Overview on causal therapies in epidermolysis bullosa and their current clinical status.
Although rare genodermatoses such as Epidermolysis bullosa have received more attention over the last years, no approved treatment options targeting causal mutations are currently available. Still, such diseases can be devastating, in some cases even associated with life-threatening secondary manifestations. Therefore, developing treatments that target disease-associated complications along with causal therapies remains the focus of current research efforts, in order to increase patient’s quality of life and potentially their life expectancy. Epidermolysis bullosa is a genodermatosis that is caused by mutations in either one of 16 genes, predominantly encoding structural components of the skin and mucosal epithelia that are crucial to give these barrier organs physical and mechanical resilience to stress. The genetic heterogeneity of the disease is recapitulated in the high variability of phenotypic expressivity observed, ranging from minor and localized blistering to generalized erosions and wound chronification, rendering certain subtypes a systemic disease that is complicated by a plethora of secondary manifestations. During the last decades, several studies have focused on developing treatments for EB patients and significant progress has been made, as reflected by numerous publications, patents, and registered trials available. Overall, strategies range from causal to symptom-relieving approaches, and include gene, RNA and cell therapies, as well as drug developments based on biologics and small molecules. In this chapter, we highlight the most recent and promising approaches that are currently being investigated in order to provide effective treatments for patients with epidermolysis bullosa in the future.
- Epidermolysis bullosa
- gene therapy
- drug development
- wound healing
- squamous cell carcinoma
Epidermolysis bullosa (EB) refers to a group of rare genodermatoses typically characterized by vulnerability of the skin to friction or trauma, leading to blistering and wounding to various extents,
Worldwide approximately 500,000 people suffer from EB, which can be classified into four major groups . Mainly dominantly inherited mutations within genes encoding keratin 5, 14 and plectin lead to EB simplex (EBS), associated with intraepidermal blister formation (Figure 1). Mutations within genes, encoding laminin-332, type XVII collagen or integrin-α6β4, are the main causes of the junctional form of EB (JEB), characterized by tissue separation within the lamina lucida of the basement membrane zone (BMZ). The severe dystrophic variant of EB (DEB) is caused exclusively by mutations within the gene
Despite advances in our understanding of the spectrum of pathologies associated with the different subtypes of EB, a systemic cure is still out of reach. However, the increasing number of trials that are being conducted reflects significant progress in clinical research, including strategies to correct and/or modulate the aberrant molecules and mechanisms underlying this devastating disease. [4, 5, 6, 7] The remarkable differences between EB-types and numerous subtypes, renders the development of therapies a complex challenge, as both inter-subtype and inter-individual differences require the development of more personalized treatments.
Major complications in EB range from itch and pain, to a predisposition to wound chronification and tumor development, with molecular contributors deriving from various sources including different tissue-associated cell types, matrix components, as well as inflammatory events. The majority of these comorbidities, in and of themselves, are not unique to EB, so that the repurposing of clinically approved treatments against such symptoms represents an attractive path for a more rapid market approval of these compounds for EB. Furthermore, new evidence arising in the study of these common conditions can be leveraged to direct therapy development in EB. Nevertheless, rigorous evaluation for safety in this specific, vulnerable patient group is warranted for any candidate therapeutic. This is especially true for cancer therapies which are associated with significant cellular toxicity and often have the adverse effect of exacerbating the wound healing deficiencies associated with the disease.
2. Therapy development for EB
2.1 Causal therapies for epidermolysis bullosa
2.1.1 Gene replacement therapies for epidermolysis bullosa
The development of causal therapies has always been a main focus of EB research (Table 1).
|EB subtype||Therapeutic goal||Therapeutic strategy||Targeted gene/protein||Status||Ref|
|Causal therapy||Gene editing||pre-clinical||[8, 9]|
|SMaRT||pre-clinical||[10, 11, 12, 13]|
|Causal therapy||cDNA replacement||clinical||[14, 15, 16, 17]|
|Read-through therapy||PTC read-through||clinical|||
|Causal therapy||cDNA replacement||clinical||[19, 20, 21, 22, 23]|
|AON||pre-clinical||[24, 25, 26, 27]|
|Gene editing||pre-clinical||[24, 28, 29, 30, 31, 32, 33, 34, 35, 36]|
|SMaRT||pre-clinical||[10, 37, 38, 39, 40]|
|Protein therapy||Protein replacement||Type VII vollagen||pre-clinical|||
|Cell therapy||Allogeneic fibroblast injection||Type VII collagen||clinical|||
|Read-through therapy||PTC read-through||clinical|||
Currently, gene replacement strategies that exploit viral vectors to introduce full-length wild type cDNA copies of the affected gene into the skin cells of patients , have advanced the furthest in clinical trials. However, this strategy relies on genomic integration of the transgene to achieve long-term restoration of gene function, which bears a low risk of genomic toxicity due to insertional mutagenesis that can result in tumor development as shown for for X-linked severe combined immunodeficiency (X-SCID) . However, no such deleterious events have been observed thus far for EB [14, 15, 16, 17, 19]. In general, cutaneous gene therapies have the advantage that grafted skin areas are easy to monitor, with developing tumors easily detected and promptly excised. Until now, transplantation of genetically corrected skin grafts represents the most auspicious approach, due to the limited number of viral vectors suitable for
To date, the most successful application of gene replacement therapy has been achieved in junctional EB (JEB) patients carrying mutations in the
Despite these successes, attempts to apply the same strategy in recessive dystrophic EB (RDEB) demonstrated no long-lasting effects. While long-term
For most patients, improved wound healing and an accurate deposition of type VII collagen within the regenerated skin could be detected. However, expression of the transgene significantly decreased over time . Possible reasons for this include the size of the
In contrast to targeting patient keratinocytes, a combined gene and cell therapy approach using patient autologous fibroblasts was recently evaluated in a phase I, open-label, single-center clinical trial in four RDEB patients . Based on previous preclinical data , patient fibroblasts were first modified
2.1.2 Gene editing strategies for epidermolysis bullosa
Gene editing platforms based on programmable nucleases have advanced at a rapid pace, such that the correction of any gene is now, at least in theory, conceivable. At their core, designer nucleases consist of DNA endonucleases, such as zinc-finger nucleases (ZNF), transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR) /CRISPR-associated protein 9 (Cas9), which are guided to specific DNA loci of interest, where they generate double-strand breaks (DSBs) and trigger the activation of DNA repair mechanisms [49, 50]. The most frequent repair pathway, termed non-homologous end joining pathway (NHEJ), relies on the introduction of small insertions and deletions (indels) at the DSB site , which can be leveraged for the inactivation of genes carrying dominant negative missense mutations [8, 52], or for the reframing of genes bearing pathogenic frameshift mutations [28, 29].
While not a genetic correction
The same strategy can also be used to reframe mutant mRNA and was recently shown by several groups to be a promising editing approach in RDEB [28, 29, 31, 32]. Leveraging recently developed algorithms to accurately predict end-joining (EJ) repair outcomes following CRISPR/Cas9-mediated cleavage, we were able to achieve significant restoration of
Of course, the holy grail of gene therapy has been to achieve a traceless repair of the disease-causing mutation. With current editing technologies, this is now attainable by invoking the high-fidelity homology directed repair (HDR) pathway. By providing an exogenous HDR donor sequence, which bears homology to the target region, the exchange of whole gene regions or individual nucleotides can be achieved [18, 24, 49, 50]. However, in comparison to EJ-based targeting strategies, gene editing efficiency is generally reduced, as homologous recombination is only active during the late S/G2 phase of the cell cycle . Nevertheless, in EB, HDR-based gene repair strategies have been successfully applied to EBS  and RDEB cells [24, 33, 35]
The application of HDR-based approaches to the correction of patient-derived induced pluripotent stem cells (iPSCs) further increases the range of therapeutic options for patients, especially as the isolation of epidermal holoclones can be a limiting factor [14, 34]. Pre-clinical studies using corrected iPSCs, that were then differentiated into keratinocytes and fibroblasts, and used to generate three-dimensional skin equivalents (HSEs) on the backs of immunodeficient mice, showed normal type VII collagen expression and restored anchoring fibrils . Alternative strategies, not based on homology-directed repair, comprise base editing, which has proven to be a suitable option for correcting pathogenic mutations in RDEB . The most recent genome editing tool, prime editing, can be used to directly write new genetic information into a selected genomic locus using a Cas9 nickase fused to an engineered reverse transcriptase (RT) domain . Via a prime editing guide RNA (pegRNA), which specifies the target site and represents the RT template encoding the desired edit, the prime editor is directed to the target locus. Here, the Cas9 nickase makes a single strand DNA break, that induces the hybridization of the nicked genomic strand to the complementary primer binding site (PBS) sequence, located within the pegRNA. A subsequent reverse transcription of the RT template, carrying the desired edit, followed by a cellular DNA repair mechanism lead to the insertion of the respective genetic modification at the target site . Prime editing potentially improves safety, efficiency and applicability, and its application to EB will undoubtedly be confirmed in appropriate disease models in the near future.
2.1.3 RNA-based therapies for epidermolysis bullosa
A promising RNA-based strategy for the restoration of functional protein expression in EB is based on the use of antisense oligonucleotides (AON) for the specific knockdown of genes or their modification via splicing interference . AONs are generally short fragments of modified DNA or RNA which, in the case of splicing modulation, hybridize to splicing elements (
Another RNA-based strategy for mRNA correction, namely RNA
2.1.4 Protein- and cell-based therapies for epidermolysis bullosa
Protein replacement strategies were recently applied in preclinical studies for RDEB. The local or intravenous injection of recombinant type VII collagen led to its homing to the dermal-epidermal junction and promoted wound healing . Another therapeutic option for RDEB is the administration of type VII collagen-expressing allogeneic fibroblasts into healing RDEB wounds. Particularly when injected intradermally allogeneic fibroblast therapy resulted in a significant decrease in wound area when compared to standard of care after 2 and 12 weeks of treatment .
2.1.5 Read-through strategies for epidermolysis bullosa
Premature stop codon (PTC) read-through strategies rely on agents that allow for the incorporation of a random amino acid at the PTC position in the mRNA. Depending on the importance of the original amino acid to protein function, as well as impact of the introduced amino acid to
In summary, numerous strategies to target the genetic cause of EB, as well as ameliorate disease-associated complications, are under intensive investigation. These act at various levels, from genes and gene products, to cellular pathways, tissue processes, and systemic events. While each strategy has distinct strengths and challenges, they all share the overarching aim of significatnly improving the QoL of patients (Figure 2).
2.1.6 Immunological aspects of causal therapy
Immunological tolerance to self-antigens result from central and peripheral tolerance mechanisms. Central tolerance in the thymus results in either negative selection of self-reactive T cells or development of self-specific suppressive regulatory T cells, both of which require expression and presentation of self-antigens to developing thymocytes. Additionally, various peripheral mechanisms of tolerance protect the body from deleterious reactions against self-tissues. These include anatomical sequestration of self-antigens, deletion of peripheral autoreactive lymphocytes, the development of functional unresponsiveness of lymphocytes (anergy) and action of regulatory T cells [67, 68].
A major risk in patients, especially those completely lacking expression of the affected protein, is that this protein is missing from the repertoire of self-antigens presented during central tolerance establishment. As the aim of causal therapies is to restore the missing protein or repair a defective (
2.2 Treating complications of EB
While gene therapy is the only curative option for EB, strategies to ameliorate symptoms are critically needed to increase patient’s QoL and prevent severe complications of the disease until causal therapies are available for all EB patients. The number of preclinical and clinical studies published, including those currently registered, reflects the great effort placed into providing such. Strategies to identify suitable candidates are diverse, but great potential lies in drug repurposing, as this facilitates timely development of potent treatments by leveraging already existing pre-clinical and clinical data. Even so, the methodological challenges inherent to conducting studies in rare disease populations can complicate the clinical evaluation of repositioning such drugs for EB .
The active components of drugs generally comprise small molecules or biologics. While low molecular weight small molecules can be derived chemically and exhibit distinct advantages regarding delivery and route of administration, biologics, which are much larger, often interfere very specifically with distinct pathomechanisms and show overall less toxicity. Functionally, small molecules are frequently designed as inhibitors of
In the context of EB several approaches addressing various complications have been reported, with the majority of primary outcomes measured being improvement of wound healing, reduction of blistering, and mitigation of itch. While some of the evaluated compounds have reached late stage clinical trials, first marketing approvals are still awaited [4, 5, 6, 7, 69].
2.2.1 Reduction of blistering
Across the various EB types, blistering of the skin may be the first clinical manifestation of skin fragility following mechanical friction or trauma. While in some patients blisters heal without scarring, in patients suffering from more severe subtypes these degenerate into wounds and are accompanied by multiple comorbidities. Thus, preventing blistering or accelerating their resolution is a logical primary outcome measure for clinical trials due to its relevance to patients, at least in distinct patient cohorts . Particularly in EBS, where patients only rarely develop wounds, reducing blister numbers will improve patients’ QoL substantially. Especially during childhood, EBS patients are prone to developing numerous blisters, which may prevent children from
For EBS, uncovering the pathways and molecular mediators underscoring the pathogenic keratin biology in cells, proved instrumental to the development of a topical formulation of the drug diacerein, which has been shown in randomized controlled trials (RCTs) to significantly reduce blister numbers in comparison to patients who received placebo [70, 71]. Moreover, during the patient follow-up period when no treatment was applied, a delayed recurrence of blisters was observed, pointing towards a long-term stabilization of the skin. Diacerein is a small molecule that interferes with the expression and signaling of the pro-inflammatory cytokine IL-1ß at various levels. Upregulation of IL-1ß, triggered by the accumulation of mutated keratins, is characteristic for distinct subtypes of EBS. However, the reciprocal effect of IL-1ß to further induce the expression of mutant keratins was also observed in patient cells. Thus, interfering with this positive feedback loop proved beneficial to stabilizing the keratinocyte’s intermediate filament network and consequently, also the patient’s skin .
2.2.2 Wound healing
EB patients develop wounds throughout their lifetime, and their management and daily care routine represent a major burden that is accompanied by substantial discomfort. There is currently no standard treatment for the treatment of non-healing or severely infected wounds in EB. Defects in wound healing, associated with infections and persistent inflammation are presumed major drivers of wound chronification, which is a major risk factor for the development of particularly aggressive squamous cell carcinomas (SCC) [2, 3]. Thus, means to improve wound healing, and thereby prevent downstream complications that severely decrease QoL and that may even be life-threatening, are urgently needed. This is also reflected by the high number of clinical trials that primarily aim to improve wound healing, either by applying drugs that modulate wound healing associated pathways (
Promising outcomes were recently reported from a trial using anti-inflammatory/immunomodulatory betulin-rich birch bark extract (Filzuvez, previously Oleogel S-10), wherein 41.3% of patients treated with Oleogel-S10 met the primary endpoint of target wound closure within 45 (± 7) days as compared to 28.9% of patients within the placebo arm. Furthermore, among the EB subtypes evaluated, patients with RDEB appeared to be particularly responsive to treatment (NCT03068780). In the context of wound healing, induction of PTC read-through as a means of triggering re-expression of genes harboring nonsense mutations, is particularly attractive in cases where the drugs being evaluated for these purposes have known antibacterial and anti-inflammatory activity. Particularly for junctional and dystrophic EB patients, clinical trials investigating the aminoglycoside antibiotic gentamicin are still ongoing [43, 73, 74] (NCT04140786, NCT03526159, NCT04644627, NCT03392909). Additionally for RDEB, the anti-inflammatory, anti-allergic immunomodulator Amlexanox, typically used against mouth ulcers, has emerged as a novel candidate in preclinical studies .
Pruritus is a particularly agonizing aspect associated with all subtypes of EB which not only impairs patients’ QoL, but also leads to additional skin damage as it provokes scratching. Even though pruritus is not a life-threatening symptom
For patients with EB, a handful of studies targeting itch have been published, the most recent being a randomized-controlled trial (RCT) evaluating the neurokinin-1 receptor (NK1R) antagonist Serlopitant in patients with any EB subtype . Serlopitant disrupts Substance P (SP) associated signaling by preventing its binding to NK1R. Expressed on multiple skin cell types, NK1R is thought to play a major role in the transmission of itch signals in the peripheral and central nervous systems . In the RCT above, 14 EB patients received serlopitant or placebo over a period of 8 weeks, and reduction of pruritus was assessed using a numeric rating scale. Even though results were not statistically significant, patients who had received the investigational drug tended to achieve a ≥ 3 point reduction in itch compared to placebo, and a positive impact on QoL was reported by the patients. However, a larger clinical trial will be needed to provide clear evidence on the efficacy of serlopitant in reducing pruritus in patients with EB .
Another agent currently being evaluated against pruritus in patients with EB-pruriginosa is the anti-interleukin-4 receptor alpha (IL-4Rα) monoclonal antibody Dupilumab [78, 79]. Already indicated for atopic dermatitis, Dupilumab inhibits both IL-4 and IL-13 signaling and modulates Th2-mediated immune mechanisms. The promising outcome of both studies, which included a total of three patients, might provide a rationale for larger RCTs in the future that extend to other subtypes of EB.
Interestingly, in a single-patient observational study aimed primarily at investigating the wound healing benefits of a low-dose topical calcipotriol ointment in DEB, a significant reduction of itch was reported as a highly patient-relevant outcome. Calcipotriol is an analogue of the active form of vitamin D3, an important skin homeostasis factor with roles in cell proliferation, differentiation, antimicrobial defense, and immune modulation. Calcipotriol has proven anti-proliferative effects in keratinocytes, which is leveraged for the treatment of plaque psoriasis. For this reason, we investigated a lower concentration with respect to antimicrobial peptide induction in DEB keratinocytes. In addition to the complete closure of a chronic wound within two weeks of treatment, we observed significant improvement in the diversity of the skin microbiota on the treated skin area, with complete clearance of
Repeated cycles of injury and subsequent persistent inflammation trigger a cascade of events leading to progressive fibrosis, followed by tissue stiffening and increased risk of tumor development in patients with dystrophic EB. Additionally, fibrotic webbing at limb extremities post-wounding ultimately leads to fusion of fingers and toes (called mitten deformities), severely limiting their use. Thus, strategies to support a normal course of wound healing are investigated to avoid or minimize deviations from deposition of a normal skin matrix . A key player in EB-associated fibrosis is TGF-ß, a pro-inflammatory cytokine whose pleiotropic effects are highly context-dependent, and which has been shown to be constitutively expressed in RDEB-skin [83, 84]. While TGF-ß1 promotes wound healing under normal conditions, excessive TGF-ß1 signaling leads to abnormal ECM deposition and scar formation, as confirmed in a type VII collagen hypomorphic mouse model [85, 86]. Thus, modulating the expression of TGF-ß1 was hypothesized to be beneficial in reducing fibrosis. In this context, losartan, an angiotensin II antagonist with anti-fibrotic effects, has been evaluated in preclinical studies, where Nystrom
In a drug repurposing approach, endoglin (CD105), a type III co-receptor for TGF-1, and raloxifene, an estrogen receptor modulator, were tested in a pre-clinical setting for their potential to attenuate RDEB-associated fibrosis. Indeed, both drugs were shown to modulate profibrotic events, rendering them potential candidates for repositioning both compounds for the treatment of patients with EB .
2.3 EB-associated squamous cell carcinoma
Cutaneous tumors are a life-threatening complication that arise especially in patients with RDEB. Owing to the repeated cycles of wounding, infection and inflammation, RDEB patients are at especially high risk of developing aggressive squamous cell carcinoma (RDEB-SCC) with high risk-features. The sites of tumor occurrence are predominantly at sites of chronic and long-term wounds , especially on the extremities , indicating that tumorigenesis is related to the pathology of RDEB. The SCCs tend to arise in early adulthood, with a reported median age of 29 years at time of diagnosis, although the youngest case reported was in a 6-year old patient . In comparison to the general population, RDEB patients have an estimated 70-fold higher risk of developing SCC , with cumulative risk rising from 7.5% at age 20 years, to 67.8% at age 35 years, to 90.1% by age 55 . Despite aggressive therapy with multiple treatment modalities, median survival time from time of first diagnosis is 4–5 years , making RDEB-SCC the primary cause of premature death in these patients. The first choice of treatment still consists of wide local excision of the tumor, and even amputation of the extremity is sometimes necessary. Radiotherapy and conventional chemotherapeutic approaches have been mostly used palliatively in EB SCC and considering their strong adverse effects (
Genomic analyses combined with transcriptomic profiling of tumors highlight cell endogenous mutagenic processes mediated by APOBEC enzymes, which are associated with an innate defense mechanism against ongoing microbial infection, as a major driver of carcinogenesis in RDEB. These observations indicate that effective wound management, which includes an antimicrobial component, could potentially lower cancer risk in these patients. Genetically, RDEB-SCC closely resembles ultraviolet (UV) light-induced SCC and SCC of the head and neck (HN-SCC), with driver mutations in known cancer-associated genes such as
2.3.1 EGFR inhibition
Cetuximab, a monoclonal antibody targeting the extracellular domain of epidermal growth factor receptor (EGFR), has been used for therapy against both HN-SCC, as well as advanced unresectable cutaneous UV-SCC [93, 94]. This agent inhibits tumor cell proliferation by blocking receptor tyrosine kinase activity upstream of known survival-, growth-, and migration- signaling cascades mediated by
2.3.2 JAK1/2 inhibition
The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway mediates cellular responses to a variety of cytokines and growth factors, including IL-6 and EGF, downstream of these ligands binding their cognate receptors. These responses include proliferation, differentiation, migration, survival, and apoptosis, and are dependent on cell- and tissue-type, as well as the context of the signal . In this respect JAK/STAT plays important roles in developmental and homeostatic processes, and is also aberrantly active in numerous cancers, including HN-SCC . Increased levels of phosphorylated STAT3, a downstream effector of JAK, were observed in RDEB-SCC cells over normal keratinocytes, providing rationale for evaluating the effect of the JAK1/2 inhibitor ruxolitinib in a murine xenograft model of human RDEB-SCC . In this preclinical study, ruxolitinib effectively reduced tumor mass, when administered either orally or topically onto tumors, because reduced STAT3 signaling led to decreased cell proliferation. These observations argue that ruxolitinib may be a promising anticancer drug for RDEB-SCC. When ruxolitinib was used with the aim to counteract the fibrotic processes in the skin of type VII collagen hypomorphic mice, a reduction of phosphorylated STAT3 in fibroblasts and SCC
2.3.3 Polo-like kinase 1 inhibition
Due to the aggressive and metastasising nature of RDEB-SCC, which is atypical of UV-induced cutaneous SCCs that arise in the general population, gene expression assays were performed to identify differentially regulated genes that might account for this difference in tumor behavior. Among the handful of genes identified was polo-like kinase 1 (PLK1) , a serine/threonine protein kinase which was over-expressed in a number of different tumors . Blocking PLK1 leads to mitotic arrest, inhibition of cell proliferation and apoptosis. Notably, cells were more sensitive to PLK1 inhibition when p53 was defective .
2.3.4 Immune checkpoint blocking
Immune checkpoints are molecules that are either able to turn on (co-stimulatory molecules) or turn off (inhibitory molecules) immune signaling, generally referring to the activation of responses in T cells. Tumors have developed mechanisms to exploit these immune checkpoint molecules in order to evade immune surveillance and escape clearance by
In the last decade, the number of clinical trials registered for the evaluation of therapies against various primary and secondary pathologies associated with the various forms of EB has risen dramatically (>70 clinical trials; clinicaltrials.gov). They reflect the progress in the optimization of previous gene therapeutic approaches, discovery and advancement of novel gene editing technologies, and the increase in our understanding of the molecular and cellular mechanisms underpinning the nature of these EB-related complications. Drug repositioning has largely been prioritized, as leveraging the existing pharmacological and safety data represents the fastest and most economical route to clinical trials, and when successful, to marketing approval for EB. To further support the development of new therapy options for rare diseases like EB, several programs, like the orphan designation program by the European Medicines Agency (EMA) have been launched.
This is good news for patients, but also creates challenges for the recruitment of sufficient participants to the various trials, due to the rarity of the disease, strict inclusion criteria, and the disinclination of patients to participate. This heightens the risk of recruitment failure and the inability to meet statistical endpoints, resulting in extended trials that come at increased costs. Surmounting these challenges will require close collaboration within the entire EB community, to establish an international patient registry, incentivize patient participation, address logistical and regulatory aspects of multi-center trials, and allow for new outcome measures and the development of statistical methods for small cohorts. In parallel, applying current state-of-the-art methods that maximize acquisition of multi-modal data from patient samples, alongside the continuing advances in artificial intelligence, will further support the development of new therapies at various levels, starting from
The authors would like to thank DEBRA Austria for their continued support and funding of our work. This work is generated within the ERN skin.
Conflict of interest
The authors declare no conflict of interest.