含有 E4 和 E2a 基因缺失的改良辅助质粒可提高 AAV 的产量。

IF 3.9 3区 医学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Human gene therapy Pub Date : 2024-09-01 Epub Date: 2024-07-31 DOI:10.1089/hum.2024.059
Laura van Lieshout, Stacy Ota, Annie Adusei, Eli Wiberg, Katrina Costa-Grant, Dimpal Lata, Serena Dollive, Marissa Stanvick, Ifeyinwa Iwuchukwu, Diane Golebiowski, Jin Yin
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引用次数: 0

摘要

在腺相关病毒(AAV)的生产过程中,使用辅助质粒来替代腺病毒感染是几十年来的普遍做法。腺病毒 E4、E2a 和 VA RNA 基因足以支持 AAV 的高效复制。为了确保所有转染 DNA 在 AAV 生产中发挥功能性作用,我们对 E4 和 E2a 基因进行了缺失,以确定是否有任何部分是可有可无的。虽然 E2a 内含子中 900 bp 的缺失没有影响,但从 E4 基因中删除开放阅读框(orf)1-4 却使 AAV 的产量翻了一番。E4∆orf1-4 的缺失与 E4orf6 转录本的减少以及 Rep 和 Cap 转录本和蛋白质水平的增加有关,这与粗裂解液中 AAV 生产率的提高相对应。这些研究的最终成果是一种辅助质粒(称为 OXB-Helper_3),它比原始对照质粒小 3.4 kb 以上,在多种囊壳血清型、基因组设计和转染平台上提高了约 2 倍的载体基因组 (VG) 生产率。
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An Improved Helper Plasmid Containing Deletions Within the E4 and E2a Genes Results in Increased Adeno-Associated Virus Productivity.

The use of a helper plasmid to replace adenovirus infection for adeno-associated virus (AAV) manufacturing has been common practice for decades. Adenovirus E4, E2a, and VA RNA genes are sufficient to support efficient AAV replication. In an effort to ensure that all transfected DNA has a functional role in AAV production, deletions were introduced to the E4 and E2a genes to determine if any portions were dispensable. Although a 900 bp deletion in the E2a intron did not have an impact, the removal of open reading frames (orf) 1-4 from the E4 gene resulted in a doubling of AAV productivity. The E4Δorf1-4 deletion was associated with a reduction in E4orf6 transcripts, along with an increase in Rep and Cap transcripts and protein levels, which corresponded to increased AAV productivity in crude lysate. The final product of these studies was a helper plasmid, termed OXB-Helper_3, that is >3.4 kb smaller than the original control plasmid and resulted in ∼2× improvement in vector genome productivity across multiple capsid serotypes, genome designs, and transfection platforms.

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来源期刊
Human gene therapy
Human gene therapy 医学-生物工程与应用微生物
CiteScore
6.50
自引率
4.80%
发文量
131
审稿时长
4-8 weeks
期刊介绍: Human Gene Therapy is the premier, multidisciplinary journal covering all aspects of gene therapy. The Journal publishes in-depth coverage of DNA, RNA, and cell therapies by delivering the latest breakthroughs in research and technologies. Human Gene Therapy provides a central forum for scientific and clinical information, including ethical, legal, regulatory, social, and commercial issues, which enables the advancement and progress of therapeutic procedures leading to improved patient outcomes, and ultimately, to curing diseases.
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