Targeted DNA Nanoparticles in Brain

大脑中的靶向 DNA 纳米颗粒

• 批准号: 7914207
• 负责人: David M. Yurek
• 金额: $ 18.8万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-15 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7914207
• 关键词: Animal Model; Animals; Antibodies; Antibody Formation; Astrocytes; Binding; Blood - brain barrier anatomy; Brain; Caliber; Cell Culture Techniques; Cell Line; Cells; Complex; DNA; Data; Dopamine; Dose; Drug Formulations; Elements; Enzymes; Gene Expression; Gene Transfer; Hippocampus (Brain); Human; Imaging Techniques; Immune response; Injection of therapeutic agent; Intracarotid; Intravenous; Lead; Ligands; Luciferases; Magnetic Resonance Imaging; Multienzyme Complexes; Neuraxis; Neurodegenerative Disorders; Neuroglia; Neurons; Non-Viral Vector; Nucleic Acids; Parkinson Disease; Pathway interactions; Pattern; Peptides; Plasmids; Process; Production; Proteins; Protocols documentation; Rattus; Reporter Genes; Research Design; Serpins; Shapes; Slice; Specificity; Techniques; Technology; Testing; Tissues; Toxic effect; Transfection; Transferrin Receptor; Translating; Tyrosine 3-Monooxygenase; Virus; base; brain cell; brain tissue; cell type; dopaminergic neuron; experience; immunogenic; immunogenicity; in vivo; nanometer; nanoparticle; nervous system disorder; non-viral gene therapy; novel; particle; plasmid DNA; promoter; protein expression; public health relevance; quantum; receptor; retinal rods; therapeutic gene; therapeutic transgene; transgene expression; translational study; uptake

DESCRIPTION (provided by applicant): The proposed studies will determine the feasibility of compacting plasmid DNA into "nanoparticles" and using these nanoparticles to deliver their payload into cells of the central nervous system (CNS) as a non-viral, gene therapy technique. DNA compacting techniques will be used to form nanoparticles containing condensed DNA plasmids with diameters in the range of 8-12 nanometers. Preliminary data shows that non-targeted DNA nanoparticles (DNPs) can be injected directly into brain and produce long-term transgene expression brain cells, primarily in astrocytes. The principle studies will focus on synthesizing DNPs that can be administered intracerebrally or systemically and increase the transfection efficiency in neurons. Recent studies have demonstrated that bioconjugated quantum rods can be targeted to the transferrin receptor (TfR) and traverse the blood-brain barrier (BBB). These particles have an average size and shape that are similar to our DNPs. As these are key determinants in particle transport, it is reasonable to postulate that DNPs modified to target the TfR may cross the BBB. In preliminary studies we have succeeded in targeting the DNPs to neurons of the hippocampus in brain slices utilizing a ligand to the serpin enzyme complex receptor (sec-R), C105Y, which has been shown to be a novel cell-penetrating peptide; and its receptor, sec-R, has been identified on neurons. Taken together, we hypothesize that unimodal targeting of DNPs to the TfR will enable them to cross the BBB and non-specifically transfect neural cells (neurons and/or glia), while bimodal targeting of DNPs to the TfR and sec-R will enable them to cross the BBB and predominantly transfect neuronal cells. To achieve dopamine neuron specificity, we will use plasmid constructs that contain the tyrosine hydroxylase (TH) promoter; TH is the rate limiting enzyme in the biosynthetic pathway for dopamine. The study design in this two year project will focus on 1) the synthesis of targeted DNPs, 2) transfection efficiency of targeted DNPs in primary neuronal cultures or cell lines, 3) in vivo tracking and transfection efficiency of targeted DNPs using MRI, microPET, and bioluminescent imaging techniques as well as immunohistochemical and protein analyses, 4) toxicity of targeted DNPs in brain, and 5) feasibility of repeated administration of DNPs while maintaining safe and stable transgene expression in brain. Successful results in these studies could then be applied to animal models of neurodegenerative disorders and possibly lead to translational studies for the treatment of neurological disorders, such as Parkinson's disease. PUBLIC HEALTH RELEVANCE: The proposed studies will use a novel nanoparticle technology that allows nucleic acids (DNA) to be compacted near their theoretical limit; this technology almost duplicates the compaction efficiency of viruses. We present preliminary data showing proof-of-concept that these nanoparticles can be used as a non-viral gene therapy for transfecting cells in the brain. In the proposed studies, we will conjugate moieties to the nanoparticles that will target receptors at the blood-brain barrier (BBB) allowing them to cross from the brain vasculature into the brain tissue. Another conjugated moiety will specifically target a novel rapid uptake mechanism that has been identified on neurons in order to increase the neuronal transfection efficient of the nanoparticles. Successful results from our animal studies could then be translated to human studies using these targeted nanoparticles as a form of non-viral, gene therapy to treat various neurological disorders.

描述（由申请人提供）：拟议的研究将确定将质粒 DNA 压缩成“纳米颗粒”并使用这些纳米颗粒将其有效负载递送到中枢神经系统（CNS）细胞中作为非病毒基因治疗技术的可行性。 DNA压缩技术将用于形成包含直径在8-12纳米范围内的浓缩DNA质粒的纳米粒子。初步数据表明，非靶向DNA纳米颗粒（DNP）可以直接注射到大脑中并产生长期转基因表达的脑细胞，主要是星形胶质细胞。主要研究将集中于合成可脑内或全身给药的 DNP，并提高神经元的转染效率。最近的研究表明，生物共轭量子棒可以靶向转铁蛋白受体（TfR）并穿过血脑屏障（BBB）。这些颗粒的平均尺寸和形状与我们的 DNP 相似。由于这些是颗粒运输的关键决定因素，因此可以合理地假设，经过修饰以靶向 TfR 的 DNP 可能会穿过 BBB。在初步研究中，我们利用丝氨酸蛋白酶抑制剂复合物受体 (sec-R) C105Y 的配体，成功将 DNP 靶向脑切片中的海马神经元，C105Y 已被证明是一种新型细胞穿透肽；其受体 sec-R 已在神经元上被发现。综上所述，我们假设 DNP 单峰靶向 TfR 将使它们能够穿过 BBB 并非特异性转染神经细胞（神经元和/或神经胶质细胞），而 DNP 双峰靶向 TfR 和 sec-R 将使它们能够穿过 BBB穿过 BBB 并主要转染神经元细胞。为了实现多巴胺神经元特异性，我们将使用含有酪氨酸羟化酶（TH）启动子的质粒构建体； TH 是多巴胺生物合成途径中的限速酶。这个为期两年的项目的研究设计将重点关注 1) 靶向 DNP 的合成，2) 原代神经元培养物或细胞系中靶向 DNP 的转染效率，3) 使用 MRI、microPET 进行体内跟踪和靶向 DNP 的转染效率，和生物发光成像技术以及免疫组织化学和蛋白质分析，4）靶向DNP在大脑中的毒性，以及5）重复施用DNP同时保持安全和稳定的转基因的可行性大脑中的表达。这些研究的成功结果可以应用于神经退行性疾病的动物模型，并可能导致治疗帕金森病等神经系统疾病的转化研究。 公共健康相关性：拟议的研究将使用一种新型纳米颗粒技术，使核酸（DNA）压缩到接近其理论极限；这项技术几乎复制了病毒的压缩效率。我们提供的初步数据表明，这些纳米颗粒可用作转染大脑细胞的非病毒基因疗法。在拟议的研究中，我们将结合部分到纳米颗粒上，这些纳米颗粒将靶向血脑屏障（BBB）上的受体，使它们能够从脑脉管系统穿过进入脑组织。另一个缀合部分将专门针对已在神经元上鉴定的新型快速摄取机制，以提高纳米颗粒的神经元转染效率。我们的动物研究的成功结果可以转化为人类研究，使用这些靶向纳米颗粒作为一种非病毒基因疗法来治疗各种神经系统疾病。