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.

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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