siRNA Microbicides

描述（由申请人提供）：我们以U19合作协议的形式寻求支持，以表征RNA干扰（RNAi）在预防和治疗由HIV-1， HSV和HPV引起的感染中的杀微生物潜力。RNAi是指通过大约19-21个核苷酸长度的单链和双链RNA对转录后和翻译后的基因进行沉默。在本提案中，我们将把我们的研究局限于被称为短干扰RNA （siRNA）的短ds RNA。在细胞导入后，siRNA被整合到RNAi沉默复合体（RISC）中，并以序列特异性的方式介导mRNA的降解，从而有效抑制蛋白质表达。通过动物模型，我们已经证明siRNA可以作为脂质体制剂引入阴道粘膜。详细的研究表明，单次阴道siRNA治疗在一周内可将目标蛋白水平降低90%。我们建议鉴定siRNA来选择性地降解涉及HIV-1、HSV和HPV传播和/或复制的病毒和/或宿主蛋白。在体外确认siRNA基因沉默效力和抗病毒活性后，将使用动物模型来确定阴道siRNA治疗后靶向蛋白敲除的动力学。虽然脂质体制剂可以立即进行这些原理验证性研究，但将投入大量精力研究siRNA的纳米胶囊制剂，以实现有效的粘膜摄取和有利的毒性特征。同时的研究将揭示对siRNA结构本身的化学修饰是否会增加分子的活性谱。因此，在2.5年的时间里，我们将鉴定出化学优化的siRNA分子，这些分子在阴道给药后作为无毒纳米胶囊在体内减少了验证的病原体相关靶点的表达。此后，所有的努力都将指向确定siRNA在HIV-1、HSV和HPV传播和感染动物模型中的体内功效。这些目标将在四个项目和两个核心中以高度协调的方式实现：项目1:HIV-1预防（Ramratnam,Mansfield），项目2:HSV预防和治疗（Herold），项目3:siRNA结构优化（Alexander, Shaw），项目4:HPV预防和治疗（Lambert, Oz, Marx），核心a：行政管理（Ramratnam），核心B：配方（McDonough）。在项目结束时，我们将生成临床前数据，以证明siRNA杀微生物剂在人类受试者中的进一步评估是合理的。

DESCRIPTION (provided by applicant): We seek support in the form of a U19 Cooperative Agreement to characterize the microbicidal potential of RNA interference (RNAi) in preventing and treating infections caused by HIV-1, HSV and HPV. RNAi refers to post-transcriptional and post-translational gene silencing by single and double stranded RNA of approximately 19-21 nucleotide length. In this proposal, we will confine our studies to short ds RNA that have been termed short interfering RNA (siRNA). Upon cellular introduction, siRNA are incorporated into the RNAi Silencing Complex (RISC) and mediate the degradation of mRNA in a sequence-specific manner leading to potent inhibition of protein expression. Using animal models, we have demonstrated that siRNA can be introduced into vaginal mucosa as liposomal formulations. Detailed studies have revealed that a single vaginal siRNA treatment reduces levels of targeted proteins by up to 90% over a one-week period. We propose to identify siRNA to selectively degrade viral and/or host proteins implicated in the transmission and/or replication of HIV-1, HSV and HPV. After in vitro confirmation of siRNA gene silencing potency and anti-viral activity, animal models will be used to define the kinetics of targeted protein knockdown following vaginal siRNA treatment. While liposomal formulations will allow the immediate pursuit of these proof-of-principle studies, considerable effort will be devoted to nanocapsule formulation of siRNA to allow for efficient mucosal uptake and favorable toxicity profile. Simultaneous investigations will reveal whether chemical modifications to the siRNA structure itself increase the activity profile of the molecules. Thus, at the 2.5 year mark, we will have identified chemically optimized siRNA molecules that reduce the expression of validated pathogen-related targets in vivo after their vaginal administration as non-toxic nanocapsules. Thereafter, all effort will be directed to defining in vivo efficacy of siRNA in animal models of HIV-1, HSV and HPV transmission and infection. These goals will be pursued in four projects and two cores in a highly coordinated manner: Project 1: HIV-1 prevention (Ramratnam,Mansfield), Project 2: HSV prevention and treatment (Herold), Project 3: siRNA structural optimization (Alexander, Shaw), Project 4: HPV prevention and treatment (Lambert, Oz, Marx), Core A: Administrative (Ramratnam), Core B: Formulation (McDonough). At the end of the project, we will have generated pre-clinical data to justify the further evaluation of siRNA microbicides in human subjects.