Exons 45-55 Skipping Using Mutation-Tailored Cocktails of Antisense Morpholinos in the DMD Gene
Exons 45-55 Skipping Using Mutation-Tailored Cocktails of Antisense Morpholinos in the DMD Gene
复制标题
DOI：
10.1016/j.ymthe.2019.07.012
复制
发表时间：
2019-11-06
影响因子：
12.4
通讯作者：
Yokota, Toshifumi
中科院分区：
文献类型：
--
作者：
Echigoya, Yusuke;Lim, Kenji Rowel Q.;Yokota, Toshifumi
肌营养不良蛋白（DMD）基因突变和随之发生的肌营养不良蛋白缺失导致杜氏肌营养不良症（DMD）。DMD的一种有前途的疗法，使用反义磷酰二胺吗啉代寡聚物（PMO）的单外显子跳跃，目前面临的主要问题在于反义药物诱导功能未定义的肌营养不良蛋白的产生，并且在具有不同突变的患者中可能不类似地有效。因此，该方法的适用性限于框外突变。在这里，使用外显子跳跃效率预测工具，我们设计了用于外显子45-55跳跃的三种不同的PMO鸡尾酒组，旨在产生具有保留功能的肌营养不良蛋白变体，如在具有框内外显子45-55缺失的温和或无症状个体中所见。其中，最有效的一组由选择的PMO组成，每个PMO在基于细胞的筛选中有效地跳过指定的外显子。这些组合PMO适合于永生化DMD患者肌细胞的不同缺失，显著诱导外显子45-55跳跃，去除3、8或10个外显子，并恢复肌营养不良蛋白，如蛋白质印迹所示。在人源化小鼠中证实了最多11个人DMD外显子的体内跳跃。这一发现表明，我们的PMO集可用于为外显子45-55跳跃创建突变定制的鸡尾酒，并治疗超过65%的携带框外或框内缺失的DMD患者。
Mutations in the dystrophin (DMD) gene and consequent loss of dystrophin cause Duchenne muscular dystrophy (DMD). A promising therapy for DMD, single-exon skipping using antisense phosphorodiamidate morpholino oligomers (PMOs), currently confronts major issues in that an antisense drug induces the production of functionally undefined dystrophin and may not be similarly efficacious among patients with different mutations. Accordingly, the applicability of this approach is limited to out-of-frame mutations. Here, using an exon-skipping efficiency predictive tool, we designed three different PMO cocktail sets for exons 45-55 skipping aiming to produce a dystrophin variant with preserved functionality as seen in milder or asymptomatic individuals with an in-frame exons 45-55 deletion. Of them, the most effective set was composed of select PMOs that each efficiently skips an assigned exon in cell-based screening. These combinational PMOs fitted to different deletions of immortalized DMD patient muscle cells significantly induced exons 45-55 skipping with removing 3,8, or 10 exons and dystrophin restoration as represented by western blotting. In vivo skipping of the maximum 11 human DMD exons was confirmed in humanized mice. The finding indicates that our PMO set can be used to create mutation-tailored cocktails for exons 45-55 skipping and treat over 65% of DMD patients carrying out-of-frame or in-frame deletions.