Cancer targeted therapy through bacterial RNAi

描述（由申请人提供）：最近发现的RNAi创造了机会，以开发一个全新的类别的药物对各种人类疾病的工作沉默致病基因。然而，基于RNAi的药物的开发受到递送和制造困难以及宿主干扰素样反应激活的阻碍。其中，交付已证明是主要障碍。到目前为止，RNAi递送已经挑战了主要集中在各种制药技术（例如siRNA修饰、纳米技术和脂质体）上的常规方法。我们假设非致病性细菌可以被工程化以同时“制造”和“递送”RNAi。初步研究表明，E.表达针对β-连环蛋白的shRNA的大肠杆菌或减毒鼠伤寒沙门氏菌可以在体外和体内诱导显著的基因沉默。这种新颖的方法提供了几个优点。最重要的是细菌与病毒的临床安全性，因为细菌不会整合到人类基因组中。这种方法还消除了siRNA制造问题，并且可以规避或减轻宿主干扰素样应答，因为siRNA是在细胞内产生的。这项SBIR提案的长期目标是开发一种基于细菌RNAi的临床相容的靶向癌症治疗。本（第1阶段）提案中概述的实验对于基于细菌的RNAi疗法的设计和开发是必要的，并分为两个特定目标。在具体目标1中，我们将使用各种体外试验来确定调节细菌介导的RNAi的关键机制，以及细菌RNAi是否缺乏宿主干扰素样反应。在具体目标2中，我们将使用两种众所周知的小鼠结肠癌模型来检查厌氧菌是否可以通过利用体内实体瘤内的缺氧环境来选择性地递送shRNA。需要这些研究的结果来优化我们后续II期研究的设计和开发。我们在第二阶段的总体目标将是完成临床上相容的基于RNAi的靶向治疗结肠癌的设计和优化，使用厌氧菌，如减毒鼠伤寒沙门氏菌。在这些研究中，减毒S.鼠伤寒沙门氏菌将被改造以产生针对结肠癌致癌基因β-连环蛋白的shRNA。然后将使用结肠癌模型评估工程化细菌在体内介导治疗性RNAi的能力。如果建立基于细菌的方法，应该有助于释放基于RNAi的干预措施对广泛疾病的巨大潜力。公共卫生相关性：最近发现的RNA干扰（RNAi），一种允许细胞中特定基因被关闭的机制，为许多疾病的治疗带来了巨大的希望，但在递送方面遇到了重大问题。最近，我们已经证明，非致病性细菌可以被工程化以激活人类细胞中的RNAi，并特异性地沉默各种结肠癌相关基因。本提案中概述的研究应使我们能够确定优化细菌介导的RNAi所需的关键要素，并实现我们的总体目标，即开发一种临床相容的、基于细菌RNAi的结肠癌靶向治疗方法。

DESCRIPTION (provided by applicant): The recent discovery of RNAi has created the opportunity to develop an entirely new class of drugs against a variety of human diseases that work by silencing disease causing genes. The development of RNAi-based drugs, however, has been hampered by difficulties in delivery and manufacturing, as well as the activation of host interferon-like responses. Of these, delivery has proved to be the major obstacle. RNAi delivery has, thus far, defied conventional approaches that focus mostly on various pharmaceutical technologies, such as siRNA modifications, nanotechnology and liposomes. We hypothesize that non-pathogenic bacteria can be engineered to simultaneously "manufacture" and "deliver" RNAi. In preliminary studies, we show that E. coli or attenuated Salmonella typhimurium expressing shRNA's directed against beta-catenin can induce significant gene silencing in vitro and in vivo. This novel approach offers several advantages. Foremost is clinical safety for bacteria versus viruses since bacteria do not integrate into the human genome. This approach also eliminates the siRNA manufacture issue, and may circumvent or mitigate host interferon-like responses since siRNA is produced intracellularly. The long-term goal of this SBIR proposal is to develop a clinically compatible targeted cancer therapy based on bacterial RNAi. The experiments outlined in this (Phase 1) proposal are necessary for the design and development of bacteria-based RNAi therapy, and are grouped into two specific aims. In Specific Aim 1, we will use various in vitro assays to identify the key mechanisms that regulate bacteria-mediated RNAi, and whether bacterial RNAi is devoid of host interferon-like responses. In Specific Aim 2, we will use two well known models of murine colon cancer to examine whether anaerobic bacteria can be used to selectively deliver shRNA's by taking advantage of the hypoxic milieu inside solid tumors in vivo. Results from these studies are needed to optimize the design and development of our subsequent Phase II studies. Our overall goal in Phase II will be to complete the design and optimization of a clinically compatible RNAi- based targeted therapy for colon cancer using anaerobic bacteria, such as attenuated Salmonella typhimurium. For these studies, attenuated S. typhimurium will be engineered to produce shRNA's directed against the colon cancer oncogene beta-catenin. The ability of the engineered bacteria to mediate therapeutic RNAi in vivo will then be assessed using colon cancer models. The bacteria-based approach, if established, should help to unlock the enormous potential for RNAi-based interventions against a wide spectrum of diseases. PUBLIC HEALTH RELEVANCE: The recent discovery of RNA interference (RNAi), a mechanism that allows specific genes to be }switched off} in cells, holds great promise for the treatment of many diseases but has met with significant problems in terms of delivery. Recently, we have shown that non-pathogenic bacteria can be engineered to activate RNAi in human cells and specifically silence various colon cancer-associated genes. The research outlined in this proposal should enable us to identify the key elements necessary for optimizing bacteria-mediated RNAi, and achieve our overall goal of developing a clinically compatible, bacterial RNAi-based, targeted therapy for colon cancer.