Uchida Kazuhisa, Ito-Kudo Emi, Higashiyama Kiyoko, Masumi-Koizumi Kyoko, Yusa Keisuke, Yuan Yuzhe
Graduate School of Science, Technology and Innovation, Kobe University, Kobe, Japan.
Hum Gene Ther. 2025 May;36(9-10):814-822. doi: 10.1089/hum.2024.264. Epub 2025 Mar 19.
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.
重组腺相关病毒（rAAVs）是人类基因治疗中广泛使用的病毒载体。然而，DNA杂质，如质粒DNA和宿主细胞DNA，仍然是最终产品质量控制的一个重大问题。我们的研究检测了纯化的rAAV1-ZsGreen1、rAAV2-ZsGreen1、rAAV5-ZsGreen1和rAAV6-ZsGreen1样本，发现通过液滴数字PCR检测，它们含有0.69-3.27%源自三种质粒的DNA杂质。这些源自质粒的杂质主要由源自pAAV质粒的杂质组成（≥98.88%），少量为pRC1、pRC2mi342、pRC5或pRC6（≤0.91%）以及pHelper（≤0.21%）质粒。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脱氧核糖核酸酶。从rAAV样本中提取的DNA杂质对核酸酶P1表现出高敏感性，但对λ外切核酸酶不敏感。同样，衣壳内包裹的宿主细胞DNA对核酸酶也表现出类似的敏感性。这些发现表明，源自质粒和宿主细胞DNA的DNA杂质以单链DNA形式被包裹进rAAV衣壳，可能通过与rAAV基因组类似的机制。
