Striatal CaV1.3 Calcium Channels: An Overlooked Antidyskinetic Target for PD

 描述(申请人提供)：先前的研究，包括我们实验室的研究表明，含有CaV1.2/1.3通道拮抗剂的皮下缓释微丸可以减少低剂量(6 mg/kg)和高剂量(12.5 mg/kg)左旋多巴诱导的运动障碍(LID)的表达，然而，这种作用是部分的，随着时间的推移而消失。这些数据表明，CaV1.3通道是一个潜在的抗运动障碍靶点，然而，范围的限制和随着时间的推移保护的丧失是否与药物限制有关仍不清楚。目前有几个问题限制了CaV1.3通道拮抗剂与药理药物一起用于帕金森病(PD)的有效性，这些问题包括：1)目前还没有可用的药理学药剂，可以选择性地沉默CaV1.3通道而不影响在心血管功能中很重要的CaV1.2通道；2)即使高浓度的现有二氢吡啶(DHP)药物也不能完全抑制CaV1.3通道；以及3)传统上使用的药物阻断会导致非连续性通道阻断，我们认为这导致了先前所有临床和临床前研究的不同或部分保护结果。我们认为，先前的研究提供了强大而必要的理论基础，即CaV1.3通道是一个潜在的抗运动障碍靶点，开发一种创新的方法来确认其可能的临床应用是必要的。为了提供明确的原则证据，没有药理学限制，我们提出了三个特定的目标(SA)，这将允许检查连续的，高效的和靶标选择性的，mRNA水平的纹状体CaV1.3通道沉默对LID的影响，使用R21机制来帮助开发和执行这些重要的研究。在SA 1中，我们将确定通过我们的重组腺相关病毒(RAAV)介导的针对CaV1.3 mRNA的短发夹状RNA(ShRNA)的表达实现纹状体CaV1.3基因和蛋白质沉默的时间进程，这将指导SA 2和3中干预的时机。在SA 2中，我们将检验这样的假设，即在左旋多巴暴露之前，纹状体CaV1.3通道的结构性沉默将提供有效和持久的LID改善。在SA 3中，我们将测试这样的假设，即在已经表达LID的受试者中，对纹状体CaV1.3通道的结构性沉默将显著降低已建立的LID的严重程度。

 DESCRIPTION (provided by applicant): Previous studies, including those in our lab have shown that subcutaneous, slow release pellets containing CaV1.2/1.3 channel antagonists can reduce the expression of levodopa-induced dyskinesias (LID) produced by low dose (6 mg/kg) and high dose levodopa (12.5 mg/kg), however, this effect is partial and lost over time. These data suggest that the CaV1.3 channel is a potential antidyskinetic target, yet whether the limitation in scope and loss of protection over time are related to pharmacological limitation remains unknown. There are several issues that limit validating the involvement of CaV1.3 channel antagonism for any use in Parkinson's disease (PD) with pharmacological agents, which includes: 1) there is no currently available pharmacological agent that can selectively silence CaV1.3 channels without impacting the CaV1.2 channels that are important in cardiovascular function; 2) CaV1.3 channels are incompletely inhibited even by high concentrations of currently available dihydropyridine (DHP) drugs ; and 3) pharmacological blockade traditionally employed results in non-continuous channel blockade, which we propose contributes to the variable or partial protective outcome of all previous clinical and preclinical studies. We posit that the previous studies provide strong and necessary rationale that the CaV1.3 channel is a potential antidyskinetic target and that development of an innovative approach to confirm its possible clinical utility is warranted. To provide unequivocal proof-of- principle evidence, devoid of pharmacological limitations, we propose three Specific Aims (SA) that will allow examination of the impact of continuous, high potency and target-selective, mRNA-level silencing of striatal CaV1.3 channel on LIDs, using the R21 mechanisms to assist in developing and executing these important studies. In SA 1, we will determine the time course of striatal CaV1.3 gene and protein silencing achieved with our recombinant adeno-associated virus (rAAV)-mediated expression of a short hairpin RNA (shRNA) designed against the CaV1.3 mRNA, which will guide the timing of interventions in SA 2 and 3. In SA 2, we will test the hypothesis that constitutive silencing of striatal CaV1.3 channels prior to levodopa exposure will provide potent and enduring amelioration of LIDs. In SA 3, we will test the hypothesis that constitutive silencing of striatal CaV1.3 channels in subjects already expressing LIDs will significantly decrease severity of established LIDs.