Li Ming-Jie, Bauer Gerhard, Michienzi Alessandro, Yee Jiing-Kuan, Lee Nan-Sook, Kim James, Li Shirley, Castanotto Daniela, Zaia John, Rossi John J
Division of Molecular Biology, Beckman Research Institute of the City of Hope, Duarte, California 91010, USA.
Mol Ther. 2003 Aug;8(2):196-206. doi: 10.1016/s1525-0016(03)00165-5.
A primary advantage of lentiviral vectors is their ability to pass through the nuclear envelope into the cell nucleus thereby allowing transduction of nondividing cells. Using HIV-based lentiviral vectors, we delivered an anti-CCR5 ribozyme (CCR5RZ), a nucleolar localizing TAR RNA decoy, or Pol III-expressed siRNA genes into cultured and primary cells. The CCR5RZ is driven by the adenoviral VA1 Pol III promoter, while the human U6 snRNA Pol III-transcribed TAR decoy is embedded in a U16 snoRNA (designated U16TAR), and the siRNAs were expressed from the human U6 Pol III promoter. The transduction efficiencies of these vectors ranged from 96-98% in 293 cells to 15-20% in primary PBMCs. A combination of the CCR5RZ and U16TAR decoy in a single vector backbone gave enhanced protection against HIV-1 challenge in a selective survival assay in both primary T cells and CD34(+)-derived monocytes. The lentiviral vector backbone-expressed siRNAs also showed potent inhibition of p24 expression in PBMCs challenged with HIV-1. Overall our results demonstrate that the lentiviral-based vectors can efficiently deliver single constructs as well as combinations of Pol III therapeutic expression units into primary hematopoietic cells for anti-HIV gene therapy and hold promise for stem or T-cell-based gene therapy for HIV-1 infection.
慢病毒载体的一个主要优点是它们能够穿过核膜进入细胞核，从而实现对非分裂细胞的转导。利用基于HIV的慢病毒载体，我们将一种抗CCR5核酶（CCR5RZ）、一种核仁定位的TAR RNA诱饵或由Pol III表达的siRNA基因导入培养细胞和原代细胞中。CCR5RZ由腺病毒VA1 Pol III启动子驱动，而人U6 snRNA Pol III转录的TAR诱饵嵌入U16 snoRNA（命名为U16TAR）中，siRNAs由人U6 Pol III启动子表达。这些载体的转导效率在293细胞中为96 - 98%，在原代PBMC中为15 - 20%。在原代T细胞和CD34(+)来源的单核细胞的选择性存活试验中，单个载体骨架中CCR5RZ和U16TAR诱饵的组合对HIV-1攻击提供了增强的保护。慢病毒载体骨架表达的siRNAs在受到HIV-1攻击的PBMC中也显示出对p24表达的有效抑制。总体而言，我们的结果表明，基于慢病毒的载体能够有效地将单个构建体以及Pol III治疗性表达单元的组合递送至原代造血细胞中用于抗HIV基因治疗，并且为基于干细胞或T细胞的HIV-1感染基因治疗带来了希望。
