Abstract：
The ABCA4 gene, involved in Stargardt disease, has a high percentage of splice-altering pathogenic variants, some of which cause complex RNA defects. Although antisense oligonucleotides (AONs) have shown promising results in splicing modulation, they have not yet been used to target complex splicing defects. Here, we performed AON-based rescue studies on ABCA4 complex splicing defects. Intron 13 variants c.1938-724A>G, c.1938-621G>A, c.1938-619A>G, and c.1938-514A>G all lead to the inclusion of different pseudo-exons (PEs) with and without an upstream PE (PE1). Intron 44 variant c.6148-84A>T results in multiple PE inclusions and/or exon skipping events. Five novel AONs were designed to target these defects. AON efficacy was assessed by in vitro splice assays using midigenes containing the variants of interest. All screened complex splicing defects were effectively rescued by the AONs. Although varying levels of efficacy were observed between AONs targeting the same PEs, for all variants at least one AON restored splicing to levels comparable or better than wildtype. In conclusion, AONs are a promising approach to target complex splicing defects in ABCA4.
摘要：
ABCA4基因,与Stargardt病有关,有很高比例的剪接改变致病变异，其中一些会导致复杂的RNA缺陷。尽管反义寡核苷酸（AON）在剪接调节中显示出有希望的结果，它们尚未用于靶向复杂的剪接缺陷。这里,我们对ABCA4复合剪接缺陷进行了基于AON的挽救研究.Intron13变体c.1938-724A>G，c.1938-621G>A，c.1938-619A>G，和c.1938-514A>G都导致包含有和没有上游PE（PE1）的不同伪外显子（PE）。内含子44变体c.6148-84A>T导致多个PE包涵体和/或外显子跳跃事件。设计了五个新的AON来针对这些缺陷。通过使用含有目的变体的midigenes的体外剪接测定来评估AON功效。所有筛选的复杂剪接缺陷均被AON有效挽救。尽管在靶向相同PE的AON之间观察到不同水平的功效，对于所有变体,至少一个AON恢复剪接至与野生型相当或更好的水平。总之,AON是针对ABCA4中复杂剪接缺陷的一种有前途的方法。
