CD4 aptamer-siRNA chimeras to prevent HIV transmission

描述（由申请人提供）： 利用RNA干扰的力量进行疾病预防的主要障碍是在体内将小RNA递送穿过细胞质膜。这对于T淋巴细胞来说是一个特别可怕的障碍，T淋巴细胞是体内的移动靶标，并且难以通过脂质转染剂转导。我们已经设计、体外转录、纯化和测试了嵌合小RNA，其设计用于将小干扰RNA（siRNA）递送到人CD4+细胞中。这些嵌合体由一段结构化的RNA组成，称为适体，它识别并结合CD4，CD4在其3 '端连接到siRNA的有义（或无活性乘客）链。然后，RNA的这条链退火成小的反义（活性链）RNA。CD4适体-siRNA嵌合体特异性进入并诱导CD4+细胞中的基因沉默，但不在CD8 T细胞中。嵌合体沉默CD4+细胞系（HeLa-CD4，Jurkat）和原代CD4+人静息或活化PBMC中的基因表达。此外，用针对CCR5和/或HIV基因的CD4适体-siRNA预处理原代T细胞和巨噬细胞以及极化的宫颈阴道组织外植体抑制HIV复制。因此，嵌合体在组织内运输，以找到并包裹完整组织中固有层内的免疫细胞。适体-siRNA嵌合体不损伤生殖器粘膜或诱导组织中IFN、IFN应答基因、IL-12、IL-6或IL-8的表达。在NOD/scid/IL2R？-/-中阴道内应用CD4适体-siRNA嵌合体后获得的初步数据用人骨髓、胎肝和胸腺重建的小鼠（BLT小鼠）显示了Cy3标记的嵌合体的摄取和宫颈阴道组织CD4+细胞中的CCR5沉默。基于这些初步结果，我们假设阴道内应用靶向HIV和/或CCR5的CD4适体-siRNA嵌合体可以预防HIV的性传播而不会引起过度毒性。此外，我们预计嵌合RNA作为适合全球使用的杀微生物剂中的活性成分是实用的，并且保护将持续约一周，这意味着局部施用将不需要在性交前进行。该提案将开始评估CD4适体-siRNA嵌合体是否可以用作局部杀微生物剂的活性成分，以预防HIV的性传播。为了实现这一目标，我们将（1）测试SiRNA-CD4适体在体外和极化外植体中用于细胞特异性摄取、基因沉默、抑制HIV感染和潜在毒性的效用;（2）优化SiRNA-CD4适体的设计;和（3）测试阴道内给予人源化BLT小鼠的SiRNA-CD4适体保护免于HIV阴道传播的有效性和持久性。在缺乏有效疫苗的情况下，局部杀微生物剂可以提供一种帮助控制全球艾滋病毒流行的方法。一种基于siRNA的杀微生物剂，一种已被证明在小鼠中抑制HSV-2传播至少一周的方法，提供了一种建立相对长期的保护以防止生殖道中的性传播的方法，以减少在性交前治疗的需要。我们建议在这项提案中研究一种新的方法，以克服扩大抑制HSV-2传播给HIV的有希望的结果的主要障碍，即使小RNA进入HIV感染的免疫细胞。

DESCRIPTION (provided by applicant): The main obstacle to harnessing the power of RNA interference for disease prevention is delivering small RNAs across the cell plasma membrane in vivo. This is an especially formidable obstacle for T lymphocytes, which are a moving target in the body and are refractory to transduction by lipid transfection agents. We have engineered, in vitro transcribed, purified and tested chimeric small RNAs designed to deliver small interfering RNAs (siRNAs) into human CD4+ cells. These chimeras are composed of a structured piece of RNA, called an aptamer, that recognizes and binds to CD4, which is linked at its 3'-end to the sense (or inactive passenger) strand of an siRNA. This strand of the RNA is then annealed to a small antisense (active strand) RNA. The CD4 aptamer-siRNA chimeras specifically enter and induce gene silencing in CD4+ cells, but not in CD8 T cells. Chimeras silence gene expression both in CD4+ cell lines (HeLa-CD4, Jurkat) and in primary CD4+ human resting or activated PBMCs. Moreover, pretreatment of primary T cells and macrophages, as well as polarized cervicovaginal tissue explants, with CD4 aptamer-siRNAs directed against CCR5 and/or HIV genes inhibits HIV replication. Therefore the chimeras traffic within tissues to find and transduce immune cells within the lamina propria in intact tissues. The aptamer-siRNA chimeras do not damage the genital mucosa or induce expression of IFNs, IFN response genes, IL-12, IL-6 or IL-8 in tissues. Preliminary data obtained after intravaginal application of CD4 aptamer-siRNA chimeras in NOD/scid/IL2R?-/- mice reconstituted with human bone marrow, fetal liver and thymus (BLT mice) show both uptake of Cy3-labeled chimeras and CCR5 silencing in cervicovaginal tissue CD4+ cells. Based on these preliminary results, we hypothesize that intravaginal application of CD4 aptamer-siRNA chimeras targeting HIV and/or CCR5 can prevent sexual transmission of HIV without causing undue toxicity. Moreover we expect that chimeric RNAs are practical as the active ingredient in a microbicide suitable for global use and that protection will last for about a week implying that topical application will not need to be performed just before intercourse. This proposal will begin to evaluate whether CD4 aptamer-siRNA chimeras can be used as the active component of a topical microbicide to prevent sexual transmission of HIV. To accomplish this goal, we will (1) test the utility of siRNA- CD4 aptamers in vitro and in polarized explants for cell-specific uptake, gene silencing, inhibition of HIV infection, and potential toxicity; (2) optimize the design of siRNA-CD4 aptamers; and (3) test siRNA-CD4 aptamers administered intravaginally to humanized BLT mice for their effectiveness and durability of protection from HIV vaginal transmission. In the absence of an effective vaccine, topical microbicides could provide a way to help control the global HIV pandemic. An siRNA-based microbicide, an approach which has been shown to inhibit HSV-2 transmission for at least a week in mice, provides a method to establish relatively long-lived protection against sexual transmission in the genital tract to obviate the need for treatment just before sexual intercourse. We propose to investigate in this proposal a novel method for overcoming the major obstacle to extending the promising results for inhibiting HSV-2 transmission to HIV, namely getting small RNAs into the immune cells that HIV infects.