Improving the helper-dependent adenoviral vector system

改进辅助依赖性腺病毒载体系统

• 批准号: 6713764
• 负责人: Philip Ng
• 金额: $ 25.99万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6713764
• 关键词: Adenoviridae; biotechnology; cell free system; gene delivery system; genetic regulatory element; helper virus; laboratory mouse; nucleic acid amplification techniques; technology /technique development; transfection /expression vector; transposon /insertion element

Helper-dependent (HD) adenoviral vectors are devoid of all viral coding sequences and can efficiently transduce a wide variety of cell types from numerous species in a cell cycle independent manner. The first efficient and currently most widely used method of generating HD vector relies on coinfection of Cre-expressing 293 cells with the HD vector and a helper virus bearing a packaging signal flanked by IoxP sites. Cre-mediated packaging signal excision renders the helper viral genome unpackagable but still able to replicated and provide all functions in trans necessary for HD vector propagation. It has become clear that HD vectors can direct long-term transgene expression in vivo with negligible toxicity and appear to be ideally suited for many gene therapy applications. However, despite their tremendous potential, several obstacles currently hinder the progress of this technology. Specifically, the current systems for generating HD vectors are technically demanding, not easily scalable and not widely available. These obstacles severely limit meaningful efficacy and safety studies of these vectors in large animal models and their ultimate use in clinical applications. Therefore, the overall objective of this proposal is to develop improved systems as well as optimized standard operating procedures to efficiently and reliably generate large quantities of HD vectors. To accomplish this, we will investigate and optimize the factors that influence HD vector amplification efficiency, develop simple methods for large scale vector production and develop systems to further reduce helper virus contamination. We will also develop improved, certifiable reagents for generating vectors suitable for clinical applications, which will be made widely available. Successful completion of these objectives would permit application of this promising technology for the treatment of a wide variety of acquired and inherited diseases.

助手依赖性（HD）腺病毒载体没有所有病毒编码序列，并且可以独立于细胞周期的方式从许多物种中有效地从众多物种中传递多种细胞类型。产生HD载体的第一种高效且目前最广泛的方法依赖于与HD载体和辅助病毒一起表达CRE的293个细胞，并带有带有IOXP位点的包装信号的辅助病毒。 CRE介导的包装信号切除可使辅助病毒基因组取消包装，但仍然能够复制并提供HD矢量传播所必需的Trans所有功能。很明显，高清矢量可以在体内引导长期的转基因表达，并可以忽略不计，并且似乎非常适合许多基因治疗应用。但是，尽管具有巨大的潜力，但有几个障碍 目前阻碍了这项技术的进步。具体而言，当前生成高清矢量的系统在技术上是要求的，不容易扩展并且不广泛可用。这些障碍严重限制了这些向量在大型动物模型中的有意义的功效和安全性研究，并在临床应用中的最终使用。因此，该提案的总体目标是开发改进的系统以及优化的标准操作程序，以有效，可靠地产生大量的HD矢量。为此，我们将调查并优化影响HD矢量扩增效率的因素，为大规模矢量生产开发简单的方法，并开发系统以进一步减少辅助病毒污染。我们还将开发改进的，可认证的试剂以生成 适用于临床应用的向量将广泛使用。这些目标的成功完成将允许将这种有前途的技术应用于治疗多种被遗传和遗传疾病的治疗。