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

Abstract

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.

In vitro high-content tissue models to address precision medicine challenges

“…Other studies have engineered colonic organoids to assess the association of hypertension and SARS-CoV-2 infections since hypertension is one of the most common comorbidities in COVID-19 patients ( Li et al, 2020a ). Human intestinal organoids infected with SARS-CoV-2 were used as a model to study infection in the gut ( Wang et al, 2022 ; Zhou et al, 2020b ), which demonstrated that the gut is also another target organ since ACE2 is highly expressed on enterocytes ( Becker et al, 2022 ; Lamers et al, 2020 ). These discoveries could be an explanation to the common gastrointestinal symptoms observed in COVID-19 patients such as diarrhea ( Ye et al, 2020 ).…”

Organoids and microphysiological systems: Promising models for accelerating AAV gene therapy studies

“…They have also begun to define the transduction pattern in organoids comprised of heterogeneous cell populations (vector tropism) and the expression profile and localization of transgene product (thereby testing the true selectivity of various “cell-specific” promoters), and they have explored some of the mechanisms of cell attachment of AAV vectors to different cell types within these human tissues. These studies have also used organoids to validate animal study outcomes in an all-human setting and to test novel approaches to gene delivery/editing to target infectious agents such as HIV and COVID-19 ( 132 , 139 ). By using iPSCs, either derived from patients or engineered via CRISPR/Cas9 gene editing, to fabricate these organoids, investigators have provided critical proof-of-concept for the value of using organoids to model a specific disease, e.g.…”

Can pulmonary RNA delivery improve our pandemic preparedness?

“…First, only one single-sequence siRNA molecule was used. Considering how fast SARS-CoV-2 escape mutations are observed when treated with shRNA [ 44 ], it seems likely that drug resistant RSV variants emerged under therapy. Therefore, in upcoming anti-viral siRNA trials, a mixture of siRNA sequences should be delivered in parallel [ 45 ].…”

“…But the question is: Can escape mutations arise also for these highly conserved regions, or would the virus lose viability or other critical characteristics if this region were to mutate? A recent study shows how fast escape mutations arise upon AAV-mediated shRNA transduction [ 44 ]. Therefore, there is still work to do for us.…”