RAPID GENERATION OF RAAV PACKAGING CELL LINES USING FACS

使用 FACS 快速生成 RAAV 包装细胞系

• 批准号: 2422987
• 负责人: JACK R BARBER
• 金额: $ 8.32万
• 依托单位: ITHERX PHARMACEUTICALS, INC.
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 1998-03-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2422987
• 关键词: Adenoviridae; biomarker; cell line; drug delivery systems; flow cytometry; gene dosage; gene expression; gene targeting; gene therapy; plasmids; tissue /cell culture; transfection /expression vector; western blottings

Gene therapy has been proposed in the treatment of diseases ranging from cancer and viral infections to metabolic disorders such as cystic fibrosis. The main hurdle to effective gene therapy remains delivery of genetic drugs at therapeutic doses. Adeno- associated viral vectors can be used as gene delivery vehicles in a number of applications. The major limitation to the use of this technology is achieving reproducible vector preparations with high infectious titer and a low ratio of defective particles. To successfully develop this technology, the entire process must be scaleable and yield consistent product free of contaminating adenovirus and wild-type AAV. Immusol plans to develop packaging cell lines for rAAV vector production using flow cytometry to continuously monitor transgene expression. Helper plasmids for rAAV vector production incorporating FACS sortable markers will be constructed and used to test for high titer vector production in transient assays. Stable packaging cell lines based on these plasmids will be developed and screened for stable expression of the necessary helper functions. Packaging cell lines with desirable titer and particle ratios will be adapted for growth in suspension culture and in low or no serum media. PROPOSED COMMERCIAL APPLICATION Immusol plans to commercially develop recombinant Adeno- associated viral vector packaging cell lines for use in gene delivery, including ribozyme delivery, for assorted diseases including infectious and metabolic disease and cancer.

基因疗法已被提出用于疾病的治疗 从癌症、病毒感染到代谢紊乱 比如囊性纤维化。有效的基因治疗的主要障碍 仍然以治疗性剂量递送基因药物。腺体- 相关的病毒载体可作为基因输送载体在 申请数量。使用此技术的主要限制是 技术正在实现可重复的载体制备，具有高度的 感染性滴度和低缺陷颗粒比率。至 成功开发这项技术，整个过程必须 可扩展且产量一致、无污染的产品 腺病毒和野生型AAV。Immusol计划开发 利用FLOW技术包装rAAV载体生产用细胞系 用细胞仪持续监测转基因表达。帮手 用于重组AAV载体生产的重组AAV表达载体 将构建标记并用于检测高滴度载体 瞬变分析中的产物。稳定的包装细胞系基于 这些质粒将被开发出来并进行稳定性筛选 必要的帮助器函数的表达式。包装细胞系 具有所需的效价和颗粒比率将被调整以适合在 悬浮培养和低血清或无血清培养。 建议的商业应用 Immusol计划商业化开发重组腺病毒- 相关的病毒载体包装细胞系用于基因传递， 包括核酶传递，用于治疗各种疾病，包括 传染病和代谢性疾病以及癌症。