Rapid development of SARS-CoV-2 specific therapeutics that leverage virus specific RNA elements

利用病毒特异性 RNA 元件快速开发 SARS-CoV-2 特异性疗法

• 批准号: 10115505
• 负责人: JEFFREY S GLENN
• 金额: $ 34.82万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-05 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10115505
• 关键词: 2019-nCoV; Attenuated; Binding; Binding Sites; Cells; Coronavirus; Development; Disease; Dose; Elements; Formulation; Genome; Goals; Grant; Immunity; In Vitro; Infection; Influenza; Influenza A virus; Lead; Length; Link; Luciferases; Lung; Maps; MicroRNAs; Mus; Mutate; Nucleic Acid Amplification Tests; Nucleotides; Oligonucleotides; Pollen; Positioning Attribute; RNA; Reagent; Reporter; Signal Transduction; Single-Stranded DNA; Structure; Technology; Testing; Therapeutic; Time; Transfection; Translations; Variant; Virus; computational suite; design; in vivo; insight; locked nucleic acid; novel; nucleic acid analog; nucleic acid delivery; prevent; screening panel; therapeutic RNA; therapeutic development; tool

ABSTRACT: Our goal is leverage our recent insights into coronavirus B conserved RNA structures, and our discovery of formulations for high efficiency lung delivery, into the rapid development of SARS-CoV-2 specific therapeutics. Using a novel suite of computational technology tools, we have identified predicted RNA secondary structures in regions conserved across coronavirus B genomes including SARS-CoV-2. We have also identified two tandem predicted microRNA 191 (miR191) binding sites within the 5’-most such structure. In our current grant on influenza A virus (IAV), we identified an RNA secondary structure conserved across all IAV isolates that is essential for in vitro packaging and in vivo disease, then designed short highly stable locked nucleic acid (LNA) oligonucleotides to bind and distort this RNA packaging signal, and demonstrated that a single dose of our lead LNA can a) provide immediate 100% protection for over 14 days from a lethal inoculum of IAV, b) provide 100% survival when administered 3 days after a lethal IAV inoculum, and c) while sufficiently attenuating the infection, enable the subsequent development of high level immunity. Moreover, we have also recently discovered that empty deproteinized pollen shells represent an outstanding vehicle for delivery of LNAs to the lung with much greater efficacy and tolerability than current formulations for nucleic acid delivery. We now hypothesize that 1) our identified RNA secondary structures in SARS-CoV-2 represent ideal candidate targets for disrupting the virus lifecycle, via structure-specific LNAs; 2) the miR191 binding sites within the 5’-most conserved RNA secondary structure reflect an essential mechanism for regulating translation of corona B viruses that is amenable to targeting by specifically designed LNAs; 3) our novel deproteinized pollen formulation represents an ideal means of delivering such LNAs to both prevent and treat established SARS-CoV-2 infections. We will test these hypotheses via the following specific aims that are to: 1) Determine which LNA gapmers from a screening panel synthesized against our identified conserved RNA secondary structure targets are most disruptive to the latter’s integrity, as assessed by SHAPE, REVI, and Mutate-and-Map; 2) Refine the sequence (total LNA length, fine nucleotide target position, and length of single stranded DNA gapmer) of the top performing LNA and test a panel of LNA analogs to identify the most potent disrupter of targeted SARS-CoV-2 conserved RNA secondary structure; 3) Determine the effect of LNAs designed to sequester miR191 in cells transfected with a SARS-CoV-2 5’ terminal RNA segment linked to a luciferase reporter; 4) Determine the effect of the identified lead LNAs (targeting conserved SARS-CoV-2 RNA secondary structure, and sequestering miR191) on cells infected with SARS-CoV-2 in vitro, and in vivo when delivered intranasally by current lung-targeting transfection reagent (i.e.JetPEi) vs. pollen shells to SARS-CoV- 2-infected mice. Successful accomplishment of our aims will yield proof-of-concept for an exciting new class of anti- SARS-CoV-2 RNA therapeutics within the short time frame of this proposal.

摘要：我们的目标是利用我们最近对冠状病毒B构成RNA结构的见解，我们 发现用于高效肺输送的公式进入SARS-COV-2特异性的快速发展 治疗。使用新颖的计算技术工具套件，我们已经确定了预测的RNA 在包括SARS-COV-2在内的冠状病毒B基因组中保守的地区的二级结构。我们有 还确定了两个串联预测的microRNA 191（miR191）结合位点，其中最多的结构中。 在我们目前对影响力病毒（IAV）的授予中，我们确定了所有的RNA二级结构 对于体外包装和体内疾病至关重要的IAV分离株，然后设计短的高度稳定锁定 核酸（LNA）寡核苷酸以结合和扭曲该RNA包装信号，并证明A A 单剂量的LNA可以a）从致命接种物中提供14天以上的立即100％保护 IAV，b）当致死IAV后3天给药时，可提供100％的存活，c） 减弱感染，使后来的高水平免疫力发展。而且，我们也 最近发现，空的脱蛋白花粉壳代表了交付的出色工具 与目前的核酸输送公式相比，LNA的效率和耐受性要大得多。 现在，我们假设1）我们在SARS-COV-2中确定的RNA二级结构代表理想 通过特定于结构的LNA破坏病毒生命周期的候选目标； 2）miR191结合位点 在5'最多配置的RNA二级结构中反映了调节的基本机制 由专门设计的LNA靶向的电晕B病毒的翻译； 3）我们的小说 脱蛋白的花粉公式代表了提供此类LNA以预防和治疗的理想方法 已建立的SARS-COV-2感染。我们将通过以下特定目的来检验这些假设：1） 确定来自筛选面板中的哪些LNA差距针对我们所鉴定的RNA合成 二级结构目标对后者的完整性最具破坏性，如形状，revi和 突变和图； 2）完善序列（总LNA长度，细核苷酸目标位置和单个长度 滞留的dna gapmer）表现的LNA并测试一组LNA类似物，以识别最有效的 靶向SARS-COV-2构成RNA二级结构； 3）确定LNA的效果 设计用于隔离用SARS-COV-2 5'末端RNA段翻译的细胞中的miR191 萤光素酶记者； 4）确定已鉴定的铅LNA的效果（靶向构成SARS-COV-2 RNA 二级结构，并在体外感染了SARS-COV-2的细胞上，并在体内进行隔离的miR191） 通过当前的肺靶向转染试剂（即JETPEI）与花粉壳的鼻内交付 两只感染的小鼠。成功实现我们的目标将为一个令人兴奋的新课程提供概念验证 在本提案的短时间内，抗SARS-COV-2 RNA治疗。