Mann Christopher J, Honeyman Kaite, McClorey Graham, Fletcher Sue, Wilton Stephen D
Australian Neuromuscular Research Institute, Centre for Neuromuscular and Neurological Disorders, University of Western Australia, Perth, Western Australia, 6907.
J Gene Med. 2002 Nov-Dec;4(6):644-54. doi: 10.1002/jgm.295.
Duchenne muscular dystrophy (DMD) is a fatal genetic disorder caused by dystrophin gene mutations that preclude synthesis of a functional protein. One potential treatment of the disorder has utilised antisense oligoribonucleotides (AOs) to induce removal of disease-associated exons during pre-mRNA processing. Induced in-frame mRNA transcripts encode a shorter but functional dystrophin. We have investigated and improved the design of AOs capable of removing exon 23, and thus the disease-causing nonsense mutation, from mRNA in the mdx mouse model of DMD.
H-2K(b)-tsA58 mdx cultures were transfected with complexes of Lipofectin and AOs. Exon skipping was detected by RT-PCR and subsequent protein production was demonstrated by Western blotting. AOs were delivered at a range of doses in order to compare relative efficiencies.
We describe effective and reproducible exon 23 skipping with several AOs, including one as small as 17 nucleotides. Furthermore, the location of a sensitive exon 23 target site has been refined, whilst minimum effective doses have been estimated in vitro. These doses are significantly lower than previously reported and were associated with the synthesis of dystrophin protein in vitro.
These results demonstrate the increasing feasibility of an AO-based therapy for treatment of DMD. By refining AO design we have been able to reduce the size and the effective dose of the AOs and have dramatically improved the efficiency of the technique.
杜氏肌营养不良症（DMD）是一种由肌营养不良蛋白基因突变引起的致命性遗传疾病，该突变导致无法合成功能性蛋白。该疾病的一种潜在治疗方法是利用反义寡核糖核苷酸（AO）在mRNA前体加工过程中诱导去除与疾病相关的外显子。诱导产生的符合读框的mRNA转录本编码一种较短但有功能的肌营养不良蛋白。我们研究并改进了能够从DMD的mdx小鼠模型的mRNA中去除外显子23从而去除致病的无义突变的AO设计。
用脂质体和AO的复合物转染H-2K(b)-tsA58 mdx细胞培养物。通过逆转录聚合酶链反应（RT-PCR）检测外显子跳跃，并通过蛋白质印迹法证明随后的蛋白质产生。以一系列剂量递送AO以比较相对效率。
我们描述了几种AO能有效且可重复地实现外显子23跳跃，包括一种短至17个核苷酸的AO。此外，已优化了外显子23敏感靶位点的位置，同时在体外估计了最小有效剂量。这些剂量显著低于先前报道的剂量，并且与体外肌营养不良蛋白的合成相关。
这些结果证明了基于AO的疗法治疗DMD的可行性不断提高。通过优化AO设计，我们能够减小AO的大小和有效剂量，并显著提高了该技术的效率。
