Antisense Oligonucleotide Design and Evaluation of Splice-Modulating Properties Using Cell-Based Assays

Abstract

Antisense oligonucleotide (AON)-based splice modulation has been proven to hold great promise as a therapeutic strategy for a number of hereditary conditions. AONs are small modified single-stranded RNA or DNA molecules that are complementary to splice enhancer or silencer target sites. Upon pre-mRNA binding, AONs will prevent or stimulate binding of the spliceosome thereby modulating splicing events. AONs can be designed and applied for different genes and genetic disorders as the specificity depends on their nucleotide sequence. Here we provide a guideline for setting up AON-based splice-modulation experiments by describing a detailed protocol to design and evaluate AONs using a combination of in silico and in vitro analyses.

Keywords: Antisense oligonucleotides; Genetic therapy; Minigene splice assay; Pre-mRNA; Splice modulation; Splicing.