Becker, PS, Taylor, JA, Trobridge, GD, Zhao, X, Beard, BC, Chien, S et al.. Preclinical correction of human Fanconi anemia complementation group A bone marrow cells using a safety-modified lentiviral vector. Gene Ther 17: 1244-1252

Division of Hematology, Department of Medicine, University of Washington, 815 Mercer Street, Seattle, WA 98109, USA.
Gene therapy (Impact Factor: 3.1). 10/2010; 17(10):1244-52. DOI: 10.1038/gt.2010.62
Source: PubMed


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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    • "The CD20-CAR gene, the iC9 gene (provided by Dr. David Spencer, GenBank NM001229), and the Δ19 gene were linked together using 2A sequence peptides [29] and cloned into the SINpWPT-GFP backbone to generate the iC9-CD20CAR-Δ19 lentiviral vector (Figure 1). Lentiviral vectors were pseudotyped with VSV-G envelope and produced by transient transfection of 293T cells [30]. "
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    ABSTRACT: Modification of T cells with chimeric antigen receptors (CAR) has emerged as a promising treatment modality for human malignancies. Integration of co-stimulatory domains into CARs can augment the activation and function of genetically targeted T cells against tumors. However, the potential for insertional mutagenesis and toxicities due to the infused cells have made development of safe methods for removing transferred cells an important consideration. We have genetically modified human T cells with a lentiviral vector to express a CD20-CAR containing both CD28 and CD137 co-stimulatory domains, a "suicide gene" relying on inducible activation of caspase 9 (iC9), and a truncated CD19 selectable marker. Rapid expansion (2000 fold) of the transduced T cells was achieved in 28 days after stimulation with artificial antigen presenting cells. Transduced T cells exhibited effective CD20-specific cytotoxic activity in vitro and in a mouse xenograft tumor model. Activation of the iC9 suicide switch resulted in efficient removal of transduced T cells both in vitro and in vivo. Our work demonstrates the feasibility and promise of this approach for treating CD20(+) malignancies in a safe and more efficient manner. A phase I clinical trial using this approach in patients with relapsed indolent B-NHL is planned.
    Full-text · Article · Dec 2013 · PLoS ONE
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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. "

    Full-text · Article · Feb 2012 · Human gene therapy
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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.
    Full-text · Article · May 2011 · Molecular Therapy
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