SBIR PA22-176 - RNA aptamers for rapid response to COVID-19 variants

摘要 由SARS-CoV-2病毒引发的新冠肺炎大流行，在全球范围内产生了前所未有的破坏性影响。 尽管疫苗接种有望结束传播，但快速的突变增加了突破 完全接种疫苗人群中的感染率。众所周知，RNA病毒具有非常高的突变率和 进化论。高突变率与毒力调节和逃避宿主的能力有关 免疫力报告迫切需要能够跟上病毒变异的治疗方法。关于分子 S的蛋白受体结合域和血管紧张素转换酶2是关键 病毒进入的介体，因此药物阻断S1RBD与ACE2的结合可能是有效的 针对SARS-CoV-2的治疗。事实上，针对S蛋白的中和抗体已经开发出来 并在临床上使用。遗憾的是，抗体工程很难跟上病毒进化的步伐。 Delta变种的传染性是以前变种的两倍，而Omicron变种表现出更多的传染性 斯派克蛋白的突变比其他变异体更多。由于风险，这些变种引起了疾控中心的担忧 免疫逃逸和传播性增加。需要有效的药物来对抗快速突变的变种 不能用抗体来满足，因为治疗性抗体的生产既耗时又昂贵。在……里面 在这种背景下，基于核酸的适体，也被称为‘化学抗体’，有可能解决这一问题 挑战。使用体外化学组合方法选择适配子，系统进化 通过指数浓缩的配体(SELEX)，并提供比抗体更好的优势，以解决 变异病毒因选育和化学生产快、易化学修饰、高 热稳定性好，免疫原性低。尽管RNA适配子对核酸酶和肾脏清除很敏感， 2‘-氟嘧啶修饰显著增加了抗性核酸酶，而多价适配子 或者结合到钉子上可以增加适配子的大小，从而增加循环时间。在我们的预赛中 研究中，我们筛选了一系列针对SARS-CoV-2野生型S1RBD蛋白的RNA适配子，并 发明了一种专利方法，以高产率和低成本产生化学修饰的血清稳定的RNA。 所选适配子对WT及其变异体(Alpha， Beta、Gamma和Omicron)，但还不是Delta变体。在本项目中，我们将解决Delta变体和 将我们当前的适配子和新的抗Delta适配子优化为双特异性形式(避免快速肾脏 净空)。我们将通过以下具体目标实现我们的目标：1)筛选和表征 抗Delta变异的适配子和形成二价结构的适配子的优化；2)评价 抗病毒活性。抗病毒能力将在伪病毒和活体传染病病毒中进行评估。 通过我们和NIAID达成的协议。除了特定的适配子，我们还将建立一个平台，并 我们对病毒突变做出快速反应的能力。在应对快速变异的传染病方面， 开发基于适体的疗法作为抗体的替代品类似于开发mrna疫苗。 优于传统灭活病毒疫苗，因为适体和mRNAs都可以筛选/设计 快点。

ABSTRACT The COVID-19 pandemic caused by SARS-CoV-2 viruses has had an unprecedented disruptive global impact. Although vaccination has been expected to end the spread, the fast mutations have increased the breakthrough infection rates in the fully vaccinated population. RNA viruses are known to have very high rates of mutation and evolution. The high rate of mutation is correlated with virulence modulation and the ability to escape host immunity posting an urgent need for treatments that can keep up with the virus mutations. On the molecular level, spike (S) protein receptor binding domain (RBD) and angiotensin-converting enzyme 2 (ACE2) are key mediators for viral entry, therefore pharmacological disruption of S1 RBD binding to ACE2 could be an effective treatment against SARS-CoV-2. Indeed, neutralization antibodies against the S protein have been developed and are used in clinics. Unfortunately, it is difficult for antibody engineering to keep up with the virus evolution. The Delta variant is twice as contagious as the previous variants, whereas the Omicron variant exhibits more mutations in the spike protein than other variants. These variants have raised CDC's concerns due to the risks of immune escape and increased transmissibility. The need for effective drugs against the fast mutating variants can not be met by antibodies because the production of therapeutic antibodies is time-consuming and costly. In this context, nucleic acid-based aptamers, also known as `chemical antibodies' have the potential to address this challenge. Aptamers are selected using an in vitro chemical combinatorial approach, systematic evolution of ligands by exponential enrichment (SELEX), and offer advantages over antibodies to address the problem of mutating viruses because of the fast selection and chemical production, easy chemical modification, high thermostability, and low immunogenicity. Although RNA aptamer is sensitive to nucleases and renal clearance, 2'-fluoro-pyrimidine modification has significantly increased the resistance nucleases, while multivalent aptamers or conjugation to PEGs can increase aptamer sizes and consequently the circulation time. In our preliminary studies, we have selected a series of RNA aptamers targeting the wild-type SARS-CoV-2 S1RBD protein and invented a proprietary approach to generate chemical-modified serum-stable RNAs at high yield and low cost. The selected aptamers show the universal inhibitory effect to RBD-ACE2 binding for WT and variants (Alpha, Beta, Gamma, and Omicron) but not the Delta variant yet. In this project, we will address the Delta variant and optimize our current aptamers and the new anti-Delta aptamer into a bispecific format (avoid rapid renal clearance). We will reach our goals through the following specific aims:1) Screening and characterization of aptamers against the Delta variant and optimization of aptamers by forming bivalent structures, and 2) Evaluation of the antiviral activity. The antiviral capability will be assessed in pseudovirus as well as live infectious viruses through our established agreement with NIAID. Beyond the specific aptamers, we will establish a platform and our ability for quick responses to virus mutations. In terms of responding to fast-mutating infectious diseases, developing aptamer-based therapeutics as an alternative to antibodies is similar to developing mRNA vaccines over conventional inactivated virus vaccines because both aptamers and mRNAs can be screened/designed quickly.