PDF(Pubmed)
Abstract：
Autosomal dominant optic atrophy (ADOA) is frequently caused by mutations in the optic atrophy 1 (OPA1) gene, with haploinsufficiency being the major genetic pathomechanism. Almost 30% of the OPA1-associated cases suffer from splice defects. We identified a novel OPA1 mutation, c.1065+5G>A, in patients with ADOA. In patient-derived fibroblasts, the mutation led to skipping of OPA1 exon 10, reducing the OPA1 protein expression by approximately 50%. We developed a molecular treatment to correct the splice defect in OPA1 using engineered U1 splice factors retargeted to different locations in OPA1 exon 10 or intron 10. The strongest therapeutic effect was detected when U1 binding was engineered to bind to intron 10 at position +18, a position predicted by bioinformatics to be a promising binding site. We were able to significantly silence the effect of the mutation (skipping of exon 10) and simultaneously increase the expression level of normal transcripts. Retargeting U1 to the canonical splice donor site did not lead to a detectable splice correction. This proof-of-concept study indicates for the first time the feasibility of splice mutation correction as a treatment option for ADOA. Increasing the amount of correctly spliced OPA1 transcripts may suffice to overcome the haploinsufficiency.
摘要：
常染色体显性遗传性视神经萎缩（ADOA）常由视神经萎缩1（OPA1）基因突变引起，单倍体不足是主要的遗传病理机制。几乎30％的OPA1相关病例患有剪接缺陷。我们发现了一个新的OPA1突变，c.1065+5G>A，ADOA患者。在患者来源的成纤维细胞中，该突变导致OPA1外显子10的跳跃,使OPA1蛋白表达减少约50%.我们使用重新定位到OPA1外显子10或内含子10中不同位置的工程U1剪接因子开发了一种分子治疗方法来纠正OPA1中的剪接缺陷。当U1结合被工程化以在位置+18处与内含子10结合时，检测到最强的治疗效果，该位置由生物信息学预测为有希望的结合位点。我们能够显著沉默突变的作用(外显子10的跳跃)并同时增加正常转录物的表达水平。将U1重新定位到规范剪接供体位点不会导致可检测的剪接校正。这项概念验证研究首次表明了剪接突变校正作为ADOA治疗选择的可行性。增加正确剪接的OPA1转录物的量可能足以克服单倍体不足。
