Nucleic Acid-Based Technologies Targeting Coronaviruses

Abstract

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently creating a global health emergency. This crisis is driving a worldwide effort to develop effective vaccines, prophylactics, and therapeutics. Nucleic acid (NA)-based treatments hold great potential to combat outbreaks of coronaviruses (CoVs) due to their rapid development, high target specificity, and the capacity to increase druggability. Here, we review key anti-CoV NA-based technologies, including antisense oligonucleotides (ASOs), siRNAs, RNA-targeting clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas), and mRNA vaccines, and discuss improved delivery methods and combination therapies with other antiviral drugs.

Keywords: RNA-targeting CRISPR-Cas; antisense oligonucleotide (ASO); coronavirus (CoV); lipid-ASO nanomicelle; mRNA vaccines; siRNA.

Figure 1

Key Nucleic Acid (NA)-Based Technologies Targeting Coronavirus (CoV). Several key NA-based strategies against CoV, including antisense oligonucleotides (ASOs), siRNA, RNA-targeting clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas) systems, and mRNA vaccines were discussed in this review. In particular, ASOs, siRNAs, and CRISPR-Cas technologies can be used as anti-CoV therapeutics through sequence-specific targeting of the RNA viral genome or host cellular genes involved in crucial viral activities (also see Figure 2, Figure 3, Figure 4). Once mRNA vaccines are delivered into the host cells, the mRNAs can be translated into viral proteins that subsequently trigger immune responses (also see Figure 5). Abbreviation: RISC, RNA-induced silencing complex.

Figure 2

Antisense Oligonucleotide (ASO) Mechanism of Action and Strategies to Combat Coronaviruses. Association of ASOs with carriers enhances their cellular delivery (1). Alternatively, lipid-modified ASOs (LASO) can self-assemble into nanomicelles (2), which have the ability to encapsulate hydrophobic drugs such as antiviral small molecules (3) and enter the cells unaided (4). Once delivered into the cells, ASOs, LASOs, and drugs can be released (5). ASOs/LASOs base-pair with the target RNA sequences, which could be host cellular mRNA or a viral RNA genome (6). The formed DNA:RNA hybrids can induce cleavage of the RNA in the heteroduplexes (through RNase H recruitment and activity), leading to degradation of the targeted sequences (7). Binding of ASOs/LASOs to the target RNA sequences also can form a steric hindrance blocking translation or modulating alternative splicing, thereby shutting down the gene expression of host genes or disrupting RNA-based viral functions such as translation, replication, and transcription (8). In addition, the conjugated drugs can exert their activities via inhibition of viral processes such as transcription/replication, host entry, and virus assembly and budding (9). The figure was modified from Robson et al. [20], with permission, and created with BioRender.

Figure 3

Mechanism of siRNA Technology and Strategies to Downregulate Coronaviruses. siRNA molecules are associated with carriers that facilitate their delivery into the cells (1). The siRNAs are recognized and loaded into the RNA-induced silencing complexes (RISC), which subsequently separate two strands of associated siRNAs and release the sense strands (3, 4). The RISC-associated antisense strand directs the complex to the target matching RNA sequences, which could be viral RNAs or host cellular transcripts (5), leading to RNA degradation catalyzed by RISC enzyme (6). As a consequence, these siRNAs can downregulate the expression of target host/viral genes crucial for viral activities and interrupt viral replication/transcription. Created with BioRender.

Figure 4

RNA-Targeting Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Systems for Coronavirus Inhibition. The adeno-associated virus (AAV) serves as a carrier to deliver vector constructs consisting of both Cas13d effector and guide RNAs (gRNAs) (1). Once inside the cell, Cas13d protein and gRNA are expressed and Cas13d protein forms a complex with gRNA (2). The spacer sequence acts as a guide to the Cas13d effector by matching to the complementary sequences in the viral RNA genome (3), thus allowing the associated Cas13d effector to cleave the viral RNA (4), therefore disrupting viral functions. Created with BioRender.

Figure 5

Basic Mechanism of Action of mRNA Vaccines. The naked mRNAs are encapsulated with a carrier such as a lipid nanoparticle (LNP), which enhances cellular delivery and stability of mRNAs. The LNP-mRNA vaccines are subsequently delivered into the body by intramuscular injection. Once LNP-mRNAs are present in the host cells, the mRNAs are released and translated by the host protein synthesis machinery. The proteasomal degradation of the generated proteins produces peptides that are subsequently associated with MHC class I molecules and presented on the surface of host antigen-presenting cells. The peptide-MHC I complexes are recognized by the CD8+ T cells, stimulating cellular immune responses. Created with BioRender. Abbreviations: MHC, major histocompatibility complex; ORF, open reading frame; UTR, untranslated region.