Therapeutic Strategy to Treat Alzheimer's Disease by VGF Delivery into Brain

通过将 VGF 输送至大脑来治疗阿尔茨海默病的治疗策略

• 批准号: 10738951
• 负责人: Takahisa Kanekiyo
• 金额: $ 60.02万
• 依托单位: NORTH DAKOTA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-16 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10738951
• 关键词: Acceleration; Age; Age Months; Age-associated memory impairment; Aging; Alzheimer' s Disease; Alzheimer' s disease patient; Alzheimer' s disease therapy; American; Amyloid; Amyloid beta-42; Amyloid beta-Protein; Animals; Astrocytes; Attenuated; Binding; Biodistribution; Biological Assay; Blood - brain barrier anatomy; Brain; Bypass; Cell Culture Techniques; Cells; Cerebrum; Chitosan; Cognitive; Complementary DNA; Complex; Coupled; Dementia; Development; Drug Kinetics; Elderly; Encapsulated; Environment; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Film; Functional disorder; Gene Delivery; Genes; Goals; Growth; Hemolysis; Homeostasis; Human; Hydration status; Impaired cognition; In Vitro; Induced pluripotent stem cell derived neurons; Intranasal Administration; Intravenous; Learning; Ligands; Liposomes; Mannose; Measures; Mediating; Medicine; Memory; Mental disorders; Micelles; Modeling; Mus; NMR Spectroscopy; Nerve; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Omega-3 Fatty Acids; Organ; Organoids; Particle Size; Pathogenesis; Pathology; Pathway Analysis; Pathway interactions; Penetration; Peptides; Phenotype; Plasmids; Play; Polymers; Prevention; Research; Role; Route; SLC2A1 gene; Surface; Techniques; Testing; Tetanus Toxin; Therapeutic; Therapeutic Effect; Thinness; Toxic effect; Transfection; Transferrin; Western Blotting; Wild Type Mouse; addiction; aged; amyloid pathology; aqueous; beta amyloid pathology; biomaterial compatibility; blood-brain barrier crossing; brain endothelial cell; brain parenchyma; cohort; cytotoxicity; design; effective therapy; gene delivery system; gene therapy; hyperphosphorylated tau; improved; in vivo; infrared spectroscopy; innovation; intravenous administration; model design; mouse model; nanomicelles; nanoparticle; nervous system disorder; neurobehavior; neurogenesis; neuronal survival; non-viral gene delivery; novel; overexpression; particle; penetratin; rabies virus glycoprotein G; receptor; self assembly; synaptic function; synaptogenesis; tau Proteins; tau-1; tetanus toxin fragment C; uptake; vector; zeta potential

SUMMARY/ABSTRACT: Alzheimer’s disease (AD) is a progressive neurodegenerative disease that has emerged as the most prevalent form of late-life dementia in humans, in which the formation and accumulation of hyperphosphorylated tau protein and amyloid-β (Aβ) are believed to play key roles in AD pathogenesis. Of note, the recent multiscale causal network analysis in Accelerated Medicines Partnership for Alzheimer’s Disease (AMP-AD) cohort identified that VGF is the only downregulated key driver for AD. VGF is synthesized by neurons in the brain where it promotes growth and survival of neurons, and is involved in neurogenesis, synaptogenesis and energy homeostasis. VGF plays a critical role in learning, memory, and pathophysiology of neurodegenerative diseases. Therefore, this proposal aims to develop a novel effective gene therapy for AD by targeting VGF. The major challenge in the field of gene therapy for AD is to design a safe vector that can cross the blood brain barrier (BBB) and target the desired cells. We propose to develop innovative and targeted nanoparticles conjugated with human VGF cDNA plasmid (pVGF) for the treatment of AD by delivering into brain after intravenous and intranasal administration. Intranasal route provides a direct entrance of CNS therapeutics to the brain and therefore this is a promising non-invasive pathway for gene to reach the brain parenchyma by bypassing the BBB. We would synthesize two types of nanoparticles- liposomal nanoparticles and ω-3 fatty acid grafted chitosan based nanomicelles. Both types of nanoparticles will be grafted with targeting ligands [transferrin (Tf), mannose (MAN), and brain and neuron specific cell penetrating peptide (CPP)]. It has been found that the Tf and GLUT-1 receptors are present on the surface of brain endothelial cells as well as on neurons. MAN is a substrate for GLUT1. In addition, the CPP will further improve the penetration of nanoparticles/nanomicelles into brain. Therefore, we propose to design liposomal nanoparticles encapsulating gene and modifying the surface of nanoparticles with Tf, MAN and CPP. Similarly, ω-3 fatty acid grafted chitosan will be also modified by grafting with Tf, MAN and CPP. These graft polymers will form self-assembled cationic nanomicelles in aqueous environment to provide selective targeting of complexed pVGF to brain. The long-term goal of the proposed research is to design a non-viral gene delivery carrier for efficient delivery of pVGF to brain through intravenous and intranasal administrations for prevention and treatment of aging-related cognitive decile including AD. We propose three specific aims to accomplish the long-term goal of the proposed research. Aim 1. Synthesize and characterize nanoparticles/nanomicelles loaded with pVGF: The CPP-liposomal nanoparticles will be synthesized using thin film hydration technique followed by insertion of Tf- and MAN- coupled micelles using post-insertion technique. We propose to use three BBB and neuron specific CPPs: (i) a non-toxic fragment of tetanus toxin known as tetanus toxin C fragment (TTC), (ii) penetratin, and (iii) rabies virus glycoprotein (RVG-9R containing a nerve binding region). For nanomicelles, we will synthesize graft polymer (GP) of chitosan with ω-3 fatty acid. The GP will be further grafted with MAN, Tf and CPP, and characterize by infrared (IR) and NMR spectroscopy. The GP will self-assemble in aqueous media to form nanomicelles. The nanoparticles/nanomicelles will be evaluated for particle size, zeta potential, encapsulation efficiency, cell uptake and uptake mechanism(s), transfection efficiency, cell cytotoxicity, and hemolysis assay. The transport efficacy of pVGF loaded nanoparticles/nanomicelles will be evaluated across an in vitro BBB model designed by combining primary human epithelial cells (HBMECs) and primary human astrocytes (HA). We will evaluate the effect of nanoparticles/nanomicelles on transfection efficiency, Aβ levels and tau-phosphorylation in the cell culture BBB model by seeding the APP Swe/Ind or MAPT P301L-overexpressing SHSY5Y cells in 24-well plates. Secretion of Aβ40 and Aβ42 in the culture supernatant, as well as intracellular accumulation in cell lysates, will be determined by ELISA. Total tau and phosphorylated tau levels in the cell lysates and culture medium will be measured by Western blot assay/ELISA. Aim 2. Evaluate the in vivo biocompatibility, organ toxicity, pharmacokinetics and VGF expression in wild type mice of varying ages: To establish successful gene therapies for AD, we will validate the nanoparticles/nanomicelles for their biocompatibility, organ toxicity, and pharmacokinetics (biodistribution) after administering intravenously or intranasally into wild type mice at 3 months of age. In addition, the VGF gene delivery will be further validated in wild-type mice at 3 and 24 months of ages. Aim 3. Assess the therapeutic effects of the nanoparticle/nanomicelle-mediated VGF gene delivery on cognitive impairment and Aβ pathology: To establish successful gene therapies for AD-related phenotypes and age-related cognitive decline, we will examine effects of VGF gene therapy through the functionalized-nanoparticles/nanomicelles on neurobehaviors, synaptic functions and/or amyloid pathology. The nanoparticles will be administered intravenously or intranasally into amyloid model 5xFAD mice and aged wild- type mice, and the effects will be assessed. For human relevance, we will also use iPSC-derived neurons and cerebral organoids from AD patients and assess the effects on neurodegeneration and Aβ/tau pathologies. Collectively, we anticipate that the proposed study will contribute towards the development of high efficiency non-viral gene delivery system to deliver pVGF into brain for successful gene therapy for AD and other neurodegenerative diseases.

总结/摘要： 阿尔茨海默病（AD）是一种进行性神经退行性疾病， 人类晚期痴呆症的一种形式，其中过度磷酸化的tau蛋白的形成和积累 β淀粉样蛋白（amyloid-β，Aβ）在AD发病机制中起重要作用。值得注意的是，最近的多尺度因果关系 阿尔茨海默病加速药物伙伴关系（AMP-AD）队列的网络分析发现， VGF是AD唯一下调的关键驱动因素。VGF是由大脑中的神经元合成的， 神经元的生长和存活，并参与神经发生、突触发生和能量稳态。VGF 在学习、记忆和神经退行性疾病的病理生理学中起关键作用。因此本 本研究旨在开发一种以VGF为靶点的有效基因治疗AD的新方法。的主要挑战 AD的基因治疗领域是设计一种能够穿过血脑屏障（BBB）并靶向 想要的细胞。我们建议开发与人VGF结合的创新和靶向纳米颗粒 cDNA质粒（pVGF）经静脉和鼻腔给药后脑内给药治疗AD 局鼻内途径提供了CNS治疗剂直接进入脑的入口，因此这是有效的。 这是一种很有前途的非侵入性途径，使基因绕过血脑屏障到达脑实质。我们 合成两种类型纳米粒-脂质体纳米粒和ω-3脂肪酸接枝壳聚糖基纳米粒 纳米胶束。两种类型的纳米颗粒都将接枝有靶向配体[转铁蛋白（Tf），甘露糖（MAN）， 和脑和神经元特异性细胞穿透肽（CPP）]。已经发现Tf和GLUT-1受体 存在于脑内皮细胞和神经元的表面。MAN是GLUT 1的底物。在 此外，CPP将进一步改善纳米颗粒/纳米胶束进入脑的渗透。所以我们 设计了一种基因包封的脂质体纳米粒，并对纳米粒表面进行了修饰， Tf、MAN和CPP。同样，ω-3脂肪酸接枝壳聚糖也将通过接枝Tf、MAN和 CPP。这些接枝聚合物将在水性环境中形成自组装阳离子纳米胶束， 复合的pVGF选择性靶向脑。拟议研究的长期目标是设计一个 通过静脉内和鼻内有效递送pVGF至脑的非病毒基因递送载体 用于预防和治疗包括AD在内的衰老相关的认知十分位数的施用。我们提出了三 具体目标是实现拟议研究的长期目标。目标1.合成和表征 加载有pVGF的纳米颗粒/纳米胶束：CPP-脂质体纳米颗粒将使用以下方法合成： 薄膜水合技术，然后使用后插入插入Tf-和MAN-偶联胶束 法我们建议使用三种BBB和神经元特异性CPP：（i）破伤风毒素的无毒片段 称为破伤风毒素C片段（TTC），（ii）穿膜蛋白，和（iii）狂犬病病毒糖蛋白（含有RVG-9 R 神经结合区）。对于纳米胶束，我们将合成ω-3脂肪酸与壳聚糖的接枝聚合物（GP）。 将GP进一步与MAN、Tf和CPP接枝，并用红外光谱和核磁共振谱进行表征。 GP将在水性介质中自组装以形成纳米胶束。纳米颗粒/纳米胶束将是 评价颗粒大小、ζ电位、包封效率、细胞摄取和摄取机制， 转染效率、细胞毒性和溶血测定。pVGF负载的转运效率 纳米颗粒/纳米胶束将在体外BBB模型中进行评估，该模型通过组合主要的 人上皮细胞（HBMEC）和原代人星形胶质细胞（HA）。我们将评估 纳米颗粒/纳米胶束对细胞培养BBB中转染效率、Aβ水平和tau磷酸化的影响 通过在24孔板中接种APP Swe/Ind或MAPT P301 L-过表达的SHSY 5 Y细胞来构建模型。分泌 Aβ40和Aβ42在培养上清液中的含量以及细胞裂解物中的细胞内蓄积将被 通过ELISA测定。将测定细胞裂解物和培养基中的总tau和磷酸化tau水平。 通过蛋白质印迹分析/ELISA测量。目标2.评价体内生物相容性、器官毒性， 在不同年龄的野生型小鼠中的药代动力学和VGF表达： 我们将验证纳米颗粒/纳米胶束的生物相容性，器官毒性， 在3 ℃下静脉内或鼻内给药至野生型小鼠后的药代动力学（生物分布） 月龄。此外，VGF基因递送将在3个月和24个月时在野生型小鼠中进一步验证 年龄。目标3.评估纳米颗粒/纳米胶束介导的VGF基因的治疗效果 对认知障碍和Aβ病理学的治疗：建立成功的AD相关基因疗法 表型和与年龄相关的认知能力下降，我们将检查VGF基因治疗的效果，通过 功能化纳米颗粒/纳米胶束对神经行为、突触功能和/或淀粉样蛋白病理学的影响。的 纳米颗粒将静脉内或鼻内给予淀粉样蛋白模型5xFAD小鼠和老龄野生小鼠， 型小鼠，并评估其效果。对于人类相关性，我们还将使用iPSC衍生的神经元， 从AD患者的脑类器官中提取并评估对神经变性和Aβ/tau病理学的影响。 总的来说，我们预计拟议的研究将有助于发展高效率的 将pVGF递送到脑中用于AD和其他疾病的成功基因治疗的非病毒基因递送系统 神经退行性疾病