Evaluation of siRNA nanoformulations and aerosol delivery for treatment of SARS-CoV-2

2019年新型冠状病毒SARS-CoV-2会导致COVID-19这一大流行病毒性疾病。这种疾病在世界范围内造成死亡，特别是在心血管疾病和动脉高血压患者中。截至2020年6月，全球报告病例近660万例，死亡39.3万人；美国有195万例病例，超过11.1万人死亡。COVID-19给已经面临癌症治疗和生存磨难的癌症患者带来了具体和重大的直接负担。除非紧急开发出一种不干扰当前癌症化疗的治疗方法，否则SARS-CoV-2将继续对上述高风险群体（包括当前和未来的癌症患者）的生命构成重大威胁，而不管癌症的类型和阶段如何。冠状病毒被阳性单链RNA基因组包裹；属于冠状病毒科。冠状病毒由至少5种主要的基本蛋白组成：编码多肽的复制酶（pp1ab）、刺突(S)、包膜(E)、膜(M)和核衣壳(N)蛋白。病毒的生命周期开始于感染，病毒粒子通过与宿主细胞受体的结合和内吞作用进入细胞（S蛋白），病毒基因组释放到细胞质中并运输到宿主细胞核（N蛋白），在那里进行复制（pp1ab）并释放新的病毒颗粒。这会导致呼吸道、肠道、肝脏和神经系统疾病。pp1ab、E和N蛋白是SARS-CoV-2独有的，不在哺乳动物细胞中天然表达，为靶向治疗COVID-19提供了潜在的机会。目前的药物和治疗策略包括阻断rna聚合酶、以mrna为基础的疫苗以诱导抗体产生、抗体治疗以及从Covid-19幸存者身上分离血浆和抗体。由于靶向对SARS-CoV-2感染和传播至关重要的蛋白质存在一些不必要的副作用，现有药物的益处有限，以及COVID-19疾病动物模型的可用性有限，我们的策略是基于对SARS-CoV-2蛋白的最大抑制（在转染单个SARS-CoV-2蛋白的模型细胞系中）使用和优先考虑siRNA候选物，以进行进一步评估。我们的目标是使用基于sirna的治疗候选药物，雾化（通过碰撞雾化器）中性磷脂1,2-二油基-锡-甘油基-3-磷脂酰胆碱纳米脂质体（DOPC）或植物源性囊泡（PDV）特异性靶向SARS-CoV-2的重要蛋白，以抑制其复制和病毒组装。我们期望这种方法将有一个强大的抗病毒RNAi反应，导致病毒清除。

The 2019 novel coronavirus, SARS-CoV-2 causes COVID-19, a pandemic viral disease. This disease has resulted in world-wide fatalities, particularly in patients with cardiovascular disease, and arterial hypertension. As of June 2020, close to 6.6 million cases and death of 393,000 were reported worldwide; 1.95 million cases and more than 111,000 deaths in the US. COVID-19 poses a specific and substantial immediate burden on cancer patients who are already facing the tribulations of cancer treatment and survivorship. SARS-CoV-2 will continue to be a major threat to the lives of the above high risk groups including current and future cancer patients irrespective of the type and stage of cancer, unless a treatment that does not interfere with current cancer chemotherapies is developed urgently. CoVs are enveloped with a positive sense, single-strand RNA genome; and belong to the Coronaviridae family. CoVs are composed of at least 5 major fundamental proteins: replicase encoding polypeptide (pp1ab), Spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins. The viral life cycle begins with the infection, entry of the virion into cells by the binding to the host cell receptor and endocytosis (S protein), release of the viral genome into the cytoplasm and its transport to the host nucleus (N protein) where it replicates (pp1ab) and new virus particles are released. This results in respiratory, enteric, hepatic, and neurologic diseases. pp1ab, E and N proteins, unique to SARS-CoV-2 and not natively expressed in mammalian cells, offer potential opportunities for therapeutic targeting to cure COVID-19. Current drug and treatment strategies include blocking of RNA-polymerase, mRNA-based vaccines to induce antibody production, antibody treatments, and isolation of plasma and antibodies from the survivors of Covid-19. Since there are several unwarranted side effects of targeting the proteins essential to SARS-CoV-2 infection and spread, limited benefits of the current drugs, and limited availability of COVID-19 disease animal models, our strategy is to use and prioritize siRNA candidates based on the greatest inhibition of SARS-CoV-2 proteins (in model cell lines transfected with individual SARS-CoV-2 proteins) for further evaluation. We aim to use the siRNA-based therapeutic candidates with either an aerosolized (through a collision nebulizer) neutral phospholipid 1,2- dioleoyl-sn-glycero-3-phosphatidylcholine nanoliposomes (DOPC) or plant-derived vesicles (PDV) to specifically target the vital proteins of SARS-CoV-2 to inhibit its replication and virus assembly. We expect this approach will have a potent anti-viral RNAi response leading to viral clearance.