TMPRSS2 as a potential target for treatments of COVID-19 and respiratory infectious viruses in lung

项目摘要 2019年12月，一种新型冠状病毒，现在被称为严重急性呼吸综合征冠状病毒2（SARS- 新型冠状病毒（COV-2）袭击了中国武汉，并引发了目前的冠状病毒病（COVID-19）大流行。目前没有 被证明有效治疗或预防COVID-19的药物或疫苗。迫切需要 确定有效的治疗选择：可以有效治愈疾病的疫苗和/或药物。虽然疫苗将 作为一个社区对抗病毒的最终方式，抗病毒药物可能会更快地开发和批准，特别是 因为一种广泛可用且有效的疫苗可能需要数年时间。抗病毒药物将极大地造福于 目前受病毒影响的人，帮助个人康复，减少死亡人数。抗病毒药物也会减少 阳性携带者的数量，从而遏制疾病的传播。 该提案旨在开发一种有效的抗病毒药物，以阻止病毒进入细胞，特别是进入肺泡型 II（AT 2）细胞，远端肺的干细胞。多亏了最近的研究，我们知道了哪扇门（一种叫做ACE 2的受体）， “钥匙”（一种称为TMPRSS 2的蛋白酶）是病毒进入细胞的关键。我们的目标是移除“钥匙”，使病毒无法打开 打开“门”，进入宿主细胞。我们将使用最近开发的三维（3D）体外肺类器官模型， 它概括了肺结构和细胞环境的许多方面，并已被用于研究呼吸 病毒，包括SARS-CoV-2。该系统比细胞系更好地代表组织，但提供了更少的好处 比组织外植体或动物模型复杂。此外，我们还生成了一组高度敏感和特异的 针对TMPRSS 2的小鼠单克隆抗体（mAb）。在初步研究中，TMPRSS 2 mAb，AL 20， 显示没有细胞毒性的迹象，具有抑制SARS-CoV-2假病毒进入细胞系的趋势。此外，委员会还认为， 我们已经鉴定了至少两种与TMPRSS 2形成复合物的丝氨酸蛋白酶抑制剂（serpins）， 与SARS-CoV-2感染率呈负相关。这些发现导致我们的假设， 靶向TMPRSS 2可以抑制SARS-CoV-2病毒进入和传播。 为了验证我们的假设，我们将首先测试AL 20阻断SARS-CoV-2进入肺中AT 2细胞的功效 类器官，并阐明潜在的机制。然后，我们将评估丝氨酸蛋白酶抑制剂对TMPRSS 2活性的影响， SARS-CoV-2病毒进入和传播。最后，为了探索将AL 20推进到人体试验的可行性，我们还建议 在可用的K18-hACE 2小鼠中人源化并测试AL 20。该转基因株表达人ACE 2，受KRT 18调节。 启动子，指导表达到肺上皮细胞，以提供必要的临床前数据，以测试的可行性， 进行人体临床试验。 这些研究将为TMPRSS 2调节SARS-CoV-2进入的机制提供重要的见解，并建议 潜在的抗COVID-19治疗候选物。拟议的工作有可能影响数百万人的生活， 受COVID-19和其他呼吸道病毒（如甲型流感）影响的个体使用TMPRSS 2进入细胞。

Project Summary In December of 2019, a novel coronavirus, now referred to as severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2), struck Wuhan, China and unleashed the current coronavirus disease (COVID-19) pandemic. There are currently no medications or vaccines proven to be effective for the treatment or prevention of COVID-19. There is an urgent need to identify effective therapeutic options: a vaccine and/or drugs that can effectively cure the disease. Although a vaccine will be the ultimate way to combat the virus as a community, antivirals are likely to be developed and approved faster, especially since a broadly available and effective vaccine is likely years away. Antivirals would hugely benefit the population that is currently affected by the virus, helping individuals recover and reducing the number of deaths. Antivirals would also reduce the number of positive carriers and thus curb the spread of the disease. This proposal aims to develop an efficient antiviral to impede the virus’ entry into cells, specifically into lung alveolar type II (AT2) cells, the stem cells of the distal lung. Thanks to recent studies, we know which “door” (a receptor called ACE2) and “key” (a protease called TMPRSS2) the virus uses to enter cells. Our goal is to remove the “key” so the virus cannot open the “door” and enter host cells. We will use a recently developed 3-dimensional (3D) in vitro lung organoid model that recapitulates many aspects of lung structure and the cellular environment and that has been used to study respiratory viruses, including SARS-CoV-2. This system represents tissues better than cell lines, but offers the benefit of being less complex than tissue explants or animal models. In addition, we have generated a panel of highly sensitive and specific mouse monoclonal antibodies (mAbs) directed against TMPRSS2. In preliminary studies, the lead TMPRSS2 mAb, AL20, shows no signs of cytotoxicity with a trend towards inhibition of SARS-CoV-2 pseudovirus entry in cell lines. Furthermore, we have identified at least two serine protease inhibitors (serpins) that form complexes with TMPRSS2, and the presence of these complexes is inversely correlated with the SARS-CoV-2 infection rate. These findings lead to our hypothesis that targeting TMPRSS2 can inhibit SARS-CoV-2 viral entry and spread. To test our hypothesis, we will first test the efficacy of AL20 for blocking the entry of SARS-CoV-2 into AT2 cells in lung organoids, and elucidate the underlying mechanisms. We will then evaluate the effects of serpins on TMPRSS2 activity and SARS-CoV-2 viral entry and spread. Finally, to explore the feasibility of advancing AL20 to human trials, we also propose to humanize and test AL20 in available K18-hACE2 mice. This transgenic strain expresses human ACE2, regulated by the KRT18 promoter that directs expression to lung epithelia, to provide the pre-clinical data necessary to test the feasibility of advancing to human clinical trials. These studies will provide critical insights into the mechanisms whereby TMPRSS2 regulates SARS-CoV-2 entry, and suggest potential therapeutic candidates against COVID-19. The proposed work has the potential to impact the lives of millions of individuals affected by COVID-19 and other respiratory viruses, such as influenza A, that use TMPRSS2 to enter cells.