2026-06-14T03:46:21Zhttps://repisalud.isciii.es/rest/oai/requestoai:repisalud.isciii.es:20.500.12105/93542024-11-29T15:24:07Zcom_20.500.12105_2173com_20.500.12105_2051col_20.500.12105_19597
Repisalud
author
Quintana-Bustamante, Oscar
author
Fañanas-Baquero, Sara
author
Orman, Israel
author
Torres-Ruiz Raul, Raul
author
Duchateau, Philippe
author
Poirot, Laurent
author
Gouble, Agnès
author
Bueren, Juan A
author
Segovia, Jose Carlos
2020-03-27T17:16:59Z
2020-03-27T17:16:59Z
2019-10-16
PLoS One. 2019;14(10):e0223775.
10.1371/journal.pone.0223775
1932-6203
1932-6203
PloS one
31618280
http://hdl.handle.net/20.500.12105/9354
Pyruvate 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.
eng
Gene editing of PKLR gene in human hematopoietic progenitors through 5' and 3' UTR modified TALEN mRNA
journal article
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URL
https://repisalud.isciii.es/bitstreams/12796fe9-1dbc-4698-bbda-f0b1857d0850/download
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GeneeditingofPKLRgeneinhuman_2019.pdf
URL
https://repisalud.isciii.es/bitstreams/acc09edd-1d54-4177-b3f4-bda39e297cd1/download
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GeneeditingofPKLRgeneinhuman_2019.pdf.txt