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

开发用于研究红细胞疾病并作为筛查平台的人体模型细胞系统

• 批准号: MR/S021140/1
• 负责人: Jan Frayne
• 金额: $ 62.46万
• 依托单位: University of Bristol
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FS021140%2F1
• 关键词: Developing; human; model; cellular; systems

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

红细胞（RBC）疾病可导致慢性贫血，是世界范围内发病率和死亡率的主要来源。其中，地中海贫血综合征（α和β地中海贫血）和镰状细胞病（SCD）代表了显著的全球健康问题和卫生服务的财政负担，没有可用于地中海贫血的药物，并且仅有2种用于SCD的药物，但不适合许多患者。主要治疗方法是红细胞输注，唯一的治愈方法是骨髓移植。因此，迫切需要新的具有成本效益的治疗方法来为最多的人提供最佳治疗。然而，研究这些疾病严重阻碍了缺乏合适和足够数量的材料从病人，缺乏合适的细胞系，准确地模拟疾病状态。虽然可以从外周血干细胞体外产生红系细胞，但是该方法受到细胞的有限扩增潜力的严重限制，因此所产生的细胞数量有限，需要重复收集，这是一种特别不适合贫血患者的方法。因此，这些疾病的小鼠模型通常用于生物学研究和药物评价，但小鼠和人类红细胞生成（RBC产生过程）之间存在根本差异。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并产生了1）第一个永生化的成人人类红细胞系（BEL-A），其重现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其细胞核以产生成熟的红细胞，提供可持续的细胞供应，我们已经广泛表征; 2）用于将突变引入BEL-A细胞的平台，通过单个或多个基因编辑创建亚系。我们现在有独特的机会利用这些工具和技术创建人类模型细胞系，RBC疾病系统，提供可持续和可再生的细胞供研究。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α地中海贫血细胞系，这些细胞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SCD细胞系。这些细胞系将为研究人类突变的细胞特异性效应提供独特的机会，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，消除患者样本之间的许多实验变量。此外，针对给定疾病的这样一系列系将有助于确定疾病机制的可变性，以及跨表型谱的药物评价等。系将经历广泛的表征以验证疾病表型，并作为数据资源以便于我们自己和他人使用系。在广泛的分析中，我们将包括比较蛋白质组学，以验证已知的目标，并确定新的失调蛋白，为未来的研究。所有数据都将在一个专门的网站上提供。这些细胞系将成为一个有价值的资源，用于广泛的应用，包括：i）进一步研究疾病表型背后的红系细胞特异性分子机制，ii）临床相关的药物评价和作用模式分析筛选工具，iii）分析基因治疗策略的试剂iv）插入，验证和功能测定从全基因组研究中鉴定的突变作为疾病严重程度的潜在调节剂。总之，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，而是一个相关的和广泛特征化的疾病系的概要，作为我们和研究界的一个现成资源，

项目成果

Reproducible immortalization of erythroblasts from multiple stem cell sources provides approach for sustainable RBC therapeutics.

• DOI: 10.1016/j.omtm.2021.06.002
• 发表时间: 2021-09-10
• 期刊: Molecular therapy. Methods & clinical development
• 作者: Daniels DE;Ferguson DCJ;Griffiths RE;Trakarnsanga K;Cogan N;MacInnes KA;Mordue KE;Andrienko T;Ferrer-Vicens I;Ramos Jiménez D;Lewis PA;Wilson MC;Canham MA;Kurita R;Nakamura Y;Anstee DJ;Frayne J
• 通讯作者: Frayne J

Bone marrow sinusoidal endothelium controls terminal erythroid differentiation and reticulocyte maturation.

• DOI: 10.1038/s41467-021-27161-3
• 发表时间: 2021-11-29
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Heil J;Olsavszky V;Busch K;Klapproth K;de la Torre C;Sticht C;Sandorski K;Hoffmann J;Schönhaber H;Zierow J;Winkler M;Schmid CD;Staniczek T;Daniels DE;Frayne J;Metzgeroth G;Nowak D;Schneider S;Neumaier M;Weyer V;Groden C;Gröne HJ;Richter K;Mogler C;Taketo MM;Schledzewski K;Géraud C;Goerdt S;Koch PS
• 通讯作者: Koch PS