Nonsequential Pre-mRNA Splicing: From Basic Understanding to Impacts on Splice-Manipulating Therapies

The pre-mRNA splicing process is an essential aspect of gene expression and function and plays a substantial role in the complexity of higher eukaryotes. The development of antisense oligonucleotides (AOs) to harness the splicing process and manipulate it to treat various inherited and acquired diseases has been boosted by its flexibility and customisation capability. As the amount of research in this space increases, certain aspects need to be considered, in particular, how nonsequential splicing of pre-mRNA can impact AO-mediated splicing manipulation. In this chapter, we reviewed literature discussing intron removal order and several examples of disease-causing mutations impacted by this phenomenon. We also compared two strategies used to study intron removal order and the occasions that they are best suited. Finally, we discuss how nonsequential splicing could facilitate or impede the development of splice-manipulating AOs and aspects to consider when analysing AO effectiveness.