Enhanced APOE2 Expression into Brain for Therapeutic Strategy for Alzheimer's Disease

增强 APOE2 在大脑中的表达，用于阿尔茨海默病的治疗策略

• 批准号: 10208342
• 负责人: Takahisa Kanekiyo
• 金额: $ 143.76万
• 依托单位: NORTH DAKOTA STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-05-15 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10208342
• 关键词: APP-PS1; Abeta clearance; Age; Age-Months; Age-associated memory impairment; Aging; Alleles; Alzheimer' s Disease; Alzheimer' s disease patient; Alzheimer' s disease therapy; American; Amyloid; Amyloid beta-Protein; Amyloid deposition; Apolipoprotein E; Astrocytes; Biodistribution; Biological Assay; Blood - brain barrier anatomy; Brain; Cells; Centenarian; Chitosan; Coculture Techniques; Cognitive; Coupled; Dementia; Development; Disease; Drug Kinetics; Elderly; Encapsulated; Endothelium; Environmental Risk Factor; Event; Film; Gene Delivery; Gene-Modified; Genes; Genetic; Genotype; Goals; Hemolysis; Homeostasis; Human; Hydration status; Impaired cognition; In Vitro; Individual; Intercellular Fluid; Knock-out; Knockout Mice; Late Onset Alzheimer Disease; Light; Lipids; Liposomes; Measures; Mediating; Micelles; Microdialysis; Modeling; Mus; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Non-Viral Vector; Organ; Particle Size; Pathogenesis; Pathology; Pathway interactions; Penetration; Peptides; Phenotype; Plasma; Play; Porifera; Predisposition; Prevention; Property; Proteins; Protons; Research; Role; Sampling; Surface; Synaptic plasticity; System; TFRC gene; Techniques; Tetanus Toxin; Therapeutic; Therapeutic Effect; Thinness; Toxic effect; Transfection; Transferrin; Wild Type Mouse; abeta accumulation; abeta deposition; aged; base; biomaterial compatibility; blood-brain barrier function; brain endothelial cell; clinical application; clinically relevant; cognitive function; cytotoxicity; design; effective therapy; gene delivery system; gene therapy; genetic risk factor; genetic variant; human old age (65+); improved; in vivo; lipid nanoparticle; model design; mouse model; nanoparticle; nervous system disorder; neurobehavior; neuroblastoma cell; neurofilament; non-viral gene delivery; overexpression; penetratin; peptide I; plasmid DNA; protective effect; rabies virus glycoprotein G; receptor; synaptic function; tau Proteins; tau phosphorylation; tau-1; tetanus toxin fragment C; therapeutic gene; therapeutically effective; transcytosis; 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. The accumulation, aggregation, and deposition of amyloid-β (Aβ) in the brain are central events in AD pathogenesis. Despite intense effort, an effective therapy for AD has yet to be established. While multiple genetic and environmental factors are involved in AD pathogenesis, the ε4 allele of the APOE gene encoding apolipoprotein E (APOE) is the strongest genetic risk factor for late-onset AD among the three human APOE genotypes (ε2, ε3, ε4). In humans, Aβ deposition is more pronounced in APOE4 carriers compared with non-carriers in both AD patients and aged healthy individuals. APOE plays a critical role in maintaining synaptic plasticity and neuronal function by controlling lipid homeostasis, with the APOE2 allele having a superior function. The ε2 allelic variant has been found to be more prevalent among centenarians and associated with decreased susceptibility to AD. Studies on the role of the APOE2 in relation to AD suggest that APOE2 is neuroprotective and positively associated with cognitive functions in aging. Therefore, increasing APOE2 levels in the brain is predicted to be an effective therapeutic strategy for AD. Development of successful strategies for treating these disorders is limited due to the protective function of blood brain barrier (BBB). Gene therapy possesses a broad potential for the treatment of numerous neurological diseases, including AD. However, the major challenge in the field of gene therapy is the design of safe non-viral vectors that can cross the BBB. The transferrin (Tf) receptors are present on the surface of brain endothelial cells. The lipid nanoparticles can be surface modified with Tf protein for targeting the brain endothelial receptors and conjugated to brain specific cell penetrating peptide (CPP) for improving their internalization into brain by overcoming receptor saturation. Therefore, we propose to design near neutral, PEGylated liposomal nanoparticles encapsulating gene and modifying the surface of nanoparticles with Tf and CPP. Furthermore, the transfection properties of chitosan will be utilized for improving the transfection of gene by facilitating endosomal escape via the proton-sponge mechanism inside the cells. The long-term goal of the proposed research is to design a non- viral gene delivery carrier for efficient delivery of plasmid DNA encoding APOE2 (pAPOE2) to brain for prevention and treatment of AD. We propose three specific aims to accomplish the long-term goal of the proposed research: Aim 1. Synthesize and characterize liposomal nanoparticles loaded with chitosan-pAPOE2 polyplexes: The brain specific CPP-liposomes will be synthesized using thin film hydration technique followed by insertion of Tf coupled micelles using post-insertion technique. We propose to use five BBB specific CPPs: (i) CGN (d- CGNHPHLAKYNGT); (ii) RDP (KSVRTWNEIIPSKGCLRVGGRCHPHVNGGGRRRRRRRRR; (iii) Rabies Virus Glycoprotein RVG-9R, (iv) a non-toxic fragment of tetanus toxin, tetanus toxin C fragment (TTC), and (v) penetratin. The liposomal nanoparticles 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 APOE2 loaded liposomal nanoparticles will be evaluated across an in vitro BBB model designed by combining human cerebral microvascular endothelial cells (hCMEC/D3), human astrocytes and APP Swe/Ind- or MAPT P301L-overexpressing human neuroblastoma cells (SHSY5Y). We will also determine the effect of liposomal nanoparticles on Aβ levels and Tau phosphorylation in the medium and cell lysates from the co-culture system. Aim 2. Evaluate the in vivo biocompatibility, organ toxicity, pharmacokinetics and APOE2 expression in mice of varying ages: To establish successful gene therapies for AD, we will validate the Tf-CPP-liposomal nanoparticles for their biocompatibility, organ toxicity, and pharmacokinetics (biodistribution) in wild type mice at 3 months of age. In addition, the APOE2 gene delivery will be further validated in APOE-knockout mice at 3 and 24 months of ages. Aim 3. Assess the therapeutic effects of the functionalized-liposome-mediated APOE2 gene delivery on cognitive impairment and Aβ and tau pathology in vivo: To establish successful gene therapies for AD-related phenotypes and age-related cognitive decline, we will examine effects of APOE2 gene therapy through the functionalized-liposomes on neurobehaviors, synaptic functions and/or amyloid and tau pathology. The liposomes will be injected into amyloid model APP/PS1 mice (3 and 6 months old), tau model PS19 mice (3 and 6 months old) and aged wild-type mice (12 and 24 months old), and the effects will be assessed 3 months after the administration. For clinical relevance, we will measure neurofilament light chain (NfL) levels in their plasma samples to assess effects on neurodegeneration. In addition, we will also measure plasma concentrations of Aβ and p-tau in respective mouse models. In addition, interstitial fluid Aβ clearance will be analyzed using in vivo microdialysis in the APP/PS1 mice at 3 months of age 1 month after the administration. Collectively, we anticipate that the proposed study will contribute towards the development of high efficiency non-viral gene delivery system to cross the BBB for successful gene therapy for neurological disorders and determine protective effects of increasing brain APOE2 on AD-related conditions.

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 人类后期痴呆的形式。淀粉样β（Aβ）在 大脑是AD发病机理中的中心事件。尽管付出了强烈的努力，但对广告的有效疗法尚未 已确立的。尽管AD发病机理涉及多个遗传和环境因素，但ε4等位基因 编码载脂蛋白E（APOE）的APOE基因是迟到AD的强遗传风险因素 三种人APOE基因型（ε2，ε3，ε4）。在人类中，Aβ沉积在APOE4载体中更为明显 与AD患者和老年健康个体的非携带者相比。 Apoe在 通过控制脂质体内稳态来维持突触可塑性和神经元功能，并使用APOE2等位基因 具有出色的功能。发现ε2等位基因变体在一半的人中更为普遍 与AD敏感性降低有关。关于APOE2在AD中的作用的研究表明 APOE2是神经保护性的，与衰老中的认知功能呈正相关。因此，增加 预计大脑中的APOE2水平是AD的有效治疗策略。成功的发展 由于血脑屏障的保护功能（BBB），治疗这些疾病的策略受到限制。基因 治疗具有广泛的治疗包括AD在内的神经系统疾病的潜力。 但是，基因治疗领域的主要挑战是可以穿越的安全非病毒载体的设计 BBB。转铁蛋白（TF）受体存在于脑内皮细胞的表面上。脂质 纳米颗粒可以用TF蛋白进行表面修饰，以靶向脑内皮受体并结合。 通过克服脑特异性细胞穿透肽（CPP），以改善其内在化。 接收器满意度。因此，我们建议设计接近中性的，卵形的脂质体纳米颗粒 用TF和CPP封装基因并修饰纳米颗粒的表面。此外，翻译 壳聚糖的特性将用于通过促进内体逃生通过 细胞内部的质子海绵机制。拟议研究的长期目标是设计非 病毒基因递送载体可有效递送编码APOE2（Papoe2）的质粒DNA到大脑以进行预防 和AD的处理。我们提出了三个具体目标，以实现拟议研究的长期目标： AIM 1。合成并表征带有壳聚糖Papoe2多流线的脂质体纳米颗粒： 大脑特异性CPP脂质体将使用薄膜水合技术合成，然后插入 使用后插入技术的TF耦合胶束。我们建议使用五个BBB特异性CPPS：（i）CGN（d- cgnhphlakyngt）; （ii）RDP（ksvrtwneiipskgclrvggrchphvngggrrrrrrrrrrrr;（iii）狂犬病病毒 糖蛋白RVG-9R，（iv）破伤风毒素，破伤风毒素C片段（TTC）和（V）的无毒片段 穿透性。将评估脂质体纳米颗粒的粒径，Zeta电位，封装 效率，细胞摄取和摄取机制，翻译效率，细胞毒性和溶血测定法。 将在体外BBB模型上评估APOE2负载的脂质体纳米颗粒的运输效率 通过结合人脑微血管内皮细胞（HCMEC/D3），人星形胶质细胞和 APP SWE/IND-或MAPT P301L过表达的人神经母细胞瘤细胞（SHSY5Y）。我们还将确定 脂质体纳米颗粒对培养基和细胞裂解物中Aβ水平和Tau磷酸化的影响 共培养系统。目标2。评估体内生物相容性，器官毒性，药代动力学和 不同年龄的小鼠中的APOE2表达：为了建立AD成功的基因疗法，我们将验证 TF-CPP-脂质体纳米颗粒的生物相容性，器官毒性和药代动力学 （生物分布）在3个月大的野生型小鼠中。此外，APOE2基因输送将进一步 在3和24个月的年龄中在Apoe-Knockout小鼠中进行了验证。目标3。评估的治疗作用 官能化 - 脂体介导的APOE2基因在认知障碍和Aβ和TAU上的递送 体内病理学：建立成功的广告相关表型和与年龄相关的认知的成功基因疗法 下降，我们将通过功能化 - 脂质体对APOE2基因治疗的影响 神经行为，突触功能和/或淀粉样蛋白和TAU病理学。脂质体将注入淀粉样蛋白 型号APP/PS1小鼠（3和6个月大），TAU型号PS19小鼠（3和6个月大）和老化的野生型小鼠 （12个月和24个月大），该影响将在管理后3个月进行评估。对于临床相关性， 我们将测量其血浆样品中的神经丝轻链（NFL）水平，以评估对影响的影响 神经变性。此外，我们还将在相对小鼠中测量Aβ和P-TAU的血浆浓度 型号。此外，将使用APP/PS1中的体内微透析分析间质流体Aβ清除率 政府后3个月1个月的小鼠。总体而言，我们预计拟议的研究将 有助于发展高效率非病毒基因输送系统以跨越BBB的发展 成功用于神经系统疾病的基因疗法，并确定增加脑APOE2的受保护作用 在广告相关条件下。