Brain Neurotropic Growth Factor Delivery to Prevent and Treat Alzheimer’s Disease

摘要： 神经退行性疾病已成为老年人痴呆症的最常见原因，并已 据报道，65岁以上的美国人中约有5%受到影响，80岁以上的人中有20%。有几个 2010年全球有3600万痴呆症患者，到2030年增加到6600万，到2030年增加到1.15亿 2050年。2010年，全球痴呆症的成本为6,040亿美元。这是全球GDP的1%，很可能这些 费用将随着痴呆症患者数量的增加而成比例增加。基因疗法已被确认为 具有治疗包括阿尔茨海默病在内的多种神经系统疾病的广泛潜力 (Ad)。AD是一种进行性神经退行性疾病，是最常见的痴呆症，由 毒性淀粉样蛋白-β(A-β)多肽在大脑中的蓄积，从而开发出有效的治疗方法 都是令人向往的。然而，基因治疗领域的主要挑战是设计安全的非病毒 可以穿越血脑屏障(BBB)的载体。已发现转铁蛋白受体是 存在于脑内皮细胞表面。脂质体是以脂质为基础的纳米颗粒，可以在表面 转铁蛋白修饰脑内皮细胞受体并与细胞结合 穿透性多肽(CPP)通过克服受体饱和而改善其在脑内的内化。 因此，我们建议将脂质体与两种配体(1)受体靶向蛋白(Tf)和(2)偶联。 一个CPP。因此，我们将通过使用TF和CPP修饰表面来设计近中性的聚乙二醇化脂质体。 此外，还将利用低分子量壳聚糖的转染性来改善 通过促进细胞内的内质粒逃逸而进行的基因转导。建议的长期目标是 研究旨在设计一种基因递送载体，用于将神经生长因子(NGF)高效地输送到脑内 防治阿尔茨海默病。我们提出了以下三个具体目标：(1)。合成和合成 壳聚糖-PDNA复合体转铁蛋白和环磷酰胺偶联脂质体的表征 用薄膜水合技术合成，然后用后胶束插入TF偶联胶束。 插入技术。根据理化性质，我们建议使用三种类型的CPP [阳离子亲水性(HIV-TAT、PasR8和R9F2)、阳离子两亲性(pVEC、穿透性和Mellittin) 和疏水性(PFVYLI、五肽QLPVM和Kaposi成纤维细胞生长因子衍生肽)。 目的基因(pGFP或pNGF)将与壳聚糖复合，以改善其转染性。 脂质体。将对脂质体的颗粒大小、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。(2)。至 用二维血脑屏障模型评价脂质体的跨屏障转运效率 通过脑共培养设计的二维血脑屏障模型评价脂质体的转运效率 培养插入物两侧的内皮细胞和原代星形胶质细胞。有必要开发一种高效的 在体外BBB模型中，向初级神经元的运输可以通过内皮屏障来调节。(3)。 目的：评价Tf-CPP-脂质体在体内的分布和转染率，并考察其在体内的作用。 Tf-CPP-脂质体介导的淀粉样变性小鼠基因治疗：建立成功的基因治疗方法 对于AD，我们将验证通过上述目的获得的TF-CPP-脂质体的分布、毒性和 尾部注射给药对小鼠脑内的转染率。最后，我们将检查NGF的影响 Tf-CPP脂质体基因治疗对淀粉样蛋白病理、神经发生、突触功能和 淀粉样蛋白模型APP/PS1小鼠不同年龄的神经行为。我们预期拟议的研究将 为开发高效的非病毒基因传递载体跨越血脑屏障和 将pNGF基因转移到期望的靶点，以成功地进行AD的基因治疗。

SUMMARY: Neuro-degenerative diseases have become the most common cause of dementia among the elderly and have been reported to affect about 5% of Americans over age of 65, and 20% over the age of 80 years. There were 36 million people living with dementia worldwide in 2010, increasing to 66 million by 2030 and 115 million by 2050. In 2010, the global cost of dementia was $604 billion. This is 1% of global GDP and it is likely that these costs will increase in proportion to the number of people with dementia. Gene therapy has been identified to possess a broad potential for the treatment of numerous neurological diseases, including Alzheimer’s disease (AD). AD is a progressive neurodegenerative disease and the most common form of dementia caused by accumulation of toxic amyloid-β (Aβ) peptides in the brain, in which the development of effective therapies have been desired. However, the major challenge in the field of gene therapy is the design of safe non-viral vectors that can cross the blood brain barrier (BBB). It has been found that the transferrin receptors are present on the surface of brain endothelial cells. The liposomes, lipid based nanoparticles, can be surface modified with transferrin (Tf) protein for targeting the brain endothelial receptors and conjugated to cell penetrating peptide (CPP) for improving their internalization into brain by overcoming receptor saturation. Therefore, we propose to conjugate the liposomes with two ligands (1) a receptor targeting protein (Tf) and (2) a CPP. Thus we will design near-neutral, PEGylated liposomes by modifying the surface with Tf and CPP. Furthermore, the transfection properties of low molecular weight chitosan will be utilized for improving the transfection of gene by facilitating endosomal escape inside the cells. The long term goal of the proposed research is to design a gene delivery carrier for efficient delivery of Nerve Growth Factor (NGF) to brain for prevention and treatment of AD. We propose the following three specific aims: (1). To synthesize and characterize Tf and CPP coupled liposomes loaded with chitosan-pDNA polyplexes: The 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 three types of CPPs based on the physico-chemical properties [cationic hydrophilic (HIV-Tat, PasR8 and R9F2), cationic amphiphilic (pVec, penetratin and Mellittin) and hydrophobic (PFVYLI, pentapeptide QLPVM and Kaposi Fibroblast Growth Factor derived peptide). The gene of interest (pGFP or pNGF) will be complexed with chitosan to improve transfection properties of liposomes. The liposomes will be evaluated for particle size, zeta potential, encapsulation efficiency, cell uptake and uptake mechanism(s), transfection efficiency, cell cytotoxicity, and hemolysis assay. (2). To evaluate the transport efficacy of liposomes across the barrier layer using 2-Dimensional (2D) BBB model: The transport efficacy of liposomes will be evaluated across 2D BBB model designed by co-culture of brain endothelial and primary astrocytes on opposite sides of culture inserts. There is a need to develop an efficient in vitro BBB model where the transport to the primary neurons can be regulated via the endothelial barrier. (3). To assess the distribution and transfection efficiency of Tf-CPP-liposomes in vivo and investigate effects of the Tf-CPP-liposome-mediated gene therapy in amyloid AD model mice: To establish successful gene therapies for AD, we will validate the Tf-CPP-liposomes obtained through above aims for their distribution, toxicity and transfection efficiency into mouse brains through tail vain injection. Finally, we will examine effects of NGF gene therapy through the Tf-CPP-liposomes on amyloid pathology, neurogenesis, synaptic functions and neurobehaviors in amyloid model APP/PS1 mice at different ages. We anticipate that the proposed study will contribute towards the development of high efficiency non-viral gene delivery vector to cross the BBB and deliver the pNGF gene to the desired target site for successful gene therapy for AD.