RNA-based therapeutics: an overview and prospectus

Abstract

The growing understanding of RNA functions and their crucial roles in diseases promotes the application of various RNAs to selectively function on hitherto "undruggable" proteins, transcripts and genes, thus potentially broadening the therapeutic targets. Several RNA-based medications have been approved for clinical use, while others are still under investigation or preclinical trials. Various techniques have been explored to promote RNA intracellular trafficking and metabolic stability, despite significant challenges in developing RNA-based therapeutics. In this review, the mechanisms of action, challenges, solutions, and clinical application of RNA-based therapeutics have been comprehensively summarized.

Fig. 1. An overview of major developments in the RNA-targeting field.

A timeline of RNA-based technological advances, drug approvals and other important events are highlighted. Rapid development in the RNA-targeting field has been applied to treat rare and common diseases. ASOs antisense oligonucleotides, RNAi RNA interference, RISC RNA-induced silencing complex, siRNA small interfering RNA, saRNA small activating RNAs, ssRNA single-stranded RNA.

Fig. 2. Types of RNA-based therapeutics and modes of action.

A Antisense oligonucleotides (ASOs) can modulate the target gene expression through two mechanisms. (①) In the occupancy-mediated degradation way, ASOs trigger the target mRNA cleavage by RNase H1 or ribozymes. The Occupancy-only mechanisms do not directly degrade target RNA. Instead, it regulates the gene expression in several ways: (②) alter RNA splicing using splice-switching ASOs to induce exon skipping or exon inclusion; (③) lead to nonsense-mediated mRNA decay (NMD); (④) inhibit or activate translation; (⑤) block the microRNAs binding to target mRNA. B RNA interference (RNAi). Long double-stranded RNA (dsRNA) and precursor microRNA (pre-miRNA) are processed by Dicer into short interfering RNA (siRNA). The antisense strand (indicated as a blue strand) of siRNA is loaded into the RNA-induced silencing complex (RISC) for RNA targeting, degradation or translation repression. C CRISPR/Cas-based RNA editing system includes two Cas nuclease categories, Cas9 and Cas13. A guide RNA(gRNA) binds to Cas9 to cleave ssRNA with (①) or without (②) the help of a protospacer-adjacent motif (PAM). (③) A single CRISPR RNA (crRNA) guides Cas13 to target specific RNA having a protospacer flanking sequence (PFS). (④) In addition to knockdown target RNA, a catalytically deactivated Cas13b (dCas9b) facilitates the A-to-I editing with ADAR. D RNA aptamers function as agonizts or delivery agents. (①) As an antagonist aptamer, Pegaptanib interacts explicitly with vascular endothelial growth factor (VEGF) to inhibit the interaction of VEGF with its receptors, thus treating macular degeneration. (②) Cell type-specific RNA aptamers deliver agents (miRNA, siRNA, shRNA, antibody and chemotherapy drugs) by linking to or conjugating. E mRNA vaccine. (①) The mRNA vaccine against SARS-CoV-2 (mRNA-1273) is delivered into antigen-presenting cells by lipid nanoparticle (LNP). (②)The mRNA encoding SARS-CoV-2 spike protein is released into the cytoplasm and translated to antigen protein by the ribosome. (③) Some antigen proteins are degraded into small peptides by the proteasome and presented to the surface of CD8+ T cells by major histocompatibility complex I (MHCI). The CD8+ cytotoxic T-cell-mediated immunity kills infected cells by secreting perforin or granzyme. (④) Other antigen proteins are degraded in the lysosome and displayed on the surface of T helper cells by MHC II. The B-cell/antibody-mediated humoral immunity uses antibodies to neutralize pathogens.

Fig. 3. Overcoming challenges in the development of RNA therapeutics.

A Common chemical modifications. RNA-based drugs often have various chemical modifications, including 5′-and 3′-end conjugates, 2′-sugar substitution and internucleoside linkage modifications. B Nanocarriers delivery strategies. Five representative nanocarriers are shown: (①) Lipid nanoparticles encapsulating nucleic acids. (②) Cationic polymers electrostatically bind to negatively-charged nucleic acids to form polyplexes. (③) Engineered exosomes with aptamers or therapeutic RNAs on the outer surface. (④) Spherical nucleic acid nanoparticle consisting of an inorganic core coated in densely packed oligonucleotides attached by chemical linkages. (⑤) Self-assembled DNA cage tetrahedron nanostructure. Oligonucleotide drugs can be incorporated into the design of the DNA cage itself. Additional targeting ligands and polyethylene glycol (PEG) can be further conjugated to the nanostructure. These nanocarriers can deliver RNA molecules through binding to the cell membrane, endocytosis, endosome escape and RNAs are released in the cytoplasm and translation to proteins or incorporated into corresponding ribonucleoprotein complexes to silence target transcripts.