title: Generation of a High Number of Healthy Erythroid Cells from Gene-Edited Pyruvate Kinase Deficiency Patient-Specific Induced Pluripotent Stem Cells.
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.
authors.affiliation: Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT), Centro de Investigaciones Biomédicas en Red de Enfermedades Raras (CIBERER), Madrid 28040, Spain; Advanced Therapies Mixed Unit, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid 28040, Spain; Center for Stem Cell and Regenerative Medicine, Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center, Houston, TX 77030, USA.
authors.forename: Zita
authors.initials: Z
authors.lastname: Garate
authors.affiliation: Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT), Centro de Investigaciones Biomédicas en Red de Enfermedades Raras (CIBERER), Madrid 28040, Spain; Advanced Therapies Mixed Unit, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid 28040, Spain. Electronic address: oscar.quintana@ciemat.es.
authors.forename: Oscar
authors.initials: O
authors.lastname: Quintana-Bustamante
authors.affiliation: Center for Stem Cell and Regenerative Medicine, Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center, Houston, TX 77030, USA.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Crane
authors.affiliation: Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow G12 8QQ, UK.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Olivier
authors.affiliation: Cellectis, Paris 75013, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Poirot
authors.affiliation: Cellectis, Paris 75013, France.
authors.forename: Roman
authors.initials: R
authors.lastname: Galetto
authors.affiliation: Agios Pharmaceuticals, Cambridge, MA 02139-4169, USA.
authors.forename: Penelope
authors.initials: P
authors.lastname: Kosinski
authors.affiliation: Agios Pharmaceuticals, Cambridge, MA 02139-4169, USA.
authors.forename: Collin
authors.initials: C
authors.lastname: Hill
authors.affiliation: Agios Pharmaceuticals, Cambridge, MA 02139-4169, USA.
authors.forename: Charles
authors.initials: C
authors.lastname: Kung
authors.affiliation: Hematology and Cell Therapy, Clinica Universidad de Navarra and CIMA, Pamplona 31008, Spain.
authors.forename: Xabi
authors.initials: X
authors.lastname: Agirre
authors.affiliation: Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT), Centro de Investigaciones Biomédicas en Red de Enfermedades Raras (CIBERER), Madrid 28040, Spain; Advanced Therapies Mixed Unit, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid 28040, Spain.
authors.forename: Israel
authors.initials: I
authors.lastname: Orman
authors.affiliation: Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT), Centro de Investigaciones Biomédicas en Red de Enfermedades Raras (CIBERER), Madrid 28040, Spain; Advanced Therapies Mixed Unit, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid 28040, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Cerrato
authors.affiliation: Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT), Centro de Investigaciones Biomédicas en Red de Enfermedades Raras (CIBERER), Madrid 28040, Spain; Advanced Therapies Mixed Unit, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid 28040, Spain.
authors.forename: Omaira
authors.initials: O
authors.lastname: Alberquilla
authors.affiliation: Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT), Centro de Investigaciones Biomédicas en Red de Enfermedades Raras (CIBERER), Madrid 28040, Spain; Advanced Therapies Mixed Unit, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid 28040, Spain.
authors.forename: Fatima
authors.initials: F
authors.lastname: Rodriguez-Fornes
authors.affiliation: JST PRESTO and Ophthalmology, Keio University, Tokyo 108-8345, Japan.
authors.forename: Noemi
authors.initials: N
authors.lastname: Fusaki
authors.affiliation: Histocompatibility and Molecular Biology Laboratory, Centro de Transfusion de Madrid, Madrid 28032, Spain.
authors.forename: Felix
authors.initials: F
authors.lastname: Garcia-Sanchez
authors.affiliation: Serviço de Hematologia, Centro Hospitalar e Universitario de Coimbra, Coimbra 3000-075, Portugal.
authors.forename: Tabita M
authors.initials: TM
authors.lastname: Maia
authors.affiliation: Serviço de Hematologia, Centro Hospitalar e Universitario de Coimbra, Coimbra 3000-075, Portugal.
authors.forename: Maria L
authors.initials: ML
authors.lastname: Ribeiro
authors.affiliation: Hospital Universitario Niño Jesús, Madrid 28009, Spain.
authors.forename: Julian
authors.initials: J
authors.lastname: Sevilla
authors.affiliation: Hematology and Cell Therapy, Clinica Universidad de Navarra and CIMA, Pamplona 31008, Spain.
authors.forename: Felipe
authors.initials: F
authors.lastname: Prosper
authors.affiliation: Agios Pharmaceuticals, Cambridge, MA 02139-4169, USA.
authors.forename: Shengfang
authors.initials: S
authors.lastname: Jin
authors.affiliation: Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow G12 8QQ, UK.
authors.forename: Joanne
authors.initials: J
authors.lastname: Mountford
authors.affiliation: Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT), Centro de Investigaciones Biomédicas en Red de Enfermedades Raras (CIBERER), Madrid 28040, Spain; Advanced Therapies Mixed Unit, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid 28040, Spain.
authors.forename: Guillermo
authors.initials: G
authors.lastname: Guenechea
authors.affiliation: Cellectis, Paris 75013, France.
authors.forename: Agnes
authors.initials: A
authors.lastname: Gouble
authors.affiliation: Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT), Centro de Investigaciones Biomédicas en Red de Enfermedades Raras (CIBERER), Madrid 28040, Spain; Advanced Therapies Mixed Unit, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid 28040, Spain.
authors.forename: Juan A
authors.initials: JA
authors.lastname: Bueren
authors.affiliation: Center for Stem Cell and Regenerative Medicine, Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center, Houston, TX 77030, USA.
authors.forename: Brian R
authors.initials: BR
authors.lastname: Davis
authors.affiliation: Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT), Centro de Investigaciones Biomédicas en Red de Enfermedades Raras (CIBERER), Madrid 28040, Spain; Advanced Therapies Mixed Unit, Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid 28040, Spain. Electronic address: jc.segovia@ciemat.es.
authors.forename: Jose C
authors.initials: JC
authors.lastname: Segovia
mesh_terms: D000483:Alleles; D000746:Anemia, Hemolytic, Congenital Nonspherocytic / Q000235:genetics* / Q000628:therapy*; D001483:Base Sequence; D002452:Cell Count; D018076:DNA, Complementary / Q000235:genetics; D041905:Erythroid Cells / Q000166:cytology* / Q000378:metabolism; D018390:Gene Targeting; D015316:Genetic Therapy; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D011770:Pyruvate Kinase / Q000172:deficiency* / Q000235:genetics*; D015323:Pyruvate Metabolism, Inborn Errors / Q000235:genetics* / Q000628:therapy*; D011995:Recombination, Genetic
chemical_list: D018076:DNA, Complementary; D011770:Pyruvate Kinase