Department of Infectious Diseases/Virology, Medical Faculty, University of Heidelberg, BioQuant BQ0030, Im Neuenheimer Feld 267, 69120 Heidelberg, Germany; Faculty of Biosciences, University of Heidelberg, 69120 Heidelberg, Germany.
Department of Infectious Diseases/Molecular Virology, Medical Faculty, Center for Integrative Infectious Diseases Research (CIID), University of Heidelberg, 69120 Heidelberg, Germany; Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, FL 32611, USA.
Mol Ther. 2022 May 4;30(5):2005-2023. doi: 10.1016/j.ymthe.2022.01.024. Epub 2022 Jan 14.
Despite rapid development and deployment of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), clinically relevant modalities to curb the pandemic by directly attacking the virus on a genetic level remain highly desirable and are urgently needed. Here we comprehensively illustrate the capacity of adeno-associated virus (AAV) vectors co-expressing a cocktail of three short hairpin RNAs (shRNAs; RNAi triggers) directed against the SARS-CoV-2 RdRp and N genes as versatile and effective antiviral agents. In cultured monkey cells and human gut organoids, our most potent vector, SAVIOR (SARS virus repressor), suppressed SARS-CoV-2 infection to background levels. Strikingly, in control experiments using single shRNAs, multiple SARS-CoV-2 escape mutants quickly emerged from infected cells within 24-48 h. Importantly, such adverse viral adaptation was fully prevented with the triple-shRNA AAV vector even during long-term cultivation. In addition, AAV-SAVIOR efficiently purged SARS-CoV-2 in a new model of chronically infected human intestinal cells. Finally, intranasal AAV-SAVIOR delivery using an AAV9 capsid moderately diminished viral loads and/or alleviated disease symptoms in hACE2-transgenic or wild-type mice infected with human or mouse SARS-CoV-2 strains, respectively. Our combinatorial and customizable AAV/RNAi vector complements ongoing global efforts to control the coronavirus disease 2019 (COVID-19) pandemic and holds great potential for clinical translation as an original and flexible preventive or therapeutic antiviral measure.
尽管针对严重急性呼吸综合征冠状病毒 2（SARS-CoV-2）的疫苗已迅速开发和部署，但通过在遗传水平上直接攻击病毒来遏制大流行的临床相关方法仍然非常需要，并且迫切需要。在这里，我们全面说明了共表达三种针对 SARS-CoV-2 RdRp 和 N 基因的短发夹 RNA（shRNA；RNAi 触发物）的腺相关病毒（AAV）载体作为多功能和有效抗病毒剂的能力。在培养的猴细胞和人类肠道类器官中，我们最有效的载体 SAVIOR（SARS 病毒抑制剂）将 SARS-CoV-2 感染抑制到背景水平。引人注目的是，在使用单个 shRNA 的对照实验中，感染细胞内的多种 SARS-CoV-2 逃逸突变体在 24-48 小时内迅速出现。重要的是，即使在长期培养过程中，三链 shRNA AAV 载体也完全阻止了这种不利的病毒适应。此外，AAV-SAVIOR 可有效清除慢性感染人肠道细胞模型中的 SARS-CoV-2。最后，使用 AAV9 衣壳的鼻内 AAV-SAVIOR 递送适度降低了 hACE2 转基因或野生型小鼠感染人或鼠 SARS-CoV-2 株的病毒载量和/或减轻了疾病症状。我们的组合和可定制的 AAV/RNAi 载体补充了全球正在进行的控制 2019 年冠状病毒病（COVID-19）大流行的努力，并具有作为原始且灵活的预防或治疗性抗病毒措施进行临床转化的巨大潜力。
