Anderson J, Akkina R
Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA.
Gene Ther. 2007 Sep;14(17):1287-97. doi: 10.1038/sj.gt.3302958. Epub 2007 Jun 28.
The CCR5 co-receptor is necessary for cellular entry by R5 tropic viral strains involved in primary HIV infection, but is dispensable for normal human physiology. Owing to its crucial role in HIV-1 infection, the CCR5 co-receptor has been the subject of many therapeutic approaches, including gene therapy. siRNA targeting was shown to be effective in downregulating CCR5 expression and conferring significant protection against HIV-1 in susceptible cells. However, complete knockdown of CCR5 expression has not been achieved and thus remains an elusive goal. In these studies, we identified new CCR5 siRNAs capable of achieving complete knockdown of the co-receptor expression. Our transfection studies have shown that longer 28-mer short hairpin siRNAs are very effective in gene downregulation as assessed by fluorescence-activated cell sorting and transcript quantitation by quantitative real-time polymerase chain reaction. These siRNAs conferred strong antiviral protection during viral challenge. To obtain stable expression, highly potent siRNA expression cassettes were introduced into lentiviral vectors. Similar high levels of CCR5 downregulation were observed in stably transduced cells with concomitant viral protection in cultured cell lines. To translate these results to a stem cell gene therapy setting, CD34 hematopoietic progenitor cells were transduced with lentiviral vectors to derive transgenic macrophages. The transgenic cells also exhibited high levels of CCR5 downregulation and viral resistance. With regard to Pol-III promoter-mediated siRNA expression, higher efficacies were obtained with U6-driven CCR5 siRNAs. However, in contrast to previous reports, no apparent cytotoxicities were observed in transgenic cells containing U6-driven siRNA constructs. Thus the above anti-CCR5 siRNAs are among the most effective demonstrated to date and are very promising candidates for clinical applications.
CCR5 共受体对于原发性 HIV 感染中涉及的 R5 嗜性病毒株进入细胞是必需的，但对正常人体生理功能并非必不可少。由于其在 HIV-1 感染中的关键作用，CCR5 共受体一直是包括基因治疗在内的多种治疗方法的研究对象。靶向 siRNA 已被证明可有效下调 CCR5 表达，并在易感细胞中对 HIV-1 提供显著保护。然而，尚未实现 CCR5 表达的完全敲低，因此仍然是一个难以实现的目标。在这些研究中，我们鉴定出了能够实现共受体表达完全敲低的新型 CCR5 siRNA。我们的转染研究表明，通过荧光激活细胞分选和定量实时聚合酶链反应进行转录本定量评估，较长的 28 聚体短发夹 siRNA 在基因下调方面非常有效。这些 siRNA 在病毒攻击期间赋予了强大的抗病毒保护。为了获得稳定表达，将高效 siRNA 表达盒引入慢病毒载体。在稳定转导的细胞中观察到类似的高水平 CCR5 下调，并在培养的细胞系中具有伴随的病毒保护作用。为了将这些结果转化为干细胞基因治疗环境，用慢病毒载体转导 CD34 造血祖细胞以产生转基因巨噬细胞。转基因细胞也表现出高水平的 CCR5 下调和病毒抗性。关于 Pol-III 启动子介导的 siRNA 表达，U6 驱动的 CCR5 siRNA 具有更高的效率。然而，与先前的报道相反，在含有 U6 驱动的 siRNA 构建体的转基因细胞中未观察到明显的细胞毒性。因此，上述抗 CCR5 siRNA 是迄今为止已证明的最有效的之一，是非常有前景的临床应用候选物。
