Preclinical correction of human Fanconi anemia complementation group A bone marrow cells using a safety-modified lentiviral vector.
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.
Becker
P S
PS
Division of Hematology, Department of Medicine, University of Washington, 815 Mercer Street, Seattle, WA 98109, USA. pbecker@u.washington.edu
Taylor
J A
JA
Trobridge
G D
GD
Zhao
X
X
Beard
B C
BC
Chien
S
S
Adair
J
J
Kohn
D B
DB
Wagner
J E
JE
Shimamura
A
A
Kiem
H-P
HP
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
D052217:Fanconi Anemia Complementation Group A Protein; D016685:Mitomycin; D000111:Acetylcysteine