluripotent stem cells (iPSCs) (59–61). The clinical use of ESCs has significant ethical and biological obstacles related to their derivation from embryos and potential for immunological rejection, respectively. These disadvantages can be circumvented by the alternative use of iPSCs, which are derived from an individual’s (autologous) somatic cells by exogenous expression of defined transcription factors and have biological characteristics similar to ESCs. Patient-specific
ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 325–331
Therapies for inherited skin fragility disorders
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Figure 2. Revertant mosaicism in skin fragility. a. Areas of revertant mosaicism on the dorsal aspect of the hand of a young man with Kindler syndrome are outlined in green. The inset shows a magnification of the marked area, to better demonstrate the contrast between the affected, atrophic and the revertant skin with normal skin texture. b. Keratinocytes were isolated from the skin of a healthy control and from the revertant epidermis of a patient with Kindler syndrome. Kindlin-1 stains positive the control cells mainly at focal adhesions. Note that in the photographed field, only one cell derived from the revertant skin of the patient expresses kindlin-1, while the others stain negative, as expected by the mutation constellation.
mutant and corrected iPSCs were generated from individuals with recessive dystrophic EB (59,60,62,63) and junctional EB (64,65) and differentiated into epidermal keratinocytes which were able to build three-dimensional skin equivalents, suggesting their full functionality (66,67). Such models can be used to study disease mechanisms or for testing pharmacological agents. Safety issues and optimization of protocols must be addressed before application of iPSCs in clinical practice, but the field is changing very rapidly (Table 2). The translation of cell-based therapies for tissue repair remains limited by several factors, including poor cell survival and limited target site retention (Table 2). Advances in nanotechnology enable the development of specifically designed delivery matrices to address these limitations and thereby improve the efficacy of cellbased therapies (68).
Revertant mosaicism Spontaneous gene repair, also called revertant mosaicism or natural gene therapy, has been documented in all main types of EB (69). The interest in this topic has strongly increased in the past 5 years because of the therapeutic potential of these patients’ own, spontaneously corrected cells. It is conceivable that revertant mosaicism is more common than initially assumed (70–72) and that patients acquire revertant cells at some point, and the frequency, diversity and functionality of the revertant cells depend on various factors, including the strength of selection (2). Fundamental scientific questions regarding the mechanisms which drive revertant mutational events remain to be addressed. Clinically, revertant skin areas demonstrate a ‘normal’ or improved texture and mechanical resistance, compared to the surrounding affected skin (Fig. 2a). The difference can be confirmed through immunostaining with antibodies specific to the mutant protein. The elucidation of the reversion mechanisms on genomic level is more labour intensive and requires laser-dissection microscopy or deep sequencing. From a pragmatic perspective, patients benefit from identification and expansion of revertant tissue which provides material for cell therapies. In an initial revertant cell therapy on a patient with mosaic collagen XVII -deficient junctional EB, functional repair was not
ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 325–331
achieved due to the low percentage of revertant cells in the graft (73). Indeed, revertant epidermis is a mosaic, which only contains a certain percentage of revertant cells that can be lost in in vitro culture conditions (Fig. 2b). This behaviour seems to be dependent on the protein which is affected in each specific EB subtype. For example, in the case of the reversion of collagen XVII mutations, stainings showed 40% revertant cells after first passage, 25% revertant cells after the second passage and a drop of revertant cells to 15%, 1% and
