New Treatment of the Brain in Niemann Pick C

摘要 尼曼-皮克C1型疾病（NPC 1）是一种破坏性的遗传性神经退行性溶酶体疾病， 由编码细胞内膜的NPC 1基因突变引起的胆积病 胆固醇转运蛋白目前，NPC 1的治疗选择很少。蛋白质替代疗法 这是不可能的，因为NPC 1蛋白是一种不溶性膜转运蛋白。人类基因治疗 NPC 1或NPC 1的长期存在的小鼠模型由于NPC 1的大尺寸而未被尝试 mRNA，这超过了普通基因治疗病毒载体的能力。相比之下，大尺寸基因可以 包封在非病毒递送系统如特洛伊木马脂质体（THL）中。THL制造 用受体特异性单克隆抗体（MAb），作为分子特洛伊木马来运送THL 进入脑细胞的核区在先前的工作中，THL介导的质粒DNA递送 在神经疾病的小鼠模型中产生了治疗效果，包括帕金森病、脑 癌症或溶酶体贮积病。假设：在目前的工作中测试的假设是，NPC 1 通过定期静脉内（IV）给药， 包封编码NPC 1基因的质粒DNA的THL。这一假设得到了先前工作的支持， 其中胚胎用野生型NPC 1基因转染的NPC 1小鼠模型和这些转基因小鼠， 小鼠与NPC 1小鼠杂交。在大脑中替换NPC 1基因有效地治愈了 NPC 1小鼠的疾病。初步数据：先前使用溶酶体贮积病小鼠模型的工作， 显示通过IV施用THL有可能实现小鼠脑中野生型基因的置换 携带编码溶酶体酶基因的表达质粒DNA。THL的目标是 对小鼠转铁蛋白受体（TfR）具有特异性的MAb。先前的工作也表明，重复，慢性IV THL的施用不引起毒性或免疫反应。具体目标：首先，THL将 制造，并且这些THL用对小鼠TfR特异性的MAb靶向，并包封THL。 编码NPC 1 cDNA的表达质粒DNA。表达质粒DNA将被工程化， 以前用于治疗性质粒DNA的方法。第二，THL的效力将在 使用人NPC 1成纤维细胞进行细胞培养。第三，将为本项目产生一群NPC 1小鼠 产生至少36只纯合子小鼠。小鼠将接受治疗12周，每周IV 从6周龄开始施用携带NPC 1 DNA的THL。治疗效果将是 通过存活率、体重和器官重量、脑组织学、外周器官组织学以及脑 NPC 1基因表达的生化参数。这项研究可以转化为人类NPC 1，因为 先前的工作表明，有可能将转基因传递到成年非人类大脑中的几乎所有细胞， 灵长类动物静脉注射THL。

Abstract Significance: Niemann-Pick Type C1 disease (NPC1) is a devastating inherited neurodegenerative lysosomal storage disease caused by mutations in the NPC1 gene, which encodes for an intracellular membrane cholesterol transporter. Currently, there are few treatment options for NPC1. Protein replacement therapy is not possible, because the NPC1 protein is an insoluble membrane transporter. Gene therapy of either human NPC1, or a long standing mouse model of NPC1, has not been attempted, owing to the large size of the NPC1 mRNA, which exceeds the capacity of common gene therapy viral vectors. In contrast, large size genes can be encapsulated in nonviral delivery systems such as Trojan horse liposomes (THLs). THLs are manufactured with a receptor-specific monoclonal antibody (MAb), which acts as a molecular Trojan horse to ferry the THL from blood into the nuclear compartment of brain cells. In prior work, THL-mediated delivery of plasmid DNA has produced therapeutic effects in mouse models of neural disease including, Parkinson's disease, brain cancer, or lysosomal storage disease. Hypothesis: The hypothesis tested in the present work is that the NPC1 gene can be effectively replaced in brain, and in peripheral organs, with regular intravenous (IV) administration of THLs encapsulating plasmid DNA encoding the NPC1 gene. This hypothesis is supported by prior work in the NPC1 mouse model wherein embryos were transfected with the wild type NPC1 gene and these transgenic mice were cross-bred with the NPC1 mouse. Replacement of the NPC1 gene in brain effectively cured the disease in the NPC1 mouse. Preliminary Data: Prior work with a lysosomal storage disease mouse model has shown it is possible to achieve replacement of the wild type gene in mouse brain with IV administration of THLs carrying the expression plasmid DNA encoding the lysosomal enzyme gene. The THLs are targeted with a MAb specific for the mouse transferrin receptor (TfR). Prior work has also shown that repeat, chronic IV administration of THLs causes no toxicity or immune reactions. Specific Aims: First, THLs will be manufactured, and these THLs are targeted with the MAb specific for the mouse TfR, and encapsulate an expression plasmid DNA encoding for the NPC1 cDNA. The expression plasmid DNA will be engineered with methods used previously for therapeutic plasmid DNA. Second, the potency of the THLs will be assessed in cell culture using human NPC1 fibroblasts. Third, a colony of NPC1 mice will be generated for this project producing at least 36 homozygote mice. The mice will undergo treatment for 12 weeks with weekly IV administration of THLs carrying the NPC1 DNA, beginning at the age of 6 weeks. Treatment efficacy will be assessed with survival, body and organ weights, brain histology, peripheral organ histology, as well as brain biochemical parameters of NPC1 gene expression. This research can be translated to human NPC1, because prior work as shown it is possible to deliver transgenes to virtually all cells in the brain of the adult non-human primate with IV administration of THLs.