Tworig Joshua, Grafton Francis, Fisher Kaylin, Hörer Markus, Reid Christopher A, Mandegar Mohammad A
Ascend Advanced Therapies CA, Inc, Alameda, CA 94501, USA.
Ascend Advanced Therapies GmbH, 82152 Planegg, Germany.
Mol Ther Methods Clin Dev. 2024 Nov 18;32(4):101384. doi: 10.1016/j.omtm.2024.101384. eCollection 2024 Dec 12.
Recombinant adeno-associated virus (rAAV) is a widely used viral vector for gene therapy. However, these vectors have limited availability due to manufacturing challenges with productivity and quality. These challenges can be addressed by better understanding the mechanisms that influence cellular responses during rAAV production. In this study, we aimed to identify targets that may enhance rAAV production using transcriptomic analyses of five cell lines with variable capacities for rAAV production. Using an intersectional approach, we measured the transcriptional responses of these cells during rAAV production and compared transcriptional profiles between high and base producers to identify possible targets for enhancing production. During rAAV production, we found transcriptional differences in cell cycle and nucleosome components contributed to proliferative capacity and DNA replication. We also saw upregulation of several core functions, including transcription, stress response, and Golgi and endoplasmic reticulum organization. Conversely, we saw consistent downregulation of other factors, including inhibitors of DNA-binding proteins and mitochondrial components. With a drug-connectivity analysis, we identified five classes of drugs that were predicted to enhance rAAV production. We also validated the efficacy of histone deacetylase and microtubule inhibitors. Our data uncover novel and previously identified pathways that may enhance rAAV production and quality to expand availability of rAAV for gene therapies.
重组腺相关病毒（rAAV）是一种广泛用于基因治疗的病毒载体。然而，由于在生产力和质量方面的制造挑战，这些载体的可用性有限。通过更好地理解在rAAV生产过程中影响细胞反应的机制，可以解决这些挑战。在本研究中，我们旨在通过对五种具有不同rAAV生产能力的细胞系进行转录组分析，确定可能提高rAAV产量的靶点。使用交叉分析方法，我们测量了这些细胞在rAAV生产过程中的转录反应，并比较了高产细胞系和基础生产细胞系之间的转录谱，以确定可能提高产量的靶点。在rAAV生产过程中，我们发现细胞周期和核小体成分的转录差异有助于增殖能力和DNA复制。我们还观察到几个核心功能的上调，包括转录、应激反应以及高尔基体和内质网组织。相反，我们观察到其他一些因子的持续下调，包括DNA结合蛋白抑制剂和线粒体成分。通过药物连接性分析，我们确定了五类预计可提高rAAV产量的药物。我们还验证了组蛋白脱乙酰酶和微管抑制剂的疗效。我们的数据揭示了可能提高rAAV产量和质量以扩大rAAV用于基因治疗的可用性的新途径和先前已确定的途径。
