Extracellular vesicles encapsulating CRISPR machinery for treatment of SARS-CoV-2 infection

摘要/摘要 SARS-CoV-2已导致全球数百万人死亡。有效的抗病毒治疗 迫切需要各种治疗方案。CRISPR介导的基因组编辑提供了一条非常有希望的途径 用于治疗各种遗传病。特别是对CRISPR-CAS13系统进行了演示 具有通过降解病毒基因组RNA抑制SARS-CoV-2和流感感染的潜力 和病毒基因的表达。然而，提供CRISPR机制来启动基因组编辑仍然是非常具有挑战性的 在活体内有效。细胞外小泡(EV)含有多种分子成分，包括脂类、信使核糖核酸、 MicroRNAs和蛋白质。大量研究表明，电动汽车通过以下方式调节细胞间的通讯 传输其封装的内容。该方案旨在构建封装CRISPR的电动汽车 机械并将EV运送到呼吸道上皮细胞，以抑制SARS-CoV-2在体内的增殖。我们会 因此，对Cas13d蛋白进行基因工程，使Cas13d/CRISPR-RNA(CrRNA)核糖核蛋白 复合体可以被封装成电动汽车。我们还将设计电动汽车的薄膜，使电动汽车瞄准 并传递Cas13d/crRNA以抑制SARS-CoV-2病毒的组装和增殖， 从而抑制新冠肺炎。这项研究将使我们了解基于电动汽车的车辆的可行性 提供基因组编辑设备以抑制SARS-CoV-2在肺上皮细胞中的增殖。这项研究将 为新冠肺炎患者提供治疗选择，以降低疾病严重程度和死亡率。

Summary/Abstract SARS-CoV-2 has caused the deaths of millions of people globally. Effective antiviral therapeutic treatment options are urgently needed. CRISPR-mediated genome editing has provided a very promising avenue for treatment of a variety of genetic diseases. Particularly, the CRISPR-Cas13 system has been demonstrated to possess the potential of inhibiting SARS-CoV-2 and influenza infections by degradation of viral genomic RNA and viral mRNA. However, it is still very challenging to deliver the CRISPR machinery to initiate genome editing efficiently in vivo. Extracellular vesicles (EVs) contain a variety of molecular components including lipids, mRNA, microRNAs, and proteins. A large body of studies has shown that EVs mediate cell-to-cell communication by transmitting their encapsulated contents. This proposal intends to construct EVs encapsulating the CRISPR machinery and deliver the EVs to respiratory epithelial cells to inhibit SARS-CoV-2 proliferation in vivo. We will therefore genetically engineer the Cas13d protein so that Cas13d/CRISPR-RNA (crRNA) ribonucleoprotein complex can be encapsulated into EVs. We will also engineer the membrane of EVs, such that EVs target respiratory epithelial cells and deliver Cas13d/crRNA for inhibiting SARS-CoV-2 viral assembly and proliferation, thereby inhibiting COVID-19. This study will allow us to understand the feasibility of an EVs-based vehicle to deliver genome editing machinery to inhibit SARS-CoV-2 proliferation in lung epithelial cells. This study will provide a treatment option for COVID-19 patients to reduce disease severity and mortality.