WO2022089606A1

WO2022089606A1 - Methods for preparing mature red blood cells in vitro

Info

Publication number: WO2022089606A1
Application number: PCT/CN2021/127603
Authority: WO
Inventors: Xiaofei GAO
Original assignee: Westlake Therapeutics (Hangzhou) Co. Limited
Priority date: 2020-10-30
Filing date: 2021-10-29
Publication date: 2022-05-05
Also published as: CN116568802A

Abstract

Provided is a method for producing red blood cells, comprising the step of: (a) providing a population of Lin- CD34- hemopoietic progenitor cells; (b) culturing the population of Lin-CD34- hemopoietic progenitor cells from (a) in a first medium comprising a combination of an aryl hydrocarbon receptor antagonist, a HIF-1 modulator and a PPAR alpha agonist; and (c) culturing the population of cells obtained from (b) in a second medium comprising a combination of a vitamin, a methylxanthine compound, and a thyroid hormone receptor agonist, thereby producing red blood cells.

Description

Methods for preparing mature red blood cells in vitro

FIELD OF THE INVENTION

The present disclosure relates to the field of erythroid culture system and method, and in particular to in vitro human CD34-erythroid culture system and method.

BACKGROUND

Red blood cells (RBCs) are the most numerous cell type in blood and account for a quarter of the total number of cells in the human body. RBCs possesses many unique characteristics that make them an attractive tool in therapeutics and diagnostics for various purposes, e.g., for in vivo delivery of natural or synthetic payloads: for example, extensive circulatory range (RBCs travel through the whole vascular system of the body) ; good biocompatibility; long circulatory half-life (the life span is approximately 120 days in humans) ; large surface-to-volume ratio; and absence of nucleus and mitochondria (no protein synthesis, no proliferation, no mutation) .

In order to meet the need for RBCs, the technology of red blood cell regeneration has developed rapidly. The current red blood cell regeneration methods are mainly based on CD34 ⁺ bone marrow hematopoietic stem cells or CD34 ⁺ umbilical cord blood hematopoietic stem cells. The extraction of CD34 ⁺ bone marrow hematopoietic stem cells will cause great physical pain and harm to cell supplier, while CD34 ⁺ umbilical cord blood hematopoietic stem cells can only be obtained during a specific period of time when the newborn is born. Accordingly, the above two methods have great limitations in clinical application.

There appears to be discordant results on the differentiation potential of CD34 ^-hematopoietic stem cells, and the absence of a suitable erythroid differentiation culture system also limits the use of CD34 ^-hematopoietic stem cells.

Therefore, there is still a need for an improved method for preparing mature red blood cells in vitro.

SUMMARY

In one aspect, the present disclosure provides a method for producing red blood cells, comprising the steps of: (a) providing a population of Lin-CD34-hemopoietic progenitor cells; (b) culturing the population of Lin-CD34-hemopoietic progenitor cells from (a) in a first medium comprising one or more additives selected from a group consisting of an aryl hydrocarbon receptor antagonist, a HIF-1 modulator, a PPAR alpha agonist, and any combination thereof, preferably a combination of an aryl hydrocarbon receptor antagonist, a HIF-1 modulator and a PPAR alpha agonist; and (c) culturing the population of cells obtained from (b) in a second medium comprising one or more additives selected from a group consisting of a vitamin, a methylxanthine compound, a thyroid hormone receptor agonist, and any combination thereof, preferably a combination of a vitamin, a methylxanthine compound, and a thyroid hormone receptor agonist, thereby producing red blood cells.

In some embodiments, in step (a) the population of Lin-CD34-hemopoietic progenitor cells has been expanded in an expansion medium such as a hematopoietic stem cell expansion medium.

In some embodiments, the expansion medium comprises fms-like tyrosine kinase 3 ligand (Flt3L) , stem cell factor (SCF) , interleukin 3 (IL-3) , and interleukin 6 (IL-6) .

In some embodiments, the expansion medium comprises about 10-100 ng/mL Flt3L, about 50-150 ng/mL SCF, about 1-20 ng/mL IL-3, and about 5-30 ng/mL IL-6.

In some embodiments, the expansion medium is a serum-free expansion medium.

In some embodiments, the population of Lin-CD34-hemopoietic progenitor cells has been expanded for about 2-5 days.

In some embodiments, the first medium is a first differentiation medium.

In some embodiments, the aryl hydrocarbon receptor antagonist selected from a group consisting of SR1, 4- (2- (Pyridin-3-yl) -9-isopropyl-9H-purin-6-ylamino) ethyl) phenol, 4- (2- (9-Isopropyl-2- (2-methyl-1H-imidazol-1-yl) -9H-purin-6-ylamino) ethyl) phenol, 4- (2- (2- (5-Chloropyridine-3-yl) -9-isopropyl-9H-purin-6-ylamino) ethyl) phenol, 4- (2- (6- (5-Fluoropyridin-3-yl) -1-isopropyl-1H-pyrazolo [3, 4-d] pyrimidin-4-ylamino) ethyl) pheno1, 4- (2- (2- (5-Fluoropyridin-3-yl) -7-isopropyl-7H-pyrrolo [2, 3-d] pyrimidin-4-ylamino) ethyl) phenol, (R) -4-2- (2- (benzo [b] thiophen-3-yl) -9-tetrahydrofuran-3-yl) -9H-purin-6-ylamino) ethyl) phenol, 2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3-yl) -9H-purin-6-ylamino) ethyl) phenol1, (R) -2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3-yl) -9H-purin-9-yl) propan-1-ol, (S) -2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3-yl) -9H-purin-9-yl) propan-1-ol, 4- (2- (6- (5-Fluoropyridin-3-yl) -1-isopropyl-1H-imidazol [4, 5-c] pyridin-4-ylamino) ethyl) phenol, 4- (2- (5- (5-Fluoropyridin-3-yl) -3-isopropyl-3H-imidazo [4, 5-c] pyridin-7-ylamino) ethyl) phenol, 3-2- (2- (benzo [b] thiophen-3-yl) -9-isopropyl-9H-purin-6-ylamino) ethyl) -1H-indol-5-yl 6- (5- ( (3aS, 4S, 6aR) -2-oxohexahydro-1H-thienol {3, 4-d} imidazol-4-yl) pentanamido) hexonoate, and 3-2- (2- (benzo [b] thiophen-3-yl) -9-isopropyl-9H-purin-6-ylamino) ethyl) -1H-indol1-5-yl 6- (tert-butoxycarbonylamino) hexonoate and any combination thereof.

In some embodiments, the aryl hydrocarbon receptor antagonist is in an amount of about 0.1 -5 μM.

In some embodiments, the HIF-1 modulator is a HIF-1 activator such as a prolyl hydroxylase inhibitor (PHI) , preferably the PHI being selected from a group consisting of FG-4592, FG-2216, FG-4539, N- ( (l-chloro-4-hydroxy-isoquinoline-3-carbonyl) -amino) -acetic acid, [ (7-Bromo-4-hydroxy-isoquinoline-3-carbonyl) -amino] -acetic acid, [ (1-Chloro-4-hydroxy-7-methoxy-isoquinoline-3-carbonyl) -amino] -acetic acid, (7-Chloro-3-hydroxy- quinoline-2-carbonyl) -amino] -acetic acid, [ (3-Hydroxy-6-isopropoxy-quinoline-2-carbonyl) -amino] -acetic acid, and [ (4-Hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl) -amino} -acetic acid, and dimethyloxalylglycine (DMOG) , and any combination thereof.

In some embodiments, the HIF-1 modulator is in an amount of about 10 -500 nM.

In some embodiments, the PPAR alpha agonist is selected from a group consisting of Fenofibrate, Clofibrate, Bezafibrate, Clinofibrate, Ciprofibrate, Etofibrate, Gemfibrate Gemfibrozil) , Pemabate ( (R) -2- {3- { [N- (benzoxazol-2-yl) -N-3- (4-methoxyphenoxy) propyl] aminomethyl} phenoxy} butyric acid) , GW-7647 (2- [ [4- [2- [ [ (cyclohexylamino) carbonyl] (4-cyclohexylbutyl) amino] ethyl] phenyl] thio] -2-methylpropanoic acid) , WY-14643 (pilinic acid) , AM3102 (CAS number: 213182-22-0) , CAY15073 (CAS number: 853652-40-1 ) , CP775146 (CAS Number: 702680-17-9) , GW9578 (CAS Number: 247923-29-1) , GW590735 (CAS Number: 622402-22-6) , Oleylethanolamide, Palmitoylethanolamide, Tesaglitazar ( (S) -2-ethoxy-3- [4- [4- [ (methylsulfonyl) oxy] phenethoxy] phenyl) propionic acid) , LY518674 (2- [4- [3- [2, 5-Dihydro-1- [ (4-methylphenyl) methyl] -5-oxo-1H-1, 2, 4-triazol-3-yl] propyl] phenoxy] -2-methyl-propionic acid) , and any combination thereof.

In some embodiments, the PPAR alpha agonist is in an amount of about 0.01 –50 μM.

In some embodiments, the first medium further comprises transferrin, insulin, IL-3, EPO, and SCF.

In some embodiments, the first medium comprises about 100-600 μg/mL transferrin, about 5-20 μg/mL insulin, about 0.5-7 ng/mL IL-3, about 1-7 U/mL EPO, and about 5-120 ng/mL SCF.

In some embodiments, step (b) comprises culturing the population of Lin-CD34-hemopoietic progenitor cells for about 7-13 days such as about 9-11 days.

In some embodiments, the culturing in step (b) results in the proliferation and/or differentiation of the population of Lin-CD34-hemopoietic progenitor cells.

In some embodiments, the second medium is a second differentiation medium.

In some embodiments, the vitamin is selected from a group of vitamin C, vitamin E, derivatives or analogues of vitamin C or E, and any combination thereof.

In some embodiments, the second medium comprises vitamin in an amount of about 1-100 μM.

In some embodiments, the second medium comprises vitamin C and/or derivatives or analogues thereof in an amount of about 1-50 μM.

In some embodiments, the second medium comprises vitamin E and/or derivatives or analogues thereof in an amount of about 1-50 μM.

In some embodiments, the methylxanthine compound is selected from a group consisting of caffeine, 3-isobutyl-1-methylxanthine (IBMX) , theophylline and theobromine, preferably in an amount of about 1-50 μM.

In some embodiments, the thyroid hormone receptor agonist is a thyroid hormone receptor α or β agonist, preferably a thyroid hormone receptor β agonist.

In some embodiments, the thyroid hormone receptor agonist is a thyroxine (T4) , tri-iodothyronine (T3) and their analogues, preferably thyroxine and its analogue, preferably in an amount of about 0.1 -10 μM.

In some embodiments, the second medium comprises a combination of vitamin C, vitamin E, caffeine and thyroxine.

In some embodiments, the second medium further comprises transferrin, insulin, and EPO, preferably further comprises about 100-600 μg/mL transferrin, about 5-20 μg/mL insulin, and about 0.5-5 U/mL EPO.

In some embodiments, step (c) comprises culturing the population of cells obtained from (b) for about 5-11 days such as about 7-9 days.

In some embodiments, the population of Lin-CD34-hemopoietic progenitor cells is isolated from a blood sample, such as a peripheral blood sample, a cord blood sample or from bone marrow.

In some embodiments, the blood sample is a human peripheral blood sample.

In some embodiments, the population of Lin-CD34-hemopoietic progenitor cells is obtained by isolating peripheral blood mononuclear cells (PBMCs) from the blood sample and removing lineage positive (Lin+) cells from the PBMCs.

In another aspect, the present disclosure provided a method of culturing Lin-CD34-hemopoietic progenitor cells, comprising culturing a population of Lin-CD34-hemopoietic progenitor cells in a medium comprising one or more additives selected from a group consisting of an aryl hydrocarbon receptor antagonist, a HIF-1 modulator, a PPAR alpha agonist, and any combination thereof, preferably a combination of an aryl hydrocarbon receptor antagonist, a HIF-1 modulator and a PPAR alpha agonist.

In some embodiments, the aryl hydrocarbon receptor antagonist is selected from a group consisting of SR1, 4- (2- (Pyridin-3-yl) -9-isopropyl-9H-purin-6-ylamino) ethyl) phenol, 4- (2- (9-Isopropyl-2- (2-methyl-1H-imidazol-1-yl) -9H-purin-6-ylamino) ethyl) phenol, 4- (2- (2- (5-Chloropyridine-3-yl) -9-isopropyl-9H-purin-6-ylamino) ethyl) phenol, 4- (2- (6- (5-Fluoropyridin-3-yl) -1-isopropyl-1H-pyrazolo [3, 4-d] pyrimidin-4-ylamino) ethyl) pheno1, 4- (2- (2- (5-Fluoropyridin-3-yl) -7-isopropyl-7H-pyrrolo [2, 3-d] pyrimidin-4-ylamino) ethyl) phenol, (R) -4-2- (2- (benzo [b] thiophen-3-yl) -9-tetrahydrofuran-3-yl) -9H-purin-6-ylamino) ethyl) phenol, 2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3-yl) -9H-purin-6-ylamino) ethyl) phenol1, (R) -2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridin- 3-yl) -9H-purin-9-yl) propan-1-ol, (S) -2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3-yl) -9H-purin-9-yl) propan-1-ol, 4- (2- (6- (5-Fluoropyridin-3-yl) -1-isopropyl-1H-imidazol [4, 5-c] pyridin-4-ylamino) ethyl) phenol, 4- (2- (5- (5-Fluoropyridin-3-yl) -3-isopropyl-3H-imidazo [4, 5-c] pyridin-7-ylamino) ethyl) phenol, 3-2- (2- (benzo [b] thiophen-3-yl) -9-isopropyl-9H-purin-6-ylamino) ethyl) -1H-indol-5-yl 6- (5- ( (3aS, 4S, 6aR) -2-oxohexahydro-1H-thienol {3, 4-d} imidazol-4-yl) pentanamido) hexonoate, and 3-2- (2- (benzo [b] thiophen-3-yl) -9-isopropyl-9H-purin-6-ylamino) ethyl) -1H-indol1-5-yl 6- (tert-butoxycarbonylamino) hexonoate and any combination thereof.

In some embodiments, the HIF-1 modulator is a HIF-1 activator such as a prolyl hydroxylase inhibitor (PHI) , preferably the PHI being selected from a group consisting of FG-4592, FG-2216, FG-4539, N- ( (l-chloro-4-hydroxy-isoquinoline-3-carbonyl) -amino) -acetic acid, [ (7-Bromo-4-hydroxy-isoquinoline-3-carbonyl) -amino] -acetic acid, [ (1-Chloro-4-hydroxy-7-methoxy-isoquinoline-3-carbonyl) -amino] -acetic acid, (7-Chloro-3-hydroxy-quinoline-2-carbonyl) -amino] -acetic acid, [ (3-Hydroxy-6-isopropoxy-quinoline-2-carbonyl) -amino] -acetic acid, and [ (4-Hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl) -amino} -acetic acid, and dimethyloxalylglycine (DMOG) , and any combination thereof.

In some embodiments, the HIF-1 modulator is in an amount of about 10 -500 nM.

In some embodiments, the medium is a differentiation medium.

In some embodiments, the medium further comprises transferrin, insulin, IL-3, EPO, and SCF.

In some embodiments, the differentiation medium comprises about 100-600 μg/mL transferrin, about 5-20 μg/mL insulin, about 0.5-7 ng/mL IL-3, about 1-7 U/mL EPO, and about 5-120 ng/mL SCF.

In some embodiments, the method comprises culturing the population of Lin-CD34-hemopoietic progenitor cells for about 7-13 days such as about 9-11 days.

In some embodiments, the culturing results in the proliferation and/or differentiation of the population of Lin-CD34-hemopoietic progenitor cells.

In another aspect, the present disclosure provides a method of producing red blood cells, comprising: culturing a population of nucleated red blood cell precursors in a medium comprising one or more additives selected from a group consisting of a vitamin, a methylxanthine compound, a thyroid hormone receptor agonist, and any combination thereof, preferably a combination of a vitamin, a methylxanthine compound, and a thyroid hormone receptor agonist, thereby producing red blood cells.

In some embodiments, the population of nucleated red blood cell precursors is at a terminal differentiation stage.

In some embodiments, the nucleated red blood cell precursors are reticulocytes.

In some embodiments, the nucleated red blood cell precursors are obtained from a population of Lin-CD34-hemopoietic progenitor cells.

In some embodiments, the medium is a differentiation medium.

In some embodiments, the medium comprises vitamin in an amount of about 1-100 μM.

In some embodiments, the medium comprises vitamin C and/or derivatives or analogues thereof in an amount of about 1-50 μM.

In some embodiments, the medium comprises vitamin E and/or derivatives or analogues thereof in an amount of about 1-50 μM.

In some embodiments, the thyroid hormone receptor agonist is thyroxine (T4) , tri-iodothyronine (T3) and their analogues, preferably thyroxine and its analogue, preferably in an amount of about 0.1 -10 μM.

In some embodiments, the medium comprises a combination of vitamin C, vitamin E, caffeine and thyroxine.

In some embodiments, the medium further comprises transferrin, insulin, and EPO, preferably further comprises about 100-600 μg/mL transferrin, about 5-20 μg/mL insulin, and about 0.5-5 U/mL EPO.

In some embodiments, the nucleated red blood cell precursors are cultured for about 5-11 days such as about 7-9 days.

In another aspect, the present disclosure provides a population of cells obtained by the method as described herein.

In another aspect, the present disclosure provides a medium supplement composition comprising one or more additives selected from a group consisting of an aryl hydrocarbon receptor antagonist, a HIF-1 modulator, a PPAR alpha agonist, and any combination thereof, preferably a combination of an aryl hydrocarbon receptor antagonist, a HIF-1 modulator and a PPAR alpha agonist. In some embodiments, the composition further comprises transferrin, insulin, IL-3, EPO, and SCF.

In another aspect, the present disclosure provides a medium supplement composition comprising a vitamin, a methylxanthine compound, and a thyroid hormone receptor agonist. In some embodiments, the composition further comprises transferrin, insulin and EPO.

In another aspect, the present disclosure provides a method for producing engineered red blood cells, comprising the steps of: (a) providing a population of Lin-CD34-hemopoietic progenitor cells; (b) culturing the population of Lin-CD34-hemopoietic progenitor cells from (a) in a first medium comprising one or more additives selected from a group consisting of an aryl hydrocarbon receptor antagonist, a HIF-1 modulator, a PPAR alpha agonist, and any combination thereof, preferably a combination of an aryl hydrocarbon receptor antagonist, a HIF-1 modulator and a PPAR alpha agonist, to induce them to differentiate into erythroid cells, and prior to or concurrently with the differentiation, introducing one or more exogenous nucleic acids into the erythroid cells; and (c) culturing the erythroid cells to induce enucleation, thereby producing the engineered red blood cells.

In some embodiments, step (c) comprises culturing the cells obtained from (b) in a second medium comprising one or more additives selected from a group consisting of a vitamin, a methylxanthine compound, a thyroid hormone receptor agonist, and any combination thereof, preferably a combination of a vitamin, a methylxanthine compound, and a thyroid hormone receptor agonist, thereby producing the engineered red blood cells.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, embodiments of the invention are illustrated by way of example. It is to be expressly understood that the description and drawings are only for the purpose of illustration and as an aid to understanding, and are not intended as a definition of the limits of the invention.

Figure 1 shows phase-specific small-molecule combination significantly increases erythroid cell expansion and differentiation efficiency in the ex vivo human CD34-erythroid culture system.

Figure 2 shows combination of small molecules synergistically enhances human erythroid progenitor production.

Figure 3 shows phase-specific small-molecule combination increases enucleation efficiency in the ex vivo human CD34-erythroid culture system.

Figure 4 shows synergism of small molecules increases erythroid cell number during phase III differentiation.

Figure 5 shows expression of erythroid genes was up-regulated in the cells cultured with phase-specific small-molecule combination in the ex vivo human CD34-erythroid culture system.

Figures 6A-6B show the effect of neuron active drugs on enhancing red blood cells production in a mouse red cell production culture system.

DETAILED DESCRIPTION

For the purposes of understanding of the present disclosure, reference will now be made to the embodiments illustrated in the drawings, and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of this disclosure is thereby intended.

In the present disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings as generally understood by a person skilled in the art. Although any methods and materials similar or equivalent to those described herein find use in the practice of the present disclosure, the preferred methods and materials are described herein. Accordingly, the terms defined herein are more fully described by reference to the Specification as a whole.

As used herein, the singular terms “a, ” “an, ” and “the” include the plural reference unless the context clearly indicates otherwise.

Unless the context requires otherwise, the terms “comprise, ” “comprises” and “comprising, ” or similar terms are intended to mean a non-exclusive inclusion, such that a recited list of elements or features does not include those stated or listed elements solely, but may include other elements or features that are not listed or stated.

The term “culturing” as used herein refers to maintaining cells in a medium with or without cell expansion or differentiation for any period of time.

The term “differentiation” as used herein refers to a process by which a less specialized cell such as a stem cell develops or matures to possess a more distinct form and function with a concomitant loss of potential. Cells that are less specialized can be differentiated into cells that are more specialized by culturing the cells under particular conditions or in specific media as known in the art.

The term “peripheral blood mononuclear cells” or “PBMCs” as used herein refers to cells with a single nucleus in peripheral blood, including various lymphocyte and monocyte. One useful method for isolation of peripheral blood mononuclear cells is Ficoll-Hypaque density gradient centrifugation.

The term “biotin-labeled antibody” as used herein refers to an antibody with biotin attached to. Biotin-labeling reaction is simple and mild and rarely inhibits antibody activity. Covalently binding biotin to an antibody is a very simple and direct labeling means.

The term “hematopoietic stem cell expansion medium” as used herein refers to a serum-free medium with addition of hematopoietic growth factors and /or other stimulators selected by users, in order to promote the expansion of hematopoietic stem /progenitor cells (HSPC) . One useful hematopoietic stem cell expansion medium may be StemSpan ^TM SFEM serum-free expansion medium.

The term “fms-like tyrosine kinase 3 ligand” or “Flt3L” as used herein, also known as FL, Flt3L and FLT3LG, refers to an alpha-helix cytokine that promotes the differentiation of a plurality of hematopoietic cell lineages. FLT3LG is structurally homologous to stem cell factor (SCF) and colony-stimulating factor 1 (CSF-1) . FLT3LG, as a growth factor, increases the number of cells by activating hematopoietic progenitor cells. In one embodiment, Flt3L may be recombinant human fms-like tyrosine kinase 3 ligand (rhFlt3L) .

The term “stem cell factor” or “SCF” as used herein, also known as Kit ligand (KITLG) , refers to a type I transmembrane glycoprotein of the SCF family. SCF is a ligand for receptor protein tyrosine kinase KIT, and plays an important role in regulating cell survival and proliferation, hematopoiesis, stem cell maintenance, cell development, migration and function. In one embodiment, SCF may be recombinant human stem cell factor (rhSCF) .

The term “interleukin 3” or “IL-3” as used herein refers to a glycoprotein belonging to the hematopoietic growth factor family, which has shown multi-lineage activity in preclinical in vitro and in vivo studies. Hematopoietic progenitor cells, with the help of IL-3 protein, proliferate and differentiate into mature red blood cells, mast cells, megakaryocytes and granulocytes. In one embodiment, IL-3 may be recombinant human interleukin 3 (rhIL-3) .

The term “interleukin 6” or “IL-6” as used herein refers to a multi-functional cytokine that regulates immune response, hematopoietic function, acute phase response and inflammatory response. It works with IL-3 to promote the proliferation of hematopoietic cells. In one embodiment, IL-6 may be recombinant human interleukin 6 (rhIL-6) .

The term “erythropoietin” or “EPO” as used herein refers to a main erythropoiesis factor, which interacts synergistically with various other growth factors (e.g., IL-3, IL-6, glucocorticoids, and SCF) for the development of erythroid lineages from pluripotent progenitor cells. Burst-forming unit-erythroid (BFU-E) cells begin to express erythropoietin receptor and are sensitive to erythropoietin. EPO is an important erythropoietic cytokine. In one embodiment, EPO may be recombinant human erythropoietin (rhEPO) .

The term “transferrin” or “holo transferrin” as used herein refers to a main ferritin in plasma and can form a complex with iron ions for the production of hemoglobin in red blood cells. In one embodiment, transferrin may be human transferrin (human holo transferrin) .

The term “CD235a” as used herein, also known as “blood glycoprotein A” , refers to a single-pass transmembrane glycoprotein expressed in mature red blood cells and erythroid precursor cells, and is a specific marker protein on the surface of red blood cells. The expression of CD235a indicates that the cells differentiated into erythroid cells. Flow cytometry analysis shows that in the SFEM (serum free expansion medium) stage, cells do not express CD235a, indicating that the cells do not enter erythroid differentiation, and after the culture medium is changed to the first differentiation medium, the cytokines in the medium induce the cells to differentiate into erythroid cells, and the cells begin to express CD235a and the proportion of cells with CD235a continues to increase as the differentiation proceeds. After the medium is changed to the second differentiation medium, since the cells has completely entered the erythroid line, almost all the cells express CD235a, indicating that almost all the cells are erythroid cells.

The term “Hoechst33342” as used herein refers to a fluorescent dye used to stain DNA. The dye can pass through cell membrane and bind to DNA. If the cell is not enucleated, the binding between the dye and DNA can be detected by flow cytometry with a positive signal. If the cell is enucleated, a negative signal will be detected by flow cytometry.

In view that CD235a is a surface marker of red blood cells, the co-expression of both CD235a and Hoechst33342 signal can be detected by flow cytometry at the same time. CD235a-positive Hoechst 33342-positive indicates erythroid nucleated cells, and CD235a-positive Hoechst 33342-negative indicates mature red blood cells. In the SFEM and first differentiation media, cells entered into erythroid differentiation from Lin ^-cells with CD235a expression increasing, but the cells do not enucleate, and thus the cells are Hoechst 33342 positive. When the cells enter the second differentiation medium, the cells begin to mature and enucleate, and Hoechst 33342 negative cells appear, indicating that the red blood cells are mature.

The present disclosure is based, at least in part, on the development of an in vitro multi-phase culturing process for differentiating human CD34-hematopoietic progenitor cells into red blood cells or enucleated precursors thereof. It was surprisingly discovered that the culturing process provided herein yielded not only a significant improvement in the extent of enucleation but also a significant increase in cell yield as compared to previously described culturing processes.

One objective of the present disclosure is to provide a method for preparing mature red blood cells in vitro using peripheral blood, and more specifically, a method for in vitro culture of a large number of red blood cells using a small amount of Lin ^-CD34 ^-cells in peripheral blood, and to establish a stable erythroid differentiation culture system and condition to solve the problem of red blood cell differentiation in vitro.

Described herein is an in vitro culturing process for producing mature enucleated red blood cells from CD34-progenitor cells (e.g., from a human subject) . This culturing process involves multiple differentiation stages (e.g., 2 stage) and optionally an expansion stage prior to the differentiation stages. In some embodiments, the total time period for the in vitro culturing process described herein can range from 15-33 days (e.g., 18-29 days or 15-25 days) . In one example, the total time period is 23 days.

In some embodiments, the present disclosure provides a 3-phase erythroid expansion and differentiation method, wherein in phase I (e.g., from day 1 to day 5) , CD34-cells are cultured in an expansion medium, while in phases II (e.g., from day 6 to day 13) and III (e.g., from day 14 to day 23) , the expanded CD34-cells are differentiated in a first and second differentiation medium, respectively, thus obtaining mature enucleated red blood cells.

In one aspect, the present disclosure provides a method for producing red blood cells, comprising the steps of: (a) providing a population of Lin ^-CD34 ^-hemopoietic progenitor cells, optionally the population of Lin ^-CD34 ^-hemopoietic progenitor cells having been expanded in an expansion medium such as a hematopoietic stem cell expansion medium; (b) culturing the population of Lin ^-CD34 ^-hemopoietic progenitor cells from (a) in a first medium comprising one or more additives selected from a group consisting of an aryl hydrocarbon receptor antagonist, a HIF-1 modulator, a PPAR alpha agonist, and any combination thereof, preferably a combination of an aryl hydrocarbon receptor antagonist, a HIF-1 modulator and a PPAR alpha agonist; and (c) culturing the population of cells obtained from (b) in a second medium comprising one or more additives selected from a group consisting of a vitamin, a methylxanthine compound, a thyroid hormone receptor agonist, and any combination thereof, preferably a combination of a vitamin, a methylxanthine compound, and a thyroid hormone receptor agonist, thereby producing red blood cells.

Lin ^-CD34 ^-hemopoietic progenitor cells

The term “lineage negative cells” or “Lin ^-cells” as used herein refers to cells that are essentially free of lineage markers. Lineage markers are characteristic of cell lineages. Exemplary lineage markers are CD3, CD14, CD16, CD19, CD41a, CD56, and D235a. In fact, lineage negative cells are essentially not stained by the lineage antibodies. Lineage negative cells comprise stem and progenitor cells. Accordingly, lineage negative cells also show stem and progenitor cell activity. Lin negative cells or a blood cell population enriched for lineage negative cells can be purified by enriching a blood cell population that is essentially free of lineage markers. For example, the lineage negative cells can be purified by depleting cells that are positive for at least one lineage marker selected from the group consisting of CD3, CD14, CD16, CD19, CD41a, CD56 and CD235a. In some embodiments, a lineage cell depletion kit can be used to perform the purification. To the contrary, lineage positive (Lin ⁺) cells are a mix of all cells expressing mature cell lineage markers. Examples of lineage positive cells include T cells, B cells, NK cells, dendritic cells, monocytes, granulocytes, erythroid cells, and their committed precursors.

CD34 is a cell surface glycoprotein and functions as a cell-cell adhesion factor. The term “Lin ^-CD34 ^-hemopoietic progenitor cells” or “Lin ^-CD34 ^-cells” as used herein refers to cells that have not entered various hematopoietic lineages, and do not express lineage and CD34 surface markers. Lin ^-CD34 ^-hemopoietic progenitor cells are well known in the art. Various techniques can be used to separate or isolate the CD34 ^-cell population from a suitable source such as bone marrow, peripheral blood or cord blood. In one embodiment, Lin ^-CD34 ^-hemopoietic progenitor cells are preferably isolated from peripheral blood as peripheral blood is known to contain no or extremely little CD34 ⁺ cells.

In one embodiment, Lin ^-CD34 ^-cells are obtained by isolating mononuclear cells from peripheral blood to separate mononuclear cells from other cells such as red blood cells and platelets, and removing lineage positive (Lin+) cells from the blood sample by e.g. using a lineage cell depletion kit. In some embodiments, the lineage Lin ⁺ can be removed by using suitable antibodies, which may be e.g. anti-human CD3 antibody, anti-human CD14 antibody, anti-human CD16 antibody, anti-human CD19 antibody, anti-human CD41a antibody, anti-human CD56 antibody and anti-human CD235a antibody. In some embodiments, the antibodies can be murine anti-human antibodies, e.g., mouse anti-human antibodies. In some further embodiments, the antibodies may be used in an amount of about 2-6 μg/ml, such as 3, 4, and 5 μg/ml.

In some embodiments, the blood sample is a peripheral blood sample, a cord blood sample or a fetal blood sample. In a further embodiment, the blood sample is a human peripheral blood sample.

In one embodiment, a method of obtaining Lin ^-CD34 ^-cells may comprise obtaining peripheral blood and isolating peripheral blood mononuclear cells (PBMCs) ; centrifuging the PBMCs and re-suspending the cells in a cell isolation buffer supplemented with bovine serum albumin and EDTA in a phosphate buffer solution; adding biotin-labeled antibodies; adding streptavidin-labeled magnetic beads; removing cells with specific surface markers under magnetic force; collecting the isolation buffer that is not adsorbed; and centrifuging the buffer to obtain Lin ^-CD34 ^-cells.

Expansion Stage

Optionally, the in vitro culturing process described herein includes an expansion stage, in which the CD34-progenitor cells are allowed to expand or proliferate. As used herein, expansion or proliferation includes any increase in cell number. For example, the expansion may include a 2-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1000-fold or higher increase in cell number. The starting cell density of the cultured population of CD34-cells can be from about 1x10 ² cells to about 1x10 ⁶ cells/mL or higher, such as about 1x10 ³ cells to about 1x10 ⁵ cells/mL.

Base media useful for expanding CD34-progenitor cells are known in the art. Various cell culture media and supplements can be used to expand the population of CD34-progenitor cells. As used herein, "expansion medium" refers to a cell culture medium that is used to expand a population of CD34-cells in an expansion stage. In some embodiments, the expansion medium comprises one or more of base media selected from the group consisting of: StemSpan ^TM media (e.g., Serum-Free Expansion Media, SFEM or SFEM II) , Dulbecco's MEM (DMEM) , IMDM, DMEM/F12, MEM, Opti-MEM, ISCOVE, HAM F12, HAM F10, M199, L15, 6M NCTC109 medium, Fischer medium, Waymouth medium, VPSFM medium, Williams medium, RPMI (e.g., RPMI-1640) , StemMACS ^TM HSC Expansion media, HPGM (Cambrex, Walkersville, MD) , StemPro-34 (Invitrogen, Carlsbad, CA) , Stemline (Sigma) and StemSpan H3000, and StemMACS HSC Expansion Media. It can be appreciated that other base media suitable for expanding CD34-cells may be used.

The base media used in the expansion medium as described above as well as the base media used in the differentiation media described below may be enriched according to the needs of the cells with additional nutrient factors such as sugars such as glucose, amino acids such as glutamine, a combination of nonessential amino acids or of essential amino acids or of peptides, acids or acid salts such as sodium pyruvate, EDTA salts, and the like.

In some embodiments, the expansion medium is serum free and/or plasma free. In some embodiments one or more of the cell culture methods provided herein are performed in the absence of serum and/or plasma. In some embodiments, the expansion medium may comprise one or more serum substitutes. Serum substitutes which are incorporated according to the invention may be selected from the group known in the art to support in vitro hematopoietic stem cell (HSC) expansion, for example, Albumax, bovine serum albumin (BSA) , transferrin (TF) , glutamine, hydrocortisone (HC) , peptone, 2-mercaptoethanol (2-ME) , insulin, polyvinylpyrrolidone (PVP) , Serum Replacement 1 (Sigma-Aldrich) , Serum Replacement 2 (Sigma-Aldrich) and/or BIT9500 (StemCell) .

It should be appreciated that the expansion of CD34-cells in vitro may be supported by the presence of several cytokines or growth factors. In some embodiments, the Lin ^-CD34 ^-cells are expanded in a hematopoietic stem cell expansion medium supplemented with a combination of cytokines. In a further embodiment, the combination of cytokines comprises fms-like tyrosine kinase 3 ligand (Flt3L) , stem cell factor (SCF) , interleukin 3 (IL-3) , and interleukin 6 (IL-6) . In a further embodiment, the combination of cytokines comprises about 10-100 ng/mL (e.g., 20, 30, 40, 50, 60, 70, 80 and 90 ng/mL) human Flt3L, about 50-150 ng/mL (e.g., 50, 60, 70, 80, 90, 100, 110, 120, 130 and 140 ng/mL) human SCF, about 1-20 ng/mL (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15 and 18 ng/mL) human IL-3, and 5-30 ng/mL (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 22, 25 and 28 ng/mL) human IL-6. In some embodiments, recombinant cytokines can be used.

In some embodiments, the expansion medium may optionally comprise one or more glucocorticoids, which are a class of corticosteroids. Exemplary corticosteroids include, without limitation, Cortisol, cortisone, prednisone, prednisolone methylprednisolone dexamethasone, betamethasone, triamcinolone. In some embodiments, the expansion medium may comprise from 10 nM to 1 mM of a corticosteroid, e.g., from 50 nM to 900 nM, from 100 nM to 800 nM, from 200 nM to 700 nM, from 300 nM to 600 nM, from 400 nM to 500 nM of a corticosteroid. In some embodiments, the expansion medium comprises dexamethasone, e.g., from 50 nM to 150nM of dexamethasone.

In one example, the expansion stage of the in vitro culturing process described herein can be performed as follows. A population of CD34-peripheral blood cells is placed in an expansion container at a cell density of 1 x 10 ⁴-l x l0 ⁶ (e.g., l x 10 ⁵) cells/mL. The CD34-cells are cultured in an expansion medium (e.g., StemSpan II medium) supplemented with a cytokine mixture of recombinant human Flt-3 ligand (e.g., 50 ng/mL -150 ng/mL) , recombinant human SCF (e.g., 50 ng/mL -150 ng/mL) , recombinant human IL-3 (e.g., 10 ng/mL -30 ng/mL) , recombinant human IL-6 (e.g., 10 ng/mL -30 ng/mL) , optionally dexamethasone (e.g., 50nM -150nM) , and 2%penicillin and streptomycin under suitable conditions (e.g., 37 ℃) for 1-6 days (e.g., 2-5 days, 3-4 days, or 5 days) .

In some embodiments, the expansion medium may be substantially free of certain supplements, such as serum, plasma, erythropoietin (EPO) , insulin, holo transferrin and/or heparin.

Differentiation Stage

The in vitro culturing process described herein may involve two differentiation stages, in which CD34-progenitor cells differentiate into mature enucleated red blood cells. In each differentiation stage, CD34-progenitor cells (either obtained from the expansion stage or collected from the original source) or cells obtained from a preceding differentiation stage can be cultured in a medium comprising one or more suitable cytokines (e.g., those described herein) under suitable conditions for a suitable period of time.

Biological properties of the cells, such as cell size and expression of surface markers, may be monitored during the course or at the end of each differentiation stage to evaluate the status of erythropoiesis. Whenever necessary, cytokines can be timely supplied and/or withdrawn at each differentiation stage to achieve optimal erythroid differentiation and/or synchronizing the cell population in culture.

At each of the differentiation stages, CD34-progenitor cells, either obtained from the expansion stage described herein or isolated from an original source (e.g., human peripheral blood) , or cells obtained from the preceding differentiation stage can be cultured in a suitable medium, such as those described above, supplemented with one or more supplements (e.g., any of the supplements described above) under suitable culturing conditions for a suitable period of time. In some embodiments, one or more of the differentiation media comprise any of the base media provided herein (e.g., Serum-Free Expansion Media, SFEM or SFEM II) , Dulbecco's MEM (DMEM) , IMDM, DMEM/F12, MEM, Opti-MEM, ISCOVE, HAM F12, HAM F10, M199, L15, 6M NCTC109 medium, Fischer medium, Waymouth medium, VPSFM medium, Williams medium, RPMI (e.g., RPMI-1640) ) . In some embodiments, one or more of the differentiation media comprise IMDM.

It should also be appreciated that any of the differentiation media may also be supplemented with other components commonly used in cell culture, e.g., serum (e.g., human serum) , plasma (e.g., human plasma) , glutamine, bovine serum albumin, one or more antibiotics (e.g., penicillin and streptomycin) , or any combination thereof. In some embodiments, any or all of the differentiation media are supplemented with serum (e.g., serum from a horse, pig, rabbit, goat, cow or human) . The amount of serum may be in any suitable amount to support the growth and/or differentiation of CD34 ^-cells in culture. In some embodiments, any or all of the differentiation media are supplemented with plasma (e.g., plasma from a horse, pig, rabbit, goat, cow or human) . The amount of plasma may be in any suitable amount to support the growth and/or differentiation of CD34 ^-cells in culture.

In some embodiments, the differentiation medium is serum free and/or plasma free. In some embodiments one or more of the cell culture methods provided herein are performed in the absence of serum and/or plasma. In some embodiments, the differentiation medium may comprise one or more serum substitutes. Serum substitutes which are incorporated according to the invention may be selected from the group known in the art, for example, Albumax, bovine serum albumin (BSA) , glutamine, hydrocortisone (HC) , peptone, 2-mercaptoethanol (2-ME) , polyvinylpyrrolidone (PVP) , Serum Replacement 1 (Sigma-Aldrich) , Serum Replacement 2 (Sigma-Aldrich) and/or BIT9500 (StemCell) .

First Differentiation

In one embodiment, the in vitro culturing process described herein comprises a step of culturing the population of Lin ^-CD34 ^-hemopoietic progenitor cells, which optionally have been expanded in an expansion medium, in a first medium (e.g., a first differentiation medium) comprising one or more additives such as small molecules. A small molecule is often an organic compound having a molecular weight equal to or less than 3.0 kD, e.g., equal to or less than 2.0 or 1.5 kD, e.g., equal to or less than 1 kD, e.g., equal to or less than 500 daltons and usually multiple carbon-carbon bonds. Small molecules often comprise one or more functional groups that mediate structural interactions with proteins, e.g., hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, and in some embodiments at least two of the functional chemical groups. A small molecule may comprise cyclic carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more chemical functional groups and/or heteroatoms. In one embodiments, small molecules useful in the present invention are selected from a group consisting of an aryl hydrocarbon receptor antagonist, a HIF-1 modulator, a PPAR alpha agonist, and any combination thereof, such as a combination of an aryl hydrocarbon receptor antagonist and a HIF-1 modulator, a combination of an aryl hydrocarbon receptor and a PPAR alpha agonist, a combination of a HIF-1 modulator and a PPAR alpha agonist, preferably a combination of an aryl hydrocarbon receptor antagonist, a HIF-1 modulator and a PPAR alpha agonist.

It is surprisingly found that a small molecule combination of an aryl hydrocarbon receptor antagonist, a HIF-1 modulator and a PPAR alpha agonist can act, in a synergistic way, to promote the proliferation and/or differentiation of Lin ^-CD34 ^-hemopoietic progenitor cells obtained from the expansion stage or collected from the original source.

Aryl hydrocarbon receptor antagonist

The term “an aryl hydrocarbon receptor antagonist” as used herein refers to an agent (such as a compound) capable of down-regulating the activity and/or expression of aryl hydrocarbon receptor and/or a downstream effector of aryl hydrocarbon receptor pathway (e.g., an agent capable of down-regulating the protein expression of aryl hydrocarbon receptor and/or the protein expression of one or more downstream effectors of aryl hydrocarbon receptor) .

Contemplated for use in the present invention are any of the compounds disclosed in U.S. Patent Application No. 2010/0183564. The description of aryl hydrocarbon receptor antagonists provided herein can be found in U.S. Patent Publication No. 2010/0183564.

Examples of aryl hydrocarbon receptor antagonists that can be used in the compositions and methods of the present invention include, but are not limited to: SR1, 4- (2- (Pyridin-3-yl) -9-isopropyl-9H-purin-6-ylamino) ethyl) phenol, 4- (2- (9-Isopropyl-2- (2-methyl-1H-imidazol-1-yl) -9H-purin-6-ylamino) ethyl) phenol, 4- (2- (2- (5-Chloropyridine-3-yl) -9-isopropyl-9H-purin-6-ylamino) ethyl) phenol, 4- (2- (6- (5-Fluoropyridin-3-yl) -1-isopropyl-1H-pyrazolo [3, 4-d] pyrimidin-4-ylamino) ethyl) pheno1, 4- (2- (2- (5-Fluoropyridin-3-yl) -7-isopropyl-7H-pyrrolo [2, 3-d] pyrimidin-4-ylamino) ethyl) phenol, (R) -4-2- (2- (benzo [b] thiophen-3-yl) -9-tetrahydrofuran-3-yl) -9H-purin-6-ylamino) ethyl) phenol, 2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3-yl) -9H-purin-6-ylamino) ethyl) phenol1, (R) -2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3-yl) -9H-purin-9-yl) propan-1-ol, (S) -2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3-yl) -9H-purin-9-yl) propan-1-ol, 4- (2- (6- (5-Fluoropyridin-3-yl) -1-isopropyl-1H-imidazol [4, 5-c] pyridin-4-ylamino) ethyl) phenol, 4- (2- (5- (5-Fluoropyridin-3-yl) -3-isopropyl-3H-imidazo [4, 5-c] pyridin-7- ylamino) ethyl) phenol, 3-2- (2- (benzo [b] thiophen-3-yl) -9-isopropyl-9H-purin-6-ylamino) ethyl) -1H-indol-5-yl 6- (5- ( (3aS, 4S, 6aR) -2-oxohexahydro-1H-thienol {3, 4-d} imidazol-4-yl) pentanamido) hexonoate, and 3-2- (2- (benzo [b] thiophen-3-yl) -9-isopropyl-9H-purin-6-ylamino) ethyl) -1H-indol1-5-yl 6- (tert-butoxycarbonylamino) hexonoate. U.S. Patent Publication 2010/0183564 describes and exemplifies these and other aryl hydrocarbon receptor antagonists that can be used in the compositions and methods of this invention (see, e.g., Table 1 and Table 2) .

In certain embodiments, an aryl hydrocarbon receptor antagonist is an organic compound, a small interference RNA (siRNA) molecule capable of down-regulating the expression of aryl hydrocarbon receptor, or an antisense oligonucleotide capable of down-regulating the expression of aryl hydrocarbon receptor (see U.S. Patent Publication No. 2010/0183564) .

One of skill in the art will readily be able to determine appropriate concentrations of an aryl hydrocarbon receptor antagonist. In some embodiments, an aryl hydrocarbon receptor antagonist is added to the medium in an amount of about 0.05 -5 μM, for example, about 0.1 -5 μM, 0.2 -4.5 μM, 0.3 -4 μM, 0.4 -3.5 μM, 0.5 -3 μM, 0.6 -2.5 μM, 0.7 -2 μM, 0.8 -1.5 μM, 0.9 -1.2 μM, 1-1.1 μM or 1μM.

HIF-1 modulator

The term “a HIF-1 modulator” as used herein refers to an agent (such as a compound) that modulates, for example, activates HIF-1 e.g., HIF-1a, in cells. In some embodiments, a HIF-1 modulator is a HIF-1 activator. As used herein, "HIF-1 activator" refers to a compound that increases (enhances, promotes) HIF-1 activity in a cell. A HIF-1 activator can act by any of a variety of mechanisms. Such mechanisms could include, for example, any mechanism that results in increased levels of HIF-1, e.g., by causing increased expression or reduced degradation of a HIF alpha subunit.

A variety of HIF-1 activators are of use in various embodiments of the invention. In some embodiments of the invention, a HIF-1 activator is a compound that inhibits hydroxylation of HIF-1. HIF-1 amino acid hydroxylations are performed by HIF hydroxylases. In some embodiments, a HIF-1 activator is a compound that inhibits synthesis, stability, or activity of a prolyl hydroxylase. Such compounds are referred to herein as prolyl hydroxylase inhibitors (PHIs) . In some embodiments, the PHI is a HIF PHI, i.e., a PHI that inhibits

PHD

1, 2, and/or 3. In some embodiments of the invention, a HIF-1 activator is a compound that inhibits FIH synthesis, stability, or activity.

A variety of PHIs are known in the art and can be used in certain embodiments of the invention. In some embodiments, a PHI binds to a HIF hydroxylase and inhibits its enzymatic activity. In some embodiments, a HIF PHI is the compound referred to as FG-4592 (Roxadustat) , FG-2216, or FG-4539. Exemplary PHIs are also described, e.g., in WO/2010022240.

In some embodiments, a HIF PHI is a structural mimetic of 2-oxoglutarate. Exemplary compounds are described e.g., in WO/2005011696. Exemplary compounds include, e.g., N- ( (l-chloro-4-hydroxy-isoquinoline-3-carbonyl) -amino) -acetic acid, [ (7-Bromo-4-hydroxy-isoquinoline-3-carbonyl) -amino] -acetic acid, [ (1-Chloro-4-hydroxy-7-methoxy-isoquinoline-3-carbonyl) -amino] -acetic acid, (7-Chloro-3-hydroxy-quinoline-2-carbonyl) -amino] -acetic acid, [ (3-Hydroxy-6-isopropoxy-quinoline-2-carbonyl) -amino] -acetic acid, and [ (4-Hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl) -amino} -acetic acid, and dimethyloxalylglycine (DMOG) .

In some embodiments, a PHI comprises an oligonucleotide such as an RNAi agent, e.g., a siRNA that inhibits expression of a HIF PHD, e.g., PHD 1, PHD2, and/or PHD3. In some embodiments, a PHI comprises a polypeptide.

One of skill in the art will readily be able to determine appropriate concentrations of a HIF-1 modulator. In some embodiments, a HIF-1 modulator is added to the medium in an amount of about 1-500 nM, for example, about 10-500 nM, 20-450 nM, 30-400 nM, 40-350 nM, 50-300 nM, 60-250 nM, 70-200 nM, 80-150 nM, 90-120 nM, 100-110 nM, or 100 nM.

PPAR alpha agonist

The term “peroxisome proliferator-activated receptor alpha agonist” or “aPPAR alpha agonist” refers to an agent (such as a compound) capable of activating peroxisome proliferator-activated receptor alpha (PPAR-α) . As used herein, a PPAR-α agonist may include any compound that is proven to be capable of selectively activating PPAR-α. Such a PPAR-α agonist may be an agonist of more than one PPAR subtype; however, a PPAR-αagonist selectively activates PPAR-α over PPAR-γ, PPAR-δ or PPAR γ/δ. It is preferred that the PPAR-α agonist is a PPAR-α agonist with at least twice the activity of any other PPAR subtype.

Several PPAR-alpha agonists are described in the art which can be used in the context of the present invention. Non-limiting examples are aliphatic compounds described in WO03004484, heterocyclic compounds described in WO03043985, arylthiazolidinedione and aryloxazolidinedione derivatives described in WO00078312 and WO00078313, bicyclic compounds described in WO05095363, phenoxyacetic acid derivative described in WO05095364, aryloxyacetic acids described in WO01060807 and U.S. Pat. No. 6,569,879, 2-aryloxy-2-arylalkanoic acids described in WO02064094, aniline derivatives described in WO04111020, benzopyrancarboxylic acid derivatives described in U.S. Pat. No. 6,645,997 and U.S. Pat. No. 6,713,508, thiazole-2-carboxamide derivatives described in WO05037804, oleoylethanolamide-like compounds described in WO05002524, heteroaryl derivatives as described in WO05049606, pyrazolyl indolyl derivatives described in WO05085235, indolyl derivatives substituted with a thiazole ring as described in WO05005423, benzannelated compounds as described in WO05049572, pyrazole phenyl derivatives as described in WO05105754, substituted heteroaryl and phenylsulfamoyl compounds as described in US2005288340, dithiolane derivatives as described in WO01025226, analogues of resveratrol such as pterostilbene as described in US200657231, phenyl derivatives as described in WO05049573, substituted phenylpropionic acid derivatives as described in U.S. Pat. No. 6,506,797 and U.S. Pat. No. 6,949,259, tetrahydroisoquinoline derivatives as described in U.S. Pat. No. 6,987,118.

In some embodiments, non-limiting examples of PPAR-alpha agonists include Fenofibrate, Clofibrate, Bezafibrate, Clinofibrate, Ciprofibrate, Etofibrate, Gemfibrate Gemfibrozil) , Pemabate ( (R) -2- {3- { [N- (benzoxazol-2-yl) -N-3- (4-methoxyphenoxy) propyl] aminomethyl} phenoxy} butyric acid) and other fibrates and amphiphilic carboxylic acids. Illustrative examples of PPAR-α agonists that can be used in the present disclosure may also include, but are not limited to, GW-7647 (2- [ [4- [2- [ [ (cyclohexylamino) carbonyl] (4-cyclohexylbutyl) amino] ethyl] phenyl] thio] -2-methylpropanoic acid) , WY-14643 (pilinic acid) , AM3102 (CAS number: 213182-22-0) , CAY15073 (CAS number: 853652-40-1 ) , CP775146 (CAS Number: 702680-17-9) , GW9578 (CAS Number: 247923-29-1) , GW590735 (CAS Number: 622402-22-6) , Oleylethanolamide, Palmitoylethanolamide, Tesaglitazar ( (S) -2-ethoxy-3- [4- [4- [ (methylsulfonyl) oxy] phenethoxy] phenyl) propionic acid) , LY518674 (2- [4- [3- [2, 5-Dihydro-1- [ (4-methylphenyl) methyl] -5-oxo-1H-1, 2, 4-triazol-3-yl] propyl] phenoxy] -2-methyl-propionic acid) , or any combination thereof.

In one embodiment, PPAR-α agonists that can be used in the present disclosure include, but are not limited to, fenofibrate, GW-7647, clofibrate, gemfibrozil, ciprofibrate, bezafibrate, or any combination thereof.

One of skill in the art will readily be able to determine appropriate concentrations of a PPAR-α agonist. In some embodiments, PPAR-α agonist is added to the medium in an amount of about 0.01 –100 μM for example, about 0.01 –50 μM, 0.05 –50 μM, 0.1 –45 μM, 1 –40 μM, 2 –35 μM, 3 –30 μM, 4 –30 μM, 5 –25 μM, 6 –20 μM, 7 –15 μM, 8 –13 μM, 9 –12 μM, 10 –11 μM and 10 μM.

In some embodiments, the CD34-cells may be cultured in a first medium (e.g., in a differentiation medium) in the presence of the additives as described above and a mixture of supplements (e.g., cytokines) including, but not limited to transferrin, insulin, IL-3, SCF, and EPO (including human EPO, EPO from other species, or EPO analogs such as Epoetin alfa, Epoetin beta) at suitable concentrations (e.g., at 37℃ under 5%CO ₂) for a suitable period of time (e.g., 2-7 days, 4-6 days, 5-7 days, or 2-5 days) . One of skill in the art will readily be able to determine appropriate concentrations of the supplements (e.g., cytokines) in the medium.

In some embodiments, the concentration of transferrin in the first medium ranges from about 100-600 μg/mL, such as 200-550 μg/mL, 300-500 μg/mL, 400-500 μg/mL, or 500 μg/mL. In some embodiments, the concentration of insulin in the first medium ranges from about 1-20 μg/mL, such as 5-20 μg/mL, 6-18 μg/mL, 7-16 μg/mL, 8-14 μg/mL, 9-12 μg/mL, or 10-11 μg/mL. In some embodiments, the concentration of IL-3 in the first medium ranges from about 0.1-10 ng/ml. In some embodiments, the concentration of IL-3 ranges from about 0.5 ng/ml to 7 ng/ml, such as from 1 ng/ml to 6.5 ng/ml, from 2 ng/ml to 6 ng/ml, from 3 ng/ml to 5.5 ng/ml, or from 4 ng/ml to 5 ng/ml. In some embodiments, the concentration of EPO in the first medium ranges from about 1-7 U/mL, such as 2-6 U/mL, 3-6 U/mL, 4-6 U/mL, or 5-6 U/mL. In some embodiments, the concentration of SCF in the first medium ranges from about 5-120 ng/mL, such as 10-115 ng/mL, 20-110 ng/mL, 30-110 ng/mL, 40-110 ng/mL, 50-110 ng/mL, 60-110 ng/mL, 70-110 ng/mL, 80-110 ng/mL, 90-110 ng/mL, 95-105 ng/mL, or 100 ng/mL.

In some embodiments, in addition to the additives as described above, the first medium may contain: fetal bovine serum (FBS) , human plasma, glutamine, BSA, transferrin, insulin, one or more antibiotics (e.g., penicillin and/or streptomycin) , IL-3, EPO, and SCF. In some embodiments, the first medium may be a medium (such as an Iscove's Modified Dulbecco's Medium (IMDM) ) containing: fetal bovine serum (FBS) , human plasma, glutamine, BSA, transferrin, insulin, one or more antibiotics (e.g., penicillin and/or streptomycin, such as Penicillin-Streptomycin) , IL-3, EPO, and SCF. In a further embodiment, the first medium may be a medium (such as an Iscove's Modified Dulbecco's Medium (IMDM) ) containing: about 5-15% (v/v) (such as about 6, 7, 8, 9, 10, 11, 12, 13, and 14%(v/v) ) FBS, about 2-10% (v/v) (such as about 3, 4, 5, 6, 7, 8, 9 and 10% (v/v) ) human plasma, about 1-4 mM (such as 2 and 3 mM) glutamine, about 5-15 mg/ml (such as about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15 mg/ml) BSA, about 100-600 μg/mL (such as 200, 300, 400, 500 and 600 μg/mL) human transferrin, about 5-20 μg/mL (such as 6, 7, 8, 9, 10, 11 and 12, 15 and 18 μg/mL) human insulin, an effective amount of one or more antibiotics (e.g., penicillin and/or streptomycin, such as Penicillin-Streptomycin) (e.g., a combination of about 80-120 U/ml penicillin and about 80-120 μg/ml streptomycin, such as a combination of about 100 U/ml penicillin and about 100 μg/ml streptomycin) , about 0.5-7 ng/mL (such as 1, 2, 3, 4, 5 and 6 ng/mL) human IL-3, about 1-7 U/mL (e.g., 1, 2, 3, 4, 5, 6 and 7 U/mL) human EPO, and about 5-120 ng/mL (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 and 110 ng/mL) human SCF. In one embodiment, recombinant human cytokines are used.

Second Differentiation

In one aspect, the present disclosure provides a method of producing red blood cells, comprising culturing a population of nucleated red blood cell precursors in a medium (e.g., a second differentiation medium) . In some embodiments, the population of nucleated red blood cell precursors is at a terminal differentiation stage, and in some embodiments, the nucleated red blood cell precursors are reticulocytes. In some embodiments, the medium comprises one or more additives selected from a group consisting of a vitamin, a methylxanthine compound, a thyroid hormone receptor agonist, and any combination thereof, such as a combination of vitamin and a methylxanthine compound, a combination of vitamin and a thyroid hormone receptor agonist, and a combination of a methylxanthine compound and a thyroid hormone receptor agonist, preferably a combination of a vitamin, a methylxanthine compound, and a thyroid hormone receptor agonist. In some embodiments, the nucleated red blood cell precursors are obtained from a population of Lin-CD34-hemopoietic progenitor cells.

In some embodiments, after the phase I differentiation as described herein or other known phase I differentiation, the obtained cells are further cultured in a second medium (e.g., a second differentiation medium) comprising one or more additives selected from a group consisting of a vitamin, a methylxanthine compound, a thyroid hormone receptor agonist, and any combination thereof, such as a combination of vitamin and a methylxanthine compound, a combination of vitamin and a thyroid hormone receptor agonist, and a combination of a methylxanthine compound and a thyroid hormone receptor agonist, preferably a combination of a vitamin, a methylxanthine compound, and a thyroid hormone receptor agonist, thereby producing red blood cells.

In one embodiment, the in vitro culturing process described herein further comprises a step of culturing a population of nucleated red blood cell precursors in a medium comprising one or more additives selected from a group consisting of a vitamin, a methylxanthine compound, a thyroid hormone receptor agonist, and any combination thereof, such as a combination of vitamin and a methylxanthine compound, a combination of vitamin and a thyroid hormone receptor agonist, and a combination of a methylxanthine compound and a thyroid hormone receptor agonist, preferably a combination of a vitamin, a methylxanthine compound, and a thyroid hormone receptor agonist, thereby producing red blood cells.

It is surprisingly found that a combination of a vitamin, a methylxanthine compound, and a thyroid hormone receptor agonist can act, in a synergistic way, to promote the enucleation /maturation of nucleated red blood cell precursors at a terminal differentiation stage for example reticulocytes differentiation. Without wishing to be bound by theory, the combination of these compounds targeting vitamin receptors, thyroid hormone receptor and caffeine receptor enhances not only cell differentiation during terminal differentiation stage but also increases cell proliferation.

Vitamin

The generic term “vitamin” as used herein may include various vitamins themselves, vitamin derivatives or analogues such as salts or esters thereof, or any combination thereof. In some embodiments, contemplated vitamins for use in the present disclosure may preferably be selected from a group of vitamin C, vitamin E, derivatives or analogues of vitamin C or E, and any combination thereof.

Derivatives or analogues of vitamin C are known in the art. In some embodiments, exemplary vitamin C derivatives or analogues may include sodium ascorbate, magnesium ascorbate, sodium ascorbyl phosphate, magnesium ascorbyl phosphate, erythorbic acid, sodium erythorbate, ascorbyl acetate, ascorbyl propionate, ascorbyl palmitate, erythorbyl acetate, erythorbyl propionate, erythorbyl palmitate, and glycosyl ascorbic acid.

Derivatives or analogues of vitamin E are also known in the art. By vitamin E derivatives it is meant derivatives of vitamin E and more particularly derivatives retaining the antioxidant properties of Vitamin E. By vitamin E analog it is meant compounds having a modified phytyl chain. In a preferred embodiment the vitamin E analogue is Trolox.

In some preferable embodiments, the medium comprises a combination of two types of vitamins: vitamin C (including its derivatives and/or analogues) and vitamin E (including its derivatives and/or analogues) .

One of skill in the art will readily be able to determine appropriate concentrations of vitamin. In some embodiments, vitamin is added to the medium in an amount of about 1 -100 μM, for example, 2 -90 μM, 3 -80 μM, 4 -70 μM, 5 -60 μM, 6 -50 μM, 7 -40μM, 8 -30 μM, 9 -20 μM, 10 -15 μM, 10 -12 μM or 10 μM. In some embodiments, vitamin C and/or its derivative or analogue are added to the medium in an amount of about 1 -50 μM, for example, 2 -45 μM, 3 -40 μM, 4 -35 μM, 5 -30 μM, 6 -25 μM, 7 –20 μM, 8 -15 μM, 9 -10 μM or 10 μM. In some embodiments, vitamin E and/or its derivative or analogue are added to the medium in an amount of about 1 -50 μM, for example, 2 -45 μM, 3 -40 μM, 4 -35 μM, 5 -30 μM, 6 -25 μM, 7 –20 μM, 8 -15 μM, 9 -10 μM or 10 μM. In some embodiments, a combination of (a) vitamin C and/or its derivative or analogue and (b) vitamin E and/or its derivative or analogue are added to the medium in an amount of about 1 -50 μM, respectively.

Methylxanthine compound

The term “methylxanthine compound” as used herein refers to a class of xanthine compounds with one or more methyl substituents, including but not limited to caffeine (1, 3, 7-trimethylxanthine) , IMBX (3-isobutyl-1-methylxanthine) , theophylline (1, 3-dimethylxanthine) , theobromine (3, 7-dimethylxanthine) or the like. Without wishing to be bound by theory, it is believed that the methylxanthine compound interact with caffeine receptor (e.g., adenosine receptor such as adenosine receptor A1) in a cell to function synergistically with one or more of other additives. In some embodiments, caffeine and IMBX are preferred and caffeine is most preferred.

One of skill in the art will readily be able to determine appropriate concentrations of a methylxanthine compound. In some embodiments, a methylxanthine compound is added to the medium in an amount of about 1 -50 μM, for example, 2 -45 μM, 3 -40 μM, 4 -35 μM, 5 -30 μM, 6 -25 μM, 7 –20 μM, 8 -15 μM, 9 -10 μM or 10 μM.

Thyroid hormone receptor agonist

The term “thyroid hormone receptor agonist” as used herein refers to an agent (such as a compound) that activates thyroid hormone (TH) receptor e.g., TH receptor alpha or beta, in cells. In preferably embodiments, the thyroid hormone receptor agonist is a thyroid hormone receptor-beta agonist that activates TH receptor-beta in cells.

In some embodiments, the thyroid hormone receptor agonist may be thyroid hormone such as thyroxine (T4) , tri-iodothyronine (T3) and their analogues. Examples of thyroid hormone analogues are also provided herein and can include 3, 5-dimethyl-4- (4’-hydroy-3’-isopropylbenzyl) -phenoxy acetic acid (GC-1) , or 3, 5-diiodothyropropionic acid (DITPA) , tetraiodothyroacetic acid (TETRAC) , and triiodothyroacetic acid (TRIAC) . Other suitable thyroid hormone analogues are described in FIG. 20 Tables A-D of US7785632B2.

Exemplary thyroid hormone receptor agonists are also described, e.g., in US10800767B2, US20170319604A1, US20090082310A1 and US7785632B2.

One of skill in the art will readily be able to determine appropriate concentrations of a thyroid hormone receptor agonist. In some embodiments, a thyroid hormone receptor agonist is added to the medium in an amount of about 0.1-20 μM, for example, about 0.1-10 μM, 0.2-9 μM, 0.3-8 μM, 0.4-7 μM, 0.5-6 μM, 0.6-5 μM, 0.7-4 μM, 0.8-3 μM, 0.9-2 μM, 1-1.5 μM, or 1 μM.

In some embodiments, the cells may be cultured in a second medium (e.g., in a second differentiation medium) in the presence of the additives as described above and a mixture of supplements (e.g., cytokines) including, but not limited to transferrin, insulin, and EPO (including human EPO, EPO from other species, or EPO analogs such as Epoetin alfa, Epoetin beta) at suitable concentrations (e.g., at 37℃ under 5%CO ₂) for a suitable period of time (e.g., 5-11 days, 6-10 days, or 7-11 days) . One of skill in the art will readily be able to determine appropriate concentrations of the supplements (e.g., cytokines) in the medium.

In some embodiments, the concentration of transferrin in the second medium ranges from about 100-600 μg/mL, such as 200-550 μg/mL, 300-500 μg/mL, 400-500 μg/mL, or 500 μg/mL. In some embodiments, the concentration of insulin in the second medium ranges from about 1-20 μg/mL, such as 5-20 μg/mL, 6-18 μg/mL, 7-16 μg/mL, 8-14 μg/mL, 9-12 μg/mL, or 10-11 μg/mL. In some embodiments, the concentration of EPO in the second medium ranges from about 0.5-7 U/mL, such as about 0.5-5 U/mL, 1-4 U/mL, 1.5-3 U/mL, 2-2.5 U/mL, or 2 U/mL.

In some embodiments, the second medium comprises about 100-600 μg/mL transferrin, about 5-20 μg/mL insulin, and about 0.5-5 U/mL EPO.

In some embodiments, in addition to the additives described above, the second medium may contain FBS, human plasma, glutamine, BSA, transferrin, insulin, one or more antibiotics (e.g., penicillin and/or streptomycin) , and EPO. In some embodiments, the second medium is a medium (such as an Iscove's Modified Dulbecco's Medium (IMDM) ) containing: FBS, human plasma, glutamine, BSA, transferrin, insulin, one or more antibiotics (e.g., penicillin and/or streptomycin) , and EPO. In a further embodiment, the second medium is a medium (such as an Iscove's Modified Dulbecco's Medium (IMDM) ) containing: about 5-15%(v/v) (such as about 6, 7, 8, 9, 10, 11, 12, 13, and 14% (v/v) ) FBS, about 2-10% (v/v) (such as about 3, 4, 5, 6, 7, 8, 9 and 10% (v/v) ) human plasma, about 1-4 mM (such as 2 and 3 mM) glutamine, about 5-15 mg/ml (such as about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15 mg/ml) BSA, about 100-600 μg/mL (such as 100, 200, 300, 400, 500 and 600 μg/mL) human transferrin, about 5-20 μg/mL (such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 15 and 18 μg/mL) human insulin, an effective amount of one or more antibiotics (e.g., penicillin and/or streptomycin, such as Penicillin-Streptomycin) (e.g., a combination of about 80-120 U/ml penicillin and about 80-120 μg/ml streptomycin, such as a combination of about 100 U/ml penicillin and about 100 μg/ml streptomycin) , and about 0.5-5 U/mL (e.g., 1, 2, 3 and 4 U/mL) human EPO. In one embodiment, recombinant human cytokines are used.

Medium supplement composition

In some aspects, the present disclosure provides a medium supplement composition, which for example is to be added in a base medium as described herein to form a differentiation medium for producing red blood cells.

In some embodiments, the present disclosure provides a medium supplement composition comprising one or more additives (such as small molecules) selected from a group consisting of an aryl hydrocarbon receptor antagonist, a HIF-1 modulator, a PPAR alpha agonist, and any combination thereof, preferably a combination of an aryl hydrocarbon receptor antagonist, a HIF-1 modulator and a PPAR alpha agonist. In some embodiments, the medium supplement composition further comprises one or more of transferrin, insulin, IL-3, EPO, and SCF, preferably a combination of transferrin, insulin, IL-3, EPO, and SCF.

In some embodiments, the present disclosure provides a medium supplement composition comprising one or more additives selected from a group consisting of a vitamin, a methylxanthine compound, a thyroid hormone receptor agonist, and any combination thereof, preferably a combination of a vitamin, a methylxanthine compound, and a thyroid hormone receptor agonist. In some embodiments, the medium supplement composition further comprises one or more of transferrin, insulin, and EPO, preferably a combination of transferrin, insulin, and EPO.

Engineered red blood cells

Also described herein are methods of preparing genetically engineered red blood cells capable of expressing proteins of interest, such as cell surface proteins or fusion proteins comprising a red blood cell transmembrane protein and an antigen binding protein that binds to an antigen, such as a toxin or an antigen of a pathogen. Such engineered red blood cells may be produced using any of the cell culture methods provided herein.

Expression vectors for producing proteins of interest (e.g., fusion proteins) may be introduced into CD34-progenitor cells. In some embodiments, the expression vectors can be designed such that they can incorporate into the genome of cells by homologous or non-homologous recombination by methods known in the art. Methods for transferring expression vectors into CD34-progenitor cells include, but are not limited to, viral mediated gene transfer, liposome mediated transfer, transformation, transfection and transduction, e.g., viral mediated gene transfer such as the use of vectors based on DNA viruses such as adenovirus, adeno-associated virus and herpes virus, as well as retroviral based vectors. Examples of modes of gene transfer include e.g., naked DNA, CaP04 precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors, adjuvant-assisted DNA, gene gun, catheters. In one example, a viral vector is used. Liposomes that also include a targeting antibody or fragment thereof can be used in the methods described herein.

A "viral vector" as used herein refers to a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro. Examples of viral vectors include retroviral vectors such as lentiviral vectors, adenovirus vectors, adeno-associated virus vectors and the like. In aspects where gene transfer is mediated by a retroviral vector, a vector construct refers to the polynucleotide comprising the retroviral genome or part thereof, and a therapeutic gene. In one embodiment, lentiviral vectors are preferred.

A gene encoding any protein, including a cell surface protein (e.g., transmembrane protein, integral membrane protein) or any of the fusion proteins can be inserted into a suitable vector (e.g., a retroviral vector such as lentiviral vectors) using methods well known in the art (Sambrook et al, Molecular Cloning, A Laboratory Mannual, 3rd Ed., Cold Spring Harbor Laboratory Press) . For example, the gene and vector can be contacted, under suitable conditions, with a restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined together with a ligase.

In some embodiments, the CD34-progenitor cells provided herein can be genetically modified using any of the methods described herein or known in the art such that they are capable of expressing a protein, such as a cell surface protein (e.g., integral membrane protein or transmembrane protein) , or a fusion protein comprising a red blood cell transmembrane protein and a peptide heterologous to the membrane protein, such as an antigen binding protein that binds to a toxin or antigen of a pathogen. A red blood cell transmembrane protein can be conjugated to another peptide (e.g., antigen binding protein) directly or via a linker. Preferably the transduction of the CD34-progenitor cells with a gene encoding a surface protein is via a viral vector such as a retroviral vector (such as lentiviral vectors) .

A cell surface protein can be a fusion protein comprising a membrane protein and at least one heterologous protein (e.g., antigen binding protein) . In some embodiments, the cell surface protein comprises a red blood cell transmembrane protein and an antigen binding protein. Such membrane proteins are known in the art. For example, red blood cell precursors express high levels of the transferrin receptor (Tfr) , which is a type II membrane protein. Any membrane proteins present in mature RBCs can be used for constructing fusion proteins. The membrane protein can be fused to an antigen binding protein at the terminus that is exposed to the extracellular or luminal space. In some examples, the terminus of the fusion protein that is exposed to cytoplasm may also be fused to a protein of interest, which can be a cytoplasmic protein.

Any of the genetically modified CD34-progenitor cells described herein can be cultured under suitable conditions allowing for differentiation into mature enucleated red blood cells, e.g., the in vitro culturing process (e.g., the second differentiation) described herein. The resultant enucleated red blood cells are capable of expressing the surface protein of interest, such as a fusion protein as described herein, which can be evaluated and confirmed by routine methodology (e.g., Western blotting or FACS analysis) .

Depending on the intended applications of the engineered red blood cells, a wide variety of proteins of interest such as a binding agent, a therapeutic agent or a detection agent can be contemplated in the present disclosure. In some embodiments, an agent may comprise a protein, a peptide (e.g., an extracellular domain of oligomeric ACE2) , an antibody or its functional antibody fragment, an antigen or epitope, a MHC-peptide complex, an enzyme (e.g., a functional metabolic or therapeutic enzyme) , a cytokine, a growth factor, a ligand, a receptor, an immunotolerance-inducing peptide, a targeting moiety or any combination thereof.

In some embodiments, a protein of interest is an enzyme such as a functional metabolic or therapeutic enzyme, e.g., an enzyme that plays a role in metabolism or other physiological processes in a mammal. In some embodiments a protein is an enzyme that plays a role in carbohydrate metabolism, amino acid metabolism, organic acid metabolism, porphyrin metabolism, purine or pyrimidine metabolism, and/or lysosomal storage. Deficiencies of enzymes or other proteins can lead to a variety of diseases, e.g., diseases associated with defects in carbohydrate metabolism, amino acid metabolism, organic acid metabolism, purine or pyrimidine metabolism, lysosomal storage disorders, and blood clotting, among others. Metabolic diseases are characterized by the lack of functional enzymes or excessive intake of metabolites. Thus, the metabolites deposition in the circulation and tissues causes tissue damage. Due to the wide distribution in human body of RBCs, the present disclosure contemplates modifying membrane proteins of RBCs with functional metabolic enzymes. The enzymes targeted RBCs will uptake metabolites in plasma of patients. Exemplary enzymes include urate oxidase for gout, phenylalanine ammonia-lyase for Phenylketonuria, acetaldehyde dehydrogenase for alcoholic hepatitis, butyrylcholinesterase for ***e metabolite, and the like. In some embodiments, red blood cells having urate oxidase conjugated thereto may be administered to a subject in need of treatment of chronic hyperuricemia, e.g., a patient with gout, e.g., gout that is refractory to other treatments.

In some embodiments, the protein of interest is an antibody, including an antibody, an antibody chain, an antibody fragment e.g., scFv, an antigen-binding antibody domain, a VHH domain, a single-domain antibody, a camelid antibody, a nanobody, an adnectin, or an anticalin. The red blood cells having antibodies attached thereto may be used as a delivery vehicle for the antibodies and/or the antibodies may serve as a targeting moiety. Exemplary antibodies include anti-tumor antibodies such as PD-1 antibodies.

In some embodiments, the protein of interest may comprise an antigen or epitopes or a binding moiety that binds to an antigen or epitope. In some embodiments an antigen is any molecule or complex comprising at least one epitope recognized by a B cell and/or by a T cell. In some embodiments an antigen is a surface protein of, e.g., a viral capsid, envelope, or coat, or bacterial, fungal, protozoal, or parasite cell. Exemplary viruses may include, e.g., coronaviruses (e.g., SARS-CoV and SARS-CoV-2) , HIV, dengue viruses, encephalitis viruses, yellow fever viruses, hepatitis virus, Ebola viruses, influenza viruses, and herpes simplex virus (HSV) 1 and 2. In some embodiments, an antigen is a tumor antigen (TA) , which can be any antigenic substance produced by cells in a tumor, e.g., tumor cells or in some embodiments tumor stromal cells (e.g., tumor-associated cells such as cancer-associated fibroblasts or tumor-associated vasculature) .

In some embodiments, an antigen is a peptide. Peptides may bind directly to MHC molecules expressed on cell surfaces, may be ingested and processed by APC and displayed on APC cell surfaces in association with MHC molecules, and/or may bind to purified MHC proteins (e.g., MHC oligomers) . In some embodiments a peptide contains at least one epitope capable of binding to an appropriate MHC class I protein and/or at least one epitope capable of binding to an appropriate MHC class II protein. In some embodiments a peptide comprises a CTL epitope (e.g., the peptide can be recognized by CTLs when bound to an appropriate MHC class I protein) .

In some embodiments, the agent may comprise a MHC-peptide complex, which may comprise a MHC and a peptide such as an antigenic peptide or an antigen for activating immune cells. In some embodiments, the antigenic peptide is associated with a disorder and is able to activate CD8 ⁺ T cells when presented by a MHC class I molecule. Class-I major histocompatibility complex (MHC-I) is presenting antigen peptides to and activating immune cells particularly CD8 ⁺ T cells, which are important for fighting against cancers, infectious diseases, etc.

In some embodiments, the protein of interest may comprise a growth factor. Growth factors include, e.g., members of the vascular endothelial growth factor (VEGF, e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D) , epidermal growth factor (EGF) , insulin-like growth factor (IGF; IGF-1, IGF-2) , fibroblast growth factor (FGF, e.g., FGF1-FGF22) , platelet derived growth factor (PDGF) , or nerve growth factor (NGF) families.

In some embodiments, the protein of interest may comprise a cytokine or the biologically active portion thereof. In some embodiments a cytokine is an interleukin (IL) e.g., any of IL-1 to IL-38 (e.g., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12) , interferons (e.g., a type I interferon, e.g., IFN-α) , and colony stimulating factors (e.g., G-CSF, GM-CSF, M-CSF) . Cytokine (such as recombinant IL-2, recombinant IL-7, recombinant IL-12) loaded RBCs is a therapeutic delivery system for increasing tumor cytotoxicity and IFN-γ production.

In some embodiments, the protein of interest may comprise a receptor or receptor fragment. In some embodiments, the receptor is a cytokine receptor, growth factor receptor, interleukin receptor, or chemokine receptor. In some embodiments a growth factor receptor is a TNFα receptor (e.g., Type I TNF-α receptor) , VEGF receptor, EGF receptor, PDGF receptor, IGF receptor, NGF receptor, or FGF receptor. In some embodiments a receptor is TNF receptor, LDL receptor, TGF receptor, or ACE2.

In some embodiments, the present disclosure provides a method for producing engineered red blood cells comprising the steps of: (a) providing a population of Lin ^-CD34 ^-hemopoietic progenitor cells; (b) culturing the population of Lin ^-CD34 ^-hemopoietic progenitor cells from (a) in a first medium comprising one or more additives selected from a group consisting of an aryl hydrocarbon receptor antagonist, a HIF-1 modulator, a PPAR alpha agonist, and any combination thereof, preferably a combination of an aryl hydrocarbon receptor antagonist, a HIF-1 modulator and a PPAR alpha agonist, to induce them to differentiate into erythroid cells, and prior to or concurrently with the differentiation, introducing one or more exogenous nucleic acids into the erythroid cells; and (c) culturing the erythroid cells to induce enucleation. In some embodiments, step (c) comprises culturing the population of cells obtained from (b) in a second medium comprising one or more additives selected from a group consisting of a vitamin, a methylxanthine compound, a thyroid hormone receptor agonist, and any combination thereof, preferably a combination of a vitamin, a methylxanthine compound, and a thyroid hormone receptor agonist, thereby producing engineered red blood cells.

In some embodiments, the expansion medium is a serum-free expansion medium.

In some embodiments, the first medium is a first differentiation medium.

In some embodiments, the HIF-1 modulator is in an amount of about 10 -500 nM.

In some embodiments, the PPAR alpha agonist is selected from a group consisting of Fenofibrate, Clofibrate, Bezafibrate, Clinofibrate, Ciprofibrate, Etofibrate, Gemfibrate Gemfibrozil) , Pemabate ( (R) -2- {3- { [N- (benzoxazol-2-yl) -N-3- (4- methoxyphenoxy) propyl] aminomethyl} phenoxy} butyric acid) , GW-7647 (2- [ [4- [2- [ [ (cyclohexylamino) carbonyl] (4-cyclohexylbutyl) amino] ethyl] phenyl] thio] -2-methylpropanoic acid) , WY-14643 (pilinic acid) , AM3102 (CAS number: 213182-22-0) , CAY15073 (CAS number: 853652-40-1 ) , CP775146 (CAS Number: 702680-17-9) , GW9578 (CAS Number: 247923-29-1) , GW590735 (CAS Number: 622402-22-6) , Oleylethanolamide, Palmitoylethanolamide, Tesaglitazar ( (S) -2-ethoxy-3- [4- [4- [ (methylsulfonyl) oxy] phenethoxy] phenyl) propionic acid) , LY518674 (2- [4- [3- [2, 5-Dihydro-1- [ (4-methylphenyl) methyl] -5-oxo-1H-1, 2, 4-triazol-3-yl] propyl] phenoxy] -2-methyl-propionic acid) , and any combination thereof.

In some embodiments, the second medium is a second differentiation medium.

In some embodiments, the blood sample is a human peripheral blood sample.

In some embodiments, the one or more exogenous nucleic acids comprises a first polynucleotide sequence encoding a urate oxidase (UOX) or a functional variant thereof, a second polynucleotide sequence encoding a urate transporter 1 (URAT1) or a functional variant thereof; or both of the first polynucleotide sequence and the second polynucleotide sequence.

In some further embodiments, the URAT1 comprises an amino acid sequence of SEQ ID NO: 1 below:

In some further embodiments, the UOX comprises an amino acid sequence of SEQ ID NO: 2 below:

In some further embodiments, the present disclosure provides a pharmaceutical composition comprising engineered red blood cells as produced by the method described herein and a physiologically acceptable excipient.

In some embodiments, the present disclosure provides use of engineered red blood cells as produced by the method described herein in the preparation of a medicament for treating a disorder associated with elevated serum uric acid levels. In some further embodiments, the disorder is gout or a hyperuricemia disease.

In some embodiments, the present disclosure provides a method for the treatment of a disorder associated with elevated serum uric acid levels in a subject comprising: a) taking a blood sample (e.g., peripheral blood sample) from e.g. the subject, b) producing engineered red blood cells by using the method as described herein; and c) infusing a therapeutically effective amount of the engineered red blood cells into the subject. In some further embodiments, the disorder is gout or a hyperuricemia disease.

In some embodiments, the present disclosure provides a method for the treatment of a disorder associated with elevated serum uric acid levels in a subject comprising infusing a therapeutically effective amount of engineered red blood cells as produced by the method described herein into the subject. In some embodiments, the method further comprises performing blood typing before the infusion to ensure the subject is compatible with the engineered red blood cell.

In some embodiments, the one or more exogenous nucleic acids comprise an expression vector (such as a lentiviral expression vector) carries a gene of interest to be expressed. In some embodiments, the gene of interest encodes a fusion protein, such as a cell surface membrane protein comprising an anchoring moiety containing at least the transmembrane region of CD235a. In some embodiments, the fusion protein contains an artificial MHC single chain molecule, and in some further embodiments, the fusion protein containing an artificial MHC single chain molecule which, from N-terminus to C-terminus, comprises an antigenic peptide, a first peptide linker, a β2-microglobulin, a second peptide linker and a MHC class I heavy chain lacking the transmembrane region and the cytoplastic region. In some embodiments, the artificial MHC single chain molecule is fused at its C-terminus to the N-terminus of the anchoring moiety, optimally with a third peptide linker. In some embodiments, the fusion protein further comprises a signal peptide, e.g., one selected from β2-microglobulin signal peptide or CD235a signal peptide or their combination. In some embodiments, the first peptide linker and the second peptide linker are rich in Gly and Ser.

In some embodiments, the antigenic peptide is associated with a disorder and is able to activate CD8+ T cells when presented by a MHC class I molecule, for example, a cancer neoantigen, or is derived from an oncoprotein or a virus protein. In some embodiments, the antigenic peptide is 8, 9, 10 or 11 amino acids in length.

In some embodiments, the fusion protein comprises an amino acid sequence of SEQ ID NO: 3 below (MHC I OT1 β CD235a protein sequence: Mouse β2-microglobulin signal peptide is indicated with a dotted underline, OT-1 peptide is indicated with a solid underline, three peptide linkers are showed in bold, Mouse β2-microglobulin is showed in italic, H2-Kb (Y84C) Heavy Chain is indicated with a double underline, and CD235a is indicated with a wavy underline. ) :

In some further embodiments, the fusion protein comprises an amino acid sequence of SEQ ID NO: 4 below (MHC I OT1 αβ CD235a protein sequence: CD235a signal peptide is indicated with a dashed underline, Mouse β2-microglobulin signal peptide is indicated with a dotted underline, OT-1 peptide is indicated with a solid underline, three peptide linkers are showed in bold, Mouse β2-microglobulin is showed in italic, H2-b (Y84C) Heavy Chain is indicated with a double underline, and CD235a is indicated with a wavy underline) :

In some embodiments, the present disclosure provides a method for the treatment of a disorder associated with an antigenic peptide in a subject comprising: a) taking a blood sample (e.g., peripheral blood sample) from e.g. the subject, b) producing engineered red blood cells by using the method as described herein; and c) infusing a therapeutically effective amount of the engineered red blood cells into the subject. In some further embodiments, the antigenic peptide is a fragment of HPV E6 or E7 protein.

In some embodiments, the one or more exogenous nucleic acids comprise an expression vector (such as a lentiviral expression vector or a non-viral vector) carries a gene of interest to be expressed. In some embodiments, the gene of interest encodes a polypeptide, α galactosidase A (α-GAL A) or a functional variant thereof, or glyoxylate reductase/hydroxypyruvate reductase (GRHPR) or a functional variant thereof. the polypeptide comprises an amino acid sequence having at least 75%sequence identity to SEQ ID NO: 5 or SEQ ID NO: 6, or the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 6. SEQ ID NO: 5 (Full-length sequence of human α galactosidase A) :

SEQ ID NO: 6 (Full-length sequence of human GRHPR) :

In some embodiments, the present disclosure provides a method of treating a subject having a lysosomal storage disease, comprising administering an effective amount of the engineered red blood cells as obtained by the method as described herein to the subject. In some embodiments, the method of treating a subject having a lysosomal storage disease comprises: a) taking a blood sample (e.g., peripheral blood sample) from e.g., the subject, b) producing engineered red blood cells by using the method as described herein; and c) infusing a therapeutically effective amount of the engineered red blood cells into the subject. In some embodiments, the lysosomal storage disease is Fabry disease and the polypeptide is αgalactosidase A (α-GAL A) or a functional variant thereof. In some embodiments, the lysosomal storage disease is type II primary hyperoxaluria (PH2) , and the polypeptide is glyoxylate reductase/hydroxypyruvate reductase (GRHPR) or a functional variant thereof.

Uses of Red Blood Cells

Any of the genetically modified CD34-progenitor cells and enucleated red blood cells (including those obtained from the in vitro culturing process described herein) , which can also be genetically modified as described herein, are within the scope of the present disclosure.

Depending on the agent of interest, enucleated red blood cells having a surface modification of an agent of interest (e.g., a fusion protein comprising a red blood cell transmembrane protein and an antigen binding protein) as described herein can be administered to a subject in need thereof for various purposes, e.g., treating a specific disease when the agent of interest is a therapeutic agent, performing a blood transfusion, detecting the presence of specific cell types when the agent of interest is capable of recognizing the target cells, eliciting desired immune responses when the agent of interest is immunogenic, and neutralizing a toxin in a subject when the agent is capable of binding to the toxin. Any suitable delivery route can be used in the methods described herein, e.g., directly into the circulatory system, e.g., intravenously or by injection or infusion, e.g., cell infusion.

The term "subject, " as used herein, refers to an individual organism, for example, an individual mammal. In some embodiments, the subject is a human.

In some embodiments, a subject receives a single dose of cells, or receives multiple doses of cells, e.g., between 2 and 5, 10, 20, or more doses, over a course of treatment. In some embodiments a dose or total cell number may be expressed as cells/kg. For example, a dose may be about 10 ³, 10 ⁴, 10 ⁵, 10 ⁶, 10 ⁷, 10 ⁸ cells/kg. In some embodiments a course of treatment lasts for about 1 week to 12 months or more e.g., 1, 2, 3 or 4 weeks or 2, 3, 4, 5 or 6 months. In some embodiments a subject may be treated about every 2-4 weeks. One of ordinary skills in the art will appreciate that the number of cells, doses, and/or dosing interval may be selected based on various factors such as the weight, and/or blood volume of the subject, the condition being treated, response of the subject, etc. The exact number of cells required may vary from subject to subject, depending on factors such as the species, age, weight, sex, and general condition of the subject, the severity of the disease or disorder, the particular cell (s) , the identity and activity of agent (s) conjugated to the cells, mode of administration, concurrent therapies, and the like.

As used herein, “treating” , “treat” or “treatment” refers to a therapeutic intervention that at least partly ameliorates, eliminates or reduces a symptom or pathological sign of a pathogen-associated disease, disorder or condition after it has begun to develop. Treatment need not be absolute to be beneficial to the subject. The beneficial effect can be determined using any methods or standards known to the ordinarily skilled artisan.

As used herein, “preventing” , “prevent” or “prevention” refers to a course of action initiated prior to infection by, or exposure to, a pathogen or molecular components thereof and/or before the onset of a symptom or pathological sign of the disease, disorder or condition, so as to prevent infection and/or reduce the symptom or pathological sign. It is to be understood that such preventing need not be absolute to be beneficial to a subject. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of the disease, disorder or condition, or exhibits only early signs for the purpose of decreasing the risk of developing a symptom or pathological sign of the disease, disorder or condition.

It will be appreciated by those skilled in the art that other variations of the embodiments described herein may also be practiced without departing from the scope of the invention. Other modifications are therefore possible.

Although the disclosure has been described and illustrated in exemplary forms with a certain degree of particularity, it is noted that the description and illustrations have been made by way of example only. Numerous changes in the details of construction and combination and arrangement of parts and steps may be made. Accordingly, such changes are intended to be included in the invention, the scope of which is defined by the claims.

EXAMPLES

In the Examples, if specific conditions are not indicated, conventional conditions or the conditions recommended by manufacturers are used. Reagents or instruments without a manufacturer information are commercially available.

Example 1

I. Material preparation

1.1 Biotinylated antibodies

Table 1.

Antibody	Final concentration
Biotinylated mouse anti-human CD3 antibody	4μg/ml
Biotinylated mouse anti-human CD14 antibody	4μg/ml
Biotinylated mouse anti-human CD16 antibody	4μg/ml
Biotinylated mouse anti-human CD19 antibody	4μg/ml
Biotinylated mouse anti-human CD41a antibody	4μg/ml
Biotinylated mouse anti-human CD56 antibody	4μg/ml
Biotinylated mouse anti-human CD235a antibody	4μg/ml

1.2 Cell expansion medium (Phase I medium)

Table 2.

1.3 First differentiation medium (Phase II medium)

Control Phase II medium (i.e., base medium in Fig. 2) is formulated according to the table 3 below:

Table 3.

Components	Final concentration
IMDM	85% (v/v)
Fetal Bovine Serum (FBS, Gibco)	10% (v/v)
Human Plasma	5% (v/v)
Glutamine	2 mM
Bovine serum albumin	10 mg/ml
Human transferrin	500 μg/ml
Recombinant human insulin	10 μg/ml
Penicillin	100 U/ml
Streptomycin
	100 μg/ml
Recombinant human interleukin 3	5 ng/ml
Recombinant human erythropoietin	6 U/ml
Recombinant human stem cell factor	100 ng/ml

The following additives are added to the control Phase II medium to formulate Phase II media: (1) 1 μM SR1 (StemRegenin1) , 100 nM FG-4592, and 10 μM fenofibrate (PPARa) ; (2) 1 μM SR1; (3) 100 nM FG-4592, and (4) 10 μM fenofibrate (PPARa) .

1.4 Second differentiation medium (Phase III medium)

Control Phase III medium (i.e., differentiation medium in Fig. 4) is formulated according to the table 4 below:

Table 4.

Components	Final concentration
IMDM	85% (v/v)
Fetal Bovine Serum (FBS, Gibco)	10% (v/v)
Human Plasma	5% (v/v)
Glutamine	2mM
Bovine serum albumin	10 mg/ml
Human transferrin	500μg/ml
Recombinant human insulin	10 μg/ml
Penicillin	100 U/ml
Streptomycin
	100 μg/ml
Recombinant human erythropoietin	2U/ml

The following additives are added to the control Phase III medium to formulate Phase III media: (1) 10 μM Caffeine, 10 μM Vitamin C, 10 μM Vitamin E, and 1 μM thyroxine (Thyroid hormone) ; (2) 10 μM Caffeine; (3) 10 μM Vitamin C; (4) 10 μM Vitamin E; and (5) 1 μM thyroxine (Thyroid hormone) .

II. Isolation of peripheral blood mononuclear cells

Human whole blood was taken and diluted with phosphate buffered saline at a rate of 1: 1. The diluted blood was centrifuged by use of a lymphocyte separation solution (LymphoprepTM, STEMCELL Technologies) and a lymphocyte separation tube, at 1200 × g for 15 minutes, and mononuclear cells were drawn with a capillary tube.

III. Collection of Lin ^-CD34 ^-cells in peripheral blood

After centrifuging the peripheral blood mononuclear cells obtained in (II) at 300 ×g for 10 minutes, the cells were resuspended at 1 × 10 ⁶ cells/ml in a cell isolation buffer added with 2%bovine serum albumin and 1 mM EDTA in a phosphate buffer solution, to obtain a cell suspension.

The biotin-labeled antibodies were added at concentrations shown in Table 1 to the cell suspension, 300 μl of streptavidin-labeled magnetic beads were added, and then the mixture was incubated at 4 ℃ for 30 minutes. Then the mixture was placed in a magnetic rack for 6-8 minutes to perform lineage cell separation, and the isolation buffer that was not adsorbed was collected and centrifuged at 300 × g for 10 minutes to obtain Lin ^-CD34 ^-cells. IV.Preparation of mature red blood cells

On Day 0, various groups of 0.1-0.5 × 10 ⁵ the Lin ^-CD34 ^-cells were seeded into a hematopoietic stem cell expansion medium as shown in Table 2. The cells were cultured to Day 4, which stage is defined as the expansion stage of Lin ^-CD34 ^-cell. On Day 5, the culture systems in the various groups were changed by changing the medium to the control Phase II medium (Table 3) and Phase II media (1) - (4) as described in “1.3 First differentiation medium (Phase II medium) ” , respectively. The cells were cultured at 37 ℃, 5%CO ₂ until Day 13, which stage is defined as the first differentiation stage of Lin ^-CD34 ^-cells. On Day 14, the culture systems were changed by changing the medium to the control Phase III medium (Table 4) and Phase III media (1) - (5) as described in “1.4 Second differentiation medium (Phase III medium) ” , respectively, and the cells were cultured at 37 ℃, 5%CO ₂ until Day 22, which stage is defined as the enucleation and maturation stage of red blood cells. During Days 0-22, the corresponding stage media were changed every 2-4 days.

V. Cell expansion number analysis

The number of cells was measured on Days 0-22, and the cells in the culture system were fully resuspended every 3-4 days. 10 μl of the cell suspension was taken and mixed with 10 μl of trypan blue staining solution, and counted by a cell counter.

VI. Analysis of erythrocyte enucleation

The proportion of CD235a-positive/Hoechst 33342-negative cells was analyzed by flow cytometry, which can reflect the enucleation of cells.

VIII. Results

Fig. 1 showed phase-specific small-molecule combination significantly increases erythroid cell expansion and differentiation efficiency in the ex vivo human CD34-erythroid culture system. Human CD34-cells were cultured by a 3-phase erythroid expansion and differentiation method as described herein. In phase I (day 0～4) , cells in both groups were cultured in the expansion medium. In phases II (day 5～13) and III (day 14～22) , cells in Group 1 were cultured in the differentiation medium without addition of the phase-specific small-molecule combination. In phase II, cells in Group 2 were cultured with proliferation medium supplemented with compound combination containing SR1, FG-4592, and fenofibrate. The cell number of Group 2 increased by at least two-fold in phase II, compared to that of Group 1. In phase III, cells in Group 2 were switched to a differentiation medium supplemented with Vitamin C, E, caffeine and thyroxine. As shown in Figs. 1 and 3, the combination of these compounds targeting vitamin receptors, thyroid hormone receptor and caffeine receptor enhances not only cell differentiation during terminal differentiation stage but also increases cell proliferation.

Fig. 2 showed the combination of small molecules synergistically enhances human erythroid progenitor production. Human CD34-cells were cultured in the expansion medium (day 0～4) , and then switched to the proliferation medium supplemented with the single compound or combination of the three compounds indicated for 6 days. BFU-E colony numbers were quantified by plating on methylcellulose 1000 cells after culturing in the conditions described above. BFU-E colonies were quantified after 12-14 days. Error bars represent mean ± S.D. from three biological replicates.

Fig. 3 showed phase-specific small-molecule combination increases enucleation efficiency in the ex vivo human CD34-erythroid culture system. Human CD34-cells were cultured by a 3-phase erythroid expansion and differentiation method as described herein. In phase II, cells in

Groups

1 and 2 were cultured with proliferation medium supplemented with compound combination containing SR1, FG-4592, and fenofibrate. In page III, Group 1 cells were cultured without addition of the phase-specific small-molecule combination; Group 2 cells were cultured with medium supplemented with phase-specific compound combination (the combination of Caffeine, Vitamin C, Vitamin E, and thyroxine) . At day 19 of the human erythroid culture, group 2 cells with combination of compounds has much higher enucleation rate compared to group 1, as indicated by the percentage of CD235a ⁺Hoechst33342 ^-cell population.

Fig. 4 showed synergism of small molecules increases erythroid cell number during phase III differentiation. Human CD34-cells were cultured by a 3-phase erythroid expansion and differentiation method as described herein. In phase II, cells in

Groups

1 and 2 were cultured with proliferation medium supplemented with compound combination containing SR1, FG-4592, and fenofibrate. In phase III, human CD34-cells were cultured in differentiation medium supplemented with or without the indicated compounds. Erythroid cell numbers were counted at the end of the culture. Combination of the four small molecules significantly increased red cell production compared to that of single compound alone.

Fig. 5 showed expression of erythroid genes was up-regulated in the cells cultured with phase-specific small-molecule combination in the ex vivo human CD34-erythroid culture system. Human CD34-cells were cultured by a 3-phase erythroid expansion and differentiation method as described herein. In phase II, cells in

Groups

1 and 2 were cultured with proliferation medium supplemented with compound combination containing SR1, FG- 4592, and fenofibrate. In phase III, Group 1 cells were cultured without addition of the phase-specific small-molecule combination; and Group 2 cells were cultured with medium supplemented with phase-specific compound combination (the combination of Caffeine, Vitamin C, Vitamin E, and thyroxine) . Total RNA was purified from cells of day 16 in culture for quantitative PCR analysis. The elevation of erythroid gene expression in group 2 demonstrates that phase-specific small-molecule combination enhances erythroid differentiation.

Example 2

This Example was performed to test the effect of neuron active drugs on enhancing red blood cells production in a mouse red cell production culture system.

BFU-E erythroid progenitor cells isolated from E14.5 mouse fetal livers were untreated or treated with 100 nM DEX (Dexmathesone) , 100 nM DEX + neuron active drugs of indicated concentration in Figs. 6A-B in serum-free erythroid liquid expansion (SFELE) medium containing rmSCF and rhEpo. Cell numbers were counted every 3 days. Only 10uM IBMX or 10uM and 50um caffeine together with DEX increases mouse red cell production by 2-3 fold more than DEX alone. As shown in Figs. 6A and 6B, only neuron active drugs having a methylxanthine-like structure enhanced red blood cells production in a mouse red cell production culture system.

Although preferred embodiments of the invention have been described herein, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims. All documents disclosed herein, including those in the following reference list, are incorporated by reference.

Claims

A method for producing red blood cells, comprising the steps of:

(a) providing a population of Lin ^-CD34 ^-hemopoietic progenitor cells;

(b) culturing the population of Lin ^-CD34 ^-hemopoietic progenitor cells from (a) in a first medium comprising one or more additives selected from a group consisting of an aryl hydrocarbon receptor antagonist, a HIF-1 modulator, a PPAR alpha agonist, and any combination thereof, preferably a combination of an aryl hydrocarbon receptor antagonist, a HIF-1 modulator and a PPAR alpha agonist; and

(c) culturing the population of cells obtained from (b) in a second medium comprising one or more additives selected from a group consisting of a vitamin, a methylxanthine compound, a thyroid hormone receptor agonist, and any combination thereof, preferably a combination of a vitamin, a methylxanthine compound, and a thyroid hormone receptor agonist, thereby producing red blood cells.
The method of claim 1, wherein in step (a) the population of Lin ^-CD34 ^-hemopoietic progenitor cells has been expanded in an expansion medium such as a hematopoietic stem cell expansion medium.
The method of claim 2, wherein the expansion medium comprises fms-like tyrosine kinase 3 ligand (Flt3L) , stem cell factor (SCF) , interleukin 3 (IL-3) , and interleukin 6 (IL-6) .
The method of claim 3, wherein the expansion medium comprises about 10-100 ng/mL Flt3L, about 50-150 ng/mL SCF, about 1-20 ng/mL IL-3, and about 5-30 ng/mL IL-6.
The method of any of claims 2-4, wherein the expansion medium is a serum-free expansion medium.
The method of any of claims 2-5, wherein the population of Lin ^-CD34 ^-hemopoietic progenitor cells has been expanded for about 2-5 days.
The method of any of claims 1-6, wherein the first medium is a first differentiation medium.
The method of any of claims 1-7, wherein the aryl hydrocarbon receptor antagonist is selected from a group consisting of SR1, 4- (2- (Pyridin-3-yl) -9-isopropyl-9H-purin-6-ylamino) ethyl) phenol, 4- (2- (9-Isopropyl-2- (2-methyl-1H-imidazol-1-yl) -9H-purin-6-ylamino) ethyl) phenol, 4- (2- (2- (5-Chloropyridine-3-yl) -9-isopropyl-9H-purin-6-ylamino) ethyl) phenol, 4- (2- (6- (5-Fluoropyridin-3-yl) -1-isopropyl-1H-pyrazolo [3, 4-d] pyrimidin-4-ylamino) ethyl) pheno1, 4- (2- (2- (5-Fluoropyridin-3-yl) -7-isopropyl-7H-pyrrolo [2, 3-d] pyrimidin-4-ylamino) ethyl) phenol, (R) -4-2- (2- (benzo [b] thiophen-3-yl) -9-tetrahydrofuran-3-yl) -9H-purin-6-ylamino) ethyl) phenol, 2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3-yl) -9H-purin-6-ylamino) ethyl) phenol1, (R) -2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3-yl) -9H-purin-9-yl) propan-1-ol, (S) -2- (6- (2- (1H-indol-3- yl) ethylamino) -2- (5-fluoropyridin-3-yl) -9H-purin-9-yl) propan-1-ol, 4- (2- (6- (5-Fluoropyridin-3-yl) -1-isopropyl-1H-imidazol [4, 5-c] pyridin-4-ylamino) ethyl) phenol, 4- (2- (5- (5-Fluoropyridin-3-yl) -3-isopropyl-3H-imidazo [4, 5-c] pyridin-7-ylamino) ethyl) phenol, 3-2- (2- (benzo [b] thiophen-3-yl) -9-isopropyl-9H-purin-6-ylamino) ethyl) -1H-indol-5-yl 6- (5- ( (3aS, 4S, 6aR) -2-oxohexahydro-1H-thienol {3, 4-d} imidazol-4-yl) pentanamido) hexonoate, and 3-2- (2- (benzo [b] thiophen-3-yl) -9-isopropyl-9H-purin-6-ylamino) ethyl) -1H-indol1-5-yl 6- (tert-butoxycarbonylamino) hexonoate and any combination thereof.
The method of any of claims 1-8, wherein the aryl hydrocarbon receptor antagonist is in an amount of about 0.1 -5 μM.
The method of any of claims 1-9, wherein the HIF-1 modulator is an HIF-1 activator such as a prolyl hydroxylase inhibitor (PHI) , preferably the PHI being selected from a group consisting of FG-4592, FG-2216, FG-4539, N- ( (l-chloro-4-hydroxy-isoquinoline-3-carbonyl) -amino) -acetic acid, [ (7-Bromo-4-hydroxy-isoquinoline-3-carbonyl) -amino] -acetic acid, [ (1-Chloro-4-hydroxy-7-methoxy-isoquinoline-3-carbonyl) -amino] -acetic acid, (7-Chloro-3-hydroxy-quinoline-2-carbonyl) -amino] -acetic acid, [ (3-Hydroxy-6-isopropoxy-quinoline-2-carbonyl) -amino] -acetic acid, and [ (4-Hydroxy-7-phenylsulfanyl-isoquinoline-3-carbonyl) -amino} -acetic acid, and dimethyloxalylglycine (DMOG) , and any combination thereof.
The method of any of claims 1-10, wherein the HIF-1 modulator is in an amount of about 10 -500 nM.
The method of any of claims 1-11, wherein the PPAR alpha agonist is selected from a group consisting of Fenofibrate, Clofibrate, Bezafibrate, Clinofibrate, Ciprofibrate, Etofibrate, Gemfibrate Gemfibrozil) , Pemabate ( (R) -2- {3- { [N- (benzoxazol-2-yl) -N-3- (4-methoxyphenoxy) propyl] aminomethyl} phenoxy} butyric acid) , GW-7647 (2- [ [4- [2- [ [ (cyclohexylamino) carbonyl] (4-cyclohexylbutyl) amino] ethyl] phenyl] thio] -2-methylpropanoic acid) , WY-14643 (pilinic acid) , AM3102 (CAS number: 213182-22-0) , CAY15073 (CAS number: 853652-40-1 ) , CP775146 (CAS Number: 702680-17-9) , GW9578 (CAS Number: 247923-29-1) , GW590735 (CAS Number: 622402-22-6) , Oleylethanolamide, Palmitoylethanolamide, Tesaglitazar ( (S) -2-ethoxy-3- [4- [4- [ (methylsulfonyl) oxy] phenethoxy] phenyl) propionic acid) , LY518674 (2- [4- [3- [2, 5-Dihydro-1- [ (4-methylphenyl) methyl] -5-oxo-1H-1, 2, 4-triazol-3-yl] propyl] phenoxy] -2-methyl-propionic acid) , and any combination thereof.
The method of any of claims 1-12, wherein the PPAR alpha agonist is in an amount of about 0.01 –50 μM.
The method of any of claims 1-13, wherein the first medium further comprises transferrin, insulin, IL-3, EPO, and SCF.
The method of claim 14, wherein the first medium comprises about 100-600 μg/mL transferrin, about 5-20 μg/mL insulin, about 0.5-7 ng/mL IL-3, about 1-7 U/mL EPO, and about 5-120 ng/mL SCF.
The method of any of claims 1-15, wherein step (b) comprises culturing the population of Lin ^-CD34 ^-hemopoietic progenitor cells for about 7-13 days such as about 9-11 days.
The method of any of claims 1-16, wherein the culturing in step (b) results in the proliferation and/or differentiation of the population of Lin ^-CD34 ^-hemopoietic progenitor cells.
The method of any of claims 1-17, wherein the second medium is a second differentiation medium.
The method of any of claims 1-18, wherein the vitamin is selected from a group of vitamin C, vitamin E, derivatives or analogues of vitamin C or E, and any combination thereof.
The method of any of claims 1-19, wherein the second medium comprises vitamin in an amount of about 1-100 μM.
The method of any of claims 1-20, wherein the second medium comprises vitamin C and/or derivatives or analogues thereof in an amount of about 1-50 μM.
The method of any of claims 1-21, wherein the second medium comprises vitamin E and/or derivatives or analogues thereof in an amount of about 1-50 μM.
The method of any of claims 1-22, wherein the methylxanthine compound is selected from a group consisting of caffeine, 3-isobutyl-1-methylxanthine (IBMX) , theophylline and theobromine, preferably in an amount of about 1-50 μM.
The method of any of claims 1-23, wherein the thyroid hormone receptor agonist is a thyroid hormone receptor α or β agonist, preferably a thyroid hormone receptor β agonist.
The method of any of claims 1-24, wherein the thyroid hormone receptor agonist is a thyroxine (T4) , tri-iodothyronine (T3) and their analogues, preferably thyroxine and its analogue, preferably in an amount of about 0.1 -10 μM.
The method of any of claims 1-25, wherein the second medium comprises a combination of vitamin C, vitamin E, caffeine and thyroxine.
The method of any of claims 1-26, wherein the second medium further comprises transferrin, insulin, and EPO, preferably further comprises about 100-600 μg/mL transferrin, about 5-20 μg/mL insulin, and about 0.5-5 U/mL EPO.
The method of any of claims 1-27, wherein step (c) comprises culturing the population of cells obtained from (b) for about 5-11 days such as about 7-9 days.
The method of any of claims 1-28, wherein the population of Lin ^-CD34 ^-hemopoietic progenitor cells is isolated from a blood sample, such as a peripheral blood sample, a cord blood sample or from bone marrow.
The method of claim 29, wherein the blood sample is a human peripheral blood sample.
The method of any of claims 28-30, wherein the population of Lin ^-CD34 ^-hemopoietic progenitor cells is obtained by isolating peripheral blood mononuclear cells (PBMCs) from the blood sample and removing lineage positive (Lin ⁺) cells from the PBMCs.
A method of culturing Lin ^-CD34 ^-hemopoietic progenitor cells, comprising culturing a population of Lin ^-CD34 ^-hemopoietic progenitor cells in a medium comprising one or more additives selected from a group consisting of an aryl hydrocarbon receptor antagonist, a HIF-1 modulator, a PPAR alpha agonist, and any combination thereof, preferably a combination of an aryl hydrocarbon receptor antagonist, a HIF-1 modulator and a PPAR alpha agonist.
The method of claim 32, wherein the aryl hydrocarbon receptor antagonist is selected from a group consisting of SR1, 4- (2- (Pyridin-3-yl) -9-isopropyl-9H-purin-6-ylamino) ethyl) phenol, 4- (2- (9-Isopropyl-2- (2-methyl-1H-imidazol-1-yl) -9H-purin-6-ylamino) ethyl) phenol, 4- (2- (2- (5-Chloropyridine-3-yl) -9-isopropyl-9H-purin-6-ylamino) ethyl) phenol, 4- (2- (6- (5-Fluoropyridin-3-yl) -1-isopropyl-1H-pyrazolo [3, 4-d] pyrimidin-4-ylamino) ethyl) pheno1, 4- (2- (2- (5-Fluoropyridin-3-yl) -7-isopropyl-7H-pyrrolo [2, 3-d] pyrimidin-4-ylamino) ethyl) phenol, (R) -4-2- (2- (benzo [b] thiophen-3-yl) -9-tetrahydrofuran-3-yl) -9H-purin-6-ylamino) ethyl) phenol, 2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3-yl) -9H-purin-6-ylamino) ethyl) phenol1, (R) -2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3-yl) -9H-purin-9-yl) propan-1-ol, (S) -2- (6- (2- (1H-indol-3-yl) ethylamino) -2- (5-fluoropyridin-3-yl) -9H-purin-9-yl) propan-1-ol, 4- (2- (6- (5-Fluoropyridin-3-yl) -1-isopropyl-1H-imidazol [4, 5-c] pyridin-4-ylamino) ethyl) phenol, 4- (2- (5- (5-Fluoropyridin-3-yl) -3-isopropyl-3H-imidazo [4, 5-c] pyridin-7-ylamino) ethyl) phenol, 3-2- (2- (benzo [b] thiophen-3-yl) -9-isopropyl-9H-purin-6-ylamino) ethyl) -1H-indol-5-yl 6- (5- ( (3aS, 4S, 6aR) -2-oxohexahydro-1H-thienol {3, 4-d} imidazol-4-yl) pentanamido) hexonoate, and 3-2- (2- (benzo [b] thiophen-3-yl) -9-isopropyl-9H-purin-6-ylamino) ethyl) -1H-indol1-5-yl 6- (tert-butoxycarbonylamino) hexonoate and any combination thereof.
The method of claim 32 or 33, wherein the aryl hydrocarbon receptor antagonist is in an amount of about 0.1 -5 μM.
The method of any of claims 32-34, wherein the HIF-1 modulator is a HIF-1 activator such as a prolyl hydroxylase inhibitor (PHI) , preferably the PHI being selected from a group consisting of FG-4592, FG-2216, FG-4539, N- ( (l-chloro-4-hydroxy-isoquinoline-3-carbonyl) -amino) -acetic acid, [ (7-Bromo-4-hydroxy-isoquinoline-3-carbonyl) -amino] -acetic acid, [ (1-Chloro-4-hydroxy-7-methoxy-isoquinoline-3-carbonyl) -amino] -acetic acid, (7-Chloro-3-hydroxy-quinoline-2-carbonyl) -amino] -acetic acid, [ (3-Hydroxy-6-isopropoxy-quinoline-2-carbonyl) -amino] -acetic acid, and [ (4-Hydroxy-7-phenylsulfanyl-isoquinoline-3- carbonyl) -amino} -acetic acid, and dimethyloxalylglycine (DMOG) , and any combination thereof.
The method of any of claims 32-35, wherein the HIF-1 modulator is in an amount of about 10 -500 nM.
The method of any of claims 32-36, wherein the PPAR alpha agonist is selected from a group consisting of Fenofibrate, Clofibrate, Bezafibrate, Clinofibrate, Ciprofibrate, Etofibrate, Gemfibrate Gemfibrozil) , Pemabate ( (R) -2- {3- { [N- (benzoxazol-2-yl) -N-3- (4-methoxyphenoxy) propyl] aminomethyl} phenoxy} butyric acid) , GW-7647 (2- [ [4- [2- [ [ (cyclohexylamino) carbonyl] (4-cyclohexylbutyl) amino] ethyl] phenyl] thio] -2-methylpropanoic acid) , WY-14643 (pilinic acid) , AM3102 (CAS number: 213182-22-0) , CAY15073 (CAS number: 853652-40-1 ) , CP775146 (CAS Number: 702680-17-9) , GW9578 (CAS Number: 247923-29-1) , GW590735 (CAS Number: 622402-22-6) , Oleylethanolamide, Palmitoylethanolamide, Tesaglitazar ( (S) -2-ethoxy-3- [4- [4- [ (methylsulfonyl) oxy] phenethoxy] phenyl) propionic acid) , LY518674 (2- [4- [3- [2, 5-Dihydro-1- [ (4-methylphenyl) methyl] -5-oxo-1H-1, 2, 4-triazol-3-yl] propyl] phenoxy] -2-methyl-propionic acid) , and any combination thereof.
The method of any of claims 32-37, wherein the PPAR alpha agonist is in an amount of about 0.01 –50 μM.
The method of any of claims 32-38, wherein the medium is a differentiation medium.
The method of any of claims 32-39, wherein the medium further comprises transferrin, insulin, IL-3, EPO, and SCF.
The method of any of claims 32-40, wherein the differentiation medium comprises about 100-600 μg/mL transferrin, about 5-20 μg/mL insulin, about 0.5-7 ng/mL IL-3, about 1-7 U/mL EPO, and about 5-120 ng/mL SCF.
The method of any of claims 32-41, wherein the method comprises culturing the population of Lin ^-CD34 ^-hemopoietic progenitor cells for about 7-13 days such as about 9-11 days.
The method of any of claims 32-42, wherein the culturing results in the proliferation and/or differentiation of the population of Lin ^-CD34 ^-hemopoietic progenitor cells.
A method of producing red blood cells, comprising: culturing a population of nucleated red blood cell precursors in a medium comprising one or more additives selected from a group consisting of a vitamin, a methylxanthine compound, a thyroid hormone receptor agonist, and any combination thereof, preferably a combination of a vitamin, a methylxanthine compound, and a thyroid hormone receptor agonist, thereby producing red blood cells.
The method of claim 44, wherein the population of nucleated red blood cell precursors is at a terminal differentiation stage.
The method of claim 44 or 45, wherein the nucleated red blood cell precursors are reticulocytes.
The method of any of claims 44-46, wherein the nucleated red blood cell precursors are obtained from a population of Lin ^-CD34 ^-hemopoietic progenitor cells.
The method of any of claims 44-47, wherein the medium is a differentiation medium.
The method of any of claims 44-48, wherein the vitamin is selected from a group of vitamin C, vitamin E, derivatives or analogues of vitamin C or E, and any combination thereof.
The method of any of claims 44-49, wherein the medium comprises vitamin in an amount of about 1-100 μM.
The method of any of claims 44-50, wherein the medium comprises vitamin C and/or derivatives or analogues thereof in an amount of about 1-50 μM.
The method of any of claims 44-51, wherein the medium comprises vitamin E and/or derivatives or analogues thereof in an amount of about 1-50 μM.
The method of any of claims 44-52, wherein the methylxanthine compound is selected from a group consisting of caffeine, 3-isobutyl-1-methylxanthine (IBMX) , theophylline and theobromine, preferably in an amount of about 1-50 μM.
The method of any of claims 44-53, wherein the thyroid hormone receptor agonist is a thyroid hormone receptor α or β agonist, preferably a thyroid hormone receptor β agonist.
The method of any of claims 44-54, wherein the thyroid hormone receptor agonist is thyroxine (T4) , tri-iodothyronine (T3) and their analogues, preferably thyroxine and its analogue, preferably in an amount of about 0.1 -10 μM.
The method of any of claims 44-55, wherein the medium comprises a combination of vitamin C, vitamin E, caffeine and thyroxine.
The method of any of claims 44-56, wherein the medium further comprises transferrin, insulin, and EPO, preferably further comprises about 100-600 μg/mL transferrin, about 5-20 μg/mL insulin, and about 0.5-5 U/mL EPO.
The method of any of claims 44-57, wherein the nucleated red blood cell precursors are cultured for about 5-11 days such as about 7-9 days.
A population of cells obtained by the method of any of claims 1-58.
A medium supplement composition comprising one or more additives selected from a group consisting of an aryl hydrocarbon receptor antagonist, a HIF-1 modulator, a PPAR alpha agonist, and any combination thereof, preferably a combination of an aryl hydrocarbon receptor antagonist, a HIF-1 modulator and a PPAR alpha agonist.
The medium supplement composition of claim 60, further comprising transferrin, insulin, IL-3, EPO, and SCF.
A medium supplement composition comprising a vitamin, a methylxanthine compound, and a thyroid hormone receptor agonist.
The medium supplement composition of claim 62, further comprising transferrin, insulin and EPO.
A method for producing engineered red blood cells, comprising the steps of:

(a) providing a population of Lin ^-CD34 ^-hemopoietic progenitor cells;

(b) culturing the population of Lin ^-CD34 ^-hemopoietic progenitor cells from (a) in a first medium comprising one or more additives selected from a group consisting of an aryl hydrocarbon receptor antagonist, a HIF-1 modulator, a PPAR alpha agonist, and any combination thereof, preferably a combination of an aryl hydrocarbon receptor antagonist, a HIF-1 modulator and a PPAR alpha agonist, to induce them to differentiate into erythroid cells, and prior to or concurrently with the differentiation, introducing one or more exogenous nucleic acids into the erythroid cells; and

(c) culturing the erythroid cells to induce enucleation, thereby producing the engineered red blood cells.
The method of claim 64, wherein step (c) comprises culturing the cells obtained from (b) in a second medium comprising one or more additives selected from a group consisting of a vitamin, a methylxanthine compound, a thyroid hormone receptor agonist, and any combination thereof, preferably a combination of a vitamin, a methylxanthine compound, and a thyroid hormone receptor agonist, thereby producing the engineered red blood cells.