Developing RNA Interference for an HIV microbicide

开发用于 HIV 杀微生物剂的 RNA 干扰

• 批准号: 6745421
• 负责人: Judy Lieberman
• 金额: $ 33.08万
• 依托单位: IMMUNE DISEASE INSTITUTE, INC.
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6745421
• 关键词: AIDS education /prevention; Macaca nemestrina; RNA interference; T lymphocyte; animal tissue; antiAIDS agent; antiinfective agents; biotechnology; clinical research; dendritic cells; epithelium; gene delivery system; human immunodeficiency virus; laboratory mouse; macrophage; small interfering RNA; technology /technique development; vagina; virus genetics

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.

描述（由申请人提供）：当无细胞或细胞相关病毒主要通过在巨噬细胞、树突细胞和活化的 T 淋巴细胞上表达的 CCR5 辅助受体感染细胞时，就会发生 HIV 的性传播。一种可用于阴道或肛门预防性传播的杀菌剂将为控制艾滋病毒的传播做出重大贡献。这项创新资助将开始探索 RNA 干扰 (RNAi) 可以构成有效抗 HIV 杀菌剂基础的假设。 RNAi 是一种古老的、进化上保守的宿主防御病毒和转座元件的方法，它使用称为小干扰 RNA (siRNA) 的小双链 RNA，通过同源 mRNA 的靶向降解，以精确的特异性沉默基因表达。人们对 RNAi 治疗病毒感染的治疗潜力感到非常兴奋。一个主要障碍是如何将 siRNA 传递到细胞中。我们的初步结果表明，双链 siRNA 可以在不转染的情况下递送至巨噬细胞和活化的 T 细胞，并导致延长的基因沉默，从而抑制从头感染以及已感染细胞中的病毒复制。这表明双链 siRNA 可能作为杀微生物剂的活性成分。 需要采取许多步骤来确定基于 siRNA 的杀微生物剂是否可行。这些包括体内将 siRNA 递送至小动物生殖器粘膜中的树突状细胞、巨噬细胞和（如果可能）淋巴细胞，并证明递送的 siRNA 能有效抑制 HIV 的产生。递送方法必须是安全的并且与适合阴道和/或肛门递送的制剂相容，并且不会引起粘膜炎症。早期的原理验证研究最终需要通过在小动物和灵长类动物中进行的正式药代动力学、毒性和功效研究来补充。这笔资助将重点测试在小鼠体外和体内将双链 siRNA 递送至巨噬细胞和树突状细胞的方法，并与 NIH 的 A. Blauvelt 合作，在女性生殖器上皮的人类离体皮肤水泡模型中进行 siRNA 递送还将研究使用猪尾猕猴 Macaca nemestrina 的 PBMC 进行体外实验，为以后在该猕猴模型中的递送、毒性和挑战研究做好准备。将比较递送方法在体外诱导抗 HIV/SHIV 感染的沉默状态的能力。具体目标是： 1. 优化在体外和雌性小鼠生殖粘膜中向小鼠巨噬细胞、树突状细胞和 T 细胞递送沉默 CCR5 的双链 siRNA 的策略； 2. 在人体皮肤水泡模型中测试siRNA递送策略； 3.测试用于沉默猪尾猕猴PBMC中的SIV gag和CCR5的siRNA和siRNA递送策略。