MODULATION OF K+ CHANNEL PROPERTIES BY ANTISENSE DNA

尽管出现了新的药物和手术方法， 癫痫发作对许多癫痫患者来说仍然不够。 药物治疗往往是不令人满意的，由于副作用， 抗惊厥药手术治疗有时可以切除病灶 癫痫组织的区域，但在这个过程中， 大脑的这一部分就丢失了。我们提出一种基因工程 技术，可用于修改离子通道在局部 大脑的各个区域。这种新颖的方法可以避免这些缺点 是当前治疗方式中固有的。 电压门控离子通道的药理学修饰是一种有效的方法。 癫痫治疗的基石主要的抗惊厥药物 苯妥英和卡马西平通过促进钠通道失活而起作用。 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 灭活应该提供抗癫痫作用。我们建议测试 这一假设通过减少钾通道的失活， 反义敲低β 1基因对培养大鼠海马神经元的影响 亚基是通道失活所必需的。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。述病毒载体 将直接适用于未来的体内效率研究， 癫痫的实验模型产生的钾离子通道 不能完全表达β 1亚单位的神经元， 功能正常，但将具有较慢的失活特性， 从而增强钾从细胞的流出。我们假设这 改变将使神经元不太可能维持快速重复的 放电：改变的神经元将具有更长的峰间期和更长的放电间隔。 最大发射频率更低。这些变化的引入， 即使是一小部分神经元也足以 阻止快速的同步放电，这是神经元的基础， 癫痫发作这个项目的成功完成可能会为 局灶性癫痫的基因疗法的进展 本提案的具体目标是：（1）制定方法， 特异性mRNA信息和蛋白表达的定量 原代培养大鼠锥体神经元β 1亚单位的研究 海马体。(2)开发将反义DNA导入 培养的锥体神经元使用寡核苷酸，和复制- 缺陷型疱疹病毒载体系统。(3)展示这些的效果 反义基因敲减技术对转录和翻译的影响 β 1亚基。(4)研究β 1亚基缺失对 动作电位的形状和发放模式的锥体神经元使用 膜片钳技术，从而验证我们的假设，抑制 失活将限制最大神经元放电率。

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.