Background/Aim: Mutations in COL7A1 affecting the expression or function of type-VII collagen lead to an impaired mechanical integrity of the skin - a hallmark of recessive dystrophic epidermolysis bullosa (RDEB). As a consequence patients suffer from severe blister formation and a high risk of erosions becoming chronic, non-healing wounds. In addition to ongoing efforts to address the genetic cause itself, we explored whether the anti-inflammatory drug diacerein could be amenable to a repurposing approach to improve wound healing. Methods: Archival tissue sections from chronic wounds were tested in immunofluorescence-stainings for signals of excessive inflammation. In a cell-culture model using RDEB-keratinocytes (KC), gene expression associated with IL-1ß pro-inflammatory signalling, particularly MMP-9, was assessed using RT-PCR. The effect of treating cells with diacerein was assessed by western blot, as well as in a functional fibrinogen-based invasion assay. Results: We found high levels of MMP-9 and its major regulator IL-1ß in granulation tissue of chronic wounds as well as at the epidermal wound edge. In addition, we were able to demonstrate that a bacterial toxin of a frequent colonizer of RDEB wounds (i.e. pseudomonas aeruginosa derived LPS) triggered a robust induction of MMP-9 in RDEB-KC in vitro. Treatment of these KCs with diacerein modulated the triggered inflammatory response, both, by reducing MMP-9 abundance as well as its activity. This is of high relevance as high expression levels and activity of MMP-9 is typically associated with impaired wound healing. Finally, in order to monitor whether diacerein was able to decrease MMP-9 activity in a functional setting, we performed a matrix-based migration assay, hypothesizing that keratinocyte motility reflects MMP-9 activity in a fibrinogen-gel-based assay. Indeed, the migratory potential of RDEB-KCs was significantly reduced upon diacerein treatment. Conclusion: Our results suggest a beneficial effect of the anti-inflammatory drug diacerein for the treatment of RDEB wounds.

Mutations within COL7A1 cause malfunction, reduction or complete absence of type VII collagen in the skin's basement membrane zone (BMZ) leading to recessive dystrophic epidermolysis bullosa (RDEB), a severe and rare skin blistering disease associated with a high risk of skin cancer as well as increased mortality. In this study, we use a 3' RNAtrans-splicing molecule (RTM) as repair molecule to develop a non-viral, non-invasive and efficient in vivo RNA therapy for RDEB. The RTMS6m, cloned into a non-viral minicircle vector (MN511A1), is capable of correcting all mutations occurring between exon 65 and exon 118 of COL7A1 via a trans-splicing reaction between the mutated mRNA and the repair molecule. We tested the specificity and efficiency of the RTM in vitro in RDEB fibroblasts and keratinocytes and detected correct trans-splicing in up to 1.5 % of COL7A1 transcripts via next generation sequencing (NGS). C7 expression was detected via immunofluorescence (IF) staining and Western Blot analysis of transfected cells. Additionally, we used liposomes to deliver the RTMS6m into RDEB skin equivalents (SEs) and were able to detect a ∼4-fold increase of C7 levels at the BMZ compared to the level of mutant C7 present in untreated RDEB SEs. In conclusion, we are able to transiently correct RDEB keratinocytes and fibroblasts in an in vitro setting and are now aiming to analyze the functionality of the 3'-RTMS6m repair molecule in a RDEB xenograft mouse model in vivo.

This study investigates the contribution of epigenetic processes to phenotypic manifestation in the monogenic disease epidermolysis bullosa (EB), focusing on DNA methylation-regulated gene expression. We aim to identify potential modifier genes capable of influencing disease expressivity in EB patients with specific interest in epigenetic patterns related to wound healing. To analyze the effect of loss of type VII collagen on the methylome, we cultured primary keratinocytes from recessive dystrophic EB patients with a COL7A1 exon 3 mutation (RDEB-KC) and healthy controls (HC-KC), for subsequent DNA-extraction and bisulfite conversion. Methylation analyses were performed using the Infinium Methylation EPIC Bead Chip array. Our analyses showed a separation of experimental groups in principal component analysis. Based on >36,000 identified significantly differentially methylated CpGs in >6,000 significantly demethylated regions (DMRs), we were able to extract hyper- (∼34%) and hypo-methylated regions (∼66%) in RDEB-KC vs. HC-KC. Among DMRs were hypermethylated promoter regions of HOXA2 and HOXD3/MIR10B/HOXD4, whose deregulation is associated with skin diseases and impaired wound healing. In addition to significant hypermethylation of the HOXA2 promoter, transcriptome data confirm significant downregulation of HOXA2 expression in RDEB-KC compared to HC-KC. Molecular signature enrichment analysis revealed that cellular processes such as inflammatory response, epithelial-to-mesenchymal transition, cell differentiation, or tissue development were enriched in differentially methylated regions. Preliminary results indicate that loss of type VII collagen has a potentially relevant impact on the methylome. Our data suggest alterations in the methylation status of genes related to wound healing.

This study aims to investigate the degree of deregulation of tumor suppressor miR-200b in recessive dystrophic epidermolysis bullosa squamous cell carcinoma (RDEB-SCC) and to gain insight into whether and how miR-200b affects RDEB-SCC pathomechanisms. We hypothesize that miR-200b expression correlates with the epithelial-to-mesenchymal transition (EMT) state in RDEB-SCCs, and consequently, that the epithelial phenotype can be restored upon reintroduction of miR-200b. MiRNA expression profiling was performed on cultured RDEB-SCCs and -keratinocytes, with subsequent confirmation of deregulated miRNAs, including miR-200b, by TaqMan-qPCR. Cell morphology was classified as cobblestone or spindle-shaped using image cytometry. Further, mobility of RDEB-SCC cells was assessed by migration assay and supernatants of miR-200b overexpressing RDEB-SCC were used to treat endothelial cells (HUVEC) in an angiogenesis assay. A lower expression of miR-200b was observed in RDEB-SCCs, although to a varying extent between isolates from different tumor biopsies. Interestingly, RDEB-SCC cells exhibited morphological differences, varying between cobblestone- and spindle-shaped phenotypes, indicative of different stages in EMT, and low miR-200b-3p levels correlated with a mesenchymal phenotype and accelerated migration. Reintroduction of miR-200b-3p into RDEB-SCC cells resulted in a phenotype change towards epithelial and a significant attenuation in migration. Additionally, a reduction in tube formation capacity of HUVECs was observed upon exposure to supernatants of miR-200b overexpressing RDEB-SCCs. We show that the abundance of miR-200b negatively correlates with an aggressive, migratory phenotype of RDEB-SCCs, providing new insights into the malignancy of RDEB-SCCs. In addition, this miRNA might be a potential therapeutic target.

In this study we aim to correct mutations within COL7A1 that cause malfunction, reduction or complete absence of type VII collagen in the skin's basement membrane zone (BMZ), leading to dystrophic epidermolysis bullosa (DEB), a severe and rare skin blistering disease associated with a high risk of skin cancer as well as increased mortality. Therefore, we use a 3'-RTMS6m repair molecule to develop a safe, non-viral, non-invasive and efficient in vivo RNA therapy for DEB. This RTM-S6m, cloned into a non-viral minicircle-GFP vector, is capable to correct all mutations occurring between exon 65 and exon 108 of COL7A1 via a trans-splicing reaction between the repair molecule and the mutated mRNA. We tested the efficiency and specificity of the RTM in vitro in DEB keratinocytes and fibroblasts and confirmed correct trans-splicing on mRNA level via qPCR analysis, NGS as well as type VII collagen expression via immunofluorescence (IF) staining of transfected cells. Additionally we used a complex of 3'-RTMS6m with liposomes to deliver the RTMS6m onto DEB skin equivalents and were able to show a partial restoration of the type VII collagen expression at the BMZ, confirmed via IF staining of cryosections. Based on the conclusion that we can efficiently correct DEB keratinocytes and fibroblasts in an in vitro setting, we are aiming to analyze the functionality of the 3'-RTMS6m repair molecule in a DEB xenograft mouse model in vivo.

We are developing an ex vivo gene editing therapy for epidermolytic ichthyosis (EI), using transcription activator-like effector nucleases (TALENs) to knockout dominant-negative mutant KRT10 alleles in keratinocytes.EI is a skin fragility disorder caused by heterozygous mutations in KRT1 or KRT10. Keratins K10 and K1 polymerise to build the intermediate filament (IF) cytoskeleton of epidermal suprabasal keratinocytes. Mutant keratins integrate into this, resulting in fragility and collapse upon mild stress, leading to IF aggregate formation and blistering of the skin. TALENs are designer nucleases that can be used to introduce frameshift mutations and inactivate targeted genes. TALENs targeting KRT10 were constructed and shown to cleave efficiently at the target site, with 52% of single cell clones displaying successful modification without selection. A clone displaying modifications on mutant alleles alone was isolated. Mutant KRT10 knockout was confirmed in these cells at the RNA and protein level. This clone was expanded for immunofluorescent analysis of differentiated monolayers under resting and stress conditions. Phenotypic restoration of the keratin network was demonstrated. This was concurrent with murine skin xenograft examination. Electron microscopy of grafts subsequently confirmed stable keratin IF structure and normal desmosome interaction in edited epidermis. TALEN off-target activity was not observed upon examination of 22 predicted sites via next-generation sequencing. Our data demonstrate efficient and safe knockout of mutant KRT10 and phenotypic restoration of IF cytoskeleton and normal epidermal structure following TALEN treatment of EI cells.

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.

Junctional epidermolysis bullosa (JEB) is a devastating disease of the skin and mucous membranes, characterized by blistering and erosions upon minor mechanical friction. Genotypically, mutations within the type XVII collagen gene underlie this phenotype. In this project, we targeted a cryptic splice site arising due to a mutation (c. 380-1 G>A) at the COL17A1 intron 6 / exon 7 junction. Utilization of this splice site leads to the loss of 16 nucleotides at the mRNA level. Subsequently, the reading frame is shifted so that a premature stop codon is generated within exon 9 and the mRNA is degraded by nonsense-mediated decay. Using three distinct antisense oligonucleotides (ASOs), we aimed to either restore the wild type splice pattern of COL17A1 or achieve skipping of exon 7, which would lead to a slightly truncated, but potentially functional type XVII collagen, since the reading frame remains intact. We transfected patient keratinocytes with either of the ASOs and analyzed overall COL17A1 expression, as well as the generation of alternative splicing products. Amplification and sequencing of the COL17A1 mRNA from exon 5 to exon 9 showed skipping of exon 7 as the most prominent result of ASO treatments in JEB keratinocytes. Additionally, alleles with a restored wild type splice pattern, as well as such showing concomitant skipping of exons 6 and 7, were identified. SqRT-PCR confirmed an increase in overall COL17A1 expression. Furthermore, full length (180 kDa) type XVII collagen was detected upon ASO treatment using Western blot analysis. Taken together, we conclude that targeting a cryptic splice site with ASOs may hold therapeutic potential for JEB and possibly other forms of EB with similar types of mutations.

Generalized-severe epidermolysis bullosa simplex (EBS-gen sev) is caused by mutations within either the keratin 14 or keratin 5 gene, phenotypically resulting in blistering and wounding of the skin and mucous membranes in response to mechanical trauma. Although first clinical trials using gene editing technologies show promising results, systemic treatment is still out of reach, especially due to the autosomal dominant inheritance and the resulting necessity in not only providing sufficient amounts of the wild type allele but also in down regulating the disease causing copy. The topically applied small molecule diacerein showed promising results in reducing EBS-gen sev patients blister numbers in a resent phase 2/3 clinical trial. In order to address the safety of this ointment, we analyzed the metabolization of a 1% diacerein ointment both in vitro and in vivo. A Franz diffusion cell setup demonstrated complete conversion into rhein within the skin. Further, uptake and bio-transformation into rhein was also observed in patients upon topical application. Rhein was detected in both urine and serum samples. Treatment of 3% of the body surface for four weeks resulted in systemic rhein levels that were approximately 150-fold lower than levels detected 24 hours after single-dose oral intake, as shown by others. In summary, our results demonstrate that the prodrug diacerein is converted into its active form rhein within the skin, thereby allowing for the exertion of its anti-inflammatory effect in EBS-gen sev patient skin.

With the ability to induce rapid and efficient repair of disease-causing mutations, CRISPR/Cas9 technology is ideally suited for gene therapy approaches for recessive and dominant monogenic disorders. In this study, we have corrected a causal missense mutation in exon 6 of the keratin 14 gene (KRT14) that results in generalized severe epidermolysis bullosa simplex (EBS-gen sev), using a double nicking strategy targeting intron 7, followed by homology-directed repair (HDR). Co-transfection experiments with a Cas9 D10A nickase (Cas9n), a predicted sgRNA pair and a minicircle donor vector harboring the homologous wild type KRT14 sequence for HDR, led to the correction of ∼ 16 % of the mutated KRT14 allele, with a general recombination efficiency of > 30 %. Restoration of normal K14 protein expression and phenotypic correction of EBS-gen sev keratinocytes was demonstrated by Western blot and immunofluorescence analysis respectively, revealing normalization of K14 and absence of disease-associated K14 aggregates within the cytoplasm upon heat shock. Next generation sequencing revealed a promising safety profile for the CRISPR/Cas9n double nicking approach with no detectable off-target activity for a set of predicted sites. In conclusion, we demonstrate a highly efficient and specific gene editing approach for KRT14, offering a causal treatment option for EBS.

CD4+ T cells, but not non-classical monocytes, are dispensable for the development of chronic lymphocytic leukemia in the TCL1-tg murine model