A bifunctional aptamer-siRNA chimera targeting ACE2 for the inhibition of SARS-CoV-2 S pseudovirus entry and replication

Abstract

The relentless evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the emergence of immune-evasive variants underscore an urgent need for novel therapeutic strategies that are resilient to viral mutations. Targeting conserved host factors essential for viral entry represents a promising approach to overcome this challenge. Here, we report the development of a bifunctional therapeutic platform targeting the primary human receptor for SARS-CoV-2, angiotensin-converting enzyme 2 (ACE2). Using systematic evolution of ligands by exponential enrichment (SELEX), we isolate a high-affinity DNA aptamer, designated AA2, that binds to human ACE2 with a dissociation constant (Kd) of 5.41 ± 1.23 nM. Molecular docking and competitive binding assays confirm that AA2 sterically hinders the interaction between the viral spike receptor-binding domain (RBD) and ACE2. Consequently, AA2 demonstrates potent neutralization of SARS-CoV-2 S pseudovirus entry into host cells. To achieve a synergistic antiviral effect, we engineer an aptamer-siRNA chimera (AsiC) by conjugating AA2 to a short interfering RNA (siRNA) targeting the GFP coding region of the pseudovirus genome. This AsiC construct significantly represses viral replication compared to aptamer or siRNA treatment alone, validating a dual mechanism of action that combines receptor blockade with targeted gene silencing. This study establishes a robust proof-of-concept for an ACE2-targeted AsiC, representing a new class of dual-function antiviral therapeutics with the potential to effectively combat current and future ACE2-dependent coronaviruses.

1. Introduction

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has persisted as a formidable public health crisis, driven by the virus's remarkable capacity for genetic mutation [1]. This high mutability has led to the sequential emergence of variants of concern (VOCs), such as Omicron, whose constellations of mutations in the spike (S) protein's receptor-binding domain (RBD) confer enhanced transmissibility and immune evasion [2]. Consequently, the efficacy of therapies targeting viral components is consistently undermined, and the utility of most monoclonal antibodies is severely compromised [3–5]. While direct-acting small-molecule antivirals, such as remdesivir and paxlovid, remain important tools, their efficacy is constrained by limited therapeutic windows and the potential for viral resistance [6,7]. Epidemiological evidence indicates that 20% of COVID-19 cases progress to severe disease requiring hospitalization, with 5%‒10% developing acute respiratory distress syndrome (ARDS), creating persistent burdens on global healthcare systems [8,9]. This reality frames the central challenge as a perpetual race against an evolving viral target, highlighting the critical need for broad-spectrum interventions.

This evolutionary pressure necessitates a strategic shift from virus-centric therapies to host-directed antivirals (HDAs), which target conserved host factors that are indispensable for the viral life cycle [10]. Human angiotensin-converting enzyme 2 (ACE2), which serves as the critical cellular receptor for coronaviruses such as SARS-CoV and SARS-CoV-2 [11,12], is a quintessential HDA target for coronaviruses. Its interaction with the viral S-protein not only facilitates viral entry [11,13] but also induces a critical conformational change in the S-protein to an early fusion intermediate state that is essential for subsequent membrane fusion [14]. Capitalizing on this mechanism, researchers have developed a novel broad-spectrum anti-coronavirus agent that simultaneously blocks viral entry and membrane fusion by inhibiting ACE2-mediated conformational changes [14].