Process Sciences Department, Biomarin Pharmaceutical Inc, Novato, CA, 94949.
Amgen Bioprocessing Center, Keck Graduate Institute, Claremont, CA, 91711.
Biotechnol Prog. 2019 Jan;35(1):e2725. doi: 10.1002/btpr.2725. Epub 2018 Oct 22.
Recombinant adeno-associated viral vectors (rAAV) are promising therapies for genetic diseases. Although current platforms for recombinant vector production can generate drug material for pre-clinical and clinical studies, rAAV biomanufacturing will eventually face commercial supply challenges if per cell vector productivity and process scalability are not improved. Because considerable efforts have traditionally focused on optimizing rAAV plasmid design, herein we investigate the impact of host cell proteins on vector production to identify proteins that may enhance rAAV yield. Using a rAAV2-GFP-producing Saccharomyces cerevisiae model in combination with the yeast Tet Hughes Collection screening library, we identified 22 gene candidates that improved rAAV DNA replication (rAAV-GFP/18s rDNA ratio) and vector yield (benzonase-resistant rAAV DNA vector genome titer) as high as 6-fold and 15-fold relative to control, respectively. The candidate proteins participate in biological processes such as DNA replication, ribosome biogenesis, and RNA and protein processing. The best five candidates (PRE4, HEM4, TOP2, GPN3, and SDO1) were further screened by generating overexpression mutants in the YPH500 yeast strain. Subsequent clone evaluation was performed to confirm the rAAV-promoting activity of selected candidates under plate-based and bioreactor-controlled fermentation conditions. Digital droplet PCR analysis of cell lysate and AVB resin-purified material confirmed HEM4 and TOP2 overexpression mutants displayed the highest per cell total rAAV DNA productivity (1.6 and 1.7-fold increase over control, respectively) and per cell vector productivity (3 and 4-fold over control, respectively). This evaluation confirmed that overexpression of HEM4 and TOP2 proteins enhanced total and benzonase-resistant rAAV DNA yield. Further studies are needed to understand their mechanism of action and to assess their potential application in molecular strategies for rAAV production. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2725, 2019.
重组腺相关病毒载体（rAAV）是治疗遗传疾病的有前途的疗法。尽管当前用于重组载体生产的平台可以为临床前和临床研究生成药物材料，但如果每个细胞的载体产率和工艺可扩展性没有提高，rAAV 生物制造最终将面临商业供应挑战。因为传统上已经投入了相当大的努力来优化 rAAV 质粒设计，所以在这里，我们研究了宿主细胞蛋白对载体生产的影响，以确定可能提高 rAAV 产量的蛋白。我们使用酿酒酵母（Saccharomyces cerevisiae）rAAV2-GFP 生产模型，结合酵母 Tet Hughes 收集筛选文库，鉴定了 22 个候选基因，它们分别将 rAAV DNA 复制（rAAV-GFP/18s rDNA 比值）和载体产量（苯甲脒抗性 rAAV DNA 载体基因组滴度）提高了 6 倍和 15 倍。候选蛋白参与 DNA 复制、核糖体生物发生以及 RNA 和蛋白质加工等生物学过程。最好的五个候选物（PRE4、HEM4、TOP2、GPN3 和 SDO1）通过在 YPH500 酵母菌株中生成过表达突变体进一步筛选。随后在平板和生物反应器控制发酵条件下进行克隆评估，以确认所选候选物的 rAAV 促进活性。细胞裂解物和 AVB 树脂纯化材料的数字液滴 PCR 分析证实，HEM4 和 TOP2 过表达突变体显示出最高的每个细胞总 rAAV DNA 生产力（分别比对照增加 1.6 倍和 1.7 倍）和每个细胞载体生产力（分别比对照增加 3 倍和 4 倍）。该评估证实，HEM4 和 TOP2 蛋白的过表达增强了总和苯甲脒抗性 rAAV DNA 的产量。需要进一步研究以了解它们的作用机制，并评估它们在 rAAV 生产的分子策略中的潜在应用。 © 2018 美国化学工程师学会生物技术进展，35：e2725，2019。
