Programmable antivirals: Targeting viral RNA secondary structures with LNAs and small molecules

可编程抗病毒药物：利用 LNA 和小分子靶向病毒 RNA 二级结构

• 批准号: 10514269
• 负责人: JEFFREY S GLENN
• 金额: $ 891.52万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-16 至 2025-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10514269
• 关键词: 2019-nCoV; Antisense Oligonucleotides; Antiviral Agents; Back; Beauty; Binding; Bioinformatics; COVID-19 therapeutics; Chikungunya virus; Chimera organism; Clinic; Clinical; Collection; Combined Modality Therapy; Cryoelectron Microscopy; Data; Development; Dose; Generations; Genetic Diseases; Genome; Hepatitis C; Human; Human Genetics; In Vitro; Influenza A virus; Interferons; Lead; Methods; Middle East Respiratory Syndrome Coronavirus; Mutation; Names; Nature; Oseltamivir; Outpatients; Pancreatic ribonuclease; Pharmaceutical Preparations; Process; RNA; RNA Viruses; RNA library; Resistance; Resistance development; Resolution; Ribonuclease H; Ribonucleases; SARS coronavirus; SARS-CoV-2 B.1.617.2; SARS-CoV-2 genome; SARS-CoV-2 infection; SARS-CoV-2 spike protein; Signal Transduction; Site; Structure; Testing; Therapeutic; Translating; Untranslated RNA; Vaccines; Virus; anti-viral efficacy; antisense nucleic acid; base; design; improved; in vivo; locked nucleic acid; next generation; novel; novel strategies; nuclease; nucleic acid-based therapeutics; nucleoside analog; pandemic coronavirus; pandemic disease; programs; recruit; remdesivir; resistant strain; respiratory virus; small molecule; synergism; therapeutic target; vaccine efficacy; viral RNA; virology

ABSTRACT: Our overall objective is to develop a novel class of outpatient therapeutics targeting highly- conserved RNA structures in the genomes of SARS-CoV-2 and other respiratory viruses of pandemic potential. Using influenza A virus (IAV) as a proof-of-concept, we previously showed that: 1) adding high-resolution RNA secondary structure target information into the design of antisense oligonucleotide (ASO)-based therapeutics can greatly enhance antiviral efficacy over simply targeting conserved primary sequence, as can incorporating into the ASO design the ability to recruit RNase H to the target site; 2) a single intranasal dose of a highly stable, locked nucleic acid (LNA) ASO designed against a universally conserved RNA structure provides 100% survival when given 14 days before, or 3 days after a lethal IAV inoculum; and 3) no resistance to our LNA has been selectable. Applying a similar approach, via a process we now term “programmable antivirals,” to SARS-CoV-2, we 1) rapidly identified highly conserved RNA structures; 2) designed LNAs against these targets and showed that cryo-electron microscopy (cryoEM) of a structure led to improved LNA design; 3) demonstrated that our lead LNAs have compelling in vitro and in vivo efficacy against reference and clinical isolates, including virus harboring mutations that reduce vaccine efficacy (e.g. delta variant). Complementarily, we have pioneered a novel strategy, named “Inforna,” to design small molecules that selectively bind viral RNA structures and inhibit noncoding RNAs involved in human genetic diseases and RNA viruses, including SARS-CoV-2, some with activities in the nM and pM range. The small molecules can be modified to recruit an endogenous nuclease, akin to the RNase H-induced degradation by LNA ASOs. We term this type of small molecule a “ribonuclease targeting chimera (RIBOTAC).” We now hypothesize that: 1) our lead LNA molecules—including one targeting a RNA structure common to SARS-CoV-1 and MERS-CoV—already represent ideal development candidates; 2) the LNAs’ antiviral potency can be further enhanced; 3) the resulting LNAs will have a high barrier to the development of resistance and be broadly active against wild-type and vaccine-resistant strains; 4) Inforna can help design small molecules and RIBOTACs against our identified target structures; 5) our therapeutics are combinable with other anti-SARS-CoV-2 agents; and 6) analogous approaches can be rapidly applied against RNA viruses of pandemic concern. We will test these hypotheses by: 1) selecting a lead (and back up) LNA anti- SARS-CoV-2 therapeutic from “second generation” LNAs informed by optimizations around current leads and cryoEM structures of their targets; 2) advancing the lead LNA therapeutic towards the clinic by expanding the in vitro and in vivo virology data package and performing requisite CMC and IND-enabling activities; 3) identifying and optimizing small molecules and RIBOTACs against the same SARS-CoV-2 RNA targets; and 4) identifying, characterizing, and targeting conserved candidate RNA structure targets in other RNA viruses of pandemic potential and developing programmable LNA and small molecule/RIBOTAC therapeutics against them.

摘要：我们的总体目标是开发一类新型的门诊治疗，以高度靶向 SARS-COV-2基因组中的保守RNA结构和其他大流行潜力的呼吸道病毒。 使用影响力A A A Impactza A病毒（IAV）作为概念验证，我们先前表明：1）添加高分辨率RNA 二级结构目标信息到基于反义寡核苷酸（ASO）治疗的设计 可以大大提高抗病毒效率，而不是简单地靶向配置的一级序列，这可以合并 进入ASO设计的能力，可以将RNase H募集到目标位点； 2）一个高度稳定的鼻内剂量 针对普遍配置的RNA结构设计的锁定核酸（LNA）ASO可提供100％的生存 在14天或致死IAV接种物后3天给予时； 3）对我们的LNA没有阻力 可选。应用类似方法，通过一个过程，我们现在将“可编程抗病毒药”称为SARS-COV-2， 我们1）迅速鉴定出高度组成的RNA结构； 2）设计针对这些目标的LNA并显示 结构的冷冻电子显微镜（冷冻）可改善LNA设计。 3）证明我们的领导 LNA具有引人注目的体外和体内效率，以针对参考和临床分离株（包括病毒） 具有降低疫苗效率的突变（例如三角洲变体）。补充，我们已经开创了 新型策略，称为“ Inforna”，设计了选择性结合病毒RNA结构并抑制的小分子 参与人类遗传疾病和RNA病毒的非编码RNA，包括SARS-COV-2，一些 NM和PM范围的活动。可以修改小分子以募集内源性核酸酶 通过LNA ASOS诱导RNase H诱导的降解。我们称这种类型的小分子为“核糖核酸酶” 靶向嵌合体（Ribotac）。”现在我们假设：1）我们的铅LNA分子 - 包括一个靶向 SARS-COV-1和MERS-COV共有的RNA结构，就代表了理想的发展候选者； 2）LNA的抗病毒效力可以进一步提高； 3）由此产生的LNA将具有很高的障碍 抗性的发展，并广泛活跃于野生型和抗疫苗的菌株； 4）Inforna可以 帮助设计小分子和Ribotacs，以针对我们确定的目标结构进行设计； 5）我们的疗法是 与其他抗SARS-COV-2代理结合； 6）可以快速应用类似方法 大流行关注的RNA病毒。我们将通过：1）选择铅（和向上）LNA抗 - 来自“第二代” LNA的SARS-COV-2疗法通过围绕当前潜在客户和 其目标的冷冻结构； 2）通过扩展铅LNA疗法向诊所推进诊所 体外和体内病毒学数据包，并进行必要的CMC和辅助活动； 3）识别 并针对相同的SARS-COV-2 RNA靶标优化小分子和ribotacs； 4）确定， 在其他大流行的RNA病毒中表征和靶向配置的候选RNA结构靶标 潜在的和开发可编程的LNA和小分子/Ribotac疗法。