Towards Novel Therapies for CACNA1A Neurological Disorders

摘要 CaV2.1孔形成1A亚基突变导致包括癫痫在内的一系列神经系统疾病 脑病(EE)、家族性偏瘫1型(FHM1)、发作性共济失调2型(EA2)、脊髓小脑 共济失调6型(SCA6)和智力残疾(ID)。ClinVar数据库包含&gt；1000 CACNA1A的条目 突变，其中大多数(437)被归类为意义未知(VUS)、致病(137)或 可能致病(61例)。为CACNA1a开发有效疗法的努力面临着几个挑战 通道病：1)导致疾病的大量突变使人们不清楚是否常见 可以找到治疗方法；2)个体突变引起的功能变化的全部范围以及这些变化之间的关系 疾病的病因尚不明确；3)缺乏针对CaV2.1功能缺陷的新疗法。 我们假设数百个不同的CACNA1A突变属于几个离散的功能基团 可以通过为每一类量身定做的新型生物工程分子来靶向。我们的长期目标是获得 深入了解CACNA1A突变如何导致一系列神经疾病和 开发能够解决功能缺陷的分子，作为潜在的治疗方法。在这里，我们提出一种 跨越单通道和全细胞钙通道生物物理学的跨学科、多层次的建议，患者- 特异性诱导多能干细胞神经元(HiPSC-神经元)--CACNA1A型小鼠神经学模型 疾病，以及矫正分子的发展。建议书的广度由协作和 结合两个实验室之间的资源，科克拉夫特实验室(哥伦比亚大学)在以下方面拥有丰富的专业知识 CAV通道的分子生理学和生物物理学及其功能调控创新工具的开发 表达；Rossignol实验室(蒙特利尔大学)在基因生成和功能表征方面拥有专业知识 研究神经疾病的CACNA1A型小鼠模型。Rossignol博士是一位临床医生兼科学家，他与一群 CACNA1A患者，因此也为该项目带来了临床医生的视角。我们都提出了三个目标 这得到了强劲的初步数据的支持。1)确定不同CACNA1A的整体功能影响 重组CaV2.1通道上的突变，并开发量身定制的方法来纠正不同类别的 突变。2)建立人IPSC神经元模型，研究CACNA1a通道病的发病机制 并评价新的潜在治疗分子的疗效。3)利用鼠标模型确定 疾病的机制和评估新的量身定做的疾病治疗方法的有效性。

SUMMARY Mutations in CaV2.1 pore-forming 1A subunit cause a spectrum of neurological diseases including epileptic encephalopathies (EE), familial hemiplegic migraine type 1 (FHM1), episodic ataxia type 2 (EA2), spinocerebellar ataxia type 6 (SCA6), and intellectual disability (ID). The ClinVar database has entries for >1000 CACNA1A mutations most of which (437) are classified as variants of unknown significance (VUS), pathogenic (137), or likely pathogenic (61). There are several challenges for efforts to develop effective therapies for CACNA1A channelopathies: 1) the large number of dmutations that give rise to disease make it unclear whether common therapies can be found; 2) the full scope of functional alterations due to individual mutations and how these relate to disease etiology are ambiguous; and 3) lack of novel therapeutics targeted to CaV2.1 functional deficiencies. We hypothesize that the hundreds of distinct CACNA1A mutations fall into a few discrete functional groups that can be targeted by novel bioengineered molecules tailored for each class. Our long-term objective is to gain an in-depth perspective on how distinct CACNA1A mutations give rise to a spectrum of neurological disorders and to develop molecules that can address the functional deficits as potential therapeutics. Here, we propose an inter-disciplinary, multi-level proposal spanning single-channel and whole-cell Ca2+ channel biophysics, patient- specific induced pluripotent stem cell neurons (hiPSC-neurons), mouse models of CACNA1A neurological disease, and development of corrective molecules. The breadth of the proposal is enabled by collaboration and combining resources between two labs− the Colecraft lab (Columbia University) has strong expertise in molecular physiology and biophysics of CaV channels and developing innovative tools to regulate their functional expression; the Rossignol lab (Montreal University) has expertise in generation and functional characterization of CACNA1A mouse models of neurological disease. Dr. Rossignol is a clinician-scientist with a cohort of CACNA1A patients who thus also brings a clinician’s perspective to the project. We propose three Aims all of which are supported by strong preliminary data. 1) Determine holistic functional impact of distinct CACNA1A mutations on recombinant CaV2.1 channels, and develop tailored approaches to correct different classes of mutations. 2) Develop human ipsc-neurons to model and elucidate mechanisms of CACNA1A channelopathies and to evaluate efficacy of novel potential therapeutic molecules. 3) Utilize mouse models to determine mechanisms of disease and evaluate efficacy of novel tailored approaches to treat disease.