Gene editing of PKLR gene in human hematopoietic progenitors through 5’ and 3’ UTR modified TALEN mRNA

Autoři: Oscar Quintana-Bustamante aff001;  Sara Fañanas-Baquero aff001;  Israel Orman aff001;  Raul Torres aff003;  Philippe Duchateau aff005;  Laurent Poirot aff005;  Agnès Gouble aff005;  Juan A. Bueren aff001;  Jose C. Segovia aff001
Působiště autorů: Division of Hematopoietic Innovative Therapies, Centro de Investigaciones Energéticas Medioambientales y Tecnológicas/Centro de Investigación Biomédica en Red de Enfermedades Raras (CIEMAT/CIBERER), Madrid, Spain aff001;  Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain aff002;  Centro Nacional de Investigaciones Oncológicas (CNIO), Madrid, Spain aff003;  Instituto Josep Carreras, Barcelona, Spain aff004;  CELLECTIS, Paris, France aff005
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article


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.

Klíčová slova:

DNA – DNA sequence analysis – Genetic loci – Nested polymerase chain reaction – Nucleases – Polymerase chain reaction – TALENs – Nuclear matrix


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