Cell compositions and related methods

Abstract

This invention relates to erythroid cells, such as reticulocyte cells, engineered to express a variant protein which comprises one or more mutations which increase the level of the variant protein in the cytosol of the cells. The invention also relates to erythroid cells, such as reticulocyte cells, genetically engineered to overexpress antioxidant proteins and / or an intrinsically disordered proteins (IDPs) in their cytosol and variants of such proteins which facilitate their overexpression in the cytosol of cells. Modification of the cells in this way enhances their storage, whether by refrigeration, freezing, lyophilisation or another storage method. The cells of the invention can be used to deliver therapeutic proteins or polypeptides.

Description

[0001] CELL COMPOSITIONS AND RELATED METHODS

[0002] TECHNICAL FIELD

[0003] This invention relates to erythroid cells, such as reticulocyte cells, engineered to express a variant protein which comprises one or more mutations which increase the level of the variant protein in the cytosol of the cells. The invention also relates to erythroid cells, such as reticulocyte cells, genetically engineered to overexpress antioxidant proteins and / or an intrinsically disordered proteins (IDPs) in their cytosol and variants of such proteins which facilitate their overexpression in the cytosol of cells. Modification of the cells in this way enhances their storage, whether by refrigeration, freezing, lyophilisation or another storage method. The cells of the invention can be used to deliver therapeutic proteins or polypeptides.

[0004] BACKGROUND

[0005] In vertebrates, including humans, the provision of a dependable and persistent supply of oxygen to the various tissues and organs of the body is critical to health. This vital task is performed by blood flowing around the circulatory system of the body. While blood contains many different components, it is specifically the red blood cells (RBCs, or erythrocytes) within blood that are responsible for oxygen capture, transport, and delivery. RBCs perform this role by means of the protein haemoglobin (Hb), which is an iron(II)-containing protein that can bind oxygen molecules in the lung and release the oxygen molecules at sites elsewhere in the body where oxygen is required.

[0006] Even short periods of oxygen deprivation can lead to harmful effects such as irreversible organ damage. Longer periods of oxygen deprivation, even just a few minutes in severe cases, can lead to death. One condition under which the body can be starved of oxygen is severe blood loss, such as during a major haemorrhage, and it is critical to restore blood volume by slowing or stopping the blood loss and by restoring the volume of circulating blood by transfusion of blood into the body.

[0007] Currently, blood transfusion is performed using stocks of donated blood. The transfusion of whole blood is not usually required or recommended. As such, donated blood is typically separated into RBCs, platelets, and plasma, as separate transfusable components. Typical usage restrictions for stocks of RBCs are that they must be refrigerated at 6 °C and must be used within 35-42 days.

[0008] These temperature and time constraints on RBC storage require planning for anticipated need and supply chain logistics. There may be instances where the supply of stored RBCs is insufficient, including situations where there are insufficient donors and / or if there is a sudden rise in demand as can occur in the aftermath of a major catastrophe. There may also be instances where maintenance of a constantly replenished cold supply chain may be difficult, such as in certain third world countries, inaccessible and / or remote environments, and / or in military operations.

[0009] One potential method to provide for a reliable long-term stock of RBCs is the lyophilisation, or freeze-drying, of RBCs. With certain cell types, the lyophilised state may survive storage at ambient temperatures and / or survive storage for longer periods of time than refrigerated cells. Efforts to freeze-dry RBCs have been under way since the 1980s but two significant problems were encountered when freeze-drying RBCs and these problems have proved difficult to overcome. The first problem is that significant proportions of the RBCs did not survive the lyophilisation and reconstitution process. The second problem was that the iron in the haemoglobin of the RBCs was being oxidised from Fe(II) to Fe(III). Haemoglobin that contains the oxidised form of iron, Fe(III), is referred to as methaemoglobin (metHb). MetHb is incapable of binding with oxygen and as such is non-functional for transport of oxygen around the body.

[0010] Investigations have been made into a variety of techniques with the aim of helping the RBCs to remain intact throughout the lyophilisation and reconstitution process and to prevent significant levels of methaemoglobin from being generated. For instance, GB 1502016B discloses some progress in designing compositions that can be used to protect RBCs. In this disclosure, a biopolymer such as PVP was found to protect the RBCs, though use of such a biopolymer is not ideal for a composition that is to be injected into the human bloodstream.

[0011] The present invention aims to address one or more of the above limitations.

[0012] SUMMARY OF THE INVENTION

[0013] The inventors have identified that reticulocyte cells have attractive characteristics, they are osmotically more stable, they have a higher abundance of natural protective proteins than red blood cells and they also naturally transport trehalose (a lyoprotectant) into the cells. This is particularly useful because reticulocytes, while different from RBCs, are the enucleated direct precursor to RBCs and therefore contain the full complement of haemoglobin. Reticulocytes therefore have the capacity to transport oxygen just as effectively as mature RBCs. In addition, the reticulocytes mature into RBCs once in the bloodstream.

[0014] As such, lyophilised reticulocyte cells could offer a new and valuable source of lyophilised compositions that can be used, for example, in blood transfusion. One observed advantage of lyophilising reticulocyte cells in the presence of a lyoprotectant, as compared with RBCs, is that this may allow higher survival rates of the reticulocyte cells to be obtained. In other words, high proportions of the reticulocytes could remain intact after reconstitution because they can import in lyoprotectant naturally. A further observed advantage is that with the correct formulation the reticulocyte cells can be stored for extended periods of time in the lyophilised form, and subsequently reconstituted, with minimal loss of cell viability as compared with reticulocyte cells that were reconstituted immediately. A further advantage of using reticulocyte cells in this way is the longevity of the cells once injected into the body. Once in the body, reticulocytes take around 1-2 days to mature to erythrocytes and from this point the erythrocyte is expected still to have its full lifetime.

[0015] In contrast to lyophilisation of mature blood cells, another advantage of lyophilising reticulocytes is that a biopolymer such as is disclosed in GB 1502016B for protecting mature RBCs during lyophilisation may not be required. Without wishing to be bound by theory, it is thought that reticulocytes are more osmotically stable and may be able to better cope with the alterations induced by lyophilisation. Reticulocytes are generally about 20% larger cells than red cells and they have greater expression of transport proteins on the cell surface than mature RBCs. Reticulocytes may also have some transport proteins that red cells have lost when they become mature. This may also help to protect the reticulocytes.

[0016] Reticulocytes may also have higher levels of antioxidant proteins because they are larger and because they are freshly made they may also have higher levels of metabolites that also support them such as ATP or NADH.

[0017] The reticulocyte cells or any other cell of the invention may be advantageously used to deliver therapeutic proteins or polypeptides via the bloodstream. The reticulocyte cells of the invention, either lyophilised or reconstituted, may also be used as clinical standards for clinical tests, for instance some clinical machines specifically measure reticulocytes and companies use cells fixed in different ways as standards.

[0018] The invention therefore provides a lyophilised composition comprising a cell population and a lyoprotectant, wherein the cell population comprises at least about 10% reticulocyte cells. The invention also provides: a pre-lyophilisation composition comprising a cell population, a lyoprotectant and a lyophilisation medium, wherein the cell population comprises at least about 10% reticulocyte cells; a method of preparing a lyophilised composition of the invention, comprising (a) suspending a cell population in a lyophilisation medium comprising a lyoprotectant, wherein the cell population comprises at least about 10% reticulocyte cells, and (b) subjecting the suspension produced in step (a) to a lyophilisation process to generate the lyophilised composition; a method of reconstituting a lyophilised composition of the invention, comprising contacting the lyophilised composition with a reconstitution medium; a reconstituted composition obtained by or obtainable by (i) a method of the invention or (ii) by reconstituting a lyophilised composition of the invention; and a reconstituted composition comprising a cell population, a lyoprotectant and a reconstitution medium, wherein the cell population comprises at least about 10% reticulocyte cells.

[0019] The inventors have surprisingly shown that reticulocyte cells are capable of taking up trehalose. In particular, before lyophilisation, reticulocyte cells can be loaded with trehalose, for instance by storing the reticulocyte cells overnight in a medium containing trehalose. The invention therefore provides a reticulocyte cell or a mature red blood cell (RBC) comprising intracellular trehalose.

[0020] The inventors have further surprisingly shown that it is possible to overexpress trehalose transporters, such as GLUT8 transporters, in erythroid cells. This requires fewer mutations that required for other cells (Flessner, Lauren B, and Kelle H Moley. "Similar [DE]XXXL[LI] motifs differentially target GLUT8 and GLUT12 in Chinese hamster ovary cells." Traffic (Copenhagen, Denmark) vol. 10,3 (2009): 324-33). The invention therefore provides an erythroid cell genetically engineered to express a trehalose transporter or a variant thereof on its surface. This enhances trehalose uptake further beyond the cells natural capacity.

[0021] The inventors have also surprisingly shown it is possible to modify proteins to increase their level (i.e., facilitate their overexpression) in the cytosol of erythroid cells. The inventors have identified several protein modifications which increase the level of the variant proteins in the cytosol. For instance, the inventors have shown that using proteins that do not possess ubiquitination sites or mutating one or more ubiquitination sites in the proteins reduce their intracellular degradation. The inventors have also shown that introducing one or more inactivating mutations in the proteins' signal peptides enhance expression and retention in the cytosol of cells. The inventors have also shown that introducing one or more inactivating mutations in the mitochondrial targeting sequence of proteins reduce their targeting to mitochondria and increase their expression in the cytosol of cells. The invention therefore provides an erythroid cell genetically engineered to express a variant protein which comprises one or more mutations which increase the level of the variant protein in the cytosol of the cell. The invention also provides: a population of erythroid cells of the invention; a variant mitochondrial enzyme protein which comprises one or more inactivating mutations in its in its mitochondrial targeting peptide; a polynucleotide which encodes a variant mitochondrial enzyme protein of the invention; a vector comprising a polynucleotide of the invention; and a pharmaceutical composition comprising an erythroid cell of the invention, a population of the invention, a variant enzyme protein of the invention, a polynucleotide of the invention or a vector of the invention and a pharmaceutically or physiologically acceptable diluent and / or carrier.

[0022] The invention also provides an erythroid cell of the invention, a population of the invention, a variant enzyme protein of the invention, a polynucleotide of the invention, a vector of the invention or a pharmaceutical composition of the invention for use in therapy.

[0023] The inventors have also surprisingly shown it is possible to overexpress antioxidant proteins and / or an intrinsically disordered proteins (IDPs) in the cytosol of erythroid cells. This is surprising because it was unexpected that antioxidant proteins and / or IDPs could be overexpressed in the cytosol of erythroid cells. The inventors have identified several protein modifications which facilitate overexpression of the proteins in the cytosol. For instance, the inventors have shown that using proteins that do not possess ubiquitination sites or mutating one or more ubiquitination sites in the proteins reduce their intracellular degradation. The inventors have also shown that introducing one or more inactivating mutations in the proteins' signal peptides enhance expression and retention in reticulocytes. The overexpression of these proteins has the potential to further enhance the storage properties of the cells, whether by refrigeration, freezing, lyophilisation or another storage method. The invention therefore provides an erythroid cell genetically engineered to overexpress an antioxidant protein and / or an intrinsically disordered protein (IDP) in its cytosol. The invention also provides: a population of erythroid cells of the invention; a variant antioxidant protein which comprises a mutation at one or more predicted ubiquitination sites; a GPxl or GPx4 variant protein which comprises a mutation at one or more predicted ubiquitination sites; a variant antioxidant protein which comprises one or more inactivating mutations in its signal peptide; a GPx4, GPx7, GPx8, Prx3 or Prx5 variant protein comprising one or more inactivating mutations in its signal peptide; a polynucleotide which encodes a variant protein of the invention; a vector comprising a polynucleotide of the invention; a pharmaceutical composition comprising a reconstituted composition of the invention, an erythroid cell of the invention, a population of the invention, a variant protein of the invention, a polynucleotide of the invention or a vector of the invention and a pharmaceutically or physiologically acceptable diluent and / or carrier; and a method of improving the ability of a cell to survive lyophilisation, the method comprising genetically engineering the cell to overexpress an antioxidant protein and / or an intrinsically disordered protein (IDP) in its cytosol.

[0024] The invention also provides a lyophilised composition of the invention, a reconstituted composition of the invention, a reticulocyte cell of the invention, a mature RBC of the invention, an erythroid cell of the invention, a cell of the invention, a population of the invention, a variant protein of the invention, a polynucleotide of the invention, a vector of the invention or a pharmaceutical composition of the invention for use in therapy.

[0025] BRIEF DESCRIPTION OF THE FIGURES

[0026] Figure 1: Following an overnight incubation with 500mM trehalose, lxlO6the cells were lysed and assessed using the Megazyme trehalose assay kit. Measurements were conducted on a spectophotometer. The term "Loaded" signifies that, before being sent to the freeze- drying facility, the cells were incubated overnight in the incubator at 37°C with 5% CO2 in PBSAG supplemented with 500mM trehalose. In contrast, the control cells were incubated overnight in PBSAG.

[0027] Figure 2A: The expression of the GLUT8 transporter in BELA cells during differentiation was quantified using flow cytometry. The GLUT8-GPS mutated transporter was c-MYC tagged to enable tracking of expression during mutation. After fixation and permeabilization, the cells were measured on flow, and two distinct expression lines were successfully generated which could be differentiated to produce reticulocytes with either "low" or "high" GLUT8-GPS at the surface. Figure 2B: To assess the functionality of the mutated GLUT8 transporter in BEL-A cells, the ability of these GLUT8-expressing cells to uptake trehalose was examined using a trehalose uptake assay. These results indicate that higher expression levels of the transporter correlate with an elevated intracellular trehalose concentration.

[0028] Figure 3 depicts the experiment involving reticulocytes derived from CD34 and cultured reticulocytes expressing the GLUT8 transporter. The samples underwent overnight (O / N) incubation at 37°C with 5% CO2 in PBSAG or PBSAG supplemented with 500mM trehalose. The next day, the cells were washed three times with PBSAG and then aliquoted into batches of 10 million cells in 500 pl of either PBSAG or PBSAG supplemented with 10% trehalose. Figure 3 displays trehalose uptake after overnight loading with 500mM trehalose. Cell assessments were conducted using a Megazyme trehalose kit, and measurements were taken using a spectrophotometer. Figure 4: Expression of c-MYC tagged GLUT8 in CD34+ derived reties using a selectable vector (blastacidin). Figure 4A: GLUT8-GPS-MYC was cloned into a selectable vector (with puromycin) generating a final reticulocyte population uniformly expressing the transporter. Figure 4B: Flow cytometry histogram of IgG isotype control (black) and c-MYC (9E10) staining in fixed and permeabilised differentiating CD34+ cells on days 7, 12, and filtered reticulocytes, for both untransduced (UT) control cells (turquoise) and expressing GLUT8- GPS-MYC (orange). Figure 4C: Trehalose concentration (pg per 1 xlO6cells) in UT and GLUT8-GPS expressing reticulocytes after overnight incubation with 10% trehalose in PBSAG (blue) or just PBSAG (red). Trehalose levels were quantified using a Megazyme assay kit. Absorbance was measured at 340 nm and converted to pg using the displayed standard curve.

[0029] Figure 5A: Protein expression of c-MYC tagged desiccation protective proteins (CAHS1, CAHS2, CAHS3, AAVLEA, PvLEA) during differentiation. BEL-A cells where transduced, cloned and a high expressing single clone was differentiated. Expression of proteins was analysed at in the expanding BEL-A cells, day 6 of differentiation and in reticulocytes using permeabilised cells and flow cytometry (with c-MYC (9E10)). Figure 5B: Protein expression during differentiation in CD34+ haematopoietic stem cells. CD34+ haematopoietic stem cells were transduced with either CAHS1-GFP, CAHS2-GFP or AavLEA-GFP and subsequently the expression level was determined by flow cytometry at day 6, 12 and day 19 of differentiation. Deformability was tested by ARCA on positively sorted reticulocytes whereby the deformability of control reticulocytes is depicted in red and reticulocytes expressing the protein of interest in blue.

[0030] Figure 6: Expression of FLAG tagged desiccation protective CAHS proteins in CD34+ derived erythroblasts and reticulocytes. FLAG tagged CAHS1,CAHS2 and CAHS3 were cloned into a selectable vector (blastacidin), transduced into CD34+ cells and then these cells were differentiated, generating reticulocytes that uniformly expressed these proteins. Representative western blots of cell lysates (lxlO6cells) obtained from the indicated transduced cells taken on days 10, 14, and filtered reticulocytes (day 20). Membranes were labelled with monoclonal antibodies against FLAG-tag (clone M2) and GAPDH (loading control).

[0031] Figure 7: Overexpression of c-MYC tagged peroxiredoxin enzymes in immortalised BEL-A erythroblasts and reticulocytes. Figure 7A: Representative western blots of cell lysates (Prxl and Prx6: lxlO6cells, Prx2: 0.25xl06cells) obtained from indicated cell lines, labelled with monoclonal antibodies against Prx-1, Prx2, Prx6 and GAPDH (loading control). Figure 7B: Flow cytometry histogram of IgG isotype control (black) and c-MYC (9E10) labelling in fixed and permeabilised BEL-A erythroblasts from untransduced (UT) control cells (turquoise) and the respective Prx transduced cell lines (orange). Figure 7C: Bar graphs illustrating Prx overexpression from indicated cell lines at day 0 (dO) and day 12 (dl2) of differentiation. Day 12 samples were gated for based on Hoechst staining to obtain data for day 12 nucleated (dl2n) cells and day 12 reticulocytes (dl2r). Mean fluorescence intensities (MFI) were normalised to background c-MYC labelling in UT control cells (n = 3, mean ± SD). Figure 7D: Flow cytometry analysis of Prx construct expression in day 12 samples (n = 3). Fixed and permeabilised cells were labelled with either an IgG isotype control (black) or c- MYC (9E10) antibody and gated for based on Hoechst staining to obtain data for day 12 nucleated (dl2n) cells and day 12 reticulocytes (dl2r).

[0032] Figure 8: Overexpression of glutathione peroxidase enzymes in immortalised BEL-A erythroblasts and reticulocytes. Figure 8A: Representative western blots of cell lysates (lxlO6cells) obtained from indicated cell lines, labelled with monoclonal antibodies against GPxl, GPx4 and GAPDH (loading control). Figure 8B: Flow cytometry histogram of IgG isotype control (black) and c-MYC (9E10) staining in fixed and permeabilised BEL-A erythroblasts from untransduced (UT) control cells (turquoise) and the respective GPx transduced cell lines (orange). Figure 8C: Flow cytometry analysis of GPx construct expression in day 12 samples (n = 3). Fixed and permeabilised cells were labelled with either an IgG isotype control (black) or c-MYC (9E10) antibody and gated for based on Hoechst staining to obtain data for day 12 nucleated (dl2n) cells and day 12 reticulocytes (dl2r). Figure 8D: Bar graphs illustrating GPx overexpression from indicated cell lines at day 0 (dO) and day 12 (dl2) of differentiation. Day 12 samples were gated for based on Hoechst staining to obtain data for day 12 nucleated (dl2n) cells and day 12 reticulocytes (dl2r). Mean fluorescence intensities (MFI) were normalised to background c-MYC labelling in UT control cells (n = 3, mean ± SD). Figure 8E: Bar graphs illustrating GPx4U73Coverexpression analyzed by intracellular flow cytometry following treatment with either 5 pM MG132, 10 pM leupeptin, 10 pM bardoxolone, a vehicle control (blue) or left untreated (UT) (grey) for a total of 6 hrs. Mean fluorescence intensities (MFI) were normalised to background c-MYC labelling in UT control cells Data is shown as the mean ± SD, n = 3, * p < 0.05, ** p < 0.01, ***p < 0.001, 2-tailed unpaired Student's t-test.

[0033] Figure 9: Overexpression of glutathione peroxidase 4 constructs in immortalised BEL-A erythroblasts and reticulocytes. Figure 9A: Flow cytometry histogram of IgG isotype control (black) and c-MYC (9E10) staining in fixed and permeabilised BEL-A erythroblasts from untransduced (UT) control cells (turquoise) and the respective GPx4 transduced cell lines. Figure 9B: A representative western blot of cell lysates (lxlO6) obtained from the indicated cell lines, labelled with monoclonal antibodies against GPx4 and GAPDH (loading control). Figure 9C: Flow cytometry analysis of GPx4 construct expression in day 12 samples (n = 3). Fixed and permeabilised cells were labelled with either an IgG isotype control (black) or c-MYC (9E10) antibody and gated for based on Hoechst staining to obtain data for day 12 nucleated (dl2n) cells and day 12 reticulocytes (dl2r). Figure 9D: Bar graphs illustrating GPx overexpression from indicated cell lines at day 0 (dO) and day 12 (dl2) of differentiation. Day 12 samples were gated for based on Hoechst staining to obtain data for day 12 nucleated (dl2n) cells and day 12 reticulocytes (dl2r). Mean fluorescence intensities (MFI) were normalised to background c-MYC labelling in UT control cells (n = 3, mean ± SD).

[0034] Figure 10: Overexpression of c-MYC tagged glutathione peroxidase 1 constructs expressed in immortalised BEL-A erythroblasts and reticulocytes. Flow cytometry histogram of IgG isotype control (black) and c-MYC (9E10) staining in fixed and permeabilised expanding BEL-A erythroblast cells from untransduced (UT) control cells (turquoise) and the respective GPxl transduced cell lines (orange). Cells were transduced with constructs selectable by puromycin. Figure 10B: Representative western blots of cell lysates (lxlO6cells) obtained from indicated cell lines, labelled with monoclonal antibodies against GPxl and GAPDH (loading control). Figure IOC: Flow cytometry histogram of fixed and permeabilised cells labelled with IgG isotype control (black) or c-MYC (9E10) on day 11 differentiating BEL-A cells gated for reticulocytes based on Hoechst staining to obtain data for day 11 nucleated (dllnucleated) cells and day 11 reticulocytes (dllreticulocytes).

[0035] Figure 11: Overexpression of glutathione peroxidase 4 (MYC-sGPx4 3'UTR 6KR) in CD34+ erythroblasts and reticulocytes. MYC tagged sGPx4 3'UTR 6KR was cloned into a selectable vector (puromycin), transduced into CD34+ cells and then these cells were differentiated, generating reticulocytes that uniformly expressed this protein. Figure 11A: Representative western blots of cell lysates (lxlO6cells) obtained from indicated cell lines on days 7, 10, and filtered reticulocytes (day 20) of CD34+ erythroid cultures. One untransduced (UT) sample and two expressing MYC-sGPx4 3'UTR 6KR(#1 and #2) were analysed. Membranes were labelled with monoclonal antibodies against GPx4 and GAPDH (loading control).

[0036] Figure 11B: Flow cytometry histogram of IgG isotype control (black) and c-MYC (9E10) staining in fixed and permeabilised differentiating CD34+ cells on days 7, 10, and filtered reticulocytes (day 20). Samples and time points correspond to the same shown in panel A, with data presented for untransduced (UT) control cells (turquoise), and MYC-GPx4 3'UTR 6KR culture #1 (blue) and #2 (orange).

[0037] Figure 12A: Schematic of the two human alanine amino transferase 2 (AAT2) constructs designed with (huAAT2 FL) and without (huAAT2 N27del) a mitochondrial targeting peptide (highlighted in yellow). Figure 12B: Human AAT2 (huAAT2) protein expression during differentiation in CD34+ haematopoietic stem cells. CD34+ haematopoietic stem cells were transduced with either full length (FL) huAAT2 or N-terminally truncated huAAT2 (N27del) where a 27 amino acid mitochondrial targeting peptide had been removed. Positively transduced cells were selected for using puromycin antibiotic and differentiated to generate reticulocytes. Protein expression level was determined by western blot on day 8 (d8), day 20 (d20) and a filtered population of pure d20 reticulocytes (d20r). Figure 12C: AAT2 protein expression for each construct in CD34-derived cultured reticulocytes (d20r) set relative to starting (d8) levels. Western blot bands were quantified and normalized to d8 starting protein expression for each of the AAT2 constructs accordingly.

[0038] Figure 13: Overexpression of glutathione peroxidase 1 constructs in CD34+ erythroblasts and reticulocytes. MYC tagged GPxlU49Cand GPxl 3'UTR were cloned into a selectable vector (puromycin), transduced into CD34+ cells and then these cells were differentiated, generating reticulocytes that uniformly expressed these proteins. Figure 13A: Representative western blots of cell lysates (lxlO6cells) obtained from indicated transduced cells on days 7, 10, 14, and filtered reticulocytes (day 20) of CD34+ erythroid cultures from two separate donors. Membranes were labelled with monoclonal antibodies against GPxl and GAPDH (loading control). Figure 13B: Flow cytometry histogram of IgG isotype control (black) and c-MYC (9E10) staining in fixed and permeabilised differentiating CD34+ cells on days 7, 10, 14, and filtered reticulocytes (day 20). Samples correspond to the same two donors and time points as shown in panel A, with data presented for untransduced (UT) control cells (turquoise), and cultures expressing MYC-GPxlU49C(orange) or MYC-GPxl 3'UTR (blue).

[0039] DESCRIPTION OF THE SEQUENCE LISTING

[0040] The sequences are identified and described in the Table below.

[0041] DETAILED DESCRIPTION

[0042] General disclosure

[0043] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

[0044] The present invention is described with respect to particular embodiments and with reference to certain Figures but the invention is not limited thereto but only by the claims. Any reference signs in the claims shall not be construed as limiting the scope. Of course, it is to be understood that not necessarily all aspects or advantages may be achieved in accordance with any particular embodiment of the invention. Thus, for example those skilled in the art will recognize that the invention may be embodied or carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other aspects or advantages as may be taught or suggested herein. The invention, both as to organization and method of operation, together with features and advantages thereof, may best be understood by reference to the following detailed description when read in conjunction with the accompanying Figures. The aspects and advantages of the invention will be apparent from and elucidated with reference to the embodiment(s) described hereinafter. Reference throughout this specification to "some embodiments" or a "preferred embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment(s) is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in some embodiments" or "in a preferred embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may do so. Similarly, it should be appreciated that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. This method of disclosure, however, is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment.

[0045] In addition as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes two or more polypeptide, reference to "a fusion protein" includes two or more such proteins, reference to "a test" includes two or more tests, reference to "a kit" refers to two or more kits, reference to "a method" includes two or more methods and the like.

[0046] In all of the discussion herein, the standard one letter codes for amino acids are used. These are as follows: alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamic acid (E), glutamine (Q), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y) and valine (V).

[0047] Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4thed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016), for definitions and terms of the art. The definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art. "About" as used herein when referring to a measurable value such as a percentage or an amount and the like, is meant to encompass variations of ± 20 % or ± 10 %, more preferably ± 5 %, even more preferably ± 1 %, and still more preferably ± 0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods. Any statement herein including the term "about" includes the same feature without the term. For instance, a variant of SEQ ID NO: 35 having at least "about" 80% identity or homology to the sequence of SEQ ID NO: 35 over its entire length includes a variant of SEQ ID NO: 35 having at least 80% identity or homology to the sequence of SEQ ID NO: 35 over its entire length.

[0048] Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", mean "including but not limited to", and do not exclude other components, integers, or steps.

[0049] Preferred features of each aspect of the invention may be as described in connection with any of the other aspects. Within the scope of this application it is expressly intended that the various aspects, embodiments, examples, and alternatives set out in the preceding paragraphs, in the claims and / or in the following description and drawings, and in particular the individual features thereof, may be taken independently or in any combination. That is, all embodiments and / or features of any embodiment can be combined in any way and / or combination, unless such features are incompatible.

[0050] In the context of the present invention, a "detectable level" may comprise a detectable level of the mRNA encoding the relevant marker, e.g. CD71, and / or the marker protein, e.g. the CD71 protein. Preferably, a "detectable level" comprises a detectable level of the protein, e.g. the CD71 protein. A "detectable level" may comprise a detectable level of the mRNA encoding the relevant marker, e.g. CD71, and a detectable level of the marker protein, e.g. the CD71 protein. A "detectable level" may comprise a detectable activity of the relevant marker, e.g. CD71 (which is a receptor for transferrin). In all instances herein, the term "level" is interchangeable with "amount", "activity" or "amount and activity".

[0051] The presence of mRNA and / or protein may be detected using any routine method in the art. Such methods include immunofluorescence, immunohistochemistry, western blotting, quantitative polymerase chain reaction (qPCR), reporter assays, enzyme-linked immunosorbent assay (ELISA), microscopy, flow cytometry, enzymatic staining, dye incorporation, chemiluminescent oxygen detection reagents, fluorometry, fluorescence in situ hybridization (FISH) and an alkaline phosphatase assay. The skilled person is capable of detecting the activity of any particular marker using known assays. Suitable assays are described in the Examples. In the context of the present invention, "genetically engineered" means that the cell or population of cells has been genetically modified to express the relevant factor, such as the trehalose transporter, antioxidant protein or intrinsically disordered protein (IDP). The genetically engineered cell or population expresses a detectable level of the relevant factor, such as the trehalose transporter, antioxidant protein or IDP. The embodiments in this paragraph equally apply to the variant proteins discussed below which comprise one or more mutations which increase their level in the cytosol of cells.

[0052] Typically, "genetically engineered" means that the genetic material of the cell or population of cells has been altered to express the relevant factor, such as the trehalose transporter, antioxidant protein or IDP. When "genetically engineered", the cell or population of cells typically comprises an exogenous polynucleotide that encodes and is configured to express the relevant factor, such as the trehalose transporter, antioxidant protein or IDP. A skilled person is capable of identifying such exogenous polynucleotides in the cell or population of cells. The exogenous polynucleotide may comprise any of the control sequences discussed below and / or may be part of any of the vectors discussed below. The embodiments in this paragraph equally apply to the variant proteins discussed below which comprise one or more mutations which increase their level in the cytosol of cells.

[0053] Prior to genetic engineering, the cell or population preferably does not express a detectable level or expresses undetectable levels of the relevant marker, such as the trehalose transporter, antioxidant protein or IDP. Prior to genetic engineering, the cell or population may express a detectable level of the relevant marker, such as the trehalose transporter, antioxidant protein or IDP. The detectable level may be low because of the cell or population's phenotype or epigenetics or because the factor is degraded or secreted from the cell. The cell or population may be genetically engineered to overexpress the relevant factor. The cell or population may be genetically engineered to express an increased level of the relevant factor. The embodiments in this paragraph equally apply to proteins before they modified to comprise one or more mutations which increase their level in the cytosol of cells as discussed below. These may be known as "unmodified" or "non-mutated" proteins.

[0054] In the context of the invention, "genetically engineered to overexpress" means the cell or population of cells has been genetically modified to express an increased level of the relevant factor, such as the antioxidant protein and / or IDP. The increased level may be an increased amount and / or an increased activity. The increased level is typically an increased detectable level. The increased level is typically compared with the level in the cell or population before the genetic engineering is conducted. The increased level is typically compared with the level in a cell or a population that has not undergone the genetic engineering, such as a corresponding, wild-type cell or population. The embodiments in this paragraph equally apply to the variant proteins discussed below which comprise one or more mutations which increase their level in the cytosol of cells. The marker may be overexpressed by any amount. The marker, such as the antioxidant protein and / or IDP, may be overexpressed by at least about 5%, such as least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, as least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100%. The marker, such as the antioxidant protein and / or IDP, may be overexpressed by at least about a factor of 2, such as least about a factor of 5, at least about a factor of 10, at least about a factor of 50, at least about a factor of 100, at least about a factor of 500, as least about a factor of 1000, at least about a factor of 5000, at least about a factor of 10,000 or more. The embodiments in this paragraph equally apply to the variant proteins discussed below which comprise one or more mutations which increase their level in the cytosol of cells. The level of a variant protein may be increased by any of the amounts in this paragraph.

[0055] The cell or population may be genetically modified to express a level of the marker, such as the antioxidant protein and / or IDP, that is increased by at least about 5%, such as least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, as least about 60%, at least about 70%, at least about 80%, at least about 90% or at least about 100%. The cell or population may be genetically modified to express a level of the marker, such as the antioxidant protein and / or IDP, that is increased by at least about a factor of 2, such as least about a factor of 5, at least about a factor of 10, at least about a factor of 50, at least about a factor of 100, at least about a factor of 500, as least about a factor of 1000, at least about a factor of 5000, at least about a factor of 10,000 or more. The embodiments in this paragraph equally apply to the variant proteins discussed below which comprise one or more mutations which increase their level in the cytosol of cells. The level of a variant protein may be increased by any of the amounts in this paragraph.

[0056] In the context of the invention, a cell or population is genetically engineered to overexpress a relevant factor, such as the antioxidant protein and / or IDP, it the cytosol. This means there an increased level of the relevant factor, such as the antioxidant protein and / or IDP, in the cytosol of the cell or the cells in the population. This may be achieved by genetically engineering the cell or population to decrease the transport / trafficking of the relevant factor to intracellular organelles, such as mitochondria, and / or to decrease the secretion of the relevant factor. The invention provides variant proteins which are modified to reduce their intracellular transport / trafficking and / or secretion. Genetically engineering cells to express these variant proteins typically results in their overexpression in the cytosol of the cells. The embodiments in this paragraph equally apply to the variant proteins discussed below which comprise one or more mutations which increase their level in the cytosol of cells.

[0057] Lyophilised composition

[0058] The present invention provides a lyophilised composition. The term "lyophilised" (or "freeze- dried") is well understood in the art. The lyophilised composition comprises a cell population and a lyoprotectant. In some embodiments, the lyophilised composition consists essentially of, or consists of, a cell population and a lyoprotectant.

[0059] In some embodiments, the lyophilised composition may comprise further agents. For instance, the lyophilised composition may comprise one or more agents selected from buffer agents, stabilisers, antimicrobial agents, antioxidants, free radical scavengers, solubilizing agents, tonicifying agents, and / or surfactants.

[0060] In some embodiments, the lyophilised composition consists essentially of, or consists of, a cell population, a lyoprotectant, and one or more agents selected from buffer agents, antioxidants, and / or free radical scavengers.

[0061] In some embodiments, the lyophilised composition does not comprise a non-cellular polymer. A "cellular polymer" is a polymer of the cells in the lyophilised composition. In other words, the lyophilised composition does not comprise any polymers that are in addition to those present in the lyophilised composition by way of being part of the cell population. In some embodiments, the lyophilised composition does not comprise a synthetic or non-biological polymer. In some embodiments, the lyophilised composition does not comprise a non-biological polymer or synthetic polymer, wherein the non-biological polymer or synthetic polymer is a polymer that enables the active loading of the cell with a cryoprotectant and / or lyoprotectant. In some embodiments, the lyophilised composition does not comprise a non-biological polymer or synthetic polymer, wherein the non-biological polymer or synthetic polymer is a polymer that renders the cell membranes permeable on a temporary basis. In some embodiments, the lyophilisation composition does not comprise a cell membrane permeabilising agent, preferably a synthetic and / or non-biological cell membrane permeabilising agent. In some embodiments, the lyophilisation composition does not comprise a polymer selected from PLP, PV-50, PL-50, PP-30, PP-50, PP-60, PP-75, polymers selected from the group consisting of polyvinylpyrrolidone (PVP) and polyvinylpyrrolidone derivatives, and dextran and dextran derivatives, proteins added to the composition and / or hydroxyethyl starch. Biopolymers PLP, PV-50, PL-50, PP-30, PP-50, PP- 60, and PP-75 can be made by the processes defined in Lynch et al (2010) Biomaterials, 31 (23) 6096-6103; Eccleston et al (2000) J Control Release; 69(2): 297-307; Chen et al (2009) J Mater Chem; 19: 4217-4224; and GB 2502016.

[0062] In some embodiments, the lyophilised composition comprises a residual water content of less than about 10 percentage by weight (wt%). The lyophilised composition preferably comprises a residual water content of less than about 7 wt%, less than about 5 wt%, less than about 2 wt% or less than about 1 wt%.

[0063] Cell population In some embodiments, the cell population accounts for all of the cells in the lyophilised composition.

[0064] The cell population comprises at least about 10% reticulocyte cells. In some embodiments, the cell population comprises at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98% or at least about 99% reticulocyte cells. For therapeutic applications, it is generally preferred that the cell population comprises at least about 60% reticulocyte cells, more preferably at least about 80% reticulocyte cells. In a particularly preferred embodiment, the cell population comprises at least about 99% reticulocyte cells. In some embodiments, the majority of the cell population comprises reticulocyte cells.

[0065] In some embodiments, the cell population comprises at least about lxlO5, at least about lxlO6, at least about lxlO7, at least about lxlO8, at least about lxlO9, or at least about lxlO10cells.

[0066] In some embodiments, the cell population comprises at least about lxlO8, at least about lxlO9, at least about lxlO10, at least about lxlO11, at least about lxlO12, at least about lxlO13, or at least about lxlO14reticulocyte cells.

[0067] The identity of the cells making up the remainder of the cell population is not particularly limited. The cells making up the remainder of the cell population can be a single cell type or can be a mixture of different cell types.

[0068] In some embodiments, the cell population comprises, consists of, or is, a mammalian cell population. The cell population may be a human, dog, cat, primate, horse, murine, rat, rodent, bovine, murine, porcine, or ovine cell population. In a preferred embodiment, the cell population comprises, consists of, or is a human cell population. In some embodiments, the cell population comprises, consists of, or is, a wild-type cell population. In some embodiments, the cell population comprises, or consists of, genetically modified cells.

[0069] In some embodiments, the cell population comprises or consists of the intact cells in the composition. "Intact cells" are generally cells that are viable. In other words, they are cells that have not been ruptured. This can be measured using routine methods. For example, the cells can be assessed on a flow cytometer, for instance as described in the Examples.

[0070] In some embodiments, the cell population comprises, consists essentially of, or consists of, a fraction of whole blood. In some embodiments, the cell population comprises, consists essentially of, or consists of, a cell fraction isolated from whole blood. As a reference, whole blood typically consists of a cell population comprising 0.5% to 2.5% reticulocyte cells in adults, and 2% to 6% reticulocyte cells in infants. In some embodiments, the cell population comprises, consists essentially of, or consists of, a RBC fraction of whole blood that has been treated to enrich the proportion of reticulocyte cells to at least about 10% reticulocyte cells.

[0071] In some embodiments, the cell population is not a fraction of whole blood. A skilled person is able to tell whether or not a cell population is a fraction of whole blood using standard means in the art. For instance, the skilled person can perform assays on the cell population to detect markers of whole blood, such as compounds found in whole blood. In some embodiments, the cell population does not comprise a fraction of whole blood. In some embodiments, the cell population does not comprise reticulocyte cells isolated from whole blood. In some embodiments, the reticulocyte cells are not isolated from whole blood. In these embodiments, the cell population may still comprise cells that are derived from a component of whole blood, such as a reticulocyte cell culture derived from a stem cell or induced pluripotent stem cell isolated from whole blood.

[0072] In some embodiments, the cell population is produced by cell culture. The cell culture can be a derivative of an erythroid progenitor cell population. The erythroid progenitor cell population is not particularly limited and can comprise any cell that is capable of maturing into a reticulocyte. The term "erythroid progenitor cell" or "erythroid progenitor" can be used to refer to cells at different stages along the differentiation / maturation pathway. The term "erythroid progenitor cell" generally refers to cells that have a cell nucleus, i.e. before enucleation has started. The erythroid progenitor cell can be a stem cell, haematopoietic stem cell, induced pluripotent stem cell (iPSC), erythroid immortalized cell line or erythroblast cell. Preferably the erythroid progenitor is a CD34+ cell, and / or a BEL-A cell. For instance, the erythroid progenitor can be a CD34+ cell derived from a stem cell or an iPSC. Where the erythroid progenitor is a non-CD34+ stem cell or iPSC or immortalised cell, the cell type or lineage can produce erythroblasts which then differentiate to reticulocytes.

[0073] Reticulocyte cells

[0074] The term "reticulocyte cell" is well understood in the field. However, to provide some detail, a "reticulocyte cell" is a cell derived from an erythroid progenitor that is enucleated but which has not yet become a mature RBC. Reticulocyte cells are generally understood to be immature RBCs which are known to be larger than mature RBCs, they are usually CD71 positive and have most membrane proteins at higher levels than the mature RBCs. They can also contain remnants of organelles and also mitochondria and these are generally lost when the reticulocyte cells mature.

[0075] Enucleated erythroid cells, including reticulocyte cells and mature RBCs, display erythroid Band 3 protein (also known as anion exchanger 1 (AE1) or solute carrier family 4 member 1 (SLC4A1)) on the erythroid cell surface. In production of enucleated erythroid cells, erythroid cells can generally be identified as such by the presence of erythroid Band 3 and by having no nucleus. Wild-type reticulocyte cells are typically Band 3 positive and CD71 positive. In contrast, mature RBCs are Band 3 positive and CD71 negative.

[0076] The reticulocyte cells preferably have a cell volume of at least about 100 fL, preferably at least about 110 fL. The reticulocyte cells preferably have a cell volume of less than about 150 fL, less than about 145 fL, less than about 135 fL, less than about 130 fL, less than about 125 fL, or less than about 120 fL. The reticulocyte cells preferably have a cell volume of from about 100 fL to about 150 fL, such as from about from about 100 fL to about 130 fL or from about from about 110 fL to about 120 fL. The reticulocyte cells can have a cell volume of about 115 fL. In contrast, mature RBCs typically have cell volumes ranging from about 50 to about 98 fL, such as from about 50 to about 80 fL or from about 86 to about 98 fL. The skilled person can determine the cell volume using standard techniques in the art, such as using microscopy to determine the cell dimensions and thus calculating the cell volume or using clinical instruments designed to measure cell volume.

[0077] The reticulocyte cells preferably express a detectable level of CD71. The reticulocyte cells are preferably CD71 positive. The reticulocyte cells preferably express detectable levels of Band 3 and CD71. The reticulocyte cells are preferably Band 3 and CD71 positive. The reticulocyte cells preferably do not comprise a cell nucleus. The reticulocyte cells preferably (a) express a detectable level of CD71 or are CD71 positive, (b) express detectable levels of Band 3 or are Band 3 positive, (c) do not comprise a cell nucleus or any combination thereof, such as (a), (b), (c), (a) and (b), (a) and (c), (b) and (c) or (a), (b) and (c).

[0078] In any of these embodiments, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98% or at least about 99% of the reticulocyte cells(a) express a detectable level of CD71 or are CD71 positive, (b) express detectable levels of Band 3 or are Band 3 positive, (c) do not comprise a cell nucleus or any combination thereof, such as (a), (b), (c), (a) and (b), (a) and (c), (b) and (c) or (a), (b) and (c).

[0079] Detectable levels are defined above. The skilled person can determine the presence or absence of CD71 and / or Band 3 on cells using standard techniques in the art, such as those described above. The skilled person can determine the presence or absence of a cell nucleus using standard techniques in the art, such as microscopy.

[0080] In some embodiments, the reticulocyte cells comprise, consist essentially of, consist of, or are, mammalian reticulocyte cells. The reticulocyte cells may be a human, dog, cat, primate, horse, murine, rat, rodent, bovine, murine, porcine, or ovine reticulocyte cells. In a preferred embodiment, the reticulocyte cells comprise, consist essentially of, or consist of, or are, human reticulocyte cells. In some embodiments, the reticulocyte cells comprise, consist essentially of, consist of, or are, wild-type reticulocyte cells. In some embodiments, the reticulocyte cells comprise, consist essentially of, consist of, or are, genetically modified reticulocyte cells. In some embodiments, the reticulocyte cells comprise, consist essentially of, consist of, or are, non-wild-type reticulocyte cells. In a particularly preferred embodiment, the reticulocyte cells are cultured reticulocyte cells.

[0081] In some embodiments, the reticulocyte cells are a fraction of whole blood. However, it is generally preferred that the reticulocyte cells are not a fraction of whole blood. In some embodiments, the reticulocyte cells are not isolated from whole blood. In embodiments where the reticulocyte cells are not a fraction of or are not isolated from whole blood, the reticulocyte cells may still be derived from a component of whole blood, such as a reticulocyte cell culture derived from a stem cell isolated from whole blood.

[0082] Normally, the percentage of reticulocytes in whole blood is generally low. One way to make a large population of reticulocytes is therefore to produce them by culturing them from primary cell sources such as adult peripheral blood, cord blood, bone marrow or from an immortalised cell source such as iPSC or cell lines such as BEL-A like lines immortalised with E6 and E7. In some embodiments, reticulocyte cells are a derivative of an erythroid progenitor cell population. The erythroid progenitor cell population is not particularly limited and can comprise any cell that is capable of maturing into a reticulocyte. The erythroid progenitor cell can be a stem cell, haematopoietic stem cell, induced pluripotent stem cell (iPSC), erythroid immortalized cell line or erythroblast cell. Preferably the erythroid progenitor is a CD34+ cell, and / or a BEL-A cell. For instance, the erythroid progenitor can be a CD34+ cell derived from a stem cell or an iPSC. Where the erythroid progenitor is a stem cell or iPSC, the cell lineage is to produce erythroblasts which then differentiate to reticulocytes.

[0083] The skilled person will understand that, in the context of the invention, reticulocyte cells or mature RBCs cannot themselves be genetically engineered because they do not contain nuclei. Such genetically engineered cells are typically derived from erythroid progenitor (also called immature or precursor cells) cells that have been genetically engineered as required.

[0084] In some embodiments, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% of the reticulocyte cells are intact. In some embodiments, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% of the reticulocyte cells are intact following reconstitution. "Intact" means that the reticulocyte cells are viable. In other words, the cells have not been ruptured. The skilled person knows how to test for the percentage of intact cells. For instance, the lyophilised composition can be reconstituted in a liquid medium and analysed by flow cytometry, gated on the intact reticulocytes based on forward / side scatter. Alternatively, the cells can be centrifuged into a pellet and the amount of free haemoglobin (released when the cells lyse) can be measured in the supernatant.

[0085] In some embodiments, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% of the reticulocyte cells survive reconstitution. In some embodiments, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% of the reticulocyte cells survive reconstitution. "Survive reconstitution" generally means that the reticulocyte cells are intact and viable. In other words, the cells have not been ruptured. The skilled person knows how to test for the percentage of intact and viable cells as described.

[0086] In some embodiments, the reticulocyte cells comprise at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, haemoglobin molecules with respect to the total number of haemoglobins plus methaemoglobin molecules. In some embodiments, the reticulocyte cells comprise less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 5%, less than about 2% or less than about 1% methaemoglobin molecules, with respect to the total number of haemoglobin and methaemoglobin molecules. In some embodiments, the reticulocyte cells comprise at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, haemoglobin molecules with respect to the number of haemoglobin molecules in a corresponding fresh cell population. 'Corresponding' means a cell population that can be used to determine the proportion of haemoglobin molecules that have been oxidised to methaemoglobin during the lyophilisation process. Generally, this means a corresponding cell population comprising fresh cells, such as a freshly prepared cell culture or freshly harvested cells, prior to lyophilisation. In some embodiments, the reticulocyte cells comprise at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99%, haemoglobin molecules with respect to the number of haemoglobin molecules in the cell population prior to lyophilisation. Spectrophotometry can be used to assess the proportions of haemoglobin and methaemoglobin. In some embodiments, the reticulocyte cells retain at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% of their oxygen carrying capacity with respect to circulating wild-type reticulocyte cells.

[0087] Lyoprotectant The lyoprotectant is an agent that can protect the reticulocyte cells during the lyophilisation process and / or during storage as the lyophilised composition. The term "lyoprotectant" is well understood in the art. The lyoprotectant can protect cell viability, such as by protecting the cells against rupturing and helping them to remain intact, and / or protect the haemoglobin within the reticulocyte cells against oxidation. In some embodiments, the lyoprotectant can protect cell viability and protect against haemoglobin oxidation.

[0088] In some embodiments, the lyoprotectant can be a water replacement effect agent. A "water displacement effect agent" is an agent that can displace water from biological tissues and materials such as cells. For instance, water displacement effect agents are capable of displacing water molecules that bind to the surface of cell membranes.

[0089] In some embodiments, the lyoprotectant comprises, consists essentially of, consists of, or is, one or more of (a) a saccharide, (b) an antioxidant, and (c) an intrinsically disordered protein (IDP), such as (a), (b), (c), (a) and (b), (a) and (c), (b) and (c) or (a), (b) and (c).

[0090] Saccharides

[0091] In some embodiments, the lyoprotectant comprises a saccharide. The saccharide may be a monosaccharide, an oligosaccharide, or a polysaccharide. In some embodiments, the saccharide is a monosaccharide or a disaccharide. In a preferred embodiment, the lyoprotectant is a disaccharide.

[0092] In some embodiments, the lyoprotectant is selected from trehalose, sucrose, fructose, glucose, mannitol, sorbitol, arabinose, and / or ribose.

[0093] In a particularly preferred embodiment, the lyoprotectant comprises, consists essentially of, consists of, or is, trehalose. At least a fraction of the trehalose is intracellular. At least a fraction of the trehalose is preferably present inside the reticulocyte cells.

[0094] In some embodiments, the reticulocyte cells are genetically engineered to express a trehalose transporter or a variant thereof on their surfaces. This facilitates the uptake of trehalose by the reticulocyte cells and is discussed in more detail below.

[0095] Antioxidants

[0096] In some embodiments, the lyoprotectant comprises, consists essentially of, consists of, or is, an antioxidant. In some embodiments, the lyoprotectant comprises, consists essentially of, consists of, or is, an antioxidant protein (or a "proteinaceous antioxidant"). In some embodiments, the lyoprotectant protein comprises or is selected from a glutathione peroxidase (GPx) protein or peroxiredoxin (Prx) protein. In some embodiments, the antioxidant is selected from glutathione peroxidase 1 (GPxl), GPx2, GPx3, GPx4, GPx5, GPx6, GPx7, GPx8, peroxiredoxin 1 (Prxl), Prx2, Prx3, Prx4, Prx5, Prx6 and variants thereof. In some embodiments, the antioxidant is selected from GPxl, GPx4, Prxl, Prx2, Prx6 and variants thereof. In some embodiments, the antioxidant is selected from GPxl, GPx4, and variants thereof. In some embodiments, the antioxidant is selected from Prxl, Prx2, Prx6 and variants thereof.

[0097] Preferred variants of the proteins are discussed below. Any of these may be used in the lyophilised composition of the invention. For instance, the antioxidant may be modified to increase its expression in the cytosol of cells, for instance by making mutations at one or more predicted ubiquitination sites, making one or more inactivating mutations in its signal peptide and / or making one or more inactivating mutations in its mitochondrial targeting peptide. The antioxidant is preferably human. The antioxidant may also be a non-human antioxidant humanised to express in human cells.

[0098] In preferred embodiments, the GPxl or variant thereof comprises or consists of the sequence shown in SEQ ID NO: 2, 4 or 6 or a variant thereof. In preferred embodiments, the GPxl or variant thereof comprises or consists of the sequence shown in SEQ ID NO: 2, 4, 6 or 55 or a variant thereof.

[0099] In preferred embodiments, the GPx4 or variant thereof comprises or consists of the sequence shown in SEQ ID NO: 8, 12, 14 or 16 or a variant thereof. In a most preferred embodiment, the GPx4 or variant thereof comprises or consists of the sequence shown in SEQ ID NO: 16 or a variant thereof.

[0100] In preferred embodiments, the Prxl or variant thereof comprises or consists of the sequence shown in SEQ ID NO: 18 or 20 or a variant thereof.

[0101] In preferred embodiments, the Prx2 or variant thereof comprises or consists of the sequence shown in SEQ ID NO: 22 or 24 or a variant thereof.

[0102] In preferred embodiments, the Prx6 or variant thereof comprises or consists of the sequence shown in SEQ ID NO: 26 or 28 or a variant thereof.

[0103] In preferred embodiments, the GPx2 or variant thereof comprises or consists of the sequence shown in SEQ ID NO: 38 or a variant thereof.

[0104] In preferred embodiments, the GPx3 or variant thereof comprises or consists of the sequence shown in SEQ ID NO: 39 or a variant thereof.

[0105] In preferred embodiments, the GPx5 or variant thereof comprises or consists of the sequence shown in SEQ ID NO: 40 or a variant thereof.

[0106] In preferred embodiments, the GPx6 or variant thereof comprises or consists of the sequence shown in SEQ ID NO: 41 or a variant thereof. In preferred embodiments, the GPx7 or variant thereof comprises or consists of the sequence shown in SEQ ID NO: 42 or a variant thereof.

[0107] In preferred embodiments, the GPx8 or variant thereof comprises or consists of the sequence shown in SEQ ID NO: 43 or a variant thereof.

[0108] In preferred embodiments, the Prx3 or variant thereof comprises or consists of the sequence shown in SEQ ID NO: 44 or a variant thereof.

[0109] In preferred embodiments, the Prx4 or variant thereof comprises or consists of the sequence shown in SEQ ID NO: 45 or a variant thereof.

[0110] In preferred embodiments, the Prx5 or variant thereof comprises or consists of the sequence shown in SEQ ID NO: 46 or a variant thereof.

[0111] The antioxidant protein may be produced separately and added to the lyophilised composition of the invention. The cell population and / or the reticulocyte cells are preferably genetically engineered to overexpress the antioxidant protein in their cytosol. This is discussed in more detail below.

[0112] Without wishing to be bound by theory, it is understood that the GPx and Prx families protect against reactive oxygen species (ROS) in general but also have a protective effect for lipid peroxidation which is relevant during freeze drying and reconstitution processes. One source of damage that the cell undergoes is stress from ROS production, particularly during reconstitution. In addition, antioxidant proteins are expected to protect haemoglobin against oxidation. A further benefit is that antioxidant proteins are also expected to help protect reticulocyte cells during storage (even without freeze drying) and also help the reticulocyte cells and subsequent mature RBCs to last longer in circulation as ROS play a role in red cell ageing. It may also have a therapeutic effect to reduce damage to wounds as ROS increases in wounds so blood cells with higher antioxidants may be protective.

[0113] Intrinsically disordered proteins (IDPs)

[0114] In some embodiments, the lyoprotectant comprises an intrinsically disordered protein (IDP). The IDP preferably has a protective effect against desiccation in its host organism. The IDP is preferably a protective IDP.

[0115] The term "intrinsically disordered protein" is well understood, and generally refers to proteins wherein the whole or a part of the protein lacks, or lacks the propensity to form, a three-dimensional structure. In some embodiments, the lyoprotectant comprises an insect and / or microanimal IDP. In some embodiments, the lyoprotectant comprises a tardigrade specific protein and / or a late embryogenesis abundant (LEA) protein or equivalent protein. Tardigrade specific proteins can also be referred to as IDPs from tardigrades. In some embodiments, the lyoprotectant comprises a cytoplasmic abundant heat soluble (CAHS) protein. CAHS proteins are understood to help micro-animals such as tardigrades survive desiccation. In some embodiments, the lyoprotectant comprises a CAHS1 and / or CAHS3 protein, such as a tardigrade CAHS1 and / or CAHS3 protein. In some embodiments, the lyoprotectant comprises one or more of (i) CAHS1, (ii) CAHS2 and (iii) CAHS3 protein, such as one or more of (i) a tardigrade CAHS1, (ii) a tardigrade CAHS2 and (iii) a tardigrade CAHS3 protein. The lyoprotectant may comprise (i), (ii), (iii), (i) and (ii), (i) and (iii), (ii) and (iii) or (i), (ii) and (iii).

[0116] In some embodiments, the lyoprotectant comprises a late embryogenesis abundant (LEA) protein. LEA proteins are understood to protect against protein aggregation during desiccation and / or osmotic stress from low temperature. In some embodiments, the lyoprotectant comprises an insect or nematode, preferably a nematode, LEA protein.

[0117] LEA proteins are characterised by a 11-mer motif and are known to reduce aggregation of proteins during dehydration. The LEA proteins can be subdivided based upon species into Group 1, Group2 and Group 3, whereby Group 3 proteins are expressed in animals. The inventors identified two Group 3 LEA proteins namely PvLEA22, which is expressed in Polypedilum vanderplanki, and AavLEA which is expressed in Aphelenchus avenae.

[0118] In some embodiments, the lyoprotectant comprises, consists essentially of, consists of, or is, a Group 3 LEA protein, In some embodiments, the lyoprotectant comprises, consists essentially of, consists of, or is, PvLEA and / or AavLEA.

[0119] In some embodiments, the IDP is selected from CAHS1, CAHS3, PvLEA, AavLEA and variants thereof. In some embodiments, the IDP comprises or consists of the sequence shown in SEQ ID NO: 29, 30, 31, 32, or 33 or a variant thereof. In some embodiments, the IDP is selected from CAHS1, CAHS2, CAHS3, PvLEA, AavLEA and variants thereof. In some embodiments, the IDP comprises or consists of the sequence shown in SEQ ID NO: 29, 30, 31, 32, 33, 49, 51 or 53 or a variant thereof.

[0120] The IDP may be produced separately and added to the lyophilised composition of the invention. The cell population and / or the reticulocyte cells are preferably genetically engineered to overexpress the IDP. This is discussed in more detail below.

[0121] General

[0122] In some embodiments, the lyoprotectant can comprise a proteinaceous lyoprotectant. In these embodiments, the reticulocyte cells can be a derivative of erythroid progenitor cells genetically engineered to express the proteinaceous lyoprotectant. In some embodiments, the lyoprotectant comprises, consists essentially of, or consists of, a single agent. In other embodiments, the lyoprotectant comprises more than one agent.

[0123] In some embodiments, the lyoprotectant comprises one or more of: a. a saccharide selected from trehalose, sucrose, fructose, glucose, mannitol, sorbitol, arabinose, and / or ribose; b. an antioxidant protein selected from GPxl, GPx2, GPx3, GPx4, GPx5, GPx6, GPx7, GPx8, Prxl, Prx2, Prx3, Prx4, Prx5, Prx6 and variants thereof; and c. an IDP selected from CAHS1, CAHS3, PVLEA, and / or AavLEA or an IDP selected from CAHS1, CAHS2, CAHS3, PVLEA, and / or AavLEA.

[0124] The lyoprotectant may comprise a, b, c, a and b, a and c, b and c, or a, b and c.

[0125] In some embodiments, the lyoprotectant is present at a concentration of greater than about 100 mM, greater than about 200 mM, greater than about 300 mM, greater than about 400 mM, or greater than about 500mM. In some embodiments, the lyoprotectant is present at a concentration of less than about 5000 mM, less than about 4000 mM, less than about 3000 mM, less than about 2000 mM, or less than about 1000 mM in the lyophilised composition. The lyoprotectant is preferably present at a concentration of greater than about 500 mM and less than about 1000 mM in the lyophilised composition. In some embodiments, the lyoprotectant is present at a concentration of greater than about 0.1 wt%, greater than about 0.5 wt%, greater than about 1 wt%, greater than about 2 wt%, greater than about 4 wt%, greater than about 6 wt% or greater than about 8 wt%, and less than about 20 wt%, less than about 18 wt%, less than about 16 wt%, less than about 14 wt%, less than about 12 wt% or less than about 10 wt%. The lyoprotectant is preferably present at a concentration greater than about 0.1 wt% and less than about 20 wt%. In some embodiments, the lyoprotectant is present at a concentration of greater than about 5 g / L, greater than about 10 g / L, greater than about 20 g / L, greater than about 50 g / L, greater than about 100 g / L, greater than about 200 g / L, or greater than about 500 g / L, and less than about 1000 g / L, less than about 900 g / L, less than about 800 g / L, less than about 700 g / L, less than about 600 g / L or less than about 550 g / L. The lyoprotectant is preferably present at a concentration of greater than about 10 g / L and less than about 550 g / L.

[0126] Therapeutic Protein or Polypeptide

[0127] In some embodiments, the reticulocyte cells further comprise or express a therapeutic protein or polypeptide. The therapeutic protein or polypeptide is not typically present in wild-type reticulocyte cells. A "therapeutic protein or polypeptide" is a protein or polypeptide with a useful therapeutic function. In other words, the reticulocyte cells can be used as a carrier of therapeutic proteins or polypeptides. An advantage of this is that, once inserted into the circulatory system of the body, the reticulocyte cells can transport the therapeutic protein or polypeptide around the entire circulatory system providing the therapeutic protein or polypeptide with rapid and systemic distribution around the body of the subject. In addition, the reticulocyte can protect the therapeutic protein or polypeptide from the body's metabolic or defensive processes, preventing the therapeutic protein or polypeptide from being degraded. In addition, for a therapeutic protein or polypeptide that is intracellular within the reticulocyte, the reticulocyte can protect against the body launching an immune response.

[0128] In some embodiments, the reticulocyte cells comprise or express a therapeutic protein or polypeptide comprising a sequence that is not naturally present in wild-type reticulocyte cells. In some embodiments, the therapeutic protein or polypeptide is an exogenous protein or polypeptide. In some embodiment, the therapeutic protein or polypeptide can be a variant of an endogenous protein or polypeptide. In some embodiments, the therapeutic protein or polypeptide can be an exogenous protein or polypeptide or a variant of an endogenous protein or polypeptide. In some embodiments, the reticulocyte cells overexpress the therapeutic protein or polypeptide. This is defined below.

[0129] In some embodiments, the therapeutic protein or polypeptide is able to exhibit a benefit, such as a therapeutic benefit, in a subject. In most embodiments, the therapeutic protein or polypeptide is not a haem-containing protein, such as haemoglobin and methaemoglobin. In some embodiments, the therapeutic protein or polypeptide is intracellular within the reticulocyte cells. In some embodiments, the therapeutic protein or polypeptide is displayed on the surfaces of the reticulocytes.

[0130] Preferably, the therapeutic protein or polypeptide is an enzyme. In a particular embodiment, the therapeutic protein is alanine amino transferase, thymidine phosphorylase, glutamine synthase, hexokinase, glucokinase, phenylalanine hydroxylase, alcohol dehydrogenase, catalase, glucose-6-phosphate dehydrogenase, adenosine deaminase, asparaginase, uricase, bacterial L-phenylalanine ammonia lyase, alanine aminotransferase, glutamate dehydrogenase, arginine deiminase, or arginase. Preferably the therapeutic protein is thymidine phosphorylase, oxalate decarboxylase, oxalate oxidase, glutamine synthase, hexokinase, glucokinase, phenylalanine hydroxylase, alcohol dehydrogenase, catalase, glucose-6-phosphate dehydrogenase, adenosine deaminase, asparaginase, uricase or bacterial L-phenylalanine ammonia lyase. In a preferred embodiment the therapeutic protein is thymidine phosphorylase.

[0131] The reticulocyte may comprise more than one type of therapeutic protein or therapeutic polypeptide. In such some embodiments, a plurality of therapeutic protein types can be used, for example, to provide a chain of enzymes in order to catalyse a sequence of reactions. As another example, a plurality of therapeutic protein types can be used to offset adverse effects of any given therapeutic protein.

[0132] The therapeutic protein or polypeptide can be exogenous or endogenous. In some embodiments, the therapeutic protein or polypeptide does not comprise an endogenous protein or endogenous therapeutic polypeptide. Where the therapeutic protein or polypeptide is endogenous, the concentration in the reticulocyte cell can be elevated through means such as suppression of loss of the therapeutic protein or polypeptide during enucleation and / or through overexpression. In some embodiments, the therapeutic protein or polypeptide can comprise an endogenous protein or polypeptide that is overexpressed. Overexpression techniques in cells such as erythroid progenitors are well known in the art and are, for instance, described in WO 2021 / 053243, the entirety of which is incorporated by reference herein.

[0133] In a preferred embodiment, the therapeutic protein or polypeptide is exogenous. "Exogenous" refers proteins or polypeptides which are not naturally expressed in the reticulocytes. This includes proteins or polypeptides which comprise one or more amino acid mutations with respect to an endogenous protein. Exogenous can also cover isoforms from other cells or species, or non-naturally occurring proteins or polypeptides, such as hybrid proteins, chimeric proteins, fusion proteins or de novo protein or polypeptide sequences. Genetic engineering techniques are discussed in more detail below.

[0134] In some embodiments, the reticulocyte cell is a derivative of an erythroid progenitor that is genetically engineered to express or overexpress the therapeutic protein or polypeptide. The definition of "genetically engineered" can be found above.

[0135] One challenge faced with expression of a therapeutic protein or polypeptide is that the therapeutic protein or polypeptide is expelled or degraded during the enucleation process that leads to the reticulocyte cell. Methods for suppressing this reduction in therapeutic protein or polypeptide are known in the art and have been outlined in WO 2021 / 053243, the entirety of which is incorporated by reference herein. In brief, ubiquitination was identified as an important method by which protein and polypeptide concentrations are reduced during enucleation. In some embodiments, ubiquitination can be inhibited through use of a ubiquitinase inhibitor. Ubiquitinase inhibitors are non-specific and can have deleterious effects beyond simply retaining the therapeutic protein or polypeptide. It has been reported that ubiquitination is an essential part of the erythroid cell maturation process (Nguyen, A.T., et al., Science, 2017, 357(6350)). As such, global inhibition of ubiquitination (using ubiquitinase inhibitors such as MG132) means many non-erythroid proteins would be retained in the developing reticulocyte or erythrocyte. As a result, many aspects of cell function would be disrupted which can adversely impact important aspects of the cell such as lifetime and immune system compatibility. In certain cases, broad ubiquitination inhibition may prevent differentiation altogether and / or lead to cell death.

[0136] In some embodiments, the therapeutic protein or polypeptide is configured such that ubiquitination of the therapeutic protein or polypeptide is hindered or prevented. As such, the inhibition occurs locally at the therapeutic protein or polypeptide rather than by blanket inhibition of ubiquitination. This is a much more targeted technique for preventing or hindering ubiquitin-mediated degradation of the therapeutic protein or polypeptide. The ubiquitination of the therapeutic protein or polypeptide can be hindered or prevented as discussed in more detail below for antioxidant proteins.

[0137] Composition before lyophilisation

[0138] The invention also provides a pre-lyophilisation composition comprising a cell population, a lyoprotective agent and a lyophilisation medium, wherein the cell population comprises at least about 10% reticulocyte cells. The reticulocyte cells may be any of those discussed above.

[0139] The lyophilisation medium can be any medium that is compatible with the reticulocyte cells. The lyophilisation medium can be an aqueous medium. In most embodiments, the lyophilisation medium comprises or consists of a buffer. In some embodiments, the lyophilisation medium comprises or consists of an aqueous buffer. In some embodiments, the pre-lyophilisation composition consists essentially of, or consists of, a cell population, a lyoprotective agent, and an aqueous buffer.

[0140] The pre-lyophilisation composition can be in the liquid or solid phase. In some embodiments, the pre-lyophilisation composition is at a temperature above the freezing temperature of the pre-lyophilisation composition. In other words, the pre-lyophilisation composition is a liquid. In some embodiments, the pre-lyophilisation composition is at a temperature below the freezing temperature of the pre-lyophilisation composition. In other words, the pre-lyophilisation composition is a solid.

[0141] The invention also provides a method of preparing a pre-lyophilisation composition, wherein the method comprises contacting a cell population comprising at least about 10% reticulocyte cells with a lyophilisation medium and a lyoprotective agent. The lyoprotectant may be any of those discussed above. The reticulocyte cells may be any of those discussed above. The lyophilisation medium may be any of those discussed above.

[0142] Method of lyophilising The invention also provides a method of preparing a lyophilised composition of the invention. The lyophilised composition comprises a cell population and a lyoprotectant, wherein the cell population comprises at least about 10% reticulocyte cells.

[0143] The method comprises (a) suspending a cell population in a lyophilisation medium comprising a lyoprotectant, wherein the cell population comprises at least about 10% reticulocyte cells. The lyoprotectant may be any of those discussed above. The reticulocyte cells may be any of those discussed above. The lyophilisation medium may be any of those discussed above.

[0144] In some embodiments, the cell population is suspended in the lyophilisation medium comprising the lyoprotectant in step (a) for at least about 1 hour. The cell population is preferably suspended in the lyophilisation medium comprising the lyoprotectant in step (a) for at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 12 hours, at least about 14 hours, at least about 18 hours, at least about 20 hours, at least about 24 hours, or at least about 48 hours. The cell population is preferably suspended in the lyophilisation medium comprising the lyoprotectant in step (a) overnight.

[0145] As explained above, the inventors have surprisingly shown reticulocyte cells are capable of taking up trehalose. Suspension of the cell population in the lyophilisation medium comprising the lyoprotectant in step (a) may comprise loading the cells with the lyoprotectant. In some embodiments, the method comprises (a) suspending a cell population in a lyophilisation medium comprising a lyoprotectant, wherein the cell population comprises at least about 10% reticulocyte cells, for at least about 1 hour or any of the time discussed above to load the cells with the lyoprotectant. The lyoprotectant is preferably selected from trehalose, sucrose, fructose, glucose, mannitol, sorbitol, arabinose, and ribose. The lyoprotectant is preferably trehalose. The reticulocyte cells are preferably genetically engineered to express a trehalose transporter or a variant thereof on their surfaces.

[0146] The method also comprises (b) subjecting the suspension produced in step (a) to a lyophilisation process to generate the lyophilised composition. Step (b) may be conducted after any of the time described above for step (a).

[0147] Lyophilisation generally involves first freezing the liquid composition to a frozen solid and then subjecting the frozen solid to reduced pressure that removes the frozen liquid by sublimation. The resulting composition is the lyophilised, or 'freeze-dried', composition.

[0148] Lyophilisation is a useful technique for removing liquids, typically aqueous liquids, from biological materials such as cells. The use of low temperature helps to pause metabolic processes and prevent degradation of biological material such as proteins. However, one potential source of damage can be ice crystal formation in the freezing of water-based liquids. Ice crystal formation can expand the volume in comparison to the liquid phase and / or generate spiky ice crystals, both of which can rupture the cell.

[0149] The lyophilisation process in step (b) can comprise a first drying stage. The first drying stage typically comprises two features, cooling to freeze a composition, and exposure of the frozen composition to reduced pressure to remove frozen water by sublimation.

[0150] The first drying stage can comprise a rapid cooling stage. In the rapid cooling stage, the pre-lyophilisation composition starts in the liquid phase, and is cooled rapidly such that water freezes in an amorphous form. In other words, the water freezes with suppression of ice crystal formation. In some embodiments, the pre-lyophilisation composition can be cooled at greater than about 0.5 °C per minute until frozen. In some embodiments, the pre- lyophilisation composition can be frozen in a freezer that is precooled to about -50 °C or lower. It is generally preferred that the freezer is precooled to about -55 °C or lower.

[0151] In the first drying stage, the frozen composition is exposed to reduced pressure. In other words, the pressure is reduced to below atmospheric pressure. The extent of the pressure drop is not particularly limited. Generally, a lower pressure will provide for more rapid water sublimation. In some embodiments, the pressure is less than about 500 microbar, less than about 250 microbar, less than about 100 microbar, less than about 50 microbar or less than about 20 microbar.

[0152] The temperature can be kept constant or can be varied. In some embodiments, the temperature is subsequently raised by between about 5 and about 10 °C to accelerate sublimation of water. For instance, rapid cooling may be conducted in a freezer precooled to about -55 °C and once the composition is frozen the temperature may be raised to about - 50 °C.

[0153] The low temperature and low pressure can be maintained until the water is removed. The low temperature and low pressure can be maintained until the majority of the water is removed. The low temperature and low pressure can be maintained until the bulk water has been removed. The low temperature and low pressure can be maintained until frozen water cannot be identified by visual inspection.

[0154] In some embodiments, the first drying stage comprises: a. cooling the suspension at greater than about 0.5 °C per minute until frozen, b. reducing the pressure to below atmospheric pressure, and c. maintaining the reduced pressure for a period of time sufficient to remove the majority of the water.

[0155] The lyophilisation process in step (b) can comprise a second drying stage. The second drying stage can be used to remove residual water. The second drying stage can comprise heating the composition above 0 °C. The second drying stage can comprise heating the composition to room temperature. The second drying stage can comprise heating the composition to at least about 1 °C, 2 °C, 5 °C, 10 °C, 15 °C, or 20 °C. The second drying stage can comprise heating the composition while maintaining low pressure.

[0156] The second drying stage can comprise: d. raising the temperature to above 0 °C, and e. maintaining the reduced pressure for a period of time sufficient to remove water.

[0157] Method of reconstitution

[0158] The invention also provides a method of reconstitution of a lyophilised composition of the invention by contacting the lyophilised composition with a reconstitution medium. The lyophilised composition comprises a cell population and a lyoprotectant, wherein the cell population comprises at least about 10% reticulocyte cells. The lyoprotectant may be any of those discussed above. The reticulocyte cells may be any of those discussed above. In all instances herein, "reconstitution" or the like is interchangeable with "rehydration" or the like.

[0159] The reconstitution medium is preferably a liquid. The reconstitution medium is preferably compatible with intravenous injection. The reconstitution medium is preferably sterile. Reconstitution can be enhanced by contacting the lyophilised composition with the reconstitution medium and then mildly agitating the composition.

[0160] The reconstitution medium preferably comprises an aqueous liquid. In some embodiments, the reconstitution medium comprises saline solution. In some embodiments, the reconstitution medium is an aqueous saline solution. In some embodiments, the reconstitution medium has a salinity of at least about 0.5%, preferably at least about 0.7%, or preferably about 0.9%. In some embodiments, the reconstitution medium has a salinity of less than about 12%, preferably less than about 5%, or more preferably less than about 2%. 'Salinity' refers to salt concentration, such as concentration of NaCI.

[0161] In some embodiments, the reconstitution medium comprises SAGM, PAGGM, AS1, AS3, human plasma, artificial plasma solution, phosphate buffered saline, or mixtures thereof. The reconstitution medium may be any of the pharmaceutically or physiologically acceptable diluents and / or carriers discussed below in relation to the pharmaceutical composition of the invention.

[0162] Reconstituted composition

[0163] The invention also provides a reconstituted composition obtained by or obtainable by the reconstituting method of the invention. The invention also provides a reconstituted composition obtained by or obtainable by reconstituting a lyophilised composition of the invention. The lyophilised composition comprises a cell population and a lyoprotectant, wherein the cell population comprises at least about 10% reticulocyte cells. The lyoprotectant may be any of those discussed above. The reticulocyte cells may be any of those discussed above.

[0164] The invention also provides a reconstituted composition comprising a cell population and a lyoprotectant in a reconstitution medium, and wherein the cell population comprises at least about 10% reticulocyte cells. The lyoprotectant may be any of those discussed above. The reticulocyte cells may be any of those discussed above. The reconstitution medium may be any of those discussed above.

[0165] Preferably, the reconstitution medium comprises a pharmaceutically acceptable salt solution or buffer. Preferably, the reconstitution medium comprises SAGM, PAGGM, AS1, AS3, human plasma, or artificial plasma solution, or physiologically compatible buffers such as phosphate buffered saline. In some embodiments, the reconstitution medium has a salinity of at least about 0.5%, preferably at least about 0.7%, or preferably about 0.9%. In some embodiments, the reconstitution medium has a salinity of less than about 12%, preferably less than about 5%, or more preferably less than about 2%. 'Salinity' refers to salt concentration, such as concentration of NaCI. Preferably, the reconstitution medium is a pharmaceutically or physiologically acceptable diluent and / or carrier discussed below with reference to the pharmaceutical compositions of the invention.

[0166] Reticulocyte cells and RBCs of the invention

[0167] The invention also provides a reticulocyte cell or a mature red blood cell (RBC) comprising intracellular trehalose. The invention also provides a reticulocyte cell or a mature red blood cell (RBC) comprising an increased amount or level of intracellular trehalose. The mature RBC is preferably matured from a reticulocyte cell. The presence of intracellular trehalose can be measured using routine methods. For instance, the cell can be lysed and the presence of trehalose can be confirmed using a spectrophotometer measurement, for instance at 240nm. An increased amount or level is typically measured compared with native or wild-type reticulocyte cells or mature RBCs. The invention also provides a population of reticulocyte cells or a mature red blood cells (RBCs) comprising intracellular trehalose. The invention also provides a population of reticulocyte cells or mature red blood cells (RBCs) comprising an increased level or amount intracellular trehalose. Populations of cells are discussed in more detail.

[0168] The reticulocyte cell or a mature RBC of the invention preferably comprises at least about 10 pg intracellular trehalose. The cell more preferably comprises at least about 15 pg, at least about 20 pg, at least about 23 pg, at least about 25 pg, at least about 30 pg, at least about 35 pg, or at least about 40 pg intracellular trehalose.

[0169] The population of reticulocyte cells or mature RBCs of the invention preferably comprises at least about 10 pg intracellular trehalose per 106cells. The population more preferably comprises at least about 15 pg, at least about 20 pg, at least about 23 pg, at least about 25 pg, at least about 30 pg, at least about 35 pg, or at least about 40 pg intracellular trehalose per 106cells.

[0170] The invention also provides a method of producing a reticulocyte cell of the invention, a mature RBC of the invention, or a population of the invention, comprising suspending a reticulocyte, a mature RBC or a population of reticulocyte cells or mature RBCs in a medium comprising trehalose. The medium may be any of the media discussed above, especially in relation to the lyophilisation method of the invention. The medium preferably comprises at least about 5% trehalose (w / v), such as at least about 10% trehalose (w / v), at least about 15% trehalose (w / v) or at least about 20% trehalose (w / v).

[0171] In some embodiments, the cell or population is suspended in the medium comprising trehalose for at least about 1 hour. The cell or population is preferably suspended in the medium comprising trehalose for at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 12 hours, at least about 14 hours, at least about 18 hours, at least about 20 hours, at least about 24 hours, or at least about 48 hours. The cell or population is preferably suspended in the medium comprising trehalose overnight.

[0172] In some embodiments, the reticulocyte or mature RBC is genetically engineered to express a trehalose transporter or a variant thereof on its surface. In some embodiments, the reticulocyte or mature RBC is derived from erythroid progenitor cell genetically engineered to express a trehalose transporter or a variant thereof on its surface. The definition of "genetically engineered" can be found above. An advantage of the trehalose transporter is that it can increase loading of the reticulocyte cells with trehalose. This can help achieve beneficial concentrations of trehalose within the reticulocyte cells more quickly and / or with lower external concentrations of trehalose. In some embodiments, the trehalose transporter is a glucose transporter (GLUT8) protein or a variant thereof. GLUT8 is a glucose transporter that plays a role in intracellular trafficking of glucose. GLUT8 has also been observed to be a trehalose transporter. GLUT8 is the sole mammalian trehalose transporter identified to date. The GLUT8 protein or variant thereof is preferably a human GLUT8 protein or a variant thereof. The GLUT8 protein or variant thereof is preferably a protein comprising or consisting of the sequence shown in SEQ ID NO: 35 or a variant thereof.

[0173] In a particularly preferred embodiment, the GLUT8 variant protein comprises a modified N- terminal region that is capable of localising the variant protein to the plasma membrane. The modified N-terminal region can comprise any number and combination of mutations. The modified N-terminal region can comprise one or more mutations, such as 2, 3, 4, 5, 6 or more mutations. The one or more mutations may be one or more substitutions, one or more deletions, one or more additions or any combination thereof.

[0174] Wild-type mammalian GLUT8 protein is internalized following its expression at the cell surface because it is normally located in lysosomes. GLUT8 features an NH2- terminus [DE]XXXL[LI] targeting motif which plays a role in directing the protein towards endosomes, lysosomes, and related organelles. Research conducted in Chinese Hamster Ovary (CHO) cells revealed that the GLUT8 transporter could be directed to the plasma membrane by both substituting the XXX residues of the lysosomally located GLUT8 transporter with GPN and altering the dileucine (LL) motif to AA (which localizes GLUT12 transporter to the plasma membrane), (Flessner and Moley "Similar [DE]XXXL[LI] Motifs Differentially Target GLUT8 and GLUT12 in Chinese Hamster Ovary Cells", Traffic (2009), vollO, p324-333). The inventors have surprisingly shown that only the GPS mutation (the human mutation corresponding to the GPN mutation in mice) is needed to direct the human GLUT8 protein to the surface of human erythroid cells, including reticulocyte cells and RBCs. The GLUT8 variant protein preferably comprises or only comprises a TPQ to GPN mutation. The mouse GLUT8 variant protein preferably comprises or only comprises a TPQ to GPN mutation. The GLUT8 variant protein preferably comprises or only comprises a mutation of the N terminal [DE]XXXL[LI] motif to [DE]GPNL[LI]. The GLUT8 variant protein preferably does not comprise a mutation of the dileucine motif (LL) in the N terminal [DE]XXXL[LI] motif. The human GLUT8 variant protein preferably comprises or only comprises a TPQ to GPS mutation. The human GLUT8 variant protein preferably comprises or only comprises a mutation of the N terminal [DE]XXXL[LI] motif to [DE]GPSL[LI]. The human GLUT8 variant protein preferably does not comprise a mutation of the dileucine motif (LL) in the N terminal [DE]XXXL[LI] motif. The GLUT8 variant protein preferably comprises or consists of the sequence shown in SEQ ID NO: 37. In some embodiments, the reticulocyte cell or the mature RBCs of the invention further comprises or expresses a therapeutic protein or polypeptide. Any of the embodiments discussed above equally apply here.

[0175] In some embodiments, the reticulocyte cell or mature RBC of the invention is genetically engineered to overexpress an antioxidant protein and / or an intrinsically disordered protein (IDP) in its cytosol. Any of the embodiments discussed below equally apply here. The reticulocyte cell or mature RBC of the invention may be genetically engineered to express one of the variant proteins of the invention.

[0176] Erythroid cells genetically engineered to express a trehalose transporter

[0177] The invention also provides an erythroid cell genetically engineered to express a trehalose transporter or a variant thereof on its surface. In some embodiments, the erythroid cell is an erythroid progenitor cell, a reticulocyte, or a mature RBC. The erythroid progenitor cell may be any of those discussed above. The definition of "genetically engineered" can be found above. The trehalose transporter may be any of the transporters discussed above, especially a glucose transporter 8 (GLUT8) protein or a variant thereof. The GLUT8 variant protein preferably comprises one of the specific mutations discussed above, especially the TPQ to GPN mutation in mouse GLUT8 or the TPQ to GPS mutation in human GLUT8. The GLUT8 variant protein preferably comprises or consists of the sequence shown in SEQ ID NO: 37.

[0178] In some embodiments, the erythroid of the invention further comprises or expresses a therapeutic protein or polypeptide. Any of the embodiments discussed above equally apply here.

[0179] In some embodiments, the erythroid cell of the invention is genetically engineered to overexpress an antioxidant protein and / or an intrinsically disordered protein (IDP) in its cytosol. Any of the embodiments discussed below equally apply here. The erythroid cell of the invention may be genetically engineered to express one of the variant proteins of the invention.

[0180] Erythroid cells genetically engineered to express increased levels of a protein in their cytosol The invention also provides an erythroid cell genetically engineered to express a variant protein which comprises one or more mutations which increase the level of the variant protein in the cytosol of the cell.

[0181] In some embodiments, the erythroid cell is an erythroid progenitor cell, a reticulocyte, or a mature RBC. The erythroid progenitor cell may be any of those discussed above. The erythroid cell is preferably human. The definition of "genetically engineered" can be found above. The increased level may be an increased amount and / or an increased activity. The increased level is typically an increased detectable level. The increased level is typically compared with the level before the genetic engineering is conducted. The increased level is typically compared with the level of the protein (i.e., the unmodified or non-mutated protein) in the cell before the genetic engineering is conducted. The level of the variant protein may be increased by any of the amounts defined above with reference to "overexpression".

[0182] The invention also provides an erythroid cell genetically engineered to express a variant protein which comprises one or more mutations which facilitate overexpression of the variant protein in the cytosol of the cell. The definition of "overexpression" and the extent to which the variant protein can be overexpressed is also defined above.

[0183] The unmodified or non-mutated protein is typically one or more of ubiquitinated in the erythroid cell, secreted from the erythroid cell or targeted to intracellular organelles, such as mitochondria, in the erythroid cell. This limits the expression of the protein in the cytosol. The variant protein includes one or more mutations to inhibit or reduce one or more of these processes and this results in an increased level of or overexpression of the variant protein in the cytosol of the erythroid cell.

[0184] The one or mutations preferably comprise (a) one or mutations at one or more predicted ubiquitination sites and / or of one or more surface lysine residues, (b) one or more inactivating mutations in its signal peptide, (c) one or more inactivating mutations in its mitochondrial targeting peptide or (d) any combination thereof. The combination in (d) may be (a) and (b), (a) and (c), (b) and (c) or (a), (b) and (c). The variant protein may comprise (a), (b), (c), (a) and (b), (a) and (c), (b) and (c) or (a), (b) and (c).

[0185] The variant protein can comprise any number and combination of one or more mutations. The variant protein can comprise 2, 3, 4, 5, 6 or more mutations. The one or more mutations may be one or more substitutions, one or more deletions, one or more additions or any combination thereof. The one or more mutations are preferably one or more deletions. Any number of amino acids may be deleted from the protein, such as at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20 or at least 25 amino acids.

[0186] In (a), mutating one or more predicted ubiquitination sites reduces intracellular degradation of the variant protein and this results in an increased level or its overexpression in the cytosol of the erythroid cell compared with the unmodified or non-mutated protein. In (a), any number of predicted ubiquitination sites may be mutated or substituted. For instance, at least 1, at least 2, at least 3, at least 4, at least 5 or at least 6 predicted ubiquitination sites may be mutated or substituted. Preferably, the variant protein comprises a mutation or substitution at all of its predicted ubiquitination sites. Predicated ubiquitination sites are defined in more detail below and all of that discussion equally applies here.

[0187] In (a), one or more surface lysine residues may act as ubiquitination sites even though they are not predicted as such (for instance as described below). Any number of surface lysine residues may be mutated or substituted. For instance, at least 1, at least 2, at least 3, at least 4, at least 5 or at least 6 surface lysine residues may be mutated or substituted. The three-dimensional structure of the protein and its surface can be determined using standard methods in the art (e.g., AlphaFold or PSIPRED).

[0188] Signal peptides are short amino acid sequences that direct proteins to the correct location within the erythroid cell or out of the erythroid cell (via the secretory pathway). Secretion of proteins means they are lost from the cytosol. During the development of a RBC, its organelles are removed and therefore any proteins directed to organelles by their signal peptides will be lost. In (b), one or more inactivating mutations in the signal peptide reduce secretion of the variant protein or trafficking of the variant protein to organelles and this results in an increased level or overexpression of the variant protein in the cytosol of the erythroid cell compared with the unmodified or non-mutated protein, especially when the organelles are lost. In (b), the variant protein can comprise any number and combination of one or more inactivating mutations. The variant protein can comprise 2, 3, 4, 5, 6 or more inactivating mutations. The one or more inactivating mutations may be any of the types of mutations described above. The variant protein preferably lacks at least a part of its signal peptide. The variant protein may lack any amount of its signal peptide. In some embodiments, the at least a part is at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20 or at least 25 amino acids. In some embodiments, the at least a part is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% of the signal peptide. The variant protein most preferably lacks the whole or complete signal peptide.

[0189] In (c), one or more inactivating mutations in the mitochondrial targeting peptide reduce trafficking of the variant protein to mitochondria and this results in an increased level or overexpression of the variant protein in the cytosol of the erythroid cell compared with the unmodified or non-mutated protein, especially when the mitochondria are lost. In (c), the variant protein can comprise any number and combination of one or more inactivating mutations. The variant protein can comprise 2, 3, 4, 5, 6 or more inactivating mutations. The one or more inactivating mutations may be any of the types of mutations described above. The variant protein preferably lacks at least a part of its mitochondrial targeting peptide. The variant protein may lack any amount of its mitochondrial targeting peptide. In some embodiments, the at least a part is at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20 or at least 25 amino acids. In some embodiments, the at least a part is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% of the signal peptide. The variant protein most preferably lacks the whole or complete mitochondrial targeting peptide. Mitochondrial targeting peptides are known in the art and can be predicted by analysing the sequence of a protein, for example as described in Example 9. An example of a mitochondrial targeting peptide is shown in positions 2-28 of SEQ ID NO: 57.

[0190] The variant protein may be a variant of any protein. The variant protein may be a variant of a protein from any of erythroid cell or species discussed above or below. The variant protein is preferably a variant of a human protein.

[0191] The variant protein may be a variant of an antioxidant protein and / or an intrinsically disordered protein (IDP) in its cytosol. The antioxidant and / or IDP any variants thereof may be any of those discussed below in connection with erythroid cells genetically engineered to overexpress an antioxidant protein and / or IDP.

[0192] The variant protein may be a variant of an enzyme. The enzyme may be any of those discussed above. The enzyme is preferably selected from hydrolases, oxidoreductases, lyases, transferases, ligases, isomerases, phosphorylases, synthases, kinases, hydroxylase, dehydrogenase, deaminases, proteases, metalloproteinases, lyases, aminotransferases, deiminases, decarboxylases, oxidases and lysosomal enzymes. The enzyme is preferably thymidine phosphorylase, alanine aminotransferase, thymidine phosphorylase, glutamine synthase, hexokinase, glucokinase, phenylalanine hydroxylase, alcohol dehydrogenase, catalase, glucose-6-phosphate dehydrogenase, adenosine deaminase, asparaginase, uricase, bacterial L-phenylalanine ammonia lyase, alanine aminotransferase, glutamate dehydrogenase, arginine deiminase, arginase, oxalate decarboxylase, oxalate oxidase, a serine protease, tissue plasminogen activator, and larondinase. The enzyme may be mitochondrial.

[0193] The enzyme is preferably alanine aminotransferase 2 (AAT2). The invention also provides an erythroid cell genetically engineered to express a variant AAT2 protein which comprises one or more mutations which increase the level of or facilitate overexpression of the variant AAT2 protein in the cytosol of the cell. The variant protein may be a variant of SEQ ID NO: 57. The variant protein may be a variant of SEQ ID NO: 57 lacking one or more of residues 2-28. The variant protein may lack any number of residues 2-28, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 residues. The variant protein preferably lacks all of residues 2-28 in SEQ ID NO: 57. The variant protein preferably comprises or consists of the sequence shown in SEQ ID NO: 59 or a variant thereof. In some embodiments, the erythroid cell is further genetically engineered to express a trehalose transporter or a variant thereof on its surface. Any of the embodiments discussed above equally apply here.

[0194] In some embodiments, the erythroid cell of the invention further comprises or expresses a therapeutic protein or polypeptide. Any of the embodiments discussed above equally apply here.

[0195] The erythroid cell may be autologous. In other words, the erythroid cell may be derived from the subject into which the cell will be administered. The erythroid cell is preferably allogeneic. In other words, the erythroid cell is preferably derived from a subject that is immunologically compatible with the subject into which the erythroid cell will be administered. The administration of autologous or allogeneic cells to subjects is well documented.

[0196] The erythroid cell may be engineered to be antibiotic resistant.

[0197] Erythroid cells genetically engineered to overexpress an antioxidant protein and / or IDP The invention also provides an erythroid cell genetically engineered to overexpress an antioxidant protein and / or an intrinsically disordered protein (IDP) in its cytosol.

[0198] In some embodiments, the erythroid cell is an erythroid progenitor cell, a reticulocyte, or a mature RBC. The erythroid progenitor cell may be any of those discussed above. The erythroid cell is preferably human.

[0199] The definition of "genetically engineered" can be found above. The definition of "overexpression" and the extent to which the antioxidant protein and / or IDP can be overexpressed is also defined above. The erythroid cell may be genetically engineered to express an increased level of the antioxidant protein and / or IDP in the cytosol of the cell. The increased level may be an increased amount and / or an increased activity. The increased level is typically an increased detectable level. The overexpression or increased level is typically compared with the expression or level in the cell or population before the genetic engineering is conducted. The invention also provides an erythroid cell genetically engineered to express an increased level of an antioxidant protein and / or IDP in its cytosol.

[0200] A key part of the invention is the overexpression of the antioxidant protein and / or IDP in the cytosol. It is important these proteins are overexpressed in the cytosol of the erythroid cells and not targeted to the mitochondria or other intracellular organelles or secreted from the cells. This allows the proteins to protect against reactive oxygen species (ROS) in general but also have a protective effect for lipid peroxidation during freeze drying and reconstitution processes as described above. The antioxidant protein may be any antioxidant protein. The antioxidant protein may be from any of the cells or species discussed above or below. The antioxidant protein is preferably human. The antioxidant protein may be a non-human antioxidant protein humanised to express in human cells. The IDP may be any of those discussed above or below. The antioxidant protein and / or the IDP may comprise any of the one or more mutations defined in (a)-(d) above.

[0201] In some embodiments, the antioxidant is superoxide dismutase or catalase or a variant thereof. In some embodiments, the antioxidant protein is modified to prevent its targeting to mitochondria. In some embodiments, the antioxidant protein comprises one or more inactivating mutations in its mitochondrial targeting peptide. The one or more inactivating mutations prevent the variant protein from being targeting to the mitochondria. The skilled person is capable of determining whether a mutation inactivates the mitochondrial targeting peptide and prevents the variant protein from being targeted to the mitochondria. The one or more inactivating mutations may be any of those discussed below with reference to the signal peptide.

[0202] In some embodiments, the antioxidant protein is a glutathione peroxidase (GPx) or a variant thereof. In some embodiments, the antioxidant protein is GPxl, GPx2, GPx3, GPx4, GPx5, GPx6, GPx7, GPx8 or a variant thereof. The antioxidant protein preferably comprises or consists of the sequence shown in SEQ ID NO: 2, 4, 6, 38, 39, 8, 10, 12, 14, 16, 40, 41, 42 or 43 or a variant thereof. The antioxidant protein preferably comprises or consists of the sequence shown in SEQ ID NO: 2, 4, 6, 38, 39, 8, 10, 12, 14, 16, 40, 41, 42, 43 or 55 or a variant thereof. The antioxidant protein is preferably GPxl, GPx4 or a variant thereof. The antioxidant protein is preferably GPxl or a variant thereof. The antioxidant protein preferably comprises or consists of the sequence shown in SEQ ID NO: 2, 4 or 6 or a variant thereof. The antioxidant protein preferably comprises or consists of the sequence shown in SEQ ID NO: 2, 4, 6 or 55 or a variant thereof. The antioxidant protein is preferably GPx4 or a variant thereof. The antioxidant protein preferably comprises or consists of the sequence shown in SEQ ID NO: 8, 10, 12, 14 or 16 or a variant thereof. The antioxidant protein most preferably comprises or consists of the sequence shown in SEQ ID NO: 16 or a variant thereof.

[0203] In some embodiments, the antioxidant protein is a peroxiredoxin (Prx) or a variant thereof. In some embodiments, the antioxidant protein is Prxl, Prx2, Prx3, Prx4, Prx5, Prx6 or a variant thereof. The antioxidant protein preferably comprises or consists of the sequence shown in SEQ ID NO: 18, 20, 22, 24, 44, 45, 46, 26 or 28 or a variant thereof. In some embodiments, the antioxidant protein is Prxl, Prx2, Prx3, or a variant thereof. The antioxidant protein preferably comprises or consists of the sequence shown in SEQ ID NO: 18, 20, 22, 24, 26 or 44 or a variant thereof. In some embodiments, the antioxidant protein is Prx2 or a variant thereof. The antioxidant protein preferably comprises or consists of the sequence shown in SEQ ID NO: 22 or 24 or a variant thereof.

[0204] In some embodiments, the antioxidant protein is modified to prevent its secretion from the cell. In some embodiments, the GPx4 variant protein, the GPx5 variant protein, the GPx7 variant protein, the GPx8 variant protein, the Prx3 variant protein or the Prx5 variant protein comprises one or more inactivating mutations in its signal peptide. In some embodiments, the GPx4 variant protein comprises one or more inactivating mutations in its signal peptide. The one or more inactivating mutations prevent the variant protein from being trafficked to intracellular organelles or secreted from the cell. The skilled person is capable of determining whether a mutation inactivates the signal peptide and prevents the variant protein from being trafficked to intracellular organelles or secreted from the cell.

[0205] The variant protein can comprise any number and combination of inactivating mutations. The variant protein can comprise one or more mutations, such as 2, 3, 4, 5, 6 or more mutations. The one or more mutations may be one or more substitutions, one or more deletions, one or more additions or any combination thereof. The one or more mutations are preferably one or more deletions. Any number of amino acids may be deleted from the signal peptide, such as at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20 or at least 25 amino acids.

[0206] The variant protein preferably lacks at least a part of its signal peptide. The variant protein may lack any amount of its signal peptide. In some embodiments, the at least a part is at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20 or at least 25 amino acids. In some embodiments, the at least a part is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% of the signal peptide.

[0207] The variant protein most preferably lacks the whole or complete signal peptide.

[0208] The GPx4 signal peptide corresponds to the first 27 amino acids (i.e., positions 1-27) in SEQ ID NO: 8. The GPx4 variant protein preferably comprises one or more mutations in these 27 amino acids. The one or more mutations may be any of those described above. The Gpx4 variant protein preferably comprises deletion of the first 27 amino acids (i.e., positions 1- 27) in SEQ ID NO: 8. This is also called short GPx4 or sGPx4.

[0209] In some embodiments, the variant antioxidant protein comprises a mutation at one or more predicted ubiquitination sites. Ubiquitination sites are locations within proteins to which ubiquitin is capable of binding and degrading the protein. Mutating one or more ubiquitination sites reduces the degradation of the variant protein within the cell. A webbased algorithm, PhosphoSitePlus (https: / / www.phosphosite.org / homeAction.action), can be used to predict ubiquitination sites. PhosphoSitePlus integrates data for post translational modifications (PMTs) that have either been published in the literature or are unpublished generated at Cell Signalling Technology (CST). Data include human, mouse, and rat proteins / isoforms. Predicted ubiquitination sites can also be identified through the curation of data published in the literature.

[0210] In preferred embodiments, the variant antioxidant protein comprises a substitution at one or more predicted ubiquitination sites. Predicted ubiquitination sites typically comprise lysine (K). The K is preferably substituted with arginine (R). This maintains the positive charge at the substituted position.

[0211] Any number of predicted ubiquitination sites may be mutated or substituted, such as at least 1, at least 2, at least 3, at least 4, at least 5 or at least 6. Preferably, the variant protein comprises a mutation or substitution at all of its predicted ubiquitination sites.

[0212] In preferred embodiments, the GPxl variant or the GPx4 variant comprises a mutation or substitution at one or more predicted ubiquitination sites or all of its predicted ubiquitination sites. In preferred embodiments, the GPxl variant comprises a mutation or substitution at one or more predicted ubiquitination sites or all of its predicted ubiquitination sites. In preferred embodiments, the GPx4 variant comprises a mutation or substitution at one or more predicted ubiquitination sites or all of its predicted ubiquitination sites.

[0213] The GPxl variant preferably comprises a mutation or substitution at one or more of K88, K148 and K166, such as K88, K148, K166, K88 and K148, K88 and K166, K148 and K166 or K88, K148 and K166. These positions are shown in SEQ ID NO: 2. The GPxl variant preferably comprises a mutation or substitution at one or more of the positions corresponding to positions K88, K148 and K166 in SEQ ID NO: 2, such as K88, K148, K166, K88 and K148, K88 and K166, K148 and K166 or K88, K148 and K166. In preferred embodiments, K is substituted with R.

[0214] The GPx4 variant preferably comprises a mutation or substation at one or more of (a) K107, (b) K126, (c) K148, (d) K162R, (e) K167 and (f) K191. These positions are shown in SEQ ID NO: 8. The GPx4 variant preferably comprises a mutation or substitution at one or more of the positions corresponding to (a) K107, (b) K126, (c) K148, (d) K162R, (e) K167 and (f) K191 in SEQ ID NO: 8. The GPx4 variant protein may comprise a mutation or substitution at any number and combination of these positions, such as (a); (b); (c); (d); (e); (f); (a) and (b); (a) and (c); (a) and (d); (a) and (e); (a) and (f); (b) and (c); (b) and (d); (b) and (e); (b) and (f); (c) and (d); (c) and (e); (c) and (f); (d) and (e); (d) and (f); (e) and (f); (a),

[0215] (b) and (c); (a), (b) and (d); (a), (b) and (e); (a), (b) and (f); (a), (c) and (d); (a), (c) and (e); (a), (c) and (f); (a), (d) and (e); (a), (d) and (f); (a), (e) and (f); (b), (c) and (d); (b),

[0216] (c) and (e); (b), (c) and (f); (b), (d) and (e); (b), (d) and (f); (b), (e) and (f); (c), (d) and (e); (c), (d) and (f); (c), (e) and (f); (d), (e) and (f); (a), (b), (c) and (d); (a), (b), (c) and

[0217] (e); (a), (b), (c) and (f); (a), (b), (d) and (e); (a), (b), (d) and (f); (a), (b), (e) and (f);

[0218] (a), (c), (d) and (e); (a), (c), (d) and (f); (a), (c), (e) and (f); (a), (d), (e) and (f); (b), (c), (d) and (e); (b), (c), (d) and (f); (b), (c), (e) and (f); (b), (d), (e) and (f); (c), (d), (e) and

[0219] (f); (a), (b), (c), (d) and (e); (a), (b), (c), (d) and (f); (a), (b), (c), (e) and (f); (a), (b), (d), (e) and (f); (a), (c), (d), (e) and (f); (b), (c), (d), (e) and (f); or (a), (b), (c), (d), (e) and (f). In preferred embodiments, K is substituted with R.

[0220] In some embodiments, the antioxidant protein comprises selenocysteine (U). In some embodiments, the GPxl, GPx2, GPx3, GPx4, GPx6 or variant thereof comprises selenocysteine (U). In some embodiments, the GPxl, GPx4, or variant thereof comprises selenocysteine (U). In some embodiments, the GPxl or variant thereof comprises selenocysteine (U) at the position corresponding to position 49 in SEQ ID NO: 2. In some embodiments, the GPx4 or variant thereof comprises selenocysteine (U) the position corresponding to position 73 in SEQ ID NO: 8. This can be achieved by expressing the antioxidant protein from a polynucleotide which comprises a 3' untranslated region (3'UTR) containing a Sec insertion sequence (SECIS). An example of a suitable 3'UTR is the final 207 nucleotides of SEQ ID NO: 15. Any SECIS may be used in the invention. The SECIS preferably comprises or consists of the sequence shown in SEQ ID NO: 47.

[0221] In other embodiments, the GPxl variant protein comprises cysteine (C) at the position corresponding to position 49 in SEQ ID NO: 2. In some embodiments, the GPxl variant protein comprises a U49C mutation. In other embodiments, the GPx4 variant protein comprises cysteine (C) the position corresponding to position 73 in SEQ ID NO: 8. In some embodiments, the GPx4 variant protein comprises a U73C mutation. This can be achieved by expressing the antioxidant protein from a polynucleotide without a 3' untranslated region (3'UTR) containing a Sec insertion sequence (SECIS).

[0222] In a preferred embodiment, the GPxl variant protein comprises a mutation at one or more predicted ubiquitination sites, such as K88, K148 and K166, and selenocysteine (U), preferably at the position corresponding to position 49 in SEQ ID NO: 2. Any of the embodiments discussed above equally apply this embodiment.

[0223] In a preferred embodiment, the GPx4 variant protein comprises (1) a mutation at one or more predicted ubiquitination sites, such as K80R, K97R, K121R, K135R, K140R, K164R, (2) one or more inactivating mutations in its signal peptide, such as deletion of its signal peptide and (3) and selenocysteine (U), preferably at the position corresponding to position 49 in SEQ ID NO: 2.

[0224] In preferred embodiments, the GPxl or variant thereof comprises or consists of the sequence shown in SEQ ID NO: 2, 4 or 6 or a variant thereof. In preferred embodiments, the GPxl or variant thereof comprises or consists of the sequence shown in SEQ ID NO: 2, 4, 6 or 55 or a variant thereof.

[0225] In preferred embodiments, the GPx4 or variant thereof comprises or consists of the sequence shown in SEQ ID NO: 12, 14 or 16 or a variant thereof. In a most preferred embodiment, the GPx4 or variant thereof comprises or consists of the sequence shown in SEQ ID NO: 16 or a variant thereof.

[0226] In preferred embodiments, the Prxl or variant thereof comprises or consists of the sequence shown in SEQ ID NO: 18 or 20 or a variant thereof.

[0227] In preferred embodiments, the Prx2 or variant thereof comprises or consists of the sequence shown in SEQ ID NO: 22 or 24 or a variant thereof.

[0228] In preferred embodiments, the Prx6 or variant thereof comprises or consists of the sequence shown in SEQ ID NO: 26 or 28 or a variant thereof.

[0229] In some embodiments, the IDP is CAHS1, CAHS3, PvLEA, AavLEA, or a variant thereof. In some embodiments, the IDP comprises or consists of the sequence shown in SEQ ID NO: 29, 30, 31, 32, or 33 or a variant thereof. In some embodiments, the IDP is CAHS1, CAHS2, CAHS3, PvLEA, AavLEA, or a variant thereof. In some embodiments, the IDP comprises or consists of the sequence shown in SEQ ID NO: 29, 30, 31, 32, 33, 49, 51 or 53 or a variant thereof. In some embodiment, the erythroid cell is human. Human cells do not express these IDPs. In some embodiment, the erythroid cell is a human reticulocyte.

[0230] In some embodiments, the erythroid cell is further genetically engineered to express a trehalose transporter or a variant thereof on its surface. Any of the embodiments discussed above equally apply here.

[0231] In some embodiments, the erythroid cell of the invention further comprises or expresses a therapeutic protein or polypeptide. Any of the embodiments discussed above equally apply here.

[0232] The invention also relates to a cell genetically engineered to overexpress an antioxidant protein and / or an intrinsically disordered protein (IDP) in its cytosol. The genetically engineered cell may be any cell. In some embodiments, the cell is mammalian. The cell may be a human, dog, cat, primate, horse, murine, rat, rodent, bovine, murine, porcine, or ovine cell. In preferred embodiments, the cell is human.

[0233] The cell may be derived from the ectoderm, endoderm, or mesoderm. The cell may be an endothelium cell. The cell may be an epithelium cell. The cell may be derived from immune system, heart, brain, vasculature, skin, intestine, lung, thyroid, reproductive organ, bladder, kidney, pancreas, oral mucosal, eye or liver. The cell may be a stem cell, such as an embryonic stem cell, induced pluripotent stem cell or mesenchymal stem cell, bone cell, such as an osteoclast, osteoblast or osteocyte, tendon cell, such as a tenoblast or tenocyte, chondrocyte, synovial cell, vascular cell, connective tissue cell, such as a fibroblast, blood cell, such as a red blood cell, immune cell, platelet, neutrophil or basophil, muscle cells, such as a skeletal muscle cell, cardiac muscle cell or smooth muscle cell, reproductive cell, such as a sperm, oocyte, duct cell or epididymal cell, secretory cell, adipocyte, liver lipocyte, epithelial cell, odontoblast, cementoblast, hormone-secreting cell, barrier cell, exocrine secretory epithelial cell, nerve cell, astrocyte, oligodendrocyte, or neuron.

[0234] The genetically engineered cell is preferably a blood cell. The blood cell may be an erythroid progenitor cell, a reticulocyte, a mature RBC, a platelet, or an immune cell. Immune cells are also known as white blood cells. The immune cell may be a neutrophil granulocyte and precursor, such as a myeloblast, promyelocyte, myelocyte, or metamyelocyte, eosinophil granulocyte and precursor, basophil granulocyte and precursor, mast cell, leukocyte, lymphocyte, helper T cell, regulatory T cell, cytotoxic T cell, natural killer T cell, natural killer cell, innate lymphoid cell (ILC), B cell, macrophage, dendritic cell, plasma cell, neutrophils, or monocytes.

[0235] In some embodiments, the cell is derived from a stem cell or an immortalised cell line. Suitable stem cells and immortalised cell lines are known in the art and are discussed above. The cell may be derived from a human induced pluripotent stem (iPS) cell. Such cells can be identified on the basis of the presence of one or more transcription factors which were used to induce pluripotency. Such transcription factors include, but are not limited to, Oct-3 / 4, Soxl, Sox2, Sox3, Soxl5, Soxl8, Klf2, Klf4, c-Myc, n-Myc, l-Myc, Nanog, LIN28 and Glisl. Such cells may also include evidence of the machinery used to deliver such transcription factors.

[0236] The cell may be autologous. In other words, the cell may be derived from the subject into which the cell will be administered. The cell is preferably allogeneic. In other words, the cell is preferably derived from a subject that is immunologically compatible with the subject into which the cell will be administered. The administration of autologous or allogeneic cells to subjects is well documented. These embodiments apply to the erythroid cells of the invention.

[0237] The erythroid cell may be engineered to be antibiotic resistant.

[0238] Variants

[0239] The invention relates to various proteins or variants thereof, including a trehalose transporter, a GLUT8 protein, GPxl, GPx2, GPx3, GPx4, GPx5, GPx6, GPx7, GPx8, Prxl, Prx2, Prx3, Prx4, Prx5, Prx6, CAHS1, CAHS3, PvLEA, AavLEA, or a variant thereof. In some embodiments, the invention relates to CAHS2 or a variant thereof. In some embodiments, a variant protein comprises or consists of a sequence having at least about 80% homology or identity to the reference sequence. For instance, a variant of GPxl preferably comprises or consists of a sequence having at least about 80% homology or identity to the sequence shown in SEQ ID NO: 2. In preferred embodiments, the variant protein comprises or consists of a sequence having at least about 82%, at least about 85%, at least about 87%, at least about 90%, at least about 92%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity to the reference sequence. Homology and / or identity isa / are typically measured over the entire length of the reference sequence, typically the parent protein.

[0240] Standard methods in the art may be used to determine homology or identity. For example, the UWGCG Package provides the BESTFIT program which can be used to calculate homology or identity, for example used on its default settings (Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology and identity or line up sequences (such as identifying equivalent residues or corresponding sequences (typically on their default settings)), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S.F et al (1990) J Mol Biol 215:403-10. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0241] Alignment of sequences using these types of software also allows the skilled person to identify corresponding positions between sequences, such as between a variant protein and its parent sequence.

[0242] Amino acid substitutions, such as conservative substitutions may be present in the variant protein, for example up to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 or more substitutions. Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties, or similar side-chain volume. The amino acids introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge to the amino acids they replace. Alternatively, the conservative substitution may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well- known in the art.

[0243] The variant protein may be modified to introduce one or more cysteines, one or more hydrophobic amino acids, one or more charged amino acids, one or more non-native amino acids, one or more polar amino acids, or one or more photoreactive amino acids. Any number and combination of such introductions may be made. The introduction is preferably by substitution or addition. One or more amino acid residues may additionally be deleted from the polypeptides described above. Up to 1, 2, 3, 4, 5, 10, 20 or 30 or more residues may be deleted.

[0244] The variant protein may comprise a fragment of the parent protein. Fragments may be at least about 50, at least about 100, at least about 150, or at least about 200 amino acids in length.

[0245] One or more amino acids may be alternatively or additionally added to the proteins described above. An extension may be provided at the amino terminal or carboxy terminal. The extension may be quite short, for example from 1 to 10 amino acids in length. Alternatively, the extension may be longer, for example up to 50 or 100 amino acids.

[0246] In preferred embodiments, the variant protein is a functional variant protein. In such embodiments, the variant protein retains the function of its parent protein. The skilled person can test functional variants using routine methods. For instance, the ability of GPxl or GPx4 variant protein to catalyse reaction converting reduced monomeric glutathione to glutathione disulfide can be tested using a routine GPx activity assay.

[0247] Any of the proteins of the invention may further comprise a methionine (M) at its N- terminus. This typically reflects the start codon (ATG) in the polynucleotide used to express the protein.

[0248] Any of the proteins of the invention may be modified to assist their identification or purification, for example by the addition of histidine residues (a his tag), aspartic acid residues (an asp tag), a streptavidin tag, a flag tag, a SUMO tag, a GST tag, a MBP tag or a myc tag. The tag is preferably a myc tag. The myc tag preferably comprises or consists of the sequence shown in positions 2-5 of SEQ ID NO: 4. The myc tag is preferably positions at the N-terminus of the protein variant immediately after the N-terminal methionine. An alternative to introducing a genetic tag is to chemically react a tag onto a native or engineered position on the protein.

[0249] Preferred variants of SEQ ID NOs: 4, 6, 10, 12, 14, 16, 20, 24, 18-33 and 55 lack the myc tag. Myc tags are defined above.

[0250] Populations

[0251] The invention also provides a population of cells of the invention. In particular, the invention provides a population of reticulocyte cells or mature red blood cells comprising intracellular trehalose. The invention also provides a population of erythroid cells genetically engineered to express a trehalose transporter or a variant thereof on their surface. The invention also provides a population of erythroid cells genetically engineered to overexpress an antioxidant protein and / or an intrinsically disordered protein (IDP) in their cytosol. The invention also provides a population of erythroid cells genetically engineered to express an increased level of an antioxidant protein and / or an intrinsically disordered protein (IDP) in their cytosol. The invention also provides a population of erythroid cells genetically engineered to express a variant protein which comprises one or more mutations which increase the level of or facilitate overexpression of the variant protein in the cytosol of the cells. The cells may be any of the cells discussed above. The population may be present in any of the media discussed above or below. The population may be present in a lyophilised composition of the invention.

[0252] The population may comprise any number of cells, such as at least about 5 x 105cells of the invention. In some embodiments, the population comprises at least about 1 x 106, at least about 2 x 106, at least about 2.5 x 106, at least about 5 x 106, at least about 1 x 107, at least about 2 x 107, at least about 5 x 107, at least about 1 x 108or at least about 2 x 108cells of the invention. In some instances, the population may comprise at least about 1.0 x 107, at least about 1.0 x 108, at least about 1.0 x 109, at least about 1.0 x 1010, at least about 1.0 x 1011or at least about 1.0 x 1012cells of the invention or even more.

[0253] In any of the populations of the invention, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% of the cells comprise intracellular trehalose and / or are genetically engineered.

[0254] Any cell or population of the invention may be isolated, substantially isolated, purified, or substantially purified. A cell or population is isolated or purified if it is completely free of any other components, such as culture medium. A cell or population is substantially isolated or substantially purified if it is mixed with carriers or diluents, such as a culture medium or a pharmaceutical composition, which will not interfere with its intended use. Other carriers and diluents are discussed in more detail below.

[0255] Variant proteins of the invention

[0256] The invention also provides a variant protein which comprises one or more mutations which increase the level of or facilitate overexpression of the variant protein in the cytosol of an erythroid cell. The one or more mutations may increase the level of the variant protein in the cytosol of the erythroid cell. The increased level may be an increased amount and / or an increased activity. The increased level is typically an increased detectable level. The increased level or overexpression is typically compared with the level or expression of the unmodified or non-mutated protein. The unmodified or non-mutated protein is typically one or more of ubiquitinated in the erythroid cell, secreted from the erythroid cell or targeted to intracellular organelles, such as mitochondria. This limits the expression of the protein in the cytosol. The variant protein includes one or more mutations to inhibit or reduce one or more of these processes and this results in an increased level or overexpression of the variant protein in the cytosol of an erythroid cell. The variant protein may comprise (a) one or mutations at one or more predicted ubiquitination sites and / or of one or more surface lysine residues, (b) one or more inactivating mutations in its signal peptide, (c) one or more inactivating mutations in its mitochondrial targeting peptide or (d) any combination thereof. The combination in (d) may be (a) and (b), (a) and (c), (b) and (c) or (a), (b) and (c). The variant protein may comprise (a), (b), (c), (a) and (b), (a) and (c), (b) and (c) or (a), (b) and (c). All of the discussion above in connection with the erythroid cells of the invention genetically engineered to express the variant protein equally apply here. The variant protein is preferably a variant enzyme protein. The enzyme may be any of those listed above. The variant enzyme protein preferably comprises one or more inactivating mutations in its mitochondrial targeting peptide. The variant protein may be a variant of human AAT2. The variant protein may be a variant of SEQ ID NO: 57. The variant protein may be a variant of SEQ ID NO: 57 lacking one or more of residues 2-28. The variant protein may lack any number of residues 2-28, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 residues. The variant protein preferably lacks all of residues 2-28 in SEQ ID NO: 57. The variant protein preferably comprises or consists of the sequence shown in SEQ ID NO: 59 or a variant thereof.

[0257] The invention also provides a variant antioxidant protein which comprises a mutation at one or more predicted ubiquitination sites. In particular, the invention provides a Gpxl or Gpx4 variant protein which comprises a mutation at one or more predicted ubiquitination sites. Any of the embodiments discussed above with reference to genetically modified cells of the invention equally apply to the variant proteins of the invention.

[0258] In a preferred embodiment, the GPxl variant protein comprises a mutation at one or more predicted ubiquitination sites, such as K88, K148 and K166. The GPxl variant protein may comprise a U49C mutation. In a preferred embodiment, the GPxl variant protein further comprises selenocysteine (U), preferably at the position corresponding to position 49 in SEQ ID NO: 2. Any of the embodiments discussed above equally apply this embodiment.

[0259] The invention also provides a variant antioxidant protein which comprises one or more inactivating mutations in its signal peptide. In particular, the invention provides a GPx4, GPx7, GPx8, Prx3 or Prx5 variant protein which comprises one or more inactivating mutations in its signal peptide. Any of the embodiments discussed above with reference to genetically modified cells of the invention equally apply to the variant proteins of the invention. The invention also provides a variant antioxidant protein which comprises (1) a mutation at one or more predicted ubiquitination sites and (2) one or more inactivating mutations in its signal peptide, such as deletion of its signal peptide.

[0260] In a preferred embodiment, the GPx4 variant protein comprises (1) a mutation at one or more predicted ubiquitination sites, such as K80R, K97R, K121R, K135R, K140R, K164R, and / or (2) one or more inactivating mutations in its signal peptide, such as deletion of its signal peptide. The GPx4 variant protein may comprise a U73C mutation. In a preferred embodiment, the GPx4 variant protein further comprises (3) and selenocysteine (U), preferably at the position corresponding to position 49 in SEQ ID NO: 2.

[0261] In preferred embodiments, the GPx4 variant proteins comprises or consists of the sequence shown in SEQ ID NO: 14 or 16 or a variant thereof. In a most preferred embodiment, the GPx4 or variant thereof comprises or consists of the sequence shown in SEQ ID NO: 16 or a variant thereof.

[0262] In any of the embodiments discussed above, the variant protein may be a variant of a human protein. The variant protein may be a variant of non-human protein humanised to express in human cells.

[0263] Polynucleotides

[0264] The invention also provides a polynucleotide which encodes a variant protein of the invention. The variant protein may be any of those described above. In a preferred embodiment, the polynucleotide comprises a 3' untranslated region (3'UTR) containing a Sec insertion sequence (SECIS). An example of a suitable 3'UTR is the final 207 nucleotides of SEQ ID NO: 15. Any SECIS may be used in the invention. The SECIS preferably comprises or consists of the sequence shown in SEQ ID NO: 47.

[0265] In a preferred embodiment, the polynucleotide comprises or consists of the sequence shown in SEQ ID NO: 15 or variant thereof. Variants are defined above with reference to the polypeptides of the invention.

[0266] It will be appreciated that the polynucleotide sequence is recombinant. Preferably, the polynucleotide sequence is isolated. In some embodiments, the polynucleotide is nonnatural.

[0267] In some embodiments, the polynucleotide may further comprise a sequence encoding a reporter gene. Suitable reporter genes include, but are not necessarily limited to, luciferase, myc, HNIS, hNET and HSVtK.

[0268] A polynucleotide, such as a nucleic acid, is a polymer comprising two or more nucleotides. The nucleotides can be naturally occurring or artificial. A nucleotide typically contains a nucleobase, a sugar and at least one linking group, such as a phosphate, 2'0-methyl, 2' methoxy-ethyl, phosphoramidate, methylphosphonate or phosphorothioate group. The nucleobase is typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines and more specifically adenine (A), guanine (G), thymine (T), uracil (U) and cytosine (C). The sugar is typically a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The sugar and the nucleobase together form a nucleoside. Preferred nucleosides include, but are not limited to, adenosine, guanosine, 5-methyluridine, uridine, cytidine, deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine. The nucleosides may be adenosine, guanosine, uridine, and cytidine.

[0269] The nucleotides are typically ribonucleotides or deoxyribonucleotides. The nucleotides may be deoxyribonucleotides. The nucleotides typically contain a monophosphate, diphosphate, or triphosphate. Phosphates may be attached on the 5' or 3' side of a nucleotide.

[0270] Nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), 5-methylcytidine monophosphate, 5-methylcytidine diphosphate, 5-methylcytidine triphosphate, 5- hydroxymethylcytidine monophosphate, 5-hydroxymethylcytidine diphosphate, 5- hydroxymethylcytidine triphosphate, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP) and deoxycytidine triphosphate (dCTP), 5-methyl-2'-deoxycytidine monophosphate, 5-methyl-2'-deoxycytidine diphosphate, 5-methyl-2'-deoxycytidine triphosphate, 5-hydroxymethyl-2'-deoxycytidine monophosphate, 5-hydroxymethyl-2'-deoxycytidine diphosphate and 5-hydroxymethyl-2'- deoxycytidine triphosphate. The nucleotides may be selected from AMP, UMP, GMP, CMP, dAMP, dTMP, dGMP or dCMP. In some embodiments, the nucleotides are selected from dAMP, dTMP, dGMP or dCMP.

[0271] The nucleotides may contain additional modifications. In particular, suitable modified nucleotides include, but are not limited to, 2'amino pyrimidines (such as 2'-amino cytidine and 2'-amino uridine), 2'-hyrdroxyl purines (such as , 2'-fluoro pyrimidines (such as 2'- fluorocytidine and 2'fluoro uridine), hydroxyl pyrimidines (such as 5'-a-P-borano uridine), 2'-0-methyl nucleotides (such as 2'-O-methyl adenosine, 2'-O-methyl guanosine, 2'-O- methyl cytidine and 2'-O-methyl uridine), 4'-thio pyrimidines (such as 4'-thio uridine and 4'- thio cytidine) and nucleotides have modifications of the nucleobase (such as 5-pentynyl-2'- deoxy uridine, 5-(3-aminopropyl)-uridine and l,6-diaminohexyl-N-5-carbamoylmethyl uridine).

[0272] One or more nucleotides in the polynucleotide(s) may be modified, for instance with a label or a tag. The label may be any suitable label which allows the nucleotides to be detected. Suitable labels include, but are not limited to, fluorescent molecules, radioisotopes, e.g.,125I,35S, enzymes, antibodies, antigens, other polynucleotides, and ligands such as biotin.

[0273] The nucleotides in the polynucleotide(s) may be attached to each other in any manner. The nucleotides may be linked by phosphate, 2'0-methyl, 2' methoxy-ethyl, phosphoramidate, methylphosphonate or phosphorothioate linkages. The nucleotides are typically attached by their sugar and phosphate groups. The nucleotides may be connected via their nucleobases as in pyrimidine dimers.

[0274] The polynucleotide(s) may comprise a deoxyribonucleic acid (DNA) or a ribonucleic acid (RIMA). The polynucleotide(s) may be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), morpholino nucleic acid or other synthetic polymers with nucleotide side chains.

[0275] Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with a different base, mixed- base and / or deoxyinosine residues (Batzer et al., 1991, Nucleic Acid Res. 19:5081; Ohtsuka et al., 1985, J. Biol. Chem. 260:2605-2608; and Rossolini et al., 1994, Mol. Cell. Probes 8:91-98).

[0276] Substitutions may be used for the practices of codon optimisation and codon wobble, both of which are known to those skilled in the art. Thus, it will be appreciated that codon- optimised and codon-wobbled isolated polynucleotide(s) are also envisaged. In an embodiment, the polynucleotide(s) is / are codon-optimised for human expression.

[0277] The polynucleotide(s) can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of an existing sequence. Direct chemical synthesis of polynucleotides can be accomplished by methods known in the art, such as the phosphotriester method of Narang et al., 1979, Meth. Enzymol. 68:90; the phosphodiester method of Brown et al., 1979, Meth. Enzymol. 68: 109; the diethylphosphoramidite method of Beaucage et al., 1981, Tetra. Lett., 22: 1859; and the solid support method of U.S. Pat. No. 4,458,066. Introducing mutations to a polynucleotide sequence by PCR can be performed as described in, e.g., PCR Technology: Principles and Applications for DNA Amplification, H. A. Erlich (Ed.), Freeman Press, NY, N.Y., 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, Calif, 1990; Mattila et al., 1991, Nucleic Acids Res. 19:967; and Eckert et al., 1991, PCR Methods and Applications 1: 17.

[0278] Vectors

[0279] The invention also provides a vector comprising a polynucleotide sequence of the invention. The polynucleotide may be any of those described above. The invention also provides a cell comprising or expressing a polynucleotide sequence of the invention or a vector of the invention. The cell may be any of those described above.

[0280] In some embodiments, the vector is an expression vector. Various expression vectors can be employed to express the polynucleotide of the invention.

[0281] Both viral-based and non-viral expression vectors can be used in the invention. Non-viral vectors and systems include plasmids, episomal vectors, typically with an expression cassette for expressing a protein or RNA, and human artificial chromosomes (see, e.g., Harrington et al., 1997, Nat Genet. 15:345). For example, non-viral vectors useful for expression in mammalian (e.g., human) cells include pThioHis A, B and C, pcDNA3.1 / His, pEBVHis A, B and C, (Invitrogen, San Diego, Calif.), MPS V vectors, and numerous other vectors known in the art for expressing other proteins and / or nucleotide sequences. Useful viral vectors include vectors based on retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Barr virus, vaccinia virus vectors and Semliki Forest virus (SFV). See, Brent et al., supra; Smith, 1995, Annu. Rev. Microbiol. 49:807; and Rosenfeld et al., 1992, Cell 68: 143.

[0282] Preferably, the vector is a retroviral, lentiviral, adenoviral, or adeno-associated viral vector. Examples of such vectors include the SFG retroviral expression vector (see Riviere et al., 1995, Proc. Natl. Acad. Sci. (USA) 92:6733-6737).

[0283] In some embodiments, the vector is a retroviral or lentiviral vector. In some embodiments, the vector is a retroviral vector. Optionally, the vector is an SFG retroviral vector. In some embodiments the vector is a lentiviral vector. Lentiviral vectors include self-inactivating lentiviral vectors (so-called SIN vectors).

[0284] Expression vectors for mammalian host cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer (see, e.g., Queen, et al., 1986, Immunol. Rev. 89:49-68), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters may be constitutive, cell typespecific, stage-specific, and / or modulatable or regulatable. Useful promoters include, but are not limited to, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIII promoter, the constitutive MPS V promoter, the tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), the constitutive CMV promoter, the SSFV promoter, the EFl alpha promoter, the phosphoglycerate kinase (PGK) promoter and promoter-enhancer combinations known in the art. The promoter may be the MSCV promoter.

[0285] Genetic engineering

[0286] The invention also provides a method of modifying a cell, comprising genetically engineering the cell to express a variant protein of the invention, a polynucleotide of the invention or a vector of the invention. The cell, variant protein, polynucleotide, or vector may be any of those discussed above.

[0287] The invention also provides a method of improving the ability of a cell to survive lyophilisation, the method comprising genetically engineering the cell to overexpress an antioxidant protein and / or an intrinsically disordered protein (IDP) in its cytosol. The invention also provides a method of improving the ability of a cell to survive lyophilisation, the method comprising genetically engineering the cell to express an increased level of an antioxidant protein and / or an intrinsically disordered protein (IDP) in its cytosol. The antioxidant protein may be any of those described above, including the various variant proteins. The IDP may be any of those described above. In a preferred embodiment, the method comprises genetically engineering the cell to express a variant protein of the invention, a polynucleotide of the invention or a vector of the invention. The cell, variant protein, polynucleotide, or vector may be any of those discussed above. The cell is preferably an erythroid cell.

[0288] The genetic engineering of cells can be carried out according to standard cloning and expression techniques, which are known in the art (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0289] The method may comprise introducing the vector and / or polynucleotide of the invention into the cell. In some embodiments, the method comprises transfecting the cell with the vector or polynucleotide of the invention to express the variant protein of the invention.

[0290] The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection, and the like.

[0291] Alternatively, the method may comprise transducing the cell with the vector and / or polynucleotide of the invention to express the variant protein of the invention. For example, a viral vector, as disclosed above, may be used for delivery of the polynucleotide.

[0292] In embodiments where the cell is an immune cell, the cell may be activated prior to, simultaneous to or after genetically engineering. Activation may be prior to genetically engineering the cell. In some embodiments, the method comprises a simultaneous activation and genetic engineering step. Activation methods are known in the art.

[0293] In some embodiments, the cells are genetically engineered to express a marker or factor which ensures that only successfully genetically engineered cells remain viable. Thus, any non-genetically engineered cells are eliminated, thereby improving the purity of the resulting cell population.

[0294] Pharmaceutical compositions

[0295] Also provided is a pharmaceutical composition comprising a reconstituted composition of the invention, a cell of the invention, a variant protein of the invention, a polynucleotide of the invention or a vector of the invention and a pharmaceutically or physiologically acceptable diluent and / or carrier. The composition, cell, variant protein, polynucleotide, or vector may be any of those discussed above. The cell may be a reticulocyte cell of the invention, an erythroid cell of the invention or any cell of the invention. The cell or cells in the population are preferably erythroid cell(s) or reticulocyte cell(s). The cell or cells in the population are preferably human.

[0296] Also provided is a pharmaceutical composition comprising an erythroid cell of the invention, a population of the invention, a variant protein of the invention, a polynucleotide of the invention or a vector of the invention and a pharmaceutically or physiologically acceptable diluent and / or carrier. The erythroid cell, population, variant protein, polynucleotide, or vector may be any of those discussed above. The erythroid cell or cells in the population are preferably reticulocyte cell(s). The erythroid cell or cells in the population are preferably human.

[0297] The carrier and / or diluent is generally selected to be suitable for the intended mode of administration and can include agents for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, colour, isotonicity, odour, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. Typically, these carriers and / or diluents include aqueous or alcoholic / aqueous solutions, emulsions, or suspensions, including saline and / or buffered media. Suitable further agents for inclusion in the pharmaceutical composition include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulphite, or sodium hydrogensulphite), buffers (such as borate, bicarbonate, Tris-HCI, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as free serum albumin, gelatin, or immunoglobulins), colouring, flavouring and diluting agents, emulsifying agents, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; sorbitan esters; polysorbates such as Polysorbate 20 or Polysorbate 80; Triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancing agents (such as sucrose or sorbitol), tonicity enhancing agents (such as alkali metal halides, such as sodium or potassium chloride, or mannitol sorbitol), delivery vehicles, excipients and / or pharmaceutical adjuvants.

[0298] The carrier and / or diluent may be a parenteral, optionally intravenous vehicle. Suitable parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's. Suitable physiologically acceptable thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatin and alginates may be included. Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer's dextrose. In some cases, one might include agents to adjust tonicity of the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in a pharmaceutical composition. For example, in many cases it is desirable that the composition is substantially isotonic. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents, and inert gases, may also be present. The precise formulation will depend on the route of administration. Additional relevant principle, methods and components for pharmaceutical formulations are well known (see, e.g., Allen, Loyd V. Ed, (2012) Remington's Pharmaceutical Sciences, 22ndEdition).

[0299] A pharmaceutical composition of the present invention can be administered by one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled person, the route and / or mode of administration will vary depending upon the desired results. Routes of administration for pharmaceutical compositions of the invention include intravenous, intramuscular, intradermal, intraperitoneal, intrapleural, subcutaneous, intratumoural, spinal, intra-bone marrow or other parenteral routes of administration, for example by injection or infusion. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intratumoural, intrapleural and intra- sternal injection and infusion. In some embodiments, the pharmaceutical composition is administered intravenously. In other embodiments, administration is intrapleural or intraperitoneal. When parenteral administration is contemplated, the pharmaceutical compositions are usually in the form of a sterile, pyrogen-free, parenterally acceptable composition. A particularly suitable vehicle for parenteral injection is a sterile, isotonic solution, properly preserved. The pharmaceutical composition can be in the form of a lyophilizate, such as a lyophilized cake.

[0300] Alternatively, the pharmaceutical composition of the invention can be administered by a nonparenteral route, such as a topical, epidermal, or mucosal route of administration, for example, intranasally, orally, vaginally, rectally, sublingually, or topically.

[0301] In some embodiments, the pharmaceutical composition is for subcutaneous administration. Typically, the pharmaceutical compositions for subcutaneous administration contain suitable stabilizers (e.g., amino acids, such as methionine, and or saccharides such as sucrose), buffering agents and tonicifying agents. Alternatively, the pharmaceutical composition may be for intra-bone marrow administration.

[0302] Therapy

[0303] The invention also provides a lyophilised composition of the invention, a reconstituted composition of the invention, a reticulocyte cell of the invention, a mature RBC of the invention, an erythroid cell of the invention, a cell of the invention, a population of the invention, a variant protein of the invention, a polynucleotide of the invention, a vector of the invention or a pharmaceutical composition of the invention for use in therapy.

[0304] The invention also provides an erythroid cell of the invention, a population of the invention, a variant protein of the invention, a polynucleotide of the invention, a vector of the invention or a pharmaceutical composition of the invention for use in therapy.

[0305] The invention also provides use of a lyophilised composition of the invention, a reconstituted composition of the invention, a reticulocyte cell of the invention, a mature RBC of the invention, an erythroid cell of the invention, a cell of the invention, a population of the invention, a variant protein of the invention, a polynucleotide of the invention, a vector of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for use in therapy.

[0306] The invention also provides use of an erythroid cell of the invention, a population of the invention, a variant protein of the invention, a polynucleotide of the invention, a vector of the invention or a pharmaceutical composition of the invention in the manufacture of a medicament for use in therapy.

[0307] The invention also provides a method of treating a subject in need thereof, comprising administering a lyophilised composition of the invention, a reconstituted composition of the invention, a reticulocyte cell of the invention, a mature RBC of the invention, an erythroid cell of the invention, a cell of the invention, a population of the invention, a variant protein of the invention, a polynucleotide of the invention, a vector of the invention or a pharmaceutical composition of the invention to the subject.

[0308] The invention also provides a method of treating a subject in need thereof, comprising administering an erythroid cell of the invention, a population of the invention, a variant protein of the invention, a polynucleotide of the invention, a vector of the invention or a pharmaceutical composition of the invention to the subject.

[0309] In some embodiments, the lyophilised composition is used in therapy or is administered after reconstitution. The lyophilised composition may be reconstituted in any of the ways discussed above. In some embodiments, the reconstituted composition is administered straight away. For instance, the reconstituted composition can be administered within about 5 minutes, within about 10 minutes, within about 15 minutes, or within about 20 minutes, of reconstitution. In other embodiments, the reconstituted composition can be stored for a period of time before use. In such embodiments, the reconstituted composition may be stored for up to about half an hour, up to about one hour, up to about two hours, up to about three hours, up to about four hours, up to about five hours, up to about six hours, up to about seven hours, up to about eight hours, up to about nine hours, up to about ten hours, up to about 15 hours, up to about 20 hours, up to about 24 hours, or up to about 48 hours, after reconstitution before use. Where the reconstituted composition is stored for a period of time before use, reconstitution can be done using a suitable storage buffer.

[0310] The therapy or treatment is preferably blood transfusion, enzyme replacement therapy, organ reconditioning, or detoxification. In some embodiments, the therapy is blood transfusion. In this embodiment, the reticulocyte cells can be natural or wild-type cells.

[0311] In some embodiments, the therapy or treatment is enzyme replacement therapy, organ reconditioning, or detoxification. In this embodiment, the reticulocyte cells can further comprise or express a therapeutic protein or polypeptide. In some embodiments, the therapy or treatment is the treatment or prevention of a RBC disease or disorder, preferably a RBC disease or disorder associated with increased reactive oxygen species (ROS) activity. Overexpression of antioxidant proteins in accordance with the invention can protect the cells of the invention from ROS activity. In some embodiments, the RBC disease or disorder is beta thalassemia, sickle cell disease, cerebella ataxia, or motor neurone disease.

[0312] In embodiments where the cell of the invention can further comprises or expresses a therapeutic protein or polypeptide, the therapy or treatment can comprises treating or preventing a disease or disorder using the therapeutic protein or polypeptide. The disease or disorder may be, for instance, an inflammatory disease or disorder, an infection, or cancer.

[0313] In the context of the present invention, an inflammatory disease is a disease or disorder which comprises the damage or destruction of healthy viable cells by inflammation. Examples of inflammatory diseases or disorders include, but are not necessarily limited to, autoimmune disease, allergy, asthma, coeliac disease, nephritis, hepatitis, reperfusion injury, graft versus host disease (GvHD), transplant rejection and infection.

[0314] Autoimmune disease may comprise rheumatoid arthritis, psoriasis, system lupus erythematosus (lupus), inflammatory bowel disease, multiple sclerosis, diabetes, Guillain- Barre syndrome, chronic inflammatory demyelinating polyneuropathy, Graves' disease, Hashimoto's thyroiditis, Myasthenia gravis, Aplastic Anaemia (AA), Vasculitis or combinations thereof.

[0315] The infection may be caused by any pathogenic agent. The pathogenic agent may be a bacterium, an archaeon, a fungus, or a virus.

[0316] Preferably, the cancer is anal cancer, bile duct cancer (cholangiocarcinoma), bladder cancer, blood cancer, bone cancer, bowel cancer, brain tumours, breast cancer, colorectal cancer, cervical cancer, endocrine tumours, eye cancer (such as ocular melanoma), fallopian tube cancer, gall bladder cancer, head and / or neck cancer, Kaposi's sarcoma, kidney cancer, larynx cancer, leukaemia, liver cancer, lung cancer, lymph node cancer, lymphoma, melanoma, mesothelioma, myeloma, neuroendocrine tumours, ovarian cancer, oesophageal cancer, pancreatic cancer, penis cancer, primary peritoneal cancer, prostate cancer, Pseudomyxoma peritonei, skin cancer, small bowel cancer, soft tissue sarcoma, spinal cord tumours, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, trachea cancer, unknown primary cancer, vagina cancer, vulva cancer or endometrial cancer. The leukaemia is preferably acute lymphoblastic leukaemia, acute myeloid leukaemia, chronic lymphocytic leukaemia, or chronic myeloid leukaemia. The lymphoma is preferably Hodgkin lymphoma or non-Hodgkin lymphoma. The cancer is preferably primary cancer or secondary cancer. The administration route may be any of those discussed above. In preferred embodiments, administration is conducted by intravenous injection.

[0317] The therapy or method typically comprises administering a therapeutically effective amount or a prophylactically effective amount of the composition, cell, population, variant protein, polynucleotide, vector, or pharmaceutical composition. A therapeutically effective amount is an amount which ameliorates one or more symptoms, such as all the symptoms, of the disease or disorder and / or abolishes one or more symptoms, such as all the symptoms, of the disease or disorder. The therapeutically effective amount preferably cures the disease or disorder. A prophylactically effective amount is an amount which prevents the onset of the disease or disorder and / or prevents the onset of one or more symptoms, such as all the symptoms, of the disease or disorder. The prophylactically effective amount preferably prevents the subject from developing the disease or disorder. Suitable amounts are discussed in more detail below.

[0318] The composition, cell, population, variant protein, polynucleotide, vector, or pharmaceutical composition may be administered to a subject that displays symptoms of disease or disorder. The composition, cell, population, variant protein, polynucleotide, vector, or pharmaceutical composition may be administered to a subject that is asymptomatic, i.e., does not display symptoms of disease or disorder. The composition, cell, population, variant protein, polynucleotide, vector, or pharmaceutical composition may be administered when the subject's disease status is unknown, or the subject is expected not to have a disease or disorder. The composition, cell, population, variant protein, polynucleotide, vector, or pharmaceutical composition may be administered to a subject that is predisposed, such as genetically predisposed, to developing the disease or disorder.

[0319] Administration of the composition, cell, population, variant protein, polynucleotide, vector, or pharmaceutical composition to the subject may reduce symptoms by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or even about 100%, when compared to an untreated subject.

[0320] The subject may be a mammal. Optionally, the subject is a human, horse, dog, or cat. In preferred embodiments, the subject is human.

[0321] In embodiments where the subject is human, the subject may be a human adult or child. In the context of the present invention, an adult will be understood to be an at least 18-year- old human. A child will be understood to be a human less than 18 years old. In some embodiments, the adult is at least 60 years old.

[0322] In embodiments wherein the therapy or treatment uses cells, the number of cells administered to the subject should take into account the route of administration, the disease or disorder being treated, the weight of the subject and / or the age of the subject. In general, from about 1 x 106to about 1 x 1011cells are administered to the subject. In one embodiment, from about 1 x 107to about 1 x IO10cells, or from about 1 x 108to about 1 x 109cells are administered to the subject.

[0323] In some embodiments, from about 1 mg / ml to about 100 mg / ml of the composition, cell, population, variant protein, polynucleotide, vector, or pharmaceutical composition may be administered to the subject. In some embodiments, from about 1 mg / ml to about 10 mg / ml of the composition, cell, population, variant protein, polynucleotide, vector, or pharmaceutical composition are administered to the subject.

[0324] The invention may be used in combination with other means of, and substances for, treating disease or disorder. In some cases, the composition, cell, population, variant protein, polynucleotide, vector, or pharmaceutical composition may be administered simultaneously, sequentially, or separately with other substances which are intended for treating the disease or disorder or ameliorating the symptoms of the disease or disorder, or for providing pain relief. The composition, cell, population, variant protein, polynucleotide, vector, or pharmaceutical composition may be used in combination with existing treatments for disease or disorder and may, for example, be simply mixed with such treatments. Thus the invention may be used to increase the efficacy of existing treatments for disease.

[0325] In some embodiments, the cell or population of the invention can be autologous with respect to the subject. For example, an erythroid progenitor can be removed from the subject, differentiated to a cell population comprising at least about 10% reticulocyte cells, lyophilised, and ultimately reconstituted and returned to the subject. Alternatively the cell population may be obtained by harvesting reticulocyte cells from the subject and lyophilising. Preferably, however, cell or population is allogeneic with respect to the subject. In this embodiment, it would be preferred that the cell or population is matched to the subject.

[0326] Preferred features of each aspect of the invention may be as described in connection with any of the other aspects. Within the scope of this application it is expressly intended that the various aspects, embodiments, examples, and alternatives set out in the preceding paragraphs, in the claims and / or in the following description and drawings, and in particular the individual features thereof, may be taken independently or in any combination. That is, all embodiments and / or features of any embodiment can be combined in any way and / or combination, unless such features are incompatible.

[0327] SEQUENCES

[0328] The following table summarises the sequences for use in the invention.

[0329]

[0330] EXAMPLES

[0331] The sequences used in these Examples are shown in the table above.

[0332] Example 1 : Reticulocytes can be preloaded with trehalose by incubation overnight

[0333] For these experiments, donated CD34+ cells were differentiated to generate cultured reticulocyte cell populations. CD34+ haematopoietic cells were isolated from donated whole blood, proliferated, and differentiated into reticulocytes over 21 days and then leukofiltered to purify the reticulocytes.

[0334] The cultured reticulocytes were concentrated by centrifugation and then the effect of using lyoprotectant agent was tested by resuspending the cells in either PBSAG buffer alone or PBSAG supplemented with 10% trehalose (wt / vol). A proportion of samples were also stored overnight in PBSAG or PBSAG supplemented with 500mM trehalose in an incubator at 37°C at 5% CO2. The reticulocytes incubated overnight in PBS containing 500mM trehalose are referred to as "Loaded". The controls and the overnight loaded samples were washed in PBSAG the next day 3 times and then stored in PBSAG alone or supplemented with 10% trehalose. The samples were stored on ice prior to lyophilisation by freeze drying as described below.

[0335] To confirm the trehalose overnight loaded reticulocytes do accumulate trehalose inside, lx 106of each reticulocyte sample were lysed and assayed for trehalose content using a commercial Megazyme trehalose assay kit, which assesses trehalose content using a spectrophotometer measurement at 240nm. The Figure 1 confirms that trehalose accumulates inside the overnight trehalose loaded cells and not in the standard PBSAG buffer stored cells which had no trehalose present.

[0336] Example 2 : A modified trehalose transporter GLUT8 can be overexpressed in erythroid cells and can be used to transport trehalose into the cells

[0337] The GLUT8 transporter, the sole mammalian trehalose transporter identified, features an NH2-terminus [DE]XXXL[LI] targeting motif which is known to be important for targeting to the lysosomes. This N terminal motif plays a role in directing the protein towards endosomes, lysosomes, and related organelles. Research conducted in HEK293 cells has revealed that by substituting the TPQ (XXX) residues in this targeting sequence of the lysosomally located GLUT8 transporter, with GPN taken from the GLUT12 transporter (which localizes the GLUT12 transporter to the plasma membrane), the subcellular location of the GLUT8 transporter can be altered to the plasma membrane. Additionally, altering the dileucine (LL) motif to AA has been reported to reposition the GLUT8 transporter to the plasma membrane. To investigate which alteration would yield the most effective plasma membrane expression in erythroid cells, the wild-type GLUT8 (SEQ ID NOs: 34 and 35), the corresponding human GPS-mutated variant (SEQ ID NOs: 36 and 37), and the LL-mutated variant were expressed in BEL-A cells which are an erythroid model that produce reticulocytes.

[0338] Flow cytometry on the cells suggested that both the LL and the GPS mutant expressed well but this was on permeabilised cells because the c-Myc tag is located on the intracellular side of the protein. Immunofluorescent imaging suggested that only the TPQ mutant was expressed to the cell surface (data not shown).

[0339] Therefore, while the LL mutant demonstrates high expression levels in expanding BEL-A cells, it is located intracellularly, rendering it unsuitable for the specific purpose in these experiments. The experiments below employ the GLUT8-GPS modified transporter which will be referred to below as "GLUT8" and / or "GLUT8-GPS".

[0340] To confirm that erythroid cells that express GLUT8 on the surface do accumulate trehalose faster than unmodified cells, stable BEL-A cells expressing GLUT8-GPS (SEQ ID NOs: 36 and 37) either "low" or "high" (Figure 2A) were incubated with 500mM trehalose for specific times and then lysed and assayed for trehalose content using a commercial Megazyme trehalose assay kit which assesses trehalose content using a spectrophotometer measurement at 340nm. The graph in Figure 2B demonstrates that trehalose does accumulate inside the cells faster when they express GLUT8-GPS, confirming that the modified GLUT8 is active at the surface in erythroblasts and can transport more trehalose than unmodified control BELA cells.

[0341] We also explored GLUT8 expression in primary erythroid cultures using a selectable vector. The GLUT8-GPS-MYC construct was cloned into puromycin selectable plasmid CD710-B-1- Puro (System Biosciences). Using this vector guarantees alongside puromycin selection ensures that the generated reticulocytes will all express the desired construct. CD34+ hematopoietic stem cells were transduced on day 3 after isolation and puromycin (lpg / mL) was added to culture between days 5-8 as shown on Figure 4A. The cells were differentiated into reticulocytes during a 20 day, 3-step culture method as previously described by Kupzig et al. Briefly, isolated CD34+ cells were seeded at lxlO5 / mL in IMDM base medium (IMDM, 3% heat inactivated AB serum, 2 mg / mL human serum albumin, 10 pg / mL insulin, 3 U / mL heparin, 3 U / mL erythropoetin and 200 pg / mL holo-transferrin). Base medium was supplemented with 40ng / mL SCF and Ing / mL IL-3 from days 0 to 8 and 40ng / mL SCF only from day 8 to 13. Thereafter, cells were kept in culture in base medium only until day 20. To obtain a purified population of reticulocytes cultures were filtered either using a 5 pm Acrodisc filter (VWR) when handling volumes lower than lOOmL or using leukofilters.

[0342] Figure 4B confirms expression of GLUT8-GPS-MYC during differentiation as assessed by flow cytometry. Cells were fixed, permeabilised, and labelled by detecting the c-MYC tag. This data highlights retention of GLUT8 expression during differentiation in primary differentiated reticulocytes.

[0343] After isolating reticulocytes, cells were incubated overnight at 37°C with either 10% trehalose in PBSAG (w / v) or just PBSAG. To confirm and quantify the trehalose uptake, 1 xlO6cells per assay were lysed and using the same Megazyme kit mentioned previously. Absorbance was measured at 340 nm and converted to pg using a standard curve as shown in Figure 4C. Figure 4C indicates increased trehalose uptake in the reticulocytes expressing GLUT8-GPS by approximately 80%. Therefore, the modified GLUT8-GPS is targeted to the surface of cultured reticulocytes and active at the surface, transporting trehalose into the cell.

[0344] Example 3: Preparation of reticulocytes expressing a modified GLUT8

[0345] For these experiments, CD34+ cells were differentiated to generate cultured reticulocyte cell populations. CD34+ haematopoietic cells were isolated from donated whole blood, proliferated, and differentiated into reticulocytes over 21 days and then leukofiltered to purify the reticulocytes. To test the effect of GLUT8 expression, CD34+ cells were transduced with lentivirus and then proliferated, differentiated and reticulocytes prepared.

[0346] To confirm the GLUT8 trehalose loaded CD34+ derived reticulocytes accumulate more trehalose inside when loaded overnight, lxlO6of each reticulocyte sample were lysed and assayed for trehalose content using a commercial Megazyme trehalose assay kit which assesses trehalose content using a spectrophotometer measurement at 240nm. The graph in Figure 3 confirms again that trehalose accumulates inside the overnight trehalose loaded cells and not in the standard PBSAG buffer stored cells which had no trehalose. It also shows again that in CD34+ derived reticulocytes expressing GLUT8-GPS, a small amount of trehalose accumulates inside reticulocytes even in just PBSAG buffer alone (perhaps produced from the cells metabolism) and the GLUT8-GPS trehalose accumulation is twice as much as unmodified reticulocytes when loaded with trehalose overnight. So GLUT8-GPS expression at the surface of reticulocytes enhances trehalose intracellular loading.

[0347] Example 4: Overexpression of natural protective proteins sourced from nature Tardigrade-specific intrinsically disordered proteins (TDPs) are essential for desiccation tolerance in tardigrades. These proteins are intrinsically disorganised proteins (IDP) and are normally upregulated upon desiccation. TDPs form non-crystalline amorphous solids (vitrify) upon desiccation, and this vitrified state mirrors their protective capabilities. Three tardigrade specific proteins were selected for expression in reticulocytes, namely cytosolic abundant heat soluble (CAHS) protein 1, CAHS protein 2 and CAHS protein 3. These proteins are not present in human reticulocytes.

[0348] Late-Embryogenesis-Abundant (LEA) proteins are expressed in insects, nematodes, and plants. LEA proteins are characterised by a 11-mer motif and are known to reduce aggregation of proteins during dehydration. The LEA proteins can be subdivided based upon species into group 1, group2 and group 3, whereby group 3 proteins are expressed in animals. Two group 3 exemplar LEA proteins namely PvLEA22, which is expressed in Polypedilum vanderplanki, and AavLEA which is expressed in Aphelenchus avenae, were selected. Again, these proteins are not present in human reticulocytes.

[0349] The erythroid cell line BEL-A was used as a model system to demonstrate it is possible to express this selection of naturally sourced desiccation protective proteins (SEQ ID NOs: 29- 33) in erythroid cells. BEL-A cells were single sorted to generate a high expressing single clone cell line. The highest 2 expressing clones of each construct were selected and terminally differentiated 4 times, and expression was analysed at day 0 (expanding BELA cells), in differentiating erythroblasts to day 8, and in reticulocytes (day 12). A representative graph of protein expression during differentiation measured detecting c-myc by flow cytometry on permeabilsed cells is shown in Figure 5A and this confirms that although some protein expression is lost during the differentiation to reticulocytes, a proportion of these exogenously expressed desiccation protective proteins can be retained. These may provide protection to a freeze-dried reticulocyte or facilitate longer storage when expression is optimised further.

[0350] These protective proteins were also expressed as Green fluorescent tagged fusion proteins in expanding and differentiating CD34+ cells to monitor expression. For these experiments CD34+ cells were transduced with the specific lentivirus construct and then the CD34+ cells proliferated, differentiated and reticulocytes prepared. Expression levels of CAHS1-GFP, CAHS2-GFP and AavLEA-GFP in expanding CD34+ haematopoietic stem cells and subsequently differentiated these to reticulocytes was measured during differentiation by flow cytometry, at day 6, 12 and day 19 (reticulocytes) see Figure 5B. To test if the expression of these proteins affected deformability, reticulocytes expressing CAHS1, CAHS2 or AavLEA were FACS sorted. The deformability of these reticulocytes was measured using the ARCA deformation device (which measures cell deformability) and compared to untransduced cells and no difference was seen between control reticulocytes (untransduced reticulocytes) and reticulocytes expressing CAHS1, CAHS2 and AavLEA (Figure 5B).

[0351] To avoid the loss of cells during FACS, new constructs were designed to allow for antibiotic selection of CD34+. As there might be future interest in co-express these natural proteins with, e.g., GLUT8 or antioxidant proteins, all 3 CAHS proteins were cloned into CD710-B-1- Blast (System Biosciences) with a c-terminal FLAG tag (SEQ ID NOs: 48-53). CD34+ hematopoietic stem cells were transduced with each specific construct on day 3 after isolation and blasticidin (lOpg / mL) was added to culture between days 5-8. The cells were differentiated into reticulocytes during a 20 day, 3-step culture method as previously described.

[0352] Expression levels of CAHS1-FLAG, CAHS2-FLAG and CAHS3-FLAG in differentiating CD34+ haematopoietic stem cells and filtered reticulocytes was assessed by western blot on cell lysates (1 xlO6) at days 10, 14 and day 20 (filtered reticulocytes) as shown on Figure 6. As detected by labelling against FLAG, the expression of CAHS1-FLAG is lowest expressed using this construct and difficult to detect in reticulocytes. CAHS2-FLAG expression decreased as cell differentiate and mature with reduced expression observed in reticulocytes. CAHS3-FLAG was the highest expressed of the 3 tested CAHS1 protective proteins and it's abundance is retained throughout differentiation in CD34+ derived reticulocytes.

[0353] Example 5: Overexpression of peroxiredoxins to protect for reactive oxygen species

[0354] Due to their oxygen transporting role and high iron content, RBCs are particularly at risk of oxidative stress in the circulation and during storage. To minimise the impact of oxidative stress, RBCs possess a comprehensive antioxidant system. These include antioxidants of low molecular weight, such as ascorbate (vitamin C) and glutathione, in addition to a series of more complex enzymatic pathways for dealing with reactive oxygen species and damage. The process of lyophilization / desiccation, storage and subsequent rehydration is also known to generate damaging reactive oxygen species which will likely compromise the survival or disrupt prolonged storage of cells. This led to the exploration of whether it is possible to further enhance the already high expression levels of antioxidant enzymes in erythroid cells using lentivirus to produce reticulocytes with further enhanced resilience to oxidative stress to improve freeze drying / storage properties. Enzymes that would help lipid peroxidation and minimize haemoglobin oxidation were specifically selected but essentially the methods used here could be applied to any antioxidant enzyme.

[0355] There are six peroxiredoxin (Prx) isoforms that exist in mammalian cells. Of these, peroxiredoxin 2 (Prx2) is considered a key player in the RBC antioxidant defense mechanism and is the third most abundant protein in RBCs. Its dual functionality as a protective haemoglobin molecular chaperone that reduces met haem production also makes it an attractive antioxidant to overexpress in reticulocytes. Prxl and Prx6 are also normally present in the cytoplasm of RBC, albeit to much lower expression levels compared to Prx2.

[0356] Expanding BEL-A cells were transduced using lentivirus for each wild type human Prx isoform which were tagged with c-MYC to distinguish its expression from endogenous protein (SEQ ID NOs: 19, 20, 23, 24, 27 and 28). The cells were expanded to generate a stable BEL-A erythroblast line expressing the individual Prx isoforms 1, 2 and 6 and then differentiated to generate reticulocytes. Figure 7A shows western blot analysis of overexpression of each protein using an isoform specific antibody in day 0 (proerythroblasts) expanding BEL-A cells. Note that the exogenously expressed protein is larger on Western blot due to the inclusion of a c-MYC tag and all are substantially expressed compared to the lower band corresponding to the endogenous Prx protein.

[0357] Figure 7B confirms expression of each protein in expanding BEL-A cells. Figures 7C and 7D show the Prx expression at the start dO (proerythroblast) and day 12 (dl2) end stage mixed cell population (orthochromatic, reties and nuclei) or reticulocytes only (dl2r) of BEL-A differentiation which was analysed by flow cytometry by permeabilization of the cells and then labelling by detecting the c-MYC tag. Reticulocytes are gated on specifically by using DNA negative population because reticulocytes do not contain a nucleus. Mean fluorescent intensities were normalized to background antibody labelling in nontransduced (UT) control cells. This data shows that expression of each Prx enzyme isoform is maintained and retained in reticulocytes with minimal loss of Prx isoform expression during differentiation.

[0358] This work establishes for the first time that the developing red blood cells can be genetically engineered to overexpress antioxidant proteins, despite endogenous versions already being very highly expressed in the cells.

[0359] Therefore it is possible to extrapolate from these Prx isoform results to also suggest that other enzymes such as superoxide dismutase or catalase which are also naturally highly abundant in red blood cells could be also overexpressed using lentivirus to enhance the antioxidant capacity of the cell. It is possible that the mitochondrial superoxide dismutase which is normally expressed but then lost when mitochondria are lost during the production of the mature red blood cell could still be successfully overexpressed and retained in the cytosol by removal of its mitochondrial targeting sequence. Other antioxidants such as glutathione peroxidases are more complicated due to the requirements for selenocysteine see below.

[0360] Example 6: Overexpression of glutathione peroxidase 1 and 4

[0361] GPxl is also considered a principal player of RBC ROS defence and is also endogenously expressed at high levels in RBCs. GPxl has been shown to associate with the RBC membrane and is therefore postulated to play a role in preventing lipid peroxidation and was therefore selected to overexpress. GPx4 has recently gained increasing attention due to its leading role in the regulation of iron-dependent ferroptosis.

[0362] Figure 8 demonstrates the overexpression of wild type, tagged glutathione peroxidase (GPx) proteins in BEL-A cells, wherein expanding BEL-A cells were transduced lentivirus for each wild type human GPxl and GPx4 cDNA tagged with c-MYC (see Figure 8A and B). The cells were expanded to generate a stable BEL-A erythroblast line and then later differentiated to generate reticulocytes. This shows that unlike Prx protein family, selecting only the known cDNA sequence for glutathione peroxidases is not enough for expression.

[0363] It is hypothesized that this was likely due to the requirement of these enzymes for a specialised amino acid, selenocysteine in the active site and demonstrated this by mutating the selenocysteine to cysteine (which would still retain antioxidant activity). Figure 8A and B shows western blot and flow cytometry analysis of the overexpression of GPx isoforms 1 and 4 in BEL-A cells, wherein expanding BEL-A cells were transduced with either wild type human GPxl, a U49C GPxl, wild type human full length GPx4, or a U73C GPx4 construct (SEQ ID NOs: 3-6 and 9-12). Figure 8B (lower panel) shows flow cytometry analysis comparing GPxl U49C and GPx4 U73C expression, respectively. This shows that the selenocysteine mutants where the selenocysteine is mutated to a cysteine enables expression. This confirms that the selenocysteine incorporation is important for GPx expression and that it is possible to circumvent this to overexpress glutathione peroxidases like GPxl and GPx4 when the selenocysteine is mutated to cysteine. Figures 8C and 8D illustrate the expression of GPxl U49C and GPx4 U73C after differentiation to reticulocytes using nontransduced cells as a negative control comparing the day 12 mixed population end point (dl2) and also the dayl2 gated on reties only (dl2r). This demonstrates that although GPxl U49C can be retained in differentiating erythroid cells to reticulocyte stage, the GPx4 U73C is lost.

[0364] The limited GPx4 U73C expression achieved in expanding BEL-As and loss of significant expression during differentiation suggests that this protein is being tightly regulated in some unknown manner. As ubiquitin-mediated proteasomal degradation and lysosome- induced degradation are considered the two main pathways of erythropoietic protein turnover, the effects of the proteosome inhibitor, MG-132, and lysosomal inhibitor, leupeptin, were investigated to established whether GPx4 U73C overexpression could be further enhanced in expanding BEL-A cells. Furthermore, specific HSC70 recognition motifs that trigger GPx4 for degradation via chaperone-mediate autophagy (CMA) have also been recently identified. Since CMA commonly facilitates the degradation of oxidized proteins, it is also possible that bardoxolone-mediated CMA inhibition may therefore also help to increase GPx4 U73C overexpression in erythroid cells. Expanding GPx4 U73C BEL-A cell lines were exposed to each inhibitor or the respective vehicle control (DMSO) for a total of 6 hours. Resultant expression levels were determined by intracellular flow cytometry and normalised to background c-MYC labelling in control BEL-A cells.

[0365] As shown in Figure 8E, differentiating BEL-A erythroblasts expressing GPx4 U73C incubated with 5 pM MG132 resulted in a significant increase in GPx4 U73C expression. No increase in expression was observed upon treatment with either 10 pM leupeptin or 10 pM bardoxolone, indicating that ubiquitin-mediated degradation is the major contributor to GPx4 degradation in differentiating erythroid cells.

[0366] Example 7: Manoeuyres to enhance glutathione peroxidase expression and retention in reticulocytes

[0367] Next, it was established whether GPx4 could in fact be even more highly overexpressed by using a new optimization strategy. As selenocysteine (Sec) containing GPx is preferential for both catalytic activity and resilience to hyperoxidation, demonstrating GPx overexpression of the unmodified protein remained a key goal for GPx cDNA designs (called here GPxSec). To attempt to incorporate the selenocysteine, the GPx4 construct sequence was extended to include the native GPx4 3' untranslated region (UTR) nucleotide sequence that contains a Sec insertion sequence (SECTS) - a mRNA structure that aids the recruitment of tRNASec. The hypothesis being that the inclusion of this sequence will enable incorporation of the selenocysteine and thus expression of GPx4 (and by extrapolation due to having the same requirements GPxl).

[0368] GPx4 is known to exist in 3 forms: a full-length long from (22 kDa) expressed from the primary transcript predominantly in spermatozoa; a short form (19 kDa) generally found in subcellular locations such as the cytoplasm, nucleus and microsome of other cell types; and a sperm-specific nuclear form, which is translated from an alternative exon located in the first intron of the GPx4 gene. Further examination of the full-length primary GPx4 transcript used to design expression constructs thus far, revealed a 25 amino acid mitochondrial leader sequence at the N-terminus. The western blot results in Figure 9B also suggested that a proportion of GPx4 protein was a lower molecular weight, so may have been cleaved like many mitochondrial signal sequences after import into mitochondria. It was therefore hypothesised that the presence of this signal sequence may limit GPx4 expression and affect retention, because the mitochondria are lost during the late stages of differentiation and during reticulocyte maturation. Therefore, the short form of GPx4 was selected to take forward for further study.

[0369] Figures 9A, 9C and 9D show that successful overexpression of GPx4Sec(SEQ ID NOs: 13 and 14) was achieved upon removal of an N-terminal sequence that is required for GPx4 mitochondrial targeting and inclusion of a 3'UTR sequence (sGPx43 UTR). However, in expression tests, loss of expression of GPx4 during differentiation to reticulocytes was still observed which was likely due to ubiquitin mediated degradation (see evidence in Example 6 above).

[0370] Degradation of the protein was blocked by mutation of 6 potential ubiquitination sites (a manoeuvre used for thymidine phosphorylase, as described, for example, in WO 2021 / 053243). The ubiquitination sites were selected based on software described in Hornbeck PV, Zhang B, Murray B, Kornhauser JM, Latham V, Skrzypek E PhosphoSitePlus, 2014: mutations, PTMs and recalibrations. Nucleic Acids Res. 2015 43 : D512-20. Through this, the potential ubiquitination sites K80, K97, K121, K135, K140, K164 were identified. These sites were all confirmed to be surface exposed using computer modelling and were not located in the active site. The sGPx43 UTRconstruct was modified to contain these 6 lysines mutated to arginine (the argine selected to retain the positive charged amino acid) but mutating potential ubiquitination sites so they cannot be ubiquitinated (K80R, K97R, K121R, K135R, K140R, K164R; called here sGPx43 UTR6KR; SEQ ID NOs: 15 and 16). The expression of sGPx43 UTR6KR matched that of wild type sGPx43 UTRoverexpression in expanding BEL-As (see Figure 9A) as measured by flow cytometry on permeabilised cells, indicating that mutagenesis of these sites is not detrimental to the overall structure and therefore expression of the enzyme. Stable cell lines were made for each of the GPx4secconstructs.

[0371] Differentiation of the GPx4SecBEL-A cell lines (see Figures 9C and 9D) revealed overall reduction in sGPx43 UTRexpression, whereby around 70% of expression was lost, predominantly in the first 4 days of differentiation. Despite this, a proportion of sGPx43 UTRoverexpression persisted in day 12 reticulocytes (see the graphs in Figure 9D).

[0372] Expression of sGPx43 UTR6KR construct version as judged by flow cytometry on permeabilsed cells, gave consistently higher expression than sGPx43 UTRthroughout differentiation (see Figure 9C and right panel). Strikingly day 12 reticulocytes, demonstrated high retention of sGPx43 UTR6KR expression (see the graphs in Figure 9D), showing that sGPx43 UTR6KR facilitates expression and retention in reticulocytes.

[0373] It has therefore been established here using GPx4 as an exemplar protein that combined removal of a signal sequence that would impede cytosolic expression or result in loss of protein during erythropoiesis can help enzyme expression and retention in reticulocytes. For peroxidases such as GPx4 inclusion of a 3'UTR sequence to ensure incorporation of selenocysteine enables expression of the protein and where detected the disruption of the ubiquitination sites on the such as the 6 sites mutated here on GPx4 protein allows for significant concentrations of the protein to persist throughout differentiation and enucleation stages such that the resultant reticulocyte (and / or erythrocyte) contain levels of glutathione peroxidase 4 in reticulocytes that were previously unobtainable by means of retaining the overexpressed protein.

[0374] Similar to GPx4, the GPxl construct sequence was also extended to include the native GPxl 3'UTR nucleotide sequence containing the SECIS region. Both MYC-GPxlU49Cand MYC-GPX1 3'UTR (SEQ ID NO 54-55) were cloned into CD710-B-1-Puro. Expanding BEL-A cell lines were transduced with lentivirus specific for each protein and selected using lpg / mL puromycin. Figure 10A confirms expression of both proteins in expanding BEL-A cells after fixing, permabilsation and labelling for c-MYC. As observed for GPx4, the U49C mutation allows for higher expression when compared with MYC-GPxl 3'UTR which requires incorporation of the selenocysteine residue. Cell lysates (1 xlO6) of expanding cells were also analysed by immunoblotting (Figure 10B) using monoclonal anti-GPxl antibodies which allowed for the detection of both endogenous and exogenous forms of GPxl. In concordance with the flow cytometry data, the expression of unmodified GPxl (MYC-GPxl 3'UTR) is lower than MYC- GPxlU49C.

[0375] The BEL-A cell lines were differentiated for 11 days and c-MYC expression was determined in both nucleated (di inucleated) and enucleated (reticulocytes, d 1 lreticulocytes) cells. The cell populations can be gated on specifically by using DNA specific stain (Hoechst) since reticulocytes do not have a nucleus. Figure IOC shows that expression of both constructs decreases during differentiation and is lower on reticulocytes than nucleated cells. On day 11 the expression of MYC-GPxlU49C(top panel) remains higher than MYC-GPxl 3'UTR. This suggests further alterations are likely necessary to retain this protein in human erythroblasts for example by blocking ubiquitination.

[0376] Example 8: Expression of glutathione peroxidase 1 and 4 in primary CD34+ derived reticulocytes.

[0377] The next objective was to express GPxl and GPx4 in primary CD34+cells. As demonstrated in Example 7, the MYC-sGPx4 3'UTR 6KR construct exhibited the highest and most sustained expression during BEL-A differentiation. This optimized construct was cloned into the CD710-B-1-Puro vector to enable antibiotic selection as it had already been done for GPxlU49Cand 3'UTR constructs.

[0378] CD34+cells were isolated from PBMCs and transduced with lentiviral vectors encoding MYC- GPxlU49C, MYC-GPxl 3'UTR, or MYC-sGPx4 3'UTR 6KR on day 3 of differentiation. Puromycin selection was applied between days 5 and 8 to enrich transduced cells. On day 20, reticulocytes were purified by filtration.

[0379] Figure 13 confirms the expression of both GPxl constructs during primary erythropoiesis using immunoblotting and flow cytometry. Cell lysates (1 xlO6) of differentiating cells on days 7, 10, 14, and 20 (filtered reticulocytes) were analysed via immunoblotting (Figure 13A) with monoclonal anti-GPxl antibodies which allowed for the detection of both endogenous and exogenous forms of GPxl. GAPDH was used as a loading control. The data from both biological replicates demonstrate that the GPxlU49Cconstruct, which eliminates the need for selenocysteine incorporation, achieves higher expression levels of exogenous GPxl compared to endogenous GPxl. In contrast, the GPxl 3'UTR construct exhibits expression levels comparable to endogenous GPxl, with both tending to decrease by the reticulocyte stage. Figure 13B presents flow cytometry data for the same samples and time points analysed in panel A. Fixed and permeabilized cells were labelled with c-MYC antibodies, confirming protein expression patterns consistent with the immunoblotting results.

[0380] Figure 11 provides complementary data on the expression of the MYC-sGPx4 3'UTR 6KR construct using a selectable vector. Immunoblotting (Figure 11A) of cell lysates (1 x 106) from days 7, 10, and 20 (filtered reticulocytes) confirms sustained expression of MYC-sGPx4 throughout erythroid differentiation. Two donors (#1 and #2) are shown, with both demonstrating robust protein expression in earlier differentiation stages and a moderate decline by the reticulocyte stage. Of note, no endogenous expression GPx4 was detected in untransfected reticulocytes at these exposures. GAPDH was used as a loading control. Flow cytometry histograms (Figure 11B) further validate these findings, showing c-MYC staining for the same samples and time points. Expression was consistent between the two replicates.

[0381] Example 9 - Expression of human AAT2 in the cytosol

[0382] Human alanine aminotransferase 2 (huAAT2) is typically located in mitochondria. Upon examination of the huAAT2 protein domains, as assigned by UniProt (Q8TD30), a 27 amino acid mitochondrial targeting sequence was identified at the N-terminus. Like GPx4, it was therefore hypothesized that the presence of this signaling sequence may limit huAAT2 expression and affect retention, as mitochondria are lost during the late stages of differentiation and during reticulocyte maturation. Therefore, a truncated variant of huAAT2 that lacked the first 27 amino acids (huAAT2 N27del) was generated for further study (Figure 12A).

[0383] Coding sequences (according to the Ensembl database) were codon optimized for mammalian expression, synthesized and cloned in the lentiviral vector CD170B-1 (Systems Biosciences) by GenScript. CD34+ hematopoietic stem cells were transduced with lentivirus particles containing the respective constructs on day 3 of differentiation and washed 3 times in PBS 24 hrs later. Positively transduced cells were selected for by supplementing cell cultures with puromycin antibiotic for 72 hrs from day 5. AAT2 overexpression was then assessed by western blotting of whole cell lysates at the indicated time points using an AAT2 specific monoclonal antibody.

[0384] Figure 12B shows the successful overexpression of both full length (FL) and truncated (N27del) huAAT2 in CD34+ hematopoietic stem cells at the start of differentiation (d8) through to the end of differentiation (d20) and in a population of purified reticulocytes (d20r). Overall, a much greater proportion of starting huAAT2 protein expression was observed to be retained in reticulocytes overexpressing huAAT2 lacking the N-terminus (N27del) compared to cells overexpressing the full-length protein (FL). On quantification of western blot band signal intensity, reticulocytes (d20r) derived from CD34+ cells overexpressing huAAT2 N27del were found to express 8.9x more AAT2 protein than those derived from stem cells overexpressing huAAT2 FL. As depicted in Figure 12C, only 7.4% of starting expression was retained in reticulocytes derived from CD34+ cells overexpressing huAAT2 FL. However, reticulocytes derived from CD34+ cells overexpressing huAAT2 N27del retained almost 70% of starting expression.

[0385] This therefore demonstrates an additional exemplar protein whereby removal of the mitochondrial signaling sequence to enhance cytosolic expression has improved enzyme retention in culture-derived reticulocytes.

[0386] EMBODIMENTS

[0387] The invention provides the following embodiments.

[0388] 1. A lyophilised composition comprising a cell population and a lyoprotectant, wherein the cell population comprises at least about 10% reticulocyte cells.

[0389] 2. A lyophilised composition according to embodiment 1, wherein the lyoprotectant comprises one or more of (a) a saccharide, (b) an antioxidant, and (c) an intrinsically disordered protein (IDP).

[0390] 3. A lyophilised composition according to embodiment 1 or 2, wherein (a) the saccharide is selected from trehalose, sucrose, fructose, glucose, mannitol, sorbitol, arabinose, and ribose, (b) the antioxidant is selected from glutathione peroxidase 1 (GPxl), GPx2, GPx3, GPx4, GPx5, GPx6, GPx7, GPx8, peroxiredoxin 1 (Prxl), Prx2, Prx3, Prx4, Prx5, Prx6 and variants thereof, and (c) the IDP is selected from cytoplasmic abundant heat soluble 1 (CAHS1), CAHS3, Polypedilum vanderplanki late embryogenesis abundant protein (PvLEA), Aphelenchus avenae LEA (AavLEA) and variants thereof.

[0391] 4. A lyophilised composition according to any one of the preceding embodiments, wherein the lyoprotectant comprises or consists of trehalose.

[0392] 5. A lyophilised composition according to embodiment 4, wherein at least a fraction of the trehalose is intracellular.

[0393] 6. A lyophilised composition according to any one of the preceding embodiments, wherein the reticulocyte cells are genetically engineered to express a trehalose transporter or a variant thereof on their surfaces.

[0394] 7. A lyophilised composition according to embodiment 6, wherein the trehalose transporter is a glucose transporter 8 (GLUT8) protein or variant thereof.

[0395] 8. A lyophilised composition according to any one of the preceding embodiments, wherein the reticulocyte cells are genetically engineered to overexpress an antioxidant protein and / or an intrinsically disordered protein (IDP) in their cytosol.

[0396] 9. A lyophilised composition according to any one of the preceding embodiments, wherein at least about 30% of the reticulocyte cells survive reconstitution and / or wherein at least about 30% of the reticulocyte cells comprise at least about 50% haemoglobin molecules with respect to the total number of haemoglobin plus methaemoglobin molecules.

[0397] 10. A lyophilised composition according to any one of the preceding embodiments, wherein the cell population comprises at least about 90% reticulocyte cells. 11. A lyophilised composition according to any one of the preceding embodiments, wherein the reticulocyte cells further comprise or express a therapeutic protein or polypeptide.

[0398] 12. A lyophilised composition according to any one of the preceding embodiments, wherein the reticulocyte cells are human.

[0399] 13. A pre-lyophilisation composition comprising a cell population, a lyoprotectant and a lyophilisation medium, wherein the cell population comprises at least about 10% reticulocyte cells.

[0400] 14. A pre-lyophilisation composition according to embodiment 13, wherein the lyoprotectant and / or the cells are as defined in any one of embodiments 1-12.

[0401] 15. A method of preparing a lyophilised composition according to any one of embodiments 1-12, comprising (a) suspending a cell population in a lyophilisation medium comprising a lyoprotectant, wherein the cell population comprises at least about 10% reticulocyte cells, and (b) subjecting the suspension produced in step (a) to a lyophilisation process to generate the lyophilised composition.

[0402] 16. A method according to embodiment 15, wherein the method comprises (a) suspending the cell population in the lyophilisation medium comprising the lyoprotectant for at least about 12 hours.

[0403] 17. A method of reconstituting a lyophilised composition according to any one of embodiments 1-12, comprising contacting the lyophilised composition with a reconstitution medium.

[0404] 18. A reconstituted composition obtained by or obtainable by (i) a method according to embodiment 17 or (ii) by reconstituting a lyophilised composition according to any one of embodiments 1-12.

[0405] 19. A reconstituted composition comprising a cell population, a lyoprotectant and a reconstitution medium, wherein the cell population comprises at least about 10% reticulocyte cells.

[0406] 20. A reconstituted composition according to embodiment 19, wherein the lyoprotectant and / or the cells are as defined in any one of embodiments 1-12.

[0407] 21. A reticulocyte cell or a mature red blood cell (RBC) comprising intracellular trehalose.

[0408] 22. A cell according to embodiment 21, wherein the cell is genetically engineered to express a trehalose transporter or a variant thereof on its surface. 23. An erythroid cell genetically engineered to express a trehalose transporter or a variant thereof on its surface.

[0409] 24. An erythroid cell according to embodiment 23, wherein the erythroid cell is an erythroid progenitor cell, a reticulocyte, or a mature red blood cell.

[0410] 25. An erythroid cell according to embodiment 23 or 24, wherein the trehalose transporter is a glucose transporter 8 (GLUT8) protein or a variant thereof.

[0411] 26. An erythroid cell according to embodiment 25, wherein the GLUT8 variant protein comprises a TPQ to GPN mutation.

[0412] 27. An erythroid cell according to embodiment 26, wherein the GLUT8 variant protein comprises or consists of the sequence shown in SEQ ID NO: 37.

[0413] 28. A cell genetically engineered to overexpress an antioxidant protein and / or an intrinsically disordered protein (IDP) in its cytosol.

[0414] 29. A cell according to embodiment 28, wherein the antioxidant protein is a glutathione peroxidase (GPx) or a variant thereof or a peroxiredoxin (Prx) or a variant thereof.

[0415] 30. A cell according to embodiment 29, wherein the GPx is GPxl, GPx2, GPx3, GPx4, GPx5, GPx6, GPx7 or GPx8 or the Prx is Prxl, Prx2, Prx3, Prx4, Prx5 or Prx6.

[0416] 31. A cell according to embodiment 30, wherein the GPx4 variant protein, the GPx5 variant protein, the GPx7 variant protein, the GPx8 variant protein, the Prx3 variant protein or the Prx5 variant protein comprises one or more inactivating mutations in its signal peptide.

[0417] 32. A cell according to embodiment 31, wherein the variant protein lacks at least a part of the signal peptide.

[0418] 33. A cell according to any one of embodiments 30-32, wherein the GPxl variant protein or the GPx4 variant protein comprises a mutation at one or more predicted ubiquitination sites.

[0419] 34. A cell according to embodiment 33, wherein the GPxl variant protein comprises a mutation at one or more of K88, K148 and K166 or the GPx4 variant protein comprises a mutation at one or more of K107, K126, K148, K162R, K167 and K191.

[0420] 35. A cell according to embodiment 34, wherein K is substituted with R. 36. A cell according to any one of embodiments 29-35, wherein the GPxl, GPx2, GPx3, GPx4, GPx6 or variant thereof is expressed from a polynucleotide which comprises a 3' untranslated region (3'UTR) containing a Sec insertion sequence (SECIS).

[0421] 37. A cell according to any one of embodiments 29-35, wherein the GPxl variant protein comprises a U49C mutation or the GPx4 variant protein comprises a U73C mutation.

[0422] 38. A cell according to any one of embodiments 29-37, wherein the GPxl or variant thereof comprises the sequence shown in SEQ ID NO: 2, 4 or 6 or a variant thereof or the GPx4 or variant thereof comprises the sequence shown in SEQ ID NO: 12, 14 or 16 or a variant thereof.

[0423] 39. A cell according to any one of embodiments 28-38, wherein the IDP is CAHS1, CAHS3, PvLEA, AavLEA, or a variant thereof.

[0424] 40. A cell according to any one of embodiments 28-39, wherein the cell is further genetically engineered to express a trehalose transporter or a variant thereof on its surface

[0425] 41. A cell according to any one of embodiments 28-40, wherein the cell further comprises or expresses a therapeutic protein or polypeptide.

[0426] 42. A cell according to any one of embodiments 28-41, wherein the cell is a blood cell.

[0427] 43. A cell according to embodiments 21-42, wherein the cell is a human cell.

[0428] 44. A cell according to any one of embodiments 21-43, wherein the cell is derived from a stem cell or an immortalised cell line.

[0429] 45. A population of cells according to any one of embodiments 21-44.

[0430] 46. A variant antioxidant protein which comprises a mutation at one or more predicted ubiquitination sites

[0431] 47. A variant protein according to embodiment 46, wherein the variant protein is a Gpxl or Gpx4 variant protein.

[0432] 48. A variant protein according to embodiment 47, wherein the GPxl variant protein comprises a mutation at one or more of K88, K148 and K166 or the GPx4 variant protein comprises a mutation at one or more of K107, K126, K148, K162R, K167 and K191.

[0433] 49. A variant protein according to embodiment 48, wherein K is substituted with R. 50. A variant protein according to any one of embodiments 47-49, wherein the GPx4 variant protein comprises one or more inactivating mutations in its signal peptide acks at least a part of its signal peptide.

[0434] 51. A variant protein according to any one of embodiments 47-50, wherein the GPx4 variant comprises the sequence shown in SEQ ID NO: 16.

[0435] 52. A variant protein according to any one of embodiments 47-49, wherein the GPxl variant protein comprises a U49C mutation or the GPx4 variant protein comprises a U73C mutation.

[0436] 53. A variant antioxidant protein which comprises one or more inactivating mutations in its signal peptide.

[0437] 54. A variant protein according to embodiment 53, wherein the variant protein lacks at least a part of the signal peptide.

[0438] 55. A variant protein according to embodiment 53 or 54, wherein the variant antioxidant protein is a GPx4, GPx7, GPx8, Prx3 or Prx5 variant protein.

[0439] 56. A polynucleotide which encodes a variant protein according to any one of embodiments 46-55.

[0440] 57. A polynucleotide according to embodiment 56, wherein the polynucleotide comprises a 3' untranslated region (3'UTR) containing a Sec insertion sequence (SECIS).

[0441] 58. A polynucleotide according to embodiment 56 or 57, wherein the polynucleotide comprises or consists of the sequence shown in SEQ ID NO: 15 or a variant thereof.

[0442] 59. A vector comprising a polynucleotide according to any one of embodiments 56-58.

[0443] 60. A pharmaceutical composition comprising a reconstituted composition according to any one of embodiments 18-20, a cell according to any one of embodiments 21-44, a population according to claim 45, a variant protein according to any one of embodiments 46-55, a polynucleotide according to any one of embodiments 56-58 or a vector according to embodiment 59 and a pharmaceutically or physiologically acceptable diluent and / or carrier.

[0444] 61. A method of improving the ability of a cell to survive lyophilisation, the method comprising genetically engineering the cell to overexpress an antioxidant protein and / or an intrinsically disordered protein (IDP) in its cytosol. . A method according to embodiment 61, wherein the antioxidant protein is as defined in any one of embodiments 29-38, the IDP is as defined in embodiment 39, or the cell is as defined in any one of embodiments 40-44. . A method according to embodiment 61 or 62, wherein the method comprises genetically engineering the cell to express a variant protein according to any one of embodiments

[0445] 46-55, a polynucleotide according to any one of embodiments 56-58 or a vector according to embodiment 59. . A lyophilised composition according to any of embodiments 1-12, a reconstituted composition according to any one of embodiments 18-20, a reticulocyte cell or mature RBC according to any one of embodiments 21-22, an erythroid cell according to any one of embodiments 23-27, a cell according to any one of embodiments 28-44, a population according to embodiment 45, a variant protein according to any one of embodiments 46- 55, a polynucleotide according to any one of embodiments 56-58, a vector according to embodiment 59 or a pharmaceutical composition according to embodiment 60 for use in therapy.

Claims

CLAIMS1. An erythroid cell genetically engineered to express a variant protein which comprises one or more mutations which increase the level of the variant protein in the cytosol of the cell.

2. An erythroid cell according to claim 1, wherein the one or mutations comprise (a) one or mutations at one or more predicted ubiquitination sites and / or one or more surface lysine residues, (b) one or more inactivating mutations in its signal peptide, (c) one or more inactivating mutations in its mitochondrial targeting peptide or (d) any combination thereof.

3. An erythroid cell according to claim 1 or 2, wherein the variant protein is a variant of an antioxidant protein and / or an intrinsically disordered protein (IDP).

4. An erythroid cell according to claim 3, wherein the antioxidant protein is a glutathione peroxidase (GPx) or a peroxiredoxin (Prx).

5. An erythroid cell according to claim 4, wherein the GPx is GPxl, GPx2, GPx3, GPx4, GPx5, GPx6, GPx7 or GPx8 or the Prx is Prxl, Prx2, Prx3, Prx4, Prx5 or Prx6.

6. An erythroid cell according to claim 5, wherein the GPx4 variant protein, the GPx5 variant protein, the GPx7 variant protein, the GPx8 variant protein, the Prx3 variant protein or the Prx5 variant protein comprises one or more inactivating mutations in its signal peptide.

7. An erythroid cell according to claim 6, wherein the variant protein lacks at least a part of the signal peptide.

8. An erythroid cell according to any one of claims 5-7, wherein the GPxl variant protein or the GPx4 variant protein comprises a mutation at one or more predicted ubiquitination sites.

9. An erythroid cell according to claim 8, wherein the GPxl variant protein comprises a mutation at one or more of K88, K148 and K166 or the GPx4 variant protein comprises a mutation at one or more of K107, K126, K148, K162R, K167 and K191.

10. An erythroid cell according to claim 9, wherein K is substituted with R.

11. An erythroid cell according to any one of claims 4-10, wherein the GPxl, GPx2, GPx3, GPx4, GPx6 or variant thereof is expressed from a polynucleotide which comprises a 3' untranslated region (3'UTR) containing a Sec insertion sequence (SECIS).

12. An erythroid cell according to any one of claims 4-10, wherein the GPxl variant protein comprises a U49C mutation or the GPx4 variant protein comprises a U73C mutation.

13. An erythroid cell according to any one of claims 4-12, wherein the GPxl or variant thereof comprises the sequence shown in SEQ ID NO: 2, 4, 6 or 55 or a variant thereof or the GPx4 or variant thereof comprises the sequence shown in SEQ ID NO: 12, 14 or 16 or a variant thereof.

14. An erythroid cell according to any one of claims 3-13, wherein the IDP is CAHS1, CAHS2, CAHS3, PvLEA, AavLEA, or a variant thereof.

15. An erythroid cell according to claim 1 or claim 2, wherein the variant protein is a variant of an enzyme.

16. An erythroid cell according to claim 15, wherein the enzyme is selected from hydrolases, oxidoreductases, lyases, transferases, ligases, isomerases, phosphorylases, synthases, kinases, hydroxylase, dehydrogenase, deaminases, proteases, metalloproteinases, lyases, aminotransferases, deiminases, decarboxylases, oxidases and lysosomal enzymes.

17. An erythroid cell according to claim 16, wherein the enzyme is thymidine phosphorylase, alanine aminotransferase, thymidine phosphorylase, glutamine synthase, hexokinase, glucokinase, phenylalanine hydroxylase, alcohol dehydrogenase, catalase, glucose-6-phosphate dehydrogenase, adenosine deaminase, asparaginase, uricase, bacterial L-phenylalanine ammonia lyase, alanine aminotransferase, glutamate dehydrogenase, arginine deiminase, arginase, oxalate decarboxylase, oxalate oxidase, a serine protease, tissue plasminogen activator, or larondinase,.

18. An erythroid cell according to any one of the preceding claims, wherein the erythroid cell is further genetically engineered to express a trehalose transporter or a variant thereof on its surface19. An erythroid cell according to any one of the preceding claims, wherein the erythroid cell further comprises or expresses a therapeutic protein or polypeptide.

20. An erythroid cell according to any one of the preceding claims, wherein the erythroid cell is a reticulocyte cell.

21. An erythroid cell according to any one of the preceding claims, wherein the erythroid cell is a human erythroid cell.

22. An erythroid cell according to any one of the preceding claims, wherein the erythroid cell is derived from a stem cell or an immortalised cell line.

23. A reticulocyte cell or a mature red blood cell (RBC) comprising intracellular trehalose.

24. A cell according to claim 23, wherein the cell is genetically engineered to express a trehalose transporter or a variant thereof on its surface.

25. An erythroid cell genetically engineered to express a trehalose transporter or a variant thereof on its surface.

26. An erythroid cell according to claim 25, wherein the erythroid cell is an erythroid progenitor cell, a reticulocyte, or a mature red blood cell.

27. An erythroid cell according to claim 25 or 26, wherein the trehalose transporter is a glucose transporter 8 (GLUT8) protein or a variant thereof.

28. An erythroid cell according to claim 27, wherein the GLUT8 variant protein comprises a TPQ to GPS mutation.

29. An erythroid cell according to claim 28, wherein the GLUT8 variant protein comprises or consists of the sequence shown in SEQ ID NO: 37.

30. A population of cells according to any one of the preceding claims.

31. A variant antioxidant protein which comprises a mutation at one or more predicted ubiquitination sites.

32. A variant protein according to claim 31, wherein the variant protein is a GPxl or GPx4 variant protein.

33. A variant protein according to claim 32, wherein the GPxl variant protein comprises a mutation at one or more of K88, K148 and K166 or the GPx4 variant protein comprises a mutation at one or more of K107, K126, K148, K162R, K167 and K191.

34. A variant protein according to claim 33, wherein K is substituted with R.

35. A variant protein according to any one of claims 32-34, wherein the GPx4 variant protein comprises one or more inactivating mutations in its signal peptide acks at least a part of its signal peptide.

36. A variant protein according to any one of claims 32-35, wherein the GPx4 variant comprises the sequence shown in SEQ ID NO: 16.

37. A variant protein according to any one of claims 32-34, wherein the GPxl variant protein comprises a U49C mutation or the GPx4 variant protein comprises a U73C mutation.

38. A variant antioxidant protein which comprises one or more inactivating mutations in its signal peptide.

39. A variant protein according to claim 38, wherein the variant protein lacks at least a part of the signal peptide.

40. A variant protein according to claim 38 or 39, wherein the variant antioxidant protein is a GPx4, GPx7, GPx8, Prx3 or Prx5 variant protein.

41. A variant mitochondrial enzyme protein which comprises one or more inactivating mutations in its in its mitochondrial targeting peptide.

42. A variant mitochondrial protein according to claim 41, wherein the variant protein lacks at least a part of the mitochondrial targeting peptide.

43. A variant mitochondrial protein according to claim 41 or 42, wherein the variant enzyme protein is a variant of the any of the enzymes in claims 16 and 17.

44. A polynucleotide which encodes a variant protein according to any one of 31-43.

45. A polynucleotide according to claim 44, wherein the polynucleotide comprises a 3' untranslated region (3'UTR) containing a Sec insertion sequence (SECIS).

46. A polynucleotide according to claim 44 or 45, wherein the polynucleotide comprises or consists of the sequence shown in SEQ ID NO: 15 or a variant thereof.

47. A vector comprising a polynucleotide according to any one of claims 44-46.

48. A pharmaceutical composition comprising an erythroid cell according to any one of claims 1-29, a population according to claim 30, a variant protein according to any one of claims 31-43, a polynucleotide according to any one of claims 44-46 or a vector according to claim 47 and a pharmaceutically or physiologically acceptable diluent and / or carrier.

49. A method of improving the ability of a cell to survive lyophilisation, the method comprising genetically engineering the cell to overexpress an antioxidant protein and / or an intrinsically disordered protein (IDP) in its cytosol.

50. A method according to claim 49, wherein the antioxidant protein is as defined in any one of claims 4-13, the IDP is as defined in claim 14, or the cell is as defined in any one of claims 18-21.

51. A method according to claim 49 or 50, wherein the method comprises genetically engineering the cell to express a variant protein according to any one of claims 31-40, a polynucleotide according to any one of claims 44-46 or a vector according to claim 47.

52. An erythroid cell according to any one of claims 1-29, a population according to claim 30, a variant protein according to any one of claims 31-43, a polynucleotide according to any one of claims 44-46, a vector according to claim 47 or a pharmaceutical composition according to claim 48 for use in therapy.

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