Antisense oligonucleotide allele-specific targeting of EFEMP1 in a patient-derived model of Doyne honeycomb retinal dystrophy

Patient-derived iPSC-RPE harbouring the R345W (c.1033C>T) heterozygous variant in EFEMP1 underwent a 7-day gymnotic treatment with ASO1.1 at the doses 0.5 µM, 1 µM, 5 µM and 10 µM, no treatment (NT) and control (CTRL) ASO conditions were included as controls. Samples underwent targeted next generation sequencing, using primers that amplified the region in EFEMP1 harbouring the R345W (c.1033C>T) heterozygous variant, allowing for the allelic discrimination of gymnotically delivered antisense oligonucleotides (ASOs).携带EFEMP1基因R345W（c.1033C>T）杂合变异的患者来源诱导多能干细胞源性视网膜色素上皮细胞（patient-derived iPSC-RPE），使用ASO1.1进行了7天裸转染处理，处理浓度分别为0.5 µM、1 µM、5 µM及10 µM；同时设置未处理组（no treatment, NT）与对照反义寡核苷酸（control ASO, CTRL）组作为对照。 对上述样本开展靶向下一代测序（targeted next generation sequencing）检测，所用引物可扩增EFEMP1基因中携带R345W（c.1033C>T）杂合变异的区域，以此实现裸递送反义寡核苷酸（gymnotically delivered antisense oligonucleotides, ASOs）的等位基因判别。