siRNA silencing of cellular entry proteins as a gene therapy strategy for Covid-19

ABSTRACT

SARS-CoV-2 remains a significant public health threat, causing severe respiratory illness in susceptible individuals. Several effective Covid-19 vaccines have been developed but novel SARS-CoV-2 variants continuously emerge that are more transmissible and have potential to evade vaccine immune responses. We are developing a novel therapy that does not depend on an immune response, based on siRNA-mediated silencing of Angiotensin-converting enzyme 2 (ACE2) receptor and Transmembrane Serine Protease 2 (TMPRSS2). SARS-CoV-2 requires these host proteins to enter respiratory epithelial cells at the cell surface, through binding and priming of its Spike protein. As a cell model for SARS-CoV-2 infection, we have utilised primary nasal epithelial cells (NHNE), as well as HEK293T cells overexpressing ACE2 and TMPRSS2. siRNA transfection in NHNE cells led to a 78%-88% knockdown of ACE2 and TMPRSS2, as determined by qRT-PCR and western blot data. TMPRSS2 knockdown in the overexpressing HEK293T cells resulted in an 87% reduction in infectivity from SARS-CoV-2 Spike-pseudotyped lentiviruses expressing a luciferase transgene, indicative of a significant reduction in virus entry (p < 0.0001 by one-way ANOVA). We are now working to confirm these results with live SARS-CoV-2 and to test lipid nanoparticle delivery of the siRNAs to air-liquid interface grown NHNEs to more accurately model the respiratory airway. This siRNA approach could provide a novel therapy for immunocompromised individuals who do not gain sufficient protection from SARS-CoV-2 vaccines. Additionally, by targeting host proteins rather than virus components, our therapy is likely to remain effective in spite of emerging SARS-CoV-2 variants that circumvent pre-existing immune responses.