Morpholino antisense oligonucleotide induced dystrophin exon 23 skipping in mdx mouse muscle
Morpholino antisense oligonucleotide induced dystrophin exon 23 skipping in mdx mouse muscle
复制标题
DOI：
10.1093/hmg/ddg196
复制
发表时间：
2003-08-01
影响因子：
3.5
通讯作者：
Wilton, SD
中科院分区：
文献类型：
--
作者：
Gebski, BL;Mann, CJ;Wilton, SD
mdx小鼠肌营养不良模型是由于肌营养不良蛋白基因23外显子的无义突变引起的。我们之前已经证明，2'- o -甲基磷硫酸反义寡核苷酸（AOs）可以在初级转录物的加工过程中诱导23号外显子的去除。这导致框内mRNA转录和随后在mdx肌肉中表达稍短的肌营养不良蛋白。AO设计的改进已经允许在纳米摩尔浓度的mdx小鼠肌肉培养中诱导有效的外显子跳变。相比之下，在培养细胞中，当在微摩尔范围内传递时，morolino AOs对β -珠蛋白基因前mrna进行剪接干预，以纠正异常剪接。morpholino化学产生一种中性分子，具有特殊的生物稳定性，但细胞传递能力差。我们提供的数据表明，mdx细胞中的外显子跳变可能是由纳米摩尔浓度的morpholino AOs诱导的，当其退火为寡核苷酸或“链”时，并以阳离子脂质体的形式递送。我们已经研究了许多牵链设计和化学成分，包括混合骨干寡核苷酸，以及它们影响morpholino AO递送和功效的能力。值得注意的是，我们在mdx肌肉体内肌内注射morpholino AO：牵链脂丛后检测到肌营养不良蛋白的合成并纠正了肌层定位。我们展示了morpholino AO的增强递送，使有利的特性能够被用于潜在的治疗结果。
The mdx mouse model of muscular dystrophy arose due to a nonsense mutation in exon 23 of the dystrophin gene. We have previously demonstrated that 2'-O-methyl phosphorothioate antisense oligonucleotides (AOs) can induce removal of exon 23 during processing of the primary transcript. This results in an in-frame mRNA transcript and subsequent expression of a slightly shorter dystrophin protein in mdx muscle. Refinement of AO design has allowed efficient exon skipping to be induced in mdx mouse muscle cultures at nanomolar concentrations. In contrast, splicing intervention by morpholino AOs has been applied to the beta-globin gene pre-mRNA in cultured cells to correct aberrant splicing when delivered in the micromolar range. The morpholino chemistry produces a neutral molecule that has exceptional biological stability but poor cellular delivery. We present data showing that exon skipping in mdx cells may be induced by morpholino AOs at nanomolar concentrations when annealed to a sense oligonucleotide or 'leash', and delivered as a cationic lipoplex. We have investigated a number of leash designs and chemistries, including mixed backbone oligonucleotides, and their ability to influence delivery and efficacy of the morpholino AO. Significantly, we detected dystrophin protein synthesis and correct sarcolemmal localisation after intramuscular injection of morpholino AO : leash lipoplexes in mdx muscle in vivo. We show enhanced delivery of a morpholino AO, enabling the advantageous properties to be exploited for potentially therapeutic outcomes.