Therapy Development for Epidermolysis Bullosa

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

A cancer stem cell-like phenotype is associated with miR-10b expression in aggressive squamous cell carcinomas

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

Identification of Rigosertib for the Treatment of Recessive Dystrophic Epidermolysis Bullosa–Associated Squamous Cell Carcinoma

PURPOSE: Squamous cell carcinoma (SCC) of the skin is the leading cause of death in patients with the severe generalized form of the genetic disease recessive dystrophic epidermolysis bullosa (RDEB). Although emerging data are identifying why patients suffer this fatal complication, therapies for treatment of RDEB SCC are in urgent need. EXPERIMENTAL DESIGN: We previously identified polo-like kinase 1 (PLK1) as a therapeutic target in skin SCC, including RDEB SCC. Here, we undertake a screen of 6 compounds originally designated as PLK1 inhibitors, and detail the efficacy of the lead compound, the multipathway allosteric inhibitor ON-01910, for targeting RDEB SCC in vitro and in vivo. RESULTS: ON-01910 (or rigosertib) exhibited significant specificity for RDEB SCC: in culture rigosertib induced apoptosis in 10 of 10 RDEB SCC keratinocyte populations while only slowing the growth of normal primary skin cells at doses 2 orders of magnitude higher. Furthermore, rigosertib significantly inhibited the growth of two RDEB SCC in murine xenograft studies with no apparent toxicity. Mechanistically, rigosertib has been shown to inhibit multiple signaling pathways. Comparison of PLK1 siRNA with MEK inhibition, AKT inhibition, and the microtubule-disrupting agent vinblastine in RDEB SCC shows that only PLK1 reduction exhibits a similar sensitivity profile to rigosertib. CONCLUSIONS: These data support a “first in RDEB” phase II clinical trial of rigosertib to assess tumor targeting in patients with late stage, metastatic, and/or unresectable SCC

Evaluating a Targeted Cancer Therapy Approach Mediated by RNA

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

Cells from discarded dressings differentiate chronic from acute wounds in patients with Epidermolysis Bullosa

Impaired wound healing complicates a wide range of diseases and represents a major cost to healthcare systems. Here we describe the use of discarded wound dressings as a novel, cost effective, accessible, and non-invasive method of isolating viable human cells present at the site of skin wounds. By analyzing 133 discarded wound dressings from 51 patients with the inherited skin-blistering disease epidermolysis bullosa (EB), we show that large numbers of cells, often in excess of 100 million per day, continually infiltrate wound dressings. We show, that the method is able to differentiate chronic from acute wounds, identifying significant increases in granulocytes in chronic wounds, and we show that patients with the junctional form of EB have significantly more cells infiltrating their wounds compared with patients with recessive dystrophic EB. Finally, we identify subsets of granulocytes and T lymphocytes present in all wounds paving the way for single cell profiling of innate and adaptive immune cells with relevance to wound pathologies. In summary, our study delineates findings in EB that have potential relevance for all chronic wounds, and presents a method of cellular isolation that has wide reaching clinical application

5′RNA Trans-Splicing Repair of COL7A1 Mutant Transcripts in Epidermolysis Bullosa

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

Evaluating a Targeted Cancer Therapy Approach Mediated by RNA trans-Splicing In Vitro and in a Xenograft Model for Epidermolysis Bullosa-Associated Skin Cancer

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