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  • Publication
    Non-viral delivery of CRISPR-Cas9 complexes for targeted gene editing via a polymer delivery system
    (Springer Nature, 2022-04-01) Ahern, Jonathan O'Keeffe; Lara-Saez, Irene; Zhou, Dezhong; Murillas Angoiti, Rodolfo; Bonafont Aragó, José; Mencía Rodríguez, Ángeles; García Díez, Marta; Manzanares, Dario; Lynch, Jennifer; Foley, Ruth; Xu, Quian; Sigen, A.; Larcher Laguzzi, Fernando; Wang, Wenxin
    Recent advances in molecular biology have led to the CRISPR revolution, but the lack of an efficient and safe delivery system into cells and tissues continues to hinder clinical translation of CRISPR approaches. Polymeric vectors offer an attractive alternative to viruses as delivery vectors due to their large packaging capacity and safety profile. In this paper, we have demonstrated the potential use of a highly branched poly(beta-amino ester) polymer, HPAE-EB, to enable genomic editing via CRISPRCas9-targeted genomic excision of exon 80 in the COL7A1 gene, through a dual-guide RNA sequence system. The biophysical properties of HPAE-EB were screened in a human embryonic 293 cell line (HEK293), to elucidate optimal conditions for efficient and cytocompatible delivery of a DNA construct encoding Cas9 along with two RNA guides, obtaining 15-20% target genomic excision. When translated to human recessive dystrophic epidermolysis bullosa (RDEB) keratinocytes, transfection efficiency and targeted genomic excision dropped. However, upon delivery of CRISPR-Cas9 as a ribonucleoprotein complex, targeted genomic deletion of exon 80 was increased to over 40%. Our study provides renewed perspective for the further development of polymer delivery systems for application in the gene editing field in general, and specifically for the treatment of RDEB.
  • Publication
    Evaluating Interaction of Cord Blood Hematopoietic Stem/Progenitor Cells with Functionally Integrated Three-Dimensional Microenvironments
    (Oxford University Press, 2018-03-01) Mokhtari, Saloomeh; Almeida De Matos Baptista, Pedro Miguel; Lamar, Zanneta; Vyas, Dipen A.; Freeman, Charles Jordan; Moran, Emma; Brovold, Matthew; Llamazares, Guillermo A.; Porada, Christopher D.; Soker, Shay; Almeida Porada, Graca
    Despite advances in ex vivo expansion of cord blood-derived hematopoietic stem/progenitor cells (CB-HSPC), challenges still remain regarding the ability to obtain, from a single unit, sufficient numbers of cells to treat an adolescent or adult patient. We and others have shown that CB-HSPC can be expanded ex vivo in two-dimensional (2D) cultures, but the absolute percentage of the more primitive stem cells decreases with time. During development, the fetal liver is the main site of HSPC expansion. Therefore, here we investigated, in vitro, the outcome of interactions of primitive HSPC with surrogate fetal liver environments. We compared bioengineered liver constructs made from a natural three-dimensional-liver-extracellular-matrix (3D-ECM) seeded with hepatoblasts, fetal liver-derived (LvSt), or bone marrow-derived stromal cells, to their respective 2D culture counterparts. We showed that the inclusion of cellular components within the 3D-ECM scaffolds was necessary for maintenance of HSPC viability in culture, and that irrespective of the microenvironment used, the 3D-ECM structures led to the maintenance of a more primitive subpopulation of HSPC, as determined by flow cytometry and colony forming assays. In addition, we showed that the timing and extent of expansion depends upon the biological component used, with LvSt providing the optimal balance between preservation of primitive CB HSPC and cellular differentiation.
  • Publication
    A persulfidation-based mechanism controls aquaporin-8 conductance
    (American Association for the Advancement of Science, 2018-05-02) Bestetti, Stefano; Medraño Fernandez, Iria; Galli, Mauro; Ghitti, Michela; Bienert, Gerd P.; Musco, Giovanna; Orsi, Andrea; Rubartelli, Anna; Sitia, Roberto
    Upon engagement of tyrosine kinase receptors, nicotinamide adenine dinucleotide phosphate (NADPH)-oxidases release H2O2 in the extracellular space. We reported previously that aquaporin-8 (AQP8) transports H2O2 across the plasma membrane and is reversibly gated during cell stress, modulating signal strength and duration. We show that AQP8 gating is mediated by persulfidation of cysteine 53 (C53). Treatment with H2S is sufficient to block H2O2 entry in unstressed cells. Silencing cystathionine beta-synthase (CBS) prevents closure, suggesting that this enzyme is the main source of H2S. Molecular modeling indicates that C53 persulfidation displaces a nearby histidine located in the narrowest part of the channel. We propose that H2O2 molecules transported through AQP8 sulfenylate C53, making it susceptible to H2S produced by CBS. This mechanism tunes H2O2 transport and may control signaling and limit oxidative stress.
  • Publication
    Gluten-sensitive enteropathy in recessive dystrophic epidermolysis bullosa
    (Oxford Academic, 2023-12) Sacedón, Rosa; Arriba, María del Carmen de; Martínez Santamaría, Lucia; Maseda, Rocío; Herráiz Gil, Sara; Jiménez, Eva; Rosales, Isabel; Quintana, Lucia; Illera, Nuria; García Díez, Marta; Butta, Nora; Fernández-Bello, Ihosvany; Lwin, Su M.; Fernández-Arquero, Miguel; León Canseco, Carlos; Mcgrath, John A.; Vicente, M. Angeles; Río Nechaevsky, Marcela del; Lucas, Raúl de; Sánchez-Ramón, Silvia; Escámez Toledano, María José; Ministerio de Ciencia e Innovación (España); Universidad Carlos III de Madrid
  • Publication
    Protein kinase C zeta Interacts with a novel binding region of Galfaq to act as a functional effector
    (Elsevier, 2016-04-29) Sánchez Fernández, Guzmán; Cabezudo, Sofia; Caballero, Álvaro; García Hoz, Carlota; Tall, Gregory G.; Klett Arroyo, Javier; Michnick, Stephen W.; Mayor Jr., Federico; Ribas, Catalina; Comunidad de Madrid; Ministerio de Educación y Ciencia (España)
    Heterotrimeric G proteins play an essential role in the initiation of G protein-coupled receptor (GPCR) signaling through specific interactions with a variety of cellular effectors. We have recently reported that GPCR activation promotes a direct interaction between Galfaq and protein kinase C zeta (PKCzeta), leading to the stimulation of the ERK5 pathway independent of the canonical effector PLCbeta. We report herein that the activation-dependent Galfaq/PKCzeta complex involves the basic PB1-type II domain of PKCzeta and a novel interaction module in Galfaq different from the classical effector-binding site. Point mutations in this Galfaq region completely abrogate ERK5 phosphorylation, indicating that Galfaq/PKCzeta association is required for the activation of the pathway. Indeed, PKCzeta was demonstrated to directly bind ERK5 thus acting as a scaffold between Galfaq and ERK5 upon GPCR activation. The inhibition of these protein complexes by G protein-coupled receptor kinase 2, a known Galfaq modulator, led to a complete abrogation of ERK5 stimulation. Finally, we reveal that Galfaq/PKCzeta complexes link Galfaq to apoptotic cell death pathways. Our data suggest that the interaction between this novel region in Galfaq and the effector PKCzeta is a key event in Galfaq signaling.
  • Publication
    The transition from linear to highly branched poly(beta-amino ester)s: Branching matters for gene delivery
    (American Association for the Advancement of Science, 2016-06-17) Zhou, Dezhong; Greiser, U.; Cutlar, L.; Gao, Y.; Wang, W.; O'Keeffe Ahern, Jonathan; McMahon, S.; Duarte Gonzalez, Blanca; Larcher Laguzzi, Fernando; Rodriguez, B.J.; Wang, Wenxin
    Nonviral gene therapy holds great promise but has not delivered treatments for clinical application to date. Lack of safe and efficient gene delivery vectors is the major hurdle. Among nonviral gene delivery vectors, poly(beta-amino ester)s are one of the most versatile candidates because of their wide monomer availability, high polymer flexibility, and superior gene transfection performance both in vitro and in vivo. However, to date, all research has been focused on vectors with a linear structure. A well-accepted view is that dendritic or branched polymers have greater potential as gene delivery vectors because of their three-dimensional structure and multiple terminal groups. Nevertheless, to date, the synthesis of dendritic or branched polymers has been proven to be a well-known challenge. We report the design and synthesis of highly branched poly(beta-amino ester)s (HPAEs) via a one-pot "A2 + B3 + C2"&-type Michael addition approach and evaluate their potential as gene delivery vectors. We find that the branched structure can significantly enhance the transfection efficiency of poly(beta-amino ester)s: Up to an 8521-fold enhancement in transfection efficiency was observed across 12 cell types ranging from cell lines, primary cells, to stem cells, over their corresponding linear poly(beta-amino ester)s (LPAEs) and the commercial transfection reagents polyethyleneimine, SuperFect, and Lipofectamine 2000...
  • Publication
    Gene editing for the efficient correction of a recurrent COL7A1 mutation in recessive dystrophic epidermolysis bullosa keratinocytes
    (Elsevier, 2016-01) Chamorro, Cristina; Mencía Rodríguez, Ángeles; Almarza, David; Duarte, Blanca; Büning, Hildegard; Sallach, Jessica; Hausser, Ingrid; Río Nechaevsky, Marcela del; Larcher Laguzzi, Fernando; Murillas, Rodolfo; Comunidad de Madrid; Ministerio de Economía y Competitividad (España)
    Clonal gene therapy protocols based on the precise manipulation of epidermal stem cells require highly efficient gene-editing molecular tools. We have combined adeno-associated virus (AAV)-mediated delivery of donor template DNA with transcription activator-like nucleases (TALE) expressed by adenoviral vectors to address the correction of the c.6527insC mutation in the COL7A1 gene, causing recessive dystrophic epidermolysis bullosa in a high percentage of Spanish patients. After transduction with these viral vectors, high frequencies of homology-directed repair were found in clones of keratinocytes derived from a recessive dystrophic epidermolysis bullosa (RDEB) patient homozygous for the c.6527insC mutation. Gene-edited clones recovered the expression of the COL7A1 transcript and collagen VII protein at physiological levels. In addition, treatment of patient keratinocytes with TALE nucleases in the absence of a donor template DNA resulted in nonhomologous end joining (NHEJ)-mediated indel generation in the vicinity of the c.6527insC mutation site in a large proportion of keratinocyte clones. A subset of these indels restored the reading frame of COL7A1 and resulted in abundant, supraphysiological expression levels of mutant or truncated collagen VII protein. Keratinocyte clones corrected both by homology-directed repair (HDR) or NHEJ were used to regenerate skin displaying collagen VII in the dermo-epidermal junction.
  • Publication
    Deregulated FADD expression and phosphorylation in T-cell lymphoblastic lymphoma
    (Impact Journals, 2016-08-18) Marín Rubio, José L.; Arriba Pérez, María del Carmen de; Cobos Fernández, María A.; González Sánchez, Laura; Ors, Inmaculada; Sastre, Isabel; Fernández Piqueras, José; Villa Morales, María; Comunidad de Madrid; Ministerio de Economía y Competitividad (España); Ministerio de Educación, Cultura y Deporte (España)
    In the present work, we show that T-cell lymphoblastic lymphoma cells exhibit a reduction of FADD availability in the cytoplasm, which may contribute to impaired apoptosis. In addition, we observe a reduction of FADD phosphorylation that inversely correlates with the proliferation capacity and tumor aggressiveness. The resultant balance between FADD-dependent apoptotic and non-apoptotic abilities may define the outcome of the tumor. Thus, we propose that FADD expression and phosphorylation can be reliable biomarkers with prognostic value for T-LBL stratification.
  • Publication
    Technological advances in fibrin for tissue engineering
    (SAGE, 2023-08-14) Sanz Horta, Raúl; Matesanz Fernandez-Arias, Ana; Gallardo, Alberto; Reinecke, Helmut; Jorcano Noval, José Luis; Acedo Gallardo, Pablo; Velasco Bayón, Diego; Elvira, Carlos; Comunidad de Madrid; Ministerio de Economía y Competitividad (España)
    Fibrin is a promising natural polymer that is widely used for diverse applications, such as hemostatic glue, carrier for drug and cell delivery, and matrix for tissue engineering. Despite the significant advances in the use of fibrin for bioengineering and biomedical applications, some of its characteristics must be improved for suitability for general use. For example, fibrin hydrogels tend to shrink and degrade quickly after polymerization, particularly when they contain embedded cells. In addition, their poor mechanical properties and batch-to-batch variability affect their handling, long-term stability, standardization, and reliability. One of the most widely used approaches to improve their properties has been modification of the structure and composition of fibrin hydrogels. In this review, recent advances in composite fibrin scaffolds, chemically modified fibrin hydrogels, interpenetrated polymer network (IPN) hydrogels composed of fibrin and other synthetic or natural polymers are critically reviewed, focusing on their use for tissue engineering.
  • Publication
    The adipokine lipocalin-2 in the context of the osteoarthritic osteochondral junction
    (Nature Research, 2016-07-07) Villalvilla, Amanda; García Martín, Adela María; Largo, Raquel; Guaillo, Oreste; Herrero Beaumont, Gabriel; Gomez, Rodolfo
    Obesity and osteoarthritis (OA) form a vicious circle in which obesity contributes to cartilage destruction in OA, and OA-associated sedentary behaviour promotes weight gain. Lipocalin-2 (LCN2), a novel adipokine with catabolic activities in OA joints, contributes to the obesity and OA pathologies and is associated with other OA risk factors. LCN2 is highly induced in osteoblasts in the absence of mechanical loading, but its role in osteoblast metabolism is unclear. Therefore, because osteochondral junctions play a major role in OA development, we investigated the expression and role of LCN2 in osteoblasts and chondrocytes in the OA osteochondral junction environment. Our results showed that LCN2 expression in human osteoblasts and chondrocytes decreased throughout osteoblast differentiation and was induced by catabolic and inflammatory factors; however, TGF-beta 1 and IGF-1 reversed this induction. LCN2 reduced osteoblast viability in the presence of iron and enhanced the activity of MMP-9 released by osteoblasts. Moreover, pre-stimulated human osteoblasts induced LCN2 expression in human chondrocytes, but the inverse was not observed. Thus, LCN2 is an important catabolic adipokine in osteoblast and chondrocyte metabolism that is regulated by differentiation, inflammation and catabolic and anabolic stimuli, and LCN2 expression in chondrocytes is regulated in a paracrine manner after osteoblast stimulation.
  • Publication
    Effects of photodynamic therapy on dermal fibroblasts from xeroderma pigmentosum and Gorlin-Goltz syndrome patients
    (Impact Journals, LLC, 2017-09-29) Zamarrón, Alicia; García Díez, Marta; Rio Nechaevsky, Marcela Andrea del; Larcher Laguzzi, Fernando; Juarranz, Angeles; Comunidad de Madrid; Ministerio de Economía y Competitividad (España)
    PDT is widely applied for the treatment of non-melanoma skin cancer premalignant and malignant lesions (actinic keratosis, basal cell carcinoma and in situ squamous cell carcinoma). In photodynamic therapy (PDT) the interaction of a photosensitizer (PS), light and oxygen leads to the formation of reactive oxygen species (ROS) and thus the selective tumor cells eradication. Xeroderma pigmentosum (XP) and Gorlin-Goltz Syndrome (GS) patients are at high risk of developing skin cancer in sun-exposed areas. Therefore, the use of PDT as a preventive treatment may constitute a very promising therapeutic modality for these syndromes. Given the demonstrated role of cancer associated fibroblasts (CAFs) in tumor progression and the putative CAFs features of some cancer-prone genodermatoses fibroblasts, in this study, we have further characterized the phenotype of XP and GS dermal fibroblasts and evaluated their response to methyl-d-aminolevulinic acid (MAL)-PDT compared to that of dermal fibroblasts obtained from healthy donors. We show here that XP/GS fibroblasts display clear features of CAFs and present a significantly higher response to PDT, even after being stimulated with UV light, underscoring the value of this therapeutic approach for these rare skin conditions and likely to other forms of skin cancer were CAFs play a major role.
  • Publication
    Genomic expression differences between cutaneous cells from red hair color individuals and black hair color individuals based on bioinformatic analysis
    (Impact Journals, 2017) Puig-Butille, Joan Anton; Giménez Xavier, Pol; Visconti, Alessia; Nsengimana, Jeremie; Garcia Garcia, Francisco; Tell Marti, Gemma; Escámez Toledano, María José; Newton-Bishop, Julia; Bataile, Veronique; Río Nechaevsky, Marcela del; Dopazo, Joaquin; Falchi, Mario; Puig, Susana; Comunidad de Madrid; European Commission; Ministerio de Economía y Competitividad (España)
    The MC1R gene plays a crucial role in pigmentation synthesis. Loss-of-function MC1R variants, which impair protein function, are associated with red hair color (RHC) phenotype and increased skin cancer risk. Cultured cutaneous cells bearing loss-of-function MC1R variants show a distinct gene expression profile compared to wild-type MC1R cultured cutaneous cells. We analysed the gene signature associated with RHC co-cultured melanocytes and keratinocytes by Protein-Protein interaction (PPI) network analysis to identify genes related with non-functional MC1R variants. From two detected networks, we selected 23 nodes as hub genes based on topological parameters. Differential expression of hub genes was then evaluated in healthy skin biopsies from RHC and black hair color (BHC) individuals. We also compared gene expression in melanoma tumors from individuals with RHC versus BHC. Gene expression in normal skin from RHC cutaneous cells showed dysregulation in 8 out of 23 hub genes (CLN3, ATG10, WIPI2, SNX2, GABARAPL2, YWHA, PCNA and GBAS). Hub genes did not differ between melanoma tumors in RHC versus BHC individuals. The study suggests that healthy skin cells from RHC individuals present a constitutive genomic deregulation associated with the red hair phenotype and identify novel genes involved in melanocyte biology.
  • Publication
    Antibiotic capture by bacterial lipocalins uncovers an extracellular mechanism of intrinsic antibiotic resistance
    (American Society for Microbiology, 2017-05-03) El-Halfawy, Omar M.; Klett Arroyo, Javier; Ingram, Rebecca J.; Loutet, Slade A.; Murphy, Michael E. P.; Martin Santamaría, Sonsoles; Valvano, Miguel A.; European Commission; Ministerio de Economía y Competitividad (España)
    The potential for microbes to overcome antibiotics of different classes before they reach bacterial cells is largely unexplored. Here we show that a soluble bacterial lipocalin produced by Burkholderia cenocepacia upon exposure to sublethal antibiotic concentrations increases resistance to diverse antibiotics in vitro and in vivo. These phenotypes were recapitulated by heterologous expression in B. cenocepacia of lipocalin genes from Pseudomonas aeruginosa, Mycobacterium tuberculosis, and methicillin-resistant Staphylococcus aureus. Purified lipocalin bound different classes of bactericidal antibiotics and contributed to bacterial survival in vivo. Experimental and X-ray crystal structure-guided computational studies revealed that lipocalins counteract antibiotic action by capturing antibiotics in the extracellular space. We also demonstrated that fat-soluble vitamins prevent antibiotic capture by binding bacterial lipocalin with higher affinity than antibiotics. Therefore, bacterial lipocalins contribute to antimicrobial resistance by capturing diverse antibiotics in the extracellular space at the site of infection, which can be counteracted by known vitamins.
  • Publication
    Classification of skin phenotypes caused by diabetes mellitus using complex scattering parameters in the millimeter-wave frequency range
    (Scientific Reports, 2017-07-19) Dornuf, Fabian; Martín Mateos, Pedro; Duarte, Blanca; Hils, Bernhard; Bonilla Manrique, Óscar Elías; Larcher Laguzzi, Fernando; Acedo Gallardo, Pablo; Krozer, Viktor
    The pathological skin phenotype caused by hyperglycemia is an important indicator for the progress of diabetes mellitus. An early detection of diabetes assures an early intervention to regulate the carbohydrate metabolism. In this publication a non-invasive detection principle based on the measurement of complex scattering parameters in the millimeter-wave frequency range is presented. The measurement principle provides evidence of the applicability for the identification of different glycemic states in animal models. The method proposed here can be used to predict diabetes status in animal models and is interesting for application on humans in view of safeness of millimeter-wave radiation. Furthermore the complex scattering parameters give important information about the anatomic varieties between the analyzed skin samples of the different mice strains. In contrast to other methods, our approach is less sensitive to skin variations between animals.
  • Publication
    Assessment of the risk and characterization of non-melanoma skin cancer in Kindler syndrome: study of a series of 91 patients
    (BMC, 2019-07-24) Guerrero Aspizua, Sara; Conti, Claudio Jorge; Escámez Toledano, María José; Castiglia, Daniele; Zambruno, Giovanna; Youssefian, Leila; Vahidenezhad, Hassan; Requena, Luis; Hin, Peter; Tadini, Gianluca; Yordanova, Ivelina; Martin, Ludovic; Uitto, Jouni; Has, Cristina; Río Nechaevsky, Marcela del; Comunidad de Madrid; European Commission; Ministerio de Economía y Competitividad (España)
    Background: Kindler Syndrome (KS) is a rare genodermatosis characterized by skin fragility, skin atrophy, premature aging and poikiloderma. It is caused by mutations in the FERMT1 gene, which encodes kindlin-1, a protein involved in integrin signalling and the formation of focal adhesions. Several reports have shown the presence of non-melanoma skin cancers in KS patients but a systematic study evaluating the risk of these tumors at different ages and their potential outcome has not yet been published. We have here addressed this condition in a retrospective study of 91 adult KS patients, characterizing frequency, metastatic potential and body distribution of squamous cell carcinoma (SCC) in these patients. SCC developed in 13 of the 91 patients. Results: The youngest case arose in a 29-year-old patient; however, the cumulative risk of SCC increased to 66.7% in patients over 60 years of age. The highly aggressive nature of SCCs in KS was confirmed showing that 53.8% of the patients bearing SCCs develop metastatic disease. Our data also showed there are no specific mutations that correlate directly with the development of SCC; however, the mutational distribution along the gene appears to be different in patients bearing SCC from SCC-free patients. The body distribution of the tumor appearance was also unique and different from other bullous diseases, being concentrated in the hands and around the oral cavity, which are areas of high inflammation in this disease. Conclusions: This study characterizes SCCs in the largest series of KS patients reported so far, showing the high frequency and aggressiveness of these tumors. It also describes their particular body distribution and their relationship with mutations in the FERMT-1 gene. These data reinforce the need for close monitoring of premalignant or malignant lesions in KS patients.
  • Publication
    Safety and early efficacy outcomes for lentiviral fibroblast gene therapy in recessive dystrophic epidermolysis bullosa
    (American Society for Clinical Investigation, 2019-01-01) Liu, Li; Guy, Alyson; Petrova, Anastasia; Abdul-Wahab, Alya; Reid, Fiona; Phillips, Rachel; Elstad, Maria; Georgiadis, Christos; Aristodemou, Sophia; Lovell, Patricia A.; Mcmillan, James R.; Mee, John; Martínez-Queipo, Magdalena; Rashidghamat, Ellie; Tziotzios, Christos; Onoufriadis, Alexandros; Chen, Mei; Chan, Lucas; Rarzaneh, Farzin; Miskinyte, Snaigune; Titeux, Matthias; Ozoemena, Linda; Pramanik, Rashida; Serrano, Sonia; Rowles, Racheal; Maurin, Clarisse; Orrin, Elizabeth; Rio Nechaevsky, Marcela Andrea Del; Tolar, Jakub; Bauer, Johann W.; Larcher Laguzzi, Fernando; Antoniou, Michael N.; Hovnanian, Alain; Thrasher, Adrian J.; Mellerio, Jemina E.; Qasim, Waseem; Mcgrath, John A:
    BACKGROUND. Recessive dystrophic epidermolysis bullosa (RDEB) is a severe form of skin fragility disorder due to mutations in COL7A1 encoding basement membrane type VII collagen (C7), the main constituent of anchoring fibrils (AFs) in skin. We developed a self-inactivating lentiviral platform encoding a codon-optimized COL7A1 cDNA under the control of a human phosphoglycerate kinase promoter for phase I evaluation. METHODS. In this single-center, open-label phase I trial, 4 adults with RDEB each received 3 intradermal injections (~1 × 106 cells/cm2 of intact skin) of COL7A1-modified autologous fibroblasts and were followed up for 12 months. The primary outcome was safety, including autoimmune reactions against recombinant C7. Secondary outcomes included C7 expression, AF morphology, and presence of transgene in the injected skin. RESULTS. Gene-modified fibroblasts were well tolerated, without serious adverse reactions or autoimmune reactions against recombinant C7. Regarding efficacy, there was a significant (P < 0.05) 1.26-fold to 26.10-fold increase in C7 mean fluorescence intensity in the injected skin compared with noninjected skin in 3 of 4 subjects, with a sustained increase up to 12 months in 2 of 4 subjects. The presence of transgene (codon-optimized COL7A1 cDNA) was demonstrated in the injected skin at month 12 in 1 subject, but no new mature AFs were detected. CONCLUSION. To our knowledge, this is the first human study demonstrating safety and potential efficacy of lentiviral fibroblast gene therapy with the presence of COL7A1 transgene and subsequent C7 restoration in vivo in treated skin at 1 year after gene therapy. These data provide a rationale for phase II studies for further clinical evaluation.
  • Publication
    Human aquaporin-11 guarantees efficient transport of H2O2 across the endoplasmic reticulum membrane
    (Elsevier, 2020-01) Bestetti, Stefano; Galli, Mauro; Sorrentino, Ilaria; Pinton, Paolo; Rimessi, Alessandro; Sitia, Roberto; Medraño Fernandez, Iria
    Hydrogen peroxide (H2O2) is an essential second intracellular messenger. To reach its targets in the cytosol, H2O2 must cross a membrane, a feat that requires aquaporins (AQP) endowed with 'peroxiporin' activity (AQP3, AQP8, AQP9). Here, we exploit different organelle-targeted H2O2-sensitive probes to show that also AQP11 efficiently conduits H2O2. Unlike other peroxiporins, AQP11 is localized in the endoplasmic reticulum (ER), accumulating partly in mitochondrial-associated ER membranes (MAM). Its downregulation severely perturbs the flux of H2O2 through the ER, but not through the mitochondrial or plasma membranes. These properties make AQP11 a potential regulator of ER redox homeostasis and signaling.
  • Publication
    Large-scale production of LGR5-positive bipotential human liver stem cells
    (The American Association for the Study of Liver Diseases, 2020-07) Schneeberger, Kerstin; Sanchez Romero, Natalia; Ye, Shicheng; Van Steenbeek, Frank G.; Oosterhoff, Loes A.; Pla Palacin, Iris; Chen, Chen; Van Wolferen, Monique E.; Van Tienderen, Gilles; Lieshout, Ruby; Colemonts Vroninks, Haaike; Schene, Imre; Hoekstra, Ruurdtje; Verstegen, Monique M. A.; Van Der Laan, Luc J. W.; Penning, Louis C.; Fuchs, Sabine A.; Clevers, Hans; De Kock, Joery; Baptista, Pedro Miguel; Spee, Bart; European Commission
    Background and Aims: The gap between patients on transplant waiting lists and available donor organs is steadily increasing. Human organoids derived from leucine‐rich repeat‐containing G protein‐coupled receptor 5 (LGR5)–positive adult stem cells represent an exciting new cell source for liver regeneration; however, culturing large numbers of organoids with current protocols is tedious and the level of hepatic differentiation is limited. Approach and Results: Here, we established a method for the expansion of large quantities of human liver organoids in spinner flasks. Due to improved oxygenation in the spinner flasks, organoids rapidly proliferated and reached an average 40‐fold cell expansion after 2 weeks, compared with 6‐fold expansion in static cultures. The organoids repopulated decellularized liver discs and formed liver‐like tissue. After differentiation in spinner flasks, mature hepatocyte markers were highly up‐regulated compared with static organoid cultures, and cytochrome p450 activity reached levels equivalent to hepatocytes. Conclusions: We established a highly efficient method for culturing large numbers of LGR5‐positive stem cells in the form of organoids, which paves the way for the application of organoids for tissue engineering and liver transplantation.
  • Publication
    Efficient CRISPR-Cas9-mediated gene ablation in human keratinocytes to recapitulate genodermatoses: modeling of Netherton syndrome
    (Elsevier, 2020-09-11) Gálvez, Victoria; Chacón Solano, Esteban; Bonafont Aragó, José; Mencía Rodríguez, Ángeles; Di, Wei-Li; Murillas, Rodolfo; Llames, Sara; Vicente, Asunción; Río Nechaevsky, Marcela del; Carretero, Marta; Larcher Laguzzi, Fernando; Comunidad de Madrid; Ministerio de Economía y Competitividad (España)
    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.
  • Publication
    Beneficial effect of systemic allogeneic adipose derived mesenchymal cells on the clinical, inflammatory and immunologic status of a patient with recessive dystrophic epidermolysis bullosa: A case report
    (Frontiers, 2020-11) Maseda, Rocio; Martínez Santamaría, Lucia; Sacedon, Rosa; Butta, Nora; Arriba Pérez, María del Carmen de; García Barcenilla, Sara; García Díez, Marta; Illera Esteban, Nuria; Pérez Conde, Isabel; Carretero Trillo, Marta; Jiménez, Eva; Melen, Gustavo; Borobia, Alberto M.; Jiménez Yuste, Víctor; Vicente, Ángeles; Río Nechaevsky, Marcela del; de Lucas Laguna, Raúl; Escámez Toledano, María José; Comunidad de Madrid; Ministerio de Economía y Competitividad (España)
    Recessive dystrophic epidermolysis bullosa (RDEB) is an incurable inherited mucocutaneous fragility disorder characterized by recurrent blisters, erosions, and wounds. Continuous blistering triggers overlapping cycles of never-ending healing and scarring commonly evolving to chronic systemic inflammation and fibrosis. The systemic treatment with allogeneic mesenchymal cells (MSC) from bone marrow has previously shown benefits in RDEB. MSC from adipose tissue (ADMSC) are easier to isolate. This is the first report on the use of systemic allogeneic ADMSC, correlating the clinical, inflammatory, and immunologic outcomes in RDEB indicating long-lasting benefits. We present the case of an RDEB patient harboring heterozygous biallelic COL7A1 gene mutations and with a diminished expression of C7. The patient presented with long-lasting refractory and painful oral ulcers distressing her quality of life. Histamine receptor antagonists, opioid analgesics, proton-pump inhibitors, and low-dose tricyclic antidepressants barely improved gastric symptoms, pain, and pruritus. Concomitantly, allogeneic ADMSC were provided as three separate intravenous injections of 106 cells/kg every 21 days. ADMSC treatment was well-tolerated. Improvements in wound healing, itch, pain and quality of life were observed, maximally at 6-9 months post-treatment, with the relief of symptoms still noticeable for up to 2 years. Remarkably, significant modifications in PBL participating in both the innate and adaptive responses, alongside regulation of levels of profibrotic factors, MCP-1/CCL2 and TGF-beta, correlated with the health improvement. This treatment might represent an alternative for non-responding patients to conventional management. It seems critical to elucidate the paracrine modulation of the immune system by MSC for their rational use in regenerative/immunoregulatory therapies.