Methods for genetic modification of hematopoietic cells

Abstract

The present invention relates generally to methods for the genetic modification of hematopoietic cells. In particular, the present invention relates to the use of prostaglandin E2 (PGE2), poloxamer, and protamine sulfate to enhance transduction by recombinant retroviral vectors. The compositions and methods of the present disclosure are particularly suitable for use in gene therapy, including the treatment of monogenic genetic diseases and disorders. [Selected Figure] Figure 1

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 712,146, filed July 30, 2018, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application is being filed electronically via EFS-Web and contains a sequence listing submitted electronically in .txt format. The .txt file contains a sequence listing entitled "ROPA_01001WO_SeqList_ST25.txt", created on July 30, 2019, and is 57 kilobytes in size. The sequence listing contained in this .txt file is a part of the specification and is incorporated herein by reference in its entirety.

[0003] FIELD OF THEINVENTION The present invention relates generally to a method for genetically modifying hematopoietic cells, and in particular to enhancing transduction by recombinant retroviral vectors using a combination of two or more transduction enhancers selected from prostaglandin E2 (PGE2), recombinant fibronectin fragments, poloxamer, and protamine sulfate. [Background technology]

[0004] 2. Background of the Invention Ex vivo gene transfer into target cells is a method used clinically for cell and gene therapy. A recombinant retroviral vector (e.g., a recombinant lentiviral vector) can be used to deliver a polynucleotide to a cell (e.g., a hematopoietic cell). Contacting a target hematopoietic cell with a recombinant retroviral (e.g., lentiviral) vector results in gene delivery to the hematopoietic cell, a method known as transduction. The hematopoietic cell may then be administered to a subject with the intention that the hematopoietic cell will engraft itself into the bone marrow of the subject.

[0005] Retroviral (e.g., lentiviral) vector transduction is often limited in efficiency, and transduction efficiency is often a major obstacle to successful gene therapy. Thus, there remains an unmet need for compositions and methods suitable for application to hematopoietic cells in a clinical context. The present disclosure provides such compositions and methods, as well as others. Summary of the Invention

[0006] The present invention relates generally to methods for genetic modification of hematopoietic cells. In particular, the present invention relates to enhancing transduction by recombinant retroviral vectors using a combination of two or more transduction enhancers, where at least two of the transduction enhancers of the combination are selected from prostaglandin E2 (PGE2), poloxamer, recombinant fibronectin fragments, and / or protamine sulfate. In some embodiments, the combination includes three or more transductions including prostaglandin E2 (PGE2), poloxamer, and protamine sulfate. In some embodiments, the combination includes four or more transductions including prostaglandin E2 (PGE2), poloxamer, recombinant fibronectin fragments, and protamine sulfate. The compositions and methods of the present disclosure are particularly suitable for gene therapy applications, including the treatment of genetic diseases and disorders caused by a single gene. Advantageously, the methods of the present disclosure result in less toxicity (longer survival) of the transduced cell population compared to transduction without the transduction enhancer.

[0007] In one aspect, the disclosure provides a method for genetically modifying a hematopoietic cell, comprising contacting the hematopoietic cell with a poloxamer, contacting the hematopoietic cell with prostaglandin E2 (PGE2) or a derivative thereof, and contacting the hematopoietic cell with a recombinant retroviral vector.

[0008] In one aspect, the disclosure provides a method for genetically modifying a hematopoietic cell, comprising providing a hematopoietic cell, contacting the hematopoietic cell with a poloxamer, contacting the hematopoietic cell with prostaglandin E2 (PGE2) or a derivative thereof, and contacting the hematopoietic cell with a recombinant retroviral vector.

[0009] In certain embodiments, the recombinant retroviral vector is a recombinant lentiviral vector.

[0010] In certain embodiments, the hematopoietic cells are engineered.

[0011] In certain embodiments, the providing step comprises enrichment for CD34+ cells.

[0012] In certain embodiments, hematopoietic cells are cultured in vessels coated with recombinant fibronectin fragments.

[0013] In some embodiments, the poloxamer is selected from the group consisting of poloxamer 288, poloxamer 335, poloxamer 338, and poloxamer 407.

[0014] In some embodiments, the poloxamer is poloxamer 338 (LentiBOOST).

[0015] In certain embodiments, the PGE2 or a derivative thereof is modified.

[0016] In certain embodiments, the PGE2 or derivative thereof is 16,16-dimethylPGE2 (dmPGE2).

[0017] In certain embodiments, the PGE2 or derivative thereof is unmodified.

[0018] In some embodiments, the method further comprises contacting the hematopoietic cells with protamine sulfate and / or recombinant fibronectin fragments. In some embodiments, the recombinant fibronectin fragments may be present in the liquid culture medium or coated on the culture dish. In some embodiments, the cells may be pretreated by culturing in a dish containing recombinant fibronectin and / or the recombinant fibronectin fragments may be present in the liquid medium during transduction.

[0019] In certain embodiments, the contacting steps are performed simultaneously or over overlapping periods of time.

[0020] In one embodiment, the concentration of PGE2 or a derivative thereof is 5 to 30 μg / mL.

[0021] In one embodiment, the concentration of PGE2 or a derivative thereof is about 10 μg / mL.

[0022] In one embodiment, the concentration of the poloxamer is 200 to 1200 μg / mL.

[0023] In one embodiment, the concentration of the poloxamer is about 1000 μg / mL.

[0024] In one embodiment, the concentration of protamine sulfate is 4 to 10 μg / mL.

[0025] In one embodiment, the concentration of protamine sulfate is about 4 μg / mL.

[0026] In one embodiment, the concentration of the recombinant fibronectin fragment, for example RetroNectin, is about 5 to about 50 ug / mL when used in liquid culture medium.

[0027] In one embodiment, the concentration of the recombinant fibronectin fragment, eg, RetroNectin, is about 20 μg / mL when used in liquid culture medium.

[0028] In a second aspect, the disclosure provides a method for enhancing recombinant retroviral vector-mediated genetic modification of hematopoietic cells, the method comprising ex vivo treating or contacting hematopoietic cells with an effective amount of PGE2 or a derivative thereof and with an effective amount of a poloxamer; and exposing or contacting the hematopoietic cells with a recombinant retroviral vector comprising a polynucleotide comprising a gene of interest, wherein the efficacy of viral transduction of the recombinant retroviral vector is enhanced compared to transduction of hematopoietic cells by the recombinant retroviral vector in the absence of PGE2 and poloxamer.

[0029] In certain embodiments, the method further comprises treating or contacting the hematopoietic cells ex vivo with an effective amount of protamine sulfate and / or a recombinant fibronectin fragment.

[0030] In certain embodiments, the gene of interest complements a defect in a gene associated with a monogenic genetic disease or disorder.

[0031] In certain embodiments, the gene of interest is selected from the group consisting of RPK, ITGB2, FANCA, FANCC, FANCG, TCIRG1, CLCN7, TNFSF11, PLEKHM1, TNFRSF11A, and OSTM1. In certain embodiments, the gene of interest encodes a protein encoded by any of these genes, or encodes a functional fragment or functional variant of any of these genes. In certain embodiments, the gene or protein is a human gene or protein.

[0032] In certain embodiments, the methods combat the clinical sequelae or ameliorate a monogenic genetic disease or disorder.

[0033] In certain embodiments, the monogenic disease or disorder is selected from the group consisting of Fanconi Anemia (including any of the complementation groups), Leukocyte Adhesion Deficiency Type 1, Pyruvate Kinase Deficiency, and Infantile Malignant Osteoporosis.

[0034] In certain embodiments, the hematopoietic cells are CD34 enriched cells, optionally hematopoietic cells, bone marrow (BM) derived cells, cord blood (CB) derived cells, or mobilized peripheral blood (mPB) cells. In certain embodiments, the hematopoietic cells are obtained from a subject to be treated with the recombinantly modified hematopoietic cells.

[0035] In a third aspect, the disclosure provides a method for recombinant retroviral vector-mediated genetic modification of hematopoietic cells, comprising the steps of: preparing CD34-enriched cells from a biological sample (optionally peripheral blood) obtained from a subject treated with G-CSF or an analogue thereof (optionally filgrastim, sargramostim, or pegfilgrastim) and / or plerixafor; and detecting an increase in the expression of a gene encoding ... and genetically modifying the CD34-enriched cells with a recombinant retroviral vector comprising a gene or a polynucleotide encoding a CD34-enriched polypeptide or a functional fragment thereof, and a eukaryotically active promoter sequence operably linked thereto; wherein the genetically modifying step comprises contacting the CD34-enriched cells with the recombinant retroviral vector, PGE2 and a poloxamer, and optionally with protamine sulfate and / or a recombinant fibronectin fragment.

[0036] The present disclosure relates to an in vitro method for recombinant retroviral vector-mediated genetic modification of hematopoietic cells, comprising: preparing CD34-enriched cells from a biological sample (optionally peripheral blood) obtained from a subject treated with G-CSF or an analog thereof (optionally filgrastim, sargramostim, or pegfilgrastim) and / or plerixafor; and genetically modifying the CD34-enriched cells with a recombinant retroviral vector for a disease or disorder selected from Fanconi anemia, leukocyte adhesion deficiency type I, pyruvate kinase deficiency, or infantile malignant osteoporosis; The retroviral vector comprises a polynucleotide encoding a Fanconi anemia complementation group (FANC) gene, ITGB2, R-type pyruvate kinase, CLCN7, OSTM1, TCIRG1, TNFSF11, PLEKHM1, TNFRSF11A, or a gene encoding a functional variant or functional fragment thereof, and a eukaryotically active promoter sequence operably linked thereto; and the genetically modifying step comprises contacting the CD34-enriched cells with PGE2 and a poloxamer, and optionally with protamine sulfate.

[0037] The present disclosure provides a method of treating a genetic disease or disorder in a subject in need thereof, comprising providing to the subject genetically modified hematopoietic cells that express a polypeptide that is missing or mutated due to the genetic disease or disorder in the subject. In certain embodiments, CD34-enriched cells obtained from a biological sample (optionally peripheral blood) obtained from a subject after treating the subject with G-CSF or an analog thereof (optionally filgrastim, sargramostim, or pegfilgrastim) and / or plerixafor are genetically modified by contacting the CD34-enriched cells with a recombinant retroviral vector that includes an expression cassette that includes a polynucleotide sequence encoding a polypeptide in the presence of at least two transduction enhancers selected from prostaglandin E2 (PGE2), poloxamer, recombinant fibronectin fragment, and / or protamine sulfate, and providing the resulting genetically modified cells to the subject. In certain embodiments, the disease or disorder is selected from Fanconi anemia, leukocyte adhesion deficiency type I, pyruvate kinase deficiency, or infantile malignant osteoporosis; and the recombinant retroviral vector comprises a polynucleotide comprising a Fanconi anemia complementation group (FANC) gene, ITGB2, R-type pyruvate kinase, CLCN7, OSTM1, TCIRG1, TNFSF11, PLEKHM1, TNFRSF11A, or a gene encoding a functional variant or functional fragment thereof, and a eukaryotically active promoter sequence operably linked thereto.

[0038] In certain embodiments, the disclosure provides a method of treating Fanconi anemia in a subject in need thereof, comprising administering hematopoietic cells produced by genetically modifying hematopoietic cells with a recombinant retroviral vector comprising a polynucleotide encoding a Fanconi anemia complementation group (FANC) gene, or a gene encoding a functional variant or functional fragment thereof, according to the methods disclosed herein.

[0039] In certain embodiments, the disclosure provides a method of treating leukocyte adhesion deficiency type I in a subject in need thereof, comprising administering hematopoietic cells produced by genetically modifying hematopoietic cells with a recombinant retroviral vector comprising a polynucleotide encoding the ITGB2 gene, or a gene encoding a functional variant or functional fragment thereof, according to the methods disclosed herein.

[0040] In certain embodiments, the disclosure provides a method of treating pyruvate kinase deficiency in a subject in need thereof, comprising administering hematopoietic cells produced by genetically modifying hematopoietic cells with a recombinant retroviral vector comprising a polynucleotide encoding an R-type pyruvate kinase gene, or a gene encoding a functional variant or functional fragment thereof, according to the methods disclosed herein.

[0041] In certain embodiments, the disclosure provides a method of treating infantile malignant osteoporosis in a subject in need thereof, comprising administering hematopoietic cells produced by genetically modifying hematopoietic cells with a recombinant retroviral vector comprising a polynucleotide encoding a CLCN7, OSTM1, TCIRG1, TNFSF11, PLEKHM1 or TNFRSF11A gene, or a gene encoding a functional variant or functional fragment thereof, according to the methods disclosed herein.

[0042] In a further related aspect, the disclosure provides a method of producing a hematopoietic cell population comprising at least 80% or at least 90% genetically modified hematopoietic cells, comprising ex vivo contacting the hematopoietic cells with a recombinant retroviral vector (optionally a lentiviral vector) comprising a polynucleotide comprising a gene of interest or encoding a polypeptide of interest, the contacting occurring in the presence of PGE2 or a derivative thereof, optionally human PGE2 or 16,16-dimethyl PGE2 (dmPGE2), and a poloxamer, optionally poloxamer 338 (LentiBOOST™). The cells may be contacted with the retroviral vector under conditions and for a time sufficient to permit transduction of the cells by the retroviral vector, e.g., for at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 12 hours, at least 16 hours, or at least 24 hours in a suitable medium. In some embodiments, the cells are transduced either once or two consecutive times, e.g., after priming, with each transduction cycle lasting 12-24 hours or 16-18 hours. In some embodiments, the cells are contacted with the retroviral vector and transduction enhancer for the same or overlapping time periods. After transduction, the cells may be prepared in a freezing mix (e.g., CryoStor CS5, BioLife Solutions, Bothell, WA) and cryopreserved for later use. In certain embodiments of this and other aspects, the poloxamer is selected from the group consisting of poloxamer 288, poloxamer 335, poloxamer 338, and poloxamer 407. In certain embodiments, the poloxamer is poloxamer 338 (LentiBOOST™). In certain embodiments, the PGE2 or derivative thereof is unmodified or modified, such as 16,16-dimethyl PGE2 (dmPGE2). In certain embodiments, the method further comprises contacting the hematopoietic cells with protamine sulfate and / or a recombinant fibronectin fragment.In some embodiments, the concentration of PGE2 or a derivative thereof is 5-30 μg / mL, or about 10 μg / mL. In some embodiments, the concentration of poloxamer is 200-1200 μg / mL, or about 1000 μg / mL. In some embodiments, the concentration of protamine sulfate is 4-10 μg / mL, or about 4 μg / mL. In some embodiments, the polynucleotide complements a defect in a gene associated with a monogenic genetic disease or disorder. In some embodiments, the polypeptide of interest is selected from the group consisting of RPK, ITGB2, FANCA, FANCC, FANCG, TCIRG1, CLCN7, TNFSF11, PLEKHM1, TNFRSF11A, and OSTM1. In some embodiments, the disease or disorder is a monogenic disease or disorder, e.g., selected from the group consisting of Fanconi Anemia, Leukocyte Adhesion Deficiency Type 1, Pyruvate Kinase Deficiency, and Infantile Malignant Osteopetrosis. In certain embodiments, the hematopoietic cells are CD34-enriched or CD34+ hematopoietic cells, optionally bone marrow (BM)-derived cells, cord blood (CB)-derived cells, or mobilized peripheral blood (mPB) cells. In some embodiments, the hematopoietic cell population has a VCN / cell of at least 1.0, at least 1.5, at least 2.0, or at least 2.5.

[0043] In a related aspect, the disclosure provides a hematopoietic cell population comprising at least 80% or at least 90% genetically modified hematopoietic cells, the hematopoietic cell population being produced by the disclosed method.

[0044] In another related aspect, the disclosure provides a method of treating a genetic disease or disorder in a subject in need thereof, comprising providing to the subject a population of hematopoietic cells comprising at least 80% or at least 90% genetically modified hematopoietic cells produced by the disclosed method, the hematopoietic cells being obtained from the subject prior to ex vivo contact with a retroviral vector, and the gene of interest encodes a functional polypeptide that is mutated or deleted in the subject due to the genetic disease or disorder. In some embodiments, the polypeptide is selected from the group consisting of RPK, ITGB2, FANCA, FANCC, FANCG, TCIRG1, CLCN7, TNFSF11, PLEKHM1, TNFRSF11A, and OSTM1. In some embodiments, the disease or disorder is a monogenic disease or disorder, e.g., selected from the group consisting of Fanconi Anemia, Leukocyte Adhesion Deficiency Type 1, Pyruvate Kinase Deficiency, and Infantile Malignant Osteopetrosis. In certain embodiments, the hematopoietic cells are CD34-enriched cells, optionally bone marrow (BM)-derived cells, cord blood (CB)-derived cells, or mobilized peripheral blood (mPB) cells.

[0045] In various embodiments of any of the aspects and embodiments disclosed herein, the cells are transduced on a dish coated with a recombinant fibronectin fragment, e.g., RetroNectin™. In various embodiments of any of the aspects and embodiments disclosed herein, the cells are pretreated by culturing on a dish coated with a recombinant fibronectin fragment, e.g., RetroNectin™, before and / or during transduction. In some embodiments, the cells are transduced in liquid medium containing Retro-Nectin. In some embodiments, the cells are pretreated by culturing on a dish coated with a recombinant fibronectin fragment and are also transduced in liquid medium in the presence of the recombinant fibronectin fragment and other TEs. That is, some embodiments expose the cells to recombinant fibronectin before exposure to other TEs during transduction, while in some embodiments, the cells are exposed to recombinant fibronectin for the same or overlapping period as the other TEs.

[0046] In various embodiments of any of the aspects and embodiments disclosed herein, the cells are transduced in the presence of prostaglandin E2 (PGE2), poloxamer (e.g., LentiBoost™), recombinant fibronectin fragments (e.g., RetroNectin™), and protamine sulfate.

[0047] [The present invention 1001] 1. A method for genetically modifying hematopoietic cells, comprising: (a) providing hematopoietic cells; (b) contacting the hematopoietic cells with a poloxamer; (c) contacting the hematopoietic cells with prostaglandin E2 (PGE2) or a derivative thereof; (d) contacting the hematopoietic cells with a recombinant retroviral vector; A method comprising: [The present invention 1002] 1001. The method of claim 1001, wherein said recombinant retroviral vector is a recombinant lentiviral vector. [The present invention 1003] The method of claim 10, wherein said hematopoietic cells are engineered. [The present invention 1004] The method of claim 1003, wherein said hematopoietic cells are CD34 enriched cells. [The present invention 1005] The method of claim 1001, wherein the poloxamer is selected from the group consisting of poloxamer 288, poloxamer 335, poloxamer 338, and poloxamer 407. [The present invention 1006] The method of the present invention 1005, wherein the poloxamer is poloxamer 338 (LentiBOOST(TM)). [The present invention 1007] 1001. The method of claim 10, wherein said PGE2 or a derivative thereof is modified. [The present invention 1008] The method of the present invention 1007, wherein the PGE2 or a derivative thereof is 16,16-dimethylPGE2 (dmPGE2). [The present invention 1009] 1001. The method of claim 10, wherein said PGE2 or derivative thereof is unmodified. [The present invention 1010] Any of the methods of the invention further comprising contacting the hematopoietic cells with protamine sulfate, and / or a recombinant fibronectin fragment, optionally RetroNectin™. [The present invention 1011] The method of any one of claims 1001 to 1010, wherein the contacting steps are performed over the same period of time or over overlapping periods of time. [The present invention 1012] The method according to any one of claims 1001 to 1011, wherein the concentration of the PGE2 or a derivative thereof is 5 to 30 µg / mL. [The present invention 1013] The method of claim 10, wherein the concentration of the PGE2 or derivative thereof is about 10 μg / mL. [The present invention 1014] Any of the methods of claims 1001 to 1013, wherein the concentration of the poloxamer is 200 to 1200 μg / mL. [The present invention 1015] The method of claim 10, wherein the concentration of the poloxamer is about 1000 μg / mL. [The present invention 1016] The method according to any one of claims 1010 to 1015, wherein the concentration of the protamine sulfate is 4 to 10 µg / mL. [The present invention 1017] The method of claim 1016, wherein the concentration of said protamine sulfate is about 4 μg / mL. [The present invention 1018] 1. A method for enhancing recombinant retroviral vector-mediated genetic modification of hematopoietic cells, comprising: (a) contacting hematopoietic cells ex vivo with an effective amount of PGE2 or a derivative thereof, optionally human PGE2 or 16,16-dimethyl PGE2 (dmPGE2), and with an effective amount of a poloxamer, optionally poloxamer 338 (LentiBOOST™); (b) contacting the hematopoietic cells with a recombinant retroviral vector comprising a polynucleotide that contains a gene of interest or that encodes a polypeptide of interest; Including, A method, wherein the efficacy of viral transduction of the recombinant retroviral vector is enhanced compared to transduction of hematopoietic cells by the recombinant retroviral vector in the absence of PGE2 or a derivative thereof and poloxamer. [The present invention 1019] The method of claim 1018, further comprising contacting said hematopoietic cells ex vivo with an effective amount of protamine sulfate and / or a recombinant fibronectin polypeptide or variant thereof. [The present invention 1020] The method of any one of claims 10 to 18, wherein said gene of interest complements a defect in a gene associated with a monogenic genetic disease or disorder. [The present invention 1021] 1020. The method of claim 1019, wherein said gene of interest or polypeptide of interest is selected from the group consisting of RPK, ITGB2, FANCA, FANCC, FANCG, TCIRG1, CLCN7, TNFSF11, PLEKHM1, TNFRSF11A and OSTM1. [The present invention 1022] The method of the present invention 1018 for preventing or ameliorating a genetic disease or disorder caused by a single gene. [The present invention 1023] The method of claim 1022, wherein said monogenic disease or disorder is selected from the group consisting of Fanconi anemia, leukocyte adhesion deficiency type 1, pyruvate kinase deficiency, and infantile malignant osteopetrosis. [The present invention 1024] The method of the present invention, wherein said hematopoietic cells are CD34 enriched cells, optionally hematopoietic cells, bone marrow (BM) derived cells, cord blood (CB) derived cells, or mobilized peripheral blood (mPB) cells. [The present invention 1025] 1. A method for recombinant retroviral vector-mediated genetic modification of hematopoietic cells, comprising: (a) preparing CD34-enriched cells from a biological sample (optionally, peripheral blood) obtained from a subject treated with G-CSF or an analog thereof (optionally, filgrastim, sargramostim, or pegfilgrastim) and / or plerixafor; (b) genetically modifying the CD34-enriched cells with a recombinant retroviral vector comprising a polynucleotide encoding a Fanconi anemia complementation group (FANC) gene, ITGB2, R-type pyruvate kinase, OSTM1, TCIRG1, or a gene encoding a functional variant or functional fragment thereof, and an active eukaryotic promoter sequence operably linked thereto; Including, The method, wherein the genetically modifying step comprises contacting the CD34-enriched cells with the recombinant retroviral vector, PGE2 and poloxamer, and optionally with protamine sulfate and / or a recombinant fibronectin polypeptide or variant thereof. [The present invention 1026] The method of claim 1025, wherein said CD34 enriched cells are selected from the group consisting of hematopoietic cells, bone marrow (BM) derived cells, cord blood (CB) derived cells, or mobilized peripheral blood (mPB) cells. [The present invention 1027] 1. A method for transducing hematopoietic cells with a lentiviral vector, comprising: Culturing the cells in a liquid medium containing a lentiviral vector. Including, The method, wherein the liquid medium comprises poloxamer and prostaglandin E2 (PGE2) or a derivative thereof. [The present invention 1028] The method of claim 1027, wherein the liquid medium comprises protamine sulfate. [The present invention 1029] 1. A method for transducing hematopoietic cells with a lentiviral vector, comprising: (a) providing a lentiviral vector in a solution comprising a poloxamer and prostaglandin E2 (PGE2) or a derivative thereof; (b) providing hematopoietic cells in a liquid culture medium; (c) adding the solution to a liquid culture medium; A method comprising: [The present invention 1030] The method of claim 1029, wherein the liquid medium comprises protamine sulfate. [The present invention 1031] The method according to any one of claims 1025 to 1030, wherein the lentivirus vector is a recombinant lentivirus vector. [The present invention 1032] 13. The method of any one of claims 1025 to 1031, wherein said hematopoietic cells are engineered. [The present invention 1033] The method according to any one of claims 1025 to 1032, wherein the hematopoietic cells are cells enriched for CD34. [The present invention 1034] Any of the methods of claims 1025 to 1033, wherein the poloxamer is selected from the group consisting of poloxamer 288, poloxamer 335, poloxamer 338, and poloxamer 407. [The present invention 1035] Any of the methods of claims 1025 to 1033, wherein the poloxamer is poloxamer 338 (LentiBOOST (trademark)). [The present invention 1036] The method of any one of claims 1025 to 1035, wherein the PGE2 or the derivative thereof is modified. [The present invention 1037] The method according to any one of claims 1025 to 1036, wherein the PGE2 or a derivative thereof is 16,16-dimethylPGE2 (dmPGE2). [The present invention 1038] The method according to any one of claims 1025 to 1037, wherein the PGE2 or a derivative thereof is unmodified. [The present invention 1039] The method according to any one of claims 1025 to 1038, wherein the concentration of the PGE2 or a derivative thereof is 5 to 30 µg / mL. [The present invention 1040] The method according to any one of claims 1025 to 1039, wherein the concentration of the PGE2 or a derivative thereof is about 10 µg / mL. [The present invention 1041] The method according to any one of claims 1025 to 1040, wherein the concentration of the poloxamer is 200 to 1200 μg / mL. [The present invention 1042] Any of the methods of claims 1025 to 1041, wherein the concentration of the poloxamer is about 1000 μg / mL. [The present invention 1043] The method according to any one of claims 1025 to 1042, wherein the concentration of the protamine sulfate is 4 to 10 µg / mL. [The present invention 1044] The method according to any one of claims 1025 to 1043, wherein the concentration of the protamine sulfate is about 4 µg / mL. [The present invention 1045] Any of the methods of claims 1025 to 1044, wherein the lentiviral vector comprises a sequence encoding a polypeptide of interest selected from the group consisting of RPK, ITGB2, FANCA, FANCC, FANCG, TCIRG1, CLCN7, TNFSF11, PLEKHM1, TNFRSF11A and OSTM1. [The present invention 1046] Any of the methods according to claims 1025 to 1045, for preventing or ameliorating a genetic disease or disorder caused by a single gene. [The present invention 1047] The method of claim 1046, wherein said monogenic disease or disorder is selected from the group consisting of Fanconi anemia, leukocyte adhesion deficiency type 1, pyruvate kinase deficiency, and infantile malignant osteopetrosis. [The present invention 1048] Any of the methods of claims 1025 to 1047, wherein the hematopoietic cells are CD34 enriched cells, optionally hematopoietic cells, bone marrow (BM) derived cells, cord blood (CB) derived cells, or mobilized peripheral blood (mPB) cells. [The present invention 1049] 1. A method for treating, ameliorating, and / or preventing a disease or disorder in a subject in need thereof, comprising: providing said subject with hematopoietic cells transduced with a retroviral vector comprising a polynucleotide comprising a sequence encoding a therapeutic protein operably linked to a promoter sequence. Including, The method, wherein the cells are transduced by contacting the cells with a recombinant retroviral vector and with two or more, three or more, or four or more transduction enhancers selected from the group consisting of prostaglandin E2 (PGE2) or a derivative thereof, poloxamer, recombinant fibronectin fragments, and protamine sulfate. [The present invention 1050] The method of claim 1049, wherein the cell is contacted with PGE2 and the poloxamer. [The present invention 1051] The method of claim 1049 or 1050, wherein said recombinant retroviral vector is a recombinant lentiviral vector. [The present invention 1052] The method according to any one of claims 1049 to 1051, wherein the hematopoietic cells are cells enriched for CD34. [The present invention 1053] The method of any one of claims 1049 to 1052, wherein the poloxamer is selected from the group consisting of poloxamer 288, poloxamer 335, poloxamer 338, and poloxamer 407. [The present invention 1054] Any of the methods of claims 1049 to 1053, wherein the poloxamer is poloxamer 338 (LentiBOOST (trademark)). [The present invention 1055] The method according to any one of claims 1049 to 1054, wherein the PGE2 or the derivative thereof is modified. [The present invention 1056] The method of the present invention, wherein the PGE2 or a derivative thereof is 16,16-dimethylPGE2 (dmPGE2). [The present invention 1057] The method of any of claims 1049 to 1054, wherein the PGE2 or a derivative thereof is unmodified. [The present invention 1058] The method according to any one of claims 1049 to 1057, comprising contacting the hematopoietic cells with protamine sulfate. [The present invention 1059] The method according to any one of claims 1049 to 1058, wherein the concentration of the PGE2 or a derivative thereof is 5 to 30 µg / mL. [The present invention 1060] The method according to any one of claims 1049 to 1059, wherein the concentration of the PGE2 or derivative thereof is about 10 µg / mL. [The present invention 1061] The method according to any one of claims 1049 to 1060, wherein the concentration of the poloxamer is 200 to 1200 μg / mL. [The present invention 1062] The method of any one of claims 1049 to 1061, wherein the concentration of the poloxamer is about 1000 μg / mL. [The present invention 1063] The method according to any one of claims 1049 to 1062, wherein the concentration of the protamine sulfate is 4 to 10 µg / mL. [The present invention 1064] The method according to any one of claims 1049 to 1063, wherein the concentration of the protamine sulfate is about 4 µg / mL. [The present invention 1065] The method of any of claims 1049 to 1064, wherein said polynucleotide complements a defect in a gene associated with a monogenic genetic disease or disorder. [The present invention 1066] 6. The method of any one of claims 1049 to 1065, wherein the therapeutic protein is selected from the group consisting of RPK, ITGB2, FANCA, FANCC, FANCG, TCIRG1, CLCN7, TNFSF11, PLEKHM1, TNFRSF11A and OSTM1. [The present invention 1067] The method according to any one of claims 1049 to 1066, wherein the disease or disorder is a genetic disease or disorder caused by a single gene. [The present invention 1068] The method of claim 1067, wherein said monogenic disease or disorder is selected from the group consisting of Fanconi anemia, leukocyte adhesion deficiency type 1, pyruvate kinase deficiency, and infantile malignant osteopetrosis. [The present invention 1069] The method according to any one of claims 1049 to 1068, wherein the hematopoietic cells are CD34 enriched cells, optionally hematopoietic cells, bone marrow (BM) derived cells, cord blood (CB) derived cells, or mobilized peripheral blood (mPB) cells. [The present invention 1070] The method of any one of claims 1049 to 1069, wherein the hematopoietic cells have a VCN / cell ratio of at least 1.0, at least 1.5, at least 2.0, or at least 2.5. [The present invention 1071] The method of any of claims 1049 to 1070, wherein said hematopoietic cells have a transduction efficiency of at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or at least 100%. [The present invention 1072] 1. A method for producing a hematopoietic cell population comprising at least 80% or at least 90% genetically modified hematopoietic cells, comprising: Contacting hematopoietic cells ex vivo with a recombinant retroviral vector (optionally a lentiviral vector) comprising a polynucleotide containing a gene of interest or encoding a polypeptide of interest. Including, The method, wherein the contacting occurs in the presence of PGE2 or a derivative thereof, optionally human PGE2 or 16,16-dimethyl PGE2 (dmPGE2), and a poloxamer, optionally poloxamer 338 (LentiBOOST(TM)). [The present invention 1073] The method of claim 1072, wherein the poloxamer is selected from the group consisting of poloxamer 288, poloxamer 335, poloxamer 338, and poloxamer 407. [The present invention 1074] Any of the methods of claims 1072 to 1073, wherein the poloxamer is poloxamer 338 (LentiBOOST (trademark)). [The present invention 1075] The method according to any one of claims 1072 to 1074, wherein the PGE2 or the derivative thereof is modified. [The present invention 1076] The method of the present invention, wherein the PGE2 or a derivative thereof is 16,16-dimethylPGE2 (dmPGE2). [The present invention 1077] The method of any one of claims 1072 to 1074, wherein said PGE2 or a derivative thereof is unmodified. [The present invention 1078] The method of any of claims 1072 to 1077, further comprising contacting the hematopoietic cells with protamine sulfate. [The present invention 1079] The method according to any one of claims 1072 to 1078, wherein the concentration of the PGE2 or a derivative thereof is 5 to 30 µg / mL. [The present invention 1080] The method of claim 1079, wherein the concentration of the PGE2 or derivative thereof is about 10 μg / mL. [The present invention 1081] Any of the methods of claims 1072 to 1080, wherein the concentration of the poloxamer is 200 to 1200 μg / mL. [The present invention 1082] Any of the methods of claims 1049 to 1081, wherein the concentration of the poloxamer is about 1000 μg / mL. [The present invention 1083] The method according to any one of claims 1072 to 1082, wherein the concentration of the protamine sulfate is 4 to 10 µg / mL. [The present invention 1084] The method of claim 1083, wherein the concentration of said protamine sulfate is about 4 μg / mL. [The present invention 1085] The method of any of claims 1072 to 1084, wherein said polynucleotide complements a defect in a gene associated with a monogenic genetic disease or disorder. [The present invention 1086] 6. The method of any of claims 1072 to 1085, wherein the polypeptide of interest is selected from the group consisting of RPK, ITGB2, FANCA, FANCC, FANCG, TCIRG1, CLCN7, TNFSF11, PLEKHM1, TNFRSF11A and OSTM1. [The present invention 1087] The method of claim 1085, wherein the disease or disorder is a genetic disease or disorder caused by a single gene. [The present invention 1088] The method of claim 1087, wherein said monogenic disease or disorder is selected from the group consisting of Fanconi anemia, leukocyte adhesion deficiency type 1, pyruvate kinase deficiency, and infantile malignant osteopetrosis. [The present invention 1089] Any of the methods of claims 1072 to 1088, wherein the hematopoietic cells are CD34-enriched cells, and are optionally bone marrow (BM)-derived cells, cord blood (CB)-derived cells, or mobilized peripheral blood (mPB) cells. [The present invention 1090] The method of any of claims 1049 to 1069, wherein the hematopoietic cell population has a VCN / cell ratio of at least 1.0, at least 1.5, at least 2.0, or at least 2.5. [The present invention 1091] A hematopoietic cell population comprising at least 80% or at least 90% genetically modified hematopoietic cells, the hematopoietic cell population being produced by any of the methods of the present inventions 1072 to 1090. [The present invention 1092] 1. A method of treating a genetic disease or disorder in a subject in need thereof, comprising: providing said hematopoietic cell population of the invention to said subject. Including, the hematopoietic cells are obtained from the subject prior to ex vivo contact with the retroviral vector; The method, wherein said gene of interest encodes a functional polypeptide which is mutated or deleted in said subject due to said genetic disease or disorder. [The present invention 1093] 1092. The method of claim 1092, wherein the polypeptide is selected from the group consisting of RPK, ITGB2, FANCA, FANCC, FANCG, TCIRG1, CLCN7, TNFSF11, PLEKHM1, TNFRSF11A and OSTM1. [The present invention 1094] The method of claim 1092, wherein the disease or disorder is a genetic disease or disorder caused by a single gene. [The present invention 1095] The method of claim 1094, wherein said monogenic disease or disorder is selected from the group consisting of Fanconi anemia, leukocyte adhesion deficiency type 1, pyruvate kinase deficiency, and infantile malignant osteopetrosis. [The present invention 1095] The method of any one of claims 1092 to 1094, wherein the hematopoietic cells are CD34-enriched cells, and are optionally bone marrow (BM)-derived cells, cord blood (CB)-derived cells, or mobilized peripheral blood (mPB) cells. Other features and advantages of the present invention will be apparent from and are encompassed by the following detailed description and claims. [Brief description of the drawings]

[0048] [Figure 1] Figure 1 shows an illustrative transduction protocol. The amount or combination of TEs can be varied. Cryopreservation is optional. Cells can be used fresh or after cryopreservation. [Diagram 2] Figures 2A and 2B show the results of VCN measurements for cells in liquid medium transduced in the presence of PGE2. For Figure 2A, the left bar shows the results for cells transduced with 2.5 x 107 TU / mL lentiviral vector, and the right bar shows the results for cells transduced with 5 x 107 TU / mL lentiviral vector, for each concentration shown. [Figure 3A]3A-3C show the results of the CFC assay (FIG. 3A), the VCN measurement (FIG. 3B), and the transduction rate (%) in the CFC assay (FIG. 3C) for cells transduced in the presence of PGE2. [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 4] FIG. 4 shows the results of VCN measurements for cells in liquid culture transduced in the presence of LentiBOOST. [Figure 5A] 5A-5C show the results of VCN measurements and % transduction rates in CFC assays for cells transduced with LentiBOOST. [Figure 5B] See legend to Figure 5A. [Figure 5C] See legend to Figure 5A. [Figure 6] FIG. 6 shows the results of VCN measurements for cells in liquid culture transduced with one or more of LentiBOOST (LB), PGE2, and protamine sulfate (PS). [Figure 7A] Figures 7A-7E show the results of CFC assays (Figure 7A), VCN measurements (Figure 7B), and % transduction results (Figure 7C) for cells transduced with one or more of LentiBOOST (LB), PGE2, and protamine sulfate (PS). Figures 7D and 7E show the effect of LB, PGE2, and PS on transduction rate (Figure 7D), and VCN (Figure 7E) of CFCs derived from different sources of CD34+, including umbilical cord blood (CB-CD34+) and mobilized peripheral blood (mPB-CD34+). [Figure 7B] See legend to Figure 7A. [Figure 7C] See legend to Figure 7A. [Figure 7D] See legend to Figure 7A. [Figure 7E] See legend to Figure 7A. [Figure 8] Figure 8 shows the expansion results of transduction with PS alone ("without TE") or with PS plus the transduction enhancers LB and PGE2 ("with TE"). VCN assays are shown for cells after 14 days in liquid culture. [Figure 9A] Figures 9A-9C show the expansion of transduction results with PS alone or with PS plus the transduction enhancers LB and PGE2. Figure 9A shows the results of a CFC assay. Figure 9B shows the results of VCN in CFU. Results are shown for early erythroid progenitor (BFU-E (burst forming unit-erythroid)) cells, granulocyte-macrophage progenitor (CFU-GM), and myeloid progenitor (CFU-GM). Figure 9C shows the transduction efficiency in CFC. [Figure 9B] See legend to Figure 9A. [Figure 9C] See legend to Figure 9A. [Figure 10] Figures 10A and 10B show the in vivo results of CD34+ cells transduced with PS alone or PS with the transduction enhancer LB and PGE2 transplanted into immunodeficient NSG mice. The percentage (%) of human CD45-positive (hCD45+) cells (Figure 10A) and VCN / cell (Figure 10B) are shown. Results are shown at 1 month (1), 2 months (2), or 3 months (3) post-transplant (mpt). [Figure 11] FIG. 11 shows VCN in liquid culture medium for LV batches of GMP with (+TE) or without (−TE) transduction enhancers LB and PGE2 at MOI 20 or MOI 50. [Figure 12] 12A-12D show colony forming units (CFU) for total cells (FIG. 12A), BFU (FIG. 12B), GM (FIG. 12C), and mixed myeloid progenitor cells (FIG. 12D) for LV batches of GMP with (+TE) or without (-TE) the transduction enhancers LB and PGE2. [Figure 13]Figures 13A and 13B show VCN in CFU for LV batches of GMP with (+TE) or without (-TE) transduction enhancers LB and PGE2 for early erythroid progenitor (BFU-E), granulocyte-macrophage progenitor (CFU-GM), and myeloid progenitor (CFU-GM) cells at MOI 20 (Figure 13A) and MOI 50 (Figure 13B). [Figure 14] Figures 14A and 14B show VCN and transduction efficiency in CFU with (+TE) or without (-TE) transduction enhancers LB and PGE2 at an MOI of 20 (Figure 14A) and an MOI of 50 (Figure 14B). [Figure 15] FIG. 15 is a schematic map of the pCCL-PGK-FANCAW-82-PRO transfer vector. [Figure 16] FIG. 16 is a schematic map of the pCCL-ChimhCD18W-82-RO transfer vector. [Figure 17] FIG. 17 is a schematic map of the pCCL-PGK-coRPKW-82-RO transfer vector. [Figure 18] FIG. 18 is a schematic map of the pRRL.PPT.EFS.tcirg1h.wpre transfer vector. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0049] Detailed Description The present disclosure provides compositions and methods for genetic modification of hematopoietic cells. In particular, the present invention relates to the use of a combination of two or more of prostaglandin E2 (PGE2), poloxamer, recombinant fibronectin fragments, and / or protamine sulfate to enhance transduction by recombinant retroviral vectors. The compositions and methods of the present disclosure are particularly suitable for gene therapy applications, including the treatment of monogenic diseases and disorders. Factors that have limited the success of gene therapy, including low transduction efficiency, are addressed by the compositions and methods provided herein.

[0050] A.Definition Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. In carrying out the present invention, methods and materials similar or equivalent to those described herein can be used, but suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Additionally, the materials, methods, and examples described herein are illustrative only and are not intended to be limiting.

[0051] The various embodiments contemplated herein will employ, unless specifically indicated to the contrary, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology and cell biology within the skill of the art, many of which are described below for purposes of illustration, and such techniques are explained more fully in the literature. For example, Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008);Short Protocols in Molecular Biology:A Compendium of Methods from Current Protocols in Molecular Biology,Greene Pub.Associates and Wiley-Interscience;Glover,DNA Cloning:A Practical Approach,vol.I&II(IRL Press,Oxford,1985);Anand,Techniques for the Analysis of Complex Genomes,(Academic Press,New York,1992);Transcription and Translation (B. Hames & S. Higgins, Eds., 1984); Perbal, A Practical Guide to Molecular Cloning (1984); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998); Current Protocols in Immunology QE Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; as well as monographs in publications such as Advances in Immunology, each of which is expressly incorporated herein by reference.

[0052] As used herein, the terms "about" or "approximately" refer to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by about 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% from the reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In certain embodiments, the terms "about" or "approximately" when preceding a numerical value refer to a value that is within a range of plus or minus 15%, 10%, 5%, or 1%.

[0053] "Transfection" refers to the introduction of naked DNA into cells by non-viral means.

[0054] "Infection" refers to the method of introducing foreign DNA into a cell using a viral vector.

[0055] "Transduction" refers to the introduction of foreign DNA into the genome of a cell using a viral vector.

[0056] "Vector copy number" or "VCN" refers to the number of copies of a vector in a sample divided by the number of cells. Typically, vector copy number is determined by quantitative polymerase chain reaction (qPCR) using a probe to the Psi sequence of the integrated provirus, and the number of cells is determined by qPCR using a probe to a human housekeeping gene that results in two copies per cell (one per chromosome).

[0057] "Transduction efficiency" refers to the percentage of cells transduced with at least one copy of the provirus. For example, 1×10 6 Cells were exposed to the virus and 0.5 × 10 6 If the cells are determined to have at least one copy of the virus in their genome, the transduction efficiency is 50%. An illustrative method for determining transduction efficiency is flow cytometry.

[0058] As used herein, the term "retrovirus" or "retroviral" refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy, which then covalently integrates the genomic DNA into the host genome. Retroviral vectors are a common vehicle for gene delivery (Miller, 2000, Nature. 357:455-460). Once the virus is integrated into the host genome, it is called a "provirus." The provirus serves as a template for RNA polymerase II and is responsible for the expression of virally encoded RNA molecules.

[0059] Illustrative retroviruses (family Retroviridae) include, but are not limited to, (1) the genus Gammaretrovirus, such as Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), and feline leukemia virus (FLV); (2) the genus Spumavirus, such as simian foamy viruses; and (3) the genus Lentivirus, such as human immunodeficiency virus-1 and simian immunodeficiency virus.

[0060] As used herein, the term "lentiviral" or "lentivirus" refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include, but are not limited to, HIV (human immunodeficiency virus; including HIV types 1 and 2); Visna-Maedi virus (VMV); Caprine arthritis-encephalitis virus (CAEV); Equine infectious anemia virus (EIAV); Feline immunodeficiency virus (FIV); Bovine immunodeficiency virus (BIV); and Simian immunodeficiency virus (SIV). In one embodiment, HIV-based vector backbones (i.e., elements of HIV cis-acting sequences) are preferred.

[0061] Retroviral vectors, and more particularly lentiviral vectors, can be used in the practice of the present invention. Thus, as used herein, the term "retroviral vector" is meant to include "lentiviral vector," and as used herein, the term "retrovirus" is meant to include "lentivirus."

[0062] The term "vector" is used herein to refer to a nucleic acid molecule that can introduce or transport another nucleic acid molecule. The introduced nucleic acid is generally combined with, e.g., inserted into, the nucleic acid molecule of the vector. A vector may contain sequences that direct autonomous replication or reverse transcription in a cell, or may contain sequences that are sufficient to allow integration into host cell DNA. Useful vectors include viral vectors. Useful viral vectors include, for example, replication-defective retroviruses and lentiviruses.

[0063] The term "viral vector" can refer to either a vector or vector particle capable of introducing a nucleic acid into a cell, or the introduced nucleic acid itself. A viral vector contains structural and / or functional genetic elements that are primarily derived from a virus. The term "retroviral vector" refers to a viral vector that contains structural and functional genetic elements or portions thereof that are primarily derived from a retrovirus. The term "lentiviral vector" refers to a viral vector that contains structural and functional genetic elements or portions thereof, including LTRs, that are primarily derived from a lentivirus. The term "hybrid" refers to a vector, LTR, or other nucleic acid that contains both retroviral sequences, e.g., lentivirus, and non-lentiviral viral sequences. In one embodiment, a hybrid vector refers to a vector or transfer plasmid that contains retroviral sequences, e.g., lentivirus, for reverse transcription, replication, integration, and / or packaging.

[0064] In certain embodiments, the terms "lentiviral vector" and "lentiviral expression vector" may be used to refer to lentiviral transfer plasmids and / or infectious lentiviral particles. When reference is made herein to elements such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc., it is understood that the sequences of these elements are present in RNA form in the lentiviral particles of the invention and in DNA form in the DNA plasmids of the invention.

[0065] According to certain embodiments, most or all of the viral vector backbone sequence is derived from a lentivirus, for example HIV-1. However, it should be understood that lentiviral sequences from many different sources can be used, and that many substitutions and alterations can be accommodated in a lentiviral sequence without impairing the ability of the transfer vector to perform the functions described herein. Furthermore, a variety of lentiviral vectors are known in the art, see Naldini et al., (1996a, 1996b, and 1998); Zufferey et al., (1997); Dull et al., 1998, U.S. Patent No. 6,013,516; and U.S. Patent No. 5,994,136, many of which may be adapted to produce the viral vector or transfer plasmid of the present invention.

[0066] As used herein, the term "polynucleotide" or "nucleic acid" generally refers to a biological polymer that includes nucleotide monomers covalently linked in a strand, such as DNA and RNA. In some embodiments, polynucleotide refers to genomic DNA (gDNA), complementary DNA (cDNA), or DNA. Polynucleotides include single-stranded and double-stranded polynucleotides, either recombinant, synthetic, or isolated. In some embodiments, polynucleotide refers to messenger RNA (mRNA), RNA, genomic RNA (gRNA), positive strand RNA (RNA(+)), negative strand RNA (RNA(-)). As used herein, the term "polyribonucleotide" or "ribonucleic acid" also refers to messenger RNA (mRNA), RNA, genomic RNA (gRNA), positive strand RNA (RNA(+)), negative strand RNA (RNA(-)). Preferably, the polynucleotides of the present invention include polynucleotides or variants having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the reference sequences described herein (see, e.g., the sequence listing), and typically the variants maintain at least one biological activity of the reference sequence. In various illustrative embodiments, viral vector and transfer plasmid polynucleotide sequences and compositions comprising the same are contemplated. In certain embodiments, polynucleotides encoding one or more polypeptide agents and / or other genes of interest are contemplated. In certain embodiments, the polynucleotides encoding polypeptide agents include, but are not limited to, the genes RPK, ITGB2, FANCA, FANCC, FANCG, TCIRG1, CLCN7, TNFSF11, PLEKHM1, TNFRSF11A and OSTM1. In some embodiments, the polynucleotide is a codon-optimized variant of any of these genes. In some embodiments, the polynucleotide encodes a human polypeptide, or a functional fragment or variant thereof, such as a polypeptide encoded by any of the disclosed genes.

[0067] As used herein, a "pseudotyped" vector refers to a vector that has a recombinant capsid or envelope protein that is different from the capsid or envelope protein of the native vector. For example, a VSVG pseudotyped lentiviral vector is a vector generated by co-expressing the envelope protein of the VSVG virus with the RNA genome of the virus in a packaging cell line in a manner that allows the incorporation of the VSVG envelope protein into the viral particle containing the RNA genome. Pseudotyped vectors may have altered tropism and / or reduced immunogenicity to make them desirable for use in gene therapy applications. It is within the skill of the artisan to alter the pseudotype of a vector as well as to generate pseudotyped vectors by producing viral particles in different packaging cell lines or by co-expressing envelope proteins (or capsid proteins) from plasmids or other DNA that code for different envelope proteins (or capsid proteins). Exemplary methods are provided in Cronin et al. Curr. Gene Ther. 5:387-398 (2005). In some embodiments, the methods of the disclosure involve the use of a pseudotyped recombinant retroviral vector (e.g., a lentiviral vector). In some embodiments, the pseudotyped recombinant retroviral vector is VSVG pseudotyped.

[0068] By "enhance" or "promote" or "increase" or "expand" generally refers to the ability of the compositions and / or methods contemplated herein to elicit, generate, or produce a greater number of transduced cells, or to elicit, generate, or produce a higher VCN in a population of transduced cells, as compared to the number of cells transduced with either a vehicle or a control molecule / composition. In one embodiment, hematopoietic stem or progenitor cells transduced with the compositions and methods contemplated herein include an increase in the number of transduced cells, or an increase in VCN in a population of transduced cells, as compared to existing transduction compositions and methods. Increased cell transduction can be confirmed using methods known in the art, such as, for example, reporter assays, RT-PCR, and cell surface protein expression, among others. An "increased" or "enhanced" amount of transduction is typically a "statistically significant" amount and can include an increase of 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold or more (e.g., 500-fold, 1000-fold) (e.g., 1.5, 1.6, 1.7, 1.8, etc., including all integers and decimals between and above 1) in the number of cells transduced by vehicle, control composition, or other transduction methods.

[0069] By "reduce" or "lower" or "lessen" or "reduce" or "alleviate" generally refers to a composition or method that results in relatively fewer transduced cells as compared to cells transduced by a composition and / or method according to the invention. A "reduced" or "reduced" amount of transduced cells is typically a "statistically significant" amount and can include a 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold or more (e.g., 500-fold, 1000-fold) (e.g., 1.5, 1.6, 1.7, 1.8, etc., including all integers and decimals between and above 1) decrease in the number of transduced cells (reference response) produced by a composition and / or method according to the invention.

[0070] "Maintain" or "preserve" or "maintain" or "no change" or "no substantial change" or "no substantial decrease" generally refers to a physiological response that is comparable to a response elicited by either a vehicle, a control molecule / composition, or a response in a particular cell. A comparable response is one that is not significantly different from or has no measurable difference from a reference response.

[0071] In the following description, certain specific details are set forth to provide a thorough understanding of the various illustrative embodiments of the invention contemplated herein. However, those skilled in the art will understand that certain illustrative embodiments may be practiced without these details. Furthermore, each individual vector or group of vectors derived from the various combinations of structures and substitutions described herein is to be understood as being disclosed by this application to the same extent as if each vector or group of vectors were described separately. That is, the selection of a particular vector structure or particular substitution is within the scope of this disclosure.

[0072] As used herein, "X-VIVO 20" or "X-VIVO" refers to X-VIVO™ 20 chemically defined, serum-free hematopoietic cell medium, available from Lonza®. Other media besides X-VIVO 20 may be used, and one of skill in the art can select a suitable medium for cell growth and transduction.

[0073] As used herein, "rhSCF" refers to recombinant human stem cell factor.

[0074] As used herein, "rhTPO" refers to recombinant human thrombopoeitin.

[0075] As used herein, "rh-FLT3-L" refers to recombinant human fms-related tyrosine kinase 3-ligand.

[0076] As used herein, "IL-3" or "rhIL-3" refers to recombinant human interleukin-3.

[0077] As used herein, "PGE2" refers to prostaglandin E2 (PGE2), also known as dinoprostone.

[0078] As used herein, "poloxamer" refers to a non-ionic triblock copolymer composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)).

[0079] As used herein, "recombinant fibronectin fragment" refers to any fragment of the protein fibronectin that promotes and enhances transduction efficiency. Without being bound by theory, it is believed that recombinant fibronectin fragments promote co-localization of lentivirus or retrovirus with target cells. An example of a recombinant fibronectin fragment is the CH296 fragment of human fibronectin, trade name RetroNectin™.

[0080] The concentrations of PGE2 or a derivative thereof, poloxamer, or protamine sulfate provided in this disclosure and claims refer to the concentration of each agent in the medium in which the cells are cultured.

[0081] As used herein, "LB" or "LentiBoost" refers to the LentiBOOST™ transduction enhancer available from Sirion Biotech®. Synonyms include poloxamer 338, F108, and Kolliphor® P338.

[0082] As used herein, "HSA" refers to human serum albumin.

[0083] As used herein, "DMSO" refers to dimethylsulfoxide.

[0084] As used herein, "CFC" refers to colony forming cells. The colony forming cell (CFC) assay is used to examine the proliferation and differentiation patterns of hematopoietic progenitor cells by their ability to form colonies in semi-solid medium. The number and morphology of colonies formed by a defined number of input cells gives preliminary information about the differentiation and proliferation potential of the progenitor cells. An exemplary assay is given in Sarma et al. Colony forming cell (CFC) assay for human hematopoietic cells. J Vis Exp. 2010 Dec 18; (46).

[0085] As used herein, "LC" refers to "liquid culture (liquid)."

[0086] As used herein, "CFU" refers to colony forming unit. CFU is understood to be synonymous with CFC, but may also be used to refer to the type of CFU that grows in semi-solid medium.

[0087] As used herein, "TU" refers to transducing units. TU / mL is a common measurement of the functional titer of a retroviral (lentiviral) preparation.

[0088] As used herein, "PS" refers to protamine sulfate.

[0089] As used herein, "TE" refers to one or more transduction enhancers.

[0090] As used herein, "CB fresh cells" refers to fresh cord blood cells.

[0091] As used herein, "MOI" refers to multiplicity of infection.

[0092] As used herein, "BFU-E" refers to early erythroblastic progenitor (BFU-E) cells, the earliest red blood cell precursors.

[0093] As used herein, "CFU-GM" refers to colony forming unit of granulocyte-macrophage progenitor cells.

[0094] As used herein, "CFU-GEMM" refers to colony forming units of bone marrow stem cells (granulocyte, erythrocyte, monocyte, megakaryocyte).

[0095] As used herein, "mPB" refers to mobilized peripheral blood cells.

[0096] As used herein, "SCGM" refers to CellGenix® SCGM Serum-Free Medium.

[0097] As used herein, the terms "administering" or "introducing" or "providing" refer to delivering a hematopoietic cell population to a subject, for example, by injecting the cell population into a subject intra-arterially or intravenously. The hematopoietic cell population may be administered in a variety of solutions, such as, for example, saline. In some embodiments, the solution used will be isotonic with respect to the subject's blood and pH buffered.

[0098] Typically, a cell is said to be "transduced" when a viral vector or viral vector particle introduces heterologous DNA (eg, a vector or its expression cassette) into the genome of the cell.

[0099] As used herein, the term "host cell" refers to a cell that has been transduced by a viral vector or viral vector particle. It will be understood that the term "host cell" refers to the originally transduced cell and its progeny.

[0100] The terms "treatment," "treating," and the like are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, for example in terms of reducing the likelihood of a disease or its symptoms occurring in a subject, completely or partially preventing a disease or its symptoms, and / or therapeutic, for example in terms of partially or completely curing adverse effects on and / or caused by a disease. As used herein, "treatment" covers any treatment of a disease in a mammal, and includes (a) preventing the onset of a disease in a subject who may be predisposed to the disease but has not yet been diagnosed as having the disease, (b) inhibiting the disease, i.e., arresting its onset, or (c) relieving the disease, i.e., causing regression of the disease. Therapeutic agents may be administered before, during, or after the onset of the disease or injury. Of particular interest is the treatment of ongoing disease, where the treatment stabilizes or reduces undesirable clinical symptoms in the patient. It is desirable that such treatment is performed prior to complete loss of function in the affected tissue. Desirably, the subject treatment will be administered during, and in some cases after, the symptomatic stage of the disease.

[0101] The terms "individual," "host," "subject," and "patient" are used interchangeably herein and refer to mammals, including, but not limited to, humans and non-human primates, including monkeys and humans; mammalian sport animals (e.g., horses); mammalian livestock animals (e.g., sheep, goats, etc.); mammalian pet animals (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0102] B. Embodiments and Modifications Various compositions and methods are described below. Although certain compositions and methods are exemplified herein, it will be understood that any of a number of alternative compositions and methods are available and suitable for use in practicing the compositions and methods disclosed herein. It will also be understood that evaluation of the expression constructs and methods disclosed herein may be performed using methods standard in the art.

[0103] 1. Transduction with a combination of transduction enhancers The present disclosure provides advantageous methods for transducing hematopoietic cells with lentiviral vectors to produce populations of hematopoietic cells having a high percentage of cells transduced by the lentiviral vector. These methods are particularly advantageous for transducing hematopoietic cells with lentiviral gene therapy vectors to correct genetic defects because they result in large numbers of cells that express a functional product of the corrected gene introduced by the lentiviral gene therapy vector.

[0104] A variety of transduction enhancers are known in the art, including polybrene, protamine sulfate, retronectin (a recombinant fibronectin fragment), and DEAE dextran. In some cases, polycationic agents such as polybrene have been employed as transduction enhancers. Denning et al. Mol Biotechnol. 2013 Mar; 53(3): 308-314. Additionally, rapamycin and cyclosporine A are used as transduction enhancers. However, in the present disclosure, a specific combination of transduction enhancers is identified that achieves significantly higher levels of lentiviral transduction of hematopoietic cells compared to each of the transduction enhancers used alone.

[0105] In one aspect, the disclosure provides a method for genetically modifying hematopoietic cells, comprising contacting the hematopoietic cells with at least two transduction enhancers selected from prostaglandin E2 (PGE2) or derivatives thereof, poloxamer, protamine sulfate, and recombinant fibronectin fragments, and contacting the hematopoietic cells with a recombinant retroviral vector. The cells may be contacted with the retroviral vector under conditions and for a time sufficient to permit transduction of the cells by the retroviral vector, for example, for at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 12 hours, or at least 16 hours in a suitable medium. In some embodiments, the cells are transduced either once or two consecutive times, for example after a pre-stimulation, with each transduction cycle lasting 12-24 hours, or 16-18 hours. In some embodiments, the cells are contacted with the retroviral vector and the transduction enhancer for the same or overlapping periods of time. In certain embodiments, the cells are contacted with the transduction enhancer and the recombinant retroviral vector simultaneously or for overlapping periods of time. In certain embodiments, the two or more transduction enhancers comprise a poloxamer. In certain embodiments, the poloxamer is poloxamer 338 (LentiBOOST™). In certain embodiments, the two or more transduction enhancers comprise or consist of a poloxamer and PGE2 or a derivative thereof. In certain embodiments, the poloxamer is poloxamer 338 (LentiBOOST™). In certain embodiments, the two or more transduction enhancers comprise or consist of a poloxamer and protamine sulfate. In certain embodiments, the poloxamer is poloxamer 338 (LentiBOOST™). In one embodiment, the two or more transduction enhancers comprise or consist of a poloxamer, PGE2 and protamine sulfate. In one embodiment, the poloxamer is poloxamer 338 (LentiBOOST™).In some embodiments, the two or more transduction enhancers include or consist of prostaglandin E2 (PGE2) or a derivative thereof, poloxamer, protamine sulfate, and a recombinant fibronectin fragment. In various embodiments of any of the aspects and embodiments disclosed herein, the cells are transduced on a dish coated with a recombinant fibronectin fragment, e.g., RetroNectin™. In various embodiments, the cells are pretreated by culturing on a dish coated with a recombinant fibronectin fragment, e.g., RetroNectin™, prior to and / or during transduction. In some embodiments, the method comprises culturing at about 2ug / cm. 2 In some embodiments, the cells are pre-stimulated by culturing on plates coated with RetroNectin™ (RN) of the same species. In some embodiments, the cells are transduced in liquid medium containing RetroNectin™. In some embodiments, the cells are pre-treated by culturing on dishes coated with recombinant fibronectin fragments and are transduced in liquid culture also in the presence of recombinant fibronectin fragments and other TEs.

[0106] In one aspect, the disclosure provides a method of genetically modifying a hematopoietic cell, comprising providing a hematopoietic cell; contacting the hematopoietic cell with at least two transduction enhancers selected from prostaglandin E2 (PGE2) or a derivative thereof, a poloxamer (e.g., poloxamer 338 (LentiBOOST™)), protamine sulfate, and a recombinant fibronectin fragment; and contacting the hematopoietic cell with a recombinant retroviral vector. In certain embodiments, the cell is contacted with the transduction enhancers and the recombinant retroviral vector simultaneously or for overlapping periods of time. In certain embodiments, the two or more transduction enhancers comprise a poloxamer. In certain embodiments, the poloxamer is poloxamer 338 (LentiBOOST™). In certain embodiments, the two or more transduction enhancers comprise a poloxamer and PGE2 or a derivative thereof. In certain embodiments, the poloxamer is poloxamer 338 (LentiBOOST™). In some embodiments, the two or more transduction enhancers comprise a poloxamer and protamine sulfate or consist of a poloxamer and protamine sulfate. In some embodiments, the poloxamer is poloxamer 338 (LentiBOOST™). In some embodiments, the two or more transduction enhancers comprise a poloxamer, PGE2 and protamine sulfate or consist of a poloxamer, PGE2 and protamine sulfate. In some embodiments, the poloxamer is , poloxamer 338 (LentiBOOST™). In one embodiment, the two or more transduction enhancers comprise or consist of prostaglandin E2 (PGE2) or a derivative thereof, poloxamer, protamine sulfate, and a recombinant fibronectin fragment. In one embodiment, the poloxamer is poloxamer 338 (LentiBOOST™).In some embodiments, the two or more transduction enhancers include or consist of prostaglandin E2 (PGE2) or a derivative thereof, poloxamer, protamine sulfate, and a recombinant fibronectin fragment. In various embodiments of any of the aspects and embodiments disclosed herein, the cells are transduced on a dish coated with a recombinant fibronectin fragment, e.g., RetroNectin™. In various embodiments, the cells are pretreated by culturing on a dish coated with a recombinant fibronectin fragment, e.g., RetroNectin™, prior to and / or during transduction. In some embodiments, the method comprises culturing at about 2ug / cm. 2 In some embodiments, the cells are pre-stimulated by culturing on plates coated with RetroNectin™ (RN) of the same species. In some embodiments, the cells are transduced in liquid medium containing Retro-Nectin™. In some embodiments, the cells are pre-treated by culturing on dishes coated with recombinant fibronectin fragments and are transduced in liquid culture also in the presence of recombinant fibronectin fragments and other TEs.

[0107] In various embodiments of the methods disclosed herein, a population of cells is cultured in the presence of a retroviral vector, one or more agents that stimulate the prostaglandin EP receptor signaling pathway, and a poloxamer having an average molecular weight of about 10,000 daltons. In certain embodiments, the cells are contacted with the transduction enhancer and the recombinant retroviral vector simultaneously or for overlapping periods of time.

[0108] In various embodiments of the methods disclosed herein, a population of cells is cultured in the presence of a retroviral vector, one or more agents that stimulate the prostaglandin EP receptor signaling pathway, and a poloxamer having an average molecular weight of polypropylene subunits greater than about 2250 daltons and containing greater than about 40% polyethylene oxide. In certain embodiments, the cells are contacted with the transduction enhancer and the recombinant retroviral vector simultaneously or for overlapping periods of time.

[0109] In certain embodiments of the methods disclosed herein, the cells are transduced in a medium that includes a combination of three transduction enhancers, e.g., poloxamer 338 (LentiBOOST), PGE2, and protamine sulfate, in liquid medium. The cells may be attached to a culture dish or the cells may not be attached to a culture dish. In certain embodiments, the cells are contacted with the transduction enhancer and the recombinant retroviral vector for simultaneous or overlapping periods of time. In certain embodiments, the method includes pre-stimulation by culturing the cells on a plate coated with a recombinant fibronectin fragment, e.g., RetroNectin™ (RN). In certain embodiments, the cells are transduced on a dish coated with a recombinant fibronectin fragment, e.g., RetroNectin™. In various embodiments, the cells are pre-treated before and / or during transduction by culturing on a dish coated with a recombinant fibronectin fragment, e.g., RetroNectin™. In one embodiment, the method comprises administering a concentration of about 2 ug / cm 2 In some embodiments, the cells are pre-stimulated by culturing on plates coated with RetroNectin™ (RN) of the same species. In some embodiments, the cells are transduced in liquid medium containing Retro-Nectin™. In some embodiments, the cells are pre-treated by culturing on dishes coated with recombinant fibronectin fragments and are transduced in liquid culture also in the presence of recombinant fibronectin fragments and other TEs.

[0110] In a related aspect, the disclosure provides a method for enhancing recombinant retroviral vector-mediated genetic modification of hematopoietic cells, comprising ex vivo treatment or contacting hematopoietic cells with an effective amount of a combination of two or more transduction enhancers, at least two of which are selected from PGE2 or a derivative thereof, poloxamer, protamine sulfate, and recombinant fibronectin fragments. In an embodiment, the poloxamer is poloxamer 338 (LentiBOOST™). In some embodiments, the method comprises contacting a cell with PGE2 or a derivative thereof and an effective amount of a poloxamer; and contacting the cell with a recombinant retroviral vector comprising a polynucleotide comprising a gene of interest, wherein the efficacy of viral transduction of the retroviral vector is enhanced compared to transduction of hematopoietic cells by the recombinant retroviral vector in the absence of the combination of transduction enhancers, e.g., PGE2 and poloxamer. In an embodiment, the two or more transduction enhancers comprise a poloxamer and PGE2 or a derivative thereof. In some embodiments, the poloxamer is poloxamer 338 (LentiBOOST™). In some embodiments, the two or more transduction enhancers comprise or consist of a poloxamer and protamine sulfate. In some embodiments, the method further comprises ex vivo treating or contacting the hematopoietic cells with an effective amount of protamine sulfate. In some embodiments, the two or more transduction enhancers comprise or consist of prostaglandin E2 (PGE2) or a derivative thereof, a poloxamer, protamine sulfate and a recombinant fibronectin fragment. In various embodiments of any of the aspects and embodiments disclosed herein, the cells are transduced on a dish coated with a recombinant fibronectin fragment, e.g., RetroNectin™.In various embodiments, the cells are pretreated prior to and / or during transduction by culturing on dishes coated with a recombinant fibronectin fragment, e.g., RetroNectin™. In one embodiment, the method comprises culturing the cells on a dish coated with a recombinant fibronectin fragment, e.g., RetroNectin™, at a concentration of about 2 ug / cm. 2 In some embodiments, the cells are pre-stimulated by culturing on plates coated with RetroNectin™ (RN) of the same species. In some embodiments, the cells are transduced in liquid medium containing Retro-Nectin™. In some embodiments, the cells are pre-treated by culturing on dishes coated with recombinant fibronectin fragments and are transduced in liquid culture also in the presence of recombinant fibronectin fragments and other TEs.

[0111] In certain embodiments of any of the methods disclosed herein, the cells are contacted with a solution or culture medium comprising two or more transduction enhancers. The solution or culture medium may further comprise a recombinant retroviral vector, or the recombinant retroviral vector may be added after the cells are contacted with the solution or culture medium comprising the transduction enhancers. In certain embodiments, the cells are present in a culture dish comprising the solution or culture medium. In certain embodiments, the culture dish is coated with a recombinant fibronectin fragment. The cells may be contacted with the retroviral vector under conditions and for a time sufficient to permit transduction of the cells by the retroviral vector, for example, for at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 12 hours, or at least 16 hours in a suitable medium. In some embodiments, the cells are transduced either once or two consecutive times, for example after a pre-stimulation, with each transduction cycle lasting 12-24 hours, or 16-18 hours. In some embodiments, the cells are contacted with the retroviral vector and the transduction enhancer for the same or overlapping periods of time.

[0112] In some embodiments of any of the methods disclosed herein, the cells are contacted with a poloxamer. In some embodiments, the poloxamer is selected from the group consisting of poloxamer 288, poloxamer 335, poloxamer 338, and poloxamer 407. In some embodiments, the poloxamer is poloxamer 338 (LentiBOOST™). LentiBOOST™ can be used at a final concentration of about 50 μg / mL to about 1,500 μg / mL, about 500 μg / mL to about 1,500 μg / mL, about 750 μg / mL to about 1,250 μg / mL, about 900 μg / mL, about 900 μg / mL, about 950 μg / mL, about 1000 μg / mL, about 1050 μg / mL, about 1100 μg / mL, or about 1150 μg / mL.

[0113] In some embodiments of any of the methods disclosed herein, the cells are contacted with PGE2 or a derivative thereof. In some embodiments, the PGE2 or a derivative thereof is modified. In some embodiments, the PGE2 or a derivative thereof is a dimethylated PGE2. In some embodiments, the dimethylated PGE2 is 16,16-dimethyl prostaglandin E2. 16,16-dimethyl prostaglandin E2 has the following structure (represented as the "skeleton structure" and also referred to as the "linear structural formula" or "truncated structural formula"): TIFF0007664829000001.tif38128Molecular formula:C 22 H 36 O 5 .

[0114] In some embodiments, the PGE2 or derivative thereof is unmodified.

[0115] In some embodiments, PGE2 or a derivative thereof can be used at a final concentration of about 1 μM to about 200 μM, about 10 μM to about 20 μM, about 20 μM to about 40 μM, about 40 μM to about 60 μM, about 60 μM to about 80 μM, about 5 μM, about 10 μM, about 15 μM, about 20 μM, about 25 μM, about 30 μM, about 35 μM, or about 40 μM. PGE2 or a derivative thereof can be used at a final concentration of about 0.3 μM to about 70 μg / ml, about 3 μg / ml to about 7 μg / ml, about 7 μg / ml to about 13 μg / ml, about 13 μg / ml to about 20 μg / ml, about 20 μg / ml to about 26 μg / ml, about 2 μg / ml, about 3 μg / ml, about 4 μg / ml, about 5 μg / ml, about 6 μg / ml, about 7 μg / ml, about 8 μg / ml, about 9 μg / ml, about 10 μg / ml, about 11 μg / ml, about 12 μg / ml, about 13 μg / ml, about 14 μg / ml, or about 15 μg / ml.

[0116] In some embodiments of any of the methods disclosed herein, the method includes contacting hematopoietic cells with protamine sulfate. Protamine sulfate can be used at a final concentration of about 4 μg / mL to about 15 μg / mL, about 5 μg / mL to about 10 μg / mL, or about 5 μg / mL, about 6 μg / mL, about 7 μg / mL, about 8 μg / mL, about 9 μg / mL, about 10 μg / mL, about 11 μg / mL, about 12 μg / mL, about 13 μg / mL, about 14 μg / mL, or about 15 μg / mL or more. In certain embodiments, protamine sulfate can be used at a final concentration of about 1 μg / mL to about 5 μg / mL, about 3 μg / mL to about 5 μg / mL, or about 1 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL, or about 5 μg / mL.

[0117] In some embodiments of any of the methods disclosed herein, the cells are contacted with poloxamer 338 and PGE2 or a derivative thereof. In certain embodiments, poloxamer 338 and PGE2 or a derivative thereof are used at a final concentration of about 900 μg / mL and about 3 μg / mL to about 7 μg / mL, respectively, or about 900 μg / mL and about 2 μg / mL, respectively, or about 900 μg / mL and about 3 μg / mL, respectively, or about 900 μg / mL and about 3 μg / mL, respectively, or about 900 μg / mL and about 4 μg / mL, respectively, or about 900 μg / mL and about 5 μg / mL, respectively, or about 900 μg / mL and about 6 μg / mL, respectively, or about 900 μg / mL and about 7 μg / mL, respectively, or about 900 μg / mL and about 8 μg / mL, respectively, or about 900 μg / mL and about 9 μg / mL, respectively. In any of the foregoing embodiments, protamine sulfate may optionally be used at a final concentration of about 3 μg / mL to about 7 μg / mL, about 1 μg / mL to about 5 μg / mL, about 3 μg / mL to about 5 μg / mL, or about 1 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL, about 5 μg / mL, or about 6 μg / mL.

[0118] In some embodiments, poloxamer 338 and PGE2 or a derivative thereof are used at final concentrations of about 950 μg / mL and about 3 μg / mL to about 7 μg / mL, respectively, or about 900 μg / mL and about 2 μg / mL, respectively, or about 950 μg / mL and about 3 μg / mL, respectively, or about 950 μg / mL and about 3 μg / mL, respectively, or about 950 μg / mL and about 4 μg / mL, respectively, or about 950 μg / mL and about 5 μg / mL, respectively, or about 950 μg / mL and about 6 μg / mL, respectively, or about 950 μg / mL and about 7 μg / mL, respectively, or about 950 μg / mL and about 8 μg / mL, respectively, or about 950 μg / mL and about 9 μg / mL, respectively. In any of the foregoing embodiments, protamine sulfate may optionally be used at a final concentration of about 3 μg / mL to about 7 μg / mL, about 1 μg / mL to about 5 μg / mL, about 3 μg / mL to about 5 μg / mL, or about 1 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL, about 5 μg / mL, or about 6 μg / mL.

[0119] In some embodiments, poloxamer 338 and PGE2 or a derivative thereof are used at a final concentration of about 1000 μg / mL and about 3 μg / mL to about 7 μg / mL, respectively, or about 1000 μg / mL and about 2 μg / mL, respectively, or about 1000 μg / mL and about 3 μg / mL, respectively, or about 1000 μg / mL and about 3 μg / mL, respectively, or about 1000 μg / mL and about 4 μg / mL, respectively, or about 1000 μg / mL and about 5 μg / mL, respectively, or about 1000 μg / mL and about 6 μg / mL, respectively, or about 1000 μg / mL and about 7 μg / mL, respectively, or about 1000 μg / mL and about 8 μg / mL, respectively, or about 1000 μg / mL and about 9 μg / mL, respectively. In any of the foregoing embodiments, protamine sulfate may optionally be used at a final concentration of about 3 μg / mL to about 7 μg / mL, about 1 μg / mL to about 5 μg / mL, about 3 μg / mL to about 5 μg / mL, or about 1 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL, about 5 μg / mL, or about 6 μg / mL.

[0120] In some embodiments, poloxamer 338 and PGE2 or a derivative thereof are used at a final concentration of about 1050 μg / mL and about 3 μg / mL to about 7 μg / mL, respectively, or about 1050 μg / mL and about 2 μg / mL, respectively, or about 1050 μg / mL and about 3 μg / mL, respectively, or about 1050 μg / mL and about 3 μg / mL, respectively, or about 1050 μg / mL and about 4 μg / mL, respectively, or about 1050 μg / mL and about 5 μg / mL, respectively, or about 1050 μg / mL and about 6 μg / mL, respectively, or about 1050 μg / mL and about 7 μg / mL, respectively, or about 1050 μg / mL and about 8 μg / mL, respectively, or about 1050 μg / mL and about 9 μg / mL, respectively. In any of the foregoing embodiments, protamine sulfate may optionally be used at a final concentration of about 3 μg / mL to about 7 μg / mL, about 1 μg / mL to about 5 μg / mL, about 3 μg / mL to about 5 μg / mL, or about 1 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL, about 5 μg / mL, or about 6 μg / mL.

[0121] In some embodiments of any of the methods disclosed herein, the cells are contacted with poloxamer 338 and PGE2 or a derivative thereof. In certain embodiments, poloxamer 338 and PGE2 or a derivative thereof are used at a final concentration of about 0.5 mg / mL and about 3 μg / mL to about 7 μg / mL, respectively, or about 0.5 mg / mL and about 2 μg / mL, respectively, or about 0.5 mg / mL and about 3 μg / mL, respectively, or about 900 μg / mL and about 3 μg / mL, respectively, or about 0.5 mg / mL and about 4 μg / mL, respectively, or about 0.5 mg / mL and about 5 μg / mL, respectively, or about 0.5 mg / mL and about 6 μg / mL, respectively, or about 0.5 mg / mL and about 7 μg / mL, respectively, or about 0.5 mg / mL and about 8 μg / mL, respectively, or about 0.5 mg / mL and about 9 μg / mL, respectively. In any of the foregoing embodiments, protamine sulfate may optionally be used at a final concentration of about 3 μg / mL to about 7 μg / mL, about 1 μg / mL to about 5 μg / mL, about 3 μg / mL to about 5 μg / mL, or about 1 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL, about 5 μg / mL, or about 6 μg / mL.

[0122] In some embodiments of any of the methods disclosed herein, the cells are contacted with poloxamer 338 and PGE2 or a derivative thereof. In certain embodiments, poloxamer 338 and PGE2 or a derivative thereof are used at a final concentration of about 1 mg / mL and about 3 μg / mL to about 7 μg / mL, respectively, or about 1 mg / mL and about 2 μg / mL, respectively, or about 1 mg / mL and about 3 μg / mL, respectively, or about 900 μg / mL and about 3 μg / mL, respectively, or about 1 mg / mL and about 4 μg / mL, respectively, or about 1 mg / mL and about 5 μg / mL, respectively, or about 1 mg / mL and about 6 μg / mL, respectively, or about 1 mg / mL and about 7 μg / mL, respectively, or about 1 mg / mL and about 8 μg / mL, respectively, or about 1 mg / mL and about 9 μg / mL, respectively. In any of the foregoing embodiments, protamine sulfate may optionally be used at a final concentration of about 3 μg / mL to about 7 μg / mL, about 1 μg / mL to about 5 μg / mL, about 3 μg / mL to about 5 μg / mL, or about 1 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL, about 5 μg / mL, or about 6 μg / mL.

[0123] In some embodiments of any of the methods disclosed herein, the cells are contacted with poloxamer 338 and PGE2 or a derivative thereof. In certain embodiments, poloxamer 338 and PGE2 or a derivative thereof are used at a final concentration of about 2 mg / mL and about 3 μg / mL to about 7 μg / mL, respectively, or about 2 mg / mL and about 2 μg / mL, respectively, or about 2 mg / mL and about 3 μg / mL, respectively, or about 900 μg / mL and about 3 μg / mL, respectively, or about 2 mg / mL and about 4 μg / mL, respectively, or about 2 mg / mL and about 5 μg / mL, respectively, or about 2 mg / mL and about 6 μg / mL, respectively, or about 2 mg / mL and about 7 μg / mL, respectively, or about 2 mg / mL and about 8 μg / mL, respectively, or about 2 mg / mL and about 9 μg / mL, respectively. In any of the foregoing embodiments, protamine sulfate may optionally be used at a final concentration of about 3 μg / mL to about 7 μg / mL, about 1 μg / mL to about 5 μg / mL, about 3 μg / mL to about 5 μg / mL, or about 1 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL, about 5 μg / mL, or about 6 μg / mL.

[0124] In some embodiments of any of the methods disclosed herein, the cells are contacted with poloxamer 338 and PGE2 or a derivative thereof. In certain embodiments, poloxamer 338 and PGE2 or a derivative thereof are used at a final concentration of about 4 mg / mL and about 3 μg / mL to about 7 μg / mL, respectively, or about 4 mg / mL and about 2 μg / mL, respectively, or about 4 mg / mL and about 3 μg / mL, respectively, or about 900 μg / mL and about 3 μg / mL, respectively, or about 4 mg / mL and about 4 μg / mL, respectively, or about 4 mg / mL and about 5 μg / mL, respectively, or about 4 mg / mL and about 6 μg / mL, respectively, or about 4 mg / mL and about 7 μg / mL, respectively, or about 4 mg / mL and about 8 μg / mL, respectively, or about 4 mg / mL and about 9 μg / mL, respectively. In any of the foregoing embodiments, protamine sulfate may optionally be used at a final concentration of about 3 μg / mL to about 7 μg / mL, about 1 μg / mL to about 5 μg / mL, about 3 μg / mL to about 5 μg / mL, or about 1 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL, about 5 μg / mL, or about 6 μg / mL.

[0125] In some embodiments of any of the methods disclosed herein, the cells are contacted with poloxamer 338 and PGE2 or a derivative thereof. In certain embodiments, the poloxamer 338 and PGE2 or a derivative thereof are at about 0.5 mg / mL to about 4 mg / mL and about 3 μg / mL to about 7 μg / mL, respectively, or about 0.5 mg / mL to about 4 mg / mL and about 2 μg / mL, respectively, or about 0.5 mg / mL to about 4 mg / mL and about 3 μg / mL, respectively, or about 900 μg / mL and about 3 μg / mL, respectively, or about 0.5 mg / mL to about 4 mg / mL and about 3 μg / mL, respectively. 0.5mg / mL to about 4mg / mL and about 5μg / mL, respectively, or about 0.5mg / mL to about 4mg / mL and about 6μg / mL, respectively, or about 0.5mg / mL to about 4mg / mL and about 7μg / mL, respectively, or about 0.5mg / mL to about 4mg / mL and about 8μg / mL, respectively, or about 0.5mg / mL to about 4mg / mL and about 9μg / mL, respectively. In any of the above embodiments, protamine sulfate may optionally be used at a final concentration of about 3μg / mL to about 7μg / mL, about 1μg / mL to about 5μg / mL, about 3μg / mL to about 5μg / mL, or about 1μg / mL, about 2μg / mL, about 3μg / mL, about 4μg / mL, about 5μg / mL, or about 6μg / mL.

[0126] In a preferred embodiment, the cells are transduced with the lentiviral vector in a medium containing poloxamer 338 at about 0.5 mg / mL to about 4 mg / mL; PGE2 at about 10 μg / mL to about 50 μg / mL; and protamine sulfate at about 1 μg / mL to about 10 μg / mL. In an attached configuration, the substrate is in some cases coated with the CH296 fragment of human fibronectin, trade name RetroNectin™, using a RetroNectin™ solution at a concentration of about 20 μg / mL to about 100 μg / mL.

[0127] In a preferred embodiment, the cells are contacted with about 0.5 mg / mL to about 4 mg / mL of poloxamer 338; about 10 μg / mL to about 50 μg / mL of PGE2; and about 1 μg / mL to about 10 μg / mL of protamine sulfate.

[0128] In a preferred embodiment, cells are transduced with lentiviral vectors in medium containing poloxamer 338 at about 1 mg / mL; PGE2 at about 10 μM; and protamine sulfate at about 4 μg / mL. In an attached configuration, the substrate is coated with the CH296 fragment of human fibronectin, in some cases under the trade name RetroNectin™, using a RetroNectin™ solution at a concentration of about 20 μg / mL. In one embodiment, the culture dish is coated with 2 ug / cm 2 In one embodiment, the method includes pre-stimulation by culturing the cells on plates coated with a recombinant fibronectin fragment, for example RetroNectin™ (RN).

[0129] In a preferred embodiment, the cells are contacted with about 1 mg / mL poloxamer 338; about 10 μM PGE2; and about 4 protamine sulfate.

[0130] Further illustrative embodiments are provided in Tables 1 and 2. Any of the combinations and concentrations of transduction enhancers shown can be used according to the disclosed methods and with other cell types. Additionally, any of the combinations shown can be further combined with recombinant fibronectin, e.g., RetroNectin™, for example, by coating the culture dishes used for transduction with RetroNectin™.

[0131] Table 1. CB cells and transduction enhancer combinations TIFF0007664829000002.tif150161

[0132] Table 2. mPB cells and transduction enhancer combinations TIFF0007664829000003.tif39161TIFF0007664829000004.tif248161

[0133] In some embodiments, the contacting steps are performed simultaneously or over overlapping periods of time.

[0134] In some embodiments, the concentration of PGE2 or a derivative thereof is 5 to 30 μg / mL.

[0135] In some embodiments, the concentration of PGE2 or a derivative thereof is about 10 μg / mL.

[0136] In some embodiments, the concentration of the poloxamer is between 200 and 1200 μg / mL.

[0137] In some embodiments, the concentration of the poloxamer is about 1000 μg / mL.

[0138] In some embodiments, the concentration of protamine sulfate is 4-10 μg / mL.

[0139] In some embodiments, the concentration of protamine sulfate is about 4 μg / mL.

[0140] In some embodiments, hematopoietic cells are cultured or grown in vessels coated with recombinant fibronectin or a fragment thereof, which enhances transduction efficiency. Recombinant fibronectin fragments (e.g., the CH296 fragment of human fibronectin, trade name RetroNectin™) promote co-localization of lentivirus or retrovirus with target cells and enhance transduction efficiency.

[0141] In some embodiments, the methods include contacting hematopoietic cells with a recombinant fibronectin fragment, a poloxamer, and PGE2.

[0142] In some embodiments, the method includes contacting a hematopoietic cell with a recombinant fibronectin fragment, a poloxamer, and protamine sulfate. In some embodiments, the method includes contacting a hematopoietic cell with a recombinant fibronectin fragment, a poloxamer, PGE2, and protamine sulfate.

[0143] In some embodiments, the methods include contacting the hematopoietic cells with a recombinant fibronectin fragment, PGE2, and protamine sulfate.

[0144] In certain embodiments of any of the methods disclosed herein, the cells are contacted with the transduction enhancer for the same or overlapping time period. In certain embodiments, the cells are also contacted with a recombinant retroviral vector, e.g., for the same or overlapping time period as the cells are contacted with the transduction enhancer. In certain embodiments, the cells are present in a vessel containing a solution or culture medium, and the transduction enhancer is present in the vessel and / or culture medium.

[0145] Prostaglandins Prostaglandins generally refer to hormone-like molecules derived from fatty acids containing 20 carbon atoms, including a five-membered carbon ring, as described herein and known in the art. Prostaglandin E2 (PGE2), also known as dinoprostone, is a naturally occurring prostaglandin used as a medicine. PGE2 has the following structure (represented as the "skeletal structure" and also called the "linear structural formula" or "truncated structural formula"): TIFF0007664829000005.tif38128PGE2 molecular formula:C 22 H 36 O 5 .

[0146] Prostaglandin E2 (PGE2) has been shown to increase levels of lentiviral transgene delivery in ex vivo cultures of CD34+ cells. Heffner et al. Mol Ther. 2018 Jan 3;26(1):320-328.

[0147] An illustrative example of an "analog" or "derivative" of PGE2 is 16,16-dimethyl PGE 2 (dmPGE 2 ), 16-16 dimethyl PGE 2 p-(p-acetamidobenzamido)phenyl ester, 11-deoxy-16,16-dimethyl PGE 2 , 9-deoxy-9-methylene-16, 16-dimethyl PGE 2 , 9-deoxy-9-methylene PGE 2 , 9-ketofluprostenol, 5-trans PGE 2 , 17-phenyl-omega-trinor PGE 2 , P.G.E. 2 Serinolamide, PGE 2 Methyl ester, 16-phenyltetrahydrofuran PGE 2 , 15(S)-15-methyl PGE 2 , 15(R)-15-methyl PGE 2 , 8-iso-15-keto PGE 2 , 8-isoPGE 2 Isopropyl ester, 20-hydroxy PGE 2 , nocloprost, sulprostone, butaprost, 15-keto PGE 2 , and 19(R)hydroxy PGE 2 These include, but are not limited to:

[0148] Also contemplated herein are prostaglandin analogs or derivatives having a structure similar to PGE2 substituted with halogen at the 9-position (see, e.g., WO 2001 / 12596, incorporated herein by reference in its entirety), as well as 2-decarboxy-2-phosphinico prostaglandin derivatives, such as those described in U.S. Patent Application Publication No. 2006 / 0247214, incorporated herein by reference in its entirety.

[0149] Poloxamer Poloxamers are non-ionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)). Poloxamers are also known by the trade names Synperonics®, Pluronics®, and Kolliphor®. Because the length of the polymer blocks can be tailored, many different poloxamers exist. Poloxamers are usually named with the letter P (for poloxamer) followed by three Arabic numerals, the first two of which, when multiplied by 100, give the approximate molecular weight of the polyoxypropylene core, and the last, when multiplied by 10, gives the percentage of polyoxyethylene content (e.g., P407 = a poloxamer with a polyoxypropylene molecular weight of 4000 g / mol and a polyoxyethylene content of 70%).

[0150] In certain embodiments, the poloxamer has polypropylene subunits with an average molecular weight of at least about 2750 daltons. In certain embodiments, the poloxamer has polypropylene subunits with an average molecular weight of at least about 3250 daltons. In certain embodiments, the poloxamer has polypropylene subunits with an average molecular weight of at least about 4000 daltons or at least about 10,000 daltons.

[0151] In certain embodiments, the poloxamer comprises at least about 50% polyethylene oxide. In certain embodiments, the poloxamer comprises at least about 60% polyethylene oxide. In certain embodiments, the poloxamer comprises at least about 70% polyethylene oxide. In certain embodiments, the poloxamer comprises at least about 80% polyethylene oxide.

[0152] In certain embodiments, the poloxamer has polypropylene subunits with an average molecular weight of at least about 2750 daltons and the poloxamer comprises at least about 40% polyethylene oxide. In certain embodiments, the poloxamer has polypropylene subunits with an average molecular weight of at least about 2750 daltons and the poloxamer comprises at least about 50% polyethylene oxide. In certain embodiments, the poloxamer has polypropylene subunits with an average molecular weight of at least about 3250 daltons and the poloxamer comprises at least about 50% polyethylene oxide.

[0153] In certain embodiments, the poloxamer is selected from the group consisting of poloxamer 288, poloxamer 335, poloxamer 338, and poloxamer 407. In one embodiment, the poloxamer is poloxamer 288. In one embodiment, the poloxamer is poloxamer 335. In one embodiment, the poloxamer is poloxamer 338. In one embodiment, the poloxamer is poloxamer 407. In certain embodiments, the recombinant retroviral vector is a recombinant lentiviral vector.

[0154] Recently, poloxamer F108 has been shown to improve transduction of hematopoietic cells. Hoefig et al. J Gene Med. 2012 Aug;14(8):549-60; US Patent No. 9,771,599. Inclusion of poloxamer in standard hematopoietic stem cell (HSC) transduction protocols results in high transduction efficiency while preserving the differentiation capacity of transduced HSCs into various hematopoietic lineages. Hauber at al. Hum Gene Ther Methods. 2018 Apr;29(2):104-113.

[0155] Recombinant fibronectin fragment The recombinant fibronectin fragment can be any fragment of the protein fibronectin, for example human fibronectin, that promotes and enhances transduction efficiency. Without being bound by theory, it is believed that the recombinant fibronectin fragment promotes co-localization of lentivirus or retrovirus with target cells. An example of a recombinant fibronectin fragment is the CH296 fragment of human fibronectin, trade name RetroNectin™.

[0156] hematopoietic cells Hematopoietic cells that can be transduced according to the methods disclosed herein include any hematopoietic cell or population thereof. In certain embodiments, the hematopoietic cells are mammalian hematopoietic cells obtained from a mammal, e.g., a human. In certain embodiments, the cells are obtained from a human to be treated with the hematopoietic cells after transduction according to the methods disclosed herein. In certain embodiments, the hematopoietic cells are enriched for CD34+ cells. In certain embodiments, the hematopoietic cells are a population of CD34-enriched cells obtained from a biological sample obtained from a subject. In one embodiment, the biological sample is a bone marrow sample. In another embodiment, the biological sample is peripheral blood. In another embodiment, the biological sample is umbilical cord blood.

[0157] In certain embodiments, the biological sample, e.g., peripheral blood, is obtained from a subject after mobilization of hematopoietic stem cells (HSCs). In one embodiment, HSCs and / or progenitor cells are mobilized by treating the subject with G-CSF or an analog thereof. HSCs and progenitor cells (HSPCs) in peripheral blood can be mobilized prior to collection of the biological sample. Peripheral blood HSCs and HSPCs can be mobilized by any method known in the art. Peripheral blood HSCs and HSPCs can be mobilized by treating the subject with any agent described herein or known in the art that increases the number of HSPCs circulating in the peripheral blood of the subject. For example, in certain embodiments, peripheral blood is mobilized by treating the subject with one or more cytokines or growth factors (e.g., G-CSF, KIT ligand (KL), IL-I, IL-7, IL-8, IL-11, Flt3 ligand, SCF, thrombopoietin, or GM-CSF (e.g., sargramostim, etc.)). Different types of G-CSF that can be used in the peripheral blood mobilization method include filgrastim and long-acting G-CSF: pegfilgrastim. In certain embodiments, peripheral blood is mobilized by treating the subject with one or more chemokines (e.g., macrophage inflammatory protein-1a (MIP1a / CCL3)), chemokine receptor ligands (e.g., chemokine receptor 2 ligands GROL3 and GR013M), chemokine receptor analogs (e.g., stromal cell-derived factor 1a (SDF-1a) protein analogs, such as CTCE-0021, CTCE-0214, or SDF-1a, such as Met-SDF-113), or chemokine receptor antagonists (e.g., chemokine (CXC motif) receptor 4 (CXCR4) antagonists, such as AMD3100). In certain embodiments, peripheral blood is mobilized by treating the subject with one or more anti-integrin signaling agents (e.g., a function-blocking anti-very late antigen 4 (VLA-4) antibody or anti-vascular cell adhesion molecule 1 (VCAM-1)). In certain embodiments, peripheral blood is mobilized by treating the subject with one or more cytotoxic drugs, such as, for example, cyclophosphamide, etoposide, or paclitaxel.In certain embodiments, peripheral blood can be mobilized by administering one or more of the agents listed above to a subject over a period of time. For example, a subject can be treated with one or more agents (e.g., G-CSF) by injection (e.g., subcutaneous, intravenous, or intraperitoneal) once or twice daily for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days before harvesting HSPCs. In certain embodiments, HSPCs are harvested within 1, 2, 3, 4, 5, 6, 7, 8, 12, 14, 16, 18, 20, or 24 hours after the last administration of the agent used to mobilize HSPCs to peripheral blood. In certain embodiments, HSCs and HSPCs are mobilized by treating the subject with two or more different types of agents as described above or known in the art, such as, for example, a growth factor (e.g., G-CSF) and a chemokine receptor antagonist (e.g., a CXCR4 receptor antagonist such as AMD3100) or a growth factor (e.g., G-CSF or KL) and an anti-integrin agent (e.g., a function-blocking VLA-4 antibody). In one embodiment, HSCs and / or progenitor cells are mobilized by treating the subject with G-CSF or an analog thereof. In one embodiment, the G-CSF is filgrastim. In one embodiment, HSCs and / or progenitor cells are mobilized by treating the subject with plerixafor. In certain embodiments, HSCs and / or progenitor cells are mobilized using a combination of filgrastim and plerixafor, filgrastim alone, or plerixafor alone. In certain embodiments, different types of recruitment agents are administered simultaneously or sequentially.For further information regarding methods of peripheral blood mobilization, see, e.g., Craddock et al., 1997, Blood 90(12):4779-4788; Jin et al., 2008, Journal of Translational Medicine 6:39; Pelus, 2008, Curr. Opin. Hematol. 15(4):285-292; Papayannopoulou et al., 1998, Blood 91(7):2231-2239; Tricot et al., 2008, Haematologica 93(11):1739-1742; and Weaver et al., 2001, Bone Marrow Transplantation 27(2):S23-S29).

[0158] In certain embodiments, peripheral blood is obtained via a syringe or catheter inserted into a vein of the subject. For example, peripheral blood can be collected using a blood component separator. Blood flows from the vein through a catheter into a blood component separator, which separates white blood cells, including HSPCs, from the remainder of the blood, which is then returned to the subject. Blood component separation can be performed for several hours or for consecutive days (e.g., 1-5 days) until sufficient HSPCs are collected.

[0159] In certain embodiments, bone marrow is obtained from the subject's posterior iliac crest by needle aspiration (see, eg, Koda et al., 1984, J. Clin Invest. 73:1377-1384).

[0160] In certain embodiments, the hematocrit of the biological sample may be determined. The hematocrit may be determined by centrifuging the sample in a processing chamber to separate the RBCs of the sample into layers so that the packed red blood cell volume may be determined. It is recognized that the sample may be combined with an anticoagulant to aid in determining the hematocrit, and such an anticoagulant may be added to the processing chamber before or during centrifugation. Alternatively, the hematocrit may be determined by measuring an optical property of the sample. For example, the sample may be analyzed using a spectrometer. It is recognized that any type of known spectroscopic method may be used to determine the hematocrit, such as Raman spectroscopy and / or light scattering.

[0161] In certain embodiments, the biological sample is depleted of red blood cells, e.g., prior to preparing one or more cell populations enriched for CD34+ cells from the biological sample. In some embodiments, the cells remaining after the depletion process are washed. In another embodiment, a non-specific IgG is added to the washed cells. In some embodiments, the non-specific IgG is flebogamma.

[0162] In some cases, two or more biological samples, including, for example, bone marrow samples obtained at various times, including other time increments, such as 1 day, 2 days, 3 days, 4 days or more apart, or 1, 2 or 3 weeks apart, or 1, 2 or 3 months apart, or years apart, are mixed together prior to CD34+ sorting.

[0163] Enrichment of CD34+ cells from hematopoietic cells In some embodiments, the hematopoietic cells are CD34 positive hematopoietic cells. Typically, CD34+ cells are prepared by high stringency enrichment of CD34. Alternatively, or in addition to high stringency enrichment, low stringency enrichment of CD34 may be performed.

[0164] As used herein, "high stringency" or "high stringency conditions" refers to a method of enriching a cell population intended to result in a substantial enrichment of cells for cells expressing a particular biological marker, e.g., CD34. For example, clinically used "high stringency" enrichment for CD34 is characterized by the fact that the enrichment is performed under conditions that result in substantial enrichment of cells for cells expressing a particular biological marker, e.g., CD34. +This results in a mean and median cell yield of 61.6% and 65.7%, and a mean and median relative purity (N=166) of 88.5% and 95.9% (Clin Lab. 2016 Jul 1;62(7):1243-1248 (PMID:28164638)). "High stringency" refers to a process that targets substantial enrichment of the relatively rare cell type CD34+, which typically comprises 0.2-2% of the cell product in mobilized leukapheresis or bone marrow harvest. High stringency enrichment of CD34+ cells from mobilized leukapheresis or bone marrow harvest targets a final CD34+ concentration that is increased from 0.2-2% to over 80%. To achieve this, the biological sample is first applied to the capture substrate, followed by repeated buffer exchanges, referred to herein as "washing", to remove weakly bound or non-specifically bound cells to the capture substrate. Typically, cells are removed from the capture substrate and reapplied with each wash cycle. The removal and reapplication can be accomplished manually by pipetting from a tube, or automatically using a pump and tube system. For example, using Quad Technologies' MagCloudz® in conjunction with a Dynabeads® magnetic cell separation system, the cell-magnetic particle complexes are separated in a tube on a magnetic stand and washed manually. Using Miltenyi Biotec's CliniMACS® system, the cell-magnetic particle complexes are applied to a magnetic column in a tube set with a preset automated program, and washed / reapplied using a valve pump system.In certain embodiments, the selection under high stringency conditions can be performed on a variety of devices, including, but not limited to, Miltenyi Biotec's MACSQuant Tyto®, Quad Technologies' MagCloudz®, GE's Sepax® cell separation system, Terumo's Elutra® cell separation system, COBE's Spectra® cell separator, SynGen's LAB® system or WASH® system, Fresenius-Kabi's Lovo®, Miltenyi Biotec's CliniMACS® system or CliniMACS Prodigy® system. The selection can be performed in the laboratory or at the point of care. Detailed methods for the preparation and enrichment of cells and cell populations, including exemplary methods for the selection of CD34+ cells under high stringency conditions, are described, for example, in International Patent Application Publication WO 2016 / 118780. Illustrative selection methods useful for high stringency selection are provided in U.S. Patent No. 8,727,132. Further illustrative selection methods are provided in International Patent Application No. PCT / US2019 / 027083, particularly in Example 1.

[0165] In a high stringency enrichment protocol, a biological sample containing CD34+ cells is labeled with a CD34 labeling agent, for example, directly bound immunomagnetic beads. The biological sample may be suspended in any suitable liquid, for example, but not limited to, phosphate buffered saline (PBS) optionally containing ethylenediaminetetraacetic acid (EDTA) at a buffer pH and isotonicity compatible with cell viability. In some cases, the liquid used also contains human serum albumin, at a suitable concentration, for example, about 2.5%. Using magnetic activated cell sorting (MACS) technology, the biological sample is applied to a column after labeling, which contains a magnetically susceptible or ferromagnetic material. Using the MACS system, the magnetically susceptible or ferromagnetic material of the column retains the target cells without affecting the ability of non-target cells to flow through and exit the column. Such magnetically susceptible or ferromagnetic materials include iron, steel, cobalt nickel, and other ferromagnetic rare earth metals in their alloys. Those skilled in the art will recognize that such materials can be easily magnetized and demagnetized. In some embodiments, the biological sample is recirculated over the magnetically susceptible or ferromagnetic material one or more times. After loading the column, the bound cells are washed and eluted and / or reloaded onto the column at a slower speed to improve the purity of the enriched fraction. Suitable washing buffers include PBS with (optional) EDTA and (optional) human serum albumin. Any components of the labeled biological sample that are removed during the washing steps are collected in a waste bag or a "non-target" bag. After suitable washing steps, the high stringency enriched cells are eluted into the target cell bag.

[0166] In low stringency enrichment protocol, the biological sample containing CD34+ cells is labeled with a CD34 labeling agent, for example, directly bound immunomagnetic beads. After labeling, the biological sample is applied to a column containing magnetically susceptible or ferromagnetic material using magnetic activated cell sorting (MACS) technology at a lower flow rate than in high stringency enrichment. As in high stringency enrichment, the magnetically susceptible or ferromagnetic material retains the target cells without affecting the ability of non-target cells to flow through and exit the column. In some embodiments, the biological sample is recirculated over the magnetically susceptible or ferromagnetic material one or more times. After loading the column, in low stringency enrichment, the bound cells are washed at a lower stringency. The bound cells are then eluted into a collection bag.

[0167] In an exemplary embodiment, low stringency enrichment is performed by modifying the standard operating method of the MACS system such that the software program of the "removal mode" intended to achieve high stringency removal (i.e. removal of target cells) results in low stringency enrichment instead. By operating the MACS system in the removal mode, the target cells in the biological sample are bound to the magnetically susceptible or ferromagnetic material in the column using a slow column loading and a washing step with a lower stringency than in the enrichment mode operation. The unlabeled cells are allowed to flow by the MACS system into a washing bag or so-called "target" bag. The removal mode program then switches the output valve to direct the liquid into the so-called "non-target" bag, and then demagnetizes the column. The target cells are recovered by the method, in which the CD34+ enriched cell population, which was enriched under low stringency conditions, is eluted into the so-called "non-target" bag by successive application of liquid over the demagnetized column.

[0168] Those skilled in the art will recognize that this low stringency enrichment method can be performed on a variety of devices, including, but not limited to, Miltenyi Biotec's MACSQuant Tyto®, Quad Technologies' MagCloudz®, GE's Sepax® Cell Separation System, Terumo's Elutra® Cell Separation System, COBE's Spectra® Cell Separator, SynGen's LAB System® or WASH® System, Fresenius-Kabi's Lovo®, Miltenyi Biotec's CliniMACS® System or CliniMACS Prodigy® System. Those skilled in the art will be able to reprogram the software of such MACS systems without undue experimentation such that the output valve directs the flow through the first binding step to a waste bag or a "non-target" bag (rather than the target bag) and directs the eluted low stringency CD34 enriched population to the "target" bag. In short, a low stringency enrichment is then carried out in separation mode, without the usual washing steps of a conventional MACS program.

[0169] As used herein, "low stringency" or "low stringency conditions" refers to a method of enriching a cell population intended to result in enrichment of cells for cells expressing a particular biological marker, e.g., CD34, in such a manner that the yield of the enriched cell population is maintained higher than that achieved by high stringency sorting, at the expense of, i.e., reduced enrichment of, cells expressing the biological marker relative to other cells in the biological sample. By definition, the fold enrichment under high stringency conditions is greater than that under low stringency conditions. The fold enrichment of cells, e.g., CD34+ cells, in a (CD34 or other marker) enriched cell population at high stringency is, in some cases, 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, or 4 times the fold enrichment of CD34+ cells in a (CD34 or other marker) enriched cell population at low stringency. In one embodiment, the fold enrichment of cells, e.g., CD34+ cells, in a (CD34 or other marker) enriched cell population at high stringency is 2-4 times higher than the fold enrichment of CD34+ cells in a (CD34 or other marker) enriched cell population at low stringency. In certain embodiments, sorting under low stringency conditions can be performed on a variety of devices, including, but not limited to, Miltenyi Biotec's MACSQuant Tyto®, Quad Technologies' MagCloudz®, GE's Sepax® cell separation system, Terumo's Elutra® cell separation system, COBE's Spectra® cell separator, SynGen's LAB® system or WASH® system, Fresenius-Kabi's Lovo®, Miltenyi Biotec's CliniMACS® system or CliniMACS Prodigy® system. Sorting can be performed in the laboratory or at the point of care.Exemplary cell enrichment methods under low stringency conditions are provided in International Patent Application No. PCT / US2019 / 027083, which is incorporated by reference in its entirety.

[0170] The population of cells enriched for CD34+ cells can be produced by sorting CD34+ cells under high stringency and / or low stringency conditions, thereby producing a CD34-enriched cell population at high stringency and / or a CD34-enriched cell population at low stringency. The method of sorting CD34+ cells can be positive selection, negative selection, or a combination thereof. In one embodiment, a biological sample obtained from a subject is divided into two samples, and one sample is used to prepare a CD34-enriched cell population at high stringency and the other sample is used to prepare a CD34-enriched cell population at low stringency. In other embodiments, a biological sample obtained from a subject is first subjected to low stringency CD34+ selection to prepare a low stringency CD34-enriched cell population, and then a portion of the low stringency CD34-enriched population is subjected to high stringency CD34+ selection to prepare a high stringency CD34-enriched cell population. Selection may be applied sequentially, such that selection of CD34-enriched cells under low stringency conditions may be applied first, followed by selection of additional CD34-enriched cells from the resulting population under high stringency conditions. In other cases, selection of CD34-enriched cells under high stringency conditions may be applied first, followed by selection of the remaining population of CD34-enriched cells under low stringency conditions. In some cases, the cell population may be divided such that low stringency or high stringency selection is applied to a fraction of cells that have previously been subjected to high stringency or low stringency selection. In some cases, a single biological sample is divided into two or more samples prior to selection of CD34-enriched cells under low or high stringency conditions.

[0171] In all cases, all possible permutations contemplate high or low stringency sorting prior to mixing or dividing the biological sample or enriched cell population, or after mixing or dividing the biological sample or enriched cell population. In one embodiment, the method comprises the steps of: + preparing a CD34 enriched cell population at high stringency from a first biological sample obtained from a subject by sorting cells; and + and preparing a low stringency CD34 enriched cell population from a second biological sample obtained from the subject by sorting the cells.

[0172] Transduced hematopoietic cells As described in more detail in the Examples, a population of hematopoietic cells transduced in the presence of a combination of transduction enhancers disclosed herein, e.g., protamine sulfate, PGE2 or a derivative thereof, and poloxamer (e.g., LentiBOOST), exhibits superior characteristics compared to hematopoietic cells transduced without the combination of transduction enhancers. In certain embodiments, the cells are also transduced in the presence of a recombinant fibronectin fragment, e.g., RetroNectin™. In certain embodiments, the transduced hematopoietic cells or populations thereof have one or more of increased VCN, increased VCN / cell, increased percentage of genetically modified CFU, and increased percentage of genetically modified CFC. In some embodiments, rescue of the gene of interest is enhanced. In certain embodiments for patients with LAD-1, CD18 expression was significantly increased after transduction with a lentiviral vector comprising the CD18 gene compared to transduction without PGE2 or poloxamer, or compared to transduction without or with only one transduction enhancer. + The percentage of cells increases.

[0173] In some embodiments of the disclosed methods, the percentage of hematopoietic cells genetically modified by the method is increased by at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, or at least 8-fold compared to the percentage of hematopoietic cells genetically modified with the same viral vector but without treating the cells with PGE2 or poloxamer, or compared to the percentage of hematopoietic cells genetically modified with the same viral vector but treating the cells with only one transduction enhancer.

[0174] In some embodiments, a method of transducing hematopoietic cells with a retroviral (e.g., lentiviral) vector results in a population of hematopoietic cells having a VCN / cell of at least 1.0, at least 1.5, at least 2.0, or at least 2.5. In some embodiments, a method of treating a subject comprises providing to the subject a population of transduced hematopoietic cells having a VCN / cell of at least 1.0, at least 1.5, at least 2.0, or at least 2.5.

[0175] In some embodiments, methods of transducing hematopoietic cells with retroviral vectors result in a population of hematopoietic cells that has a transduction efficiency of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%. In some embodiments, methods of treating a subject include providing to the subject a population of transduced hematopoietic cells that has a transduction efficiency of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%.

[0176] In some embodiments, the method of transducing hematopoietic cells with a retroviral vector results in a population of hematopoietic cells in which the percentage of colony forming cells is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% transduced. In some embodiments, the method of treating a subject comprises providing to the subject a population of transduced hematopoietic cells in which the percentage of colony forming cells is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% transduced.

[0177] In some embodiments, the disclosure provides populations of hematopoietic cells transduced with a recombinant retroviral vector (e.g., a lentiviral vector) having a VCN / cell of at least 1.0, at least 1.5, at least 2.0, or at least 2.5. In some embodiments, the disclosure provides populations of hematopoietic cells transduced with a recombinant retroviral vector (e.g., a lentiviral vector) having a transduction efficiency of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%.

[0178] In some embodiments, the present disclosure provides a method for producing a cellular endothelial cell that is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% ... or at least 75% endothelial cell that is at least 75% endothelial cell that is at least 75% endothelial cell that is at least 75% endothelial cell that is at least 75% endothelial cell that is at least 75% endothelial cell that is at least 75% endothelial cell that is at least 75% endothelial cell that is at least 75% endothelial cell that is at A method for producing a population of hematopoietic cells, 5% of which are genetically modified, is provided, comprising ex vivo contacting the hematopoietic cells with a recombinant retroviral vector (optionally a lentiviral vector) comprising a polynucleotide comprising a gene of interest or encoding a polypeptide of interest, the contacting occurring in the presence of PGE2 or a derivative thereof, optionally human PGE2 or 16,16-dimethyl PGE2 (dmPGE2), and a poloxamer, optionally poloxamer 338 (LentiBOOST™). The cells may be contacted with the retroviral vector under conditions and for a time sufficient to permit transduction of the cells by the retroviral vector, for example, at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, or at least 12 hours in a suitable medium. In some embodiments, the cells are contacted with the retroviral vector and the transduction enhancer for the same or overlapping periods of time.

[0179] Advantageously, the methods of the disclosure result in reduced toxicity (longer survival) of the transduced cell population compared to transduction without the transduction enhancer. In some embodiments, the disclosure provides methods of producing a population of hematopoietic cells, where toxicity is reduced by at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, or at least 35% compared to transduction without the transduction enhancer.

[0180] Vectors and Expression Cassettes Any convenient recombinant retroviral vector that finds use in delivering polynucleotide sequences to mammalian cells is encompassed by the recombinant retroviral vector of the present disclosure. For example, the vector can contain single-stranded or double-stranded nucleic acid, e.g., single-stranded or double-stranded DNA. For example, the recombinant retroviral vector can be DNA. The vector can contain single-stranded or double-stranded RNA, including modified forms of RNA. In another embodiment, the recombinant retroviral vector can be RNA, e.g., mRNA or modified mRNA.

[0181] In certain embodiments, the recombinant retroviral vector may be a viral vector derived from a virus, e.g., an adenovirus, an adeno-associated virus, a lentivirus (LV), a herpesvirus, an alphavirus, or a retrovirus, e.g., Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), Murine mammary tumor virus (MuMTV), Gibbon ape leukemia virus (GaLV), Feline leukemia virus (FLV), Spumavirus, Friend murine leukemia virus, Murine stem cell virus (MSCV) or Rous sarcoma virus (RSV). Although embodiments involving the use of LV are described in more detail below, it is expected that those skilled in the art will recognize that similar knowledge and techniques in the art can also be used to maintain non-LV recombinant retroviral vectors. In some embodiments, the recombinant retroviral vector is a self-limiting LV.

[0182] In certain embodiments, the viral vector is a lentiviral vector, hi some embodiments, it is a pseudotyped lentiviral vector, for example a VSVG pseudotyped lentiviral vector.

[0183] In particular, certain methods disclosed herein involve transducing two populations of stem or progenitor cells, e.g., hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (also referred to herein as "hematopoietic precursors"), with a recombinant retroviral vector encoding and / or expressing a polypeptide preparation, e.g., FANCA, one population prepared by selection under high stringency conditions and the other population prepared by selection under low stringency conditions. In one embodiment, the cell population is enriched for CD34+ cells. In one embodiment, the HSCs or hematopoietic precursors are from a subject with reduced or absent protein activity from one or more FANCA-encoded proteins. In one embodiment, the subject has FA-A. In one embodiment, the endogenous FANCA gene of the HSCs is deleted and / or mutated.

[0184] In one embodiment, transduction of a cell with a recombinant retroviral vector results in the integration into the cell genome of an expression cassette comprising a promoter operably linked to a polynucleotide sequence encoding a therapeutic agent within the recombinant retroviral vector, hi some embodiments, transduction of a cell with a recombinant retroviral vector results in the expression of a therapeutic agent, e.g., a biologically active FANCA protein.

[0185] For example, biologically active FANCA protein forms part of the FA core complex. In some embodiments, the FANCA gene is delivered via a viral vector. In one embodiment, the FANCA gene is delivered via a lentiviral vector. In some embodiments, the lentiviral vector is PGK-FANCA.WPRE*LV. After transduction of bone marrow (BM) cells or stem or progenitor cells from FA-A patients with the FANCA lentiviral vector (LV), the therapeutic vector is intended to be integrated into the genome of the cells. Upon integration, the therapeutic protein (e.g., human FANCA protein) is expressed by the cells. The transduced FA cells are genetically corrected and are thereby able to activate the FA pathway by monoubiquitination of FANCD2 and FANCI. These proteins can then migrate to areas of DNA damage and cooperate with other DNA repair proteins to promote DNA repair in these cells as occurs in healthy cells.

[0186] As discussed herein, the subject methods and compositions find use in expressing a transgene, e.g., FANCA, in the cells of an animal. For example, the subject compositions may be used in research, e.g., to determine the effect a gene has on cell viability and / or function. As another example, the subject compositions may be used in medicine, e.g., to treat a disease such as FA.

[0187] In some embodiments, the subject methods result in a therapeutic effect, such as, for example, preventing the onset of a disease, halting the progression of a disease, or reversing the progression of a disease. For example, in one embodiment, the disease is Fanconi anemia (FA). In another embodiment, the disease or disorder is bone marrow failure (BMF). In one embodiment, the disease is thrombocytopenia. In another embodiment, the disease is leukopenia. In one embodiment, the disease is pancytopenia. In one embodiment, the disease is neutropenia. In another embodiment, the disease is anemia. In some embodiments, the subject methods include detecting that a therapeutic effect has been achieved. One of skill in the art will recognize that such measures of therapeutic efficacy will be applicable to the particular disease that has been altered, and will recognize appropriate detection methods to use to measure therapeutic efficacy.

[0188] Thus, the present invention provides a method of treating FA or one or more of the hematological manifestations of FA. In one embodiment, the hematological manifestations of FA are selected from one or more of bone marrow failure (BMF), thrombocytopenia, leukopenia, pancytopenia, neutropenia, and anemia. In a particular embodiment, the hematological manifestation is BMF, which appears in childhood in most FA patients. In one embodiment, the hematological manifestation is thrombocytopenia. In another embodiment, the hematological manifestation is leukopenia. In one embodiment, the hematological manifestation is pancytopenia. In one embodiment, the hematological manifestation is neutropenia. In another embodiment, the hematological manifestation is anemia. In one embodiment, the hematological manifestation is a combination of two or more of BMF, thrombocytopenia, leukopenia, pancytopenia, neutropenia, and anemia.

[0189] Additional LV vectors that can be used in accordance with the methods disclosed herein include, but are not limited to, those prepared using the transfer vectors disclosed below.

[0190] 2. Use of Transduced Hematopoietic Cells In some embodiments, hematopoietic cells transduced according to the methods disclosed herein are used for gene therapy. In certain embodiments, hematopoietic cells are transduced with a vector comprising a gene of interest. The transduced cells can be provided to a subject in need thereof, for example, to treat a genetic disease or disorder in the subject. In certain embodiments, the gene of interest complements a defect in a gene associated with a monogenic genetic disease or disorder. In some embodiments, the subject comprises a mutation in an endogenous gene of interest. In some embodiments, the gene of interest provided to the subject is codon-optimized, for example, to enhance expression in mammalian cells.

[0191] In certain embodiments, the gene of interest is selected from the group consisting of Fanconi Anemia Complementation Group A (FANCA), Complementation Group C (FANCC), and Complementation Group G (FANCG). In certain embodiments, the gene of interest is Red-cell type Pyruvate Kinase (RPK). In certain embodiments, the gene of interest is Integrin beta 2 (ITGB2), and / or the gene of interest encodes a protein encoded by any of the genes disclosed herein or functional fragments or functional variants thereof.

[0192] In certain embodiments, the methods prevent or ameliorate a monogenic genetic disease or disorder.

[0193] In certain embodiments, the monogenic disease or disorder is selected from the group consisting of Fanconi Anemia, Leukocyte Adhesion Deficiency Type 1, Pyruvate Kinase Deficiency, and Infantile Malignant Osteoporosis.

[0194] Namely, methods are provided for treating monogenic diseases or disorders including, but not limited to, Fanconi anemia, leukocyte adhesion deficiency type 1, pyruvate kinase deficiency, and infantile malignant osteopetrosis. In certain embodiments, the methods comprise providing to a subject in need thereof hematopoietic cells transduced with a retroviral vector comprising a polynucleotide comprising a sequence encoding a therapeutic protein operably linked to a promoter sequence, the cells being transduced according to the methods disclosed herein, e.g., in the presence of two or more transduction enhancers as disclosed herein.

[0195] In some embodiments, the invention includes a cell comprising a gene expression cassette, gene transfer cassette, or recombinant retroviral vector, e.g., any of those disclosed herein. In related embodiments, the cell has been transduced with a recombinant retroviral vector comprising the expression cassette or has the expression cassette integrated into the genome of the cell, and the transduction has been carried out according to the methods disclosed herein. In some embodiments, the cell is a cell used to produce a recombinant retroviral vector, e.g., a packaging cell.

[0196] In certain embodiments, the cells are cells that are to be delivered to a subject to provide the subject with a gene product encoded by the expression cassette. That is, in certain embodiments, the cells are autologous to the subject to be treated or obtained from the subject to be treated. In other embodiments, the cells are allogeneic to the subject to be treated or obtained from a donor other than the subject to be treated. In certain embodiments, the cells are mammalian cells, e.g., human cells. In certain embodiments, the cells are blood cells, erythrocytes, hematopoietic progenitor cells, e.g., bone marrow cells that are lineage-depleted bone marrow cells, hematopoietic stem cells (e.g., CD34+), or committed hematopoietic erythroid progenitor cells. In certain embodiments, the cells are CD34+ cells obtained from the subject to be treated with the cells after transduction of the cells with a recombinant retroviral vector disclosed herein. In certain embodiments, the cells are CD34+ FA cells obtained from a subject diagnosed with FA. The present disclosure further includes populations of hematopoietic cells (optionally CD34 enriched cells) transduced according to the methods disclosed herein.

[0197] In some embodiments, the methods disclosed herein result in a therapeutic effect, such as, for example, preventing the onset of a disease, halting the progression of a disease, or reversing the progression of a disease. For example, in one embodiment, the disease is BMF. In one embodiment, the disease is thrombocytopenia. In another embodiment, the disease is leukopenia. In one embodiment, the disease is pancytopenia. In one embodiment, the disease is neutropenia. In another embodiment, the disease is anemia. In some embodiments, the subject method includes detecting that a therapeutic effect has been achieved. One skilled in the art will recognize that such a measurement of therapeutic efficacy will be applicable to the particular disease that has been modified, and will recognize the appropriate detection method to use to measure therapeutic efficacy.

[0198] Expression of the transgene using the subject transgene is expected to be robust. Thus, in some instances, expression of the transgene, such as by measuring therapeutic efficacy, e.g., by measuring levels of the gene product, may be observed within 2 months, e.g., within 4 weeks, 3 weeks, or 2 weeks, e.g., 1 week after administration, of the subject compositions. Expression of the transgene is also expected to persist over time. Thus, in some instances, expression of the transgene, such as by measuring therapeutic efficacy, e.g., by measuring levels of the gene product, may be observed for 2 months or more, e.g., 4 months or more, 6 months or more, 8 months or more, or 10 months or more, and in some instances for 1 year or more, e.g., 2 years, 3 years, 4 years, or 5 years, and in certain instances for 5 years or more, of the subject compositions.

[0199] In certain embodiments, the method includes detecting expression of the transgene in a cell or in a subject, where expression is enhanced compared to expression from a polynucleotide cassette that does not include one or more of the improved elements of the present disclosure. Typically, expression will be enhanced by 2-fold or more, e.g., 3-fold, 4-fold, or 5-fold or more, and in some cases 10-fold, 20-fold, or 50-fold or more, e.g., 100-fold, compared to expression from a reference, or control, polynucleotide cassette, e.g., as known in the art, and this is evidenced, e.g., by earlier detection, higher levels of gene product, a stronger functional effect on the cell, etc.

[0200] In some embodiments, the dose of cells a patient receives by infusion can be obtained from a transduction process. In various preferred embodiments, the dose is at least about 1×10 cells per kilogram of patient body weight. 1 pieces, 1×10 2 pieces, 1×10 3 pieces, 1×10 4 pieces, 1×10 5 pieces, 1×10 6 pieces, 1×10 7 pieces, 1×108 or more high stringency CD34 enriched cells are infused into the patient. In various preferred embodiments, at least about 1×10 1 pieces, 1×10 2 pieces, 1×10 3 pieces, 1×10 4 pieces, 1×10 5 pieces, 1×10 6 pieces, 1×10 7 pieces, 1×10 8 10 or more low stringency CD34 enriched cells are infused into the patient. In some embodiments, 1×10 6 pieces~4×10 6 In another embodiment, 3×10 high stringency CD34 enriched cells are infused into the patient per kilogram of patient body weight. 5 pcs and 4×10 6 high stringency CD34 enriched cells are infused into the patient. In some embodiments, 1×10 high stringency CD34 enriched cells are infused into the patient per kilogram of patient body weight. 6 pieces~4×10 6 In another embodiment, 3×10 high stringency CD34 enriched cells are infused into the patient per kilogram of patient body weight. 5 pcs and 4×10 6 A number of high stringency CD34 enriched cells are infused into the patient. In some embodiments, the cells will be infused into the patient in a single dose. In other embodiments, the cells will be infused into the patient in multiple doses (e.g., populations of high stringency and low stringency CD34 enriched cells are administered one or more times over time). The transduced cells may be infused immediately after the transduction process is completed. In certain embodiments, the transduced cells are stored or frozen until use, while in some embodiments, the transduced cells are provided to the subject immediately or shortly after transduction of the cells, for example within 1 hour, 2 hours, 4 hours, or 8 hours.

[0201] Upon integration, a therapeutic protein (e.g., human FANCA protein) is expressed by the cells. The transduced FA cells are genetically corrected and are thereby able to activate the FA pathway by monoubiquitination of FANCD2 and FANCI. These proteins migrate to areas of DNA damage and cooperate with other DNA repair proteins to promote DNA repair in these cells as occurs in healthy cells.

[0202] As described in more detail in the Examples, in vitro preclinical data with BM samples from human FA patients have already demonstrated the efficacy of FANCA LV to correct the phenotype of these cells.

[0203] In one embodiment, at least 1 x 10 per kilogram of patient body weight 5 ~4×10 5 Modified CD34 + The cells (e.g., FANCA transduced HSCs) are administered to restore hematopoietic cells in untreated FA patients. In some embodiments, the transduced cells are infused or administered to the patient immediately after transduction. In other embodiments, the transduced cells are frozen prior to infusion or administration to treated or untreated patients.

[0204] Genetic correction of HSCs from FA patients followed by autologous transplantation of these cells (hematopoietic gene therapy) is a good alternative for FA patients, especially for those patients who lack an HLA-homogenous sibling for allogeneic transplantation. In one embodiment, hematopoietic gene therapy is the preferred treatment regimen for patients who lack an HLA-homogenous sibling. In another embodiment, hematopoietic gene therapy is the preferred treatment regimen for patients who have an HLA-homogenous sibling.

[0205] Fanconi anemia (FA) is an autosomal recessive disease (except for complementation group FA-B, which is X-linked), and the median survival of patients is approximately 24 years (Butturini A, et al. (1994) Blood 84:1650-1655; Kutler DI, et al. (2003) Blood 101:1249-1256). At birth, these patients generally have normal blood counts. The first hematological abnormality detected in these patients is often macrocytosis, which usually evolves with thrombocytopenia, anemia, and pancytopenia. Bone marrow failure (BMF) is usually observed in these patients after 5 to 10 years, with the mean age of onset of hematological disorders being 7 years. Approximately 80% of FA patients will have evidence of BMF within the first decade of life. Based on epidemiological studies to date, unless a malignant episode precedes growth failure, nearly all patients with FA will develop BMF by the age of 40 years, which is the leading cause of death in these patients. Due to the complex clinical manifestations of FA, management of these patients is primarily focused on improving the clinical manifestations of: bone marrow failure (BMF), myeloid leukemia, and solid tumors.

[0206] In certain embodiments, the disclosure provides a method of treating Fanconi Anemia (FA) in a subject in need thereof, comprising administering hematopoietic cells transduced with a recombinant retroviral vector comprising a polynucleotide encoding a Fanconi Anemia Complementation Group (FANC) gene, or a gene encoding a functional variant or functional fragment thereof, according to the methods disclosed herein. In some embodiments, the gene encodes FANCA.

[0207] In some embodiments, the methods disclosed herein are used to transduce hematopoietic cells with lentiviral vectors produced using pCCL-PGK-FANCAW-82-PRO transfer vector, for example to generate cell populations for the treatment of FA. pCCL-PGK-FANCAW-82-PRO is a lentiviral vector based on the pCCL transfer plasmid used in the third generation lentiviral vector system. The pCCL transfer plasmid is a lentiviral vector containing a chimeric CMV-HIV 5'LTR and a vector backbone containing a simian virus 40 polyadenylation sequence and an (enhancer-less) origin of replication sequence downstream of the HIV 3'LTR, which replaces most of the human sequences remaining from the HIV integration site. In pCCL, the CMV enhancer and promoter (nucleotides -673 to -1 relative to the transcription start; GenBank accession number K03104) are joined to the R region of HIV-1. The vector uses the PGK promoter linked to a codon-optimized FANCA gene containing upstream RRE and cPPT / CTS elements and a downstream wPRE element (Figure 15).

[0208] The resulting lentiviral vector is used to transduce autologous CD34+ hematopoietic stem cells ("HSCs"), thereby complementing the genetic defect. Briefly, HSCs are mobilized by treating the patient with G-CSF, plerifaxor, or a combination of G-CSF and plerifaxor. HSCs are then harvested by apheresis from the patient's peripheral blood. CD34+ cells are enriched by magnetic capture (e.g., with Miltenyi Biotec's CliniMACS system), and the CD34+ enriched cells are transduced ex vivo according to the methods disclosed herein with lentiviral particles previously generated by transient transfection of a third generation lentiviral vector system containing the pCCL-PGK-FANCAW-82-RO transduction plasmid. In some embodiments, the cells are transduced in the presence of PGE2 and poloxamer. In some embodiments, the poloxamer is poloxamer 338 (LentiBOOST™). The transduced HSCs are then implanted into the patient by infusion to reconstitute the HSC niche with FANCA-expressing cells.

[0209] The sequence of the FANCA expression cassette in pCCL-PGK-FANCAW-82-PRO(5'-3') is as follows: The coding sequence for FANCA is shown in bold capital letters. The WPRE sequence is underlined. TIFF0007664829000006.tif124160TIFF0007664829000007.tif218160TIFF0007664829000008.tif101160

[0210] The FANCA protein sequence encoded by this polypeptide is provided as SEQ ID NO: 2. Vectors for expression of FANCA that are useful in the present invention include, but are not limited to, those disclosed in International Patent Application WO 2018 / 049273, the disclosure of which is incorporated herein in its entirety.

[0211] Leukocyte adhesion deficiency type 1 (LAD-1) is a rare autosomal recessive disorder caused by mutations in the ITGB2 gene, which encodes CD18, a protein present in several cell surface receptor complexes present on leukocytes, including lymphocyte function-associated antigen type 1 (LFA-1), complement receptor type 3 (CR-3), and complement receptor type 4 (CR-4). LFA-1 deficiency renders neutrophils unable to adhere to and migrate out of blood vessels, which can lead to increased neutrophil counts. It also impairs lymphocyte functions of immune cell interaction, immune recognition, and cell killing.

[0212] In certain embodiments, the disclosure provides a method of treating leukocyte adhesion deficiency type I (LAD-1) in a subject in need thereof, comprising administering hematopoietic cells transduced with a recombinant retroviral vector comprising a polynucleotide encoding the ITGB2 gene, or a gene encoding a functional variant or functional fragment thereof, according to the methods disclosed herein.

[0213] In some embodiments, the methods disclosed herein are used to transduce hematopoietic cells with lentiviral vectors produced using pCCL-ChimhCD18W-82-RO transfer vector, for example to generate cell populations for the treatment of LAD-1. pCCL-ChimhCD18W-82-RO is a lentiviral transfer vector based on the pCCL transfer plasmid used in the third generation lentiviral vector system. The pCCL transfer plasmid is a lentiviral vector containing a chimeric CMV-HIV 5'LTR and a vector backbone containing a simian virus 40 polyadenylation sequence and an (enhancer-less) origin of replication sequence downstream of the HIV 3'LTR, which replaces most of the human sequences remaining from the HIV integration site. In pCCL, the CMV enhancer and promoter (nucleotides -673 to -1 relative to the transcription start; GenBank accession number K03104) are joined to the R region of HIV-1. The vector uses the Chim promoter linked to the codon-optimized ITGB2 gene containing upstream RRE and cPPT / CTS elements and a downstream wPRE element (Figure 16). The Chim promoter is a fusion of the c-Fes promoter and the CTSG minimal 5' flanking region (the TATA box of the CTSG promoter has been mutated to restrict transcription initiation to only the c-Fes minimal promoter).

[0214] Lentiviral particles are generated by transient transfection of a third generation lentiviral vector system containing a LAD-1 transduction plasmid. The lentiviral particles are used to transduce autologous CD34+ hematopoietic stem cells (HSCs), thereby complementing the genetic defect. Briefly, HSCs are mobilized by treating the patient with G-CSF, plerifaxor, or a combination of G-CSF and plerifaxor. HSCs are then harvested by apheresis from the patient's peripheral blood. CD34+ cells are enriched by magnetic capture (e.g., with Miltenyi Biotec's CliniMACS system), and the CD34+ enriched cells are transduced ex vivo with lentiviral particles. The transduction process incorporates the use of transduction enhancers, specifically polyaxamers (LentiBOOST licensed by Rocket from Sirion Biotech GmbH for both clinical and commercial use) and PGE2 (commercially available from LGM Pharma). The transduced HSCs are then implanted into the patient by infusion, where they reconstitute the HSC niche with LAD-1 expressing cells.

[0215] The resulting lentiviral vector is used to transduce autologous CD34+ hematopoietic stem cells ("HSCs") to complement the genetic defect. Briefly, HSCs are mobilized by treating the patient with G-CSF, plerifaxor, or a combination of G-CSF and plerifaxor. HSCs are then harvested by apheresis from the patient's peripheral blood. CD34+ cells can be enriched by magnetic capture (e.g., with Miltenyi Biotec's CliniMACS system), and the CD34+ enriched cells are transduced ex vivo according to the methods disclosed herein with lentiviral particles previously generated by transient transfection of a third generation lentiviral vector system containing the pCCL-ChimhCD18W-82-RO transduction plasmid. In some embodiments, the cells are transduced in the presence of PGE2 and poloxamer. In some embodiments, the poloxamer is poloxamer 338 (LentiBOOST™). The transduced HSCs are then implanted into the patient by infusion to reconstitute the HSC niche with ITGB2-expressing cells.

[0216] The sequence of the ITGB2 expression cassette sequence in pCCL-ChimhCD18W-82-RO(5'-3') is as follows: The coding sequence for ITGB2, also known as CD18, is shown in bold capital letters. The WPRE sequence is underlined. TIFF0007664829000009.tif212158

[0217] The human ITGB2 protein sequence is provided as SEQ ID NO:4.

[0218] Pyruvate kinase deficiency (PKD) is a monogenic metabolic disease caused by mutations in the PKLR gene that impairs the energetic balance in erythrocytes, thereby causing a highly variable hemolytic anemia that can be fatal during the neonatal period.

[0219] In certain embodiments, the disclosure provides a method of treating pyruvate kinase deficiency in a subject in need thereof, comprising administering hematopoietic cells transduced with a recombinant retroviral vector comprising a polynucleotide encoding an R-type pyruvate kinase gene, or a gene encoding a functional variant or functional fragment thereof, according to the methods disclosed herein.

[0220] In some embodiments, the methods disclosed herein are used to transduce hematopoietic cells with lentiviral vectors produced using pCCL-PGK-coRPKW-82-RO transfer vector, for example to generate cell populations for the treatment of IMO. pCCL-PGK-coRPKW-82-RO (PKD plasmid) is based on the pCCL transfer plasmid used in third generation lentiviral vector systems. The pCCL transfer plasmid is a lentiviral vector containing a chimeric CMV-HIV 5'LTR and a vector backbone containing a simian virus 40 polyadenylation sequence and an (enhancer-less) origin of replication sequence downstream of the HIV 3'LTR, which replaces most of the human sequences remaining from the HIV integration site. In pCCL, the CMV enhancer and promoter (nucleotides -673 to -1 relative to the transcription start site; GenBank accession number K03104) are joined to the R region of HIV-1. The PKD plasmid contains the PGK promoter linked to the codon-optimized PKLR gene containing upstream RRE and cPPT / CTS elements and a downstream wPRE element (Figure 1). Lentiviral vector particles are generated by transient transfection of a third generation lentiviral vector system containing the PKD plasmid (Figure 17).

[0221] The lentiviral vector particles can then be used to transduce autologous CD34+ hematopoietic stem cells ("HSCs"), thereby complementing the genetic defect. Briefly, HSCs are mobilized by treating the patient with G-CSF, plerifaxor, or a combination of G-CSF and plerifaxor. HSCs are then harvested by apheresis from the patient's peripheral blood. CD34+ cells can be enriched by magnetic capture (e.g., with Miltenyi Biotec's CliniMACS system), and the CD34+ enriched cells are transduced ex vivo with lentiviral vector particles according to the methods disclosed herein. In certain embodiments, the cells are transduced in the presence of PGE2 and poloxamer. In certain embodiments, the poloxamer is poloxamer 338 (LentiBOOST™). The transduced HSCs are then implanted into the patient by infusion to reconstitute the HSC niche with PKLR-expressing cells.

[0222] The sequence of the PKLR expression cassette in pCCL-PGK-coRPKW-82-RO(5'-3') is as follows: The PKLR coding sequence is in bold capital letters, the PGK promoter is in italics, and the WPRE sequence is underlined. TIFF0007664829000010.tif94160TIFF0007664829000011.tif137159

[0223] The PKLR protein sequence encoded by this polypeptide is provided as SEQ ID NO: 6. Further PKLR polynucleotide sequences useful in the present invention include SEQ ID NOs: 7-9. Vectors for expression of PKLR useful in the present invention include, but are not limited to, those disclosed in International Patent Application No. PCT / US2019 / 041465, the disclosure of which is incorporated herein in its entirety.

[0224] Infantile malignant osteopetrosis (IMO) is a rare recessive genetic disorder characterized by increased bone mass driven by dysfunctional osteoclasts. The disease is most often caused by mutations in the TCIRG1 gene, which encodes a subunit of the V-type ATPase involved in the ability of osteoclasts to resorb bone. Richter et al. show that osteoclast function can be restored by lentiviral vector-mediated expression of TCIRG1, but the exact threshold for restoration of resorption as well as cellular response to vector-mediated TCIRG1 expression is unknown.

[0225] In one embodiment, the disclosure provides a method of treating infantile malignant osteoporosis in a subject in need thereof, comprising administering hematopoietic cells transduced with a recombinant retroviral vector comprising a polynucleotide encoding a CLCN7, OSTM1, T cell immunoregulatory factor 1, H+ transporting ATPase V0 subunit a3 (TCIRG1), TNFSF11, PLEKHM1 or TNFRSF11A gene, or a gene encoding a functional variant or functional fragment thereof, according to the methods disclosed herein.

[0226] In some embodiments, the methods disclosed herein are used to transduce hematopoietic cells with lentiviral vectors produced using the pCCL.PPT.EFS.tcirg1h.wpre transfer vector, for example to generate cell populations for the treatment of IMO. pCCL.PPT.EFS.tcirg1h.wpre is a lentiviral transfer vector based on the pCCL transfer plasmid used in third generation lentiviral vector systems. The pCCL transfer plasmid is a lentiviral vector containing a chimeric CMV-HIV 5'LTR and a vector backbone containing a simian virus 40 polyadenylation sequence and an (enhancer-less) origin of replication sequence downstream of the HIV 3'LTR, which replaces most of the human sequences remaining from the HIV integration site. In pCCL, the CMV enhancer and promoter (nucleotides -673 to -1 relative to the transcription start; GenBank accession number K03104) are joined to the R region of HIV-1. The vector uses the EFS promoter (a short, intronless form of the EF1 alpha promoter) linked to a codon-optimized TCIRG1 gene containing upstream RRE and cPPT / CTS elements and a downstream wPRE element (Figure 18).

[0227] The resulting lentiviral vector is used to transduce autologous CD34+ hematopoietic stem cells ("HSCs"), thereby complementing the genetic defect. In some embodiments, HSCs are mobilized by treating the patient with G-CSF, plerifaxor, or a combination of G-CSF and plerifaxor. HSCs are then harvested from the patient's peripheral blood by apheresis. CD34+ cells can be enriched by magnetic capture (e.g., with Miltenyi Biotec's CliniMACS system), and the CD34+ enriched cells can be transduced ex vivo according to the methods disclosed herein with lentiviral particles previously generated by transient transfection of a lentiviral vector system containing the pCCLL.PPT.EFS.tcirg1h.wpre transfer plasmid. In some embodiments, the cells are transduced in the presence of PGE2 and a poloxamer. In some embodiments, the poloxamer is poloxamer 338 (LentiBOOST™). The transduced HSCs are then implanted into the patient by injection to generate TCIRG1-expressing osteoclasts.

[0228] The sequence of the TCIRG1 expression cassette sequence in pCCL.PPT.EFS.tcirg1h.wpre(5'-3') is as follows: The coding sequence for TCIRG1, also known as CD18, is shown in bold capital letters. The WPRE sequence is underlined. TIFF0007664829000012.tif178160TIFF0007664829000013.tif95159

[0229] The TCIRG1 protein sequence encoded by this polypeptide is provided as SEQ ID NO:11.

[0230] In some cases, recombinant retroviral vectors provide transgenes for genes other than those associated with a disease or disorder, or repair genes other than those associated with a disease or disorder. For example, but not limited to, recombinant retroviral vectors can upregulate or downregulate immune effector genes, change cell surface markers, provide alternative MHC molecules, or encode immunoglobulin genes. In particular, in some cases, recombinant retroviral vectors are intended to provide for the use of allogeneic transplantation or transplantation with mismatched donors, such as by altering immune markers (e.g., HLA or MHC genes) or by causing the expression of immune effector genes.

[0231] The coding sequence to be expressed in the cell can be any polynucleotide sequence, e.g., a gene or a cDNA, that encodes a gene product, e.g., a polypeptide or an RNA-based therapeutic (siRNA, antisense, ribozyme, shRNA, etc.). The coding sequence can be heterologous to the promoter sequence to which it is operably linked, i.e., not naturally operably associated with it. Alternatively, the coding sequence can be endogenous to the promoter sequence to which it is operably linked, i.e., effectively associated with that promoter. The gene product can act endogenously in the mammalian cell, or the gene product can act exogenously, e.g., secreted. For example, when the transgene is a therapeutic gene, the coding sequence can be any gene that encodes a desired gene product or a functional fragment or variant thereof that can be used as a therapy to treat a disease or disorder. In various embodiments, the transgene encodes human FANCA.

[0232] In some embodiments, the transgene coding sequence is modified or "codon optimized" to enhance expression by replacing less frequently occurring codons with more frequently occurring codons. The coding sequence is the portion of the mRNA sequence that codes for amino acids for translation. During translation, each of the 61 trinucleotide codons is translated into one of the 20 amino acids, resulting in degeneracy or redundancy in the genetic code. However, different cell types and different animal species utilize tRNAs (each carrying an anticodon) that code for the same amino acids at different frequencies. If a gene sequence contains codons that are rarely represented by the corresponding tRNA, the ribosomal translation machinery may slow down, preventing efficient translation. Expression can be improved for a particular species through "codon optimization," in which the coding sequence is altered to code for the same protein sequence but utilizes highly represented codons and / or codons utilized by highly expressed human proteins (Cid-Arregui et al., 2003; J. Virol. 77:4928). In one aspect of the invention, the coding sequence of the transgene is modified to replace codons expressed at low frequency in mammals or primates with codons expressed at high frequency in primates. For example, in some embodiments, the coding sequence encoded by the transgene encodes a polypeptide having at least 85% sequence identity to a polypeptide encoded by a sequence disclosed above or herein, such as at least 90% sequence identity, such as at least 95% sequence identity, at least 98% identity, at least 99% identity, and at least one codon of the coding sequence has a higher tRNA frequency in humans than the corresponding codon in the sequence disclosed above or herein.

[0233] In further embodiments, the transgene coding sequence is modified to enhance expression by terminating or removing open reading frames (ORFs) that do not code for the desired transgene. An open reading frame (ORF) is a nucleic acid sequence that follows a start codon and does not contain a stop codon. The ORF may be in forward or reverse orientation and may be "in frame" or "out of frame" relative to the gene of interest. Such open reading frames have the potential to be expressed in an expression cassette flanking the gene of interest and may lead to unwanted adverse effects. In one aspect of the invention, the coding sequence of the transgene is modified to remove open reading frames by further altering the codon frequency. This can be done by removing the start codon (ATG) and introducing a stop codon (TAG, TAA or TGA) into the reverse-oriented ORF or out of frame, while preserving the amino acid sequence and maintaining codons that are highly utilized in the gene of interest (i.e., avoiding codons with a frequency of less than 20%). In the present disclosure, the transgene coding sequence may be optimized by either codon optimization and removal of non-transgenic ORFs, or by using both techniques. As will be apparent to one skilled in the art, it is preferable to remove or minimize non-transgenic ORFs after codon optimization in order to remove ORFs introduced during codon optimization.

[0234] Additionally, as will be appreciated by those of skill in the art, the expression cassettes and recombinant retroviral vectors may optionally contain other elements, including, but not limited to, restriction sites to facilitate cloning, and regulatory elements related to the particular recombinant retroviral vector.

[0235] In some aspects of the invention, the subject polynucleotide cassettes are used to deliver genes to cells, e.g., to determine the effect the gene has on cell viability and / or cell function, to treat cellular disorders, etc. In various embodiments, delivery of the viral vector to a cell by transduction can occur in vivo, ex vivo, or in vitro. Thus, in some aspects of the invention, a composition that provides for expression of a transgene in a mammalian cell is a recombinant retroviral vector, which comprises a polynucleotide cassette, e.g., a gene transfer cassette, of the present disclosure.

[0236] Recombinant retroviral vectors comprising a polynucleotide cassette of the present disclosure may be produced using standard methodologies.

[0237] For example, in the case of LV viral particles, an LV expression vector according to the invention is introduced into the producer cells, which may be followed by the introduction of an LV helper construct that contains an LV coding region that can be expressed in the producer cells and that complements the absence of LV helper functions in the LV vector. This is followed by the introduction of a helper virus and / or additional vector into the producer cells that provides accessory functions that can support efficient LV production. The producer cells are then cultured to produce LV. These steps are carried out using standard methodologies. In a particular embodiment, the plasmids shown in Figures 38-41 are used to produce recombinant retroviral vectors.

[0238] Methods for Producing Viral Vectors Recombinant retroviral vectors comprising a polynucleotide cassette of the present disclosure may be produced using standard methodologies.

[0239] For example, in the case of LV viral particles, an LV expression vector according to the invention is introduced into the producer cells, which may be followed by the introduction of an LV helper construct, which comprises an LV coding region that can be expressed in the producer cells and preserves the absence of LV helper functions in the LV vector. This is followed by the introduction of a helper virus and / or additional vector into the producer cells, which provides accessory functions that can support efficient LV production. The producer cells are then cultured to produce LV. These steps are carried out using standard methodologies. In a particular embodiment, the plasmids shown in Figures 38-41 are used to produce recombinant retroviral vectors.

[0240] Any suitable method of producing viral vectors for delivery of the subject polynucleotide cassettes can be used, including but not limited to those described in the Examples below. For contacting mammalian cells in vitro or in vivo, any concentration of infectious viral vector suitable for effectively transducing mammalian cells can be prepared. For example, viral particles can be prepared at concentrations of 10 per ml. 8 The concentration of infectious units or more can be prepared, for example, at 1×10 per mL. 8 infectious units; 5 x 10 per mL 8 infectious units; 10 per mL 9 infectious units; 5 x 10 per mL 9 infectious units; 10 per mL 10 infectious units; 5 x 10 per mL 10 infectious units; 10 per mL 11 infectious units; 5 x 10 per mL 11 infectious units; 10 per mL 12 infectious units; 5 x 10 per mL 12 infectious units; 10 per mL 13 infectious units; 1.5 x 10 per mL 13 infectious units; 3 x 10 per mL 13 infectious units; 5 x 10 per mL13 infectious units; 7.5 x 10 per mL 13 infectious units; 9 x 10 per mL 13 infectious units; 1 x 10 per mL 14 Infectious units, 5 x 10 per mL 14 Infectious units, typically 1 x 10 per mL 15 The infection units are as follows:

[0241] In preparing the subject LV recombinant retroviral vectors, any host cell that produces LV viral particles may be used, including, for example, mammalian cells (e.g., HEK 293T cells). The host cell can also be a packaging cell in which the LV gag / pol and Rev genes are stably maintained in the host cell or producer cell in which the LV vector genome is stably maintained and packaged. LV vectors are purified and prepared using standard techniques known in the art.

[0242] Pharmaceutical Compositions and Formulations The present invention includes pharmaceutical compositions and formulations comprising the cell populations described herein and pharma- ceutically acceptable carriers, diluents or excipients. The subject cell populations can be combined with pharma- ceutically acceptable carriers, diluents and agents that are generally safe, non-toxic, and useful for preparing formulations that include excipients that are desirable and acceptable for use in primates. Examples of such excipients, carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Supplementary active compounds can also be included in the formulation. The solutions or suspensions used in the formulations may include sterile diluents such as water for injection, saline solution, dimethylsulfoxide (DMSO), fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating compounds such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, or phosphates; surfactants to prevent aggregation, such as Tween 20; and compounds to adjust tonicity, such as sodium chloride or glucose. The pH can be adjusted using acids or bases, such as hydrochloric acid or sodium hydroxide. In certain embodiments, the formulations are sterile.

[0243] In some embodiments, the cell population is manufactured according to Current Good Manufacturing Practices. Manufactured according to Current Good Manufacturing Practices means that the formulation prepared for administration is sufficiently safe to be permitted for administration to human subjects under governing regulations and government approvals. Generally, governing regulations and approvals will mandate that the formulation meet pre-approved criteria for identity, strength, quality, and purity. The criteria include numerical limits, ranges, or other suitable measurements of test results used to determine whether a formulation meets Current Good Manufacturing Practices. Specifications describe analytical procedures used to test for compliance with the criteria. The formulation can be evaluated in lots. A lot is a specific amount of formulation that is tested to ensure compliance with the criteria.

[0244] The formulations can be included in a container, pack, or dispenser, eg, a syringe, eg, a prefilled syringe, together with instructions for administration.

[0245] If necessary or beneficial, the formulation can include a local anesthetic such as lidocaine to ease pain at the site of the injection.

[0246] The therapeutically effective amount of cells in the formulation is 10 2 Beyond cells, 10 3 Beyond cells, 10 4 Beyond cells, 10 5 Beyond cells, 10 6 Beyond cells, 10 7 Beyond cells, 10 8 Beyond cells, 10 9 Beyond cells, 10 10 More than 10 cells 11 The therapeutically effective amount of cells in the formulation can be greater than 10 3 Less than 10 cells 4 Less than 10 cells 5 Less than 10 cells 6Less than 10 cells 7 Less than 10 cells 8 Less than 10 cells 9 Less than 10 cells 10 Less than 10 cells 11 Less than 10 cells 12 The therapeutically effective amount of cells in the formulation can be less than 10 3 cells ~10 12 cells, 10 4 cells ~10 11 cells, 10 5 cells ~10 10 cells, 10 8 cells ~10 12 cells, 10 9 cells ~10 12 cells, 10 8 cells ~10 10 cells, or 10 9 cells ~10 11 It may be a cell.

[0247] In the formulations disclosed herein, the cells are generally in a volume of 1 liter or less, 500 mL or less, 250 mL or less, or 100 mL or less. Thus, the density of cells administered is typically less than 10 4 >10 cells / mL 7 More than 10 cells / mL 8 More than cells / mL.

[0248] The formulations disclosed herein can be prepared for administration, for example, by injection, infusion, perfusion or lavage. The therapeutically effective amount administered is 10 2 Beyond cells, 10 3 Beyond cells, 10 4 Beyond cells, 10 5 Beyond cells, 10 6 Beyond cells, 10 7 Beyond cells, 10 8 Beyond cells, 10 9 Beyond cells, 10 10 More than 10 cells 11 In certain embodiments, the minimum dose is 2×10 cells / kg of subject body weight. 6The therapeutically effective amount administered is 10 3 Less than 10 cells 4 Less than 10 cells 5 Less than 10 cells 6 Less than 10 cells 7 Less than 10 cells 8 Less than 10 cells 9 Less than 10 cells 10 Less than 10 cells 11 Less than 10 cells 12 In certain embodiments, the maximum dose may include less than 2×10 cells per kg of subject body weight. 12 The therapeutically effective amount of cells to be administered is 10 3 cells ~10 12 cells, 10 4 cells ~10 11 cells, 10 5 cells ~10 10 cells, 10 8 cells ~10 12 cells, 10 9 cells ~10 12 cells, 10 8 cells ~10 10 cells, or 10 9 cells ~10 11 It may be a cell.

[0249] In some embodiments, the pharmaceutical compositions provided herein comprise a therapeutically effective amount of a cell population disclosed herein in admixture with pharma- ceutically acceptable carriers and / or excipients, such as saline, phosphate buffered saline, phosphate, and amino acids, polymers, polyols, sugars, buffers, preservatives, and other proteins. Examples of amino acids, polymers, and sugars include octylphenoxypolyethoxyethanol compounds, polyethylene glycol monostearate compounds, polyoxyethylene sorbitan fatty acid esters, sucrose, fructose, dextrose, maltose, glucose, mannitol, dextran, sorbitol, inositol, galactitol, xylitol, lactose, trehalose, bovine or human serum albumin, citrate, acetate, Ringer's and Hank's solutions, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene, and glycol. Preferably, the formulation is stable at 4° C. for at least 6 months. In certain embodiments, the cell population is freshly prepared from an in vivo source, hi certain embodiments, the cell population is frozen for storage prior to or after formulation into a pharmaceutical composition.

[0250] In some embodiments, the pharmaceutical compositions provided herein comprise a buffer, such as phosphate buffered saline (PBS) or sodium phosphate / sodium sulfate, Tris buffer, glycine buffer, sterile water, and other buffers known to those of skill in the art, such as those described in Good et al. (1966) Biochemistry 5: 467. The pH of the buffer in which the pharmaceutical composition contains the tumor suppressor gene contained in the adenoviral vector delivery system may be in the range of 6.5 to 7.75, preferably 7 to 7.5, and most preferably 7.2 to 7.4.

[0251] All publications mentioned herein are incorporated herein by reference as if to disclose and describe the methods and / or materials for which the publications are cited. To the extent there is a conflict with an incorporated publication, it is understood that the present disclosure supersedes any disclosure.

[0252] It is further noted that the claims may be drafted to exclude optional elements. As such, this specification is intended to act as a predicate for the use of exclusive terminology such as "solely," "only," and the like, or for the use of a "negative" limitation in connection with the recitation of claim elements.

[0253] The publications discussed herein are provided merely for their disclosure prior to the filing date of the present application. Further, the publication dates provided may be different from the actual publication dates, which may need to be independently confirmed.

[0254] The disclosure is further described in the following examples, which do not limit the scope of the disclosure described in the claims. EXAMPLES

[0255] Example 1 Transduction of hematopoietic cells with protamine sulfate and PGE2 Transduction of hematopoietic cells with retroviral vectors for clinical use remains challenging. In particular, it is unclear from the literature which transduction enhancer or combinations are likely to be the most effective or have the greatest magnitude of effect achievable. This example establishes that the combination of protamine sulfate and PGE2 increases transduction efficiency.

[0256] An exemplary protocol for transduction of hematopoietic cells with lentiviral vectors as carried out in the following examples is provided in Figure 1, however, in the various experiments described in the examples, the transduction medium contained no TE, one TE, or various combinations of TE. 2The transduction medium contained the contents of the priming medium (X-VIVO™ 20 medium combined with 100 ng / mL rhSCF, 100 ng / mL rhTPO, 100 ng / mL rh-FLT3-L, and 20 ng / mL IL-3) and 4 μg / mL protamine sulfate.

[0257] In one embodiment, hematopoietic cells were transduced with VSVG pseudotyped LVs expressing a GFP (green fluorescent protein) transgene reporter. Cells were cultured at 2.5×10 in liquid culture in the presence of various concentrations of PGE2. 7 TU / mL or 5 x 10 7 Cells were transduced with either lentiviral vector at 100 TU / mL and assayed for VCN after 14 days in liquid culture (Figure 2A). The results show that increasing concentrations of PGE2 (10 μg / mL, 30 μg / mL, or 50 μg / mL) increased transduction efficiency, as measured by vector copy number / cell.

[0258] In a separate experiment, cells were cultured at 1 × 10 7 TU / mL or 1×10 8 Cells were transduced with either lentiviral vector at TU / mL and assayed for VCN after 14 days in liquid culture (Figure 2B). At both vector concentrations, PGE2 increased the transduction efficiency, determined as VCN / cell. Colony forming unit (CFU) assays were performed with transduced cells (Figure 3A, CFC = colony forming cells), and VCN and transduction rate (%) were analyzed in single colonies (Figures 3B and 3C). The highest concentration of PGE2 tested transduced up to approximately 75% of the cells (Figure 3C).

[0259] Example 2 Transduction of hematopoietic cells with protamine sulfate and poloxamer F108 This example establishes that protamine sulfate and poloxamer F108 (also known as poloxamer 338) increase transduction efficiency. Tests following the general protocol shown in Figure 1 were performed in liquid culture with poloxamer F108 (LentiBOOST™). Results for VCN in liquid culture are shown in Figure 4, and for analysis of CFU assays (Figure 5A) and VCN in isolated single CFUs (Figures 5B and 5C) in Figures 5A-5C. Concentrations of 0.5 mg / mL, 1 mg / mL, 2 mg / mL, and 4 mg / mL of LentiBOOST™ increased the transduction efficiency of VSVG pseudotyped LVs expressing a GFP transgene reporter. With PGE2, the highest concentration of PGE2 tested was able to transduce up to about 75% of the cells (Figure 5C).

[0260] Example 3 Transduction of hematopoietic cells with protamine sulfate, PGE2, and poloxamer F108 This example establishes that, surprisingly, the combination of protamine sulfate, PGE2 and LentiBOOST™ increased the transduction efficiency above that observed with either PGE2 or LentiBOOST™ alone. A study was performed according to the protocol shown in Figure 1 and used to compare protamine sulfate (PS), LentiBOOST™ (LB), a combination of LB and PGE2, a combination of PS and LB, or a combination of PS, LB and PGE2. LB was used at a concentration of 1 mg / mL, PGE2 at a concentration of 10 ug / mL, and PS at 4 ug / mL. The results obtained for VCN in transduced cells after 14 days in liquid culture are shown in Figure 6. Figure 7A shows the CFU assay in transduced cells derived from CB and mPB. Also shown are VCN in isolated colonies in the presence of a combination of LB, PGE2 and PS (Figure 7B, Figure 7E) and transduction efficiency (Figure 7C, Figure 7D). As shown in FIG. 7C, more than 80% of the cells are transduced by the vector under these conditions.

[0261] Example 4 Methods for scale-up and in vivo testing of transduction enhancers This example demonstrates that the protocol established in Examples 1-3 can be implemented into therapeutic vectors and scaled up to produce sufficient vectors for clinical trials using cord blood as input. Scale-up of the transduction procedure was performed using CD34 enriched cord blood (CB) cells and LV produced from pCCL-PGK-FANCAW-82-PRO, specifically including LB, PGE2 and PS, without transduction enhancers (except PS, which was used in all experiments) or with transduction enhancers (+TE). LB was used at a concentration of 1 mg / mL, PGE2 at a concentration of 10 ug / mL, and PS at 4 ug / mL. VCN after 14 days in liquid culture is shown in FIG. 8. The results of the CFU assay (Figure 9A), VCN results on isolated single CFU (Figure 9B) and transduction efficiency results on CFU (Figure 9C) on cells transduced in vitro before transplantation are shown in Figures 9A-9C. The results are shown in Figure 9B for cells of early erythroid progenitor cells (BFU-E (burst forming unit-erythroid)), granulocyte-macrophage progenitor cells (CFU-GM) and myeloid progenitor cells (CFU-GM). Figures 10A and 10B show the in vivo results. Cord blood CD34 + Cells were transduced in the presence or absence of transduction enhancers (TE is a combination of LB, PGE2 and PS) and the overnight multiplicity of infection (MOI) of therapeutic LV was 50. LB was used at a concentration of 1mg / mL, PGE2 was used at a concentration of 10ug / mL and PS was used at 4ug / mL. The transduced cells were then harvested, washed and transfected with 1-2.5x10 6 Suspended at a density of 1.3-1.6 x 10 cells / mL. 5 A range of transduced cells were transplanted intravenously into 1.5 Gy irradiated immunodeficient NSG mice as a xenogeneic model of human hematopoiesis. +) Cell percentage (%) (FIG. 10A) and VCN / cell (FIG. 10B) were assessed in bone marrow cells from transplanted animals 1 month after transplant (1 mpt), 2 months after transplant (2 mpt), or 3 months after transplant (3 mpt).

[0262] Example 5 LV production and transduction of MPB under Good Manufacturing Practice (GMP) conditions This example demonstrates that the protocol established in Examples 1-4 can be implemented into therapeutic vectors and scaled up to produce sufficient vectors for clinical trials using mobilized peripheral blood as input. GMP LV production was performed and used in the next set of experiments performed with CD34+ hematopoietic stem cells purified from mobilized peripheral blood (mPB) from healthy donors. The LV vector was VSVG pseudotyped LV expressing the FANCA transgene, which was produced using the pCCL-PGK-FANCAW-82-PRO transduction vector. Figure 11 shows the VCN / cells in transduced cells after 14 days at two different MOIs in liquid culture with (+TE) or without (-TE) the transduction enhancers PS, LB, and PGE2. LB was used at a concentration of 1 mg / mL, PGE2 at 10 ug / mL, and PS at 4 ug / mL. Figures 12A-12D show colony forming unit (CFU) assay results for whole cells, BFU, GM, and mixed myeloid progenitors for GMP003 with (+TE) or without (-TE) transduction enhancers PS, LB, and PGE2 at two different MOIs. Figures 13A and 13B show VCN for isolated single CFU for GMP003 with (+TE) or without (-TE) transduction enhancers PS, LB, and PGE2 at MOI 20 or MOI 50. Results are shown for burst forming unit-erythroid (BFU-E) cells, granulocyte-macrophage progenitors (CFU-GM), and myeloid progenitors (CFU-GM). Figures 14A and 14B show the transduction efficiency and VCN in CFU for cells of early erythroid progenitors (BFU-E), granulocyte-macrophage progenitors (GM), and myeloid progenitors (Mixed), with (+TE) or without (-TE) the transduction enhancers PS, LB, and PGE2.

Claims

1. 1. A method for ex vivo genetic modification of hematopoietic cells, comprising: (a) contacting a hematopoietic cell with a poloxamer; (b) contacting the hematopoietic cells with prostaglandin E2 (PGE2) or a derivative thereof; (c) contacting the hematopoietic cells with a recombinant fibronectin fragment; (d) contacting the hematopoietic cells with a recombinant retroviral vector; A method comprising:

2. The method of claim 1 , wherein the recombinant retroviral vector is a recombinant lentiviral vector.

3. 3. The method of claim 1 or 2, wherein the poloxamer is selected from the group consisting of poloxamer 288, poloxamer 335, poloxamer 338, and poloxamer 407.

4. The method according to claim 1 or 2, wherein the PGE2 or a derivative thereof is 16,16-dimethyl PGE2 (dmPGE2) and / or unmodified PGE2.

5. The method of claim 1 or 2, wherein the recombinant fibronectin fragment is RetroNectin™.

6. The method of any one of claims 1 to 5, further comprising contacting the hematopoietic cells with protamine sulfate.

7. 10. The method of claim 1 or 6, wherein the contacting steps are performed for the same period of time or for overlapping periods of time.

8. a) the concentration of the PGE2 or a derivative thereof is 5 to 30 μg / mL; b) the concentration of the poloxamer is between 200 and 1200 μg / mL; and / or c) the concentration of the recombinant fibronectin fragment is 5 to 50 μg / mL; The method according to any one of claims 1 to 7.

9. 7. The method of claim 6, wherein the concentration of the protamine sulfate is 4 to 10 μg / mL.

10. 10. The method of any one of claims 1 to 9, wherein the hematopoietic cells are CD34 enriched cells, bone marrow (BM) derived cells, cord blood (CB) derived cells, or mobilized peripheral blood (mPB) cells.

11. 1. A method for enhancing recombinant retroviral vector-mediated genetic modification of hematopoietic cells, comprising: (a) contacting hematopoietic cells ex vivo with an effective amount of PGE2 or a derivative thereof and with an effective amount of a poloxamer; (b) contacting the hematopoietic cells with a recombinant fibronectin fragment; (c) contacting the hematopoietic cells with a recombinant retroviral vector comprising a polynucleotide containing a gene of interest or encoding a polypeptide of interest; Including, the efficacy of viral transduction of said recombinant retroviral vector is enhanced compared to the transduction of hematopoietic cells by said recombinant retroviral vector in the absence of PGE2 or a derivative thereof, poloxamer, and recombinant fibronectin fragment; method.

12. The method of claim 11, wherein the hematopoietic cells are CD34-enriched cells, bone marrow (BM)-derived cells, cord blood (CB)-derived cells, or mobilized peripheral blood (mPB) cells.

13. 13. The method of claim 11 or 12, wherein the gene of interest complements a defect in a gene associated with a monogenic genetic disease or disorder.

14. The monogenic disease or disorder is selected from the group consisting of Fanconi anemia, leukocyte adhesion deficiency type 1, pyruvate kinase deficiency, and infantile malignant osteopetrosis; and 14. The method of claim 13, wherein the gene of interest or polypeptide of interest is selected from the group consisting of RPK, ITGB2, FANCA, FANCC, FANCG, TCIRG1, CLCN7, TNFSF11, PLEKHM1, TNFRSF11A, and OSTM1, or functional variants or fragments thereof.

15. The method of any one of claims 11 to 14, further comprising contacting the hematopoietic cells ex vivo with an effective amount of protamine sulfate.

16. 1. A method for producing a hematopoietic cell population comprising at least 80% genetically modified hematopoietic cells, comprising: Contacting hematopoietic cells ex vivo with a recombinant lentiviral vector comprising a polynucleotide containing a gene of interest or encoding a polypeptide of interest. Including, The method, wherein the contacting occurs in the presence of PGE2 or a derivative thereof, a poloxamer, and a recombinant fibronectin fragment, and the hematopoietic cell population has a VCN / cell of at least 1.

0.

17. a) the poloxamer is selected from the group consisting of poloxamer 288, poloxamer 335, poloxamer 338, and poloxamer 407; b) the PGE2 or derivative thereof is 16,16-dimethyl PGE2 (dmPGE2) or unmodified PGE2; and / or c) the recombinant fibronectin fragment is RetroNectin™; 17. The method of claim 16.

18. a) the concentration of the PGE2 or a derivative thereof is 5 to 30 μg / mL; b) the concentration of the poloxamer is between 200 and 1200 μg / mL; and / or c) the concentration of the recombinant fibronectin fragment is 5 to 50 μg / mL; 18. The method according to claim 16 or 17.

19. The method of any one of claims 16 to 18, further comprising contacting the hematopoietic cells with protamine sulfate.

20. 20. The method of claim 19, wherein the concentration of protamine sulfate is 4 to 10 μg / mL.

21. The method of any one of claims 16 to 20, wherein the polynucleotide complements a deficiency in a gene associated with a monogenic genetic disease or disorder.

22. the monogenic disease or disorder is selected from the group consisting of Fanconi anemia, leukocyte adhesion deficiency type 1, pyruvate kinase deficiency, and infantile malignant osteopetrosis; and / or 22. The method of claim 21, wherein the polypeptide of interest is selected from the group consisting of RPK, ITGB2, FANCA, FANCC, FANCG, TCIRG1, CLCN7, TNFSF11, PLEKHM1, TNFRSF11A, and OSTM1.

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