Nam Jung-Soo, Lee Ji-Eun, Lee Kwang-Hee, Yang Yeji, Kim Soo-Hyun, Bae Gyu-Un, Noh Hohsuk, Lim Kwang-Il
Department of Medical and Pharmaceutical Sciences, Sookmyung Women's University, Seoul 04310, Korea.
Department of Chemical and Biological Engineering, Sookmyung Women's University, Seoul 04310, Korea.
Mol Ther Methods Clin Dev. 2018 Nov 13;12:58-70. doi: 10.1016/j.omtm.2018.11.001. eCollection 2019 Mar 15.
The unique ability of retroviruses to integrate genes into host genomes is of great value for long-term expression in gene therapy, but only when integrations occur at safe genomic sites. To reap the benefit of using retroviruses without severe detrimental effects, we developed several murine leukemia virus (MLV)-based gammaretroviral vectors with safer integration patterns by perturbing the structure of the integrase via insertion of DNA-binding zinc-finger domains (ZFDs) into an internal position of the enzyme. ZFD insertion significantly reduced the inherent, strong MLV integration preference for genomic regions near transcriptional start sites (TSSs), which are the most dangerous spots. The altered retroviral integration pattern was related to increased formation of residual primer-binding site sequences at the 3' end of proviruses. Several ZFD insertion mutants showed lower frequencies of integrations into the TSS genome regions when having the residual primer-binding site sequences in the proviruses. Our findings not only can extend the use of retroviruses in biomedical applications, but also provide a glimpse into the mechanisms underlying retroviral integration.
逆转录病毒将基因整合到宿主基因组中的独特能力对于基因治疗中的长期表达具有重要价值，但前提是整合发生在安全的基因组位点。为了在不产生严重有害影响的情况下利用逆转录病毒的优势，我们通过将DNA结合锌指结构域（ZFDs）插入整合酶的内部位置来扰乱其结构，从而开发了几种基于鼠白血病病毒（MLV）的γ逆转录病毒载体，使其具有更安全的整合模式。ZFD插入显著降低了MLV对转录起始位点（TSSs）附近基因组区域固有的强烈整合偏好，而这些区域是最危险的位点。逆转录病毒整合模式的改变与原病毒3'端残留引物结合位点序列的形成增加有关。当原病毒中存在残留引物结合位点序列时，几种ZFD插入突变体显示出整合到TSS基因组区域的频率较低。我们的发现不仅可以扩展逆转录病毒在生物医学应用中的使用，还能让我们初步了解逆转录病毒整合的潜在机制。
