Efficient small nucleic acids to combat human respiratory viruses

反义程序已经开发了很长时间，用于人类的治疗用途，也用于作物保护。它们的目的是抑制（“沉默”）细胞或致病性靶RNA或调节其功能。中心功能组分是小核酸（NA），例如小干扰RNA（siRNA）或反义DNA-寡核苷酸（阿索），其主要将核酸内切酶引导至靶标。在研究和（前）临床试验取得相当大的进展后，这一概念现在经历了复兴。第一批批准的疗法令人印象深刻地证明siRNA和阿索药理学是安全的和商业上可行的。正在开发的配方，提高疗效，安全性和药理学的小NA。然而，反义程序仍然遇到相当大的技术限制。由于它们的高结构复杂性，还不可能可靠地鉴定靶RNA中可被NA接近的区域，并且在下文中被称为“可接近位点”或α-位点。因此，siRNA或阿索试剂的设计依赖于不确定的计算机预测或经验测试。迫切需要解决这个问题。我们的实验室表征RNA结合蛋白（Rbps）和RNA基序参与复制的人类，动物和植物病原性RNA病毒。在植物中，我们研究了以RNA沉默为中心的抗病毒免疫应答。在这里，我们开发了一种实验方法，“eNA筛选”，它可靠地鉴定了体外siRNA和ASO，这些siRNA和ASO能够与复杂结构的RNA分子（如植物病毒的mRNA或基因组RNA）的α位点相关联。在体外和在植物中，这些靶RNA的蛋白质表达和/或复制可以用以这种方式鉴定的eNA有效地抑制（e因此代表有效）。这是第一次，siRNA和ASO可以在“试管”中鉴定，然后以高特异性和效率在体内使用。因此，eNA筛选技术有可能显着提高反义程序的效力和安全性。在拟议的项目中，我们希望研究在eNA筛选条件下复杂RNA分子的结构是如何形成的，并定义a-位点的特征。此外，我们将将该筛查应用于人类呼吸道病毒的两个例子，即甲型流感病毒和呼吸道合胞病毒。对于抗病毒应用，我们认为使用靶向病毒RNA中许多α位点的eNA的多价组合特别重要。因此，快速复制的病毒和变异体（准种）可以被消灭，并防止其逃逸。通过专门使用高效的eNA，我们的长期目标是开发耐受性良好的口服/鼻腔治疗方法，以对抗几乎没有或没有抗病毒物质和/或疫苗的病毒。这些可能有助于减少感染早期阶段的病毒载量，抑制病毒传播，降低严重疾病进展和大流行的风险。

Antisense procedures have long been in development, for therapeutic use in humans, and also for crop protection. They aim to inactivate (‘silence’) cellular or pathogenic target RNAs or to modulate their function. Central functional components are small nucleic acids (NAs) such as small interfering RNAs (siRNAs) or antisense DNA-oligonucleotides (ASO), which mostly direct endonucleases to the targets. After considerable progress in research and (pre)clinical trials, the concept now experiences a revival. First approved therapies impressively demonstrate that siRNA and ASO pharmacology is safe and commercially viable. Formulations are being developed, which improve the efficacy, safety and pharmacology of small NA.Nevertheless, antisense procedures still encounter considerable technical limitations. Due to their high structural complexity, it was yet impossible to reliably identify regions in target RNAs that are accessible for NAs and are referred to as ‘accessible sites’ or a-sites in the following. Consequently, the design of siRNA or ASO agents relied on uncertain in silico predictions or empirical tests. There is an urgent need to solve this problem. Our laboratory characterizes RNA-binding proteins (Rbps) and RNA motifs involved in the replication of human, animal and plant pathogenic RNA viruses. In plant, we study the antiviral immune response, which is centrally based on RNA silencing. Here, we developed an experimental method, the ‘eNA screen’, which reliably identifies in vitro siRNAs and ASOs that are capable to associate to a-sites of complex structured RNA molecules such as mRNAs or genomic RNAs of plant viruses. Both in vitro and in planta, protein expression and/or replication of these target RNAs can be efficiently inhibited with eNAs identified in this way (e thus stands for efficient). For the first time, siRNAs and ASOs can be identified in the ‘test-tube’ and then be used in vivo with high specificity and efficiency. The eNA screen technology thus has the potential to significantly increase the potency and safety of antisense procedures.In the proposed project, we want to investigate how structures of complex RNA molecules are formed under eNA screen conditions and define the characteristics of a-sites. Moreover, we will apply the screen to two examples of human respiratory viruses, influenza A and respiratory syncytial virus. For antiviral applications, we consider it particularly important to use multivalent combinations of eNAs targeting many a-sites in viral RNAs. Thus, fast-replicating viruses and also variants (quasispecies) could be fought and escape prevented. By exclusively using highly efficient eNAs, our long-term goal is to develop well-tolerated oral/nasal treatments against viruses for which little or no antiviral substances and/or vaccines are available. These could help to reduce the viral load in early stages of infection, inhibit viral spread and reduce the risk of severe disease progression and pandemics.