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AAV产量,生物活性和粒子异质性受到基因组大小和非编码DNA元素的影响

Gina Blahetek¹, Benjamin Lindner², Martin Oti^3,4

¹Global Drug Discovery Sciences, Boehringer Ingelheim Pharma GmbH & Co. KG, 88397 Biberach, Germany.

Molecular therapy. Methods & clinical development

|July 1, 2025

概括

腺相关病毒 (AAV) 载体生产质量取决于基因组大小. 通过添加DNA序列来优化AAV载体基因组可以改善生产,但需要仔细选择序列以保持载体的完整性和生物活性.

科学领域:

分子生物学分子生物学
基因治疗基因治疗
生物技术是生物技术.

背景情况:

腺相关病毒 (AAV) 载体是基因疗法的领先平台.
有效的AAV生产需要最佳的载体完整性,包括全尺寸的基因组包装和高的满/空比.
目前的AAV生产方法产生了充满的,空的和部分填充的囊的混合物.

研究的目的:

研究AAV基因组大小如何影响包装效率,基因组完整性,体比率和生物活性.
为了评估插入非编码填充序列对AAV矢量特征的影响.
确定用于治疗应用的"正确大小"AAV基因组的最佳策略.

主要方法:

设计的单链CMV-EGFP-多A表达带,尺寸从2-5kb.
在表达卡塞特上游或下游插入非编码的stuffer序列.
分析了AAV产量,生物活性,满/空比例和基因组完整性,使用不同的磁带大小.

主要成果:

随着表达卡塞特大小的增加,AAV产量和生物活性系统地下降.
观察到过度填充的磁带<2.5 kb,而基因组切断和部分填充增加了>4.5 kb.
意想不到的是,第二个填充序列并没有导致类似的生产力和生物活性损失.

结论:

插入非编码DNA是一种可行的方法,可以优化AAV基因组大小的制造 (CMC) 方面.
用于基因组"正确大小"的特定DNA序列对于保持AAV质量和生物活性至关重要.
需要仔细选择序列,以确保在基因传递中保持一致的AAV载体性能.

关键词:

美国的FTT.萨穆克斯MPMP 的意思在 UTR 中使用 UTR.完全/空的比率是完全/空的比例.质量测光仪的质量测光仪.纳米孔长读序列测序没有编码的DNA.监管部门的监管机构.矢量设计的设计方式

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