Human angiotensin-converting enzyme 2-specific antisense oligonucleotides reduce infection with SARS-CoV-2 variants.

BACKGROUND: The Spike protein mutation severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to decreased protective effect of various vaccines and mAbs, suggesting that blocking SARS-CoV-2 infection by targeting host factors would make the therapy more resilient against virus mutations. Angiotensin-converting enzyme 2 (ACE2) is the host receptor of SARS-CoV-2 and its variants, as well as many other coronaviruses. Downregulation of ACE2 expression in the respiratory tract may prevent viral infection. Antisense oligonucleotides (ASOs) can be rationally designed on the basis of sequence data, require no delivery system, and can be administered locally. OBJECTIVE: We sought to design ASOs that can block SARS-CoV-2 by downregulating ACE2 in human airway. METHODS: ACE2-targeting ASOs were designed using a bioinformatic method and screened in cell lines. Human primary nasal epithelial cells cultured at the air-liquid interface and humanized ACE2 mice were used to detect the ACE2 reduction levels and the safety of ASOs. ASO-pretreated nasal epithelial cells and mice were infected and then used to detect the viral infection levels. RESULTS: ASOs reduced ACE2 expression on mRNA and protein level in cell lines and in human nasal epithelial cells. Furthermore, they efficiently suppressed virus replication of 3 different SARS-CoV-2 variants in human nasal epithelial cells. In vivo, ASOs also downregulated human ACE2 in humanized ACE2 mice and thereby reduced viral load, histopathologic changes in lungs, and increased survival of mice. CONCLUSIONS: ACE2-targeting ASOs can effectively block SARS-CoV-2 infection. Our study provides a new approach for blocking SARS-CoV-2 and other ACE2-targeting virus in high-risk populations.

Preventing ATP Degradation by ASO-Mediated Knockdown of CD39 and CD73 Results in A2aR-Independent Rescue of T Cell Proliferation.

The adenosine axis contributes to the suppression of antitumor immune responses. The ectonucleotidase CD39 degrades extracellular adenosine triphosphate (ATP) to adenosine monophosphate (AMP), which is degraded to adenosine by CD73. Adenosine binds to, e.g., the A2a receptor (A2aR), which reportedly suppresses effector immune cells. We investigated effects of ATP, AMP, and adenosine analogs on T cell proliferation, apoptosis, and proinflammatory cytokine secretion. CD39 and CD73 expression were suppressed using antisense oligonucleotides (ASOs), and A2aR was blocked using small molecules. Addition of ATP to T cells reduced proliferation and induced apoptosis. Intriguingly, those effects were reverted by suppression of CD39 and/or CD73 expression but not A2aR inhibition. Adenosine analogs did not suppress proliferation but inhibited secretion of proinflammatory cytokines. Here, we suggest that suppression of T cell proliferation is not directly mediated by A2aR but by intracellular downstream metabolites of adenosine, as blockade of the equilibrative nucleoside transporter (ENT) or adenosine kinase rescued proliferation and prevented induction of apoptosis. In conclusion, adenosine might primarily affect cytokine secretion directly via adenosine receptors, whereas adenosine metabolites might impair T cell proliferation and induce apoptosis. Therefore, inhibition of CD39 and/or CD73 has evident advantages over A2aR blockade to fully revert suppression of antitumor immune responses by the adenosine axis.