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.

氨基糖苷类抗生素被发现可以抑制mdx小鼠中有缺陷的dystrophin基因中的无义突变，这表明可能是治疗Duchenne肌营养不良症(DMD)的方法。然而，庆大霉素治疗存在以下问题：1)由于耳毒性和肾毒性，不能给DMD患者给予能够诱导通读的大剂量庆大霉素。2、由于庆大霉素的半衰期较短，不能保持高浓度的庆大霉素诱导通读。3、很难找到适用于庆大霉素治疗的患者，因为：1)只有5%到13%的DMD患者有dystrophin基因的无义突变，2)由于dystrophin基因很长(14kb)，寻找基因的无义突变是具有挑战性的，3)氨基糖苷类药物诱导的通读效率取决于无义突变的种类。为了解决问题1和2，我们使用了混合脂质体。在…给药的mdx小鼠340 mg/kg以上的复合脂质体包裹庆大霉素2周，未见耳肾毒性。相比之下，只有注射相同剂量的庆大霉素的MDX小鼠表现出耳毒性。此外，注射庆大霉素复合脂质体的mdx鼠的抗肌萎缩蛋白阳性纤维的效率高于单独注射庆大霉素的mdx鼠，注射庆大霉素的mdx鼠的血清CK低于单独注射庆大霉素的mdx鼠。为了解决这一问题，我们建立了一套适合氨基糖苷类药物治疗的DMD患者筛选系统，分离了9例DMD患者的成纤维细胞，用AdMyoD诱导分化为肌源性细胞系，并与庆大霉素联合应用。用体外抗肌营养不良蛋白染色和Western blotting检测抗肌营养不良蛋白在庆大霉素作用下肌管的表达。结果表明，庆大霉素治疗存在TGA无义突变的DMD患者远比TAA和TAG无义突变的DMD患者有效。较少

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