From Antisense RNA to RNA Modification: Therapeutic Potential of RNA-Based Technologies

Abstract

Therapeutic oligonucleotides interact with a target RNA via Watson-Crick complementarity, affecting RNA-processing reactions such as mRNA degradation, pre-mRNA splicing, or mRNA translation. Since they were proposed decades ago, several have been approved for clinical use to correct genetic mutations. Three types of mechanisms of action (MoA) have emerged: RNase H-dependent degradation of mRNA directed by short chimeric antisense oligonucleotides (gapmers), correction of splicing defects via splice-modulation oligonucleotides, and interference of gene expression via short interfering RNAs (siRNAs). These antisense-based mechanisms can tackle several genetic disorders in a gene-specific manner, primarily by gene downregulation (gapmers and siRNAs) or splicing defects correction (exon-skipping oligos). Still, the challenge remains for the repair at the single-nucleotide level. The emerging field of epitranscriptomics and RNA modifications shows the enormous possibilities for recoding the transcriptome and repairing genetic mutations with high specificity while harnessing endogenously expressed RNA processing machinery. Some of these techniques have been proposed as alternatives to CRISPR-based technologies, where the exogenous gene-editing machinery needs to be delivered and expressed in the human cells to generate permanent (DNA) changes with unknown consequences. Here, we review the current FDA-approved antisense MoA (emphasizing some enabling technologies that contributed to their success) and three novel modalities based on post-transcriptional RNA modifications with therapeutic potential, including ADAR (Adenosine deaminases acting on RNA)-mediated RNA editing, targeted pseudouridylation, and 2′-O-methylation.

Drug delivery systems for RNA therapeutics

“…First, its small number of nucleotides enables scientists to use solid-phase synthesis to manufacture siRNA with site-specific chemical modifications, usually in the phosphate backbone and sugar rings. A whole suite of chemistries has been developed for a variety of ribose modifications, such as 2′-O-methyl (2′-OMe), 2′-methoxyethyl (2′-MOE), 2′-fluor (2′-F), locked nucleic acid oligonucleotides, constrained ethyl oligonucleotides (cEt), and tricyclo-DNA oligonucleotides (tc-DNA) 38 . Complementing these modifications are phosphate backbone modifications including phosphorothioates, phosphorodiamidate morpholino oligonucleotides (PMO), peptide nucleic acid oligonucleotides (PNA), and nucleobase modifications, such as 5-methylcytosine (m5C).…”

The Critical Contribution of Pseudouridine to mRNA COVID-19 Vaccines

“…Ψ is derived from uridine via a base-specific isomerization reaction called pseudouridylation ( Figure 1 ), in which the nucleobase rotates 180° around the N3-C6 axis, resulting in the change of nucleobase-sugar bond (from N1-C1′ bond to C5-C1′ bond). The resulting C-C bond allows the nucleobase to rotate more freely ( Adachi et al, 2021 ). In addition, Ψ can provide an extra hydrogen bond donor (in the N1H) in the major groove while keeping the hydrogen bond donor and acceptor (same as in its original uridine) in the Watson-Crick face.…”

“…Recently, new generations of LNPs were developed and used to deliver patisiran®, an RNAi-based drug approved in 2018, which generated optimism for RNA therapeutics delivery ( Hoy, 2018 ). Indeed, with the approval of patisiran®, there was a mounting belief that LNPs could become enabling technologies for multiple RNA modalities ( Adachi et al, 2021 ). This was a major accomplishment and a scientific breakthrough, and, in fact, current mRNA vaccines are delivered with LNPs that are prepared by mixing four lipids in the presence of ethanol in very specific conditions ( Jeffs et al, 2005 ; Buschmann et al, 2021 ).…”

Gene Therapy in Hemophilia: Recent Advances

“…Gene therapy treatments based on siRNA (small interference RNA) are also being developed. These therapies are intended to modify the translation of messenger RNA, inhibiting or modifying production of the protein without affecting the encoding gene [ 29 ].…”