278 results on '"Juan A. Bueren"'
Search Results
2. Preclinical safety and efficacy of lentiviral-mediated gene therapy for leukocyte adhesion deficiency type I
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Cristina Mesa-Núñez, Carlos Damián, María Fernández-García, Begoña Díez, Gayatri Rao, Jonathan D. Schwartz, Ken M. Law, Julián Sevilla, Paula Río, Rosa Yáñez, Juan A. Bueren, and Elena Almarza
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leukocyte adhesion deficiency type I ,LAD-I ,CD18 ,β2-integrins ,primary immunodeficiencies ,gene therapy ,Genetics ,QH426-470 ,Cytology ,QH573-671 - Abstract
Leukocyte adhesion deficiency type I (LAD-I) is a primary immunodeficiency caused by mutations in the ITGB2 gene, which encodes for the CD18 subunit of β2-integrins. Deficient expression of β2-integrins results in impaired neutrophil migration in response to bacterial and fungal infections. Using a lentiviral vector (LV) that mediates a preferential myeloid expression of human CD18 (Chim.hCD18-LV), we first demonstrated that gene therapy efficiently corrected the phenotype of mice with severe LAD-I. Next, we investigated if the ectopic hCD18 expression modified the phenotypic characteristics of human healthy donor hematopoietic stem cells and their progeny. Significantly, transduction of healthy CD34+ cells with the Chim.hCD18-LV did not modify the membrane expression of CD18 nor the adhesion of physiological ligands to transduced cells. Additionally, we observed that the repopulating properties of healthy CD34+ cells were preserved following transduction with the Chim.hCD18-LV, and that a safe polyclonal repopulation pattern was observed in transplanted immunodeficient NOD scid gamma (NSG) mice. In a final set of experiments, we demonstrated that transduction of CD34+ cells from a severe LAD-I patient with the Chim.hCD18-LV restores the expression of β2-integrins in these cells. These results offer additional preclinical safety and efficacy evidence supporting the gene therapy of patients with severe LAD-I.
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- 2022
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3. Gene therapy restores the transcriptional program of hematopoietic stem cells in Fanconi anemia
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Miren Lasaga, Paula Río, Amaia Vilas-Zornoza, Nuria Planell, Susana Navarro, Diego Alignani, Beatriz Fernández-Varas, Daniel Mouzo, Josune Zubicaray, Roser M. Pujol, Eileen Nicoletti, Jonathan D. Schwartz, Julián Sevilla, Marina Ainciburi, Asier Ullate-Agote, Jordi Surrallés, Rosario Perona, Leandro Sastre, Felipe Prosper, David Gomez-Cabrero, and Juan A. Bueren
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Diseases of the blood and blood-forming organs ,RC633-647.5 - Abstract
Clinical trials have shown that lentiviral-mediated gene therapy can ameliorate bone marrow failure (BMF) in nonconditioned Fanconi anemia (FA) patients resulting from the proliferative advantage of corrected FA hematopoietic stem and progenitor cells (HSPC). However, it is not yet known if gene therapy can revert affected molecular pathways in diseased HSPC. Single-cell RNA sequencing was performed in chimeric populations of corrected and uncorrected HSPC co-existing in the BM of gene therapy-treated FA patients. Our study demonstrates that gene therapy reverts the transcriptional signature of FA HSPC, which then resemble the transcriptional program of healthy donor HSPC. This includes a down-regulated expression of TGF-β and p21, typically up-regulated in FA HSPC, and upregulation of DNA damage response and telomere maintenance pathways. Our results show for the first time the potential of gene therapy to rescue defects in the HSPC transcriptional program from patients with inherited diseases; in this case, in FA characterized by BMF and cancer predisposition.
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- 2023
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4. Specific correction of pyruvate kinase deficiency-causing point mutations by CRISPR/Cas9 and single-stranded oligodeoxynucleotides
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Sara Fañanas-Baquero, Matías Morín, Sergio Fernández, Isabel Ojeda-Perez, Mercedes Dessy-Rodriguez, Miruna Giurgiu, Juan A. Bueren, Miguel Angel Moreno-Pelayo, Jose Carlos Segovia, and Oscar Quintana-Bustamante
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pyruvate kinase deficiency ,point mutation ,ssODN ,CRISPR/Cas9 ,precise gene editing ,allele specificity ,Biotechnology ,TP248.13-248.65 ,Genetics ,QH426-470 - Abstract
Pyruvate kinase deficiency (PKD) is an autosomal recessive disorder caused by mutations in the PKLR gene. PKD-erythroid cells suffer from an energy imbalance caused by a reduction of erythroid pyruvate kinase (RPK) enzyme activity. PKD is associated with reticulocytosis, splenomegaly and iron overload, and may be life-threatening in severely affected patients. More than 300 disease-causing mutations have been identified as causing PKD. Most mutations are missense mutations, commonly present as compound heterozygous. Therefore, specific correction of these point mutations might be a promising therapy for the treatment of PKD patients. We have explored the potential of precise gene editing for the correction of different PKD-causing mutations, using a combination of single-stranded oligodeoxynucleotides (ssODN) with the CRISPR/Cas9 system. We have designed guide RNAs (gRNAs) and single-strand donor templates to target four different PKD-causing mutations in immortalized patient-derived lymphoblastic cell lines, and we have detected the precise correction in three of these mutations. The frequency of the precise gene editing is variable, while the presence of additional insertions/deletions (InDels) has also been detected. Significantly, we have identified high mutation-specificity for two of the PKD-causing mutations. Our results demonstrate the feasibility of a highly personalized gene-editing therapy to treat point mutations in cells derived from PKD patients.
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- 2023
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5. Improved efficacy of mesenchymal stromal cells stably expressing CXCR4 and IL-10 in a xenogeneic graft versus host disease mouse model
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Rosario Hervás-Salcedo, María Fernández-García, Miriam Hernando-Rodríguez, Cristian Suárez-Cabrera, Juan A. Bueren, and Rosa M. Yáñez
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mesenchymal stromal cells ,graft vs host disease ,CXCR4 ,IL10 ,stem cell therapy ,immunomodulation ,Immunologic diseases. Allergy ,RC581-607 - Abstract
Previous clinical trials have shown that mesenchymal stromal cells (MSCs) can modulate graft versus host disease (GvHD) after allogeneic hematopoietic transplantation, although with variable efficacy. To improve the anti-GvHD effect of these cells, adipose tissue derived-human MSCs (Ad-MSCs) were transduced with a lentiviral vector conferring stable expression of CXCR4, a molecule involved in cell migration to inflamed sites, and IL-10, a cytokine with potent anti-inflammatory properties. In vitro experiments showed that the expression of these molecules in Ad-MSCs (named CXCR4-IL10-MSCs) efficiently enhanced their migration towards SDF-1α and also improved their immunomodulatory properties compared to unmodified Ad-MSCs (WT-MSCs). Moreover, using a humanized GvHD mouse model, CXCR4-IL10-MSCs showed improved therapeutic effects, which were confirmed by histopathologic analysis in the target organs. Additionally, compared to WT-MSCs, CXCR4-IL10-MSCs induced a more marked reduction in the number of pro-inflammatory Th1 and Th17 cells, a higher polarization towards an anti-inflammatory T cell profile (CD3+-IL10+ cells), and increased the number of regulatory T and B cells. Our in vitro and in vivo studies strongly suggest that CXCR4-IL10-MSCs should constitute an important new generation of MSCs for the treatment of GvHD in patients transplanted with allogeneic hematopoietic grafts.
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- 2023
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6. Clinically relevant gene editing in hematopoietic stem cells for the treatment of pyruvate kinase deficiency
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Sara Fañanas-Baquero, Oscar Quintana-Bustamante, Daniel P. Dever, Omaira Alberquilla, Rebeca Sanchez-Dominguez, Joab Camarena, Isabel Ojeda-Perez, Mercedes Dessy-Rodriguez, Rolf Turk, Mollie S. Schubert, Annalisa Lattanzi, Liwen Xu, Jose L. Lopez-Lorenzo, Paola Bianchi, Juan A. Bueren, Mark A. Behlke, Matthew Porteus, and Jose-Carlos Segovia
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hematopoietic stem cell ,gene editing ,hemolytic anemia ,pyruvate kinase deficiency ,gene therapy ,CRISPR-Cas9 ,Genetics ,QH426-470 ,Cytology ,QH573-671 - Abstract
Pyruvate kinase deficiency (PKD), an autosomal-recessive disorder, is the main cause of chronic non-spherocytic hemolytic anemia. PKD is caused by mutations in the pyruvate kinase, liver and red blood cell (PKLR) gene, which encodes for the erythroid pyruvate kinase protein (RPK). RPK is implicated in the last step of anaerobic glycolysis in red blood cells (RBCs), responsible for the maintenance of normal erythrocyte ATP levels. The only curative treatment for PKD is allogeneic hematopoietic stem and progenitor cell (HSPC) transplant, associated with a significant morbidity and mortality, especially relevant in PKD patients. Here, we address the correction of PKD through precise gene editing at the PKLR endogenous locus to keep the tight regulation of RPK enzyme during erythropoiesis. We combined CRISPR-Cas9 system and donor recombinant adeno-associated vector (rAAV) delivery to build an efficient, safe, and clinically applicable system to knock in therapeutic sequences at the translation start site of the RPK isoform in human hematopoietic progenitors. Edited human hematopoietic progenitors efficiently reconstituted human hematopoiesis in primary and secondary immunodeficient mice. Erythroid cells derived from edited PKD-HSPCs recovered normal ATP levels, demonstrating the restoration of RPK function in PKD erythropoiesis after gene editing. Our gene-editing strategy may represent a lifelong therapy to correct RPK functionality in RBCs for PKD patients.
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- 2021
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7. Improved collection of hematopoietic stem cells and progenitors from Fanconi anemia patients for gene therapy purposes
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Julián Sevilla, Susana Navarro, Paula Rio, Rebeca Sánchez-Domínguez, Josune Zubicaray, Eva Gálvez, Eva Merino, Elena Sebastián, Carmen Azqueta, José A. Casado, José C. Segovia, Omaira Alberquilla, Massimo Bogliolo, Francisco J. Román-Rodríguez, Yari Giménez, Lise Larcher, Rocío Salgado, Roser M. Pujol, Raquel Hladun, Ana Castillo, Jean Soulier, Sergi Querol, Jesús Fernández, Jonathan Schwartz, Nagore García de Andoín, Ricardo López, Albert Catalá, Jordi Surralles, Cristina Díaz-de-Heredia, and Juan A. Bueren
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Fanconi anemia ,HSPC collection ,gene therapy ,Mozobil ,mobilization ,leukapheresis ,Genetics ,QH426-470 ,Cytology ,QH573-671 - Abstract
Difficulties in the collection of hematopoietic stem and progenitor cells (HSPCs) from Fanconi anemia (FA) patients have limited the gene therapy in this disease. We have investigated (ClinicalTrials.gov, NCT02931071) the safety and efficacy of filgrastim and plerixafor for mobilization of HSPCs and collection by leukapheresis in FA patients. Nine of eleven enrolled patients mobilized beyond the threshold level of 5 CD34+ cells/μL required to initiate apheresis. A median of 21.8 CD34+ cells/μL was reached at the peak of mobilization. Significantly, the oldest patients (15 and 16 years old) were the only ones who did not reach that threshold. A median of 4.27 million CD34+ cells/kg was collected in 2 or 3 aphereses. These numbers were markedly decreased to 1.1 million CD34+ cells/kg after immunoselection, probably because of weak expression of the CD34 antigen. However, these numbers were sufficient to facilitate the engraftment of corrected HSPCs in non-conditioned patients. No procedure-associated serious adverse events were observed. Mobilization of CD34+ cells correlated with younger age, higher leukocyte counts and hemoglobin values, lower mean corpuscular volume, and higher proportion of CD34+ cells in bone marrow (BM). All these values offer crucial information for the enrollment of FA patients for gene therapy protocols.
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- 2021
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8. Preclinical studies of efficacy thresholds and tolerability of a clinically ready lentiviral vector for pyruvate kinase deficiency treatment
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Susana Navarro, Oscar Quintana-Bustamante, Rebeca Sanchez-Dominguez, Sergio Lopez-Manzaneda, Isabel Ojeda-Perez, Aida Garcia-Torralba, Omaira Alberquilla, Kenneth Law, Brian C. Beard, Antonella Bastone, Michael Rothe, Mariela Villanueva, Juan C. Ramirez, Sara Fañanas-Baquero, Virginia Nieto-Romero, Andrea Molinos-Vicente, Sonia Gutierrez, Eileen Nicoletti, María García-Bravo, Juan A. Bueren, Jonathan D. Schwartz, and Jose-Carlos Segovia
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hematopoiesis ,gene therapy ,lentiviral vectors ,erythroid metabolic diseases ,pyruvate kinase deficiency ,biodistribution ,Genetics ,QH426-470 ,Cytology ,QH573-671 - Abstract
Pyruvate kinase deficiency (PKD) is a rare autosomal recessive disorder caused by mutations in the PKLR gene. PKD is characterized by non-spherocytic hemolytic anemia of variable severity and may be fatal in some cases during early childhood. Although not considered the standard of care, allogeneic stem cell transplantation has been shown as a potentially curative treatment, limited by donor availability, toxicity, and incomplete engraftment. Preclinical studies were conducted to define conditions to enable consistent therapeutic reversal, which were based on our previous data on lentiviral gene therapy for PKD. Improvement of erythroid parameters was identified by the presence of 20%–30% healthy donor cells. A minimum vector copy number (VCN) of 0.2−0.3 was required to correct PKD when corrected cells were transplanted in a mouse model for PKD. Biodistribution and pharmacokinetics studies, with the aim of conducting a global gene therapy clinical trial for PKD patients (RP-L301-0119), demonstrated that genetically corrected cells do not confer additional side effects. Moreover, a clinically compatible transduction protocol with mobilized peripheral blood CD34+ cells was optimized, thus facilitating the efficient transduction on human cells capable of repopulating the hematopoiesis of immunodeficient mice. We established conditions for a curative lentiviral vector gene therapy protocol for PKD.
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- 2021
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9. Upregulation of NKG2D ligands impairs hematopoietic stem cell function in Fanconi anemia
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José A. Casado, Antonio Valeri, Rebeca Sanchez-Domínguez, Paula Vela, Andrea López, Susana Navarro, Omaira Alberquilla, Helmut Hanenberg, Roser Pujol, José-Carlos Segovia, Jordi Minguillón, Jordi Surrallés, Cristina Díaz de Heredia, Julián Sevilla, Paula Rio, and Juan A. Bueren
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Immunology ,Stem cells ,Medicine - Abstract
Fanconi anemia (FA) is the most prevalent inherited bone marrow failure (BMF) syndrome. Nevertheless, the pathophysiological mechanisms of BMF in FA have not been fully elucidated. Since FA cells are defective in DNA repair, we hypothesized that FA hematopoietic stem and progenitor cells (HSPCs) might express DNA damage–associated stress molecules such as natural killer group 2 member D ligands (NKG2D-Ls). These ligands could then interact with the activating NKG2D receptor expressed in cytotoxic NK or CD8+ T cells, which may result in progressive HSPC depletion. Our results indeed demonstrated upregulated levels of NKG2D-Ls in cultured FA fibroblasts and T cells, and these levels were further exacerbated by mitomycin C or formaldehyde. Notably, a high proportion of BM CD34+ HSPCs from patients with FA also expressed increased levels of NKG2D-Ls, which correlated inversely with the percentage of CD34+ cells in BM. Remarkably, the reduced clonogenic potential characteristic of FA HSPCs was improved by blocking NKG2D–NKG2D-L interactions. Moreover, the in vivo blockage of these interactions in a BMF FA mouse model ameliorated the anemia in these animals. Our study demonstrates the involvement of NKG2D–NKG2D-L interactions in FA HSPC functionality, suggesting an unexpected role of the immune system in the progressive BMF that is characteristic of FA.
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- 2022
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10. Enhanced anti-inflammatory effects of mesenchymal stromal cells mediated by the transient ectopic expression of CXCR4 and IL10
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Rosario Hervás-Salcedo, María Fernández-García, Miriam Hernando-Rodríguez, Oscar Quintana-Bustamante, Jose-Carlos Segovia, Marcio Alvarez-Silva, Mariano García-Arranz, Pablo Minguez, Victoria del Pozo, Marta Rodríguez de Alba, Damián García-Olmo, Carmen Ayuso, María Luisa Lamana, Juan A. Bueren, and Rosa María Yañez
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Mesenchymal stromal cells ,CXCR4 ,IL10 ,mRNA-modified MSCs ,Inflammation ,MSC homing ,Medicine (General) ,R5-920 ,Biochemistry ,QD415-436 - Abstract
Abstract Background Mesenchymal stromal cells (MSCs) constitute one of the cell types most frequently used in cell therapy. Although several studies have shown the efficacy of these cells to modulate inflammation in different animal models, the results obtained in human clinical trials have been more modest. Here, we aimed at improving the therapeutic properties of MSCs by inducing a transient expression of two molecules that could enhance two different properties of these cells. With the purpose of improving MSC migration towards inflamed sites, we induced a transient expression of the C-X-C chemokine receptor type 4 (CXCR4). Additionally, to augment the anti-inflammatory properties of MSCs, a transient expression of the anti-inflammatory cytokine, interleukin 10 (IL10), was also induced. Methods Human adipose tissue-derived MSCs were transfected with messenger RNAs carrying the codon-optimized versions of CXCR4 and/or IL10. mRNA-transfected MSCs were then studied, first to evaluate whether the characteristic phenotype of MSCs was modified. Additionally, in vitro and also in vivo studies in an LPS-induced inflamed pad model were conducted to evaluate the impact associated to the transient expression of CXCR4 and/or IL10 in MSCs. Results Transfection of MSCs with CXCR4 and/or IL10 mRNAs induced a transient expression of these molecules without modifying the characteristic phenotype of MSCs. In vitro studies then revealed that the ectopic expression of CXCR4 significantly enhanced the migration of MSCs towards SDF-1, while an increased immunosuppression was associated with the ectopic expression of IL10. Finally, in vivo experiments showed that the co-expression of CXCR4 and IL10 increased the homing of MSCs into inflamed pads and induced an enhanced anti-inflammatory effect, compared to wild-type MSCs. Conclusions Our results demonstrate that the transient co-expression of CXCR4 and IL10 enhances the therapeutic potential of MSCs in a local inflammation mouse model, suggesting that these mRNA-modified cells may constitute a new step in the development of more efficient cell therapies for the treatment of inflammatory diseases.
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- 2021
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11. Generation of dyskeratosis congenita-like hematopoietic stem cells through the stable inhibition of DKC1
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Carlos Carrascoso-Rubio, Hidde A. Zittersteijn, Laura Pintado-Berninches, Beatriz Fernández-Varas, M. Luz Lozano, Cristina Manguan-Garcia, Leandro Sastre, Juan A. Bueren, Rosario Perona, and Guillermo Guenechea
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Dyskeratosis congenita ,DKC1 gene ,Bone marrow failure disorders ,Hematopoietic stem cells ,Short hairpin RNA ,Lentiviral vectors ,Medicine (General) ,R5-920 ,Biochemistry ,QD415-436 - Abstract
Abstract Dyskeratosis congenita (DC) is a rare telomere biology disorder, which results in different clinical manifestations, including severe bone marrow failure. To date, the only curative treatment for the bone marrow failure in DC patients is allogeneic hematopoietic stem cell transplantation. However, due to the toxicity associated to this treatment, improved therapies are recommended for DC patients. Here, we aimed at generating DC-like human hematopoietic stem cells in which the efficacy of innovative therapies could be investigated. Because X-linked DC is the most frequent form of the disease and is associated with an impaired expression of DKC1, we have generated DC-like hematopoietic stem cells based on the stable knock-down of DKC1 in human CD34+ cells with lentiviral vectors encoding for DKC1 short hairpin RNAs. At a molecular level, DKC1-interfered CD34+ cells showed a decreased expression of TERC, as well as a diminished telomerase activity and increased DNA damage, cell senescence, and apoptosis. Moreover, DKC1-interfered human CD34+ cells showed defective clonogenic ability and were incapable of repopulating the hematopoiesis of immunodeficient NSG mice. The development of DC-like hematopoietic stem cells will facilitate the understanding of the molecular and cellular basis of this inherited bone marrow failure syndrome and will serve as a platform to evaluate the efficacy of new hematopoietic therapies for DC.
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- 2021
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12. In Vitro and In Vivo Genetic Disease Modeling via NHEJ-Precise Deletions Using CRISPR-Cas9
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Sergio López-Manzaneda, Isabel Ojeda-Pérez, Nerea Zabaleta, Aída García-Torralba, Omaira Alberquilla, Raúl Torres, Rebeca Sánchez-Domínguez, Laura Torella, Emmanuel Olivier, Joanne Mountford, Juan C. Ramírez, Juan A. Bueren, Gloria González-Aseguinolaza, and Jose-Carlos Segovia
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Genetics ,QH426-470 ,Cytology ,QH573-671 - Abstract
The development of advanced gene and cell therapies for the treatment of genetic diseases requires reliable animal and cellular models to test their efficacy. Moreover, the availability of the target human primary cells of these therapies is reduced in many diseases. The development of endonucleases that can cut into specific sites of the cell genome, as well as the repair of the generated break by non-homologous end-joining, results in a variety of outcomes, insertions, deletions, and inversions that can induce the disruption of any specific gene. Among the many methods that have been developed for gene editing, CRISPR-Cas9 technology has become one of the most widely used endonuclease tools due to its easy design and its low cost. It has also been reported that the use of two guides, instead of just the one required, reduces the outcomes of non-homologous end joining mainly to the precise genomic sequences between the cutting sites of the guides used. We have explored this strategy to generate useful cellular and animal models. Different distances between the two guides have been tested (from 8 to 500 bp apart), and using the optimal range of 30–60 bp we have obtained a human primary cellular model of a genetic disease, pyruvate kinase deficiency, where the availability of the target cells is limited. We have also generated an in vivo model of glycolate oxidase (GO) deficiency, which is an enzyme involved in the glyoxylate metabolism following the same strategy. We demonstrate that the use of two-guide CRISPR-Cas9-induced non-homologous end joining is a feasible and useful tool for disease modeling, and it is most relevant to those diseases in which it is difficult to get the cells that will be genetically manipulated.
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- 2020
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13. Mesenchymal stem/stromal cell-based therapy for the treatment of rheumatoid arthritis: An update on preclinical studies
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Mercedes Lopez-Santalla, Juan A. Bueren, and Marina I. Garin
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Rheumatoid arthritis ,Mesenchymal stem/stromal cells ,Animal models ,Protocols ,Improvements in MSC-based therapy ,Medicine ,Medicine (General) ,R5-920 - Abstract
Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by synovial inflammation and progressive joint destruction and is a primary cause of disability worldwide. Despite the existence of numerous anti-rheumatic drugs, a significant number of patients with RA do not respond or are intolerant to current treatments. Mesenchymal stem/stromal cell (MSCs) therapy represents a promising therapeutic tool to treat RA, mainly attributable to the immunomodulatory effects of these cells. This review comprises a comprehensive analysis of the scientific literature related to preclinical studies of MSC-based therapy in RA to analyse key aspects of current protocols as well as novel approaches which aim to improve the efficacy of MSC-based therapy.
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- 2021
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14. Enhanced Susceptibility of Galectin-1 Deficient Mice to Experimental Colitis
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Raquel Fernandez-Perez, Mercedes Lopez-Santalla, Rebeca Sánchez-Domínguez, Omaira Alberquilla, Irene Gutiérrez-Cañas, Yasmina Juarranz, Juan A. Bueren, and Marina I. Garin
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galectin-1 ,inflammatory bowel disease ,immune regulation ,DSS ,cell therapy ,regulatory T cells ,Immunologic diseases. Allergy ,RC581-607 - Abstract
Galectin-1 is a β-galactoside-binding lectin, ubiquitously expressed in stromal, epithelial, and different subsets of immune cells. Galectin-1 is the prototype member of the galectin family which shares specificity with β-galactoside containing proteins and lipids. Immunomodulatory functions have been ascribed to endogenous galectin-1 due to its induction of T cell apoptosis, inhibitory effects of neutrophils and T cell trafficking. Several studies have demonstrated that administration of recombinant galectin-1 suppressed experimental colitis by modulating adaptive immune responses altering the fate and phenotype of T cells. However, the role of endogenous galectin-1 in intestinal inflammation is poorly defined. In the present study, the well-characterized acute dextran sulfate sodium (DSS)-induced model of ulcerative colitis was used to study the function of endogenous galectin-1 during the development of intestinal inflammation. We found that galectin-1 deficient mice (Lgals1−/− mice) displayed a more severe intestinal inflammation, characterized by significantly elevated clinical scores, than their wild type counterparts. The mechanisms underlying the enhanced inflammatory response in colitic Lgals1−/− mice involved an altered Th17/Th1 profile of effector CD4+ T cells. Furthermore, increased frequencies of Foxp3+CD4+ regulatory T cells in colon lamina propria in Lgals1−/− mice were found. Strikingly, the exacerbated intestinal inflammatory response observed in Lgals1−/− mice was alleviated by adoptive transfer of wild type Foxp3+CD4+ regulatory T cells at induction of colitis. Altogether, these data highlight the importance of endogenous galectin-1 as a novel determinant in regulating T cell reactivity during the development of intestinal inflammation.
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- 2021
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15. The Current Status of Mesenchymal Stromal Cells: Controversies, Unresolved Issues and Some Promising Solutions to Improve Their Therapeutic Efficacy
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David García-Bernal, Mariano García-Arranz, Rosa M. Yáñez, Rosario Hervás-Salcedo, Alfonso Cortés, María Fernández-García, Miriam Hernando-Rodríguez, Óscar Quintana-Bustamante, Juan A. Bueren, Damián García-Olmo, Jose M. Moraleda, José C. Segovia, and Agustín G. Zapata
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MSC bioengineering ,MSC homing ,MSC immunomodulation ,MSC preconditioning ,MSC therapeutic efficacy ,Biology (General) ,QH301-705.5 - Abstract
Mesenchymal stromal cells (MSCs) currently constitute the most frequently used cell type in advanced therapies with different purposes, most of which are related with inflammatory processes. Although the therapeutic efficacy of these cells has been clearly demonstrated in different disease animal models and in numerous human phase I/II clinical trials, only very few phase III trials using MSCs have demonstrated the expected potential therapeutic benefit. On the other hand, diverse controversial issues on the biology and clinical applications of MSCs, including their specific phenotype, the requirement of an inflammatory environment to induce immunosuppression, the relevance of the cell dose and their administration schedule, the cell delivery route (intravascular/systemic vs. local cell delivery), and the selected cell product (i.e., use of autologous vs. allogeneic MSCs, freshly cultured vs. frozen and thawed MSCs, MSCs vs. MSC-derived extracellular vesicles, etc.) persist. In the current review article, we have addressed these issues with special emphasis in the new approaches to improve the properties and functional capabilities of MSCs after distinct cell bioengineering strategies.
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- 2021
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16. Natural estrogens enhance the engraftment of human hematopoietic stem and progenitor cells in immunodeficient mice
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Sara Fañanas-Baquero, Israel Orman, Federico Becerra Aparicio, Silvia Bermudez de Miguel, Jordi Garcia Merino, Rosa Yañez, Yolanda Fernandez Sainz, Rebeca Sánchez, Mercedes Dessy-Rodríguez, Omaira Alberquilla, David Alfaro, Agustin Zapata, Juan A. Bueren, Jose Carlos Segovia, and Oscar Quintana-Bustamante
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Diseases of the blood and blood-forming organs ,RC633-647.5 - Abstract
Hematopoietic Stem and Progenitor Cells are crucial in the maintenance of lifelong production of all blood cells. These Stem Cells are highly regulated to maintain homeostasis through a delicate balance between quiescence, self-renewal and differentiation. However, this balance is altered during the hematopoietic recovery after Hematopoietic Stem and Progenitor Cell Transplantation. Transplantation efficacy can be limited by inadequate Hematopoietic Stem Cells number, poor homing, low level of engraftment, or limited self-renewal. As recent evidences indicate that estrogens are involved in regulating the hematopoiesis, we sought to examine whether natural estrogens (estrone or E1, estradiol or E2, estriol or E3 and estetrol or E4) modulate human Hematopoietic Stem and Progenitor Cells. Our results show that human Hematopoietic Stem and Progenitor Cell subsets express estrogen receptors, and whose signaling is activated by E2 and E4 on these cells. Additionally, these natural estrogens cause different effects on human Progenitors in vitro. We found that both E2 and E4 expand human Hematopoietic Stem and Progenitor Cells. However, E4 was the best tolerated estrogen and promoted cell cycle of human Hematopoietic Progenitors. Furthermore, we identified that E2 and, more significantly, E4 doubled human hematopoietic engraftment in immunodeficient mice without altering other Hematopoietic Stem and Progenitor Cells properties. Finally, the impact of E4 on promoting human hematopoietic engraftment in immunodeficient mice might be mediated through the regulation of mesenchymal stromal cells in the bone marrow niche. Together, our data demonstrate that E4 is well tolerated and enhances human reconstitution in immunodeficient mice, directly by modulating human Hematopoietic Progenitor properties and indirectly by interacting with the bone marrow niche. This application might have particular relevance to ameliorate the hematopoietic recovery after myeloablative conditioning, especially when limiting numbers of Hematopoietic Stem and Progenitor Cells are available.
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- 2020
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17. Measles virus envelope pseudotyped lentiviral vectors transduce quiescent human HSCs at an efficiency without precedent
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Camille Lévy, Fouzia Amirache, Anais Girard-Gagnepain, Cecilia Frecha, Francisco J. Roman-Rodríguez, Ornellie Bernadin, Caroline Costa, Didier Nègre, Alejandra Gutierrez-Guerrero, Lenard S. Vranckx, Isabelle Clerc, Naomi Taylor, Lars Thielecke, Kerstin Cornils, Juan A. Bueren, Paula Rio, Rik Gijsbers, François-Loïc Cosset, and Els Verhoeyen
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Specialties of internal medicine ,RC581-951 - Abstract
Abstract: Hematopoietic stem cell (HSC)–based gene therapy trials are now moving toward the use of lentiviral vectors (LVs) with success. However, one challenge in the field remains: efficient transduction of HSCs without compromising their stem cell potential. Here we showed that measles virus glycoprotein–displaying LVs (hemagglutinin and fusion protein LVs [H/F-LVs]) were capable of transducing 100% of early-acting cytokine-stimulated human CD34+ (hCD34+) progenitor cells upon a single application. Strikingly, these H/F-LVs also allowed transduction of up to 70% of nonstimulated quiescent hCD34+ cells, whereas conventional vesicular stomatitis virus G (VSV-G)–LVs reached 5% at the most with H/F-LV entry occurring exclusively through the CD46 complement receptor. Importantly, reconstitution of NOD/SCIDγc−/− (NSG) mice with H/F-LV transduced prestimulated or resting hCD34+ cells confirmed these high transduction levels in all myeloid and lymphoid lineages. Remarkably, for resting CD34+ cells, secondary recipients exhibited increasing transduction levels of up to 100%, emphasizing that H/F-LVs efficiently gene-marked HSCs in the resting state. Because H/F-LVs promoted ex vivo gene modification of minimally manipulated CD34+ progenitors that maintained stemness, we assessed their applicability in Fanconi anemia, a bone marrow (BM) failure with chromosomal fragility. Notably, only H/F-LVs efficiently gene-corrected minimally stimulated hCD34+ cells in unfractionated BM from these patients. These H/F-LVs improved HSC gene delivery in the absence of cytokine stimulation while maintaining their stem cell potential. Thus, H/F-LVs will facilitate future clinical applications requiring HSC gene modification, including BM failure syndromes, for which treatment has been very challenging up to now.
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- 2017
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18. Direct Conversion of Fibroblasts to Megakaryocyte Progenitors
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Julian Pulecio, Oriol Alejo-Valle, Sandra Capellera-Garcia, Marianna Vitaloni, Paula Rio, Eva Mejía-Ramírez, Ilaria Caserta, Juan A. Bueren, Johan Flygare, and Angel Raya
- Subjects
transdifferentiation ,lineage conversion ,Fanconi anemia ,thrombocytopenia ,platelets ,Biology (General) ,QH301-705.5 - Abstract
Current sources of platelets for transfusion are insufficient and associated with risk of alloimmunization and blood-borne infection. These limitations could be addressed by the generation of autologous megakaryocytes (MKs) derived in vitro from somatic cells with the ability to engraft and differentiate in vivo. Here, we show that overexpression of a defined set of six transcription factors efficiently converts mouse and human fibroblasts into MK-like progenitors. The transdifferentiated cells are CD41+, display polylobulated nuclei, have ploidies higher than 4N, form MK colonies, and give rise to platelets in vitro. Moreover, transplantation of MK-like murine progenitor cells into NSG mice results in successful engraftment and further maturation in vivo. Similar results are obtained using disease-corrected fibroblasts from Fanconi anemia patients. Our results combined demonstrate that functional MK progenitors with clinical potential can be obtained in vitro, circumventing the use of hematopoietic progenitors or pluripotent stem cells.
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- 2016
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19. Generation of a High Number of Healthy Erythroid Cells from Gene-Edited Pyruvate Kinase Deficiency Patient-Specific Induced Pluripotent Stem Cells
- Author
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Zita Garate, Oscar Quintana-Bustamante, Ana M. Crane, Emmanuel Olivier, Laurent Poirot, Roman Galetto, Penelope Kosinski, Collin Hill, Charles Kung, Xabi Agirre, Israel Orman, Laura Cerrato, Omaira Alberquilla, Fatima Rodriguez-Fornes, Noemi Fusaki, Felix Garcia-Sanchez, Tabita M. Maia, Maria L. Ribeiro, Julian Sevilla, Felipe Prosper, Shengfang Jin, Joanne Mountford, Guillermo Guenechea, Agnes Gouble, Juan A. Bueren, Brian R. Davis, and Jose C. Segovia
- Subjects
Medicine (General) ,R5-920 ,Biology (General) ,QH301-705.5 - Abstract
Pyruvate kinase deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene. Erythrocytes from PKD patients show an energetic imbalance causing chronic non-spherocytic hemolytic anemia, as pyruvate kinase defects impair ATP production in erythrocytes. We generated PKD induced pluripotent stem cells (PKDiPSCs) from peripheral blood mononuclear cells (PB-MNCs) of PKD patients by non-integrative Sendai viral vectors. PKDiPSCs were gene edited to integrate a partial codon-optimized R-type pyruvate kinase cDNA in the second intron of the PKLR gene by TALEN-mediated homologous recombination (HR). Notably, we found allele specificity of HR led by the presence of a single-nucleotide polymorphism. High numbers of erythroid cells derived from gene-edited PKDiPSCs showed correction of the energetic imbalance, providing an approach to correct metabolic erythroid diseases and demonstrating the practicality of this approach to generate the large cell numbers required for comprehensive biochemical and metabolic erythroid analyses.
- Published
- 2015
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20. Terapias avanzadas en enfermedades raras
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Cristina Fillat, Marcela del Río, Lucía Martínez-Santamaría, and Juan A. Bueren
- Subjects
terapia génica ,terapia celular ,ingeniería de tejidos ,enfermedades raras ,General Works - Abstract
Las terapias avanzadas comprenden un grupo de medicamentos biológicos basados en la terapia génica, la terapia celular y la ingeniería de tejidos. En este artículo se presentan algunos conceptos básicos, se describen las estrategias más prometedoras y se detallan los resultados de diferentes ensayos clínicos para un grupo significativo de enfermedades, especialmente las enfermedades hereditarias monogénicas. Se hace un mayor hincapié en la terapia génica ex vivo en enfermedades del sistema hematopoyético, al ser el grupo de enfermedades pioneras que además han orientado la investigación en el campo. Destacamos también algunos ejemplos de éxito de terapia génica in vivo y señalamos la contribución de la terapia celular y de la ingeniería de tejidos al grupo de enfermedades raras de la piel. Pretendemos con ello dar una visión de la situación de las terapias avanzadas en enfermedades raras y señalamos algunas de las vías de futuro orientadas al desarrollo de tratamientos más eficaces y seguros.
- Published
- 2018
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21. Biodistribution and Efficacy of Human Adipose-Derived Mesenchymal Stem Cells Following Intranodal Administration in Experimental Colitis
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Mercedes Lopez-Santalla, Pablo Mancheño-Corvo, Amelia Escolano, Ramon Menta, Olga DelaRosa, Jose Luis Abad, Dirk Büscher, Juan M. Redondo, Juan A. Bueren, Wilfried Dalemans, Eleuterio Lombardo, and Marina I. Garin
- Subjects
adipose-derived mesenchymal stem cells ,intranodal therapy ,colitis ,biodistribution ,efficacy ,immunomodulation ,Immunologic diseases. Allergy ,RC581-607 - Abstract
Mesenchymal stem cells (MSCs) have a large potential in cell therapy for treatment of inflammatory and autoimmune diseases, thanks to their immunomodulatory properties. The encouraging results in animal models have initiated the translation of MSC therapy to clinical trials. In cell therapy protocols with MSCs, administered intravenously, several studies have shown that a small proportion of infused MSCs can traffic to the draining lymph nodes (LNs). This is accompanied with an increase of different types of regulatory immune cells in the LNs, suggesting the importance of migration of MSCs to the LNs in order to contribute to immunomodulatory response. Intranodal (IN), also referred as intralymphatic, injection of cells, like dendritic cells, is being proposed in the clinic for the treatment of cancer and allergy, showing that this route of administration is clinically safe and efficient. In this study, we investigated, for the first time, the biodistribution and the efficacy of Luciferase+ adipose-derived MSCs (Luci-eASCs), infused through the inguinal LNs (iLNs), in normal mice and in inflamed mice with colitis. Most of the Luci-eASCs remain in the iLNs and in the adipose tissue surrounding the inguinal LNs. A small proportion of Luci-eASCs can migrate to other locations within the lymphatic system and to other tissues and organs, having a preferential migration toward the intestine in colitic mice. Our results show that the infused Luci-eASCs protected 58% of the mice against induced colitis. Importantly, a correlation between the response to eASC treatment and a higher accumulation of eASCs in popliteal, parathymic, parathyroid, and mesenteric LNs were found. Altogether, these results suggest that IN administration of eASCs is feasible and may represent an effective strategy for cell therapy protocols with human adipose-derived MSCs in the clinic for the treatment of immune-mediated disorders.
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- 2017
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22. Intralymphatic Administration of Adipose Mesenchymal Stem Cells Reduces the Severity of Collagen-Induced Experimental Arthritis
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Eleuterio Lombardo, Pablo Mancheño-Corvo, Mercedes Lopez-Santalla, Ramon Menta, Olga DelaRosa, Francisca Mulero, Borja del Rio, Cristina Ramirez, Dirk Büscher, Juan A. Bueren, Juan Lopez-Belmonte, Wilfried Dalemans, and Marina I. Garin
- Subjects
adipose mesenchymal stem cells ,intralymphatic route ,collagen-induced arthritis ,efficacy ,immunomodulation ,Immunologic diseases. Allergy ,RC581-607 - Abstract
Mesenchymal stem cells (MSCs) are multipotent stromal cells with immunomodulatory properties. They have emerged as a very promising treatment for autoimmunity and inflammatory diseases such as rheumatoid arthritis. Previous studies have demonstrated that MSCs, administered systemically, migrate to lymphoid tissues associated with the inflammatory site where functional MSC-induced immune cells with a regulatory phenotype were increased mediating the immunomodulatory effects of MSCs. These results suggest that homing of MSCs to the lymphatic system plays an important role in the mechanism of action of MSCs in vivo. Thus, we hypothesized that direct intralymphatic (IL) (also referred as intranodal) administration of MSCs could be an alternative and effective route of administration for MSC-based therapy. Here, we report the feasibility and efficacy of the IL administration of human expanded adipose mesenchymal stem cells (eASCs) in a mouse model of collagen-induced arthritis (CIA). IL administration of eASCs attenuated the severity and progression of arthritis, reduced bone destruction and increased the levels of regulatory T cells (CD25+Foxp3+CD4+ cells) and Tr1 cells (IL10+CD4+), in spleen and draining lymph nodes. Taken together, these results indicate that IL administration of eASCs is very effective in modulating established CIA and may represent an alternative treatment modality for cell therapy with eASCs.
- Published
- 2017
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23. Comparative Analysis between the In Vivo Biodistribution and Therapeutic Efficacy of Adipose-Derived Mesenchymal Stromal Cells Administered Intraperitoneally in Experimental Colitis
- Author
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Mercedes Lopez-Santalla, Pablo Mancheño-Corvo, Amelia Escolano, Ramon Menta, Olga Delarosa, Juan M. Redondo, Juan A. Bueren, Wilfried Dalemans, Eleuterio Lombardo, and Marina I. Garin
- Subjects
adipose-derived mesenchymal stem cells ,intraperitoneal therapy ,biodistribution ,efficacy ,colitis ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
Mesenchymal stem cells (MSCs) have emerged as a promising treatment for inflammatory diseases. The immunomodulatory effect of MSCs takes place both by direct cell-to-cell contact and by means of soluble factors that leads to an increased accumulation of regulatory immune cells at the sites of inflammation. Similar efficacy of MSCs has been described regardless of the route of administration used, the inflammation conditions and the major histocompatibility complex context. These observations raise the question of whether the migration of the MSCs to the inflamed tissues is a pre-requisite to achieve their beneficial effect. To address this, we examined the biodistribution and the efficacy of intraperitoneal luciferase-expressing human expanded adipose-derived stem cells (Luci-eASCs) in a mouse model of colitis. Luci-eASC-infused mice were stratified according to their response to the Luci-eASC treatment. According to the stratification criteria, there was a tendency to increase the bioluminescence signal in the intestine at the expense of a decrease in the bioluminescence signal in the liver in the “responder” mice. These data thus suggest that the accumulation of the eASCs to the inflamed tissues is beneficial for achieving an optimal modulation of inflammation.
- Published
- 2018
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24. Data from Gefitinib and Afatinib Show Potential Efficacy for Fanconi Anemia–Related Head and Neck Cancer
- Author
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Jordi Surrallés, Jordi Minguillón, Diego Arango, Juan A. Bueren, Thomas Helleday, Jordi Carreras-Puigvert, Adriana Lasa, Enrique Lerma, Alan González, Pau Riera, Carlos Carrascoso-Rubio, Rocío Nieto, Maria José Ramírez, Llorenç Rovirosa, José Antonio Casado, Águeda Martínez-Barriocanal, and Helena Montanuy
- Abstract
Purpose:Fanconi anemia rare disease is characterized by bone marrow failure and a high predisposition to solid tumors, especially head and neck squamous cell carcinoma (HNSCC). Patients with Fanconi anemia with HNSCC are not eligible for conventional therapies due to high toxicity in healthy cells, predominantly hematotoxicity, and the only treatment currently available is surgical resection. In this work, we searched and validated two already approved drugs as new potential therapies for HNSCC in patients with Fanconi anemia.Experimental Design:We conducted a high-content screening of 3,802 drugs in a FANCA-deficient tumor cell line to identify nongenotoxic drugs with cytotoxic/cytostatic activity. The best candidates were further studied in vitro and in vivo for efficacy and safety.Results:Several FDA/European Medicines Agency (EMA)-approved anticancer drugs showed cancer-specific lethality or cell growth inhibition in Fanconi anemia HNSCC cell lines. The two best candidates, gefitinib and afatinib, EGFR inhibitors approved for non–small cell lung cancer (NSCLC), displayed nontumor/tumor IC50 ratios of approximately 400 and approximately 100 times, respectively. Neither gefitinib nor afatinib activated the Fanconi anemia signaling pathway or induced chromosomal fragility in Fanconi anemia cell lines. Importantly, both drugs inhibited tumor growth in xenograft experiments in immunodeficient mice using two Fanconi anemia patient–derived HNSCCs. Finally, in vivo toxicity studies in Fanca-deficient mice showed that administration of gefitinib or afatinib was well-tolerated, displayed manageable side effects, no toxicity to bone marrow progenitors, and did not alter any hematologic parameters.Conclusions:Our data present a complete preclinical analysis and promising therapeutic line of the first FDA/EMA-approved anticancer drugs exerting cancer-specific toxicity for HNSCC in patients with Fanconi anemia.
- Published
- 2023
25. Supplementary Figure 5 from Gefitinib and Afatinib Show Potential Efficacy for Fanconi Anemia–Related Head and Neck Cancer
- Author
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Jordi Surrallés, Jordi Minguillón, Diego Arango, Juan A. Bueren, Thomas Helleday, Jordi Carreras-Puigvert, Adriana Lasa, Enrique Lerma, Alan González, Pau Riera, Carlos Carrascoso-Rubio, Rocío Nieto, Maria José Ramírez, Llorenç Rovirosa, José Antonio Casado, Águeda Martínez-Barriocanal, and Helena Montanuy
- Abstract
Figure S5. FACS gating strategy to analyze white blood cell population in WT and Fanca-deficient mice.
- Published
- 2023
26. Supplementary Figure 8 from Gefitinib and Afatinib Show Potential Efficacy for Fanconi Anemia–Related Head and Neck Cancer
- Author
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Jordi Surrallés, Jordi Minguillón, Diego Arango, Juan A. Bueren, Thomas Helleday, Jordi Carreras-Puigvert, Adriana Lasa, Enrique Lerma, Alan González, Pau Riera, Carlos Carrascoso-Rubio, Rocío Nieto, Maria José Ramírez, Llorenç Rovirosa, José Antonio Casado, Águeda Martínez-Barriocanal, and Helena Montanuy
- Abstract
Figure S8. Additional data on genotoxicity in blood samples from WT or Fanca-deficient mice, by gefitinib and afatinib treatment.
- Published
- 2023
27. Supplementary Figure 2 from Gefitinib and Afatinib Show Potential Efficacy for Fanconi Anemia–Related Head and Neck Cancer
- Author
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Jordi Surrallés, Jordi Minguillón, Diego Arango, Juan A. Bueren, Thomas Helleday, Jordi Carreras-Puigvert, Adriana Lasa, Enrique Lerma, Alan González, Pau Riera, Carlos Carrascoso-Rubio, Rocío Nieto, Maria José Ramírez, Llorenç Rovirosa, José Antonio Casado, Águeda Martínez-Barriocanal, and Helena Montanuy
- Abstract
Figure S2. Additional data on gefitinib and afatinib in vivo mouse xenograft experiments. Weight control.
- Published
- 2023
28. Supplementary Fig. S3 from Relevance of the Fanconi anemia pathway in the response of human cells to trabectedin
- Author
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Juan A. Bueren, Federico Gago, Beatriz Albella, Juan José Vaquero, Juan Carlos Tercero, Laura Pérez, Alberto Domingo, Verónica García-Hernández, Esther Marco, Paula Río, and José A. Casado
- Abstract
Supplementary Fig. S3 from Relevance of the Fanconi anemia pathway in the response of human cells to trabectedin
- Published
- 2023
29. Data from Relevance of the Fanconi anemia pathway in the response of human cells to trabectedin
- Author
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Juan A. Bueren, Federico Gago, Beatriz Albella, Juan José Vaquero, Juan Carlos Tercero, Laura Pérez, Alberto Domingo, Verónica García-Hernández, Esther Marco, Paula Río, and José A. Casado
- Abstract
Trabectedin (Yondelis; ET-743) is a potent anticancer drug that binds to DNA by forming a covalent bond with a guanine in one strand and one or more hydrogen bonds with the opposite strand. Using a fluorescence-based melting assay, we show that one single trabectedin-DNA adduct increases the thermal stability of the double helix by >20°C. As deduced from the analysis of phosphorylated H2AX and Rad51 foci, we observed that clinically relevant doses of trabectedin induce the formation of DNA double-strand breaks in human cells and activate homologous recombination repair in a manner similar to that evoked by the DNA interstrand cross-linking agent mitomycin C (MMC). Because one important characteristic of this drug is its marked cytotoxicity on cells lacking a functional Fanconi anemia (FA) pathway, we compared the response of different subtypes of FA cells to MMC and trabectedin. Our data clearly show that human cells with mutations in FANCA, FANCC, FANCF, FANCG, or FANCD1 genes are highly sensitive to both MMC and trabectedin. However, in marked contrast to MMC, trabectedin does not induce any significant accumulation of FA cells in G2-M. The critical relevance of FA proteins in the response of human cells to trabectedin reported herein, together with observations showing the role of the FA pathway in cancer suppression, strongly suggest that screening for mutations in FA genes may facilitate the identification of tumors displaying enhanced sensitivity to this novel anticancer drug. [Mol Cancer Ther 2008;7(5):1309–18]
- Published
- 2023
30. Supplementary Figure 3 from Gefitinib and Afatinib Show Potential Efficacy for Fanconi Anemia–Related Head and Neck Cancer
- Author
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Jordi Surrallés, Jordi Minguillón, Diego Arango, Juan A. Bueren, Thomas Helleday, Jordi Carreras-Puigvert, Adriana Lasa, Enrique Lerma, Alan González, Pau Riera, Carlos Carrascoso-Rubio, Rocío Nieto, Maria José Ramírez, Llorenç Rovirosa, José Antonio Casado, Águeda Martínez-Barriocanal, and Helena Montanuy
- Abstract
Figure S3. Additional immunohistochemistry images from tumor xenografts.
- Published
- 2023
31. Supplementary Figure 7 from Gefitinib and Afatinib Show Potential Efficacy for Fanconi Anemia–Related Head and Neck Cancer
- Author
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Jordi Surrallés, Jordi Minguillón, Diego Arango, Juan A. Bueren, Thomas Helleday, Jordi Carreras-Puigvert, Adriana Lasa, Enrique Lerma, Alan González, Pau Riera, Carlos Carrascoso-Rubio, Rocío Nieto, Maria José Ramírez, Llorenç Rovirosa, José Antonio Casado, Águeda Martínez-Barriocanal, and Helena Montanuy
- Abstract
Figure S7. Additional data on gefitinib and afatinib toxicity analysis in WT and Fanca-deficient mice.
- Published
- 2023
32. Supplementary Figure 6 from Gefitinib and Afatinib Show Potential Efficacy for Fanconi Anemia–Related Head and Neck Cancer
- Author
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Jordi Surrallés, Jordi Minguillón, Diego Arango, Juan A. Bueren, Thomas Helleday, Jordi Carreras-Puigvert, Adriana Lasa, Enrique Lerma, Alan González, Pau Riera, Carlos Carrascoso-Rubio, Rocío Nieto, Maria José Ramírez, Llorenç Rovirosa, José Antonio Casado, Águeda Martínez-Barriocanal, and Helena Montanuy
- Abstract
Figure S6. Additional data on gefitinib and afatinib toxicity analysis in WT and Fanca-deficient mice.
- Published
- 2023
33. Supplementary Figure 1 from Gefitinib and Afatinib Show Potential Efficacy for Fanconi Anemia–Related Head and Neck Cancer
- Author
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Jordi Surrallés, Jordi Minguillón, Diego Arango, Juan A. Bueren, Thomas Helleday, Jordi Carreras-Puigvert, Adriana Lasa, Enrique Lerma, Alan González, Pau Riera, Carlos Carrascoso-Rubio, Rocío Nieto, Maria José Ramírez, Llorenç Rovirosa, José Antonio Casado, Águeda Martínez-Barriocanal, and Helena Montanuy
- Abstract
Figure S1. Additional data from high content screening and validation
- Published
- 2023
34. Supplementary Text from Gefitinib and Afatinib Show Potential Efficacy for Fanconi Anemia–Related Head and Neck Cancer
- Author
-
Jordi Surrallés, Jordi Minguillón, Diego Arango, Juan A. Bueren, Thomas Helleday, Jordi Carreras-Puigvert, Adriana Lasa, Enrique Lerma, Alan González, Pau Riera, Carlos Carrascoso-Rubio, Rocío Nieto, Maria José Ramírez, Llorenç Rovirosa, José Antonio Casado, Águeda Martínez-Barriocanal, and Helena Montanuy
- Abstract
Supplementary Text
- Published
- 2023
35. Role of Hospital Exemption in Europe: position paper from the Spanish Advanced Therapy Network (TERAV)
- Author
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Fermín Sánchez-Guijo, Cristina Avendaño-Solá, Lina Badimón, Juan A. Bueren, Josep M. Canals, Joaquim Delgadillo, Julio Delgado, Cristina Eguizábal, María-Eugenia Fernández-Santos, Damián García-Olmo, Gloria González-Aseguinolaza, Manel Juan, Francisco Martín, Rosario Mata, Nuria Montserrat, Antonio Pérez-Martínez, José A. Pérez-Simón, Felipe Prósper, Álvaro Urbano-Ispizua, Agustín G. Zapata, Anna Sureda, and José M. Moraleda
- Subjects
Transplantation ,Hematology - Abstract
Instituto de Salud Carlos III, European Union - NextGenerationEU, Recovery, Transformation and Resilience Plan RD21/0017/0001
- Published
- 2023
36. Natural gene therapy by reverse mosaicism leads to improved hematology in <scp>Fanconi</scp> anemia patients
- Author
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Cristina Beléndez, Jonathan D. Schwartz, Monica Lopez, Estela Carrasco, Gayatri R Rao, Jordi Minguillón, Judith Balmaña, Roser Pujol, Eileen Nicoletti, José A. Casado, María José Ramírez, Massimo Bogliolo, Jordi Surrallés, Albert Català, Julián Sevilla, Cristina Díaz de Heredia, Susana Navarro, Juan A. Bueren, Paula Río, Bienvenida Argilés, Juan Pablo Trujillo-Quintero, and Isabel Badell
- Subjects
Adult ,Male ,Oncology ,medicine.medical_specialty ,Adolescent ,SOMATIC MOSAICISM ,Young Adult ,03 medical and health sciences ,0302 clinical medicine ,Fanconi anemia ,Internal medicine ,medicine ,Humans ,Progenitor cell ,Child ,Hematology ,Mosaicism ,business.industry ,Bone marrow failure ,Hematopoietic stem cell ,Genetic Therapy ,medicine.disease ,Leukemia ,Fanconi Anemia ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,CELLS ,Cohort ,Bone marrow ,business ,030215 immunology - Abstract
Fanconi anemia (FA) is characterized by chromosome fragility, bone marrow failure (BMF) and predisposition to cancer. As reverse genetic mosaicism has been described as "natural gene therapy" in patients with FA, we sought to evaluate the clinical course of a cohort of FA mosaic patients followed at referral centers in Spain over a 30-year period. This cohort includes patients with a majority of T cells without chromosomal aberrations in the DEB-chromosomal breakage test. Relative to non-mosaic FA patients, we observed a higher proportion of adult patients in the cohort of mosaics, with a later age of hematologic onset and a milder evolution of (BMF). Consequently, the requirement for hematopoietic stem cell transplant (HSCT) was also lower. Additional studies allowed us to identify a sub-cohort of mosaic FA patients in whom the reversion was present in bone marrow (BM) progenitor cells leading to multilineage mosaicism. These multilineage mosaic patients are older, have a lower percentage of aberrant cells, have more stable hematology and none of them developed leukemia or myelodysplastic syndrome when compared to non-mosaics. In conclusion, our data indicate that reverse mosaicism is a good prognostic factor in FA and is associated with more favorable long-term clinical outcomes.
- Published
- 2021
37. Interim Results from an Ongoing Phase 1/2 Study of Lentiviral-mediated Ex-vivo Gene Therapy for Pediatric Patients with Severe Leukocyte Adhesion Deficiency-I (LAD-I)
- Author
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Claire Booth, Julián Sevilla, Gayatri R. Rao, Maria Chitty Lopez, Elena Almarza, Dayna Terrazas, Josune Zubicaray, Marta González Vicent, Kritika Chetty, Grainne O'Toole, Jinhua Xu-Bayford, Eileen Nicoletti, Augustine Fernandes, Caroline Kuo, Satiro de Oliveira, Theodore B. Moore, Grace Choi, Miriam Zeini, Cristina Mesa-Núñez, Adrian J. Thrasher, Juan A. Bueren, Jonathan D. Schwartz, and Donald B. Kohn
- Subjects
Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
38. Lentiviral-mediated Gene Therapy for Adults and Children with Severe Pyruvate Kinase Deficiency: Results from an Ongoing Global Phase 1 Study
- Author
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Ami J. Shah, José Luis López Lorenzo, Julián Sevilla, Susana Navarro, Lucía Llanos, Begoña Pérez de Camino Gaisse, Sol Sanchez, Josune Zubicaray, Bert Glader, May Chien, Oscar Quintana Bustamante, Miriam Zeini, Grace Choi, Eileen Nicoletti, Gayatri R. Rao, Maria Grazia Roncarolo, Juan A. Bueren, Jonathan D. Schwartz, and José C. Segovia
- Subjects
Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
39. Adenine base editing is an efficient approach to restore function in FA patient cells without double-stranded DNA breaks
- Author
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Sebastian M. Siegner, Alexandra Clemens, Laura Ugalde, Laura Garcia-Garcia, Juan A. Bueren, Paula Rio, Mehmet E. Karasu, and Jacob E. Corn
- Abstract
Fanconi Anemia (FA) is a debilitating genetic disorder with a wide range of severe symptoms including bone marrow failure and predisposition to cancer. CRISPR-Cas genome editing manipulates genotypes by harnessing DNA repair and has been proposed as a potential cure for FA. But FA is caused deficiencies in DNA repair itself, preventing the use of editing strategies such as homology directed repair. Recently developed base editing (BE) systems do not rely on double stranded DNA breaks and might be used to target mutations in FA genes, but this remains to be tested. Here we develop a proof of concept therapeutic base editing strategy to address two of the most prevalent FANCA mutations in patient cells. We find that optimizing adenine base editor construct, vector type, guide RNA format, and delivery conditions lead to very effective genetic modification in multiple FA patient backgrounds. Optimized base editing restored FANCA expression, molecular function of the FA pathway, and phenotypic resistance to crosslinking agents. ABE8e mediated editing in primary hematopoietic stem and progenitor cells from an FA patient was both genotypically effective and restored FA pathway function, indicating the potential of base editing strategies for future clinical application in FA.
- Published
- 2022
40. In Vitro and In Vivo Genetic Disease Modeling via NHEJ-Precise Deletions Using CRISPR-Cas9
- Author
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Joanne C. Mountford, Rebeca Sanchez-Dominguez, Isabel Ojeda-Perez, Raul M. Torres, Gloria González-Aseguinolaza, Juan C. Ramirez, Aída Garcia-Torralba, Nerea Zabaleta, Sergio López-Manzaneda, Laura Torella, José C. Segovia, Emmanuel Olivier, Juan A. Bueren, and Omaira Alberquilla
- Subjects
0301 basic medicine ,lcsh:QH426-470 ,Disease ,Computational biology ,Genome ,03 medical and health sciences ,Endonuclease ,0302 clinical medicine ,Genome editing ,Genetics ,medicine ,CRISPR ,lcsh:QH573-671 ,Molecular Biology ,Gene ,biology ,lcsh:Cytology ,medicine.disease ,lcsh:Genetics ,030104 developmental biology ,030220 oncology & carcinogenesis ,biology.protein ,Molecular Medicine ,Original Article ,Cellular model ,Pyruvate kinase deficiency - Abstract
The development of advanced gene and cell therapies for the treatment of genetic diseases requires reliable animal and cellular models to test their efficacy. Moreover, the availability of the target human primary cells of these therapies is reduced in many diseases. The development of endonucleases that can cut into specific sites of the cell genome, as well as the repair of the generated break by non-homologous end-joining, results in a variety of outcomes, insertions, deletions, and inversions that can induce the disruption of any specific gene. Among the many methods that have been developed for gene editing, CRISPR-Cas9 technology has become one of the most widely used endonuclease tools due to its easy design and its low cost. It has also been reported that the use of two guides, instead of just the one required, reduces the outcomes of non-homologous end joining mainly to the precise genomic sequences between the cutting sites of the guides used. We have explored this strategy to generate useful cellular and animal models. Different distances between the two guides have been tested (from 8 to 500 bp apart), and using the optimal range of 30–60 bp we have obtained a human primary cellular model of a genetic disease, pyruvate kinase deficiency, where the availability of the target cells is limited. We have also generated an in vivo model of glycolate oxidase (GO) deficiency, which is an enzyme involved in the glyoxylate metabolism following the same strategy. We demonstrate that the use of two-guide CRISPR-Cas9-induced non-homologous end joining is a feasible and useful tool for disease modeling, and it is most relevant to those diseases in which it is difficult to get the cells that will be genetically manipulated., Graphical Abstract, A CRISPR-Cas9 system generates precise deletions (up to 90% efficiency) when two guides are used simultaneously by means of non-homologous end joining. The PAM-in/PAM-in Cas9 orientation is the best combination to generate precise deletions. This gene-editing procedure facilitates the generation of homogeneously genetically defined cell and animal models.
- Published
- 2020
41. Cell Therapy With Mesenchymal Stem Cells Induces an Innate Immune Memory Response That Attenuates Experimental Colitis in the Long Term
- Author
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Rosario Hervas-Salcedo, Juan A. Bueren, María Fernández-García, Marina I. Garin, and Mercedes Lopez-Santalla
- Subjects
medicine.medical_treatment ,Cell- and Tissue-Based Therapy ,Inflammation ,Mesenchymal Stem Cell Transplantation ,stem cell therapy ,Inflammatory bowel disease ,Time ,Cell therapy ,Mice ,Immune system ,Animals ,Antigens, Ly ,Medicine ,Myeloid Cells ,Intestinal Mucosa ,AcademicSubjects/MED00260 ,sustain protection ,CD11b Antigen ,Innate immune system ,business.industry ,Mesenchymal stem cell ,Gastroenterology ,Original Articles ,General Medicine ,Stem-cell therapy ,Colitis ,medicine.disease ,Eccojc/1000 ,Immunity, Innate ,Mice, Inbred C57BL ,Disease Models, Animal ,medicine.anatomical_structure ,Cancer research ,Bone marrow ,medicine.symptom ,business ,Immunologic Memory - Abstract
Background and Aims Mesenchymal stem cells [MSCs] are used in preclinical and clinical studies for treatment of immune-mediated disorders, thanks to their immunomodulatory properties. Cell therapy with MSCs induces multiple effects in the immune system which ultimately lead to increase in the number of immune cells with regulatory phenotype. In this study, we investigated whether the beneficial effects of MSC therapy are maintained in the long term in a clinically relevant mouse model of colitis. Methods A single dose of adipose-derived MSCs [aMSCs] was infused into dextran sulphate sodium [DSS]-induced colitic mice during the induction phase of the disease. Following a latency period of 12 weeks, mice were re-challenged with a second 7-day cycle of DSS. Results DSS-induced colitic mice treated with aMSCs showed significant reduction in their colitic disease activity index during the second DSS challenge when compared with non-aMSC treated DSS-induced colitic mice. Strikingly, the long-term protection induced by aMSC therapy was also observed in Rag-1-/- mice where no adaptive immune memory cell responses take place. Increased percentages of Ly6G+CD11b+ myeloid cells were observed 12 weeks after the first inflammatory challenge in the peritoneal cavity, spleen, and bone marrow of DSS-induced colitic mice that were infused with aMSCs. Interestingly, upon re-challenge with DSS, these animals showed a concomitant increase in the regulatory/inflammatory macrophage ratio in the colon lamina propria. Conclusions Our findings demonstrate for the first time that MSC therapy can imprint an innate immune memory-like response in mice which confers sustained protection against acute inflammation in the long term.
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- 2020
42. Targeted gene therapy into a safe harbor site in human hematopoietic progenitor cells
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Fátima Rodríguez-Fornés, Oscar Quintana-Bustamante, M. Luz Lozano, Guillermo Guenechea, Juan A. Bueren, and José C. Segovia
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0301 basic medicine ,Transcription activator-like effector nuclease ,Reporter gene ,Genetic enhancement ,CD34 ,Nucleofection ,Transfection ,Biology ,Cell biology ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,030220 oncology & carcinogenesis ,Genetics ,Molecular Medicine ,Stem cell ,Progenitor cell ,Molecular Biology - Abstract
Directed gene therapy mediated by nucleases has become a new alternative to lead targeted integration of therapeutic genes in specific regions in the genome. In this work, we have compared the efficiency of two nuclease types, TALEN and meganucleases (MN), to introduce an EGFP reporter gene in a specific site in a safe harbor locus on chromosome 21 in an intergenic region, named here SH6. The efficiency of targeted integration mediated by SH6v5-MN and SH6-TALEN in HEK-293H cells was up to 16.3 and 15.0%. A stable expression was observed both in the pool of transfected cells and in established pseudoclones, with no detection of off-target integrations by Southern blot. In human hematopoietic stem and progenitor CD34+ cells, the nucleofection process preserved the viability and clonogenic capacity of nucleofected cells, reaching up to 3.1% of specific integration of the transgene in colony forming cells when the SH6-TALEN was used, although no expression of the transgene could be found in these cells. Our results show the possibility to specifically integrate genes at the SH6 locus in CD34+ progenitor cells, although further improvements in the efficacy of the procedure are required before this approach could be used for the gene editing of hematopoietic stem cells in patients with hematopoietic diseases.
- Published
- 2020
43. The Qi Site of Cytochrome b is a Promiscuous Drug Target in Trypanosoma cruzi and Leishmania donovani
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Ian H. Gilbert, Sandra Carvalho, Stephen Thompson, Lorna MacLean, Fabio Zuccotto, Susan Wyllie, Timothy J. Miles, Lalitha Sastry, Ignacio Cotillo, Manu De Rycker, Juan A. Bueren-Calabuig, Sonia Moniz, Rachel Milne, Albane Kessler, Maria L. Marco, Julio Martin, Richard J. Wall, Alan H. Fairlamb, Juan Cantizani-Perez, Michael G. Thomas, Stephen Patterson, and Sujatha Manthri
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0301 basic medicine ,Phenotypic screening ,Trypanosoma cruzi ,030106 microbiology ,Leishmania donovani ,Computational biology ,Biology ,Article ,drug target ,03 medical and health sciences ,parasitic diseases ,Drug Discovery ,medicine ,Humans ,Chagas Disease ,Antiparasitic Agents ,Cytochrome b ,Drug discovery ,Cytochromes b ,medicine.disease ,biology.organism_classification ,3. Good health ,High-Throughput Screening Assays ,030104 developmental biology ,Infectious Diseases ,Visceral leishmaniasis ,Mechanism of action ,cytochrome b ,Coenzyme Q – cytochrome c reductase ,Leishmaniasis, Visceral ,medicine.symptom ,mechanism of action - Abstract
Available treatments for Chagas' disease and visceral leishmaniasis are inadequate, and there is a pressing need for new therapeutics. Drug discovery efforts for both diseases principally rely upon phenotypic screening. However, the optimization of phenotypically active compounds is hindered by a lack of information regarding their molecular target(s). To combat this issue we initiate target deconvolution studies at an early stage. Here, we describe comprehensive genetic and biochemical studies to determine the targets of three unrelated phenotypically active compounds. All three structurally diverse compounds target the Qi active-site of cytochrome b, part of the cytochrome bc1 complex of the electron transport chain. Our studies go on to identify the Qi site as a promiscuous drug target in Leishmania donovani and Trypanosoma cruzi with a propensity to rapidly mutate. Strategies to rapidly identify compounds acting via this mechanism are discussed to ensure that drug discovery portfolios are not overwhelmed with inhibitors of a single target.
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- 2020
44. Optimised molecular genetic diagnostics of Fanconi anaemia by whole exome sequencing and functional studies
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Rafael Fernández-Delgado, Massimo Bogliolo, Jordi Surrallés, Inmaculada Pérez de Soto, Fatima Bañez, Christopher Bauser, Cristina Beléndez-Bieler, Joaquín Dopazo, Eva M. Galvez, Raquel Sáez-Villaverde, Laura Rosiñol, Antonio Molinés, José Moraleda Jimenez, Miriam Aza-Carmona, Neda Stjepanovic, Gregorio de la Mata, Núria Muñoz-Subirana, Albert Català, Juan Miguel Bergua Burgues, Maria Marín, Leonort Senent, Ines Hernadez, Cristina Diaz-Heredia, Bienvenida Argilés, A. Figuera, Judith Reina-Castillón, Estela Carrasco, Macarena Gonzalez, Marta García, José A. Casado, José Nieto, Julián Sevilla, Luis A. Pérez-Jurado, Elena Cela, Ricardo López Almaraz, Isabel Cuesta, Antonio Escudero Soto, Raquel Portugal, José Manue Vagace, Benjamín Rodríguez-Santiago, Tobias Paprotka, Isabel Badell, Inés Hernando, Raquel Hladun, Cristina Vicho, Marta Barragaño, Anna Carrió, Pia Gallano, Francisco Lendínez, José Miguel Cosuelo, Roser Pujol, Marcos López-Sánchez, Ana Ruiz-Llobet, María Tapia, Phil Ancliff, Juan Antonio Muñoz, Monica Lopez, María Luisa Antelo, Alexandra Regueiro, Alberto Valiente, F.M. Garcia, Juan A. Bueren, Paula Río, Beatriz Arrizabalaga, Ana Maria Galera-Miñarro, Maria Carmen Garcia-Pardos, Judith Balmaña, and Lidia Gonzalez-Quereda
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Male ,0301 basic medicine ,haematology (incl blood transfusion) ,DNA Copy Number Variations ,DNA Repair ,DNA repair ,Mutation, Missense ,Single-nucleotide polymorphism ,Biology ,Polymorphism, Single Nucleotide ,Cell Line ,Gene Knockout Techniques ,03 medical and health sciences ,0302 clinical medicine ,FANCE ,hemic and lymphatic diseases ,Exome Sequencing ,FANCD2 ,Genetics ,Humans ,genetics ,Genetic Predisposition to Disease ,Copy-number variation ,Genetics (clinical) ,Exome sequencing ,Fanconi Anemia Complementation Group A Protein ,Point mutation ,clinical genetics, genetics, haematology (incl blood transfusion) ,FANCA ,DNA-Binding Proteins ,Fanconi Anemia ,030104 developmental biology ,030220 oncology & carcinogenesis ,Female ,clinical genetics - Abstract
PurposePatients with Fanconi anaemia (FA), a rare DNA repair genetic disease, exhibit chromosome fragility, bone marrow failure, malformations and cancer susceptibility. FA molecular diagnosis is challenging since FA is caused by point mutations and large deletions in 22 genes following three heritability patterns. To optimise FA patients’ characterisation, we developed a simplified but effective methodology based on whole exome sequencing (WES) and functional studies.Methods68 patients with FA were analysed by commercial WES services. Copy number variations were evaluated by sequencing data analysis with RStudio. To test FANCA missense variants, wt FANCA cDNA was cloned and variants were introduced by site-directed mutagenesis. Vectors were then tested for their ability to complement DNA repair defects of a FANCA-KO human cell line generated by TALEN technologies.ResultsWe identified 93.3% of mutated alleles including large deletions. We determined the pathogenicity of three FANCA missense variants and demonstrated that two FANCA variants reported in mutations databases as ‘affecting functions’ are SNPs. Deep analysis of sequencing data revealed patients’ true mutations, highlighting the importance of functional analysis. In one patient, no pathogenic variant could be identified in any of the 22 known FA genes, and in seven patients, only one deleterious variant could be identified (three patients each with FANCA and FANCD2 and one patient with FANCE mutations)ConclusionWES and proper bioinformatics analysis are sufficient to effectively characterise patients with FA regardless of the rarity of their complementation group, type of mutations, mosaic condition and DNA source.
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- 2019
45. Preclinical safety and efficacy of lentiviral-mediated gene therapy for leukocyte adhesion deficiency type I
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Cristina Mesa-Núñez, Carlos Damián, María Fernández-García, Begoña Díez, Gayatri Rao, Jonathan D. Schwartz, Ken M. Law, Julián Sevilla, Paula Río, Rosa Yáñez, Juan A. Bueren, and Elena Almarza
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Genetics ,Molecular Medicine ,Molecular Biology - Abstract
Leukocyte adhesion deficiency type I (LAD-I) is a primary immunodeficiency caused by mutations in the
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- 2021
46. The European Society of Gene and Cell Therapy: A Nearly 30-Year Endeavor to Make Gene Therapy a Clinical Reality
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Juan A. Bueren, Alberto Auricchio, Elizabeth M. Wilson, Hildegard Büning, Axel Schambach, Buning, H., Wilson, E., Bueren, J., Schambach, A., and Auricchio, A.
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Sirolimus ,2019-20 coronavirus outbreak ,Coronavirus disease 2019 (COVID-19) ,business.industry ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,Genetic enhancement ,Cell- and Tissue-Based Therapy ,Coronary Artery Disease ,Genetic Therapy ,Virology ,Clinical reality ,Cell therapy ,Genetics ,Molecular Medicine ,Medicine ,Humans ,business ,Molecular Biology ,Gene - Abstract
no abstract available
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- 2021
47. Elucidation of Ligand-Dependent Modulation of Disorder-Order Transitions in the Oncoprotein MDM2.
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Juan A. Bueren-Calabuig and Julien Michel
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- 2015
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48. Gene Therapy Restores the Transcriptional Program of Hematopoietic Stem Cells in Fanconi Anemia
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Miren Lasaga, Paula Río, Amaia Vilas-Zornoza, Nuria Planell, Susana Navarro, Diego Alignani, Beatriz Fernández-Varas, Josune Zubicaray, Roser M. Pujol, Eileen Nicoletti, Jonathan D. Schwartz, Julián Sevilla, Marina Ainciburi, Asier Ullate-Agote, Jordi Surrallés, Rosario Perona, Leandro Sastre, Felipe Prosper, David Gomez-Cabrero, and Juan A. Bueren
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medicine.anatomical_structure ,DNA damage ,Fanconi anemia ,DNA repair ,Genetic enhancement ,medicine ,Cancer research ,Hematopoietic stem cell ,DNA Repair Pathway ,Progenitor cell ,Biology ,medicine.disease ,Gene - Abstract
SUMMARY PARAGRAPH Fanconi anemia (FA) is a monogenic inherited disease associated with mutations in genes that encode for proteins participating in the FA/BRCA DNA repair pathway. Mutations in FA genes result in chromosomal instability and cell death, leading to cancer risks and progressive cell mortality, most notably in hematopoietic stem and progenitor cells (HSPC). Recently, we showed the first clinical evidence that gene therapy confers engraftment and proliferative advantage of gene-corrected HSPCs in FA patients1. Despite this and many other gene therapy advances, the question of whether the molecular pathways affected in monogenic diseases can be reverted by lentiviral-mediated gene therapy has never been addressed. This is even more challenging in DNA repair syndromes such as FA since in these cases, transcriptional defects in affected cells might not be restored due to DNA damage accumulated prior to gene therapy. Using single-cell RNA sequencing in HSPCs from FA-A patients previously treated by ex vivo gene therapy, we demonstrate that lentiviral-mediated gene therapy prior to severe bone marrow failure not only restores the expression of the defective gene, but also induces a long-term correction of the transcriptional program in FA HSPCs, which then acquire a signature characteristic of healthy HSPCs. Our results reveal new molecular evidence showing the potential of gene therapy to fully rescue phenotypic defects in FA, a devastating HSPC disease characterized by defective DNA repair.
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- 2021
49. Mechanism of allosteric activation of human mRNA cap methyltransferase (RNMT) by RAM: insights from accelerated molecular dynamics simulations
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Juan A. Bueren-Calabuig, Victoria H. Cowling, Andrei V. Pisliakov, and Marcus G Bage
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Protein Conformation, alpha-Helical ,S-Adenosylmethionine ,Transcription, Genetic ,Gene Expression ,RNA polymerase II ,Primary transcript ,01 natural sciences ,Substrate Specificity ,chemistry.chemical_compound ,Transcription (biology) ,Cloning, Molecular ,0303 health sciences ,010304 chemical physics ,biology ,Drug discovery ,Nucleic Acid Enzymes ,RNA-Binding Proteins ,Translation (biology) ,S-Adenosylhomocysteine ,Recombinant Proteins ,Cell biology ,Thermodynamics ,RNA Polymerase II ,Protein Binding ,RNA Caps ,Allosteric regulation ,Genetic Vectors ,Guanosine ,Molecular Dynamics Simulation ,03 medical and health sciences ,Allosteric Regulation ,0103 physical sciences ,Genetics ,Escherichia coli ,Humans ,Protein Interaction Domains and Motifs ,Amino Acid Sequence ,Binding site ,030304 developmental biology ,Messenger RNA ,Binding Sites ,Sequence Homology, Amino Acid ,RNA ,Cooperative binding ,Methyltransferases ,Kinetics ,chemistry ,biology.protein ,Protein Conformation, beta-Strand ,Sequence Alignment - Abstract
The RNA guanine-N7 methyltransferase (RNMT) in complex with RNMT-activating miniprotein (RAM) catalyses the formation of a N7-methylated guanosine cap structure on the 5′ end of nascent RNA polymerase II transcripts. The mRNA cap protects the primary transcript from exonucleases and recruits cap-binding complexes that mediate RNA processing, export and translation. By using microsecond standard and accelerated molecular dynamics simulations, we provide for the first time a detailed molecular mechanism of allosteric regulation of RNMT by RAM. We show that RAM selects the RNMT active site conformations that are optimal for binding of substrates (AdoMet and the cap), thus enhancing their affinity. Furthermore, our results strongly suggest the likely scenario in which the cap binding promotes the subsequent AdoMet binding, consistent with the previously suggested cooperative binding model. By employing the network community analyses, we revealed the underlying long-range allosteric networks and paths that are crucial for allosteric regulation by RAM. Our findings complement and explain previous experimental data on RNMT activity. Moreover, this study provides the most complete description of the cap and AdoMet binding poses and interactions within the enzyme’s active site. This information is critical for the drug discovery efforts that consider RNMT as a promising anti-cancer target.
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- 2019
50. Molecular basis for recognition of the Group A Carbohydrate backbone by the PlyC streptococcal bacteriophage endolysin
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C. M. Scholte, Vincent A. Fischetti, Daniel C. Nelson, Helge C. Dorfmueller, Natalia Korotkova, Juan A. Bueren-Calabuig, Fabio Zuccotto, A. Arunrao Pohane, H. King, and S. Ajay Castro
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Bacteriophage ,Cell wall ,chemistry.chemical_compound ,biology ,chemistry ,Lytic cycle ,Biochemistry ,Lysin ,Glycosyl ,Peptidoglycan ,biology.organism_classification ,Epitope ,Binding domain - Abstract
Endolysins are peptidoglycan (PG) hydrolases that function as part of the bacteriophage (phage) lytic system to release progeny phage at the end of a replication cycle. Notably, endolysins alone can produce lysis without phage infection, which offers an attractive alternative to traditional antibiotics. Endolysins from phage that infect Gram-positive bacterial hosts contain at least one enzymatically active domain (EAD) responsible for hydrolysis of PG bonds and a cell wall binding domain (CBD) that binds a cell wall epitope, such as a surface carbohydrate, providing some degree of specificity for the endolysin. Whilst the EADs typically cluster into conserved mechanistic classes with well-defined active sites, relatively little is known about the nature of the CBDs and only a few binding epitopes for CBDs have been elucidated. The major cell wall components of many streptococci are the polysaccharides that contain the polyrhamnose (pRha) backbone modified with species-specific and serotype-specific glycosyl side chains. In this report, using molecular genetics, microscopy, flow cytometry and lytic activity assays, we demonstrate the interaction of PlyCB, the CBD subunit of the streptococcal PlyC endolysin, with the pRha backbone of the cell wall polysaccharides, Group A Carbohydrate (GAC) and serotype c-specific carbohydrate (SCC) expressed by the Group A Streptococcus and Streptococcus mutans, respectively. Molecular dynamics simulations reveal a previously unidentified binding pocket that is regulated by a gatekeeper residue and uncover that a previously reported inactive PlyC mutant is locked into a 9closed9 conformation. Docking studies with the short GAC backbone oligosaccharides expose potential protein-carbohydrate interactions and are consistent with PlyCB binding to the unmodified pRha or pRha decorated with the GAC side chains.
- Published
- 2021
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