Developing RNA Interference for and HIV microbicide

描述(申请人提供)：当无细胞或细胞相关病毒主要通过表达在巨噬细胞、树突状细胞和激活的T淋巴细胞上的CCR5辅助受体感染细胞时，艾滋病毒发生性传播。一种可用于阴道或肛门以防止性传播的杀微生物剂将对控制艾滋病毒的传播作出重大贡献。这一创新的拨款将开始探索这样一个假设，即RNA干扰(RNAi)可以形成有效的抗HIV微生物剂的基础。RNAi是一种古老的、进化上保守的针对病毒和转座元件的宿主防御系统，它使用称为小干扰RNAs(SiRNA)的小双链RNAs，通过靶向降解同源mRNAs来抑制基因表达。对于RNAi治疗病毒感染的治疗潜力，已经有很多令人兴奋的事情。一个主要的障碍是如何将siRNA输送到细胞中。我们的初步结果表明，双链siRNAs可以在不经转染的情况下传递给巨噬细胞和激活的T细胞，并导致长时间的基因沉默，从而抑制新感染和已感染细胞中的病毒复制。这表明双链siRNAs可能是杀菌剂中的活性成分。 需要许多步骤来确定基于siRNA的杀微生物剂是否可能。其中包括体内向小动物生殖器粘膜中的树突状细胞、巨噬细胞和淋巴细胞(如果可能)传递siRNAs，以及证明传递siRNAs有效地抑制艾滋病毒的产生。分娩方法必须是安全的，并与一种适用于阴道和/或肛门分娩的配方相兼容，该配方不会引起粘膜炎症。早期的原则验证研究最终需要在小动物和灵长类动物身上进行正式的药代动力学、毒性和疗效研究。这笔拨款将集中在测试方法，在小鼠体内和体外向巨噬细胞和树突状细胞传递双链siRNA，并与美国国立卫生研究院的A.Blauvelt合作，在人类女性生殖器上皮体外皮肤水泡模型中，也将研究使用猪尾猕猴的PBMCs进行体外传递的siRNA，为以后在这个猕猴模型中传递、毒性和挑战研究做准备。将比较不同的给药方法在体外诱导抵抗HIV/SHV感染的沉默状态的能力。其具体目的是：1.优化策略，在体外和雌性小鼠生殖器粘膜中向小鼠巨噬细胞、树突状细胞和T细胞传递沉默CCR5的双链siRNAs；2.在人类皮肤水泡模型中测试siRNA传递策略；3.测试siRNAs和siRNA传递策略，以沉默猪尾猕猴PBMC中的SIV Gag和CCR5。

DESCRIPTION (provided by applicant): Sexual transmission of HIV occurs when cell-free or cell-associated virus infects cells primarily via the CCR5 coreceptor, expressed on macrophages, dendritic cells and activated T lymphocytes. A microbicide that could be used vaginally or anally to prevent sexual transmission would make a substantial contribution to controlling the spread of HIV. This innovative grant will begin to explore the hypothesis that RNA interference (RNAi) can form the basis of an effective anti-HIV microbicide. RNAi is an ancient, evolutionarily conserved, host defense against viruses and transposable elements, which uses small doublestranded RNAs, called small interfering RNAs (siRNA), to silence gene expression with exquisite specificity by targeted degradation of homologous mRNAs. There has been a lot of excitement about the therapeutic potential of RNAi to treat viral infection. A major obstacle is how to deliver siRNAs into cells. Our preliminary results suggest that duplex siRNAs can be delivered to macrophages and activated T cells without transfection and lead to prolonged gene silencing, which can inhibit de novo infection as well as viral replication in already infected cells. This suggests that duplex siRNAs might serve as the active component in a microbicide. Many steps are needed to determine whether an siRNA-based microbicide is possible. These include in vivo delivery of siRNAs to dendritic cells, macrophages, and (if possible) lymphocytes in the genital mucosa of small animals, and demonstration that delivered siRNAs effectively inhibit HIV production. Delivery methods have to be safe and compatible with a formulation suitable for vaginal and/or anal delivery that does not induce inflammation at the mucosa. Early proof-of-principle studies eventually need to be complemented by formal pharmacokinetics, toxicity and efficacy studies in small animals and primates. This grant will focus on testing methods to deliver duplex siRNA to macrophages and dendritic cells in vitro and in vivo in mice and in collaboration with A. Blauvelt of the NIH in a human ex vivo skin blister model for the female genital epithelium, siRNA delivery in vitro using PBMCs from the pig-tailed macaque Macaca nemestrina will also be studied, in preparation for later delivery, toxicity and challenge studies in this macaque model. Delivery methods will be compared for their ability to induce a silenced state resistant to HIV/SHIV infection in vitro. The specific aims are to: 1. optimize strategies to deliver duplex siRNAs that silence CCR5 to mouse macrophages, dendritic cells and T cells in vitro and in the female mouse genital mucosa; 2. test siRNA delivery strategies in the human skin blister model; 3. test siRNAs and siRNA delivery strategies for silencing SIV gag and CCR5 in PBMCs from pig-tailed macaques.