Product for Therapy and Methods

Abstract

A method of making an erythroid cell comprising elevated levels of a target protein or polypeptide, the method comprising: a) provision of an erythroid progenitor which is able to express the target protein or polypeptide; b) expression of the target protein or polypeptide; and c) maturation of the erythroid progenitor into the erythroid cell; wherein during maturation of the erythroid progenitor into the erythroid cell, the target protein or polypeptide is configured and/or inhibited such that ubiquitination of the target protein or polypeptide is hindered or prevented. Erythroid cells, pharmaceutical compositions and methods of use related thereto, and a method of screening for proteins or polypeptides degraded by ubiquitination during maturation of an erythroid progenitor are also provided.

Claims

1. A method of making a reticulocyte comprising elevated levels of a target protein or polypeptide, the method comprising: a) provision of an erythroid progenitor which is able to express the target protein or polypeptide; b) expression of the target protein or polypeptide; and c) maturation of the erythroid progenitor into the reticulocyte; wherein during maturation of the erythroid progenitor into the reticulocyte, the target protein or polypeptide is configured and/or inhibited such that ubiquitination of the target protein or polypeptide is hindered or prevented.

2. A method according to claim 1, wherein ubiquitination of the target protein or polypeptide is hindered or prevented by: i. where the target protein or polypeptide comprises a ubiquitination site, provision of an inhibitor of the target protein or polypeptide ubiquitination site during maturation of the erythroid progenitor into the reticulocyte; ii. where the target protein or polypeptide is a variant of a source protein or polypeptide with a ubiquitination site, provision of an erythroid progenitor which is able to express a target protein or polypeptide comprising a mutation that renders ubiquitination hindered or prevented with respect to the source protein or polypeptide; and/or iii. provision of an erythroid progenitor which is able to express an exogenous protein or polypeptide having no ubiquitination site.

3. A method according to claim 1, comprising the further step of: d) red cell development, in particular maturation of the reticulocyte into an erythrocyte; wherein during maturation to the erythrocyte, the target protein or polypeptide is configured and/or inhibited such that ubiquitination is hindered or prevented, preferably hindered or prevented by any of (i)-(iii) of claim 2.

4. A method according to any preceding claim, wherein the target protein comprises an endogenous protein and/or the target polypeptide comprises an endogenous polypeptide.

5. A method according to any of claims 1-3, wherein the target protein comprises an overexpressed endogenous protein or an exogenous protein and/or the target polypeptide comprises an overexpressed endogenous polypeptide or an exogenous polypeptide.

6. A method according to any preceding claim, wherein the inhibitor is a natural substrate or a natural product of the target protein or polypeptide or is a reversible inhibitor of a natural substrate or natural product of the target protein or polypeptide.

7. A method according to any preceding claim, wherein the erythroid progenitor is a stem cell, haematopoietic stem cell, induced pluripotent cell, erythroid immortalized cell line or erythroblast cell, preferably a CD34+ cell, CD34− cell, or a BEL-A cell.

8. A method according to any preceding claim, wherein the target protein or polypeptide is an enzyme.

9. A method according to any preceding claim, wherein the target protein is thymidine phosphorylase, glutamine synthase, hexokinase/glucokinase, phenylalanine hydroxylase, alcohol dehydrogenase, catalase, glucose-6-phosphate dehydrogenase, adenosine deaminase, L-asparaginase, uricase, bacterial L-phenylalanine ammonia lyase, alanine aminotransferase, glutamate dehydrogenase, arginine deiminase, or arginase.

10. A method according to any preceding claim, wherein the target protein is thymidine phosphorylase, and the thymidine phosphorylase ubiquitination site is inhibited by thymidine, deoxyuridine, thymine, uridine, 2-deoxy ribose 1-phosphate, or derivatives or analogs of these, preferably thymidine.

11. A method according to any of claims 2 to 10, wherein the exogenous protein or polypeptide having no ubiquitination site is a non-eukaryotic protein or polypeptide, preferably a bacterial protein or polypeptide.

12. A method according to any preceding claim, wherein the reticulocyte or erythrocyte is an isolated reticulocyte or isolated erythrocyte.

13. An erythroid cell comprising a target protein or polypeptide, wherein: i) where the target protein or polypeptide comprises a ubiquitination site, the erythroid cell further comprises an inhibitor of the target protein or polypeptide ubiquitination site; and/or ii) where the target protein or polypeptide is a variant of a source protein or polypeptide with a ubiquitination site, the target protein or polypeptide comprises a mutation that renders ubiquitination hindered or prevented with respect to the source protein or polypeptide; and/or iii) the target protein or polypeptide comprises an exogenous protein or polypeptide having no ubiquitination site.

14. An erythroid cell according to claim 13, wherein the erythroid cell is an erythroid progenitor cell.

15. An erythroid cell according to claim 13, wherein the erythroid cell is an enucleated erythroid cell.

16. An erythroid cell according to any of claims 13-15, wherein the target protein comprises an endogenous protein and/or the target polypeptide comprises an endogenous polypeptide.

17. An erythroid cell according to any of claims 13-15, wherein the target protein comprises an overexpressed endogenous protein or an exogenous protein and/or the target polypeptide comprises an overexpressed endogenous polypeptide or an exogenous polypeptide.

18. An erythroid cell according to any of claims 13-17, wherein the inhibitor is a natural substrate or a natural product of the target protein or polypeptide or is a reversible inhibitor of a natural substrate or natural product of the target protein or polypeptide.

19. An erythroid cell according to any of claims 13-18, wherein the target protein is an enzyme.

20. An erythroid cell according to any of claims 13-19, wherein the target protein is thymidine phosphorylase, glutamine synthase, hexokinase/glucokinase, phenylalanine hydroxylase, alcohol dehydrogenase, catalase, glucose-6-phosphate dehydrogenase, adenosine deaminase, L-asparaginase, uricase, bacterial L-phenylalanine ammonia lyase, alanine aminotransferase, glutamate dehydrogenase, arginine deiminase, or arginase.

21. An erythroid cell according to any of claims 13-20, wherein the target protein is thymidine phosphorylase and the thymidine phosphorylase ubiquitination site is inhibited by thymidine, deoxyuridine, thymine, uridine and/or 2-deoxy ribose 1-phosphate, or derivatives or analogues of these, preferably thymidine.

22. An erythroid cell according to any of claims 13-21, wherein the erythroid cell is an isolated erythroid cell.

23. An erythroid cell according to any of claims 13-22, wherein the target protein or polypeptide that has no ubiquitination site is a non-eukaryotic protein, preferably a bacterial protein.

24. An erythroid cell obtainable by the method of any of claims 1-12.

25. A pharmaceutical composition comprising an erythroid cell according to any one of claims 13-24 and a pharmaceutically acceptable carrier, excipient and/or adjuvant.

26. A pharmaceutical composition according to claim 25, comprising an erythroid cell stored in an erythroid cell storage buffer comprising the inhibitor of the target protein or polypeptide ubiquitination site.

27. A pharmaceutical composition comprising an erythroid cell comprising a target protein or polypeptide comprising a ubiquitination site and an inhibitor of the target protein or polypeptide ubiquitination site and a pharmaceutically acceptable carrier, excipient and/or adjuvant.

28. A pharmaceutical composition according to claim 27, wherein the erythroid cell is stored in an erythroid cell storage buffer comprising the inhibitor of the target protein or polypeptide ubiquitination site.

29. A pharmaceutical composition comprising an erythroid cell comprising a target protein or polypeptide, wherein the target protein or polypeptide is a variant of a source protein comprising a ubiquitination site, and wherein the target protein or polypeptide comprises a mutation that renders ubiquitination hindered or prevented with respect to the source protein or polypeptide and a pharmaceutically acceptable carrier, excipient and/or adjuvant.

30. A pharmaceutical composition according to claim 28 or 29, wherein the erythroid cell is an enucleated erythroid cell, preferably a reticulocyte or erythrocyte.

31. An erythroid cell according to any of claims 13-24, or a pharmaceutical composition according to any of claims 25-30, for use in therapy.

32. An erythroid cell or a pharmaceutical composition for use according to claim 31, for use in enzyme replacement therapy, organ reconditioning or detoxification, preferably for the treatment of Mitochondrial Neurogastrointestinal Encephalomyopathy, Hyperammonemia, Hyperglycaemia, phenylalanine hydroxylase enzyme deficiency, alcohol toxicity/detoxification, catalase deficiency and/or preventing cell damage by reactive oxygen species, G6PD deficiency, adenosine deaminase deficiency, acute lymphoblastic leukemia cancer, hyperuricemia, phenylketonuria/phenylalanine hydroxylase enzyme deficiency, or hyperargininaemia.

33. Use of an erythroid cell according to any of claims 13-24, or a pharmaceutical composition according to any of claims 25-30, for the manufacture of a medicament for use in therapy.

34. A method of treatment of a subject in need thereof, the method comprising administering a pharmaceutically effective amount of an erythroid cell according to any of claims 13-24, or a pharmaceutically effective amount of a pharmaceutical composition according to any of claims 25-30.

35. A method of screening for a protein or polypeptide that is degraded by ubiquitination during maturation of an erythroid progenitor, the method comprising expressing a test protein in an erythroid progenitor and: (a) identifying if the test protein amount is elevated when the erythroid progenitor is matured with ubiquitinase activity hindered or prevented, as compared with the test protein amount when the erythroid progenitor is matured without ubiquitinase activity hindered or prevented; (b) identifying if a test protein is labelled when a labelled ubiquitin construct is provided during maturation of the erythroid progenitor; or (c) identifying if the test protein is ubiquitinated through anti-ubiquitin antibody labelling or by mass spectrometry.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0074] One or more embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0075] FIG. 1A shows an example of endogenous thymidine phosphorylase (TP) expression measured by flow cytometry in isolated CD34+ stem cells, isolated reticulocytes and isolated red blood cells from blood donations whereby the dark grey depicts the IgG isotype control and light grey the TP expression. FIG. 1B shows quantification of the flow cytometry graphs (N=3). FIG. 1C shows TP activity as measured in isolated CD34+ stem cells, isolated reticulocytes, isolated red blood cells and as a control isolated platelet from blood donations, tested by a spectrophotometer assay. FIG. 1D shows an example of endogenous thymidine phosphorylase (TP) expression measured by flow cytometry in in vitro cultured day 8 (proerythroblast), day 12 (basophilic erythroblast) and reticulocytes whereby the dark grey depicts the IgG isotype control and light grey is the TP expression. FIG. 1E shows quantification of the flow cytometry graphs (N=3). FIG. 1F shows TP activity as measured in in vitro cultured day 8 (proerythroblast), day 12 (basophilic erythroblast) and reticulocytes. FIG. 1G shows an example of endogenous thymidine phosphorylase (TP) expression measured by flow cytometry in expanding BEL-A, Day 6 differentiated (polychromatic) and BEL-A derived reticulocytes whereby the dark grey depicts the IgG isotype control and light grey the TP expression. FIG. 1H shows quantification of the flow cytometry graphs (N=3).

[0076] FIG. 2 shows the increase in TP expression by exogenous overexpression of TP in cultured CD34+ derived erythroid progenitors expressing TP cells (cTP; FIG. 2A) and expanding BEL-A expressing TP cells (bTP; FIG. 2B), the TP activity in units per cTP cells (FIG. 2C) and the deformability of cTP derived reticulocytes (FIG. 2D)

[0077] FIG. 3 shows the effect of degradation inhibitors on TP expression in cTP cells (FIG. 3A) and a 3D model showing the result of modelling of human TP and location of ubiquitination sites which would need to be mutated (FIG. 3B);

[0078] FIG. 4 shows expression levels of mutated TP in cTP cells (FIG. 4A; cTP-mut) and bTP cells (FIG. 4B; bTP-mut) and activity levels of mutated TP in cTP cells (FIG. 4C; cTP-mut) and bTP cells (FIG. 4D; bTP-mut);

[0079] FIG. 5 shows expression levels of TP in the presence of thymidine supplementation in cTP cells (FIG. 5A) and bTP cells (FIG. 5B);

[0080] FIG. 6 shows a schematic of cell differentiation from a hematopoietic stem cell to a red blood cell;

[0081] FIG. 7 shows a schematic of cell differentiation lineages, starting from a hematopoietic stem cell and from a BEL-A pro-erythroblast respectively, exemplifying stages in vitro where a viral vector can be added.

[0082] FIG. 8 shows a bar graph showing overexpression of glutamine synthase during differentiation using lentivirus as assessed by flow cytometry across two different cultures as well as a bar graph showing glutamine synthase expression levels in the presence and absence of MG132.

[0083] FIG. 9 shows a bar graph showing overexpression of adenosine deaminase (tagged with c-Myc) during differentiation using lentivirus as assessed by flow cytometry across two different cultures as well as a Western blot showing expression in in vitro derived reticulocytes.

[0084] FIG. 10 shows a bar graph showing overexpression of L-asparaginase (tagged with c-Myc) using lentivirus during differentiation as assessed by flow cytometry across two different cultures as well as a Western blot showing expression in in vitro derived reticulocytes.

[0085] FIG. 11 shows a bar graph showing expression of uricase (tagged with c-Myc) using lentivirus during differentiation as assessed by flow cytometry across two different cultures as well as a Western blot showing expression in in vitro derived reticulocytes.

DETAILED DESCRIPTION

Example 1—Endogenous TP Expression in Erythroid Progenitors, Reticulocytes and Erythrocytes

[0086] We first confirmed the base line endogenous expression and thymidine phosphorylase (TP) activity levels in isolated haematopoietic CD34.sup.+ stem cells (i.e. isolated from blood), standard donor derived reticulocytes and erythrocytes. Expression was assessed by flow cytometry on fixed and permeabilised cells and TP activity was determined using a spectrophotometer-based assay whereby the difference in absorbance levels of thymine, in the presence of TP, after 30 minutes at 37° C. was measured. As expected, both assays confirmed a low endogenous expression and activity of TP in CD34.sup.+ haematopoietic stem cells and reticulocytes and no TP expression or activity in red blood cells (FIGS. 1A, 1B and 1C). Freshly isolated platelets from peripheral blood were included as a positive control for the activity assay (1C) as these blood cells contain TP. Next, we examined the endogenous TP expression and activity in erythroid cells differentiated in vitro from CD34.sup.+ haematopoietic stem cells. Previously, we have reported on the different stages of erythroid maturation in our in vitro culturing system (Griffiths, R. E., et al., Blood, 2012, 119(26), p. 6296-306). We refer herein to the days in culture and their approximate stage of differentiation in brackets based on this knowledge. FIGS. 1D, 1E and 1F shows that the expression of endogenous TP and activity on day 8 (pro-erythroblast) day and day 12 (polychromatic erythroblast) is low. This also demonstrates that the expression and activity of unmodified filtered in vitro cultured reticulocytes is comparable to donor isolated endogenous reticulocytes. We next tested endogenous TP expression in the erythroid cell line, BEL-A, an erythroid cell line with the capacity to differentiate into reticulocytes, that are comparable to in vitro cultured reticulocytes (Trakarnsanga, K., et al., Nat Commun, 2017. 8: p. 14750). The advantage of using BEL-A cells is these provide a sustainable source of cells that can be genetically modified and stored frozen with the modification maintained indefinitely (Hawksworth, J., et al., EMBO Mol Med, 2018. 10(6); Trakarnsanga, K., et al., Nat Commun, 2017. 8: p. 14750), whereas CD34+ derived cultures are finite and need to be reinitiated each time. Expanding BEL-A cells (which are comparable to proerythroblast erythroblasts) have a low endogenous TP expression FIGS. 1G and 1H, and the BEL-A derived reticulocytes exhibit no measurable TP expression (FIG. 1H), comparable to CD34+ derived cultured reticulocytes (FIG. 1E) and in vivo isolated reticulocytes (FIG. 1B).

Example 2—Exogenous Overexpression of Thymidine Phosphorylase in In Vitro CD34+ Cells and BEL-A Derived Cells Using Lentivirus

[0087] Cultured erythroid progenitors expressing TP cells (cTP) and expanding BEL-A expressing TP cells (bTP) were created by stably transducing the cells with lentivirus expressing human TP cDNA. Subclones were created from the polyclonal bTP population by blind single cell sorting using FACS. Day 6 cTP (pro-erythroblast) and expanding bTP cells (pro-erythroblast) exhibited a 25- and 45-fold increase respectively, in TP enzyme expression by flow cytometry compared to endogenous expression (FIGS. 2A and 2B). The activity assay confirmed the presence of active enzyme with an approximate concentration of 8.6×10.sup.−9 unit per polychromatic cTP cell, which is comparable to the natural endogenous expression of approximately 10 freshly isolated platelets.

[0088] The cTP cells were differentiated and TP expression was measured at day 10 (basophilic erythrocytes) day 14 (polychromatic erythrocytes) day 16 (orthochromatic erythrocytes) and in filtered reticulocytes (see FIG. 2A). bTP expression was measured during differentiation at day 4 (basophilic erythrocytes) day 6 (polychromatic erythrocytes) day 10 (orthochromatic erythrocytes) and in reticulocytes (see FIG. 2B). Though expression in cTP and bTP reticulocytes was observed to be 6 and 12-fold increased compared to endogenous levels, a striking decline of expression is observed during terminal differentiation. The TP activity measured in filtered cTP reticulocytes was 4.4×10.sup.−9U/Cell (see FIG. 2C). The deformability of the cTP derived reticulocytes was measured using an Automated Rheoscope Cell analyser (ARCA). This showed that cTP reticulocytes are comparable to the unmodified cTP control reticulocytes in both size and deformability (FIG. 2D).

Example 3—Thymidine Phosphorylase is Degraded by the Ubiquitin Degradation Pathway in Erythroblasts

[0089] The substantial loss of TP enzyme expression during differentiation could mean that TP is being actively degraded during terminal differentiation. To test whether exogenous TP degradation during differentiation is due to ubiquitination or lysosomal degradation, we subjected the day 14 (orthochromatic erythrocytes) cTP cells to an ubiquitination inhibitor MG132, or a lysosomal degradation inhibitor, leupeptin (Tsubuki, S., et al., J Biochem, 1996, 119(3), p. 572-6; Hershko, A. and A. Ciechanover, Annu Rev Biochem, 1982, 51, p. 335-64). TP expression was first measured on day 14 and then 24 hours after incubation with inhibitors or the vehicle control. While leupeptin did not impair degradation, inhibition of degradation was observed upon addition of MG132 (FIG. 3A). This indicates that ubiquitination during differentiation is a significant cause of human TP protein degradation.

Example 4—Modelling of Human TP and Mutagenesis of the Ubiquitination Site

[0090] Examination of the human crystal structure of the TP dimer (2J0F.pdb), showed that the protein is composed of two homodimers, each consisting of an 6 α-helixes α-domain and an α/β-domain which consists of antiparallel β-sheet surrounded by α-helixes (Norman, R. A., et al., Structure, 2004, 12(1), p. 75-84). These domains can rotate 8° relative to each other upon substrate binding. Without substrate present, TP is in an open conformation, while binding to thymidine and phosphate causes the enzyme to close. Kinetic studies conducted on E. coli and rabbit TP protein have shown that there is a sequential mechanism of binding, whereby the substrate thymidine is the first to bind, and 2-dR-1-P the last to be released (Krenitsky, T. A., J Biol Chem, 1968, 243(11), p. 2871-5).

[0091] Human and mouse TP proteins are 81.2% identical and sequence comparison confirmed that 2 known ubiquitination sites located at residues 115 and 221 in mouse TP enzyme are conserved in human TP structure. Examination of the structures suggested that both conserved lysines are an integral part of the thymidine binding site and therefore the alteration of these important residues could affect TP activity or stability because the active site is altered (FIG. 3B). The two lysine residues in human TP were exchanged for arginine residue to preserve the active site structure but remove the ubiquitation sites, making TP-mut. This enzyme was expressed in both day 3 in vitro cultured erythrocytes (cTP-mut) and expanding BEL-A cells (bTP-mut) and the cells subsequently differentiated. The TP-mut expression levels achieved were comparable to day 6 cTP and expanding bTP cells (see FIGS. 4A and 4B) but no TP activity was detected in the cTP-mut reticulocyte (FIG. 4C). The mutations compromise the enzyme activity and stability. This provides proof of concept that mutation of ubiquitination sites can be used to prevent protein degradation through the enucleation process, but that care must be taken to ensure any mutation does not destroy the desired protein activity. Therefore, either the enzyme active site would need to be reengineered to retain activity but remove the ubiquitin sites or an alternate means of disrupting TP ubiquitination are required.

Example 5—Thymidine Phosphorylase Degradation is Reduced by Thymidine Supplementation

[0092] After further examination of the molecular human TP structure we observed that the 2 ubiquitination sites that correspond with murine TP, would only be available for ubiquitination in the absence of substrate. We therefore hypothesised that supplementation of the culture media with TP enzyme substrate thymidine may result in a closed TP structure, reducing its degradation by obscuring access to the lysine's ubiquitination sites in the active site. However, previous reports have shown that addition of thymidine can arrest cell cycle and also inhibited cell growth in K562 cells at a concentration of 1 mM (Anisimov, A. G., et al., Izv Akad Nauk Ser Biol, 2003(3), p. 275-84; Thomas, D. B. and C. A. Lingwood, Cell, 1975, 5(1), p. 37-42). To determine if increased thymidine concentrations in our culture media could prevent TP degradation, media was daily supplemented with 0.5 mM thymidine starting at different points during the culture process. We confirmed an increase in cell death and inhibition of differentiation when thymidine was added early during in vitro culture e.g. Day 0 of differentiation, data not shown. To circumvent this, 0.5 mM thymidine supplementation was added daily during differentiation, from day 14 (polychromatic erythroblast stage) in cTP cells and day 6 (polychromatic erythroblast stage) in bTP cells of differentiation, which is approximately the point overexpressed human TP is normally degraded. This manoeuvre doubled TP enzyme abundance in both cTP reticulocytes and bTP reticulocytes compared to cells produced using the standard differentiation media (FIGS. 5A and 5B) which was not supplemented with thymidine.

Example 6—Investigating Retention of Further Enzymes

[0093] To investigate the degree to which exogenous enzyme expression is retained during erythropoiesis, four different enzymes were expressed in CD34+ haematopoietic stem cells (using lentivirus) and subsequently differentiated to reticulocytes: glutamine synthase (FIG. 8), adenosine deaminase (FIG. 9), L-asparaginase (FIG. 10), and uricase (FIG. 11). The exogenous proteins Uricase (a bacterial enzyme), human L-asparaginase (ASP) and human adenine deaminase (ADA) were tagged with c-Myc to facilitate measurement of expression during differentiation by flow cytometry and western blot. GAPDH is an endogenous enzyme which was also blotted for in some cases. For human glutamine synthetase (GS) an antibody specific for this protein was used to detect expression by flow cytometry. The bar graphs in FIGS. 8-11 show expression of indicated enzymes during differentiation as assessed by flow cytometry across two different cultures. Western blots illustrate expression of the enzymes in in vitro derived reticulocytes. To explore whether ubiquitination enhances GS enzyme retention at the end of the culture, MG132 (a ubiquitin inhibitor) was added at day 15 to the GS (over)expressing erythroblasts. Expression was measured at day 19 by flow cytometry.

Example 7—Identification of Further Enzymes Compatible with Invention

[0094] The inventors conducted sequence site scans for ubiquitin consensus sequences on key enzymes according to the method below. For alanine aminotransferase, site scan for consensus sequences suggest ubiquitin sites on both human isoforms. For glutamate dehydrogenase, site scan suggests ubiquitin sites on one of two enzymes. For arginine deiminase, there are 6 human isoforms and site scan identified that 3 isoforms as having ubiquitin consensus sites. For arginase, one of two isoforms was identified as having a ubiquitin consensus site.

Material and Methods

[0095] Antibodies

[0096] Monoclonal thymidine phosphorylase antibody (clone P-GF.44C) was used at 1/10 dilution (Thermo scientific). Secondary antibodies used were APC-conjugated monoclonal anti-mouse IgG1 or polyclonal anti-IgG (Biolegend) or Alexa647-anti-human (Jackson Laboratories) and used at 1:50 (v/v).

[0097] BEL-A Cell Culture

[0098] BEL-A cells were cultured as previously described (Trakarnsanga, K., et al., Nat Commun, 2017, 8, p. 14750). In brief, cells were maintained in expansion medium StemSpan SFEM (Stem Cell Technologies) supplemented with 50 ng/mL SCF milteny, 3 U/mL EPO (Roche, Welwyn Garden City, UK), 1 μM dexamethasone (Sigma-Aldrich) and 1 μg/mL doxycycline (Sigma-Aldrich)] at 1-3×105 cells/mL. Complete medium changes were performed every 48 hours. Differentiation was induced as previously described: cells were seeded at 1.5×10.sup.5/mL in differentiation medium (Iscove's modified Dulbecco's medium (IMDM), Source BioScience, Nottingham UK) containing 3% (v/v) AB Serum (Sigma-Aldrich, Poole UK), 2 mg/ml HSA (Irvine Scientific, Newtown Mount Kennedy, Ireland), 10 μg/ml Insulin (Sigma-Aldrich), 3U/ml heparin (Sigma-Aldrich), 500 μg/ml transferrin (Sanquin Blood Supply, Netherlands) and 3U/ml Epo (Roche, Welwyn Garden City, UK) supplemented with 1 ng/mL IL-3 (R&D Systems, Abingdon UK), 10 ng/mL SCF and 1 μg/mL doxycycline. After 2 days, cells were reseeded at 3×10.sup.5/ml in fresh medium. On differentiation day 4, cells were reseeded at 5×10.sup.5/ml in fresh medium without doxycycline. On differentiation day 6, a complete media change was performed, and cells were reseeded at 1×10.sup.6/mL. On day 8, cells were transferred to differentiation medium (containing no SCF, IL-3 or doxycycline) and maintained at 1×10.sup.6/mL with complete medium changes every 2 days until day 12.

[0099] CD34 Cell Culture

[0100] As previously described (Griffiths, R. E., et al., Blood, 2012. 119(26), p. 6296-306), CD34+ haemopoietic stem cells (HSC) here isolated from human blood donor mononuclear cells or from thawed cryopreserved cord blood units by magnetic bead separation according to manufacturer's instructions (Miltenyi Biotech Ltd, Bisley UK). CD34+ cells were grown at a density of 2×10.sup.5 cells/ml using a base medium consisting of IMDM (Source BioScience) containing 3% (v/v) AB Serum, 2 mg/ml HSA, 10 μg/ml Insulin, 3U/ml heparin, 500 μg/ml transferrin and 3U/ml Epo. In the first stage (days 0-10) this was supplemented with 10 ng/ml stem cell factor and 1 ng/ml IL-3 and in the second stage (days 11-13) with 10 ng/ml SCF. In the final stage to day 19, only the base medium was used. 10 μM leupeptin (Sigma-Aldrich) or 5 μM MG132 (Sigma-Aldrich) was added to 1×10.sup.6 differentiating erythroblasts for 24 hours at 37° C., 5% CO.sub.2. Cells where fixed with paraformaldehyde and analysed by flow cytometry.

[0101] Lentiviral Transduction

[0102] Human TP cDNA sequence was ordered and cloned in the XLG3 vector by Genscript (Genscript, Leiden NL). The original TP sequence was mutated at sites 115 and 221 from a lysine to arginine, thereby creating the TP-mut Lentivirus was prepared according to previously published protocols (Satchwell et al Haematologica, 2015 100; 133-142 Doi:10.3324/haematol.2014.114538). For transduction of BEL-A and CD34+ haematopoietic cells, virus was added to 2×10.sup.5 cells in 2 ml medium in the presence of 8 μg/ml polybrene for 24 h. Cells were washed three times and resuspended in fresh medium.

[0103] Flow Cytometry and FACS

[0104] For flow cytometry on undifferentiated BEL-As, 1×10.sup.5 cells were fixed 1% paraformaldehyde, 0.0075% glutaraldehyde and permeabilised with 0.1% Triton X-100, resuspended in PBSAG (PBS+1 mg/ml BSA, 2 mg/ml glucose)+1% BSA were labelled with primary antibody for 30 minutes at 4° C. Cells were washed in PBSAG, incubated for 30 min at 4° C. with appropriate APC-conjugated secondary antibody, and washed and data acquired on a MacsQuant VYB Analyser using a plate reader. Reticulocytes were identified by gating upon Hoechst-negative population. For FACS sorting of cells, a BDInflux Cell Sorter was used to isolate single clones, by sorting the propidium iodide-negative population into 96-well plates.

[0105] TP Activity Assay

[0106] The cells were resuspended 1×10.sup.6 cells in lysis buffer (50 mM Tris-HCl, pH 7.2, 1% (w/v) triton X-100, 2 mM phenylmethylsulfonyl fluoride (PMSF), 0.02% (v/v) 2-mercaptoethanol). The lysate was centrifuged at 16000 g for 30 min at 4° C. and then add 176 mM thymidine and 5× TP reaction buffer (0.5 M Tris-arsenate, pH 6.5) to the supernatant. The controls used were lysis buffer alone or lysis buffer containing known concentrations of purified TP protein (Sigma-Aldrich, Poole UK). The reaction was incubated at 37° C. for 30 min and then terminated by addition of 0.3M NaOH. The absorbance measured using a spectrophotometer at 299 nm and compared to the TP enzyme standard concentration curve. (Martí, R., L. C. López, and M. Hirano, Methods in Molecular Biology (Clifton, N.J.), 2012. 837: p. 121-133)

[0107] Reticulocyte Deformability Measurements Using ARCA

[0108] 1×10.sup.6 reticulocytes were resuspended in 200 μl polyvinylpyrrolidone solution (PVP viscosity 28.1; Mechatronics Instruments, The Netherlands). Samples were assayed in an ARCA (Dobbe, J. G. G., et al., Measurement of the distribution of red blood cell deformability using an automated rheoscope. 2002. 50(6), p. 313-325) which consists of a plate-plate optical shearing stage (model CSS450) mounted on a Linkam imaging station assembly and temperature controlled using Linksys32 software (Linkam Scientific Instruments, Surrey, UK). The microscope was equipped with an LMPlanFL 50× with a 10.6 mm working distance objective (Olympus, Essex, UK) illuminated by a X-1500 stroboscope (PerkinElmer, The Netherlands) through a band-pass interference filter (CWL 420 nm, FWHM 10 nm; Edmund Optics, Poppleton, UK). Images were acquired using a uEye camera (UI-2140SE-M-GL; IDS GmbH, Obersulm, Germany). At least 1,000 cell images per sample were acquired and analysed using bespoke ARCA software.

[0109] TP Modelling

[0110] Ubiquitination sites were predicted from mUbiSiDa—Database of mammalian protein ubiquitination sites (http://202.195.183.4:8000/BroGo3_data.php?name=0016154). Which provided details of the mouse ubiquitination sites analysed in Wagner et al (Mol Cell Proteomics, 2012, 11(12), p. 1578-85). Clustal Omega (DOI: 10.1093/nar/gkz268) was used to align human and mouse sequences. Protein structures were visualized, and image produced with UCSF chimera software (DOI: 10.1002/jcc.20084).

[0111] Ubiquitin site consensus sequence identification. The full protein sequence was subjected to a search for ubiquitin consensus sequences using two sequence search programs. The first is UbPred (Radivojac, P., Vacic, V., Haynes, C., Cocklin, R. R., Mohan, A., Heyen, J. W., Goebl, M. G., and Iakoucheva, L. M. Identification, Analysis and Prediction of Protein Ubiquitination Sites. Proteins: Structure, Function, and Bioinformatics. 78(2):365-380. (2010)) which can be accessed online at http://www.ubpred.org/ and the second is PhosphoSite (“Hornbeck P V, Zhang B, Murray B, Kornhauser J M, Latham V, Skrzypek E PhosphoSitePlus, 2014: mutations, PTMs and recalibrations. Nucleic Acids Res. 2015 43:D512-20.”) which can be accessed online at https://www.phosphosite.org/ (the latter provides all potential post translational sites—including acetylation and ubiquitination).