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

总结/摘要 SARS-CoV-2已在全球造成数百万人死亡。有效的抗病毒治疗 迫切需要治疗方案。CRISPR介导的基因组编辑提供了一个非常有前途的途径 用于治疗各种遗传疾病。特别地，CRISPR-Cas 13系统已经被证明是 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mRNA。然而，交付CRISPR机器来启动基因组编辑仍然非常具有挑战性 在体内有效。细胞外囊泡（EV）含有多种分子组分，包括脂质、mRNA， microRNA和蛋白质。大量的研究表明，EV通过以下方式介导细胞间的通讯： 传送其封装的内容物。该提案旨在构建封装CRISPR的EV 机械化并将EV递送至呼吸道上皮细胞以抑制体内SARS-CoV-2增殖。我们将 因此，基因工程化Cas 13 d蛋白，使得Cas 13 d/CRISPR-RNA（crRNA）核糖核蛋白 复合物可以被封装到EV中。我们还将设计电动汽车的膜， 呼吸道上皮细胞并递送抑制SARS-CoV-2病毒组装和增殖的Cas 13 d/crRNA， 从而抑制COVID-19。这项研究将使我们了解电动汽车的可行性， 提供基因组编辑机制，以抑制SARS-CoV-2在肺上皮细胞中的增殖。本研究将 为COVID-19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.