WO2021083278A1

WO2021083278A1 - Engineering red blood cells for treating gout and hyperuricemia diseases

Info

Publication number: WO2021083278A1
Application number: PCT/CN2020/124849
Authority: WO
Inventors: Xiaofei GAO; Xiaoqian NIE; Yanjie HUANG; Hui Li
Original assignee: Westlake Therapeutics (Hangzhou) Co. Limited
Priority date: 2019-10-29
Filing date: 2020-10-29
Publication date: 2021-05-06
Also published as: CN114746546A

Abstract

Provided are engineered red blood cells comprising a urate oxidase (UOX) or a functional variant thereof; a urate transporter 1 (URAT1) or a functional variant thereof; or a UOX or a functional variant thereof and a URAT1 or a functional variant thereof. Provided are the uses of the engineered red blood cells in treating hyperuricemia associated diseases.

Description

Engineering Red Blood Cells for Treating Gout and Hyperuricemia Diseases

FIELD OF THE INVENTION

The present disclosure relates to engineering red blood cells, and in particular, to engineering red blood cells carrying URAT1 and/or UOX. The present disclosure also relates to methods for treating hyperuricemia associated diseases.

BACKGROUND

Gout is the most common form of inflammatory arthritis in adults, especially in men, with a prevalence ranging from 1%to 4%globally [1, 2] . Gout occurs when monosodium urate crystal (MSU) deposited in tissues, causing inflammation and intense pain of a gout attack. The biologic precursor to gout is elevated serum uric acid (UA) levels (i.e., hyperuricemia) . Although hyperuricemia is the strongest single risk factor for the development of gout and is universally present in patients with gout, not all individuals with hyperuricemia develop gout. Recent work has emphasized the importance of the innate immune response [2] .

Conventional urate-lowering agents such as anti-inflammatory drugs (colchicine) , xanthine oxidase inhibitors (allopurinol, febuxostat) or uricosuric agents (probenecid, benzbromarone) induce very slow reduction in UA deposits, not allowing for the rapid resolution of tophi for all patients with gout and are mainly used at the early stage [3] .

Urate oxidase (UOX, uricase) is a liver enzyme that metabolizes UA into allantoin, a more water-soluble compound, which is easily excreted by the kidney [2, 3] . All mammals produce UOX, except humans and certain primates. Indeed, during evolution, UOX was inactivated in humans primarily due to missense and frame-shift mutations in the gene encoding this enzyme [2, 4] . Uricase undeniably represents a valuable treatment option for chronic tophaceous gout when conventional urate-lowering agents may not be used.

Rasburicase, a recombinant UOX from A. flavus, was approved by EMEA in 2001

and the Food and Drug Administration (FDA) in 2002

for tumor lysis syndrome [5, 6] . This agent significantly reduces serum UA levels and acts faster than allopurinol. The recommended dose is 0.2 mg/kg in children and adults [7, 8] . However, its biological half-life is short---only 21h, so rasburicase is given by infusion once daily for ≤ 7 days [7] . In addition, recent studies have shown that repeated UOX injections could cause anaphylactic reactions with the production of antibodies that neutralize UOX enzyme activity [9] .

Pegloticase, a recombinant porcine UOX, with a baboon C-terminal sequence, is a modified pegylated recombinant UOX developed to be the first non-immunogenic biologic for treating the hyperuricemia of refractory gout, approved by the FDA for patients with chronic gout [3, 10] . A 6-month study versus placebo showed that pegloticase (infused at 8 mg every 2 weeks) , induced a significant decrease in plasma UA in about 40%of the patients (associated with a tendency for tophi dissolution) [3] . However, the remaining patients had no response, which was correlated with the formation of pegloticase antibodies and infusion reactions [3] . More than 10%of the patients treated with pegloticase had adverse events such as kidney stones, joint pain, anaemia, muscle spasms, dyspnea, headache, nausea, and fever [7] .

Available recombinant UOX (rasburicase, pegloticase) drugs are potent hypouricemic agents for gout. However, there are several limitations for the current therapy. First, UOX has significant immunogenicity and it may induce severe allergic reactions [11] . Conjugating therapeutic enzymes to PEG may reduce immune responses in patients. However, studies showed that many patients treated with PEG-conjugated enzymes developed anti-PEG antibodies. Moreover, PEG may adversely affect the activity of the conjugated enzyme, leading to reduced efficacy in the treatment [12, 13] . Second, the therapeutic enzymes may become inactivated or eliminated in vivo due to short half-life, limited bioavailability, and/or interactions with plasma proteins [14] . Third, production and purification of the enzymes tend to be time-consuming, and thus treatments with enzyme replacement therapy are very costly. The cost of treatment (calculated on an annual basis) is estimated to be about € 7200 for rasburicase and € 41, 240 for pegloticase [3] . Therefore, we need new gout therapy that is more efficacious and safer.

Engineered RBCs (eRBCs) are attractive carriers for the introduction of novel therapeutics, and have the following benefits: (1) red blood cells are the most abundant cells in the blood, occupying a quarter of all human cells, and are widely distributed to the human body through circulation; (2) red blood cells have a long life span (about 120 days for humans and 50 days for mice) . Furthermore, old or damaged red blood cells can be cleared by macrophages in the liver and spleen without causing immune responses; (3) red blood cells are biocompatible, especially when using autologous red blood cells; (4) mature red blood cells have no nuclei, mitochondria, or other organelles. Thus, any modification made to the DNA of RBC precursors is eliminated upon their enucleation and cannot lead to abnormal growth or tumorigenicity after their transfusion into a recipient; and (5) red blood cells protect the drug substance from premature inactivation and/or degradation, as well as the organism from the toxic effects of the drug [15-17] .

A major regulator of serum urate is renal excretion of UA. In humans, net reabsorption of UA into the blood predominates owing to less excretion of UA than is filtered at the glomerulus. URAT1, a urate–anion exchanger, can transport UA from the urinary lumen, which is an important step for UA reabsorption [2, 18] .

A significant reduction in immunogenicity, the prolonging half-life of UOX and rapid resolution of tophi are the key objectives in the development of new gout therapy.

SUMMARY

In one aspect, the present disclosure provides an engineered red blood cell (eRBC) comprising:

a urate oxidase (UOX) or a functional variant thereof;.

a urate transporter 1 (URAT1) or a functional variant thereof; or

a UOX or a functional variant thereof and a URAT1 or a functional variant thereof.

In some embodiments, the URAT1 comprises an amino acid sequence of SEQ ID NO: 2.

In some embodiments, the UOX comprises an amino acid sequence of SEQ ID NO: 4.

In another aspect, the present disclosure provides a method for producing engineered red blood cells, which comprises:

1) collecting lineage negative cells (Lin-cells) from a blood sample or Lin-CD34+ cells from a bone marrow sample,

2) expanding the Lin-or Lin-CD34+ cells;

3) culturing the expanded Lin-or Lin-CD34+ cells to induce them to differentiate into erythroid cells; and, concurrently with the differentiation, introducing one or more exogenous nucleic acids into the expanded Lin-cells or Lin-CD34+ cells;

4) culturing the erythroid cells to induce enucleation,

wherein 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 embodiments, the UOX comprises an amino acid sequence of SEQ ID NO: 4.

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

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

In some embodiments, the Lin-cells are Lin-CD34-cells.

In some embodiments, the step 1) includes isolating peripheral blood mononuclear cells (PBMCs) from the peripheral blood sample and isolating Lin-CD34-cells from the PBMCs.

In some embodiments, the step 1) includes removing lineage positive (Lin+) cells from the peripheral blood sample by using a lineage cell depletion kit.

In some embodiments, the step 2) including culturing the Lin-cells in a hematopoietic stem cell expansion medium supplemented with a combination of cytokines, and the combination of cytokines comprise fms-like tyrosine kinase 3 ligand (Flt3L) , stem cell factor (SCF) , interleukin 3 (IL-3) , and interleukin 6 (IL-6) .

In some embodiments, the combination of cytokines comprise 50 ng/mL human Flt3L, 50 ng/mL human SCF, 10 ng/mL human IL-3, and 10 ng/mL human IL-6.

In some embodiments, the hematopoietic stem cell expansion medium is StemSpan ^TM SFEM serum-free expansion medium.

In some embodiments, the step 2) including culturing the Lin-cells at 37℃ under 5%CO ₂ for about 5 days.

In some embodiments, the step 3) comprising culturing the expanded Lin-cells in a first differentiation medium supplemented with cytokines related to erythroid development.

In some embodiments, the first differentiation medium is an Iscove's Modified Dulbecco's Medium (IMDM) containing: fetal bovine serum (FBS) or human serum, human plasma, glutamine, BSA, transferrin, insulin, Penicillin-Streptomycin, IL-3, EPO, and SCF.

In some embodiments, the first differentiation medium is an Iscove's Modified Dulbecco's Medium (IMDM) containing: 10-15%FBS or human serum, 5-10%human plasma, 1-4 mM glutamine, 1-2%BSA, 300-600 μg/mL human transferrin, 8-13 μg/mL human insulin, 2%Penicillin-Streptomycin, 3-5 ng/mL human IL-3, 4-7 U/mL human EPO, and 100 ng/mL human SCF.

In some embodiments, wherein the step 3) comprising culturing the expanded Lin-CD34-cells at 37℃ under 5%CO ₂ for about 9 days.

In some embodiments, the step 4) comprising culturing the erythroid cells in a second differentiation medium, wherein the second differentiation medium is an Iscove's Modified Dulbecco's Medium (IMDM) containing: FBS or human serum, human plasma, glutamine, BSA, transferrin, insulin, Penicillin-Streptomycin, and EPO.

In some embodiments, the second differentiation medium is an Iscove's Modified Dulbecco's Medium (IMDM) containing: 15%FBS or human serum, 5-10%human plasma, 1-4 mM glutamine, 1-2%BSA, 300-600 μg/mL human transferrin, 8-13 μg/mL human insulin, 2%Penicillin-Streptomycin, and 1-5 U/mL human EPO.

In some embodiments, the step 4) comprising culturing erythroid cells at 37℃ under 5%CO ₂ for about 7 days.

In some embodiments, in the step 3) the introduction of the one or more exogenous nucleic acids into the expanded Lin-cells is carried out on the 1st day of culturing the expanded Lin-cells in the first differentiation medium.

In some embodiments, the exogenous nucleic acid is an expression vector.

In some embodiments, the exogenous nucleic acid is a lentiviral expression vector.

In another aspect, the present disclosure provides a pharmaceutical composition comprising the engineered red blood cells and a physiologically acceptable excipient.

In another aspect, the present disclosure provides uses of the engineered red blood cells in the preparation of a medicament for treating a disorder associated with elevated serum uric acid levels.

In some embodiments, the disorder is gout or a hyperuricemia disease.

In another aspect, the present disclosure provides a method for the treatment of a disorder associated with elevated serum uric acid levels in a subject, which comprises:

a) taking a blood sample or a bone marrow sample from the subject,

b) producing engineered red blood cells by using the method described above; and

c) infusing a therapeutically effective amount of the engineered red blood cells into the subject.

In some embodiments, the disorder is gout or a hyperuricemia disease

In another aspect, the present disclosure provides a method for the treatment of a disorder associated with elevated serum uric acid levels in a subject, which comprises infusing a therapeutically effective amount of the engineered red blood cell 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.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows the RBC engineering strategy for gout and hyperuricemia therapy.

Fig. 2 depicts experimental scheme of engineering hematopoietic stem and/or progenitor cells to express URAT1 and UOX, and subsequently differentiating the engineered hematopoietic stem and/or progenitor cells in vitro into mature red blood cells carrying both functional proteins.

Fig. 3 depicts representative analysis that can be used to test the functions of engineered RBCs (eRBCs) expressing URAT1 and UOX in vitro, as well as their functions in vivo using established animal models for gout.

Fig. 4 shows a workflow of eRBC generation from mouse embryos. Mouse erythroid progenitors were isolated from fetal livers at embryonic 14.5 days. Cells were then infected with MSCV-URAT1/UOX-copGFP before induced into terminally differentiated mouse red cells expressing URAT1 or UTRA1 and UOX.

Fig. 5 shows that eRBCs expressing URAT1 and/or UOX have undergone normal erythroid differentiation. Here shows an example of FACS analysis of eRBCs generated from Lin-PBMCs of a gout patient at day 8 of the in vitro differentiation. The following are the markers used in the FACS analyses: CD235a, CD117, CD71, Hoechst33342, and GFP (the reporter gene indicating expression of URAT1 and/or UOX) . CD235a, CD117 and CD71 are used to track the progression of erythroid development. Hoechst33342 stains DNA, and therefore, Hoechst 33342 negative cells are enucleated cells. The channels used to detect corresponding signals: Y585-PE: PI; B525-FITC: GFP; R660-APC: CD235a; B650-PC5.5: CD71; UV450: Hoechst33342. (A) . eRBC-URAT1 at day 8 of the in vitro erythroid differentiation; (B) . eRBC-URAT1+UOX at day 8 of the in vitro erythroid differentiation.

Fig. 6 Both eRBC-URAT1 and eRBC-URAT1+UOX generated from Lin-PBMCs of the gout patients reduced the UA concentration in vitro efficiently. (A) . UA concentration of the medium in which eRBCs were cultured. 10 ⁵ eRBCs, eRBC-URAT1 or eRBC-URAT1+UOX were cultured in the medium with 465 μM UA (mimicking UA concentration of a gout patient) for 24 hours. After the incubation, the UA concentration in the medium was determined by a coupled enzyme reaction (Abcam) . (B) . The consumption rates of UA by eRBC-URAT1 and eRBC-UOX+URAT1 were calculated based on the results from Fig. 6A.

FIG. 7 shows tissue distribution of engineered RBCs in NSG mouse by live imaging. DIR-labeled RBCs were injected into NSG mouse via intravenous injection and the subsequent distributions of the labeled red blood cells were examined by in vivo imaging at 1 h, 5 h, 48 h, and 5 days after the injection of red blood cells. Mouse #1: no treatment ; Mouse #2: 2 μM DIR only in PBS; Mouse #3: 1x10 ⁸ human red blood cells differentiated from PBMCs in vitro and labeled with 2 μM DIR; Mouse #4: 1x10 ⁸ human red blood cells infected with vector only differentiated from PBMCs in vitro and labeled with 2 μM DIR; Mouse #5: 1x10 ⁸ human red blood cells transducing with lentiviral vectors encoding phenylalanineammonialyase (PAL) and differentiated from PBMCs in vitro and labeled with 2 μM DIR; Mouse #6: 5x10 ⁷ human red blood cells transducing with lentiviral vectors encoding glyoxylate and hydroxypyruvate reductase (GRHPR) and differentiated from PBMCs in vitro and labeled with 2 μM DIR.

FIG. 8 shows an example of images of tissue samples after the injection of red blood cells. DIR-stained red blood cells were intravenously injected into each mouse. Seven days after the injection, the mice were sacrificed, and the tissue samples were imaged. Mouse #1: no treatment ; Mouse #2: 2 μM DIR only in PBS; Mouse #4: 1x10 ⁸ human red blood cells infected with vector only differentiated from PBMCs in vitro and labeled with 2 μM DIR; Mouse #5: 1x10 ⁸ human red blood cells transducing with lentiviral vectors encoding phenylalanineammonialyase (PAL) and differentiated from PBMCs in vitro and labeled with 2 μM DIR; Mouse #6: 5x10 ⁷ human red blood cells transducing with lentiviral vectors encoding glyoxylate and hydroxypyruvate reductase (GRHPR) and differentiated from PBMCs in vitro and labeled with 2 μM DIR.

Fig. 9 shows that engineered mouse eRBC-URAT1+UOX reduced UA concentration in a hyperuricemia and gout mouse model. A representative functional assay of mouse eRBC-URAT1 and eRBC-UOX+URAT1 in vivo. (A) . Hyperuricemia was induced by intraperitoneal injection of MSU crystal suspension at the dose of 250 mg/kg, and the serum UA concentration was analyzed by a coupled enzyme reaction; (B) . Hyperuricemia was induced 3 days after 2x10 ⁸ eRBC-URAT1 or eRBC-URAT1+UOX cells or PBS (Blank) were intravenously injected into the mice. UA concentration in the serum was measured to assess the therapeutic effects of the eRBCs.

DETAILED DESCRIPTION

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art. Any methods, devices and materials similar or equivalent to those described herein can be used in the practice of this invention. The following definitions are provided to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element" means one element or more than one element.

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 CD1c, CD3, CD11c, CD14, CD15, CD16, CD20, CD41, CD56, CD203c, CD235a, and/or BDCA2. 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-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 CD1c, CD3, CD11c, CD14, CD15, CD16, CD20, CD41, CD56, CD203c, CD235a, and/or BDCA2. In some cases, 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.

The term “functional variant” means any variant comprising substitution, deletion or addition of one or a few to plural amino acids to UOX or URAT1, provided that the variant substantially retains the same function as UOX or URAT1 possesses.

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 “pharmaceutically acceptable excipient” as used herein refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, carrier, manufacturing aid (e.g., lubricant, talc magnesium) , solvent or encapsulating material, involved in carrying or transporting a therapeutic compound for administration to a subject. Each excipient should be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject.

fms-like tyrosine kinase 3 ligand (Flt3L) or FMS-like tyrosine kinase 3 ligand (Flt-3 ligand) , also known as FL, Flt3L and FLT3LG, is a protein which promotes HSCs differentiation to various hematopoietic lineages. FLT3LG is structurally homologous to stem cell factor (SCF) and colony stimulating factor 1 (CSF-1) . FLT3LG increases the number of cells by activating hematopoietic progenitor cells.

Stem cell factor (SCF) or Kit ligand (KITLG) , also known as stem cell factor (SCF) , belongs to the SCF family of type I transmembrane glycoproteins. KITLG is a ligand for the receptor-type protein tyrosine kinase KIT. SCF plays an important role in regulating cell survival and proliferation, hematopoiesis, stem cell maintenance, cell development, migration and function.

Interleukin 3 (IL-3) is a glycoprotein belonging to the hematopoietic growth factor family, which exhibits multi-lineage activity in preclinical in vitro and in vivo studies. Hematopoietic progenitor cells proliferate and differentiate into mature red blood cells, mast cells, megakaryocytes and granulocytes with the help of IL-3 protein.

Interleukin-6 (IL-6) is a multifunctional cytokine that regulates immune response, hematopoietic function, acute phase response and inflammatory response. Synergistically promotes hematopoietic cell proliferation with IL-3.

Erythropoietin (Epo) is a major erythropoietin that interacts with various other growth factors (IL-3, IL-6, glucocorticoids, and SCF) that develop erythroid lineages from pluripotent progenitor cells. Explosive forming unit-erythrocyte (BFU-E) cells begin to express erythropoietin receptors and are sensitive to erythropoietin. It is an important erythroid hematopoietic cytokine.

Holo human transferrin is a major ferritin in plasma, which forms a complex with iron ions for hemoglobin production in red blood cells.

CD235a, also known as blood glycoprotein A, is a single transmembrane glycoprotein dominantly expressed on the surface of both mature red blood cells and erythroid precursor cells, is a lineage specific marker for red blood cells.

CD117 is mast cell/stem cell growth factor receptor, expressed on the surface of hematopoietic stem cells and other cells.

CD71, also known as Transferrin receptor 1, is a transmembrane glycoprotein composed of two disulfide-bonded monomers linked by two disulfide bonds. Each monomer binds to a full transferrin molecule to produce an iron-transferrin-transferrin receptor complex that enters the cell by endocytosis.

Propidium Iodide (PI) binds to double-stranded DNA. PI cannot cross intact plasma membrane and therefore will only be present in DNA of cells where the plasma membrane has been compromised/permeabilized.

Hoechst33342 is a fluorescent dye used for DNA staining of living cells.

copGFP is a green fluorescent protein, which is a commonly used fluorescent reporter.

We have established an in vitro RBC regeneration culture technology, which can increase proliferation of erythroid progenitors and enable efficient and stable production of enucleated RBCs. Normal human RBCs can be produced in culture from hematopoietic stem and progenitor cells, but the source of hematopoietic stem cells limits the clinical application, such as bone marrow (BM) aspirates, mobilized peripheral blood, and umbilical cord blood (UCB) . We generate a series of novel methods using lineage negative CD34 negative peripheral blood mononuclear cells (Lin-CD34-PBMCs) or lineage negative CD34 positive hematopoietic stem cells (Lin-CD34+ HSCs) to induce erythroid cell proliferation and differentiation, which lays a strong foundation for the eRBC therapy.

We use RBCs as new carriers to deliver UOX to the patient, via designing eRBCs to express both URAT1 and UOX, which can increase UA consumption rate and further improve the consumption efficiency of UA (Fig. 1 and Fig. 2) . A series of experiments have been carried out to confirm the in vitro and in vivo functions of eRBCs carrying URAT1 or carrying both URAT1 and UOX (Fig. 3) .

EXAMPLES

Example 1. Lentivirus preparation

Design of MSCV-URAT1-copGFP and MSCV-UOX-copGFP plasmids: the cDNA sequences of URAT1 (Genbank, AY358183.1) and UOX (Genbank, X61765.1) are listed as followings. The synthesized DNA of the genes were cloned in the lentiviral vector MSCV.

The DNA sequence of URAT1: (SEQ ID NO: 1)

The amino acid sequence of URAT1: (SEQ ID NO: 2)

The DNA sequence of UOX: (SEQ ID NO: 3)

The amino acid sequence of UOX: (SEQ ID NO: 4)

The lentivirus expressing URAT1 and UOX genes was produced by using MSCV-URAT1-copGFP (or MSCV-UOX-copGFP) , pSPAX2, VSVG at the ratio of 2: 1: 1. Using 6-well plate as an example, 2 μg MSCV-URAT1-copGFP (or MSCV-UOX-copGFP) , 1 μg pSPAX2 and 1 μg VSVG were transfected by using calcium phosphate into HEK 293T cells for generating virus. 12 hours post transfection, calcium phosphate was replaced by fresh DMEM medium containing 10%FBS. The supernatant was collected at 48 hours and 72 hours post transfection and were filtered through a 0.45 μm filters.

The filtered supernatants are subjected to ultracentrifugation and concentrated at a speed of 70000 RCF for 2 hours at a temperature of 4℃. After centrifugation, the supernatant was removed and pellets containing viral particles were resuspended using differentiation stage 1 medium (described herein blow) . The virus titer was quantified by using p24 ELISA kit, and stored at -80℃.

Example 2. Human red blood cells engineered to express URAT1 and UOX

Mature red blood cells are differentiated from early erythroid progenitor cells derived from hematopoietic stem cells. However, due to the difficulty in obtaining hematopoietic stem cells from human bone marrows, we have developed an in vitro human erythroid differentiation culture system by using Lin-CD34+ HSCs or Lin-CD34-peripheral blood mononuclear cells (PBMCs) to generate mature human red cells. The detailed steps are as follows.

1) The peripheral blood from gout patients or healthy subjects with matched blood types or umbilical cord blood was diluted 1: 1 with phosphate buffer, and PBMCs or perenrichment HSCs were isolated by centrifugation at 1200 x g for 15 minutes, using a lymphocyte separation solution (LymphoprepTM, STEMCELL Technologies) .

2) Lin-CD34-PBMCs from peripheral blood were isolated by using a Human Lineage Cell Depletion Set-DM kit (BD Biosciences) ; Lin-CD34+ HSCs from cord blood samples were isolated by using EasySep ^TM Human Cord Blood CD34 Positive Selection Kit II (Stemcell Technologies) .

3) The isolated Lin-CD34-PBMCs or Lin-CD34+ HSCs cells were cultured in hematopoietic stem cell expansion medium (StemSpanTM SFEM, STEMCELL Technologies) supplemented with penicillin-streptomycin (Gibco) and a combination of cytokines containing 50 ng/ml recombinant human fms-like tyrosine kinase 3 ligand (Flt3L) , 50 ng/ml recombinant human stem cell factor (SCF) , 10 ng/ml recombinant human interleukin 3 (IL-3) , 10 ng/ml recombinant human interleukin 6 (IL-6) or StemSpanTM CC100 (STEMCELL Technologies) . Cells were cultured at 37℃ under 5%CO ₂ .

4) At day 5 during the culture, the cultured cells were switched to the differentiation stage 1 medium consisting of IMDM (Iscove's Modified Dulbecco's Medium, Sigma-Aldrich) , 10-15%fetal bovine serum (FBS, Gibco) or human serum, 5-10%human plasma, 1-4 mM glutamine, 1-2%BSA (Albumin from bovine serum) , 300-600 μg/mL human transferrin (holo human transferrin, Sigma -Aldrich) , 8-13 μg/mL recombinant human insulin (Sigma-Aldrich) , 2%Penicillin-Streptomycin (Gibco) , 3-5 ng/mL recombinant human interleukin III (rhIL) -3, Peprotech) , 4-7 U/mL recombinant human erythropoietin (rhEpo, Amgen) , 100 ng/mL recombinant human stem cell factor (rhSCF, Peprotech) . Cells were cultured_at 37℃ under 5%CO ₂.

5) At day 6 during the culture, cells were infected with lentivirus expressing URAT1, or URAT1 and UOX. Cultured cells were resuspended with the differentiation stage 1 medium at a density of 1 x 10 ⁶ /mL and infected with the virus at the concentration of 5×10 ⁷ TU/ml-5×10 ⁸ TU/ml with polybrene (Merck, 10ng/mL) . Centrifugal infection was performed using a horizontal rotor centrifuge for 90 min at a speed of 500 x g and 32℃.

6) 12 hours post infection, medium was replaced with fresh differentiation stage 2 medium at 37 ℃under 5%CO ₂. The differentiation stage 2 medium was consisting of IMDM (Iscove's Modified Dulbecco's Medium, Sigma-Aldrich) , 15%fetal bovine serum (FBS, Gibco) or 15%autologous serum, 5-10%human plasma (Plasma) , 1-4 mM glutamine, 1-2%BSA (Albumin from bovine serum) , 300-600 μg/mL human transferrin (holo human transferrin, Sigma-Aldrich) , 8-13 μg/mL recombinant human insulin (Sigma-Aldrich) , 2%Penicillin-Streptomycin (Gibco) , 1-5 U/mL recombinant human erythropoietin (rhEpo, Amgen) . Cells were cultured another 7 days, during which the medium was changed with fresh differentiation stage 2 medium every 2 days. The expression of CD235a, CD117, CD71, Hoechst 33342 on cells during the final differentiation in vitro were determined by flow cytometry.

During the culturing, we performed FACS analyses for CD235a, CD117, CD71, Hoechst33342, and GFP (the reporter gene indicating expression of URAT1 and/or UOX) . A typical result was showed in Fig 5.

Example 3. Analysis of URAT1 and UOX expression on engineered RBCs.

Total RNA was isolated from 10 ⁶ eRBC-URAT1+UOX and eRBC-control, using RNeasy mini kit (QIAGEN) according to the manufacturer’s protocol. cDNA was synthesized using a PrimeScript RT Reagent Kit with gDNA Eraser (Takara) with oligo (dT) primers. qRT-PCR was performed using Terra ^TM qPCR Direct TB Green (Takara) . The following are the primer sequences used for qRT-PCR:

URAT1 forward primer: 5’-TCCAGACGGTGTACGAGATG-3’ (SEQ ID NO: 5)

URAT1 reverse primer: 5’-GGATGTCCACGACACCAATGA-3’ (SEQ ID NO: 6)

UOX forward primer: 5’-CGGTGTCCAGACGGTGTACG-3’ (SEQ ID NO: 7)

URAT1 reverse primer: 5’–GGTCCAGCGGTGGCAGACAAT-3’ (SEQ ID NO: 8)

18s forward primer: 5’-GCTTAATTTGACTCAACACGGGA-3’ (SEQ ID NO: 9)

18s reverse primer: 5’-AGCTATCAATCTGTCAATCCTGTC-3’ (SEQ ID NO: 10)

Example 4. Analysis of function of eRBC-URAT1 or URAT1+UOX in vitro

eRBCs were incubated with serum from gout patients. The content of UA in the patient serum was measured at different time points to determine the consumption rates of UA by the engineered red blood cells in vitro. The results were showed in Fig. 6.

Example 5. Tissue distribution and the lifespan of human eRBCs in NSG mice

DIR labelled eRBCs were injected into the immune-deficient NSG mice through intravenous injection, and tissue distribution and survival of the labelled eRBCs cells in the mouse was analysed by live imaging, flow cytometry, immunohistochemistry, etc. Some results were showed in Fig. 7 and Fig. 8.

Example 6. Analysis of the function of eRBC-URAT1 or URAT1+UOX to consume the UA in vivo

Engineering mouse red blood cells to express URAT1 and UOX (Fig. 4)

1. Isolating mouse fetal liver cells (FLC) from embryos at E13.5-14.5 days as described previously [19] .

3. Single-cell suspension solution was collected by pipetting up and down, which were subsequently filtered by 25μm strainers (BD Falcon 35-2235) .

4. Cells were centrifuged at 1500 RPM for 5 min, and re-suspend by the red cell lysis buffer (Ammonium Chloride Solution from Stemcell) for 10 mins at 4℃.

5. Cells were centrifuged again at 1500 RPM for 5 min. The lysis buffer was removed and cells were resuspended with 10 mL PBS-2%FBS.

6. ChromPure Rat IgG (Jackson ImmunoResearch, #012-000-003) at 50 μL/mouse were added and incubated with cells at 4℃ for 5min.

7. Biotinylated anti-mouse TER119 antibodies (BD Pharmingen, #553672) at 1 μL/1*10 ⁶ cells were added to cells and incubated at 4℃ for 15 min.

8. Ms Lineage Panel (Fisher Scientific (Thermo Fisher Scientific) #BDB559971 ) were added to cells at the concentration of 2 μL /1*10 ⁶ cells and incubated at 4C for 15 min.

9. Cells were washed with 10 times volume of PBS and were centrifuged at 1500 RPM for 5 min at 4℃.

10. Streptavidin Particles Plus -DM (magnetic beads) (BD Pharmigen , #557812) (5μL/1*10 ⁶ cells) were added to cells and incubated at 4C for 30min. Afterwards, streptavidin coated magnetic beads were removed by using the magnetic holder.

11. The remaining cells, which were lineage negative mouse cells, were transferred to new tubes.

14. Cells were seeded into a 24-well plate at the concentration of 2*10 ⁵/mL at 37℃. Cells were infected with lentivirus as described previously.

15. After the infection, cells were cultured into the differentiated medium for 44-48 hours for the maturation.

Mouse model of hyperuricemia

The model of hyperuricemia was induced by intraperitoneal injection of 250mg/kg of MSU crystal suspension as described [20] . Blood were collected from tail vein at 0, 2, 5, 8, 24h, analysing their serum UA concentration.

The function of mouse eRBC-URAT1+UOX in hyperuricemia mice

2x10 ⁸ functional mouse eRBCs were intravenously injected into mice. 3 days after injection, these mice were induced hyperuricemia by intraperitoneal injection of 250mg/kg of MSU crystal suspension, analysing their serum UA concentration at 0, 2, 5, 8, 24h, 48h.

Here, we assessed the therapeutic functions of eRBC-URAT1+UOX in our mouse model of hyperuricemia. We injected 2×10 ⁸ eRBCs-URAT1+UOX intravenously 3 days before MSU injection. The eRBCs-URAT1+UOX significantly alleviated the elevated serum UA in the mouse model (Fig. 9) .

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Claims

An engineered red blood cell (eRBC) comprising:

a urate oxidase (UOX) or a functional variant thereof;.

a urate transporter 1 (URAT1) or a functional variant thereof; or

a UOX or a functional variant thereof and a URAT1 or a functional variant thereof.
The engineered red blood cell of claim 1, wherein the URAT1 comprises an amino acid sequence of SEQ ID NO: 2.
The engineered red blood cell of claim 1 or claim 2, wherein the UOX comprises an amino acid sequence of SEQ ID NO: 4.
A method for producing engineered red blood cells comprising:

1) collecting lineage negative cells (Lin-cells) from a blood sample or Lin-CD34+ cells from a bone marrow sample,

2) expanding the Lin-cells or Lin-CD34+ cells;

3) culturing the expanded Lin-cells or Lin-CD34+ cells to induce them to differentiate into erythroid cells; and, concurrently with the differentiation, introducing one or more exogenous nucleic acids into the expanded Lin-cells or Lin-CD34+ cells; and

4) culturing the erythroid cells to induce enucleation,

wherein 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.
The method of claim 4, wherein the URAT1 comprises an amino acid sequence of SEQ ID NO: 2.
The method of claim 4 or claim 5, wherein the UOX comprises an amino acid sequence of SEQ ID NO: 4.
The method of any one of claims 4-6, wherein the blood sample is a peripheral blood sample, a cord blood sample or a fetal blood sample.
The method of any one of claims 4-7, wherein the blood sample is a human peripheral blood sample.
The method of any one of claims 4-8, wherein the Lin-cells are Lin-CD34-cells.
The method of any one of claims 4-9, wherein the step 1) includes isolating peripheral blood mononuclear cells (PBMCs) from the peripheral blood sample and isolating Lin-CD34-cells from the PBMCs.
The method of any one of claims 4-10, wherein the step 1) comprising removing lineage positive (Lin+) cells from the peripheral blood sample by using a lineage cell depletion kit.
The method of any one of claims 4-11, wherein the step 2) including culturing the Lin-cells in a hematopoietic stem cell expansion medium supplemented with a combination of cytokines, and wherein combination of cytokines comprise fms-like tyrosine kinase 3 ligand (Flt3L) , stem cell factor (SCF) , interleukin 3 (IL-3) , and interleukin 6 (IL-6) .
The method of any one of claims 4-12, wherein the combination of cytokines comprise 50 ng/mL human Flt3L, 50 ng/mL human SCF, 10 ng/mL human IL-3, and 10 ng/mL human IL-6.
The method of any one of claims 4-13, wherein the hematopoietic stem cell expansion medium is StemSpan ^TM SFEM serum-free expansion medium.
The method of any one of claims 4-14, wherein the step 2) including culturing the Lin-cells at 37℃ under 5%CO ₂ for about 5 days.
The method of any one of claims 4-15, wherein the step 3) comprising culturing the expanded Lin-cells in a first differentiation medium supplemented with cytokines related to erythroid development.
The method of any one of claims 4-16, wherein the first differentiation medium is an Iscove's Modified Dulbecco's Medium (IMDM) containing: fetal bovine serum (FBS) or human serum, human plasma, glutamine, BSA, transferrin, insulin, Penicillin-Streptomycin, IL-3, EPO, and SCF.
The method of any one of claims 4-17, wherein the first differentiation medium is an Iscove's Modified Dulbecco's Medium (IMDM) containing: 10-15%FBS or human serum, 5-10%human plasma, 1-4 mM glutamine, 1-2%BSA, 300-600 μg/mL human transferrin, 8-13 μg/mL human insulin, 2%Penicillin-Streptomycin, 3-5 ng/mL human IL-3, 4-7 U/mL human EPO, and 100 ng/mL human SCF.
The method of any one of claims 4-18, wherein the step 3) comprising culturing the expanded Lin-CD34-cells at 37℃ under 5%CO ₂ for about 9 days.
The method of any one of claims 4-19, wherein the step 4) comprising culturing the erythroid cells in a second differentiation medium, wherein the second differentiation medium is an Iscove's Modified Dulbecco's Medium (IMDM) containing: FBS or human serum, human plasma, glutamine, BSA, transferrin, insulin, Penicillin-Streptomycin, and EPO.
The method of any one of claims 4-20, wherein the second differentiation medium is an Iscove's Modified Dulbecco's Medium (IMDM) containing: 15%FBS or human serum, 5-10%human plasma, 1-4 mM glutamine, 1-2%BSA, 300-600 μg/mL human transferrin, 8-13 μg/mL human insulin, 2%Penicillin-Streptomycin, and 1-5 U/mL human EPO.
The method of any one of claims 4-21, wherein the step 4) comprising culturing erythroid cells at 37℃ under 5%CO ₂ for about 7 days.
The method of any one of claims 4-22, wherein, in the step 3) , the introduction of the one or more exogenous nucleic acids into the expanded Lin-cells is carried out on the 1st day of culturing the expanded Lin-cells in the first differentiation medium.
The method of any one of claims 4-23, wherein the exogenous nucleic acid is an expression vector.
The method of any one of claims 4-24, wherein the exogenous nucleic acid is a lentiviral expression vector.
A pharmaceutical composition comprising an engineered red blood cell of any one of claims 1-3 and a physiologically acceptable excipient.
Use of engineered red blood cells of any one of claims 1-3 in the preparation of a medicament for treating a disorder associated with elevated serum uric acid levels.
The use of claim 27, wherein the disorder is gout or a hyperuricemia disease.
A method for the treatment of a disorder associated with elevated serum uric acid levels in a subject comprising:

a) taking a blood sample or a bone marrow sample from the subject,

b) producing engineered red blood cells by using a method of any one of the claims 4-24; and

c) infusing a therapeutically effective amount of the engineered red blood cells into the subject.
The method of claim 29, wherein the disorder is gout or a hyperuricemia disease
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 the engineered red blood cell of any one of claims 1-3 into the subject.
The method of claim 31 further comprising performing blood typing before the infusion to ensure the subject is compatible with the engineered red blood cell.