Read-Through for Nonsense Mutations in Type XVII Collagen-Deficient Junctional Epidermolysis Bullosa

The project is funded by E-Rare-3 Joint Translational Call 2017 (to CH, MR, and FLL) and Bundesministerium für Bildung und Forschung 01GM1805 (to CH). IC received a fellowship from the European Academy of Dermatology and Venereology. We thank Blanca Duarte for skin-equivalent grafting procedures. The funding sources for this study : ERA-NET E-RARE JTC 2017 (MutaEB) (CH, MR, and FLL), (Grant AC17/00054 [FLL]) cofunded with European Regional Development Funds, BMBF 01GM1805, EADV (IC

Efficient CRISPR-Cas9-mediated gene ablation in human keratinocytes to recapitulate genodermatoses: modeling of Netherton syndrome

Current efforts to find specific genodermatoses treatments and define precise pathogenesis mechanisms require appropriate surrogate models with human cells. Although transgenic and gene knockout mouse models for several of these disorders exist, they often fail to faithfully replicate the clinical and histopathological features of the human skin condition. We have established a highly efficient method for precise deletion of critical gene sequences in primary human keratinocytes, based on CRISPR-Cas9-mediated gene editing. Using this methodology, in the present study we generated a model of Netherton syndrome by disruption of SPINK5. Gene-edited cells showed absence of LEKTI expression and were able to recapitulate a hyperkeratotic phenotype with most of the molecular hallmarks of Netherton syndrome, after grafting to immunodeficient mice and in organotypic cultures. To validate the model as a platform for therapeutic intervention, we tested an ex vivo gene therapy approach using a lentiviral vector expressing SPINK5. Re-expression of SPINK5 in an immortalized clone of SPINK5-knockout keratinocytes was capable of reverting from Netherton syndrome to a normal skin phenotype in vivo and in vitro. Our results demonstrate the feasibility of modeling genodermatoses, such as Netherton syndrome, by efficiently disrupting the causative gene to better understand its pathogenesis and to develop novel therapeutic approaches

Correction of recessive dystrophic epidermolysis bullosa by homology-directed repair-mediated genome editing

Genome-editing technologies that enable the introduction of precise changes in DNA sequences have the potential to lead to a new class of treatments for genetic diseases. Epidermolysis bullosa (EB) is a group of rare genetic disorders characterized by extreme skin fragility. The recessive dystrophic subtype of EB (RDEB), which has one of the most severe phenotypes, is caused by mutations in COL7A1. In this study, we report a gene-editing approach for ex vivo homology-directed repair (HDR)-based gene correction that uses the CRISPR-Cas9 system delivered as a ribonucleoprotein (RNP) complex in combination with donor DNA templates delivered by adeno-associated viral vectors (AAVs). We demonstrate sufficient mutation correction frequencies to achieve therapeutic benefit in primary RDEB keratinocytes containing different COL7A1 mutations as well as efficient HDR-mediated COL7A1 modification in healthy cord blood-derived CD34+ cells and mesenchymal stem cells (MSCs). These results are a proof of concept for HDR-mediated gene correction in different cell types with therapeutic potential for RDEB.This work was supported by Spanish grants PI17/01747, PI20/00615, AC17/00054 (MutaEB-E-rare), and CIBERER ER18TRL714 from the Instituto de Salud Carlos III and grant SAF2017-86810-R from the Ministry of Economy and Competitiveness , all co-funded with European Regional Development Funds , and Avancell-CM grant ( S2017/BMD-3692 ). Authors are indebted to Almudena Holguín and Nuria Illera for grafting experiments, and to Jesus Martínez and Edilia De Almeida for animal maintenance and care

Identification and functional characterization of mutation-independent disease-severity modulators in recessive dystrophic epidermolysis bullosa

Mención Internacional en el título de doctorGenodermatoses are rare inherited skin diseases, mostly monogenic, with a relevant social, economic, and clinical burden. Among them, epidermolysis bullosa (EB) comprises a group of skin fragility disorders, defined by blistering upon minimal trauma, with heterogeneous pathogenesis and clinical manifestations. Recessive dystrophic EB (RDEB) is the most severe form of the disease, caused by mutations in the COL7A1 gene that encodes the main component of the anchoring fibrils that attach the epithelium to the underneath dermal matrix. Patients with RDEB suffer from chronic mucocutaneous blisters driving perpetual cycles of inflammation and fibrosis that support metastatic cutaneous squamous cell carcinomas. There is currently no cure for the disease, but several approaches are being developed in preclinical and clinical phases. Considering that RDEB involves cutaneous and extracutaneous manifestations it is presumed necessary the combination of diverse therapies (e.g. cell, gene, and symptom-relief therapies) to improve clinical manifestations and even find a definitive cure. To this end, it is necessary to increase the understanding of the pathomechanisms underlying the primary defect in RDEB, aiming to find new disease modifiers with therapeutic potential. In Chapter 1, we used RNA-Seq-based technologies to study altered signaling pathways and gene expression changes that could promote disease progression and carcinogenesis in RDEB and two other cancer-prone genodermatoses with premature aging and chronic inflammation as common hallmarks. Transcriptomic profile of dermal fibroblasts derived from patients with RDEB, Kindler EB (Kindler Syndrome, KS), and Xeroderma pigmentosum C (XPC) was analyzed. Inter-disease comparison pointed to a common activated and synthetic phenotype in all genodermatoses, independent of the primary causal deficient gene. This common signature shares some similarities with that found in myofibroblasts and cancer-associated fibroblasts (CAFs), characterized by aberrant expression of extracellular matrix (ECM) proteins, TGF-β signaling, and oxidative stress. Additionally, the pipeline allowed us to describe and validate in patients’ serum samples periostin as a novel biomarker in RDEB. In Chapter 2, to investigate modulating factors of the disparate disease course and severity in RDEB, we took advantage of an atypical clinical case of two siblings with equal COL7A1 genotype but discordant phenotypes. Transcriptomic and mechanistic analyses pointed towards oxidative stress, hyper-responsiveness to TGF-β, and higher fibroblasts' contractile capacity as facilitating players of severe fibrotic features. Furthermore, we disclose a novel and natural TGF-β inhibitor, PRELP, that could explain, at least in part, the observed differences in skin fragility and fibrosis. Finally, we tested the beneficial therapeutic effects of antioxidants and PRELP in in vitro models, observing a reduction of the pro-fibrotic and contractile phenotype characteristic of RDEB fibroblasts (RDEBFs). This doctoral thesis sheds light on mutation-independent mechanisms that affect severity and progression in RDEB, necessary info to develop reliable biomarkers, and effective symptom-relief or adjuvant therapies to improve patients’ quality of life. These findings could also have significance for other diseases with similar pathomechanisms, both rare and common disorders (e.g. KS, XPC, fibrosis, and cancer).Programa de Doctorado en Ciencia y Tecnología Biomédica por la Universidad Carlos III de MadridPresidente: Teresa Odorisio.- Secretario: Luisa María Botella Cubells.- Vocal: Ángeles Juarranz de la Fuent

Estrutura vegetal e flora associada ao manguezal de Mata de Limón, Puntarenas, Costa Rica

La estructura de los manglares se ve afectada negativamente por diferentes factores y sus variaciones llegan a causar estrés fisiológico. El objetivo de la investigación fue describir el estado de la estructura vegetal y la flora asociada al humedal de Mata de Limón. En el año 2014, se establecieron seis parcelas de monitoreo, en donde se determinó el número de árboles, la altura, el diámetro a la altura del pecho (DAP) y la especie, para calcular el índice de complejidad y de valor de importancia. Se encontraron seis de las siete especies de flora nuclear identificadas en los manglares de Costa Rica. El índice de complejidad general fue de 4.97, Avicennia germinans tuvo el mayor valor de importancia, presente en cinco parcelas. Además, se reconocieron 26 organismos de vegetación asociada, distribuidos en 17 familias. La presencia de los diferentes mangles está determinada por la dinámica natural de diversos factores presentes en el ecosistema, sin embargo, debido al desarrollo de las actividades humanas su condición se puede ver alterada. Dadas las perturbaciones que ha enfrentado el humedal de Mata de Limón, como la construcción de la línea del tren, de la carretera y el puerto de Caldera; su conformación vegetal presenta una evidente heterogeneidad, de manera que las zonas más alejadas de la ocupación humana poseen mayor complejidad y presencia florística típica del ecosistema de manglar.Mangrove forests can be negatively affected by different factors, and variations in these factors can cause physiological stress. The objective of this investigation was to describe the state of the vegetation structure and associated flora of the Mata de Limón mangrove forest. In the year 2014, six plots were established and measurements were carried out in each plot to determine the number of trees, heights, diameters at breast height and species of mangrove to calculate indicators of complexity and importance. Six of the seven nuclear species of Costa Rican mangrove forests were found. The general complexity index was 4.97, and Avicennia germinans had the highest importance value, being present in five of the six plots. In addition, 26 species of associated vegetation were identified, distributed in 17 families. Presence of mangrove forests is determined by the natural dynamics of diverse factors in an ecosystem, but human activities can alter its. The mangrove forest of Mata de Limón has been affected by factors such as the construction of train tracks, roads and the Caldera seaport; its vegetative composition is therefore heterogeneous, with the areas farthest from human interaction displaying greater complexity and a higher presence of floral species typically present in mangrove ecosystems.A estrutura dos manguezais está negativamente afetada por diferentes fatores e suas variações chegam a causar estresse fisiológico. O objetivo da pesquisa foi descrever o estado da estrutura vegetal e a flora associada ao pântano de Mata de Limón. Em 2014, foram estabelecidas seis parcelas de monitoramento, nas quais foram determinados o número de árvores, a altura, o diâmetro à altura do peito (DAP) e a espécie, para calcular o índice de complexidade e de valor de importância. Foram encontrados seis das sete espécies de flora nuclear identificadas nos manguezais da Costa Rica. O índice de complexidade geral foi de 4.97, Avicennia germinans teve o maior valor de importância, presente em cinco parcelas. Além disso, foram reconhecidos 26 organismos de vegetação associada, distribuídos em 17 famílias. A presença dos diferentes manguezais está determinada pela dinâmica natural de diversos fatores presentes no ecossistema, entretanto, devido ao desenvolvimento das atividades humanas, sua condição pode-se ver alterada. Dadas as perturbações enfrentadas pelo pântano de Mata de Limón, como a construção da linha do trem, da rodovia e do porto de Caldera, sua conformação vegetal apresenta uma evidente heterogeneidade, de maneira que as zonas mais distantes da ocupação humana possuem maior complexidade e presença florística típica do ecossistema de manguezal

Caracterização do substrato e da água intersticial do manguezal Mata de Limón, Puntarenas, Costa Rica

Los manglares son sistemas que mediante la interacción marino terrestre varían durante el tiempo y son altamente productivos. El objetivo del estudio fue describir la granulometría, parámetros fisicoquímicos y nutrientes del humedal de Mata de Limón. En el año 2014, se establecieron seis parcelas de monitoreo para analizar los sedimentos, nutrientes y parámetros fisicoquímicos. Se encontró mayor presencia de arenas, en especial las gruesas. Los valores de pH fueron muy similares, la mayoría cercanos a 8; mientras que la oxidación-reducción mostró una predominancia a procesos reductivos con un mínimo de -54 y un máximo de -3 mV. En cuanto a los nutrientes la concentración más alta de amonio fue de 29.2 μmol/L, en tanto que la más baja fue de cero. En el caso del nitrato, la superior fue de 30.53 μmol/L, y la inferior de 1.01 μmol/L. Tanto para los fosfatos como los silicatos, la mayor fue de 21.59 y 174.95 μmol/L, y la menor de 0.71 y 19.66 μmol/L respectivamente. El humedal de Mata de Limón está conformado mayormente por arenas; los parámetros fisicoquímicos están afectados por variaciones espaciotemporales, y un alto contenido de nutrientes. Sin embargo, es perturbado por presiones antropogénicas, causando inestabilidad en las variables evaluadas, lo que incrementa su complejidad; por el contrario, el sistema fluvial corresponde a un patrón más estable con cambios predecibles al interior del ecosistema.Mangroves are systems that, through land-sea interaction, vary in time and are highly productive. The objective of the study was to describe the granulometry, physicochemical parameters, and nutrients of the Mata de Limón Wetlands. In 2014, six monitoring plots were established to analyze the sediments, nutrients and physicochemical parameters. A greater presence of sand was found, especially thick sand. The pH values were very similar, most of them close to 8, while oxidation-reduction values showed a predominance of reductive processes with a minimum value of -54 and a maximum value of -3 mV. Regarding nutrients, the maximum concentration of ammonium was 29.2 μmol/L, while the minimum was zero. In the case of nitrate, the highest was 30.53 μmol/L and the lowest was 1.01 μmol/L. For both phosphates and silicates, the maximum values were 21.59 and 174.95 μmol/L, and the minimum were 0.71 and 19.66 μmol/L, respectively. The Mata de Limón Wetlands are comprised mainly by sand; the physicochemical parameters are affected by spatiotemporal variations and a high quantity of nutrients. Nevertheless, it is disturbed by anthropogenic pressures, causing instability in the evaluated variables, which increases their complexity; on the contrary, the river system corresponds to a more stable pattern with predictable changes to the interior of the ecosystem.Os manguezais são sistemas que, por meio da interação marinho-terrestre, variam ao longo do tempo e são altamente produtivos. O objetivo do estudo foi descrever a granulometria, os parâmetros físico-químicos e os nutrientes da área úmida da Mata de Limón. Em 2014, seis parcelas de monitoramento foram estabelecidas para analisar sedimentos, nutrientes e parâmetros físico-químicos. Foi encontrada uma maior presença de areias, especialmente areias grossas. Os valores de pH foram muito semelhantes, em sua maioria próximos a 8, enquanto a oxidação-redução mostrou uma predominância de processos redutivos com um mínimo de -54 e um máximo de -3 mV. Em relação aos nutrientes, a maior concentração de amônio foi de 29.2 µmol/L, enquanto a menor foi zero. No caso do nitrato, o valor mais alto foi de 30.53 µmol/L e o mais baixo foi de 1.01 µmol/L. Para fosfatos e silicatos, o valor mais alto foi de 21.59 e 174.95 µmol/L, e o mais baixo foi de 0.71 e 19.66 µmol/L, respectivamente. A zona úmida da Mata de Limón consiste principalmente de areias; os parâmetros físico-químicos são afetados por variações espaço-temporais e por um alto teor de nutrientes. No entanto, ele é perturbado por pressões antropogênicas, causando instabilidade nas variáveis avaliadas, o que aumenta sua complexidade; em contraste, o sistema fluvial corresponde a um padrão mais estável com mudanças previsíveis no ecossistema

Characterization of the substrate and interstitial water of Mata de Limón mangrove, Puntarenas, Costa Rica

Los manglares son sistemas que mediante la interacción marino terrestre varían durante el tiempo y son altamente productivos. El objetivo del estudio fue describir la granulometría, parámetros fisicoquímicos y nutrientes del humedal de Mata de Limón. En el año 2014, se establecieron seis parcelas de monitoreo para analizar los sedimentos, nutrientes y parámetros fisicoquímicos. Se encontró mayor presencia de arenas, en especial las gruesas. Los valores de pH fueron muy similares, la mayoría cercanos a 8; mientras que la oxidación-reducción mostró una predominancia a procesos reductivos con un mínimo de -54 y un máximo de -3 mV. En cuanto a los nutrientes la concentración más alta de amonio fue de 29.2 µmol/L, en tanto que la más baja fue de cero. En el caso del nitrato, la superior fue de 30.53 µmol/L, y la inferior de 1.01 µmol/L. Tanto para los fosfatos como los silicatos, la mayor fue de 21.59 y 174.95 µmol/L, y la menor de 0.71 y 19.66 µmol/L respectivamente. El humedal de Mata de Limón está conformado mayormente por arenas; los parámetros fisicoquímicos están afectados por variaciones espaciotemporales, y un alto contenido de nutrientes. Sin embargo, es perturbado por presiones antropogénicas, causando inestabilidad en las variables evaluadas, lo que incrementa su complejidad; por el contrario, el sistema fluvial corresponde a un patrón más estable con cambios predecibles al interior del ecosistemaMangroves are systems that, through land-sea interaction, vary in time and are highly productive. The objective of the study was to describe the granulometry, physicochemical parameters, and nutrients of the Mata de Limón Wetlands. In 2014, six monitoring plots were established to analyze the sediments, nutrients and physicochemical parameters. A greater presence of sand was found, especially thick sand. The pH values were very similar, most of them close to 8, while oxidation-reduction values showed a predominance of reductive processes with a minimum value of -54 and a maximum value of -3 mV. Regarding nutrients, the maximum concentration of ammonium was 29.2 µmol/L, while the minimum was zero. In the case of nitrate, the highest was 30.53 µmol/L and the lowest was 1.01 µmol/L. For both phosphates and silicates, the maximum values were 21.59 and 174.95 µmol/L, and the minimum were 0.71 and 19.66 µmol/L, respectively. The Mata de Limón Wetlands are comprised mainly by sand; the physicochemical parameters are affected by spatiotemporal variations and a high quantity of nutrients. Nevertheless, it is disturbed by anthropogenic pressures, causing instability in the evaluated variables, which increases their complexity; on the contrary, the river system corresponds to a more stable pattern with predictable changes to the interior of the ecosystem.Universidad Naiconal, Costa RicaEscuela de Ciencias Biológica

Structure and associated flora of the Mata de Limón mangrove forest Puntarenas, Costa Rica

La estructura de los manglares se ve afectada negativamente por diferentes factores y sus variaciones llegan a causar estrés fisiológico. El objetivo de la investigación fue describir el estado de la estructura vegetal y la flora asociada al humedal de Mata de Limón. En el año 2014, se establecieron seis parcelas de monitoreo, en donde se determinó el número de árboles, la altura, el diámetro a la altura del pecho (DAP) y la especie, para calcular el índice de complejidad y de valor de importancia. Se encontraron seis de las siete especies de flora nuclear identificadas en los manglares de Costa Rica. El índice de complejidad general fue de 4.97, Avicennia germinans tuvo el mayor valor de importancia, presente en cinco parcelas. Además, se reconocieron 26 organismos de vegetación asociada, distribuidos en 17 familias. La presencia de los diferentes mangles está determinada por la dinámica natural de diversos factores presentes en el ecosistema, sin embargo, debido al desarrollo de las actividades humanas su condición se puede ver alterada. Dadas las perturbaciones que ha enfrentado el humedal de Mata de Limón, como la construcción de la línea del tren, de la carretera y el puerto de Caldera; su conformación vegetal presenta una evidente heterogeneidad, de manera que las zonas más alejadas de la ocupación humana poseen mayor complejidad y presencia florística típica del ecosistema de manglarMangrove forests can be negatively affected by different factors, and variations in these factors can cause physiological stress. The objective of this investigation was to describe the state of the vegetation structure and associated flora of the Mata de Limón mangrove forest. In the year 2014, six plots were established and measurements were carried out in each plot to determine the number of trees, heights, diameters at breast height and species of mangrove to calculate indicators of complexity and importance. Six of the seven nuclear species of Costa Rican mangrove forests were found. The general complexity index was 4.97, and Avicennia germinanshad the highest importance value, being present in five of the six plots. In addition, 26 species of associated vegetation were identified, distributed in 17 families. Presence of mangrove forests is determined by the natural dynamics of diverse factors in an ecosystem, but human activities can alter its. The mangrove forest of Mata de Limón has been affected by factors such as the construction of train tracks, roads and the Caldera seaport; its vegetative composition is therefore heterogeneous, with the areas farthest from human interaction displaying greater complexity and a higher presence of floral species typically present in mangrove ecosystemsEscuela de Ciencias Biológica

Fibroblast activation and abnormal extracellular matrix remodelling as common hallmarks in three cancer-prone genodermatoses

Altres ajuts: CIBERER; European Regional Development Funds; Comunidad de Madrid (AvanCell-CM S2017/BMD-3692); Fundació La Marato de TV3 (201331-30); CERCA Programme, Generalitat de Catalunya; Fundación Científica de la Asociación Española Contra el Cancer.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