Optimized spliceosome-mediated RNA trans-splicing for cellular imaging and therapy

In contrast to canonical splicing, where exons of the same transcript are joined after intron excision, trans-splicing, an alternative form of splicing, combines exons from distinct transcripts and has inspired an artificial exon replacement strategy – spliceosome-mediated pre-RNA trans-splicing (SMaRT). Central to this technique is the rational design of the RNA molecule, which binds to the target transcript during splicing level and delivers exons with the desired modifications into the vicinity of the spliceosome. Trans-splicing maintains endogenous gene regulation and allows the replacement of larger or smaller gene parts, making it a promising research and therapeutic tool. Its breakthrough is burdened by its poor efficiency, but studies show that it can be increased with small optimization of the trans-splicing construct design. In this research project, we want to use trans-splicing in two areas of interest of our research group: microscopic visualization of NLRP3 inflammasome and in the correction of mutations associated with CTNNB1 syndrome. In terms of inflammasome, we intend to use trans-splicing as a new means for fluorescent labeling of endogenous inflammasome proteins. This will be achieved by designing trans-splicing constructs that will replace the terminal exons in the transcripts with exon and fluorescent protein fusions. For easier visualization, we will also gain tempo-spatial control over the complex assembly using optogenetic constructs. We will then monitor the complex assembly by 3D SIM microscopy after light stimulation of the generated stable cell lines that will express these constructs. In terms of CTNNB1 mutations, we would like to use trans-splicing as a means of replacing mutated CTNNB1 exons. To this end, we want to first optimize the expression of trans-splicing constructs. Efficacy will then be tested with various reporter systems and later using viral delivery of trans-splicing constructs into cell and mouse models of CTNNB1 syndrome. Trans-splicing-based intervention could represent the basis for new treatment strategies.