Dvelopment of multi-component peptide vehicles for in vivo neuronal gene therapy

用于体内神经元基因治疗的多组分肽载体的开发

• 批准号: 7898848
• 负责人: Ester J Kwon
• 金额: $ 0.97万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-23 至 2010-06-11
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7898848
• 关键词: Address; Adult; Affect; Animal Model; Antibodies; Area; Binding; Biological Assay; Brain; Bromodeoxyuridine; Bypass; Cell Proliferation; Cell Survival; Cells; Disease; Dose; Drug Formulations; Endocytic Vesicle; Ensure; Enzyme-Linked Immunosorbent Assay; Ependymal Cell; Evaluation; Exhibits; Fibroblast Growth Factor 2; Gene Delivery; Gene Transfer; Genes; Growth Factor; HIV Envelope Protein gp41; Huntington Disease; Immunohistochemistry; In Vitro; Individual; Injection of therapeutic agent; Interleukin-2; Intraventricular; Intraventricular Injections; Label; Lateral; Luciferases; Lytic; Mediating; Mediator of activation protein; Nerve Degeneration; Neuraxis; Neurodegenerative Disorders; Neurons; PC12 Cells; Penetration; Peptides; Plasmids; Polyethylene Glycols; Polymers; Population; Proliferating; Property; Public Health; Reporter Genes; Research; Route; Serologic tests; Slice; Sodium Chloride; Specificity; Stroke; System; Toxicity Tests; Transfection; Ventricular; Viral; Work; base; cell type; design; gene therapy; in vivo; lateral ventricle; nerve stem cell; non-viral gene delivery; non-viral gene therapy; particle; subventricular zone; therapeutic gene; trafficking; treatment strategy; uptake; vector

DESCRIPTION (provided by applicant): Controlled proliferation of neural progenitor cells (NPCs) is a potential strategy for the treatment of neurodegenerative conditions such as Huntington's Disease and stroke. The subventricular zone (SVZ) of the adult mammalian brain harbors an NPC population that has been shown to migrate throughout the brain and mature into new neurons. It has been shown that stimulating the proliferation and differentiation of NPCs through the delivery of therapeutic genes encoding mitogenic factors such as fibroblast growth factor 2 (FGF2) can cause increased recruitment to damaged areas in the brain. The work described herein proposes to develop a multi-component nonviral gene delivery vehicle for FGF2 delivery to NPCs of the SVZ. The delivery vehicle will incorporate two bioactive peptides: (1) a targeting peptide to maximize uptake of particles and (2) a lytic peptide to mediate endosomal escape. In addition, vehicles will be modified with polyethylene glycol (PEG) for salt stability, which is required for vehicles administered in vivo. The proposed work is outlined in the aims below: Aim 1: Vehicles that incorporate both Tet1, HGP, and PEG will be synthesized. Vehicle formulations will be screened for desired physicochemicaj properties and optimized for transfection efficiency and binding in vitro. Materials will also be tested for toxicity using a cell viability assay. Aim 2: Optimal plasmid and polymer amount will be determined in vivo by intraventricular administration of vehicles. Formulations from Aim 1 will be used to deliver reporter gene constructs. Bulk expression will be quantified in brain lysate and distribution of expression will be determined by immunolabeling of brain slices. Aim 3: The optimal vehicle found in Aim 2 will be used to deliver FGF2. Quantification and localization of FGF2 expression will be done using ELISA and immunolabeling. FGF2-mediated proliferation and differentiation in brain slices will be identified by BrdU+/NeuN+ labeling and quantified by serology. This research investigates a potential strategy for the treatment of neurodegenerative diseases. It has relevance to public health since neurodegenerative diseases affects over 20 million individuals worldwide.

描述（由申请人提供）：神经祖细胞（NPC）的受控增殖是治疗神经退行性疾病（例如亨廷顿病和中风）的潜在策略。成年哺乳动物大脑的室下区（SVZ）藏有NPC群体，该群体已被证明迁移到整个大脑并成熟为新的神经元。已经表明，通过递送编码促有丝分裂因子如成纤维细胞生长因子2（FGF 2）的治疗性基因来刺激NPC的增殖和分化可以引起脑中受损区域的募集增加。本文所述的工作提出开发用于将FGF 2递送至SVZ的NPC的多组分非病毒基因递送载体。递送载体将掺入两种生物活性肽：（1）靶向肽以使颗粒的摄取最大化，和（2）裂解肽以介导内体逃逸。此外，将用聚乙二醇（PEG）对溶媒进行改性，以获得盐稳定性，这是制备所需的。 体内施用的媒介物。拟议工作的目标概述如下： 目的1：将合成包含Tet 1、HGP和PEG的载体。溶剂制剂将 筛选所需的物理化学性质，并优化转染效率和结合。 体外还将使用细胞活力试验检测材料的毒性。 目的2：通过脑室内施用以下化合物在体内确定最佳质粒和聚合物量： 车辆.来自目标1的制剂将用于递送报告基因构建体。批量表达将是 在脑裂解物中定量，并通过脑切片的免疫标记来确定表达的分布。 目标3：目标2中发现的最佳载体将用于递送FGF 2。量化和定位 将使用ELISA和免疫标记进行FGF 2表达。FGF 2介导的增殖和 通过BrdU+/NeuN+标记鉴定脑切片中的分化，并通过血清学定量。 这项研究探讨了治疗神经退行性疾病的潜在策略。它有 神经退行性疾病影响着全世界2 000多万人。