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

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。淀粉样蛋白-β(A-β)在人体内的积累、聚集和沉积 脑是阿尔茨海默病发病的中心事件。尽管付出了巨大的努力，但治疗阿尔茨海默病的有效方法尚未找到 已经成立了。阿尔茨海默病的发病与多种遗传和环境因素有关，ε-4等位基因在AD发病中起重要作用。 编码载脂蛋白E(ApoE)的载脂蛋白E基因是晚发性阿尔茨海默病最强的遗传危险因素 3种人载脂蛋白E基因(ε2、ε3、ε4)。在人类中，β沉积在载脂蛋白4携带者中更加明显 AD患者和老年健康人与非携带者的比较。APOE在以下方面发挥了关键作用 APOE2等位基因通过控制脂质稳态来维持突触可塑性和神经元功能 具有优越功能的。已发现ε2等位基因变异在百岁老人中更普遍， 与AD易感性降低有关。关于载脂蛋白2与阿尔茨海默病相关的研究表明 APOE2具有神经保护作用，与衰老时的认知功能呈正相关。因此，增加 脑内APOE2水平被预测为治疗AD的有效策略。成功的发展 由于血脑屏障(BBB)的保护作用，治疗这些疾病的策略有限。基因 治疗方法在治疗包括阿尔茨海默病在内的多种神经系统疾病方面具有广阔的潜力。 然而，基因治疗领域的主要挑战是设计可以交叉的安全的非病毒载体。 BBB。转铁蛋白(Tf)受体存在于脑内皮细胞表面。脂类 纳米粒表面可修饰转铁蛋白，用于靶向脑血管内皮细胞受体并进行偶联 通过克服脑特异性细胞穿透肽(CPP)促进其内化进入大脑 受体饱和。因此，我们建议设计近中性的聚乙二醇化脂质体纳米粒 对基因进行包裹，并用转移因子和环磷酰胺修饰纳米颗粒的表面。此外，转染法 壳聚糖的性质将被用来通过促进内质粒逃逸来提高基因的转染率 细胞内的质子-海绵机制。拟议研究的长期目标是设计一种非 高效脑内携带APOE2(PAPOE2)基因的病毒载体 以及AD的治疗。我们提出了三个具体目标，以实现拟议研究的长期目标： 目的1.制备壳聚糖-pAPOE2复合体脂质体纳米粒并对其进行表征： 脑特异性CPP脂质体将采用薄膜水合技术合成，然后插入 使用后插入技术的TF偶联胶束。我们建议使用五种特定于BBB的CPP：(I)CGN(d- CGNHPHLAKYNGT)；(Ii)RDP(KSVRTWNEIIPSKGCLRVGGGRCHPHVNGGGRRRRRRRRRR；(Iii)狂犬病病毒 糖蛋白RVG-9R，(Iv)破伤风毒素的无毒片段，破伤风毒素C片段(TTC)，和(V) 穿透力强。脂质体纳米粒的颗粒大小、Zeta电位、包封性将进行评估 效率、细胞摄取和摄取机制(S)、转染率、细胞毒性和溶血试验。 APOE2脂质体纳米粒的转运效率将通过体外血脑屏障模型进行评估 由人脑微血管内皮细胞(hCMEC/D3)、人脑星形胶质细胞和 APP-Swe/Ind-或MAPT P301L-高表达人神经母细胞瘤细胞(SHSY5Y)我们还将确定 脂质体纳米粒对培养上清液和细胞裂解物中Aβ水平和Tau磷酸化的影响 共同文化体系。目的：评价该制剂的体内生物相容性、器官毒性、药代动力学和生物相容性。 APOE2在不同年龄小鼠中的表达：为了建立成功的AD基因治疗，我们将验证 Tf-CPP-脂质体纳米粒的生物相容性、器官毒性和药代动力学 (生物分布)在3月龄野生型小鼠体内。此外，APOE2基因的传递将进一步 在3个月和24个月龄的APOE基因敲除小鼠中得到验证。目的3.评估该疗法的治疗效果 功能化脂质体介导载脂蛋白2基因治疗认知障碍及Aβ和tau的研究 体内病理学：为AD相关表型和年龄相关认知建立成功的基因治疗 ，我们将通过功能化脂质体检测APOE2基因治疗的效果。 神经行为、突触功能和/或淀粉样蛋白和tau病理。脂质体将被注入淀粉样蛋白 模型APP/PS1小鼠(3月龄和6月龄)、tau模型PS19小鼠(3月龄和6月龄)和老龄野生型小鼠 (12个月和24个月大)，效果将在服药3个月后评估。就临床意义而言， 我们将测量他们血浆样本中神经丝轻链(NFL)的水平，以评估对 神经退行性变。此外，我们还将分别测量小鼠血浆中Aβ和p-tau的浓度 模特们。此外，将在APP/PS1中使用活体微透析法分析间质性液体A的β清除情况 3个月龄小鼠，给药后1个月。总的来说，我们预计拟议的研究将 有助于开发高效的非病毒基因传递系统，以跨越血脑屏障 神经系统疾病的成功基因治疗及增加脑内载脂蛋白2的保护作用 与AD相关的病症。

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.