Aims: COVID-19 remains a severe concern for global health and long-COVID symptoms are on the rise. Despite the vaccinations that can efficiently prevent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, there are still no effective treatments of the disease once people are infected. Elevated expression of the entry receptor angiotensin-converting enzyme 2 (ACE2) on cardiac cells of heart patients make them more susceptible to SARS-CoV-2 infection. The human transcriptome is mostly composed of non-coding RNA transcripts, which are not translated into proteins. Such non-coding RNAs are now known to be essential and functional molecules, which are crucial for development and even regulation of non-communicable diseases, but also of infectious diseases. In this project, we aim to investigate a specific subtype of non-coding RNAs, namely covalently closed, circular RNA molecules in the context of SARS-CoV-2 infection and propagation in the cardiovascular system.

Methods & results: To do so, we infected human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) with SARS-CoV-2 particles followed by RNA sequencing and detailed bioinformatics analysis to select circRNAs, which are significantly upregulated after SARS-CoV-2 infection in hiPSC-CMs. We validated these candidates in an SARS-CoV-2 infection timeline in hiPSC-CMs and confirmed their expression in 3D ex-vivo human lung and human heart slices via qPCR. Following this in a preventive circRNA based therapeutic approach we modulated the circRNA expression by the application of siRNAs prior to SARS-CoV-2 infection in hiPSC-CMs and observed reduced viral entry and viral load in the cardiomyocytes via qPCR, immunostaining and plaque assay. In order to explore the underlying mechanisms we performed RNA sequencing and pathway analysis after circRNAs knockdown in SARS-CoV-2 infected cardiomyocytes. Furthermore, we produced in vitro transcribed circRNA products to achieve overexpression of the selected candidate to further modulate the target prior to SARS-CoV-2 infection in cardiomyocytes.

Conclusion: Taken together, the data generated in this study will help to establish the role of circRNAs as potential therapeutic targets in the treatment of acute COVID-19.