Brain DNA Therapeutics with Trojan Horse Liposomes

摘要 Niemann Pick Type C1（NPC 1）是一种破坏性的脑退行性孤儿病。 由编码细胞内膜胆固醇转运蛋白的NPC 1基因突变引起。 目前尚无获批的NPC 1治疗方法。一种潜在的治疗方法是基因治疗， 递送编码NPC 1转运蛋白的基因。然而，NPC 1基因对于载体来说太大， 目前用于基因治疗的病毒载体的骨架。另一种方法是NPC 1的非病毒基因治疗， 这就是这个项目的目标。然而，限制脑用质粒DNA药物开发的因素是， 是血脑屏障（BBB）递送技术。质粒DNA治疗剂的脑递送是可能的 使用特洛伊木马脂质体（THL）技术。一个质粒DNA，大到20 kb， 包封在100-150 nm脂质体的内部。脂质体的表面与几个 聚乙二醇（PEG），这一过程称为聚乙二醇化。1-2% PEG的尖端 链与受体特异性单克隆抗体（MAb）缀合，所述抗体靶向受体介导的 在血脑屏障上的转运系统，如人胰岛素受体（HIR）。HIRMAb结合内源性 血脑屏障上的胰岛素受体结合内源性胰岛素，触发受体介导的转运进入大脑 脑细胞上的受体，以触发受体介导的内吞作用进入脑细胞，并且HIRMAb 导致质粒DNA分流到脑细胞的核区室。THL技术已经 在过去的15年里，在研发阶段开发，并已减少到在多种动物中的实践 神经疾病模型，包括溶酶体贮积症、实验性帕金森病、脑 癌症和基因传递到视网膜。假设：在目前的工作中测试的假设是， THL的制造可以从研发阶段推进到商业阶段， NPC 1的临床试验这是通过对THL生产的拟议修改实现的： 加压挤出机，其可以产生大体积的脂质体，和（B）THL的制剂，其为 冻干粉末，在输注当天用生理盐水复溶。初步数据：可行性研究 使用C-5机械挤出机表明，THL可以成功地用该装置制造， 高水平的DNA包封和THL的最终直径约为120 nm。具体目标： 首先，将产生HIRMAb和编码人NPC 1 cDNA的质粒DNA以支持THL 生产规模比过去的研发生产规模扩大100倍。第二，包裹DNA的HIRMAb-THL 将用于支持灵长类动物的初步研究每批HIRMAb、质粒DNA和THL将在 采用多种检测方法和规定的验收标准进行测定。第三，成人剂量范围研究 将对恒河猴进行测定，以确定血浆药代动力学、脑中的NPC 1基因递送 和外周器官，抗药物抗体反应和组织组织学，每周输注3次剂量的THL。如果 如果成功，这项工作将为第一个使用THL技术的人类治疗提供基础， 试图将尼曼匹克C1型突变基因（一种常染色体隐性遗传基因）传递到大脑， 进行性、致死性神经退行性疾病，目前尚无治疗方法。

Abstract Significance: Niemann Pick Type C1 (NPC1) is a devastating degenerative orphan disease of the brain caused by mutations in the NPC1 gene, which encodes for an intracellular membrane cholesterol transporter. There is currently no approved treatment for NPC1. A potentially curative treatment is gene therapy aimed at delivery of a gene encoding the NPC1 transporter. However, the NPC1 gene is too large for the vector backbone of current viral vectors for gene therapy. An alternative approach is non-viral gene therapy of NPC1, which is the goal of this project. However, the limiting factor in the drug development of plasmid DNA for brain is the blood-brain barrier (BBB) delivery technology. Brain delivery of plasmid DNA therapeutics is possible with the use of the Trojan horse liposome (THL) technology. A plasmid DNA, as large as 20 kb, can be encapsulated in the interior of a 100-150 nm liposome. The surface of the liposome is conjugated with several thousand strands of polyethyleneglycol (PEG), a process called pegylation. The tips of 1-2% of the PEG strands is conjugated with a receptor-specific monoclonal antibody (MAb) that targets a receptor-mediated transport system on the BBB, such as the human insulin receptor (HIR). The HIRMAb binds the endogenous insulin receptor on the BBB, to trigger receptor-mediated transport into brain, binds the endogenous insulin receptor on brain cells, to trigger receptor-mediated endocytosis into cells of the brain, and the HIRMAb causes triage of the plasmid DNA to the nuclear compartment of brain cells. The THL technology has been developed at the R&D stage over the last 15 years, and has been reduced to practice in multiple animal models of neural disease, including a lysosomal storage disorder, experimental Parkinson's disease, brain cancer, and gene delivery to the retina. Hypothesis: The hypothesis tested in the present work is that the manufacturing of THLs can be advanced from the R&D stage to a commercial stage that can support human clinical trials of NPC1. This is enabled by the proposed modifications of THL production: (a) use of a large pressurized extruder that can produce liposomes in large volumes, and (b) formulation of the THLs as a freeze-dried power to be reconstituted in saline on the day of infusion. Preliminary Data: Feasibility studies using the C-5 mechanical extruder show that THLs can be successfully manufactured with this device, with high levels of DNA encapsulation and final diameters of the THLs of approximately 120 nm. Specific Aims: First, the HIRMAb and a plasmid DNA encoding the human NPC1 cDNA will be produced to support THL manufacturing at a 100X scale-up over past R&D production. Second, HIRMAb-THLs encapsulating the DNA will be produced to support an initial primate study. Each lot of HIRMAb, plasmid DNA, and THL will be assayed with multiple test methods with defined acceptance criteria. Third, a dose-ranging study in adult Rhesus monkeys will be performed to determine the plasma pharmacokinetics, NPC1 gene delivery in brain and peripheral organs, anti-drug antibody response, and tissue histology, at 3 doses of THLs infused weekly. If successful, this work will provide the basis for the first human therapeutic using the THL technology, which will seek to deliver to brain the gene that is mutated in Niemann Pick type C1, an autosomal recessive, progressive, lethal neurodegenerative disease, for which there is no current therapy.