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）：当无细胞或与细胞相关的病毒主要通过CCR5共肽感染细胞时发生HIV的性传播，该病毒在巨噬细胞，树突状细胞和活化的T淋巴细胞上表达。可以通过阴道或肛门用于防止性传播的杀菌剂将为控制艾滋病毒的传播做出重大贡献。这种创新的授予将开始探讨RNA干扰（RNAi）可以构成有效抗HIV杀菌剂的基础的假设。 RNAi是一种古老的，进化保守的宿主防御病毒和转座元素，它使用小型双驱动的RNA，称为小型干扰RNA（siRNA），通过有针对性的同源mRNA的靶向降解来使基因表达沉默。 RNAi治疗病毒感染的治疗潜力引起了很多兴奋。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，双链体可以传递到巨噬细胞并活化的T细胞而无需转染并导致延长基因沉默，这可以抑制已感染细胞中的从头感染以及病毒复制。这表明双工siRNA可以用作微生物中的活性成分。 需要许多步骤来确定是否可能基于siRNA的杀菌剂。其中包括在小动物的生殖器粘膜中体内递送siRNA向树突状细胞，巨噬细胞和（如果可能的话）淋巴细胞，以及递送siRNA的证明可有效抑制HIV的产生。递送方法必须安全且与适合于阴道和/或肛门递送的配方兼容，该配方不会在粘膜处诱发炎症。最终，早期的原则研究最终需要与小动物和灵长类动物的正式药代动力学，毒性和效力研究相辅相成。 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并在这个猕猴模型中进行挑战研究。将在体外诱导对HIV/SHIV感染的抗性状态的能力，比较递送方法。具体的目的是：1。优化策略以在体外和雌性小鼠生殖器粘膜中，将CCR5沉默的双链体siRNA沉默到小鼠巨噬细胞，树突状细胞和T细胞； 2。在人类皮肤水泡模型中测试siRNA递送策略； 3。从猪尾猕猴中测试siRNA和siRNA输送策略，用于在PBMC中沉默的SIV SIV GAG和CCR5。