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： 阿尔茨海默病 (AD) 是一种进行性神经退行性疾病，已成为最流行的疾病 人类晚年痴呆的一种形式，β-淀粉样蛋白 (Aβ) 在体内积累、聚集和沉积。 大脑是 AD 发病机制的核心事件，尽管付出了巨大的努力，但尚未找到针对 AD 的有效治疗方法。 虽然 AD 发病机制涉及多种遗传和环境因素，但 ε4 等位基因 编码载脂蛋白 E (APOE) 的 APOE 基因是晚发性 AD 最强的遗传风险因素 三种人类 APOE 基因型（ε2、ε3、ε4） 在人类中，Aβ 沉积在 APOE4 携带者中更为明显。 与非携带者相比，APOE 在 AD 患者和老年健康个体中都发挥着关键作用。 通过 APOE2 等位基因控制脂质稳态来维持突触可塑性和神经元功能 已发现 ε2 等位基因变体在百岁老人中更为普遍。 APOE2 在 AD 中的作用研究表明，与 AD 易感性降低有关。 APOE2 具有神经保护作用，并且与衰老过程中的认知功能呈正相关。 大脑中的 APOE2 水平预计将成为成功开发 AD 的有效治疗策略。 由于血脑屏障（BBB）基因的保护功能，治疗这些疾病的策略受到限制。 疗法在治疗包括 AD 在内的多种神经系统疾病方面具有广泛的潜力。 然而，基因治疗领域的主要挑战是设计能够交叉的安全非病毒载体。 BBB。转铁蛋白（Tf）受体存在于脑内皮细胞的表面。 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缀合 大脑特异性细胞穿透肽（CPP），通过克服 因此，我们建议设计近中性的聚乙二醇化脂质体纳米颗粒。 封装基因并用Tf和CPP修饰纳米粒子的表面此外，转染。 壳聚糖的特性将用于通过促进内体逃逸来改善基因的转染 该研究的长期目标是设计一种非质子海绵机制。 病毒基因递送载体，用于将编码 APOE2 (pAPOE2) 的质粒 DNA 有效递送至大脑进行预防 我们提出了三个具体目标来实现拟议研究的长期目标： 目标 1. 合成并表征负载有壳聚糖-pAPOE2 复合物的脂质体纳米颗粒： 大脑特异性 CPP 脂质体将使用薄膜水合技术合成，然后插入 我们建议使用五种 BBB 特异性 CPP：(i) CGN (d-)。 CGNHPHLAKYNGT)；(ii) RDP (KSVRTWNEIIPSKGCLRVGGRCHPHVNGGGRRRRRRRRR；(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) 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、转染效率、细胞毒性和溶血测定。 将通过体外 BBB 模型评估负载 APOE2 的脂质体纳米颗粒的转运功效 结合人脑微血管内皮细胞（hCMEC/D3）、人星形胶质细胞和 我们还将确定 APP Swe/Ind- 或 MAPT P301L- 过表达的人神经母细胞瘤细胞 (SHSY5Y)。 脂质体纳米粒子对培养基和细胞裂解物中 Aβ 水平和 Tau 磷酸化的影响 目标 2. 评估体内生物相容性、器官毒性、药代动力学和 APOE2 在不同年龄小鼠中的表达：为了建立成功的 AD 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个月龄的野生型小鼠中，APOE2基因传递将进一步进行。 在 3 个月和 24 个月大的 APOE 敲除小鼠中得到验证。目标 3. 评估治疗效果。 功能化脂质体介导的 APOE2 基因递送对认知障碍以及 Aβ 和 tau 的影响 体内病理学：建立针对 AD 相关表型和年龄相关认知的成功基因疗法 下降，我们将通过功能化脂质体检查 APOE2 基因治疗对 神经行为、突触功能和/或淀粉样蛋白和 tau 蛋白病理学将脂质体注射到淀粉样蛋白中。 APP/PS1 模型小鼠（3 和 6 个月大）、tau 模型 PS19 小鼠（3 和 6 个月大）和老年野生型小鼠 （12 和 24 个月大），给药后 3 个月评估效果以了解临床相关性。 我们将测量他们血浆样本中的神经丝轻链 (NfL) 水平，以评估对 此外，我们还将测量各小鼠中 Aβ 和 p-tau 的血浆浓度。 此外，将使用 APP/PS1 中的体内微透析来分析间质液 Aβ 清除率。 总的来说，我们预计所提出的研究将在给药后 1 个月时对 3 个月大的小鼠进行。 为开发高效非病毒基因传递系统以跨越 BBB 做出贡献 成功治疗神经系统疾病的基因疗法并确定增加大脑 APOE2 的保护作用 与 AD 相关的情况。