Transkingdom Gene Silencing

描述（由申请人提供）： RNA干扰（RNAi）是一种高度保守的转录后基因沉默机制，其在治疗遗传性、表观遗传性和感染性疾病方面的潜力有望给医学带来革命性的变化。然而，释放这种潜力的努力在交付方面遇到了挑战，这已被证明是翻译给人类的主要绊脚石。近年来，我们发现编码RNAi的细菌可以在体内外诱导哺乳动物细胞中有效的基因沉默（transkingdom RNAi，tkRNAi）。这一基本观察结果不仅对体内微生物组与宿主细胞之间的相互作用具有重要意义，而且还为基因功能研究和治疗人类疾病提供了体内递送RNAi的临床兼容手段。这种新颖的方法提供了几个优点。最重要的是临床安全性，因为细菌不会将遗传物质整合到人类基因组中。tkRNAi还消除了化学合成siRNA的需要，并且可以减轻宿主免疫应答，因为沉默RNA在靶细胞内产生。此外，减毒细菌的需要以及经修饰或突变的细菌载体的环境释放风险可以通过已经开发的基于细菌的干预措施的技术来解决。为了将这一基础科学观察转化为临床应用，我们设计了非致病性E。大肠杆菌或减毒鼠伤寒沙门氏菌编码针对β-连环蛋白的RNAi，并证明口服这些细菌可介导肠上皮细胞中有效和特异性的基因β-连环蛋白沉默，并对治疗APC min/+小鼠的息肉病具有显著功效。我们进一步设计了一种优化的基于tkRNAi的治疗方法，用于治疗家族性腺瘤性息肉病（FAP）患者。与业界合作，这种tkRNAi治疗剂已获得FDA批准进行Ia/II期试验。这是FDA批准的第一个口服基因沉默疗法，也是第一个基因沉默疗法。 FAP患者的靶向治疗。该建议旨在阐明和表征跨王国基因沉默的分子过程和机制。具体来说，我们将检验我们的假设，即细菌介导的RNAi可以有效和安全地沉默哺乳动物细胞中的靶基因，足以纠正疾病表型。我们将使用APCmin/+小鼠作为我们的体内模型。为了验证这一假设，我们将：1）研究跨王国基因沉默的分子过程和机制; 2）全面分析tkRNAi介导的肠道β-连环蛋白沉默的分子机制; 3）研究开发下一代tkRNAi技术的可能性。这些研究的成功完成不仅将显著增强我们对微生物组与宿主细胞之间相互作用的理解，而且对于开发和使用基于tkRNAi的治疗剂以满足FAP患者的紧急未满足的医疗需求以及使用跨王国基因沉默治疗其他人类疾病也至关重要。

DESCRIPTION (provided by applicant): The discovery of RNA interference (RNAi), a highly conserved post-transcriptional mechanism of gene silencing, promises to revolutionize medicine due to its potential to treat genetic, epigenetic and infectious disease. Efforts to unleash this potential, however, have met with challenges in delivery which have proven to be the major stumbling block for translation to humans. Recently we have discovered that bacteria encoding RNAi can induce potent gene silencing in mammalian cells (termed transkingdom RNAi, tkRNAi) in vitro and in vivo. This fundamental observation not only has significant implications for the interaction between the microbiome and host cells in vivo, but also offers a clinically compatible means to deliver RNAi in vivo for gene function research and for treating human diseases. This novel approach offers several advantages. Foremost is clinical safety since bacteria do not integrate genetic material into the human genome. tkRNAi also eliminates the need to chemically synthesize siRNA, and may mitigate host immune responses since the silencing RNA are produced inside target cells. Furthermore, the need for attenuation of bacteria, and the risk of environmental release of modified or mutated bacterial vectors can be addressed by technologies already developed for bacteria-based interventions. To translate this basic science observation toward clinical application, we have engineered non-pathogenic E. coli or attenuated Salmonella typhimurium to encode RNAi against beta-catenin, and demonstrated that oral administration of these bacteria can mediate potent and specific gene beta-catenin silencing in intestinal epithelial cells and has significant efficacy for treating polyposis in APCmin/+ mice. We further designed an optimized tkRNAi-based therapy for treating Familial Adenatomous Polyposis (FAP) patients. In collaboration with industry, this tkRNAi therapeutic has received FDA clearance for phase Ia/II trials. This is the first oral gene silencing therapy to be cleared by FDA, and also the first gene targeted therapy for FAP patients. This proposal is designed to elucidate and characterize the molecular processes and mechanisms of transkingdom gene silencing. Specifically, we will examine our hypothesis that bacteria-mediated RNAi can silence a target gene effectively and safely in mammalian cells sufficient for correcting disease phenotypes. We will use APCmin/+ mice as our in vivo model. To test this hypothesis, we will: 1) investigate the molecular processes and mechanism of transkingdom gene silencing; 2) comprehensively analyze the molecular mechanism of tkRNAi-mediated silencing of beta-catenin in the intestine, and 3) examine the possibility of developing a next generation tkRNAi technology. The successful completion of these studies will not only significantly enhance our understanding of interactions between the microbiome and host cells, but also be critical for developing and employing a tkRNAi-based therapeutic to meet urgent unmet medical needs of FAP patients and for using transkingdom gene silencing for treating other human diseases.