Xu, L, O’Malley, T, Sands, MS, Wang, B, Meyerrose, T, Haskins, ME et al.. In vivo transduction of hematopoietic stem cells after neonatal intravenous injection of an amphotropic retroviral vector in mice. Mol Ther 10: 37-44

Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA.
Molecular Therapy (Impact Factor: 6.23). 08/2004; 10(1):37-44. DOI: 10.1016/j.ymthe.2004.04.010
Source: PubMed


Hematopoietic stem cells (HSC) are important targets for gene therapy. Most protocols involve ex vivo modification, in which HSC are transduced in vitro and injected into the recipient. An in vivo delivery method might simplify HSC gene therapy. We previously demonstrated that iv injection of an amphotropic retroviral vector (RV) into newborn mice resulted in long-term expression from hepatocytes. The goal of this study was to determine if HSC were also transduced. After neonatal administration of 1 x 10(10) transducing units/kg of RV, peripheral blood cells had approximately 0.1 copy of RV per cell for up to 22 months. At 18 months, RV sequences were detected in T, B, and myeloid cells from bone marrow (BM). Unfractionated BM was transplanted into naive recipients after total body irradiation. Recipients maintained similar levels of the RV in their blood cells for 10 months, at which time RV sequences were present at the same integration site in all lineages of cells from BM. We conclude that neonatal iv injection of RV results in transduction of HSC in mice, which might be used for BM-directed gene therapy. Transduction of blood cells after liver-directed neonatal gene therapy might have adverse effects in patients, although no leukemias developed here.

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    • "Trans-expression of retroviral structural proteins from non vector-homologous plasmids avoids the production of replication competent retrovirus (RCR) [4,5]. Many retroviral vectors are derived from murine leukemia virus (MLV) in both, ecotropic and amphotropic versions [6,7]. Lentiviral vectors based on HIV may offer advantages because of their lower insertion frequency in crucial loci involved in cell growth regulation and their ability to transduce non-dividing cells [8,9]. "
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    ABSTRACT: Retroviral vectors are widely used tools for gene delivery and gene therapy. They are useful for gene expression studies and genetic manipulation in vitro and in vivo. Many retroviral vectors are derived from the mouse gammaretrovirus, murine leukemia virus (MLV). These vectors have been widely used in gene therapy clinical trials. XMRV, initially found in prostate cancer tissue, was the first human gammaretrovirus described. We developed a new retroviral vector based on XMRV called pXC. It was developed for gene transfer to human cells and is produced by transient cotransfection of LNCaP cells with pXC and XMRV-packaging plasmids. We demonstrated that pXC mediates expression of inserted transgenes in cell lines. This new vector will be a useful tool for gene transfer in human and non-human cell lines, including gene therapy studies.
    Full-text · Article · Jun 2011 · Virology Journal
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    • "This study demonstrates that neonatal intravenous injection of 10 10 TU/kg of this -RV, which was sufficient to result in expression in 23 9% of hepatocytes and 0.24 0.20 copy of -RV DNA per cell in liver, did not increase the risk of malignancy at 1.75 years over that observed in control mice of the same strain that did not receive the -RV. Although the hematopoietic tumors that developed in -RVtreated mice had 0.04 copy of -RV per cell, this was likely due to contamination of the tumor with normal hematopoietic cells, as this value was lower than the value of 0.10 copy per cell that we previously observed in peripheral blood and spleen after neonatal intravenous injection of a similar -RV into mice (Xu et al., 2004). If integration adjacent to an oncogene were driving the development of the tumor, the -RV DNA copy number should be 0.5 copy per cell, as histological evaluation of these tumors suggests that 50% of the cells were abnormal (data not shown). "
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    ABSTRACT: Neonatal intravenous injection of gammaretroviral vectors (gamma-RVs) with an intact long terminal repeat (LTR) and an internal liver promoter can result in long-term expression in liver cells and correction of mucopolysaccharidosis. Some expression also occurs in blood cells and brain, which likely derives from the LTR, and may contribute to clinical efficacy. The goal of this project was to determine whether neonatal gene therapy with an LTR-intact gamma-RV would induce tumors in mice. Fifty-one normal newborn C57BL/6 mice were injected intravenously at 10(10) transducing units/kg with a gamma-RV expressing canine beta-glucuronidase (GUSB) cDNA. This resulted in transduction of 23 +/- 9% of hepatocytes as determined by histochemical staining, and 0.24 +/- 0.20 copy of gamma-RV DNA per cell in liver as determined by real-time polymerase chain reaction. Serum GUSB activity was stable for 1.75 years after transduction at 705 +/- 119 units/ml. Ninety-six percent of mice survived for the duration of evaluation, which was similar to the survival rate for 65 control mice that were not injected with gamma-RV. One gamma-RV-treated mouse (2%) developed a small (diameter, 2 mm) liver adenoma, which was similar to the frequency of liver adenomas (2%) or hepatocellular carcinoma (2%) in untreated mice. Although 22% of gamma-RV-treated mice developed hematopoietic tumors, none contained high gamma-RV DNA copy numbers, and the frequency was similar to that in the control group (22%). We conclude that neonatal intravenous injection of an LTR-intact gamma-RV does not have a high risk of inducing cancer in mice.
    Full-text · Article · Nov 2008 · Human gene therapy
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    • "Blood-derived cells could be the source of DNA and RNA sequences in other organs in dogs, as hematopoietic cells were transduced in mice with a similar neonatal gene therapy approach [33], and blood cells can migrate into other organs. Therefore, peripheral WBCs were evaluated for RV DNA and RNA sequences, and for GUSB activity. "
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    ABSTRACT: Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disease due to deficient activity of beta-glucuronidase (GUSB) that results in accumulation of glycosaminoglycans in many organs. We have previously reported that neonatal intravenous injection of a gamma retroviral vector (RV) expressing canine GUSB resulted in transduction of hepatocytes, high levels of GUSB modified with mannose 6-phosphate in blood, and reduction in disease manifestations in the heart, bone, and eye. However, it was unclear if liver was the only site of expression, and the effect upon other organs was not assessed. We demonstrate here that blood cells from these RV-treated MPS VII dogs had substantial copies of RV DNA, and expressed the RNA at 2% of the level found in liver. Therefore, expression of GUSB in blood cells may synergize with uptake of GUSB from blood to reduce storage in organs. The RV-treated dogs had marked biochemical and pathological evidence of reduction in storage in liver, thymus, spleen, small intestines, and lung, and partial reduction of storage in kidney tubules. The brain had 6% of normal GUSB activity, and biochemical and pathological evidence of reduction in storage in neurons and other cell types. Thus, this neonatal gene therapy approach is effective and might be used in humans if it proves to be safe. Both secretion of enzyme into blood by hepatocytes, and expression in blood cells that migrate into organs, may contribute to correction of disease.
    Full-text · Article · Feb 2006 · Molecular Genetics and Metabolism
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