Department of Microbiology, New York University School of Medicine, New York, NY, USA.
Gene Ther. 2013 May;20(5):514-20. doi: 10.1038/gt.2012.61. Epub 2012 Aug 16.
Lentiviral vectors are widely used for the stable expression of genes and small hairpin RNA (shRNA)-mediated knockdown and are currently under development for clinical use in gene therapy. Pseudotyping of the vectors with VSV-G allows them to infect a wide range of cell types. However, myeloid cells, such as dendritic cells and macrophages, are relatively refractory to lentiviral vector transduction as a result of the myeloid-specific restriction factor, SAMHD1. SIVmac/HIV-2 and related viruses relieve the SAMHD1-mediated restriction by encoding Vpx, a virion-packaged accessory protein that induces the degradation of SAMHD1 upon infection. HIV-1 does not encode Vpx and cannot package the protein. We report the development of an HIV-1-based lentiviral vector in which the Vpx packaging motif has been placed in the p6 region of the Gag/Pol expression vector that is used to generate the lentiviral vector virions. The virions package Vpx in high copy number and infect myeloid cells with a two-log increase in titer. Transduction of dendritic cells with an shRNA against transportin-3 resulted in >90% knockdown of the encoding mRNA. The system can be applied to any HIV-based lentiviral vector and is useful for laboratory and clinical applications where the efficient transduction of myeloid cells is required.
慢病毒载体被广泛用于基因的稳定表达和短发夹 RNA(shRNA)介导的敲低，目前正处于基因治疗的临床应用开发阶段。用 VSV-G 对载体进行假型化可以使它们感染广泛的细胞类型。然而，由于髓系特异性限制因子 SAMHD1 的存在，髓系细胞（如树突状细胞和巨噬细胞）对慢病毒载体转导相对耐受。SIVmac/HIV-2 和相关病毒通过编码 Vpx 来缓解 SAMHD1 介导的限制，Vpx 是一种病毒包装的辅助蛋白，在感染时诱导 SAMHD1 的降解。HIV-1 不编码 Vpx，也不能包装该蛋白。我们报告了一种基于 HIV-1 的慢病毒载体的开发，其中 Vpx 包装基序已被放置在 Gag/Pol 表达载体的 p6 区域，该载体用于生成慢病毒载体病毒颗粒。这些病毒颗粒以高拷贝数包装 Vpx，并使髓系细胞感染的效价增加两个对数级。用针对转运蛋白-3 的 shRNA 转导树突状细胞可导致编码 mRNA 的敲低超过 90%。该系统可应用于任何基于 HIV 的慢病毒载体，适用于需要有效转导髓系细胞的实验室和临床应用。
