Search

Background Epidermolysis bullosa (EB) is a rare, genetically and clinically heterogeneous group of skin fragility disorders. No cure is currently available, but many novel and repurposed treatments are upcoming. For adequate evaluation and comparison of clinical studies in EB, well-defined and consistent consensus-endorsed outcomes and outcome measurement instruments are necessary. Objectives To identify previously reported outcomes in EB clinical research, group these outcomes by outcome domains and areas and summarize respective outcome measurement instruments. Methods A systematic literature search was performed in the databases MEDLINE, Embase, Scopus, Cochrane CENTRAL, CINAHL, PsycINFO and trial registries covering the period between January 1991 and September 2021. Studies were included if they evaluated a treatment in a minimum of three patients with EB. Two reviewers independently performed the study selection and data extraction. All identified outcomes and their respective instruments were mapped onto overarching outcome domains. The outcome domains were stratified according to subgroups of EB type, age group, intervention, decade and phase of clinical trial. Results The included studies (n = 207) covered a range of study designs and geographical settings. A total of 1280 outcomes were extracted verbatim and inductively mapped onto 80 outcome domains and 14 outcome areas. We found a steady increase in the number of published clinical trials and outcomes reported over the past 30 years. The included studies mainly focused on recessive dystrophic EB (43%). Wound healing was reported most frequently across all studies and referred to as a primary outcome in 31% of trials. Great heterogeneity of reported outcomes was observed within all stratified subgroups. Moreover, a diverse range of outcome measurement instruments (n = 200) was identified. Conclusions We show substantial heterogeneity in reported outcomes and outcome measurement instruments in EB clinical research over the past 30 years. This review is the first step towards harmonization of outcomes in EB, which is necessary to expedite the clinical translation of novel treatments for patients with EB.

Mutations in the COL7A1 gene lead to malfunction, reduction or complete absence of type VII collagen (C7) in the skin’s basement membrane zone (BMZ), impairing skin integrity. In epidermolysis bullosa (EB), more than 800 mutations in COL7A1 have been reported, leading to the dystrophic form of EB (DEB), a severe and rare skin blistering disease associated with a high risk of developing an aggressive form of squamous cell carcinoma. Here, we leveraged a previously described 3′-RTMS6m repair molecule to develop a non-viral, non-invasive and efficient RNA therapy to correct mutations within COL7A1 via spliceosome-mediated RNA trans-splicing (SMaRT). RTM-S6m, cloned into a non-viral minicircle-GFP vector, is capable of correcting all mutations occurring between exon 65 and exon 118 of COL7A1 via SMaRT. Transfection of the RTM into recessive dystrophic EB (RDEB) keratinocytes resulted in a trans-splicing efficiency of ~1.5% in keratinocytes and ~0.6% in fibroblasts, as confirmed on mRNA level via next-generation sequencing (NGS). Full-length C7 protein expression was primarily confirmed in vitro via immunofluorescence (IF) staining and Western blot analysis of transfected cells. Additionally, we complexed 3′-RTMS6m with a DDC642 liposomal carrier to deliver the RTM topically onto RDEB skin equivalents and were subsequently able to detect an accumulation of restored C7 within the basement membrane zone (BMZ). In summary, we transiently corrected COL7A1 mutations in vitro in RDEB keratinocytes and skin equivalents derived from RDEB keratinocytes and fibroblasts using a non-viral 3′-RTMS6m repair molecule.

Chondroitin sulfate (CS) proteoglycan 4 (CSPG4) is a cell surface proteoglycan that is currently under investigation as a marker of cancer malignancy, and as a potential target of anticancer drug treatment. CSPG4 acts as a driver of tumourigenesis by regulating turnover of the extracellular matrix (ECM) to promote tumour cell invasion, migration as well as inflammation and angiogenesis. While CSPG4 has been widely studied in certain malignancies, such as melanoma, evidence is emerging from global gene expression studies, which suggests a role for CSPG4 in squamous cell carcinoma (SCC). While relatively treatable, lack of widely agreed upon diagnostic markers for SCCs is problematic, especially for clinicians managing certain patients, including those who are aged or infirm, as well as those with underlying conditions such as epidermolysis bullosa (EB), for which a delayed diagnosis is likely lethal. In this review, we have discussed the structure of CSPG4, and quantitatively analysed CSPG4 expression in the tissues and pathologies where it has been identified. The aim of this review has been to collate the information available from functional studies and recent transcriptome analysis to determine the usefulness of CSPG4 expression as a diagnostic marker and therapeutic target in management of malignant SCC.

Continuous exposure of the skin to environmental, mechanical and chemical stress necessitates constant self‐renewal of the epidermis to maintain its barrier function. This self‐renewal ability is attributed to epidermal stem cells (EPSCs), which are long‐lived, multipotent cells located in the basal layer of the epidermis. Epidermal homeostasis – coordinated proliferation and differentiation of EPSCs – relies on fine‐tuned adaptations in gene expression which in turn are tightly associated with specific epigenetic signatures and metabolic requirements. In this review, we will briefly summarize basic concepts of EPSC biology and epigenetic regulation with relevance to epidermal homeostasis. We will highlight the intricate interplay between mitochondrial energy metabolism and epigenetic events – including miRNA‐mediated mechanisms – and discuss how the loss of epigenetic regulation and epidermal homeostasis manifests in skin disease. Discussion of inherited epidermolysis bullosa (EB) and disorders of cornification will focus on evidence for epigenetic deregulation and failure in epidermal homeostasis, including stem cell exhaustion and signs of premature ageing. We reason that the epigenetic and metabolic component of epidermal homeostasis is significant and warrants close attention. Charting epigenetic and metabolic complexities also represents an important step in the development of future systemic interventions aimed at restoring epidermal homeostasis and ameliorating disease burden in severe skin conditions.

Background Hereditary epidermolysis bullosa (EB) comprises a heterogeneous group of rare genodermatoses, which are caused by mutations in genes involved in the maintenance of the structural and functional integrity of dermo-epidermal adhesion in various stratified epithelia. In severe variants, generalized skin disease, extracutaneous manifestations and multi-organ involvement cause considerable morbidity and mortality. Causal and early treatment by re-expression of a respective mutated gene is the major long-term goal in therapy development. However, characterization and targeted modulation of pathogenic molecular cascades in EB also holds great promise as a symptom-relieving approach to ameliorate phenotype, complications and quality of life. Small molecules are chemical structures of less than 900 Da that can diffuse across cell membranes and interfere with target biomolecules, thus influencing their function at different levels. They constitute the vast majority of active components of all approved drugs. Methods We performed PubMed and Google Scholar search for publications and screened FDA- and EMA-hosted clinical trial registries to identify studies using small molecule-based drugs for epidermolysis bullosa. Upon detailed analysis this resulted in the identification of a total of 84 studies. Results We identified 52 publications and 32 registered trials that investigate small molecules for their safety and efficacy as treatment for different aspects of epidermolysis bullosa. Further, a total of 38 different small molecules clinically used in EB were found. Most frequent outcome measures concerned wound healing, reduction in blister numbers, as well as reduction of itch and pain, predominantly for EBS and RDEB. Conclusion We provide a comprehensive summary of the current status of clinical small molecule development for EB and discuss prospects and limitations in orphan drug development for rare conditions like EB.

Despite a significant rise in the incidence of cutaneous squamous cell carcinoma (SCC) in recent years, most SCCs are well treatable. However, against the background of pre-existing risk factors such as immunosuppression upon organ transplantation, or conditions such as recessive dystrophic epidermolysis bullosa (RDEB), SCCs arise more frequently and follow a particularly aggressive course. Notably, such SCC types display molecular similarities, despite their differing etiologies. We leveraged the similarities in transcriptomes between tumors from organ transplant recipients and RDEB-patients, augmented with data from more common head and neck (HN)-SCCs, to identify drugs that can be repurposed to treat these SCCs. The in silico approach used is based on the assumption that SCC-derived transcriptome profiles reflect critical tumor pathways that, if reversed towards healthy tissue, will attenuate the malignant phenotype. We determined tumor-specific signatures based on differentially expressed genes, which were then used to mine drug-perturbation data. By leveraging recent efforts in the systematic profiling and cataloguing of thousands of small molecule compounds, we identified drugs including selumetinib that specifically target key molecules within the MEK signaling cascade, representing candidates with the potential to be effective in the treatment of these rare and aggressive SCCs.

Conventional anti-cancer therapies based on chemo- and/or radiotherapy represent highly effective means to kill cancer cells but lack tumor specificity and, therefore, result in a wide range of iatrogenic effects. A promising approach to overcome this obstacle is spliceosome-mediated RNA trans-splicing (SMaRT), which can be leveraged to target tumor cells while leaving normal cells unharmed. Notably, a previously established RNA trans-splicing molecule (RTM44) showed efficacy and specificity in exchanging the coding sequence of a cancer target gene (Ct-SLCO1B3) with the suicide gene HSV1-thymidine kinase in a colorectal cancer model, thereby rendering tumor cells sensitive to the prodrug ganciclovir (GCV). In the present work, we expand the application of this approach, using the same RTM44 in aggressive skin cancer arising in the rare genetic skin disease recessive dystrophic epidermolysis bullosa (RDEB). Stable expression of RTM44, but not a splicing-deficient control (NC), in RDEB-SCC cells resulted in expression of the expected fusion product at the mRNA and protein level. Importantly, systemic GCV treatment of mice bearing RTM44-expressing cancer cells resulted in a significant reduction in tumor volume and weight compared with controls. Thus, our results demonstrate the applicability of RTM44-mediated targeting of the cancer gene Ct-SLCO1B3 in a different malignancy.

Psoriasis is an inflammatory skin disease characterized by increased neo-vascularization, keratinocyte hyperproliferation, a pro-inflammatory cytokine milieu and immune cell infiltration. Diacerein is an anti-inflammatory drug, modulating immune cell functions, including expression and production of cytokines, in different inflammatory conditions. Therefore, we hypothesized that topical diacerein has beneficial effects on the course of psoriasis. The current study aimed to evaluate the effect of topical diacerein on imiquimod (IMQ)-induced psoriasis in C57BL/6 mice. Topical diacerein was observed to be safe without any adverse side effects in healthy or psoriatic animals. Our results demonstrated that diacerein significantly alleviated the psoriasiform-like skin inflammation over a 7-day period. Furthermore, diacerein significantly diminished the psoriasis-associated splenomegaly, indicating a systemic effect of the drug. Remarkably, we observed significantly reduced infiltration of CD11c+ dendritic cells (DCs) into the skin and spleen of psoriatic mice with diacerein treatment. As CD11c+ DCs play a pivotal role in psoriasis pathology, we consider diacerein to be a promising novel therapeutic candidate for psoriasis.

Mutations within the

Documento escrito por un elevado número de autores/as, solo se referencia el/la que aparece en primer lugar y los/as autores/as pertenecientes a la UC3M. Junctional epidermolysis bullosa (JEB) is a debilitating hereditary skin disorder caused by mutations in genes encoding laminin-332, type XVII collagen (C17), and integrin-α6β4, which maintain stability between the dermis and epidermis. We designed patient-specific Cas9-nuclease- and -nickase-based targeting strategies for reframing a common homozygous deletion in exon 52 of COL17A1 associated with a lack of full-length C17 expression. Subsequent characterization of protein restoration, indel composition, and divergence of DNA and mRNA outcomes after treatment revealed auspicious efficiency, safety, and precision profiles for paired nicking-based COL17A1 editing. Almost 46% of treated primary JEB keratinocytes expressed reframed C17. Reframed COL17A1 transcripts predominantly featured 25- and 37-nt deletions, accounting for >42% of all edits and encoding C17 protein variants that localized accurately to the cell membrane. Furthermore, corrected cells showed accurate shedding of the extracellular 120-kDa C17 domain and improved adhesion capabilities to laminin-332 compared with untreated JEB cells. Three-dimensional (3D) skin equivalents demonstrated accurate and continuous deposition of C17 within the basal membrane zone between epidermis and dermis. Our findings constitute, for the first time, gene-editing-based correction of a COL17A1 mutation and demonstrate the superiority of proximal paired nicking strategies based on Cas9 D10A nickase over wild-type Cas9-based strategies for gene reframing in a clinical context. We thank Drs. Andreas Traweger and Andrea Wagner for providing access to their cryotome. We further want to thank Dr. Ludwig Aigner as well as Karin Roider, MSc, for providing access to the microscope core facility. The MiSeq Sequencing was performed by the Next-Generation Sequencing Facility at Vienna BioCenter Core Facilities (VBCF), member of the Vienna BioCenter (VBC), Austria. F.L., B.D., and M.G. are supported by grant PI20/00615 from Spanish ISCIII co-financed by European Regional Development Fund. B.L. is supported by grant 20204-WISS/225/241/17-2021 from the Land Salzburg. This work was funded by the LHF Charitable Trust (LHFCT), the Austrian Science Fund (P 32769-B), the Paracelsus Medical University Salzburg (PMU-FFF) (A-20/02/041-KOU), and DEBRA Austria. This work is generated within the ERN skin.

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.

Intermediate junctional epidermolysis bullosa caused by mutations in the COL17A1 gene is characterized by the frequent development of blisters and erosions on the skin and mucous membranes. The rarity of the disease and the heterogeneity of the underlying mutations renders therapy developments challenging. However, the high number of short in-frame exons facilitates the use of antisense oligonucleotides (AON) to restore collagen 17 (C17) expression by inducing exon skipping. In a personalized approach, we designed and tested three AONs in combination with a cationic liposomal carrier for their ability to induce skipping of COL17A1 exon 7 in 2D culture and in 3D skin equivalents. We show that AON-induced exon skipping excludes the targeted exon from pre-mRNA processing, which restores the reading frame, leading to the expression of a slightly truncated protein. Furthermore, the expression and correct deposition of C17 at the dermal–epidermal junction indicates its functionality. Thus, we assume AON-mediated exon skipping to be a promising tool for the treatment of junctional epidermolysis bullosa, particularly applicable in a personalized manner for rare genotypes.

Background Cutaneous squamous cell carcinomas (cSCC) are the primary cause of premature deaths in patients suffering from the rare skin-fragility disorder recessive dystrophic epidermolysis bullosa (RDEB), which is in marked contrast to the rarely metastasizing nature of these carcinomas in the general population. This remarkable difference is attributed to the frequent development of chronic wounds caused by impaired skin integrity. However, the specific molecular and cellular changes to malignancy, and whether there are common players in different types of aggressive cSCCs, remain relatively undefined. Methods MiRNA expression profiling was performed across various cell types isolated from skin and cSCCs. Microarray results were confirmed by qPCR and by an optimized in situ hybridization protocol. Functional impact of overexpression or knock-out of a dysregulated miRNA was assessed in migration and 3D-spheroid assays. Sample-matched transcriptome data was generated to support the identification of disease relevant miRNA targets. Results Several miRNAs were identified as dysregulated in cSCCs compared to control skin. These included the metastasis-linked miR-10b, which was significantly upregulated in primary cell cultures and in archival biopsies. At the functional level, overexpression of miR-10b conferred the stem cell-characteristic of 3D-spheroid formation capacity to keratinocytes. Analysis of miR-10b downstream effects identified a novel putative target of miR-10b, the actin- and tubulin cytoskeleton-associated protein DIAPH2. Conclusion The discovery that miR-10b mediates an aspect of cancer stemness – that of enhanced tumor cell adhesion, known to facilitate metastatic colonization – provides an important avenue for future development of novel therapies targeting this metastasis-linked miRNA.

BackgroundCutaneous squamous cell carcinomas (cSCC) are the primary cause of premature deaths in patients suffering from the rare skin-fragility disorder recessive dystrophic epidermolysis bullosa, which is in marked contrast to the rarely metastasizing nature of these carcinomas in the general population. This remarkable difference is attributed to the frequent development of chronic wounds caused by an impaired skin integrity. However, the specific molecular and cellular changes to malignancy, and whether there are common players in different types of aggressive cSCCs, remain relatively undefined.MethodsMiRNA expression profiling was performed across various cell types isolated from skin and cSCCs. Microarray results were confirmed by qPCR and by an optimized in situ hybridization protocol. Functional impact of overexpression of a dysregulated miRNA was assessed in migration and 3D spheroid assays. Sample-matched transcriptome data was generated to support the identification of disease relevant miRNA targets.ResultsSeveral miRNAs were identified as dysregulated in cSCCs as compared to controls. These included the metastasis-linked miR-10b, which was significantly upregulated in primary cell cultures and in archival biopsies. At the functional level, overexpression of miR-10b conferred the stem cell-characteristic of 3D-spheroid formation capacity to keratinocytes, and impaired their mobility. Analysis of miR-10b downstream effects identified a novel putative target of miR-10b, the actin- and tubulin cytoskeleton-associated protein DIAPH2.ConclusionThe discovery that miR-10b confers an aspect of cancer stemness – that of enhanced tumor cell adhesion, known to facilitate metastatic colonization - provides an important avenue for future development of novel therapies targeting this metastasis-linked miRNA.

Mutations within the COL7A1 gene underlie the inherited recessive subtype of the blistering skin disease dystrophic epidermolysis bullosa (RDEB). Although gene replacement approaches for genodermatoses are clinically advanced, their implementation for RDEB is challenging and requires endogenous regulation of transgene expression. Thus, we are using spliceosome-mediated RNA trans-splicing (SMaRT) to repair mutations in COL7A1 at the mRNA level. Here, we demonstrate the capability of a COL7A1-specific RNA trans-splicing molecule (RTM), initially selected using a fluorescence-based screening procedure, to accurately replace COL7A1 exons 1 to 64 in an endogenous setting. Retroviral RTM transduction into patient-derived, immortalized keratinocytes resulted in an increase in wild-type transcript and protein levels, respectively. Furthermore, we revealed accurate deposition of recovered type VII collagen protein within the basement membrane zone of expanded skin equivalents using immunofluorescence staining. In summary, we showed for the first time the potential of endogenous 5′ trans-splicing to correct pathogenic mutations within the COL7A1 gene. Therefore, we consider 5′ RNA trans-splicing a suitable tool to beneficially modulate the RDEB-phenotype, thus targeting an urgent need of this patient population.

Summary Background Generalized severe epidermolysis bullosa simplex (EBS‐gen sev) is a genetic blistering skin disease in which autosomal dominant mutations in either the keratin KRT5 or KRT14 genes lead to impaired function of the intermediate filament cytoskeleton in the basal epidermis. Here we present an ex vivo RNA trans‐splicing‐based therapeutic approach to correct the phenotype. Objectives To correct a mutation within exon 1 of the KRT14 gene, using a 5′‐trans‐splicing approach, where any mutation within the first seven exons could be replaced by a single therapeutic molecule. Methods A therapeutic RNA trans‐splicing molecule containing wild‐type exons 1–7 was stably transduced into an EBS patient‐derived keratinocyte line. Trans‐splicing was confirmed via reverse‐transcriptase polymerase chain reaction, Western blotting and immunofluorescence microscopy. Skin equivalents generated from corrected keratinocytes were grafted onto nude mice and analysed about 8 weeks post‐transplantation for regular epidermal stratification, trans‐splicing‐induced green fluorescent protein expression and blistering. Results Transplanted skin equivalents generated from trans‐splicing‐corrected patient keratinocytes showed a stable and blister‐free epidermis. KRT14 correction disrupted EBS‐gen sev‐associated proinflammatory signalling, as shown at the mRNA and protein levels. Disruption of the pathogenic feedback loop in addition to overall downregulation of KRT14 expression highlighted the effect of KRT14 correction on the EBS pathomechanism. Conclusions Our data demonstrate that trans‐splicing‐mediated mRNA therapy is an effective method for the correction of dominantly inherited KRT14 mutations at the transcriptional level. This results in the rescue of the EBS‐gen sev phenotype and stabilization of the epidermis in a xenograft mouse model., What's already known about this topic? RTM163, described in this study, was previously used in a transient in vitro transfection system, where the ability to correct KRT14 at the mRNA level was demonstrated. What does this study add? In this study, we stably transduced RTM163 in a second patient‐derived keratinocyte line.Successful trans‐splicing was confirmed in this cell line.The expression of disease‐related marker genes, which are characteristically deregulated in epidermolysis bullosa simplex, were analysed.For the first time this study showed that RNA trans‐splicing molecule‐transduced patient keratinocytes can differentiate into a phenotypically normal and blister‐free epidermis in a xenograft mouse model. What is the translational message? This study shows the feasibility of using spliceosome‐mediated RNA trans‐splicing to generate a stable and blister‐free epidermis in vivo.Combined with pre‐existing ex vivo gene therapeutic methods, this might be a valid option for future treatments of dominantly inherited genodermatoses. Linked Comment: Bremer and van den Akker. Br J Dermatol 2019; 180:17–19.

A characteristic feature of the skin blistering disease epidermolysis bullosa simplex is keratin filament (KF) network collapse caused by aggregation of the basal epidermal keratin type II (KtyII) K5 and its type I partner keratin 14 (K14). Here, we examine the role of keratin phosphorylation in KF network rearrangement and cellular functions. We detect phosphorylation of the K5 head domain residue T150 in cytoplasmic epidermolysis bullosa simplex granules containing R125C K14 mutants. Expression of phosphomimetic T150D K5 mutants results in impaired KF formation in keratinocytes. The phenotype is enhanced upon combination with other phosphomimetic K5 head domain mutations. Remarkably, introduction of T150D K5 mutants into KtyII-lacking (KtyII(-/-)) keratinocytes prevents keratin network formation altogether. In contrast, phosphorylation-deficient T150A K5 leads to KFs with reduced branching and turnover. Assembly of T150D K5 is arrested at the heterotetramer stage coinciding with increased heat shock protein association. Finally, reduced cell viability and elevated response to stressors is noted in T150 mutant cells. Taken together, our findings identify T150 K5 phosphorylation as an important determinant of KF network formation and function with a possible role in epidermolysis bullosa simplex pathogenesis.

Dystrophic epidermolysis bullosa (DEB) is a blistering skin disease caused by mutations in the gene COL7A1 encoding collagen VII. DEB can be inherited as recessive DEB (RDEB) or dominant DEB (DDEB) and is associated with a high wound burden. Perpetual cycles of wounding and healing drive fibrosis in DDEB and RDEB, as well as the formation of a tumor-permissive microenvironment. Prolonging wound-free episodes by improving the quality of wound healing would therefore confer substantial benefit for individuals with DEB. The collagenous domain of collagen VII is encoded by 82 in-frame exons, which makes splice-modulation therapies attractive for DEB. Indeed, antisense oligonucleotide–based exon skipping has shown promise for RDEB. However, the suitability of antisense oligonucleotides for treatment of DDEB remains unexplored. Here, we developed QR-313, a clinically applicable, potent antisense oligonucleotide specifically targeting exon 73. We show the feasibility of topical delivery of QR-313 in a carbomer-composed gel for treatment of wounds to restore collagen VII abundance in human RDEB skin. Our data reveal that QR-313 also shows direct benefit for DDEB caused by exon 73 mutations. Thus, the same topically applied therapeutic could be used to improve the wound healing quality in RDEB and DDEB.

Chemical UV-filters are frequently applied as active ingredients in sunscreen to protect from detrimental effects of UV radiation. Regardless, many of these compounds are not well characterized concerning their capacity to counteract UV induced reactive oxygen species (ROS). Intracellular ROS release is an early event upon UV exposure and a crucial trigger of reaction cascades that may provoke adverse effects both in short- and long-term. We report a strategy to assess the capacity of UV-filters (ecamsule, oxybenzone and menthyl anthranilate) to counteract UVA/UVB stress in the human keratinocyte HaCaT and the wildtype Fibs E6/E7 fibroblast cell lines. The reduction of ROS levels was taken as primary endpoint. The effect of treatment on the cells' metabolic activity was analyzed as an indicator of viability post-treatment, to investigate potential immediate and late (photo)toxicity. Additionally, the compounds' antioxidative capacity was investigated using an azo-based radical generator. Established antioxidants, quercetin and N-acetylcysteine, were used as controls. Data showed remarkable differences in the mode of action of the chemical UV-filters, ranging from protective to pro-oxidative properties, indicating the need for more detailed mode of action-based investigations. Certainly, additional consideration and evaluation will be necessary to further extrapolate these in vitro data for the assessment of in vivo exposure situations. However, the presented approach enables parallel investigations of photoprotective and phototoxic effects of UV-filters, and thus can complement and extent existing in vitro testing strategies.

In recent years, RNA trans-splicing has emerged as a suitable RNA editing tool for the specific replacement of mutated gene regions at the pre-mRNA level. Although the technology has been successfully applied for the restoration of protein function in various genetic diseases, a higher trans-splicing efficiency is still desired to facilitate its clinical application. Here, we describe a modified, easily applicable, fluorescence-based screening system for the generation and analysis of antisense molecules specifically capable of improving the RNA reprogramming efficiency of a selected KRT14-specific RNA trans-splicing molecule. Using this screening procedure, we identified several antisense RNAs and short rationally designed oligonucleotides, which are able to increase the trans-splicing efficiency. Thus, we assume that besides the RNA trans-splicing molecule, short antisense molecules can act as splicing modulators, thereby increasing the trans-splicing efficiency to a level that may be sufficient to overcome the effects of certain genetic predispositions, particularly those associated with dominantly inherited diseases.

Spliceosome-mediated RNA trans-splicing has become an emergent tool for the repair of mutated pre-mRNAs in the treatment of genetic diseases. RNA trans-splicing molecules (RTMs) are designed to induce a specific trans-splicing reaction via a binding domain for a respective target pre-mRNA region. A previously established reporter-based screening system allows us to analyze the impact of various factors on the RTM trans-splicing efficiency in vitro. Using this system, we are further able to investigate the potential of antisense RNAs (AS RNAs), presuming to improve the trans-splicing efficiency of a selected RTM, specific for intron 102 of COL7A1. Mutations in the COL7A1 gene underlie the dystrophic subtype of the skin blistering disease epidermolysis bullosa (DEB). We have shown that co-transfections of the RTM and a selected AS RNA, interfering with competitive splicing elements on a COL7A1-minigene (COL7A1-MG), lead to a significant increase of the RNA trans-splicing efficiency. Thereby, accurate trans-splicing between the RTM and the COL7A1-MG is represented by the restoration of full-length green fluorescent protein GFP on mRNA and protein level. This mechanism can be crucial for the improvement of an RTM-mediated correction, especially in cases where a high trans-splicing efficiency is required.

Due to its unique immunological properties, the skin is an attractive target tissue for allergen-specific immunotherapy. In our current work, we combined a dendritic cell targeting approach with epicutaneous immunization using an ablative fractional laser to generate defined micropores in the upper layers of the skin. By coupling the major birch pollen allergen Bet v 1 to mannan from S. cerevisiae via mild periodate oxidation we generated hypoallergenic Bet-mannan neoglycoconjugates, which efficiently targeted CD14+ dendritic cells and Langerhans cells in human skin explants. Mannan conjugation resulted in sustained release from the skin and retention in secondary lymphoid organs, whereas unconjugated antigen showed fast renal clearance. In a mouse model, Bet-mannan neoglycoconjugates applied via laser-microporated skin synergistically elicited potent humoral and cellular immune responses, superior to intradermal injection. The induced antibody responses displayed IgE-blocking capacity, highlighting the therapeutic potential of the approach. Moreover, application via micropores, but not by intradermal injection, resulted in a mixed TH1/TH17-biased immune response. Our data clearly show that applying mannan-neoglycoconjugates to an organ rich in dendritic cells using laser-microporation is superior to intradermal injection. Due to their low IgE binding capacity and biodegradability, mannan neoglycoconjugates therefore represent an attractive formulation for allergen-specific epicutaneous immunotherapy.

Epidermolysis bullosa simplex (EBS) is a rare genetic, blistering skin disease for which there is no cure. Treatments that address the pathophysiology of EBS are needed.Compare the impact of 1% diacerein cream with placebo in reducing the number of blisters in EBS.In a randomized, placebo-controlled, phase 2/3 trial we used a 1% diacerein topical formulation to treat defined skin areas in 17 patients. In a 2-period crossover trial, 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. The prespecified primary end point was the proportion of patients with a reduction of number of blisters by more than 40% from baseline in selected areas over the treatment episode.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). This effect was still significant after the follow-up. Changes in absolute blister numbers were significant for the diacerein group only. No adverse effects were observed.Low patient numbers and no invasive data acquisition because of clinical burden in children.This trial provides evidence of the impact of 1% diacerein cream in the treatment of EBS.

In the treatment of genetic disorders, repairing defective pre-mRNAs by RNA trans-splicing has become an emerging alternative to conventional gene therapy. Previous studies have made clear that the design of the binding domains of the corrective RNA trans-splicing molecules (RTMs) is crucial for their optimal functionality. We established a reporter-based screening method that allows for selection of highly functional RTMs from a large pool of variants. The efficiency and functionality of the screen were validated in the COL7A1 gene, in which mutations are the cause of the skin disease dystrophic epidermolysis bullosa. Comparison of RTMs containing different binding domains hybridizing to COL7A1 intron 64/exon 65 revealed highly different trans-splicing efficiencies. Isolated RTMs were then adapted for endogenous trans-splicing in a recessive dystrophic epidermolysis bullosa (RDEB) keratinocyte cell line expressing reduced levels of COL7A1 mRNA. Our results confirm the applicability and relevance of prescreening reporter RTMs, as significant levels of endogenous COL7A1 mRNA repair were seen with RTMs identified as being highly efficient in our screening system.

Spliceosome-mediated RNA trans-splicing (SMaRT) is an RNA-based technology to reprogram genes for diagnostic and therapeutic purposes. For the correction of genetic diseases, SMaRT offers several advantages over traditional gene-replacement strategies. SMaRT protocols have recently been used for in vitro phenotypic correction of a variety of genetic disorders, ranging from epidermolysis bullosa to neurodegenerative diseases. In vivo studies are currently bringing trans-splicing RNA therapy toward clinical application. In this review, we summarize the progress made toward the medical use of SMaRT and provide an outlook on its upcoming applications.

The major challenge to a successful gene therapy of autosomal dominant genetic diseases is a highly efficient and specific knock-down or repair of the disease-causing allele. In epidermolysis bullosa simplex-type Dowling-Meara (EBS-DM), a single amino acid exchange in exon 1 of the keratin 14 gene (K14) triggers a severe skin phenotype, characterized by blistering of the skin and mucous membranes after minor trauma. We chose spliceosome-mediated RNA trans-splicing to specifically replace exons 1-7 of the K14 gene. In this approach, the mutated coding region is replaced by an RNA-trans-splicing molecule (RTM) that incorporates a binding domain (BD) and the wild-type sequence of K14. Since the BD is crucial for the trans-splicing functionality, we developed a fluorescence-based RTM screen consisting of an RTM library containing random BDs. Co-transfection of the library with a target molecule enabled us to identify highly functional RTMs. The best RTMs were adapted for endogenous trans-splicing in an EBS-DM patient cell line. In this cell line, we were able to detect functional, efficient and correct trans-splicing on RNA and protein levels. Scratch assays confirmed phenotypic reversion in vitro. Owing to concomitant knock-down and repair of the mutated allele, we assume that trans-splicing is a promising tool for the treatment of autosomal dominant genetic disease.

Keratin filaments form obligatory heterodimers consisting of one type I and one type II keratin that build the intermediate filaments. In keratinocytes, type II keratin 6 (K6) interacts with type I keratin 16 (K16). We previously showed that the intermediate filament protein K16 is up-regulated in aged human skin. Here, we report that there is an obvious imbalance of K16 to K6 mRNA in in vivo and in vitro aging, which possibly leads to cellular effects. To unveil a possible biological function of K16 overexpression we investigated the migration potential of keratinocytes having up-regulated K16 expression in vitro. Two cell lines were established by transfection of human keratinocytes (HaCaT cells) with K16 or control vectors and subsequent fluorescence-activated cell sorting. By performing migration assays we were able to show a 90% reduction in the migration ability of the K16-overexpressing keratinocytes. In addition, a delay in wound closure associated with K16-overexpressing cells was shown by scratch assays. Transient overexpression of K6A in K16-overexpressing keratinocytes partially corrected the cell-migration defect. By real-time PCR we excluded co-regulation of the annotated interaction partner, K6, in the K16 cell line. Finally, we observed a decreased level of tyrosine phosphorylation in K16-overexpressing cells. Taken together, these data highlight the possibility of a physiological role for K6/K16 heterodimers in keratinocyte cell migration, in addition to the heterodimer's known functions in cell differentiation and mechanical resilience.

The efficient treatment of hereditary disorders, especially of those caused by dominant-negative mutations still remains an obstacle to be overcome. Allele specificity is a critical aspect that must be addressed by silencing therapies such as small interfering RNA, which has the potential risk of also reducing expression of the normal allele. To overcome this hurdle, we used spliceosome-mediated RNA trans-splicing (SMaRT) to replace mRNA exon segments in an in vitro disease model. In this model, a heterozygous insertion of a leucine codon into exon 9 of the plectin gene (PLEC1) leads to aggregation of plectin peptide chains and subsequent protein degradation recapitulating, together with a nonsense mutation on the other allele, the blistering skin disease epidermolysis bullosa simplex with muscular dystrophy (EBS-MD). Transient transfection of EBS-MD fibroblasts with a 5' pre-trans-splicing molecule encoding wild-type exons 2-9 led to specific replacement of the mutated 5' portion of the endogenous PLEC1 transcript through trans-splicing. This treatment reduced the levels of mutant mRNA and restored a wild-type pattern of plectin expression as revealed by immunofluorescence microscopy. When EBS-MD fibroblasts were transfected with retroviral constructs, the level of full-length plectin protein in the corrected fibroblasts increased by 58.7%. Thus, SMaRT may be a promising new tool for treatment of autosomal-dominant genetic diseases.

Background Regenerative medicine is strictly dependent on stem cells as a source for a high diversity of somatic cells. However, the isolation of such from individuals suffering from severe genetic skin blistering diseases like epidermolysis bullosa (EB) is often associated with further organ damage. Methods Stem cells were isolated from 112 urine samples from 21 different healthy donors, as well as from 33 urine samples from 25 donors with EB. The cultivation of these cells was optimized by testing different media formulations and pre-coating of culture vessels with collagen. The identity of cells was confirmed by testing marker expression, differentiation potential and immune-modulatory properties. Results We provide here an optimized protocol for the reproducible isolation of mesenchymal stem cells from urine, even from small volumes as obtained from patients with EB. Furthermore, we offer a basic characterization of those urine-derived stem cells (USCs) from healthy donors, as well as from patients with EB, and demonstrate their potential to differentiate into chondrocytes, osteoblasts and adipocytes, as well as their immune-modulatory properties. Conclusions Thus, USCs provide a novel and non-invasive source of stem cells, which might be applied for gene-therapeutic approaches to improve medical conditions of patients with EB. Electronic supplementary material The online version of this article (doi:10.1186/s13104-015-1686-7) contains supplementary material, which is available to authorized users.

Summary Background Several fungal species are known to cause severe respiratory and cutaneous allergicdiseases. Extracts from several allergenic fungi are used for in vivo and in vitro tests, as standardpreparations are still not available.Objective The aims are to define the pattern of in vivo and in vitro IgE reactivity to fungal species inan allergic population with respiratory symptoms; to determine the influence of different extractpreparations on diagnostic results; and to evaluate whether there exists a relationship between thediagnostic pattern of reactivity and the pattern of specific IgE reactivity in immunoblots.Methods Skin prick tests were applied to a cohort of 4962 respiratory subjects, aged 3–80 years.Fungal extracts from Alternaria, Aspergillus, Candida, Cladosporium, Penicillium, Saccharomyces,and Trichophyton were used, along with extracts from pollens, mites, and animal dander.Demographical and diagnostic data were recorded. IgE detection was carried out with the sameallergenic extracts plus Malassezia. Comparative skin tests and IgE detection were carried out usingextracts from three commercial suppliers. IgE immunoblots were carried out with the same panel ofcommercial fungal extracts and were compared with in-house extracts. Data analysis was carried outby grouping the population on the basis of their reactivity to a single, to two or to more than two,mould species.Results Nineteen percent of the allergic population reacted to at least one fungal extract by meansof the skin test. Alternaria and Candida accounted for the largest number of positive tests, and alongwith Trichophyton they were the main sensitizers in the subset of patients with an isolatedsensitization. The prevalence of skin test reactivity increased for these three fungi in the subsets withtwo associated reactivities and, furthermore, in the subset showing reactivity to more than twomould species. In the latter group, a steady increase of the skin test reactivity was recorded for all theother fungal sources, suggesting a clustered reactivity. Comparative skin and IgE testing withdifferent groups of subjects with a simple pattern of skin reactivity resulted in sensitivity differencesbetween in vivo and in vitro tests, whereas discrepant results were recorded in the subsets of patientswith multiple fungi sensitization. Although hampered by the limited reliability of fungal extracts, IgEimmunoblots revealed differing patterns of reactivity when sera from the three subsets were used.This suggests a link between the diagnostic reactivity pattern and the IgE sensitization to extracts’components. Age and gender distribution differed among the Alternaria-, Candida-, andTrichophyton-sensitized subjects, but not in the subset with more than two fungi sensitizations.Conclusions The preliminary assessment of a new classification of the mould-sensitized populationhas been reached. The limiting quality of fungal extracts requires future studies using an allergenicmolecule-based approach. The diagnostic process and the definition of the reactivity pattern wouldthus be easy, and it could lead to a novel specific immunotherapy approach.Keywords allergens, comparative study, epidemiology, fungal allergy diagnosis, fungal extract, IgEdetection, immunoblotting, prevalence, skin tests.Submitted 18 November 2002; revised 4 June 2003; accepted 28 July 2003