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

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

Thalassemia syndromes and sickle cell disease (SCD) represent a significant global health problem and financial burden to health services. Studying these diseases is severely impeded by paucity of suitable and adequate quantities of material from patients, and lack of suitable cell lines. Mouse models of the diseases are therefore routinely used for biological studies and drug evaluation, but fundamental differences exist between mouse and human erythropoiesis. New approaches and human systems for these disorders are therefore essential.
We have recently 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 we have extensively characterised 2) a platform for CRISPR genome editing of BEL-A cells, creating sublines with single or multiple gene edits.
We now have 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 BEL-A cells via CRISPR. We aim to create a range of beta and alpha thalassemia lines with mutations associated with different disease severity, and a SCD line. Such a compendium of lines for a disease will also help determine variability in disease mechanisms, as well as evaluation of drugs etc across spectra of phenotypes. Importantly all lines will have a constant genetic background, and the same as the founder line, negating the many experimental variables between patient samples
Lines will undergo extensive characterisation using a wide range of approaches both to validate disease phenotype and to provide a data resource to facilitate application of the lines by ourselves and others, including comparative proteomics to also identify novel dysregulated proteins.

Studying the molecular defects behind many RBC diseases is severely impeded by paucity of suitable, and adequate quantities of material from anemic patients, and lack of suitable cell lines. Of such diseases the thalassemia syndromes and sickle cell disease represent a significant global health problem and financial burden to health services.
Impact to academic researchers and health service professionals will thus come from the opportunity to create human model cellular systems for these diseases utilising our BEL-A line (a previously unavailable resource) and genome editing (as well as our immortalisation methodology) and from application of the lines themselves for (i) further delineating the molecular basis of disease pathophysiology (ii) as clinically relevant screening tools for the validation and evaluation of drugs and their mode of action (iii) testing vectors and their permutations for gene therapy strategies (iv) insertion, verification and functional determination of SNPS identified from GWAS as potential genetic modifiers of disease severity, overall facilitating advancement in scientific knowledge.
Impact will also stem from our use of multiplex quantitative proteomics to identify novel dysregulated proteins in the disease cells, along with pathway and gene set enrichment analysis tools to determine molecular mechanisms/pathways effected and to identify upstream regulators.
Impact will further stem from use of the lines by industry. We have already received requests from three pharmaceutical companies to use the lines in drug development work. Provision of the lines to such companies will be considered in discussion with the University of Bristol Research and Enterprise Development (RED) team on a one-by-one basis. We have not ruled out charging commercial organisations, reinvesting income into infrastructure to support distribution to non-commercial organisations.
As our aim is to make the lines easily accessible for researchers, following discussion with RED we do not plan to patent. This will not affect their impactfullness.
Furthermore, our approaches can be used to generate lines for other RBC diseases creating further impact. Indeed, we have already had requests from researchers for specific disease lines.
To date there are not drugs available for the thalassemia's and just 2 for SCD, but not suitable for many patients. New drugs and therapeutic strategies are therefore desperately required. Our proposed disease lines will have impact on health and wellbeing by enabling greater understanding of the diseases and by providing screening platforms from drugs and reagents expediating the development of novel therapeutics.
The lines will also have economic impact as will reduce the number of model mice presently used to study these diseases, producing significant financial savings.
Impact will stem from publications in high-profile peer-reviewed journals; invitations to disseminate data at conferences both home and abroad; our long-standing collaboration with National Health Service Blood & Transplant (NHSBT) in Bristol, Oxford and Cambridge, presenting data at research seminars and at the annual NHSBT R&D conference; formation of new national and international collaborations; further funding applications.
The funded researcher will receive high-class training in multi-disciplinary research essential for modern medically orientated research in academia, healthcare or industry. Such impact will also derive from skill enhancement of the applicants. We also anticipate considerable impact generated through collaborative links already established nationally and internationally and with our project partners their groups and institutes.
Impact to the public and wider community of young researchers will incur by our continued public engagement activities and commitment to training international and national postgraduate students, undergraduates and sixth form pupils