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

总结： 神经退行性疾病已成为老年痴呆症的最常见原因， 据报道，65岁以上的美国人中约有5%受到影响，80岁以上的美国人中约有20%受到影响。有 2010年全球痴呆症患者为3600万人，到2030年将增加到6600万人，到2020年将增加到1.15亿人。 2050. 2010年，全球痴呆症的成本为6040亿美元。这是全球GDP的1%，很可能这些国家 费用将随着痴呆症患者人数的增加而增加。基因治疗已被确定为 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，是由阿尔茨海默病引起的最常见的痴呆形式。 毒性淀粉样蛋白-β（Aβ）肽在大脑中的积累，其中开发有效的治疗方法 被渴望。然而，基因治疗领域的主要挑战是设计安全的非病毒载体。 可以穿过血脑屏障（BBB）的载体。已经发现转铁蛋白受体是 存在于脑内皮细胞的表面。脂质体，基于脂质的纳米颗粒，可以是表面活性剂。 用转铁蛋白（Tf）蛋白修饰，用于靶向脑内皮受体，并与细胞 穿透肽（CPP），通过克服受体饱和来改善它们向脑内的内化。 因此，我们建议将脂质体与两种配体缀合：（1）受体靶向蛋白（Tf）和（2） a CPP。因此，我们将通过用Tf和CPP修饰表面来设计近中性的PEG化脂质体。 此外，低分子量壳聚糖的转染性质将用于改善细胞的增殖。 通过促进细胞内的内体逃逸来转染基因。拟议的长期目标 研究是设计一种基因递送载体，用于将神经生长因子（NGF）有效地递送到大脑， AD的预防和治疗。我们提出以下三个具体目标：（1）。不能合成和 表征负载有壳聚糖-pDNA聚合复合物的Tf和CPP偶联的脂质体：CPP-脂质体将 使用薄膜水合技术合成，然后使用后处理插入Tf偶联胶束， 插入技术我们建议根据物理化学性质使用三种类型的CPP [阳离子亲水性（HIV-Tat，PasR 8和R9 F2），阳离子两亲性（pVec，穿透素和Mellittin） 和疏水性（PFVYLI、五肽QLPVM和卡波西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转染特性。 脂质体。将评价脂质体的粒度、zeta电位、包封率、细胞粘附率、细胞粘附率和细胞粘附率。 摄取和摄取机制、转染效率、细胞毒性和溶血测定。（二）、到 使用二维（2D）BBB模型评估脂质体穿过屏障层的转运功效： 将通过脑共培养设计的2D BBB模型评价脂质体的转运功效 内皮细胞和原代星形胶质细胞在培养插入物的相对侧。有必要制定一个有效的 体外BBB模型，其中向原代神经元的转运可以通过内皮屏障调节。（三）、 评价转铁蛋白CPP脂质体在体内的分布和转染效率， 脂质体介导转铁蛋白磷酸肽基因治疗淀粉样AD小鼠的实验研究 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 通过尾静脉注射将转染效率提高到小鼠脑中。最后，我们将研究神经生长因子的影响， 通过Tf-CPP-脂质体的基因治疗对淀粉样蛋白病理学、神经发生、突触功能和 在不同年龄的淀粉样蛋白模型APP/PS1小鼠的神经行为。我们预计，拟议的研究将 有助于开发高效的非病毒基因递送载体以穿过BBB， 将pNGF基因递送到所需的靶位点，用于成功的AD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.