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Recessive dystrophic epidermolysis bullosa is a severe skin fragility disease caused by loss of functional type VII collagen at the dermal-epidermal junction. A frameshift mutation in exon 80 of COL7A1 gene, c.6527insC, is highly prevalent in the Spanish patient population. We have implemented geneediting strategies for COL7A1 frame restoration by NHEJ-induced indels in epidermal stem cells from patients carrying this mutation. TALEN nucleases designed to cut within the COL7A1 exon 80 sequence were delivered to primary patient keratinocyte cultures by non-integrating viral vectors. After genotyping a large collection of vector-transduced patient keratinocyte clones with high proliferative potential, we identified a significant percentage of clones with COL7A1 reading frame recovery and Collagen VII protein expression. Skin equivalents generated with cells from a clone lacking exon 80 entirely were able to regenerate phenotypically normal human skin upon their grafting onto immunodeficient mice. These patientderived human skin grafts showed Collagen VII deposition at the basement membrane zone, formation of anchoring fibrils, and structural integrity when analyzed 12 weeks after grafting. Our data provide a proof-of-principle for recessive dystrophic epidermolysis bullosa treatment through ex vivo gene editing based on removal of pathogenic mutationcontaining, functionally expendable COL7A1 exons in patient epidermal stem cells. The study was mainly supported by DEBRA International—funded by DEBRA Austria (grant termed as Larcher 1). Additional funds come from Spanish grants SAF2013-43475-R and SAF2017-86810-R from the Ministry of Economy and Competitiveness and PI14/00931 and PI17/01747 from the Instituto de Salud Carlos III, all of them co-funded with European Regional Development Funds (ERDF).

Summary Background Basal epidermolysis bullosa simplex (EBS) is a group of blistering geno-dermatoses mostly caused by mutations in the keratin genes, KRT5 and KRT14.Recessive mutations represent about 5% of all EBS mutations, being common andspecific in populations with high consanguinity, where affected patients showsevere phenotypes.Objectives To accomplish the first mutational analysis in patients of Spanish originwith EBS and to delineate a comprehensive genotype–phenotype correlation.Methods Twenty-one EBS families were analysed. Immunofluorescence mapping atthe dermoepidermal junction level was performed on skin biopsies from patients.Mutation screening of the entire coding sequences of KRT5 and KRT14 in genomicDNA was assessed by polymerase chain reaction and direct sequencing.Results KRT5 or KRT14 causative mutations were identified in 18 of the 21 EBSfamilies. A total of 14 different mutations were disclosed, of which 12 weredominant missense mutations and two truncating recessive mutations. Five of the14 mutations were novel including three dominant in KRT5 (p.V186E, p.T321Pand p.A428T) and two recessive in KRT14 (p.K116X and p.K250RfsX8). The twopatients with EBS carrying homozygous recessive mutations were affected bysevere phenotypes and belonged to consanguineous families. All five familieswith the EBS Dowling–Meara subtype carried recurrent mutations affecting thehighly conserved ends of the a-helical rod domain of K5 and K14. The sevenmutations associated with the localized EBS subtype were widely distributedalong the KRT5 and KRT14 genes. Two families with mottled pigmentation carriedthe P25L mutation in KRT5, commonly associated with this subtype.Conclusions This study further confirms the genotype–phenotype correlation estab-lished for EBS in other ethnic groups, and is the first in a Mediterranean country(excluding Israel). This study adds two novel recessive mutations to the worldwiderecord to date, which includes a total of 14 mutations. As in previous reports, therecessive mutations resulted in a lack of keratin K14, giving rise to a generalizedand severe presentation.

Over the past few years, whole skin xenotransplantation models that mimic different aspects of psoriasis have become available. However, these models are strongly constrained by the lack of skin donor availability and homogeneity. We present in this study a bioengineering-based skin-humanized mouse model for psoriasis, either in an autologous version using samples derived from psoriatic patients or, more importantly, in an allogeneic context, starting from skin biopsies and blood samples from unrelated healthy donors. After engraftment, the regenerated human skin presents the typical architecture of normal human skin but, in both cases, immunological reconstitution through intradermal injection in the regenerated skin using in vitro-differentiated T1 subpopulations as well as recombinant IL-17 and IL-22 Th17 cytokines, together with removal of the stratum corneum barrier by a mild abrasive treatment, leads to the rapid conversion of the skin into a bona fide psoriatic phenotype. Major hallmarks of psoriasis were confirmed by the evaluation of specific epidermal differentiation and proliferation markers as well as the mesenchymal milieu, including angiogenesis and infiltrate. Our bioengineered skin-based system represents a robust platform to reliably assess the molecular and cellular mechanisms underlying the complex interdependence between epidermal cells and the immune system. The system may also prove suitable to assess preclinical studies that test the efficacy of novel therapeutic treatments and to predict individual patient response to therapy.

Summary Background Dystrophic epidermolysis bullosa (DEB) is a genodermatosis caused by mutations in COL7A1. The clinical manifestations are highly variable from nail dystrophy to life-threatening blistering, making early molecular diagnosis and prognosis of utmost importance for the affected families. Mutation identification is mandatory for prenatal testing. Objectives To conduct the first mutational analysis of COL7A1 in a Spanish cohort, to assess mutation consequences at protein/mRNA level and to establish genotype–phenotype correlations. Methods Forty-nine Spanish patients with DEB were studied. Antigen mapping was performed on patient skin biopsies. COL7A1 mutation screening in genomic DNA was performed by polymerase chain reaction (PCR) and direct sequencing. Mutation consequences were determined by reverse transcriptase-PCR. Results Eight patients belonged to three unrelated families with dominant DEB. Forty-one were affected with recessive DEB (RDEB). Specifically, 27 displayed the severe generalized subtype, eight the other generalized subtype and six a localized phenotype (two pretibial, three acral and one inversa). Thirty-five mutations were identified, 20 of which are novel. The pathogenic mutation c.6527insC accounted for 46·3% of Spanish RDEB alleles. A consistent genotype–phenotype correlation was established. Conclusions Although the COL7A1 database indicates that most DEB mutations are family specific, the pathogenic mutation c.6527insC was highly recurrent in our cohort. This level of recurrence for a single genetic defect has never previously been reported for COL7A1. Our findings are essential to the clinicians caring for patients with DEB in Spain and in the large population of Spanish descendants in Latin America. They also provide geneticists a molecular clue for a priority mutation screening strategy.

SPARC (secreted protein, acidic and rich in cysteine), a matricellular glycoprotein, modulates the interactions of cells with the extracellular matrix. Studies in null-mice revealed a role of SPARC in wound healing. Here we examined the effect of SPARC in a skin-humanized mouse wound healing model. This model is based in the regeneration of human skin onto the back of nude mice by transplantation of a dermo-epidermal equivalent. The regenerated human skin was excisionally wounded with biopsy punches. At the moment of wounding an adenoviral vector encoding the cDNA for SPARC was intradermally injected. We are currently assessing the effects of in vivo gene transfer of SPARC at the wound site in the healing process. Criticals events of wound healing including reepithelialization, regeneration of dermoepidermal junction and dermal remodeling were studied at different time points postwounding.

Antimicrobial peptides are major components of the innate defense system that present microbicidal activity against gram positive and negative bacteria, yeast, and some enveloped viruses. The human cathelicidin LL-37 is expressed in skin upon wounding and, in addition to its role in antimicrobial defense, it has also been previously demonstrated that this peptide might be involved in reepithelization. We have generated an adenoviral vector containing the complete sequence of LL-37 along with an Ires-GFP expression cassette. The efficacy of this vector was probed by testing the conditioned medium from adenoviral transduced keratinocytes in preventing bacterial growth. We have studied the in vitro effects of LL-37 on HK migration and proliferation. In addition, we have efficiently transduced human skin grafts (in immunodeficient mice) using an in vivo gene transfer approach and we are currently testing the involvement of this peptide in wound repair.

Using a recently described skin-humanized model based on the engraftment of human bioengineered skin equivalents onto immunodeficient mice, we compared the efficacy of different in vivo gene transfer strategies aimed at delivering growth factors to promote skin wound healing. The approaches involving transient delivery of keratinocyte growth factor (KGF) to wounds performed in the engrafted human skin included (1) KGF gene transfer by intradermal adenoviral injection; (2) KGF gene transfer by adenoviral vector immobilized in a fibrin carrier; and (3) KGF-adenoviral gene-transferred human fibroblasts embedded in a fibrin matrix. All delivery systems achieved KGF protein overproduction at the wound site, with a concomitant re-epithelialization enhancement. However, although direct gene delivery strategies exhibited variability in terms of the number of successfully transduced humanized mice, the use of genetically modified fibroblast-containing matrix as an in situ protein bioreactor was highly reproducible, leading to a significant improvement of the overall healing process. This latter approach appeared to be the most reliable means to deliver growth factors to wounds and also avoided the potential danger of scoring cases of faulty administration as therapeutic failures and direct exposure to viral vectors. The combined use of cell and gene therapy appears a robust tool to aid healing in a clinical context.

Cutaneous diabetic wounds greatly affect the quality of life of patients, causing a substantial economic impact on the healthcare system. The limited clinical success of conventional treatments is mainly attributed to the lack of knowledge of the pathogenic mechanisms related to chronic ulceration. Therefore, management of diabetic ulcers remains a challenging clinical issue. Within this context, reliable animal models that recapitulate situations of impaired wound healing have become essential. In this study, we established a new in vivo humanised model of delayed wound healing in a diabetic context that reproduces the main features of the human disease. Diabetes was induced by multiple low doses of streptozotocin in bioengineered human-skin-engrafted immunodeficient mice. The significant delay in wound closure exhibited in diabetic wounds was mainly attributed to alterations in the granulation tissue formation and resolution, involving defects in wound bed maturation, vascularisation, inflammatory response and collagen deposition. In the new model, a cell-based wound therapy consisting of the application of plasma-derived fibrin dermal scaffolds containing fibroblasts consistently improved the healing response by triggering granulation tissue maturation and further providing a suitable matrix for migrating keratinocytes during wound re-epithelialisation. The present preclinical wound healing model was able to shed light on the biological processes responsible for the improvement achieved, and these findings can be extended for designing new therapeutic approaches with clinical relevance. This work was supported by grants from the Science and Innovation Ministry of Spain (SAF2010-16976), from the European VI Framework Programme (LSHB-CT-512102), from Comunidad de Madrid (S2010/BMD-2420; CELLCAM) and from Fundacion Ramon Areces (CIVP16A1864).

Background Skin diseases are a major health problem. Some of the most severe conditions involve genetic disorders, including cancer. Several of these human diseases have been modelled in genetically modified mice, thus becoming a highly valuable preclinical tool for the treatment of these pathologies. However, development of three-dimensional models of skin using keratinocytes from normal and/or genetically modified mice has been hindered by the difficulty to subculture murine epidermal keratinocytes. Methods We have generated a murine epidermal cell line by serially passaging keratinocytes isolated from the back skin of adult mice. We have termed this cell line COCA. Cell culture is done in fully defined media and does not require feeder cells or any other coating methods. Results COCA retained its capacity to differentiate and stratify in response to increased calcium concentration in the cell culture medium for more than 75 passages. These cells, including late passage, can form epidermis-like structures in three-dimensional in vitro models with a well-preserved pattern of proliferation and differentiation. Furthermore, these cells form epidermis in grafting assays in vivo, and do not develop tumorigenic ability. Conclusions We propose that COCA constitutes a good experimental system for in vitro and in vivo skin modelling. Also, cell lines from genetically modified mice of interest in skin biology could be established using the method we have developed. COCA keratinocytes would be a suitable control, within a similar background, when studying the biological implications of these alterations.

The human antimicrobial peptide LL-37 plays an important role in host defense against infection. In addition to its antimicrobial action, other activities have been described in eukaryotic cells that may contribute to the healing response. In this study, we demonstrated that in vitro human cathelicidin activates migration of the human keratinocyte cell line HaCaT, involving phenotypic changes related to actin dynamics and associated to augmented tyrosine phosphorylation of proteins involved in focal adhesion complexes, such as focal adhesion kinase and paxillin. Other events involved in the LL-37 response were the induction of the Snail and Slug transcription factors, activation of matrix metalloproteinases and activation of the mitogen-activated protein kinase , and phosphoinositide 3-kinase/Akt signaling pathways. These signaling events could be mediated not only through the transactivation of EGFR but also through the induction of G-protein-coupled receptor FPRL-1 expression in these cells. Finally, by in vivo adenoviral transfer of the antimicrobial peptide to excisional wounds in ob/ob mice, we demonstrated that LL-37 significantly improved re-epithelialization and granulation tissue formation. The protective and regenerative activities of LL-37 support its therapeutic potential to promote wound healing.

Top of pageAbstract Leptin deficiencies are involved in rare cases of inherited human obesity and in various forms of lipodystrophy. Previous results from our laboratory showed the feasibility of leptin deficiency correction in the ob/ob mice model using skin grafts from transgenic mice expressing leptin from the epidermis. In spite of epidermal stem cell targeting, several attempts to achieve permanent correction of leptin-deficient ob/ob mice through oncortetrovirus-transduced human keratinocyte (HK) grafts resulted unsuccessful. When human keratinocytes were transduced with a leptin-ires GFP-encoding HIV- derived lentivirus, leptin levels in culture were 3-fold higher that those achieved with oncoretrovirus-transduced HK. Consistently, immunodeficient ob/ob mice orthotopically grafted with lentivirus- transduced HK as part of bioengineered skins, showed therapeutic serum leptin levels. Phenotypic correction of engrafted ob/ob (immunodeficient) mice included weight loss, decreased food intake and normalization of blood glucose. Permanent correction (longer than 22 weeks post-grafting) was achieved in two animals. Skin regenerated form leptin lentivirus-transduced HK displayed histological features similar to those of native human skin. This is the first report of the long-sought sustained correction of a systemic protein deficiency using orthotopic grafting of ex vivo genetically modified human keratinocytes acting as a bioreactor.