MODULATION OF K+ CHANNEL PROPERTIES BY ANTISENSE DNA

反义 DNA 对 K 通道特性的调节

• 批准号: 2771889
• 负责人: SCOTT J SHERMAN
• 金额: $ 8.96万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-30 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2771889
• 关键词: action potentials; antisense nucleic acid; disease /disorder model; electrophysiology; epilepsy; gene targeting; herpes simplex virus 1; hippocampus; immunocytochemistry; laboratory rat; messenger RNA; method development; molecular cloning; neuropharmacology; potassium channel; pyramidal cells; tissue /cell culture; transfection; transfection /expression vector; voltage /patch clamp

Despite the emergence of new drugs and surgical methods, the control of seizures remains inadequate for many patients with epilepsy. Pharmacological treatment is often unsatisfactory due to side effects of anti-convulsant drugs. Surgical treatment can sometimes remove a focal area of epileptogenic tissue, but in the process, the normal functions of that portion of the brain are lost. We propose a genetic engineering technique that could be used to modify the ionic channels in localized areas of the brain. This novel approach could circumvent the drawbacks inherent in current therapeutic modalities. The pharmacological modification of voltage-gated ionic channels is a cornerstone of epilepsy treatment. The major anti-convulsant drugs phenytoin and carbamazepine act by promoting sodium channel inactivation. Potassium channels act in opposition to sodium channels and are responsible for the repolarization phase of the action potential. We hypothesize that enhancement of potassium channel function by blockade of inactivation should provide an anti-epileptic effect. We propose to test this hypothesis by reducing the inactivation of potassium channels in cultured rat hippocampal neurons by antisense knockdown of the beta1 subunit which is necessary for channel inactivation. The antisense knockdown will be carried by direct exposure to oligonucleotides and by transfection with the herpes simplex viral vector system. The viral vector will be directly applicable to future in vivo studies of efficiency in experimental models of epilepsy. The potassium channels produced by neurons unable to fully express the beta1 subunit are expected to functional normally, but will have a slower inactivation properties, thereby enhancing potassium efflux from the cell. We hypothesize that this change will render the neuron less likely to sustain rapid repetitive firing: the altered neurons will have a longer interspike interval and a slower maximum frequency of firing. The introduction of these changes in even a small proportion of a pool of neurons could be sufficient to prevent the rapid synchronous firing that constitutes the neurons basis of a seizure. The successful completion of this project might set the stage of the development of a genetic therapy for focal epilepsies. The specific aims of this proposal are to: (1) Develop methods for the quantification of specific mRNA message and protein expression of the beta1 subunit in primary cultures of pyramidal neurons from rat hippocampus. (2) Develop methods of introducing antisense DNA into cultured pyramidal neurons using oligonucleotides, and the replication- deficient herpes viral vector system. (3) Demonstrate the effect of these antisense knockdown techniques on the transcription and translation of the beta1 subunit. (4) Study the effect of loss of the beta1 subunit on the action potential shape and firing patterns of pyramidal neurons using patch clamp techniques, thereby testing our hypothesis that inhibition of inactivation will limit the maximal neuronal firing rate.

尽管出现了新的药物和手术方法，但控制 对于许多癫痫患者来说，癫痫发作仍然不够。 由于药物的副作用，药物治疗常常不能令人满意。 抗惊厥药物。手术治疗有时可以去除病灶 癫痫组织的区域，但在此过程中，其正常功能 大脑的那部分丢失了。我们提出基因工程 可用于修饰局部离子通道的技术 大脑的区域。这种新颖的方法可以克服缺点 当前治疗方式所固有的。 电压门控离子通道的药理学修饰是 癫痫治疗的基石。主要抗惊厥药物 苯妥英和卡马西平通过促进钠通道失活起作用。 钾通道与钠通道相反， 负责动作电位的复极化阶段。我们 假设通过阻断钾通道功能来增强钾通道功能 灭活应提供抗癫痫作用。我们建议测试 这一假设是通过减少钾通道的失活来实现的。 通过反义敲低 beta1 培养大鼠海马神经元 通道失活所必需的亚基。反义 敲低将通过直接暴露于寡核苷酸和通过 用单纯疱疹病毒载体系统转染。病毒载体 将直接应用于未来的体内效率研究 癫痫实验模型。产生的钾通道 无法完全表达 beta1 亚基的神经元预计 功能正常，但失活速度较慢， 从而增强钾从细胞中的流出。我们假设这 变化将使神经元不太可能维持快速重复 放电：改变的神经元将具有更长的放电间隔和 较慢的最大发射频率。这些变化的引入 即使神经元池中的一小部分也足以 防止构成神经元基础的快速同步放电 癫痫发作。该项目的成功完成可能奠定基础 局灶性癫痫基因疗法的发展。 本提案的具体目标是： (1) 制定方法 特定 mRNA 信息和蛋白质表达的量化 大鼠锥体神经元原代培养物中的β1亚基 海马体。 (2) 开发反义DNA导入方法 使用寡核苷酸培养锥体神经元，并复制 疱疹病毒载体系统缺陷。 (3) 展示这些效果 反义敲低技术对转录和翻译的影响 β1 亚基。 (4) 研究β1亚基缺失对 使用锥体神经元的动作电位形状和放电模式 膜片钳技术，从而检验我们的假设，即抑制 失活将限制最大神经元放电率。