RNA therapeutics are expected to expand the range of druggable targets from proteins to RNAs and DNAs. Cell surface, extracellular, and intracellular proteins remain favorable targets for the development of small-molecule and protein (e.g., antibody) therapeutics, as well as RNA aptamer drugs (1). Actually, the majority of human genome sequences transcribed as functional ncRNAs largely outnumbered mRNAs to be translated into proteins. Both mRNAs and ncRNAs can be directly targeted by RNA drugs such as ASO/asRNAs, miRNAs, and siRNAs (2). Once introduced into cells, mRNA therapeutics (3) may be developed for protein replacement therapy or vaccination. In addition, gRNAs (4) could be used along with other elements to directly edit the target gene sequences for the treatment of particular diseases.

The tRNA/pre-miRNA–based RNA bioengineering technology for the production of biological RNAi agents (BERA) for research and development. After the design of a target BERA/sRNA (e.g., miRNA, siRNA, and asRNA, etc.), the corresponding coding sequence is cloned into a vector. Expression of target BERA in bacterial culture is readily verified through RNA gel electrophoresis, and BERA can be purified to a high degree of homogeneity using different methods [e.g., anion exchange fast protein liquid chromatography (FPLC)]. Purity of isolated BERA is determined by HPLC analysis and endotoxin pyrogen testing. These bioengineered RNA molecules should better recapitulate the properties of natural RNAs to exert biological or pharmacological actions, as both are produced and folded in living cells. Indeed, BERA/sRNAs can be selectively processed to warhead sRNAs for the modulation of target gene expression in human cells and control of diseases in animal models. WT, wild type.

Common approaches for the formulation and delivery of RNA therapeutics. RNA drugs administered intravenously need to be protected against excessive degradation by serum RNases and overcome the cell membrane barriers to gain access to intracellular targets. After entering cells through endocytosis or other mechanisms, RNA therapeutics are released in the cytoplasm, translocated, and incorporated into corresponding ribonucleoprotein complexes to silence target transcripts. As chemical modifications largely improve the metabolic stability and PK properties, RNA drug may be encapsulated in nanoparticle using different systems such as LNP and LPP. In addition, conjugation of a trivalent GalNAc to the RNA drug or use of antibody can improve the efficiency of delivery to targeted cells.

Chemical structures of some small-molecule antibiotics that are known to target bacterial ribosomal RNAs to inhibit protein synthesis for the treatment of infections. Among them, aminoglycosides (e.g., streptomycin, neomycin, and paromomycin) and tetracyclines (e.g., tetracycline and tigecycline) directly act on 16S rRNAs, whereas natural and semisynthetic macrolides (e.g., erythromycin, spiramycin, and telithromycin) as well as synthetic oxazolidinones (e.g., linezolid and eperezolid) bind to 23S rRNAs to exert antimicrobial activities.

Interactions of representative small-molecule antibiotics with specific nucleotide residues of individual rRNAs within ribosomal subunits. (A) Paromomycin binds to 16S rRNA in the decoding region A-site on the 30S subunit. Specifically, ring I stacks against the G1491 and interacts with A1408 through hydrogen bonding. Ring I also penetrates into the RNA helix and facilitates the flipping out of A1493. Ring II forms tight interactions with both nucleobases and the backbone of the RNA, whereas ring III just makes weak contacts. (B) Linezolid binds to 23S rRNA in the A-site cleft near PTC of the 50S subunit to interfere with tRNA accommodation. In particular, the O4 of ring-C interacts with U2585 via hydrogen bonding. Although G2576 stacks directly onto G2505, other residues clustered around U2504 form the binding pocket linezolid; among them, C2452 interacts with U2504 through the indicated hydrogen bonds. (C) Telithromycin binds to 23S rRNA in the NPET of the 50S subunit through its 3-keto group and hydrophobic interactions.

Chemical structures of some small-molecule compounds under clinical or preclinical studies that act on various types of RNAs for the treatment of different diseases. (A) Ataluren targets rRNA to promote insertion of near-cognate tRNAs at the site of the dystrophin gene toward nonsense suppression for the treatment of Duchenne muscular dystrophy. (B) Netilmicin and anthraquinone derivatives targeting HIV TAR RNA motif; benzimidazole derivative Isis-11 and diaminopiperidine analog acting on HIV IRES; MTDB as an inhibitor of SAR-CoV RNA pseudoknot; and DPQ identified to attenuate influenza A promoter RNA for the control of virus infections. (C) Synthetic ribocils and natural roseoflavin mimic FMN riboswitch ligand to modulate target gene expression to exert antimicrobial activities. (D) Risdiplam and branaplam, interacting with SMN2 pre-mRNAs to switch splicing and thus elevate the expression of functional SMN protein for the treatment of spinal muscular atrophy amenable to SMN deficiency. (E) Targarprimir-96 and targarpremir-210 that block the biogenesis of oncogenic miRNAs through direct binding to pri-miR-96 and pre-miR-210, respectively, to elicit antitumoral activities.