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Brain DNA Therapeutics with Trojan Horse Liposomes

特洛伊木马脂质体的脑 DNA 治疗

基本信息

批准号：
9351580
负责人：
William M Pardridge
金额：
$ 64.73万
依托单位：
LIPOGENE COMPANY, INC.
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-15 至 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9351580
关键词：
Adult Alpha-galactosidase Animal Model Antibody Response Binding Biological Assay Biomedical Technology Blood - brain barrier anatomy Brain Brain Diseases Brain Pathology Caliber Cell Compartmentation Clinical Chemistry Clinical Trials Complementary DNA Confocal Microscopy DNA DNA delivery Data Data Reporting Development Devices Disease Disease model Dose Drug Kinetics Encapsulated Equus caballus Experimental Models Experimental Parkinsonism Feasibility Studies Formulation Freeze Drying Future Galactosidase Galanin Gene Delivery Gene Transduction Agent Genes Goals Grant Histocytochemistry Histology Hour Human Infusion procedures Inherited Injectable Insulin Receptor Intracellular Membranes Intravenous Liposomes Liver Macaca mulatta Malignant neoplasm of brain Mechanics Mediating Methodology Methods Modification Monkeys Monoclonal Antibodies Mutate Mutation National Institute of Neurological Disorders and Stroke Neuraxis Neurodegenerative Disorders Nuclear Organ Parkinson Disease Peripheral Pharmaceutical Preparations Phase Plasma Plasmids Primates Process Production Rare Diseases Reaction Recommendation Research Retina Safety Saline Small Business Innovation Research Grant Stainless Steel Surface System Technology Testing Therapeutic Therapeutic Effect Therapeutic Uses Tissues Transgenes Transgenic Organisms Triage Vertebral column Viral Vector Work base beta-Galactosidase brain cell cholesterol transporters clinical application curative treatments drug development gene therapy in vivo large scale production neural model new technology non-viral gene therapy nonhuman primate novel therapeutics plasmid DNA pressure receptor receptor mediated endocytosis reconstitution research and development scale up small molecule technology development therapeutic gene therapeutic transgene transcytosis transgene expression vector virtual

项目摘要

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.

抽象的意义：尼曼皮克 C1 型 (NPC1) 是一种毁灭性的大脑退行性疾病由 NPC1 基因突变引起，该基因编码细胞内膜胆固醇转运蛋白。目前尚无批准的 NPC1 治疗方法。一种潜在的治疗方法是基因疗法，旨在传递编码 NPC1 转运蛋白的基因。然而，NPC1 基因对于载体而言太大当前用于基因治疗的病毒载体的骨干。另一种方法是 NPC1 的非病毒基因治疗，这是这个项目的目标。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。质粒 DNA 疗法的大脑递送是可能的使用特洛伊木马脂质体（THL）技术。长达 20 kb 的质粒 DNA 可以封装在 100-150 nm 脂质体的内部。脂质体表面缀合有多种数千股聚乙二醇 (PEG)，这一过程称为聚乙二醇化。 1-2% PEG 的提示链与受体特异性单克隆抗体（MAb）缀合，该抗体靶向受体介导的 BBB 上的转运系统，例如人胰岛素受体 (HIR)。 HIRMAb 结合内源性 BBB 上的胰岛素受体，触发受体介导的转运进入大脑，与内源性胰岛素结合脑细胞上的受体，触发受体介导的脑细胞内吞作用，以及 HIRMAb 导致质粒 DNA 分类到脑细胞的核区室。 THL技术已经过过去 15 年的研发阶段开发，并已在多种动物中得到实践神经疾病模型，包括溶酶体贮积症、实验性帕金森病、大脑癌症和基因传递到视网膜。假设：本工作中检验的假设是 THL 的制造可以从研发阶段推进到可以支持人类的商业阶段 NPC1的临床试验。这是通过对 THL 生产提出的修改来实现的：(a) 使用大量可以大量生产脂质体的加压挤出机，以及 (b) 将 THL 配制为冻干粉末在输注当天用盐水重新配制。初步数据：可行性研究使用 C-5 机械挤出机表明，可以使用该设备成功制造 THL，并且高水平的 DNA 封装和 THL 的最终直径约为 120 nm。具体目标：首先，将生产 HIRMAb 和编码人 NPC1 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了 100 倍。其次，HIRMAb-THLs 封装 DNA 将用于支持初步灵长类动物研究。每批 HIRMAb、质粒 DNA 和 THL 将被使用多种测试方法进行测定，并确定验收标准。三、成人剂量范围研究将用恒河猴来测定血浆药代动力学、NPC1 基因在大脑中的传递每周输注 3 剂 THL，观察外周器官、抗药物抗体反应和组织组织学。如果成功后，这项工作将为使用 THL 技术的第一个人类治疗奠定基础，这将试图将 Niemann Pick C1 型（一种常染色体隐性遗传）突变的基因传递到大脑，进行性、致命的神经退行性疾病，目前尚无治疗方法。