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

描述(申请人提供)：神经前体细胞的受控增殖是一种潜在的治疗神经退行性疾病的策略，如亨廷顿病和中风。成年哺乳动物大脑的脑室下区(SVZ)容纳着NPC群体，这些群体已被证明在整个大脑中迁移并成熟为新的神经元。已有研究表明，通过传递编码有丝分裂因子的治疗性基因，如成纤维细胞生长因子2(FGF2)，刺激神经干细胞的增殖和分化，可导致脑内受损区域的募集增加。本文描述的工作建议开发一种多组分的非病毒基因递送载体，用于将FGF2递送到SVZ的NPC。给药载体将包含两种生物活性多肽：(1)靶向多肽，以最大限度地吸收颗粒；(2)裂解多肽，介导内体逃逸。此外，车辆将使用聚乙二醇盐稳定性(聚乙二醇)进行改性，这是 体内给药的载体。拟议的工作概述如下： 目标1：将合成同时整合Tet1、HGP和PEG3的载体。汽车配方将是 筛选所需的物理化学特性，并针对转染率和结合进行优化 体外培养。材料还将使用细胞活性测试进行毒性测试。 目的2：通过脑室给药，在体内确定最适质粒和聚合物量 车辆。Aim 1的配方将用于传递报告基因构建物。批量表达式将为 在脑裂解液中的定量和表达的分布将通过脑片的免疫标记来确定。 目标3：在目标2中找到的最佳交通工具将用于交付FGF2。定量化与本土化 FGF2的表达将使用ELISA法和免疫标记法。成纤维细胞生长因子2介导的增殖和 脑片的分化将通过BrdU+/Neun+标记和血清学定量来鉴定。 这项研究调查了一种治疗神经退行性疾病的潜在策略。它有 这与公共卫生有关，因为神经退行性疾病影响到全世界2000多万人。

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.