Translating preclinical studies of mitochondrial disease mice towards monitoring disease progression in patients

总体目标：使用患者特异性变体MRPL 44（L156 R）建立线粒体疾病的新型小鼠模型，并利用多组学构建线粒体疾病的综合病理生理学。线粒体疾病是一种具有异质性表现的遗传性多系统遗传性疾病。线粒体有自己独立的蛋白质合成机制，线粒体核糖体（mitoribosomes）是其中的一部分。线粒体疾病的新出现的疾病组是线粒体中的突变;通常影响由线粒体DNA编码的蛋白质的产生。因此，在线粒体突变患者中发现的常见生化缺陷是氧化磷酸化复合物的缺陷。MRPL 44是一种大亚基蛋白，其变异体L156 R最初被发现在婴儿心肌病患者中是致病性的。到目前为止，有9名MRPL 44突变患者，所有患者都携带至少一个L156 R等位基因，使其成为关键的目标变体。此外，严重缺乏线粒体疾病的稳健动物模型，这被证明是治疗方法开发的障碍。因此，我们希望首先使用关键的患者特异性变体MRPL 44（L156 R）建立一个敲入小鼠模型，该模型准确模拟患者表型，然后使用多组学深度表型分析小鼠，观察RNA，蛋白质和代谢物的所有差异。这将有助于建立线粒体疾病的发病机制，并找到候选生物标志物，这是监测疾病进展的宝贵工具，特别是在线粒体疾病中。 第一阶段：MRPL 44（L156 R）小鼠作为线粒体疾病的准确动物模型的验证。由于心脏和肝脏的分子表型已经确定，我们将在骨骼肌和大脑中展示这些分子表型。将使用4个不同年龄组（1个月、3个月、6个月和12个月）验证疾病的进展性质。 第二阶段：使用组学技术对MRPL 44（L156 R）小鼠进行深度表型分析和表征。将使用不同组织进行更大队列的RNA测序。此外，将对不同年龄组的关键组织进行串联质谱分析，以进行蛋白质组学和代谢组学分析。这将用于寻找候选生物标志物。 第三阶段：在线粒体疾病患者样本中验证从模型中鉴定的生物标志物。这将需要使用人体组织，并将遵守人体组织法。 伦理考虑：虽然该项目使用小鼠作为主要研究对象，但该项目的这一部分不需要伦理批准，因为已经收集了小鼠的样本，并且没有活的小鼠。该项目主要包括小鼠组织的分子分析。人体组织法不包括细胞系。

Overall Aim: Establish a novel mice model of mitochondrial disease using patient specific variant MRPL44(L156R) and leverage multi-omics to build a comprehensive pathophysiology of mitochondrial disorders. Mitochondrial disorders are inherited multi-systemic genetic disorder with heterogeneous presentation. The mitochondria have their own separate protein synthesis mechanism which mitochondrial ribosomes (mitoribosomes) are part of. New emerging disease group of mitochondrial diseases are mutation in the mitoribosome; typically affecting production of proteins encoded by the mitochondrial DNA. Thus, a common biochemical deficiency found in patients of mitoribosome mutations are deficiency in the complexes of oxidative phosphorylation. MRPL44 is a protein of the large subunit and its variant L156R was found to be pathogenic originally in infantile cardiomyopathy patient. To date, there are 9 patients of MRPL44 mutations and all of the patients carry at least one allele of the L156R making it a key variant of interest. Additionally, there profound lack of robust animal models of mitochondrial disorders which is proving to be an hinderance in the development of therapeutics. Therefore, we want to first make a knock-in mice model using key patient specific variant MRPL44(L156R) that is accurately emulating patient phenotypes and then characterise the mice using multi-omics deep phenotyping looking at all the differences in the RNA, protein and metabolites. This will help to establish the mechanism of pathogenesis of mitochondrial disorders and find candidate biomarkers which is invaluable tool for monitoring the progression of disease which is particularly relevant in mitochondrial disorders. MethodsPhase one: validation of the MRPL44(L156R) mice as an accurate animal model of mitochondrial disorders. As the molecular phenotypes of the heart and liver were established already, we will demonstrate these molecular phenotypes in skeletal muscle and brain. The progressive nature of the disease will be validated using 4 different age groups: 1m, 3m, 6m and 12m. Phase two: deep phenotyping and characterisation of the MRPL44(L156R) mice using omics technology. RNA sequencing of a bigger cohort using different tissues will be performed. Moreover, Tandem-mass spectrometry will be performed on the key tissues in different age groups for proteomic and metabolomic analysis. This will be used to find candidate biomarkers. Phase three: the validation of biomarkers identified from the model in mitochondrial disease patient samples. This will require use of human tissues and will be done complying to the human tissue act. Ethical considerations: Although, this project uses mice as the subject of the primary investigation, this arm of the project does not require ethical approvals as the samples from the mice are already collected and there are no alive mice. This project will mainly consist of molecular analysis of mice tissues. Cell lines are not covered by human tissue act.