| Project/Area Number |
17209051
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Orthopaedic surgery
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| Research Institution | Research Institute, Osaka Medical Center for Cancer and Cardiovascular Disaeses |
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Principal Investigator |
TAKAHASHI Katsuhito Research Institute, Osaka Medical Center for Cancer and Cardiovascular Disaeses, Research Institute, Director (40211338)
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| Co-Investigator(Kenkyū-buntansha) |
YOSHIKAWA Hideki Osaka University, Graduate School of Medicine, 教授 (60191558)
YAMAMURA Hisako Osaka Medical Center for Cancer and Cardiovascular Diseases, Research Institute, Associate Director (50342994)
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| Project Period (FY) |
2005 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥30,160,000 (Direct Cost: ¥23,200,000、Indirect Cost: ¥6,960,000)
Fiscal Year 2006: ¥11,570,000 (Direct Cost: ¥8,900,000、Indirect Cost: ¥2,670,000)
Fiscal Year 2005: ¥18,590,000 (Direct Cost: ¥14,300,000、Indirect Cost: ¥4,290,000)
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| Keywords | Herpes Virus / Calponin / Sarcoma / Gene Therapy / GMP-grade / GMP |
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| Research Abstract |
It is necessary to perform efficacy testing and biological evaluation to develop oncolytic viral agents for cancer gene therapy. There are three steps in the procedures of purification and production of the viral agents in the GMP-grade, that is 1) construction of the master cell bank and their biological evaluation, 2) generation of the master viral seed stoch and their biological evaluation and 3) purified viral production and their biological evaluation. Of those, the construction of the master cell bank is the most important step for generation of the viral stock safely and efficiently. In this research, our goals were to establish the primary cultured sarcoma cells from various patients with leiomyosarcoma to perform efficacy testing in the preclinical settings, and to produce the seed cell stock for contraction of the master cell bank of viral producing cells.
We established 9 clones of the primary cell cultures of the human leiomyosarcoma cells from surgical specimens and charact
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erized them by evaluating their calponin expression and replication rates. We then carried out the efficacy testing of d12.CALP△RR both in vitro and in vivo using transplantation models in the SCID mice. The full length of ICP4 gene (3897-bp), in which sequence was optimally arranged for maximum protein production in the Vero cells derived from African Green Monkey, was chemically synthesized. The frequency of codon usage and the GC content were optimized. We placed the intrinsic expression regulatory element of the ICP4 gene in the upstream region of the ICP4 gene and SV40 promoter-directed neo-resistant gene (795-bp) with polyA in their down stream region. The synthesized DNA construct, finishing the endotoxin and aseptic examination, was prepared. We transfected the construct to Vero cells with known passages of 129 which were tested with various biological examination. Cells stably expressiong ICP4 mRNA at various levels were isolated and cloned by selection with G418. The ICP4 expression was verified by both the quantitative PCR and Western blot. All procedures were performed in the state-of-the art Cell Vector Processing Isolator which enables production of GMP-grade cells and viruses and equipped within our department.
In the case of production of oncolytic viruses as anti-cancer agents in the clinical grade, it is important to unify their compliant by purifying viral DNA as homogenous as possible from a single clone of the genomic DNA. For this purpose, we constructed BACmid system which could clone the whole genome of HSV-1. We purified genomic DNA of d12.CALP△RR from the above mentioned ICP4-expressing Vero designer cells. We then proceeded the experiments for cloning of the dl2.CALP△RR DNA genome into the BACmid vector, and for determining sequences of the whole genome DNA of d12.CALP△RR. Less
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