Recombinant AAV Genome Size Effect on Viral Vector Production, Purification, and Thermostability

IF 4.6 2区医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL

Molecular Therapy-Methods & Clinical Development Pub Date : 2024-01-17 DOI:10.1016/j.omtm.2024.101188

Nermin Ibreljic, Benjamin E. Draper, Carl W. Lawton

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Abstract

Adeno-associated virus (AAV) has shown great promise as a viral vector for gene therapy in clinical applications. This work studied the effect of the genome size on AAV production, purification, and thermostability by producing AAV2-GFP using suspension adapted HEK293 cells via triple transfection using AAV plasmids containing the same green fluorescent protein (GFP) transgene with DNA stuffers for variable size AAV genomes consisting of 1.9, 3.4, and 4.9 kb (ITR to ITR). Production was performed at the small and large shake flask scales and the results showed that the 4.9 kb GFP genome had significantly reduced encapsidation compared to other genomes. The large shake flask productions were purified by AEX chromatography and the results suggest that the triple transfection condition significantly impacts the AEX retention time and resolution between the full and empty capsid peaks. Charge detection mass spectrometry (CD-MS) was performed on all AEX full capsid peak samples showing a wide distribution of empty, partial, full length, and co-packaged DNA in the capsids. The AEX purified samples were then analyzed by differential scanning fluorimetry (DSF) and the results suggest that sample formulation may improve the thermostability of AAV genome ejection melting temperature regardless of the packaged genome content.

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重组 AAV 基因组大小对病毒载体生产、纯化和耐热性的影响

腺相关病毒（AAV）作为基因治疗的病毒载体在临床应用中显示出巨大的前景。这项研究利用悬浮适配的 HEK293 细胞，通过使用含有相同绿色荧光蛋白（GFP）转基因的 AAV 质粒进行三重转染，生产 AAV2-GFP，基因组大小对 AAV 生产、纯化和热稳定性的影响，AAV 基因组由 1.9、3.4 和 4.9 kb（ITR 到 ITR）组成。结果表明，与其他基因组相比，4.9 kb GFP 基因组的包被率明显降低。采用 AEX 色谱法纯化了大型摇瓶生产的产品，结果表明，三重转染条件对 AEX 保留时间以及全囊壳峰和空囊壳峰之间的分辨率有显著影响。对所有 AEX 全包囊峰样品进行了电荷检测质谱分析（CD-MS），结果显示包囊中广泛分布着空 DNA、部分 DNA、全长 DNA 和共包装 DNA。然后用差示扫描荧光定量法（DSF）对 AEX 纯化样品进行了分析，结果表明，无论包装的基因组含量如何，样品配方都可以提高 AAV 基因组弹射熔解温度的恒温性。

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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.