Brain DNA Therapeutics with Trojan Horse Liposomes

摘要 意义：Niemann Pick C1型(NPC1)是一种毁灭性的大脑退行性孤儿疾病 这是由NPC1基因突变引起的，NPC1基因编码细胞膜胆固醇转运蛋白。 目前还没有批准的治疗NPC1的方法。一种潜在的治愈方法是针对以下目标的基因疗法 NPC1转运体编码基因的传递。然而，NPC1基因对于载体来说太大了 目前用于基因治疗的病毒载体的主干。另一种方法是NPC1的非病毒基因治疗， 这就是这个项目的目标。然而，脑用质粒DNA药物开发的限制因素 是血脑屏障(BBB)输送技术。脑内注射质粒DNA疗法是可能的 使用特洛伊木马脂质体(THL)技术。一个大到20kb的质粒DNA可以是 被封装在100-150 nm脂质体的内部。脂质体的表面与几个 上千股聚乙二醇化，这一过程称为聚乙二醇化。小贴士1-2%的聚乙二醇 Strands与受体特异性的单抗(Mab)结合，该抗体针对受体介导的 血脑屏障上的运输系统，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，触发受体介导的脑内转运，与内源性胰岛素结合 脑细胞上的受体，以触发受体介导的内吞作用进入脑细胞，以及HIRMAb 导致将质粒DNA分类到脑细胞的核间室。THL技术已经被 在过去的15年中，在研发阶段开发，并已简化为在多种动物上实践 神经疾病的模型，包括溶酶体储存障碍，实验性帕金森病，大脑 癌症，以及基因输送到视网膜。假设：目前工作中检验的假设是 THLS的制造可以从研发阶段推进到商业阶段，可以支持人类 NPC1的临床试验。这是由于对THL生产的拟议修改：(A)使用大量 可大量生产脂质体的加压挤出机，以及(B)THLS的配方 冻干粉在输液当天用生理盐水重新配制。初步数据：可行性研究 使用C-5机械挤出机表明，使用该设备可以成功地制造THL，具有 高水平的DNA包裹体和最终直径约120 nm的THL。具体目标： 首先，将生产HIRMAb和编码人NPC1基因的质粒DNA以支持Th1 与过去的研发生产相比，制造规模扩大了100倍。第二，包裹DNA的HIRMAb-THLS 将用于支持灵长类动物的初步研究。每批HIRMAb、质粒DNA和Thl将被 用多种测试方法进行分析，并定义验收标准。第三，成人的剂量范围研究 将对恒河猴进行血浆药代动力学、NPC1基因脑内转运的测定 外周器官、抗药物抗体反应和组织学检查，每周3次。如果 这项工作的成功将为第一次使用THL技术的人类治疗提供基础，这将 寻求将Niemann Pick C1型突变的基因传递给大脑，C1型是一种常染色体隐性遗传， 进行性的、致命的神经退行性疾病，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.