Aiming at determination of the structure of oncolytic HSV-1 targeting to sarcoma and contruction of seed cell stock of virus-producing Vero designer cells for clinical application

旨在确定针对肉瘤的溶瘤HSV-1的结构以及构建用于临床应用的产病毒Vero设计细胞的种子细胞库

• 批准号: 17209051
• 负责人: TAKAHASHI Katsuhito
• 金额: $ 19.3万
• 依托单位: Research Institute, Osaka Medical Center for Cancer and Cardiovascular Disaeses
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17209051/
• 关键词: Herpes Virus; Calponin; Sarcoma; Gene Therapy; GMP-grade; GMP

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 … More 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

因此，开发用于肿瘤基因治疗的溶瘤病毒制剂需要进行有效性检测和生物学评价。GMP级病毒制剂的纯化和生产程序分为3个步骤，即1）主细胞库的构建及其生物学评价，2）主病毒种子库的生成及其生物学评价，3）纯化病毒的生产及其生物学评价。其中，主细胞库的构建是安全有效地生成病毒储备液的最重要步骤。本研究的目的是建立平滑肌肉瘤原代培养细胞系，用于临床前的有效性检测，并为病毒产生细胞的主细胞库的建立提供种子细胞库。我们建立了9个人平滑肌肉瘤原代细胞系克隆， ...更多信息 通过评估它们的钙调蛋白表达和复制率来评估它们。在SCID小鼠移植模型上进行了d12.CALP△RR的体外和体内疗效试验。化学合成了全长ICP 4基因（3897-bp），其中序列经过优化排列，以在源自非洲绿色猴的Vero细胞中产生最大蛋白质产量。优化了密码子使用频率和GC含量。我们将ICP 4基因的内在表达调控元件置于ICP 4基因的上游区域，并将SV 40启动子指导的新抗性基因（795-bp）与polyA置于其下游区域。制备合成的DNA构建体，完成内毒素和无菌检查。我们将构建体转染到Vero细胞中，已知传代129次，用各种生物学检查进行测试。通过G418筛选，分离并克隆了在不同水平稳定表达ICP 4 mRNA的细胞。通过定量PCR和Western blot验证ICP 4的表达。所有操作均在本部门配备的最先进的细胞载体分离器（Cell Vector Processing Isolator）中进行，该分离器可生产GMP级细胞和病毒。在生产临床级抗癌药物溶瘤病毒的情况下，重要的是通过从基因组DNA的单一克隆中纯化尽可能同质的病毒DNA来统一其合规性。为此，我们构建了能够克隆HSV-1全基因组的BACmid系统。我们从上述表达ICP 4的Vero设计细胞中纯化了d12.CALP△RR的基因组DNA。将d12.CALP △RR基因组DNA克隆到BACmid载体上，并测定了d12.CALP△RR全基因组DNA的序列。少

项目成果

Effects of hl-calponin on the contractile properties of bladder vs. vascular smooth muscle in SM-B null mice

hl-钙调蛋白对 SM-B 缺失小鼠膀胱与血管平滑肌收缩特性的影响

• 发表时间: 2006
• 期刊: J.Physiol.(London) 577
• 作者: Babu GJ;Celia G;Rhee AY;Yamamura H;Takahashi K;Brozovick FV;Osol G;Periassamy M
• 通讯作者: Periassamy M

Stimulation of cyclooxygenase-2 expression by bone-derived transforming growth factor-beta enhances bone metastasis in breast cancer

骨源性转化生长因子-β 刺激环氧合酶-2 表达可增强乳腺癌骨转移

• 发表时间: 2006
• 期刊: Cancer Research 66
• 作者: Hiraga T;Myoui A.;Choi M.E.;Yoshikawa H.;Yoneda T.
• 通讯作者: Yoneda T.

増殖型HSVベクターの開発-腫瘍溶解性ウイルスによる肉腫の標的遺伝子療法

增殖HSV载体的开发-利用溶瘤病毒进行肉瘤的靶向基因治疗

• 发表时间: 2006
• 期刊: 日本臨床 64
• 作者: 高橋 克仁;山村 倫子
• 通讯作者: 山村 倫子

Development of a novel cell-targeted therapy for leiomyosarcoma and uterine myoma by tumor-selective replicating and attenuated HSV-1 d12CALP△RR

通过肿瘤选择性复制和减毒HSV-1 d12CALP△RR开发针对平滑肌肉瘤和子宫肌瘤的新型细胞靶向疗法

• 发表时间: 2005
• 作者: Yamamura H. Kamiura S.;Takahashi K.
• 通讯作者: Takahashi K.

Loss of HB-EGF in smooth muscle or endothelial cell lineages causes heart malformation.

平滑肌或内皮细胞谱系中 HB-EGF 的缺失会导致心脏畸形。

• 发表时间: 2006
• 期刊: Biochem Biophys Res Commun 350
• 作者: Nanba D;Kinugasa Y;Morimoto C;Koizumi M;Yamamura H;Takahashi K;Takakura N;Mekada E;Hashimoto K;Higashiyama S
• 通讯作者: Higashiyama S