SUPPRESSION OF HIV-1 IN VITRO BY PUTATIVE TRIPLE HELIX OLIGONUCLEOTIDES

这份报告描述了设计用于 补充HIV-1前病毒基因组上的特定靶点并形成 “三螺旋”结构，可通过阻断病毒的 目标双链DNA的主要凹槽。我们设计了几个ON 与HIV-1基因组中的嘌呤富集区互补，目标是 抑制艾滋病毒感染的能力。这些国家统计局针对的艾滋病毒基因包括 GAG、POL、TAT、NEF和VIF。我们测试了这些国家安全局的进入能力 并抑制HIV-1(MN)感染H9淋巴细胞系。 On和病毒同时直接加入细胞培养基中。英国国家统计局是 在初始处理后10分钟内出现在处理细胞的细胞核中。 用10到50微米浓度的下列任一种药物处理感染细胞 ONS显著降低了病毒水平，表现为：1)50%-90% P24捕获酶联免疫吸附试验检测细胞外p24抗原下降 通过检测到细胞内p24的伴随减少 免疫荧光标记和FACSCAN分析，3)类似的减少 相对病毒DNA含量(通过细胞裂解产物的DNA聚合酶链式反应检测) 未经治疗的受感染细胞。细胞的生长和活力不受 这些都是低浓度的。在最初的研究中，被感染的细胞被 用50um的GAG特异性抗原产生的p24抗原比感染的少85% 用50um的非特异性的嘧啶处理的细胞，具有类似的碱基 组成(对照)。关于非特异性ONS的进一步研究在 进步。原代人外周血的初步研究 淋巴细胞也显示出HIV特异性三重抑制病毒的作用 螺旋ONS。这些ONS显示出作为抗艾滋病毒药物的前景。 正在进行的研究正在解决1)对病毒治疗的影响 转录本，2)ON在细胞和细胞核中的持久性，以及3) ONS与HIV序列形成三螺旋复合体的能力。

This report describes pyrimidine oligonucleotides (ONs) designed to complement a specific target on the HIV-1 proviral genome and form a "triple helix" structure which could affect virus activity by blocking the major groove of the target double-stranded DNA. We designed several ONs complementary to purine-rich regions within the HIV-1 genome with the goal of suppressing HIV infection. The HIV genes targeted by these ONs include gag, pol, tat, nef and vif. We tested the ability of these ONs to enter the cells and suppress HIV-1 (MN) infection of the H9 lymphocyte cell line. ON and virus were added directly to cell media at the same time. ONs were present in the nuclei of treated cells within 10 mins of initial treatment. Treatment of infected cells with 10 to 50 uM concentrations of any of the ONs significantly reduced virus levels as demonstrated by: 1) a 50-90% decrease in extracellular p24 antigen as measured by p24 capture ELISA, 2) a concomitant decrease in intracellular p24 as detected by immunofluorescence labeling and FACSCAN analysis, 3) a similar reduction in amounts of viral DNA (detected by DNA PCR assay of cell lysates) relative to untreated infected cells. Cell growth and viability was not affected at these low ON concentrations. In an initial study, infected cells treated with 50 uM of GAG specific ON produced 85% less p24 antigen than infected cells treated with 50 uM of a nonspecific, pyrimidine ON with similar base composition (control). Further studies with nonspecific ONs are in progress. Preliminary studies with primary human peripheral blood lymphocytes also demonstrated suppression of virus by HIV-specific triple helix ONs. These ONs show promise as anti-HIV agents. Ongoing studies are addressing 1) the effects of ON treatment on viral transcripts, 2) the persistence of ON in cells and nuclei, and 3) the ability of the ONs to form triple helix complexes with HIV sequences.