Mixture design as a tool for improving full-to-empty particle ratios across various GOIs in rAAV production.

IF 4.5 3区 医学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY
Konstantina Tzimou, Pol Hulsbus-Andreu, Ece Bahar Yildirim, Lars K Nielsen, Jesús Lavado-García
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引用次数: 0

Abstract

Optimization of recombinant adeno-associated virus (rAAV) production is essential for effective gene therapy applications. However, multiple factors affect the rAAV productivity in mammalian cells, and often they interact with each other, making the optimization process highly challenging. In our previous work, we showed how coupling mixture design (MD) with face-centered central composite design (FCCD) was the most suitable design of experiments (DOE) approach for optimizing rAAV2 productivity and cell viability. In this study, we built on this method and demonstrate that combining MD with FCCD can be used to optimize the percentage of full capsids in rAAV2 upstream preparation. Additionally, we investigate the influence of the gene of interest (GOI) on the optimal conditions for viral particle production and packaging efficiency. By integrating MD and FCCD methodologies, we achieved an improvement of almost 100-fold in Log(Vp) in the case of egfp-expressing rAAV, and a 12-fold increase in bdnf-expressing full rAAV capsids, suggesting that this combined approach is a versatile and effective strategy for optimizing rAAV production processes. These findings emphasize the need for a comprehensive understanding of the factors influencing rAAV production to enhance the efficiency and efficacy of viral vector applications in gene therapy.

混合设计是提高rAAV生产中各种goi的满空颗粒比的工具。
优化重组腺相关病毒(rAAV)的生产是有效的基因治疗应用的必要条件。然而,在哺乳动物细胞中,有多种因素影响rAAV的产生,并且它们之间经常相互作用,这使得优化过程非常具有挑战性。在我们之前的工作中,我们展示了耦合混合设计(MD)与面心中心复合设计(FCCD)是优化rAAV2生产力和细胞活力的最合适的实验设计(DOE)方法。在本研究中,我们以该方法为基础,证明了将MD与FCCD相结合可以优化rAAV2上游制备中完整衣壳的百分比。此外,我们还研究了感兴趣基因(GOI)对病毒颗粒生产和包装效率的最佳条件的影响。通过整合MD和FCCD方法,我们实现了表达egfp的rAAV的Log(Vp)提高近100倍,表达bdnf的完整rAAV衣壳的Log(Vp)提高12倍,这表明这种组合方法是优化rAAV生产过程的通用有效策略。这些发现强调需要全面了解影响rAAV产生的因素,以提高病毒载体在基因治疗中的应用效率和效果。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Gene Therapy
Gene Therapy 医学-生化与分子生物学
CiteScore
9.70
自引率
2.00%
发文量
67
审稿时长
4-8 weeks
期刊介绍: Gene Therapy covers both the research and clinical applications of novel therapeutic techniques based on a genetic component. Over the last few decades, significant advances in technologies ranging from identifying novel genetic targets that cause disease through to clinical studies, which show therapeutic benefit, have elevated this multidisciplinary field to the forefront of modern medicine.
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