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

摘要/摘要： 阿尔茨海默病(AD)是一种进行性神经退行性疾病，已成为最常见的 人类晚年痴呆的一种形式，其中过度磷酸化的tau蛋白的形成和积累 而淀粉样蛋白-β(A-β)被认为在AD的发病机制中起关键作用。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)队列中的网络分析发现 VGF是AD的唯一下调的关键驱动因素。VGF是由大脑中的神经元合成的，在那里它促进 神经元的生长和存活，并参与神经发生、突触形成和能量稳态。VGF 在学习、记忆和神经退行性疾病的病理生理学中起着关键作用。因此，这 该提案旨在通过靶向VGF来开发一种新的有效的阿尔茨海默病基因疗法。面临的主要挑战 阿尔茨海默病的基因治疗领域是设计一种能够跨越血脑屏障和靶点的安全载体 所需的单元格。我们建议开发与人血管内皮生长因子偶联的创新和靶向纳米颗粒 经静脉和鼻腔给药治疗阿尔茨海默病的基因工程载体 行政管理。鼻腔途径提供了中枢神经系统治疗药物直接进入大脑的途径，因此这是 一种有希望的非侵入性途径，使基因绕过血脑屏障到达脑实质。我们会 两种纳米粒子的合成--脂质体纳米粒和ω-3脂肪酸接枝壳聚糖纳米粒 纳米胶束。这两种类型的纳米颗粒都将与靶向配体[转铁蛋白(Tf)，甘露糖(MAN)， 和脑和神经元特异性细胞穿透肽(CPP)]。已发现Tf和GLUT-1受体 存在于脑内皮细胞和神经元的表面。人是GLUT1的底物。在……里面 此外，CPP还将进一步提高纳米颗粒/纳米胶束在大脑中的渗透率。因此，我们 建议设计包埋基因的脂质体纳米粒并对其表面进行修饰 Tf、MAN和CPP。类似地，ω-3脂肪酸接枝壳聚糖也将通过与TF、MAN和 CPP。这些接枝聚合物将在水环境中形成自组装的阳离子纳米胶束，从而提供 将复杂的pVGF选择性靶向脑内。拟议研究的长期目标是设计一种 非病毒基因载体通过静脉和鼻腔高效地将pVGF输送到脑内 预防和治疗包括阿尔茨海默病在内的与衰老相关的认知衰退的管理。我们提出三个建议 具体目标是实现拟议研究的长期目标。目的1.合成和表征 负载pVGF的纳米粒/纳米胶束：CPP-脂质体纳米粒的合成将使用 薄膜水化技术，然后使用插入后插入Tf-和人偶联胶束 技术。我们建议使用三种血脑屏障和神经元特异性CPP：(I)破伤风毒素的无毒片段 称为破伤风毒素C片段(TTC)，(Ii)穿透性，和(Iii)狂犬病病毒糖蛋白(RVG-9R，包含 神经结合区)。对于纳米胶束，我们将合成壳聚糖与ω-3脂肪酸的接枝聚合物(GP)。 该GP将进一步接枝MAN、Tf和CPP，并用红外光谱和核磁共振光谱对其进行表征。 GP将在水介质中自组装形成纳米胶束。纳米粒子/纳米胶束将是 评价颗粒大小、Zeta电位、包封率、细胞摄取和摄取机制(S)， 转染率、细胞毒性和溶血试验。PVGF载药后的转运效果 纳米粒子/纳米胶束将通过体外血脑屏障模型进行评估，该模型由初级 人上皮细胞(HBMECs)和原代人星形胶质细胞(HA)。我们将评估这些措施的效果 纳米粒/纳米胶束对人血脑屏障细胞转染率、A-β水平和tau-磷酸化的影响 将高表达SHSY5Y的APP Swe/Ind或MAPT P301L细胞接种于24孔板中建立模型。分泌物 培养上清液中Aβ40和Aβ42的数量，以及细胞裂解物中的细胞内积累 用酶联免疫吸附试验测定。细胞裂解产物和培养液中的总tau和磷酸化tau水平将是 采用Western印迹分析/ELISA法进行检测。目的2.评价其体内生物相容性、器官毒性、 成功基因在不同年龄野生型小鼠体内的药代动力学和VGF表达 对于AD的治疗，我们将验证纳米粒/纳米胶束的生物兼容性、器官毒性和 野生型小鼠静脉或鼻腔给药后的药代动力学(生物分布) 几个月大的孩子。此外，VGF基因的传递将在3个月和24个月的野生型小鼠身上得到进一步验证 年代久远。目的3.评价纳米粒/纳米胶束介导的血管生长因子基因的治疗效果 认知障碍与β病理：为AD相关疾病建立成功的基因治疗 表型和年龄相关的认知功能减退，我们将通过 功能化纳米颗粒/纳米胶束对神经行为、突触功能和/或淀粉样蛋白病理的影响。这个 纳米粒将通过静脉或鼻腔注射到淀粉样蛋白模型5xFAD小鼠和老年野生小鼠体内。 输入小鼠，效果将得到评估。为了与人类相关，我们还将使用IPSC衍生的神经元和 并评估其对神经退行性变和Aβ/tau病理的影响。 总的来说，我们预计拟议的研究将有助于发展高效率 非病毒基因递送系统将pVGF送入脑内，成功治疗AD等 神经退行性疾病。

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.