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门诊治疗药物，针对高度 SARS-CoV-2 和其他具有大流行潜力的呼吸道病毒的基因组中存在保守的 RNA 结构。 使用甲型流感病毒 (IAV) 作为概念验证，我们之前表明：1) 添加高分辨率 RNA 二级结构靶标信息纳入反义寡核苷酸 (ASO) 疗法的设计 与简单地靶向保守的一级序列相比，可以大大增强抗病毒功效，因为可以将 将 RNase H 招募到目标位点的能力纳入 ASO 设计中； 2) 高度稳定的单次鼻内剂量， 针对普遍保守的 RNA 结构设计的锁核酸 (LNA) ASO 可提供 100% 的存活率 在致命 IAV 接种前 14 天或接种后 3 天接种； 3）我们的 LNA 没有遇到任何阻力 可选择的。通过我们现在称之为“可编程抗病毒药物”的过程，对 SARS-CoV-2 应用类似的方法， 我们 1) 快速鉴定高度保守的 RNA 结构； 2) 针对这些目标设计了 LNA 并展示了 结构的冷冻电子显微镜 (cryoEM) 改进了 LNA 设计； 3）证明我们的领先地位 LNA 对参考和临床分离株（包括病毒）具有引人注目的体外和体内功效 含有降低疫苗功效的突变（例如 delta 变体）。作为补充，我们开创了 名为“Infona”的新策略，旨在设计选择性结合病毒 RNA 结构并抑制病毒的小分子。 与人类遗传疾病和 RNA 病毒有关的非编码 RNA，包括 SARS-CoV-2，其中一些具有 活性在 nM 和 pM 范围内。小分子可以被修饰以招募内源性核酸酶，类似于 RNase H 诱导的 LNA ASO 降解。我们将这种类型的小分子称为“核糖核酸酶” 靶向嵌合体（RIBOTAC）。”我们现在假设：1) 我们的先导 LNA 分子——包括一种靶向 SARS-CoV-1 和 MERS-CoV 共有的 RNA 结构——已经代表了理想的开发候选者； 2）LNA的抗病毒效力可以进一步增强； 3）由此产生的LNA将对 产生抗药性并广泛积极对抗野生型和疫苗抗性菌株； 4）信息可以 帮助根据我们确定的目标结构设计小分子和 RIBOTAC； 5）我们的治疗方法是 可与其他抗 SARS-CoV-2 药物联合使用； 6）类似的方法可以快速应用于 大流行关注的RNA病毒。我们将通过以下方式测试这些假设：1）选择一个主导（和备份）LNA 抗 来自“第二代”LNA 的 SARS-CoV-2 治疗药物通过围绕当前先导化合物的优化而获得信息 其目标的冷冻电镜结构； 2) 通过扩大治疗范围，将领先的 LNA 治疗推向临床 体外和体内病毒学数据包并执行必要的 CMC 和 IND 支持活动； 3）识别 针对相同的 SARS-CoV-2 RNA 靶点优化小分子和 RIBOTAC； 4）识别， 表征并针对大流行的其他 RNA 病毒中保守的候选 RNA 结构靶标 潜力并正在开发针对它们的可编程 LNA 和小分子/RIBOTAC 疗法。