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

基本信息

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

项目摘要

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

开发用于癌症基因治疗的溶瘤病毒制剂需要进行功效测试和生物学评价，GMP级病毒制剂的纯化和生产过程分为三个步骤，即1）主细胞的构建。病毒库及其生物学评价，2）主病毒种子库的产生及其生物学评价，3）纯化病毒的生产及其生物学评价，其中，主细胞库的构建是病毒产生最重要的步骤。安全高效地库存。研究中，我们的目标是建立来自不同平滑肌肉瘤患者的原代培养肉瘤细胞，以在临床前环境中进行功效测试，并生产用于收缩病毒产生细胞的主细胞库的种子细胞储备。我们建立了 9 个克隆来自手术标本的人类平滑肌肉瘤细胞的原代细胞培养物和特征通过评估其钙调蛋白表达和复制率进行了功效测试。 d12.CALP△RR 在 SCID 小鼠移植模型中的体外和体内 ICP4 基因的全长（3897-bp），其中序列经过优化排列，以在源自非洲绿猴的 Vero 细胞中获得最大的蛋白质产量。，是化学合成的，我们将ICP4基因的内在表达调控元件放置在ICP4基因和SV40的上游区域。下游区域带有polyA的启动子导向的neo抗性基因（795-bp），完成内毒素和无菌检查后，我们将构建体转染至已知传代的129代Vero细胞中并进行测试。通过G418选择稳定表达ICP4 mRNA的细胞通过定量PCR和Western印迹验证。我们部门配备了最先进的细胞载体处理隔离器，能够生产 GMP 级细胞和病毒。在生产临床级抗癌药物的溶瘤病毒时，重要的是要统一为此，我们构建了 BACmid 系统，可以克隆 HSV-1 的整个基因组。从上述表达ICP4的Vero设计细胞中获得d12.CALP△RR，然后我们进行了将dl2.CALP△RR DNA基因组克隆到BACmid载体中的实验，并确定了d12.CALP全基因组DNA的序列。 △RR。

项目成果

期刊论文数量（30）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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

DOI：
发表时间：
2006
期刊：
J.Physiol.(London) 577
影响因子：
0
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

DOI：
发表时间：
2006
期刊：
Cancer Research 66
影响因子：
0
作者：
Hiraga T;Myoui A.;Choi M.E.;Yoshikawa H.;Yoneda T.
通讯作者：
Yoneda T.

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。

DOI：
发表时间：
2006
期刊：
Biochem Biophys Res Commun 350
影响因子：
0
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

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