title: Lentiviral-Mediated Gene Therapy in Fanconi Anemia-A Mice Reveals Long-Term Engraftment and Continuous Turnover of Corrected HSCs.
abstract: Fanconi anemia is a DNA repair-deficiency syndrome mainly characterized by cancer predisposition and bone marrow failure. Trying to restore the hematopoietic function in these patients, lentiviral vector-mediated gene therapy trials have recently been proposed. However, because no insertional oncogenesis studies have been conducted so far in DNA repair-deficiency syndromes such as Fanconi anemia, we have carried out a genome-wide screening of lentiviral insertion sites after the gene correction of Fanca(-/-) hematopoietic stem cells (HSCs), using LAM-PCR and 454-pyrosequencing. Our studies first demonstrated that transduction of Fanca(-/-) HSCs with a lentiviral vector designed for clinical application efficiently corrects the phenotype of Fanconi anemia repopulating cells without any sign of toxicity. The identification of more than 6,500 insertion sites in primary and secondary recipients showed a polyclonal pattern of reconstitution, as well as a continuous turnover of corrected Fanca(-/-) HSC clones, without evidences of selection towards specific common integration sites. Taken together our data show, for the first time in a DNA repair-deficiency syndrome, that lentiviral vector-mediated gene therapy efficiently corrects the phenotype of affected HSCs and promotes a healthy pattern of clonal turnover in vivo. These studies will have a particular impact in the development of new gene therapy trials in patients affected by DNA repair syndromes, particularly in Fanconi anemia.
authors.forename: F Javier
authors.initials: FJ
authors.lastname: Molina-Estevez
authors.forename: Ali
authors.initials: A
authors.lastname: Nowrouzi
authors.forename: M Luz
authors.initials: ML
authors.lastname: Lozano
authors.forename: Anne
authors.initials: A
authors.lastname: Galy
authors.forename: Sabine
authors.initials: S
authors.lastname: Charrier
authors.forename: Christof
authors.initials: C
authors.lastname: von Kalle
authors.forename: Guillermo
authors.initials: G
authors.lastname: Guenechea
authors.affiliation: CIEMAT, Edificio 70, Avenida Complutense 40, 28040 Madrid. Spain. juan.bueren@ciemat.es.
authors.forename: Juan A
authors.initials: JA
authors.lastname: Bueren
authors.forename: Manfred
authors.initials: M
authors.lastname: Schmidt
mesh_terms: D000818:Animals; D002460:Cell Line; D004260:DNA Repair / Q000235:genetics; D005199:Fanconi Anemia / Q000235:genetics / Q000628:therapy*; D052217:Fanconi Anemia Complementation Group A Protein / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000166:cytology; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D018345:Mice, Knockout; D016133:Polymerase Chain Reaction; D014161:Transduction, Genetic / Q000379:methods
chemical_list: C120277:Fanca protein, mouse; D052217:Fanconi Anemia Complementation Group A Protein