Therapeutic applications of trans-splicing

Abstract

Background: RNA trans-splicing joins exons from different pre-mRNA transcripts to generate a chimeric product. Trans-splicing can also occur at the protein level, with split inteins mediating the ligation of separate gene products to generate a mature protein.

Sources of data: Comprehensive literature search of published research papers and reviews using Pubmed.

Areas of agreement: Trans-splicing techniques have been used to target a wide range of diseases in both in vitro and in vivo models, resulting in RNA, protein and functional correction.

Areas of controversy: Off-target effects can lead to therapeutically undesirable consequences. In vivo efficacy is typically low, and delivery issues remain a challenge.

Growing points: Trans-splicing provides a promising avenue for developing novel therapeutic approaches. However, much more research needs to be done before developing towards preclinical studies.

Areas timely for developing research: Increasing trans-splicing efficacy and specificity by rational design, screening and competitive inhibition of endogenous cis-splicing.

Keywords: trans-splicing; cancer; gene therapy; genetic disease; infectious disease; ribozyme-mediated trans-splicing; spliceosome-mediated RNA trans-splicing (SMaRT); split intein-mediated trans-splicing.

Fig. 1

Schematic depiction of different types of pre-mRNA splicing. (a) Cis-splicing. Exons from a single pre-mRNA molecule are ligated together to generate a mature linear transcript. (b) Intergenic trans-splicing. Exons from different genes are joined to produce a chimeric molecule. (c) Intragenic trans-splicing. Exons from different pre-mRNAs of the same gene are spliced together, resulting in exon duplication and sense-antisense fusion. (d) SL trans-splicing. SL exon is spliced onto multiple genes of a polycistronic pre-mRNA, resulting in a number of mature transcripts containing a common 5′ sequence.

Fig. 2

Schematic depiction of different types of exon replacement. * indicates a mutation within the targeted exon.

Fig. 3

Schematic depiction of protein trans-splicing mediated by split inteins. The DNA sequence coding for the desired product is split into two pieces, and the N-intein and C-intein coding sequences added to each gene fragment. The two genetic constructs are co-delivered to target cells and undergo transcription and translation to produce two precursor polypeptides. The N-intein and C-intein self-assemble, excise themselves out of the polypeptide, and ligate the flanking regions together to generate the mature full-length protein.

Fig. 4

Mechanism of action of the HSV-tk/GCV suicide system. HSV-tk is delivered to and specifically expressed in target cells, followed by application of the inactive prodrug ganciclovir (GCV). HSV-tk and other endogenous enzymes phosphorylate GCV to produce the activated drug, which induces DNA replication chain termination and consequently cell death.