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种新型的门诊治疗药物， SARS-CoV-2和其他具有大流行潜力的呼吸道病毒基因组中的保守RNA结构。 使用甲型流感病毒（IAV）作为概念验证，我们先前表明：1）添加高分辨率RNA 将二级结构靶信息引入基于反义寡核苷酸（阿索）的治疗剂的设计中 可以大大增强抗病毒功效，而不是简单地靶向保守的一级序列， 将RNA酶H募集到靶位点的能力引入到阿索设计中; 2）单次鼻内剂量的高度稳定的， 针对普遍保守的RNA结构设计的锁核酸（LNA）阿索提供100%存活 当在致死性IAV接种物之前14天或之后3天给予时;以及3）对我们的LNA没有抗性， 可选的。通过我们现在称之为“可编程抗病毒药物”的过程，将类似的方法应用于SARS-CoV-2， 我们1）快速鉴定高度保守的RNA结构; 2）针对这些靶标设计LNA，并显示 结构的低温电子显微镜（cryoEM）导致了LNA设计的改进; 3）证明了我们的领先优势 LNA对参考和临床分离株（包括病毒）具有令人信服的体外和体内疗效 携带降低疫苗效力的突变（例如δ变体）。作为补充，我们开创了一个 一种名为“Inforna”的新策略，用于设计选择性结合病毒RNA结构并抑制 参与人类遗传疾病和RNA病毒的非编码RNA，包括SARS-CoV-2，其中一些具有 在nM和pM范围内的活性。小分子可以被修饰以募集内源性核酸酶，类似于 RNA酶H诱导的LNA ASO降解。我们称这种小分子为“核糖核酸酶 靶向嵌合体（RIBOTAC）。我们现在假设：1）我们的领导LNA分子-包括一个靶向 SARS-CoV-1和MERS-CoV共有的RNA结构已经代表了理想的开发候选物; 2)LNAs的抗病毒效力可以进一步增强; 3）所得LNAs将对 产生耐药性，并对野生型和疫苗耐药菌株具有广泛活性; 4）Inforna可 帮助设计针对我们确定的靶结构的小分子和RIBOTAC; 5）我们的治疗方法 可与其他抗SARS-CoV-2药物联合使用;以及6）类似方法可快速应用于 RNA病毒的大流行的关注。我们将通过以下方式测试这些假设：1）选择一个引导（和备份）LNA抗- SARS-CoV-2治疗来自“第二代”LNA，通过围绕当前电极导线的优化和 cryoEM结构的目标; 2）推进领先的LNA治疗走向临床，通过扩大 体外和体内病毒学数据包，并执行必要CMC和IND使能活动; 3）识别 并针对相同的SARS-CoV-2 RNA靶标优化小分子和RIBOTAC;以及4）鉴定， 表征和靶向大流行性感冒的其他RNA病毒中的保守候选RNA结构靶标 潜在的和开发针对它们的可编程LNA和小分子/RIBOTAC治疗剂。