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方法，针对高度... SARS-CoV-2和其他可能大流行的呼吸道病毒基因组中保守的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 反义寡核苷酸治疗药物设计中的二级结构靶向信息 通过简单地靶向保守的初级序列，可以极大地增强抗病毒效果，就像整合 在ASO设计中将RNaseH招募到靶部位的能力；2)单次鼻腔剂量高度稳定的， 针对普遍保守的RNA结构设计的锁定核酸(LNA)ASO可提供100%的存活率 在接种致死IAV前14天或接种后3天；3)对我们的LNA没有抵抗力 可选。对SARS-CoV-2病毒应用类似的方法，通过我们现在称为“可编程抗病毒药物”的过程， 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 结构的冷冻电子显微镜(CryoEM)导致了LNA设计的改进；3)证明了我们的领先地位 LNAs在体外和体内对参考和临床分离株，包括病毒，具有令人信服的效果 含有降低疫苗效力的突变(例如，Delta变体)。作为补充，我们开创了一种 一种名为“Inforna”的新策略，用于设计选择性结合病毒RNA结构并抑制病毒的小分子 涉及人类遗传病和RNA病毒的非编码RNA，包括SARS-CoV-2，其中一些带有 NM和PM范围内的活动。小分子可以被修饰以招募一种内源核酸酶，类似于 LNA ASOS对核糖核酸酶H的降解作用。我们把这种小分子称为“核糖核酸酶”。 靶向嵌合体(RIBOTAC)。我们现在假设：1)我们的主要LNA分子-包括一个靶向 SARS常见的RNA结构--CoV-1和MERS-CoV--已经是理想的研发候选者； 2)LNAs的抗病毒效力可以进一步增强；3)由此产生的LNAs将具有很高的屏障 对野生型和疫苗耐药株具有广泛的抗药性；4)Informna可以 帮助针对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；以及6)类似方法可迅速应用于 引起大流行关注的RNA病毒。我们将通过以下方式检验这些假设：1)选择一个领先的(和备份的)LNA反 来自第二代LNA的SARS-CoV-2治疗方法，通过围绕当前导线和 2)通过扩大LNA治疗的靶点，将LNA引导疗法推向临床 体外和体内病毒学数据包并执行必要的CMC和IND使能活动；3)识别 并针对相同的SARS-CoV-2RNA靶点优化小分子和RIBOTAC；以及4)识别， 鉴定和靶向其他大流行RNA病毒中保守的候选RNA结构靶点 潜在的和开发可编程的LNA和小分子/RIBOTAC疗法来对抗它们。