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.

Antisense oligonucleotide-based treatment of retinitis pigmentosa caused by USH2A exon 13 mutations

“…The length of USH2A exon 13 is well conserved between human (642 nucleotides) and zebrafish (648 nucleotides), and the (spacing between) cysteine residues that are essential for EGF-lam domain formation are identical ( Figure 2 A). Following the previously published guidelines for AON design, 19 , 20 six antisense phosphorodiamidate morpholino oligomers (PMOs) were designed to target the zebrafish ush2a exon 13 splice acceptor site, splice donor site, or exonic splice enhancer (ESE) motifs ( Figure S1 A; Table 1 ). The exon-skipping potential of the PMOs was first investigated by injecting the individual PMOs into the yolk of 1- to 2-cell-stage ush2a rmc1 embryos ( Figure S1 B).…”

Antisense Oligonucleotide Screening to Optimize the Rescue of the Splicing Defect Caused by the Recurrent Deep-Intronic ABCA4 Variant c.4539+2001G>A in Stargardt Disease

“…Previously, we showed that the variants c.4539+2001G>A and c.4539+2028C>T cause the insertion of a 345-nt pseudoexon in a retina-specific manner. Four AONs (AON1 to AON4) targeting this region were designed according to previously described guidelines [35,36,39] and were assessed in PPCs. Our results showed that two AONs (AON1 and AON4) were able to restore correct ABCA4 splicing by skipping the pseudoexon in a mutation-dependent manner.…”

“…When analyzing the groups, we found correlations with the Tm and the GC content. Previously published guidelines [35,36,39] have indicated that Tm should be above 48 °C and the GC content between 40% and 60%. Based on our analyses, it seems that if the temperature is higher than 51 °C and the GC content close to 60%, the chances of designing an AON with a good efficacy are higher.…”

Whole genome sequencing for USH2A-associated disease reveals several pathogenic deep-intronic variants that are amenable to splice correction

“…AONs were designed according to previously published guidelines 26 , 27 and purchased from Eurogentec (Seraing, Belgium) with 2′-O-MOE modifications and a complete phosphorothiorate backbone. For each PE, at least two AONs were designed complementary to splice acceptor or donor sites, or exonic splice enhancer regions.…”