RNA exon editing: Splicing the way to treat human diseases

Abstract

RNA exon editing is a therapeutic strategy for correcting disease-causing mutations by inducing trans-splicing between a synthetic RNA molecule and an endogenous pre-mRNA target, resulting in functionally restored mRNA and protein. This approach enables the replacement of exons at the kilobase scale, addresses multiple mutations with a single therapy, and maintains native gene expression without changes to DNA. For genes larger than 5 kb, RNA exon editors can be delivered in a single vector despite AAV capacity limitations because only mutated exons need to be replaced. While correcting mutations by trans-splicing has been previously demonstrated, prior attempts were hampered by low efficiency or lack of translation in preclinical models. Advances in synthetic biology, next-generation sequencing, and bioinformatics, with a deeper understanding of mechanisms controlling RNA splicing, have triggered a re-emergence of trans-splicing and the development of new RNA exon editing molecules for treating human disease, including the first application in a clinical trial (this study was registered at ClinicalTrials.gov [NCT06467344]). Here, we provide an overview of RNA splicing, the history of trans-splicing, previously reported therapeutic applications, and how modern advances are enabling the discovery of RNA exon editing molecules for genetic targets unable to be addressed by conventional gene therapy and gene editing approaches.

Keywords: MT: RNA and epigenetic editing Special Issue; RNA exon editing; gene therapy; rare diseases; spliceosome; trans-splicing.

Graphical abstract

Figure 1

Spliceosome-mediated RNA trans-splicing Simple illustration depicting trans-splicing between two pre-mRNA transcripts mediated by the spliceosome.

Figure 2

Mechanism of cis- and trans-splicing (A) RNA cis-splicing involves the splicing and removal of introns to generate a mRNA transcript, involving two steps of transesterification reactions to form a lariat intermediate and the ligated exon product. The exons are indicated as gray ovals, and the intron is depicted as a gray line. (B) 5′ trans-splicing involves a 5′ exon editor that contains 5′ replacement CDS (teal ovals) and a binding domain that base-pairs with the endogenous intron of the target pre-mRNA (gray). Transesterification reaction between the branchpoint nucleotide in the pre-mRNA and the 5′ splice site in the exon editor, followed by a second transesterification reaction generates the Y-branch intermediate and a ligated exon product that is a fusion of the exon editor CDS with native exon. (C) 3′ trans-splicing involves a 3′ exon editor that contains a binding domain that base-pairs with the endogenous intron of the target pre-mRNA and 3′ replacement CDS (teal ovals). Transesterification between the branchpoint nucleotide in the exon editor and the 5′ splice site in the upstream native exon, followed by a second transesterification reaction generates the Y-branch intermediate and a ligated exon product that is a fusion of the native exon with the exon editor CDS.