Abstract
Alternative pre-mRNA splicing can be cell-type specific and results in the generation of different protein isoforms from a single gene. Deregulation of canonical pre-mRNA splicing by disease-associated variants can result in genetic disorders. Antisense oligonucleotides (AONs) offer an attractive solution to modulate endogenous gene expression through alteration of pre-mRNA splicing events. Relevant in vitro models are crucial for appropriate evaluation of splicing modifying drugs. In this chapter, we describe how to investigate the splicing modulating activity of AONs in an in vitro skeletal muscle model, applied to Pompe disease. We also provide a detailed description of methods to visualize and analyze gene expression in differentiated skeletal muscle cells for the analysis of muscle differentiation and splicing outcome. The methodology described here is relevant to develop treatment options using AONs for other genetic muscle diseases as well, including Duchenne muscular dystrophy, myotonic dystrophy, and facioscapulohumeral muscular dystrophy.
Highlights
Pre-mRNA splicing is a highly conserved process in eukaryotes that plays a role in pre-mRNA processing
Genetic variations can lead to defects in pre-mRNA splicing that cause human disease [3]
Modulation of pre-mRNA splicing can be directed to correct aberrant splicing, to skip protein coding variants, to restore the reading frame, or to prevent expression of toxic gene products. This is possible by targeting antisense oligonucleotides (AONs) toward canonical splice sites or to cis-acting regulatory elements such as cryptic splice sites or splicing silencers/enhancers [4]
Summary
Pre-mRNA splicing is a highly conserved process in eukaryotes that plays a role in pre-mRNA processing. Modulation of pre-mRNA splicing can be directed to correct aberrant splicing, to skip protein coding variants, to restore the reading frame, or to prevent expression of toxic gene products. This is possible by targeting antisense oligonucleotides (AONs) toward canonical splice sites or to cis-acting regulatory elements such as cryptic splice sites or splicing silencers/enhancers [4]. These methods can be used to analyze splicing correction in vitro to develop RNA-based therapies for muscle disorders We have used this strategy to test AONs for Pompe disease [12] and describe the methodology here in detail
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
