| Project/Area Number |
05670488
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Gastroenterology
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| Research Institution | Sapporo Medical University |
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Principal Investigator |
KOSHITA Yoshikazu Sapporo Medical University, 医学部, 助手 (10231309)
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| Co-Investigator(Kenkyū-buntansha) |
NEDA Hiroshi Sapporo Medical University, 医学部, 講師 (80237809)
山内 尚文 札幌医科大学, 医学部, 助手 (40274930)
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| Project Period (FY) |
1993 – 1995
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| Project Status |
Completed (Fiscal Year 1995)
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| Budget Amount *help |
¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 1995: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1994: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 1993: ¥700,000 (Direct Cost: ¥700,000)
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| Keywords | gene therapy / receptor-mediated / BPV vector / cell targeting / transferrin / BPDベクター / 細胞ターゲッティング / 牛パピローマウイルス / ベクター / 高コピー数 / 染色体外複製 / チミジンキナーゼ遺伝子 |
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| Research Abstract |
A bovine papilloma virus (BPV) vector has the characteristic to replicate extrachromosomally with a rather high copy number in recipent cells (predominantly rodent cells) and was considered difficult to transfect human cells. We reported that transfection of a BPV vector with a linear form (pYK8N2L) into human hepatoma (Mahlavu) cells resulted in stable expression of the gene with a high copy number (Acta Hepatologica Japonica, 33 ; 600,1992). We have disclosed that this can be applied to other human tumor cell lines, the uterine cervical cancer (HeLa) and the colon cancer (M7609). Further, we have identified that a single s. c. injection of the pYK8N2L into an established M7609 tumor in a nude mouse allows to express the gene at least for 3 weeks. We have developped a new targeted gene delivery method into tumor cells utilizing transferrin receptors (Tf-R) that are known to be expressed on actively proliferating tumor cells (N Y Acad Sci, 336,1994). In this method, aDNA vector (BAG containing a beta-gal gene) combined with the ligand for Tf-R (Tf) through streptoavidin-biotin can be transduced into human hepatoblastoma cells (HepG2) and M7609 via Tf-R with transfection efficiencies 10 and 15%, respectively, to express beta-gal to the same extent regardless of presense of lysosomal inhibitor, chloroquine (CQ). The pulse chase study suggested that 30% of Tf-BAG was degraded in lysosome and all or a part of the remaining 70% might go to nucleus to express beta-gal. Our method is better than the similar method by Cotten using polylysine as a linker, because their conjugate needs cytotoxic CQ for gene expression. These observations support that a BPV vector combined with Tf could be a good targeted delivery system for cancer cells in a patient with hepatoma.
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