Newer Treatment Modalities in Epidermolysis Bullosa.

The term epidermolysis bullosa (EB) refers to a group of hereditary skin blistering diseases. The group is clinically and genetically heterogeneous, but all EB forms are associated with mechanically induced skin blistering and fragility. The causative gene mutations of most EB types are known. The current international consensus classification contains four main types: EB simplex (EBS), junctional EB (JEB), dystrophic EB (DEB), and Kindler syndrome (KS). The classification is based on the morphological level of blister formation. In EBS, the split is intra-epidermal, in JEB along the basement membrane and in DEB below the basement membrane. In Kindler syndrome, the dermal-epidermal junction is disorganized, and blisters can occur on all three levels. Each major EB type has further subtypes which may differ in terms of their genetic, biological or clinical characteristics. Traditionally, EB treatments have been symptomatic, but increasing understanding of disease etio-pathogenesis is facilitating development of novel evidence-based therapy approaches. First gene- and cell-based therapies are being tested at preclinical level and in clinical trials. New knowledge on secondary disease mechanisms has led to development and clinical testing of urgently needed symptom-relief therapies using small molecules and biologicals.

Introduction

The EB family: A broad spectrum of clinical subtypes

EB is clinically heterogeneous. All EB types manifest with trauma-induced skin blistering and fragility, but the individual types vary in terms of severity and associated extracutaneous manifestations [Figure 1].[123] Mutations in at least 20 different genes can cause EB [Table 1]. Molecular genetic diagnostics have lead to revelation of the genetic heterogeneity of the EB, and modern high-throughput technologies, such as chip-based diagnostics or exome sequencing, will probably uncover a few more causative genes in ultra-rare EB forms.[456] In recent years, the EBS group has expanded significantly, with very severe, but also several extremely mild subtypes that exhibit only minimal skin fragility [Table 1].[46] The JEB group has grown with a syndromal subtype, which exhibits renal, lung, and skin involvement and is caused by mutations in the integrin alpha3 gene.[7] As far as known today, all forms of dystrophic EB are caused by mutations in the collagen VII gene, making DEB ideal subtype for the investigative studies to explore disease pathogenesis and targeted therapies.

Figure 1

Clinical manifestations in epidermolysis bullosa. Upper panel: Junctional EB with mechanically induced blisters, wounds, and loss of nails in the right foot. Lower panel: Moderate dystrophic EB with mechanically induced skin fragility, inflammation, scarring, joint contractures and loss of nails in the left hand

Table 1

Major EB types and their molecular causes

Major EB type	Gene	Affected protein	Mode of inheritance
EBS	DSP	Desmoplakin	AR
JEB	PKP1	Plakophilin 1	AR
	JUP	Plakoglobin	AR
	KRT5	Keratin 5	AD
	KRT14	Keratin 14	AD, AR
	PLEC	Plectin	AR
	KLHL24	Kelch-like protein	AD
	DST	BPAG1	AR
	EXPH 5	Exophilin 5	AR
	CD151	Tetraspanin 24	AR
	TGM5	Transglutaminase 5	AR
	ITGB4	a6b4 Integrin	AR
	COL17A1	Collagen XVII	AR
	LAMA3 LAMB3	Laminin-332	AR
	LAMC2	Laminin-332	AR
	COL17A1	Laminin-332	AR
	ITGA3	Collagen XVII	AR
	ITGA6	a3b1 Integrin	AR
	ITGB4	a6b4 Integrin	AR
		a6b4 Integrin	AR
DEB	COL7A1	Collagen VII	ADAR
KS	FERMT1	Kindlin-1	AR

AR=Autosomal recessive; AD=Autosomal dominant

Diagnosis and clinical management

Diagnostics and management of the various EB forms are complex and require interdisciplinary collaboration. Molecular diagnostics is strongly recommended for all the patients with EB, because a precise diagnosis and knowledge of the hereditary mode are important for prognosis, genetic counselling, prenatal diagnosis, and for the planning of personalized therapies. However, if molecular diagnostics are not available, a recently developed clinical diagnostic matrix is suitable for predicting the EB type and even subtypes.[8] For molecular diagnostics, a two-step procedure has proven helpful. Traditionally, immunofluorescence mapping of a skin biopsy is performed first, followed by a mutation analysis of the candidate gene(s) that were determined on the basis on the immunofluorescence pattern. The introduction of next generation sequencing technologies has rendered immunofluorescence staining of a skin biopsy unnecessary as a first step. However, it is often needed for validation of the genetic findings of uncertain significance.[6] Similarly, although transmission electron microscopy of a skin biopsy is nowadays rarely used for diagnostics any more, it can be of great value for determining the consequences of mutations in EB and for skin research.

The basics of topical treatment of EB include avoiding trauma, good skin care, disinfection, and careful wound management (http://www.debra-international.org/clinical-guidelines). However, since many EB forms represent a systemic disease rather than a sole skin disorder and since chronic skin fragility has a high personal, medical and socio-economic influence on the lives of patients and their families, modern clinical management of EB is based on interdisciplinary and multi-professional care[9] (http://www.debra-international.org/clinical-guidelines/complete-eb-guidelines.html). Depending on the severity and the affection of different organ systems, including EB-associated skin cancer, care providers include dermatologists, pediatricians, dentists, ophthalmologists, gastroenterologists, surgeons, oncologists, and other medical specialists, primary care physicians and other relevant health professionals, including physical therapists, ergotherapists, nutritionists, psychologists, social workers, etc.

Expert centers on EB exist in many countries; they are networked internationally in the EB-Clinical Network “EB-Clinet” (www.EB-Clinet.org) and collaborate closely with the patient advocacy groups (www.debra-international.org). The centers provide information, advice and an extensive range of diagnostic and clinical management services, including help for precise diagnosis, genetic counselling, interdisciplinary examinations and preparation of the management plan, which can then be implemented by physicians close to home.

Newer therapy approaches

The rapid scientific developments in recent years make various therapeutic strategies for the treatment of EB appear promising. The scientific and clinical EB communities work closely together in determining priorities. Several patient advocacy groups are remarkably active and successful in fund raising; they also fund research projects (www.debra-international.org). Obviously, severe forms of EB have the highest unmet medical need, and most research efforts focus on therapies for these forms. Both curative and symptom-relief therapies are being developed, as delineated below. A number of therapies are in preclinical development and the first ones have already reached clinical trial stage, either at Phase1/2 or 3 [Table 2]. For more details, the reader is referred to recent reviews on this topic.[101112]

Table 2

Currently recruiting clinical therapy trials for EB

Therapy	Investigational Drug	EB Type	Trial identification Nr.
Therapies with curative aim
Gene therapy	Transplantation surgery of genetically corrected cultured epidermal autograft (ATMP)	JEB with COL17A1 mutations	ClinicalTrials.gov Identifier: NCT03490331
	Genetically corrected cultured epidermal autograft (ATMP)	RDEB*	ClinicalTrials.gov Identifier: NCT02984085
	FCX-007, Genetically modified autologous human dermal fibroblasts	RDEB*	ClinicalTrials.gov Identifier: NCT02810951
	KB103, a non-integrating, replication-incompetent herpes simplex virus vector expressing human collagen VII protein	DEB	ClinicalTrials.gov Identifier: NCT03536143
Antisense oligonucleotide	QR-313, an antisense oligonucleotide (AON)	DEB with mutations in exon 73 of COL7A1	ClinicalTrials.gov Identifier: NCT03605069
PTC read-through	Gentamicin, intravenous	RDEB	ClinicalTrials.gov Identifier: NCT03012191
Cell therapy	Serial mesenchymal stem cell (MSC) infusions from a related donor	All EB types	ClinicalTrials.gov Identifier: NCT02582775
	Allogeneic stem cell transplantation and “off-the-shelf” mesenchymal stem cells	All EB types	ClinicalTrials.gov Identifier: NCT01033552
Symptom-relief therapies
Anti-fibrotic	Losartan, systemic	RDEB*	EudraCT Number: 2015-003670-32
Anti-inflammatory	Diacerein, topical	EBS	ClinicalTrials.gov Identifier: NCT03154333
	Pharmacokinetics, safety of diacerein after maximum use	EBS	ClinicalTrials.gov Identifier: NCT03472287
	Oleogel, topical	All EB types	ClinicalTrials.gov Identifier: NCT03068780
	BPM31510 3.0% Cream, topical	All EB types	ClinicalTrials.gov Identifier: NCT02793960
	Sirolimus, topical	EBS	ClinicalTrials.gov Identifier: NCT03016715

*RDEB=Recessive DEB

Therapies with curative intention

Gene therapies: At the beginning of the “therapeutic era” for EB more than 10 years ago, the most obvious consideration was to replace a defective gene with gene therapy. An approach using retrovirus-mediated gene correction in keratinocytes and grafting has indeed been successful in individual cases of JEB,[131415] and a similar approach is being developed and tested for DEB[16] [Table 2]. However, it turns out that this form of gene therapy brings with its complex issues and questions relating to technological development of gene vectors, oncogenic potential and future risk of malignancy, duration of therapeutic effects, etc. Current research on cure of EB focuses on different approaches including the use of gene-corrected patient's own iPS cells, gene editing technologies, and polymer-mediated DNA delivery systems.[1718192021] It is important to note that no gene therapy approaches have been approved for EB; all these therapy developments are currently experimental and in in vitro or preclinical state, with a few exceptions that are tested in early clinical trials on individuals with EB.

An interesting option is the so called “natural gene therapy” which is based on use of cells or tissue derived from revertant mosaic patches in the patient's skin. Revertant mosaicsm, a relatively common phenomenon in human genetic disorders, is based on the fact that in individual cells the inherited mutation is compensated bya second, somatic mutation that restores the expression of the lacking protein and reverts the disease phenotype in the cell. Revertant cells proliferate clonally, and mosaic skin patches can be found in all EB types. The genetic mechanisms of the reversion have been characterized,[22] and a successful clinical application of small split thickness grafts derived from revertant skin was reported in an individual with JEB.[23]

Cell therapies: have not only led more quickly to clinical studies, but also here the therapeutic context is more complex than initially anticipated. Intradermal injections of fibroblasts improved the dermal-epidermal adhesion in the DEB mouse model for several months,[24] but pilot treatments of patients revealed this therapeutic modality to be very painful and not well tolerated[25] – and not consistently efficacious. Indeed, one study found similar effects on wound healing with both fibroblast injections and vehicle injections.[26] A systemic therapy for severe DEB by bone marrow transplantation improved symptoms in some patients, but did not cure DEB. The rate of adverse effects was high and some patients died from complications of bone marrow transplantation.[272829] Intravenous infusions with mesenchymal stromal stem cells (MSC) from the bone marrow relieved symptoms such as itch and improved the general well-being of children, but did not increase collagen VII in the skin.[2930] Current clinical trials are studying the effects of different kinds of MSC in adults with EB [Table 2]. All in all, the number of individuals treated with different cell therapy approaches is very low and, therefore, the percentage of patients with clinical benefits cannot be reliably estimated. More carefully designed and monitored studies will be needed before efficacy and cost-benefit relations can be determined.

Protein therapy: Protein replacement therapies have been successfully developed for a number of genetic diseases[31] and might have potential for EB, too.[32] A protein therapy approach using intravenous or intradermal recombinant collagen VII injections for treatment of DEB seemed promising at preclinical level,[3233] but further development and clinical assessment are still required before its suitability for treatment of patients can be determined.

Antisense oligonucleotides: The rationale here is that antisense oligonucleotide treatment of cells can lead to skipping of the mutated exons at the RNA level, and thus restore the synthesis of a nearly normal protein that lacks a small fragment encoded by the deleted exon.[34] Diseases caused by collagen gene mutations are especially suitable candidates for this strategy, since most exons in these genes are in-frame and their deletion does not cause major structural changes at the protein level. In the context of EB, the collagen VII gene and its disease, DEB, have been of particular interest. Preclinical testing in mice suggested that antisense oligonucleotide-based exon skipping can improve skin stability by partially functional collagen VII.[35] A Phase 1/2 multicenter clinical trial tests this approach in DEB [Table 2].

PTC read-through: This approach is based on the fact that approximately 10% of genetic diseases are caused by mutations that generate premature termination codons (PTC) and lead to nonsense-mediated mRNA decay.[36] If the PTC could be ignored by the transcription machinery, a full-length polypeptide with a minor modification and, hopefully, with adequate function, can be synthesized. Aminoglycoside antibiotics are known inducers of PTC read-through and have been tested for many genes. The efficiency of the read-through depends on the local nucleotide microenvironment of the mutations, and a careful selection of suitable mutations is an essential prerequisite for successful treatment. Gentamicins have demonstrated some efficacy for COL7A1 and LAMB3 mutations in vitro,[3738] and a pilot clinical study on five DEB patients treated with topical gentamicin showed increased collagen VII in the skin.[39] Topical application may circumvent the renal and ototoxicity of gentamicin, although the capacity of aminoglycosides to induce contact dermatitis may pose a challenge. A minor gentamicin component, gentamicin B1, could be an alternative because it is a potent inducer of the PTC read-through and has low toxicity.[40] Currently, a clinical trial is testing intravenous gentamicin for its efficacy in RDEB [Table 2]. Also, Amlexanox, an anti-inflmmatory drug approved in some countries can induce PTC read-through and was shown to enhance collagen VII protein synthesis in vitro in DEB cells carrying nonsense mutations.[41]

Symptom relief therapies

During the past few years, the complexity of issues relating to development and clinical implementation of curative therapies for EB has become obvious.[101129] The realization that such therapies will not enter the clinical routine until in years, combined with pressure from patient advocacy groups to finally obtain some efficacious treatments, has pushed the search for ways to ameliorate symptoms. Indeed, better understanding of molecular and cellular disease mechanisms in EB has identified novel therapeutic targets and allowed design of symptom relief therapies.[11] Such treatments do not aim at a cure but at better functionality and improved quality of life of the affected individuals.

Topical therapies: Several different topical therapies have been tested in clinical trials for EB, all of them based on evidence from in vitro studies. Diacerein, an anti-inflammatory compound isolated from rhubarb root[42] has been shown to increase skin stability in EBS.[43] In classical EBS, keratin 5 and 14 mutations cause aggregation of the intermediate filament cytoskeleton and cell fragility.[1] This, in turn, leads to an inflammatory response, e.g., via interleukin-1β signaling. In vitro testing indicated that diacerein can counteract the inflammatory process and stabilize the cells, and a subsequent, small, placebo-controlled phase 2/3 clinical study with diacerein cream demonstrated reduction of blister counts in >40% of the participants.[43] A further, international multicenter study is currently ongoing [Table 2]. Another topical agent with anti-inflammatory properties is a betulin-based oleogel derived from birch bark. In vitro and in vivo, betulin enhanced keratinocyte differentiation.[44] A small, open, blindly evaluated phase 2 pilot study in patients with DEB indicated that betulin-based oleogel promotes re-epithelialization and enhances wound healing.[45] An ongoing international Phase 3 study tests the safety and efficacy of betulin-based oleogel in larger cohorts of all types of EB. An ongoing trial tests topical 2% sirolimus, an mTOR inhibitor, as treatment of plantar blistering in patients with EBS. The goal is to downregulate the expression of mutated keratins and thereby to improve blistering and hyperkeratosis [Table 2]. An already completed trial (ClinicalTrials.gov Identifier NCT01538862) used subcutaneous injections of granulocyte colony stimulating factor (GCSF) as therapy in seven individuals with DEB. Some improvement (approximately 30%) was observed relating to counts of active blisters and erosions, and to the surface area of non-healing erosions.

Systemic therapies: Evidence is accumulating that inflammation plays an important role in EB and aggravates the phenotypes. Recent publications have reported findings of increased cytokine levels in the skin and serum of individuals with EBS.[4647] Based on the high levels of Th17 cells/cytokines in lesional EBS skin, three patients received apremilast, which led to improvement of skin blistering.[47] It would be interesting to extend the analyses to larger EBS cohorts and also to other EB types. A multicenter placebo-controlled clinical trial that tested oral Epigallocatechin 3 gallate (Polyphenon E), a proteinase inhibitor, as treatment for DEB in 17 individuals came to the conclusion that this compound was well tolerable but not more effective than placebo.[48]

Since progressive soft tissue fibrosis is a major systemic feature in DEB[14950] and a prerequisite for development of secondary squamous cell carcinoma,[351] small molecule drugs which reduce inflammation and fibrosis may be useful to postpone mitten deformities, joint contractures, and internal strictures.[11] Losartan is an approved drug for treatment of hypertension, but it has also been shown to be beneficial in some rare diseases manifesting with secondary fibrosis.[5253] The rationale is that as an AT-1 receptor antagonist losartan counteracts TGFβ signaling in a context- and disease-specific manner.[54] In DEB, inflammation and TGFβ- signaling are major drivers of fibrosis[49505155] and, therefore, losartan seems promising as a symptom-relief therapy. In vitro, it efficiently inhibited TGFβ-signaling in DEB fibroblasts, and in the DEB mouse model losartan treatment reduced TGFβ activity, inflammation and fibrosis in the skin in vivo. Macroscopically, this manifested in significant delay in the development of mitten deformities.[56] As a clinical development, an ongoing Phase 1/2 trial (REFLECT) explores safety and tolerability of losartan in children with moderate-to-severe DEB. The trial also collects first information on the efficacy of losartan to improve specific clinical symptoms and quality of life (Table 2; D. Kiritsi, personal communication).

HMGB1: Tamai et al. took a different approach to reduce systemic fibrosis in DEB employing a HMGB1-derived peptide. Under physiological and pathological conditions HMGB1 (high mobility group box 1) has a number of different functions.[57] In the context of EB it increases a specific bone marrow-derived mesenchymal stem cell population that migrates to the circulation, homes to damaged skin, and suppresses inflammation.[58] At preclinical level, in the DEB mouse model, the HMGB1-derived peptide was effective against both skin fibrosis and gastrointestinal strictures.[59] It will be intriguing to follow the progress of this therapeutic approach into clinical trials.

Conclusions and Outlook

Taken together, based on the above information, we can be optimistic that the repertoire of potential DEB therapies will continue to grow. Different evidence-based approaches are being considered and currently tested at preclinical and clinical levels for most severe EB types. Future research investigations must provide better understanding of disease mechanisms in all EB types and identify the most suitable therapeutic targets. It is unlikely, that there will be a “one-size-fits-all” therapy for each major EB type. Rather, the treatments will be based on individual mutations constellations, disease mechanisms, and phenotypic characteristics. Furthermore, it seems probable that combinations of different therapeutic principles, e.g., interval therapies using combined or alternating cell and drug therapies, to combat the causes and secondary disease features of EB will bring the best results.

Financial support and sponsorship

The author's work has been supported by Debra International and Debra Austria.

Conflicts of interest

There are no conflicts of interest.