Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095, USA.
J Gene Med. 2010 Mar;12(3):255-65. doi: 10.1002/jgm.1440.
We recently expressed a potent and noncytotoxic short hairpin (sh)RNA directed against chemokine (c-c motif) receptor 5 (CCR5) using lentiviral mediated transduction of CD34+ hematopoietic progenitor cells (HPCs) and demonstrated the stable reduction of CCR5 expression in T-lymphocytes.
In the present study, we further assessed the activity of the shRNA through HPC transduction and differentiation into macrophages derived from fetal liver CD34+ (FL-CD34+) HPCs. Transduced lentiviral vector encoding the human CCR5 shRNA was stably maintained in FL-CD34+ cells and in the terminally differentiated macrophages using macrophage colony-stimulating factor, granulocyte macrophage colony-stimulating factor, interleukin-3 and stem cell factor.
Quantitative real-time polymerase chain reaction for CCR5 mRNA indicated over 90% reduction of CCR5 mRNA levels in CCR5 shRNA-transduced population. The cells with knockdown of CCR5 expression acquired resistance to R5 tropic HIV-1 NFN-SX strain. We also developed a novel approach utilizing a mCherry-CCR5 chimeric reporter to assess the effectiveness of CCR5 target down-regulation in macrophages directly. Both the shRNA and the reporter were maintained throughout HPC differentiation to macrophages without apparent cytotoxicity.
The present study demonstrates a novel method to simply and directly assess the function of small interfering RNA and the effective inhibition of HIV-1 infection by a potential potent shRNA to CCR5 delivered into macrophages derived from HPCs.
我们最近使用慢病毒介导的转导 CD34+造血祖细胞（HPC）表达了一种针对趋化因子（c-c 基序）受体 5（CCR5）的有效且非细胞毒性的短发夹（sh）RNA，并证明了 T 淋巴细胞中 CCR5 表达的稳定降低。
在本研究中，我们通过 HPC 转导和分化为来自胎儿肝脏 CD34+（FL-CD34+）HPC 的巨噬细胞进一步评估了 shRNA 的活性。转导的慢病毒载体编码人 CCR5 shRNA，在使用巨噬细胞集落刺激因子、粒细胞巨噬细胞集落刺激因子、白细胞介素 3 和干细胞因子的 FL-CD34+细胞和终末分化的巨噬细胞中稳定维持。
CCR5 mRNA 的定量实时聚合酶链反应表明，CCR5 shRNA 转导群体中的 CCR5 mRNA 水平降低了 90%以上。表达 CCR5 下调的细胞对 R5 嗜性 HIV-1 NFN-SX 株获得了抗性。我们还开发了一种新方法，利用 mCherry-CCR5 嵌合报告基因来直接评估巨噬细胞中 CCR5 靶基因下调的有效性。shRNA 和报告基因在整个 HPC 分化为巨噬细胞的过程中均得以维持，且没有明显的细胞毒性。
本研究证明了一种新的方法，可以简单直接地评估小干扰 RNA 的功能，以及通过潜在有效的 CCR5 shRNA 直接递送至源自 HPC 的巨噬细胞中，有效地抑制 HIV-1 感染。
