title: Targeted gene therapy and cell reprogramming in Fanconi anemia.
abstract: Gene targeting is progressively becoming a realistic therapeutic alternative in clinics. It is unknown, however, whether this technology will be suitable for the treatment of DNA repair deficiency syndromes such as Fanconi anemia (FA), with defects in homology-directed DNA repair. In this study, we used zinc finger nucleases and integrase-defective lentiviral vectors to demonstrate for the first time that FANCA can be efficiently and specifically targeted into the AAVS1 safe harbor locus in fibroblasts from FA-A patients. Strikingly, up to 40% of FA fibroblasts showed gene targeting 42 days after gene editing. Given the low number of hematopoietic precursors in the bone marrow of FA patients, gene-edited FA fibroblasts were then reprogrammed and re-differentiated toward the hematopoietic lineage. Analyses of gene-edited FA-iPSCs confirmed the specific integration of FANCA in the AAVS1 locus in all tested clones. Moreover, the hematopoietic differentiation of these iPSCs efficiently generated disease-free hematopoietic progenitors. Taken together, our results demonstrate for the first time the feasibility of correcting the phenotype of a DNA repair deficiency syndrome using gene-targeting and cell reprogramming strategies.
authors.affiliation: Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain.
authors.forename: Paula
authors.initials: P
authors.lastname: Rio
authors.affiliation: Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain.
authors.forename: Rocio
authors.initials: R
authors.lastname: Baños
authors.affiliation: San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Angelo
authors.initials: A
authors.lastname: Lombardo
authors.affiliation: Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain.
authors.forename: Oscar
authors.initials: O
authors.lastname: Quintana-Bustamante
authors.affiliation: Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain.
authors.forename: Lara
authors.initials: L
authors.lastname: Alvarez
authors.affiliation: Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain.
authors.forename: Zita
authors.initials: Z
authors.lastname: Garate
authors.affiliation: San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Pietro
authors.initials: P
authors.lastname: Genovese
authors.affiliation: Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain.
authors.forename: Elena
authors.initials: E
authors.lastname: Almarza
authors.affiliation: Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain.
authors.forename: Antonio
authors.initials: A
authors.lastname: Valeri
authors.affiliation: Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain.
authors.forename: Begoña
authors.initials: B
authors.lastname: Díez
authors.affiliation: Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain.
authors.forename: Susana
authors.initials: S
authors.lastname: Navarro
authors.affiliation: NIMGenetics SL, Madrid, Spain.
authors.forename: Yaima
authors.initials: Y
authors.lastname: Torres
authors.affiliation: Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain Universidad Autónoma Barcelona/CIBERER, Barcelona, Spain.
authors.forename: Juan P
authors.initials: JP
authors.lastname: Trujillo
authors.affiliation: Division of Epithelial Biomedicine, CIEMAT/CIBERER, Madrid, Spain.
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Murillas
authors.affiliation: Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain.
authors.forename: Jose C
authors.initials: JC
authors.lastname: Segovia
authors.affiliation: NIMGenetics SL, Madrid, Spain.
authors.forename: Enrique
authors.initials: E
authors.lastname: Samper
authors.affiliation: Universidad Autónoma Barcelona/CIBERER, Barcelona, Spain.
authors.forename: Jordi
authors.initials: J
authors.lastname: Surralles
authors.affiliation: Sangamo BioSciences Inc., Richmond, CA, USA.
authors.forename: Philip D
authors.initials: PD
authors.lastname: Gregory
authors.affiliation: Sangamo BioSciences Inc., Richmond, CA, USA.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Holmes
authors.affiliation: San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy Vita Salute San Raffaele University, Milan, Italy juan.bueren@ciemat.es naldini.luigi@hsr.it.
authors.forename: Luigi
authors.initials: L
authors.lastname: Naldini
authors.affiliation: Division of Hematopoietic Innovative Therapies, CIEMAT/CIBERER, Madrid, Spain Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain juan.bueren@ciemat.es naldini.luigi@hsr.it.
authors.forename: Juan A
authors.initials: JA
authors.lastname: Bueren
mesh_terms: D002478:Cells, Cultured; D065150:Cellular Reprogramming; D005199:Fanconi Anemia / Q000235:genetics* / Q000628:therapy*; D052217:Fanconi Anemia Complementation Group A Protein / Q000235:genetics*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D018390:Gene Targeting; D015316:Genetic Therapy / Q000379:methods; D006410:Hematopoiesis; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism
chemical_list: D052217:Fanconi Anemia Complementation Group A Protein
keywords: Fanconi anemia; cell reprogramming; gene‐targeting; iPSCs; zinc finger nucleases