Developing human model cellular systems for studying Red Blood Cell diseases and as screening platforms

红细胞疾病可导致慢性贫血，是全世界发病率和死亡率的主要来源。其中地中海贫血综合征(α地中海贫血和β地中海贫血)和镰状细胞病(SCD)是一个重大的全球健康问题和卫生服务的经济负担，没有治疗地中海贫血的药物，只有2种治疗SCD的药物，但不适合许多患者。主要的治疗方法是红细胞输注，唯一有效的治疗方法是骨髓移植。因此，迫切需要新的具有成本效益的治疗方法，以便为最多的人提供最佳的治疗方法。然而，由于缺乏来自患者的适当和足够数量的材料，以及缺乏准确模拟疾病状态的合适的细胞系，对这些疾病的研究受到严重阻碍。虽然外周血干细胞可以在体外生成红系细胞，但这种方法受到细胞扩增潜力的限制，因此产生的细胞数量有限，需要重复采集，这一方法特别不适合贫血患者。因此，小鼠的疾病模型通常用于生物学研究和药物评价，但小鼠和人类的红细胞生成(红细胞产生的过程)之间存在根本的差异。因此，治疗这些疾病的新方法和人类系统是必不可少的。我们最近开发了方法学，并产生了1)第一个永生化的成人红系细胞系(BEL-A)，它概括了正常的成人红细胞生成，细胞表达正常水平的成人血红蛋白，经历正常发育，并排出它们的核以产生成熟的红细胞，提供了我们广泛描述的可持续的细胞供应；2)将突变引入BEL-A细胞的平台，创建具有单基因或多基因编辑的亚系。我们现在有独特的机会利用这些工具和技术创建RBC疾病的人类模型细胞系统，为研究提供可持续和可复制的细胞供应。疾病突变将被引入BEL-A细胞的基因组中。我们计划创建8个β地中海贫血和5个阿尔法地中海贫血品系，这些品系具有与不同疾病严重程度和不同作用模式相关的突变，以及一个SCD品系。这些品系将提供独特的机会来研究人类突变对细胞的特定影响，并在具有恒定遗传背景的人类细胞环境中评估药物和试剂，从而消除患者样本之间的许多实验变量。此外，针对特定疾病的这样一系列线条将有助于确定疾病机制中的可变性，以及跨表型光谱的药物评估等。线条将进行广泛的表征，以验证疾病表型，并作为数据来源，方便我们和他人使用这些线条。在进行的范围广泛的分析中，我们将包括比较蛋白质组学，以验证已知的靶点和识别新的失调蛋白质，用于未来的研究。所有数据将在一个专门的网站上提供。这些系列将是广泛应用的宝贵资源，包括：i)进一步研究疾病表型背后的红系细胞特定分子机制；ii)用于药物评估和作用模式分析的临床相关筛选工具；iii)分析用于基因治疗策略的试剂；iv)插入、验证和功能确定从全基因组研究中确定的潜在疾病严重程度修饰因素的突变。总而言之，我们建议的目的不仅是建立地中海贫血综合征和SCD的急需的人类细胞模型系统，而是一份相关的和广泛描述的疾病线的概要，作为我们自己和研究界随时可用的资源

Red blood cell (RBC) diseases can result in chronic anaemia and are a major source of morbidity and mortality worldwide. Among these the thalassemia syndromes (alpha and beta thalassemia) and sickle cell disease (SCD) represent a significant global health problem and financial burden to health services with no drugs available for thalassemia and just 2 for SCD, but unsuitable for many patients. The mainstay therapy is RBC transfusion, with the only curative treatment bone marrow transplant.Thus, new cost-effective treatments are desperately required to deliver optimal therapies to the greatest number of people. However, studying these diseases is severely impeded by paucity of suitable and adequate quantities of material from patients, and lack of suitable cell lines that accurately mimic the disease state. Although erythroid cells can be generated in vitro from peripheral blood stem cells, the approach is severely limited by the restricted expansion potential of the cells and thus number of cells generated, with repeat collections required, a particularly unsuitable approach for anaemic patients. Mouse models of the diseases are therefore routinely used for both biological studies and drug evaluation, but fundamental differences exist between mouse and human erythropoiesis (the process of RBC production). New approaches and human systems for these disorders are therefore essential.We have recently developed methodology and generated 1) the first immortalised adult human erythroid cell line (BEL-A) that recapitulates normal adult erythropoiesis, with cells expressing normal levels of adult haemoglobin, undergoing normal development and expelling their nuclei to produce mature red cells, providing a sustainable supply of cells which we have extensively characterised; 2) a platform for introducing mutations into the BEL-A cells, creating sublines with single or multiple gene edits.We now have the unique opportunity to exploit these tools and technologies to create lines as human model cellular systems of RBC diseases, providing a sustainable and reproducible supply of cells for study.Disease mutations will be introduced into the genome of BEL-A cells. We plan to create eight beta thalassemia and five alpha thalassemia lines with mutations associated with different disease severity and with different mode of action, as well as a SCD line. The lines will provide the unique opportunity to study cell specific effects of human mutations and evaluate drugs and reagents in a human cellular context with a constant genetic background, removing the many experimental variables between patient samples. Furthermore, such a range of lines for a given disease will help determine variability in disease mechanisms, as well as evaluation of drugs etc across spectra of phenotypes.Lines will undergo extensive characterisation to validate disease phenotype and as a data resource to facilitate use of the lines by ourselves and others. Amongst the wide range of analyses performed we will include comparative proteomics both to validate known targets and to identify novel dysregulated proteins, for future studies. All data will be made available on a dedicated website.The lines will be a valuable resource for a wide range of applications including, i) further investigation into erythroid cell specific molecular mechanisms underlying the disease phenotypes, ii) clinically relevant screening tools for drug evaluation and analysis of mode of action, iii) analysing reagents for gene therapy strategies iv) insertion, verification and functional determination of mutations identified from genome-wide studies as potential modifiers of disease severity.In summary the aim of our proposal is to create not just much needed human cellular model systems of the thalassemia syndromes and SCD, but a compendium of associated and extensively characterised disease lines as a readily available resource for ourselves and the research community