Ex vivo and in vivo suppression of SARS-CoV-2 with combinatorial AAV/RNAi expression vectors.

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.