| Project/Area Number |
11671014
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Hematology
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| Research Institution | Jichi Medical School |
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Principal Investigator |
HANAZONO Yutaka Assistant Professor, Division of Genetic Therapeutics, Center for Molecular Medicine, Jichi Medical School, 医学部, 講師 (70251246)
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| Project Period (FY) |
1999 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1999: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | hematopoietic stem cells / gene therapy / retroviral vector / Inverse PCR / cynomolgus monkeys / Primate model / 骨髄移植 / 遺伝子標識 / GFP |
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| Research Abstract |
Hematopoietic stem cells (HSCs) are desirable targets for gene therapy because of their self-renewal and multilineage differentiation ability. Retroviral vectors are extensively used for HSC gene therapy. However, the initial human trials of HSC gene marking and therapy showed that the gene transfer efficiency into human HSCs with retroviral vectors was very low in contrast to the much higher efficiency observed in murine experiments. The more quiescent nature of human HSCs and the lower density of retroviral receptors on them hindered the efficient gene transfer with retroviral vectors. Since nonhuman primates have marked similarity to humans in all aspects including the HSC biology, their models are considered to be important to evaluate and improve gene transfer into human HSCs. We have established a nonhuman primate cynomolgus macaque model for autologous HSC transplantation. Using this model, relatively high levels (5-15%) of gene-modified hematopoietic progenitor cells has been a
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chieved after gene transfer into HSCs and their autologous transplantation. This has been made possible by improving ex vivo transduction conditions such as introduction of Flt-3 ligand and specific fibronectin fragment (CH-296) into ex vivo culture during transduction and the use of retroviral vectors pseudotyped with the gibbon ape leukemia virus envelope. Our novel strategy of using the selective amplifier gene (also designated the molecular growth switch) is now being tested to further increase the fraction of gene-modified cells using the nonhuman primate model.
Although, in mice, the entire hematopoiesis can be reconstituted by a single or a few HSCs, little data about clonality of hematopoiesis in larger animals have been available. Relatively high levels of stable retroviral gene transfer described above have allowed us to conduct clonal analysis of hematopoiesis. We are currently using inverse PCR to characterize the specific retrovirally-marked clones (by the unique insertion site flanking the provirus) contributing to hematopoiesis over time. Less
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