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

UK - Department for Business, Energy and Industrial Strategy

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

Disclaimer: The data for this page has been produced from IATI data published by UK - Department for Business, Energy and Industrial Strategy. Please contact them (Show Email Address) if you have any questions about their data.

Project Data Last Updated: 27/08/2020

IATI Identifier: GB-GOV-13-FUND--GCRF-MR_S021140_1

Description

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

Objectives

The overall objective of our proposal is to create not just much needed human cellular model systems of the thalassemia syndromes and sickle cell disease (SCD), but a compendium of associated and extensively characterised disease lines complemented by the founder BEL-A line. The lines will provide a readily available and reproducible resource for ourselves and the hematology/hematopoiesis community for further delineating the underlying molecular mechanisms of disease, for developing new therapeutic strategies and as screening platforms for the effect, efficacy and action of new drugs and reagents. The thalassemia syndromes and SCD represent a significant global health problem and financial burden to health services and are of particular interest to many researchers and health service professionals. However, the underlying molecular mechanisms of the disease phenotypes are not fully understood, and therapy options limited. Studying the molecular defects behind such RBC diseases is severely impeded by paucity of suitable, and adequate quantities of material from patients, and lack of suitable cell lines. Therefore, new approaches and human model cellular systems for these disorders are essential. We have recently developed methodology and created 1) the first immortalised adult human erythroid cell line (BEL-A) that recapitulates normal erythropoiesis, expresses normal levels of adult globin, terminally differentiates and enucleates to generate mature reticulocytes, providing a sustainable supply of cells; 2) a platform for CRISPR-Cas9 genome editing of 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. Specific Objectives of Proposal: i) Generation of human disease lines by genome editing the BEL-A line. Disease mutations will be introduced into the BEL-A line using CRISPR-Cas9. We plan to create eight different beta thalassemia and five different alpha thalassemia lines with mutations associated with different disease severity and with different mode of action, as well as a SCD line. 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 and in a human cellular milieu. Importantly, all lines will have a constant genetic background, and the same as the founder line removing the many experimental variables between patient samples. ii) Validation and characterisation of disease lines Lines will undergo extensive characterisation both to validate disease phenotype and as a data resource to facilitate future studies and applications. Examples include globin expression levels and profile, subunit ratios and solubility, oxygen association/dissociation, expansion, differentiation, morphological analysis, ROS levels and apoptosis, sickling of SCD cells, abundance of proteins reported aberrantly expressed, response to reagents that increase gamma globin and to perturbation of factors involved in gamma globin silencing. iii) Multiplex comparative proteomics will be used to both validate known targets and to identify novel dysregulated proteins, along with pathway and gene set enrichment analysis tools to determine molecular mechanisms/pathways effected and upstream regulators. We will also compare the profile of such proteins across lines with mutations associated with differing disease severity for the respective thalassemia. Identified proteins will be followed up by western blot qPCR etc and overexpression or knockdown in disease and control lines to verify involvement in disease phenotype. iv) Generation of immortalised erythroid lines from patient CD34+ cells. Lines can be created directly using our immortalisation methodology as an alternative approach for risk mitigation.

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