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：
9252089
负责人：
William M Pardridge
金额：
$ 49.96万
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 2018-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9252089
关键词：
Adult Animal Model Antibody Response Binding Biological Assay Biomedical Technology Blood - brain barrier anatomy Brain Brain Diseases Brain Pathology Caliber Cells Clinical Chemistry Clinical Trials Complementary DNA Confocal Microscopy DNA DNA delivery Data Data Reporting Development Devices Disease Dose Drug Kinetics Encapsulated Equus caballus Experimental Models Experimental Parkinsonism Feasibility Studies Formulation Freeze Drying Future Galactosidase Gene Delivery Gene Expression Genes Goals Grant Histocytochemistry Histology Hour Human Infusion procedures Inherited 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 Oncogenes 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 Staging Stainless Steel Surface System Technology Testing Therapeutic Therapeutic Effect Therapeutic Uses Tissues Transgenes Transgenic Organisms Triage Vertebral column Viral Vector Work abstracting base beta-Galactosidase brain cell cholesterol transporters clinical application curative treatments drug development gene therapy in vivo intravenous administration large scale production new technology non-viral gene therapy nonhuman primate novel therapeutics plasmid DNA pressure receptor receptor mediated endocytosis reconstitution relating to nervous system research and development scale up small molecule technology development therapeutic gene transcytosis transgene expression vector

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：Niemann Pick型C1（NPC1）是大脑中毁灭性的退化性孤儿疾病由NPC1基因中的突变引起，该突变编码为细胞内膜胆固醇转运蛋白。目前尚无批准的NPC1治疗方法。潜在的治疗方法是针对的基因治疗递送编码NPC1转运蛋白的基因。但是，NPC1基因对于向量太大目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该项目的目标。但是，脑质粒DNA的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。质粒DNA，最大至20 kb，可以是封装在100-150 nm脂质体的内部中。脂质体的表面与几个千线聚乙烯甘油（PEG），这一过程称为pegylation。 1-2％的钉子的尖端链与受体介导的受体特异性单克隆抗体（MAB）结合 BBB上的运输系统，例如人类胰岛素受体（HIR）。 Hirmab结合内源性 BBB上的胰岛素受体，触发受体介导的转运到大脑，结合内源性胰岛素脑细胞上的受体，以触发受体介导的内吞作用到大脑的细胞中，并发麻导致质粒DNA分类为脑细胞的核区室。 THL技术已经在过去的15年中，在研发阶段开发，已减少到多动物的练习神经疾病的模型，包括溶酶体储存障碍，实验性帕金森氏病，大脑癌症和基因输送到视网膜。假设：在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。这是通过提议的THL生产的修改来实现的：（a）使用大型加压挤出机可以在大体积中产生脂质体，（b）THL作为A 在输液当天，将在盐水中重构的冻干能力。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，并使用大约120 nm的THL的高水平DNA封装和最终直径。具体目的：首先，将产生编码人NPC1 cDNA的杂虫和质粒DNA以支持THL 在过去的研发生产中以100倍的规模制造。其次，封装DNA的Hirmab-thls 将生产以支持初始的灵长类动物研究。每片Hirmab，质粒DNA和THL都会用具有定义的接受标准的多种测试方法测定。第三，成人的剂量范围研究将进行恒河猴，以确定血浆药代动力学，NPC1基因在脑中的递送和周围器官，抗药物抗体反应和组织学，每周注入3剂THLS。如果成功，这项工作将为使用THL技术的第一种人类治疗提供基础，这将寻求向大脑递送在Niemann Pick型C1中突变的基因，这是一种常染色体隐性，进行性致命的神经退行性疾病，目前没有治疗。