以中樞神經為標的之基因治療於球狀細胞腦白質退化症之應用

CNS-targeted Gene Therapy in Globoid Cell Leukodystrophy

林達雄(Dar-Shong Lin)

指導教授 ： 劉宣良

國立臺北科技大學/工程學院/工程科技研究所/博士(2013年)

摘要

溶酶體储積性疾病為溶酶體酵素缺乏，導致受質無法代謝，儲積於臟器或(及)神經系統引發疾病。球狀細胞大腦白質失養症 (globoid-cell leukodystrophy; GLD)，為隱性遺傳之先天性溶酶體储積性神經去髓鞘疾病，其乳糖腦苷脂酶 (galactosylcerebrosidase: GALC) 基因突變，造成神經系統白質部位神經髓鞘脫失。約90% 的GLD 患者為嬰兒期致死型，在6 個月大前發病，出現躁動不安、抽搐、錐體症候、眼神經萎縮、心智生長遲緩，於2 歲前便死亡，至今尚無有效的治療。目前有一自然突變的 twitcher 小鼠，在臨床與病理上，類似此嬰兒期致死型，可作為疾病治療模型。本研究藉由GLD的疾病模型twitcher小鼠，研發適當的基因治療傳輸策略，以達到將治療基因全面性散佈至中樞神經系統的目的。我們先前應用AAV-GALC載體來治療新生twitcher 小鼠，經單獨腦內注射AAV或者合併幹細胞移植治療的小鼠，腦部 GALC 酵素活性高達正常值的2~3倍，並且其壽命顯著延長，但所增加的壽命與增加的酵素活性並不成比例。分析其可能原因包括小腦、腦幹、與脊髓未能得到充分治療。此一研究中，我們進一步結合大腦與小腦的基因治療，藉由軸突運輸，將轉殖基因與蛋白，送至腦內其他未注射的部位，達成的轉殖基因與蛋白於中樞神經全面性散佈的成效。經治療的小鼠，其體內的毒性累積物質psychosine顯著的下降，腦內的發炎反應減經、神經髓鞘與小腦的Purkinje cells 保存良好、且壽命延長；而且轉殖基因與蛋白全面性的散佈於中樞神經系統。這項研究確立了中樞神經全面性治療的良好策略，除了適用於GLD的治療，還可以應用於中樞神經退化性疾病及小腦退化性疾病的基因治療。

關鍵字

溶酶體储積性疾病 ； 球狀細胞腦白質退化症 ； 基因治療 ； 神經髓鞘

並列摘要

Globoid cell leukodystrophy (GLD) is a devastating lysosomal storage disease caused by deficiency of the enzyme galactocerebrosidase (GALC). Currently, there is no definite cure for the GLD. Several attempts with CNS-directed gene therapy in twitcher mice (a murine model of GLD) demonstrated restricted expression of GALC activity in CNS and failure of therapeutic efficacy in cerebellum and spinal cord, resulting in various degrees of correction of biochemical, pathological and clinical phenotype. More recently, twitcher mice receiving a combination of hematopoietic and viral vector gene transfer therapies were not protected from neurodegneration and axonopathy in both cerebellum and spinal cord. This evidence indicates the requirement of sufficient and widespread GALC expression in CNS and rescue of cerebellum and spinal cord in the therapeutic intervention of murine model of GLD. In this study, we have optimized intracranial delivery of AAV2/5-GALC to the neocortex, hippocampus and cerebellum, instead of the thalamus as was previously conducted, of twitcher mice. The CNS-targeted AAV2/5 gene transfer effectively dispersed GALC transgen along the neuraxis of CNS as far as the lumbar spinal cord, and reduced the accumulation of psychosine in the CNS of twitcher mice. Most importantly, the treated twitcher mice were protected from loss of oligodendrocytes and purkinje cells, axonopathy and marked gliosis, and had significantly improved neuromotor function and prolonged lifespan. Collectively, our study addresses that this severe GLD murine model can be rescued by CNS-targeted AAV5 gene transfer and supports the development of AAV-mediated intervention as a potential therapeutic approach for GLD.

並列關鍵字

lysosomal storage disease ； Globoid cell leukodystrophy ； gene therapy

參考文獻

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