A precision medicine approach to Rett Syndrome

摘要 已知MeCP 2在神经发育障碍Rett综合征（RTT）中的致病性， 近20年;然而，目前没有可行的干预战略。造成这种缺乏的一个因素是， 在临床前开发期间依赖于雄性Mecp 2-/y敲除小鼠，尽管 事实上，RTT几乎只影响女性，她们是x连锁MECP 2等位基因（Mecp 2 +/-）的嵌合体。 此外，RTT患者很少具有导致MeCP 2蛋白丢失的突变，而是具有错义或缺失突变。 使关键功能结构域亚纯型的截短突变。为了解决这种结构效度上的缺陷， 我们对9例RTT患者尸检的小脑和运动皮层样本进行了RNA测序， 识别可能从翻译相关的位置开始的潜在的可用药靶点。这种方法 导致我们关注毒蕈碱型乙酰胆碱受体（mAChRs），其中5种亚型中有4种具有显著的 中断表达。特别令人感兴趣的是mAChR 1（CHRM 1，M1）亚型，长期以来一直是 被认为是神经精神疾病的可行治疗靶点。令人兴奋的是，我们最近使用了44个临时的 来自RTT尸检的皮质样品证实CHRM 1表达的降低是一种稳健的、高度的 RTT病理生理学在人类中的渗透方面。然后我们建立了一个M1阳性 变构调节剂（PAM），VU 0453595（VU 595），显著改善社交和呼吸（呼吸暂停） Mecp 2 +/-小鼠中的表型。在目标1中，我们建议通过测试M1的影响来扩展这些初步数据 在小鼠中使用结构不同的M1 PAM，急性和急性增强对一组完整的RTT样表型的作用， 慢性给药模式和多种药理学模式。有趣的是，如果RTT尸检表达数据 通过MECP 2突变进行分组，然后观察到不仅M1而且几乎所有M1的签名表达模式。 检测所有临床前靶基因。由于基因表达的变化通常是靶点选择的基本原理， 在受体功能正常的情况下，神经传递的调节增加了神经传导的风险。 这些发现对于RTT中的精准医学需求具有重要意义。在 目的2，我们将使用携带常见RTT突变（T158 M，R168 X，R255 X和R306 C）的小鼠来确定 表达模式是否可用于预测M1 PAM功效。我们将把这些实验与 RTT尸检样本的转录和蛋白质组学分析，以量化 RTT患者亚群的髓质。最后，在我们现有的数据集中，我们考虑了VU 595对 呼吸暂停是一个突出的发现，因为呼吸功能障碍是RTT早期致死的预测因素， 因为呼吸暂停代表高度可转换的结果测量。在目标3中，我们建议从机制上 通过结合全身体积描记术和体内循环， 在Mecp 2基因敲入小鼠中进行伏安法实验，以确定M1 PAM的作用机制，以及 定义M1在RTT亚群的呼吸回路中的作用位置和方式。

ABSTRACT The pathogenic nature of MeCP2 in the neurodevelopmental disorder Rett syndrome (RTT) has been known for almost 20 years; however, there currently no viable intervention strategies. One contributing factor to the paucity of treatment options is the reliance on male Mecp2-/y knockout mice during preclinical development, despite the fact that RTT almost exclusively affects females, who are mosaic for the x-linked MECP2 allele (Mecp2+/-). Furthermore, RTT patients rarely have mutations that result in loss of MeCP2 protein, rather have missense or truncating mutations that render key functional domains hypomorphic. To address this failing in construct validity, we performed RNA-sequencing on cerebellar and motor cortex samples from 9 RTT patient autopsies to identifying potentially druggable targets which might begin from a place of translational relevance. This approach led us to focus on muscarinic acetylcholine receptors (mAChRs), where 4 of the 5 subtypes had significantly disrupted expression. Of particular interest was the mAChR1 (CHRM1, M1) subtype, which has long been considered a viable therapeutic target for neuropsychiatric diseases. Excitingly, we recently used 44 temporal cortex samples from RTT autopsies to confirm that decreased CHRM1 expression is a robust and highly penetrant aspect of RTT pathophysiology in humans. We then established that administration of an M1 positive allosteric modulator (PAM), VU0453595 (VU595), significantly improves social and respiratory (apnea) phenotypes in Mecp2+/- mice. In Aim 1, we propose to expand these preliminary data by testing the effects of M1 potentiation against a full battery of RTT-like phenotypes in mice, using structurally distinct M1 PAMs, acute and chronic dosing paradigms, and multiple modes of pharmacology. Interestingly, if RTT autopsy expression data is binned by MECP2 mutation, then signature expression patterns are observed, not only for M1 but for virtually all preclinical target genes tested. As changes in gene expression are often the rationale for target selection, and modulation of neurotransmission in contexts where receptor function is normal carries an increased risk for adverse effects, these findings have important implications regarding the need for precision medicine in RTT. In Aim 2, we will use mice carrying common RTT mutations (T158M, R168X, R255X, and R306C) to determine whether expression patterns can be used to predict M1 PAM efficacy. We will couple these experiment with transcript and proteomic analysis of RTT autopsy samples to quantify global gene expression patterns in the medulla of RTT patient sub-populations. Finally, of our existing data set, we consider the effect of VU595 on apneas to be a salient finding, both because respiratory dysfunction is predictive of early lethality in RTT and because apneas represent a highly translatable outcome measure. In Aim 3, we propose to mechanistically dissect the role of M1 in RTT respiratory phenotypes by coupling whole body plethysmography and in vivo cyclic voltammetry experiments in Mecp2 knock-in mice to determine the M1 PAM mechanism of action, as well as define where and how M1 functions in the respiratory circuit across RTT sub-populations.