ABCA4是Stargardt疾病（STGD1）中的一个基因，在其中包含50个外显子，其中17个外显子具有三个核苷酸的倍数。目前尚未确定在框内外显子剪接跳跃的影响。已经在Usher综合征相关基因中研究过反义寡核苷酸（AONs），以诱导携带严重变异的框内外显子的跳跃，并减轻其与疾病相关的效应。在鉴定一位携带新的外显子17典型剪接位点变异的STGD1患者后，检查了缺少由外显子17编码的22个氨基酸的ABCA4的活性，并设计了能够诱导外显子17跳跃的AONs。 一位STGD1患者是复合杂合子，具有剪接变异c.2653+1G>A，该变异被预测会导致外显子17的框内跳跃，以及零突变[c.735T>G，p.(Tyr245*)]。使用多模式成像和完整的眼科检查对这位患者的临床特征进行了研究。在HEK293T细胞中使用野生型和突变中间基因进行了c.2653+1G>A的异常剪接研究。通过全反式视黄醛激活的ATP酶活性测定法，分析了缺失由外显子17编码的Asp864-Gly885的突变型ABCA4蛋白的剩余活性，以及其亚细胞定位。为了诱导外显子17的跳跃，研究了40种AONs在WT WERI-Rb-1细胞和3D人类视网膜器官样品中的影响。 患者晚发性的STGD1表明c.2653+1G>A没有完全的有害效应。体外剪接实验证实了这种变异导致了没有外显子17的ABCA4转录本。与WT ABCA4相比，ABCA4 Asp864_Gly863的突变型保持稳定，并保留了58%全反式视黄醛激活的ATP酶活性。这个序列位于ABCA4的跨膜结构域6和核苷酸结合域1之间的非结构化连接区。设计了AONs，可能减少在外显子17中携带的致病性变异的病理性。最佳的AON在视网膜器官样品中实现了59%的外显子17跳跃。 ABCA4的外显子17缺失不会导致蛋白活性的消失，并且当与零等位基因共基因时，不会引起严重的STGD1表型。通过应用AONs，可以通过外显子跳跃来减轻外显子17中的严重变异的效应，从而在STGD1患者中产生部分ABCA4活性。 © 2023. BioMed Central Ltd., 由Springer Nature的一部分。

ABCA4, the gene implicated in Stargardt disease (STGD1), contains 50 exons, of which 17 contain multiples of three nucleotides. The impact of in-frame exon skipping is yet to be determined. Antisense oligonucleotides (AONs) have been investigated in Usher syndrome-associated genes to induce skipping of in-frame exons carrying severe variants and mitigate their disease-linked effect. Upon the identification of a STGD1 proband carrying a novel exon 17 canonical splice site variant, the activity of ABCA4 lacking 22 amino acids encoded by exon 17 was examined, followed by design of AONs able to induce exon 17 skipping.A STGD1 proband was compound heterozygous for the splice variant c.2653+1G>A, that was predicted to result in in-frame skipping of exon 17, and a null variant [c.735T>G, p.(Tyr245*)]. Clinical characteristics of this proband were studied using multi-modal imaging and complete ophthalmological examination. The aberrant splicing of c.2653+1G>A was investigated in vitro in HEK293T cells with wild-type and mutant midigenes. The residual activity of the mutant ABCA4 protein lacking Asp864-Gly885 encoded by exon 17 was analyzed with all-trans-retinal-activated ATPase activity assay, along with its subcellular localization. To induce exon 17 skipping, the effect of 40 AONs was examined in vitro in WT WERI-Rb-1 cells and 3D human retinal organoids.Late onset STGD1 in the proband suggests that c.2653+1G>A does not have a fully deleterious effect. The in vitro splice assay confirmed that this variant leads to ABCA4 transcripts without exon 17. ABCA4 Asp864_Gly863del was stable and retained 58% all-trans-retinal-activated ATPase activity compared to WT ABCA4. This sequence is located in an unstructured linker region between transmembrane domain 6 and nucleotide-binding domain-1 of ABCA4. AONs were designed to possibly reduce pathogenicity of severe variants harbored in exon 17. The best AON achieved 59% of exon 17 skipping in retinal organoids.Exon 17 deletion in ABCA4 does not result in the absence of protein activity and does not cause a severe STGD1 phenotype when in trans with a null allele. By applying AONs, the effect of severe variants in exon 17 can potentially be ameliorated by exon skipping, thus generating partial ABCA4 activity in STGD1 patients.© 2023. BioMed Central Ltd., part of Springer Nature.