A robust and flexible baculovirus-insect cell system for AAV vector production with improved yield, capsid ratios and potency

IF 4.6 2区医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-03-04 DOI:10.1016/j.omtm.2024.101228

Yoko Marwidi, Hoang-Oanh B. Nguyen, David Santos, Tenzin Wangzor, Sumita Bhardwaj, Gabriel Ernie, Gregg Prawdzik, Garrett Lew, David Shivak, Michael Trias, Jada Padilla, Hung Tran, Kathleen Meyer, Richard Surosky, Alex Michael Ward

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引用次数: 0

Abstract

Manufacturing of adeno-associated viruses (AAV) for gene and cell therapy applications has increased significantly and spurred development of improved mammalian and insect cell-based production systems. We developed a baculovirus-based insect cell production system—the SGMO Helper—with a novel gene architecture and greater flexibility to modulate the expression level and content of individual Rep and Cap proteins. In addition, we incorporated modifications to the AAV6 capsid sequence that improves yield, capsid integrity, and potency. Production of recombinant AAV 6 (rAAV6) using the SGMO Helper had improved yields compared to the Bac-RepCap helper from the Kotin lab. SGMO Helper-derived rAAV6 is resistant to a previously described proteolytic cleavage unique to baculovirus-insect cell production systems and has improved capsid ratios and potency, and , compared with rAAV6 produced using Bac-RepCap. Next-generation sequencing sequence analysis demonstrated that the SGMO Helper is stable over six serial passages and rAAV6 capsids contain comparable amounts of non-vector genome DNA as rAAV6 produced using Bac-RepCap. AAV production using the SGMO Helper is scalable using bioreactors and has improved yield, capsid ratio, and potency. Our studies demonstrate that the SGMO Helper is an improved platform for AAV manufacturing to enable delivery of cutting-edge gene and cell therapies.

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用于生产 AAV 向量的稳健而灵活的杆状病毒-昆虫细胞系统，可提高产量、囊壳比率和效力

用于基因和细胞治疗的腺相关病毒（AAV）的生产量大幅增加，促进了基于哺乳动物和昆虫细胞的改良生产系统的开发。我们开发了一种基于杆状病毒的昆虫细胞生产系统--SGMO Helper，它具有新颖的基因结构和更大的灵活性，可以调节单个 Rep 蛋白和 Cap 蛋白的表达水平和含量。此外，我们还对 AAV6 的囊膜序列进行了修改，从而提高了产量、囊膜完整性和效力。与科廷实验室的 Bac-RepCap 助手相比，使用 SGMO 助手生产重组 AAV6（rAAV6）的产量有所提高。与使用 Bac-RepCap 生产的 rAAV6 相比，SGMO Helper 衍生的 rAAV6 能抵抗先前描述的一种特有于杆状病毒-昆虫细胞生产系统的蛋白酶裂解，并且提高了囊膜比率和效力。下一代测序序列分析表明，SGMO Helper 经过六次连续传代后是稳定的，而且 rAAV6 荚膜中的非载体基因组 DNA 含量与使用 Bac-RepCap 生产的 rAAV6 相当。使用 SGMO Helper 生产 AAV 可通过生物反应器进行扩展，并能提高产量、囊壳比率和效力。我们的研究表明，SGMO Helper 是一种改进的 AAV 生产平台，可用于提供最先进的基因和细胞疗法。

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来源期刊

Molecular Therapy-Methods & Clinical Development Biochemistry, Genetics and Molecular Biology-Molecular Biology

CiteScore

9.90

自引率

4.30%

发文量

163

审稿时长

12 weeks

期刊介绍： The aim of Molecular Therapy—Methods & Clinical Development is to build upon the success of Molecular Therapy in publishing important peer-reviewed methods and procedures, as well as translational advances in the broad array of fields under the molecular therapy umbrella. Topics of particular interest within the journal''s scope include: Gene vector engineering and production, Methods for targeted genome editing and engineering, Methods and technology development for cell reprogramming and directed differentiation of pluripotent cells, Methods for gene and cell vector delivery, Development of biomaterials and nanoparticles for applications in gene and cell therapy and regenerative medicine, Analysis of gene and cell vector biodistribution and tracking, Pharmacology/toxicology studies of new and next-generation vectors, Methods for cell isolation, engineering, culture, expansion, and transplantation, Cell processing, storage, and banking for therapeutic application, Preclinical and QC/QA assay development, Translational and clinical scale-up and Good Manufacturing procedures and process development, Clinical protocol development, Computational and bioinformatic methods for analysis, modeling, or visualization of biological data, Negotiating the regulatory approval process and obtaining such approval for clinical trials.