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对B型冠状病毒保守RNA结构的最新见解，以及我们的 高效肺部给药配方的发现，进入SARS-CoV-2特异性的快速发展 治疗学。使用一套新的计算技术工具，我们已经识别出预测的RNA 包括SARS-CoV-2在内的B型冠状病毒基因组中保守区域的二级结构。我们有 还在5‘-大多数这样的结构中发现了两个串联预测的microRNA191(MiR191)结合位点。 在我们目前对甲型流感病毒(IAV)的资助中，我们发现了一个在所有病毒中都保守的RNA二级结构 认为IAV分离株对体外包装和体内疾病是必不可少的，然后设计出高度稳定的短锁定 核酸(LNA)寡核苷酸结合并扭曲这种RNA包装信号，并证明了 我们的单剂铅LNA可以提供100%的即时保护，持续14天以上不受致命性接种 在接种致死的IAV后3天给予IAV，b)提供100%的存活率，以及c)在足够的情况下 减弱感染，使后续发展高水平的免疫力。此外，我们还 最近发现，空脱蛋白花粉壳是一种出色的传递 与目前用于核酸输送的制剂相比，LNA对肺的有效性和耐受性要高得多。 我们现在假设：1)我们在SARS-CoV-2中发现的RNA二级结构代表理想的结构 通过结构特异的LNA干扰病毒生命周期的候选靶点；2)miR191结合位点 在5‘-保守的RNA二级结构中，反映了一种基本的调节机制 翻译可通过特殊设计的LNA靶向的冠状病毒B；3)我们的小说 去蛋白花粉制剂是一种理想的传递这种LNAs的方法，既可以预防，也可以治疗 已确诊的SARS-CoV-2感染。我们将通过以下具体目标来检验这些假设：1) 根据我们鉴定的保守RNA，从筛选小组中确定哪些LNA缺口 二级结构目标对后者的完整性最具破坏性，如SHAPE、REVI和 突变和作图；2)精炼序列(LNA总长度、细核苷酸靶点位置、单核苷酸长度 最高性能的LNA)并测试一组LNA类似物以确定最有效的 靶向SARS-CoV-2保守RNA二级结构的干扰物；3)确定LNAs的作用 目的是在感染SARS-CoV-2 5‘末端RNA片段的细胞中隔离miR191，该片段与 荧光素酶报告；4)确定已鉴定的前导LNAs的作用(靶向保守的SARS-CoV-2RNA 在体外和体内感染SARS-CoV-2的细胞上，当 通过当前的肺靶向转染剂(即JetPEi)与花粉壳将其经鼻递送至SARS-CoV- 2感染小鼠。我们目标的成功实现将为一个令人兴奋的新阶层提供概念验证 在这项提议的短时间框架内进行抗SARS-CoV-2 RNA治疗。

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.