Targeted immunoliposomes for cell-type specific gene therapy of epilepsy

用于癫痫细胞类型特异性基因治疗的靶向免疫脂质体

• 批准号: 7496563
• 负责人: Emilio Rafael Garrido Sanabria
• 金额: $ 14.33万
• 依托单位: UNIV/TEXAS BROWNSVILLE & SOUTHMOST COLL
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-15 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7496563
• 关键词: Affect; Antibodies; Antiepileptic Agents; Antineoplastic Agents; Attenuated; Axon; Biological Assay; Blood - brain barrier anatomy; Calcium; Calcium-Activated Potassium Channel; Cancerous; Cell surface; Cells; Chronic; Code; Collaborations; Complementary DNA; Complex; Copyright; DNA Vaccines; Development; Dyes; Electrophysiology (science); Endocytosis; Engineering; Epilepsy; Epileptogenesis; Eukaryotic Cell; Excision; Exhibits; Exocytosis; FM 4-64; Face; Future; Gene Delivery; Gene Transduction Agent; Gene Transfer; Genes; Genetic; Genetic Materials; Genetic Vectors; Glutamates; Goals; Hippocampus (Brain); Image; Immunofluorescence Immunologic; Immunoliposome; In Vitro; Interneurons; Ion Channel; Knowledge; Laboratories; Lasers; Laws; Lead; Legal patent; Ligands; Lipids; Liposomes; Malignant - descriptor; Mediating; Membrane; Modeling; Molecular; Molecular Biology; Monoclonal Antibodies; Nanotechnology; Neurologic; Neurons; Non-Viral Vector; Nucleic Acids; Outcome; Pathogenesis; Patients; Pharmaceutical Preparations; Pharmacologic Substance; Phenotype; Play; Population; Potassium; Presynaptic Terminals; Principal Investigator; Protein Overexpression; Reporter; Reporter Genes; Research; Research Personnel; Resistance; Role; Scanning; Standards of Weights and Measures; Surface; Synapses; Synaptic Membranes; System; Technology; Temporal Lobe Epilepsy; Testing; Texas; Therapeutic; Therapeutic Studies; Time; Trademark; Transcriptional Activation; Transfection; Transferrin Receptor; Tumor Antigens; Universities; Up-Regulation; Vesicle; Viral; Viral Genes; Writing; cDNA Expression; cell type; drug development; enhanced green fluorescent protein; gene therapy; hippocampal pyramidal neuron; immunocytochemistry; in vivo; innovation; large-conductance calcium-activated potassium channels; mossy fiber; multidisciplinary; nanosized; nervous system disorder; neuroligin 1; neuronal excitability; neurotransmitter release; next generation; novel; novel strategies; patch clamp; plasmid DNA; postsynaptic; presynaptic; receptor mediated endocytosis; research study; therapeutic gene; therapeutic target; thought control; transgene expression; trend; vector; voltage

DESCRIPTION (provided by applicant): Epilepsy is a devastating neurological channelopathy affecting 2-3 percent of the population. In the face of major breakthroughs in antiepileptic drug development, a large contingent of patients remains pharmacoresistant. Gene therapy is a promising strategy to correct at the molecular level alterations of ion channel responsible for hyperexcitable neuronal and network phenotypes. The current knowledge of epileptogenesis indicate that 'therapeutic antiepileptic genes' need to be specifically transfected to a distinct neuronal phenotype (i.e., such as glutamatergic neurons). Targeted non-viral gene vectors such immunoliposomes have been used to selectively deliver drugs and genetic material to cancerous cells spearing the healthy ones. Immunoliposomes are cationic lipid vesicles with an external targeting moiety (antibodies directed against a cell surface molecule), which may facilitate entry of the complex into neurons via receptor-mediated endocytosis. This application proposes to develop cationic immunoliposomes as gene delivery nanovehicles that can selectively target glutamatergic neurons and deliver 'therapeutic' cDNA for expression and up-regulation of the large conductance calcium-activated potassium (BK) channel (alpha-pore forming subunit). BK channels are located in axon and presynaptic terminals in the hippocampus and it is thought that they control glutamate release. Gene therapy-mediated up-regulation of BK channels may ameliorate excessive release of glutamate in epilepsy. In Specific Aim 1, we will explore whether targeted immunoliposomes will selectively transfect cDNA coding for BK channel and reporter enhanced green fluorescent protein (EGFP) to hippocampal glutamatergic neurons. Transfections will be characterized 'in vitro' (i.e., primary hippocampal cultures). Selectivity and efficiency of liposomal vectors will be compared with other non-targeted transfection approaches by using immunocytochemistry, viability assays and laser scanning confocal imaging. Functional analysis of immunoliposome-mediated BK channel cDNA transfection will be assessed using visualized patch-clamp recordings to detect and analyze spontaneous miniature postsynaptic excitatory currents (mEPSC). Direct imaging of endocytosis by membrane-selective FM-dyes will assess whether introduction of exogenous BK channels affect synaptic release of glutamate. The long-term goal is to develop innovative targeted non-viral vectors to deliver 'therapeutic genes' for the treatment of neurological disorders as epilepsy. In future studies, the therapeutic potential of targeted immunoliposomes containing 'antiepileptic genes' will be explored 'in vivo' in a model of chronic epilepsy. This multidisciplinary application involves collaborations among junior investigators at The University of Texas at Brownsville with expertise in molecular biology of BK channels, transfection, electrophysiology, epilepsy and liposome technology. The outcome of this exploratory research may lead to a novel non-viral strategy for cell-type specific CNS gene therapy. Relevance: This multidisciplinary nanotechnology project aims to develop an innovative strategy for the gene therapy of pharmacologically resistant epilepsy. The proposed experiments will characterize 'in vitro' a novel delivery system (immunoliposomes) consisting of lipid nanosized vesicles (liposomes) exhibiting a surface recognition molecule (antibody) that can selectively target a specific neuronal population that release the neurotransmitter glutamate (glutamatergic neurons). Completion of this application may provide a revolutionary targeted gene therapy strategy by selectively delivering 'therapeutic' genetic materials to dysfunctional glutamatergic neurons, which are involved in the pathogenesis of major neurological disorders such epilepsy.

描述(由申请人提供)：癫痫是一种破坏性的神经通道病，影响2-3%的人口。面对抗癫痫药物开发的重大突破，大量患者仍对药物耐药。基因治疗是在分子水平上纠正导致神经元和网络表型过度兴奋的离子通道改变的一种有前景的策略。目前对癫痫发生的认识表明，治疗性抗癫痫基因需要特异性地转染到不同的神经元表型(如谷氨酸能神经元)。靶向的非病毒基因载体，如免疫脂质体，已经被用来选择性地将药物和遗传物质输送到癌细胞，使健康的癌细胞存活。免疫脂质体是具有外靶向部分(针对细胞表面分子的抗体)的阳离子脂泡，可通过受体介导的内吞作用促进复合体进入神经元。这项应用建议开发阳离子免疫脂质体作为基因传递纳米载体，它可以选择性地靶向谷氨酸能神经元，并传递用于表达和上调大电导钙激活钾(BK)通道(α-孔形成亚单位)的治疗性cDNAs。BK通道位于海马区的轴突和突触前终末，被认为控制谷氨酸的释放。基因治疗上调BK通道可能改善癫痫患者谷氨酸的过度释放。在特定的目标1中，我们将探索靶向免疫脂质体是否会选择性地将编码BK通道和报告增强型绿色荧光蛋白(EGFP)的cDNAs转染到海马谷氨酸能神经元。转基因将被定性为‘体外’(即，原代海马区培养)。通过免疫细胞化学、活性检测和激光扫描共聚焦成像，将脂质体载体的选择性和效率与其他非靶向转染法进行比较。免疫脂质体介导的BK通道c DNA的功能分析将使用可视化的膜片钳记录来检测和分析自发的微小突触后兴奋性电流(MEPSC)。通过膜选择性FM染料的直接内吞成像将评估外源性BK通道的引入是否影响谷氨酸的突触释放。长期目标是开发创新的靶向非病毒载体，为癫痫等神经疾病的治疗提供“治疗基因”。在未来的研究中，含有“抗癫痫基因”的靶向免疫脂质体的治疗潜力将在慢性癫痫模型中进行“体内”探索。这一多学科的应用涉及德克萨斯大学布朗斯维尔分校的初级研究人员之间的合作，他们拥有BK通道的分子生物学、转基因、电生理学、癫痫和脂质体技术方面的专业知识。这一探索性研究的结果可能导致一种新的非病毒策略，用于细胞型特异性中枢神经系统的基因治疗。相关性：这一多学科纳米技术项目旨在为药物耐药性癫痫的基因治疗开发一种创新策略。拟议的实验将描述一种新型的传递系统(免疫脂质体)，该系统由脂质纳米囊泡(脂质体)组成，具有表面识别分子(抗体)，可以选择性地靶向释放神经递质谷氨酸(谷氨酸能神经元)的特定神经元群体。这一应用的完成可能会提供一种革命性的靶向基因治疗策略，通过选择性地向功能障碍的谷氨酸能神经元输送“治疗性”遗传物质，谷氨酸能神经元参与了癫痫等主要神经疾病的发病机制。