Ex vivo Engineering of Red Blood Cells
Authors: Frayne Jan , Daniels Deborah E , Frayne Jan , Satchwell Timothy J , Hawksworth Joseph , Singleton Belinda K , Andrienko Tatyana N , Wilson Marieangela C , Kupzig Sabine , Meinders Marjolein , Kurita Ryo , Nakamura Yukio , Ferrer Vicens Ivan , Griffiths Rebecca , Cogan Nicola , Trakarnsanga Kongtana , Anstee David J. , Toye Ashley Mark 
Published: November 2019

Culture systems for human in vitro erythropoiesis are now well established. Using our 3-stage feeder-free erythroid culture system we can efficiently differentiate erythroid cells from adult and cord blood (CB) CD34+ cells with >105 fold expansion, enucleation rates of up to 95% and producing packed reticulocyte yields of >12ml post leukofiltration1. The final preparations for a first in man clinical trial of adult cultured reticulocytes produced under good manufacturing practice (RESTORE) are underway.
Although we have shown that it is possible to modify the CD34+ derived cells using lentivirus for reengineering or additions to the medium, the finite proliferative capacity of CD34+ cells in culture currently limits yield. We therefore took the alternative approach of immortalising early erythroid cells differentiated from adult bone marrow (BM) CD34+ cells, creating the BEL-A (Bristol Erythroid Line Adult) line2, a sustainable erythroid cell source that recapitulates normal adult erythropoiesis, terminally differentiating to generate enucleated reticulocytes that express normal levels of adult globin.
We have created a further 13 lines from BM, adult peripheral blood, CB and iPSC CD34+ cells, demonstrating reproducibility of the approach and its application to create lines from more accessible stem cell sources. Analysis of surface marker and globin profiles demonstrate the lines follow a similar differentiation profile to their respective primary cell source, with comparative proteomics confirming cell source representation of the lines. Lines always established at the pro-erythroblast/early basophilic stage, even when later stage erythroid cells were present in populations.
As well as proof of principal as an alternative transfusion product and improved tools for studying erythropoiesis, such lines have far reaching additional applications, a number of which we are now exploring:
Diagnostic and 'Universal' transfusion products:
Presently serological testing by blood group reference laboratories relies on donated blood, which represent a finite resource and for some blood group phenotypes can be difficult to source. We used CRISPR-Cas9 gene editing to remove blood group antigens in order to generate a sustainable bank of cell lines with useful blood group phenotypes for diagnostic purposes3. Building on this, with the aim of developing a more compatible "universal" transfusion product to meet the needs of chronically transfused patients and those with rare blood group phenotypes, we used combinatorial gene targeting to create sublines deficient in multiple antigens responsible for the most common transfusion incompatibilities (ABO [Bombay], Rh [Rhnull], Kell [K0], Duffy [Fynull], GPB [S-s-U-]). Individual and multiple blood group knockout lines retained the ability to undergo terminal differentiation and enucleation, also illustrating the capacity for coexistence of multiple rare blood group phenotypes within viable reticulocytes3.
Cytokine independent lines
Cytokines represent a substantial cost contribution to erythroid culture systems. We therefore exploited activating mutations found in patient c-Kit and EPOR that cause hypersensitivity to ligand, to create cytokine independent lines thus increasing economic viability of cultured red cells. Bi-allelic EPOR or c-kit edits were introduced into BEL-A with confirmation and exploration of mechanism on differentiation in the absence, or with substantially reduced levels of cytokines.
Model disease systems
In addition to potential therapeutic applications we are also creating lines as model cellular disease systems for studying molecular mechanisms and as drug screening platforms, via CRISPR-Cas9 gene editing of BEL-A and by directly immortalising patient stem cells. To date we have made b-thalassemia major, HbE thalassemia and lines with KLF1 mutations. Furthermore, we have shown BEL-A reticulocytes support invasion and growth of Plasmodium falciparum and are utilising the line to study mechanisms of malaria parasite invasion4. Kupzig S, Parsons SF, Curnow E, Anstee DJ, Blair A. Superior survival of ex vivo cultured human reticulocytes following transfusion into mice. 2016;102:476-483Trakarnsanga K, Griffiths RE, Wilson MC, et al. An immortalized adult human erythroid line facilitates sustainable and scalable generation of functional red cells. Nat. Commun. 2017;8:14750Hawksworth J, Satchwell TJ, Meinders M, et al. Enhancement of red blood cell transfusion compatibility using CRISPR-mediated erythroblast gene editing. EMBO Mol. Med. 2018; 10:e8454Satchwell TJ, Wright K, Haydn-Smith K, et al. Genetic manipulation of cell line derived reticulocytes enables dissection of host malaria invasion requirements. Nat. Comm. 2019;10:3806

Disclosures
No relevant conflicts of interest to declare.