Targeted immunoliposomes for cell-type specific gene therapy of 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，仍有大量患者存在抗药性。基因治疗是一种很有前途的策略，以纠正在分子水平上改变的离子通道负责过度兴奋的神经元和网络表型。癫痫发生的现有知识表明，“治疗性抗癫痫基因”需要特异性转染到不同的神经元表型（即，如神经元）。靶向非病毒基因载体如免疫脂质体已被用于选择性地将药物和遗传物质递送到与健康细胞分离的癌细胞。免疫脂质体是具有外部靶向部分（针对细胞表面分子的抗体）的阳离子脂质囊泡，其可以促进复合物通过受体介导的内吞作用进入神经元。本申请提出开发阳离子免疫脂质体作为基因递送纳米载体，其可以选择性地靶向多巴胺能神经元并递送用于表达和上调大电导钙激活钾（BK）通道（α-孔形成亚基）的“治疗性”cDNA。BK通道位于海马体的轴突和突触前末梢中，并且被认为它们控制谷氨酸的释放。基因治疗介导的BK通道上调可能改善癫痫中谷氨酸的过度释放。在具体目标1中，我们将探讨靶向免疫脂质体是否会选择性地将编码BK通道的cDNA和报告增强型绿色荧光蛋白（EGFP）转染到海马神经元。转染将被表征为“体外”（即，原代海马培养物）。脂质体载体的选择性和效率将通过使用免疫细胞化学、活力测定和激光扫描共聚焦成像与其他非靶向转染方法进行比较。免疫脂质体介导的BK通道cDNA转染的功能分析将使用可视化膜片钳记录来评估，以检测和分析自发的微小突触后兴奋电流（mEPSC）。通过膜选择性FM染料的内吞作用的直接成像将评估外源性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，以提供用于治疗癫痫等神经系统疾病的“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。这种多学科应用涉及德克萨斯大学布朗斯维尔的初级研究人员之间的合作，他们具有BK通道分子生物学、转染、电生理学、癫痫和脂质体技术方面的专业知识。这项探索性研究的结果可能会导致一种新的非病毒策略的细胞类型特异性中枢神经系统基因治疗。相关性：这个多学科的纳米技术项目旨在为耐药性癫痫的基因治疗开发一种创新策略。拟议的实验将表征“体外”一种新型的递送系统（免疫脂质体），该系统由脂质纳米囊泡（脂质体）组成，表现出表面识别分子（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。该申请的完成可能提供一种革命性的靶向基因治疗策略，通过选择性地将“治疗性”遗传物质递送到功能障碍的神经元，其参与主要神经系统疾病如癫痫的发病机制。

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.