The comparison of traditional and RNA therapeutics will be covered in this passage. To facilitate understanding of current trend in RNA therapeutic development, we will discuss the underlying mechanisms and varieties of RNA-based drugs on the market or in clinical stages. The future directions and potential clinical applications of RNA therapies will also be covered.

Most drugs currently on the market are small molecules and proteins/antibodies. These traditional drugs mainly act on the corresponding protein targets, inhibit or alter the pathological processes caused by these targets and elicit pharmacological effects for the treatment of human diseases. These protein targets are usually enzymes, receptors, ion channels, transporters, and kinases. However, only about 22% of proteins are “druggable” targets with active binding sites suitable for small molecules. The size and stability issues plus the complicated synthetic process limit the applications of protein-based therapeutics.

In contrast to conventional drugs, RNA based therapeutics can target not only proteins, but also transcripts and genes that are undruggable for small molecule or protein drugs. In addition to a broader range of targets, RNA drugs exhibit other advantageous features (Table1). Although native RNA is unstable, the modified RNA drugs during synthesis, which are subsequently encapsulated in delivery vehicles, are stable after administration as an injection. For example, Inclisiran, a synthetic siRNA targeting PCSK9 mRNA, provides durable effect over 6 months after just a single injection. The rapid and cost-effective development cycle, as evidenced by the development and approval of COVID-19 mRNA vaccines, are significantly different from the tremendous screening process and ADMET studies required for small molecule and protein drug development. Manufacturing and preparation process of RNA drugs are relatively simple and fast. All these advantages make RNA drugs ideal candidates for the development of novel RNA therapeutics.

Based on their structural characteristics and mechanisms of action involved RNA molecules, RNA therapeutics can be divided into two categories:
1) mRNAs that are translated into proteins;
2) non-coding RNAs that regulate gene transcription in the cell.
These non-coding RNA therapeutics consist of siRNA, microRNA (miRNA), ASO, aptamer, ribozyme, guide RNA (gRNA), etc. (Table 2). These RNA drugs exhibit different structural features and mechanisms of action.

ASO, siRNA and miRNA drugs bind to mRNA or pre-mRNA through the principle of complementary base pairing, thereby downregulating expression of related proteins by silencing target gene (Figure 2). mRNA molecules encoding certain peptides or proteins enter cells into the cytoplasm and induce their expression for protein replacement therapy or vaccination. Aptamers are short single-stranded oligonucleotides that bind to their targets through their unique tertiary structure rather than their sequence. The different structures of RNA aptamers enable them to adapt to different types of targets.

RNA therapeutics have changed the landscape of drug development. The broader spectrum of drug targets, simplicity and efficiency in development and manufacturing, and other advantages will lead to the development and approvals of more RNA therapeutics.

To date, the main indications for FDA-approved RNA drugs are rare diseases. For example, Nusinersen was the first drug to treat spinal muscular atrophy (AMD). Nusinersen is an ASO that increases levels of the full-length survival motor neuron (SMN) by modulating pre-mRNA splicing of SMN2 gene. The siRNA drug Givosiran was approved for the treatment of acute intermittent porphyria, followed by another siRNA drug, Lumasiran, for the treatment of primary hyperoxaluria type 1 (PH1). Viltolarsen, an ASO drug, was approved to treat Duchenne muscular dystrophy (DMD).

Other RNA drugs for rare diseases are detailed in our previous article (link). It is expected that this indication list will gradually expand to more diseases including tumors, neurological diseases, metabolic diseases, and other diseases in the future ( Table3 )

Despite the advantages of RNA therapeutics, several hurdles remain in delivering the RNA drugs to the site of therapeutic action and across the hydrophobic cell membrane into cytoplasm. But RNA is negatively charged large molecules. For example, molecular weight of single stranded ASOs is about 4~10 kDa while the molecular weight of double stranded siRNAs is about 14 kDa. In addition, naked RNAs can be rapidly degraded by nuclease in blood and activate recognition by some immune systems such as TLR3/7/8.

To overcome the barriers to efficient RNA delivery, viral and non-viral vectors (liposomes) have been developed to protect oligonucleotides from degradation and maximize delivery efficiency to the target cells. Lipid nanoparticle (LNP) are typically composed of cationic lipids, cholesterol, PEG-lipids, and phospholipids, which can mask the negative charges of RNA and avoid nuclease degradation. Chemical modifications can also improve the efficiency of RNA delivery. The incorporation of 2' chemical modifications (2'-F, 2' -OME and 2'-MOE, etc.) significantly improve RNA stability and overall half-life.

The unique higher-order structures folded from RNA primary sequence can interact with small molecules or proteins. These structures include secondary (e.g., helices, stems, loops, and bulges), tertiary (e.g., junctions, pseudoknot, and motif), and quaternary complexes. Several RNA-targeted small molecules have been approved, including Telithromycin that binds to ribosomal RNA (rRNA), ribosome-targeting Ataluren for the treatment of DMD, and Risdiplam targeting pre-mRNA of SMN2 for the treatment of spinal muscular atrophy.

Notably, RNA is more structurally related to DNA. Many originally designed and identified RNA-targeting small molecules have later been found to bind to DNA as well. Therefore, structure modification and optimization are necessary to improve selectivity for RNA to development of RNA-targeting small molecules.