Bai J, Rossi J, Akkina R
Department of Pathology, Colorado State University, Fort Collins, Colorado 80523, USA.
AIDS Res Hum Retroviruses. 2001 Mar 20;17(5):385-99. doi: 10.1089/088922201750102427.
HIV-1 infection of susceptible cells is mediated by the specific interaction of viral envelope glycoproteins with the cell surface CD4 receptor and a chemokine coreceptor, CCR5 or CXCR4. Individuals with a CCR5 genetic defect show resistance to HIV-1 infection, indicating that downregulation of CCR5 expression on target cells can prevent viral infection. In previous studies we demonstrated the utility of an anti-CCR5 ribozyme targeted to a single cleavage site in downregulating CCR5 expression and consequently providing resistance to viral infection. To improve on the level of downregulation we designed a construct containing an anti-CCR5 ribozyme heterotrimer (R5RbzTM) targeted to three different cleavage sites in CCR5 mRNA. In vitro tests showed that the anti-CCR5 ribozyme heterotrimer could effectively cleave the CCR5 RNA substrates to yield products of the expected sizes. This construct was introduced into various retroviral vectors for stable gene transduction. HOS.CD4/R5 cells stably transduced with this anti-CCR5 heterotrimer showed a marked reduction in the surface expression of CCR5 and a concomitant 70% reduction in macrophage-tropic viral infection. In addition, a retroviral vector containing the anti-CCR5 ribozyme heterotrimer and an anti-HIV-1 tat-rev ribozyme heterodimer was constructed. This construct also showed a similar inhibition of CCR5 surface expression and reduced infectability by the macrophage-tropic HIV-1 vector in HOS.CD4/R5 cells. The trimeric and multimeric ribozyme constructs were transduced into CD34+ hematopoietic progenitor cells to determine their effects on lineage-specific differentiation. We show that multivalent ribozyme gene-transduced hematopoietic progenitors differentiated normally into mature macrophages that bear CD14 and CD4 surface markers. Macrophages containing the transgenes expressed ribozymes, and showed resistance to M-tropic HIV-1 infection. These results provide strong support for the use of the trimeric anti-CCR5 ribozyme approach in a gene therapy setting for the treatment of HIV infection.
HIV-1对易感细胞的感染是由病毒包膜糖蛋白与细胞表面CD4受体以及趋化因子共受体CCR5或CXCR4之间的特异性相互作用介导的。具有CCR5基因缺陷的个体对HIV-1感染表现出抗性，这表明靶细胞上CCR5表达的下调可以预防病毒感染。在先前的研究中，我们证明了靶向单个切割位点的抗CCR5核酶在下调CCR5表达并因此提供对病毒感染的抗性方面的效用。为了提高下调水平，我们设计了一种构建体，其包含靶向CCR5 mRNA中三个不同切割位点的抗CCR5核酶异源三聚体（R5RbzTM）。体外试验表明，抗CCR5核酶异源三聚体可以有效切割CCR5 RNA底物，产生预期大小的产物。该构建体被引入各种逆转录病毒载体以进行稳定的基因转导。用这种抗CCR5异源三聚体稳定转导的HOS.CD4/R5细胞显示CCR5的表面表达显著降低，同时巨噬细胞嗜性病毒感染减少70%。此外，构建了一种包含抗CCR5核酶异源三聚体和抗HIV-1 tat-rev核酶异源二聚体的逆转录病毒载体。该构建体在HOS.CD4/R5细胞中也显示出对CCR5表面表达的类似抑制以及巨噬细胞嗜性HIV-1载体感染性的降低。将三聚体和多聚体核酶构建体转导到CD34+造血祖细胞中，以确定它们对谱系特异性分化的影响。我们表明，多价核酶基因转导的造血祖细胞正常分化为带有CD14和CD4表面标志物的成熟巨噬细胞。含有转基因的巨噬细胞表达核酶，并对M嗜性HIV-1感染表现出抗性。这些结果为在基因治疗环境中使用三聚体抗CCR5核酶方法治疗HIV感染提供了有力支持。
