Development of aiRNA technology

描述(由申请人提供)： RNA干扰(RNAi)的发现有望给医学带来革命性的变化，因为它在治疗遗传、表观遗传和传染病方面具有无限的潜力。由合成的短干扰RNA(SiRNA)介导的RNAi理论上可用于靶向任何疾病基因。然而，siRNA介导的沉默是高度可变的，哺乳动物细胞中的许多基因都被低效沉默。此外，已报道siRNA能够激活宿主干扰素样炎性反应，并介导非靶基因沉默，这两个方面都对RNAi在体内的应用提出了重大挑战。我们发现了不对称干扰RNA(AiRNA)，这是一种新的专有RNAi技术，从根本上不同于传统的siRNA及其变体。在我们的初步研究中，我们发现在体外，与siRNA相比，aiRNA具有更好的基因沉默特性(有效性、效价、持续时间、作用开始)。此外，aiRNAi机械地取消了正义链介导的靶外沉默，并显著降低了干扰素反应。在一项初步研究中，我们还证明了针对β-连环蛋白的aiRNA可以在体内靶向HT29结肠癌异种移植瘤，并介导显著的基因沉默。较小的尺寸和较高的效力应该会显著降低合成成本，并且与siRNA相比可能提供体内递送优势。这项SBIR建议的长期目标是优化用于基因沉默研究和开发RNAi疗法的aiRNA技术。在第一阶段，我们将进行优化aiRNA技术所需的关键实验。具体来说，我们将进行体外研究，以优化aiRNA结构的设计(目标1)。我们将研究aiRNA介导的基因沉默的特异性和非靶点效应，并将它们与(目标2)进行比较。我们还将确定aiRNA在体内的基因沉默特性(目标3)。需要这些研究的结果来帮助我们随后的第二阶段研究的设计和开发。我们在第二阶段的总体目标将是利用在第一阶段开发的优化的aiRNA开发和测试一种临床兼容的结肠癌靶向治疗方法。在这些研究中，将合成针对选定的结肠癌靶基因的优化的aiRNA。然后，将使用异种移植和同基因结肠癌模型来评估每个aiRNA在体内介导治疗性RNAi的能力。AiRNA的进一步发展可能有助于释放基于RNAi的针对广泛疾病的干预措施的巨大潜力。。 公共卫生相关性： 最近发现的RNA干扰(RNAi)是一种允许特定基因在细胞内被关闭的自然过程，有望给医学带来革命性的变化；然而，基于合成siRNA(短干扰RNA)的治疗方法的开发遇到了重大问题，包括诱导非特异性效应、合成成本、有限的疗效和体内给药困难。最近，我们发现了波士顿生物医学公司开发的一种新的专有RNAi技术--不对称干扰RNA(AiRNA)，与siRNA相比，它具有更优越的基因沉默特性(有效性、效价、特异性、持续时间、作用开始)。这项建议中概述的研究旨在进一步研究aiRNA，长期目标是优化这一用于基因沉默研究和开发RNAi疗法的新技术。。

DESCRIPTION (provided by applicant): The discovery of RNA interference (RNAi) promises to revolutionize medicine due to its unlimited potential to treat genetic, epigenetic and infectious diseases. RNAi mediated by synthetic short interfering RNA (siRNA) can theoretically be employed to target any disease gene. However, siRNA-mediated silencing is highly variable with many genes in mammalian cells being inefficiently silenced. In addition, siRNA have been reported to activate host interferon-like inflammatory responses, and mediate off-target gene silencing, both of which significantly challenge the application of RNAi in vivo. We have discovered aiRNA (asymmetric interfering RNA), a novel proprietary RNAi technology that is fundamentally different from conventional siRNA and its variations. In our preliminary studies, we have found that aiRNA has superior gene silencing properties (efficacy, potency, duration, onset of action) compared to siRNA in vitro. Moreover, aiRNAi mechanistically abolished sense strand mediated off-target silencing, and significantly reduced interferon responses. In a pilot study, we have also demonstrated that aiRNA directed against beta-catenin can target HT29 colon cancer xenografts in vivo and mediate significant gene silencing. The smaller size and higher potency of aiRNA should significantly reduce the synthesis cost, and may provide an in vivo delivery advantage compared to siRNA. The long-term goal of this SBIR proposal is to optimize aiRNA technology for gene silencing research and for developing RNAi therapeutics. In Phase I we will perform key experiments necessary for the optimization of aiRNA technology. Specifically, we will perform in vitro studies to optimize the design of aiRNA structures (Aim 1). We will examine the specificity and off-target effects of aiRNA-mediated gene silencing and compare them to (Aim 2). We will also determine the gene silencing properties of aiRNA in vivo (Aim 3). Results from these studies are needed to aid the design and development of our subsequent Phase II studies. Our overall goal in Phase II will be to develop and test a clinically compatible targeted therapy for colon cancer using the optimized aiRNA developed in Phase I. For these studies, optimized aiRNA directed against selected colon cancer target genes will be synthesized. The ability of each aiRNA to mediate therapeutic RNAi in vivo will then be assessed using xenograft and syngeneic colon cancer models. Further development of aiRNA may help to unleash the enormous potential of RNAi-based interventions against a wide spectrum of diseases. . PUBLIC HEALTH RELEVANCE: The recent discovery of RNA interference (RNAi), a natural process that allows specific genes to be "switched off" in cells, promises to revolutionize medicine; however, the development of therapies based on synthetic siRNA (short interfering RNA) has met with significant problems including the induction of non-specific effects, synthesis cost, limited efficacy, delivery difficulty in vivo. Recently, we have discovered that aiRNA (asymmetric interfering RNA), a novel proprietary RNAi technology developed at Boston Biomedical Inc., has superior gene silencing properties (efficacy, potency, specificity, duration, onset of action ) compared to siRNA. The research outlined in this proposal is designed to further investigate aiRNA with the long-term goal of optimizing this novel technology for gene silencing research and for developing RNAi therapeutics. .