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Targeted SMN Exon Skipping: A Useful Control to Assess In Vitro and In Vivo Splice-Switching Studies.

ABSTRACT: The literature surrounding the use of antisense oligonucleotides continues to grow, with new disease and mechanistic applications constantly evolving. Furthermore, the discovery and advancement of novel chemistries continues to improve antisense delivery, stability and effectiveness. For each new application, a rational sequence design is recommended for each oligomer, as is chemistry and delivery optimization. To confirm oligomer delivery and antisense activity, a positive control AO sequence with well characterized target-specific effects is recommended. Here, we describe splice-switching antisense oligomer sequences targeting the ubiquitously expressed human and mouse SMN and Smn genes for use as control AOs for this purpose. We report two AO sequences that induce targeted skipping of SMN1/SMN2 exon 7 and two sequences targeting the Smn gene, that induce skipping of exon 5 and exon 7. These antisense sequences proved effective in inducing alternative splicing in both in vitro and in vivo models and are therefore broadly applicable as controls. Not surprisingly, we discovered a number of differences in efficiency of exon removal between the two species, further highlighting the differences in splice regulation between species.

SUBMITTER: Flynn LL

PROVIDER: S-EPMC8156830 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The molecular basis of the skipping of constitutive exons in many messenger RNAs is not fully understood. A well-studied example is exon 9 of the human cystic fibrosis transmembrane conductance regulator gene (CFTR), in which an abbreviated polypyrimidine tract between the branch point A and the 3' splice site is associated with increased exon skipping and disease. However, many exons, both in CFTR and in other genes and have short polypyrimidine tracts in their 3' splice sites, yet they are not skipped. Inspection of the 5' splice sites immediately up- and downstream of exon 9 revealed deviations from consensus sequence, so we hypothesized that this exon may be inherently vulnerable to skipping. To test this idea, we constructed a CFTR minigene and replicated exon 9 skipping associated with the length of the polypyrimidine tract upstream of exon 9. We then mutated the flanking 5' splice sites and determined the effect on exon skipping. Conversion of the upstream 5' splice site to consensus by replacing a pyrimidine at position +3 with a purine resulted in increased exon skipping. In contrast, conversion of the downstream 5' splice site to consensus by insertion of an adenine at position +4 resulted in a substantial reduction in exon 9 skipping, regardless of whether the upstream 5' splice site was consensus or not. These results suggested that the native downstream 5' splice site plays an important role in CFTR exon 9 skipping, a hypothesis that was supported by data from sheep and mouse genomes. Although CFTR exon 9 in sheep is preceded by a long polypyrimidine tract (Y(14)), it skips exon 9 in vivo and has a nonconsensus downstream 5' splice site identical to that in humans. On the other hand, CFTR exon 9 in mice is preceded by a short polypyrimidine tract (Y(5)) but is not skipped in vivo. Its downstream 5' splice site differs from that in humans by a 2-nt insertion, which, when introduced into the human CFTR minigene, abolished exon 9 skipping. Taken together, these observations place renewed emphasis on deviations at 5' splice sites in nucleotides other than the invariant GT, particularly when such changes are found in conjunction with other altered splicing sequences, such as a shortened polypyrimidine tract. Thus, careful inspection of entire 5' splice sites may identify constitutive exons that are vulnerable to skipping.

Dataset Information

Targeted SMN Exon Skipping: A Useful Control to Assess In Vitro and In Vivo Splice-Switching Studies.

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