A novel approach of the gentamicin therapy for Duchenne muscular dystrophy using hybrid liposome and establishment of a system to identify the patients eligible for the treatment.

已发现氨基糖苷类抗生素抑制mdx小鼠中缺陷性肌营养不良蛋白基因中的无义突变，这表明可能治疗杜氏肌营养不良症（DMD）。然而，庆大霉素治疗存在以下问题：1、由于耳毒性和肾毒性，我们不能给DMD患者高剂量的庆大霉素以诱导通读。2、由于庆大霉素的半衰期短，不能保持高浓度的庆大霉素以诱导通读。3、很难找到适用于这种治疗的患者，因为：1）只有5 - 13%的DMD患者在肌营养不良蛋白基因中存在无义突变，2）由于肌营养不良蛋白cDNA非常长（14 kb），因此在基因中发现无义突变具有挑战性，3）氨基糖苷类诱导的通读效率取决于无义突变的种类。Mdx小鼠，腹腔内给予 ...更多信息 340 mg/kg庆大霉素复合脂质体连续给药2周，未见耳毒性和肾毒性。相比之下，只有庆大霉素注射相同剂量的mdx小鼠显示耳毒性。此外，注射包封庆大霉素的混合脂质体的mdx小鼠中抗肌萎缩蛋白阳性纤维的效率高于单独注射庆大霉素的小鼠，并且注射包封庆大霉素的脂质体的mdx小鼠中血清CK低于单独注射庆大霉素的小鼠。因此，结果表明，包封庆大霉素的混合脂质体对DMD患者比单独的庆大霉素更有效。为了解决问题3，我们开发了一种用于鉴定符合氨基糖苷类药物治疗资格的候选物的系统，分离来自9名肌营养不良蛋白基因无义突变的DMD患者的成纤维细胞，通过AdMyoD诱导分化为肌源性谱系，并暴露于庆大霉素。通过体外肌营养不良蛋白染色和蛋白质印迹分析监测庆大霉素暴露的肌管中肌营养不良蛋白的表达。结果表明，庆大霉素治疗对具有无义突变TGA的DMD患者比具有无义突变TAA和TAG的患者更有效。少

Aminoglycoside antibiotics have been found to suppress nonsense mutations located in the defective dystrophin gene in mdx mice, suggesting a possible treatment for Duchenne muscular dystrophy (DMD). However, the gentamicin therapy has the following problems.1,We cannot give a high dose of gentamicin which is able to induces read-through to DMD patients, because of oto- and nephrotoxicity.2,The high concentration of gentamicin cannot be kept to induce read-through, because the half-life of gentamicin is short.3,It is difficult to find patients that are applicable for this therapy, because : 1)only 5 to 13% of DMD patients have nonsense mutations in the dystrophin gene, 2)it is challenging to find nonsense mutations in the gene because dystrophin cDNA is very long (14kb), and 3)the efficiency of aminoglycoside-induced read-through is dependent on the kind of nonsense mutation.In order to solve the problem 1 and 2, we used hybrid liposomes. Mdx mice, which were intraperitoneally given at … More 340mg/kg of hybrid liposome encapsulated gentamicin for 2 weeks, did not show oto- and nephrotoxicity. In contrast, only gentamicin injected mdx mice at the same dose show ototoxicity. In addition, the efficiency of dystrophin positive fibers in the mdx mice injected hybrid liposome encapsulated gentamicin was higher than only gentamicin injected mice, and serum CK in the mdx mice injected the liposome encapsulated gentamicin was lower than gentamicin alone. Therefore, the results show that the hybrid liposome encapsulated gentamicin is more effective for DMD patients than genamicin alone.In order to solve the problem 3, we have developed a system for identifying candidates that qualify for aminoglycoside therapy, fibroblasts from 9 DMD patients with nonsense mutation of dystrophin gene were isolated, induced to differentiate to myogenic lineage by AdMyoD, and exposed with gentamicin. The dystrophin expression in gentamicin exposed myotubes was monitored by in vitro dystrophin staining and western blotting analysis. The results showed that the gentamicin therapy is far more effective for DMD patients that have nonsense mutation TGA than for patients that have nonsense mutation TAA and TAG. Less