{"title":"Encapsulation of a Single-Stranded Form of DNA Impurities into the Capsid of a Recombinant Adeno-Associated Virus.","authors":"Kazuhisa Uchida, Emi Ito-Kudo, Kiyoko Higashiyama, Kyoko Masumi-Koizumi, Keisuke Yusa, Yuzhe Yuan","doi":"10.1089/hum.2024.264","DOIUrl":null,"url":null,"abstract":"<p><p>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.</p>","PeriodicalId":13007,"journal":{"name":"Human gene therapy","volume":" ","pages":""},"PeriodicalIF":3.9000,"publicationDate":"2025-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Human gene therapy","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1089/hum.2024.264","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
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.
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.