Allamand Valérie, Bidou Laure, Arakawa Masayuki, Floquet Célia, Shiozuka Masataka, Paturneau-Jouas Marion, Gartioux Corine, Butler-Browne Gillian S, Mouly Vincent, Rousset Jean-Pierre, Matsuda Ryoichi, Ikeda Daishiro, Guicheney Pascale
Inserm, U582, Paris, France.
J Gene Med. 2008 Feb;10(2):217-24. doi: 10.1002/jgm.1140.
The most common form of congenital muscular dystrophy is caused by a deficiency in the alpha2 chain of laminin-211, a protein of the extracellular matrix. A wide variety of mutations, including 20 to 30% of nonsense mutations, have been identified in the corresponding gene, LAMA2. A promising approach for the treatment of genetic disorders due to premature termination codons (PTCs) is the use of drugs to force stop codon readthrough.
Here, we analyzed the effects of two compounds on a PTC in the LAMA2 gene that targets the mRNA to nonsense-mediated RNA decay, in vitro using a dual reporter assay, as well as ex vivo in patient-derived myotubes.
We first showed that both gentamicin and negamycin promote significant readthrough of this PTC. We then demonstrated that the mutant mRNAs were strongly stabilized in patient-derived myotubes after administration of negamycin, but not gentamicin. Nevertheless, neither treatment allowed re-expression of the laminin alpha2-chain protein, pointing to problems that may have arisen at the translational or post-translational levels.
Taken together, our results emphasize that achievement of a clinical benefit upon treatment with novel readthrough-inducing agents would require several favourable conditions including PTC nucleotide context, intrinsic and induced stability of mRNA and correct synthesis of a full-length active protein.
先天性肌营养不良最常见的形式是由细胞外基质蛋白层粘连蛋白-211的α2链缺乏引起的。在相应基因LAMA2中已鉴定出多种突变，包括20%至30%的无义突变。一种有前景的治疗由于过早终止密码子（PTC）导致的遗传疾病的方法是使用药物促使终止密码子通读。
在此，我们使用双报告基因检测体外分析了两种化合物对LAMA2基因中一个靶向mRNA进行无义介导的RNA降解的PTC的影响，以及在患者来源的肌管中进行了离体分析。
我们首先表明庆大霉素和奈氏霉素都能显著促进该PTC的通读。然后我们证明，在给予奈氏霉素后，患者来源的肌管中的突变mRNA得到了强烈稳定，但给予庆大霉素后则没有。然而，两种处理都不能使层粘连蛋白α2链蛋白重新表达，这表明可能在翻译或翻译后水平出现了问题。
综上所述，我们的结果强调，使用新型通读诱导剂治疗要取得临床益处需要几个有利条件，包括PTC的核苷酸背景、mRNA的固有稳定性和诱导稳定性以及全长活性蛋白的正确合成。
