Development of novel therapy and elucidation of pathophysiology for genetic leukodystrophy

我们正在为遗传性脑白质营养不良开发基因和细胞疗法。我们关注的主要疾病是克拉布病。这种疾病是由半乳糖苷酶的遗传缺陷引起的，并且可以获得Krabbe病的真实小鼠模型。使用这种小鼠模型，我们尝试了几种基因治疗方法。首先，我们将携带细菌LacZ基因的腺病毒载体注入正常小鼠胚胎的侧脑室。LacZ表达在整个大脑中观察到，这种表达在出生后持续超过100天。表达LacZ基因的主要细胞类型为神经元和星形胶质细胞。然而，没有少突胶质细胞表达LacZ。虽然这种方法对粘多糖样沉积症VII型小鼠非常有效，但Krabbe病的小鼠模型未能通过这种方法治愈。这种失败可能是由于Krabbe病的主要受累细胞是少突胶质细胞。所以我们测试了逆转录病毒载体而不是腺病毒载体。结果表明，不仅神经元和星形胶质细胞表达LacZ，而且胶质细胞也表达LacZ，并且表达期超过100天，表达量没有明显下降。这一观察结果有力地表明，逆转录病毒载体转染神经干细胞。因此，我们将病毒载体注射到新生小鼠脑室下区，这是已知的许多神经干细胞存在。在这种情况下，主要是星形胶质细胞和少突胶质细胞表达LacZ。由此观察，我们产生了表达半乳糖苷酶的逆转录病毒载体，并将其注射到新生ticker小鼠的脑室下区，这是Krabbe病的真实小鼠模型。逆转录病毒转导可使twitcher小鼠少突软骨细胞的异常形态得到恢复。这表明新生儿基因治疗对Krabbe病神经受累的治疗是可行的。

We are developing gene and cell therapy for genetic leukodystrophies. The main disease, which we focused on, was Krabbe disease. This disease is caused by a genetic deficiency of galactocerebrosidase and the authentic murine model of Krabbe disease is available. Using this murine model, we tried several gene therapy approaches. First, we injected adenoviral vector, which carries bacterial LacZ gene into lateral ventricles of normal mouse embryo. LacZ expression was observed throughout the brain and this expression was persisted more than 100 days after birth. Main cell type, which expressed LacZ gene was neuron and astorocytes. However, no oligodendrocytes expressed LacZ. Although this approach was very effective for mucopolysaccharidosis type VII mice, mouse model of Krabbe disease failed to be cured by this method. This failure may be due that the main affected cell in Krabbe disease is oligodendrocyte. So we tested retroviral vector instead of adenovirus vector. As a result, not only neurons and astrocytes, but also olingodendrocytes expressed LacZ, and the expression period was more than 100 days without significant decrement of expression. This observation strongly indicated that retrovirus vector transdused neural stem cell. So we injected retorovirus vector into subventricular zone of newborn mouse brain, which is known that many neural stem cells exist. In this case, mainly, astorcyte and oligidendrocyte expressed LacZ. From this observation, we generated retrovirus vector which expressed galactocerebrosidase and injected subventricular zone of newborn twitcher mouse, which is authentic mouse model of Krabbe disease. The abnormal morphology of twitcher mouse oligodedrocyte was restored by retrovirus transduction. This indicated that neonatal gene therapy may be feasible for treatment of neural involvement of Krabbe disease.