title: Preclinical correction of human Fanconi anemia complementation group A bone marrow cells using a safety-modified lentiviral vector.
abstract: One of the major hurdles for the development of gene therapy for Fanconi anemia (FA) is the increased sensitivity of FA stem cells to free radical-induced DNA damage during ex vivo culture and manipulation. To minimize this damage, we have developed a brief transduction procedure for lentivirus vector-mediated transduction of hematopoietic progenitor cells from patients with Fanconi anemia complementation group A (FANCA). The lentiviral vector FancA-sW contains the phosphoglycerate kinase promoter, the FANCA cDNA, and a synthetic, safety-modified woodchuck post transcriptional regulatory element (sW). Bone marrow mononuclear cells or purified CD34(+) cells from patients with FANCA were transduced in an overnight culture on recombinant fibronectin peptide CH-296, in low (5%) oxygen, with the reducing agent, N-acetyl-L-cysteine (NAC), and a combination of growth factors, granulocyte colony-stimulating factor (G-CSF), Flt3 ligand, stem cell factor, and thrombopoietin. Transduced cells plated in methylcellulose in hypoxia with NAC showed increased colony formation compared with 21% oxygen without NAC (P<0.03), showed increased resistance to mitomycin C compared with green fluorescent protein (GFP) vector-transduced controls (P<0.007), and increased survival. Thus, combining short transduction and reducing oxidative stress may enhance the viability and engraftment of gene-corrected cells in patients with FANCA.
authors.affiliation: Division of Hematology, Department of Medicine, University of Washington, 815 Mercer Street, Seattle, WA 98109, USA. pbecker@u.washington.edu
authors.forename: P S
authors.initials: PS
authors.lastname: Becker
authors.forename: J A
authors.initials: JA
authors.lastname: Taylor
authors.forename: G D
authors.initials: GD
authors.lastname: Trobridge
authors.forename: X
authors.initials: X
authors.lastname: Zhao
authors.forename: B C
authors.initials: BC
authors.lastname: Beard
authors.forename: S
authors.initials: S
authors.lastname: Chien
authors.forename: J
authors.initials: J
authors.lastname: Adair
authors.forename: D B
authors.initials: DB
authors.lastname: Kohn
authors.forename: J E
authors.initials: JE
authors.lastname: Wagner
authors.forename: A
authors.initials: A
authors.lastname: Shimamura
authors.forename: H-P
authors.initials: HP
authors.lastname: Kiem
mesh_terms: D000111:Acetylcysteine / Q000378:metabolism / Q000494:pharmacology; D001854:Bone Marrow Cells / Q000166:cytology / Q000378:metabolism; D005199:Fanconi Anemia / Q000473:pathology / Q000628:therapy*; D052217:Fanconi Anemia Complementation Group A Protein / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D016685:Mitomycin / Q000494:pharmacology; D014161:Transduction, Genetic
chemical_list: D052217:Fanconi Anemia Complementation Group A Protein; D016685:Mitomycin; D000111:Acetylcysteine