Epidermolysis bullosa: Advances in research and treatment

Abstract Epidermolysis bullosa (EB) is the umbrella term for a group of rare inherited skin fragility disorders caused by mutations in at least 20 different genes. There is no cure for any of the subtypes of EB resulting from different mutations, and current therapy only focuses on the management of wounds and pain. Novel effective therapeutic approaches are therefore urgently required. Strategies include gene‐, protein‐ and cell‐based therapies. This review discusses molecular procedures currently under investigation at the EB House Austria, a designated Centre of Expertise implemented in the European Reference Network for Rare and Undiagnosed Skin Diseases. Current clinical research activities at the EB House Austria include newly developed candidate substances that have emerged out of our translational research initiatives as well as already commercially available medications that are applied in off‐licensed indications. Squamous cell carcinoma is the major cause of death in severe forms of EB. We are evaluating immunotherapy using an anti‐PD1 monoclonal antibody as a palliative treatment option for locally advanced or metastatic squamous cell carcinoma of the skin unresponsive to previous systemic therapy. In addition, we are evaluating topical calcipotriol and topical diacerein as potential agents to improve the healing of skin wounds in EBS patients. Finally, the review will highlight the recent advancements of gene therapy development for EB.

a Kindler syndrome-like phenotype (early blistering subsiding with age, multi-systemic involvement including nephropathy). [9] The same group also revealed homozygous missense mutations in PLOD3 encoding lysyl hydroxylase 3 (LH3) to cause widespread connective tissue abnormalities including extensive joint contractures, skeletal abnormalities, reduced growth and sublamina densa skin blistering similar to that in recessive dystrophic EB. [10] Pathogenetically, a deficient glycosylation activity is implicated to alter post-translational modifications of type VII and other collagens causing deleterious changes in deposition and organization of extracellular matrix, such as a deficient extracellular assembly of anchoring fibrils that compromises their stability and renders them subject to degradation.
These molecular aberrations finally impair the structural and functional integrity within the highly specialized interfaces, which are crucial for cell adhesion, proliferation and differentiation, tissue repair, and barrier function. [2,3,5] Consequently, this leads to characteristic diminished resistance to mechanical stress and shearing forces with subsequent cell and tissue damage.
The index genes involved are also partly expressed in other epithelialized (gastrointestinal, respiratory, urogenital tract) or mesenchymal (skeletal muscle) organs. Apart from secondary extracutaneous involvement, this also explains the occurrence of primary extracutaneous manifestations and relevant complications especially in the severe forms of EB, making them a multisystem disease with significant morbidity and mortality. [11] Type (homo-vs heterozygosity), number (monogenic, digenic inheritance) and location of mutation(s) within the gene or gene segment, as well as the spectrum of subsequent quantitative (absence, reduction) or qualitative (gradual loss of function) disruption of protein expression, result in considerable genetic heterogeneity with complex genotype-phenotype correlations. [12,13] In addition to the primary structural-functional genetic defect, secondary epigenetic and biochemical (eg differentially regulated expression of a host of other genes involved in the maintenance and function of this microenvironment, induction of inflammatory cascades) or environmental factors also have a major impact on the individual phenotype. [12,13] This corresponds to a considerably broad spectrum of clinical manifestations and severity, ranging from limited moderate blistering on primarily mechanically exposed sites such as hands and feet that manifests at times only in late childhood or adolescence, to extensive, generalized, fatal (multiorgan) involvement with (seemingly spontaneous) blistering affecting most of epithelialized tissues already at birth. Common causes of (early) death include malnutrition, infections, organ failure and skin cancer. [14] Currently, no general curative therapy is available for all EB types. In light of the morbidity as well as lethality of numerous subvariants as well as the current availability of largely only symptomatic treatment options, causative therapeutic approaches are therefore urgently required. Such strategies include gene-, protein-and cell-based therapies. [15] This review discusses molecular procedures currently under investigation at the EB House Austria (www.eb-haus.org), a designated Centre of Expertise implemented in the European Reference Network for Rare and Undiagnosed Skin Diseases (ERN Skin) with currently 56 partners (http://skin. ern-net.eu/), and at other centres. Albeit still largely experimental and facing major hurdles including safety and efficiency to overcome, these techniques should contribute to determine curative perspectives.

| CLINI C AL RE S E ARCH
As stated above, the severe forms of EB are not restricted to the skin but affect the entire body. Several approaches have therefore been developed in the quest for a systemic therapy. Attempts using allogeneic bone marrow transplantation (BMT) with or without infusion of mesenchymal stem cells (MSCs) have shown some transient beneficial clinical response in a small number of patients with RDEB [16][17][18][19] but not in patients with JEB. [20] The mortality rates that come with such serious interventions are considerable, and the initially high expectations for the approach have not been met.
Several substances that elicit premature termination codon (PTC) read-through showed promising results in vitro for specific RDEBand JEB-associated mutations. [21][22][23] The promise of PTC readthrough substances, which often fall into the small molecule category of drugs available for repurposing, is to express a previously absent protein. The antibiotic gentamicin has already been tested in a few patients with PTC mutations and seemed to improve the skin phenotype for a few months when applied topically or via intradermal injections in some patients. [24] Clinical studies to test the effect of intravenous injections of gentamicin are currently conducted for RDEB (NCT03392909) and JEB (NCT03526159).
Systemic recombinant human type VII collagen protein administration is another therapeutic approach that is pursued for the treatment of RDEB patients. It has previously shown promising results in a mouse model for RDEB [25] and is currently being tested in a phase I/II randomized controlled single-blind clinical trial (NCT03752905) at Stanford University.
Current clinical research activities at the EB House Austria include the development of novel therapies that have emerged out of our translational research initiatives, as well as commercially available medications that are used for "off-label" uses.

| Nivolumab in locally advanced/metastatic squamous cell carcinoma
In a prospective, multi-centre, phase II trial (Eudra CT-No. 2016-002811-16), we are evaluating the administration of nivolumab (an anti-PD1 monoclonal antibody) for the palliative treatment of patients with DEB who have locally advanced or metastatic squamous cell carcinomas of the skin (SCCS) that have been unresponsive to other systemic therapies. The target population also includes patients suffering from generalized severe recessive dystrophic epidermolysis bullosa, in which SCCS, owed to a highly aggressive course with early metastatic spread, is the primary cause of death with a cumulative risk of 70% to die by age 45. [26] For both EB and non-EB SCCS, evidence on overall clinical effectiveness of standard chemotherapeutic agents (with rather poor tolerability among older patients) as well as systemic therapies targeting the epidermal growth factor receptor (EGFR) pathway remains mostly limited and is often of short duration. [26][27][28] Immunosuppression plays a major role in the pathogenesis of SCCS, [29][30][31][32] which is evident especially in solid organ transplant patients as well as severe EB forms along with malnutrition, anaemia and chronic infections. [33,34] Tumor emergence and progression may further depend upon acquisition of traits that allow cancer cells to evade immune surveillance and an effective immune response. [35][36][37] Against this background, current immunotherapy efforts attempt to break the apparent tolerance of the immune system to tumor cells and antigens by either introducing cancer antigens by therapeutic vaccination or by modulating regulatory checkpoints of the immune system. [38][39][40] As for the latter, blockade of programmed death receptor-1 (PD-1) was shown to impair downregulation of T-cell activation upon PD-1 binding [28,29] and proved clinical activity in a variety of tumor types, including melanoma, renal cell cancer and non-small cell lung cancer. [41] Plausible molecular features of SCCS, [42][43][44] consistent data from animal studies [45] as well as beneficial evidence from clinical trials in advanced head and neck SCC (HNSCC), [46,47] and, recently, locally advanced/metastatic SCCS [48][49][50] support the therapeutic scope of PD-1 inhibition. Objective response was reported to be approximately 50% in early phase I and II trials, [49] leading to the approval of the PD-1 inhibitor cemiplimab for the treatment of patients with metastatic or locally advanced SCCS who are not candidates for curative surgery or curative radiation.
Despite obvious progress, current knowledge to correlate clinical effectiveness of checkpoint inhibitors with the tumor immune microenvironment is still limited in SCCS and should be subject to further investigation. [48,[51][52][53][54][55] Our trial seeks to determine the objective response rate of immunotherapy with nivolumab in index patients with using Response Criteria in Solid Tumors version 1.1 (RECIST1.1) per site assessment up to 2 years as the primary objective. Treatment efficacy will be correlated with the number of tumor-infiltrating lymphocytes (TILs) and CD8-positive lymphocytes at baseline.

| Topical calcipotriol in dystrophic epidermolysis bullosa
Enhanced tissue fragility, persistent wounds and inflammation in EB predispose to complications including infections, sepsis and the aforementioned development of aggressive SCCS. [1,56] Common wound colonizers in EB, such as Staphylococcus sp., Streptococcus sp., Candida sp. and Pseudomonas aeruginosa, [57] with loss of microbial diversity, may contribute to prolonged inflammation, delayed wound healing and chronic wounding. [58] Most recently, a role for innate immune sensing of microbial products, such as bacterial flagellin via toll-like receptor 5, in promoting wounding-and inflammation-induced skin tumorigenesis was demonstrated, [59] highlighting that local antisepsis, topical antibiotics and local wound care are critically important in wound management and possibly in cancer prevention in EB.
All currently existing approaches for the treatment of bacterial infected wounds have some disadvantages. Antiseptic baths, which are recommended for dystrophic EB patients, are often time-consuming and exhausting as well as painful for the patients since all dressings have to be carefully removed. Despite beneficial results, silver-containing creams (eg Flammazine) should not be applied for more than maximal 2-4 weeks due to emerging silver toxicities attributable to systemic absorption. Finally, long-lasting application of antibiotic ointments for the treatment of infected wounds may lead to multi-resistant bacterial strains, such as methicillin-resistant

Staphylococcus aureus.
Antimicrobial peptides (AMPs) form part of the body's innate immune response, serve as potent antibacterial substances that control pathogenic infections and activate the innate and adaptive immune system. [60] Cathelicidin (human cationic antimicrobial protein 18, hCAP18) is a prominent AMP in human epithelial cells, which serves to augment host defense, and appears to play a role in tissue repair and wound closure. [61][62][63][64] In response to cutaneous infections, hCAP18 is upregulated in skin and shows direct antimicrobial, antiviral and antifungal activity. [64] Cathelicidin is directly regulated by vitamin D. [65] Sun exposure, leading to production of the prehormone in the skin, serves as a distinct source of vitamin D. In EB patients, limited sun exposure due to wound dressings and reduced outdoor activity may lead to vitamin D deficiency, [66] resulting in reduced cathelicidin production and lowered antimicrobial defenses.
Our preliminary data on hCAP18 mRNA levels in skin tissue samples taken from RDEB patients and RDEB patient-derived keratinocyte cell lines support this hypothesis. [67] We also observed an increase in hCAP18 expression at transcript and protein level in RDEB keratinocytes by treatment with distinct concentrations of the active vitamin D3 analogue, calcipotriol, which is already used routinely in the clinical treatment of psoriasis. [67,68] This correlated with reduced colony formation upon incubation of E coli with supernatants from cultured calcipotriol-treated RDEB cells compared to controls while retaining any anti-proliferative effect of calcipotriol detrimental to wound healing. [67,69] Based on our results including a promising single-patient observation study, [67] we are currently investigating the potential of improved wound healing in dystrophic EB via increased in vivo expression of cathelicidin and thus enhanced antimicrobial defense by topical administration of calcipotriol in a double-blind, placebo-controlled crossover study (EudraCT-No 2016-001967-35). Notably, locally produced cathelicidin can serve as a chemoattractant for innate immune cells including neutrophils, which produce and store vast amounts of AMPs and release them at sites of infection, [70] underlining the utility of this approach in diseases characterized by increased susceptibility to bacterial infections. The immunomodulatory effects should foster the restoration of microbial diversity and inflammatory balance in EB skin. Moreover, increased production of LL-37 (the active peptide form of cathelicidin) upon vitamin D treatment has previously been reported in chronic and non-healing wounds. [62] [73] This mechanism is suspected to increase the expression of the dominantly interfering mutated K14 allele, thereby potentially aggravating the EBS-gen sev phenotype as compared to milder subtypes of EBS caused by nonsense mutations within the same gene.

| Topical diacerein in EB simplex
Additionally, blister formation was found to be related to matrix metalloproteinase (MMP)-9 and chemokine CXCL8/IL8, whose expression is also dependent on the IL-1β signalling pathway, that thereby modulates the tissue microenvironment. [74,75] In this context, previous studies revealed high levels of IL-1β in blister fluid and serum samples from EBS patients [76,77] and significantly upregulated IL-1β in a mouse model for EBS. [78] In summary, studies on EBS-gen sev keratinocytes' transcriptomes and proteomes revealed a complex landscape of dysregulation, triggered by a single, heterogeneous point mutation within either of the basal keratins, and prominently affecting downstream transcriptional targets of the IL-1ß-induced stress pathway.
Therapeutic approaches to inhibit IL-1β could thus be beneficial to patients through reduction of downstream cellular inflammation.
Indeed, treatment of EBS-gen sev patient keratinocytes with diacerein, a small molecule component of rhubarb root and reported IL-1β inhibitor, not only reduced the expression levels of K14 and IL-1β and the phosphorylation levels of JNK, but also stabilized the intermediate filament network upon heat shock. [73] Additionally, the invasive potential of EBS-gen sev keratinocytes decreased upon IL-1ß inhibition in functional assays, presumably because of reduced matrix metalloproteinase expression and increased expression of cell-junction proteins. Our data are in line with the former demonstration of reduced overall K14 expression levels upon mRNA correction via spliceosome-mediated RNA trans-splicing (SMaRT), which shifts the expression ratio of wild-type vs mutated K14 alleles towards wild type. [79] Based on our in vitro data, we launched a double-blind, randomized, placebo-controlled phase 1 pilot study of topical diacerein 1% cream in 5 patients with EBS-gen sev. [80] Topical  [81] In this 2-period crossover trial, 17 patients were randomized to either placebo or diacerein for a 4-week treatment and a 3-month follow-up in period 1. After a washout, patients were crossed over during period 2. Of the patients receiving diacerein, 86% in episode 1% and 37.5% in episode 2 met the primary end point (vs 14% and 17% with placebo, respectively), which was prespecified as the proportion of patients with a reduction

| Gene therapy
So far, only a small number of EB patients have undergone gene therapy. These individuals received the classical treatment approach termed replacement gene therapy. The treatment, which is suitable for recessive forms of EB, relies on ex vivo viral transfer of a functional TA B L E 1 Facts from JEB gene replacement therapies Genetically corrected epidermal grafts LAMB3 • Showed regeneration of morphologically normal, fully differentiated and functional, mechanically stable and non-blistering transgenic skin • Can replace up to 80% of a patient's diseased skin [84] • Showed sustained synthesis of the transgenic protein [131] • Harbour EpSCs which expanded and ensured long-term or permanent regeneration [84] • Despite the risk of insertional mutagenesis tumorigenicity has not been observed within a follow-up period of currently more than 12 y ( [83,84,131] , and unpublished observations) • Following regeneration, there was no evidence of an immune response against the newly expressed protein (unpublished observations) TA B L E 2 Current strategies of molecular therapy in EB  [133][134][135] Preclinical studies [86,[88][89][90][91]93,94,[96][97][98][99]103,104,110] Austria, China, France, Italy, Spain, UK, USA Gene silencing siRNA: knockdown of the mutant allele without silencing the wildtype allele Preclinical studies [136] France "Natural gene therapy"

Revertant mosaicism
Cultivation/graftingof cells in which the inherited mutation is corrected by a spontaneous genetic event (revertant cells) JEB: Phase I-in vivo grafting of revertant epidermal sheets in two patients (COL17A1 mutation, LAMB3) [137,138]

Fibroblasts
Injection of WT allogeneic fibroblasts, to substitute the missing protein (C7 is synthetized by keratinocytes and fibroblasts) RDEB: Phase I-single intradermal injections of allogeneic normal fibroblasts to 5 patients [140] 2008, UK Increased type VII collagen at the DEJ at 2 wk and at 3 mo following injection and increased anchoring fibrils (with altered morphology) RDEB: Phase II-intradermal injections of allogeneic fibroblasts in five patients, randomized controlled trial (RCT) [141] 2013, Australia All injected wounds (vehicle and allogeneic fibroblast) healed more rapidly compared to controls RDEB: Phase II-single intradermal injection of allogeneic fibroblasts into the margins of 26 chronic erosions in 11 patients (RCT) [142] 2013, UK All injected wounds healed initially (28 d) more rapidly than non-injected wounds, without any difference between the cultured allogenic fibroblasts and vehicle RDEB: Phase I-intradermal injections of allogeneic mesenchymal stromal cells in 2 patients [143] 2010, Chile At week 12, increased healing tendency compared to control sites. Clinical improvement lasted 4-6 mo RDEB: Phase I-intravenous infusions of bone marrow-derived non-hematopoietic MSCs in 14 patients [144] 2015, Egypt Clinical improvement peaked in most of the patients after 3 mo; 2 patients showed improvement for at least 1 y RDEB: Phase I/II-intravenous allogeneic mesenchymal stromal cells in 10 children (EBSTEM) [145] 2013, UK cDNA copy of the defective endogenous gene into keratinocytes and/or fibroblasts isolated from skin biopsies, which are then grafted back onto the skin. The approach has first been applied in a patient with junctional EB caused by mutations in LAMB3 [82] and has since been used in two additional JEB patients. [83,84] [85] While replacement gene therapy is incapable to overcome dominant-negative interference in the majority of EBS or DDEB forms,

| FROM REPL ACEMENT G ENE THER APY TO PRECIS I ON MED I CINE
Considerations to advance the above-described strategy of cell and gene therapy include the employment of designer nucleases such as clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) capable of mediating gene editing to correct distinct mutations at their endogenous loci. Instead of replacing the function of the mutant gene by adding a wild-type cDNA, this technology allows the potentially traceless repair of the genetic mutation itself, eliminating the risk of insertional mutagenesis resulting from random transgene integrations.

| Gene editing
Whereas retroviral transduction of a cDNA is highly efficient, the random integration of the transgene remains a risk. This risk can be eliminated by using the next-generation gene therapy technology relying on designer nucleases. The nucleases are engineered to target the locus of interest specifically. They are transiently transfected and capable of inducing deletion or repair of disease-causing mutations.
Most efforts to develop gene editing-based therapies for EB have focused on the correction of RDEB or DDEB mutations in COL7A1, [86][87][88][89][90][91][92][93][94] , whereas JEB (LAMA3 [95] and LAMB3 [96] ) and EBS (KRT5 [97] , KRT14 [98] ) were less frequently reported. In contrast to replacement gene therapies, where one vector can be used to treat all EB patients with recessive mutations in a specific gene, most designer nuclease-mediated approaches that aim to repair gene defects are patient-or rather mutation-specific. The endogenous DNA damage repair mechanisms triggered and harnessed by the technology require that designer nucleases target specific DNA sequences in close proximity to the mutation site. For genes such as COL7A1 with a size of >30 kb and disease-causing mutations spread almost along the entire length of the gene, this is challenging. Among the first targets chosen from our laboratory and others to develop efficient nucleases were therefore hot-spot mutations such as the c.6527insC in exon 80 of COL7A1, which is highly prevalent in the Spanish dystrophic EB patient population. [86,88,94,99] Dominant-negative mutations such as those common in EBS can be corrected in different ways. In addition to a mutation-specific repair approach for a KRT14 mutation, [98] we developed another treatment approach based on deletion of a mutant keratin 5 allele while leaving the wild-type allele intact and taking over the function. [97] The latter study demonstrated that one gene-specific designer nuclease could be employed to inactivate any EBS-causing mutant KRT5 allele by inducing insertions or deletions (indels) via the errorprone non-homologous end-joining (NHEJ) repair pathway causing a frameshift at a site within the target gene that is independent on the location of the disease-causing mutation.
First attempts of gene editing mutations associated with EB used meganucleases. [89] These were soon replaced by approaches employing the more specific transcription activator-like effector nucleases (TALEN). [86,91,97,99]  nicking approaches based on single-strand DNA breaks (nicks) at the targeting site with the intention to reduce off-target site effects. [88,98] Compared to the extremely high numbers of off-target site modifications inherent to replacement gene therapies resulting from multiple random integrations in the treated cells, off-target mutations are rare or non-existent upon designer nuclease treatment. [100] While most gene editing approaches published to date for EB used viral transfer of the nuclease or the repair template or both, [86 ,87,89,92,94-96,101-105] we are focusing on the development of non-viral delivery [88,98] and traceless targeting [97] approaches with a view to translation into the clinic. Although non-viral keratinocyte transfection is less efficient and, instead of nearly 100% viral transduction efficiency, reaches maximally 70% efficiency, it avoids the risk of insertional mutagenesis. Novel safer non-viral transfer methods are currently being developed. [106][107][108] Like replacement gene therapy, the majority of the current gene editing approaches aim for ex vivo therapy often including a selection step before grafting of corrected cells. We showed that plasmid DNA can be delivered and expressed following gene gun delivery into the skin of mice. [109] Wu et al [110] demonstrated data where they successfully targeted EpSC in vivo by electroporation of RNPs, consisting of CRISPR/Cas9 and gRNA, in an RDEB mouse model.
Gene therapy targeting keratinocytes and/or fibroblasts [87,101] allows treatment of patches or larger areas of the skin. The severe types of EB, however, also involve internal organs, demanding a systemic approach. targets RNA rather than DNA, the corrections are not permanent and the risk of mutagenesis is low. The transient nature of the correction at mRNA level however means that the treatment has to be repeated to result in an improvement of the disease phenotype.

| RNA editing
In the case of collagen type VII with a predicted half-life of several weeks, [112] the effect of an application can be relatively long lasting.
Some EB-causing mutations reside in exons that are non-essential for the function of the protein that is encoded by the gene. [119] Within COL7A1, exons 79, 73 and 80 are dispensable and can, in the presence of specifically designed antisense oligonucleotides (ASOs), be skipped during pre-mRNA splicing. [120][121][122] Furthermore, ASOs can be employed to modulate and improve SMaRT efficiency. [123,124] With approximately 21 nucleotides in length, ASOs are relatively small and can be chemically modified to increase their stability. ASO therapeutics has recently been approved in the United States for Duchenne muscular dystrophy and in the United States and Europe for spinal muscular atrophy. An exon 73-skipping ASO trial for RDEB is currently recruiting patients in the United States.

| CON CLUS I ON S AND FUTURE CHALLENG E S OF EB RE S E ARCH
Epidermolysis bullosa research has advanced considerably in the past decade (Table 3), and wound and pain management has improved.
Inclusion of several clinical disciplines in addition to dermatology has proven essential in order to provide comprehensive care for EB patients. Novel highly efficient cancer therapies give hope in the battle to fight the aggressive SCCs in the most severe EB forms. Guidelines on oral health care, [125] pain management, [126] wound care [127][128][129] and cancer management [130]

ACK N OWLED G EM ENTS
We thank DEBRA Austria for funding.

CO N FLI C T O F I NTE R E S T
JWB owns shares of the company Diaderm, which receives licence payments from Castle Creek Pharma (CCP). CCP is conducting a phase 2/3 study with a diacerein-containing ointment (NCT03154333). JWB was holder of the EMA orphan drug designation on diacerein in epidermolysis bullosa.

AUTH O R CO NTR I B UTI O N S
CP, JR and ML wrote the original draft. CP, JR and ML and JWB reviewed and edited the manuscript. All authors have read and approved the final manuscript.