通过低cis三重转染产生大量和高质量的复合腺相关病毒
Hao Liu1, Yue Zhang1, Mitchell Yip1
1Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA 01605, USA.
Molecular therapy. Methods & clinical development
|April 1, 2024
概括
一种新的低-cis三重传染法显著减少了用于复合腺相关病毒 (rAAV) 基因疗法生产的等离子体DNA使用. 这种方法提高了rAAV的产量和纯度,同时改善了体内安全性.
科学领域:
- 分子生物学分子生物学
- 基因治疗制造业 基因治疗制造业
- 生物技术是生物技术.
背景情况:
- 基于复合腺相关病毒 (rAAV) 的基因疗法正在迅速进入临床和商业应用.
- 使用HEK293细胞的标准三重转化方法是rAAV制造的主要平台.
- 标准方法面临的局限性包括高等离子体DNA成本和下游净化由于等离子体骨干污染的挑战.
研究的目的:
- 开发一种更高效,更具成本效益的rAAV生产方法.
- 克服标准三重转染的局限性,特别是减少等离子体DNA的使用,提高rAAV纯度.
- 与标准方法相比,评估使用新方法产生的rAAV的功效和安全性.
主要方法:
- 在HEK293细胞中开发用于rAAV生产的"低cis"三重转化技术.
- 与标准协议相比,转基因等离子体输入减少了10到100倍.
- 对rAAV产量的表征,纯度 (减少等离子体骨干污染),体内功效 (小鼠模型) 和DNA/RNA在组织中的持久性.
主要成果:
- 低cis方法显著降低了转基因等离子体的需求 (10至100倍).
- 在包装产量抑制转基因方面实现了高达10倍的改进,并在rAAV批次中减少了等离子体骨干污染.
- 在体内研究表明,低-cis rAAV的效力相当或优于低-cis rAAV,在小鼠组织中等离子体DNA和RNA的持久性降低.
结论:
- 低cis三重转化是一种具有成本效益和可扩展的方法,用于生产高质量的rAAV.
- 这种方法提高了rAAV产量和纯度,解决了当前制造工艺的关键局限性.
- 由此产生的rAAV在体内表现出有利的特征,包括减少污染DNA/RNA的持续性,这表明安全性得到改善.
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