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

摘要：我们的目标是利用我们最近对冠状病毒B保守RNA结构的了解， 发现用于高效肺部递送的制剂，进入SARS-CoV-2特异性 治疗学使用一套新的计算技术工具，我们已经确定了预测的RNA 包括SARS-CoV-2在内的冠状病毒B基因组中保守区域的二级结构。我们有 还鉴定了在最5 '端的这种结构内的两个串联预测的microRNA 191（miR 191）结合位点。 在我们目前对甲型流感病毒（IAV）的资助中，我们确定了一种RNA二级结构， IAV分离株是体外包装和体内致病所必需的，然后设计了短的高度稳定的锁定 核酸（LNA）寡核苷酸结合和扭曲这种RNA包装信号，并证明， 单剂量我们的铅LNA可以a）提供超过14天的立即100%保护，免受致命接种物的侵害 B）当在致死IAV接种物后3天施用时提供100%的存活，以及c）当足够 减轻感染，使随后的高水平免疫力的发展。此外，我们还 最近发现，空的脱蛋白花粉壳代表了一种出色的载体， 与目前的核酸递送制剂相比，LNAs具有更高的功效和耐受性。 我们现在假设：1）我们在SARS-CoV-2中鉴定的RNA二级结构代表了理想的 通过结构特异性LNA破坏病毒生命周期的候选靶标; 2）miR 191结合位点 在5 '-最保守的RNA二级结构中，反映了调节 冠状B病毒的翻译，其适合于通过专门设计的LNA靶向; 3）我们的新的 脱蛋白花粉制剂代表了递送这种LNA以预防和治疗疾病的理想手段 确定的SARS-CoV-2感染。我们将通过以下具体目标来测试这些假设：1） 确定筛选组中针对我们鉴定的保守RNA合成的LNA缺口体 二级结构目标对后者的完整性破坏最大，如SHAPE，REVI和 2）优化序列（总LNA长度、精细核苷酸靶位置和单个寡核苷酸的长度）; 并测试一组LNA类似物，以鉴定最有效的 靶向SARS-CoV-2保守RNA二级结构的破坏剂; 3）确定LNA的作用 设计用于在用SARS-CoV-2 5'末端RNA片段转染的细胞中隔离miR 191，所述RNA片段连接到 荧光素酶报告基因; 4）确定鉴定的前导LNA（靶向保守的SARS-CoV-2 RNA）的作用 二级结构，和隔离miR 191）在体外感染SARS-CoV-2的细胞上， 通过当前肺靶向转染试剂（即JetPEi）鼻内递送与花粉壳相比， 2-感染的老鼠我们的目标的成功实现将产生一个令人兴奋的新一类的概念验证， 抗SARS-CoV-2 RNA治疗在这个建议的短时间内。

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.