Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, Switzerland.
Institute of Laboratory Medicine, Triemli Hospital, Zurich, Switzerland.
Nucleic Acids Res. 2020 May 21;48(9):4658-4671. doi: 10.1093/nar/gkaa229.
Erythropoietic protoporphyria (EPP) is a rare genetic disease in which patients experience acute phototoxic reactions after sunlight exposure. It is caused by a deficiency in ferrochelatase (FECH) in the heme biosynthesis pathway. Most patients exhibit a loss-of-function mutation in trans to an allele bearing a SNP that favors aberrant splicing of transcripts. One viable strategy for EPP is to deploy splice-switching oligonucleotides (SSOs) to increase FECH synthesis, whereby an increase of a few percent would provide therapeutic benefit. However, successful application of SSOs in bone marrow cells is not described. Here, we show that SSOs comprising methoxyethyl-chemistry increase FECH levels in cells. We conjugated one SSO to three prototypical targeting groups and administered them to a mouse model of EPP in order to study their biodistribution, their metabolic stability and their FECH splice-switching ability. The SSOs exhibited distinct distribution profiles, with increased accumulation in liver, kidney, bone marrow and lung. However, they also underwent substantial metabolism, mainly at their linker groups. An SSO bearing a cholesteryl group increased levels of correctly spliced FECH transcript by 80% in the bone marrow. The results provide a promising approach to treat EPP and other disorders originating from splicing dysregulation in the bone marrow.
红细胞生成性原卟啉症（EPP）是一种罕见的遗传性疾病，患者在阳光照射后会出现急性光毒性反应。它是由于血红素生物合成途径中的亚铁螯合酶（FECH）缺乏引起的。大多数患者表现为功能丧失突变，与携带 SNP 的等位基因发生反式作用，该 SNP 有利于转录物的异常剪接。EPP 的一种可行策略是使用剪接转换寡核苷酸（SSO）来增加 FECH 的合成，从而增加几个百分点就会带来治疗益处。然而，SSO 在骨髓细胞中的成功应用尚未描述。在这里，我们表明，包含甲氧基乙氧基化学的 SSO 会增加细胞中的 FECH 水平。我们将一个 SSO 连接到三个原型靶向基团上，并将它们施用于 EPP 小鼠模型，以研究它们的生物分布、代谢稳定性和 FECH 剪接转换能力。SSO 表现出不同的分布模式，在肝脏、肾脏、骨髓和肺部的积累增加。然而，它们也经历了大量的代谢，主要是在它们的连接基团上。带有胆固醇基团的 SSO 可使骨髓中正确剪接的 FECH 转录本水平增加 80%。这些结果为治疗 EPP 和其他源自骨髓中剪接失调的疾病提供了一种有前途的方法。
