Preclinical correction of human Fanconi anemia complementation group A bone marrow cells using a safety-modified lentiviral vector

P. S. Becker, J. A. Taylor, G. D. Trobridge, X. Zhao, B. C. Beard, S. Chien, J. Adair, D. B. Kohn, J. E. Wagner, A. Shimamura, H. P. Kiem

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30 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)1244-1252
Number of pages9
JournalGene therapy
Volume17
Issue number10
DOIs
StatePublished - Oct 1 2010

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Keywords

  • Hhypoxia
  • gene therapy
  • mitomycin C
  • reducing agent

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics

Cite this

Becker, P. S., Taylor, J. A., Trobridge, G. D., Zhao, X., Beard, B. C., Chien, S., Adair, J., Kohn, D. B., Wagner, J. E., Shimamura, A., & Kiem, H. P. (2010). Preclinical correction of human Fanconi anemia complementation group A bone marrow cells using a safety-modified lentiviral vector. Gene therapy, 17(10), 1244-1252. https://doi.org/10.1038/gt.2010.62