Recombinant adeno-associated viral (rAAV) vector-based gene therapy has been adapted for use in more than 100 clinical trials. This is mainly because of its excellent safety profile, ability to target a wide range of tissues, stable transgene expression, and significant clinical benefit. However, the major challenge is to produce a high-titer, high-potency vector to achieve a better therapeutic effect. Even though the three plasmid-based transient transfection method is currently being used for AAV production in many clinical trials, there are complications associated with scalability and it is not cost-effective. Other methods require either large-scale production of two herpes simplex viruses, rHSV-RepCap and rHSV-GOI (gene of interest), with high titers, or a stable cell line with high titer wild-type adenovirus infection. Both of these options make the process even more complex. To address this issue, we have developed a stable cell line-based production with the use of only one rHSV-RepCap virus. Using this new methodology in small-scale production, we achieved ∼1–6 E + 04 vg/cell of AAV9 in the top producer clones. Large-scale production in 10-CS (10-Cell Stack) of one of the top producing clones resulted in ∼1–2 E + 13 vg/10-CS with 50% of full capsid ratio after purification. This method could potentially be adapted to suspension cells. The major advantage of this novel methodology is that by using the rHSV-RepCap virus, high titer AAV can be produced with any GOI containing a stable adherent or suspension producer cell line. The use of this AAV production platform could be beneficial for the treatment of many diseases.

中文翻译：

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用于 AAV 基因治疗制造的新颖且可扩展的病毒载体上游工艺的详细方案


基于重组腺相关病毒 (rAAV) 载体的基因治疗已应用于 100 多项临床试验。这主要是因为其优异的安全性、靶向广泛组织的能力、稳定的转基因表达以及显着的临床效益。然而，主要的挑战是生产高滴度、高效力的载体以达到更好的治疗效果。尽管基于三种质粒的瞬时转染方法目前在许多临床试验中用于 AAV 生产，但存在与可扩展性相关的并发症，并且不具有成本效益。其他方法需要大规模生产两种高滴度单纯疱疹病毒：rHSV-RepCap 和 rHSV-GOI（目的基因），或者需要具有高滴度野生型腺病毒感染的稳定细胞系。这两种选择都会使过程变得更加复杂。为了解决这个问题，我们开发了一种基于细胞系的稳定生产，仅使用一种 rHSV-RepCap 病毒。在小规模生产中使用这种新方法，我们在顶级生产者克隆中实现了〜1-6 E + 04 vg/细胞的AAV9。产量最高的克隆之一的 10-CS（10 细胞堆叠）大规模生产产生了~1–2 E + 13 vg/10-CS，纯化后全衣壳比例为 50%。该方法有可能适用于悬浮细胞。这种新颖方法的主要优点是，通过使用 rHSV-RepCap 病毒，可以使用任何含有稳定贴壁或悬浮生产细胞系的 GOI 来生产高滴度 AAV。该AAV生产平台的使用可能有益于许多疾病的治疗。