Poly-L-aspartic Acid Enhances and Prolongs Gentamicin-mediated Suppression of the CFTR-G542X Mutation in a Cystic Fibrosis Mouse Model
Poly-L-aspartic Acid Enhances and Prolongs Gentamicin-mediated Suppression of the CFTR-G542X Mutation in a Cystic Fibrosis Mouse Model
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DOI：
10.1074/jbc.m806728200
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发表时间：
2009-03-13
影响因子：
4.8
通讯作者：
Bedwell, David M.
中科院分区：
文献类型：
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作者：
Du, Ming;Keeling, Kim M.;Bedwell, David M.
氨基糖苷类药物如庆大霉素能够抑制提前终止突变的翻译终止，导致蛋白质表达和功能的部分恢复。这一观察结果导致研究表明，这种方法可能为患有遗传性疾病的患者提供可行的治疗方法，例如由过早终止突变引起的囊性纤维化。虽然氨基糖苷类药物治疗有时会产生有害的副作用，但一些研究表明，聚阴离子如聚-L-天冬氨酸（PAA）的共同给药既可以降低毒性，又可以增加细胞内氨基糖苷类药物的浓度。在目前的研究中，我们研究了庆大霉素与PAA的联合给药如何影响培养细胞和囊性纤维化小鼠模型中过早终止密码子的通读。在培养的细胞中使用双荧光素酶通读报告系统，我们发现，相对于用相同浓度的庆大霉素单独处理的细胞，庆大霉素与PAA的共施用增加了20-40%的通读。使用Cftr（-/-）hCFTR-G542 X小鼠模型，我们发现PAA也增加了庆大霉素诱导的体内无义抑制。庆大霉素停药后，PAA显着延长的时间间隔，在此期间可以检测到通读，如短路电流测量和免疫荧光。由于使用庆大霉素抑制致病无义突变需要长期给药，PAA降低毒性并增加通读水平和持续时间的能力对这种有前景的治疗方法具有重要意义。
Aminoglycosides such as gentamicin have the ability to suppress translation termination at premature stop mutations, leading to a partial restoration of protein expression and function. This observation led to studies showing that this approach may provide a viable treatment for patients with genetic diseases such as cystic fibrosis that are caused by premature stop mutations. Although aminoglycoside treatment is sometimes associated with harmful side effects, several studies have shown that the co-administration of polyanions such as poly-L-aspartic acid (PAA) can both reduce toxicity and increase the intracellular aminoglycoside concentration. In the current study we examined how the co-administration of gentamicin with PAA influenced the readthrough of premature stop codons in cultured cells and a cystic fibrosis mouse model. Using a dual luciferase readthrough reporter system in cultured cells, we found that the co-administration of gentamicin with PAA increased readthrough 20-40% relative to cells treated with the same concentration of gentamicin alone. Using a Cftr(-/-) hCFTR-G542X mouse model, we found that PAA also increased the in vivo nonsense suppression induced by gentamicin. Following the withdrawal of gentamicin, PAA significantly prolonged the time interval during which readthrough could be detected, as shown by short circuit current measurements and immunofluorescence. Because the use of gentamicin to suppress disease-causing nonsense mutations will require their long term administration, the ability of PAA to reduce toxicity and increase both the level and duration of readthrough has important implications for this promising therapeutic approach.