Gene editing constitutes a novel approach for precisely correcting disease-causing gene mutations. Frameshift mutations in COL7A1 causing recessive dystrophic epidermolysis bullosa are amenable to open reading frame restoration by non-homologous end joining repair-based approaches. Efficient targeted deletion of faulty COL7A1 exons in polyclonal patient keratinocytes would enable the translation of this therapeutic strategy to the clinic. In this study, using a dual single-guide RNA (sgRNA)-guided Cas9 nuclease delivered as a ribonucleoprotein complex through electroporation, we have achieved very efficient targeted deletion of COL7A1 exon 80 in recessive dystrophic epidermolysis bullosa (RDEB) patient keratinocytes carrying a highly prevalent frameshift mutation. This ex vivo non-viral approach rendered a large proportion of corrected cells producing a functional collagen VII variant. The effective targeting of the epidermal stem cell population enabled long-term regeneration of a properly adhesive skin upon grafting onto immunodeficient mice. A safety assessment by next-generation sequencing (NGS) analysis of potential off-target sites did not reveal any unintended nuclease activity. Our strategy could potentially be extended to a large number of COL7A1 mutation-bearing exons within the long collagenous domain of this gene, opening the way to precision medicine for RDEB.

Background Recessive dystrophic epidermolysis bullosa (RDEB), Kindler syndrome (KS) and xeroderma pigmentosum complementation group C (XPC) are three cancer‐prone genodermatoses whose causal genetic mutations cannot fully explain, on their own, the array of associated phenotypic manifestations. Recent evidence highlights the role of the stromal microenvironment in the pathology of these disorders. Objectives To investigate, by means of comparative gene expression analysis, the role played by dermal fibroblasts in the pathogenesis of RDEB, KS and XPC. Methods We conducted RNA‐Seq analysis, which included a thorough examination of the differentially expressed genes, a functional enrichment analysis and a description of affected signalling circuits. Transcriptomic data were validated at the protein level in cell cultures, serum samples and skin biopsies. Results Interdisease comparisons against control fibroblasts revealed a unifying signature of 186 differentially expressed genes and four signalling pathways in the three genodermatoses. Remarkably, some of the uncovered expression changes suggest a synthetic fibroblast phenotype characterized by the aberrant expression of extracellular matrix (ECM) proteins. Western blot and immunofluorescence in situ analyses validated the RNA‐Seq data. In addition, enzyme‐linked immunosorbent assay revealed increased circulating levels of periostin in patients with RDEB. Conclusions Our results suggest that the different causal genetic defects converge into common changes in gene expression, possibly due to injury‐sensitive events. These, in turn, trigger a cascade of reactions involving abnormal ECM deposition and underexpression of antioxidant enzymes. The elucidated expression signature provides new potential biomarkers and common therapeutic targets in RDEB, XPC and KS. What's already known about this topic? Recessive dystrophic epidermolysis bullosa (RDEB), Kindler syndrome (KS) and xeroderma pigmentosum complementation group C (XPC) are three genodermatoses with high predisposition to cancer development. Although their causal genetic mutations mainly affect epithelia, the dermal microenvironment likely contributes to the physiopathology of these disorders. What does this study add? We disclose a large overlapping transcription profile between XPC, KS and RDEB fibroblasts that points towards an activated phenotype with high matrix‐synthetic capacity. This common signature seems to be independent of the primary causal deficiency, but reflects an underlying derangement of the extracellular matrix via transforming growth factor‐β signalling activation and oxidative state imbalance. What is the translational message? This study broadens the current knowledge about the pathology of these diseases and highlights new targets and biomarkers for effective therapeutic intervention. It is suggested that high levels of circulating periostin could represent a potential biomarker in RDEB.

Recessive dystrophic epidermolysis bullosa (RDEB) is a genetic extracellular matrix disease caused by deficiency in type VII collagen (Col VII). The disease manifests with devastating mucocutaneous fragility leading to progressive fibrosis and metastatic squamous cell carcinomas. Although Col VII abundance is considered the main predictor of symptom course, previous studies have revealed the existence of mutation-independent mechanisms that control disease progression. Here, to investigate and validate new molecular modifiers of wound healing and fibrosis in a natural human setting, and toward development of disease-modulating treatment of RDEB, we performed gene expression profiling of primary fibroblast from RDEB siblings with marked phenotypic variations, despite having equal COL7A1 genotype. Gene enrichment analysis suggested that severe RDEB was associated with enhanced response to TGF-β stimulus, oxidoreductase activity, and cell contraction. Consistently, we found an increased response to TGF-β, higher levels of basal and induced reactive oxygen species (ROS), and greater contractile ability in collagen lattices in RDEB fibroblasts (RDEBFs) from donors with severe RDEB vs mild RDEB. Treatment with antioxidants allowed a reduction of the pro-fibrotic and contractile phenotype. Importantly, our analyses revealed higher expression and deposition in skin of the relatively uncharacterized small leucine-rich extracellular proteoglycan PRELP/prolargin associated with milder RDEB manifestations. Mechanistic investigations showed that PRELP effectively attenuated fibroblasts' response to TGF-β1 stimulus and cell contractile capacity. Moreover, PRELP overexpression in RDEBFs enhanced RDEB keratinocyte attachment to fibroblast-derived extracellular matrix in the absence of Col VII. Our results highlight the clinical relevance of pro-oxidant status and hyper-responsiveness to TGF-β in RDEB severity and progression. Of note, our study also reveals PRELP as a novel and natural TGF-β antagonist with a likely dermo-epidermal pro-adhesive capacity.

The role of stroma is fundamental in the development and behavior of epithelial tumors. In this regard, limited growth of squamous cell carcinomas (SCC) or cell-lines derived from them has been achieved in immunodeficient mice. Moreover, lack of faithful recapitulation of the original human neoplasia complexity is often observed in xenografted tumors. Here, we used tissue engineering techniques to recreate a humanized tumor stroma for SCCs grafted in host mice, by combining CAF (cancer associated fibroblasts)-like cells with a biocompatible scaffold. The stroma was either co-injected with epithelial cell lines derived from aggressive SCC or implanted 15 days before the injection of the tumoral cells, to allow its vascularization and maturation. None of the mice injected with the cell lines without stroma were able to develop a SCC. In contrast, tumors were able to grow when SCC cells were injected into previously established humanized stroma. Histologically, all of the regenerated tumors were moderately differentiated SCC with a well-developed stroma, resembling that found in the original human neoplasm. Persistence of human stromal cells was also confirmed by immunohistochemistry. In summary, we provide a proof of concept that humanized tumor stroma, generated by tissue engineering, can facilitate the development of epithelial tumors in immunodeficient mice.