Encapsulation of a Single-Stranded Form of DNA Impurities into the Capsid of a Recombinant Adeno-Associated Virus.

IF 3.9 3区 医学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Kazuhisa Uchida, Emi Ito-Kudo, Kiyoko Higashiyama, Kyoko Masumi-Koizumi, Keisuke Yusa, Yuzhe Yuan
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引用次数: 0

Abstract

Recombinant adeno-associated viruses (rAAVs) are widely used viral vectors in human gene therapy. However, DNA impurities, such as plasmid DNA and host cell DNA, remain a significant quality control concern for final products. Our study examined purified rAAV1-ZsGreen1, rAAV2-ZsGreen1, rAAV5-ZsGreen1, and rAAV6-ZsGreen1 samples and found that they contained 0.69-3.27% DNA impurities derived from three plasmids, as detected by droplet digital PCR. These plasmid-derived impurities primarily consisted of those derived from the pAAV plasmid (≥98.88%), with small amounts of pRC1, pRC2mi342, pRC5, or pRC6 (≤0.91%), and pHelper (≤0.21%) plasmids. To determine the DNA strand form of these impurities within the capsids, we used two different DNases with distinct substrate specificities. The extracted DNA impurities from the rAAV samples exhibited high sensitivity to nuclease P1 but not to lambda exonuclease. Similarly, host cell DNA encapsulated within the capsids revealed similar sensitivities to the nucleases. These findings indicate that DNA impurities derived from the plasmids and host cell DNA are encapsulated into rAAV capsids as single-stranded DNA, likely through a mechanism similar to that of the rAAV genome.

将单链形式的DNA杂质封装到重组腺相关病毒的衣壳中。
重组腺相关病毒(raav)是广泛应用于人类基因治疗的病毒载体。然而,DNA杂质,如质粒DNA和宿主细胞DNA,仍然是最终产品的重要质量控制问题。本研究对纯化的rAAV1-ZsGreen1、rAAV2-ZsGreen1、rAAV5-ZsGreen1和rAAV6-ZsGreen1样品进行了检测,发现它们含有0.69-3.27%的来自三种质粒的DNA杂质。这些质粒来源的杂质主要来自pAAV质粒(≥98.88%),少量的pRC1、pRC2mi342、pRC5或pRC6质粒(≤0.91%)和pHelper质粒(≤0.21%)。为了确定衣壳内这些杂质的DNA链形式,我们使用了两种具有不同底物特异性的不同DNA酶。从rAAV样品中提取的DNA杂质对核酸酶P1具有高敏感性,但对lambda外切酶不敏感。同样,包裹在衣壳内的宿主细胞DNA对核酸酶也表现出类似的敏感性。这些发现表明,来自质粒和宿主细胞DNA的DNA杂质可能通过类似于rAAV基因组的机制,以单链DNA的形式被封装到rAAV衣壳中。
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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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