The use of stem cells for patient stratification approaches in motor neurone disease

肌萎缩侧索硬化症（Amyotrophic Lateral Sclerosis，ALS）是一种导致上下运动神经元死亡的神经退行性疾病。ALS的治疗方法一直被证明是困难的，这是由于该疾病令人印象深刻的遗传和表型异质性。通过使用患者来源的干细胞，我们进行了药物筛选，以测试三种不同抗氧化药物的神经保护作用。这项研究表明，三种药物中没有一种在所有患者培养物中具有神经保护作用，相反，我们确定了特定的药物应答者和无应答者，表明不同的药物可以在不同的患者中实现神经保护。药物治疗前后患者细胞的基因表达谱鉴定了能够区分患者应答者的特定基因特征。这种方法的主要限制是将皮肤成纤维细胞重编程为干细胞以评估特定药物对患者源性神经元的神经保护作用的可行性。这种方法在现实生活中的临床环境中是不可行的，因此我们的目标是确定是否可以在外周血单核细胞（PBMC）中识别出应答基因签名。在这个项目中，我们将特别关注一种神经保护药物，即M102，该药物将于2023年进入临床试验（由MRC资助的I期试验）。 目的：1.在患者源性神经元上测试M102并识别患者应答者和非应答者; 2.进行RNA-Seq并基于共培养结果证实应答者与非应答者的RNA标签生物标志物; 3：确定鉴定从相同供体分离的PBMC中的应答者的外周转录标签的存在。新奇：尽管非常需要，但尚未建立用于神经变性疾病的患者分层方法。这项研究在方法上是新颖的，因为它结合了一种新的细胞重编程方法，可以更可靠地模拟患者表型，转录组学和患者生物样品，如PBMC。 时间：由于临床试验将于2023年开始，通过分析外周生物样本的转录组学来了解患者分层的真实的潜力至关重要，因为它将支持未来的患者分层平台。 实验方法：我们将1。在20个体外ALS患者来源的神经元系中测试M102以鉴定患者应答者和非应答者; 2.在药物治疗之前和之后对相同的20个患者细胞系进行RNA-Seq，以基于共培养结果证实先前鉴定的应答者与非应答者的RNA特征生物标志物; 3.对相同的20名未接受药物治疗的供体的PBMC进行RNA-Seq，以确定是否存在识别应答者的外周转录特征，并且理想情况下至少部分与第2点中体外识别的特征重叠。

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease resulting in the death of the upper and lower motor neurons. Therapeutic approaches towards ALS have always proven difficult due to the impressive genetic and phenotypic heterogeneity of the disease. By using patient-derived stem cells, we ran a drug screening to test the neuroprotective effect of three different anti-oxidant drugs. This study revealed that none of the three drugs was neuroprotective in all patient cultures, instead, we identified specific drug responders and non-responders, indicating that neuroprotection can be achieved with different drugs in different patients. Gene expression profiling of patients cells before and after drug treatment identified a specific gene signature able to discriminate patient responders. The main limitation of this approach is the feasibility of reprogramming skin fibroblasts into stem cells to assess the neuroprotective effect of specific drugs on patient-derived neurons. This approach would not be feasible in a real-life clinical setting; hence we aim to determine whether the responder gene signature can be identified in peripheral blood mononuclear cell (PBMC). For this project we will focus specifically on one neuroprotective drug, namely M102 that is due to enter clinical trial in 2023 (Phase I trial funded by the MRC). Objectives: 1. Test M102 on patient-derived neurons and identify patient responders and non-responders; 2. Perform RNA-Seq and corroborate the RNA signature biomarker of responders vs non-responders based on the co-culture results; 3: Determine the presence of a peripheral transcriptional signature that identifies responders in PBMCs isolated from the same donors Novelty: Patient stratification approaches for neurodegenerative diseases are not established, although greatly needed. This study is novel in its approach, as it combines a novel methodology for cell reprogramming, which models more reliably patient phenotypes, transcriptomics and patient biosamples such as PBMCs. Timeliness: With a clinical trial due to start in 2023, understanding the real potential of patient stratification through the analysis of transcriptomics of peripheral biosamples is of primary importance, as it will support a future platform for patient stratification. Experimental Approach: We will 1. test M102 in 20 in vitro ALS patient-derived neuron lines to identify patient responders and non-responders; 2. Perform RNA-Seq on the same 20 patient cell lines before and after drug treatment to corroborate the previously identified RNA signature biomarker of responders vs non-responders based on the co-culture results; 3. Perform RNA-Seq on the PBMCs of the same 20 drug naïve donors to determine the presence of a peripheral transcriptional signature that identifies responders and ideally overlaps, at least in part, with the signature identified in vitro in point 2.