Department of Internal Medicine, University of Iowa, Iowa City, Iowa 52242, USA.
Mol Ther. 2010 May;18(5):947-54. doi: 10.1038/mt.2010.20. Epub 2010 Feb 16.
Hepatitis B virus (HBV) chronically infects 350-400 million people worldwide and causes >1 million deaths yearly. Current therapies prevent new viral genome formation, but do not target pre-existing viral genomic DNA, thus curing only approximately 1/2 of patients. We targeted HBV DNA for cleavage using zinc-finger nucleases (ZFNs), which cleave as dimers. Co-transfection of our ZFN pair with a target plasmid containing the HBV genome resulted in specific cleavage. After 3 days in culture, 26% of the target remained linear, whereas approximately10% was cleaved and misjoined tail-to-tail. Notably, ZFN treatment decreased levels of the hepatitis C virus pregenomic RNA by 29%. A portion of cleaved plasmids are repaired in cells, often with deletions and insertions. To track misrepair, we introduced an XbaI restriction site in the spacer between the ZFN sites. Targeted cleavage and misrepair destroys the XbaI site. After 3 days in culture, approximately 6% of plasmids were XbaI-resistant. Thirteen of 16 clones sequenced contained frameshift mutations that would lead to truncations of the viral core protein. These results demonstrate, for the first time, the possibility of targeting episomal viral DNA genomes using ZFNs.
乙型肝炎病毒 (HBV) 在全球范围内慢性感染 3.5 亿至 4 亿人，并导致每年超过 100 万人死亡。目前的治疗方法可以阻止新的病毒基因组形成，但不能针对已存在的病毒基因组 DNA，因此只能治愈大约一半的患者。我们使用锌指核酸酶 (ZFN) 靶向 HBV DNA 进行切割，ZFN 以二聚体形式切割。我们的 ZFN 对与含有 HBV 基因组的靶质粒共转染导致了特异性切割。在培养 3 天后，约 26%的靶标仍保持线性，而约 10%被切割并错误连接尾对尾。值得注意的是，ZFN 处理使丙型肝炎病毒前基因组 RNA 的水平降低了 29%。细胞中部分切割的质粒会进行修复，通常会出现缺失和插入。为了跟踪错误修复，我们在 ZFN 位点之间的间隔区引入了一个 XbaI 限制酶切位点。靶向切割和错误修复会破坏 XbaI 位点。在培养 3 天后，约 6%的质粒对 XbaI 具有抗性。对 16 个克隆中的 13 个进行测序，发现含有移码突变，这将导致病毒核心蛋白的截断。这些结果首次证明了使用 ZFN 靶向游离病毒 DNA 基因组的可能性。
