Shi Bingjie, Li Juan, Shi Xuanling, Jia Wenxu, Wen Yi, Hu Xiongbing, Zhuang Fengfeng, Xi Jianzhong, Zhang Linqi
*Comprehensive AIDS Research Center and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, School of Medicine, Tsinghua University, Beijing, China; †Department of Biomedical Engineering, Peking University, Beijing, China; and ‡ViewSolid Biotech, Beijing, China.
J Acquir Immune Defic Syndr. 2017 Feb 1;74(2):229-241. doi: 10.1097/QAI.0000000000001190.
Transcription activator-like effector nuclease (TALEN) represents a valuable tool for genomic engineering due to its single-nucleotide precision, high nuclease activity, and low cytotoxicity. We report here systematic design and characterization of 28 novel TALENs targeting multiple regions of CCR5 gene (CCR5-TALEN) which encodes the co-receptor critical for entry of human immunodeficiency virus type I (HIV-1). By systemic characterization of these CCR5-TALENs, we have identified one (CCR5-TALEN-515) with higher nuclease activity, specificity, and lower cytotoxicity compared with zinc-finger nuclease (CCR5-ZFN) currently undergoing clinical trials. Sequence analysis of target cell line GHOST-CCR5-CXCR4 and human primary CD4 T cells showed that the double-strand breaks at the TALEN targeted sites resulted in truncated or nonfunctional CCR5 proteins thereby conferring protection against HIV-1 infection in vitro. None of the CCR5-TALENs had detectable levels of off-target nuclease activity against the homologous region in CCR2 although substantial level was identified for CCR5-ZFN in the primary CD4 T cells. Our results suggest that the CCR5-TALENs identified here are highly functional nucleases that produce protective genetic alterations to human CCR5. Application of these TALENs directly to the primary CD4 T cells and CD34 hematopoietic stem cells (HSCs) of infected individuals could help to create an immune system resistant to HIV-1 infection, recapitulating the success of "Berlin patient" and serving as an essential first step towards a "functional" cure of AIDS.
转录激活样效应核酸酶（TALEN）因其单核苷酸精确性、高核酸酶活性和低细胞毒性，成为基因组工程中的一种重要工具。我们在此报告了针对CCR5基因多个区域的28种新型TALEN（CCR5-TALEN）的系统设计与特性研究，CCR5基因编码对I型人类免疫缺陷病毒（HIV-1）进入至关重要的共受体。通过对这些CCR5-TALEN的系统特性研究，我们鉴定出一种（CCR5-TALEN-515），与目前正在进行临床试验的锌指核酸酶（CCR5-ZFN）相比，其具有更高的核酸酶活性、特异性和更低的细胞毒性。对靶细胞系GHOST-CCR5-CXCR4和人原代CD4 T细胞的序列分析表明，TALEN靶向位点的双链断裂导致CCR5蛋白截短或无功能，从而在体外赋予对HIV-1感染的抗性。尽管在原代CD4 T细胞中鉴定出CCR5-ZFN存在相当程度的脱靶核酸酶活性，但所有CCR5-TALEN对CCR2同源区域均未检测到可检测水平的脱靶核酸酶活性。我们的结果表明，这里鉴定出的CCR5-TALEN是高度功能性的核酸酶，可对人类CCR5产生保护性基因改变。将这些TALEN直接应用于受感染个体的原代CD4 T细胞和CD34造血干细胞（HSC），可能有助于创建对HIV-1感染具有抗性的免疫系统，重现“柏林病人”的成功，并作为迈向艾滋病“功能性”治愈的关键第一步。
