EPISOMAL VECTORS FOR COLON CANCER GENE THERAPY

结肠癌基因治疗的特殊载体

• 批准号: 6137584
• 负责人: MARK J COOPER
• 金额: $ 13.59万
• 依托单位: COPERNICUS THERAPEUTICS, INC.
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-20 至 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6137584
• 关键词: Alphaherpesvirinae; DNA replication; animal mortality; athymic mouse; carcinoembryonal antigen; colon neoplasms; ganciclovir; gene expression; gene targeting; gene therapy; genetic promoter element; liposomes; neoplasm /cancer remission /regression; neoplastic cell; plasmids; simian virus 40; thymidine kinase; tissue /cell culture; transfection /expression vector

DESCRIPTION: (Applicant's Abstract) The applicant's laboratory has recently developed a novel SV40-based episomal expression vector for human gene therapy applications that is predicted to be safe while permitting high copy replication in human cells. This episomal plasmid utilizes a specifically designed SV40 large T antigen mutant to drive extrachromosomal replication in human cells while eliminating the undesired ability of large T antigen to bind and inactivate host p53 and RB tumor suppressor gene proteins. Because this vector replicates to thousands of copies by 2-3 days after gene transfer, the expression system is predicted to safely express target genes at higher levels per cell than any currently published gene therapy vector. To further modify this vector to include appropriate safety and replication-control elements, the applicant proposes to develop a novel "colon cancer-specific" expression system which will amplify extrachromosomally and express target genes only in tumor cells. Specific Aim 1 is to develop a noninfectious, safety-modified SV40 based episomal expression system to permit external control of tumor-specific vector amplification and gene expression. An SV40-based episomal expression system will be constructed which employs both tissue-specific and inducible promoters to limit expression of mutant T antigen, and hence vector amplification, to colon tumor cells. Specific Aim 2 is to determine the effectiveness of the SV40-based episomal vector system encoding herpes simplex virus thymidine kinase to kill tumor cells in vitro. After exposure to GCV, the specificity and efficiency of tumor cell kill will be evaluated. In Specific Aim 3 a preclinical colon cancer model will be employed to evaluate the ability of SV40-based episomes to eradicate established tumor xenografts in nude mice using liposome-mediated gene transfer. Mice will be followed for tumor regression and survival. In summary, this application supports key, initial "proof-of-principle" experiments to evaluate "tumor-specific" episomal vectors and gene transfer systems for gene therapy of colon cancer.

描述：（申请人摘要）申请人的实验室最近 开发了一种新型基于 SV40 的人类基因附加型表达载体 预计安全同时允许高拷贝的治疗应用 在人体细胞中复制。 该附加型质粒利用了一种特殊的 设计了 SV40 大 T 抗原突变体来驱动染色体外复制 在人体细胞中，同时消除大 T 抗原的不良能力 结合宿主 p53 和 RB 肿瘤抑制基因蛋白并使其失活。 因为 该载体在基因生成后 2-3 天复制到数千个拷贝 转移，预计表达系统能够安全表达目的基因 每个细胞的水平高于任何目前发表的基因治疗载体。 进一步修改该载体以包括适当的安全性和 复制控制元件，申请人建议开发一种新颖的 “结肠癌特异性”表达系统将放大 染色体外并仅在肿瘤细胞中表达靶基因。 具体的 目标 1 是开发一种基于 SV40 的非传染性、安全性改良的游离型 表达系统允许对肿瘤特异性载体进行外部控制 扩增和基因表达。 基于 SV40 的附加型表达系统 将构建同时采用组织特异性和诱导性的 启动子限制突变T抗原的表达，因此载体 扩增至结肠肿瘤细胞。 具体目标 2 是确定 基于 SV40 的附加型载体系统编码疱疹的有效性 单纯病毒胸苷激酶在体外杀死肿瘤细胞。 接触后 对于GCV，将评估肿瘤细胞杀伤的特异性和效率。 在具体目标 3 中，将采用临床前结肠癌模型来 评估基于 SV40 的附加体根除已形成肿瘤的能力 使用脂质体介导的基因转移在裸鼠中进行异种移植。 老鼠将会 随后进行肿瘤消退和存活。 总结一下，这个应用 支持关键的初步“原理验证”实验来评估 用于基因治疗的“肿瘤特异性”附加型载体和基因转移系统 结肠癌。