Trojan horse gene therapy of inclusion body disease

描述(申请人提供)：除了拉福拉氏病，还有许多单基因缺陷储存疾病，对成年携带者的孩子有破坏性的影响(例如尼曼-皮克病、泰-萨克斯病、法布里病和许多其他溶酶体储存障碍)。这些疾病的一个共同特征是，酶缺乏(以及未消化的代谢物在细胞内积累)会导致进行性神经恶化和过早死亡。在所有病例中，突变的基因和酶缺乏都是已知的。但由于外源基因在整个大脑中的传递和表达存在问题，这些潜在的救命基因的副本在研究实验室里处于休眠状态。大分子疗法本身并不能穿过大脑毛细血管，导致人们认为血脑屏障(BBB)可能是一个无法解决的问题。病毒载体传递基因疗法出现的并发症进一步表明，需要设计新的方法来通过血脑屏障传递大分子基因。我们建议使用最近开发的免疫脂质体BBB递送系统，通过拉福拉病基因敲除小鼠的BBB，通过静脉给药，成功地递送正常基因。如果这种非病毒传递系统能够在动物模型中治疗这种疾病，基于免疫脂质体的治疗这种致命的包涵体疾病的方法可能被开发出来用于临床。其目的是：(1)制备聚乙二醇化免疫脂质体(PIL)，并建立a)外源基因和b)正常EPM2a/laforin基因的BBB递送，以承载显著的包涵体负荷的小鼠。(2)证实静脉注射外源基因后经胎盘和胎儿脑部传递外源基因。对大坝的管理。(3)确定一次静脉注射后，Laforin基因均匀地输送到基因敲除小鼠的脑内，并确定Lafora-身体负荷的变化。(4)多次静脉注射的最佳治疗方案。在基因敲除小鼠中注射抑制Lafora的身体负担达6-12个月。血脑屏障完整性和毛细血管紧密连接的检查将显示没有微血管病变，即使在慢性PIL治疗后也是如此。

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