Identifying the transcriptomic basis underlying individualised drug response: moving towards personalised medicine in motor neurone disease (MND) usin

个体的遗传特征决定了我们对药物的反应，因此需要确定参与治疗反应的分子机制。这将允许根据患者的基因将最有效的药物分配给不同的患者群体。虽然这种方法用于哮喘和癌症，但对于受神经退行性疾病影响的患者，尚未发现遗传谱和治疗之间的匹配。运动神经元病（MND）是最常见的成人发病运动障碍，其特征是运动神经元的进行性丧失和死亡。该导师等率先开展的体外研究表明，来自MND患者的星形胶质细胞可导致野生型运动神经元死亡（Re et al 2014; Meyer et al 2014）。目前，利鲁唑是唯一获准用于治疗精神疾病的药物。其作用机制尚不清楚，人们认为利鲁唑可能通过多种途径发挥作用。此外，在我们实验室进行的药物筛选已经确定了3种有效的Nrf2激活剂，Nrf2是一种通过与抗氧化反应元件（ARE）相互作用驱动多种细胞保护基因表达的转录因子（Mead et al . 2013）。我们现在已经筛选了增加剂量的这3种ARE激活剂，以及利鲁唑与患者星形胶质细胞和运动神经元共培养系统。患者星形胶质细胞通常导致运动神经元存活减少50%。我们发现，用这4种化合物处理星形胶质细胞可以抑制它们对运动神经元的毒性，从而导致运动神经元存活率的增加，这取决于供体基因亚型。本研究的主要目的是：(1)确定利鲁唑和are激活剂对星形胶质细胞基因表达的作用模式；(2)将特定药物的作用机制与供体基因型联系起来。研究策略：我们将从人类诱导的神经祖细胞（iNPCs）中获得星形胶质细胞，并将其分化为先前描述的星形胶质细胞（Meyer et al . 2014）。我们将使用来自携带MND相关突变的患者（即SOD1， C9orf72和TDP43），散发性ALS患者和对照组的星形胶质细胞。我们将筛选三种不同浓度的利鲁唑以及三种ARE激活剂的三种浓度。我们将确定每种治疗的最有效浓度，并在药物治疗前后进行rna测序。学生将通过与研究所内的大型生物信息学小组合作，发展重要的生物信息学技能。我们将确定1。通过比较治疗和未治疗样品（患者和对照组）之间差异表达的转录本来确定每种药物的作用模式。通过比较不同组间参与药物反应的转录本，了解每种药物对不同突变患者的不同作用机制。确定具有明确基因突变的患者中参与特定药物反应的转录本组也将有助于我们更好地对散发患者进行分类，散发患者占所有ALS病例的95%。为了验证我们的筛选，我们还将对另外10个零星样本进行rna测序，通过观察它们的转录组谱来预测它们对哪种ARE激活剂更敏感，并在我们的共培养试验中测试结果。

Individual genetic characteristics determine how we respond to drugs, thus the need to identify the molecular mechanisms involved in treatment response. This will allow to assign the most effective drug to different groups of patients depending on their genetics. While this approach is used in asthma and cancer, no match between genetic profile and therapy has been identified for patients affected by neurodegenerative disorders.Motor neurone disease (MND) is the most common adult onset motor disorder, characterized by progressive loss of motoneurons and death. In vitro studies pioneered by the supervisor and others have shown that astrocytes from MND patients cause wild-type motoneuron death (Re et al 2014; Meyer et al 2014). At present, Riluzole is the only drug licensed for the treatment of MND. The mechanism(s) of action is/are unclear and it is thought that Riluzole might be active through various pathways. Moreover, a drug screening performed in our laboratories has identified 3 potent activators of Nrf2, a transcription factor driving the expression of multiple cytoprotective genes via its interaction with the antioxidant response element (ARE) (Mead et al 2013). We have now screened increasing doses of these 3 ARE activators, as well Riluzole in a co-culture system with patient astrocytes and motoneurons. Patient astrocytes typically lead to a decrease in motoneuron survival by 50%. We found that treatment of astrocytes with these 4 compounds dampens their toxicity towards motoneurons, thus leading to an increase in motoneuron survival, depending on the donor genetic subtype. The main aims of this study are 1) to identify modes of riluzole and ARE activators action on gene expression in astrocytes and 2) correlate the mechanism of action of specific drugs with donor genotype.Research strategy: We will derive astrocytes from human induced neural progenitors (iNPCs) and differentiate them to astrocytes as previously described (Meyer et al 2014). We will use astrocytes from patients carrying mutations associated with MND, namely SOD1, C9orf72 and TDP43, sporadic ALS patients and controls. We will screen 3 different concentrations of Riluzole as well as 3 concentrations of each of the 3 ARE activators.We will identify the most effective concentration of each treatment and perform RNA-sequencing before and after drug treatment. The student will develop significant bioinformatics skills by working with the large bioinformatics group within the Institute. We will determine 1. The modes of action of each drug by comparing differentially expressed transcripts between treated and untreated samples, both patients and controls.2. The different mechanism of action of each drug on groups of patients carrying different mutations by comparing the transcripts involved in drug response between those groups.Identifying groups of transcripts involved in specific drug response in patients with defined genetic mutations will also help us better categorise sporadic patients, accounting for 95% of all ALS cases.To validate our screening, we will also perform RNA-Sequencing of 10 additional sporadic samples, predict which ARE activator they should be more responsive to by looking at their transcriptomic profile and test the results in our co-culture assay.