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

脑神经生长因子输送可预防和治疗阿尔茨海默病

基本信息

批准号：
9170894
负责人：
Takahisa Kanekiyo
金额：
$ 38.81万
依托单位：
NORTH DAKOTA STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-01 至 2021-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9170894
关键词：
AIDS/HIV problem APP-PS1 Accounting Affect Age Aged, 80 and over Alzheimer&apos s Disease Alzheimer&apos s disease model American Amyloid Amyloid beta-Protein Astrocytes Automobile Driving Awareness Biological Assay Blood - brain barrier anatomy Brain Caring Cells Central Nervous System Diseases Chitosan Coculture Techniques Complex Coupled Degenerative Disorder Dementia Development Disease Disease Progression Elderly Encapsulated Fibroblast Growth Factor Film Gene Delivery Genes Goals Growth Factor Gene HIV Health Care Costs Hemolysis Hydration status In Vitro Incidence Injection of therapeutic agent Life Ligands Lipids Liposomes Mediating Micelles Modeling Molecular Weight Mus Nerve Growth Factors Neurodegenerative Disorders Neurons Neurosciences Non-Viral Vector Particle Size Penetration Peptides Porifera Prevalence Prevention Property Proteins Protons Reporting Research Services Side Site Surface TFRC gene Tail Techniques Testing Time Toxic effect Transfection Transferrin amyloid pathology base brain endothelial cell chemical property clinical application cost cytotoxicity design effective therapy gene therapy improved in vivo interest model design nanoparticle nervous system disorder neurobehavior neurogenesis neurotropic non-viral gene delivery novel strategies penetratin prevent receptor social synaptic function therapeutic gene transcytosis two-dimensional uptake vector zeta potential

AIDS/HIV problem APP-PS1 Accounting Affect Age Aged, 80 and over Alzheimer&apos s Disease Alzheimer&apos s disease model American Amyloid Amyloid beta-Protein Astrocytes Automobile Driving Awareness Biological Assay Blood - brain barrier anatomy Brain Caring Cells Central Nervous System Diseases Chitosan Coculture Techniques Complex Coupled Degenerative Disorder Dementia Development Disease Disease Progression Elderly Encapsulated Fibroblast Growth Factor Film Gene Delivery Genes Goals Growth Factor Gene HIV Health Care Costs Hemolysis Hydration status In Vitro Incidence Injection of therapeutic agent Life Ligands Lipids Liposomes Mediating Micelles Modeling Molecular Weight Mus Nerve Growth Factors Neurodegenerative Disorders Neurons Neurosciences Non-Viral Vector Particle Size Penetration Peptides Porifera Prevalence Prevention Property Proteins Protons Reporting Research Services Side Site Surface TFRC gene Tail Techniques Testing Time Toxic effect Transfection Transferrin amyloid pathology base brain endothelial cell chemical property clinical application cost cytotoxicity design effective therapy gene therapy improved in vivo interest model design nanoparticle nervous system disorder neurobehavior neurogenesis neurotropic non-viral gene delivery novel strategies penetratin prevent receptor social synaptic function therapeutic gene transcytosis two-dimensional uptake vector zeta potential

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项目摘要

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.

概括：神经脱生性疾病已成为老年痴呆症的最常见原因，并且据报道，有65岁以上的美国人中约有5％，在80岁以上的美国人中有20％。有 2010年全球有3,600万人居住在全球痴呆症，到2030年增加到6600万人，1.15亿 2050年。2010年，全球痴呆症成本为6004亿美元。这是全球GDP的1％，可能是成本将与痴呆症患者数量成比例增加。基因治疗已被确定为在包括阿尔茨海默氏病在内的众多神经系统疾病的治疗中具有广泛的潜力（广告）。 AD是一种进行性神经退行性疾病，是由在大脑中积累有毒的淀粉样蛋白β（Aβ）肽，其中有效疗法的发展已经需要。但是，基因疗法领域的主要挑战是安全非病毒的设计可以越过血脑屏障（BBB）的向量。已经发现转铁蛋白受体是存在于脑内皮细胞表面。脂质体，基于脂质的纳米颗粒，可以表面用转铁蛋白（TF）蛋白质修饰，用于靶向脑内皮接收器并连接到细胞穿透胡椒（CPP），通过克服接收器满意度来改善其内在化对大脑的内在化。因此，我们建议将脂质体与两个配体（1）靶向蛋白（TF）和（2）的配体共轭（1） CPP。我们将通过用TF和CPP修饰表面来设计近乎中性的，卵形的脂质体。此外，低分子量壳聚糖的翻译特性将用于改善通过促进细胞内部内体逃生来转染基因。提议的长期目标研究是设计一个基因输送载体，以有效地递送神经生长因子（NGF）到大脑预防和治疗AD。我们提出以下三个特定目标：（1）。合成和表征带有壳聚糖 - pDNA多聚体的TF和CPP耦合脂质体：CPP-脂质体将使用薄膜水合技术合成，然后使用后 - 插入技术。我们建议根据物理化学特性使用三种类型的CPP [阳离子亲水性（HIV-TAT，PASR8和R9F2），阳离子两亲性（PVEC，渗透素和梅利汀）和疏水性（Pfvyli，五肽QLPVM和Kaposi成纤维细胞生长因子衍生肽）。感兴趣的基因（PGFP或PNGF）将与壳聚糖复合，以提高翻译特性脂质体。将评估脂质体的粒径，Zeta电位，封装效率，细胞摄取和吸收机制，转化效率，细胞毒性和溶血测定法。（2）。到使用二维（2D）BBB模型评估脂质体在整个屏障层的运输效率：脂质体的运输效率将在由大脑共文化设计的2D BBB模型上进行评估培养物插入物的相对侧的内皮和原代星形胶质细胞。有必要提高高效在体外BBB模型中，可以通过内皮屏障调节到原发性神经元的转运。（3）。评估体内TF-CPP-脂质体的分布和转化效率，并研究淀粉样蛋白AD模型小鼠中TF-CPP-脂质体介导的基因疗法：建立成功的基因治疗对于AD，我们将验证通过上述分布，毒性和通过尾声注射将其转染效率转染到小鼠大脑中。最后，我们将检查NGF的影响通过TF-CPP脂质体在淀粉样病理学，神经发生，突触功能和淀粉样蛋白模型APP/PS1小鼠在不同年龄的淀粉样蛋白模型APP/PS1小鼠中的神经毛虫。我们预计拟议的研究将有助于发展高效率非病毒基因递送载体越过BBB和将PNGF基因传递到所需的目标位点，以成功地进行AD基因治疗。