Methods for gene modification of hematopoietic cells

Abstract

To provide a method for gene modification of hematopoietic cells, which is particularly suitable for gene therapy applications including the treatment of monogenic genetic diseases and disorders.SOLUTION: The present invention provides a method of genetic modification of 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; and (d) contacting the hematopoietic cells with a recombinant retroviral vector.SELECTED DRAWING: 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 THE INVENTION The present invention relates generally to methods for genetically modifying hematopoietic cells, and more particularly 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] Background of the Invention Ex vivo gene transfer into target cells is a clinically used method for cell and gene therapy. Recombinant retroviral vectors (e.g., recombinant lentiviral vectors) can be used to deliver polynucleotides to cells (e.g., hematopoietic cells). Contacting target hematopoietic cells with the recombinant retroviral (e.g., lentiviral) vector results in gene delivery to the hematopoietic cells, a process known as transduction. Hematopoietic cells may then be administered to a subject with the intention that the hematopoietic cells will engraft themselves into the subject's bone marrow.

[0005] Because retroviral (e.g., lentiviral) vector transduction efficiency is often limited, 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 generally relates to methods for genetically modifying hematopoietic cells. In particular, the present invention relates to enhancing transduction by a recombinant retroviral vector 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 use in gene therapy, including the treatment of monogenic genetic diseases and disorders. Advantageously, the methods of the present disclosure result in lower 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, the method 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 hematopoietic cells, the method comprising providing hematopoietic cells, contacting the hematopoietic cells with a poloxamer, contacting the hematopoietic cells with prostaglandin E2 (PGE2) or a derivative thereof, and contacting the hematopoietic cells 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, PGE2 or a derivative thereof is modified.

[0016] In one embodiment, the PGE2 or derivative thereof is 16,16-dimethyl PGE2 (dmPGE2).

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

[0018] In certain embodiments, the method further comprises contacting the hematopoietic cells with protamine sulfate and / or a recombinant fibronectin fragment. In certain embodiments, the recombinant fibronectin fragment may be present in the liquid culture medium or coated on the culture dish. In certain embodiments, the cells may be pretreated by culturing in a dish containing recombinant fibronectin, and / or the recombinant fibronectin fragment 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 μg / mL when used in a 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 present 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 poloxamer; and exposing or contacting the hematopoietic cells with a recombinant retroviral vector comprising a polynucleotide comprising a gene of interest, wherein the viral transduction efficacy 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 recombinant fibronectin fragments.

[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 method combats the clinical sequelae or ameliorates 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 present disclosure provides a recombinant retroviral vector-mediated method for 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 detecting an expression of a gene encoding one of the Fanconi anemia complementation group (FANC) genes, ITGB2, R-type pyruvate kinase, OSTM1, TCIRG1, CLCN7, OSTM1, or a functional barrier thereof. and genetically modifying the CD34-enriched cells with a recombinant retroviral vector comprising a gene or a polynucleotide encoding a CD34-enriched protein 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 provides an in vitro recombinant retroviral vector-mediated method for genetically modifying 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 1, 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 poloxamer, and optionally, with protamine sulfate.

[0037] The present disclosure provides methods for 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 deleted or mutated due to the genetic disease or disorder. 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 containing an expression cassette comprising a polynucleotide sequence encoding the polypeptide in the presence of at least two transduction enhancers selected from prostaglandin E2 (PGE2), poloxamer, a recombinant fibronectin fragment, and / or protamine sulfate, and the resulting genetically modified cells are provided to the subject. In one embodiment, the disease or disorder is selected from Fanconi anemia, leukocyte adhesion deficiency type 1, 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 present 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 present disclosure provides a method of treating leukocyte adhesion deficiency type 1 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 present 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 present 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 for producing a hematopoietic cell population comprising at least 80% or at least 90% genetically modified hematopoietic cells, comprising contacting the hematopoietic cells ex vivo with a recombinant retroviral vector (optionally a lentiviral vector) comprising a polynucleotide comprising a gene of interest or encoding a polypeptide of interest, 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™). The cells may be contacted with the retroviral vector under conditions and for a time sufficient to allow 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, 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 twice consecutively, 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 specific 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 present 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 methods 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, the cell population produced by the disclosed methods, the hematopoietic cells being obtained from the subject prior to ex vivo contact with a retroviral vector, and the gene of interest encoding 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, 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, cells are pretreated before and / or during transduction by culturing on a dish coated with a recombinant fibronectin fragment, e.g., RetroNectin™. In some embodiments, cells are transduced in liquid medium containing RetroNectin. In some embodiments, cells are pretreated by culturing on a dish coated with a recombinant fibronectin fragment and are also transduced in liquid culture in the presence of the recombinant fibronectin fragment and another TE. That is, in some embodiments, cells are exposed to recombinant fibronectin before exposure to the other TE during transduction, while in some embodiments, cells are exposed to recombinant fibronectin for the same or overlapping period as the other TE.

[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] 1001. The method of claim 1001, wherein said hematopoietic cells are engineered. [The present invention 1004] 1004. The method of claim 1003, wherein said hematopoietic cells are CD34 enriched cells. [The present invention 1005] 1001. 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 claim 1005, wherein the poloxamer is poloxamer 338 (LentiBOOST™). [The present invention 1007] 1001. The method of claim 10, wherein said PGE2 or a derivative thereof is modified. [The present invention 1008] 1007. The method of the present invention, wherein the PGE2 or a derivative thereof is 16,16-dimethyl PGE2 (dmPGE2). [The present invention 1009] 1001. The method of the present invention, wherein said PGE2 or derivative thereof is unmodified. [The present invention 1010] Any of the aforementioned 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 or overlapping time periods. [The present invention 1012] The method of 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] The method of any one 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 1014, 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] 1016. 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 containing a gene of interest or encoding a polypeptide of interest; Including, A method in which 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 recombinant fibronectin polypeptide or a 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] 1019. 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] 1023. The method of claim 1022, wherein 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. [The present invention 1024] 1018. The method of claim 1018, 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 a variant thereof. [The present invention 1026] 1025. 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 of 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] 1027. The method of claim 1027, wherein the liquid medium comprises protamine sulfate. [The present invention 1029] 1. A method of 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] 1029. The method of claim 1029, wherein the liquid medium comprises protamine sulfate. [The present invention 1031] The method of any one of claims 1025 to 1030, wherein the lentiviral vector is a recombinant lentiviral vector. [The present invention 1032] 1032. The method of any one of claims 1025 to 1031, wherein said hematopoietic cells are engineered. [The present invention 1033] The method of any one of claims 1025 to 1032, wherein the hematopoietic cells are cells enriched for CD34. [The present invention 1034] 1036. The method of any one of claims 1025 to 1033, wherein the poloxamer is selected from the group consisting of poloxamer 288, poloxamer 335, poloxamer 338, and poloxamer 407. [This 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. [This invention 1037] The method of any one of claims 1025 to 1036, wherein the PGE2 or a derivative thereof is 16,16-dimethyl PGE2 (dmPGE2). [The present invention 1038] The method of any one of claims 1025 to 1037, wherein the PGE2 or a derivative thereof is unmodified. [This invention 1039] The method of 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 of any one of claims 1025 to 1039, wherein the concentration of the PGE2 or a derivative thereof is about 10 µg / mL. [This invention 1041] The method of any one of claims 1025 to 1040, wherein the concentration of the poloxamer is 200 to 1200 μg / mL. [The present invention 1042] 1025 to 1041, wherein the concentration of the poloxamer is about 1000 μg / mL. [This invention 1043] The method of any one of claims 1025 to 1042, wherein the concentration of the protamine sulfate is 4 to 10 µg / mL. [This invention 1044] The method of any of claims 1025 to 1043, wherein the concentration of the protamine sulfate is about 4 µg / mL. [This invention 1045] 10. The method of any 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 of the present invention 1025 to 1045 for preventing or ameliorating a genetic disease or genetic disorder caused by a single gene. [This invention 1047] 1046. 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. [This 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. [This invention 1049] 1. A method of 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] 1049. The method of claim 1049, wherein said cell is contacted with PGE2 and said poloxamer. [This invention 1051] 1049. The method of claim 1050, wherein said recombinant retroviral vector is a recombinant lentiviral vector. [This invention 1052] The method of any one of claims 1049 to 1051, wherein the hematopoietic cells are CD34-enriched cells. [This invention 1053] 1052. 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. [This invention 1054] Any of the methods of claims 1049 to 1053, wherein the poloxamer is poloxamer 338 (LentiBOOST (trademark)). [This invention 1055] The method of any one of claims 1049 to 1054, wherein the PGE2 or the derivative thereof is modified. [This invention 1056] 1055. The method of the present invention, wherein the PGE2 or a derivative thereof is 16,16-dimethyl PGE2 (dmPGE2). [This invention 1057] The method of any one of claims 1049 to 1054, wherein the PGE2 or derivative thereof is unmodified. [This invention 1058] The method of any of claims 1049 to 1057, comprising contacting the hematopoietic cells with protamine sulfate. [This invention 1059] The method of 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 of any of claims 1049 to 1059, wherein the concentration of the PGE2 or derivative thereof is about 10 µg / mL. [This invention 1061] The method of any one of claims 1049 to 1060, wherein the concentration of the poloxamer is 200 to 1200 μg / mL. [This invention 1062] 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 of any of claims 1049 to 1062, wherein the concentration of the protamine sulfate is 4 to 10 µg / mL. [This invention 1064] The method of any of claims 1049 to 1063, wherein the concentration of said protamine sulfate is about 4 µg / mL. [This invention 1065] 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] 1066. The method of any of claims 1049 to 1065, wherein said therapeutic protein is selected from the group consisting of RPK, ITGB2, FANCA, FANCC, FANCG, TCIRG1, CLCN7, TNFSF11, PLEKHM1, TNFRSF11A and OSTM1. [This 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] 1067. 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. [This invention 1069] Any of the methods 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] 1069. 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. [This invention 1071] 1070. The method of any one of claims 1049 to 1070, wherein the 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%. [This 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) containing a polynucleotide comprising 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™). [This 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. [This invention 1074] Any of the methods of claims 1072 to 1073, wherein the poloxamer is poloxamer 338 (LentiBOOST (trademark)). [This invention 1075] The method of any one of claims 1072 to 1074, wherein the PGE2 or the derivative thereof is modified. [This invention 1076] 1075. The method of the present invention, wherein the PGE2 or a derivative thereof is 16,16-dimethyl PGE2 (dmPGE2). [This invention 1077] The method of any one of claims 1072 to 1074, wherein the PGE2 or derivative thereof is unmodified. [This invention 1078] The method of any of claims 1072 to 1077, further comprising contacting the hematopoietic cells with protamine sulfate. [This invention 1079] The method of 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] 1079. The method of claim 1079, wherein the concentration of the PGE2 or derivative thereof is about 10 μg / mL. [This invention 1081] The method of any one of claims 1072 to 1080, wherein the concentration of the poloxamer is 200 to 1200 μg / mL. [This invention 1082] The method of any one of claims 1049 to 1081, wherein the concentration of the poloxamer is about 1000 μg / mL. [This invention 1083] The method of any one of claims 1072 to 1082, wherein the concentration of the protamine sulfate is 4 to 10 µg / mL. [This invention 1084] 1083. The method of claim 1083, wherein the concentration of said protamine sulfate is about 4 μg / mL. [This invention 1085] 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. [This invention 1086] 1085. The method of any of claims 1072 to 1085, wherein said polypeptide of interest is selected from the group consisting of RPK, ITGB2, FANCA, FANCC, FANCG, TCIRG1, CLCN7, TNFSF11, PLEKHM1, TNFRSF11A and OSTM1. [This invention 1087] The method of claim 1085, wherein the disease or disorder is a genetic disease or disorder caused by a single gene. [This invention 1088] 1087. 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. [This invention 1089] Any of the methods of claims 1072 to 1088, wherein the hematopoietic cells are CD34-enriched cells, and optionally bone marrow (BM)-derived cells, cord blood (CB)-derived cells, or mobilized peripheral blood (mPB) cells. [The present invention 1090] 1069. The method of any one 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. [This 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. [This 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 present invention to said subject. Including, the hematopoietic cells are obtained from the subject prior to contacting them ex vivo with a retroviral vector; The method, wherein said gene of interest encodes a functional polypeptide that is mutated or deleted in said subject due to said genetic disease or disorder. [This 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. [This invention 1094] The method of claim 1092, wherein the disease or disorder is a genetic disease or disorder caused by a single gene. [This invention 1095] 1094. 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. [This invention 1095] Any of the methods 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 explanation 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. [Figure 2] 2A and 2B show the results of VCN measurements for cells in liquid medium transduced in the presence of PGE2. For Fig. 2A, at each concentration shown, the left bar shows the results for cells transduced with 2.5 x 107 TU / mL of lentiviral vector, and the right bar shows the results for cells transduced with 5 x 107 TU / mL of lentiviral vector. [Figure 3A] 3A to 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 on 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 rates (Figure 7C) for cells transduced with one or more of LentiBOOST (LB), PGE2, and protamine sulfate (PS). Figures 7D and 7E show the effects of LB, PGE2, and PS on transduction rates (Figure 7D) and VCN (Figure 7E) for CFCs derived from different sources of CD34+, including 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 PS with 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 using PS alone or PS with the transduction enhancers LB and PGE2. Figure 9A shows the results of a CFC assay. Figure 9B shows the results of VCN in terms of CFU. Results are shown for early erythroid progenitor cells (BFU-E (burst-forming unit-erythroid)), granulocyte-macrophage progenitor cells (CFU-GM), and myeloid progenitor cells (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 enhancers 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] Figures 12A-12D show colony-forming units (CFU) for total cells (Figure 12A), BFU (Figure 12B), GM (Figure 12C), and mixed myeloid progenitor cells (Figure 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 cells of early erythroid progenitors (BFU-E), granulocyte-macrophage progenitors (CFU-GM), and myeloid progenitors (CFU-GM) at an MOI of 20 (Figure 13A) and an MOI of 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] Figure 18 is a schematic map of the pRRL.PPT.EFS.tcirg1h.wpre transfer vector. DETAILED DESCRIPTION OF THE INVENTION

[0049] Detailed Description The present disclosure provides compositions and 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 of prostaglandin E2 (PGE2), poloxamer, recombinant fibronectin fragments, and / or protamine sulfate. 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 defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, 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. Furthermore, the materials, methods, and examples described herein are illustrative only and 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. 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, NYSee, for example, "Current Protocols in Immunology" (QE Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, eds., 1998); "Annual Review of Immunology" (QE Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, eds., 1991); "Advances in Immunology" (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 term "about" or "approximately" refers to an amount, 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% of the reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In certain embodiments, the term "about" or "approximately," when preceding a numerical value, refers to the value plus or minus a range of 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 cells 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 x 10 6 Cells were exposed to the virus and 0.5 × 10 6 If 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 tool for gene delivery (Miller, 2000, Nature. 357:455-460). Once a 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 virus; and (3) the genus Lentivirus, such as human immunodeficiency virus-1 and simian immunodeficiency virus.

[0060] As used herein, the terms "lentiviral" or "lentivirus" refer 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, an HIV-based vector backbone (i.e., elements of HIV cis-acting sequences) is 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 linked to, 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 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. Viral vectors contain structural and / or functional genetic elements that are primarily derived from viruses. The term "retroviral vector" refers to a viral vector that contains structural and functional genetic elements, or portions thereof, that are primarily derived from retroviruses. The term "lentiviral vector" refers to a viral vector that contains structural and functional genetic elements, or portions thereof, that are primarily derived from lentiviruses, including LTRs. The term "hybrid" refers to a vector, LTR, or other nucleic acid that contains both retroviral, e.g., lentiviral, sequences and non-lentiviral viral sequences. In one embodiment, a hybrid vector refers to a vector or transfer plasmid that contains retroviral, e.g., lentiviral, sequences 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 lentiviral particles of the invention and in DNA form in DNA plasmids of the invention.

[0065] According to certain embodiments, most or all of the viral vector backbone sequence is derived from a lentivirus, e.g., HIV-1. However, it should be understood that lentiviral sequences from many different sources can be used, and that numerous substitutions and alterations can be made in a given 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 Nos. 6,013,516; and 5,994,136, many of which may be adapted to produce the viral vectors or transfer plasmids of the present invention.

[0066] As used herein, the term "polynucleotide" or "nucleic acid" generally refers to a biological polymer comprising 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, whether recombinant, synthetic, or isolated. In some embodiments, polynucleotide refers to messenger RNA (mRNA), RNA, genomic RNA (gRNA), positive-strand RNA (RNA(+)), or 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(+)), or negative-strand RNA (RNA(-)). Preferably, polynucleotides of the 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 set forth herein (see, e.g., the Sequence Listing), and typically, the variants retain 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 therapeutics and / or other genes of interest are contemplated. In particular embodiments, the polynucleotides encode polypeptide therapeutics, including, but 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 functional variant thereof, such as a polypeptide encoded by any of the disclosed genes.

[0067] As used herein, a "pseudotyped" vector refers to a vector having a recombinant capsid or envelope protein that differs from the capsid or envelope protein of the native vector. For example, a VSVG-pseudotyped lentiviral vector is a vector generated by coexpressing the envelope protein of the VSVG virus with the viral RNA genome in a packaging cell line in a manner that allows the VSVG envelope protein to be incorporated into viral particles containing the RNA genome. Pseudotyped vectors may have altered tropism and / or reduced immunogenicity, making them desirable for use in gene therapy applications. It is within the skill of one in the art 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 coexpressing envelope proteins (or capsid proteins) from plasmids or other DNA encoding 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 pseudotyped recombinant retroviral vectors (e.g., lentiviral vectors). In some embodiments, the pseudotyped recombinant retroviral vectors are 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, 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 comprise an increased number of transduced cells or an increased VCN in a population of transduced cells, compared to existing transduction compositions and methods. Increased cell transduction can be confirmed using methods known in the art, such as 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 decimal points 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 "reducing" or "alleviating" generally refers to a composition or method that results in relatively fewer transduced cells 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 decimal points 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 the 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, the reference response.

[0071] In the following description, certain specific details are set forth to provide a thorough understanding of various illustrative embodiments of the invention contemplated herein. However, one of ordinary skill in the art will understand that certain illustrative embodiments may be practiced without these details. Furthermore, it should be understood that individual vectors or groups of vectors derived from the various combinations of structures and substitutions described herein are 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 substitutions 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 culture medium, available from Lonza®. Media other than 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 thrombopoietin.

[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 nonionic 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 lentiviruses or retroviruses with target cells. An example of a recombinant fibronectin fragment is the CH296 fragment of human fibronectin, commercially known as 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 dimethyl sulfoxide.

[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 given number of input cells provide 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."

[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 measure 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 erythroid progenitor (BFU-E) cells, the earliest red blood cell precursor cells.

[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 (granulocytes, erythroids, monocytes, megakaryocytes).

[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 can 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 (e.g., 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 generally used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, e.g., 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, e.g., partially or completely curing adverse effects on and / or resulting from 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 development; or (c) relieving the disease, i.e., causing regression of the disease. Therapeutic agents can be administered before, during, or after the onset of a disease or injury. Of particular interest is the treatment of an ongoing disease, where treatment stabilizes or alleviates undesirable clinical symptoms in the patient. Desirably, such treatment is administered prior to complete loss of function in the affected tissue. Desirably, the subject's 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 pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0102] B. Embodiments and Modifications Various compositions and methods are described below. While particular compositions and methods are exemplified herein, it will be understood that any of several 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 Transduction Enhancer Combinations 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 a genetic defect because they result in a large number of cells that express a functional product of the corrected gene introduced by the lentiviral gene therapy vector.

[0104] Various 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 cyclosporin A have been used as transduction enhancers. However, the present disclosure identifies specific combinations of transduction enhancers that achieve significantly higher levels of lentiviral transduction of hematopoietic cells compared to each transduction enhancer used alone.

[0105] In one aspect, the present 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 a derivative thereof, poloxamer, protamine sulfate, and a recombinant fibronectin fragment, 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 allow 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, or at least 16 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 to 24 hours or 16 to 18 hours. In some embodiments, the cells are contacted with the retroviral vector and the transduction enhancer for the same or overlapping time periods. In certain embodiments, cells are contacted with the transduction enhancer and the recombinant retroviral vector simultaneously or for overlapping periods. In some embodiments, the two or more transduction enhancers comprise a poloxamer. 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 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 poloxamer is poloxamer 338 (LentiBOOST™). In some embodiments, the two or more transduction enhancers comprise or consist of a poloxamer, PGE2, and protamine sulfate. In some embodiments, the poloxamer is poloxamer 338 (LentiBOOST™).In certain embodiments, 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 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 certain embodiments, the method comprises culturing at a concentration of about 2 μg / cm. 2 In some embodiments, the cells are pre-stimulated by culturing them on plates coated with RetroNectin™ (RN). In some embodiments, the cells are transduced in liquid medium containing RetroNectin™. In some embodiments, the cells are pre-treated by culturing them on dishes coated with recombinant fibronectin fragments and then transduced in liquid culture in the presence of recombinant fibronectin fragments and other TEs.

[0106] In one aspect, the disclosure provides a method for genetically modifying hematopoietic cells, the method comprising providing hematopoietic cells; contacting the hematopoietic cells 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 cells with a recombinant retroviral vector. In certain embodiments, the cells are 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 and 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 certain embodiments, 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 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 certain embodiments, the method comprises culturing at a concentration of about 2 μg / cm. 2 In some embodiments, cells are pre-stimulated by culturing them on plates coated with RetroNectin™ (RN). In some embodiments, cells are transduced in liquid medium containing RetroNectin™. In some embodiments, cells are pre-treated by culturing them on dishes coated with recombinant fibronectin fragments and then transduced in liquid culture 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 a polypropylene subunit average molecular weight 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, cells are transduced in a medium containing a combination of three transduction enhancers, e.g., poloxamer 338 (LentiBOOST), PGE2, and protamine sulfate, in a liquid medium. The cells may be attached to a culture dish, or the cells may be non-attached to the culture dish. In certain embodiments, the cells are contacted with the transduction enhancer and the recombinant retroviral vector simultaneously or for overlapping periods. In some embodiments, the method includes pre-stimulation by culturing the cells on a plate coated with a recombinant fibronectin fragment, e.g., RetroNectin™ (RN). In some 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 prior to 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 dose of about 2 ug / cm 2 In some embodiments, cells are pre-stimulated by culturing them on plates coated with RetroNectin™ (RN). In some embodiments, cells are transduced in liquid medium containing RetroNectin™. In some embodiments, cells are pre-treated by culturing them on dishes coated with recombinant fibronectin fragments and then transduced in liquid culture in the presence of recombinant fibronectin fragments and other TEs.

[0110] In a related aspect, the present disclosure provides a method for enhancing recombinant retroviral vector-mediated genetic modification of hematopoietic cells, comprising treating or contacting hematopoietic cells ex vivo with an effective amount of a combination of two or more transduction enhancers, wherein at least two of the transduction enhancers are selected from 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 method comprises contacting cells with PGE2 or a derivative thereof and an effective amount of a poloxamer; and contacting the cells with a recombinant retroviral vector comprising a polynucleotide comprising a gene of interest, wherein the viral transduction efficacy 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 one 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 a poloxamer and protamine sulfate, or consist of a poloxamer and protamine sulfate. In some embodiments, the method further comprises treating or contacting the hematopoietic cells ex vivo with an effective amount of protamine sulfate. In some embodiments, the two or more transduction enhancers comprise prostaglandin E2 (PGE2) or a derivative thereof, a poloxamer, protamine sulfate, and a recombinant fibronectin fragment, 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 them on dishes coated with a recombinant fibronectin fragment, e.g., RetroNectin™. In certain embodiments, the method comprises culturing the cells at a concentration of about 2 ug / cm. 2 In some embodiments, cells are pre-stimulated by culturing them on plates coated with RetroNectin™ (RN). In some embodiments, cells are transduced in liquid medium containing RetroNectin™. In some embodiments, cells are pre-treated by culturing them on dishes coated with recombinant fibronectin fragments and then transduced in liquid culture in the presence of recombinant fibronectin fragments and other TEs.

[0111] In certain embodiments of any of the methods disclosed herein, cells are contacted with a solution or culture medium containing two or more transduction enhancers. The solution or culture medium may further contain a recombinant retroviral vector, or the recombinant retroviral vector may be added after the cells have been contacted with the solution or culture medium containing the transduction enhancers. In certain embodiments, the cells are present in a culture dish containing 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 allow 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, or at least 16 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 to 24 hours or 16 to 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, cells are contacted with PGE2 or a derivative thereof. In some embodiments, PGE2 or a derivative thereof is modified. In some embodiments, PGE2 or a derivative thereof is 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"): TIFF2025038075000002.tif38128Molecular formula:C 22 H 36 O5.

[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, 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 final concentrations 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 final concentrations 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 final concentrations 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, 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 final concentrations 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, 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 final concentrations 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, 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 final concentrations 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, 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 final concentrations 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, cells are contacted with poloxamer 338 and PGE2 or a derivative thereof. In certain embodiments, poloxamer 338 and PGE2 or a derivative thereof are at concentrations of 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, respectively.

[0042] 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.

[0126] In a preferred embodiment, cells are transduced with lentiviral vectors in 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 adherent configuration, the substrate is coated with the CH296 fragment of human fibronectin, sometimes referred to as RetroNectin™, using a RetroNectin™ solution at a concentration of about 20 μg / mL to about 100 μg / mL.

[0127] In a preferred embodiment, 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 adherent configuration, the substrate is coated with the CH296 fragment of human fibronectin, sometimes tradenamed RetroNectin™, using a RetroNectin™ solution at a concentration of about 20 μg / mL. In one embodiment, the culture dish is coated with 2 μg / cm 2 In certain embodiments, 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, cells are contacted with about 1 mg / mL poloxamer 338; about 10 μM PGE2; and about 4 μM protamine sulfate.

[0130] Tables 1 and 2 provide further illustrative embodiments. 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™, by coating the culture dishes used for transduction with RetroNectin™.

[0131] Table 1. CB cells and transduction enhancer combinations TIFF2025038075000003.tif150161

[0132] Table 2. mPB cells and transduction enhancer combinations TIFF2025038075000004.tif39161TIFF2025038075000005.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 in a vessel coated with recombinant fibronectin or a fragment thereof, which enhances transduction efficiency. Recombinant fibronectin fragments (e.g., the CH296 fragment of human fibronectin, available under the 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 methods include contacting hematopoietic cells with a recombinant fibronectin fragment, a poloxamer, and protamine sulfate. In some embodiments, the methods include contacting hematopoietic cells with a recombinant fibronectin fragment, a poloxamer, PGE2, and protamine sulfate.

[0143] In some embodiments, the methods include contacting 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 the recombinant retroviral vector, e.g., for the same or overlapping time period as when 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 pharmaceutical. PGE2 has the following structure (represented as the "skeletal structure" and also referred to as the "linear structural formula" or "truncated structural formula"): TIFF2025038075000006.tif38128PGE2 molecular formula:C 22 H 36 O5.

[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] Illustrative examples of "analogs" or "derivatives" of PGE2 include 16,16-dimethyl PGE2 (dmPGE2), 16-16 dimethyl PGE2 p-(p-acetamidobenzamido)phenyl ester, 11-deoxy-16,16-dimethyl PGE2, 9-deoxy-9-methylene-16,16-dimethyl PGE2, 9-deoxy-9-methylene PGE2, 9-ketofluprostenol, 5-trans PGE2, 17-phenyl-omega-trinor PGE2, PGE2 serine, Examples of suitable PGE2 inhibitors include, but are not limited to, 15-hydroxy-15-keto-PGE2, 15(S)-15-methyl-PGE2, 15(R)-15-methyl-PGE2, 8-iso-15-keto-PGE2, 8-iso-PGE2 isopropyl ester, 20-hydroxy-PGE2, nocloprost, sulprostone, butaprost, 15-keto-PGE2, and 19(R)-hydroxy-PGE2.

[0148] Also contemplated herein are analogs or derivatives of prostaglandins 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 nonionic 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 typically named using the letter P (for poloxamer) followed by three Arabic numerals; the first two digits, when multiplied by 100, give the approximate molecular weight of the polyoxypropylene core, and the last digit, when multiplied by 10, gives the percentage 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 hematopoietic cell transduction. Hoefig et al. J Gene Med. 2012 Aug;14(8):549-60; U.S. 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 potential 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, e.g., 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, commercially known as 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 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, a 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 circulating HSPCs in the subject's peripheral blood. 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)). Different types of G-CSF that can be used in peripheral blood mobilization methods include filgrastim and long-acting G-CSF: pegfilgrastim. In certain embodiments, peripheral blood is mobilized by treating a 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 and CTCE-0214, or SDF-1a, such as Met-SDF-113), or chemokine receptor antagonists (e.g., chemokine (C-X-C 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 an 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 prior to collection of HSPCs. In certain embodiments, HSPCs are collected within 1, 2, 3, 4, 5, 6, 7, 8, 12, 14, 16, 18, 20, or 24 hours after the final administration of the agent used to mobilize HSPCs into peripheral blood. In certain embodiments, HSCs and HSPCs are mobilized by treating a subject with two or more different types of agents 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 a 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 a 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 mobilizing 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 subject's vein. For example, peripheral blood can be collected using a blood component separator. Blood flows from the vein through a catheter into the blood component separator, which separates white blood cells, including HSPCs, from the remainder of the blood, which is then returned to the subject's body. Blood component separation can be performed for several hours or over 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 fine needle aspiration (see, eg, Koda et al., 1984, J. Clin Invest. 73:1377-1384).

[0160] In certain embodiments, the hematocrit value of a biological sample may be determined. The hematocrit value 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 should be appreciated that the sample may be combined with an anticoagulant to aid in determining the hematocrit value, and that such an anticoagulant may be added to the processing chamber before or during centrifugation. Alternatively, the hematocrit value may be determined by measuring the optical properties of the sample. For example, the sample may be analyzed using a spectrometer. It should be appreciated that any type of known spectroscopic method for determining hematocrit value may be used, 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 other embodiments, 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 even 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 for CD34. Alternatively, or in addition to high-stringency enrichment, low-stringency enrichment for 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" CD34 enrichment involves the enrichment of cells for cells expressing a particular biological marker, e.g., CD34. +This resulted in a mean cell yield of 61.6% and a median of 65.7%, and a mean relative purity of 88.5% and a median of 95.9% (N=166) (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 increases from 0.2-2% to over 80%. To achieve this, a biological sample is initially applied to the capture substrate, followed by repeated buffer exchanges, referred to herein as "washing," to remove weakly or nonspecifically bound cells. Typically, cells are removed from the capture substrate and reapplied with each wash cycle. This removal and reapplication can be accomplished manually by pipetting from a tube or automatically using a pump and tubing system. For example, using Quad Technologies' MagCloudz® in conjunction with the Dynabeads® magnetic cell separation system, cell-magnetic particle complexes are separated in a tube on a magnetic stand and washed manually. Using Miltenyi Biotec's CliniMACS® system, cell-magnetic particle complexes are applied to a magnetic column in a tubing set using a preset automated program, followed by washing / reapplication using a valve and pump system.In certain embodiments, sorting under high stringency conditions can be performed using 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. Detailed methods for preparing and enriching cells and cell populations, including exemplary methods for sorting CD34+ cells under high stringency conditions, are described, for example, in International Patent Application Publication No. 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, such as directly coupled immunomagnetic beads. The biological sample can be suspended in any suitable liquid, such as, 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 also contains human serum albumin at a suitable concentration, such as about 2.5%. Using magnetically activated cell sorting (MACS) technology, the biological sample, after labeling, is applied to a column containing a magnetically susceptible or ferromagnetic material. Using the MACS system, the magnetically susceptible or ferromagnetic material of the column retains 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. 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, eluted, and / or reloaded onto the column at a slower rate to improve the purity of the enriched fraction. Suitable wash buffers include PBS containing (optionally) EDTA and (optionally) human serum albumin. Any components of the labeled biological sample that are removed during the wash step are collected in a waste bag or a "non-target" bag. After a suitable wash step, the highly stringently enriched cells are eluted into the target cell bag.

[0166] In a low-stringency enrichment protocol, a biological sample containing CD34+ cells is labeled with a CD34-labeling agent, such as directly bound immunomagnetic beads. Using magnetically activated cell sorting (MACS) technology, the biological sample is then applied to a column containing a magnetically susceptible or ferromagnetic material at a lower flow rate than in high-stringency enrichment. As in high-stringency enrichment, the magnetically susceptible or ferromagnetic material retains 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 achieved by modifying the standard operating method of the MACS system, with the software program for "removal mode," intended to achieve high-stringency depletion (i.e., removal of target cells), to instead result in low-stringency enrichment. Operation of the MACS system in removal mode causes target cells in a biological sample to bind to a magnetically susceptible or ferromagnetic material in the column using slow column loading and a wash step with lower stringency than operation in enrichment mode. Unlabeled cells are then passed through the MACS system into a wash bag or so-called "target" bag. The removal mode program then switches the output valve to direct fluid into a so-called "non-target" bag, after which the column is demagnetized. Continued application of fluid over the demagnetized column results in the elution of a CD34+ enriched cell population, enriched under low-stringency conditions, into the so-called "non-target" bag, using this method to recover the target cells.

[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. Without undue experimentation, those skilled in the art will be able to reprogram the software of such MACS systems so that the output valve directs the flow through the initial 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, 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 as to maintain a higher yield of the enriched cell population than 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, fold enrichment under high stringency conditions is greater than fold enrichment 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 may, in some cases, be 2, 2.25, 2.5, 2.75, 3, 3.25, 3.5, 3.75, or 4 times greater than 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 greater 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 herein by reference in its entirety.

[0170] A 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 high stringency CD34-enriched cell population and / or a low stringency CD34-enriched cell population. The method for selecting 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 high stringency CD34-enriched cell population and the other sample is used to prepare a low stringency CD34-enriched cell population. 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. Selections 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, a cell population may be divided such that low or high stringency selection is applied to a fraction of cells previously subjected to high 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 are contemplated for high or low stringency sorting before combining or dividing the biological sample or enriched cell population, or after combining or dividing the biological sample or enriched cell population. In certain embodiments, the method involves the selection of CD34 under high stringency conditions. + preparing a CD34 enriched cell population at high stringency from a first biological sample obtained from the 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, populations 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 a poloxamer (e.g., LentiBOOST), exhibit 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 levels are significantly higher after transduction with a lentiviral vector comprising the CD18 gene compared to transduction without PGE2 or poloxamer, or compared to transduction with no transduction enhancer or 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, methods of transducing hematopoietic cells with retroviral (e.g., lentiviral) vectors result 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, methods of treating a subject include 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 having 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 having 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, methods of transducing hematopoietic cells with retroviral vectors result 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, methods of treating a subject include 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 present disclosure provides a population 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 present disclosure provides a population 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 soluble cellulose membrane comprising 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%.

[0009] Provided is a method for producing a population of hematopoietic cells, 5% of which are 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, 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™). 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, 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 time periods.

[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 for producing a population of hematopoietic cells, wherein 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 vectors of the present disclosure. For example, the vector can contain single- or double-stranded nucleic acid, e.g., single- or double-stranded DNA. For example, the recombinant retroviral vector can be DNA. The vector can contain single- or double-stranded RNA, including modified forms of RNA. In another example, the recombinant retroviral vector can be RNA, e.g., mRNA or modified mRNA.

[0181] In certain embodiments, the recombinant retroviral vector can be a viral vector derived from a virus, e.g., an adenovirus, adeno-associated virus, lentivirus (LV), herpesvirus, alphavirus, or 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). While 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 recombinant retroviral vectors encoding and / or expressing a polypeptide preparation, e.g., FANCA, where one population is prepared by selection under high stringency conditions and the other population is prepared by selection under low stringency conditions. In one embodiment, the cell populations are 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 integration into the cell's 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 expression of the therapeutic agent, e.g., a biologically active FANCA protein.

[0185] For example, biologically active FANCA protein forms part of the FA core complex. In one embodiment, the FANCA gene is delivered via a viral vector. In one embodiment, the FANCA gene is delivered via a lentiviral vector. In one embodiment, the lentiviral vector is PGK-FANCA.WPRE*LV. After transducing bone marrow (BM) cells or stem or progenitor cells from an FA-A patient with the FANCA lentiviral vector (LV), the therapeutic vector is intended to be integrated into the cell's genome. Upon integration, the therapeutic protein (e.g., human FANCA protein) is expressed by the cell. The transduced FA cells are genetically corrected and are thereby able to activate the FA pathway by monoubiquitinating FANCD2 and FANCI. These proteins can then migrate to areas of DNA damage and, in cooperation with other DNA repair proteins, 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 the 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 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. Those skilled in the art will recognize that such measures of therapeutic efficacy will be applicable to the particular disease being modified, and will recognize appropriate detection methods to use to measure therapeutic efficacy.

[0188] Thus, the present invention provides methods for treating FA or one or more of the hematological manifestations of FA. In one embodiment, the hematological manifestation of FA is selected from one or more of bone marrow failure (BMF), thrombocytopenia, leukopenia, pancytopenia, neutropenia, and anemia. In a specific 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 in gene therapy. In certain embodiments, the hematopoietic cells are transduced with a vector containing 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 contains 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 method prevents or ameliorates 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 transduced according to the methods disclosed herein, e.g., in the presence of two or more transduction enhancers disclosed herein.

[0195] In certain 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 was carried out according to the methods disclosed herein. In certain 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 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 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 a 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 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 methods include detecting that a therapeutic effect has been achieved. Those skilled in the art will recognize that measuring such therapeutic efficacy will be applicable to the particular disease that has been modified, and will recognize appropriate detection methods to use to measure therapeutic efficacy.

[0198] Transgene expression using a subject transgene is expected to be robust. Thus, in some instances, transgene expression, such as by measuring therapeutic efficacy, e.g., by measuring gene product levels, may be observed within two months after administration of a subject composition, e.g., within four weeks, three weeks, or two weeks after administration, e.g., one week after administration. Transgene expression is also expected to persist over time. Thus, in some instances, transgene expression, such as by measuring therapeutic efficacy, e.g., by measuring gene product levels, may be observed for two or more months, e.g., four or more, six or more, eight or more, or ten or more months, and in some instances, one or more years, e.g., two, three, four, or five years, and in certain instances, five or more years, after administration of a subject composition.

[0199] In certain embodiments, the method includes detecting expression of the transgene in a cell or in a subject, wherein expression is enhanced compared to expression from a polynucleotide cassette that does not include one or more 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, as evidenced, for example, 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 of cells a patient receives by infusion can be at least about 1 x 10 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 x 10 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, 1×10 8 In some embodiments, 1 x 10 or more low stringency CD34 enriched cells are infused into the patient. 6 pieces~4×10 6 In another embodiment, 3 x 10 high stringency CD34 enriched cells are infused into the patient per kilogram of patient body weight. 5 pcs and 4 x 10 6 In some embodiments, 1 x 10 high stringency CD34 enriched cells are infused into the patient. 6 pieces~4×10 6 In another embodiment, 3 x 10 high stringency CD34 enriched cells are infused into the patient per kilogram of patient body weight. 5 pcs and 4 x 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 complete. In certain embodiments, the transduced cells are stored or frozen until use, while in some embodiments, the cells are provided to the subject immediately or shortly after transduction, e.g., 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. Transduced FA cells are genetically corrected and are thereby able to activate the FA pathway by monoubiquitinating FANCD2 and FANCI. These proteins migrate to areas of DNA damage and, in concert with other DNA repair proteins, promote DNA repair in these cells, just 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 Corrected 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 patients who lack an HLA-homologous sibling for allogeneic transplantation. In one embodiment, hematopoietic gene therapy is the preferred treatment regimen for patients who lack an HLA-homologous sibling. In another embodiment, hematopoietic gene therapy is the preferred treatment regimen for patients who have an HLA-homologous sibling.

[0205] Fanconi anemia (FA) is an autosomal recessive disorder (except for complementation group FA-B, which is X-linked), and the median survival time for 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 hematologic abnormality detected in these patients is often macrocytosis, which usually evolves into 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 the hematologic disorder being 7 years. Approximately 80% of FA patients will develop 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, 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 present disclosure provides methods 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 the pCCL-PGK-FANCAW-82-PRO transfer vector, e.g., 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 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 the simian virus 40 polyadenylation sequence and (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 linked to the HIV-1 R region. 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 collected from the patient's peripheral blood by apheresis. 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 transfer plasmid. In some embodiments, 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 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. TIFF2025038075000007.tif124160TIFF2025038075000008.tif218160TIFF2025038075000009.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 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, potentially resulting in increased neutrophil numbers. It also impairs immune cell interactions, immune recognition, and lymphocyte killing functions.

[0212] In certain embodiments, the present disclosure provides a method of treating leukocyte adhesion deficiency type 1 (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 the pCCL-ChimhCD18W-82-RO transfer vector, e.g., 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 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 the simian virus 40 polyadenylation sequence and (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 linked to the HIV-1 R region. 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, plerifax, or a combination of G-CSF and plerifax. HSCs are then harvested from the patient's peripheral blood by apheresis. 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 is 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 transplanted 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 collected 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 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 transfer plasmid. In some embodiments, 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 infusion to reconstitute the HSC niche with ITGB2-expressing cells.

[0216] The sequence of the ITGB2 expression cassette 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. TIFF2025038075000010.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 disrupts the energetic balance in red blood cells, thereby causing hemolytic anemia that is highly variable and can be fatal during the neonatal period.

[0219] In certain embodiments, the present 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 the pCCL-PGK-coRPKW-82-RO transfer vector, e.g., 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 the simian virus 40 polyadenylation sequence and (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 linked to the HIV-1 R region. 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 collected 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 are transduced ex vivo with lentiviral vector particles according to the methods disclosed herein. In one embodiment, the cells are transduced in the presence of PGE2 and a poloxamer. In one embodiment, the poloxamer is poloxamer 338 (LentiBOOST™). The transduced HSCs are then transplanted 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 shown in bold capital letters. The PGK promoter is italicized, and the WPRE sequence is underlined. TIFF2025038075000011.tif94160TIFF2025038075000012.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 by reference in its entirety.

[0224] Infantile malignant osteopetrosis (IMO) is a rare recessive genetic disorder characterized by increased bone mass caused 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. have shown that osteoclast function can be restored by lentiviral vector-mediated expression of TCIRG1, but the precise threshold for restoration of resorption as well as the cellular response to vector-mediated TCIRG1 expression remains unknown.

[0225] In one embodiment, the present 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, e.g., 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 the simian virus 40 polyadenylation sequence and (enhancer-less) origin of replication sequence downstream of the HIV 3' LTR, replacing 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 linked to the HIV-1 R region. 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 collected 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, 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. TIFF2025038075000013.tif178160TIFF2025038075000014.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 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, alter cell surface markers, provide alternative MHC molecules, or encode immunoglobulin genes. In particular, in some cases, recombinant retroviral vectors are intended to provide for use in allogeneic transplants or transplants with mismatched donors, for example, by altering immune markers (e.g., HLA or MHC genes) or by causing expression of immune effector genes.

[0231] The coding sequence to be expressed in a cell can be any polynucleotide sequence, e.g., a gene or cDNA, that encodes a gene product, e.g., a polypeptide or RNA-based therapeutic (e.g., 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, if the transgene is a therapeutic gene, the coding sequence can be any gene encoding 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, transgene coding sequences are 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 encodes the amino acids for translation. During translation, each of the 61 trinucleotide codons is translated into one of 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 encode the same amino acid 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 encode the same protein sequence but utilize 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 that are expressed at low frequency in mammals or primates with codons that are 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 described above or disclosed herein, e.g., at least 90% sequence identity, e.g., 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 described above or disclosed herein.

[0233] In a further embodiment, the transgene coding sequence is modified to enhance expression by terminating or removing open reading frames (ORFs) that do not encode 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. ORFs 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 may be expressed in an expression cassette flanking the gene of interest and may lead to unwanted adverse effects. In one aspect of the present invention, the transgene coding sequence is modified to remove open reading frames by further altering 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 ORF, while preserving the amino acid sequence and maintaining highly utilized codons in the gene of interest (i.e., avoiding codons with a frequency of less than 20%). In the present disclosure, transgene coding sequences can be optimized by either codon optimization and removal of non-transgene ORFs, or by using both techniques. As will be apparent to one skilled in the art, it is preferable to remove or minimize non-transgene ORFs after codon optimization in order to remove ORFs introduced during codon optimization.

[0234] Additionally, as will be appreciated by those skilled in the art, 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 the cell by transduction can occur in vivo, ex vivo, or in vitro. Thus, in some aspects of the invention, a composition that results in 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 the polynucleotide cassettes 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 present invention can be introduced into producer cells, followed by the introduction of an LV helper construct, which contains an LV coding region that can be expressed in the producer cells and complements the absence of LV helper functions in the LV vector. This can be followed by the introduction of a helper virus and / or additional vector into the producer cells, which provides accessory functions capable of supporting efficient LV production. The producer cells are then cultured to produce LV. These steps are carried out using standard methodologies. In a specific 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 the polynucleotide cassettes 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 present invention can be introduced into producer cells, followed by the introduction of an LV helper construct, which contains 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 can be followed by the introduction of a helper virus and / or additional vector into the producer cells, which provides accessory functions capable of supporting efficient LV production. The producer cells are then cultured to produce LV. These steps are carried out using standard methodologies. In a specific embodiment, the plasmids shown in Figures 38-41 are used to produce recombinant retroviral vectors.

[0240] Any suitable method for 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, infectious viral vectors can be prepared at any concentration suitable for effectively transducing mammalian cells. For example, viral particles can be prepared at concentrations of 10 per ml. 8 can be prepared at a concentration of 1 x 10 infectious units or more per mL, for example, 1 x 10 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 may also be a packaging cell in which the LV gag / pol and Rev genes are stably maintained in the host 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 pharmaceutically acceptable carriers, diluents, or excipients. The subject cell populations can be combined with pharmaceutically acceptable carriers, diluents, and agents that are generally safe, non-toxic, and useful for preparing formulations containing 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 formulations. The solutions or suspensions used in the formulations may include sterile diluents such as water for injection, saline solution, dimethyl sulfoxide (DMSO), fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial compounds such as benzyl alcohol or methylparabens; 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. 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 in accordance with Current Good Manufacturing Practices. Manufactured in accordance with 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 mandate that the formulation meet pre-approved criteria for identity, strength, quality, and purity. Acceptance criteria include numerical limits, ranges, or other suitable measurements of test results used to determine whether a formulation meets the Good Manufacturing Practices. Specifications describe analytical procedures used to test for compliance with the acceptance criteria. Each lot of the formulation can be evaluated. A lot is a specific amount of formulation that is tested to ensure compliance with the acceptance criteria.

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

[0245] Where 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 cells / mL, more than 10 7 cells / mL or more than 10 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 x 10 cells per kg of subject body weight. 6The therapeutically effective amount to be 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 is 2 x 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 pharmaceutically 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 solution and Hank's solution, cysteine, arginine, carnitine, alanine, glycine, lysine, valine, leucine, polyvinylpyrrolidone, polyethylene, and glycols. 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 before 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 skilled 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 can 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 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 serve 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 solely 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 present disclosure is further described in the following examples, which do not limit the scope of the disclosure as set forth in the claims. [Example]

[0255] Example 1 Transduction of hematopoietic cells by 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 thereof are likely to be 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 performed in the following examples is provided in Figure 1; in the various experiments described in the examples, the transduction medium contained no TE, one TE, or various combinations of TE. For priming, 2 ug / cm 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 experiment, hematopoietic cells were transduced with VSVG-pseudotyped LVs expressing a GFP (green fluorescent protein) transgene reporter. Cells were grown at 2.5 x 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 10 μg / mL and assayed for VCN after 14 days in liquid culture (Figure 2A). The results show that increasing the concentration of PGE2 (10 μg / mL, 30 μg / mL, or 50 μg / mL) increases transduction efficiency, as measured by vector copy number / cell.

[0258] In another experiment, cells were grown at 1 × 10 in liquid culture medium in the presence of PGE2. 7 TU / mL or 1 x 10 8 Cells were transduced with either lentiviral vector at 1000 TU / mL and assayed for VCN after 14 days in liquid culture (Figure 2B). At both vector concentrations, PGE2 increased the transduction efficiency, as determined by VCN / cell. Colony-forming unit (CFU) assays were performed using transduced cells (Figure 3A, CFC = colony-forming cells), and VCN and transduction efficiency (%) were analyzed for single colonies (Figures 3B and 3C). The highest concentration of PGE2 tested transduced up to approximately 75% of cells (Figure 3C).

[0259] Example 2 Transduction of hematopoietic cells by protamine sulfate and poloxamer F108 This example establishes that protamine sulfate and poloxamer F108 (also known as poloxamer 338) increase transduction efficiency. Studies were performed in liquid culture using poloxamer F108 (LentiBOOST™) according to the general protocol shown in Figure 1. Results for VCN in liquid culture are shown in Figure 4, and results for CFU assay analysis (Figure 5A) and isolated single CFU VCN (Figures 5B and 5C) are shown 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. When combined with PGE2, the highest concentration of PGE2 tested transduced up to approximately 75% of cells (Figure 5C).

[0260] Example 3 Transduction of hematopoietic cells by protamine sulfate, PGE2, and poloxamer F108 This example establishes that, surprisingly, the combination of protamine sulfate, PGE2, and LentiBOOST™ increased transduction efficiency beyond that observed with either PGE2 or LentiBOOST™ alone. Experiments were performed according to the protocol shown in Figure 1 and were 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 10 μg / mL, and PS at 4 μg / mL. Results obtained for VCN in transduced cells after 14 days in liquid culture are shown in Figure 6. Figure 7A shows CFU assays 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 (Figures 7B and 7E), as well as transduction efficiency (Figures 7C and 7D). As shown in Figure 7C, over 80% of the cells are transduced by the vector under these conditions.

[0261] Example 4 Methods for scaling up transduction enhancers and testing them in vivo This example demonstrates that the protocol established in Examples 1-3 can be adapted for therapeutic vectors and scaled up to produce sufficient vectors for clinical trials using umbilical cord blood as input. Scale-up of the transduction procedure was performed using CD34-enriched cord blood (CB) cells and LVs produced from pCCL-PGK-FANCAW-82-PRO without transduction enhancers (other than PS, which was used in all experiments) or with transduction enhancers (+TE), specifically including LB, PGE2, and PS. LB was used at a concentration of 1 mg / mL, PGE2 at 10 μg / mL, and PS at 4 μg / mL. The VCN after 14 days of liquid culture is shown in Figure 8. Figures 9A-9C show the results of a CFU assay (Figure 9A), VCN results for isolated single CFU (Figure 9B), and transduction efficiency results for CFU (Figure 9C) for cells transduced in vitro before transplantation. Figure 9B shows the results for 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 a transduction enhancer (TE, a combination of LB, PGE2, and PS), with an overnight multiplicity of infection (MOI) of 50 for therapeutic LV. LB was used at a concentration of 1 mg / mL, PGE2 at 10 μg / mL, and PS at 4 μg / mL. The transduced cells were then harvested, washed, and transfected at 1–2.5 × 10 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. +The percentage (%) of VCN cells (Figure 10A) and VCN / cell (Figure 10B) were assessed in bone marrow cells from transplanted animals at 1 month post-transplant (1 mpt), 2 months post-transplant (2 mpt), or 3 months post-transplant (3 mpt).

[0262] Example 5 LV production and MPB transduction under Good Manufacturing Practice (GMP) conditions This example demonstrates that the protocol established in Examples 1-4 can be adapted for 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 a subsequent set of experiments using CD34+ hematopoietic stem cells purified from mobilized peripheral blood (mPB) from healthy donors. The LV vector was a VSVG-pseudotyped LV expressing the FANCA transgene, which was generated using the pCCL-PGK-FANCAW-82-PRO transduction vector. Figure 11 shows the VCN / cell ratios for transduced cells at two MOIs after 14 days 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 μg / mL, and PS at 4 μg / mL. Figures 12A-12D show colony-forming unit (CFU) assay results for whole cells, BFUs, GM, and mixed myeloid progenitors for GMP003 at two different MOIs with (+TE) or without (-TE) the transduction enhancers PS, LB, and PGE2. Figures 13A and 13B show VCN for isolated single CFUs for GMP003 at MOI 20 or MOI 50 with (+TE) or without (-TE) the transduction enhancers PS, LB, and PGE2. Results are shown for early erythroid progenitor (BFU-E (burst-forming unit-erythroid)), granulocyte-macrophage progenitor (CFU-GM), and myeloid progenitor (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.

[0263] Sequence information SEQUENCE LISTING <110> CENTER OF ENERGY INVESTIGATIONS, MEDIOAMBIENTALES Y TECHNOLOGICAL, OA, MP CONSORCIO CENTRO DE INVESTIGACION BIOMEDICA EN RED FUNDACION INSTITUTO DE INVESTIGACION SANITARIA FUNDACION JIMENEZ DIAZ SPACECRAFT SEVEN, LLC <120> METHODS FOR GENE MODIFICATION OF HEMATOPOIETIC CELLS <150> US 62 / 712,146 <151> 2018-07-30 <160> 11 <170> PatentIn version 3.5 <210> 1 <211> 5554 <212> DNA <213> Artificial Sequence <220> <223> pCCL-PGK-FANCAW-82-PRO vector sequence <400> 1 ggggttgggg ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc 60 tgggcgtggt tccgggaaac gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc 120 cgttcgcagc gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc 180 tgctccgccc ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac 240 ggaagccgca cgtctcacta gtaccctcgc agacggacag cgccagggag caatggcagc 300 gcgccgaccg cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag 360 cggccgggaa ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct 420 gcccgcgcgg tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct 480 cgttgaccga atcaccgacc tctctcccca gggggatccc ccgggctgca ggaattcatg 540 tccgactcgt gggtcccgaa ctccgcctcg ggccaggacc cagggggccg ccggagggcc 600 tgggccgagc tgctggcggg aagggtcaag agggaaaaat ataatcctga aagggcacag 660 aaattaaagg aatcagctgt gcgcctcctg cgaagccatc aggacctgaa tgcccttttg 720 cttgaggtag aaggtccact gtgtaaaaaa ttgtctctca gcaaagtgat tgactgtgac 780 agttctgagg cctatgctaa tcattctagt tcatttatag gctctgcttt gcaggatcaa 840 gcctcaaggc tgggggttcc cgtgggtatt ctctcagccg ggatggttgc ctctagcgtg 900 ggacagatct gcacggctcc agcggagacc agtcaccctg tgctgctgac tgtggagcag 960 agaaagaagc tgtcttccct gttagagttt gctcagtatt tattggcaca cagtatgttc 1020 tcccgtcttt ccttctgtca agaattatgg aaaatacaga gttctttgtt gcttgaagcg 1080 gtgtggcatc ttcacgtaca aggcattgtg agcctgcaag agctgctgga aagccatccc 1140 gacatgcatg ctgtgggatc gtggctcttc aggaatctgt gctgcctttg tgaacagatg 1200 gaagcatcct gccagcatgc tgacgtcgcc agggccatgc tttctgattt tgttcaaatg 1260 tttgttttga ggggatttca gaaaaactca gatctgagaa gaactgtgga gcctgaaaaa 1320 atgccgcagg tcacggttga tgtactgcag agaatgctga tttttgcact tgacgctttg 1380 gctgctggag tacaggagga gtcctccact cacaagatcg tgaggtgctg gttcggagtg 1440 ttcagtggac acacgcttgg cagtgtaatt tccacagatc ctctgaagag gttcttcagt 1500 cataccctga ctcagatact cactcacagc cctgtgctga aagcatctga tgctgttcag 1560 atgcagagag agtggagctt tgcgcggaca caccctctgc tcacctcact gtaccgcagg 1620 ctctttgtga tgctgagtgc agaggagttg gttggccatt tgcaagaagt tctggaaacg 1680 caggaggttc actggcagag agtgctctcc tttgtgtctg ccctggttgt ctgctttcca 1740 gaagcgcagc agctgcttga agactgggtg gcgcgtttga tggcccaggc attcgagagc 1800 tgccagctgg acagcatggt cactgcgttc ctggttgtgc gccaggcagc actggagggc 1860 ccctctgcgt tcctgtcata tgcagactgg ttcaaggcct cctttgggag cacacgaggc 1920 taccatggct gcagcaagaa ggccctggtc ttcctgttta cgttcttgtc agaactcgtg 1980 ccttttgagt ctccccggta cctgcaggtg cacattctcc acccacccct ggttcccagc 2040 aagtaccgct ccctcctcac agactacatc tcattggcca agacacggct ggccgacctc 2100 aaggtttcta tagaaaacat gggactctac gaggatttgt catcagctgg ggacattact 2160 gagccccaca gccaagctct tcaggatgtt gaaaaggcca tcatggtgtt tgagcatacg 2220 gggaacatcc cagtcaccgt catggaggcc agcatattca ggaggcctta ctacgtgtcc 2280 cacttcctcc ccgccctgct cacacctcga gtgctcccca aagtccctga ctcccgtgtg 2340 gcgtttatag agtctctgaa gagagcagat aaaatccccc catctctgta ctccacctac 2400 tgccaggcct gctctgctgc tgaagagaag ccagaagatg cagccctggg agtgagggca 2460 gaacccaact ctgctgagga gcccctggga cagctcacag ctgcactggg agagctgaga 2520 gcctccatga cagaccccag ccagcgtgat gttatatcgg cacaggtggc agtgatttct 2580 gaaagactga gggctgtcct gggccacaat gaggatgaca gcagcgttga gatatcaaag 2640 attcagctca gcatcaacac gccgagactg gagccacggg aacacattgc tgtggacctc 2700 ctgctgacgt ctttctgtca gaacctgatg gctgcctcca gtgtcgctcc cccggagagg 2760 cagggtccct gggctgccct cttcgtgagg accatgtgtg gacgtgtgct ccctgcagtg 2820 ctcacccggc tctgccagct gctccgtcac cagggcccga gcctgagtgc cccacatgtg 2880 ctggggttgg ctgccctggc cgtgcacctg ggtgagtcca ggtctgcgct cccagaggtg 2940 gatgtgggtc ctcctgcacc tggtgctggc cttcctgtcc ctgcgctctt tgacagcctc 3000 ctgacctgta ggacgaggga ttccttgttc ttctgcctga aattttgtac agcagcaatt 3060 tcttactctc tctgcaagtt ttcttcccag tcacgagata ctttgtgcag ctgcttatct 3120 ccaggcctta ttaaaaagtt tcagttcctc atgttcagat tgttctcaga ggcccgacag 3180 cctctttctg aggaggacgt agccagcctt tcctggagac ccttgcacct tccttctgca 3240 gactggcaga gagctgccct ctctctctgg acacacagaa ccttccgaga ggtgttgaaa 3300 gaggaagatg ttcacttaac ttaccaagac tggttacacc tggagctgga aattcaacct 3360 gaagctgatg ctctttcaga tactgaacgg caggacttcc accagtgggc gatccatgag 3420 cactttctcc ctgagtcctc ggcttcaggg ggctgtgacg gagacctgca ggctgcgtgt 3480 accattcttg tcaacgcact gatggatttc caccaaagct caaggagtta tgaccactca 3540 gaaaattctg atttggtctt tggtggccgc acaggaaatg aggatattat ttccagattg 3600 caggagatgg tagctgacct ggagctgcag caagacctca tagtgcctct cggccacacc 3660 ccttcccagg agcacttcct ctttgagatt ttccgcagac ggctccaggc tctgacaagc 3720 gggtggagcg tggctgccag ccttcagaga cagagggagc tgctaatgta caaacggatc 3780 ctcctccgcc tgccttcgtc tgtcctctgc ggcagcagct tccaggcaga acagcccatc 3840 actgccagat gcgagcagtt cttccacttg gtcactg agatgagaa cttctgctcc 3900 cacggaggtg cctgacaca ggacacact gcccactct tcaggggct cctgaacgcc 3960 tgtctgcgga gcagagaccc ctccctgatg gtcgacttca tactggccaa gtgccagacg 4020 aaatgcccct taattttgac ctctgctctg gtgtggtggc cgagcctgga gcctgtgctg 4080 ctctgccggt ggaggagaca ctgccagagc ccgctgcccc gggaactgca gaagctacaa 4140 gaaggccggc agtttgccag cgatttcctc tccctgagg ctgcctcccc agcacccac 4200 ccggactggc tctcagctgc tgcactgcac tttgcgattc aacaagtcag ggaagaaaac 4260 atcaggaagc agctaagaa gctggactgc gagagagagg agctattggt ttccttttc 4320 ttctctcct tgatgggcct gctgtcgtca catctgacct caatagcac cacagacctg 4380 ccaaaggctt tccacgtttg tgcagcaatc ctcgagtgtt tagagagag gaagatatcc 4440 tggctggcac tcttcagtt gagagagt gacctcaggc tggggcggct cctcctccgt 4500 gtggccccgg atcagcacac caggctgctg cctttcgctt tttacagtct tctctcctac 4560 ttccatgaag acgcggccat cagggaagag gccttcctgc atgttgctgt ggacatgtac 4620 ttgaagctgg tccagctctt cgtggctggg gatacaagca cagtttcacc tccagctggc 4680 aggagcctgg agctcaaggg tcagggcaac cccgtggaac tgataacaaa agctcgtctt 4740 tttctgctgc agttaatacc tcggtgcccg aaaaagagct tctcacacgt ggcagagctg 4800 ctggctgatc gtggggactg cgacccagag gtgagcgccg ccctccagag cagacagcag 4860 gctgcccctg acgctgacct gtcccaggag cctcatctct tctgatgaga attcgatatc 4920 aagcttatcg ataccgtcga atcccccggg ctgcaggaat tcgagcatct taccgccatt 4980 tattcccata tttgttctgt ttttcttgat ttgggtatac atttaaatgt taataaaaca 5040 aaatggtggg gcaatcattt acatttttag ggatatgtaa ttactagttc aggtgtattg 5100 ccacaagaca aacatgttaa gaaactttcc cgttatttac gctctgttcc tgttaatcaa 5160 cctctggatt acaaaatttg tgaaagattg actgatattc ttaactatgt tgctcctttt 5220 acgctgtgtg gatatgctgc tttaatgcct ctgtatcatg ctattgcttc cgtacggct 5280 ttcgttttct cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc 5340 gttgtccgtc aacgtggcgt ggtgtgctct gtgtttgctg acgcaacccc cactggctgg 5400 ggcattgcca ccacctgtca actcctttct gggactttcg ctttccccct cccgatcgcc 5460 acggcagaac tcatcgccgc ctgccttgcc cgctgctgga caggggctag gttgctgggc 5520 actgaaatt ccgtggtgtt gtcggggaag ggcc 5554 <210> 2 <211> 1455 <212> PRT <213> Homo sapiens <400> 2 Put Ser Asp Ser Trp Val Pro Asn Ser Ala Ser Gly Gln Asp Pro Gly 1 5 10 15 Gly Arg Arg Arg Ala Trp Ala Glu Leu Leu Ala Gly Arg Val Lys Arg 20 25 30 Glu Lys Tyr Asn Pro Glu Arg Ala Gln Lys Leu Lys Glu Ser Ala Val 35 40 45 Arg Leu Leu Arg Ser His Gln Asp Leu Asn Ala Leu Leu Leu Glu Val 50 55 60 Glu Gly Pro Leu Cys Lys Lys Leu Ser Leu Ser Lys Val Ile Asp Cys 65 70 75 80 Asp Ser Ser Glu Ala Tyr Ala Asn His Ser Ser Ser Phe Ile Gly Ser 85 90 95 Ala Leu Gln Asp Gln Ala Ser Arg Leu Gly Val Pro Val Gly Ile Leu 100 105 110 Ser Ala Gly Met Val Ala Ser Ser Val Gly Gln Ile Cys Thr Ala Pro 115 120 125 Ala Glu Thr Ser His Pro Val Leu Leu Thr Val Glu Gln Arg Lys Lys 130 135 140 Leu Ser Ser Leu Leu Glu Phe Ala Gln Tyr Leu Leu Ala His Ser Met 145 150 155 160 Phe Ser Arg Leu Ser Phe Cys Gln Glu Leu Trp Lys Ile Gln Ser Ser 165 170 175 Leu Leu Leu Glu Ala Val Trp His Leu His Val Gln Gly Ile Val Ser 180 185 190 Leu Gln Glu Leu Leu Glu Ser His Pro Asp Met His Ala Val Gly Ser 195 200 205 Trp Leu Phe Arg Asn Leu Cys Cys Leu Cys Glu Gln Met Glu Ala Ser 210 215 220 Cys Gln His Ala Asp Val Ala Arg Ala Met Leu Ser Asp Phe Val Gln 225 230 235 240 Met Phe Val Leu Arg Gly Phe Gln Lys Asn Ser Asp Leu Arg Arg Thr 245 250 255 Val Glu Pro Glu Lys Met Pro Gln Val Thr Val Asp Val Leu Gln Arg 260 265 270 Met Leu Ile Phe Ala Leu Asp Ala Leu Ala Ala Gly Val Gln Glu Glu 275 280 285 Ser Ser Thr His Lys Ile Val Arg Cys Trp Phe Gly Val Phe Ser Gly 290 295 300 His Thr Leu Gly Ser Val Ile Ser Thr Asp Pro Leu Lys Arg Phe Phe 305 310 315 320 Ser His Thr Leu Thr Gln Ile Leu Thr His Ser Pro Val Leu Lys Ala 325 330 335 Ser Asp Ala Val Gln Met Gln Arg Glu Trp Ser Phe Ala Arg Thr His 340 345 350 Pro Leu Leu Thr Ser Leu Tyr Arg Arg Leu Phe Val Met Leu Ser Ala 355 360 365 Glu Glu Leu Val Gly His Leu Gln Glu Val Leu Glu Thr Gln Glu Val 370 375 380 His Trp Gln Arg Val Leu Ser Phe Val Ser Ala Leu Val Val Cys Phe 385 390 395 400 Pro Glu Ala Gln Gln Leu Leu Glu Asp Trp Val Ala Arg Leu Met Ala 405 410 415 Gln Ala Phe Glu Ser Cys Gln Leu Asp Ser Met Val Thr Ala Phe Leu 420 425 430 Val Val Arg Gln Ala Ala Leu Glu Gly Pro Ser Ala Phe Leu Ser Tyr 435 440 445 Ala Asp Trp Phe Lys Ala Ser Phe Gly Ser Thr Arg Gly Tyr His Gly 450 455 460 Cys Ser Lys Lys Ala Leu Val Phe Leu Phe Thr Phe Leu Ser Glu Leu 465 470 475 480 Val Pro Phe Glu Ser Pro Arg Tyr Leu Gln Val His Ile Leu His Pro 485 490 495 Pro Leu Val Pro Ser Lys Tyr Arg Ser Leu Leu Thr Asp Tyr Ile Ser 500 505 510 Leu Ala Lys Thr Arg Leu Ala Asp Leu Lys Val Ser Ile Glu Asn Met 515 520 525 Gly Leu Tyr Glu Asp Leu Ser Ser Ala Gly Asp Ile Thr Glu Pro His 530 535 540 Ser Gln Ala Leu Gln Asp Val Glu Lys Ala Ile Met Val Phe Glu His 545 550 555 560 Thr Gly Asn Ile Pro Val Thr Val Met Glu Ala Ser Ile Phe Arg Arg 565 570 575 Pro Tyr Tyr Val Ser His Phe Leu Pro Ala Leu Leu Thr Pro Arg Val 580 585 590 Leu Pro Lys Val Pro Asp Ser Arg Val Ala Phe Ile Glu Ser Leu Lys 595 600 605 Arg Ala Asp Lys Ile Pro Ser Leu Tyr Ser Thr Tyr Cys Gln Ala 610 615 620 Cys Ser Ala Ala Glu Glu Lys Pro Glu Asp Ala Ala Leu Gly Val Arg 625 630 635 640 Ala Glu Pro Asn Ser Ala Glu Glu Pro Leu Gly Gln Leu Thr Ala Ala 645 650 655 Leu Gly Glu Leu Arg Ala Ser Met Thr Asp Pro Ser Gln Arg Asp Val 660 665 670 Ile Ser Ala Gln Val Ala Val Ile Ser Glu Arg Leu Arg Ala Val Leu 675 680 685 Gly His Asn Glu Asp Asp Ser Ser Val Glu Ile Ser Lys Ile Gln Leu 690 695 700 Ser Ile Asn Thr Pro Arg Leu Glu Pro Arg Glu His Ile Ala Val Asp 705 710 715 720 Leu Leu Leu Thr Ser Phe Cys Gln Asn Leu Met Ala Ala Ser Ser Val 725 730 735 Ala Pro Pro Glu Arg Gln Gly Pro Trp Ala Ala Leu Phe Val Arg Thr 740 745 750 Met Cys Gly Arg Val Leu Pro Ala Val Leu Thr Arg Leu Cys Gln Leu 755 760 765 Leu Arg His Gln Gly Pro Ser Leu Ser Ala Pro His Val Leu Gly Leu 770 775 780 Ala Ala Leu Ala Val His Leu Gly Glu Ser Arg Ser Ala Leu Pro Glu 785 790 795 800 Val Asp Val Gly Pro Pro Ala Pro Gly Ala Gly Leu Pro Val Pro Ala 805 810 815 Leu Phe Asp Ser Leu Leu Thr Cys Arg Thr Arg Asp Ser Leu Phe Phe 820 825 830 Cys Leu Lys Phe Cys Thr Ala Ala Ile Ser Tyr Ser Leu Cys Lys Phe 835 840 845 Ser Ser Gln Ser Arg Asp Thr Leu Cys Ser Cys Leu Ser Pro Gly Leu 850 855 860 Ile Lys Lys Phe Gln Phe Leu Met Phe Arg Leu Phe Ser Glu Ala Arg 865 870 875 880 Gln Pro Leu Ser Glu Glu Asp Val Ala Ser Leu Ser Trp Arg Pro Leu 885 890 895 His Leu Pro Ser Ala Asp Trp Gln Arg Ala Ala Leu Ser Leu Trp Thr 900 905 910 His Arg Thr Phe Arg Glu Val Leu Lys Glu Glu Asp Val His Leu Thr 915 920 925 Tyr Gln Asp Trp Leu His Leu Glu Leu Glu Ile Gln Pro Glu Ala Asp 930 935 940 Ala Leu Ser Asp Thr Glu Arg Gln Asp Phe His Gln Trp Ala Ile His 945 950 955 960 Glu His Phe Leu Pro Glu Ser Ser Ala Ser Gly Gly Cys Asp Gly Asp 965 970 975 Leu Gln Ala Ala Cys Thr Ile Leu Val Asn Ala Leu Met Asp Phe His 980 985 990 Gln Ser Ser Arg Ser Tyr Asp His Ser Glu Asn Ser Asp Leu Val Phe 995 1000 1005 Gly Gly Arg Thr Gly Asn Glu Asp Ile Ile Ser Arg Leu Gln Glu 1010 1015 1020 Met Val Ala Asp Leu Glu Leu Gln Gln Asp Leu Ile Val Pro Leu 1025 1030 1035 Gly His Thr Pro Ser Gln Glu His Phe Leu Phe Glu Ile Phe Arg 1040 1045 1050 Arg Arg Leu Gln Ala Leu Thr Ser Gly Trp Ser Val Ala Ala Ser 1055 1060 1065 Leu Gln Arg Gln Arg Glu Leu Leu Met Tyr Lys Arg Ile Leu Leu 1070 1075 1080 Arg Leu Pro Ser Ser Val Leu Cys Gly Ser Ser Phe Gln Ala Glu 1085 1090 1095 Gln Pro Ile Thr Ala Arg Cys Glu Gln Phe Phe His Leu Val Asn 1100 1105 1110 Ser Glu Met Arg Asn Phe Cys Ser His Gly Gly Ala Leu Thr Gln 1115 1120 1125 Asp Ile Thr Ala His Phe Phe Arg Gly Leu Leu Asn Ala Cys Leu 1130 1135 1140 Arg Ser Arg Asp Pro Ser Leu Met Val Asp Phe Ile Leu Ala Lys 1145 1150 1155 Cys Gln Thr Lys Cys Pro Leu Ile Leu Thr Ser Ala Leu Val Trp 1160 1165 1170 Trp Pro Ser Leu Glu Pro Val Leu Leu Cys Arg Trp Arg Arg His 1175 1180 1185 Cys Gln Ser Pro Leu Pro Arg Glu Leu Gln Lys Leu Gln Glu Gly 1190 1195 1200 Arg Gln Phe Ala Ser Asp Phe Leu Ser Pro Glu Ala Ala Ser Pro 1205 1210 1215 Ala Pro Asn Pro Asp Trp Leu Ser Ala Ala Ala Leu His Phe Ala 1220 1225 1230 Ile Gln Gln Val Arg Glu Glu Asn Ile Arg Lys Gln Leu Lys Lys 1235 1240 1245 Leu Asp Cys Glu Arg Glu Glu Leu Leu Val Phe Leu Phe Phe Phe 1250 1255 1260 Ser Leu Met Gly Leu Leu Ser Ser His Leu Thr Ser Asn Ser Thr 1265 1270 1275 Thr Asp Leu Pro Lys Ala Phe His Val Cys Ala Ala Ile Leu Glu 1280 1285 1290 Cys Leu Glu Lys Arg Lys Ile Ser Trp Leu Ala Leu Phe Gln Leu 1295 1300 1305 Thr Glu Ser Asp Leu Arg Leu Gly Arg Leu Leu Leu Arg Val Ala 1310 1315 1320 Pro Asp Gln His Thr Arg Leu Leu Pro Phe Ala Phe Tyr Ser Leu 1325 1330 1335 Leu Ser Tyr Phe His Glu Asp Ala Ala Ile Arg Glu Glu Ala Phe 1340 1345 1350 Leu His Val Ala Val Asp Met Tyr Leu Lys Leu Val Gln Leu Phe 1355 1360 1365 Val Ala Gly Asp Thr Ser Thr Val Ser Pro Pro Ala Gly Arg Ser 1370 1375 1380 Leu Glu Leu Lys Gly Gln Gly Asn Pro Val Glu Leu Ile Thr Lys 1385 1390 1395 Ala Arg Leu Phe Leu Leu Gln Leu Ile Pro Arg Cys Pro Lys Lys 1400 1405 1410 Ser Phe Ser His Val Ala Glu Leu Leu Ala Asp Arg Gly Asp Cys 1415 1420 1425 Asp Pro Glu Val Ser Ala Ala Leu Gln Ser Arg Gln Gln Ala Ala Ala 1430 1435 1440 Pro Asp Ala Asp Leu Ser Gln Glu Pro His Leu Phe 1445 1450 1455 <210> 3 <211> 3903 <212> DNA <213> Artificial Sequence <220> <223> pCCL-ChimhCD18W-82-RO vector sequence <400> 3 cgcgtctgcc agctttcttg ctttgctgga gtattctgga atttgatggg ttgaggggttc 60 tggacacaat gccccaagcc ccttccttgt tgtgctgggt tcctatttct gctctcggca 120 ctgacttagc agctgctcaa gagctcacca tgttggcttg gattacacgg tctcacccac 180 atctccggca gtttgtgggc aaacttcctg agcagccttg ggtgatgaaa cctttcatgg 240 tagcaggaga atgggactgt gaattctcaa tcccctgtcc ccaccccttc cttcctctct 300 cagggccttg ctgtctagga ggagggagca cagcagcaac tgactgggca gcctttcagg 360 aaaggctagc ccgggctcga tcgagaagct tgataattc gtgaggtggg gagggctggg 420 accagggttc cctctttctc ttctgcggtg gccctggcct ggtgctagga ctgcgcgcct 480 cccctcagta cccgcggaca ccctgggctt cctgggccc agcatctgcc tggggcctcg 540 ccctgggctc cccctcctga cccccacctt gcgccccttc ccggtgttcc cggggcgctg 600 ccgggccctg gggcctgcgg ggcgcgggcg gctcttggct gggccattct ttcccggccc 660 cctcctccct tccgtttccg tggccgtgcg gccggctaga ggctgcggcc cagcgcggag 720 caggggggct ggcaggcgtc ggggcggtcg ggccggtccc gcccgcccct tcccctccac 780 aggcccgccc cggggcctgg gccaactgaa accgcgggag gaggaagcgc ggaatcagga 840 actggccggg gtccgcaccg ggcctgagtc ggtccgaggc cgtcccagga gcagctgccc 900 gaagggcgaa ttgggggatc ccccgggcta atgccaactt tgtacaaaaa agcaggctcc 960 accatgctgg gcctgcgccc cccacttctc gccctggtgg ggctgctctc cctcgggtgc 1020 gtcctctctc aggagtgcac gaagttcaag gtcagcagct gccgggaatg catcgagtcg 1080 gggcccggct gcacctggtg ccagaagctg aacttcacag ggccggggga tcctgactcc 1140 attcgctgcg acacccggcc acagctgctc atgaggggct gtgcggctga cgacatcatg 1200 gaccccacaa gcctcgctga aacccaggaa gaccacaatg ggggccagaa gcagctgtcc 1260 ccacaaaaag tgacgcttta cctgcgacca ggccaggcag cagcgttcaa cgtgaccttc 1320 cggcgggcca agggctaccc catcgacctg tactatctga tggacctctc ctactccatg 1380 cttgatgacc tcaggaatgt caagaagcta ggtggcgacc tgctccgggc cctcaacgag 1440 atcaccgagt ccggccgcat tggcttcggg tccttcgtgg acaagaccgt gctgccgttc 1500 gtgaacacgc accctgataa gctgcgaaac ccatgcccca acaaggagaa agagtgccag 1560 cccccgtttg ccttcaggca cgtgctgaag ctgaccaaca actccaacca gtttcagacc 1620 gaggtcggga agcagctgat ttccggaaac ctggatgcac ccgagggtgg gctggacgcc 1680 atgatgcagg tcgccgcctg cccggaggaa atcggctggc gcaacgtcac gcggctgctg 1740 gtgtttgcca ctgatgacgg cttccatttc gcgggcgacg ggaagctggg cgccatcctg 1800 acccccaacg acggccgctg tcacctggag gacaacttgt acaagaggag caacgaattc 1860 gactacccat cggtgggcca gctggcgcac aagctggctg aaaaacat ccagcccatc 1920 ttcgcggtga ccagtaggat ggtgaagacc tacgagaaac tcaccgagat catccccaag 1980 tcagccgtgg gggagctgtc tgaggactcc agcaatgtgg tccatctcat taagaatgct 2040 tacaataaac tctcctccag ggtattcctg gatcacaacg ccctccccga caccctgaaa 2100 gtcacctacg actccttctg cagcaatgga gtgacgcaca ggaaccagcc cagaggtgac 2160 tgtgatggcg tgcagatcaa tgtcccgatc accttccagg tgaaggtcac ggccacagag 2220 tgcatccagg agcagtcgtt tgtcatccgg gcgctgggct tcacggacat agtgaccgtg 2280 caggtccttc cccagtgtga gtgccggtgc cgggaccaga gcagagaccg cagcctctgc 2340 catggcaagg gcttcttgga gtgcggcatc tgcaggtgtg acactggcta cattgggaaa 2400 aactgtgagt gccagacaca gggccggagc agccaggagc tggaaggaag ctgccggaag 2460 gacaacaact ccatcatctg ctcagggctg ggggactgtg tctgcgggca gtgcctgtgc 2520 cacaccagcg acgtccccgg caagctgata tacgggcagt actgcgagtg tgacaccatc 2580 aactgtgagc gctacaacgg ccaggtctgc ggcggcccgg ggagggggct ctgcttctgc 2640 gggaagtgcc gctgccaccc gggctttgag ggctcagcgt gccagtgcga gaggaccact 2700 gagggctgcc tgaacccgcg gcgtgttgag tgtagtggtc gtggccggtg ccgctgcaac 2760 gtatgcgagt gccattcagg ctaccagctg cctctgtgcc aggagtgccc cggctgcccc 2820 tcaccctgtg gcaagtacat ctcctgcgcc gagtgcctga agttcgaaaa gggccccttt 2880 gggaagaact gcagcgcggc gtgtccgggc ctgcagctgt cgaacaaccc cgtgaagggc 2940 aggacctgca aggagaggga ctcagagggc tgctgggtgg cctacacgct ggagcagcag 3000 gacgggatgg accgctacct catctatgtg gatgagagcc gagagtgtgt ggcaggcccc 3060 aacatcgccg ccatcgtcgg gggcaccgtg gcaggcatcg tgctgatcgg cattctcctg 3120 ctggtcatct ggaaggctct gatccacctg agcgacctcc gggagtacag gcgctttgag 3180 aaggagaagc tcaagtccca gtggaacaat gataatcccc ttttcaagag cgccaccacg 3240 acggtcatga accccaagtt tgctgagagt taggacccag ctttcttgta caaagttggc 3300 attaggaatt cgagcatctt accgccattt attcccatat ttgttctgtt tttcttgatt 3360 tgggtataca tttaaatgtt aataaaacaa aatggtgggg caatcattta catttttagg 3420 gatatgtaat tactagttca ggtgtattgc cacaagacaa acatgttaag aaactttccc 3480 gttatttacg ctctgttcct gttaatcaac ctctggatta caaaatttgt gaaagattga 3540 ctgatattct taactatgtt gctcctttta cgctgtgtgg atatgctgct ttaatgcctc 3600 tgtatcatgc tattgcttcc cgtacggctt tcgttttctc ctccttgtat aaatcctggt 3660 tgctgtctct ttatgaggag ttgtggcccg ttgtccgtca acgtggcgtg gtgtgctctg 3720 tgtttgctga cgcaaccccc actggctggg gcattgccac cacctgtcaa ctcctttctg 3780 ggactttcgc tttccccctc ccgatcgcca cggcagaact catcgccgcc tgccttgccc 3840 gctgctggac aggggctagg ttgctgggca ctgataattc cgtggtgttg tcggggaagg 3900 gcc 3903 <210> 4 <211> 769 <212> PRT <213> Homo sapiens <400> 4 Met Leu Gly Leu Arg Pro Pro Leu Leu Ala Leu Val Gly Leu Leu Ser 1 5 10 15 Leu Gly Cys Val Leu Ser Gln Glu Cys Thr Lys Phe Lys Val Ser Ser 20 25 30 Cys Arg Glu Cys Ile Glu Ser Gly Pro Gly Cys Thr Trp Cys Gln Lys 35 40 45 Leu Asn Phe Thr Gly Pro Gly Asp Pro Asp Ser Ile Arg Cys Asp Thr 50 55 60 Arg Pro Gln Leu Leu Met Arg Gly Cys Ala Ala Asp Asp Ile Met Asp 65 70 75 80 Pro Thr Ser Leu Ala Glu Thr Gln Glu Asp His Asn Gly Gly Gln Lys 85 90 95 Gln Leu Ser Pro Gln Lys Val Thr Leu Tyr Leu Arg Pro Gly Gln Ala 100 105 110 Ala Ala Phe Asn Val Thr Phe Arg Arg Ala Lys Gly Tyr Pro Ile Asp 115 120 125 Leu Tyr Tyr Leu Met Asp Leu Ser Tyr Ser Met Leu Asp Asp Leu Arg 130 135 140 Asn Val Lys Lys Leu Gly Gly Asp Leu Leu Arg Ala Leu Asn Glu Ile 145 150 155 160 Thr Glu Ser Gly Arg Ile Gly Phe Gly Ser Phe Val Asp Lys Thr Val 165 170 175 Leu Pro Phe Val Asn Thr His Pro Asp Lys Leu Arg Asn Pro Cys Pro 180 185 190 Asn Lys Glu Lys Glu Cys Gln Pro Pro Phe Ala Phe Arg His Val Leu 195 200 205 Lys Leu Thr Asn Asn Ser Asn Gln Phe Gln Thr Glu Val Gly Lys Gln 210 215 220 Leu Ile Ser Gly Asn Leu Asp Ala Pro Glu Gly Gly Leu Asp Ala Met 225 230 235 240 Met Gln Val Ala Ala Cys Pro Glu Glu Ile Gly Trp Arg Asn Val Thr 245 250 255 Arg Leu Leu Val Phe Ala Thr Asp Asp Gly Phe His Phe Ala Gly Asp 260 265 270 Gly Lys Leu Gly Ala Ile Leu Thr Pro Asn Asp Gly Arg Cys His Leu 275 280 285 Glu Asp Asn Leu Tyr Lys Arg Ser Asn Glu Phe Asp Tyr Pro Ser Val 290 295 300 Gly Gln Leu Ala His Lys Leu Ala Glu Asn Asn Ile Gln Pro Ile Phe 305 310 315 320 Ala Val Thr Ser Arg Met Val Lys Thr Tyr Glu Lys Leu Thr Glu Ile 325 330 335 Ile Pro Lys Ser Ala Val Gly Glu Leu Ser Glu Asp Ser Ser Asn Val 340 345 350 Val His Leu Ile Lys Asn Ala Tyr Asn Lys Leu Ser Ser Arg Val Phe 355 360 365 Leu Asp His Asn Ala Leu Pro Asp Thr Leu Lys Val Thr Tyr Asp Ser 370 375 380 Phe Cys Ser Asn Gly Val Thr His Arg Asn Gln Pro Arg Gly Asp Cys 385 390 395 400 Asp Gly Val Gln Ile Asn Val Pro Ile Thr Phe Gln Val Lys Val Thr 405 410 415 Ala Thr Glu Cys Ile Gln Glu Gln Ser Phe Val Ile Arg Ala Leu Gly 420 425 430 Phe Thr Asp Ile Val Thr Val Gln Val Leu Pro Gln Cys Glu Cys Arg 435 440 445 Cys Arg Asp Gln Ser Arg Asp Arg Ser Leu Cys His Gly Lys Gly Phe 450 455 460 Leu Glu Cys Gly Ile Cys Arg Cys Asp Thr Gly Tyr Ile Gly Lys Asn 465 470 475 480 Cys Glu Cys Gln Thr Gln Gly Arg Ser Ser Gln Glu Leu Glu Gly Ser 485 490 495 Cys Arg Lys Asp Asn Asn Ser Ile Ile Cys Ser Gly Leu Gly Asp Cys 500 505 510 Val Cys Gly Gln Cys Leu Cys His Thr Ser Asp Val Pro Gly Lys Leu 515 520 525 Ile Tyr Gly Gln Tyr Cys Glu Cys Asp Thr Ile Asn Cys Glu Arg Tyr 530 535 540 Asn Gly Gln Val Cys Gly Gly Pro Gly Arg Gly Leu Cys Phe Cys Gly 545 550 555 560 Lys Cys Arg Cys His Pro Gly Phe Glu Gly Ser Ala Cys Gln Cys Glu 565 570 575 Arg Thr Thr Glu Gly Cys Leu Asn Pro Arg Arg Val Glu Cys Ser Gly 580 585 590 Arg Gly Arg Cys Arg Cys Asn Val Cys Glu Cys His Ser Gly Tyr Gln 595 600 605 Leu Pro Leu Cys Gln Glu Cys Pro Gly Cys Pro Ser Pro Cys Gly Lys 610 615 620 Tyr Ile Ser Cys Ala Glu Cys Leu Lys Phe Glu Lys Gly Pro Phe Gly 625 630 635 640 Lys Asn Cys Ser Ala Ala Cys Pro Gly Leu Gln Leu Ser Asn Asn Pro 645 650 655 Val Lys Gly Arg Thr Cys Lys Glu Arg Asp Ser Glu Gly Cys Trp Val 660 665 670 Only Tyr Thr With Glu Gln Gln Asp Gly Met Asp Arg Tyr With Tyr 675,680,685 Val Asp Glu Ser Arg Glu Cys Val Ala Gly Pro Asn Ile Ala Ala Ala Ile 690,695,700 Val Gly Gly Thr Val Ala Gly Ile Val Leu Ile Gly Ile Leu Leu Leu 705 710 715 720 Val And Trp Lys Ala Leu And His Leu Ser Asp Leu Arg Glu Tyr Arg 725 730 735 Arg Phe Glu Lys Glu Lys Leu Lys Ser Gln Trp Asn Asn Asp Asn Pro 740,745,750 Leu Phe Lys Ser Ala Thr Thr Thr Val Met Asn Pro Lys Phe Ala Glu 755,760,765 Ser <210> 5 <211> 2890 <212> DNA <213> Artificial Sequence <220> <223> pCCL-PGK-coRPKW-82-RO vector sequence <400> 5 ggggttgggg tgcgccttt tccaggcag ccctggttt gcgcagggac gcggctgctc 60 tggcgtggt tccgggaac gcagcggcgc cgaccctggg tctcgcacat tctcacgtc 120 cgttcgcagc gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc 180 tgctccgccc ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac 240 ggaagccgca cgtctcacta gtaccctcgc agacggacag cgccagggag caatggcagc 300 gcgccgaccg cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag 360 cggccgggaa ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct 420 gcccgcgcgg tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct 480 cgttgaccga atcaccgacc tctctcccca gggggatccg tcgacaccgg tgccaccatg 540 agcatccagg aaaatatcag ctctctgcag ctgcggtcct gggtgtccaa gagccagaga 600 gacctggcca agagcatcct gatcggagcc cctggcggac cagccggata cctgagaagg 660 gctagcgtgg cccagctgac ccaggaactg ggcaccgcct ttttccagca gcagcagctg 720 ccagccgcca tggccgacac ctttctggaa cacctgtgcc tgctggacat cgactctgag 780 cccgtggccg ccagaagcac cagcatcatt gccaccatcg gccctgccag cagaagcgtg 840 gagcggctga aagagatgat caagccggc atgaatatcg cccggctgaa cttctcccac 900 ggcagccacg agtaccacgc agagagcatt gccaacgtcc gggaggccgt ggagagcttt 960 gccggcagcc ccctgagcta cagacccgtg gccattgccc tggacaccaa gggccccgag 1020 atcagaacag gaattctgca gggagggcct gagagcgagg tggagctggt gaagggcagc 1080 caagtgctgg tgaccgtgga ccccgcctt agaaccagag gcaacgccaa cacagtgtgg 1140 gtggactacc ccaacatcgt gcgggtggtg cctgtgggcg gcagaatcta catcgacgac 1200 ggcctgatca gcctggtggt gcagaagatc ggacctgagg gcctggtgac ccaggtcgag 1260 aatggcggcg tgctggggcag cagaaagggc gtgaatctgc caggcgccca ggtggacctg 1320 cctggcctgt ctgagcagga cgtgagagac ctgagatttg gcgtggagca cggcgtggac 1380 atcgtgttcg ccagcttcgt gcggaaggcc tctgatgtgg ccgccgtgag agccgctctg 1440 ggccctgaag gccacggcat caagatcatc agaagatcg agaacacga gggcgtgaag 1500 cggttcgacg agatcctgga agtgtccgac ggcatcatgg tggccagagg cgacctgggc 1560 atcgagatcc ccgccgagaa ggtgttcctg gcccagaaaa tgatgatcgg acggtgcaac 1620 ctggccggca aacctgtggt gtgcgccacc cagatgctgg aaagcatgat caccaagccc 1680 agacccacca gagccgagac aagcgacgtg gccaacgccg tgctggatgg cgctgactgc 1740 atcatgctgt ccggcgagac agccaagggc aacttccccg tggaggccgt gaagatgcag 1800 cacgccattg ccagagaagc cgaggccgcc gtgtaccacc ggcagctgtt cgaggaactg 1860 cggagagccg cccctctgag cagagatccc accgaagtga ccgccatcgg agccgtggaa 1920 gccgccttca agtgctgcgc cgctgcaatc atcgtgctga ccaccacagg cagaagcgcc 1980 cagctgctgt ccagatacag acccagagcc gccgtgatcg ccgtgacaag atccgcccag 2040 gccgctagac aggtccacct gtgcagaggc gtgttccccc tgctgtaccg ggagcctccc 2100 gaggccatct gggccgacga cgtggacaga cgggtgcagt tcggcatcga gagcggcaag 2160 ctgcggggct tcctgagagt gggcgacctg gtgatcgtgg tgacaggctg gcggcctggc 2220 agcggctaca ccaacatcat gagggtgctg tccatcagct gaccgcggtc tagaggatcc 2280 cccgggctgc aggaattcga gcatcttacc gccatttatt cccatatttg ttctgtttt 2340 cttgatttgg gtatacattt aaatgttaat aaaacaaaat ggtggggcaa tcatttacat ttttagggat atgtaattac tagttcaggt gtattgccac aagacaaaca tgttaagaaa ctttcccgtt atttacgctc tgttcctgtt aatcaacctc tggattacaa aatttgtgaa agattgactg atattcttaa ctatgttgct ccttttacgc tgtgtggata tgctgcttta atgcctctgt atcatgctat tgcttcccgt acggctttcg ttttctcctc cttgtataaa 2640 tcctggttgc tgtctcttta tgaggagttg tggcccgttg tccgtcaacg tggcgtggtg 2700 tgctctgtgt ttgctgacgc aacccccact ggctggggca ttgccaccac ctgtcaactc 2760 ctttctggga ctttcgcttt cccctcccg atcgccacgg cagaactcat cgccgcctgc 2820. cttgcccgct gctggacagg ggctaggttg ctgggcactg ataattccgt ggtgttgtcg gggaaggggcc 2890 <210> 6 <211> 574 <212> PRT <213> Homo sapiens <400> 6 Met Ser Ile Gln Glu Asn Ile Ser Ser Leu Gln Leu Arg Ser Trp Val 1 5 10 15 Ser Lys Ser Gln Arg Asp Leu Ala Lys Ser Ile Leu Ile Gly Ala Pro 20 25 30 Gly Gly Pro Ala Gly Tyr Leu Arg Arg Ala Ser Val Ala Gln Leu Thr 35 40 45 Gln Glu Leu Gly Thr Ala Phe Phe Gln Gln Gln Gln Leu Pro Ala Ala 50 55 60 Met Ala Asp Thr Phe Leu Glu His Leu Cys Leu Leu Asp Ile Asp Ser 65 70 75 80 Glu Pro Val Ala Ala Arg Ser Thr Ser Ile Ile Ala Thr Ile Gly Pro 85 90 95 Ala Ser Arg Ser Val Glu Arg Leu Lys Glu Met Ile Lys Ala Gly Met 100 105 110 Asn Ile Ala Arg Leu Asn Phe Ser His Gly Ser His Glu Tyr His Ala 115 120 125 Glu Ser Ile Ala Asn Val Arg Glu Ala Val Glu Ser Phe Ala Gly Ser 130 135 140 Pro Leu Ser Tyr Arg Pro Val Ala Ile Ala Leu Asp Thr Lys Gly Pro 145 150 155 160 Glu Ile Arg Thr Gly Ile Leu Gln Gly Gly Pro Glu Ser Glu Val Glu 165 170 175 Leu Val Lys Gly Ser Gln Val Leu Val Thr Val Asp Pro Ala Phe Arg 180 185 190 Thr Arg Gly Asn Ala Asn Thr Val Trp Val Asp Tyr Pro Asn Ile Val 195 200 205 Arg Val Val Pro Val Gly Gly Arg Ile Tyr Ile Asp Asp Gly Leu Ile 210 215 220 Ser Leu Val Val Gln Lys Ile Gly Pro Glu Gly Leu Val Thr Gln Val 225 230 235 240 Glu Asn Gly Gly Val Leu Gly Ser Arg Lys Gly Val Asn Leu Pro Gly 245 250 255 Ala Gln Val Asp Leu Pro Gly Leu Ser Glu Gln Asp Val Arg Asp Leu 260 265 270 Arg Phe Gly Val Glu His Gly Val Asp Ile Val Phe Ala Ser Phe Val 275 280 285 Arg Lys Ala Ser Asp Val Ala Ala Val Arg Ala Ala Leu Gly Pro Glu 290 295 300 Gly His Gly Ile Lys Ile Ile Ser Lys Ile Glu Asn His Glu Gly Val 305 310 315 320 Lys Arg Phe Asp Glu Ile Leu Glu Val Ser Asp Gly Ile Met Val Ala 325 330 335 Arg Gly Asp Leu Gly Ile Glu Ile Pro Ala Glu Lys Val Phe Leu Ala 340 345 350 Gln Lys Met Met Ile Gly Arg Cys Asn Leu Ala Gly Lys Pro Val Val 355 360 365 Cys Ala Thr Gln Met Leu Glu Ser Met Ile Thr Lys Pro Arg Pro Thr 370 375 380 Arg Ala Glu Thr Ser Asp Val Ala Asn Ala Val Leu Asp Gly Ala Asp 385 390 395 400 Cys Ile Met Leu Ser Gly Glu Thr Ala Lys Gly Asn Phe Pro Val Glu 405 410 415 Ala Val Lys Met Gln His Ala Ile Ala Arg Glu Ala Glu Ala Ala Val 420 425 430 Tyr His Arg Gln Leu Phe Glu Glu Leu Arg Arg Ala Ala Pro Leu Ser 435 440 445 Arg Asp Pro Thr Glu Val Thr Ala Ile Gly Ala Val Glu Ala Ala Phe 450 455 460 Lys Cys Cys Ala Ala Ala Ile Ile Val Leu Thr Thr Thr Gly Arg Ser 465,470,475,480 Ala Gln Leu Leu Ser Arg Tyr Arg Pro Arg Ala Ala Val Ile Ala Val 485 490 495 Thr Arg Ser Ala Gln Ala Ala Arg Gln Val His Leu Cys Arg Gly Val 500 505 510 Phe Pro Leu Leu Tyr Arg Glu Pro Pro Glu Ala Ile Trp Ala Asp Asp 515 520 525 Val Asp Arg Arg Val Gln Phe Gly Ile Glu Ser Gly Lys Leu Arg Gly 530 535 540 Phe Leu Arg Val Gly Asp Leu Val Ile Val Val Thr Gly Trp Arg Pro 545,550,555,560 Gly Ser Gly Tyr Thr Asn Ile Met Arg Val Leu Ser Ile Ser 565 570 <210> 7 <211> 1233 <212> DNA <213> Artificial Sequence <220> <223> The PKLR polynucleotide sequeunce <400> 7 atggtctgct tcagactgtt ccctgtccct ggatctggtc tggtgcttgt gtgcttggtg 60 ctgggtgctg tgagatccta tgcccttgag ctgaacctga ctgactcaga aaatgccact 120 tgcctgtatg ccaagtggca gatgaacttc actgtgagat atgagactac caacaagacc 180 tacaagactg tgaccatctc agaccatggc actgtcacct acaatggatc aatctgtggt 240 gatgatcaga atggcccaaa gatagcagtg cagtttgggc ccggtttttc ctggattgct 300 aacttcacca aggcagcctc cacctacagc attgactcag tcagcttcag ctacaacact 360 ggggataaca ccaccttccc tgacgcagag gacaagggaa tccttactgt ggacgaactc 420 ctggcaatca gaatccccct taacgacctg ttcagatgca actccctttc aacccttgaa 480 aagaatgatg tggtgcaaca ctattgggac gtcctggtgc aagcctttgt gcagaatggg 540 acagtgagta ccaacgagtt cctctgtgac aaggacaaga ccagcactgt ggcccccact 600 atccacacca ctgtgcccag ccctaccact acccccaccc ctaaagagaa gccagaagct 660 ggaacctact cagtcaacaa tggaaatgac acatgcctcc ttgccaccat gggactgcag 720 ctgaacatca ctcaggacaa ggtggcctca gtgattaaca tcaaccctaa caccactcat 780 agcactggga gctgcagatc acatacagct ctgctgaggc tcaactcctc caccatcaag 840 tacctggact ttgtgtttgc tgtgaat gagacaggt tctacctcaa ggagtgac atttccatgt acctggtcaa tggttcagtg ttctctattg ccaacaacaa tctgagctac tgggatgcac ccctgggatc ctcctacatg tgcaacaagg agcagactgt gagtgtgtca ggtgcttttc agatcaacac ttttgacctg agggtgcagc ccttcaatgt gactcaggga aagtactcca ctgcacaaga gtgttccttg gatgatgaca ctatcctcat ccccattatt gtgggagctg gactgtcagg attgattata gtggattgtga ttgcttatgt gattggaagg agaaagagct atgctggcta ccagaccctg will be <210> 8 <211> 1233 <212> DNA <213> Artificial Sequence <220> <223> PKLR polynucleotide sequence <400> 8 atggtgtgct ttagctgtt tcctgtgcct ggttcagggc tggtcctggt ctgtctggtg ctgggggctg tcagaagcta tgccttggag ctgaacctca ctgatagtga aaatgccact tgtctgtatg ctaagtggca gatgaacttc actgtgagat atgaaaccac caacaagact tacaaaacag tgaccatctc agatcatgga actgtgacct acaacggcag cattttgtgga 240 gacgaccaga acggaccaaa aatcgctgtc caatttgggc ctggattctc ctggattgcc 300 aatttcacta aagctgcctc cacatattca attgactcag tgtccttctc ctacaacact 360 ggggacaaca ctactttccc tgatgctgaa gataagggaa tcttgacagt ggatgagctg 420 ctggctatca ggatcccttt gaatgacctg tttaggtgta attcactgag cactctggag 480 aagaacgacg tggtgcagca ctactgggac gtgctggtgc aggcctttgt gcagaacggc 540 actgtgtcca ccaacgaatt cctgtgtgat aaggacaaaa cttccactgt ggcacctaca 600 attcacacta ctgtgccttc acctaccacc actccaactc caaaggaaaa gcctgaagca 660 ggaacctact ctgtgaacaa tggcaatgat acctgtctgt tggccaccat gggcctccaa 720 ctgaacatta ctcaggacaa ggtggcctca gtgattaaca ttaaccccaa cactacccac 780 tccactggca gctgtagatc acacacagcc ttgctcagac tgaatagcag caccatcaag 840 tatttggatt ttgtgtttgc agtgaagaat gaaaacaggt tctacctgaa ggaagtcaac 900 atctcaatgt acctggtgaa cggctcagtg ttcagcattg ccaacaacaa cctctcctat tgggacgctc cactggggag cagctacatg tgtaacaagg aacagactgt gtcagtgtca ggagccttcc agattaacc ctttgatctg agggtccaac cctttaatgt cactcaagga aagtatagca ctgcccagga gtgctccctg gatgatgaca ccattctgat tccaatcatt gtgggtgcag gactttctgg gcttattatt gtgattgtga ttgcctatgt gattggcaga aggaaatcct atgcagggta ccaaactctg taa <210> 9 <211> 1233 <212> DNA <213> Artificial Sequence <220> <223> PKLR polynucleotide sequence <400> 9 atggtctgtt ttaggctgtt ccctgtccct ggttcaggac tggtcttagt gtgtctggtg cttggagctg tcagaagcta tgccctggag ctgaacctga ctgactcaga aaatgccact tgcctgtatg ccaagtggca gatgaacttc actgtcagat atgaaaccac caacaagacc father tgaccatctc agaccatggc actgtgactt acaatgggtc aatttgtgga gatgaccaga atggccctaa gatagctgtc cagtttggtc caggattcag ctggattgcc 300 aacttcacca aggcagccag cacctacagc attgactctg tgtccttctc ctacaacaca 360 ggagacaaca ccactttccc tgatgcagag gacaaaggta tcctgactgt ggatgagttg 420 ctggcaatca ggatcccact gaacgatctg ttcaggtgca actcactgtc cactctggaa 480 aagaatgatg tggtgcagca ctattgggat gtgctagtcc aggcctttgt ccagaatggg 540 actgtgtcaa ctaatgagtt cctgtgtgac aaggacaaga caagcactgt agccccact 600 atccatacca footcctag ccccaccact actccaaccc ccaaggagaa gcctgaggct 660 ggcacctact cagtgaacaa tgggaatgac acctgtttgc tggccactat gggactccaa 720 ctgaacatca cccaggacaa agtggcctct gtgatcaata tcaatcccaa caccacccac 780 agcactgggt cctgcagaag ccacactgcc ctcctgaggc tcaactcatc aactatcaag 840 tacttggatt ttgtgtttgc agtgaagaat gagaacagat tctacctcaa agaggtcaac 900 atttcaatgt acctggtgaa tgggagtgtg ttctccattg ctaacaacaa cctgagctac 960 tgggatgccc ctctgggctc ctcatacatg tgcaacaagg aacagactgt gagtgtgtca ggggccttcc agatcaacac ttttgacctg agagtgcagc cctttaatgt gacacaggga aagtacagca ctgctcagga gtgcagcctg gatgatgaca ctatcctgat ccctatcatt gtgggggcag gcctgtctgg actcattatt gtgattgtga ttgcctatgt gatagggaga aggaagtctt atgctggata ccagaccctg taa <210> 10 <211> 3384 <212> DNA <213> Artificial Sequence <220> <223> pRRL.PPT.EFS.tcirg1h.wpre vector sequence <400> 10 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgac cggtgcctag agaaggtggc gcggggtaaa ctgggaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca 180 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtgtcgtga cggcgggatcc gccaccatgg gctccatgtt tcggagcgag gaggtggccc tggtccagct 300 ctttctgccc acagcggctg cctacacctg cgtgagtcgg ctgggcgagc tgggcctcgt 360 ggagttcaga gacctcaacg cctcggtgag cgccttccag agacgctttg tggttgatgt 420 tcggcgctgt gaggagctgg agaagacctt caccttcctg caggaggagg tgcggcgggc 480 tgggctggtc ctgcccccgc caaaggggag gctgccggca cccccacccc gggacctgct 540 gcgcatccag gaggagacgg agcgcctggc ccaggagctg cgggatgtgc ggggcaacca 600 gcaggccctg cgggcccagc tgcaccagct gcagctccac gccgccgtgc tacgccaggg 660 ccatgaacct cagctggcag ccgcccacac agatggggcc tcagagagga cgcccctgct 720 ccaggccccc ggggggccgc accaggacct gagggtcaac tttgtggcag gtgccgtgga 780 gccccacaag gcccctgccc tagagcgcct gctctggagg gcctgcagag gcttcctcat 840 tgccagcttc agggagctgg agcagccgct ggagcacccc gtgacgggcg agccagccac 900 gtggatgacc ttcctcatct cctactgggg tgagcagatc ggacagaaga tccgcaagat 960 cacggactgc ttccactgcc acgtcttccc gtttctgcag caggaggagg cccgcctcgg 1020 ggccctgcag cagctgcaac agcagagcca ggagctgcag gaggtcctcg gggagacaga gcggttcctg agccaggtgc taggccgggt gctgcagctg ctgccgccag ggcaggtgca 1140 ggtccacaag atgaaggccg tgtacctggc cctgaaccag tgcagcgtga gcaccacgca caagtgcctc attgccgagg cctggtgctc tgtgcgagac ctgcccgccc tgcaggaggc 1260 cctgcgggac agctcgatgg aggagggagt gagtgccgtg gctcaccgca tcccctgccg 1320. ggacatgccc cccacactca tccgcaccaa ccgcttcacg gccagcttcc agggcatcgt ggatgcctac ggcgtgggcc gctaccagga ggtcaacccc gctccctaca ccatcatcac cttccccttc ctgtttgctg tgatgttcgg ggatgtgggc cacgggctgc tcatgttcct cttcgccctg gccatggtcc ttgcggagaa ccgaccggct gtgaaggccg cgcagaacga gatctggcag actttcttca ggggccgcta cctgctcctg cttatgggcc tgttctccat 1620 ctacaccggc ttcatctaca acgagtgctt cagtcgcgcc accagcatct tcccctcggg 1680 ctggagtgtg gccgccatgg ccaaccagtc tggctggagt gatgcattcc tggcccagca 1740. cacgatgctt accctggacc ccaacgtcac cggtgtcttc ctgggaccct acccctttgg 1800 catcgatcct atttggagcc tggctgccaa ccacttgagc ttcctcaact ccttcaagat 1860 gaagatgtcc gtcatcctgg gcgtcgtgca catggccttt ggggtggtcc tcggagtctt 1920 caaccacgtg cactttggcc agaggcaccg gctgctgctg gagacgctgc cggagctcac 1980 cttcctgctg ggactcttcg gttacctcgt gttcctagtc atctacaagt ggctgtgtgt 2040 ctgggctgcc agggccgcct cggcccccag catcctcatc cacttcatca acatgttcct 2100 cttctcccac agccccagca acaggctgct ctacccccgg caggaggtgg tccaggccac 2160 gctggtggtc ctggccttgg ccatggtgcc catcctgctg cttggcacac ccctgcacct 2220 gctgcaccgc caccgccgcc gcctgcggag gaggcccgct gaccgacagg aggaaaacaa 2280 ggccgggttg ctggacctgc ctgacgcatc tgtgaatggc tggagctccg atgaggaaaa 2340 ggcagggggc ctggatgatg aaggaggc cgagctcgtc ccctccgagg tgctcatgca 2400 ccaggccatc cacaccatcg agttctgcct gggctgcgtc tccaacaccg cctctacct 2460 gcgcctgtgg gccctgagcc tggcccacgc ccagctgtcc gaggttctgt gggccatggt 2520 gatgcgcata ggcctgggcc tgggccggga ggtgggcgtg gcggctgtgg tgctggtccc 2580 catctttgcc gcctttgccg tgatgaccgt ggctatcctg ctggtgatgg agggactctc 2640 agccttcctg cacgccctgc ggctgcactg ggtggaattc cagaacaagt tctactcagg 2700 cacgggctac aagctgagtc ccttcacctt cgctgccaca gatgactagt aagtcgacgg 2760 atcccccggg ctgcaggaat tcgagcatct taccgccatt tatacccata tttgttctgt 2820 ttttcttgat ttgggtatac atttaaatgt tataaaaca aaatggtggg gcaatcattt 2880 acatttttag ggatatgtaa ttactagttc aggtgtattg ccacaagaca aacatgttaa 2940 gaaactttcc cgttatttac gctctgttcc tgttaatcaa cctctggatt acaaaatttg 3000 tgaaagattg actgatattc ttaactatgt tgctcctttt acgctgtgtg gatatgctgc 3060 tttaatgcct ctgtatcatg ctattgcttc cgtacggct ttcgttttct cctccttgta 3120 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtccgtc aacgtggcgt 3180 ggtgtgctct gtgtttgctg acgcaacccc cactggctgg ggcattgcca ccacctgtca 3240 actcctttct gggactttcg ctttccccct cccgatcgcc acggcagaac tcatcgccgc 3300 ctgccttgcc cgctgctgga caggggctag gttgctgggc actgaaatt ccgtggtgtt 3360 gtcggggaag ctgacgtcct ttcg 3384 <210> 11 <211> 830 <212> PRT <213> Homo sapiens <400> 11 Met Gly Ser Met Phe Arg Ser Glu Glu Val Ala Leu Val Gln Leu Phe 1 5 10 15 Leu Pro Thr Ala Ala Ala Tyr Thr Cys Val Ser Arg Leu Gly Glu Leu 20 25 30 Gly Leu Val Glu Phe Arg Asp Leu Asn Ala Ser Val Ser Ala Phe Gln 35 40 45 Arg Arg Phe Val Val Asp Val Arg Arg Cys Glu Glu Leu Glu Lys Thr 50 55 60 Phe Thr Phe Leu Gln Glu Glu Val Arg Arg Ala Gly Leu Val Leu Pro 65 70 75 80 Pro Pro Lys Gly Arg Leu Pro Ala Pro Pro Pro Arg Asp Leu Leu Arg 85 90 95 Ile Gln Glu Glu Thr Glu Arg Leu Ala Gln Glu Leu Arg Asp Val Arg 100 105 110 Gly Asn Gln Gln Ala Leu Arg Ala Gln Leu His Gln Leu Gln Leu His 115 120 125 Ala Ala Val Leu Arg Gln Gly His Glu Pro Gln Leu Ala Ala Ala His 130 135 140 Thr Asp Gly Ala Ser Glu Arg Thr Pro Leu Leu Gln Ala Pro Gly Gly 145 150 155 160 Pro His Gln Asp Leu Arg Val Asn Phe Val Ala Gly Ala Val Glu Pro 165 170 175 His Lys Ala Pro Ala Leu Glu Arg Leu Leu Trp Arg Ala Cys Arg Gly 180 185 190 Phe Leu Ile Ala Ser Phe Arg Glu Leu Glu Gln Pro Leu Glu His Pro 195 200 205 Val Thr Gly Glu Pro Ala Thr Trp Met Thr Phe Leu Ile Ser Tyr Trp 210 215 220 Gly Glu Gln Ile Gly Gln Lys Ile Arg Lys Ile Thr Asp Cys Phe His 225 230 235 240 Cys His Val Phe Pro Phe Leu Gln Gln Glu Glu Ala Arg Leu Gly Ala 245 250 255 Leu Gln Gln Leu Gln Gln Gln Ser Gln Glu Leu Gln Glu Val Leu Gly 260 265 270 Glu Thr Glu Arg Phe Leu Ser Gln Val Leu Gly Arg Val Leu Gln Leu 275 280 285 Leu Pro Pro Gly Gln Val Gln Val His Lys Met Lys Ala Val Tyr Leu 290 295 300 Ala Leu Asn Gln Cys Ser Val Ser Thr Thr His Lys Cys Leu Ile Ala 305 310 315 320 Glu Ala Trp Cys Ser Val Arg Asp Leu Pro Ala Leu Gln Glu Ala Leu 325 330 335 Arg Asp Ser Ser Met Glu Glu Gly Val Ser Ala Val Ala His Arg Ile 340 345 350 Pro Cys Arg Asp Met Pro Pro Thr Leu Ile Arg Thr Asn Arg Phe Thr 355 360 365 Ala Ser Phe Gln Gly Ile Val Asp Ala Tyr Gly Val Gly Arg Tyr Gln 370 375 380 Glu Val Asn Pro Ala Pro Tyr Thr Ile Ile Thr Phe Pro Phe Leu Phe 385 390 395 400 Ala Val Met Phe Gly Asp Val Gly His Gly Leu Leu Met Phe Leu Phe 405 410 415 Ala Leu Ala Met Val Leu Ala Glu Asn Arg Pro Ala Val Lys Ala Ala 420 425 430 Gln Asn Glu Ile Trp Gln Thr Phe Phe Arg Gly Arg Tyr Leu Leu Leu 435 440 445 Leu Met Gly Leu Phe Ser Ile Tyr Thr Gly Phe Ile Tyr Asn Glu Cys 450 455 460 Phe Ser Arg Ala Thr Ser Ile Phe Pro Ser Gly Trp Ser Val Ala Ala 465 470 475 480 Met Ala Asn Gln Ser Gly Trp Ser Asp Ala Phe Leu Ala Gln His Thr 485 490 495 Met Leu Thr Leu Asp Pro Asn Val Thr Gly Val Phe Leu Gly Pro Tyr 500 505 510 Pro Phe Gly Ile Asp Pro Ile Trp Ser Leu Ala Ala Asn His Leu Ser 515 520 525 Phe Leu Asn Ser Phe Lys Met Lys Met Ser Val Ile Leu Gly Val Val 530 535 540 His Met Ala Phe Gly Val Val Leu Gly Val Phe Asn His Val His Phe 545 550 555 560 Gly Gln Arg His Arg Leu Leu Leu Glu Thr Leu Pro Glu Leu Thr Phe 565 570 575 Leu Leu Gly Leu Phe Gly Tyr Leu Val Phe Leu Val Ile Tyr Lys Trp 580 585 590 Leu Cys Val Trp Ala Ala Arg Ala Ala Ser Ala Pro Ser Ile Leu Ile 595 600 605 His Phe Ile Asn Met Phe Leu Phe Ser His Ser Pro Ser Asn Arg Leu 610 615 620 Leu Tyr Pro Arg Gln Glu Val Val Gln Ala Thr Leu Val Val Leu Ala 625 630 635 640 Leu Ala Met Val Pro Ile Leu Leu Leu Gly Thr Pro Leu His Leu Leu 645 650 655 His Arg His Arg Arg Arg Leu Arg Arg Arg Pro Ala Asp Arg Gln Glu 660 665 670 Glu Asn Lys Ala Gly Leu Leu Asp Leu Pro Asp Ala Ser Val Asn Gly 675 680 685 Trp Ser Ser Asp Glu Glu Lys Ala Gly Gly Leu Asp Asp Glu Glu Glu 690 695 700 Ala Glu Leu Val Pro Ser Glu Val Leu Met His Gln Ala Ile His Thr 705 710 715 720 Ile Glu Phe Cys Leu Gly Cys Val Ser Asn Thr Ala Ser Tyr Leu Arg 725 730 735 Leu Trp Ala Leu Ser Leu Ala His Ala Gln Leu Ser Glu Val Leu Trp 740 745 750 Ala Met Val Met Arg Ile Gly Leu Gly Leu Gly Arg Glu Val Gly Val 755 760 765 Ala Ala Val Val Leu Val Pro Ile Phe Ala Ala Phe Ala Val Met Thr 770 775 780 Val Ala Ile Leu Leu Val Met Glu Gly Leu Ser Ala Phe Leu His Ala 785 790 795 800 Leu Arg Leu His Trp Val Glu Phe Gln Asn Lys Phe Tyr Ser Gly Thr 805 810 815 Gly Tyr Lys Leu Ser Pro Phe Thr Phe Ala Ala Thr Asp Asp 820 825 830

Claims

1. 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:

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

3. The method of claim 1 , wherein the hematopoietic cells are engineered.

4. The method of claim 3 , wherein the hematopoietic cells are CD34 enriched cells.

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

6. 6. The method of claim 5, wherein the poloxamer is poloxamer 338 (LentiBOOST™).

7. The method of claim 1 , wherein the PGE2 or derivative thereof is modified.

8. The method according to claim 7, wherein the PGE2 or a derivative thereof is 16,16-dimethyl PGE2 (dmPGE2).

9. The method of claim 1 , wherein the PGE2 or derivative thereof is unmodified.

10. 2. The method of any one of the preceding claims, further comprising contacting the hematopoietic cells with protamine sulfate, and / or a recombinant fibronectin fragment, optionally RetroNectin™.

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

12. The method according to any one of claims 1 to 11, wherein the concentration of the PGE2 or derivative thereof is 5 to 30 µg / mL.

13. The method of claim 12, wherein the concentration of the PGE2 or derivative thereof is about 10 μg / mL.

14. 14. The method of any one of claims 1 to 13, wherein the concentration of the poloxamer is from 200 to 1200 μg / mL.

15. 15. The method of claim 14, wherein the concentration of the poloxamer is about 1000 μg / mL.

16. The method according to any one of claims 10 to 15, wherein the concentration of the protamine sulfate is 4 to 10 µg / mL.

17. 17. The method of claim 16, wherein the concentration of protamine sulfate is about 4 μg / mL.

18. 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 said recombinant retroviral vector is enhanced compared to transduction of hematopoietic cells by said recombinant retroviral vector in the absence of PGE2 or a derivative thereof and poloxamer.

19. The method of claim 18, further comprising contacting the hematopoietic cells ex vivo with an effective amount of protamine sulfate and / or a recombinant fibronectin polypeptide or variant thereof.

20. 20. The method of claim 18, wherein the gene of interest complements a defect in a gene associated with a monogenic genetic disease or disorder.

21. 20. The method of claim 19, 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.

22. 20. The method of claim 18, wherein a genetic disease or disorder caused by a single gene is prevented or ameliorated.

23. 23. The method of claim 22, wherein 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.

24. 19. The method of claim 18, 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.

25. 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.

26. 26. The method of claim 25, wherein the 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.

27. 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.

28. 28. The method of claim 27, wherein the liquid medium comprises protamine sulfate.

29. 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:

30. 30. The method of claim 29, wherein the liquid medium comprises protamine sulfate.

31. The method of any one of claims 25 to 30, wherein the lentiviral vector is a recombinant lentiviral vector.

32. The method of any one of claims 25 to 31, wherein the hematopoietic cells are engineered.

33. The method of any one of claims 25 to 32, wherein the hematopoietic cells are CD34 enriched cells.

34. 34. The method of any one of claims 25 to 33, wherein the poloxamer is selected from the group consisting of poloxamer 288, poloxamer 335, poloxamer 338, and poloxamer 407.

35. 34. The method of any one of claims 25 to 33, wherein the poloxamer is poloxamer 338 (LentiBOOST™).

36. The method according to any one of claims 25 to 35, wherein the PGE2 or a derivative thereof is modified.

37. The method according to any one of claims 25 to 36, wherein the PGE2 or a derivative thereof is 16,16-dimethyl PGE2 (dmPGE2).

38. The method according to any one of claims 25 to 37, wherein the PGE2 or derivative thereof is unmodified.

39. The method according to any one of claims 25 to 38, wherein the concentration of the PGE2 or derivative thereof is 5 to 30 μg / mL.

40. The method according to any one of claims 25 to 39, wherein the concentration of the PGE2 or derivative thereof is about 10 μg / mL.

41. 41. The method of any one of claims 25 to 40, wherein the concentration of the poloxamer is from 200 to 1200 μg / mL.

42. 42. The method of any one of claims 25 to 41, wherein the concentration of the poloxamer is about 1000 μg / mL.

43. 43. The method of any one of claims 25 to 42, wherein the concentration of protamine sulfate is 4 to 10 μg / mL.

44. 44. The method of any one of claims 25 to 43, wherein the concentration of protamine sulfate is about 4 μg / mL.

45. 45. The method of any one of claims 25 to 44, 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.

46. The method of any one of claims 25 to 45, for preventing or ameliorating a genetic disease or disorder caused by a gene.

47. 47. The method of claim 46, wherein 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.

48. 48. The method of any one of claims 25 to 47, 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.

49. 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.

50. 50. The method of claim 49, wherein the cell is contacted with PGE2 and the poloxamer.

51. 51. The method of claim 49 or claim 50, wherein the recombinant retroviral vector is a recombinant lentiviral vector.

52. The method of any one of claims 49 to 51, wherein the hematopoietic cells are CD34 enriched cells.

53. 53. The method of any one of claims 49-52, wherein the poloxamer is selected from the group consisting of poloxamer 288, poloxamer 335, poloxamer 338, and poloxamer 407.

54. 54. The method of any one of claims 49 to 53, wherein the poloxamer is poloxamer 338 (LentiBOOST™).

55. The method according to any one of claims 49 to 54, wherein the PGE2 or derivative thereof is modified.

56. The method of claim 55, wherein the PGE2 or derivative thereof is 16,16-dimethyl PGE2 (dmPGE2).

57. The method according to any one of claims 49 to 54, wherein the PGE2 or derivative thereof is unmodified.

58. 58. The method of any one of claims 49 to 57, comprising contacting the hematopoietic cells with protamine sulfate.

59. The method according to any one of claims 49 to 58, wherein the concentration of the PGE2 or derivative thereof is 5 to 30 μg / mL.

60. 60. The method according to any one of claims 49 to 59, wherein the concentration of the PGE2 or derivative thereof is about 10 μg / mL.

61. 61. The method of any one of claims 49 to 60, wherein the concentration of the poloxamer is from 200 to 1200 μg / mL.

62. 62. The method of any one of claims 49-61, wherein the concentration of the poloxamer is about 1000 μg / mL.

63. 63. The method of any one of claims 49 to 62, wherein the concentration of protamine sulfate is 4 to 10 μg / mL.

64. 64. The method of any one of claims 49 to 63, wherein the concentration of protamine sulfate is about 4 μg / mL.

65. 65. The method of any one of claims 49 to 64, wherein the polynucleotide complements a defect in a gene associated with a monogenic genetic disease or disorder.

66. 66. The method of any one of claims 49 to 65, wherein the therapeutic protein is selected from the group consisting of RPK, ITGB2, FANCA, FANCC, FANCG, TCIRG1, CLCN7, TNFSF11, PLEKHM1, TNFRSF11A and OSTM1.

67. The method according to any one of claims 49 to 66, wherein the disease or disorder is a genetic disease or disorder caused by a gene.

68. 68. The method of claim 67, wherein 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.

69. 69. The method of any one of claims 49 to 68, 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.

70. The method of any one of claims 49 to 69, wherein the hematopoietic cells have a VCN / cell of at least 1.0, at least 1.5, at least 2.0, or at least 2.

5.

71. 71. The method of any one of claims 49 to 70, wherein the 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%.

72. 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 said 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™).

73. 73. The method of claim 72, wherein the poloxamer is selected from the group consisting of poloxamer 288, poloxamer 335, poloxamer 338, and poloxamer 407.

74. 74. The method of any one of claims 72-73, wherein the poloxamer is poloxamer 338 (LentiBOOST™).

75. 75. The method of any one of claims 72 to 74, wherein the PGE2 or derivative thereof is modified.

76. The method of claim 75, wherein the PGE2 or a derivative thereof is 16,16-dimethyl PGE2 (dmPGE2).

77. 75. The method of any one of claims 72 to 74, wherein the PGE2 or derivative thereof is unmodified.

78. 78. The method of any one of claims 72-77, further comprising contacting the hematopoietic cells with protamine sulfate.

79. The method according to any one of claims 72 to 78, wherein the concentration of the PGE2 or derivative thereof is 5 to 30 μg / mL.

80. 80. The method of claim 79, wherein the concentration of PGE2 or a derivative thereof is about 10 μg / mL.

81. 81. The method of any one of claims 72 to 80, wherein the concentration of the poloxamer is from 200 to 1200 μg / mL.

82. 82. The method of any one of claims 49-81, wherein the concentration of the poloxamer is about 1000 μg / mL.

83. 83. The method of any one of claims 72 to 82, wherein the concentration of protamine sulfate is 4 to 10 μg / mL.

84. 84. The method of claim 83, wherein the concentration of protamine sulfate is about 4 μg / mL.

85. 85. The method of any one of claims 72 to 84, wherein the polynucleotide complements a defect in a gene associated with a monogenic genetic disease or disorder.

86. 86. The method of any one of claims 72 to 85, wherein the polypeptide of interest is selected from the group consisting of RPK, ITGB2, FANCA, FANCC, FANCG, TCIRG1, CLCN7, TNFSF11, PLEKHM1, TNFRSF11A and OSTM1.

87. 86. The method of claim 85, wherein the disease or disorder is a genetic disease or disorder caused by a single gene.

88. 88. The method of claim 87, wherein 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.

89. 89. The method of any one of claims 72 to 88, wherein the hematopoietic cells are CD34 enriched cells, optionally bone marrow (BM) derived cells, cord blood (CB) derived cells, or mobilized peripheral blood (mPB) cells.

90. 70. The method of any one of claims 49 to 69, wherein 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.

91. A hematopoietic cell population comprising at least 80% or at least 90% genetically modified hematopoietic cells, produced by the method of any one of claims 72 to 90.

92. 1. A method of treating a genetic disease or disorder in a subject in need thereof, comprising: Providing the hematopoietic cell population of claim 91 to the 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.

93. 93. The method of claim 92, wherein the polypeptide is selected from the group consisting of RPK, ITGB2, FANCA, FANCC, FANCG, TCIRG1, CLCN7, TNFSF11, PLEKHM1, TNFRSF11A and OSTM1.

94. 93. The method of claim 92, wherein the disease or disorder is a monogenic genetic disease or disorder.

95. 95. The method of claim 94, wherein 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.

95. 95. The method of any one of claims 92 to 94, wherein the hematopoietic cells are CD34 enriched cells, optionally bone marrow (BM) derived cells, cord blood (CB) derived cells, or mobilized peripheral blood (mPB) cells.

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