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.

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.

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.

(1) Background and Objective: MicroRNAs (miRs) are biomarkers for assessing the extent of cardiac remodeling after myocardial infarction (MI) and important predictors of clinical outcome in heart failure. Overexpression of miR-30d-5p appears to have a cardioprotective effect. The aim of the present study was to demonstrate whether miR-30d-5p could be used as a potential therapeutic target to improve post-MI adverse remodeling. (2) Methods and Results: MiR profiling was performed by next-generation sequencing to assess different expression patterns in ischemic vs. healthy myocardium in a rat model of MI. MiR-30d-5p was significantly downregulated (p < 0.001) in ischemic myocardium and was selected as a promising target. A mimic of miR-30d-5p was administered in the treatment group, whereas the control group received non-functional, scrambled siRNA. To measure the effect of miR-30d-5p on infarct area size of the left ventricle, the rats were randomized and treated with miR-30d-5p or scrambled siRNA. Histological planimetry was performed 72 h and 6 weeks after induction of MI. Infarct area was significantly reduced at 72 h and at 6 weeks by using miR-30d-5p (72 h: 22.89 ± 7.66% vs. 35.96 ± 9.27%, p = 0.0136; 6 weeks: 6.93 ± 4.58% vs. 12.48 ± 7.09%, p = 0.0172). To gain insight into infarct healing, scratch assays were used to obtain information on cell migration in human umbilical vein endothelial cells (HUVECs). Gap closure was significantly faster in the mimic-treated cells 20 h post-scratching (12.4% more than the scrambled control after 20 h; p = 0.013). To analyze the anti-apoptotic quality of miR-30d-5p, the ratio between phosphorylated p53 and total p53 was evaluated in human cardiomyocytes using ELISA. Under the influence of the miR-30d-5p mimic, cardiomyocytes demonstrated a decreased pp53/total p53 ratio (0.66 ± 0.08 vs. 0.81 ± 0.17), showing a distinct tendency (p = 0.055) to decrease the apoptosis rate compared to the control group. (3) Conclusion: Using a mimic of miR-30d-5p underlines the cardioprotective effect of miR-30d-5p in MI and could reduce the risk for development of ischemic cardiomyopathy.

Generalized severe epidermolysis bullosa simplex (EBS-gen sev) is caused by mutations within either the KRT5 or KRT14 gene, phenotypically resulting in blistering and wounding of the skin and mucous membranes after minor mechanical friction. In a clinical phase 2/3 trial, diacerein has recently been shown to significantly reduce blister numbers upon topical application. In this study we addressed basic pharmacokinetic parameters of locally applied diacerein in vitro and in vivo. Ex vivo experiments using a Franz diffusion cell confirmed the uptake and bio-transformation of diacerein to rhein in a porcine skin model. Rhein, the active metabolite of diacerein, was also detected in both urine and serum samples of two EBS-gen sev patients who topically applied a 1% diacerein ointment over a period of 4 weeks. The accumulated systemic levels of rhein in EBS-gen sev patients were lower than reported levels after oral application. These preliminary findings point towards the uptake and prolonged persistance of diacerein / rhein within the intended target organ - the skin. Further, they imply an acceptable safety profile at the systemic level. DRKS. DRKS00005412 . Registered 6 November 2013.

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.