A precision medicine approach to Rett Syndrome

摘要 已知MeCP2在神经发育障碍Rett综合征(RTT)中的致病本质 近20年；然而，目前还没有可行的干预战略。造成这种匮乏的一个因素 治疗选择之一是在临床前发展期间依赖雄性MeCP2/y基因敲除小鼠，尽管 事实上，RTT几乎只影响女性，她们是x连锁的MECP2等位基因(MeCP2+/-)的嵌合体。 此外，RTT患者很少有导致MeCP2蛋白丢失的突变，而是有错义或 截断使关键功能域变得次形的突变。为了解决构造有效性方面的这一缺陷， 我们对9例RTT患者尸检的小脑和运动皮质样本进行了RNA测序 确定可能从具有翻译相关性的地方开始的潜在可用药目标。这种方法 使我们专注于M胆碱受体(MAChRs)，其中5个亚型中有4个亚型显著 表达受阻。特别感兴趣的是mAChR1(CHRM1，M1)亚型，它长期以来一直是 被认为是治疗神经精神疾病的可行靶点。令人兴奋的是，我们最近使用了44个时间 RTT尸检的皮质样本证实CHRM1表达降低是强有力的和高度的 人类RTT病理生理学的穿透性方面。然后我们确定了M1阳性的管理 变构调节剂(PAM)VU0453595(VU595)显著改善社会和呼吸系统(呼吸暂停) MeCP2+/-小鼠的表型。在目标1中，我们建议通过测试M1的效果来扩展这些初步数据 在小鼠中，使用结构不同的M1 PAM，急性和非特异性地增强对一组RTT样表型的增强 慢性给药模式，以及多种药理学模式。有趣的是，如果RTT尸检数据 被MECP2突变绑定，然后观察到特征表达模式，不仅对于M1，而且对于虚拟 所有临床前靶基因均已测试。因为基因表达的变化通常是选择目标的理由，并且 在受体功能正常的情况下调节神经传递会增加患高血压的风险 除了不良反应，这些发现对RTT中精准药物的必要性具有重要的意义。在……里面 目的2，我们将使用携带常见RTT突变(T158M、R168X、R255X和R306C)的小鼠来确定 表达模式是否可以用来预测M1 PAM的疗效。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全球基因表达模式 RTT患者亚群的髓质。最后，在我们现有的数据集中，我们考虑VU595对 呼吸暂停是一个突出的发现，因为呼吸功能障碍预示着RTT的早期死亡，以及 因为呼吸暂停是一种高度可翻译的结果衡量标准。在目标3中，我们建议机械地 结合全身体积图和活体循环分析M1在RTT呼吸表型中的作用 MeCP2基因敲除小鼠的伏安实验以确定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.