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慢病毒载体因其在多种分裂和非分裂细胞中高效稳定表达的优势，已成为细胞和基因治疗领域的重要工具，但其在临床应用中可能因同源或非同源重组产生复制型慢病毒(Replication-competent lentivirus,RCL)，带来潜在安全性风险。本研究通过反向遗传学技术对HIV-1 NL4-3株进行改造，删除辅助蛋白基因(Vif、Vpr、Vpu和Nef)，并插入NanoLuc荧光报告基因，成功构建了遗传稳定的HIV弱毒株NL4-3-dA-NLuc。HIV-1 p24抗原检测、滴度测定及病毒基因组测序结果均显示病毒拯救成功。进一步研究结果表明，该弱毒株保留了母本病毒的CXCR4感染嗜性，但复制能力显著降低。在连续传5代后病毒基因组未突变，表现出良好遗传稳定性，且传代细胞可见明显细胞病变效应，可稳定检测到HIV-1 p24抗原及NanoLuc报告基因表达。RCL检测感染灵敏度验证实验显示，NL4-3-dA-NLuc株作为阳性对照病毒的RCL最低检测限为5TCID50。本研究为RCL检测方法的优化提供了一种遗传稳定且定量灵敏的阳性对照病毒，为基因治疗产品的安全性评估提供了重要技术支持，有望推动国内RCL检测技术的规范化和广泛应用。
Abstract：Lentiviral vectors have become essential tools in cell and gene therapy due to their ability to mediate efficient and stable gene expression in both dividing and non-dividing cells. However, their clinical use carries potential safety risks, as homologous or non-homologous recombination events may lead to the emergence of replication-competent lentiviruses(RCLs). In this study, an attenuated HIV-1 strain(NL4-3-dA-NLuc) was constructed using reverse genetics by deleting four accessory protein genes(Vif, Vpr, Vpu, and Nef) from the HIV-1 NL4-3 backbone and inserting the NanoLuc luciferase reporter gene. Successful viral rescue was confirmed by HIV-1 p24 antigen detection, viral titration, and full-genome sequencing. Further analysis demonstrated that the attenuated strain retained the CXCR4 tropism of the parental virus but exhibited markedly reduced replicative capacity. After five serial passages, the viral genome remained mutation-free, indicating high genetic stability. Infected cells exhibited clear cytopathic effects and showed stable expression of both the HIV-1 p24 antigen and the NanoLuc reporter gene. Sensitivity testing of the RCL assay revealed that the NL4-3-dANLuc strain, used as a positive control virus, had a minimum detectable infectious dose of 5 TCID50. This study provides a genetically stable and quantitatively sensitive positive control virus for optimizing RCL detection methods. It offers valuable technical support for the safety assessment of gene therapy products and is expected to promote the standardization and broader adoption of RCL detection protocols in China.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.250124
中图分类号:R373.9
引用信息:
[1]房恩岳,常宇桐,何畦嘉,等.复制型慢病毒检测用HIV弱毒株的构建及生物学特性研究[J].病毒学报,2025,41(05):1393-1402.DOI:10.13242/j.cnki.bingduxuebao.250124.
基金信息:
中国质检院基本科研业务费项目（项目号：2024JK023），题目：市场监管领域生物安全风险监测与预警支撑研究;中国质检院基本科研业务费项目（项目号：2023JK004），题目：关于复制型慢病毒检测用HIV弱毒株构建的研究~~