WO2021073607A1 - Ingénierie des globules rouges pour le traitement de maladies lysosomales - Google Patents

Ingénierie des globules rouges pour le traitement de maladies lysosomales Download PDF

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WO2021073607A1
WO2021073607A1 PCT/CN2020/121522 CN2020121522W WO2021073607A1 WO 2021073607 A1 WO2021073607 A1 WO 2021073607A1 CN 2020121522 W CN2020121522 W CN 2020121522W WO 2021073607 A1 WO2021073607 A1 WO 2021073607A1
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polypeptide
red blood
hematopoietic stem
lin
seq
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WO2021073607A9 (fr
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Xiaofei GAO
Xiaoqian NIE
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Westlake Therapeutics (Hangzhou) Co. Limited
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    • C12Y302/01022—Alpha-galactosidase (3.2.1.22)
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    • C12N9/2465—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)
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    • C12Y101/01081—Hydroxypyruvate reductase (1.1.1.81)
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    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the disclosure relates to methods for producing red blood cells comprising (i) engineering hematopoietic stem and/or progenitor cells to express a polypeptide such as an enzyme involved in a lysosomal storage disease, and (ii) differentiating the engineered hematopoietic stem and/or progenitor cells in vitro into mature red blood cells, wherein the mature red blood cells express the polypeptide.
  • aspects of the disclosure further relate to compositions comprising these red blood cells as well as therapeutic uses of these red blood cells.
  • the uses comprise treating a subject suffering from a lysosomal storage disease such as Fabry disease or type II primary hyperoxaluria.
  • Lysosomal storage diseases are a group of rare metabolic diseases that are characterized by the lack of functional individual lysosomal enzymes. Most LSDs are inherited as autosomal recessive or X-linked traits and typically present in infancy and childhood, although adult-onset forms also occur. Examples of LSDs include Fabry disease, Gaucher disease, and type II primary hyperoxaluria (PH2) .
  • ERT enzyme replacement therapy
  • ERT ERT for treating LSDs.
  • patients often develop immune responses or allergic reactions after infusion of the enzymes.
  • antibody responses vary with respect to the specific enzyme molecule and the individual patient, significant immune responses have been seen in patients treated with ERT.
  • Conjugating therapeutic enzymes to PEG may reduce immune responses in patients.
  • studies showed that many patients treated with PEG-conjugated enzymes develop anti-PEG antibodies.
  • PEG may adversely affect the activity of the conjugated enzyme, leading to reduced efficacy in the treatment.
  • prior ERT used recombinant enzymes expressed in vitro, which typically lack proper glycosylation modifications that are essential for enzyme uptake and intracellular transport.
  • the therapeutic enzymes may become inactivated or eliminated in vivo due to short half-life, limited bioavailability, and/or interactions with plasma proteins.
  • production and purification of the enzymes tend to be time-consuming, and thus treatments with ERT are very costly. For example, it currently costs an average of $180,000 per year to treat a patient having type I Gaucher disease, and an average of $215,000 per year to treat a patient having Fabry disease.
  • any successful application of ERT requires careful selection and use of appropriate carriers to deliver a therapeutic enzyme to the patient.
  • Some known delivery carriers include antibodies, soluble and biodegradable polymers, polysaccharides and other carriers, microcapsules, microparticles, lipoproteins, liposomes, and red blood cells.
  • red blood cells to safely and effectively deliver therapeutic polypeptides such as enzymes to treat lysosomal storage diseases such as Fabry disease and type II primary hyperoxaluria.
  • the disclosure relates to methods for producing red blood cells comprising (i) genetically engineering hematopoietic stem and/or progenitor cells to express an enzyme involved in a lysosomal storage disease, and (ii) differentiating the engineered hematopoietic stem and/or progenitor cells in vitro into mature red blood cells, wherein the mature red blood cells expresses the enzyme.
  • aspects of the disclosure further relate to compositions comprising these red blood cells as well as uses of these red blood cells for treating lysosomal storage diseases such as Fabry disease and type II primary hyperoxaluria.
  • Embodiment 1 A method of treating a subject having a lysosomal storage disease, comprising administering an effective amount of red blood cells to a subject having a lysosomal storage disease, wherein the red blood cells are engineered to express a polypeptide, and wherein expression of the polypeptide contributes to treating the lysosomal storage disease.
  • Embodiment 2 The method of embodiment 1, wherein the lysosomal storage disease is Fabry disease, and wherein the polypeptide is ⁇ galactosidase A ( ⁇ -GAL A) or a functional variant thereof.
  • Fabry disease Fabry disease
  • polypeptide is ⁇ galactosidase A ( ⁇ -GAL A) or a functional variant thereof.
  • Embodiment 3 The method of embodiment 2, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 75%sequence identity to SEQ ID NO: 1.
  • Embodiment 4 The method of any one of embodiments 2-3, wherein the polypeptide is encoded by a nucleic acid sequence having at least 75%sequence identity to SEQ ID NO: 2.
  • Embodiment 5 The method of embodiment 1, where the lysosomal storage disease is type II primary hyperoxaluria (PH2) , and wherein the polypeptide is glyoxylate reductase/hydroxypyruvate reductase (GRHPR) or a functional variant thereof.
  • PH2 type II primary hyperoxaluria
  • GSHPR glyoxylate reductase/hydroxypyruvate reductase
  • Embodiment 6 The method of embodiment 5, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least 75%sequence identity to SEQ ID NO: 3.
  • Embodiment 7 The method of any one of embodiments 5-6, wherein the polypeptide is encoded by a nucleic acid sequence having at least 75%sequence identity to SEQ ID NO: 4.
  • Embodiment 8 The method of any one of embodiments 1-7, wherein the red blood cells are differentiated from Lin - hematopoietic stem and/or progenitor cells that are engineered to express the polypeptide.
  • Embodiment 9 The method of embodiment 8, wherein the Lin - hematopoietic stem and/or progenitor cells comprise a viral vector, optionally a murine stem cell virus vector (MSCV) , that encodes the polypeptide.
  • a viral vector optionally a murine stem cell virus vector (MSCV) , that encodes the polypeptide.
  • MSCV murine stem cell virus vector
  • Embodiment 10 The method of embodiment 8, wherein the Lin - hematopoietic stem and/or progenitor cells comprise a non-viral vector that encodes the polypeptide.
  • Embodiment 11 The method of any one of embodiments 8-10, wherein the red blood cells are differentiated from Lin - and CD34 + hematopoietic stem and/or progenitor cells.
  • Embodiment 12 A method of producing red blood cells comprising the following steps: (a) engineering Lin - hematopoietic stem and/or progenitor cells to express a polypeptide; and (b) differentiating the engineered Lin - hematopoietic stem and/or progenitor cells in vitro into mature red blood cells, wherein the polypeptide is expressed in the mature red blood cells.
  • Embodiment 13 The method of embodiment 12, wherein expression of the polypeptide provides an enzyme for enzyme replacement therapy.
  • Embodiment 14 The method of any one of embodiments 12-13, wherein the polypeptide is ⁇ galactosidase A ( ⁇ -GAL A) or a functional variant thereof.
  • Embodiment 15 The method of embodiment 14, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 75%sequence identity to SEQ ID NO: 1.
  • Embodiment 16 The method of any one of embodiments 14-15, wherein the polypeptide is encoded by a nucleic acid sequence having at least 75%sequence identity to SEQ ID NO: 2.
  • Embodiment 17 The method of any one of embodiments 12-13, where the polypeptide is glyoxylate reductase/hydroxypyruvate reductase (GRHPR) or a functional variant thereof.
  • GSHPR glyoxylate reductase/hydroxypyruvate reductase
  • Embodiment 18 The method of embodiment 17, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least 75%sequence identity to SEQ ID NO: 3.
  • Embodiment 19 The method of any one of embodiments 17-18, wherein the polypeptide is encoded by a nucleic acid sequence having at least 75%sequence identity to SEQ ID NO: 4.
  • Embodiment 20 The method of any one of embodiments 12-19, wherein the Lin - hematopoietic stem and/or progenitor cells of step (a) are produced by: (1) isolating peripheral blood mononuclear cells (PBMCs) from a subject; (2) isolating Lin - hematopoietic stem and/or progenitor cells from the PBMCs, optionally by an immune-selective method; and (3) culturing the isolated Lin - hematopoietic stem and/or progenitor cells in a stem cell culture medium.
  • PBMCs peripheral blood mononuclear cells
  • Embodiment 21 The method of embodiment 20, wherein the stem cell culture medium comprises rhIL-3 and/or rhSCF.
  • Embodiment 22 The method of any one of embodiments 12-21, wherein step (a) comprises contacting the Lin - hematopoietic stem and/or progenitor cells with a viral vector, optionally a murine stem cell virus vector (MSCV) , that encodes the polypeptide.
  • a viral vector optionally a murine stem cell virus vector (MSCV)
  • MSCV murine stem cell virus vector
  • Embodiment 23 The method of any one of embodiments 12-21, wherein step (a) comprises contacting the Lin - hematopoietic stem and/or progenitor cells with a non-viral vector that encodes the polypeptide.
  • Embodiment 24 The method of any one of embodiments 12-23, wherein step (b) comprises: (1) culturing the engineered Lin - hematopoietic stem and/or progenitor cells in a differentiation culture medium; (2) detecting the expression level of CD235a, CD117, CD71, and/or the polypeptide; and optionally (3) measuring an enzymatic activity of the polypeptide.
  • Embodiment 25 The method of embodiment 24, wherein the differentiation culture medium comprises rhEpo.
  • Embodiment 26 The method of any one of embodiments 12-25, wherein the Lin - hematopoietic stem and/or progenitor cells in step (a) comprise Lin - and CD34 + hematopoietic stem and/or progenitor cells.
  • Embodiment 27 A red blood cell produced by the method of any one of embodiments 12-26.
  • Embodiment 28 A composition comprising the red blood cell of embodiment 27.
  • Embodiment 29 A method of treating a subject having a lysosomal storage disease, comprising administering an effective amount of the red blood cell of embodiment 27 or the composition of embodiment 28 to the subject.
  • Embodiment 30 A method of engineering Lin - hematopoietic stem and/or progenitor cells, comprising contacting Lin - hematopoietic stem and/or progenitor cells with a vector that encodes a polypeptide, optionally the polypeptide is ⁇ galactosidase A ( ⁇ -GAL A) or a functional variant thereof, or glyoxylate reductase/hydroxypyruvate reductase (GRHPR) or a functional variant thereof.
  • ⁇ -GAL A ⁇ galactosidase A
  • GSHPR glyoxylate reductase/hydroxypyruvate reductase
  • Embodiment 31 The method of embodiment 30, wherein the vector is a viral vector, optionally a murine stem cell virus vector (MSCV) .
  • MSCV murine stem cell virus vector
  • Embodiment 32 The method of embodiment 30, wherein the vector is a non-viral vector.
  • Embodiment 33 The method of any one of embodiments 30-32, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 75%sequence identity to SEQ ID NO: 1.
  • Embodiment 34 The method of embodiment 33, wherein the polypeptide is encoded by a nucleic acid sequence having at least 75%sequence identity to SEQ ID NO: 2.
  • Embodiment 35 The method of any one of embodiments 30-32, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 3 or an amino acid sequence having at least 75%sequence identity to SEQ ID NO: 3.
  • Embodiment 36 The method of embodiment 35, wherein the polypeptide is encoded by a nucleic acid sequence having at least 75%sequence identity to SEQ ID NO: 4.
  • Embodiment 37 The method of any one of embodiments 30-36, wherein the Lin - hematopoietic stem and/or progenitor cells are produced by: (1) isolating peripheral blood mononuclear cells (PBMCs) from a subject; (2) isolating Lin - hematopoietic stem and/or progenitor cells from the PBMCs, optionally by an immune-selective method; and (3) culturing isolated Lin - hematopoietic stem and/or progenitor cells in a stem cell culture medium.
  • PBMCs peripheral blood mononuclear cells
  • Embodiment 38 The method of embodiment 37, wherein the stem cell culture medium comprises rhIL-3 and/or rhSCF.
  • Embodiment 39 The method of any one of embodiment 30-38, wherein the Lin - hematopoietic stem and/or progenitor cells comprise Lin - and CD34 + hematopoietic stem and/or progenitor cells.
  • Embodiment 40 A red blood cell expressing a polypeptide, wherein the polypeptide is ⁇ galactosidase A ( ⁇ -GAL A) or a functional variant thereof, or glyoxylate reductase/hydroxypyruvate reductase (GRHPR) or a functional variant thereof.
  • ⁇ -GAL A ⁇ galactosidase A
  • GSHPR glyoxylate reductase/hydroxypyruvate reductase
  • Embodiment 41 The red blood cell of embodiment 40, wherein the polypeptide comprises an amino acid sequence having at least 75%sequence identity to SEQ ID NO: 1 or SEQ ID NO: 3, or the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
  • Embodiment 42 The method of embodiment 41, wherein the polypeptide is encoded by a nucleic acid sequence having at least 75%sequence identity to SEQ ID NO: 2 or SEQ ID NO: 4.
  • Embodiment 43 The red blood cell of any one of embodiments 40-42, wherein the red blood cell is differentiated from a Lin - hematopoietic stem and/or progenitor cell that is engineered to express the polypeptide.
  • Embodiment 44 The red blood cell of embodiment 43, wherein the Lin - hematopoietic stem and/or progenitor cell comprises a viral vector, optionally a murine stem cell virus vector (MSCV) , that encodes the polypeptide.
  • a viral vector optionally a murine stem cell virus vector (MSCV) , that encodes the polypeptide.
  • MSCV murine stem cell virus vector
  • Embodiment 45 The red blood cell of embodiment 43, wherein the Lin - hematopoietic stem and/or progenitor cell comprises a non-viral vector that encodes the polypeptide.
  • Embodiment 46 The red blood cell of any one of embodiments 43-45, wherein the red blood cell is differentiated from a Lin - and CD34 + hematopoietic stem and/or progenitor cell.
  • Embodiment 47 A method for treating a subject having Fabry disease or type II primary hyperoxaluria (PH2) , comprising administering the red blood cell of any one of embodiments 40-46 to the subject.
  • a method for treating a subject having Fabry disease or type II primary hyperoxaluria (PH2) comprising administering the red blood cell of any one of embodiments 40-46 to the subject.
  • Embodiment 48 Use of the red blood cell of any one of embodiments 40-46 for treating Fabry disease or type II primary hyperoxaluria (PH2) .
  • Embodiment 49 A Lin - hematopoietic stem and/or progenitor cell engineered to express a polypeptide, wherein the polypeptide is ⁇ galactosidase A ( ⁇ -GAL A) or a functional variant thereof, or glyoxylate reductase/hydroxypyruvate reductase (GRHPR) or a functional variant thereof.
  • ⁇ -GAL A ⁇ galactosidase A
  • GSHPR glyoxylate reductase/hydroxypyruvate reductase
  • Embodiment 50 The Lin - hematopoietic stem and/or progenitor cell of embodiment 49, wherein the polypeptide comprises an amino acid sequence having at least 75%sequence identity to SEQ ID NO: 1 or SEQ ID NO: 3, or the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
  • Embodiment 51 The Lin - hematopoietic stem and/or progenitor cell of embodiment 50, wherein the polypeptide is encoded by a nucleic acid sequence having at least 75%sequence identity to SEQ ID NO: 2 or SEQ ID NO: 4.
  • Embodiment 52 The Lin - hematopoietic stem and/or progenitor cell of any one of embodiments 49-51, wherein the Lin - hematopoietic stem and/or progenitor cell comprises a Lin - and CD34 + hematopoietic stem and/or progenitor cell.
  • Embodiment 53 A method for producing a red blood cell, comprising differentiating the engineered Lin - hematopoietic stem and/or progenitor cell of any one of embodiment s 49-52 in vitro into a mature red blood cell, wherein the polypeptide is expressed in the mature red blood cell.
  • FIG. 1 depicts general steps of engineering hematopoietic stem and/or progenitor cells to express an enzyme involved in an LSD, and subsequently differentiating the engineered hematopoietic stem and/or progenitor cells in vitro into mature red blood cells carrying the enzyme.
  • FIG. 2 depicts representative assays that can be used to test the enzyme activities in vitro, as well as enzymes functions in vivo using established animal models for LSDs.
  • FIG. 3 shows an example of FACS analyses of differentiated red blood cells.
  • the following labeling markers are used: CD235a, CD117, CD71, Hoechst33342, and GFP.
  • FIG. 4 shows tissue distribution of engineered RBCs in NSG mouse by live imaging. Dir-stained RBCs were injected into NSG mouse via tail vein 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 uM Dir only in PBS; Mouse #3: 1x10 8 human red blood cells differentiated from PBMCs in vitro and labeled with 2 uM Dir; Mouse #4: 1x10 8 human red blood cells infected with vector only differentiated from PBMCs in vitro and labeled with 2 uM Dir; Mouse #5: 1x10 8 human red blood cells infected with vector cloned with enzyme1 gene differentiated from PBMCs in vitro and labeled with 2 uM Dir and Mouse #6: 5x10 7 human red blood cells infected with vector cloned with enzyme2 gene differentiated from PBMCs in vitro and labeled with 2 uM Dir.
  • FIG. 5 shows an example of images of tissue samples after the injection of red blood cells.
  • Dir-stained red blood cells were intravenously injected into the tail vein of each mouse. seven days after the injection, the mice were sacrificed, and the tissue samples were imaged.
  • Mouse #4 1x10 8 human red blood cells infected with vector only differentiated from PBMCs in vitro and labeled with 2 uM Dir;
  • Mouse #5 1x10 8 human red blood cells infected with vector cloned with enzyme1 gene differentiated from PBMCs in vitro and labeled with 2 uM Dir and
  • Mouse #6 5x10 7 human red blood cells infected with vector cloned with enzyme2 gene differentiated from PBMCs in vitro and labeled with 2 uM Dir.
  • FIG. 6 shows FACS analysis of red blood cells engineered to express ⁇ -GAL A, after in vitro differentiation for 13 days.
  • the following labeling markers are used: CD235a, CD117, CD71, Hoechst33342, and GFP.
  • Y585-PE PI
  • B525-FITC GFP
  • R660-APC CD235a
  • B650-PC5.5 CD71
  • UV450 Hochest33342.
  • FIG. 7 shows FACS analysis of protein expression levels in red blood cells engineered to express ⁇ -GAL A, after in vitro differentiation for 18 days.
  • the following labeling markers are used: CD235a, CD117, CD71, Hoechst33342, and GFP.
  • Y585-PE PI
  • B525-FITC GFP
  • R660-APC CD235a
  • B650-PC5.5 CD71
  • UV450 Hochest33342.
  • FIG. 8 shows a representative ⁇ Gal A enzyme activity assay, using proteins extracted from in vitro-differentiated red blood cells.
  • FIG. 9 shows Western blot analyses of expression levels of hGRHPR in red blood cells infected with MSCV control vector or MSCV-hGRHPR-His after in vitro differentiation for 9 days.
  • Blank red blood cells without viral infection
  • MSCV red blood cells infected with MSCV
  • MSCV-hGRHPR-His red blood cells infected with MSCV-hGRHPR-His.
  • FIG. 10 shows FACS analyses of red blood cells engineered to express GRHPR, after in vitro differentiation for 13days.
  • the following labeling markers are used: CD235a, CD117, CD71, Hoechst33342, and GFP.
  • Y585-PE PI
  • B525-FITC GFP
  • R660-APC CD235a
  • B650-PC5.5 CD71
  • UV450 Hochest33342.
  • FIG. 11 shows FACS analyses of red blood cells engineered to express GRHPR, after in vitro differentiation for 18 days.
  • the following labeling markers are used: CD235a, CD117, CD71, Hoechst33342, and GFP.
  • Y585-PE PI
  • B525-FITC GFP
  • R660-APC CD235a
  • B650-PC5.5 CD71
  • UV450 Hochest33342.
  • FIG. 12 shows a representative GRHPR enzyme activity assay, using proteins extracted from red blood cells engineered to express GRHPR, after in vitro differentiation for 10 days.
  • FIG. 13 shows a representative GRHPR enzyme activity assay, using proteins extracted from red blood cells engineered to express GRHPR, after in vitro differentiation for 18 days.
  • a “functional variant” of a polypeptide is a polypeptide sequence which is not identical to the full-length polypeptide sequence yet retains the same or substantially the same function as the full-length polypeptide sequence.
  • a functional variant can possess more, fewer, or the same number of residues as the corresponding native molecule, and/or can contain one or more amino acid substitutions.
  • Methods for determining a particular protein’s functions are well-known. For instance, the enzymatic function of a polypeptide can be determined using an established assay kit. Some enzyme assays are disclosed in Examples 1 and 2.
  • LSDs are life-threatening diseases caused by insufficient activity of housekeeping enzymes required for the catabolism of materials that arise from the normal turnover of body constituents.
  • LSDs include Gaucher disease (glucocerebrosidase deficiency) , Fabry disease ( ⁇ galactosidase deficiency) , Hunter disease (iduronate-2-sulfatase deficiency) , Hurler disease (alpha-L iduronidase deficiency) , and type II primary hyperoxaluria (glyoxylate reductase/hydroxypyruvate reductase deficiency) .
  • hematopoietic stem and/or progenitor cells are characterized by the presence of suitable cell surface antigens and their ability to give rise to mature blood cells, such as red blood cells, platelets, and white blood cells.
  • suitable cell surface antigens such as red blood cells, platelets, and white blood cells.
  • Some Lin - hematopoietic stem and/or progenitor cells disclosed in Examples 1 and 2 have the capacity or a limited capacity for self-renewal, but they can be differentiated into mature red blood cells in vitro.
  • the hematopoietic stem and/or progenitor cells can be found in bone marrow and/or peripheral blood mononuclear cells.
  • red blood cells and “mature red blood cells” are used interchangeably and refer to terminally differentiated cells derived from hematopoietic stem and/or progenitor cells. They lack a nucleus and most cellular organelles. In human, red blood cells are produced in the bone marrow in response to blood hypoxia which is mediated by release of erythropoietin (EPO) by the kidney. EPO causes an increase in the number of erythroid precursor cells and induces these cells to proliferate and differentiate into mature red blood cells.
  • EPO erythropoietin
  • red blood cells After approximately 120 days of circulation, since red blood cells do not contain a nucleus or any other regenerative capabilities, they are removed from circulation by either the phagocytic activities of macrophages in the liver, spleen and lymph nodes, or by hemolysis in the plasma. Following macrophage engulfment, functional enzymes in RBCs are released into macrophages.
  • nucleic acid and “polynucleotide” are used interchangeably and refer to a polymer of deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) in either single or double stranded form.
  • DNA deoxyribonucleic acids
  • RNA ribonucleic acids
  • nucleic acid or polynucleotide encompasses polynucleotides containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • polypeptide, ” “peptide, ” and “protein” are used interchangeably to refer to a polymer of amino acid residues.
  • the term also applies to amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of corresponding naturally occurring amino acids.
  • sequence identity relates to the similarity of amino acid or nucleic acid sequences. Sequence identity is determined by the percentage of identical residues. Standard methods for the alignment of sequences are known in the art. For example, the Blast algorithm available from various public sources such as the NCBI.
  • vector refers to a vehicle into which the cDNA encoding a protein may be inserted.
  • non-viral vector e.g., plasmid
  • viral vector refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector refers to a viral vector in which additional DNA segments can be ligated into the viral genome.
  • suitable viral vectors include retroviral vectors (including lentiviral vectors) , adenoviral vectors, adeno-associated viral vectors, and hybrid vectors. See, e.g., the MSCV vector used in Examples 1 and 2.
  • a “vector” includes one or more regulatory sequences known in the art to control protein expression.
  • the term “treating” or “treatment” refers to preventing or alleviating a condition, slowing the onset or rate of development of a condition, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or ending symptoms associated with a condition, generating a complete or partial regression of a condition, curing a condition, or some combination thereof.
  • the term “treating” or “treatment” may refer to providing a functional enzyme as ERT that contributes to treating an LSD patient such that at least one or more symptoms of the LSD is ameliorated.
  • administer refers to the placement of a composition, such as a composition of red blood cells, into a subject by a method or route that results in at least partial localization of the composition at a desired site or tissue location.
  • a composition may be administered to a subject by intramuscular injection, subcutaneous injection, or intravenous injection.
  • the term “effective amount” refers to the amount of therapeutic agent, pharmaceutical composition, or pharmaceutical formulation, sufficient to reduce at least one or more symptom (s) of a disease or disorder, or to provide a desired effect.
  • an “effective amount” may vary according to factors such as the disease state, age, sex, and weight of the LSD patient and the ability of the medicaments to elicit a desired response in the LSD patient.
  • the term “subject” refers to an animal, for example a human, to whom treatment with methods and compositions according to this disclosure is provided.
  • red blood cells As an in vivo drug transport carrier, red blood cells have one or more of 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 the macrophage in the liver and spleen and will not cause significant 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.
  • 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.
  • LSDs comprise rare metabolic diseases that are characterized by the lack of functional individual lysosomal enzymes. Examples of LSDs include Fabry disease (Example 1) and type II primary hyperoxaluria (PH2) (Example 2) .
  • LSDs have devastating effects on those afflicted with them.
  • a traditional treatment is ERT where a functional enzyme is given to the patient, usually through intravenous injection in large doses.
  • novel drug carries including engineered red blood cells, that can be used to safely and effectively deliver therapeutic polypeptides such as enzymes for enzyme replace therapy.
  • the red blood cells according to this disclosure are engineered to express an enzyme involved in LSD, wherein expression of the enzyme contributes to treating an LSD patient.
  • an enzyme involved in LSD wherein expression of the enzyme contributes to treating an LSD patient.
  • Certain embodiments of red blood cells according to this disclosure are further summarized in the following paragraphs. This list is exemplary and not exhaustive of all of the red blood cell embodiments.
  • the methods of engineering red blood cells according to this disclosure can be adapted to deliver other polypeptides for therapy of LSDs.
  • the red blood cells according to this disclosure are engineered to express a polypeptide involved in Fabry disease.
  • the polypeptide is ⁇ galactosidase A ( ⁇ -GAL A) or a functional variant thereof.
  • the red blood cells according to this disclosure express a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 1, which is the full-length sequence of human ⁇ galactosidase A.
  • the red blood cells according to this disclosure express a polypeptide having the amino acid sequence of SEQ ID NO: 1.
  • the red blood cells according to this disclosure express a polypeptide that is encoded by a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 2. In some embodiments, the red blood cells according to this disclosure express a polypeptide that is encoded by the nucleic acid sequence of SEQ ID NO: 2.
  • red blood cells engineered to express glyoxylate reductase/hydroxypyruvate reductase
  • the red blood cells according to this disclosure are engineered to express a polypeptide involved in type II primary hyperoxaluria (PH2) .
  • the polypeptide is glyoxylate reductase/hydroxypyruvate reductase (GRHPR) or a functional variant thereof.
  • the red blood cells according to this disclosure express a polypeptide comprising an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 3, which is the full-length sequence of human GRHPR.
  • the red blood cells according to this disclosure express a polypeptide having the amino acid sequence of SEQ ID NO: 3.
  • the red blood cells according to this disclosure express a polypeptide that is encoded by a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 4. In some embodiments, the red blood cells according to this disclosure express a polypeptide that is encoded by the nucleic acid sequence of SEQ ID NO: 4.
  • CD34 + bone marrow hematopoietic stem cells or CD34 + umbilical cord hematopoietic stem cells from a subject, for example, an LSD patient.
  • CD34 + umbilical cord hematopoietic stem cells can only be obtained during a specific time period after a baby is born and are difficult to isolate, causing significant obstacles in the clinical application of these hematopoietic stem cells.
  • the red blood cells according to this disclosure are in vitro differentiated from hematopoietic stem and/or progenitor cells that are isolated from peripheral blood mononuclear cells (PBMCs) from a subject, for example, an LSD patient.
  • PBMCs peripheral blood mononuclear cells
  • the isolated hematopoietic stem and/or progenitor cells are then cultured in a stem cell medium for a period of time.
  • This approach allows for about 7-10 times replication of hematopoietic stem and/or progenitor cells, which replication is about 10 to 50-fold higher than previously reported.
  • the approach produces a large number of mature red blood cells suitable for delivering an LSD enzyme for ERT.
  • the method according to this disclosure allows for large-scale red blood cell engineering to deliver therapeutic polypeptides including enzymes.
  • the data indicates that when administered into a subject, these red blood cells have the same metabolic rate as normal mature red blood cells that are not engineered to express an enzyme.
  • This method can be adapted to engineer red blood cells to express another therapeutic enzyme, greatly expanding the application of engineering red blood cells for treating LSDs.
  • the red blood cells according to this disclosure are in vitro differentiated from hematopoietic stem and/or progenitor cells that are engineered to express an enzyme involved in LSD.
  • the hematopoietic stem and/or progenitor cells are Lin - hematopoietic stem and/or progenitor cells.
  • the hematopoietic stem and/or progenitor cells are Lin - and CD34 + hematopoietic stem and/or progenitor cells.
  • the hematopoietic stem and/or progenitor cells are isolated from peripheral blood mononuclear cells (PBMCs) from a subject. Methods of isolating PBMCs from a subject are known in the art. In some embodiments, the subject suffers from an LSD and the in vitro differentiated red blood cells are administered to the same subject for treating the LSD. In some embodiments, the hematopoietic stem and/or progenitor cells are isolated from PBMCs using an immune-selective method routinely practiced in the art. For example, the hematopoietic stem and/or progenitor cells may be Lin - and CD34 + hematopoietic stem and/or progenitor cells and are isolated using Lin and CD34 immune-selective beads.
  • PBMCs peripheral blood mononuclear cells
  • the hematopoietic stem and/or progenitor cells are engineered to express an an enzyme involved in an LSD through transfection/infection methods known in the art.
  • the isolated hematopoietic stem and/or progenitor cells are cultured in a stem cell culture medium that allows for self-renew of the hematopoietic stem and/or progenitor cells. See, e.g., Examples 1 and 2.
  • the stem cell culture medium contains rhIL-3 and/or rhSCF.
  • the isolated hematopoietic stem and/or progenitor cells are cultured in a stem cell culture medium for about 3, 4, 5, 6, 7, or 8 days before transfection/infection with a vector/virus encoding the enzyme involved in an LSD. In some embodiments, the isolated hematopoietic stem and/or progenitor cells are cultured in a stem cell culture medium for about 4 days before transfection/infection with a vector/virus encoding an enzyme involved in an LSD. In some embodiments, the isolated hematopoietic stem and/or progenitor cells are cultured in a stem cell culture medium for about 5 days before transfection/infection with a vector/virus encoding an enzyme involved in an LSD.
  • the isolated hematopoietic stem and/or progenitor cells are cultured in a stem cell culture medium for about 6 days before transfection/infection with a vector/virus encoding an enzyme involved in an LSD. In some embodiments, the isolated hematopoietic stem and/or progenitor cells are cultured in a stem cell culture medium for about 7 days before transfection/infection with a vector/virus encoding an enzyme involved in an LSD.
  • the incubation of the isolated hematopoietic stem and/or progenitor cells with the stem cell culture medium allows for about 5-20 times replication of the hematopoietic stem and/or progenitor cells. In some embodiments, the incubation of the isolated hematopoietic stem and/or progenitor cells with the stem cell culture medium allows for about 7-20 times replication of the hematopoietic stem and/or progenitor cells. In some embodiments, the incubation of the isolated hematopoietic stem and/or progenitor cells with the stem cell culture medium allows for about 9-20 times replication of the hematopoietic stem and/or progenitor cells.
  • the incubation of the isolated hematopoietic stem and/or progenitor cells with the stem cell culture medium allows for about 5-15 times replication of the hematopoietic stem and/or progenitor cells. In some embodiments, the incubation of the isolated hematopoietic stem and/or progenitor cells with the stem cell culture medium allows for about 7-15 times replication of the hematopoietic stem and/or progenitor cells. In some embodiments, the incubation of the isolated hematopoietic stem and/or progenitor cells with the stem cell culture medium allows for about 9-15 times replication of the hematopoietic stem and/or progenitor cells.
  • the incubation of the isolated hematopoietic stem and/or progenitor cells with the stem cell culture medium allows for about 5-10 times replication of the hematopoietic stem and/or progenitor cells. In some embodiments, the incubation of the isolated hematopoietic stem and/or progenitor cells with the stem cell culture medium allows for about 7-10 times replication of the hematopoietic stem and/or progenitor cells.
  • the hematopoietic stem and/or progenitor cells are engineered to express an enzyme involved in an LSD.
  • the enzyme is ⁇ -GAL A or a functional variant thereof.
  • the enzyme has an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 1, which is the full-length sequence of human ⁇ galactosidase A.
  • the enzyme is a polypeptide having the amino acid sequence of SEQ ID NO: 1.
  • the enzyme is encoded by a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 2.
  • the enzyme is encoded by the nucleic acid sequence of SEQ ID NO: 2.
  • the enzyme is GRHPR or a functional variant thereof.
  • the enzyme has an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 3, which is the full-length sequence of human GRHPR.
  • the enzyme is a polypeptide having the amino acid sequence of SEQ ID NO: 3.
  • the enzyme is encoded by a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 4. In some embodiments, the enzyme is encoded by the nucleic acid sequence of SEQ ID NO: 4.
  • a DNA sequence encoding the enzyme is introduced into the hematopoietic stem and/or progenitor cells through a viral vector using techniques known in the art.
  • the hematopoietic stem and/or progenitor cells are infected with a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector.
  • the viral vector is a murine stem cell virus vector (MSCV) . See Examples 1 and 2.
  • a DNA sequence encoding the enzyme is introduced into the hematopoietic stem and/or progenitor cells through a non-viral vector, for example, a plasmid, using techniques known in the art.
  • a non-viral vector for example, a plasmid
  • Transfection of cells by non-viral vectors can be achieved by the use of cationic liposomes, DNA-protein complexes such as poly-lysine-DNA complexes, or other means known in the art.
  • the hematopoietic stem and/or progenitor cells are cultured in a differentiation culture medium for about 10-21 days, about 10-20 days, about 10-19 days, about 10-18 days, about 10-17 days, about 10-16 days, about 10-15 days, about 10-14 days, about 10-13 days, about 10-12 days, about 10-11 days, about 21 days, about 20 days, about 19 days, about 18 days, about 17 days, about 16 days, about 15 days, about 14 days, about 13 days, about 12 days, or about 11 days, so that the hematopoietic stem and/or progenitor cells are differentiated into mature red blood cells expressing the enzyme involved in an LSD.
  • the differentiation culture medium contains rhEpo, which induces these cells to proliferate and differentiate into mature red blood cells.
  • the cultured cells are periodically collected and analyzed for expression levels of suitable makers indicating the differentiation and maturation of red blood cells.
  • the cells are analyzed for expression levels of CD235a, CD117, CD71, and Hoechst 33342.
  • the cultured cells may be periodically collected and analyzed for expression levels and activities of the enzyme involved in an LSD, using established assays known in the art.
  • enzyme levels can be monitored by detection of the enzyme protein in the cellular lysate (e.g., sonication) .
  • Enzyme activities can be monitored by a suitable substrate assay.
  • a substrate assay can comprise, for example, assessing the activity of an enzyme using a synthetic specific substrate, which upon the direct action of the enzyme releases a fluorophore, which can be easily quantified. See Examples 1 and 2.
  • the present disclosure provides methods of producing red blood cells that are engineered to express a polypeptide involved in an LSD.
  • the LSD is Fabry disease and the polypeptide is ⁇ galactosidase A ( ⁇ -GAL A) or a functional variant thereof.
  • the polypeptide has an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 1, which is the full-length sequence of human ⁇ galactosidase A.
  • the polypeptide has the amino acid sequence of SEQ ID NO: 1.
  • the polypeptide is encoded by a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 2. In some embodiments, the polypeptide is encoded by the nucleic acid sequence of SEQ ID NO: 2.
  • the LSD is type II primary hyperoxaluria and the polypeptide is GRHPR or a functional variant thereof.
  • the polypeptide has an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 3, which is the full-length sequence of human GRHPR.
  • the polypeptide has the amino acid sequence of SEQ ID NO: 3.
  • the polypeptide is encoded by a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 4. In some embodiments, the polypeptide is encoded by the nucleic acid sequence of SEQ ID NO: 4.
  • the methods of producing red blood cells comprise the following steps: (a) engineering isolated hematopoietic stem and/or progenitor cells to express a polypeptide; and (b) differentiating the engineered hematopoietic stem and/or progenitor cells in vitro into mature red blood cells, wherein the polypeptide is expressed in the red blood cells.
  • the hematopoietic stem and/or progenitor cells may be Lin - and CD34 + hematopoietic stem and/or progenitor cells.
  • the methods further comprise (1) isolating peripheral blood mononuclear cells (PBMCs) from a subject using approaches known in the art; (2) isolating hematopoietic stem and/or progenitor cells from the PBMCs using an immune-selective method; and (3) culturing the isolated hematopoietic stem and/or progenitor cells in a stem cell culture medium.
  • PBMCs peripheral blood mononuclear cells
  • the hematopoietic stem and/or progenitor cells may be Lin - and CD34 + hematopoietic stem and/or progenitor cells and can be isolated using Lin and CD34 immune- selective beads.
  • the stem cell culture medium contains rhIL-3 and/or rhSCF that stimulate (s) the self-renew of the isolated hematopoietic stem and/or progenitor cells.
  • the engineering step of (a) of the methods of producing red blood cells comprises transfecting/infecting the isolated hematopoietic stem and/or progenitor cells with a vector/virus that encodes the polypeptide involved in an LSD.
  • the vector is a viral vector, for example a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector.
  • the viral vector is a murine stem cell virus vector (MSCV) .
  • the vector is a non-viral vector, for example, a plasmid.
  • the vector includes one or more regulatory sequences known in the art to control the expression of the polypeptide.
  • the in vitro differentiation step of (b) of the methods of producing red blood cells comprises culturing the hematopoietic stem and/or progenitor cells that express the polypeptide involved in an LSD in a differentiation culture medium containing rhEpo, for about 10-21 days, about 10-20 days, about 10-19 days, about 10-18 days, about 10-17 days, about 10-16 days, about 10-15 days, about 10-14 days, about 10-13 days, about 10-12 days, about 10-11 days, about 21 days, about 20 days, about 19 days, about 18 days, about 17 days, about 16 days, about 15 days, about 14 days, about 13 days, about 12 days, or about 11 days, so that the hematopoietic stem and/or progenitor cells are differentiated into mature red blood cells.
  • the in vitro differentiation process can be monitored by analyzing expression levels of suitable makers indicating the stages of differentiation and maturation of red blood cells. See, e.g., Examples 1 and 2.
  • the methods according to this disclosure produce red blood cells that express a polypeptide encoded by the vector/virus transfected/infected to the hematopoietic stem and/or progenitor cells.
  • the polypeptide is an enzyme involved in an LSD, and the activity of the polypeptide can be measured using established enzyme activity assays known in the art.
  • the red blood cells according to this disclosure can be preserved in a suitable medium known in the art.
  • the red blood cells according to this disclosure can be cryopreserved in a suitable medium and stored in aliquots. Once opened, an aliquot can be reserved for the exclusive use of one subject suffering from an LSD.
  • the present disclosure also provides methods of engineering hematopoietic stem and/or progenitor cells, comprising transfecting/infecting isolated hematopoietic stem and/or progenitor cells with a vector/virus that encodes a polypeptide, wherein the polypeptide is ⁇ -GAL A or a functional variant thereof, or GRHPR or a functional variant thereof.
  • the hematopoietic stem and/or progenitor cells are Lin - hematopoietic stem and/or progenitor cells isolated from PBMCs from a subject suffering from an LSD.
  • the hematopoietic stem and/or progenitor cells are Lin - and CD34 + hematopoietic stem and/or progenitor cells isolated from PBMCs from a subject suffering from an LSD.
  • the polypeptide is introduced to the hematopoietic stem and/or progenitor cells through a vector encoding the polypeptide, where the vector can be a viral vector or a non-viral vector.
  • the vector is a viral vector, for example a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector.
  • the viral vector is a murine stem cell virus vector (MSCV) .
  • the vector is a non-viral vector, for example, a plasmid.
  • the vector includes one or more regulatory sequences known in the art to control the expression of the polypeptide.
  • compositions comprising red blood cells according to this disclosure.
  • the composition is a pharmaceutical composition comprising red blood cells provided herein and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers for use in the pharmaceutical compositions are known in the art and may include, for example, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, anesthetics, suspending/dispending agents, sequestering or chelating agents, diluents, adjuvants, excipients, and/or other non-toxic auxiliary substances.
  • compositions comprising red blood cells include liquid preparations for administration, including sterile suspensions and emulsions. Such compositions may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like.
  • a suitable carrier diluent, or excipient
  • the compositions can contain auxiliary substances such as wetting or emulsifying agents, pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the preparation desired.
  • compositions of this disclosure are conveniently provided as liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions or viscous compositions, which may be buffered to a selected pH.
  • Liquid preparations are normally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific tissues.
  • suitable carriers and other additives will depend on the exact route of administration and the nature of the particular dosage form, e.g., liquid dosage form, e.g., whether the composition is to be formulated into a solution, a suspension, gel, or another liquid form, such as a time release form or liquid-filled form.
  • a pharmaceutically acceptable preservative or cell stabilizer can be employed to increase the life of the compositions. If preservatives are necessary, it is well within the purview of the skilled artisan to select agents that will not affect the viability or efficacy of the red blood cells of this disclosure.
  • the present disclosure also provides for methods for treating a subject suffering from an LSD, comprising administering to the subject a therapeutically effective amount of the red blood cells disclosed herein.
  • the LSD is Fabry disease.
  • the red blood cells express a polypeptide that is ⁇ galactosidase A ( ⁇ -GAL A) or a functional variant thereof.
  • the polypeptide has an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 1, which is the full-length sequence of human ⁇ galactosidase A.
  • the polypeptide has the amino acid sequence of SEQ ID NO: 1.
  • the polypeptide is encoded by a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 2.
  • the polypeptide is encoded by the nucleic acid sequence of SEQ ID NO: 2.
  • the LSD is type II primary hyperoxaluria.
  • the red blood cells express a polypeptide that is GRHPR or a functional variant thereof.
  • the polypeptide has an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 3, which is the full-length sequence of human GRHPR.
  • the polypeptide has the amino acid sequence of SEQ ID NO: 3.
  • the polypeptide is encoded by a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity to SEQ ID NO: 4. In some embodiments, the polypeptide is encoded by the nucleic acid sequence of SEQ ID NO: 4.
  • the red blood cells of the present invention When administering the red blood cells of the present invention, it will generally be formulated in a unit dosage injectable form (solution, suspension, or emulsion) .
  • the pharmaceutical formulations suitable for injection include sterile aqueous solutions and dispersions.
  • the carrier can be a solvent or dispersing medium containing, for example, water, saline, phosphate buffered saline, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like) and suitable mixtures thereof.
  • various additives which enhance the stability, sterility, and isotonicity of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added.
  • a therapeutically effective amount of the red blood cells disclosed herein may vary according to factors such as the disease state, age, sex, and weight of the subject and the ability of the medicaments to elicit a desired response in the subject.
  • the administration dosage may change over the course of treatment.
  • a medical professional having ordinary skill in the art may determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician could start doses at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. A physician also knows when it is needed to re-administer the red blood cells after the removal of functional red blood cells from the last administration.
  • the red blood cells disclosed herein may be administered by any suitable route known in the art, such as subcutaneous, intraperitoneal, intravenous, intramuscular, or intradermal injection.
  • a medical professional having ordinary skill in the art may readily determine the appropriate dosing route.
  • the red blood cells can be administered via localized injection, including catheter administration.
  • the efficacy of the methods for treatment can be assessed by examining the enzymatic activities of the expressed polypeptide and and/or pathological conditions of tissues such as kidney affected by the defective enzyme in the LSD patient.
  • tissue such as kidney affected by the defective enzyme in the LSD patient.
  • LSD it is known to the skilled artisan as to how to examine the efficacy of the methods for treatment after administering the red blood cells according to this disclosure.
  • Example 1 Engineering Hematopoietic Stem and/or Progenitor Cells to Express ⁇ Galactosidase A for Treating Fabry Disease
  • Fabry disease (OMIM 301500) is an X-linked lysosomal storage disorder caused by mutations in the GLA gene encoding the lysosomal enzyme ⁇ -galactosidase A.
  • the defect in the gene causes a loss or reduction of alpha-galactosidase activity in lysosomes and results in accumulation of globotriaosylceramide (Gb3) , which in turn leads to damage of various cell types including capillary endothelial cells, renal cells, cardiomyocytes, and nerve cells.
  • Gb3 globotriaosylceramide
  • Patients suffering from Fabry disease show a variety of progressive clinical symptoms, many of which first appear in early childhood, including neuropathic pain and/or gastrointestinal (GI) problems. As the patients grow older, they often develop terminal organ failure or life-threatening cardiovascular or cerebrovascular conditions. The life expectancy of the patients is generally around 50 years.
  • ERT drugs Two ERT drugs have been approved for treating Fabry disease. The first is (agalsidase alfa) and the second is (agalsidase beta) . Each of these drugs provides for the functional ⁇ -galactosidase A to the patient via intravenous administration of a recombinant ⁇ -galactosidase A.
  • This Example provides for using ERT to treat a Fabry disease patient.
  • the hematopoietic stem and/or progenitor cells from a subject suffering from Fabry disease were isolated and engineered to express a functional ⁇ -galactosidase A.
  • the engineered hematopoietic stem and/or progenitor cells were in vitro differentiated into mature red blood cells expressing ⁇ -galactosidase A.
  • the in vitro and in vivo functions of these red blood cells can be examined using approaches as described in FIG. 2.
  • the method disclosed in this Example allows for about 7-10 times replication of hematopoietic stem and/or progenitor cells, and after in vitro differentiation, this method produces a large number of mature red blood cells suitable for delivering functional ⁇ -galactosidase A for ERT. Furthermore, when administered into the subject, these red blood cells can circulate in the subject’s body for a long time (about 120 days in human) and function to specifically metabolize its target Gb3. Alternatively, during the liver clearance step, the old and/or damaged red blood cells carrying the enzyme (i.e., ⁇ -galactosidase A) can be targeted to macrophages for removal, restoring the liver’s metabolic activities.
  • the enzyme i.e., ⁇ -galactosidase A
  • ⁇ -GAL A cDNA (X05790.1; SEQ ID NO: 2) , which encodes the polypeptide of human ⁇ -GAL A (SEQ ID NO: 1)
  • MSCV murine stem cell virus
  • the calcium phosphate was removed by medium exchange 12 hours after the transfection.
  • the supernatant was collected at 48 hours and 72 hours after the transfection and the then filtered through a 0.45 ⁇ m filter.
  • the filtered supernatant was subject to ultracentrifugation at a speed of 70000 RCF for 2 hours at a temperature of 4 °C. After ultracentrifugation, the supernatant was removed, and the pellet was re-suspended using stage 1 culture medium as disclosed in section 1.3, and the virus titer was quantified using ELISA known in the art. The virus was stored at -80 °C until use.
  • PBMCs Peripheral blood mononuclear cells
  • Lymphoprep TM lymphocyte separation solution
  • the hematopoietic stem and/or progenitor cells were isolated using the Human Lineage Cell Depletion Set -DM (BD Biosciences) to obtain Lin - and CD34 + hematopoietic stem and/or progenitor cells, using techniques known in the art, for example an immune-selective method.
  • the isolated Lin - and CD34 + hematopoietic stem and/or progenitor cells were then immediately cultured in a stem cell culture medium (StemSpan TM SFEM, STEMCELL Technologies) containing a cytokine combination (StemSpan TM CC110 S, TEMCELL Technologies) and Penicillin-Streptomycin (Gibco) .
  • the cell culturing was carried for 4 days at 37 °C under 5%CO 2 , to allow the expansion of hematopoietic stem and/or progenitor cells.
  • the state 1 culture medium contains Iscove's Modified Dulbecco's Medium (IMDM, Sigma-Aldrich) , 10-15%fetal bovine serum (FBS, Gibco) , 5-10%human plasma (Plasma) , 1-4 mM glutamine, 1-2%albumin from bovine serum (BSA) , 300-600 ⁇ g/ml holo human transferrin (Sigma-Aldrich) , 8-13 ⁇ g/ml recombinant human insulin (Sigma-Aldrich) , 2%Penicillin-Streptomycin (Gibco) , 3-5 ng/ml recombinant human interleukin 3 (rhIL-3, Peprotech) , 4-7 U/ml recombinant human erythropoietin (rhEpo, Amgen) ,
  • IMDM Iscove's Modified Dulbecco's Medium
  • FBS fetal bovine serum
  • the cultured cells were infected with the MSCV- ⁇ Gal A virus prepared in section 1.2.
  • the cells were re-suspended at a density of 1x10 5 /mL using state 1 culture medium.
  • the virus dosage was determined based on a final virus concentration at 5 ⁇ 10 7 TU/ml -5 ⁇ 10 8 TU/ml, and depending on the infection volume, polybrene at 10 ⁇ g/ml was added to the concentrated virus solution for 5 minutes. After a brief incubation, the virus solution was added to the cells.
  • a centrifugal infection was performed using a horizontal rotor centrifuge at 500xg speed, 32 °C temperature, and for 90 minutes.
  • the medium was changed to fresh state 1 culture medium 12 hours after infection, and the cells were further cultured at 37 °C under 5%CO 2 .
  • the culture medium was changed to stage 2 culture medium and the cells were cultured at 37 °C under 5%CO 2 for about 7 days.
  • the state 2 culture medium contains Iscove's Modified Dulbecco's Medium (IMDM, Sigma-Aldrich) , 15%fetal bovine serum (FBS, Gibco) , 5-10%human plasma (Plasma) , 1-4 mM glutamine, 1-2%albumin from bovine serum (BSA) , 300-600 ⁇ g/ml holo human transferrin (Sigma-Aldrich) , 8-13 ⁇ g/ml recombinant human insulin (Sigma- Aldrich) , 2%Penicillin-Streptomycin (Gibco) , and 1-5 U/ml recombinant human erythropoietin (rhEpo, Amgen) .
  • IMDM Iscove's Modified Dulbecco's Medium
  • FBS fetal bovine serum
  • the culture medium was changed every 2-4 days, and the expression levels of CD235a, CD117, CD71, Hoechst 33342, and GFP were determined by flow cytometry, as shown in FIG. 3.
  • CD235a is blood glycophorin A, which is a single transmembrane glycoprotein expressed in mature red blood cells and erythroid precursor cells.
  • CD225a is a marker on the surface of red blood cells.
  • CD117 is a mast cell/stem cell growth factor receptor, which is expressed on the surface of hematopoietic stem cells and other cells.
  • CD71 is transferrin receptor 1, which is a transmembrane glycoprotein composed of two disulfide-bonded monomers linked by two disulfide bonds. Each monomer binds to a transferrin molecule to produce an iron-transferrin-transferrin receptor complex that enters the cell by endocytosis.
  • Hoechst33342 is an established fluorescent dye used to stain cellular DNA.
  • GFP denotes green fluorescent protein. This protein is encoded by a fluorescent reporter gene that is operably linked to MSCV- ⁇ Gal A via T2A, and is therefore co-expressed with ⁇ Gal A. The strength of the GFP signal suggests the expression level of ⁇ Gal A. For example, a positive GFP signal indicates that ⁇ Gal A is expressed in the infected cells.
  • FIG. 6 shows FACS analyses of protein expression levels in red blood cells engineered to express ⁇ -GAL A, after in vitro differentiation for 10 days.
  • the following labeling markers are used: CD235a, CD117, CD71, Hoechst33342, and GFP.
  • FIG. 7 shows FACS analyses of protein expression levels in red blood cells engineered to express ⁇ -GAL A, after in vitro differentiation for 18 days.
  • the following labeling markers are used: CD235a, CD117, CD71, Hoechst33342, and GFP.
  • ⁇ -GAL A The expression level of ⁇ -GAL A in red blood cells was examined by Western blot and flow cytometry.
  • the in vitro function of red blood cells was analyzed by charactering the Gal A enzyme activity (Biovision) . After extracting the ⁇ Gal A enzyme from red blood cells, the enzyme activity was detected by a ⁇ Gal A enzyme activity detection kit (Biovision) .
  • the substrate of ⁇ Gal A is coupled with the fluorophore 4-MU, such that upon hydrolysis od the substrate by ⁇ Gal A, the fluorophore 4-MU is exposed, producing a detectable fluorescent signal (Ex/EM: 360/44) .
  • FIG. 8 shows a representative ⁇ Gal enzyme activity assay, using proteins extracted from in vitro-differentiated red blood cells.
  • the data indicates that the in vitro differentiation process successfully produced red blood cells expressing ⁇ Gal A having strong enzymatic activities. Furthermore, there was a dose-dependent increase of enzyme activity for the ⁇ Gal A extracted from the intro differentiated red blood cells.
  • red blood cells were intravenously injected into the tail vein of each immuno-deficient mouse, and the distributions of the red blood cells were analyzed by live imaging (FIG. 4) and/or immunohistochemistry (FIG. 5) routinely used in the art.
  • the survival of red blood cells expressing ⁇ -GAL A was compared to wild type red blood cells that do not express ⁇ -GAL A.
  • the in vivo function of the red blood cells can be examined using a Fabry disease mouse model ( ⁇ -Gal A KO mouse) .
  • the red blood cells are administered into the ⁇ -Gal A KO mouse, and the in vivo efficacy can be monitored by checking the levels of Gb3 and/or pathological conditions of tissues such as kidney.
  • Example 2 Engineering Hematopoietic Stem and/or Progenitor Cells to Express GRHPR for Treating Type II Primary Hyperoxaluria
  • Type II primary hyperoxaluria (PH2) (OMIM 260000, 604296) is an autosomal recessive disease caused by pathogenic variants in the glyoxylate reductase/hydroxypyruvate reductase (GRHPR) gene.
  • GRHPR glyoxylate reductase/hydroxypyruvate reductase
  • LDH lactic dehydrogenase
  • oxalate can combine with calcium to form calcium oxalate, a hard compound that is the main component of kidney and bladder stones.
  • GRHPR is expressed in all tissues, with the highest level in liver.
  • the disease is characterized by the accumulation of calcium oxalate in the kidney and urinary tract, which generally leads to the development of kidney stones and progressive kidney failure.
  • the clinical manifestations of PH2 are generally milder than those of type 1 primary hyperoxaluria, and approximately 10%of primary hyperoxaluria patients are diagnosed with PH2.
  • This Example provides for using ERT to treat a PH2 patient.
  • the hematopoietic stem and/or progenitor cells from a subject were isolated and engineered to express a functional GRHPR.
  • the engineered hematopoietic stem and/or progenitor cells were in vitro differentiated into mature red blood cells expressing GRHPR.
  • the in vitro and in vivo functions of these red blood cells can be examined using approaches as described in FIG. 2.
  • the method disclosed in this Example allows for about 7-10 times replication of hematopoietic stem and/or progenitor cells, and after in vitro differentiation, this method produces a large number of mature red blood cells suitable for delivering functional ⁇ -galactosidase A for ERT. Furthermore, when administered into the subject, these red blood cells can circulate in the subject’s body for a long time (about 120 days in human) and function to specifically metabolize its target glyoxylate. Alternatively, during the liver clearance step, the old and/or damaged red blood cells carrying the enzyme (i.e., GRHPR) can be targeted to macrophages for removal, restoring the liver’s metabolic activities.
  • GRHPR enzyme
  • the GRHPR cDNA (AGXT, NM_012203.1; SEQ ID NO: 4) , which encodes the polypeptide of human GRHPR (SEQ ID NO: 3) , was inserted into a murine stem cell virus (MSCV) vector using standard molecular cloning techniques known in the art.
  • MSCV murine stem cell virus
  • the filtered supernatant was subject to ultracentrifugation at a speed of 70000 RCF for 2 hours at a temperature of 4 °C. After ultracentrifugation, the supernatant was removed, and the pellet was re-suspended using stage 1 culture medium as disclosed in section 2.3, and the virus titer was quantified using ELISA known in the art. The virus was stored at -80 °C until use.
  • the whole blood was collected from a patient suffering from Fabry disease.
  • the peripheral blood mononuclear cells were isolated from the whole blood in the presence of a lymphocyte separation solution (Lymphoprep TM , STEMCELL Technologies) , using techniques known in the art.
  • the hematopoietic stem and/or progenitor cells were isolated using the Human Lineage Cell Depletion Set -DM (BD Biosciences) to obtain Lin - and CD34 + hematopoietic stem and/or progenitor cells, using techniques known in the art, for example an immune-selective method.
  • the isolated Lin - and CD34 + hematopoietic stem and/or progenitor cells were then immediately cultured in a stem cell culture medium (StemSpan TM SFEM, STEMCELL Technologies) containing a cytokine combination (StemSpan TM CC110 S, TEMCELL Technologies) and Penicillin-Streptomycin (Gibco) .
  • the cell culturing was carried for 4 days at 37 °C under 5%CO 2 , to allow the expansion of hematopoietic stem and/or progenitor cells.
  • the state 1 culture medium contains Iscove's Modified Dulbecco's Medium (IMDM, Sigma-Aldrich) , 10-15%fetal bovine serum (FBS, Gibco) , 5-10%human plasma (Plasma) , 1-4 mM glutamine, 1-2%albumin from bovine serum (BSA) , 300-600 ⁇ g/ml holo human transferrin (Sigma-Aldrich) , 8-13 ⁇ g/ml recombinant human insulin (Sigma-Aldrich) , 2%Penicillin-Streptomycin (Gibco) , 3-5 ng/ml recombinant human interleukin 3 (rhIL-3, Peprotech) , 4-7 U/ml recombinant human erythropoietin (rhEpo, Amgen) ,
  • IMDM Iscove's Modified Dulbecco's Medium
  • FBS fetal bovine serum
  • the cultured cells were infected with the MSCV-hGRHPR virus prepared in section 2.2.
  • the cells were re-suspended at a density of 1x10 6 using state 1 culture medium.
  • the virus dosage was determined based on a final virus concentration at 5 ⁇ 10 7 TU/ml -5 ⁇ 10 8 TU/ml, and depending on the infection volume, polybrene at 10 ⁇ g/ml was added to the concentrated virus solution for 5 minutes. After a brief incubation, the virus solution was added to the cells.
  • a centrifugal infection was performed using a horizontal rotor centrifuge at 500xg speed, 32 °C temperature, and for 90 minutes.
  • the medium was changed to fresh state 1 culture medium 12 hours after infection, and the cells were further cultured at 37 °C under 5%CO 2 .
  • the culture medium was changed to stage 2 culture medium and the cells were cultured at 37 °C under 5%CO 2 for about 7 days.
  • the state 2 culture medium contains Iscove's Modified Dulbecco's Medium (IMDM, Sigma-Aldrich) , 15%fetal bovine serum (FBS, Gibco) , 5-10%human plasma (Plasma) , 1-4 mM glutamine, 1-2%albumin from bovine serum (BSA) , 300-600 ⁇ g/ml holo human transferrin (Sigma-Aldrich) , 8-13 ⁇ g/ml recombinant human insulin (Sigma-Aldrich) , 2%Penicillin-Streptomycin (Gibco) , and 1-5 U/ml recombinant human erythropoietin (rhEpo, Amgen) .
  • IMDM Iscove's Modified Dulbecco's Medium
  • FBS fetal bovine serum
  • the culture medium was changed every 2-4 days, and the expression levels of CD235a, CD117, CD71, Hoechst 33342, and GFP were determined by flow cytometry, as shown in FIG. 3.
  • CD235a is blood glycophorin A, which is a single transmembrane glycoprotein expressed in mature red blood cells and erythroid precursor cells.
  • CD225a is a marker on the surface of red blood cells.
  • CD117 is a mast cell/stem cell growth factor receptor, which is expressed on the surface of hematopoietic stem cells and other cells.
  • CD71 is transferrin receptor 1, which is a transmembrane glycoprotein composed of two disulfide-bonded monomers linked by two disulfide bonds. Each monomer binds to a transferrin molecule to produce an iron-transferrin-transferrin receptor complex that enters the cell by endocytosis.
  • Hoechst33342 is an established fluorescent dye used to stain cellular DNA.
  • GFP denotes green fluorescent protein. This protein is encoded by a fluorescent reporter gene that is operably linked to MSCV-hGRHPR via T2A, and is therefore co-expressed with GRHPR. The strength of the GFP signal suggests the expression level of ⁇ Gal A. For example, a positive GFP signal indicates that GRHPR is expressed in the infected cells.
  • FIG. 10 shows FACS analyses of protein expression levels in red blood cells engineered to express hGRHPR, after in vitro differentiation for 10 days.
  • the following labeling markers are used: CD235a, CD117, CD71, Hoechst33342, and GFP.
  • FIG. 11 shows FACS analyses of protein expression levels in red blood cells engineered to express hGRHPR, after in vitro differentiation for 18 days.
  • the following labeling markers are used: CD235a, CD117, CD71, Hoechst33342, and GFP.
  • FIG. 9 shows Western blot analyses of expression levels of GRHPR in red blood cells infected with MSCV control vector or MSCV-hGRHPR. The data shows that GRHPR is expressed in red blood cells differentiated from hematopoietic stem and/or progenitor cells infected with MSCV-hGRHPR.
  • FIG. 12 shows a representative GRHPR enzyme activity assay, using proteins extracted from red blood cells that have been in vitro differentiated for 10 days. The data indicates that the in vitro differentiation process successfully produced red blood cells expressing functional GRHPR.
  • FIG. 13 shows a representative GRHPR enzyme activity assay, using proteins extracted from red blood cells that have been in vitro differentiated for 18 days. The data indicates that those enucleated mature red blood cells (filtered through a 10 ⁇ m filter) at the end of the in vitro differentiation process (i.e., after in vitro differentiation for 18 days) successfully produced red blood cells expressing functional GRHPR.
  • red blood cells were intravenously injected into the tail vein of each immuno-deficient mouse, and the distributions of the red blood cells were analyzed by live imaging (FIG. 4) and/or immunohistochemistry (FIG. 5) routinely used in the art.
  • the survival of red blood cells expressing GRHPR was compared to wild type red blood cells that do not express GRHPR.
  • the in vivo function of the red blood cells can be examined using a PH2 disease mouse model (GRHPR KO mouse) .
  • the red blood cells are administered into the GRHPR KO mouse, and the in vivo efficacy can be monitored by checking the levels of glyoxylate and/or pathological conditions of tissues such as kidney.

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Abstract

L'invention concerne un procédé de traitement d'un sujet ayant une maladie de stockage lysosomal, comprenant l'administration de globules rouges ou de cellules souches et/ou progénitrices hématopoïétiques Lin - génétiquement modifiées pour exprimer un polypeptide, le polypeptide pouvant être un polypeptide α-Gal A ou GRHPR. L'invention concerne également un procédé de production de globules rouges à partir de cellules souches et/ou progénitrices hématopoïétiques Lin - in vitro, des globules rouges produits par le procédé, et des compositions comprenant les globules rouges.
PCT/CN2020/121522 2019-10-17 2020-10-16 Ingénierie des globules rouges pour le traitement de maladies lysosomales WO2021073607A1 (fr)

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Citations (4)

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WO2017103612A1 (fr) * 2015-12-16 2017-06-22 Ucl Business Plc Traitement et/ou prévention de maladies de surcharge lysosomale
WO2018170239A1 (fr) * 2017-03-15 2018-09-20 The Regents Of The University Of California Méthodes de traitement de troubles du stockage lysosomal
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