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dc.contributor.authorQuintana-Bustamante, Oscar
dc.contributor.authorFañanas-Baquero, Sara
dc.contributor.authorOrman, Israel
dc.contributor.authorTorres-Ruiz Raul, Raul 
dc.contributor.authorDuchateau, Philippe
dc.contributor.authorPoirot, Laurent
dc.contributor.authorGouble, Agnès
dc.contributor.authorBueren, Juan A
dc.contributor.authorSegovia, Carlos
dc.identifier.citationPLoS One. 2019;14(10):e0223775.es_ES
dc.descriptionTheauthorswouldliketothankMiguelA.MartinforthecarefulmaintenanceofNSGmice,andRebecaSa ́nchezandOmairaAlberquillafortheirtechnicalassistanceinflowcytometry.TheauthorsalsothankFundacio ́n Botı ́n forpromotingtranslationalresearchattheHemato-poieticInnovativeTherapiesDivisionoftheCIEMATes_ES
dc.description.abstractPyruvate Kinase Deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene, which encodes the erythroid specific Pyruvate Kinase enzyme. Erythrocytes from PKD patients show an energetic imbalance and are susceptible to hemolysis. Gene editing of hematopoietic stem cells (HSCs) would provide a therapeutic benefit and improve safety of gene therapy approaches to treat PKD patients. In previous studies, we established a gene editing protocol that corrected the PKD phenotype of PKD-iPSC lines through a TALEN mediated homologous recombination strategy. With the goal of moving toward more clinically relevant stem cells, we aim at editing the PKLR gene in primary human hematopoietic progenitors and hematopoietic stem cells (HPSCs). After nucleofection of the gene editing tools and selection with puromycin, up to 96% colony forming units showed precise integration. However, a low yield of gene edited HPSCs was associated to the procedure. To reduce toxicity while increasing efficacy, we worked on i) optimizing gene editing tools and ii) defining optimal expansion and selection times. Different versions of specific nucleases (TALEN and CRISPR-Cas9) were compared. TALEN mRNAs with 5' and 3' added motifs to increase RNA stability were the most efficient nucleases to obtain high gene editing frequency and low toxicity. Shortening ex vivo manipulation did not reduce the efficiency of homologous recombination and preserved the hematopoietic progenitor potential of the nucleofected HPSCs. Lastly, a very low level of gene edited HPSCs were detected after engraftment in immunodeficient (NSG) mice. Overall, we showed that gene editing of the PKLR gene in HPSCs is feasible, although further improvements must to be done before the clinical use of the gene editing to correct PKD.es_ES
dc.relation.isversionofPublisher's versiones_ES
dc.subject.mesh3' Untranslated Regions es_ES
dc.subject.mesh5' Untranslated Regions es_ES
dc.subject.meshAnimals es_ES
dc.subject.meshCells, Cultured es_ES
dc.subject.meshGene Editing es_ES
dc.subject.meshHEK293 Cells es_ES
dc.subject.meshHematopoietic Stem Cells es_ES
dc.subject.meshHumans es_ES
dc.subject.meshMice es_ES
dc.subject.meshPyruvate Kinase es_ES
dc.subject.meshTranscription Activator-Like Effector Nucleases es_ES
dc.titleGene editing of PKLR gene in human hematopoietic progenitors through 5' and 3' UTR modified TALEN mRNAes_ES
dc.rights.licenseAtribución-NoComercial-CompartirIgual 4.0 Internacional*
dc.relation.publisherversion 10.1371/journal.pone.0223775es_ES
dc.identifier.journalPloS onees_ES
dc.repisalud.orgCNIOCNIO::Unidades técnicas::Unidad de Citogenética Moleculares_ES

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Atribución-NoComercial-CompartirIgual 4.0 Internacional
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