Cellular compositions and related methods

Abstract

The present invention relates to erythroid cells, such as reticulocytes, engineered to express a variant protein comprising one or more mutations that increase the level of the variant protein in the cytosol of the cell. The invention also relates to erythroid cells, such as reticulocytes, genetically engineered to overexpress an antioxidant protein and / or an intrinsically disordered protein (IDP) in their cytosol, as well as variants of such proteins that facilitate their overexpression in the cytosol of the cell. Modifying the cells in this way enhances their storage, whether by refrigeration, freezing, lyophilization, or another storage method. The cells of the invention can be used to deliver a therapeutic protein or polypeptide.

Description

Technical Field

[0001] This invention relates to erythroid cells, such as reticulocytes, engineered to express variant proteins comprising one or more mutations that increase the level of said variant protein in the cytosol of the cell. The invention also relates to erythroid cells, such as reticulocytes, genetically engineered to overexpress antioxidant proteins and / or intrinsically disordered proteins (IDPs) in their cytosol, and variants of such proteins that promote their overexpression in the cytosol. Modification of said cells in this manner, whether by refrigeration, freezing, lyophilization, or another storage method, enhances their storage capacity. The cells of this invention can be used for the delivery of therapeutic proteins or peptides. Background Technology

[0002] In vertebrates, including humans, a reliable and sustained supply of oxygen to the body's various tissues and organs is essential for health. This vital task is performed by blood, which flows throughout the body's circulatory system. While blood contains many different components, specifically, it is red blood cells (RBCs, or erythrocytes) that are responsible for capturing, transporting, and delivering oxygen. RBCs perform this function with the help of hemoglobin (Hb), an iron (II) protein that binds oxygen molecules in the lungs and releases them to other oxygen-dependent parts of the body.

[0003] Even short periods of oxygen deprivation can have 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 situation where the body may experience oxygen deprivation is severe blood loss, such as during massive hemorrhage, in which case it is crucial to restore blood volume by slowing or stopping the bleeding and by transfusing blood to the body to restore circulating blood volume.

[0004] Currently, blood transfusions are performed using reserves of donated blood. Whole blood transfusions are generally unnecessary or not recommended. Therefore, donated blood is typically separated into RBCs, platelets, and plasma as separate transfusionable components. Typical limitations of RBC reserves are that they must be refrigerated at 6°C and used within 35–42 days.

[0005] These temperature and time constraints on RBC storage necessitate planning for anticipated demand and supply chain logistics. There may be situations where the supply of stored RBCs is insufficient, including shortages of donors and / or a sudden surge in demand following a major disaster. There may also be situations where maintaining a constantly replenishing cold supply chain can be difficult, such as in certain third-world countries, in inaccessible and / or remote environments, and / or during military operations.

[0006] One potential method for providing a reliable long-term RBC stockpile is lyophilization or freeze-drying of RBCs. For some cell types, the lyophilized state may survive at ambient temperature and / or for a longer period than cryopreserved cell storage. Lyophilization of RBCs has been a focus since the 1980s, but two major problems have been encountered and proven difficult to overcome. The first problem is that a large proportion of RBCs fail to survive the lyophilization and remodeling process. The second problem is that iron in RBC hemoglobin is oxidized from Fe(II) to Fe(III). Hemoglobin containing the oxidized form of iron (Fe(III)) is called methemoglobin (metHb). MetHb cannot bind oxygen and therefore has no function in the transport of oxygen throughout the body.

[0007] Various technologies have been investigated to help RBCs remain intact throughout the lyophilization and remodeling process and to prevent the formation of significant levels of methemoglobin. For example, GB 1502016B discloses some advances in designing compositions that can be used to protect RBCs. In that disclosure, biopolymers such as PVP were found to protect RBCs, although the use of such biopolymers is not ideal for compositions intended for injection into the human bloodstream.

[0008] The present invention aims to address one or more of the limitations mentioned above. Summary of the Invention

[0009] The inventors have determined that reticulocytes possess attractive characteristics: they are more osmotically stable, have a higher abundance of natural protective proteins than erythrocytes, and naturally transport trehalose (a lyophilization protectant) into the cell. This is particularly useful because, although reticulocytes differ from RBCs, they are enucleated direct precursors of RBCs and therefore contain all hemoglobin. Thus, reticulocytes have the same efficient oxygen-carrying capacity as mature RBCs. Furthermore, reticulocytes mature into RBCs once they enter the bloodstream.

[0010] Therefore, lyophilized reticulocytes can provide a new and valuable source for lyophilized compositions that can be used, for example, in blood transfusions. One observed advantage of lyophilized reticulocytes in the presence of a lyophilization protectant compared to RBCs is that it allows for a higher reticulocyte viability. In other words, a high proportion of reticulocytes can remain intact after reconstitution because they can be naturally infused with the lyophilization protectant. Another observed advantage is that, with the right formulation, reticulocytes can be stored in lyophilized form for an extended period and subsequently reconstituted with minimal loss of cell viability compared to immediately reconstituted reticulocytes. Yet another advantage of using reticulocytes in this manner is the long lifespan of the cells once injected into the body. Once in the body, reticulocytes require approximately 1–2 days to mature into erythrocytes, from which point the erythrocytes are expected to retain their full lifespan.

[0011] Another advantage of lyophilizing reticulocytes compared to lyophilizing mature blood cells is that it may not require biopolymers such as those disclosed in GB 1502016B for protecting mature RBCs during lyophilization. Not wanting to be bound by theory, it is assumed that reticulocytes are more osmotically stable and may be better able to cope with lyophilization-induced alterations. Reticulocytes are typically about 20% the size of erythrocytes, and they have greater expression of transport proteins on their cell surface than mature RBCs. Reticulocytes may also possess some transport proteins that erythrocytes lose during maturation. This may also contribute to the protection of reticulocytes. Reticulocytes may also have higher levels of antioxidant proteins; because they are larger, and because they are newly generated, they may also have higher levels of metabolites that also support them, such as ATP or NADH.

[0012] The reticulocytes or any other cells of the present invention can be advantageously used for the delivery of therapeutic proteins or peptides via the bloodstream. The reticulocytes of the present invention, whether lyophilized or reconstituted, can also be used as clinical standards for clinical testing, for example, some clinical instruments are specifically designed to measure reticulocytes, and companies use cells fixed in different ways as standards.

[0013] Therefore, the present invention provides a lyophilized composition comprising a cell population and a lyophilization protectant, wherein the cell population comprises at least about 10% reticulocytes. The present invention also provides: - A pre-lyophilized composition comprising a cell population, a lyophilization protectant, and a lyophilization medium, wherein the cell population comprises at least about 10% reticulocytes; - A method for preparing the lyophilized composition of the present invention, comprising (a) suspending a cell population in a lyophilization medium containing a lyophilization protectant, wherein the cell population contains at least about 10% reticulocytes, and (b) subjecting the suspension generated in step (a) to a lyophilization process to generate the lyophilized composition. - A method for reconstructing the lyophilized composition of the present invention, comprising contacting the lyophilized composition with a reconstruction medium; - A reconstructed composition, which is obtained or can be obtained by (i) the method of the present invention or (ii) by reconstructing the lyophilized composition of the present invention; and - A reconstitution composition comprising a cell population, a lyophilization protectant, and a reconstitution medium, wherein the cell population comprises at least about 10% reticulocytes.

[0014] The inventors have surprisingly demonstrated that reticulocytes are capable of taking up trehalose. Specifically, trehalose can be loaded onto reticulocytes prior to freeze-drying, for example, by storing the reticulocytes overnight in a trehalose-containing medium. Therefore, the present invention provides reticulocytes or mature red blood cells (RBCs) containing intracellular trehalose.

[0015] The inventors have further demonstrated, surprisingly, that overexpression of trehalose transporters (such as GLUT8) in erythroid cells is possible. This requires far fewer mutations than 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). Therefore, this invention provides genetically engineered erythroid cells to express trehalose transporters or variants thereof on their surface. This further enhances trehalose uptake beyond the cell’s natural capacity.

[0016] The inventors have also surprisingly demonstrated that it is possible to modify proteins to increase their levels in the cytosol of erythroid cells (i.e., promote their overexpression). The inventors have identified several protein modifications that increase the levels of variant proteins in the cytosol. For example, the inventors have demonstrated that using proteins without ubiquitination sites or mutating one or more ubiquitination sites in said proteins reduces their intracellular degradation. The inventors have also demonstrated that introducing one or more inactivating mutations into the signal peptide of a protein enhances its expression and retention in the cytosol of the cell. The inventors have also demonstrated that introducing one or more inactivating mutations into the mitochondrial targeting sequence of a protein reduces its targeting to mitochondria and increases its expression in the cytosol of the cell. Therefore, the present invention provides genetically engineered erythroid cells to express variant proteins containing one or more mutations that increase the levels of said variant proteins in the cytosol of the cells. The present invention also provides: - An erythroid cell population of the present invention; - A variant mitochondrial enzyme protein containing one or more inactivating mutations in its mitochondrial targeting peptide; - A polynucleotide encoding a variant mitochondrial enzyme protein of the present invention; - A carrier comprising the polynucleotide of the present invention; and - A pharmaceutical composition comprising erythroid cells of the present invention, populations of the present invention, variant enzyme proteins of the present invention, polynucleotides of the present invention or carriers of the present invention, and pharmaceutically or physiologically acceptable diluents and / or carriers.

[0017] The present invention also provides erythroid cells of the present invention, populations of the present invention, variant enzyme proteins of the present invention, polynucleotides of the present invention, carriers of the present invention, or pharmaceutical compositions of the present invention for use in therapy.

[0018] The inventors have also surprisingly demonstrated that it is possible to overexpress antioxidant proteins and / or intrinsically disordered proteins (IDPs) in the cytosol of erythroid cells. This is surprising because the overexpression of antioxidant proteins and / or IDPs in the cytosol of erythroid cells is unexpected. The inventors have identified several protein modifications that promote the overexpression of said proteins in the cytosol. For example, the inventors have demonstrated that using proteins without ubiquitination sites or mutating one or more ubiquitination sites in said proteins reduces their intracellular degradation. The inventors have also demonstrated that introducing one or more inactivating mutations into the signal peptide of a protein enhances its expression and retention in reticulocytes. Overexpression of these proteins has the potential to further enhance cell storage properties, whether by cryopreservation, freezing, lyophilization, or other storage methods. Therefore, the present invention provides erythroid cells that are genetically engineered to overexpress antioxidant proteins and / or intrinsically disordered proteins (IDPs) in their cytosol. The present invention also provides: - An erythroid cell population of the present invention; - A variant antioxidant protein containing mutations at one or more predicted ubiquitination sites; - A GPx1 or GPx4 variant protein containing a mutation at one or more predicted ubiquitination sites; - A variant antioxidant protein containing one or more inactivating mutations in its signal peptide; - A GPx4, GPx7, GPx8, Prx3, or Prx5 variant protein, which contains one or more inactivating mutations in its signal peptide; - A polynucleotide encoding a variant protein of the present invention; - A carrier comprising the polynucleotide of the present invention; - A pharmaceutical composition comprising the reconstituted composition of the present invention, erythroid cells of the present invention, populations of the present invention, variant proteins of the present invention, polynucleotides of the present invention or carriers of the present invention, and pharmaceutically or physiologically acceptable diluents and / or loading agents; and - A method for improving the ability of cells to survive in lyophilization, the method comprising genetically engineering the cells to overexpress antioxidant proteins and / or intrinsically disordered proteins (IDPs) in their cytosol.

[0019] The present invention also provides a lyophilized composition of the present invention, a reconstructed composition of the present invention, reticulocytes of the present invention, mature RBCs of the present invention, erythroid cells of the present invention, cells of the present invention, populations of the present invention, variant proteins of the present invention, polynucleotides of the present invention, carriers of the present invention, or pharmaceutical compositions of the present invention for use in therapy. Attached Figure Description

[0020] Figure 1 After incubating overnight with 500 mM trehalose, 1×10 6 Cell lysis was performed and evaluated using the Megazyme Trehalose Assay Kit. Measurements were taken on a spectrophotometer. The term "loaded" refers to cells being incubated overnight in PBSAG supplemented with 500 mM trehalose at 37°C with 5% CO2 before being sent to the freeze-drying facility. In contrast, control cells were incubated overnight in PBSAG.

[0021] Figure 2A Flow cytometry was used to quantify the expression of the GLUT8 transporter in BELA cells during differentiation. The GLUT8-GPS mutant transporter was tagged with c-MYC to allow for tracking of expression during mutation. After fixation and permeabilization, cells were measured by flow cytometry, and two distinct expression lines were successfully generated, which could be differentiated to produce reticulocytes with “low” or “high” GLUT8-GPS on their surface. Figure 2B To assess the functionality of the mutant GLUT8 transporter in BEL-A cells, a trehalose uptake assay was used to examine the ability of these GLUT8-expressing cells to take up trehalose. These results indicated that higher levels of the transporter expression were associated with elevated intracellular trehalose concentrations.

[0022] Figure 3 Experiments involving CD34-derived reticulocytes and cultured reticulocytes expressing the GLUT8 transporter were described. Samples were incubated overnight (O / N) at 37°C with 5% CO2 in PBSAG or PBSAG supplemented with 500 mM trehalose. The next day, cells were washed three times with PBSAG and then divided into batches of 10 million cells each in 500 μl PBSAG or PBSAG supplemented with 10% trehalose. Figure 3 Trehalose uptake after overnight loading with 500 mM trehalose is demonstrated. Cell evaluation was performed using the Megazyme Trehalose Kit, and measurements were taken using a spectrophotometer.

[0023] Figure 4: Expression of GLUT8 with a c-MYC tag in CD34+ reticulocytes using an alternative vector (blastacidin). Figure 4A GLUT8-GPS-MYC was cloned into an optional vector (with puromycin) to generate a final reticulocyte population that uniformly expressed the transporter protein. Figure 4BFlow cytometry histograms of IgG isotype control (black) and c-MYC (9E10) staining in fixed and permeabilized CD34+ cells on days 7 and 12 of differentiation, for both untransduced (UT) control cells (turquoise) and cells expressing GLUT8-GPS-MYC (orange). Figure 4C : Trehalose concentration (µg / 1×10⁻¹) in UT and reticulocytes expressing GLUT8-GPS after overnight incubation with 10% trehalose in PBSAG (blue) or PBSAG alone (red). 6 (cells). Trehalose levels were quantified using the Megazyme assay kit. Absorbance was measured at 340 nm and converted to μg using the standard curve shown.

[0024] Figure 5A Protein expression of c-MYC-tagged desiccant protective proteins (CAHS1, CAHS2, CAHS3, AAVLEA, PvLEA) during differentiation. BEL-A cells were transduced, cloned, and differentiated into monoclonal clones with high expression. Protein expression was analyzed in expanding BEL-A cells, cells on day 6 of differentiation, and in reticulocytes using permeabilized cells and flow cytometry (using c-MYC (9E10)). Figure 5B Protein expression during differentiation in CD34+ hematopoietic stem cells. CD34+ hematopoietic stem cells were transduced with CAHS1-GFP, CAHS2-GFP, or AavLEA-GFP, and expression levels were subsequently determined by flow cytometry on days 6, 12, and 19 of differentiation. Deformability was tested on positively sorted reticulocytes using ARCA, with red representing the deformability of control reticulocytes and blue representing reticulocytes expressing the target protein.

[0025] Figure 6 Expression of FLAG-tagged desiccation-protective CAHS proteins in CD34+ erythroblasts and reticulocytes. FLAG-tagged CAHS1, CAHS2, and CAHS3 were cloned into an optional vector (blastcin), transduced into CD34+ cells, and then differentiated to generate reticulocytes uniformly expressing these proteins. Cell lysates (1×10⁻⁶) were obtained from indicated transduced cells obtained on day 10 and day 14, and from filtered reticulocytes (day 20). 6 Representative protein blots (from individual cells). The membranes were labeled with a monoclonal antibody against the FLAG tag (clone M2) and GAPDH (loaded control).

[0026] Figure 7: Overexpression of c-MYC-tagged peroxiredoxin enzyme in immortalized BEL-A erythroblasts and reticulocytes. Figure 7A: Cell lysates obtained from the indicated cell lines labeled with monoclonal antibodies against Prx-1, Prx2, Prx6, and GAPDH (loaded control) (Prx1 and Prx6: 1 × 10⁻⁶). 6 Cells, Prx2: 0.25 × 10⁻⁶ 6 Representative protein blots from untransduced (UT) control cells (turquoise) and corresponding Prx-transduced cell lines (orange) in fixed and permeabilized BEL-A erythroblasts, showing IgG isotype control (black) and c-MYC (9E10) labeling. Figure 7B: Flow cytometry histograms of IgG isotype control (black) and c-MYC (9E10) labeling from fixed and permeabilized BEL-A erythroblasts (UT) from untransduced (UT) control cells (turquoise) and corresponding Prx-transduced cell lines (orange). Figure 7C: Bar graphs showing Prx overexpression from indicated cell lines at day 0 (d0) and day 12 (d12) of differentiation. Day 12 samples were gated based on Hoechst staining to obtain data on day 12 nucleated (d12n) cells and day 12 reticulocytes (d12r). Mean fluorescence intensity (MFI) was normalized to background c-MYC labeling 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 permeabilized cells were labeled with IgG isotype control (black) or c-MYC (9E10) antibody and gated based on Hoechst staining to obtain data on nucleated (d12n) cells and reticulocytes (d12r) on day 12.

[0027] Figure 8: Overexpression of glutathione peroxidase in immortalized BEL-A erythroblasts and reticulocytes. Figure 8A: Cell lysates (1 × 10⁻⁶) obtained from the indicated cell lines labeled with monoclonal antibodies against GPx1, GPx4, and GAPDH (loaded control). 6Representative protein blots from untransduced (UT) control cells (turquoise) and corresponding GPx-transduced cell lines (orange) in fixed and permeabilized BEL-A erythroblasts, with IgG isotype control (black) and c-MYC (9E10) staining. Figure 8C: Flow cytometry analysis of GPx construct expression in samples at day 12 (n=3). Fixed and permeabilized cells were labeled with IgG isotype control (black) or c-MYC (9E10) antibody and gated based on Hoechst staining to obtain data on nucleated (d12n) cells and reticulocytes (d12r) at day 12. Figure 8D: Bar graphs showing GPx overexpression from indicated cell lines at day 0 (d0) and day 12 (d12) of differentiation. Day 12 samples were gated using Hoechst staining to obtain data on day 12 nucleated (d12n) cells and day 12 reticulocytes (d12r). Mean fluorescence intensity (MFI) was normalized to background c-MYC labeling in UT control cells (n=3, mean ± SD). Figure 8E: GPx4 cells analyzed by intracellular flow cytometry after 6 hours of treatment with 5 μM MG132, 10 μM leupeptin, 10 μM bardoxolone, mediator control (blue), or untreated (UT) (grey). U73C Bar graph of overexpression. Mean fluorescence intensity (MFI) was normalized to background c-MYC markers in UT control cells. Data are presented as mean ± SD, n=3. p < 0.05, p < 0.01, p < 0.001, Two-tailed unpaired Student's t-test.

[0028] Figure 9: Overexpression of the glutathione peroxidase 4 construct in immortalized BEL-A erythroblasts and reticulocytes. Figure 9A: Flow cytometry histograms of IgG isotype control (black) and c-MYC (9E10) staining in fixed and permeabilized BEL-A erythroblasts from untransduced (UT) control cells (turquoise) and the corresponding GPx4 transduced cell lines. Figure 9B: Cell lysates (1×10⁻⁶) obtained from the indicated cell lines labeled with monoclonal antibodies against GPx4 and GAPDH (loaded control). 6Representative protein blots of GPx4 constructs in samples from day 12. Figure 9C: Flow cytometry analysis of GPx4 construct expression in day 12 samples (n=3). Fixed and permeabilized cells were labeled with IgG isotype control (black) or c-MYC (9E10) antibody and gated based on Hoechst staining to obtain data on nucleated (d12n) cells and reticulocytes (d12r) on day 12. Figure 9D: Bar graphs showing GPx overexpression from indicated cell lines at day 0 (d0) and day 12 (d12) of differentiation. Day 12 samples were gated based on Hoechst staining to obtain data on nucleated (d12n) cells and reticulocytes (d12r) on day 12. Mean fluorescence intensity (MFI) was normalized to background c-MYC labeling in UT control cells (n=3, mean ± SD).

[0029] Figure 10 Overexpression of the c-MYC-tagged glutathione peroxidase 1 construct in immortalized BEL-A erythroblasts and reticulocytes. Flow cytometry histograms of IgG isotype control (black) and c-MYC (9E10) staining in fixed and permeabilized expanded BEL-A erythroblasts from untransduced (UT) control cells (turquoise) and the corresponding GPx1-transduced cell line (orange). Cells transduced using a construct selectable by puromycin. Figure 10 B: Cell lysates (1 × 10⁻⁶) obtained from the indicated cell lines labeled with monoclonal antibodies against GPx1 and GAPDH (loaded control). 6 A representative protein blot (of a cell). Figure 10 C: Flow cytometry histogram of fixed and permeabilized BEL-A cells labeled with IgG isotype control (black) or c-MYC (9E10) on day 11 differentiating, the BEL-A cells being gated to reticulocytes based on Hoechst staining to obtain information about nucleated (d11) cells on day 11. 有核 ) cells and day 11 reticulocytes (d11) 网织红细胞 (Data).

[0030] Figure 11: Overexpression of glutathione peroxidase 4 (MYC-sGPx4 3'UTR 6KR) in CD34+ erythroblasts and reticulocytes. The MYC-tagged sGPx4 3'UTR 6KR was cloned into an optional vector (puromycin), transduced into CD34+ cells, and then differentiated to generate reticulocytes uniformly expressing this protein. Figure 11A Cell lysates (1×10⁻⁶) obtained from indicator cell lines of CD34+ erythroid culture on days 7 and 10, and filtered reticulocytes (day 20).6 Representative protein blots were obtained from 100 cells. One untransduced (UT) sample and two samples expressing MYC-sGPx4 3'UTR 6KR (samples 1 and 2) were analyzed. The membranes were labeled with monoclonal antibodies against GPx4 and GAPDH (loaded control). Figure 11B Flow cytometry histograms of IgG isotype control (black) and c-MYC (9E10) staining in fixed and permeabilized CD34+ cells on days 7 and 10 of differentiation, and in filtered reticulocytes (day 20). Samples and time points correspond to the same samples and time points shown in Figure A, and data are presented for untransduced (UT) control cells (turquoise) and MYC-GPx4 3'UTR 6KR cultures 1 (blue) and 2 (orange).

[0031] Figure 12 A: Schematic diagram of two human alanine aminotransferase 2 (AAT2) constructs with (huAAT2 FL) and without (huAAT2 N27del) mitochondrial targeting peptides (highlighted in yellow). Figure 12 B: Human AAT2 (huAAT2) protein expression during differentiation in CD34+ hematopoietic stem cells. CD34+ hematopoietic stem cells were transduced with full-length (FL) huAAT2 or N-terminally truncated huAAT2 (N27del) (with 27 amino acids of the mitochondrial-targeting peptide removed). Positively transduced cells were selected and differentiated to generate reticulocytes using puromycin antibiotics. Protein expression levels were determined by Western blotting on day 8 (d8), day 20 (d20), and on a filtered, pure d20 reticulocyte population (d20r). Figure 12 C: AAT2 protein expression for each construct relative to the initiation (d8) level in cultured reticulocytes (d20r) derived from CD34. Protein blot bands were quantified and normalized accordingly to d8 initiation protein expression for each AAT2 construct.

[0032] Figure 13: Overexpression of the glutathione peroxidase 1 construct in CD34+ erythroblasts and reticulocytes. GPx1 with the MYC tag... U49C The GPx1 3'UTR was cloned into an optional vector (puromycin), transduced into CD34+ cells, and then differentiated into reticulocytes that uniformly express these proteins. Figure 13A Cell lysates (1×10⁻⁶) obtained from indicated transduced cells and filtered reticulocytes (day 20) from CD34+ erythroid cultures from two separate donors on days 7, 10, and 14. 6Representative protein blots (from 100 cells) were obtained. The membranes were labeled with monoclonal antibodies against GPx1 and GAPDH (with a loading control). Figure 13B Flow cytometry histograms of IgG isotype control (black) and c-MYC (9E10) staining in fixed and permeabilized CD34+ cells at days 7, 10, and 14 of differentiation, and in filtered reticulocytes (day 20). Samples correspond to the same two donors and time points as shown in Figure A, which presents untransduced (UT) control cells (turquoise) and MYC-GPx1 expressing cells. U49C Data for cultures (orange) or MYC-GPx1 3'UTR (blue).

[0033] Sequence List Description

[0034] The sequences are identified and described in the table below. Detailed Implementation

[0035] General Public

[0036] All publications, patents, and patent applications cited herein, whether above or below, are incorporated herein by reference in their entirety. All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference. Where any cited publication, patent, or patent application conflicts with any disclosure contained herein, this specification is intended to substitute for and / or give precedence to any such conflicting content.

[0037] The invention has been described with reference to specific embodiments and certain accompanying drawings, but is not limited thereto; rather, it is limited only by the claims. No reference numerals in the claims should be construed as limiting the scope. It should be understood, of course, that not all aspects or advantages may be achieved according to any particular embodiment of the invention. Therefore, for example, those skilled in the art will recognize that the invention may be embodied or practiced in a manner that achieves or optimizes one or more advantages as taught herein, without necessarily achieving other aspects or advantages that may be taught or suggested herein.

[0038] When read in conjunction with the accompanying drawings, the organization and operation of the invention, as well as its features and advantages, can be best understood by referring to the following detailed description. Aspects and advantages of the invention will become apparent and elucidated by referring to one or more embodiments described below. References to “some embodiments” or “preferred embodiments” throughout this specification mean that a particular feature, structure, or characteristic described in connection with said embodiment is included in at least one embodiment of the invention. Therefore, the phrases “some embodiments” or “preferred embodiments” appearing throughout this specification do not necessarily refer to the same embodiment, but may refer to the same embodiment. Similarly, it should be understood that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment or its description in order to simplify this disclosure and aid in understanding one or more aspects of the various inventive aspects. However, this method of disclosure should not be construed as reflecting an intention that the claimed invention requires more features than those expressly recited in each claim. Rather, as reflected in the appended claims, inventive aspects lie in fewer than all features of a single embodiment disclosed above.

[0039] Furthermore, unless otherwise expressly specified, the singular forms “a (a, an)” and “described” as used in this specification and the appended claims include a plurality of indicators. Thus, for example, a reference to “polypeptide” includes two or more polypeptides, a reference to “fusion protein” includes two or more such proteins, a reference to “test” includes two or more tests, a reference to “kit” includes two or more kits, a reference to “method” includes two or more methods, and so on.

[0040] Throughout the discussion in this article, the standard single-letter codes for amino acids are used. They 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).

[0041] Unless explicitly defined herein, all terms used herein have the same meaning as understood by one of ordinary skill in the art. For definitions and terminology in this field, practitioners may specifically refer to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th Edition, 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). The definitions provided herein should not be construed as having a scope less than that understood by one of ordinary skill in the art.

[0042] When referring to measurable values ​​such as percentages or quantities, the term “about” as used herein is intended to cover variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% relative to a specified value, as such variations are suitable for performing the disclosed methods. Any statement including the term “about” herein includes the same characteristics without that term. For example, a variant of SEQ ID NO: 35 having at least “about” 80% identity or homology with the sequence SEQ ID NO: 35 over its entire length includes a variant of SEQ ID NO: 35 having at least 80% identity or homology with the sequence SEQ ID NO: 35 over its entire length.

[0043] Throughout this specification and claims, the terms “comprise” and “contain”, as well as variations thereof (e.g., “comprising” and “comprises”), mean “including but not limited to” and do not exclude other components, integers, or steps.

[0044] Preferred features of each aspect of the invention may be described as in conjunction with any other aspect. Within the scope of this application, it is expressly intended that the various aspects, embodiments, examples, and alternatives set forth in the foregoing paragraphs, claims, and / or the following description and drawings, particularly their respective features, may be employed independently or in any combination. That is, all embodiments and / or features of any embodiment may be combined in any manner and / or combination unless such features are incompatible.

[0045] In the context of this invention, "detectable level" may include the detectable level of mRNA encoding a relevant biomarker (e.g., CD71) and / or the detectable level of a biomarker protein (e.g., CD71 protein). Preferably, "detectable level" includes the detectable level of a protein (e.g., CD71 protein). "Detectable level" may include the detectable level of both mRNA encoding a relevant biomarker (e.g., CD71) and the detectable level of a biomarker protein (e.g., CD71 protein). "Detectable level" may include the detectable activity of a relevant biomarker (e.g., CD71, which is a receptor for transferrin). Throughout this document, the term "level" is used interchangeably with "amount," "activity," or "amount and activity."

[0046] The presence of mRNA and / or protein can be detected using any conventional method in the art. Such methods include immunofluorescence, immunohistochemistry, Western blotting, quantitative polymerase chain reaction (qPCR), reporter gene assays, enzyme-linked immunosorbent assays (ELISA), microscopy, flow cytometry, enzyme staining, dye incorporation, chemiluminescent oxygen detection reagents, fluorescence assays, fluorescence in situ hybridization (FISH), and alkaline phosphatase assays. Those skilled in the art can use known assays to detect the activity of any specific biomarker. Suitable assays are described in the examples.

[0047] In the context of this invention, "genetically engineered" means that cells or cell populations have been genetically modified to express relevant factors, such as trehalose transporters, antioxidant proteins, or intrinsically disordered proteins (IDPs). Genetically engineered cells or populations express detectable levels of relevant factors, such as trehalose transporters, antioxidant proteins, or IDPs. The embodiments described in this paragraph also apply to the variant proteins discussed below, which contain one or more mutations that increase their levels in the cytosol of the cells.

[0048] Generally, "genetically engineered" means that the genetic material of a cell or cell population has been altered to express a relevant factor, such as a trehalose transporter, antioxidant protein, or IDP. When "genetically engineered," the cell or cell population typically contains exogenous polynucleotides encoding and configured to express the relevant factor, such as a trehalose transporter, antioxidant protein, or IDP. Those skilled in the art can identify such exogenous polynucleotides in the cell or cell population. Exogenous polynucleotides may contain any control sequences discussed below, and / or may be part of any vector discussed below. The embodiments described in this paragraph also apply to variant proteins discussed below, which contain one or more mutations that increase their level in the cytosol of the cell.

[0049] Prior to genetic engineering, cells or populations preferably do not express detectable levels of relevant biomarkers, such as trehalose transporters, antioxidant proteins, or IDPs, or express undetectable levels. Prior to genetic engineering, cells or populations may express detectable levels of relevant biomarkers, such as trehalose transporters, antioxidant proteins, or IDPs. Detectable levels may be low due to the phenotype or epigenetics of the cells or population, or due to degradation or secretion of the factors from the cells. Cells or populations may be genetically engineered to overexpress relevant factors. Cells or populations may be genetically engineered to express increased levels of relevant factors. The embodiments described in this paragraph also apply to proteins discussed below before they are modified to include one or more mutations that increase their levels in the cytosol of cells. These may be referred to as “unmodified” or “non-mutated” proteins.

[0050] In the context of this invention, "genetically engineered to overexpress" means that cells or cell populations have been genetically modified to express increased levels of a relevant factor, such as antioxidant proteins and / or IDPs. The increased level can be an increased amount and / or increased activity. The increased level is typically an increased detectable level. The increased level is usually compared to the level in the cells or population before genetic engineering. The increased level is also typically compared to the level in cells or populations that have not undergone genetic engineering (such as corresponding wild-type cells or populations). The embodiments described in this paragraph also apply to variant proteins discussed below, which contain one or more mutations that increase their level in the cytosol of cells.

[0051] Biomarkers may be overexpressed in any amount. Biomarkers (such as antioxidant proteins and / or IDPs) may be overexpressed at least about 5%, such as 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%, or at least about 100%. Biomarkers (such as antioxidant proteins and / or IDPs) may be overexpressed at least about 2-fold, such as at least about 5-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, at least about 1000-fold, at least about 5000-fold, at least about 10,000-fold, or more. The embodiments described in this paragraph also apply to the variant proteins discussed below, which contain one or more mutations that increase their level in the cytosol of cells. The level of the variant protein may be increased by any amount described in this paragraph.

[0052] Cells or populations may be genetically modified to express biomarkers, such as antioxidant proteins and / or IDPs, at levels increased by at least about 5% (e.g., 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%, or at least about 100%). Cells or populations may be genetically modified to express biomarkers, such as antioxidant proteins and / or IDPs, at levels increased by at least about 2-fold (e.g., at least about 5-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 500-fold, at least about 1000-fold, at least about 5000-fold, at least about 10,000-fold, or more). The embodiments described in this paragraph also apply to variant proteins discussed below, which contain one or more mutations that increase their levels in the cytosol of cells. The level of variant proteins may be increased by any amount described in this paragraph.

[0053] In the context of this invention, cells or populations are genetically engineered to overexpress relevant factors, such as antioxidant proteins and / or IDPs, in their cytosol. This means an increase in the levels of relevant factors (such as antioxidant proteins and / or IDPs) in the cytosol of cells or populations. This can be achieved by genetically engineering cells or populations to reduce the transport / transport of relevant factors to intracellular organelles (such as mitochondria) and / or to reduce the secretion of relevant factors. This invention provides variant proteins modified to reduce their intracellular transport / transport and / or secretion. Genetically engineering cells to express these variant proteins typically results in their overexpression in the cytosol of the cells. The embodiments described in this paragraph are equally applicable to the variant proteins discussed below, which contain one or more mutations that increase their levels in the cytosol of the cells.

[0054] lyophilized composition

[0055] This invention provides lyophilized compositions. The term "lyophilized" (or "freeze-dried") is well understood in the art. The lyophilized compositions comprise cell populations and lyophilization protectants. In some embodiments, the lyophilized compositions consist essentially of cell populations and lyophilization protectants, or of the same.

[0056] In some embodiments, the lyophilized composition may contain additional reagents. For example, the lyophilized composition may contain one or more reagents selected from buffers, stabilizers, antimicrobial agents, antioxidants, free radical scavengers, solubilizers, tonicating agents, and / or surfactants.

[0057] In some embodiments, the lyophilized composition consists essentially of a cell population, a lyophilization protectant, and one or more reagents selected from buffers, antioxidants, and / or free radical scavengers, or is composed of the same.

[0058] In some embodiments, the lyophilized composition does not contain non-cellular polymers. "Cellular polymers" are polymers of cells in the lyophilized composition. In other words, the lyophilized composition does not contain any polymers other than those present in the lyophilized composition as part of a cell population. In some embodiments, the lyophilized composition does not contain synthetic or non-biological polymers. In some embodiments, the lyophilized composition does not contain non-biological or synthetic polymers, wherein the non-biological or synthetic polymers are polymers that enable active loading of cells with cryoprotectants and / or lyophilization protectants. In some embodiments, the lyophilized composition does not contain non-biological or synthetic polymers, wherein the non-biological or synthetic polymers are polymers that temporarily permeate cell membranes. In some embodiments, the lyophilization composition does not contain cell membrane permeators, preferably synthetic and / or non-biological cell membrane permeators. In some embodiments, the lyophilization composition does not contain polymers 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. The biopolymers PLP, PV-50, PL-50, PP-30, PP-50, PP-60, and PP-75 can be prepared by 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 GB2502016.

[0059] In some embodiments, the lyophilized composition contains less than about 10% by weight (wt%) of residual water. Preferably, the lyophilized composition contains less than about 7 wt%, less than about 5 wt%, less than about 2 wt%, or less than about 1 wt% of residual water.

[0060] Cell population

[0061] In some implementations, the cell population comprises all the cells in the lyophilized composition.

[0062] The cell population comprises at least about 10% reticulocytes. 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% reticulocytes. For therapeutic applications, it is generally preferred that the cell population comprises at least about 60% reticulocytes, more preferably at least about 80%. In a particularly preferred embodiment, the cell population comprises at least about 99% reticulocytes. In some embodiments, the majority of the cell population comprises reticulocytes.

[0063] In some implementations, the cell population comprises at least about 1 × 10⁻⁶ cells. 5 At least approximately 1×10 6 At least approximately 1×10 7 At least approximately 1×10 8 At least approximately 1×10 9 Or at least about 1×10 10 Each cell.

[0064] In some implementations, the cell population comprises at least about 1 × 10⁻⁶ cells. 8 At least approximately 1×10 9 At least approximately 1×10 10 At least approximately 1×10 11 At least approximately 1×10 12 At least approximately 1×10 13 Or at least about 1×10 14 One reticulocyte.

[0065] The identity of the cells that make up the remainder of the cell population is not particularly restricted. The cells that make up the remainder of the cell population can be a single cell type or a mixture of different cell types.

[0066] In some embodiments, the cell population comprises, consists of, or is a mammalian cell population. The cell population can be a human, dog, cat, primate, horse, rat, rat, rodent, cattle, pig, or sheep 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, consists of, or is a wild-type cell population.

[0067] In some embodiments, the cell population comprises, or is composed of, intact cells in the composition. "Intact cells" are generally viable cells. In other words, they are cells that have not yet ruptured. This can be measured using conventional methods. For example, cells can be evaluated on a flow cytometer, as described in the examples.

[0068] In some embodiments, the cell population comprises fractions of whole blood, substantially composed of or consisting of them. In some embodiments, the cell population comprises cell fractions isolated from whole blood, substantially composed of or consisting of them. For reference, whole blood typically consists of a cell population containing 0.5% to 2.5% reticulocytes in adults and 2% to 6% reticulocytes in infants. In some embodiments, the cell population comprises RBC fractions of whole blood, substantially composed of or consisting of them, said RBC fractions having been processed to enrich the proportion of reticulocytes to at least about 10%.

[0069] In some embodiments, the cell population is not a fraction of whole blood. Those skilled in the art can determine whether a cell population is a fraction of whole blood using standard methods in the art. For example, those skilled in the art can assay the cell population to detect markers of whole blood, such as compounds found in whole blood. In some embodiments, the cell population does not contain a fraction of whole blood. In some embodiments, the cell population does not contain reticulocytes isolated from whole blood. In some embodiments, the reticulocytes are not isolated from whole blood. In these embodiments, the cell population may still contain cells derived from whole blood components, such as reticulocyte cultures derived from stem cells or induced pluripotent stem cells isolated from whole blood.

[0070] In some embodiments, cell populations are generated through cell culture. The cell culture can be a derivative of an erythroid progenitor population. The erythroid progenitor population is not particularly limited and can contain any cells capable of maturing into reticulocytes. The terms "erythroid progenitor" or "erythroid progenitor" can be used to refer to cells at different stages along the differentiation / maturation pathway. The term "erythroid progenitor" generally refers to a cell with a nucleus (i.e., before enucleation has begun). Erythroid progenitors can be stem cells, hematopoietic stem cells, induced pluripotent stem cells (iPSCs), immortalized erythroid cell lines, or erythroblasts. Preferably, the erythroid progenitor is a CD34+ cell and / or a BEL-A cell. For example, the erythroid progenitor can be a CD34+ cell derived from stem cells or iPSCs. In cases where the erythroid progenitor is a non-CD34+ stem cell or iPSC or immortalized cell, the cell type or lineage can produce erythroblasts, which then differentiate into reticulocytes.

[0071] reticulocytes

[0072] The term "reticulocyte" is well understood in the art. However, for the sake of detail, "reticulocyte" refers to a cell derived from enucleated but not yet mature erythroid progenitor cells. Reticulocytes are generally understood as immature RBCs, known to be larger than mature RBCs, they are typically CD71 positive, and they have higher levels of most membrane proteins than mature RBCs. They may also contain organelle remnants and mitochondria, which are usually lost during reticulocyte maturation.

[0073] Enucleated erythroid cells, including reticulocytes and mature RBCs, display the erythrocyte band 3 protein (also known as anion exchanger 1 (AE1) or solute carrier family 4 member 1 (SLC4A1)) on their surface. In the production of enucleated erythroid cells, they are typically distinguished by the presence of erythrocyte band 3 and by the absence of a nucleus. Wild-type reticulocytes are typically band 3 positive and CD71 positive. In contrast, mature RBCs are band 3 positive and CD71 negative.

[0074] Reticulocytes preferably have a cell volume of at least about 100 fL, more preferably at least about 110 fL. Reticulocytes 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. Reticulocytes preferably have a cell volume of about 100 fL to about 150 fL (such as about 100 fL to about 130 fL or about 110 fL to about 120 fL). Reticulocytes may have a cell volume of about 115 fL. In contrast, mature RBCs typically have a cell volume in the range of about 50 to about 98 fL (such as about 50 to about 80 fL or about 86 to about 98 fL). Those skilled in the art can determine cell volume using standard techniques in the art, such as using microscopy to determine cell size and thus calculate cell volume, or using clinical instruments designed to measure cell volume.

[0075] Reticulocytes preferably express detectable levels of CD71. Reticulocytes are preferably CD71 positive. Reticulocytes preferably express detectable levels of band 3 and CD71. Reticulocytes are preferably band 3 and CD71 positive. Reticulocytes preferably do not contain a nucleus. Reticulocytes preferably (a) express detectable levels of CD71 or are CD71 positive, (b) express detectable levels of band 3 or are band 3 positive, (c) do not contain a nucleus, or any combination thereof, such as (a), (b), (c), (a) and (b), (a) and (c), (b) and (c), or (a), (b) and (c).

[0076] 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 reticulocytes (a) express a detectable level of CD71 or are CD71 positive, (b) express a detectable level of band 3 or are band 3 positive, (c) do not contain 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).

[0077] The detectable level has been defined above. A person skilled in the art can use standard techniques in the art (such as those described above) to determine the presence or absence of CD71 and / or band 3 on cells. A person skilled in the art can use standard techniques in the art (such as microscopy) to determine the presence or absence of a cell nucleus.

[0078] In some embodiments, the reticulocytes comprise mammalian reticulocytes, are substantially composed of mammalian reticulocytes, or are mammalian reticulocytes. The reticulocytes can be human, canine, cat, primate, horse, rat, rat, rodent, bovine, swine, or sheep reticulocytes. In a preferred embodiment, the reticulocytes comprise human reticulocytes, are substantially composed of human reticulocytes, or are composed of human reticulocytes or are human reticulocytes. In some embodiments, the reticulocytes comprise wild-type reticulocytes, are substantially composed of wild-type reticulocytes, or are wild-type reticulocytes. In some embodiments, the reticulocytes comprise genetically modified reticulocytes, are substantially composed of genetically modified reticulocytes, or are genetically modified reticulocytes. In some embodiments, the reticulocytes comprise non-wild-type reticulocytes, are substantially composed of non-wild-type reticulocytes, or are composed of or are entirely non-wild-type reticulocytes. In a particularly preferred embodiment, the reticulocytes are cultured reticulocytes.

[0079] In some embodiments, reticulocytes are fractions of whole blood. However, it is generally preferred that reticulocytes are not fractions of whole blood. In some embodiments, reticulocytes are not isolated from whole blood. In embodiments where reticulocytes are not fractions of whole blood or are not isolated from whole blood, reticulocytes may still be derived from components of whole blood, such as reticulocyte cultures derived from stem cells isolated from whole blood.

[0080] Typically, the percentage of reticulocytes in whole blood is generally low. Therefore, one way to prepare a large population of reticulocytes is by culturing them from primary cell sources (such as adult peripheral blood, cord blood, bone marrow) or from immortalized cell sources (such as iPSCs, or cell lines such as BEL-A cell lines immortalized with E6 and E7). In some embodiments, reticulocytes are derivatives of a population of erythroid progenitor cells. The erythroid progenitor cell population is not particularly limited and may contain any cells capable of maturing into reticulocytes. Erythroid progenitor cells can be stem cells, hematopoietic stem cells, induced pluripotent stem cells (iPSCs), immortalized erythroid cell lines, or erythroblasts. Preferably, the erythroid progenitor cells are CD34+ cells and / or BEL-A cells. For example, erythroid progenitor cells can be CD34+ cells derived from stem cells or iPSCs. In the case where the erythroid progenitor cells are stem cells or iPSCs, the cell lineage produces erythroblasts, which then differentiate into reticulocytes.

[0081] Those skilled in the art will understand that, in the context of this invention, reticulocytes or mature RBCs themselves cannot be genetically engineered because they do not contain a nucleus. Such genetically engineered cells are typically derived from erythroid progenitor cells (also known as immature cells or precursor cells) that have been genetically engineered as needed.

[0082] 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 reticulocytes are intact. In some embodiments, after reconstitution, 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 reticulocytes are intact. "Intact" means that the reticulocytes are viable. In other words, the cells have not ruptured. Those skilled in the art know how to test the percentage of intact cells. For example, the lyophilized composition can be reconstituted in a liquid medium and analyzed by flow cytometry, gating intact reticulocytes based on forward / lateral scattering. Alternatively, the cells can be centrifuged into a precipitate, and the amount of free hemoglobin (released upon cell lysis) can be measured in the supernatant.

[0083] 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 reticulocytes survive during remodeling. "Surviving during remodeling" generally means that the reticulocytes are intact and viable. In other words, the cells have not ruptured. Those skilled in the art know how to test the percentage of intact and viable cells.

[0084] In some embodiments, reticulocytes contain 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 total number of hemoglobin and methemoglobin molecules. In some embodiments, reticulocytes contain 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% of methemoglobin molecules relative to the total number of hemoglobin and methemoglobin molecules. In some embodiments, reticulocytes contain 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 number of hemoglobin molecules relative to the corresponding fresh cell population. "Corresponding" means a cell population that can be used to determine the proportion of hemoglobin molecules oxidized to methemoglobin during the freeze-drying process. Typically, this refers to a corresponding cell population containing fresh cells prior to freeze-drying, such as freshly prepared cell cultures or freshly harvested cells. In some embodiments, the reticulocytes contain 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 hemoglobin molecules relative to the cell population prior to freeze-drying. Spectrophotometry can be used to assess the ratio of hemoglobin to methemoglobin. In some embodiments, the reticulocytes 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 the oxygen-carrying capacity relative to circulating wild-type reticulocytes.

[0085] Lyophilization protectant

[0086] A lyophilization protectant is an agent that can protect reticulocytes during the lyophilization process and / or during storage as a lyophilized composition. The term "lyophilization protectant" is well understood in the art. Lyophilization protectants can protect cell viability, such as by protecting cells from rupture and helping them remain intact, and / or by protecting hemoglobin within reticulocytes from oxidation. In some embodiments, lyophilization protectants can protect cell viability and prevent hemoglobin oxidation.

[0087] In some implementations, the lyophilization protectant can be a water displacement effector. A "water displacement effector" is a reagent that can displace water from biological tissues and materials, such as cells. For example, a water displacement effector can displace water molecules bound to the surface of a cell membrane.

[0088] In some embodiments, the lyophilization protectant comprises one or more of the following, is substantially composed of one or more of the following, is composed of one or more of the following or is one or more of the following: (a) sugar, (b) antioxidant and (c) intrinsically disordered protein (IDP), such as (a), (b), (c), (a) and (b), (a) and (c), (b) and (c), or (a), (b) and (c).

[0089] carbohydrate

[0090] In some embodiments, the lyophilization protectant comprises a sugar. The sugar can be a monosaccharide, oligosaccharide, or polysaccharide. In some embodiments, the sugar is a monosaccharide or a disaccharide. In a preferred embodiment, the lyophilization protectant is a disaccharide.

[0091] In some embodiments, the freeze-drying protectant is selected from trehalose, sucrose, fructose, glucose, mannitol, sorbitol, arabinose, and / or ribose.

[0092] In a particularly preferred embodiment, the lyophilization protectant comprises trehalose, is substantially composed of trehalose, or is composed of or is trehalose. At least a portion of the trehalose is intracellular. At least a portion of the trehalose is preferably present inside reticulocytes.

[0093] In some implementations, reticulocytes are genetically engineered to express trehalose transporter proteins or variants thereof on their surface. This promotes trehalose uptake by reticulocytes, which is discussed in more detail below.

[0094] antioxidants

[0095] In some embodiments, the lyophilization protectant comprises an antioxidant, is substantially composed of an antioxidant, or is an antioxidant. In some embodiments, the lyophilization protectant comprises an antioxidant protein (or "protein antioxidant"), is substantially composed of an antioxidant protein (or "protein antioxidant"), or is an antioxidant protein (or "protein antioxidant"). In some embodiments, the lyophilization protectant protein comprises or is selected from glutathione peroxidase (GPx) protein or peroxidase (Prx) protein. In some embodiments, the antioxidant is selected from glutathione peroxidase 1 (GPx1), GPx2, GPx3, GPx4, GPx5, GPx6, GPx7, GPx8, peroxidase 1 (Prx1), Prx2, Prx3, Prx4, Prx5, Prx6, and variants thereof. In some embodiments, the antioxidant is selected from GPx1, GPx4, Prx1, Prx2, Prx6, and variants thereof.

[0096] Preferred variants of the protein are discussed below. Any of these can be used in the lyophilized compositions of the present invention. For example, antioxidants can be modified to increase their expression in the cytosol of cells, for example by mutation at one or more predicted ubiquitination sites, by one or more inactivating mutations in their signal peptide, and / or by one or more inactivating mutations in their mitochondrial targeting peptide. Antioxidants are preferably human. Antioxidants can also be non-human antioxidants that have been humanized for expression in human cells.

[0097] In a preferred embodiment, GPx1 or a variant thereof comprises, or consists of, the sequence shown in SEQ ID NO: 2, 4 or 6, or a variant thereof. In a preferred embodiment, GPx1 or a variant thereof comprises, or consists of, the sequence shown in SEQ ID NO: 2, 4, 6 or 55, or a variant thereof.

[0098] In a preferred embodiment, GPx4 or a variant thereof comprises, or consists of, the sequence shown in SEQ ID NO: 8, 12, 14 or 16, or a variant thereof. In the most preferred embodiment, GPx4 or a variant thereof comprises, or consists of, the sequence shown in SEQ ID NO: 16, or a variant thereof.

[0099] In a preferred embodiment, Prx1 or a variant thereof comprises, or consists of, the sequence shown in SEQ ID NO: 18 or 20 or a variant thereof.

[0100] In a preferred embodiment, Prx2 or a variant thereof comprises, or consists of, the sequence shown in SEQ ID NO: 22 or 24, or a variant thereof.

[0101] In a preferred embodiment, Prx6 or a variant thereof comprises, or consists of, the sequence shown in SEQ ID NO: 26 or 28, or a variant thereof.

[0102] In a preferred embodiment, GPx2 or a variant thereof comprises, or consists of, the sequence shown in SEQ ID NO: 38 or a variant thereof.

[0103] In a preferred embodiment, GPx3 or a variant thereof comprises, or consists of, the sequence shown in SEQ ID NO: 39 or a variant thereof.

[0104] In a preferred embodiment, GPx5 or a variant thereof comprises, or consists of, the sequence shown in SEQ ID NO: 40 or a variant thereof.

[0105] In a preferred embodiment, GPx6 or a variant thereof comprises, or consists of, the sequence shown in SEQ ID NO: 41 or a variant thereof.

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

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

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

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

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

[0111] Antioxidant proteins can be produced independently and added to the lyophilized compositions of the present invention. Cell populations and / or reticulocytes are preferably genetically engineered to overexpress antioxidant proteins in their cytosol. This will be discussed in more detail below.

[0112] Without being bound by theory, it should be understood that the GPx and Prx families generally protect against reactive oxygen species (ROS) and also offer protection against lipid peroxidation associated with freeze-drying and remodeling processes. One source of cell damage is stress from ROS-induced stress, particularly during remodeling. Furthermore, antioxidant proteins are expected to protect hemoglobin from oxidation. Another benefit is that antioxidant proteins are also expected to help protect reticulocytes during storage (even without freeze-drying) and also help reticulocytes and subsequent mature RBCs persist in circulation for a longer period, as ROS play a role in erythrocyte senescence. It may also have a therapeutic effect in reducing wound damage, as ROS increases in wounds, so blood cells with higher antioxidant levels may be protective.

[0113] Intrinsic disordered proteins (IDPs)

[0114] In some embodiments, the lyophilization protectant 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 "inherently disordered protein" is well understood and generally refers to a protein in which all or part of the protein lacks a three-dimensional structure or lacks the tendency to form a three-dimensional structure. In some embodiments, the freeze-drying protectant comprises insect and / or microanimal IDPs. In some embodiments, the freeze-drying protectant comprises tardigrade-specific proteins and / or late embryogenesis abundant (LEA) proteins or equivalent proteins. Tardigrade-specific proteins may also be referred to as tardigrade-derived IDPs.

[0116] In some embodiments, the lyophilization protectant comprises cytoplasm-rich heat-soluble (CAHS) proteins. CAHS proteins are known to help microorganisms such as tardigrades survive dehydration. In some embodiments, the lyophilization protectant comprises CAHS1 and / or CAHS3 proteins, such as tardigrade CAHS1 and / or CAHS3 proteins. In some embodiments, the lyophilization protectant comprises one or more of (i) CAHS1, (ii) CAHS2, and (iii) CAHS3 proteins, such as tardigrade CAHS1, (ii) tardigrade CAHS2, and (iii) tardigrade CAHS3 proteins. The lyophilization protectant may comprise (i), (ii), (iii), (i) and (ii), (i) and (iii), (ii) and (iii), or (i), (ii) and (iii).

[0117] In some embodiments, the freeze-drying protectant comprises late embryogenesis-rich (LEA) proteins. LEA proteins are understood to prevent protein aggregation during drying and / or osmotic stress from low temperatures. In some embodiments, the freeze-drying protectant comprises insect or nematode (preferably nematode) LEA proteins.

[0118] LEA proteins are characterized by an 11-mer motif and are known to reduce protein aggregation during dehydration. LEA proteins can be subdivided into Group 1, Group 2, and Group 3 based on species, with Group 3 proteins expressed in animals. The inventors identified two Group 3 LEA proteins, found in *Chironoptera vannamei* (a species of mosquito). Polypedilum vanderplanki PvLEA22 expressed in ) and in oat nematode ( Aphelenchus avenae AavLEA is expressed in ).

[0119] In some embodiments, the lyophilization protectant comprises Group 3 LEA proteins, is substantially composed of Group 3 LEA proteins, or is a Group 3 LEA protein. In some embodiments, the lyophilization protectant comprises PvLEA and / or AavLEA, is substantially composed of PvLEA and / or AavLEA, or is a PvLEA and / or AavLEA.

[0120] 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 variants 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 variants thereof.

[0121] IDP can be generated separately and added to the lyophilized composition of the present invention. Cell populations and / or reticulocytes are preferably genetically engineered to overexpress IDP. This will be discussed in more detail below.

[0122] General Instructions

[0123] In some embodiments, the lyophilization protectant may comprise a protein-based lyophilization protectant. In these embodiments, the reticulocytes may be derivatives of erythroid progenitor cells that have been genetically engineered to express the protein-based lyophilization protectant.

[0124] In some embodiments, the lyophilization protectant comprises a single reagent, and is essentially composed of or consists of a single reagent. In other embodiments, the lyophilization protectant comprises more than one reagent.

[0125] In some implementations, the lyophilization protectant comprises one or more of the following: a. Sugars selected from trehalose, sucrose, fructose, glucose, mannitol, sorbitol, arabinose and / or ribose; b. Antioxidant proteins selected from GPx1, GPx2, GPx3, GPx4, GPx5, GPx6, GPx7, GPx8, Prx1, Prx2, Prx3, Prx4, Prx5, Prx6 and their variants; and c. An IDP selected from CAHS1, CAHS3, PPLEA and / or AavLEA, or an IDP selected from CAHS1, CAHS2, CAHS3, PPLEA and / or AavLEA.

[0126] The freeze-drying protectant may contain a, b, c, a and b, a and c, b and c, or a, b and c.

[0127] In some embodiments, the lyophilization protectant is present at a concentration greater than about 100 mM, greater than about 200 mM, greater than about 300 mM, greater than about 400 mM, or greater than about 500 mM. In some embodiments, the lyophilization protectant is present in the lyophilized composition at a concentration 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. Preferably, the lyophilization protectant is present in the lyophilized composition at a concentration greater than about 500 mM and less than about 1000 mM. In some embodiments, the lyophilization protectant is present at concentrations 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%. Preferably, the lyophilization protectant is present at a concentration greater than about 0.1 wt% and less than about 20 wt%. In some embodiments, the lyophilization protectant is present at concentrations 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. Preferably, the lyophilization protectant is present at a concentration greater than about 10 g / L and less than about 550 g / L.

[0128] Therapeutic proteins or peptides

[0129] In some implementations, reticulocytes further contain or express therapeutic proteins or peptides. These therapeutic proteins or peptides are typically not present in wild-type reticulocytes. A “therapeutic protein or peptide” is a protein or peptide with a useful therapeutic function. In other words, reticulocytes can be used as a carrier of therapeutic proteins or peptides. The advantage of this is that, once inserted into the body's circulatory system, reticulocytes can transport the therapeutic protein or peptide throughout the circulatory system, thereby providing rapid and systemic distribution of the therapeutic protein or peptide throughout the subject's body. Furthermore, reticulocytes can protect the therapeutic protein or peptide from the body's metabolic or defense processes, thus preventing its degradation. Additionally, for intracellular therapeutic proteins or peptides within the reticulocyte, the reticulocyte can prevent the body from initiating an immune response.

[0130] In some embodiments, reticulocytes contain or express therapeutic proteins or peptides with sequences not naturally present in wild-type reticulocytes. In some embodiments, the therapeutic protein or peptide is a foreign protein or peptide. In some embodiments, the therapeutic protein or peptide may be a variant of an endogenous protein or peptide. In some embodiments, the therapeutic protein or peptide may be a foreign protein or peptide, or a variant of an endogenous protein or peptide. In some embodiments, reticulocytes overexpress the therapeutic protein or peptide. This is defined below.

[0131] In some embodiments, the therapeutic protein or peptide is capable of demonstrating benefits, such as therapeutic benefits, in subjects. In most embodiments, the therapeutic protein or peptide is not a heme-containing protein, such as hemoglobin or methemoglobin. In some embodiments, the therapeutic protein or peptide is intracellular within reticulocytes. In some embodiments, the therapeutic protein or peptide is displayed on the surface of reticulocytes.

[0132] Preferably, the therapeutic protein or polypeptide is an enzyme. In a specific embodiment, the therapeutic protein is alanine aminotransferase, thymidine phosphorylase, glutamine synthase, hexokinase, glucokinase, phenylalanine hydroxylase, alcohol dehydrogenase, catalase, glucose-6-phosphate dehydrogenase, adenosine deaminase, asparaginase, uricase, bacterial L-phenylalanine aminolysase, 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 aminolysase. In a preferred embodiment, the therapeutic protein is thymidine phosphorylase.

[0133] Reticulocytes may contain more than one type of therapeutic protein or therapeutic peptide. In some such embodiments, multiple types of therapeutic proteins may be used, for example, to provide an enzyme chain to catalyze a series of reactions. As another example, multiple types of therapeutic proteins may be used to counteract the adverse effects of any given therapeutic protein.

[0134] Therapeutic proteins or peptides may be exogenous or endogenous. In some embodiments, the therapeutic protein or peptide does not contain an endogenous protein or endogenous therapeutic peptide. When the therapeutic protein or peptide is endogenous, its concentration in reticulocytes can be increased by means such as inhibiting the loss of the therapeutic protein or peptide during enucleation and / or by overexpression. In some embodiments, the therapeutic protein or peptide may contain an overexpressed endogenous protein or peptide. Overexpression techniques in cells (such as erythroid progenitor cells) are well known in the art and are described, for example, in WO2021 / 053243, the full text of which is incorporated herein by reference.

[0135] In a preferred embodiment, the therapeutic protein or peptide is exogenous. "Exogenous" means a protein or peptide that is not naturally expressed in reticulocytes. This includes proteins or peptides containing one or more amino acid mutations relative to endogenous proteins. Exogenous can also encompass isotypes from other cells or species, or non-naturally occurring proteins or peptides, such as hybrid proteins, chimeric proteins, fusion proteins, or de novo protein or peptide sequences. Genetic engineering techniques are discussed in more detail below.

[0136] In some implementations, reticulocytes are genetically engineered derivatives of erythroid ancestors to express or overexpress therapeutic proteins or peptides. The definition of "genetically engineered" can be found above.

[0137] One challenge in the expression of therapeutic proteins or peptides is that these proteins or peptides are expelled or degraded during the enucleation process that produces reticulocytes. Methods for inhibiting this reduction of therapeutic proteins or peptides are known in the art and have been outlined in WO 2021 / 053243, the full text of which is incorporated herein by reference. Simply put, ubiquitination has been identified as an important method for reducing protein and peptide concentrations during enucleation. In some embodiments, ubiquitination can be inhibited by using ubiquitinase inhibitors. Ubiquitinase inhibitors are nonspecific and may have detrimental effects beyond simply preserving therapeutic proteins or peptides. Ubiquitination has been reported as an important part of the erythroid cell maturation process (Nguyen, AT et al., Science, 2017, 357(6350)). Therefore, complete inhibition of ubiquitination (using ubiquitinase inhibitors such as MG132) means that many non-erythroid proteins will remain in developing reticulocytes or erythrocytes. Therefore, many aspects of cellular function will be disrupted, which can adversely affect important aspects of the cell, such as lifespan and immune system compatibility. In some cases, widespread ubiquitination inhibition can completely prevent differentiation and / or lead to cell death.

[0138] In some embodiments, the therapeutic protein or peptide is configured to inhibit or prevent ubiquitination of the therapeutic protein or peptide. Therefore, inhibition occurs locally at the therapeutic protein or peptide, rather than through a general inhibition of ubiquitination. This is a more targeted technique for preventing or preventing ubiquitin-mediated degradation of therapeutic proteins or peptides. Ubiquitination of therapeutic proteins or peptides can be inhibited or prevented as discussed in more detail below with respect to antioxidant proteins.

[0139] The composition before freeze drying

[0140] The present invention also provides a pre-lyophilized composition comprising a cell population, a lyophilization protectant, and a lyophilization medium, wherein the cell population comprises at least about 10% reticulocytes. The reticulocytes may be any of those discussed above.

[0141] The lyophilization medium can be any medium compatible with reticulocytes. The lyophilization medium can be an aqueous medium. In most embodiments, the lyophilization medium contains or is composed of a buffer. In some embodiments, the lyophilization medium contains or is composed of an aqueous buffer. In some embodiments, the pre-lyophilized composition consists essentially of a cell population, a lyophilization protectant, and an aqueous buffer, or is composed of the latter.

[0142] The pre-lyophilized composition may be in a liquid or solid phase. In some embodiments, the pre-lyophilized composition is at a temperature higher than the freezing temperature of the pre-lyophilized composition. In other words, the pre-lyophilized composition is a liquid. In some embodiments, the pre-lyophilized composition is at a temperature lower than the freezing temperature of the pre-lyophilized composition. In other words, the pre-lyophilized composition is a solid.

[0143] The present invention also provides a method for preparing a pre-lyophilized composition, wherein the method comprises contacting a cell population comprising at least about 10% reticulocytes with a lyophilization medium and a lyophilization protectant. The lyophilization protectant may be any of those discussed above. The reticulocytes may be any of those discussed above. The lyophilization medium may be any of those discussed above.

[0144] freeze-drying method

[0145] The present invention also provides a method for preparing the lyophilized composition of the present invention. The lyophilized composition comprises a cell population and a lyophilization protectant, wherein the cell population comprises at least about 10% reticulocytes.

[0146] The method includes (a) suspending a cell population in a lyophilization medium containing a lyophilization protectant, wherein the cell population comprises at least about 10% reticulocytes. The lyophilization protectant may be any of those discussed above. The reticulocytes may be any of those discussed above. The lyophilization medium may be any of those discussed above.

[0147] In some embodiments, in step (a), the cell population is suspended in a lyophilization medium containing a lyophilization protectant for at least about 1 hour. Preferably, in step (a), the cell population is suspended in a lyophilization medium containing a lyophilization protectant 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. Preferably, in step (a), the cell population is suspended in a lyophilization medium containing a lyophilization protectant overnight.

[0148] As explained above, the inventors have surprisingly demonstrated that reticulocytes are capable of taking up trehalose. Suspending the cell population in a lyophilization medium containing a lyophilization protectant in step (a) may include loading the cells with the lyophilization protectant. In some embodiments, the method includes (a) suspending the cell population in a lyophilization medium containing a lyophilization protectant (wherein the cell population comprises at least about 10% reticulocytes) for at least about 1 hour or any time discussed above to load the cells with the lyophilization protectant. The lyophilization protectant is preferably selected from trehalose, sucrose, fructose, glucose, mannitol, sorbitol, arabinose, and ribose. Trehalose is preferred as the lyophilization protectant. The reticulocytes are preferably genetically engineered to express a trehalose transporter protein or a variant thereof on their surface.

[0149] The method further includes (b) subjecting the suspension generated in step (a) to a lyophilization process to produce a lyophilized composition. Step (b) may be performed after any time specified for step (a).

[0150] Freeze-drying typically involves first freezing a liquid composition into a frozen solid, and then subjecting the frozen solid to reduced pressure by sublimation to remove the frozen liquid. The resulting composition is a freeze-dried or "lyophilized" composition.

[0151] Freeze-drying is a useful technique for removing liquids (typically aqueous liquids) from biological materials such as cells. The use of low temperatures helps to pause metabolic processes and prevent the degradation of biological materials such as proteins. However, a potential source of damage may be ice crystal formation during the freezing of water-based liquids. Compared to the liquid phase, ice crystal formation can cause volume expansion and / or the formation of sharp ice crystals, both of which can lead to cell rupture.

[0152] The freeze-drying process in step (b) may include a first drying stage. The first drying stage typically includes two features: cooling to freeze the composition, and exposing the frozen composition to reduced pressure to remove the frozen water by sublimation.

[0153] The first drying stage may include a rapid cooling stage. In the rapid cooling stage, the pre-lyophilized composition begins in a liquid phase and is rapidly cooled, causing the water to freeze in an amorphous form. In other words, the water freezes while inhibiting ice crystal formation. In some embodiments, the pre-lyophilized composition may be cooled at a rate greater than about 0.5°C / min until frozen. In some embodiments, the pre-lyophilized composition may be frozen in a freezer pre-cooled to about -50°C or lower. Typically, it is preferred to pre-cool the freezer to about -55°C or lower.

[0154] 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 degree of pressure drop is not particularly limited. Generally, lower pressure will provide faster water sublimation. In some embodiments, the pressure is less than about 500 microbars, less than about 250 microbars, less than about 100 microbars, less than about 50 microbars, or less than about 20 microbars.

[0155] The temperature can be kept constant or it can be varied. In some embodiments, the temperature is then raised by about 5°C to about 10°C to accelerate the sublimation of water. For example, rapid cooling can be performed in a freezer pre-cooled to about -55°C, and once the composition is frozen, the temperature can be raised to about -50°C.

[0156] It can maintain low temperature and low pressure until water is removed. It can maintain low temperature and low pressure until most water is removed. It can maintain low temperature and low pressure until a large amount of water is removed. It can maintain low temperature and low pressure until chilled water is no longer identifiable by visual inspection.

[0157] In some implementations, the first drying stage includes: a. Cool the suspension at a rate greater than approximately 0.5°C per minute until it freezes. b. Reduce the pressure to below atmospheric pressure, and c. Maintain the pressure for a period of time sufficient to remove most of the water.

[0158] The freeze-drying process in step (b) may include a second drying stage. The second drying stage may be used to remove residual water. The second drying stage may include heating the composition to above 0°C. The second drying stage may include heating the composition to room temperature. The second drying stage may include heating the composition to at least about 1°C, 2°C, 5°C, 10°C, 15°C, or 20°C. The second drying stage may include heating the composition while maintaining low pressure.

[0159] The second drying stage may include: d. Raising the temperature above 0°C, and e. Maintain the pressure reduction for a sufficient period of time to remove the water.

[0160] Refactoring methods

[0161] The present invention also provides a method for reconstructing the lyophilized composition by contacting it with a reconstructing medium. The lyophilized composition comprises a cell population and a lyophilization protectant, wherein the cell population comprises at least about 10% reticulocytes. The lyophilization protectant can be any of those discussed above. The reticulocytes can be any of those discussed above. In all instances herein, “reconstruction” and the like are interchangeable with “rehydration”.

[0162] 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 lyophilized composition with the reconstitution medium and then gently stirring the composition.

[0163] The reconstruction medium preferably comprises an aqueous liquid. In some embodiments, the reconstruction medium comprises an aqueous salt solution. In some embodiments, the reconstruction medium is an aqueous salt solution. In some embodiments, the reconstruction 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 reconstruction 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 the concentration of NaCl.

[0164] In some implementations, the reconstitution medium comprises SAGM, PAGGM, AS1, AS3, human plasma, artificial plasma solution, phosphate-buffered saline, or mixtures thereof.

[0165] The reconstitution medium can be any pharmaceutically or physiologically acceptable diluent and / or carrier discussed below with respect to the pharmaceutical compositions of the present invention.

[0166] Reconstructed Composition

[0167] The present invention also provides reconstructed compositions that are obtained or available through the reconstructing method of the present invention. The present invention also provides reconstructed compositions that are obtained or available through reconstructing the lyophilized compositions of the present invention. The lyophilized composition comprises a cell population and a lyophilization protectant, wherein the cell population comprises at least about 10% reticulocytes. The lyophilization protectant may be any of those discussed above. The reticulocytes may be any of those discussed above.

[0168] The present invention also provides a reconstructed composition comprising a cell population and a lyophilization protectant in a reconstructed medium, wherein the cell population comprises at least about 10% reticulocytes. The lyophilization protectant may be any of those discussed above. The reticulocytes may be any of those discussed above. The reconstructed medium may be any of those discussed above.

[0169] Preferably, the reconstitution medium comprises a pharmaceutically acceptable saline solution or buffer. Preferably, the reconstitution medium comprises SAGM, PAGGM, AS1, AS3, human plasma or artificial plasma solution, or a physiologically compatible buffer, 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 the concentration of NaCl. Preferably, the reconstitution medium is a pharmaceutically or physiologically acceptable diluent and / or carrier discussed below with reference to the pharmaceutical compositions of the present invention.

[0170] The reticulocytes and RBCs of the present invention

[0171] This invention also provides reticulocytes or mature erythrocytes (RBCs) containing intracellular trehalose. This invention also provides reticulocytes or mature erythrocytes (RBCs) containing an increased amount or level of intracellular trehalose. Mature RBCs are preferably derived from mature reticulocytes. The presence of intracellular trehalose can be measured using conventional methods. For example, cells can be lysed, and the presence of trehalose can be confirmed using a spectrophotometer (e.g., at 240 nm). The increased amount or level is typically measured by comparison with natural or wild-type reticulocytes or mature RBCs. This invention also provides populations of reticulocytes or mature erythrocytes (RBCs) containing intracellular trehalose. This invention also provides populations of reticulocytes or mature erythrocytes (RBCs) containing an increased level or amount of intracellular trehalose. The cell populations are discussed in more detail.

[0172] The reticulocytes or mature RBCs of the present invention preferably contain at least about 10 pg of intracellular trehalose. More preferably, the cells contain 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 of intracellular trehalose.

[0173] The reticulocyte or mature RBC population of the present invention preferably contains 10 6 At least approximately 10 μg of intracellular trehalose per cell. The population more preferably contains approximately 10 μg of intracellular trehalose per 10 cells. 6At least about 15 μg, at least about 20 μg, at least about 23 μg, at least about 25 μg, at least about 30 μg, at least about 35 μg, or at least about 40 μg of intracellular trehalose per cell.

[0174] The present invention also provides a method for producing the reticulocytes, mature RBCs, or populations of the present invention, comprising suspending the reticulocytes, mature RBCs, or populations of reticulocytes or mature RBCs in a medium containing trehalose. The medium can be any medium discussed above, particularly those related to the freeze-drying method of the present invention. The medium preferably contains 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).

[0175] In some embodiments, the cells or populations are suspended in a trehalose-containing medium for at least about 1 hour. Preferably, the cells or populations are suspended in a trehalose-containing medium 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. Preferably, the cells or populations are suspended in a trehalose-containing medium overnight.

[0176] In some embodiments, reticulocytes or mature RBCs are genetically engineered to express trehalose transporter protein or a variant thereof on their surface. In some embodiments, the reticulocytes or mature RBCs are derived from erythroid progenitor cells that have been genetically engineered to express trehalose transporter protein or a variant thereof on their surface. The definition of “genetically engineered” can be found above. The advantage of trehalose transporter protein is that it can increase the loading of trehalose onto reticulocytes. This can help achieve a beneficial trehalose concentration within the reticulocyte more quickly and / or at a lower external trehalose concentration.

[0177] 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 the intracellular transport of glucose. GLUT8 has also been observed to be a trehalose transporter. GLUT8 is the only mammalian trehalose transporter identified to date. The GLUT8 protein or a variant thereof is preferably the human GLUT8 protein or a variant thereof. The GLUT8 protein or a variant thereof is preferably a protein comprising the sequence shown in SEQ ID NO: 35 or a variant thereof, or composed of such a sequence.

[0178] In a particularly preferred embodiment, the GLUT8 variant protein includes a modified N-terminal region capable of localizing the variant protein to the plasma membrane. The modified N-terminal region may contain any number and combination of mutations. The modified N-terminal region may contain 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.

[0179] In wild-type mammals, the GLUT8 protein is internalized after expression on the cell surface because it is normally located in lysosomes. GLUT8 is characterized by an NH2-terminal [DE]XXXL[LI] targeting motif, which plays a role in directing the protein to endosomes, lysosomes, and related organelles. Studies in Chinese hamster ovary (CHO) cells have revealed that the GLUT8 transporter can be directed to the plasma membrane by simultaneously replacing the XXX residue of the lysosomal GLUT8 transporter with GPN and changing the dileucine (LL) motif to AA (which positions the 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), Vol. 10, pp. 324-333). The inventors have surprisingly shown that only a GPS mutation (corresponding to the human mutation of GPN in mice) is required to guide the human GLUT8 protein to the surface of human erythroid cells (including reticulocytes and RBCs). The GLUT8 variant protein preferably contains or only contains the TPQ to GPN mutation. The mouse GLUT8 variant protein preferably contains or only contains the TPQ to GPN mutation. The GLUT8 variant protein preferably contains or only contains the N-terminal [DE]XXXL[LI] motif to [DE]GPNL. The [LI] mutation. The GLUT8 variant protein preferably does not contain the mutation of the dileucine motif (LL) in the N-terminal [DE]XXXL[LI] motif. The human GLUT8 variant protein preferably contains or only contains the mutation from TPQ to GPS. The human GLUT8 variant protein preferably contains or only contains the mutation from the N-terminal [DE]XXXL[LI] motif to [DE]GPSL[LI]. The human GLUT8 variant protein preferably does not contain the mutation of the dileucine motif (LL) in the N-terminal [DE]XXXL[LI] motif. The GLUT8 variant protein preferably contains, or consists of, the sequence shown in SEQ ID NO: 37.

[0180] In some embodiments, the reticulocytes or mature RBCs of the present invention further comprise or express therapeutic proteins or peptides. Any embodiments discussed above are equally applicable here.

[0181] In some embodiments, the reticulocytes or mature RBCs of the present invention are genetically engineered to overexpress antioxidant proteins and / or intrinsically disordered proteins (IDPs) in their cytosol. Any embodiments discussed below are equally applicable. The reticulocytes or mature RBCs of the present invention may be genetically engineered to express one of the variant proteins of the present invention.

[0182] Erythroid cells genetically engineered to express trehalose transporter

[0183] This invention also provides erythroid cells genetically engineered to express trehalose transporter protein or variants thereof on their surface. In some embodiments, the erythroid cells are erythroid progenitor cells, reticulocytes, or mature RBCs. Erythroid progenitor cells can be any of those discussed above. The definition of “genetically engineered” can be found above. The trehalose transporter protein can be any transporter protein discussed above, particularly glucose transporter protein 8 (GLUT8) protein or variants thereof. The GLUT8 variant protein preferably contains one of the specific mutations discussed above, particularly the TPQ-GPN mutation in mouse GLUT8 or the TPQ-GPS mutation in human GLUT8. The GLUT8 variant protein preferably contains, or is composed of, the sequence shown in SEQ ID NO: 37.

[0184] In some embodiments, the erythroid cells of the present invention further comprise or express therapeutic proteins or peptides. Any embodiments discussed above are equally applicable here.

[0185] In some embodiments, the erythroid cells of the present invention are genetically engineered to overexpress antioxidant proteins and / or intrinsically disordered proteins (IDPs) in their cytosol. Any embodiments discussed below are equally applicable. The erythroid cells of the present invention may be genetically engineered to express one of the variant proteins of the present invention.

[0186] Erythroid cells genetically engineered to express increased levels of proteins in their cytosol

[0187] The present invention also provides genetically engineered erythroid cells to express variant proteins, the variant proteins comprising one or more mutations that increase the level of the variant protein in the cytosol of the cells.

[0188] In some implementations, the erythroid cells are erythroid progenitor cells, reticulocytes, or mature RBCs. Erythroid progenitor cells can be any of those discussed above. Human erythroid cells are preferred.

[0189] The definition of "genetically engineered" can be found above. Increased levels can be increased quantity and / or increased activity. Increased levels are typically increased detectable levels. Increased levels are usually compared to levels before genetic engineering. Increased levels are usually compared to levels of the protein in the cell before genetic engineering (i.e., unmodified or unmutated protein). The level of the variant protein can increase by any amount defined above as "overexpression".

[0190] The present invention also provides genetically engineered erythroid cells to express a variant protein comprising one or more mutations that promote overexpression of the variant protein in the cytosol of the cells. The definition of “overexpression” and the extent to which a variant protein can be overexpressed are also defined above.

[0191] Unmodified or non-mutated proteins typically undergo one or more of the following processes: ubiquitination in erythroid cells, secretion from erythroid cells, or targeting to organelles (such as mitochondria) within erythroid cells. This limits protein expression in the cytosol. Variant proteins consist of one or more mutations that suppress or reduce one or more of these processes, leading to increased or overexpressed levels of the variant protein in the erythroid cytosol.

[0192] The one or more mutations preferably include (a) one or more 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. The combination in (d) can be (a) and (b), (a) and (c), (b) and (c), or (a), (b) and (c). Variant proteins may contain (a), (b), (c), (a) and (b), (a) and (c), (b) and (c), or (a), (b) and (c).

[0193] Variant proteins may contain any number and combination of one or more mutations. Variant proteins may contain 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.

[0194] In (a), mutating one or more predicted ubiquitination sites reduces intracellular degradation of the variant protein and results in increased levels or overexpression of it in the cytosol of erythroid cells compared to unmodified or non-mutated proteins. In (a), any number of predicted ubiquitination sites may be mutated or substituted. For example, at least one, at least two, at least three, at least four, at least five, or at least six predicted ubiquitination sites may be mutated or substituted. Preferably, the variant protein comprises mutations or substitutions at all of its predicted ubiquitination sites. Predicted ubiquitination sites are defined in more detail below, and all such discussions apply equally to this.

[0195] In (a), one or more surface lysine residues may act as ubiquitination sites, even if they are not so predicted (e.g., as described below). Any number of surface lysine residues may be mutated or substituted. For example, at least one, at least two, at least three, at least four, at least five, or at least six 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).

[0196] A signal peptide is a short amino acid sequence that guides a protein to the correct location inside or outside erythroid cells (via the secretion pathway). Protein secretion means their loss from the cytosol. During RBC development, their organelles are removed, and therefore any proteins guided to organelles via their signal peptides are lost. In (b), one or more inactivating mutations in the signal peptide reduce the secretion of the variant protein or its transport to organelles, and result in an increased or overexpressed level of the variant protein in the erythroid cytosol, especially during organelle loss, compared to the unmodified or unmutated protein. In (b), the variant protein may contain any number and combination of one or more inactivating mutations. The variant protein may contain 2, 3, 4, 5, 6, or more inactivating mutations. The one or more inactivating mutations may be any of the mutation types described above. The variant protein preferably lacks at least a portion of its signal peptide. The variant protein may lack any amount of its signal peptide. In some embodiments, the at least portion comprises 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 portion comprises 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%, or at least about 90% of the signal peptide. The variant protein preferably lacks the entire or complete signal peptide.

[0197] In (c), one or more inactivating mutations in the mitochondrial-targeting peptide reduce the transport of the variant protein into the mitochondria and result in increased or overexpressed levels of the variant protein in the cytosol of erythroid cells, especially in cases of mitochondrial loss, compared to the unmodified or non-mutated protein. In (c), the variant protein may contain any number and combination of one or more inactivating mutations. The variant protein may contain 2, 3, 4, 5, 6 or more inactivating mutations. The one or more inactivating mutations may be any of the mutation types described above. The variant protein preferably lacks at least a portion of its mitochondrial-targeting peptide. The variant protein may lack any amount of its mitochondrial-targeting peptide. In some embodiments, the at least portion 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 portion 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%, or at least about 90% of the signal peptide. The variant protein preferably lacks the whole or complete mitochondrial targeting peptide. Mitochondrial targeting peptides are known in the art and can be predicted by analyzing the protein sequence, for example as described in Example 9. Examples of mitochondrial targeting peptides are shown in positions 2-28 of SEQ ID NO: 57.

[0198] Variant proteins can be variants of any protein. Variant proteins can be variants of proteins from any erythroid cell or species discussed above or below. Variant proteins are preferably variants of human proteins.

[0199] Variant proteins can be variants of antioxidant proteins and / or intrinsically disordered proteins (IDPs) in their cytosol. Any variant of antioxidants and / or IDPs can be any of those discussed below in conjunction with genetically engineered erythroid cells that overexpress antioxidant proteins and / or IDPs.

[0200] Variant proteins can be variants of enzymes. The enzyme can be any of those discussed above. The enzyme is preferably selected from hydrolases, oxidoreductases, lyases, transferases, ligases, isomerases, phosphorylases, synthases, kinases, hydroxylases, dehydrogenases, 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 aminotransferase, alanine aminotransferase, glutamate dehydrogenase, arginine deiminase, arginase, oxalate decarboxylase, oxalate oxidase, serine protease, tissue plasminogen activator, and larondinase. The enzyme can be mitochondrial.

[0201] The enzyme is preferably alanine aminotransferase 2 (AAT2). The present invention also provides genetically engineered erythroid cells to express a variant AAT2 protein, said variant AAT2 protein comprising one or more mutations that increase the level of the variant AAT2 protein in the cytosol of the cell or promote its overexpression. 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 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 residues 2-28 of SEQ ID NO: 57. The variant protein preferably comprises, or consists of, the sequence shown in SEQ ID NO: 59 or a variant thereof.

[0202] In some embodiments, erythroid cells are further genetically engineered to express trehalose transporter protein or a variant thereof on their surface. Any embodiments discussed above are also applicable here.

[0203] In some embodiments, the erythroid cells of the present invention further comprise or express therapeutic proteins or peptides. Any embodiments discussed above are equally applicable here.

[0204] Erythroid cells can be autologous. In other words, erythroid cells can be derived from the subject to whom the cells will be administered. Erythroid cells are preferably allogeneic. In other words, erythroid cells are preferably derived from a subject who is immunocompatible with the subject to whom the erythroid cells will be administered. The administration of autologous or allogeneic cells to subjects is well documented.

[0205] Erythroid cells can be engineered to be resistant to antibiotics.

[0206] Genetically engineered erythroid cells to overexpress antioxidant proteins and / or IDP

[0207] The present invention also provides an erythroid cell that has been genetically engineered to overexpress antioxidant proteins and / or intrinsic disordered proteins (IDPs) in its cytosol.

[0208] In some implementations, the erythroid cells are erythroid progenitor cells, reticulocytes, or mature RBCs. Erythroid progenitor cells can be any of those discussed above. Human erythroid cells are preferred.

[0209] The definition of “genetically engineered” can be found above. The definition of “overexpression” and the extent to which antioxidant proteins and / or IDPs can be overexpressed are also defined above. Erythroid cells can be genetically engineered to express increased levels of antioxidant proteins and / or IDPs in the cell’s cytosol. The increased level can be an increased amount and / or increased activity. The increased level is typically an increased detectable level. Overexpression or increased levels are usually compared to expression or levels in the cells or population prior to genetic engineering. This invention also provides genetically engineered erythroid cells to express increased levels of antioxidant proteins and / or IDPs in their cytosol.

[0210] A key aspect of this invention is the overexpression of antioxidant proteins and / or IDPs in the cytosol. Importantly, these proteins are overexpressed in the cytosol of erythroid cells and do not target mitochondria or other intracellular organelles or are secreted from the cell. This allows the proteins to resist reactive oxygen species (ROS) overall, but also provides protection against lipid peroxidation during the freeze-drying and remodeling processes described above.

[0211] Antioxidant proteins can be any type of antioxidant protein. Antioxidant proteins can originate from any cell or species discussed above or below. Antioxidant proteins are preferably human. Antioxidant proteins can also be non-human antioxidant proteins that have been humanized for expression in human cells. IDPs can be any of those IDPs discussed above or below. Antioxidant proteins and / or IDPs may contain any one or more mutations defined in (a)-(d) above.

[0212] In some embodiments, the antioxidant is superoxide dismutase or catalase or a variant thereof. In some embodiments, the antioxidant protein is modified to prevent it from targeting mitochondria. In some embodiments, the antioxidant protein contains one or more inactivating mutations in its mitochondrial-targeting peptide. These one or more inactivating mutations prevent the variant protein from being targeted to mitochondria. Those skilled in the art can determine whether the mutation inactivates the mitochondrial-targeting peptide and prevents the variant protein from being targeted to mitochondria. The one or more inactivating mutations can be any of those discussed below with reference to signal peptides.

[0213] In some embodiments, the antioxidant protein is glutathione peroxidase (GPx) or a variant thereof. In some embodiments, the antioxidant protein is GPx1, GPx2, GPx3, GPx4, GPx5, GPx6, GPx7, GPx8 or a variant thereof. The antioxidant protein preferably comprises, or is composed of, the sequence shown in SEQ ID NO: 2, 4, 6, 38, 39, 8, 10, 12, 14, 16, 40, 41, 42 or 43. The antioxidant protein preferably comprises, or is composed of, the sequence shown in SEQ ID NO: 2, 4, 6, 38, 39, 8, 10, 12, 14, 16, 40, 41, 42, 43 or 55. The antioxidant protein is preferably GPx1, GPx4 or a variant thereof. The antioxidant protein is preferably GPx1 or a variant thereof. The antioxidant protein preferably comprises, or is composed 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.

[0214] In some embodiments, the antioxidant protein is a peroxidase (Prx) or a variant thereof. In some embodiments, the antioxidant protein is Prx1, Prx2, Prx3, Prx4, Prx5, Prx6, or a variant thereof. The antioxidant protein preferably comprises, or is composed 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 Prx1, Prx2, Prx3, or a variant thereof. The antioxidant protein preferably comprises, or is composed 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 is composed of, the sequence shown in SEQ ID NO: 22 or 24, or a variant thereof.

[0215] In some embodiments, the antioxidant protein is modified to prevent its secretion from the cell. In some embodiments, the GPx4 variant, GPx5 variant, GPx7 variant, GPx8 variant, Prx3 variant, or Prx5 variant contains one or more inactivating mutations in its signal peptide. In some embodiments, the GPx4 variant contains one or more inactivating mutations in its signal peptide. These one or more inactivating mutations prevent the variant protein from being transported to intracellular organelles or secreted from the cell. Those skilled in the art can determine whether the mutation inactivates the signal peptide and prevents the variant protein from being transported to intracellular organelles or secreted from the cell.

[0216] Variant proteins may contain any number and combination of inactivating mutations. A variant protein may contain 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.

[0217] The variant protein preferably lacks at least a portion of its signal peptide. The variant protein may lack any amount of its signal peptide. In some embodiments, the at least portion 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 portion 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%, or at least about 90% of the signal peptide.

[0218] Variant proteins are preferably lacking in whole or complete signal peptides.

[0219] The GPx4 signal peptide corresponds to the first 27 amino acids (i.e., positions 1-27) of SEQ ID NO: 8. GPx4 variant proteins preferably contain one or more mutations in these 27 amino acids. The one or more mutations can be any of those described above. GPx4 variant proteins preferably contain a deletion of the first 27 amino acids (i.e., positions 1-27) of SEQ ID NO: 8. This is also referred to as short GPx4 or sGPx4.

[0220] In some implementations, the variant antioxidant protein contains mutations at one or more predicted ubiquitination sites. A ubiquitination site is a location within a protein where ubiquitin can bind and degrade the protein. Mutating one or more ubiquitination sites reduces the degradation of the variant protein within the cell. The network-based algorithm PhosphoSitePlus (https: / / www.phosphosite.org / homeAction.action) can be used to predict ubiquitination sites. PhosphoSitePlus integrates published or unpublished post-translational modification (PMT) data generated by Cell Signalling Technology (CST). The data includes human, mouse, and rat proteins / isotypes. Predicted ubiquitination sites can also be identified by compiling published data from the literature.

[0221] In a preferred embodiment, the variant antioxidant protein comprises substitutions at one or more predicted ubiquitination sites. These predicted ubiquitination sites typically contain lysine (K). K is preferably substituted with arginine (R). This maintains the positive charge at the substituted site.

[0222] Any number of predicted ubiquitination sites may be mutated or substituted, such as at least one, at least two, at least three, at least four, at least five, or at least six. Preferably, the variant protein contains mutations or substitutions at all of its predicted ubiquitination sites.

[0223] In a preferred embodiment, the GPx1 variant or the GPx4 variant contains a mutation or substitution at one or more predicted ubiquitination sites or all of them. In a preferred embodiment, the GPx1 variant contains a mutation or substitution at one or more predicted ubiquitination sites or all of them. In a preferred embodiment, the GPx4 variant contains a mutation or substitution at one or more predicted ubiquitination sites or all of them.

[0224] The GPx1 variant preferably contains mutations or substitutions 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 sites are shown in SEQ ID NO: 2. The GPx1 variant preferably contains mutations or substitutions at one or more of the positions corresponding to 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 a preferred embodiment, K is substituted with R.

[0225] The GPx4 variant preferably contains mutations or substitutions at one or more of the following sites: (a) K107, (b) K126, (c) K148, (d) K162R, (e) K167, and (f) K191. These sites are shown in SEQ ID NO: 8. The GPx4 variant preferably contains mutations or substitutions at one or more of the following sites: (a) K107, (b) K126, (c) K148, (d) K162R, (e) K167, and (f) K191 in SEQ ID NO: 8.GPx4 variant proteins can contain mutations or substitutions at any number and combination of the following positions: (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), (b) and (c); (a), (b) and (c) (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), (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) (a), (b), (c) and (d); (d), (e) and (f); (a), (b), (c) and (d); (a), (b), (c) and (e); (a), (b), (c) and (f); (a), (b), (d) and (e); (a), (b), (d) and (f); (a), (b), (e) and (f); (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) (a), (b), (c), (e), and (f); (b), (d), (e), and (f); (c), (d), (e), and (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), (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 a preferred embodiment, K is replaced by R.

[0226] In some embodiments, the antioxidant protein comprises selenocysteine ​​(U). In some embodiments, GPx1, GPx2, GPx3, GPx4, GPx6, or variants thereof comprise selenocysteine ​​(U). In some embodiments, GPx1, GPx4, or variants thereof comprise selenocysteine ​​(U). In some embodiments, GPx1 or variants thereof comprises selenocysteine ​​(U) at position 49 corresponding to position 49 in SEQ ID NO: 2. In some embodiments, GPx4 or variants thereof comprises selenocysteine ​​(U) at position 73 corresponding to position 73 in SEQ ID NO: 8. This can be achieved by expressing the antioxidant protein from a polynucleotide comprising a 3' untranslated region (3'UTR) containing a Sec insert 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 this invention. The SECIS preferably comprises, or consists of, the sequence shown in SEQ ID NO: 47.

[0227] In other embodiments, the GPx1 variant protein contains a cysteine ​​(C) at position 49 corresponding to SEQ ID NO: 2. In some embodiments, the GPx1 variant protein contains the U49C mutation. In other embodiments, the GPx4 variant protein contains a cysteine ​​(C) at position 73 corresponding to SEQ ID NO: 8. In some embodiments, the GPx4 variant protein contains the U73C mutation. This can be achieved by expressing the antioxidant protein from a polynucleotide that does not contain the 3' untranslated region (3'UTR) containing the Sec insert sequence (SECIS).

[0228] In a preferred embodiment, the GPx1 variant protein contains a mutation at one or more predicted ubiquitination sites (such as K88, K148, and K166) and preferably a selenocysteine ​​(U) at position 49 in SEQ ID NO: 2. Any of the embodiments discussed above are equally applicable to this embodiment.

[0229] 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 the deletion of its signal peptide), and (3) a selenocysteine ​​(U) preferably at position 49 in SEQ ID NO: 2.

[0230] In a preferred embodiment, GPx1 or a variant thereof comprises, or consists of, the sequence shown in SEQ ID NO: 2, 4 or 6, or a variant thereof. In a preferred embodiment, GPx1 or a variant thereof comprises, or consists of, the sequence shown in SEQ ID NO: 2, 4, 6 or 55, or a variant thereof.

[0231] In a preferred embodiment, GPx4 or a variant thereof comprises, or consists of, the sequence shown in SEQ ID NO: 12, 14 or 16, or a variant thereof. In the most preferred embodiment, GPx4 or a variant thereof comprises, or consists of, the sequence shown in SEQ ID NO: 16, or a variant thereof.

[0232] In a preferred embodiment, Prx1 or a variant thereof comprises, or consists of, the sequence shown in SEQ ID NO: 18 or 20 or a variant thereof.

[0233] In a preferred embodiment, Prx2 or a variant thereof comprises, or consists of, the sequence shown in SEQ ID NO: 22 or 24, or a variant thereof.

[0234] In a preferred embodiment, Prx6 or a variant thereof comprises, or consists of, the sequence shown in SEQ ID NO: 26 or 28, or a variant thereof.

[0235] 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 embodiments, the erythroid cells are human. Human cells do not express these IDPs. In some embodiments, the erythroid cells are human reticulocytes.

[0236] In some embodiments, erythroid cells are further genetically engineered to express trehalose transporter protein or a variant thereof on their surface. Any embodiments discussed above are also applicable here.

[0237] In some embodiments, the erythroid cells of the present invention further comprise or express therapeutic proteins or peptides. Any embodiments discussed above are equally applicable here.

[0238] This invention also relates to a cell genetically engineered to overexpress antioxidant proteins and / or intrinsically disordered proteins (IDPs) in its cytosol. The genetically engineered cell can be any type of cell. In some embodiments, the cell is mammalian. The cell can be human, canine, cat, primate, horse, mouse, rat, rodent, bovine, pig, or sheep cells. In a preferred embodiment, the cell is human.

[0239] Cells can originate from the ectoderm, endoderm, or mesoderm. Cells can be endothelial cells. Cells can be epithelial cells. Cells can originate from the immune system, heart, brain, vascular system, skin, intestines, lungs, thyroid gland, reproductive organs, bladder, kidneys, pancreas, oral mucosa, eyes, or liver. Cells can be stem cells (such as embryonic stem cells, induced pluripotent stem cells, or mesenchymal stem cells), bone cells (such as osteoclasts, osteoblasts, or osteocytes), tendon cells (such as tenoblasts or tendon cells), chondrocytes, synovial cells, vascular cells, connective tissue cells (such as fibroblasts), blood cells (such as erythrocytes, immune cells, platelets, neutrophils, or basophils), muscle cells (such as skeletal muscle cells, cardiomyocytes, or smooth muscle cells), germ cells (such as sperm, oocytes, tubule cells, or epididymal cells), secretory cells, adipocytes, hepatic adipocytes, epithelial cells, odontoblasts, cementoblasts, hormone-secreting cells, barrier cells, exocrine epithelial cells, nerve cells, astrocytes, oligodendrocytes, or neurons.

[0240] Genetically engineered cells are preferably blood cells. Blood cells can be erythroid progenitor cells, reticulocytes, mature RBCs, platelets, or immune cells. Immune cells are also known as white blood cells. Immune cells can be neutrophils and precursors (such as myeloblasts, premyelocytes, myelocytes, or postmyelocytes), eosinophils and precursors, basophils and precursors, mast cells, white blood cells, lymphocytes, helper T cells, regulatory T cells, cytotoxic T cells, natural killer T cells, natural killer cells, innate lymphocytes (ILCs), B cells, macrophages, dendritic cells, plasma cells, neutrophils, or monocytes.

[0241] In some embodiments, the cells are derived from stem cells or immortalized cell lines. Suitable stem cells and immortalized cell lines are known in the art and have been discussed above. The cells may be derived from human induced pluripotent stem (iPS) cells. Such cells can be identified based on the presence of one or more transcription factors used to induce pluripotency. Such transcription factors include, but are not limited to, Oct-3 / 4, Sox1, Sox2, Sox3, Sox15, Sox18, Klf2, Klf4, c-Myc, n-Myc, l-Myc, Nanog, LIN28, and Glis1. Such cells may also include evidence of mechanisms for delivering such transcription factors.

[0242] The cells can be autologous. In other words, the cells can be derived from the subject to whom the cells will be administered. The cells are preferably allogeneic. In other words, the cells are preferably derived from a subject who is immunocompatible with the subject to whom the cells will be administered. The administration of autologous or allogeneic cells to subjects is well documented. These embodiments are applicable to the erythroid cells of the present invention.

[0243] Erythroid cells can be engineered to be resistant to antibiotics.

[0244] variants

[0245] This invention relates to various proteins or variants thereof, including trehalose transporters, GLUT8 protein, GPx1, GPx2, GPx3, GPx4, GPx5, GPx6, GPx7, GPx8, Prx1, Prx2, Prx3, Prx4, Prx5, Prx6, CAHS1, CAHS3, PvLEA, AavLEA, or variants thereof. In some embodiments, this invention relates to CAHS2 or variants thereof. In some embodiments, the variant protein comprises, or is composed of, a sequence having at least about 80% homology or identity with a reference sequence. For example, variants of GPx1 preferably comprise, or are composed of, a sequence having at least about 80% homology or identity with the sequence shown in SEQ ID NO: 2. In a preferred embodiment, 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 with the reference sequence. Homology and / or identity are typically measured over the entire length of the reference sequence (typically the parent protein).

[0246] Homology or identity can be determined using standard methods in the art. For example, the UWGCG package provides the BESTFIT program, which can be used to calculate homology or identity, for example, using its default settings (Devereux et al. (1984) Nucleic Acids Research 12, pp. 387-395). The PILEUP and BLAST algorithms can be used to calculate homology and identity or aligned sequences (such as identifying equivalent residues or corresponding sequences (usually on their default settings)), for example as described in Altschul SF (1993) J Mol Evol 36:290-300; Altschul, SF et al. (1990) J MolBiol 215:403-10. Software for performing BLAST analysis is publicly available from the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0247] Using these types of software for sequence alignment also allows technicians to identify corresponding positions between sequences, such as the correspondence between a variant protein and its parent sequence.

[0248] Amino acid substitutions, such as conservative substitutions, can be present in variant proteins, 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 having similar chemical structures, similar chemical properties, or similar side-chain volumes. The introduced amino acids may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge as the amino acids they replace. Alternatively, conservative substitutions may introduce another aromatic or aliphatic amino acid to replace a pre-existing aromatic or aliphatic amino acid. Conservative amino acid variations are well known in the art.

[0249] Variant proteins can be modified to introduce one or more cysteine ​​residues, one or more hydrophobic amino acids, one or more charged amino acids, one or more non-natural amino acids, one or more polar amino acids, or one or more photoreactive amino acids. Such introductions can be made in any number and combination. Introduction is preferably made by substitution or addition.

[0250] One or more amino acid residues may be deleted from the above-mentioned polypeptide. A maximum of 1, 2, 3, 4, 5, 10, 20, or 30 or more residues may be deleted.

[0251] Variant proteins may contain fragments of the parent protein. The fragments may be at least about 50, at least about 100, at least about 150, or at least about 200 amino acids in length.

[0252] Alternatively, one or more amino acids may be added to the protein described above. An extension may be provided at the amino terminus or carboxyl terminus. The extension may be relatively short, for example, 1 to 10 amino acids in length. Alternatively, the extension may be longer, for example, up to 50 or 100 amino acids.

[0253] In a preferred embodiment, the variant protein is a functional variant protein. In such embodiments, the variant protein retains the function of its parent protein. Those skilled in the art can test the functional variant using conventional methods. For example, a conventional GPx activity assay can be used to test the ability of the GPx1 or GPx4 variant protein to catalyze the conversion of reduced monomeric glutathione to glutathione disulfide.

[0254] Any protein of the present invention may further include a methionine (M) at its N-terminus. This generally reflects the start codon (ATG) in the polynucleotide used to express the protein.

[0255] Any protein of the present invention can be modified to aid in its identification or purification, for example by adding histidine residues (his tags), aspartic acid residues (asp tags), streptavidin tags, flag tags, SUMO tags, GST tags, MBP tags, or myc tags. 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 located at the N-terminus of a protein variant immediately following the N-terminal methionine residue. An alternative method for introducing a genetic tag is to chemically react the tag to a natural or engineered site on the protein.

[0256] Preferred variants of SEQ ID NO: 4, 6, 10, 12, 14, 16, 20, 24, 18-33 and 55 lack the myc tag. The Myc tag is defined above.

[0257] group

[0258] This invention also provides cell populations of the invention. Specifically, this invention provides populations of reticulocytes or mature erythrocytes containing intracellular trehalose. This invention also provides populations of erythroid cells genetically engineered to express trehalose transporters or variants thereof on their surface. This invention also provides populations of erythroid cells genetically engineered to overexpress antioxidant proteins and / or intrinsically disordered proteins (IDPs) in their cytosol. This invention also provides populations of erythroid cells genetically engineered to express increased levels of antioxidant proteins and / or intrinsically disordered proteins (IDPs) in their cytosol. This invention also provides populations of erythroid cells genetically engineered to express variant proteins comprising one or more mutations that increase the level of the variant protein in the cytosol of the cell or promote its overexpression. The cells can be any cells discussed above. The populations can be present in any media discussed above or below. The populations can be present in the lyophilized compositions of the invention.

[0259] The population may contain any number of cells, such as at least about 5 × 10⁻⁶. 5 The present invention comprises cells. In some embodiments, the population contains at least about 1 × 10⁻⁶ cells. 6 At least approximately 2×10 6 At least approximately 2.5 × 10 6 At least approximately 5×10 6 At least approximately 1×10 7 At least approximately 2×10 7 At least approximately 5×10 7 At least approximately 1×10 8 Or at least about 2×10 8 The present invention comprises cells. In some cases, the population may contain at least about 1.0 × 10⁻⁶ cells. 7 At least approximately 1.0 × 10 8 At least approximately 1.0 × 10 9 At least approximately 1.0 × 10 10 At least approximately 1.0 × 10 11 Or at least about 1.0 × 10 12 One or more cells of the present invention.

[0260] In any group of the present 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 contain intracellular trehalose and / or are genetically engineered.

[0261] Any cells or populations of the present invention may be isolated, substantially isolated, purified, or substantially purified. If a cell or population contains absolutely no other components (such as culture medium), it is isolated or purified. If a cell or population is mixed with a carrier or diluent (such as culture medium or pharmaceutical composition) that will not interfere with its intended use, it is substantially isolated or substantially purified. Other carriers and diluents are discussed in more detail below.

[0262] Variant proteins of the present invention

[0263] This invention also provides one or more mutant proteins comprising increasing the level of the variant protein in the erythroid cell cytosol or promoting its overexpression. The one or more mutations can increase the level of the variant protein in the erythroid cell cytosol. The increased level can be an increased amount and / or increased activity. The increased level is typically an increased detectable level. The increased level or overexpression is typically compared to the level or expression of unmodified or non-mutated proteins.

[0264] Unmodified or non-mutated proteins typically undergo one or more of the following processes: ubiquitination in erythroid cells, secretion from erythroid cells, or targeting to organelles (such as mitochondria). This limits protein expression in the cytosol. Variant proteins include one or more mutations that inhibit or reduce one or more of these processes, resulting in increased or overexpressed levels of the variant protein in the erythroid cytosol. Variant proteins may contain (a) one or more mutations at one or more predicted ubiquitination sites and / or one or more surface lysine residues, (b) one or more inactivating mutations in their signal peptide, (c) one or more inactivating mutations in their mitochondrial targeting peptide, or (d) any combination thereof. The combination in (d) can be (a) and (b), (a) and (c), (b) and (c), or (a), (b) and (c). Variant proteins may contain (a), (b), (c), (a) and (b), (a) and (c), (b) and (c), or (a), (b) and (c). All the discussion above in connection with the erythroid cells of the present invention, which are genetically engineered to express variant proteins, also applies here. The variant protein is preferably a variant enzyme protein. The enzyme can be any of those listed above. The variant enzyme protein preferably contains one or more inactivating mutations in its mitochondrial targeting peptide. The variant protein can be a variant of human AAT2. The variant protein can be a variant of SEQ ID NO: 57. The variant protein can 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 residues 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. The variant protein preferably lacks all residues 2-28 of SEQ ID NO: 57. The variant protein preferably comprises, or consists of, the sequence shown in SEQ ID NO: 59 or a variant thereof.

[0265] The present invention also provides a variant antioxidant protein comprising a mutation at one or more predicted ubiquitination sites. In particular, the present invention provides a Gpx1 or Gpx4 variant protein comprising a mutation at one or more predicted ubiquitination sites. Any embodiments discussed above with reference to the genetically modified cells of the present invention are equally applicable to the variant proteins of the present invention.

[0266] In a preferred embodiment, the GPx1 variant protein contains a mutation at one or more predicted ubiquitination sites (such as K88, K148, and K166). The GPx1 variant protein may contain a U49C mutation. In a preferred embodiment, the GPx1 variant protein further contains a selenocysteine ​​(U) preferably at position 49 in SEQ ID NO: 2. Any of the embodiments discussed above are equally applicable to this embodiment.

[0267] The present invention also provides a variant antioxidant protein comprising one or more inactivating mutations in its signal peptide. In particular, the present invention provides a GPx4, GPx7, GPx8, Prx3, or Prx5 variant protein comprising one or more inactivating mutations in its signal peptide. Any embodiments discussed above with reference to the genetically modified cells of the present invention are equally applicable to the variant proteins of the present invention.

[0268] The present invention also provides variant antioxidant proteins comprising (1) a mutation at one or more predicted ubiquitination sites, and (2) one or more inactivating mutations in their signal peptide, such as the deletion of their signal peptide.

[0269] 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) a selenocysteine ​​(U) preferably at position 49 in SEQ ID NO: 2.

[0270] In a preferred embodiment, the GPx4 variant protein comprises, or consists of, the sequence shown in SEQ ID NO: 14 or 16, or a variant thereof. In the most preferred embodiment, GPx4 or a variant thereof comprises, or consists of, the sequence shown in SEQ ID NO: 16, or a variant thereof.

[0271] In any of the embodiments discussed above, the variant protein may be a variant of a human protein. The variant protein may also be a variant of a non-human protein that has been humanized for expression in human cells.

[0272] Polynucleotides

[0273] The present invention also provides a polynucleotide encoding a variant protein of the invention. The variant protein can be any of those described above. In a preferred embodiment, the polynucleotide comprises a 3' untranslated region (3'UTR) containing a Sec insert sequence (SECIS). An example of a suitable 3'UTR is the final 207 nucleotides of SEQ ID NO: 15. Any SECIS can be used in the present invention. SECIS preferably comprises, or consists of, the sequence shown in SEQ ID NO: 47.

[0274] In a preferred embodiment, the polynucleotide comprises, or consists of, the sequence shown in SEQ ID NO: 15 or a variant thereof. Variants of the polypeptides of the present invention are defined above with reference to them.

[0275] It should be understood that the polynucleotide sequence is recombinant. Preferably, the polynucleotide sequence is isolated. In some embodiments, the polynucleotide is non-natural.

[0276] In some implementations, the polynucleotide may further comprise a sequence encoding a reporter gene. Suitable reporter genes include, but are not limited to, luciferase, myc, HNIS, hNET, and HSVtK.

[0277] Polynucleotides (such as nucleic acids) are polymers containing two or more nucleotides. Nucleotides can be naturally occurring or artificial.

[0278] Nucleotides typically contain a nucleobase, a sugar, and at least one linking group, such as a phosphate group, 2'O-methyl, 2'methoxy-ethyl, aminophosphate group, methylphosphonate group, or thiophosphate group. The nucleobase is usually 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 usually a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The sugar and nucleobase together form a nucleoside. Preferred nucleosides include, but are not limited to, adenosine, guanosine, 5-methyluridine, uridine, cytidine, deoxyadenosine, deoxyguanosine, thymine, deoxyuridine, and deoxycytidine. Nucleosides can be adenosine, guanosine, uridine, and cytidine.

[0279] Nucleotides are typically ribonucleotides or deoxyribonucleotides. Nucleotides can be deoxyribonucleotides. Nucleotides usually contain monophosphate, diphosphate, or triphosphate. Phosphate esters can be attached to the 5' or 3' side of the nucleotide.

[0280] 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), and deoxyadenosine diphosphate (dGMP). ADP), 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 nucleotide can be selected from AMP, UMP, GMP, CMP, dAMP, dTMP, dGMP, or dCMP. In some embodiments, the nucleotide is selected from dAMP, dTMP, dGMP, or dCMP.

[0281] Nucleotides may contain additional modifications. In particular, suitable modified nucleotides include, but are not limited to, 2'-aminopyrimidines (such as 2'-aminocytidine and 2'-aminouridine), 2'-hydroxypurines (such as 2'-fluoropyrimidines (such as 2'-fluorocytidine and 2'-fluorouridine)), hydroxypyrimidines (such as 5'-α-P-boranouridine), 2'-O-methylnucleotides (such as 2'-O-methyladenosine, 2'-O-methylguanosine, 2'-O-methylcytidine and 2'-O-methyluridine), 4'-thiopyrimidines (such as 4'-thiouridine and 4'-thiocytidine), and nucleotides with nucleobase modifications (such as 5-pentynyl-2'-deoxyuridine, 5-(3-aminopropyl)uridine, and 1,6-diaminohexyl-N-5-carbamoylmethyluridine).

[0282] One or more nucleotides in a polynucleotide can be modified, for example, with a label or tag. The label can be any suitable label that allows the detection of the nucleotide. Suitable labels include, but are not limited to, fluorescent molecules, radioactive isotopes (e.g.,...). 125 I, 35 S), enzymes, antibodies, antigens, other polynucleotides, and ligands (such as biotin).

[0283] Nucleotides in polynucleotides can attach to each other in any way. Nucleotides can be linked by phosphate ester bonds, 2'O-methyl bonds, 2'methoxy-ethyl bonds, aminophosphate bonds, methylphosphonate bonds, or thiophosphate bonds. Nucleotides are typically linked by their sugar and phosphate groups. Nucleotides can also be linked via their nucleobases, as in pyrimidine dimers.

[0284] Polynucleotides may contain deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Polynucleotides may be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threonine nucleic acid (TNA), locked nucleic acid (LNA), morpholino nucleic acid, or other synthetic polymers with nucleotide side chains.

[0285] Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses its conserved modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as explicitly indicated sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with different bases, mixed bases, 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. Probes8:91-98).

[0286] This replaces practices that can be used for codon optimization and codon wobble, both of which are known to those skilled in the art. Therefore, it should be understood that separate polynucleotides for codon optimization and codon wobble are also contemplated. In one embodiment, the polynucleotide is codon-optimized for human expression.

[0287] Polynucleotides can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of existing sequences. Direct chemical synthesis of polynucleotides can be achieved 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 diethylphosphite method of Beaucage et al., 1981, Tetra. Lett., 22:1859; and the solid support method of U.S. Patent No. 4,458,066. Introducing mutations into polynucleotide sequences via PCR can be performed as described, for example, in PCR Technology: Principles and Applications for DNA Amplification, HA Erlich (ed.), Freeman Press, NY, NY, 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, PCRMethods and Applications 1:17.

[0288] carrier

[0289] The present invention also provides vectors comprising the polynucleotide sequences of the present invention. The polynucleotides may be any of those described above. The present invention also provides cells comprising or expressing the polynucleotide sequences of the present invention or the vectors of the present invention. The cells may be any of those described above.

[0290] In some implementations, the vector is an expression vector. Various expression vectors can be used to express the polynucleotides of the present invention.

[0291] Both viral and non-viral expression vectors can be used in this invention. Non-viral vectors and systems include plasmids, episome vectors (typically having expression cassettes for expressing proteins or RNA), and human artificial chromosomes (see, for example, Harrington et al., 1997, Nat Genet. 15:345). For example, non-viral vectors that can be used 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 many 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, and herpesviruses; vectors based on SV40, papillomaviruses, HBP Epstein Barrvirus, vaccinia virus vectors, and Semliki Forest virus (SFV). See Brent et al., above; Smith, 1995, Annu. Rev. Microbiol. 49:807; and Rosenfeld et al., 1992, Cell 68: 143.

[0292] Preferably, the vector is a retrovirus, lentivirus, adenovirus, or adeno-associated virus vector. Examples of such vectors include the SFG retroviral expression vector (see Riviere et al., 1995, Proc. Natl. Acad. Sci. (USA) 92:6733-6737).

[0293] 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-inactivated lentiviral vectors (so-called SIN vectors).

[0294] Expression vectors for mammalian host cells may include expression control sequences, such as origin of replication, promoters, and enhancers (see, for example, Queen et al., 1986, Immunol. Rev. 89:49-68), as well as necessary processing information sites, such as ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. These expression vectors typically contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters can be constitutive, cell type-specific, stage-specific, and / or regulated or modulotropic. Useful promoters include, but are not limited to, metallothionein promoters, constitutive adenovirus major late promoters, dexamethasone-inducible MMTV promoters, SV40 promoters, MRP polIII promoters, constitutive MPS V promoters, tetracycline-inducible CMV promoters (such as human immediate early CMV promoters), constitutive CMV promoters, SSFV promoters, EF1α promoters, phosphoglycerate kinase (PGK) promoters, and promoter-enhancer combinations known in the art. The promoter can be the MSCV promoter.

[0295] Genetic engineering

[0296] The present invention also provides a method for modifying cells, comprising genetically engineering cells to express variant proteins, polynucleotides, or vectors of the present invention. The cells, variant proteins, polynucleotides, or vectors can be any of those discussed above.

[0297] The present invention also provides a method for improving the ability of cells to survive lyophilization, the method comprising genetically engineering cells to overexpress antioxidant proteins and / or intrinsically disordered proteins (IDPs) in their cytosol. The present invention also provides a method for improving the ability of cells to survive lyophilization, the method comprising genetically engineering cells to express increased levels of antioxidant proteins and / or intrinsically disordered proteins (IDPs) in their cytosol. Antioxidant proteins can be any of those described above, including various variant proteins. IDPs can be any of those described above. In a preferred embodiment, the method comprises genetically engineering cells to express variant proteins of the present invention, polynucleotides of the present invention, or vectors of the present invention. Cells, variant proteins, polynucleotides, or vectors can be any of those discussed above. Cells are preferably erythroid cells.

[0298] The genetic engineering of cells can be performed according to standard cloning and expression techniques known in the art (e.g., as described in Sambrook, J., Fritsh, EF and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

[0299] The method may include introducing the vector and / or polynucleotide of the present invention into cells. In some embodiments, the method includes transfecting cells with the vector or polynucleotide of the present invention to express the variant protein of the present invention.

[0300] The various forms of the term "transfection" are intended to encompass a range of techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-glucan transfection, etc.

[0301] Alternatively, the method may include transducing cells with the vectors and / or polynucleotides of the present invention to express variant proteins of the present invention. For example, viral vectors as disclosed above may be used to deliver polynucleotides.

[0302] In embodiments where the cells are immune cells, the cells can be activated before, during, or after genetic engineering. Activation can occur before the cells are genetically engineered. In some embodiments, the method includes simultaneous activation and genetic engineering steps. Activation methods are known in the art.

[0303] In some implementations, the cells are genetically engineered to express markers or factors that ensure only successfully genetically engineered cells remain viable. This eliminates any non-genetically engineered cells, thereby improving the purity of the resulting cell population.

[0304] Pharmaceutical Composition

[0305] Pharmaceutical compositions are also provided comprising the reconstituted composition of the present invention, the cells of the present invention, the variant proteins of the present invention, the polynucleotides of the present invention, or the carriers of the present invention, and pharmaceutically or physiologically acceptable diluents and / or carriers. The composition, cells, variant proteins, polynucleotides, or carriers may be any of those discussed above. The cells may be reticulocytes of the present invention, erythroid cells of the present invention, or any cells of the present invention. One or more cells in the population are preferably erythroid cells or reticulocytes. One or more cells in the population are preferably human.

[0306] Pharmaceutical compositions are also provided comprising the erythroid cells of the present invention, the population of the present invention, the variant protein of the present invention, the polynucleotide of the present invention, or the carrier of the present invention, and a pharmaceutically or physiologically acceptable diluent and / or carrier. The erythroid cells, population, variant protein, polynucleotide, or carrier may be any of those discussed above. One or more erythroid cells in the population are preferably reticulocytes. One or more erythroid cells in the population are preferably human.

[0307] Carriers and / or diluents are typically selected to suit the intended mode of application and may include agents for adjusting, maintaining, or preserving, for example, the pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption, or permeation of the composition. Typically, these carriers and / or diluents comprise aqueous or alcoholic / aqueous solutions, emulsions, or suspensions, including saline and / or buffer media.

[0308] Suitable additional agents included in the pharmaceutical composition include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobial agents, antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite), buffers (such as borates, bicarbonates, Tris-HCl, citrates, phosphates, or other organic acids), fillers (such as mannitol or glycine), chelating agents (such as ethylenediaminetetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrin), proteins (such as free serum albumin, gelatin, or immunoglobulins), colorants, flavorings and diluents, emulsifiers, and hydrophilic polymers (such as polyvinylpyrrolidone). Low molecular weight peptides, 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 glycerol, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; dehydrated sorbitol esters; polysorbates, such as polysorbate 20 or polysorbate 80; Triton; tromethamine; lecithin; cholesterol or tyloxapal), stability enhancers (such as sucrose or sorbitol), tension enhancers (such as alkali metal halides, such as sodium chloride or potassium chloride, or mannitol sorbitol), delivery mediators, excipients, and / or drug adjuvants.

[0309] The carrier and / or diluent may be a parenteral or optional intravenous medium. Suitable parenteral media include sodium chloride solution, Ringer's dextrose, glucose and sodium chloride, and lactated Ringer's. Suitable physiologically acceptable thickeners may be included, such as carboxymethyl cellulose, polyvinylpyrrolidone, gelatin, and alginate. Intravenous media include fluid and nutritional supplements, as well as electrolyte supplements, such as those based on Ringer's dextrose. In some cases, agents that adjust the composition's tonicity may be included in the pharmaceutical composition, such as sugars, polyols like mannitol, sorbitol, or sodium chloride. For example, in many cases, it is desirable for the composition to be substantially isotonic. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may also be present. The exact formulation will depend on the route of administration. Other relevant principles, methods and components of pharmaceutical formulations are well known (see, for example, Allen, Loyd V., ed. (2012) Remington's Pharmaceutical Sciences, 22nd edition).

[0310] The pharmaceutical compositions of the present invention can be administered via one or more routes of administration using one or more of a variety of methods known in the art. As those skilled in the art will understand, the route and / or mode of administration will vary depending on the desired outcome. Routes of administration for the pharmaceutical compositions of the present invention include intravenous, intramuscular, intradermal, intraperitoneal, intrapleural, subcutaneous, intratumoral, spinal, intramedullary, or other parenteral administration routes, such as by injection or infusion. As used herein, the phrase “parenteral administration” refers to a mode of administration other than enteral and local administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrasheathal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, spinal, epidural, intratumoral, intrapleural, and intrasternal injection and infusion. In some embodiments, the pharmaceutical composition is administered intravenously. In other embodiments, administration is intrapleural or intraperitoneal. When considering parenteral administration, the pharmaceutical composition is generally in a sterile, pyrogen-free, parenteral acceptable composition form. A particularly suitable medium for parenteral injection is a properly preserved sterile isotonic solution. Pharmaceutical compositions may be in the form of lyophilized products, such as lyophilized cakes.

[0311] Alternatively, the pharmaceutical compositions of the present invention may be administered via non-parenteral routes, such as local, epidermal or mucosal administration, for example intranasal, oral, vaginal, rectal, sublingual or local administration.

[0312] In some embodiments, the pharmaceutical composition is intended for subcutaneous administration. Typically, pharmaceutical compositions for subcutaneous administration contain suitable stabilizers (e.g., amino acids such as methionine, and / or sugars such as sucrose), buffers, and tension agents. Alternatively, the pharmaceutical composition may be intended for intramedullary administration.

[0313] therapy

[0314] The present invention also provides a lyophilized composition of the present invention, a reconstructed composition of the present invention, reticulocytes of the present invention, mature RBCs of the present invention, erythroid cells of the present invention, cells of the present invention, populations of the present invention, variant proteins of the present invention, polynucleotides of the present invention, carriers of the present invention, or pharmaceutical compositions of the present invention for use in therapy.

[0315] The present invention also provides erythroid cells of the present invention, populations of the present invention, variant proteins of the present invention, polynucleotides of the present invention, carriers of the present invention, or pharmaceutical compositions of the present invention for use in therapy.

[0316] The present invention also provides the use of the lyophilized composition of the present invention, the reconstructed composition of the present invention, the reticulocytes of the present invention, the mature RBCs of the present invention, the erythroid cells of the present invention, the cells of the present invention, the populations of the present invention, the variant proteins of the present invention, the polynucleotides of the present invention, the carriers of the present invention, or the pharmaceutical compositions of the present invention in the manufacture of a medicament for therapeutic purposes.

[0317] The present invention also provides the use of the erythroid cells of the present invention, the populations of the present invention, the variant proteins of the present invention, the polynucleotides of the present invention, the carriers of the present invention, or the pharmaceutical compositions of the present invention in the manufacture of medicaments for therapeutic purposes.

[0318] The present invention also provides methods for treating subjects in need, comprising administering to the subject the lyophilized composition of the present invention, the reconstituted composition of the present invention, the reticulocytes of the present invention, the mature RBCs of the present invention, the erythroid cells of the present invention, the cells of the present invention, the population of the present invention, the variant protein of the present invention, the polynucleotide of the present invention, the carrier of the present invention, or the pharmaceutical composition of the present invention.

[0319] The present invention also provides a method of treating a subject in need, comprising administering to the subject erythroid cells of the present invention, the population of the present invention, the variant protein of the present invention, the polynucleotide of the present invention, the carrier of the present invention, or the pharmaceutical composition of the present invention.

[0320] In some embodiments, the lyophilized composition is used for therapeutic purposes or administered after reconstitution. The lyophilized composition may be reconstituted in any of the ways discussed above. In some embodiments, the reconstituted composition is administered immediately. For example, the reconstituted composition may be administered within about 5 minutes, about 10 minutes, about 15 minutes, or about 20 minutes after reconstitution. In other embodiments, the reconstituted composition may 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. In the case of storing the reconstituted composition for a period of time before use, a suitable storage buffer may be used for reconstitution.

[0321] The therapy or treatment is preferably a blood transfusion, enzyme replacement therapy, organ reconditioning, or detoxification. In some embodiments, the therapy is a blood transfusion. In this embodiment, the reticulocytes can be natural or wild-type cells.

[0322] In some embodiments, the therapy or treatment is enzyme replacement therapy, organ repair, or detoxification. In this embodiment, reticulocytes may further contain or express therapeutic proteins or peptides.

[0323] In some embodiments, the therapy or treatment is to treat or prevent RBC disease or condition, preferably RBC disease or condition associated with increased reactive oxygen species (ROS) activity. Overexpression of the antioxidant protein according to the invention can protect the cells of the invention from the effects of ROS activity. In some embodiments, the RBC disease or condition is β-thalassemia, sickle cell disease, cerebellar ataxia, or motor neuron disease.

[0324] In embodiments of the invention where the cells may further comprise or express therapeutic proteins or peptides, the therapy or treatment may include using the therapeutic protein or peptide to treat or prevent a disease or condition. A disease or condition may be, for example, an inflammatory disease or condition, an infection, or cancer.

[0325] In the context of this invention, inflammatory disease is a disease or condition that includes damage to or destruction of healthy, surviving cells through inflammation. Examples of inflammatory diseases or conditions include, but are not limited to, autoimmune diseases, allergies, asthma, celiac disease, nephritis, hepatitis, reperfusion injury, graft-versus-host disease (GvHD), transplant rejection, and infections.

[0326] Autoimmune diseases may include rheumatoid arthritis, psoriasis, systemic lupus erythematosus (lupus), inflammatory bowel disease, multiple sclerosis, diabetes, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, Graves' disease, Hashimoto's thyroiditis, myasthenia gravis, aplastic anemia (AA), vasculitis, or a combination thereof.

[0327] Infections can be caused by any pathogen. Pathogens can be bacteria, archaea, fungi, or viruses.

[0328] Preferably, the cancers are anal cancer, cholangiocarcinoma, bladder cancer, leukemia, bone cancer, intestinal cancer, brain tumors, breast cancer, colorectal cancer, cervical cancer, endocrine tumors, eye cancer (such as ocular melanoma), fallopian tube cancer, gallbladder cancer, head and / or neck cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, lymph node cancer, lymphoma, melanoma, mesothelioma, myeloma, neuroendocrine tumors, ovarian cancer, esophageal cancer, pancreatic cancer, penile cancer, primary peritoneal cancer, prostate cancer, and pseudomyxoma peritoneum. Pseudomyxoma peritonei Skin cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, testicular cancer, thymic cancer, thyroid cancer, tracheal cancer, cancer of unknown primary origin, vaginal cancer, vulvar cancer, or endometrial cancer. Leukemia is preferably acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, or chronic myeloid leukemia. Lymphoma is preferably Hodgkin lymphoma or non-Hodgkin lymphoma. Cancer is preferably primary or secondary cancer.

[0329] The route of administration can be any of those discussed above. In a preferred embodiment, administration is by intravenous injection.

[0330] The therapy or method typically comprises administering a therapeutically effective or preventatively effective amount of a composition, cell, population, variant protein, polynucleotide, carrier, or pharmaceutical composition. A therapeutically effective amount is an amount that improves one or more symptoms (such as all symptoms) of a disease or condition and / or eliminates one or more symptoms (such as all symptoms) of a disease or condition. A therapeutically effective amount preferably cures the disease or condition. A preventatively effective amount is an amount that prevents the onset of a disease or condition and / or prevents the onset of one or more symptoms (such as all symptoms) of a disease or condition. A preventatively effective amount preferably prevents the subject from developing a disease or condition. Suitable amounts are discussed in more detail below.

[0331] The composition, cell, population, variant protein, polynucleotide, carrier, or pharmaceutical composition may be administered to subjects exhibiting symptoms of a disease or condition. The composition, cell, population, variant protein, polynucleotide, carrier, or pharmaceutical composition may be administered to asymptomatic subjects (i.e., those not exhibiting symptoms of a disease or condition). The composition, cell, population, variant protein, polynucleotide, carrier, or pharmaceutical composition may be administered when the subject's disease state is unknown or when the subject is not expected to have a disease or condition. The composition, cell, population, variant protein, polynucleotide, carrier, or pharmaceutical composition may be administered to subjects susceptible to a disease or condition (such as a genetic predisposition).

[0332] When administered to said subjects, the composition, cells, populations, variant proteins, polynucleotides, carriers, or pharmaceutical compositions reduced 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%, compared to untreated subjects.

[0333] The subject can be a mammal. Optionally, the subject is a human, horse, dog, or cat. In a preferred embodiment, the subject is a human.

[0334] In embodiments where the subject is a human being, the subject can be an adult or a child. In the context of this invention, an adult will be understood as a person at least 18 years of age. A child will be understood as a person under 18 years of age. In some embodiments, an adult is at least 60 years of age.

[0335] In implementation plans for the use of cells in therapies or treatments, the number of cells administered to the subject should take into account the route of administration, the disease or condition being treated, the subject's weight, and / or the subject's age. Typically, approximately 1 × 10⁻⁶ cells are administered to the subject. 6 To approximately 1×10 11 Cells. In one implementation, approximately 1 × 10⁶ cells are administered to the subject. 7 To approximately 1×10 10 One cell, or approximately 1 × 10⁻⁶ 8 To approximately 1×10 9 Each cell.

[0336] In some embodiments, a composition, cell, population, variant protein, polynucleotide, carrier, or pharmaceutical composition of about 1 mg / ml to about 100 mg / ml may be administered to the subject. In some embodiments, a composition, cell, population, variant protein, polynucleotide, carrier, or pharmaceutical composition of about 1 mg / ml to about 10 mg / ml may be administered to the subject.

[0337] This invention can be used in combination with other means and substances used to treat diseases or conditions. In some cases, compositions, cells, populations, variant proteins, polynucleotides, carriers, or pharmaceutical compositions can be administered simultaneously, sequentially, or separately with other substances intended to treat a disease or condition, improve symptoms of a disease or condition, or provide pain relief. Compositions, cells, populations, variant proteins, polynucleotides, carriers, or pharmaceutical compositions can be used in combination with existing treatments for diseases or conditions, and can, for example, be simply mixed with such treatments. Therefore, this invention can be used to enhance the efficacy of existing disease treatments.

[0338] In some embodiments, the cells or populations of the present invention may be autologous to the subject. For example, erythroid progenitor cells may be taken from the subject, differentiated into a cell population containing at least about 10% reticulocytes, lyophilized, and ultimately reconstructed and returned to the subject. Alternatively, a cell population may be obtained by harvesting reticulocytes from the subject and lyophilizing them. However, preferably, the cells or populations are allogeneic relative to the subject. In this embodiment, it is preferable that the cells or populations are matched to the subject.

[0339] Preferred features of each aspect of the invention may be described as in conjunction with any other aspect. Within the scope of this application, it is expressly intended that the various aspects, embodiments, examples, and alternatives set forth in the foregoing paragraphs, claims, and / or the following description and drawings, particularly their respective features, may be employed independently or in any combination. That is, all embodiments and / or features of any embodiment may be combined in any manner and / or combination unless such features are incompatible.

[0340] sequence

[0341] The following table summarizes the sequences used in this invention.

[0342]

[0343] Example

[0344] The sequences used in these embodiments are shown in the table above.

[0345] Example 1: Reticulocytes can be pre-loaded with trehalose by incubation overnight.

[0346] For these experiments, donated CD34+ cells were differentiated to generate a population of cultured reticulocytes. CD34+ hematopoietic cells were isolated from donated whole blood, proliferated, and differentiated into reticulocytes within 21 days, followed by leukofiltration to purify the reticulocytes.

[0347] Reticulocytes cultured by centrifugation were concentrated, and the effectiveness of the lyophilization protectant was tested by suspending the cells in PBSAG buffer alone or in PBSAG supplemented with 10% trehalose (wt / vol). A proportion of samples were also stored overnight in PBSAG or PBSAG supplemented with 500 mM trehalose in an incubator at 37°C and 5% CO2. Reticulocytes incubated overnight in PBS containing 500 mM trehalose were referred to as “loaded”. Control and overnight loaded samples were washed three times with PBSAG the following day and then stored in PBSAG alone or supplemented with 10% trehalose. The samples were stored on ice and then lyophilized as described below.

[0348] To confirm that trehalose did indeed accumulate inside reticulocytes loaded with trehalose overnight, 1×10⁻⁶ cells were lysed from each reticulocyte sample. 6 Cells were analyzed, and trehalose content was determined using the commercial Megazyme Trehalose Assay Kit, which uses a spectrophotometer at 240 nm to assess trehalose content. Figure 1 This confirmed that trehalose accumulates in cells loaded with trehalose overnight, rather than in cells stored in standard PBSAG buffer where trehalose is absent.

[0349] Example 2: The modified trehalose transporter GLUT8 can be overexpressed in erythroid cells and can be used to transport trehalose into cells.

[0350] The GLUT8 transporter is the only identified mammalian trehalose transporter, notable for its NH2-terminal [DE]XXXL[LI] targeting motif, known to be important for lysosome targeting. This N-terminal motif plays a role in directing proteins to endosomes, lysosomes, and related organelles. Studies in HEK293 cells have revealed that the subcellular location of the GLUT8 transporter can be altered to the plasma membrane by replacing the TPQ (XXX) residues in this targeting sequence of the lysosome-localized GLUT8 transporter with a GPN derived from the GLUT12 transporter (which localizes the GLUT12 transporter to the plasma membrane). Furthermore, it has been reported that changing the dileucine (LL) motif to AA can relocate the GLUT8 transporter to the plasma membrane. To investigate which alterations would produce the most efficient plasma membrane expression in erythroid cells, wild-type GLUT8 (SEQ ID NO: 34 and 35), the corresponding human GPS mutant variants (SEQ ID NO: 36 and 37), and the LL mutant variant were expressed in BEL-A cells (an erythroid model that produces reticulocytes).

[0351] Flow cytometry on cells showed that both the LL and GPS mutants were well expressed, but this was on permeabilized cells because the c-Myc tag is located on the intracellular side of the protein. Immunofluorescence imaging showed that only the TPQ mutant was expressed on the cell surface (data not shown).

[0352] Therefore, although the LL mutant exhibits high expression levels in expanded BEL-A cells, its intracellular location makes it unsuitable for the specific purposes of these experiments. The following experiments utilize a GLUT8-GPS-modified transporter, which will be referred to below as "GLUT8" and / or "GLUT8-GPS".

[0353] To confirm that erythroid cells expressing GLUT8 on their surface do indeed accumulate trehalose faster than unmodified cells, stable BEL-A cells expressing "low" or "high" GLUT8-GPS (SEQ ID NO: 36 and 37) were compared. Figure 2A The trehalose was incubated with 500 mM trehalose for a specific time, and then lysed and its content was determined using the commercial Megazyme Trehalose Assay Kit, which uses a spectrophotometer at 340 nm to assess the trehalose content. Figure 2B The charts in the figure confirm that trehalose does indeed accumulate faster inside cells when cells express GLUT8-GPS, confirming that modified GLUT8 is active on the surface of erythroblasts and can transport more trehalose than unmodified control BELA cells.

[0354] We also explored GLUT8 expression in primary erythroid cultures using an optional vector. The GLUT8-GPS-MYC construct was cloned into the puromycin-selective plasmid CD710-B-1-Puro (System Biosciences). This vector, along with puromycin selection, ensured that the generated reticulocytes fully expressed the desired construct. CD34+ hematopoietic stem cells were transduced on day 3 post-isolation, and puromycin (1 µg / mL) was added to the culture between days 5 and 8. Figure 4A As shown. Cells were differentiated into reticulocytes during a 20-day, 3-step culture method as previously described by Kupzig et al. Simply put, isolated CD34+ cells were cultured at 1 × 10⁻⁶ cells per cell line. 5 Cells were seeded at a density of 1 / mL in IMDM basal medium (IMDM, 3% heat-inactivated AB serum, 2 mg / mL human serum albumin, 10 μg / mL insulin, 3 U / mL heparin, 3 U / mL erythropoietin, and 200 μg / mL holo-transferrin). The basal medium was supplemented with 40 ng / mL SCF and 1 ng / mL IL-3 from days 0 to 8, and with only 40 ng / mL SCF from days 8 to 13. Thereafter, cells were kept in basal medium alone until day 20. To obtain a purified population of reticulocytes, the culture was filtered using a 5 µm Acrodisc filter (VWR) (when handling volumes less than 100 mL) or a leukocyte filter.

[0355] Figure 4B GLUT8-GPS-MYC expression during differentiation was confirmed as assessed by flow cytometry. Cells were fixed, permeabilized, and labeled by detecting the c-MYC tag. These data highlight the preservation of GLUT8 expression during primary reticulocyte differentiation.

[0356] After isolating reticulocytes, the cells were incubated overnight at 37°C with 10% trehalose (w / v) in PBSAG or PBSAG alone. To confirm and quantify trehalose uptake, 1 × 10⁻⁶ cells were lysed for each assay. 6 Cells were analyzed using the same Megazyme kit mentioned earlier. Absorbance was measured at 340 nm using [method / method - details omitted]. Figure 4C The standard curve shown is converted to µg. Figure 4C The expression of GLUT8-GPS indicates an approximately 80% increase in trehalose uptake in reticulocytes. Therefore, the modified GLUT8-GPS is targeted to the surface of cultured reticulocytes and is active on the surface, thereby transporting trehalose into the cells.

[0357] Example 3: Preparation of reticulocytes expressing modified GLUT8

[0358] For these experiments, CD34+ cells were differentiated to generate a population of cultured reticulocytes. CD34+ hematopoietic cells were isolated from donated whole blood, proliferated, and differentiated into reticulocytes within 21 days, followed by leukocyte filtration to purify the reticulocytes. To test the role of GLUT8 expression, CD34+ cells were transduced with lentivirus, then proliferated, differentiated, and reticulocytes were prepared.

[0359] To confirm that GLUT8-loaded trehalose-derived reticulocytes accumulated more trehalose during overnight loading, 1×10⁻⁶ sclerosings were performed on each reticulocyte sample. 6 Cells were analyzed, and trehalose content was determined using the commercial Megazyme Trehalose Assay Kit, which uses a spectrophotometer at 240 nm to assess trehalose content. Figure 3 The graphs further confirm that trehalose accumulates intracellularly in cells loaded with trehalose overnight, rather than in cells stored in standard PBSAG buffer without trehalose. It also reiterates that in CD34+ reticulocytes expressing GLUT8-GPS, even in PBSAG buffer alone, small amounts of trehalose accumulate intracellularly (likely due to cellular metabolism), and the accumulation of GLUT8-GPS trehalose is twice that of unmodified reticulocytes when trehalose is loaded overnight. Therefore, GLUT8-GPS expression at the reticulocyte surface enhances intracellular trehalose loading.

[0360] Example 4: Overexpression of natural protective proteins derived from nature

[0361] Tardigrade-specific intrinsically disorganized proteins (TDPs) are crucial for tardigrade tolerance to desiccation. These proteins are intrinsically disorganized proteins (IDPs) and are typically upregulated during drying. TDPs form a non-crystalline amorphous solid (vitrification) upon drying, and this vitrification reflects their protective capabilities. Three tardigrade-specific proteins were selected for expression in reticulocytes: cytosol-rich heat-soluble (CAHS) protein 1, CAHS protein 2, and CAHS protein 3. These proteins are not present in human reticulocytes.

[0362] Late embryogenesis-rich (LEA) proteins are expressed in insects, nematodes, and plants. LEA proteins are characterized by an 11-mer motif and are known to reduce protein aggregation during dehydration. LEA proteins can be subdivided into Group 1, Group 2, and Group 3 based on species, with Group 3 proteins expressed in animals. Two exemplary Group 3 LEA proteins were selected: PvLEA22 expressed in *Chironoptera vannamei* and AavLEA expressed in *C. oatus*. Similarly, these proteins are not present in human reticulocytes.

[0363] The erythroid cell line BEL-A was used as a model system to demonstrate the possibility of expressing this selected naturally derived desiccant protein (SEQ ID NO: 29-33) in erythroid cells. BEL-A cells were single-sorted to generate monoclonal cell lines with high expression. The two clones with the highest expression in each construct were selected and terminally differentiated four times. Expression was analyzed on day 0 (BELA cells during expansion), in differentiated erythroblasts up to day 8, and in reticulocytes (day 12). A representative graph of protein expression during differentiation, measured by flow cytometry on permeabilized cells, is shown. Figure 5A This confirms that despite some loss of protein expression during reticulocyte differentiation, a certain proportion of these exogenously expressed desiccant proteins can still be retained. These may provide protection for freeze-dried reticulocytes or facilitate longer storage when expression is further optimized.

[0364] These protective proteins are also expressed as green fluorescently labeled fusion proteins in expanding and differentiating CD34+ cells for monitoring expression. For these experiments, CD34+ cells were transduced with a specific lentiviral construct, and then the CD34+ cells were proliferated, differentiated, and reticulocytes were prepared. On days 6, 12, and 19 (reticulocytes), the expression levels of CAHS1-GFP, CAHS2-GFP, and AavLEA-GFP in expanding CD34+ hematopoietic stem cells and subsequently differentiated from them in reticulocytes were measured by flow cytometry during differentiation. See [link to relevant documentation]. Figure 5B To test whether the expression of these proteins affects deformability, reticulocytes expressing CAHS1, CAHS2, or AavLEA were sorted using FACS. The deformability of reticulocytes was measured using an ARCA deformability apparatus (which measures cell deformability) and compared with untransduced cells. No differences were observed between control reticulocytes (untransduced reticulocytes) and reticulocytes expressing CAHS1, CAHS2, and AavLEA. Figure 5B ).

[0365] To avoid cell loss during FACS, novel constructs were designed to allow antibiotic selection for CD34+ cells. Since there may be future interest in co-expressing these native proteins with, for example, GLUT8 or antioxidant proteins, all three CAHS proteins were cloned into CD710-B-1-Blast (System Biosciences) with a C-terminal FLAG tag (SEQ ID NO: 48-53). CD34+ hematopoietic stem cells were transduced with each specific construct on day 3 post-isolation, and blastomycin (10 µg / mL) was added to the culture between days 5 and 8. As previously described, cells were differentiated into reticulocytes during a 20-day, three-step culture process.

[0366] like Figure 6 As shown above, on days 10, 14, and 20 (filtered reticulocytes), the cell lysate (1×10⁻⁶) was analyzed. 6 Western blotting was used to assess the expression levels of CAHS1-FLAG, CAHS2-FLAG, and CAHS3-FLAG in differentiating CD34+ hematopoietic stem cells and filtered reticulocytes. As detected by anti-FLAG labeling, CAHS1-FLAG expression was lowest in this construct and difficult to detect in reticulocytes. CAHS2-FLAG expression decreased with cell differentiation and maturation, with reduced expression observed in reticulocytes. CAHS3-FLAG was the most abundant of the three CAHS1 protective proteins tested, and its abundance remained constant throughout the differentiation process from CD34+ reticulocytes.

[0367] Example 5: Overexpression of peroxide reductase to resist reactive oxygen species

[0368] Due to their oxygen transport function and high iron content, RBCs are particularly at risk of oxidative stress during circulation and storage. To minimize the effects of oxidative stress, RBCs possess a comprehensive antioxidant system. These include low-molecular-weight antioxidants such as ascorbic acid (vitamin C) and glutathione, as well as a range of more complex enzymatic pathways for combating reactive oxygen species and damage. It is known that lyophilization / drying, storage, and subsequent rehydration processes also generate destructive reactive oxygen species, which can likely impair cell survival or disrupt prolonged cell storage. This prompted us to explore whether it is possible to use lentiviruses to further enhance the already high expression levels of antioxidant enzymes in erythroid cells to produce reticulocytes with further enhanced resistance to oxidative stress, thereby improving lyophilization / storage properties. Enzymes that contribute to lipid peroxidation and minimize hemoglobin oxidation were specifically selected, but the methods used here are essentially applicable to any antioxidant enzyme.

[0369] Six peroxidase (Prx) isoforms exist in mammalian cells. Among them, peroxidase 2 (Prx2) is considered a key player in the antioxidant defense mechanism of RBCs and is the third most abundant protein in RBCs. Furthermore, its dual function as a protective hemoglobin molecular chaperone that reduces methemoglobin production makes it an attractive antioxidant for overexpression in reticulocytes. Prx1 and Prx6 are also commonly found in RBC cytosol, although their expression levels are much lower than that of Prx2.

[0370] For each wild-type human Prx isotype (SEQ ID NO: 19, 20, 23, 24, 27, and 28) with a c-MYC tag to distinguish its expression from endogenous protein expression, BEL-A cells were amplified using lentiviral transduction. Cells were expanded to generate stable BEL-A erythroblast lines expressing single Prx isotypes 1, 2, and 6, and then differentiated to generate reticulocytes. Figure 7A shows Western blot analysis of overexpression of each protein in BEL-A cells amplified on day 0 (proerythroblasts) using isotype-specific antibodies. Note that due to the inclusion of the c-MYC tag, exogenously expressed proteins are larger on the Western blot and are all substantially expressed compared to the lower bands corresponding to endogenous Prx proteins.

[0371] Figure 7B confirms the expression of each protein in expanded BEL-A cells. Figures 7C and 7D show Prx expression in mixed cell populations (orthochromatic, reticulocytes, and nucleated cells) or reticulocytes only (d12r) at the onset (d0) (proerythroids) and end-stage of BEL-A differentiation, analyzed by flow cytometry via permeabilization followed by c-MYC tag detection. Reticulocytes were specifically gated using a DNA-negative population because they lack a nucleus. Mean fluorescence intensity was normalized to background antibody markers in untransduced (UT) control cells. This data shows that expression of each Prx enzyme isoform was maintained and preserved in reticulocytes with minimal loss of Prx isoform expression during differentiation.

[0372] This work establishes for the first time that developing red blood cells can be genetically engineered to overexpress antioxidant proteins, even though endogenous forms are already highly expressed in the cells.

[0373] Therefore, these Prx isotype results suggest that other enzymes, such as superoxide dismutase or catalase, which are also naturally abundant in erythrocytes, can also enhance cellular antioxidant capacity through lentiviral overexpression. It is possible that normally expressed mitochondrial superoxide dismutase, which is lost during mitochondrial loss and subsequently lost during the production of mature erythrocytes, can still be successfully overexpressed and retained in the cytosol by removing its mitochondrial targeting sequence. Other antioxidants, such as glutathione peroxidase, are more complex due to their requirement for selenocysteine, as discussed below.

[0374] Example 6: Overexpression of glutathione peroxidase 1 and 4

[0375] GPx1 is also considered a major player in RBC ROS defense and is expressed at high endogenous levels in RBCs. GPx1 has been shown to be associated with the RBC membrane and is therefore hypothesized to play a role in preventing lipid peroxidation, thus being selected for overexpression. GPx4 has recently gained increasing attention due to its dominant role in the regulation of iron-dependent ferroptosis.

[0376] Figure 8 illustrates the overexpression of tagged wild-type glutathione peroxidase (GPx) protein in BEL-A cells, where expanding BEL-A cells were transduced with lentiviruses carrying c-MYC-tagged wild-type human GPx1 and GPx4 cDNAs, respectively (see Figures 8A and 8B). Cells were expanded to generate stable BEL-A erythroblast lines, which were then later further differentiated to generate reticulocytes. This demonstrates that, unlike the Prx protein family, selecting only the known cDNA sequence of glutathione peroxidase is insufficient for expression.

[0377] This is likely due to the requirement of a specific amino acid, selenocysteine, at the active site of these enzymes, a hypothesis confirmed by mutating selenocysteine ​​to cysteine ​​(which retains its antioxidant activity). Figures 8A and 8B show Western blot and flow cytometry analyses of overexpression of GPx isoforms 1 and 4 in BEL-A cells, with BEL-A cells being transduced and expanded using wild-type human GPx1, U49C GPx1, wild-type full-length GPx4, or U73C GPx4 constructs (SEQ ID NO: 3-6 and 9-12). Figure 8B (bottom panel) shows flow cytometry analysis comparing GPx1 U49C and GPx4 U73C expression. This indicates that the selenocysteine ​​mutant, which mutates selenocysteine ​​to cysteine, can achieve expression. This confirms that selenocysteine ​​incorporation is important for GPx expression, and that it is possible to circumvent this by mutating selenocysteine ​​to cysteine ​​to overexpress glutathione peroxidases such as GPx1 and GPx4. Figures 8C and 8D show the expression of GPx1 U49C and GPx4 U73C after differentiation into reticulocytes using untransduced cells as a negative control, comparing the mixed population endpoint at day 12 (d12) and the reticulocyte-gated endpoint at day 12 (d12r). This confirms that while GPx1 U49C can be preserved during the differentiation of erythroid cells into reticulocytes, GPx4 U73C is lost.

[0378] The limited GPx4 U73C expression achieved in expanded BEL-A cells and the significant loss of expression during differentiation suggest that this protein is tightly regulated in some unknown way. Since ubiquitin-mediated proteasome degradation and lysosome-induced degradation are considered two major pathways for erythropoietic protein turnover, the roles of proteasome inhibitors (MG-132) and lysosome inhibitors (leucopeptide) were investigated to determine whether GPx4 U73C overexpression could be further enhanced in expanded BEL-A cells. Furthermore, specific HSC70 recognition motifs that trigger GPx4 degradation via molecular chaperone-mediated autophagy have recently been identified. Since CMA typically promotes the degradation of oxidative proteins, bardosorone-mediated CMA inhibition may also contribute to increased GPx4 U73C overexpression in erythroid cells. Expanded GPx4 U73CBEL-A cell lines were exposed to each inhibitor or the corresponding mediator control (DMSO) for a total of 6 hours. The expression levels were determined by intracellular flow cytometry and normalized to the background c-MYC markers in control BEL-A cells.

[0379] As shown in Figure 8E, incubation with 5 μM MG132 in differentiating BEL-A erythroids expressing GPx4 U73C resulted in a significant increase in GPx4 U73C expression. No increase in expression was observed after treatment with 10 μM leuproprotein or 10 μM bardoxolone, indicating that ubiquitin-mediated degradation is a major contributing factor to GPx4 degradation in differentiating erythroid cells.

[0380] Example 7: Procedures to enhance glutathione peroxidase expression and retention in reticulocytes

[0381] Next, we determined whether it was actually possible to achieve even higher levels of GPx4 overexpression using novel optimization strategies. Since GPx containing selenocysteine ​​(Sec) is beneficial for both catalytic activity and resistance to excessive oxidation, we confirmed that overexpression of the unmodified protein GPx still resulted in GPx cDNA (here referred to as GPx). Sec The key design objective was to extend the GPx4 construct sequence to include a Sec insert sequence (SECIS) to attempt incorporating selenocysteine ​​(which facilitates tRNA recruitment). Sec The natural GPx4 3' untranslated region (UTR) nucleotide sequence (mRNA structure). It is hypothesized that the inclusion of this sequence would enable the incorporation of selenocysteine ​​and thus the expression of GPx4 (and by inference, GPx1 would also be expressed due to the same requirement).

[0382] GPx4 is known to exist in three forms: a full-length (22 kDa) form primarily expressed in sperm by the primary transcript; a short (19 kDa) form typically found in subcellular locations in other cell types, such as the cytoplasm, nucleus, and microsomes; and a sperm-specific nuclear form derived from a substitute exon located in the first intron of the GPx4 gene. Further examination of the full-length primary GPx4 transcript used to design expression constructs has revealed a mitochondrial leader sequence of 25 amino acids at the N-terminus. Western blot results in Figure 9B also indicate that a proportion of the GPx4 protein has a low molecular weight and may therefore be cleaved upon ingestion into the mitochondria, similar to many mitochondrial signaling sequences. Therefore, it is hypothesized that the presence of this signaling sequence may limit GPx4 expression and affect retention, as mitochondria are lost during late differentiation and reticulocyte maturation. Thus, the short form of GPx4 was chosen for further investigation.

[0383] Figures 9A, 9C, and 9D show the N-terminal sequence required for removing the GPx4 mitochondrial targeting sequence and including the 3'UTR sequence (sGPx4). 3’UTR After that, GPx4 was implemented. SecSuccessful overexpression of (SEQ ID NO: 13 and 14). However, loss of GPx4 expression was still observed during reticulocyte differentiation in expression tests, which may be due to ubiquitin-mediated degradation (see evidence in Example 6 above).

[0384] Protein degradation was blocked by mutations in six potential ubiquitination sites (a strategy for thymidine phosphorylases, e.g., as described in WO 2021 / 053243). Ubiquitination sites were selected using software described in Hornbeck PV, Zhang B, Murray B, Kornhauser JM, Latham V, Skrzypek E, PhosphoSitePlus, 2014: mutations, PTMsand recalibrations. Nucleic Acids Res. 2015 43:D512-20. Potential ubiquitination sites K80, K97, K121, K135, K140, and K164 were identified in this manner. Computer modeling confirmed that these sites were surface-exposed and not located within the active site. sGPx4 3’UTR The construct was modified to contain these six lysine residues mutated to arginine (arginine was chosen to retain the positively charged amino acid), but the potential ubiquitination sites were mutated, making them unusable for ubiquitination (K80R, K97R, K121R, K135R, K140R, K164R; referred to here as sGPx4). 3’UTR 6KR; SEQ ID NO: 15 and 16). As measured by flow cytometry of permeabilized cells, sGPx4 in amplifying BEL-A. 3’UTR 6KR expression and wild-type sGPx4 3’UTR The overexpression matched (see Figure 9A), indicating that mutagenesis at these sites is harmless to the overall structure of the enzyme and therefore its expression. For each GPx4 sec The construct was used to prepare a stable cell line.

[0385] GPx4 Sec Differentiation of the BEL-A cell line (see Figures 9C and 9D) revealed sGPx4 3’UTR Overall expression was reduced, with approximately 70% loss, primarily within the first four days of differentiation. Nevertheless, a certain proportion of sGPx4... 3’UTR Overexpression persisted in reticulocytes on day 12 (see graph in Figure 9D).

[0386] sGPx4, as determined by flow cytometry on permeabilized cells. 3’UTRThe expression of the 6KR construct version consistently exceeded that of sGPx4 throughout the differentiation process. 3’UTR The expression of sGPx4 is shown in Figure 9C and the right panel. Notably, reticulocytes on day 12 exhibited sGPx4. 3’UTR High retention of 6KR expression (see graph in Figure 9D), showing sGPx4 3’UTR 6KR promotes expression and retention in reticulocytes.

[0387] Therefore, GPx4 has been used here as an example protein to identify that the combined removal of signal sequences that would hinder cytosol expression or lead to protein loss during erythropoiesis can help the expression and retention of enzymes in reticulocytes. For peroxidases such as GPx4, the inclusion of a 3'UTR sequence ensures the proper incorporation of selenocysteine, thereby enabling the expression of the protein; furthermore, when ubiquitination sites of such proteins (e.g., the six mutated sites on the GPx4 protein in this study) are detected to be disrupted, the protein can be maintained at a significant concentration during cell differentiation and enucleation, thereby enabling the glutathione peroxidase 4 levels in the final reticulocytes (and / or erythrocytes) to reach levels previously unattainable by retaining overexpressed proteins.

[0388] Similar to GPx4, the GPx1 construct sequence was also extended to include the native GPx1 3'UTR nucleotide sequence containing the SECIS region. MYC-GPx1 U49C The MYC-GPX1 3'UTR (SEQ ID NO 54-55) was cloned into CD710-B-1-Puro. BEL-A cell lines were amplified using lentiviral transduction specific to each protein, and selection was performed using 1 µg / mL puromycin.

[0389] Figure 10 A confirmed the expression of both proteins in expanded BEL-A cells after fixation, permeabilization, and c-MYC labeling. As observed for GPx4, the U49C mutation allowed for higher expression compared to the MYC-GPx1 3'UTR, which requires the incorporation of selenocysteine ​​residues. Further analysis was performed using Western blotting (…). Figure 10 B) Analyze cell lysates (1×10⁻⁶) of amplified cells using a monoclonal anti-GPx1 antibody that allows detection of both endogenous and exogenous forms of GPx1. 6 Consistent with flow cytometry data, expression of unmodified GPx1 (MYC-GPx1 3'UTR) was lower than that of MYC-GPx1. U49C .

[0390] BEL-A cell lines were differentiated for 11 days, and nucleated cells (d11) were further differentiated. 有核) and enucleated cells (reticulocytes, d11) 网织红细胞 c-MYC expression was determined in both cases. Since reticulocytes lack a nucleus, the cell population can be specifically gated using DNA-specific staining (Hoechst). Figure 10 C showed that the expression of both constructs decreased during differentiation, and expression on reticulocytes was lower than that on nucleated cells. On day 11, MYC-GPx1... U49C The expression of (above) remains higher than that of MYC-GPx1 3'UTR. This suggests that further modifications may be needed to retain this protein in human erythroblasts, for example, by blocking ubiquitination.

[0391] Example 8: Expression of glutathione peroxidase 1 and 4 in primary CD34+ derived reticulocytes.

[0392] The next goal is to improve upon the original CD34. + GPx1 and GPx4 were expressed in cells. As demonstrated in Example 7, the MYC-sGPx43'UTR 6KR construct exhibited the highest and most persistent expression during BEL-A differentiation. This optimized construct was cloned into the CD710-B-1-Puro vector to enable antibiotic selection, as had been performed on GPx1. U49C Just like the 3'UTR construct does.

[0393] CD34 was isolated from PBMCs on day 3 of differentiation. + Cells and encoding MYC-GPx1 U49C Transduction was performed using lentiviral vectors containing either MYC-GPx1 3'UTR or MYC-sGPx4 3'UTR 6KR. Puromycin was applied between days 5 and 8 for selection to enrich the transduced cells. Reticulocytes were purified by filtration on day 20.

[0394] Figure 13 shows the expression of the two GPx1 constructs during primary erythropoiesis, confirmed using Western blotting and flow cytometry. The expression was confirmed by Western blotting (…). Figure 13A The study analyzed cell lysates (1×10⁻⁶) from differentiating cells on days 7, 10, and 14, and from day 20 (filtered reticulocytes), using monoclonal anti-GPx1 antibodies that allow the detection of both endogenous and exogenous forms of GPx1. 6 (Number of samples). GAPDH was used as a loading control. Data from two biological replicates confirmed that eliminating the need for GPx1 incorporation of selenocysteine... U49CThe construct achieved a higher level of exogenous GPx1 expression compared to endogenous GPx1. In contrast, the GPx1 3'UTR construct exhibited a level of expression comparable to endogenous GPx1, with both tending to decrease at the reticulocyte stage. Figure 13B Flow cytometry data from the same samples and time points analyzed in Figure A are presented. Fixed and permeabilized cells were labeled with c-MYC antibody, confirming protein expression patterns consistent with Western blotting results.

[0395] Figure 11 provides supplementary data on the expression of the MYC-sGPx4 3'UTR 6KR construct using an optional vector. Cell lysates (1 × 10⁻⁶) from days 7, 10, and 20 (filtered reticulocytes) 6 Immunoblotting (number) Figure 11A The sustained expression of MYC-sGPx4 throughout erythroid differentiation was confirmed. Two donors (1 and 2) are shown, both exhibiting robust protein expression in the early differentiation stages and a modest decrease towards the reticulocyte stage. Notably, endogenous expression of GPx4 was not detected in untransfected reticulocytes in these exposures. GAPDH was used as a loading control. Flow cytometry histograms ( Figure 11B These findings were further validated, showing that c-MYC staining at the same samples and time points resulted in consistent expression between the two replicates.

[0396] Example 9 – Expression of human AAT2 in cytosol

[0397] Human alanine aminotransferase 2 (huAAT2) is normally located in mitochondria. By examining the huAAT2 protein domain assigned by UniProt (Q8TD30), a mitochondrial-targeting sequence with 27 amino acids was identified at the N-terminus. Similar to GPx4, it was hypothesized that the presence of this signaling sequence might limit huAAT2 expression and affect retention, as mitochondria are lost during late differentiation and reticulocyte maturation. Therefore, a truncated variant of huAAT2 lacking the first 27 amino acids (huAAT2 N27del) was generated for further investigation. Figure 12 A).

[0398] Codon optimization for mammalian expression was performed based on the coding sequence from the Ensembl database. The resulting lentiviral vector, CD170B-1 (Systems Biosciences), was synthesized by GenScript. CD34+ hematopoietic stem cells were transduced with lentiviral particles containing the corresponding construct on day 3 of differentiation, and washed three times in PBS after 24 hours. Positively transduced cells were selected by supplementing the cell culture with puromycin antibiotic for 72 hours starting on day 5. AAT2 overexpression was then assessed by Western blotting of whole-cell lysates using an AAT2-specific monoclonal antibody at indicated time points.

[0399] Figure 12 B shows successful overexpression of both full-length (FL) and truncated (N27del) huAAT2 in CD34+ hematopoietic stem cells from the onset of differentiation (d8) to the end of differentiation (d20) and in the purified reticulocyte population (d20r). Overall, a much larger proportion of initiating huAAT2 protein expression (N27del) was observed to be retained in reticulocytes overexpressing the N-terminus-deficient huAAT2 compared to cells overexpressing the full-length protein (FL). Quantitative analysis of Western blot signal intensity revealed that reticulocytes derived from CD34+ cells overexpressing huAAT2 N27del (d20r) expressed 8.9-fold higher levels of AAT2 protein than reticulocytes derived from stem cells overexpressing huAAT2 FL. Figure 12 As shown in Figure C, reticulocytes derived from CD34+ cells overexpressing huAAT2 FL retained only 7.4% of the initial expression. However, reticulocytes derived from CD34+ cells overexpressing huAAT2 N27del retained nearly 70% of the initial expression.

[0400] Therefore, this confirms another exemplary protein in which removing mitochondrial signaling sequences to enhance cytoplasmic expression improves enzyme retention in cultured reticulocytes.

[0401] Implementation Plan

[0402] The present invention provides the following implementation scheme.

[0403] 1. A lyophilized composition comprising a cell population and a lyophilization protectant, wherein the cell population comprises at least about 10% reticulocytes.

[0404] 2. The lyophilized composition according to embodiment 1, wherein the lyophilization protectant comprises one or more of (a) sugars, (b) antioxidants and (c) intrinsically disordered proteins (IDPs).

[0405] 3. The freeze-dried composition according to embodiment 1 or 2, wherein (a) the sugar is selected from trehalose, sucrose, fructose, glucose, mannitol, sorbitol, arabinose, and ribose; (b) the antioxidant is selected from glutathione peroxidase 1 (GPx1), GPx2, GPx3, GPx4, GPx5, GPx6, GPx7, GPx8, peroxidase 1 (Prx1), Prx2, Prx3, Prx4, Prx5, Prx6, and variants thereof; and (c) the IDP is selected from cytosol-rich heat-soluble 1 (CAHS1), CAHS3, late embryogenesis-rich protein of Chironomida vannamei (PvLEA), LEA of Nematoda oatella (AavLEA), and variants thereof.

[0406] 4. The freeze-dried composition according to any one of the foregoing embodiments, wherein the freeze-drying protectant comprises or is composed of trehalose.

[0407] 5. The freeze-dried composition according to embodiment 4, wherein at least a portion of the trehalose is intracellular.

[0408] 6. The freeze-dried composition according to any one of the foregoing embodiments, wherein the reticulocytes are genetically engineered to express trehalose transporter protein or a variant thereof on their surface.

[0409] 7. The freeze-dried composition according to embodiment 6, wherein the trehalose transporter is glucose transporter 8 (GLUT8) protein or a variant thereof.

[0410] 8. The lyophilized composition according to any one of the foregoing embodiments, wherein the reticulocytes are genetically engineered to overexpress antioxidant proteins and / or intrinsic disordered proteins (IDPs) in their cytosol.

[0411] 9. The freeze-dried composition according to any one of the foregoing embodiments, wherein at least about 30% of the reticulocytes survive during remodeling, and / or wherein at least about 30% of the reticulocytes contain at least about 50% hemoglobin molecules relative to the total number of hemoglobin plus methemoglobin molecules.

[0412] 10. The freeze-dried composition according to any one of the foregoing embodiments, wherein the cell population comprises at least about 90% reticulocytes.

[0413] 11. The lyophilized composition according to any one of the foregoing embodiments, wherein the reticulocytes further comprise or express therapeutic proteins or peptides.

[0414] 12. The freeze-dried composition according to any one of the foregoing embodiments, wherein the reticulocytes are human.

[0415] 13. A pre-lyophilized composition comprising a cell population, a lyophilization protectant, and a lyophilization medium, wherein the cell population comprises at least about 10% reticulocytes.

[0416] 14. The pre-lyophilized composition according to embodiment 13, wherein the lyophilization protectant and / or the cells are as defined in any one of embodiments 1-12.

[0417] 15. A method for preparing a lyophilized composition according to any one of embodiments 1-12, comprising (a) suspending a cell population in a lyophilization medium containing a lyophilization protectant, wherein the cell population comprises at least about 10% reticulocytes, and (b) subjecting the suspension generated in step (a) to a lyophilization process to generate the lyophilized composition.

[0418] 16. The method according to embodiment 15, wherein the method comprises (a) suspending the cell population in the lyophilization medium containing the lyophilization protectant for at least about 12 hours.

[0419] 17. A method for reconstructing a lyophilized composition according to any one of embodiments 1-12, comprising contacting the lyophilized composition with a reconstruction medium.

[0420] 18. A reconstituted composition which is obtained or obtainable by (i) the method according to embodiment 17 or (ii) by reconstituted lyophilized composition according to any one of embodiments 1-12.

[0421] 19. A reconstitution composition comprising a cell population, a lyophilization protectant, and a reconstitution medium, wherein the cell population comprises at least about 10% reticulocytes.

[0422] 20. The reconstituted composition according to embodiment 19, wherein the lyophilization protectant and / or the cells are as defined in any one of embodiments 1-12.

[0423] 21. A reticulocyte or mature erythrocyte (RBC) containing intracellular trehalose.

[0424] 22. The cell according to embodiment 21, wherein the cell is genetically engineered to express trehalose transporter protein or a variant thereof on its surface.

[0425] 23. An erythroid cell that has been genetically engineered to express trehalose transporter protein or a variant thereof on its surface.

[0426] 24. Erythroid cells according to embodiment 23, wherein the erythroid cells are erythroid progenitor cells, reticulocytes or mature erythrocytes.

[0427] 25. Erythroid cells according to embodiment 23 or 24, wherein the trehalose transporter is glucose transporter 8 (GLUT8) protein or a variant thereof.

[0428] 26. Erythroid cells according to embodiment 25, wherein the GLUT8 variant protein comprises a mutation from TPQ to GPN.

[0429] 27. The erythroid cells according to embodiment 26, wherein the GLUT8 variant protein comprises, or is composed of, the sequence shown in SEQ ID NO:37.

[0430] 28. A cell that has been genetically engineered to overexpress antioxidant proteins and / or intrinsically disordered proteins (IDPs) in its cytosol.

[0431] 29. The cell according to embodiment 28, wherein the antioxidant protein is glutathione peroxidase (GPx) or a variant thereof, or peroxidase (Prx) or a variant thereof.

[0432] 30. The cell according to embodiment 29, wherein the GPx is GPx1, GPx2, GPx3, GPx4, GPx5, GPx6, GPx7 or GPx8, or the Prx is Prx1, Prx2, Prx3, Prx4, Prx5 or Prx6.

[0433] 31. The 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.

[0434] 32. The cell according to embodiment 31, wherein the variant protein lacks at least a portion of the signal peptide.

[0435] 33. A cell according to any one of embodiments 30-32, wherein the GPx1 variant protein or the GPx4 variant protein contains a mutation at one or more predicted ubiquitination sites.

[0436] 34. The cell according to embodiment 33, wherein the GPx1 variant protein contains a mutation at one or more of K88, K148 and K166, or the GPx4 variant protein contains a mutation at one or more of K107, K126, K148, K162R, K167 and K191.

[0437] 35. The cell according to embodiment 34, wherein K is replaced by R.

[0438] 36. The cell according to any one of embodiments 29-35, wherein the GPx1, GPx2, GPx3, GPx4, GPx6 or variants thereof are expressed by a polynucleotide comprising a 3' untranslated region (3'UTR) containing a Sec insert sequence (SECIS).

[0439] 37. The cell according to any one of embodiments 29-35, wherein the GPx1 variant protein contains the U49C mutation, or the GPx4 variant protein contains the U73C mutation.

[0440] 38. The cell according to any one of embodiments 29-37, wherein the GPx1 or a variant thereof comprises the sequence shown in SEQ ID NO: 2, 4 or 6 or a variant thereof, or the GPx4 or a variant thereof comprises the sequence shown in SEQ ID NO: 12, 14 or 16 or a variant thereof.

[0441] 39. The cell according to any one of embodiments 28-38, wherein the IDP is CAHS1, CAHS3, PvLEA, AavLEA or a variant thereof.

[0442] 40. The cell according to any one of embodiments 28-39, wherein the cell is further genetically engineered to express trehalose transporter protein or a variant thereof on its surface.

[0443] 41. The cell according to any one of embodiments 28-40, wherein the cell further comprises or expresses a therapeutic protein or polypeptide.

[0444] 42. The cell according to any one of embodiments 28-41, wherein the cell is a blood cell.

[0445] 43. The cells according to embodiments 21-42, wherein the cells are human cells.

[0446] 44. The cell according to any one of embodiments 21-43, wherein the cell is derived from stem cells or immortalized cell lines.

[0447] 45. A population of cells according to any one of embodiments 21-44.

[0448] 46. ​​A variant antioxidant protein comprising mutations at one or more predicted ubiquitination sites.

[0449] 47. The variant protein according to embodiment 46, wherein the variant protein is a Gpx1 or Gpx4 variant protein.

[0450] 48. The variant protein according to embodiment 47, wherein the GPx1 variant protein contains a mutation at one or more of K88, K148 and K166, or the GPx4 variant protein contains a mutation at one or more of K107, K126, K148, K162R, K167 and K191.

[0451] 49. The variant protein according to embodiment 48, wherein K is replaced by R.

[0452] 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 and lacks at least a portion of its signal peptide.

[0453] 51. The variant protein according to any one of embodiments 47-50, wherein the GPx4 variant comprises the sequence shown in SEQ ID NO: 16.

[0454] 52. The variant protein according to any one of embodiments 47-49, wherein the GPx1 variant protein contains the U49C mutation, or the GPx4 variant protein contains the U73C mutation.

[0455] 53. A variant antioxidant protein comprising one or more inactivating mutations in its signal peptide.

[0456] 54. The variant protein according to embodiment 53, wherein the variant protein lacks at least a portion of the signal peptide.

[0457] 55. The variant protein according to embodiment 53 or 54, wherein the variant antioxidant protein is a GPx4, GPx7, GPx8, Prx3 or Prx5 variant protein.

[0458] 56. A polynucleotide encoding a variant protein according to any one of embodiments 46-55.

[0459] 57. The polynucleotide according to embodiment 56, wherein the polynucleotide comprises a 3' untranslated region (3'UTR) containing a Sec insert sequence (SECIS).

[0460] 58. The 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.

[0461] 59. A vector comprising a polynucleotide according to any one of embodiments 56-58.

[0462] 60. A pharmaceutical composition comprising a reconstituted composition according to any one of embodiments 18-20, cells 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 carrier according to embodiment 59, and a pharmaceutically or physiologically acceptable diluent and / or carrier.

[0463] 61. A method for improving the ability of cells to survive in lyophilization, the method comprising genetically engineering the cells to overexpress antioxidant proteins and / or intrinsically disordered proteins (IDPs) in their cytosol.

[0464] 62. The 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.

[0465] 63. The method according to embodiment 61 or 62, wherein the method comprises genetically engineering the cells to express 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.

[0466] 64. A lyophilized composition according to any one of embodiments 1-12, a reconstituted composition according to any one of embodiments 18-20, reticulocytes or mature RBCs according to any one of embodiments 21-22, erythroid cells according to any one of embodiments 23-27, cells 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 carrier according to embodiment 59, or a pharmaceutical composition according to embodiment 60, for use in a therapy.

Claims

1. A genetically engineered erythroid cell to express a variant protein, wherein the variant protein comprises one or more mutations that increase the level of the variant protein in the cytosol of the cell.

2. The erythroid cells of claim 1, wherein the one or more mutations comprise (a) one or more 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. The erythroid cells 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. The erythroid cells according to claim 3, wherein the antioxidant protein is glutathione peroxidase (GPx) or peroxidase (Prx).

5. The erythroid cells according to claim 4, wherein the GPx is GPx1, GPx2, GPx3, GPx4, GPx5, GPx6, GPx7 or GPx8, or the Prx is Prx1, Prx2, Prx3, Prx4, Prx5 or Prx6.

6. The erythroid cells of 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. The erythroid cells of claim 6, wherein the variant protein lacks at least a portion of the signal peptide.

8. Erythroid cells according to any one of claims 5-7, wherein the GPx1 variant protein or the GPx4 variant protein contains a mutation at one or more predicted ubiquitination sites.

9. The erythroid cells of claim 8, wherein the GPx1 variant protein contains a mutation at one or more of K88, K148, and K166, or the GPx4 variant protein contains a mutation at one or more of K107, K126, K148, K162R, K167, and K191.

10. The erythroid cells of claim 9, wherein K is replaced by R.

11. Erythroid cells according to any one of claims 4-10, wherein the GPx1, GPx2, GPx3, GPx4, GPx6 or variants thereof are expressed by a polynucleotide comprising a 3' untranslated region (3'UTR) containing a Sec insert sequence (SECIS).

12. Erythroid cells according to any one of claims 4-10, wherein the GPx1 variant protein contains the U49C mutation, or the GPx4 variant protein contains the U73C mutation.

13. Erythroid cells according to any one of claims 4-12, wherein the GPx1 or a variant thereof comprises the sequence shown in SEQ ID NO: 2, 4, 6 or 55 or a variant thereof, or the GPx4 or a variant thereof comprises the sequence shown in SEQ ID NO: 12, 14 or 16 or a variant thereof.

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

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

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

17. The erythroid cells 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 aminolysin, alanine aminotransferase, glutamate dehydrogenase, arginine deiminase, arginase, oxalate decarboxylase, oxalate oxidase, serine protease, tissue plasminogen activator, or laronidase.

18. Erythroid cells according to any one of the preceding claims, wherein the erythroid cells are further genetically engineered to express trehalose transporter protein or a variant thereof on their surface.

19. Erythroid cells according to any one of the preceding claims, wherein the erythroid cells further comprise or express therapeutic proteins or polypeptides.

20. The erythroid cells according to any one of the preceding claims, wherein the erythroid cells are reticulocytes.

21. Erythroid cells according to any one of the preceding claims, wherein the erythroid cells are human erythroid cells.

22. Erythroid cells according to any one of the preceding claims, wherein the erythroid cells are derived from stem cells or immortalized cell lines.

23. A reticulocyte or mature erythrocyte (RBC) containing intracellular trehalose.

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

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

26. The erythroid cells according to claim 25, wherein the erythroid cells are erythroid progenitor cells, reticulocytes, or mature erythrocytes.

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

28. The erythroid cells of claim 27, wherein the GLUT8 variant protein comprises a TPQ-GPS mutation.

29. The erythroid cells of claim 28, wherein the GLUT8 variant protein comprises, or is composed 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 comprising mutations at one or more predicted ubiquitination sites.

32. The variant protein of claim 31, wherein the variant protein is a GPx1 or GPx4 variant protein.

33. The variant protein of claim 32, wherein the GPx1 variant protein contains a mutation at one or more of K88, K148 and K166, or the GPx4 variant protein contains a mutation at one or more of K107, K126, K148, K162R, K167 and K191.

34. The variant protein of claim 33, wherein K is replaced by R.

35. The 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 and lacks at least a portion of its signal peptide.

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

16.

37. The variant protein according to any one of claims 32-34, wherein the GPx1 variant protein contains the U49C mutation, or the GPx4 variant protein contains the U73C mutation.

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

39. The variant protein of claim 38, wherein the variant protein lacks at least a portion of the signal peptide.

40. The variant protein of 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 comprising one or more inactivating mutations in its mitochondrial targeting peptide.

42. The variant mitochondrial protein of claim 41, wherein the variant protein lacks at least a portion of the mitochondrial targeting peptide.

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

44. A polynucleotide encoding a variant protein according to any one of claims 31-43.

45. The polynucleotide of claim 44, wherein the polynucleotide comprises a 3' untranslated region (3'UTR) containing a Sec insert sequence (SECIS).

46. ​​The polynucleotide of claim 44 or 45, wherein the polynucleotide comprises, or is composed 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 erythroid cells 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 carrier according to claim 47, and a pharmaceutically or physiologically acceptable diluent and / or carrier.

49. A method for improving the ability of cells to survive in lyophilization, the method comprising genetically engineering the cells to overexpress antioxidant proteins and / or intrinsically disordered proteins (IDPs) in their cytosol.

50. The method of 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. The method according to claim 49 or 50, wherein the method comprises genetically engineering the cells 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. Erythroid cells according to any one of claims 1-29, the population according to claim 30, the variant protein according to any one of claims 31-43, the polynucleotide according to any one of claims 44-46, the carrier according to claim 47, or the pharmaceutical composition according to claim 48, for use in therapy.

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