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

Division of Hematology, Department of Medicine, University of Washington, 815 Mercer Street, Seattle, WA 98109, USA.
Gene therapy (Impact Factor: 4.2). 10/2010; 17(10):1244-52. DOI: 10.1038/gt.2010.62
Source: PubMed

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.

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    • "Importantly, the overall survival and quality of life for individuals receiving this therapy have been superior compared with those who received other therapies, such as HCT (Fischer et al., 2010). Thus, the understanding already obtained from the gene therapy trials of different hematopoietic genetic diseases (Sheridan, 2011), the clinical observation in mosaic FA patients that self-corrected FA HSCs can expand and restore the hematopoietic compartment (Lo Ten Foe et al., 1997; Waisfisz et al., 1999; Gregory et al., 2001; Gross et al., 2002; Mankad et al., 2006), and the preclinical studies of FA gene therapy with lentiviral vectors (Galimi et al., 2002; Jacome et al., 2009; Muller et al., 2008; Becker et al., 2010; Gonzalez-Murillo et al., 2010), have collectively established a platform for a state-ofthe-art clinical trial of FA gene therapy. Adrian Thrasher (University College London, London, UK) described current activities using self-inactivating gammaretroviral vectors for X-linked SCID and the use of lentiviral vectors incorporating myeloid-specific promoters for the treatment of patients with chronic granulomatous disease. "
    Human gene therapy 02/2012; 23(2):141-4. DOI:10.1089/hum.2011.237 · 3.62 Impact Factor
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    ABSTRACT: Survival rates after allogeneic hematopoietic cell transplantation (HCT) for Fanconi anemia (FA) have increased dramatically since 2000. However, the use of autologous stem cell gene therapy, whereby the patient's own blood stem cells are modified to express the wild-type gene product, could potentially avoid the early and late complications of allogeneic HCT. Over the last decades, gene therapy has experienced a high degree of optimism interrupted by periods of diminished expectation. Optimism stems from recent examples of successful gene correction in several congenital immunodeficiencies, whereas diminished expectations come from the realization that gene therapy will not be free of side effects. The goal of the 1st International Fanconi Anemia Gene Therapy Working Group Meeting was to determine the optimal strategy for moving stem cell gene therapy into clinical trials for individuals with FA. To this end, key investigators examined vector design, transduction method, criteria for large-scale clinical-grade vector manufacture, hematopoietic cell preparation, and eligibility criteria for FA patients most likely to benefit. The report summarizes the roadmap for the development of gene therapy for FA.
    Molecular Therapy 05/2011; 19(7):1193-8. DOI:10.1038/mt.2011.78 · 6.43 Impact Factor
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    ABSTRACT: The Sleeping Beauty (SB) transposon system can insert defined sequences into chromosomes to direct the extended expression of therapeutic genes. Our goal is to develop the SB system for nonviral complementation of Fanconi anemia (FA), a rare autosomal recessive disorder accompanied by progressive bone marrow failure. We used a CytoPulse electroporation system (CytoPulse, Glen Burnie, MD, USA) to introduce SB transposons into human lymphoblastoid cells (LCL) derived from both Fanconi anemia type C (FA-C) defective and normal patients. Correction of the FA-C defect was assessed by resistance to mitomycin C, a DNA-crosslinking agent. Culture of both cell types with the antioxidant N-acetyl- l-cysteine improved cell viability after electroporation. Co-delivery of enhanced green fluorescent protein (GFP) transposon with SB100X transposase-encoding plasmid supported a 50- to 90-fold increase in stable GFP expression compared to that observed in the absence of SB100X for normal LCL, but in FA-C defective LCL SB100X enhancement of stable GFP-expression was a more moderate five- to 13-fold. SB-mediated integration and expression of the FA-C gene was demonstrated by the emergence of a mitomycin C-resistant population bearing characteristic transposon-chromosome junction sequences and exhibiting a mitomycin dose response identical to that of normal LCL. The SB transposon system achieved stable expression of therapeutic FA-C genes, complementing the genetic defect in patient-derived cells by nonviral gene transfer.
    The Journal of Gene Medicine 09/2011; 13(9):462-9. DOI:10.1002/jgm.1589 · 2.47 Impact Factor
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