Trojan horse gene therapy of inclusion body disease

描述（由申请人提供）：除拉福拉病外，还有许多单基因缺陷性胆固醇沉着病对成年携带者的子女具有破坏性影响（例如尼曼-皮克病、泰-萨克斯病、法布里病和许多其他溶酶体胆固醇沉着病）。这些疾病的一个共同特征是酶缺乏（以及未消化代谢物的细胞内积累）导致进行性神经功能恶化和早期死亡。在所有情况下，突变的基因和酶缺乏症是已知的。但是，由于在整个大脑中传递和表达外源基因的问题，这些潜在的救命基因的副本在研究实验室中处于休眠状态。单独的大分子治疗剂不能穿过脑毛细血管，导致认为血脑屏障（BBB）可能是一个无法解决的问题。病毒载体传递基因疗法的并发症进一步表明，需要设计新的方法来传递大分子基因穿过血脑屏障。我们建议使用最近开发的免疫脂质体血脑屏障传递系统，成功地提供一个正常的基因治疗通过血脑屏障敲除小鼠Lafora病，通过静脉注射。如果这种非病毒传递系统可以在动物模型中治疗这种疾病，那么可以开发一种基于免疫脂质体的治疗这种致命的包涵体疾病的方法用于临床。其目标是：（1）制备聚乙二醇化免疫脂质体（PIL）并在携带显著包涵体负荷的小鼠中建立a）外源基因和B）正常EPM 2a/laforin基因的BB B递送。(2)证明母体i. v.给药后外源基因的经胎盘和胎脑递送。(3)确认单次静脉注射后laforin基因向敲除小鼠大脑的均匀递送，并确定Lafora体负荷如何变化。以及（4）开发多次静脉注射的最佳治疗方案，其在基因敲除小鼠中抑制Lafora身体负担6-12个月。BBB完整性和毛细血管紧密连接的检查将显示即使在慢性PIL治疗后也没有微血管病理学

DESCRIPTION (provided by applicant): In addition to Lafora's Disease, there are numerous single-gene defect storage diseases that have devastating effects on the children of adult carriers (e.g. Niemann-Pick disease, Tay-Sachs disease, Fabry disease, and many other Lysosomal Storage disorders). A common feature of these disorders is that an enzyme deficiency (and the intracellular accumulation of undigested metabolites) leads to progressive neurologic deterioration and early death. In all cases, the mutated gene and enzyme deficiency is known. But copies of the potentially life-saving genes sit dormant in research laboratories because of problems in delivery and expressing an exogenous gene throughout the brain. Large molecule therapeutics alone do not cross the brain capillaries, leading to the perception that the blood-brain barrier (BBB) may be an insoluble problem. Complications seen with viral vector-delivery of gene therapies further suggest that new methods need to be devised for the delivery of large-molecule genes across the BBB. We propose to use recently developed immunoliposome BBB delivery systems to successfully deliver a normal gene therapeutically through the BBB of knock-out mice with Lafora's Disease, via intravenous administration. If this non-viral delivery system can treat the disease in animal models, an immunoliposome-based cure for this fatal inclusion body disorder could be developed for clinical use. The aims are: (1) To prepare pegylated immunoliposomes (PIL) and establish BBB delivery of a) an exogenous gene and b) delivery of the normal EPM2a/laforin gene, in mice bearing a significant inclusion body burden. (2) To demonstrate transplacental and fetal brain delivery of an exogenous gene after i.v. administration to the dam. (3) To confirm uniform delivery of the laforin gene to the brain of knock-out mice after a single intravenous injection, and determine how Lafora-body burdens change. And (4) to develop an optimal therapeutic regimen of multiple i.v. injections which suppresses Lafora body burdens for 6-12 months in knock-out mice. Examinations of BBB integrity and capillary tight-junctions will show the absence of microvascular pathology, even after chronic PIL treatments