Transient transfection of mammalian cells using plasmid DNA is a standard method to produce adeno-associated virus (AAV) vectors allowing for flexible and scalable manufacture. Typically, three plasmids are used to encode the necessary components to facilitate vector production; however, a dual-plasmid system, termed pDG, was introduced over 2 decades ago demonstrating two components could be combined resulting in comparable productivity to triple transfection. We have developed a novel dual-plasmid system, pOXB, with an alternative arrangement of sequences that results in significantly increased AAV vector productivity and percentage of full capsids packaged in comparison to the pDG dual design and triple transfection. Here, we demonstrate the reproducibility of these findings across seven recombinant AAV genomes and multiple capsid serotypes as well as the scalability of the pOXB dual-plasmid transfection at 50-L bioreactor scale. Purified drug substance showed a consistent product quality profile in line with triple-transfected vectors, except for a substantial improvement in intact genomes packaged using the pOXB dual- transfection system. Furthermore, pOXB dual- and triple-transfection-based vectors performed consistently . The pOXB dual plasmid represents an innovation in AAV manufacturing resulting in significant process gains while maintaining the flexibility of a transient transfection platform.

中文翻译：

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新型双质粒平台提供可扩展的转染，从而提高多种 AAV 血清型和基因组的生产率和包装


使用质粒 DNA 瞬时转染哺乳动物细胞是生产腺相关病毒 (AAV) 载体的标准方法，可实现灵活且可扩展的生产。通常，三个质粒用于编码必要的组件以促进载体生产；然而，二十多年前引入了一种称为 pDG 的双质粒系统，证明两种成分可以组合，从而产生与三重转染相当的生产力。我们开发了一种新型双质粒系统 pOXB，与 pDG 双重设计和三重转染相比，它具有另一种序列排列，可显着提高 AAV 载体的生产力和包装的完整衣壳的百分比。在这里，我们证明了这些发现在七个重组 AAV 基因组和多种衣壳血清型中的重现性，以及 pOXB 双质粒转染在 50 L 生物反应器规模下的可扩展性。纯化的原料药表现出与三重转染载体一致的产品质量特征，除了使用 pOXB 双转染系统包装的完整基因组有显着改善。此外，基于 pOXB 双转染和三转染的载体表现一致。 pOXB 双质粒代表了 AAV 制造领域的一项创新，可带来显着的工艺收益，同时保持瞬时转染平台的灵活性。