Eva Maria Murauer

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.

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 | POSTER

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.

The Duchenne muscular dystrophy (DMD) gene has a complex expression pattern regulated by multiple tissue-specific promoters and by alternative splicing (AS) of the resulting transcripts. Here, we used an RNAi-based approach coupled with DMD-targeted RNA-seq to identify RNA-binding proteins (RBPs) that regulate splicing of its skeletal muscle isoform (Dp427m) in a human muscular cell line. A total of 16 RBPs comprising the major regulators of muscle-specific splicing events were tested. We show that distinct combinations of RBPs maintain the correct inclusion in the Dp427m of exons that undergo spatio-temporal AS in other dystrophin isoforms. In particular, our findings revealed the complex networks of RBPs contributing to the splicing of the two short DMD exons 71 and 78, the inclusion of exon 78 in the adult Dp427m isoform being crucial for muscle function. Among the RBPs tested, QKI and DDX5/DDX17 proteins are important determinants of DMD exon inclusion. This is the first large-scale study to determine which RBP proteins act on the physiological splicing of the DMD gene. Our data shed light on molecular mechanisms contributing to the expression of the different dystrophin isoforms, which could be influenced by a change in the function or expression level of the identified RBPs.

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.

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, 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.Methods: MiRNA expression profiling was performed across various cell types isolated from skin and cSCCs. Microarray results were confirmed by qPCR and by an optimizedin situhybridization 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.Results: Several 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.Conclusion: The 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.

Human skin contains a population of memory T cells that support tissue homeostasis and provide protective immunity. The study of human memory T cells is often restricted to in vitro studies and to human PBMC serving as primary cell source. Because the tisse environment impacts the phenotype and function of memory T cells, it is crucial to study these cells within their tissue. Here we utilized immunodeficient NOD- scid IL2rγ ^null (NSG) mice that carried in vivo- generated engineered human skin (ES). ES were generated from human keratinocytes and fibroblasts and is initially devoid of skin-resident immune cells. Upon adoptive transfer of human PBMC this reductionist system allowed to study human T cell recruitment from a circulating pool of T cells into non-inflamed human skin in vivo . Circulating human memory T cells preferentially infiltrated ES and showed diverse functional profiles of T cells found in fresh human skin. The chemokine and cytokine microenvironment of ES closely resembled that of non-inflamed human skin. Upon entering the ES T cells assumed a resident memory T cell-like phenotype in the absence of infection, and a proportion of these cutaneous T cells can be locally activated upon injection of monocyte derived dendritic cells (moDCs) that presented Candida albicans . Interestingly, we found that CD69 ⁺ memory T cells produced higher levels of effector cytokines in response to Candida albicans , compared to CD69 ^- T cells. Overall, this model has broad utility in many areas of human skin immunology research, including the study of immune-mediated skin diseases.

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.