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.

摘要/摘要： 阿尔茨海默氏病（AD）是一种进行性神经退行性疾病，已成为最普遍的 人类痴呆症的形式，其中热磷酸化tau蛋白的形成和积累 据信淀粉样蛋白-β（Aβ）在AD发病机理中起关键作用。值得注意的是，最近的多阶段因果关系 阿尔茨海默氏病加速药物合作伙伴关系（AMP-AD）的网络分析确定 VGF是AD的唯一下调的关键驱动程序。 VGF是由大脑中促进的神经元合成的 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 在神经退行性疾病的学习，记忆和病理生理学中起关键作用。因此，这个 提案旨在通过靶向VGF开发一种新型的有效基因疗法来进行AD。主要挑战 AD的基因疗法领域是设计一个可以越过血脑屏障（BBB）和目标的安全载体 所需的细胞。我们建议开发与人VGF结合的创新和靶向纳米颗粒 通过在静脉内和鼻内后输送到大脑中，用于治疗AD的cDNA质粒（PVGF） 行政。鼻内路线可直接进入中枢神经系统治疗到大脑，因此是 基因通过绕过BBB来达到脑实质的有希望的非侵入性途径。我们会的 合成两种类型的纳米颗粒 - 脂质体纳米颗粒和ω-3脂肪酸接枝壳聚糖 纳米细胞。两种类型的纳米颗粒都将用靶向配体[转铁蛋白（TF），甘露糖（Man），，， 以及脑和神经元特异性细胞穿透肽（CPP）]。已经发现TF和Glut-1受体 存在于脑内皮细胞以及神经元的表面上。人是GLUT1的底物。在 此外，CPP将进一步改善纳米颗粒/纳米细胞在大脑中的渗透。因此，我们 设计脂质体纳米颗粒封装基因并用纳米颗粒表面的提案 TF，男人和CPP。同样，ω-3脂肪酸接枝壳聚糖也将通过与TF，MAN和MAN和 CPP。这些移植物聚合物将在水性环境中形成自组装的阳离子纳米细胞以提供 选择性靶向复合的PVGF对大脑。拟议研究的长期目标是设计一个 非病毒基因递送载体，通过静脉内和鼻内有效地递送PVGF 预防和治疗与衰老相关的认知十分序的管理。我们提出了三个 具体目的是实现拟议研究的长期目标。目标1。合成和表征 纳米颗粒/纳米细胞加载了PVGF：将使用CPP-脂质体纳米颗粒合成 薄膜水合技术，然后使用插入后插入TF-和man偶联胶束 技术。我们建议使用三个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）渗透素和（iii）狂犬病病毒糖蛋白（含有RVG-9R） 神经结合区域）。对于纳米细胞，我们将用ω-3脂肪酸合成壳聚糖的移植聚合物（GP）。 GP将被人，TF和CPP进一步移植，并以红外（IR）和NMR光谱法进行特征。 GP将在水性培养基中自组装以形成纳米细胞。纳米颗粒/纳米细胞将 评估粒径，Zeta电位，包封效率，细胞吸收和吸收机制， 转染效率，细胞毒性和溶血测定法。 PVGF负载的运输效率 纳米颗粒/纳米细胞将在通过组合初级的体外BBB模型上进行评估 人上皮细胞（HBMEC）和原代人星形胶质细胞（HA）。我们将评估 细胞培养BBB中转化效率，Aβ水平和tau磷酸化的纳米颗粒/纳米细胞 通过在24孔板中播种App SWE/IND或MAPT P301L过表达的SHSY5Y细胞的模型。分泌 在培养上清液中的Aβ40和Aβ42的以及细胞内裂解物的细胞内积累将是 由Elisa确定。细胞裂解物和培养基中的总tau和磷酸化的tau水平将是 通过Western印迹测定/ELISA测量。目标2。评估体内生物相容性，器官毒性， 不同年龄的野生型小鼠的药代动力学和VGF表达：建立成功的基因 AD疗法，我们将验证纳米颗粒/纳米细胞的生物相容性，器官毒性和 在静脉内或胸前施用在3中的野生型小鼠后，药代动力学（生物分布）3 几个月。此外，在3和24个月的野生型小鼠中，VGF基因输送将进一步验证 年龄。目标3。评估纳米颗粒/纳米胶介导的VGF基因的治疗作用 认知障碍和Aβ病理学的递送：建立与AD相关的成功基因疗法 表型和与年龄相关的认知下降，我们将检查VGF基因治疗的影响 在神经行为，突触功能和/或淀粉样病理学上的官能化纳米颗粒/纳米细胞。这 纳米颗粒将静脉内或胸腔内施用到淀粉样蛋白型5XFAD小鼠中 类型小鼠，将评估效果。对于人类的相关性，我们还将使用IPSC衍生的神经元和 来自AD患者的大脑器官，并评估对神经退行性的影响和Aβ/TAU病理的影响。 总体而言，我们预计拟议的研究将有助于高效的发展 非病毒基因输送系统，将PVGF输送到大脑中，以成功地用于AD和其他 神经退行性疾病。