Cancer targeted therapy through bacterial RNAi

描述（由申请人提供）：最近RNAi的发现为开发一种全新的药物类别创造了机会，这种药物通过沉默致病基因来治疗各种人类疾病。然而，基于rnai的药物的开发一直受到递送和制造的困难以及宿主干扰素样反应的激活的阻碍。其中，交付已被证明是主要障碍。到目前为止，RNAi递送已经违背了传统的方法，这些方法主要集中在各种制药技术上，如siRNA修饰、纳米技术和脂质体。我们假设非致病性细菌可以同时“制造”和“传递”RNAi。在初步研究中，我们发现表达针对β -连环蛋白的shRNA的大肠杆菌或减毒鼠伤寒沙门菌可以在体外和体内诱导显著的基因沉默。这种新颖的方法有几个优点。首先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。这种方法也消除了siRNA制造问题，并且可以规避或减轻宿主干扰素样反应，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的临床兼容的靶向癌症治疗方法。本（第一阶段）提案中概述的实验对于设计和开发基于细菌的RNAi疗法是必要的，并且分为两个特定目标。在Specific Aim 1中，我们将使用各种体外实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Specific Aim 2中，我们将使用两种众所周知的小鼠结肠癌模型来研究厌氧细菌是否可以利用体内实体瘤内的缺氧环境选择性地递送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II期的总体目标是完成设计和优化一种临床兼容的基于RNAi的结肠癌靶向治疗方法，使用厌氧菌，如减毒鼠伤寒沙门氏菌。在这些研究中，将对减毒鼠伤寒沙门氏菌进行工程改造，以产生针对结肠癌致癌基因β -连环蛋白的shRNA。工程细菌在体内介导治疗性RNAi的能力将使用结肠癌模型进行评估。这种基于细菌的方法，如果建立起来，应该有助于释放基于rna的干预措施的巨大潜力，以对抗广泛的疾病。公共卫生相关性：最近发现的RNA干扰（RNAi）是一种允许细胞中特定基因被“关闭”的机制，它为治疗许多疾病带来了巨大的希望，但在递送方面遇到了重大问题。最近，我们已经证明，非致病性细菌可以通过工程设计激活人类细胞中的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，并实现我们开发临床兼容的、基于细菌RNAi的结肠癌靶向治疗的总体目标。

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.