Methods of enveloped virus transduction

Abstract

The present disclosure relates generally to the manufacturing of gene therapy products, and specifically to methods of transducing a cell with an enveloped virus in a cell culture, methods of increasing an enveloped virus transduction efficiency of a cell in a cell culture and / or methods of increasing potency of an enveloped virus during transduction of a cell in a cell culture, the method contacting a plurality of cells in a cell culture medium with the enveloped virus and a transduction enhancer of Formula (I) or a stereoisomer, a racemate, a pharmaceutically acceptable salt or a solvate thereof.

Description

[0001] METHODS OF ENVELOPED VIRUS TRANSDUCTION

[0002] RELATED APPLICATION DATA

[0003] The present application claims priority from United States Provisional Patent Application No. 63 / 663,530 filed 24 June 2024 entitled “Methods of enveloped virus transduction”. The entire contents of this application are hereby incorporated by reference.

[0004] FIELD

[0005] The present disclosure relates generally to the manufacturing of gene therapy products, and specifically to methods of transducing a cell with an enveloped virus in a cell culture, methods of increasing an enveloped virus transduction efficiency of a cell in a cell culture, methods of increasing potency of an enveloped virus during transduction of a cell in a cell culture and / or methods of increasing transduction efficiency of a cell that differentiates into a myeloid cell in a cell culture, the method including contacting a plurality of cells in a cell culture medium with the enveloped virus and a transduction enhancer of Formula (I) or a stereoisomer, a racemate, a pharmaceutically acceptable salt or a solvate thereof.

[0006] BACKGROUND

[0007] Hematopoietic cell transplantation (HCT) is the administration of hematopoietic stem and progenitor cells (HSPCs) to patients with a variety of acquired and inherited malignant and non-malignant disorders to establish marrow and immune function. These disorders include hematologic malignancies (e.g., leukaemia, lymphoma, and myeloma), non-malignant acquired bone marrow disorders (e.g., aplastic anaemia), and genetic diseases associated with abnormal haematopoiesis and function (e.g., thalassemia, sickle cell anaemia, and severe combined immunodeficiency). HCT may also be used to support patients undergoing high-dose chemotherapy for the treatment of certain solid tumors for whom hematologic toxicity would otherwise limit drug administration (e.g., germ cell tumours, soft tissue sarcomas, and neuroblastoma).

[0008] Autologous CD34+ HSPCs can be genetically modified using lentivirus, making them an attractive target for gene therapy in the treatment of various genetic disorders and blood-borne diseases. HSPCs are mobilized and apheresate is collected from a patient. CD34+ cells are enriched ex vivo, and transduced with a lentiviral vector. Cultured and transduced CD34+ cells are harvested and formulated for infusion back into the patient. Retroviruses, e.g., lentiviruses are one of the most studied viral vectors for gene therapy. Retroviruses in general are RNA-based viruses which integrate their genetic information into the target cell chromosomes permanently. The advantages of retroviruses include long-term transgene expression in target cells, a low immunogenic potential, and the ability to transduce into dividing and non-dividing cells.

[0009] Lentiviruses are genetically engineered and usually based on human immunodeficiency virus 1 (HIV-1). To increase safety, modern vectors contain only those HIV genes which are necessary for infection and gene delivery, but the genes necessary for replication and virulence factors have been removed. Often, the envelope protein of HIV-1 is exchanged with that of another virus to allow infection of a wide range of target cells, e.g., VSV-G protein from Vesicular stomatitis Indiana virus (VSV).

[0010] To produce lentiviruses, cells such as human embryonic kidney cells HEK 293T are transfected with 3-4 plasmids. These include the transfer plasmid with the gene of interest and several packaging plasmids encoding, vesicular stomatitis G protein (VSV- G), and essential viral proteins responsible for gene integration or self-assembly. These plasmids can be transiently transfected into the cells, or a producer cell line is created with stable integration of the plasmids with inducible promoters, in which lentivirus production can be induced. Once the virus production has been induced, the release of the virus occurs by budding after successful assembly within the cells. The lentivirus is harvested from the producer cells and subsequently purified and concentrated in the downstream process, where it can then be used to subsequently transduce a population of cells suitable for use in gene therapy.

[0011] Low transduction efficiency of target cells by lentiviruses is a common pitfail of current gene therapy approaches. One strategy to circumvent these limitations is the addition of cofactors or transduction enhancers to optimize transduction protocols like cationic polymers (e.g., polybrene) or fibronectin fragments (e.g., retronectin). However, the use of fibronectin or fibronectin fragments requires coating of the culture plates and preloading of viral supernatants onto immobilized fibronectin fragments. These two steps are difficult to standardize and can lead to some saturation of target cell transduction depending on the concentrations of fibronectin fragments and viral supernatants used. Other widely used transduction enhancers include semen-derived enhancer of virus infection (SEVI), rapamycin, prostaglandin E2, dimethylprostaglandin E2 (dmPGE2), cyclosporin H, cyclosporin A, caraphenol A, rapamycin, staurosporine, polybrene and protamine sulfate. However, each of these compounds has its own limitations as a transduction enhancer, including increased nonspecific binding of vector particles to the cell surface, decreased cell proliferation and induction of cellular senescence. Thus, there is a need in the art for an improved process for transducing target cells with an enveloped virus (i.e., lentivirus) in a cell culture system, e.g., for gene therapy.

[0012] SUMMARY

[0013] In work leading up to the present invention, the inventors sought to produce a method for improving transduction of target cells with an enveloped virus, e.g., for gene therapy, at commercial scale and suitable for regulatory requirements.

[0014] The process of transducing a cell (e.g., a HSPC) with an enveloped virus in a cell culture produced by the inventors includes contacting the cells with a transduction enhancer of Formula (I) (e.g., nebivolol). The inventors found that transducing cells in the presence of this transduction enhancer increased the transduction efficiency, as well as potency, of the enveloped virus. The inventors further found that the addition of a poloxamer further increased the transduction efficiency and potency of the enveloped virus. Surprisingly, the inventors found that transducing cells in the presence of the transduction enhancer resulted in an increase in the transduction of cells along the myeloid cell lineage, without a reduction in the transduction of cells along the erythroid lineage.

[0015] Accordingly, the findings by the inventors have provided methods of transducing a cell with an enveloped virus.

[0016] The present disclosure provides a method of transducing a cell with an enveloped virus in a cell culture, the method comprising culturing a plurality of cells in a cell culture medium and contacting the cells with the enveloped virus and a transduction enhancer, wherein the transduction enhancer comprises a compound of Formula (I) or a stereoisomer, a racemate, a pharmaceutically acceptable salt or a solvate thereof:

[0017] Formula (I) wherein

[0018] X1and X2are independently selected from O, N, S or CH2;

[0019] L1and L2are independently absent or selected from -Ci-ealkyl-, -Ci ealkcnyl-^ or -Ci ealkynyl-, each of which may be optionally substituted with one or more R4or R5,

[0020] R1is selected from H, -Ci-ealkyl-, -C2-6alkenyl-;>or -C2-6alkynyl-;

[0021] R2and R3are independently selected from H, halogen, or OH; and R4and R5are independently selected from halogen, OH, -Ci-4alkyl-, and -Ci- 4alkoxy-

[0022] The present disclosure also provides a method of increasing an enveloped virus transduction efficiency of a cell in a cell culture, the method comprising culturing a plurality of cells in a cell culture medium and contacting the cells with the enveloped virus and a transduction enhancer, wherein the transduction enhancer comprises a compound of Formula (I) or a stereoisomer, a racemate, a pharmaceutically acceptable salt or a solvate thereof:

[0023] Formula (I) wherein

[0024] X1and X2are independently selected from O, N, S or CH2;

[0025] L1and L2are independently absent or selected from -Ci-ealkyl-, -C2-6alkenyl-;>or -C2-6alkynyl-, each of which may be optionally substituted with one or more R4or R5,

[0026] R1is selected from H, -Ci-ealkyl-, -C2-6alkenyl-;>or -C2-6alkynyl-;

[0027] R2and R3are independently selected from H, halogen, or OH; and

[0028] R4and R5are independently selected from halogen, OH, -Ci-4alkyl-, and -Ci- 4alkoxy-

[0029] The present disclosure further provides a method of increasing potency of an enveloped virus during transduction of a cell in a cell culture, the method comprising culturing a plurality of cells in a cell culture medium and contacting the cells with the enveloped virus and a transduction enhancer, wherein the transduction enhancer comprises a compound of Formula (I) or a stereoisomer, a racemate, a pharmaceutically acceptable salt or a solvate thereof:

[0030] X1and X2are independently selected from O, N, S or CH2; L1and L2are independently absent or selected from -Ci-ealkyl-, -C2-6alkenyl-;>or -C2-6alkynyl-, each of which may be optionally substituted with one or more R4or R5,

[0031] R1is selected from H, -Ci-ealkyl-, -C2-6alkenyl-;>or -C2-6alkynyl-;

[0032] R2and R3are independently selected from H, halogen, or OH; and

[0033] R4and R5are independently selected from halogen, OH, -Ci-4alkyl-, and -Ci- 4alkoxy-

[0034] The present disclosure provides a method of increasing transduction efficiency of a cell that differentiates into a myeloid cell in a cell culture, the method comprising culturing a plurality of cells in a cell culture medium and contacting the cells with the enveloped virus and a transduction enhancer, wherein the transduction enhancer comprises a compound of Formula (I) or a stereoisomer, a racemate, a pharmaceutically acceptable salt or a solvate thereof:

[0035] Formula (I) wherein

[0036] X1and X2are independently selected from O, N, S or CH2;

[0037] L1and L2are independently absent or selected from -Ci-ealkyl-, -Ci ealkcnyl-^ or -Ci ealkynyl-, each of which may be optionally substituted with one or more R4or R5,

[0038] R1is selected from H, -Ci-ealkyl-, -C2-6alkenyl-;>or -C2-6alkynyl-;

[0039] R2and R3are independently selected from H, halogen, or OH; and

[0040] R4and R5are independently selected from halogen, OH, -Ci-4alkyl-, and -Ci- 4alkoxy-

[0041] The present disclosure also provides a method of increasing vector copy number in a cell in a cell culture, the method comprising culturing a plurality of cells in a cell culture medium and contacting the cells with the enveloped virus and a transduction enhancer, wherein the transduction enhancer comprises a compound of Formula (I) or a stereoisomer, a racemate, a pharmaceutically acceptable salt or a solvate thereof:

[0042] Formula (I) wherein

[0043] X1and X2are independently selected from O, N, S or CH2;

[0044] L1and L2are independently absent or selected from -Ci-ealkyl-, -Ci ealkcnyl-^ or -Ci ealkynyl-, each of which may be optionally substituted with one or more R4or R5,

[0045] R1is selected from H, -Ci-ealkyl-, -C2-6alkenyl-;>or -C2-6alkynyl-;

[0046] R2and R3are independently selected from H, halogen, or OH; and

[0047] R4and R5are independently selected from halogen, OH, -Ci-4alkyl-, and -Ci- 4alkoxy-

[0048] In one example, the cell is a cell that differentiates into an erythroid cell.

[0049] In one example, the transduction of cells that differentiate into erythroid cells is not reduced.

[0050] In one example of any method described herein:

[0051] L1has a structure *-L3CH(R4)-, and L2has a structure *-L4CH(R5)-, wherein

[0052] L3and L4are independently absent or selected from -Ci-4alkyl-, -C2-4alkenyl-;>or -C2-4alkynyl-; and

[0053] * indicates a single bond to the central N.

[0054] In one example, L3and L4are both -Ci alkylin one example, X1and X2are both O.

[0055] In one example, R4and R5are both OH.

[0056] In one example, R1is H.

[0057] In one example, R2and R3are both F.

[0058] In one example, the compound of Formula (I) has the following structure:

[0059] In one example, the compound of Formula (I) is nebivolol and / or has the following structure:

[0060] In one example, the compound of Formula (I) is L-nebivolol and / or has the following structure:

[0061] In one example, the compound of Formula (I) is D-nebivolol and / or has the following structure:

[0062] In one example, the transduction enhancer comprises and / or substantially consists of: or pharmaceutically acceptable salts or solvates thereof.

[0063] In one example, the transduction enhancer comprises and / or substantially consists of a racemate of: or pharmaceutically acceptable salts of solvates thereof, in a ratio of between about 4:6 to about 6:4, or between about 4.5:5.5 to about 5.5:4.5, or about 1: 1.

[0064] In one example, the ratio is between about 4:6 to about 6:4. In another example, the ratio is between about 4.5:5.5 to about 5.5:4.5. In a further example, the ratio is between about 1: 1.

[0065] In one example, the transduction enhancer comprises and / or substantially consists of L-nebivolol or:

[0066] In one example, the compound is in the cell culture medium at a concentration of at least 1 pM. In another example, the compound is in the cell culture medium at a concentration of at least 3 pM. In a further example, the compound is in the cell culture medium at a concentration of at least 5 pM. In one example, the compound is in the cell culture medium at a concentration of at least 10 pM. In another example, the compound is in the cell culture medium at a concentration of between 1 pM and 20 pM. For example, the compound is in the cell culture medium at a concentration of about 1 pM, or about 1.5 pM, or about 2 pM, or about 2.5 pM, or about 3 pM, or about 3.5 pM, or about 4 pM, or about 4.5 pM, or about 5 pM. In one example, the compound is in the cell culture medium at a concentration of about 1 pM. In another example, the compound is in the cell culture medium at a concentration of about 3 pM. In a further example, the compound is in the cell culture medium at a concentration of about 5 pM. In one example, the compound is in the cell culture medium at a concentration of about 5 pM, or about 6 pM, or about 7 pM, or about 8 pM, or about 9 pM, or about 10 pM, or about 11 pM, or about 12 pM, or about 13 pM, or about 14 pM, or about 15 pM, or about 16 pM, or about 17 pM, or about 18 pM, or about 19 pM, or about 20 pM. In one example, the compound is in the cell culture medium at a concentration of about 10 pM.

[0067] In one example, the cell culture further comprises a water-soluble non-ionic triblock copolymer. For example, the water-soluble non-ionic triblock copolymer has an average molecular weight (in g / mol) greater than about 9800, 9900, 10000, 10100, 10200, 10300, 10400, 10500, 10600, 10700, 10800, 10900, 11000, 11100, 11200, 11300, 11400, 11500, 11600, 11700, 11800, 11900, 12000, 12100, 12200, 12300, 12400, 12500, 12600, 12700, 12800, 12900, 13000, 13100, 13200, 13300, 13400, 13500, 13600, 13700, 13800, 13900, 14000, 14100, 14200, 14300, 14400, 14500, 14600, 14700, 14800, 14900 or 15000.

[0068] In one example, the water-soluble non-ionic triblock copolymer has an average molecular weight greater than about 12000 g / mol.

[0069] In one example, the water-soluble non-ionic triblock copolymer has an average molecular weight of about 12600 g / mol.

[0070] In one example, the water-soluble non-ionic triblock copolymer has a viscosity (in Pa-s) of between about 2.0 and about 3.5.

[0071] In one example, the water-soluble non-ionic triblock copolymer is a poloxamer.

[0072] In one example, the poloxamer has an average polyoxypropylene content (in g / mol) of about or greater than about 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,

[0073] 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100,

[0074] 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500,

[0075] 4600, 4700, 4800, 4900 or 5000. For example, the poloxamer has an average polyoxypropylene content of about or greater than about 3000 g / mol.

[0076] In one example, the poloxamer has an average polyoxyethylene content (in % w / w) of about or greater than about 30, 40, 50, 60, 70, 80 or 90. For example, the poloxamer has an average polyoxyethylene content of about or greater than about 70% w / w.

[0077] In one example, the poloxamer is selected from the group consisting of poloxamer 105, poloxamer 123, poloxamer 124, poloxamer 182, poloxamer 184, poloxamer 188, poloxamer 234, poloxamer 235, poloxamer 237, poloxamer 238, poloxamer 288, poloxamer 333, poloxamer 334, poloxamer 335, poloxamer 338, poloxamer 403, poloxamer 407, and combinations thereof. For example, the poloxamer is selected from the group consisting of poloxamer 108, poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 288, poloxamer 388, poloxamer 407, and combinations thereof. In one example, the poloxamer is poloxamer 338. In another example, the poloxamer is poloxamer 407.

[0078] In one example, the poloxamer is in the cell culture medium at a concentration of at least 0.01 mg / mL. In another example, the poloxamer is in the cell culture medium at a concentration of between about 0.01 mg / mL and 10 mg / mL. In a further example, the poloxamer is in the cell culture medium at a concentration of between about 0.01 mg / mL and 5 mg / mL. For example, the poloxamer is in the cell culture medium at a concentration of between 0.1 mg / mL and 10 mg / mL. In one example, the poloxamer is in the cell culture medium at a concentration of between 0.1 mg / mL and 5 mg / mL. In one example, the poloxamer is in the cell culture medium at a concentration of at least 0.1 mg / mL. For example, the poloxamer is in the cell culture medium at a concentration of at least 0.5 mg / mL. In one example, the poloxamer is in the cell culture medium at a concentration of about 1 mg / mL.

[0079] In one example, the method comprises contacting the cells with the transduction enhancer for a period of time prior to contacting the cells with the enveloped virus.

[0080] In one example, the method comprises contacting the cells with the transduction enhancer for a period of at least 30 minutes prior to contacting the cells with the enveloped virus. For example, the method comprises contacting the cells with the transduction enhancer for a period of about 30 minutes, or about 35 minutes, or about 40 minutes, or about 45 minutes, or about 50 minutes, or about 55 minutes, or about 60 minutes prior to contacting the cells with the enveloped virus. In another example, the method comprises contacting the cells with the transduction enhancer for a period of between about 30 minutes and 1 hour 30 minutes. For example, the method comprises contacting the cells with the transduction enhancer for about 1 hour prior to contacting the cells with the enveloped virus.

[0081] In one example, the method comprises contacting the cells with the enveloped virus and a transduction enhancer simultaneously.

[0082] In one example, the method comprises contacting the cells with the enveloped virus for a period of between 15 and 30 hours. For example, the method comprises transducing the cells with the enveloped virus for a period of between 15 and 30 hours. In one example, the method comprises contacting the cells with the enveloped virus for a period of between 16 and 24 hours. For example, the method comprises transducing the cells with the enveloped virus for a period of between 16 and 24 hours. In one example, the method comprises contacting the cells with the enveloped virus for a period of about 16 hours, or about 17 hours, or about 18 hours, or about 19 hours, or about 20 hours, or about 21 hours, or about 22 hours, or about 23 hours, or about 24 hours.

[0083] In one example, the cell culture medium further comprises protamine sulphate.

[0084] In one example, the cell culture medium further comprises one or more cytokines selected from the group consisting of stem cell factor (SCF), thrombopoietin (TPO), flt3 / flk2 ligand (Flt3L) and combinations thereof. In one example, the cell culture medium further comprises SCF. In another example, the cell culture medium further comprises TPO. In a further example, the cell culture medium further comprises Flt3L. In another example, the cell culture medium further comprises SCF, TPO and Flt3L. In one example, the one or more cytokines is in the cell culture medium at a concentration of between about 10 ng / mL and 1 mg / ml. For example, the one or more cytokines is in the cell culture medium at a concentration of at least 10 ng / mL. In one example, the one or more cytokines is in the cell culture medium at a concentration of at least 20 ng / mL, or at least 50 ng / mL. For example, the one or more cytokines is in the cell culture medium at a concentration of between about 50 ng / mL and 500 ng / mL. In one example, the one or more cytokines is in the cell culture medium at a concentration of about 60 ng / mL, or about 70 ng / mL, or about 80 ng / mL, or about 90 ng / mL, or about 100 ng / mL. In one example, the one or more cytokines is in the cell culture medium at a concentration of about 120 ng / mL, or about 140 ng / mL, or about 160 ng / mL, or about 180 ng / mL, or about 200 ng / mL. In one example, the one or more cytokines is in the cell culture medium at a concentration of about 100 ng / mL, or about 200 ng / mL, or about, or about 300 ng / mL, or about, or about 400 ng / mL, or about 500 ng / mL, or about 600 ng / mL, or about 700 ng / mL, or about 800 ng / mL, or about 900 ng / mL, or about 1000 ng / mL.

[0085] In one example, the one or more cytokines is in the cell culture medium at a concentration of about 100 ng / pL. In one example, the cell culture medium further comprises about 100 ng / pL SCF. In another example, the cell culture medium further comprises about 100 ng / pL TPO. In a further example, the cell culture medium further comprises about 100 ng / pL Flt3L. In another example, the cell culture medium further comprises about 100 ng / pL of each of SCF, TPO and Flt3L.

[0086] In one example, the cell culture medium further comprises human serum albumin (HSA).

[0087] In one example, the HSA is in the cell culture medium at a concentration of between about 0.1 % to 10 %. In one example, the HSA is in the cell culture medium at a concentration of between about 1 % to 5 %. For example, the HSA is in the cell culture medium at a concentration of about 1%. In another example, the HSA is in the cell culture medium at a concentration of about 2%. In a further example, the HSA is in the cell culture medium at a concentration of about 3%. In one example, the HSA is in the cell culture medium at a concentration of about 4%. In another example, the HSA is in the cell culture medium at a concentration of about 5%.

[0088] In one example, the cells are pre- stimulated for a period of between 16 and 24 hours in cell culture medium before transduction with the enveloped virus. In one example, the cells are pre- stimulated for a period of about 16 hours, or about 17 hours, or about 18 hours, or about 19 hours, or about 20 hours, or about 21 hours, or about 22 hours, or about 23 hours, or about 24 hours. In one example, the cells are pre-stimulated in a cell culture medium comprising the one or more cytokines. For example, the cells are pre- stimulated in a cell culture medium comprising one or more cytokines at a concentration of between about 10 ng / mL and 1 mg / ml. In one example, the cells are pre- stimulated in a cell culture medium comprising the one or more cytokines at a concentration of about 100 ng / pL. In one example, the cells are pre- stimulated in a cell culture medium comprising about 100 ng / pL SCF. In another example, the cells are pre- stimulated in a cell culture medium comprising about 100 ng / pL TPO. In a further example, the cells are pre- stimulated in a cell culture medium comprising about 100 ng / pL Flt3L. In another example, the cells are pre-stimulated in a cell culture medium comprising about 100 ng / pL of each of SCF, TPO and Flt3L.

[0089] In one example, the plurality of cells are in the cell culture medium at a density of between about 1 x 105cells / mL and 1 x 1010cells / mL. For example, the plurality of cells are in the cell culture medium at a density of between about 1 x 105cells / mL and 1 x 107cells / mL. In one example, the plurality of cells are in the cell culture medium at a density of between about 1.0 x 106cells / mL and 4.0 x 106cells / mL. For example, the plurality of cells are in the cell culture medium at a density of about 2 x 106cells / mL.

[0090] In one example, enveloped virus is added to the cell culture at a multiplicity of infection (MOI) of at least 0.03. For example, enveloped virus is added to the cell culture at an MOI of at least 0.1. In another example, enveloped virus is added to the cell culture at an MOI of at least 1. In another example, enveloped virus is added to the cell culture at an MOI of about 0.03 to about 10.

[0091] In one example, virus is added to the cell culture at a ratio of viral particles per volume of culture media. For example, virus is added to the cell culture at greater than about 1 x 103transduction units per mL (TU / mL). For example, virus is added to the cell culture at about 1 x 103TU / mL to about 1 x 107TU / mL. For example, virus is added to the cell culture at about 1 x 103TU / mL, about 1 x 104TU / mL, about 1 x 105TU / mL, about 1 x 106TU / mL, or about 1 x 107TU / mL. In one example, virus is added to the cell culture at about 1 x 105TU / mL to about 1 x 106TU / mL. For example, virus is added to the cell culture at about 1 x 105TU / mL, or about 2 x 105TU / mL, about 3 x 105TU / mL, about 4 x 105TU / mL, about 5 x 105TU / mL, about 6 x 105TU / mL, about 7 x 105TU / mL, about 8 x 105TU / mL, about 9 x 105TU / mL, or about 1 x 106TU / mL.

[0092] In one example, the cell culture is operated for a period of at least 24 hours. For example, about 24 hours, or about 26 hours, or about 28 hours, or about 30 hours, or about 32 hours, or about 34 hours, or about 36 hours, or about 40 hours, or about 44 hours, or about 48 hours, or about 56 hours, or about 64 hours, or about 72 hours, or about 80 hours.

[0093] In one example, the method further comprises harvesting one or more cells transduced with the enveloped virus.

[0094] In one example, the method further comprises washing the harvested transduced cells with a formulation medium. For example, the formulation medium comprises 0.9% saline and / or human serum albumin.

[0095] In one example, the method further comprises expanding the one or more transduced cells in a myeloid expansion medium to produce a population of transduced myeloid lineage cells.

[0096] In one example, the method comprises expanding the population of transduced myeloid lineage cells in a myeloid expansion medium to produce a population of expanded transduced myeloid lineage cells.

[0097] In one example, the one or more transduced cells is initially seeded in the expansion medium at a density of between about 1 x 105cells / mL and 1 x 107cells / mL. For example, the one or more transduced cells is initially seeded in the expansion medium at a density of between about 1 x 105cells / mL and 1 x 106cells / mL. In one example, the one or more transduced cells is initially seeded in the expansion medium at a density of about 0.5 x 106cells / mL. For example, the one or more transduced cells is initially seeded in the expansion medium at a density of between about 1 x 105cells / mL and 1 x 107cells / mL, or about 0.1 x 106cells / mL and 1 x 107cells / mL, or about 0.5 x 106cells / mL and 1 x 107cells / mL, or about 0.5 x 106cells / mL and 5 x 106cells / mL, or about 0.5 x 106cells / mL and 2.5 x 106cells / mL of expansion medium. In one example, the one or more transduced cells is initially seeded in the expansion medium at a density of 1 x 105cells / mL and 1 x 107cells / mL. In one example, the one or more transduced cells is initially seeded in the expansion medium at a density of 0.5 x 106cells / mL to 5.0 x 106cells / mL. In one example, the one or more transduced cells is initially seeded in the expansion medium at a density of between 0.8 x 106cells / mL and 1.2 x 106. In one example, the one or more transduced cells is initially seeded in the expansion medium at a density of between 1 x 106cells / mL and 2.5 x 106. In one example, the one or more transduced cells is initially seeded in the expansion medium at a density of between 1.5 x 106cells / mL and 2 x 106. In one example, the one or more transduced cells is initially seeded in the expansion medium at a density of about 1 x 105cells / mL, or about 2 x 105cells / mL, or about 3 x 105cells / mL, or about 4 x 105cells / mL, or about 5 x 105cells / mL, or about 6 x 105cells / mL, or about 7 x 105cells / mL, or about 8 x 105cells / mL, or about 9 x 105cells / mL, or about 10 x 105cells / mL of expansion medium. In one example, the one or more transduced cells is initially seeded in the expansion medium at a density of about 1 x 106cells / mL, or about 2 x 106cells / mL, or about 3 x 106cells / mL, or about 4 x 106cells / mL, or about 5 x 106cells / mL, or about 6 x 106cells / mL, or about 7 x 106cells / mL, or about 8 x 106cells / mL, or about 9 x 106cells / mL, or about 10 x 106cells / mL of expansion medium. In one example, the one or more transduced cells is initially seeded in the expansion medium at a density of about 0.5 x 106cells / mL. In one example, the one or more transduced cells is initially seeded in the expansion medium at a density of about 1 x 106cells / mL. In one example, the one or more transduced cells is initially seeded in the expansion medium at a density of about 1.5 x 106cells / mL. In one example, the one or more transduced cells is initially seeded in the expansion medium at a density of about 1.8 x 106cells / mL. In one example, the one or more transduced cells is initially seeded in the expansion medium at a density of about 2 x 106cells / mL. In one example, the one or more transduced cells is initially seeded in the expansion medium at a density of 2.5 x 106cells / mL. In one example, the one or more transduced cells is initially seeded in the expansion medium at a density of 3.0 x 106cells / mL. In one example, the one or more transduced cells is initially seeded in the expansion medium at a density of 3.5 x 106cells / mL. In one example, the one or more transduced cells is initially seeded in the expansion medium at a density of 4.0 x 106cells / mL. In one example, the one or more transduced cells is initially seeded in the expansion medium at a density of 4.5 x 106cells / mL. In one example, the one or more transduced cells is initially seeded in the expansion medium at a density of 5.0 x 106cells / mL. In one example, the one or more transduced cells is initially seeded in the expansion medium at a density of about 1 x 107cells / mL, or about 2 x 107cells / mL, or about 3 x 107cells / mL, or about 4 x 107cells / mL, or about 5 x 107cells / mL, or about 6 x 107cells / mL, or about 7 x 107cells / mL, or about 8 x 107cells / mL, or about 9 x 107cells / mL, or about 10 x 107cells / mL of expansion medium. In one example, the one or more transduced cells is initially seeded in the expansion medium at a density of about 1 x 108cells / mL, or about 2 x 108cells / mL, or about 3 x 108cells / mL, or about 4 x 108cells / mL, or about 5 x 108cells / mL, or about 6 x 108cells / mL, or about 7 x 108cells / mL, or about 8 x 108cells / mL, or about 9 x 108cells / mL, or about 10 x 108cells / mL of expansion medium. In one example, the one or more transduced cells is initially seeded in the expansion medium at a density of about 1 x 109cells / mL, or about 2 x 109cells / mL, or about 3 x 109cells / mL, or about 4 x 109cells / mL, or about 5 x 109cells / mL, or about 6 x 109cells / mL, or about 7 x 109cells / mL, or about 8 x 109cells / mL, or about 9 x 109cells / mL, or about 10 x 109cells / mL of expansion medium. In one example, the method results in a transduction efficiency of at least 20%. For example, the method results in a transduction efficiency of at least 30%. In another example, the method results in a transduction efficiency of at least 40%. In another example, the method results in a transduction efficiency of at least 50%. In another example, the method results in a transduction efficiency of at least 60%. In another example, the method results in a transduction efficiency of at least 70%. In a further example, the method results in a transduction efficiency of at least 80%. For example, the method results in a transduction efficiency of at least 90%.

[0098] In one example, the method results in at least a 1.5-fold increase in potency of the enveloped virus. For example, about 1.5-fold, or about 1.6-fold, or about 1.7-fold, or about 1.8-fold, or about 1.96-fold. In another example, the method results in at least a 2- fold increase in potency of the enveloped virus. For example, about 2.1-fold, or about 2.2-fold, or about 2.3-fold, or about 2.4-fold, or about 2.5-fold. In a further example, the method results in at least a 2.5-fold increase in potency of the enveloped virus. For example, about a 2.5-fold increase.

[0099] In one example, the method further comprises seeding a population of between 100 and 500 transduced cells in a differentiation medium for a period of at least 14 days to produce one or more colony forming units (CFUs) of differentiated transduced cells. For example, the method comprises seeding a population of between 100 and 250 transduced cells. In one example, the method comprises seeding a population of about 125 transduced cells. In another example, the method comprises seeding a population of about 250 transduced cells.

[0100] In one example, the cells differentiate into myeloid or erythroid lineage. For example, the cells differentiate into myeloid lineage. In another example, the cells differentiate into erythroid lineage.

[0101] In one example, the method further comprises isolating one or more CFUs.

[0102] In one example, the method increases the vector copy number (VCN) in an isolated CFU of myeloid or erythroid transduced cells as determined by ddPCR, compared to a cell transduced in the absence of the transduction enhancer of the present disclosure.

[0103] In one example, the method increases the bulk VCN in myeloid cells that have been cultured in myeloid expansion medium.

[0104] In one example, the method increases the VCN by at least 10% compared to a cell transduced in the absence of the transduction enhancer of the present disclosure. For example, the method increases the VCN by at least 20%, compared to a cell transduced in the absence of the transduction enhancer of the present disclosure. In one example, the method increases the transduction efficiency of a cell that differentiates into a myeloid cell, compared to a cell transduced in the absence of the transduction enhancer of the present disclosure.

[0105] In one example, the transduction of cells that differentiate into an erythroid cell is not reduced.

[0106] In one example, the enveloped virus comprises a transgene introduced into its genome.

[0107] In one example, the method results in an increase in expression of the transgene by the cell by at least about 10% or more than a method in the absence of the transduction enhancer.

[0108] In one example, the transduced cell has a viability of at least 75% for at least 24 hours, or for at least 48 hours, or for at least 72 hours after transduction with the enveloped virus.

[0109] In one example, the cell culture has a volume of between about 100 uL and 500 mL. In one example, the cell culture has a volume of between about 125 uL and 2 mL. For example, the cell culture has a volume of greater than about 125 uL, or about 250uL, or about 500uL, or about ImL, or about 2 mL. In one example, the cell culture has a volume of between about 2 mL and 30 mL. For example, the cell culture has a volume of greater than about 2 mL, or about 3 mL, or about 5 mL, or about 8 mL, or about 10 mL, or about 15 mL, or about 20 mL, or about 25 mL, or about 30 mL. In one example, the cell culture has a volume of between about 30 mL and 500 mL. In one example, the cell culture has a volume of between about 30 mL and 300 mL. For example, the cell culture has a volume of greater than about 30 mL, or about 50 mL, or about 80 mL, or about 100 mL, or about 250 mL, or about 300 mL. For example, the cell culture has a volume of about 30 mL. In another example, the cell culture has a volume of about 50 mL. In one example, the cell culture has a volume of about 80 mL. In a further example, the cell culture has a volume of about 100 mL. In one example, the cell culture has a volume of about 150 mL. In another example, the cell culture has a volume of about 200 mL. In a further example, the cell culture has a volume of about 250 mL. In another example, the cell culture has a volume of about 300 mL. In one example, the cell culture has a volume of about 400 mL. In another example, the cell culture has a volume of about 500 mL.

[0110] In one example, the cell culture is at a pH of between 6.0 and 8.0 and / or at a temperature of between 35-39°C and / or < 5% dissolved carbon dioxide (CO2)

[0111] In one example, the cell culture is operated at a pH of between 6.0 and 8.0. In one example, the cell culture is at a pH of between about 6.5 and 7.5. For example, the pH is between about 6.90 and about 7.3. In one example, the pH is about 7.1. In one example, the pH is about 6.5. In one example, the pH is about 6.6. In one example, the pH is about 6.7. In one example, the pH is about 6.8. In one example, the pH is about 6.9. In one example, the pH is about 7.0. In one example, the pH is about 7.1. In one example, the pH is about 7.2. In one example, the pH is about 7.3. In one example, the pH is about 7.4. In one example, the pH is about 7.5.

[0112] In one example, the cell culture is operated with a CO2 level of between <5%. For example, the cell culture is operated with about 1% CO2. In another example, the cell culture is operated with about 2% CO2. In a further example, the cell culture is operated with about 3% CO2. In a further example, the cell culture is operated with about 4% CO2. In a further example, the cell culture is operated with about 5% CO2.

[0113] In one example, the suspension cell culture is operated at a temperature of between about 35 °C and 39 °C. For example, the suspension cell culture is at a temperature of about 35 °C, or about 35.5 °C, or about 36 °C, or about 36.5 °C, or about 37 °C, or about 37.5 °C, or about 38 °C, or about 38.5 °C, or about 39 °C. In one example, the suspension cell culture is at a temperature of between about 36.5 °C and about 37.5 °C. For example, the suspension cell culture is at a temperature of about 37.0 °C. In one example, the suspension cell culture is at a temperature of between about 38 °C and about 39 °C. For example, the suspension cell culture is at a temperature of about 38.5 °C.

[0114] In one example, the suspension cell culture is at a pH of between 6.0 and 8.0 and / or at a temperature of between 35-39 °C. In one example, the suspension cell culture is at a pH of between 6.0 and 8.0 and / or at a temperature of between 37-38.5 °C. For example, the suspension cell culture is at a pH of between about 6.8 and about 7.1 and / or at a temperature of between about 37 °C and about 38.5 °C. In one example, the suspension cell culture is at a pH of between about 6.8 and about 7.1 and at a temperature of between about 37 °C and about 38.5 °C. In one example, the suspension cell culture is at a pH of about 6.9 to about 7.0 and at a temperature of about 37.0 °C.

[0115] In one example, the method further comprises formulating the population of transduced cells with a pharmaceutically acceptable excipient. For example, the method further comprises formulating the population of harvested transduced cells with a cryopreservation medium. In one example, the cryopreservation medium is an animal protein-free, serum free cryopreservation medium. In one example, the cryopreservation medium comprises DMSO. For example, the cryopreservation medium comprises 1% to 10% DMSO. In one example, the cryopreservation medium comprises about 5% DMSO. In another example, the cryopreservation medium comprises about 10 % DMSO. In one example, the cell is a hematopoietic stem cell (HSC) or a hematopoietic progenitor cell (HPC). For example, the cell is a HSC. In another example, the cell is a HPC. For example, the HSC or HPC is a CD34 positive (CD34+) cell.

[0116] In one example, the HSC or HPC are of an erythroid lineage (burst-forming unit- erythroid; BFU-E) and / or a myeloid lineage (colony forming unit that generates granulocyte, erythrocyte, monocyte, megakaryocyte cells; CGU-GEMM). In one example, the HSC or HPC are of an erythroid lineage. In another example, the HSC or HPC are of a myeloid lineage.

[0117] In one example, the enveloped virus is a retrovirus. For example, the retrovirus is a lentivirus. For example, the lentivirus is HIV or a derivative thereof.

[0118] The present disclosure provides a genetically modified cell expressing a transgene, wherein the genetically modified cell is transduced with an enveloped virus comprising the transgene by a method of the present disclosure.

[0119] The present disclosure also provides a method of producing a genetically modified cell for gene therapy, the method comprising transducing a cell with an enveloped virus comprising a polynucleotide expressing a transgene by a method of the present disclosure, thereby producing a genetically modified cell.

[0120] The present disclosure further provides a composition comprising a population of genetically modified cells of the disclosure for use as a medicament.

[0121] BRIEF DESCRIPTION OF THE DRAWINGS

[0122] Figure 1 is a schematic representation showing the effect of lentiviral transduction in the presence of poloxamers 388 (F108) and 407 (F127) on transgene expression and vector copy number. GFP expression 72 hours following transduction with a GFP expressing lentiviral vector at MOI 0.5 in the presence of F108 or F127.

[0123] Figure 2 is a schematic representation showing effect of lentiviral transduction in the presence of nebivolol on transgene expression. (A) GFP expression 48 hours post transduction in the presence of nebivolol alone or in combination with F127. Expression data from (B) HSC brew media and (C) stem cell growth medium (SCGM) showed the effect was independent of media type.

[0124] Figure 3 is a schematic representation showing the effect of lentiviral transduction in the presence of nebivolol, and L- and D- enantiomers on (A-B) cell viability and (C-D) growth.

[0125] Figure 4 is a schematic representation showing (A) the e)ffect of lentiviral transduction in the presence of lOpM nebivolol with or without F127 on transgene expression 72 hours post-transduction. Expression data from (B) HSC brew media and (C) stem cell growth media showed the effect was independent of media type. Statistics performed: Ordinary one- ANOVA with Tukey’s multiple comparisons, ns: not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0126] Figure 5 is a schematic representation showing the effect of Multiplicity of infection (MOI) curves in cells transduced in the presence of 10 pM Nebivolol and / or F127.

[0127] Figure 6 is a schematic representation showing effect of transduction in the presence of 10 pM nebivolol, L-nebivolol, D-nebivolol, or racemic-nebivolol with or without Fl 27 at a multiplicity of infection of 1.

[0128] Figure 7 is a schematic representation showing the effect of timing of administration of nebivolol and F127 during transduction on transgene expression. Statistics performed: Repeated measures one-way ANOVA with Sidak’s Multiple Comparisons, ns: not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, 0.0001.

[0129] Figure 8 is a schematic representation showing the effect of transduction in the presence of 10 pM nebivolol and Fl 27 or Fl 08 at a multiplicity of infection of 1.

[0130] Figure 9 is a schematic representation showing effect of transduction in the presence of 10 pM Nebivolol or L-Nebivolol on (A) vector copy number and (B) transduction efficiency in erythroid and myeloid colony forming units. Statistics performed: Repeated measures two-way ANOVA with Tukey’s Multiple Comparisons, ns: not significant; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0131] Figure 10 is a schematic representation showing effect of transduction enhancers at large scale in the presence of Fl 27 with and without 10 pM nebivolol at a multiplicity of infection of 3.

[0132] DETAILED DESCRIPTION

[0133] General

[0134] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter. Thus, as used herein, the singular forms “a”, “an” and “the” include plural aspects unless the context clearly dictates otherwise. For example, reference to “a” includes a single as well as two or more; reference to “an” includes a single as well as two or more; reference to “the” includes a single as well as two or more and so forth. Those skilled in the art will appreciate that the present disclosure is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

[0135] The present disclosure is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the present disclosure.

[0136] Any example of the present disclosure herein shall be taken to apply mutatis mutandis to any other example of the disclosure unless specifically stated otherwise. Stated another way, any specific example of the present disclosure may be combined with any other specific example of the disclosure (except where mutually exclusive).

[0137] Any example of the present disclosure disclosing a specific feature or group of features or method or method steps will be taken to provide explicit support for disclaiming the specific feature or group of features or method or method steps.

[0138] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (for example, molecular biology, microbiology, virology).

[0139] Unless otherwise indicated, the conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, peptide synthesis in solution, solid phase peptide synthesis, and immunology utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al. Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present). The term “and / or”, e.g., “X and / or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

[0140] The term “about”, unless stated to the contrary, refers to + / - 20%, more for example + / - 10%, of the designated value. For the avoidance of doubt, the term “about” followed by a designated value is to be interpreted as also encompassing the exact designated value itself (for example, “about 10” also encompasses 10 exactly).

[0141] As used herein the term “from” in the shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source (i.e., includes recombinantly obtained).

[0142] Throughout this specification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0143] All publications cited herein are hereby incorporated by reference in their entirety. Where reference is made to a URL or other such identifier or address, it is understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet. Reference thereto evidences the availability and public dissemination of such information.

[0144] Any discussion of documents, acts, materials, devices, articles or the like that has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

[0145] Selected Definitions

[0146] As used herein, the term “enveloped virus” refers to DNA and RNA viruses that have a viral envelope. Envelopes are typically derived from host cell membranes (e.g., phospholipids and proteins), but may include viral glycoproteins on the surface of the envelope. Enveloped viruses also comprise a “capsid”, which is a protein layer between the envelope and viral genome. In one example, the enveloped virus is a retrovirus. For example, the enveloped virus is a lentivirus, e.g., human immunodeficiency virus.

[0147] As used herein, the term “cell culture medium” will be understood to encompass the fluid or medium in which cells are grown for the purpose of transducing with an enveloped virus. The fluid or medium does not comprise the cells (e.g., the cells may have been removed, e.g., by centrifugation and / or removal of supernatant).

[0148] As used herein, the term “cell culture” will be understood to refer to the collective of the cell culture fluid or medium and the cultured cells.

[0149] As used herein, the term “transduction” or “transducing” or “transduce” or “transduced” will be understood to mean the process by which a virus (e.g., lentivirus) stably introduces genetic material (e.g., a transgene) into a target cell’s genome (e.g., haematopoietic stem cell).

[0150] The compounds of the present disclosure may contain chiral (asymmetric) centers or the molecule as a whole may be chiral. The individual stereoisomers (enantiomers and diastereoisomers) and mixtures of these are within the scope of the present disclosure.

[0151] The term “halo” or “halogen” whether employed alone or in compound words such as haloalkyl, represents fluorine, chlorine, bromine or iodine. Further, when used in compound words such as haloalkyl, the alkyl may be partially halogenated or fully substituted with halogen atoms which may be independently the same or different. Examples of haloalkyl groups include fluoromethyl, chloromethyl, bromomethyl, iodomethyl, fluoropropyl, fluorobutyl, difluoromethyl difluoroethyl, trifluoromethyl and trifluoroethyl groups. Further examples of haloalkyl groups include -CF3, -CCI3, and - CH2CF3, -CF2CF3 and -CH2CHFCI.

[0152] As used herein, the term “alkyl” whether used alone, or in compound words such as haloalkyl, cycloalkyl, alkylcycloalkyl, alkylcarbocyclyl, heteroalkyl, alkylheterocyclyl, alkylheteroaryl, alkylamide, alkylphosphonate and alkylaryl, represents straight chain (i.e. linear) or branched chain hydrocarbon groups. Examples of alkyl groups include methyl, ethyl, n-propyl, iso-propyl, n-butyl, t-butyl, i-butyl, secbutyl, pentyl, hexyl, heptyl, octyl, nonyl and decyl groups. In one example, the alkyl group is of 1 to 20 carbon atoms (i.e. Ci-2oalkyl). In another examples, the alkyl is a group of 1 to 10 carbon atoms (i.e. Ci-ioalkyl). In another example, the alkyl group is of 1 to 6 carbon atoms (i.e. Ci-ealkyl).

[0153] As used herein, the term “heteroalkyl” represents straight chain (i.e. linear) or branched chain hydrocarbon groups which are analogous to an alkyl group, but in which one or more carbon atoms is / are replaced by one or more heteroatoms selected from nitrogen, sulfur, and oxygen.

[0154] As used herein, the term “alkenyl” represents straight (i.e. linear) or branched chain unsaturated hydrocarbon groups containing at least one carbon-carbon double bond. Examples of alkenyl groups include ethylene, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl and decenyl groups. In one example, the alkenyl group is of 2 to 20 carbon atoms (i.e. C2-2oalkenyl). In another example, the alkenyl is a group 2 to 10 carbon atoms (i.e. C2-ioalkenyl). In another example, the alkenyl group is of 2 to 6 carbon atoms (i.e. C2-6alkenyl).

[0155] As used herein, the term “alkynyl” represents straight (i.e. linear) or branched chain unsaturated hydrocarbon groups containing at least one carbon-carbon triple bond. Examples of alkenyl groups include , ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl and decynyl groups. In one example, the alkynyl group is of 2 to 20 carbon atoms (i.e. C2-2oalkynyl). In one example, the alkynyl group is of 2 to 10 carbon atoms (i.e. C2-ioalkynyl). In another examples, the alkynyl group is of 2 to 6 carbon atoms (i.e. C2-6alkynyl).

[0156] As used herein, the term “haloalkyl” represents to an alkyl group having at least one halogen substituent, where “alkyl” and “halogen” are as described above. For example, the haloalkyl group may have at least one, two or three halogen substituents. Examples of haloalkyl groups include fluoromethyl, chloromethyl, bromomethyl, iodomethyl, fluoropropyl, fluorobutyl, difluoromethyl difluoroethyl, trifluoromethyl and trifluoroethyl groups. Further examples of haloalkyl groups include -CF3, -CCI3, and - CH2CF3, -CF2CF3 and -CH2CHFCI. In one example, the haloalkyl group is of 1 to 20 carbon atoms (i.e. Ci-2ohaloalkyl). In one example, the haloalkyl group is of 1 to 10 carbon atoms (i.e. Ci-iohaloalkyl). In another example, the haloalkyl group is of 1 to 6 carbon atoms (i.e. Ci-ehaloalkyl).

[0157] As used herein, the terms “carbocyclyl” and “carbocycle” whether used alone, or in compound words such as alkylcarbocyclyl, represents a monocyclic or polycyclic ring system wherein the ring atoms are all carbon atoms, e.g., of about 3 to about 20 carbon atoms, and which may be aromatic, non-aromatic, saturated, or unsaturated, and may be substituted and / or contain fused rings. In one example, the carbocyclyl group is of 3 to 20 carbon atoms (i.e. C3-20-membered carbocyclyl). In another example, the carbocyclyl group is of 3 to 10 carbon atoms (i.e. Cs io-membered carbocyclyl). Examples of such groups include aryl groups such as phenyl, naphthyl, anthracenyl or fluorenyl, saturated groups such as cycloalkyl and cycloalkenyl groups e.g. cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl groups, or fully or partially hydrogenated phenyl, naphthyl and fluorenyl. It will be appreciated that the polycyclic ring system includes bicyclic and tricyclic ring systems.

[0158] As used herein, the term “cycloalkyl” whether used alone, or in compound words such as alkylcycloalkyl, refers to a monocyclic or polycyclic carbocyclic ring system of varying sizes, e.g., from about 3 to about 20 carbon atoms, e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl. It will be appreciated that the polycyclic ring system includes bicyclic and tricyclic ring systems.

[0159] As used herein, the term “heterocyclyl” whether used alone or in compound words such as alkylheterocyclyl, refers to a monocyclic or polycyclic ring system wherein the ring atoms are provided by at least two different elements, typically a combination of carbon and one or more of nitrogen, sulfur, and oxygen, and wherein the ring system may be aromatic such as a “heteroaryl” group, non-aromatic, saturated, or unsaturated, and may be substituted and / or contain fused rings. Heterocyclyl groups containing a suitable nitrogen atom include the corresponding N-oxides. In one example, the heterocyclyl group is of 3 to 20 atoms (i.e. 3-20-membered heterocyclyl). In another example, the heterocyclyl group is of 3 to 10 atoms (i.e. 3-10-membered heterocyclyl). The heteroatom may preferably be N, O or S. Examples of monocyclic non-aromatic heterocyclyl groups include aziridinyl, azetidinyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, tetrahydrofuranyl, tetrahydropyranyl, morpholinyl, thiomorpholinyl and azepanyl. Examples of bicyclic heterocyclyl groups in which one of the rings is non-aromatic include dihydrobenzofuranyl, indanyl, indolinyl, isoindolinyl, tetrahydroisoquinolinyl, tetrahydroquinolyl, and benzoazepanyl. Examples of monocyclic aromatic heterocyclyl groups (also referred to as monocyclic heteroaryl groups) include furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, pyridyl (e.g. the radical derived from pyridine), triazolyl, triazinyl, pyridazyl, isothiazolyl, isoxazolyl, pyrazinyl, pyrazolyl, and pyrimidinyl. Examples of bicyclic aromatic heterocyclyl groups (also referred to as bicyclic heteroaryl groups) include quinoxalinyl, quinazolinul, pyridopyrazinyl, benzoxazolyl, benzothiophenyl, benzimidazolyl, naphthyridinyl, quinolinyl, benzofuranyl, indolyl, benzothiazolyl, oxazolyl[4,5-b]pyridyl, pyridopyrimidinyl, isoquinolinyl, and benzohydroxazole. It will be appreciated that the polycyclic ring system includes bicyclic and tricyclic ring systems.

[0160] As will be understood, an “aromatic” group means a cyclic group having 4m+2 % electrons, where m is an integer equal to or greater than 1. As used herein, “aromatic” is used interchangeably with “aryl” to refer to an aromatic group, regardless of the valency of aromatic group.

[0161] As used herein, the term “aryl” whether used alone, or in compound words such as alkylaryl, represents a monocyclic (e.g. phenyl) or polycyclic (e.g. naphthyl) aromatic carbocyclic ring system. In one example, the aryl group is of 3 to 20 carbon atoms (i.e., an aromatic 3-20 membered carbocyclyl). In another example, the aryl group is of 3 to 10 carbon atoms (i.e., an aromatic 3-10 membered carbocyclyl). Examples of aryl groups include, but are not limited to, phenyl, naphthyl, anthracenyl or fluorenyl. It will be appreciated that the polycyclic ring system includes bicyclic and tricyclic ring systems. Related to the term aryl, the term “aralkyl” as used herein refers to an alkyl group wherein a hydrogen atom is replaced by an aryl group as a substituent. Examples of alkylaryl groups include, but are not limited to, an optionally substituted benzyl (e.g. -CH2- phenyl).

[0162] As used herein, the term “heteroaryl” whether used alone, or in compound words such as alkylheteroaryl, represents a monocyclic or polycyclic aromatic ring system wherein the ring atoms are provided by at least two different elements, typically a combination of carbon and one or more of nitrogen, sulfur, and oxygen, and may be substituted and / or contain fused rings. Heteroaryl groups containing a suitable nitrogen atom include the corresponding N-oxides. In one example, the heteroaryl group is of 3 to 20 atoms (i.e. 3-20-membered heteroaryl). In another example, the heteroaryl group is of 3 to 10 atoms (i.e. 3-10-membered heteroaryl). Examples of monocyclic heteroaryl groups include furanyl, thienyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, pyridyl, triazolyl, triazinyl, pyridazyl, isothiazolyl, isoxazolyl, pyrazinyl, pyrazolyl, and pyrimidinyl. Examples of bicyclic heteroaryl groups include quinoxalinyl, quinazolinul, pyridopyrazinyl, benzoxazolyl, benzothiophenyl, benzimidazolyl, naphthyridinyl, quinolinyl, benzofuranyl, indolyl, benzothiazolyl, oxazolyl[4,5- b]pyridyl, pyridopyrimidinyl, isoquinolinyl, and benzohydroxazole. All regioisomers are contemplated, e.g. 2-pyridyl, 3-pyridyl and 4-pyridyl. It will be appreciated that the polycyclic ring system includes bicyclic and tricyclic ring systems.

[0163] As used herein, the term “saturated” refers to a group where all available valence bonds of the backbone atoms are attached to other atoms Representative examples of saturated groups include, but are not limited to, butyl, cyclohexyl, piperidine, and the like.

[0164] As used herein, the term “unsaturated” refers to a group where at least one valence bond of two adjacent backbone atoms is not attached to other atoms. Representative examples include, but are not limited to, alkenes (e.g., -CH2-CH2CH=CH), phenyl, pyrrole, and the like.

[0165] As used herein, the term “optionally substituted” means that a functional group is either substituted or unsubstituted, at any available position.

[0166] As used herein, the term “substituted” refers to a group having one or more hydrogens or other atoms removed from a carbon or suitable heteroatom and replaced with a further group (i.e., substituent). As used herein, the term “unsubstituted” refers to a group that does not have any further groups attached thereto or substituted therefore.

[0167] The present disclosure relates to compounds of Formula (1) and pharmaceutically acceptable salts thereof. Salts may be formed in the case of embodiments of the compound of Formula (1), which contain a suitable acidic or basic group. Suitable salts of the compound of Formula (1) include those formed with organic or inorganic acids or bases.

[0168] As used herein, the phrase “pharmaceutically acceptable salt” refers to pharmaceutically acceptable organic or inorganic salts. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmacol Sci, 1977, 66: 1-19, the contents of which is incorporated herein by reference. Exemplary acid addition salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., 1,1'- methylene-bis-(2-hydroxy-3-naphthoate)) salts. Exemplary base addition salts include, but are not limited to, ammonium salts, alkali metal salts, for example those of potassium and sodium, alkaline earth metal salts, for example those of calcium and magnesium, and salts with organic bases, for example dicyclohexylamine, N-methyl-D-glucomine, morpholine, thiomorpholine, piperidine, pyrrolidine, a mono-, di- or tri-lower alkylamine, for example ethyl-, tert-butyl-, diethyl-, diisopropyl-, triethyl-, tributyl- or dimethyl -propylamine, or a mono-, di- or trihydroxy lower alkylamine, for example mono-, di- or triethanolamine. A pharmaceutically acceptable salt may involve the inclusion of another molecule such as an acetate ion, a succinate ion or other counterion. The counterion may be any organic or inorganic moiety that stabilizes the charge on the parent compound. Furthermore, a pharmaceutically acceptable salt may have more than one charged atom in its structure. Instances where multiple charged atoms are part of the pharmaceutically acceptable salt can have multiple counter ions. Hence, a pharmaceutically acceptable salt can have one or more charged atoms and / or one or more counterion. It will also be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the present disclosure since these may be useful as intermediates in the preparation of pharmaceutically acceptable salts or may be useful during storage or transport. In one example, the compound of Formula (1) is an acetate salt. Those skilled in the art of organic and / or medicinal chemistry will appreciate that many organic compounds can form complexes with solvents in which they are reacted or from which they are precipitated or crystallized. These complexes are known as "solvates". For example, a complex with water is known as a "hydrate". As used herein, the phrase “pharmaceutically acceptable solvate” or “solvate” refer to an association of one or more solvent molecules and a compound of the present disclosure. Examples of solvents that form pharmaceutically acceptable solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. It will be understood that the present disclosure encompasses solvated forms, including hydrates, of the compounds of Formula (1) and salts thereof.

[0169] Those skilled in the art of organic and / or medicinal chemistry will appreciate that the compounds of Formula (1) and salts thereof may be present in amorphous form, or in a crystalline form. It will be understood that the present disclosure encompasses all forms and polymorphs of the compounds of Formula (1) and salts thereof.

[0170] As used herein, the term “stereoisomer” refers to compounds having the same molecular Formula and sequence of bonded atoms (i.e., atom connectivity), though differ in the three-dimensional orientations of their atoms in space. As used herein, the term “enantiomers” refers to two compounds that are stereoisomers in that they are non- superimposable mirror images of one another. Relevant stereocenters may be denoted with (R)- or (S)- configuration. The stereochemical definitions and conventions used herein are generally consistent with Parker et al., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York and Eliel et al., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994. Many of the organic compounds exist in optically active forms, that is, they have the ability to rotate the plane of polarized light. In the description of an optically active compound, the prefixes D and E or R and S are used to indicate the absolute configuration of the molecule around its chiral centre or centres.

[0171] As used herein, the term "diastereomer" refers to a stereoisomer with two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, for example, melting points, boiling points, spectral properties, or biological activities. The diastereomer mixture can be separated by high resolution analytical procedures, such as electrophoresis and chromatography, such as HPLC.

[0172] As used herein, the term "racemate" or “racemic mixture” refers to a mixture substantially comprising two isomers (for example, stereoisomers) of a compound that is not "enantiomerically pure", including mixtures such as, without limitation, in a ratio of about 1: 1, about 6:4, about 7:3, about 8:2, about 8.2: 1.5 or about 9: 1. In some embodiments, “racemate" or "racemic mixture" refers to a substantially equimolar mixture or an equimolar mixture of enantiomers (viz. mixtures in a substantially 1: 1 ratio).

[0173] Mixtures of stereoisomers, including racemates, resulting can be resolved by methods known to those skilled in the art, for example, by separation of the diastereomeric salts thereof. Racemic products can also be resolved by chiral chromatography, for example, high performance liquid chromatography (HPLC) using a chiral adsorbent. Preferred enantiomers can also be prepared by asymmetric syntheses. See, for example, Jacques, et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Principles of Asymmetric Synthesis (2nd ed. Robert E. Gawley, Jeffrey Aube, Elsevier, Oxford, UK, 2012); Eliel, EL Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); Wilen, SH Table of Resolving Agents and Optical Resolutions p. 268 (EL Eliel, Ed., Univ. Of Notre Dame Press, Notre Dame, IN 1972); Chiral Separation Techniques: A Practical Approach (Subramanian, G. Ed., Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim, Germany, 2007.

[0174] Transduction of Target Cells with Enveloped Viruses

[0175] The present disclosure provides methods of transducing a cell with an enveloped virus in a cell culture, methods of increasing an enveloped virus transduction efficiency of a cell culture and / or methods of increasing potency of an enveloped virus during transduction of a cell in cell culture, the method comprising culturing a plurality of cells in a cell culture medium and contacting the cells with the enveloped virus and a transduction enhancer, wherein the transduction enhancer comprises a compound of Formula (I) or a stereoisomer, a racemate, a pharmaceutically acceptable salt or a solvate thereof:

[0176] Formula (I) wherein

[0177] X1and X2are independently selected from O, N, S or CH2;

[0178] L1and L2are independently absent or selected from -Ci-ealkyl-, -Ci ealkcnyl-^ or -Ci ealkynyl-, each of which may be optionally substituted with one or more R4or R5,

[0179] R1is selected from H, -Ci-ealkyl-, -C2-6alkenyl-;>or -C2-6alkynyl-; R2and R3are independently selected from H, halogen, or OH; and

[0180] R4and R5are independently selected from halogen, OH, -Ci-4alkyl-, and -Ci- 4alkoxy-

[0181] Transduction Enhancers

[0182] As described herein, the cell is contacted with a transduction enhancer during a method of producing a genetically modified cell. For example, methods of producing a genetically modified cell of the disclosure comprise culturing the cell in the presence of a transduction enhancer, or contacting the cell with a transduction enhancer before, during, or after contacting the cell with a vector.

[0183] As used herein, the term “transduction enhancer” refers to a compound of Formula (I) or a stereoisomer, a racemate, a pharmaceutically acceptable salt or a solvate thereof that enhances introduction of the genetic material (e.g., a transgene) into the target cell’s genome (e.g., haematopoietic stem cell).

[0184] The present disclosure provides a method of transducing a cell, wherein the transduction enhancer comprises a compound of Formula (I) or a stereoisomer, a racemate, a pharmaceutically acceptable salt or a solvate thereof: wherein

[0185] X1and X2are independently selected from O, N, S or CH2;

[0186] E1and L2are independently absent or selected from -Ci-ealkyl-, -Ci ealkcnyl-^ or -Ci ealkynyl-, each of which may be optionally substituted with one or more R4or R5,

[0187] R1is selected from H, -Ci-ealkyl-, -C2-6alkenyl-;>or -C2-6alkynyl-;

[0188] R2and R3are independently selected from H, halogen, or OH; and

[0189] R4and R5are independently selected from halogen, OH, -Ci-4alkyl-, and -Ci- 4alkoxy-.

[0190] In some embodiments, L1is absent. In some embodiments, L1is selected from - Ci-6alkyl-, -C2-6alkenyl-;>or -C2-6alkynyl-, optionally substituted with one or more R4or R5. In some embodiments, L1is selected from -C 1 -ealkyl-, optionally substituted with one or more R4or R5. In some embodiments, L1is selected from -Ci-salkyl-, optionally substituted with one or more R4or R5. In some embodiments, L1is selected from -Ci- 4alkyl-, optionally substituted with one or more R4or R5. In some embodiments, L1is selected from -Ci-3alkyl-, optionally substituted with one or more R4or R5. In some embodiments, L1is selected from -Ci-ialkyl-, optionally substituted with one or more R4or R5. In some embodiments, L1is selected from -Cialkyl-, optionally substituted with one or more R4or R5.

[0191] In some embodiments, L2is absent. In some embodiments, L2is selected from - Ci-6alkyl-, -C2-6alkcnyl-5or -Ci ealkynyl-, optionally substituted with one or more R4or R5. In some embodiments, L2is selected from -Ci-ealkyl-, optionally substituted with one or more R4or R5. In some embodiments, L2is selected from -Ci-salkyl-, optionally substituted with one or more R4or R5. In some embodiments, L2is selected from -Ci- 4alkyl-, optionally substituted with one or more R4or R5. In some embodiments, L2is selected from -Ci-3alkyl-, optionally substituted with one or more R4or R5. In some embodiments, L2is selected from -Ci-2alkyl-, optionally substituted with one or more R4or R5. In some embodiments, L2is selected from -Cialkyl-, optionally substituted with one or more R4or R5.

[0192] In some embodiments, L1and L2are absent. In some embodiments, L1and L2are independently selected from -Ci -ealkyl-, -Ci ealkcnyl-^ or -Ci ealkynyl-, optionally substituted with one or more R4or R5. In some embodiments, L1and L2are independently selected from — C i -ealky 1—, optionally substituted with one or more R4or R5. In some embodiments, L1and L2are independently selected from -Ci-salkyl-, optionally substituted with one or more R4or R5. In some embodiments, L1and L2are independently selected from -Ci^alkyl-, optionally substituted with one or more R4or R5. In some embodiments, L1and L2are independently selected from -Ci-3alkyl-, optionally substituted with one or more R4or R5. In some embodiments, L1and L2are independently selected from -Ci-2alkyl-, optionally substituted with one or more R4or R5. In some embodiments, L1and L2are-Cialkyl-, optionally substituted with one or more R4or R5. In some embodiments, L1and L2are the same.

[0193] In some embodiments, L1has the structure (I-A):

[0194] *-L3CH(R4)- (I-A) wherein

[0195] L3is absent or selected from -Ci^alkyl-, -C2-4alkenyl-;>or -C2-4alkynyl-; and

[0196] * indicates a single bond to the central N.

[0197] In some embodiments, L2has the structure (I-B): *-L4CH(R5)- (I-B) wherein

[0198] L4is absent or selected from -Ci-4alkyl-, -Ci^alkenyl-, or -Ci-4alkynyl-; and

[0199] * indicates a single bond to the central N.

[0200] In some embodiments, L1has the structure (I-A) and, and L2has the structure (I B):

[0201] *-L3CH(R4)- (I-A)

[0202] *-L4CH(R5)- (I-B) wherein

[0203] L3and L4are independently absent or selected from -Ci-4alkyl-, -Ci ^alkenyl-, or -C2-4alkynyl-; and

[0204] * indicates a single bond to the central N.

[0205] In some embodiments, L3is -Ci^alkyl-. In some embodiments, L3is -Ci-3alkyl- . In some embodiments, L3is -Ci-2alkyl-. In some embodiments, L3is -Cialkyl-. In some embodiments, L4is -Cialkyl- In some embodiments, L4is -Ci-3alkyl-. In some embodiments, L4is -Ci-2alkyl-. In some embodiments, L4is -Cialkyl-. In some embodiments, L3and L4are the same. In some embodiments, L3and L4are -Ci ^alkylin some embodiments, L3and L4are both -Cialkyl-.

[0206] In some embodiments, X1and X2are independently selected from O, N or CH2. In some embodiments, X1and X2are independently selected from O or N. In some embodiments, X1and X2are the same. In some embodiments, X1and X2are the same, and are selected from O or N. In some embodiments, X1and X2are both O.

[0207] In some embodiments, R4is selected from halogen, OH, -Ci^alkyl-, and -Ci- 4alkoxy-. In some embodiments, R4is selected from halogen, OH, -Ci^alkyl-, and -Ci- 2alkoxy-. In some embodiments, R4is selected from halogen, OH, -Cialkyl-, and - Cialkoxy-. In some embodiments, R4is selected from halogen or OH. In some embodiments, R4is selected from F, Cl, Br or OH. In some embodiments, R4is OH.

[0208] In some embodiments, R5is selected from halogen, OH, -Ci^alkyl-, and - Ci-4alkoxy-. In some embodiments, R5is selected from halogen, OH, -Ci-2alkyl-, and - Ci-2alkoxy-. In some embodiments, R5is selected from halogen, OH, -Cialkyl-, and - Cialkoxy-. In some embodiments, R5is selected from halogen or OH. In some embodiments, R5is selected from F, Cl, Br or OH. In some embodiments, R5is OH. In some embodiments, R4and R5are independently selected from halogen, OH, -Ci-4alkyl-, and -Ci-4alkoxy-. In some embodiments, R4and R5are independently selected from halogen, OH, -Ci-ialkyl-, and -Ci-ialkoxy-. In some embodiments, R4and R5are independently selected from halogen, OH, -Ci alkyl-, and -Ci alkoxy-. In some embodiments, R4and R5are independently selected from halogen or OH. In some embodiments, R4and R5are independently selected from F, Cl, Br or OH. In some embodiments, R4and R5are the same. In some embodiments, R4and R5are both OH.

[0209] R1is selected from H or -Ci-ealkyl-. R1is selected from H, or -Ci-salkyl-. R1is selected from H or -Ci-4alkyl-. R1is selected from H or -Ci-3alkyl-. R1is selected from H or -Ci-2alkyl-. R1is selected from H or -Cialkyl-. In some embodiments, R1is H.

[0210] In some embodiments, R2is selected from H, halogen, or OH. In some embodiments, R2is selected from H, F, Cl, Br, or OH. In some embodiments, R2is selected from F, Cl, Br, or OH. In some embodiments, R2is selected from F, Cl or Br. In some embodiments, R2is F. In some embodiments, R3is selected from H, halogen, or OH. In some embodiments, R3is selected from H, F, Cl, Br, or OH. In some embodiments, R3is selected from F, Cl, Br, or OH. In some embodiments, R3is selected from F, Cl or Br. In some embodiments, R3is F. In some embodiments, R2and R3are independently selected from H, halogen, or OH. In some embodiments, R2and R3are independently selected from H, F, Cl, Br or OH. In some embodiments, R2and R3are independently selected from F, Cl, Br or OH. In some embodiments, R2and R3are independently selected from F, Cl or Br. In some embodiments, R2and R3are the same. In some embodiments, R2and R3are both F.

[0211] In some embodiments, the compound of Formula (I) has the following structure:

[0212] In some embodiments, the compound of Formula (I) is nebivolol. It will be understood by the person skilled in the art that nebivolol has four asymmetric carbon centres (chiral centre), which give rise to the possibility of 16 theoretical isomers — these being RRRR, RRRS, RRSS, RSSS, SSSS, SRRR, SSRR, SSSR, SRSR, RSRS, SRRS, RSSR, SRSS, RSRR, RRSR, and SSRS. It will be understood that owing to the plane of symmetry, some of these isomers are identical, or exist in the meso form.

[0213] In some embodiments, the compound of Formula (I) has a structure selected from the group consisting of:

[0214]

[0215] In some embodiments, the transduction enhancer comprises a racemate of any two enantiomers of the following compound: or pharmaceutically acceptable salts or solvates thereof.

[0216] In some embodiments, the compound of Formula (I) is L-nebivolol. In some embodiments, the compound of Formula (I) is:

[0217] In some embodiments, the compound of Formula (I) is L-nebivolol and / or has the following structure:

[0218] In some embodiments, the transduction enhancer comprises and / or substantially consists of L-nebivolol and R-nebivolol, or pharmaceutically acceptable salts or solvates thereof. In some embodiments, the transduction enhancer comprises a racemate of L- nebivolol and R-nebivolol, or pharmaceutically acceptable salts or solvates thereof. In some embodiments, the transduction enhancer comprises and / or substantially consists of a racemate of L-nebivolol and R-nebivolol, or pharmaceutically acceptable salts or solvates thereof.

[0219] In some embodiments, the transduction enhancer comprises and / or substantially consists of the SRRR and RSSS isomers of nebivolol, or pharmaceutically acceptable salts or solvates thereof. In some embodiments, the transduction enhancer comprises a racemate of the SRRR and RSSS isomers of nebivolol, or pharmaceutically acceptable salts or solvates thereof. In some embodiments, the transduction enhancer comprises and / or substantially consists of a racemate of the SRRR and RSSS isomers of nebivolol, or pharmaceutically acceptable salts or solvates thereof.

[0220] In some embodiments, the transduction enhancer comprises and / or substantially consists of: or pharmaceutically acceptable salts or solvates thereof.

[0221] In some embodiments, the transduction enhancer comprises a racemate of: or pharmaceutically acceptable salts or solvates thereof.

[0222] In some embodiments, the transduction enhancer comprises and / or substantially consists of a racemate of: or pharmaceutically acceptable salts or solvates thereof.

[0223] In some embodiments, the transduction enhancer and / or racemate comprises L- nebivolol and R-nebivolol in a ratio of between about 4:6 to about 6:4. In some embodiments, the transduction enhancer and / or racemate comprises L-nebivolol and R- nebivolol in a ratio of between about 4.5:5.5 to about 5.5:4.5. In some embodiments, the racemate comprises L-nebivolol and R-nebivolol in a ratio of about 1: 1 (viz. is a substantially equimolar racemate).

[0224] In some embodiments, the transduction enhancer comprises and / or substantially consists of L-nebivolol or:

[0225] In some embodiments, the transduction enhancer substantially consists of L- nebivolol or:

[0226] Compounds of Formula (I), some of which are known in the art, and may be sourced from a chemical supplier or a contract research organisation offering services in small molecule synthesis, for example Nebivolol hydrochloride, D-Nebivolol, L- Nebivolol, and racemic-Nebivolol are available from Sigma Aldrich. Alternatively, compounds of Formula (I) may be synthesised in accordance with the common general principles of chemical synthesis known in the art, and / or by adaptation of a known synthetic method of a compound related to the desired compound. Various routes for the synthesis of nebivolol (including specific stereoisomer(s) thereof) and nebivolol analogues, along with analysis thereof, are known, see for example: Chandrasekhar et al. Tetrahedron (2000) 56(34), 6339-6344; Khandavalli et al., J. Org. Chem. (2015) 80(8), 3965-3973; Carreno et al., Europ. J. Org. Chem. (2008) 2008(12), 2035-2038; Rao et al., Journal of Chromatographic Science (2014) 52(9), 1051-1058; and Kancherla et al., British Journal of Pharmaceutical Research (2016) 14(6), 1-13.

[0227] Target Cells

[0228] The present disclosure also provides a genetically modified cell (or population thereof) expressing a transgene, wherein the genetically modified cell is transduced with an enveloped virus comprising the transgene by a method of disclosure.

[0229] As used herein, the term “cell” refers to any type of cell that can contain the expression vector. For example, the cell is modified to express an enveloped virus (e.g., a retrovirus or lentivirus) comprising a transgene.

[0230] Methods of genetically modifying cells with an enveloped virus will be apparent to the skilled person and / or described herein. For example, the lentivirus (comprising the polynucleotide encoding the transgene of interest introduced into its genome) is harvested from the producer cells and applied to the cells to be modified, resulting in expression of the transgene of the disclosure by the modified cell.

[0231] In one example, the cell is a eukaryotic cell, for example, a human cell.

[0232] In one example, the cell is a cultured cell or a primary cell, i.e., isolated directly from an organism (e.g., a human).

[0233] Suitable cells for use in the present disclosure will be apparent to the skilled person and / or are disclosed herein. In one example, the cell is a hematopoietic cell, such as hematopoietic progenitor or hematopoietic stem cell, a monocyte, a macrophage, a peripheral blood mononuclear cell, a CD4+ T lymphocyte, a CD8+ T lymphocyte, or a dendritic cell. In one example, the cell is a hematopoietic stem cell (HSC) or a hematopoietic progenitor cell (HPC). For example, the cell is an HSC. In another example, the cell is an HPC.

[0234] As used herein, the term “hematopoietic stem cell” (HSC) refers to an immature cell that can differentiate into all types of hematopoietic cells (i.e., blood cells), including white blood cells, red blood cells (including myeloid (e.g., monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells), and lymphoid lineages (e.g., T-cells, B-cells, NK-cells), and platelets.

[0235] As used herein, the term “hematopoietic progenitor cell” (HPC) refers to an intermediate cell type in blood development. HPCs are immature cells that develop from HSCs.

[0236] HSCs and HPCs for use in the present disclosure are found in the peripheral blood and the bone marrow. The cells can be allogeneic, autologous, or from a matched sibling.

[0237] In one example, the cell is allogeneic or autologous. In one example, the cell is allogeneic. In another example, the cell is autologous.

[0238] The term “allogeneic” in reference to an HSC / HPC refers to cells that are collected or obtained from another person to the subject to be or receiving treatment (i.e., a different individual).

[0239] The term “autologous” in reference to an HSC / HPC refers to cells collected or obtained from the subject to be or receiving treatment (i.e., the same individual).

[0240] In one example, the HSC or HPC are of an erythroid lineage (burst-forming unit- erythroid; BFU-E) and / or a myeloid lineage (colony forming unit that generates granulocyte, erythrocyte, monocyte, megakaryocyte cells; CGU-GEMM). For example, the HSC or HPC are of BFU-E lineage. In another example, the HSC or HPC are of CGU-GEMM lineage. In a further example, the HSCs and HPCs are of BFU-E lineage and CGU-GEMM lineage.

[0241] In one example, the cell is a cultured cell, a primary cell, or a cell from a cultured cell line, or a cell obtained from a mammal. In one example, the cell is a cultured cell. In another example, the cell is a primary cell. In a further example, the cell is a cultured cell line. In one example, the cell is a cell obtained from a mammal (e.g., a human subject). In one example, the cell has been previously obtained from the subject (i.e., the human subject). In one example, the cells have been obtained from a tissue sample or biopsy. In another example, the cells have been obtained from the subject by apheresis. It will be apparent to the skilled person that if obtained from a mammal, the cell can be obtained from numerous sources, including but not limited to blood, bone marrow, lymph node, the thymus, or other tissues or fluids. Cells for use in the present disclosure can also be enriched for or purified. Such methods may involve removing or substantially reducing the amount of, erythrocytes, platelets, serum and / or plasma in a sample. Methods disclosed herein may be performed on isolated cells, or a sample containing the target cells in addition to other cells.

[0242] Cell Manufacturing Process

[0243] In one example, the HSCs or HPCs are obtained from the subject by apheresis.

[0244] Methods of performing apheresis will be apparent to the skilled person and / or described herein. For example, apheresate is collected via a standard leukapheresis collection procedure and processed using a fluorescence-activated cell sorting (FACS) system or a magnetic bead separation system, or using a cell processing platform (such as the CliniMACS Prodigy® platform or Cytiva Sepax system).

[0245] It will be apparent to the skilled person that in order to obtain sufficient cells, apheresate may be collected on one or more consecutive days and pooled before isolation of the CD34+cell population. In another example, isolated CD34+cell populations from individual apheresates collected on one or more consecutive days may be pooled.

[0246] In one example, the cells are washed and a CD34+cell population is selected. For example, the CD34+cell population is isolated. In one example, the selected CD34+cells are pre-stimulated and transduced with lentiviral vector. In another example, the cultured and transduced CD34+cells are harvested, washed, and centrifuged, and the cell pellet is resuspended in a cryopreservation medium containing DMSO (such as CryoStor® CS5) and frozen. In one example, the cell products are sampled and tested for endotoxin, sterility, and other quality control measures. After testing, the cryopreserved formulated transduced cells are thawed and administered to a patient.

[0247] CD 34+ cell isolation

[0248] In one example, the HSC or HPC is a CD34 positive (CD34+) cell. For example, the HSC is a CD34+HSC. In another example, the HPC is a CD34+HPC.

[0249] Methods of isolating CD34+cells will be apparent to the skilled person and / or described herein. Exemplary methods of isolating CD34+cells include standard density gradient centrifugation and pre-enrichment of CD34+cells with antibodies that bind to mature blood cells or immunomagnetic separation with nanobeads coupled to CD34 antibodies. An exemplary CD34+isolation method comprises a cell selection program run on the CliniMACS Prodigy. For example, apheresis product is loaded from a Product Bag into the centrifuge chamber. A series of platelet washes are performed using Process Buffer (CliniMACS / EDTA buffer with 0.5% HSA added). CD34 Reagent (CliniMACS) and IVIG are added to the chamber with the cells and incubated. The cells are washed with Process Buffer and loaded onto the magnetic separation column. Enriched CD34+cells are eluted with Elution Buffer into a target bag. The target bag containing the selected cells is removed and placed back on the Prodigy as the cell source for the culture / transduction program (next step), along with a new tubing set.

[0250] Pre-stimulation and transduction cell culture

[0251] In one example, the isolated CD34+cells are pre-stimulated and transduced with lentiviral vector, e.g., a vector comprising the polynucleotide encoding the transgene of interest.

[0252] Methods of pre- stimulating and transducing CD34+cells will be apparent to the skilled person and / or described herein. Methods of the disclosure are applicable to transducing target cells with enveloped viruses at both small- and large-scale productions.

[0253] In one example, a HSC transduction program is run on the CliniMACS Prodigy. For example, the CD34+enriched fraction is transferred into a culture chamber at a cell concentration of between about 1E6 cells / mL and 4E6 cells / mL (for example, 2E6 cells / mL) and the media is exchanged to Pre-stimulation / Transduction Medium, and cytokines are added (100 ng / mL stem cell factor (SCF), 100 ng / mL Flt3 ligand (Flt-3L), 100 ng / mL thrombopoietin (TPO)). In one example, the pre-stimulation mixture is incubated for 16-24 hours at 37°C. CO2 can be provided, for example at 5%. In one example, the cell concentration is adjusted to 2E6 cells / mL by either addition or removal of Pre-stimulation / Transduction Medium. In one example, to initiate the CD34+cell transduction, the required volume of thawed vector product is supplemented with transduction enhancers and added to the cells. In one example, vector is added to the cells at an MOI of 10. In another example, vector is added at 1 x 106TU / mL. The transduction mixture is incubated at 37°C for 16-24 hours. After the overnight incubation, cell harvesting and washing is performed.

[0254] In one example, the CD34+enriched fraction is transferred into a pre-stimulation and transduction culture chamber in a volume of between 50 to 300 mL. For example, the CD34+enriched fraction is transferred into a culture chamber in a volume of about 50 mL, or about 60 mL, or about 70 mL, or about 80 mL, or about 90 mL, or about 100 mL. In another example, the CD34+enriched fraction is transferred into a culture chamber in a volume of about 100 mL, or about 120 mL, or about 140 mL, or about 160 mL, or about 180 mL, or about 200 mL. In a further example, the CD34+enriched fraction is transferred into a culture chamber in a volume of about 200 mL, or about 220 mL, or about 240 mL, or about 260 mL, or about 280 mL, or about 300 mL.

[0255] In one example, the pre- stimulation and transduction cell culture is operated for a period of at least 16 hours. For example, the pre- stimulation and transduction cell culture is operated for a period of between about 16 and 50 hours. In one example, the prestimulation and transduction cell culture is operated for a period of between 16 and 48 hours, for example, about 16 hours, or about 18 hours, or about 20 hours, or about 22 hours, or about 24 hours, or about 28 hours, or about 30 hours or about 32 hours or about 36 hours, or about 40 hours, or about 44 hours, or about 48 hours. In one example, the pre- stimulation and transduction cell culture is operated for at least 16 hours. For example, the pre- stimulation and transduction cell culture is operated for about 20 hours. In a further example, the pre-stimulation and transduction cell culture is operated for at least 24 hours. For example, the pre-stimulation and transduction cell culture is operated for about 28 hours. In one example, the pre-stimulation and transduction cell culture is operated for at least 30 hours. In one example, the pre-stimulation and transduction cell culture is operated for at least 32 hours. For example, the pre-stimulation and transduction cell culture is operated for a period of 36 hours. In one example, the prestimulation and transduction cell culture is operated for at least 36 hours. In one example, the pre-stimulation and transduction cell culture is operated for a period of 40 hours. In another example, the pre-stimulation and transduction cell culture is operated for a period of 44 hours. In a further example, the pre-stimulation and transduction cell culture is operated for a period of 48 hours.

[0256] In one example, the pre-stimulation cell culture is operated for a period of at least 16 hours. For example, the pre-stimulation cell culture is operated for a period of between about 16 and 24 hours. In one example, the pre-stimulation cell culture is operated for a period of about 16 hours, or about 18 hours, or about 20 hours, or about 22 hours, or about 24 hours.

[0257] In one example, the transduction cell culture is operated for a period of at least 16 hours. For example, the transduction cell culture is operated for a period of between about 16 and 24 hours. In one example, the transduction cell culture is operated for a period of about 16 hours, or about 18 hours, or about 20 hours, or about 22 hours, or about 24 hours. In one example, the pre-stimulation and transduction cell culture is operated at a temperature that permits transduction of target cells with the enveloped virus. For example, the pre-stimulation and transduction cell culture has a temperature conventionally used in the state of the art for transducing cells. In one example, the prestimulation and transduction cell culture is at a temperature of between 35-39 °C. For example, at a temperature of 37 ± 0.5 °C.

[0258] It will be apparent to the skilled person from the disclosure herein that the transduction enhancer described herein contacts the target cells in the cell culture prior to, or at the same time (i.e., simultaneously), as contacting the target cells with the enveloped virus.

[0259] In one example, the target cells are pre-treated with the transduction enhancer prior to contacting with the enveloped virus.

[0260] In one example, the target cells (i.e., CD34+HPC and / or CD34+HSC) are contacted with the enveloped virus and the transduction enhancer simultaneously. In one example, the enveloped virus and the transduction enhancer are added to the cell culture in the same solution. In another example, the enveloped virus and the transduction enhancer are added to the cell culture in separate solutions. In a further example, the enveloped virus is added to the cell culture immediately followed by addition of the transduction enhancer. In one example, the transduction enhancer is added to the cell culture immediately followed by addition of the enveloped virus.

[0261] The skilled person will appreciate that simultaneous administration does not require that the enveloped virus and transduction enhancer are added to the cell culture at exactly at the same time, only that they are added in such a manner that they have an overlapping effect on the cells (e.g., they are both active in the cell culture at the same time).

[0262] Cell Culture Medium

[0263] In one example, the cells are cultured in a cell culture medium for the purpose of being transduced with an enveloped virus. The cell culture medium may comprise recombinant or animal-derived proteinaceous compounds. For example, the cell culture medium may comprise glutamine, L-ananyl-L-glutamine, 5-oxoproline, hydroxyproline, recombinant albumin, human pharmaceutical-grade albumin, insulin, transferrin, serotransferrin, haptoglobin, and / or alpha- IB -glycoprotein. The cell culture medium may comprise charged ions or metals, such as calcium, iron, cobalt, and zinc.

[0264] In one example the cell culture medium comprises a commercially available base medium. In another example, the cell culture medium comprises a commercially available base medium that has been supplemented with one or more additives. In one example, the cell culture medium comprises a commercially available base medium that has been supplemented with commercially available supplement medium.

[0265] In one example, the cells are cultured in a cell culture medium comprising X- VIVO 10 medium (available from Lonza), HSC Brew (with or without HSC Brew Supplement) (available from Miltenyi Biotec), StemPro-34 (with or without StemPro-34 Supplement), StemPro HSC (with or without StemPro-HSC Expansion Supplement) (available from ThermoFisher), or stem cell growth medium (SCGM) (available from CellGenix).

[0266] In one example, the cell culture is supplemented with one or more supplements selected from the group consisting of protamine sulphate, one or more cytokines, human serum albumin (HSA) and combinations thereof. In one example, the cell culture is supplemented with one or more supplements selected from the group consisting of protamine sulphate, stem cell factor (SCF), thrombopoietin (TPO), flt3 / flk2 ligand (Flt3L), human serum albumin (HSA) and combinations thereof.

[0267] In one example, the cell culture is supplemented with HSA. For example, the cell culture is supplemented with between 0.1% and 3% HSA. In one example, the cell culture is supplemented with 0.1%, or 0.2%, or 0.3%, or 0.4% or 0.5% HSA. In another example, the cell culture is supplemented with 0.6%, or 0.7%, or 0.8%, or 0.9%, or 1.0% HSA. In another example, the cell culture is supplemented with 1.1%, or 1.2%, or 1.3%, or 1.4%, or 1.5% HSA. In a further example, the cell culture is supplemented with between 1.5% and 2.5% HSA. For example, the cell culture is supplemented with 1.5%, or 1.6%, or 1.7%, or 1.8%, or 1.9%, or 2.0% HSA. In another example, the cell culture is supplemented with 2.0% HSA. In one example, the cell culture is supplemented with 2.1%, or 2.2%, or 2.3%, or 2.4%, or 2.5% HSA. In another example, the cell culture is supplemented with 2.6%, or 2.7%, or 2.8%, or 2.9%, or 3.0% HSA.

[0268] In one example, the cell culture medium is supplemented with one or more cytokines selected from the group consisting of stem cell factor (SCF), thrombopoietin (TPO), flt3 / flk2 ligand (Flt3L) and combinations thereof. In one example, the cell culture medium is supplemented with SCF, TPO, Flt3L. In one example, the cell culture medium is supplemented with SCF, TPO, Flt3L at a concentration of between 1 ng / mL and 1000 ng / mL each. For example, each of the cytokines (i.e., SCF, TPO, Flt3L) are supplemented into the cell culture medium at a concentration of between 1 ng / mL and 500 ng / mL. In one example, the cell culture medium is supplemented with SCF, TPO, Flt3L at a concentration of between 1 ng / mL and 400 ng / mL each, or between 1 ng / mL and 300 ng / mL each, or between 1 ng / mL and 200 ng / mL each, or between 1 ng / mL and 100 ng / mL each. Ine on example, the cell culture medium is supplemented with SCF, TPO, Flt3L at a concentration of about 5 ng / mL each, or about 10 ng / mL each, or about 20 ng / mL each, or about 30 ng / mL each, or about 50 ng / mL each, or about 80 ng / mL each, or about 100 ng / mL each. In another example, the cell culture medium is supplemented with SCF, TPO, Flt3L at a concentration of between 50 ng / mL and 150 ng / mL each. For example, the cell culture medium is supplemented with SCF, TPO, Flt3L at a concentration of about 50 ng / mL each, or about 60 ng / mL each, or about 70 ng / mL each, or about 80 ng / mL each, or about 90 ng / mL each, or about 100 ng / mL each. In another example, the cell culture medium is supplemented with SCF, TPO, Flt3L at a concentration of about 100 ng / mL each. In one example, the cell culture medium is supplemented with SCF, TPO, Flt3L at a concentration of about 110 ng / mL each, or about 120 ng / mL each, or about 130 ng / mL each, or about 140 ng / mL each, or about 150 ng / mL each. In another example, the cell culture medium is supplemented with SCF, TPO, Flt3L at a concentration of between 250 ng / mL and 350 ng / mL each. For example, the cell culture medium is supplemented with SCF, TPO, Flt3L at a concentration of about 250 ng / mL each, or about 260 ng / mL each, or about 270 ng / mL each, or about 280 ng / mL each, or about 290 ng / mL each, or about 300 ng / mL each. In another example, the cell culture medium is supplemented with SCF, TPO, Flt3L at a concentration of about 300 ng / mL each. In one example, the cell culture medium is supplemented with SCF, TPO, Flt3L at a concentration of about 310 ng / mL each, or about 320 ng / mL each, or about 330 ng / mL each, or about 340 ng / mL each, or about 350 ng / mL each.

[0269] In one example, the cell culture medium is supplemented with a water-soluble non-ionic triblock copolymer. In some embodiments, the water-soluble non-ionic triblock copolymer has a general formula of ABA or BAB, wherein A is a hydrophilic block and B is a hydrophobic block.

[0270] In some embodiments, the tri-block copolymer (e.g. poloxamer) has an average molecular weight (in g / mol) of about or greater than about 9800, 9900, 10000, 10100, 10200, 10300, 10400, 10500, 10600, 10700, 10800, 10900, 11000, 11100, 11200, 11300, 11400, 11500, 11600, 11700, 11800, 11900, 12000, 12100, 12200, 12300, 12400, 12500, 12600, 12700, 12800, 12900, 13000, 13100, 13200, 13300, 13400, 13500, 13600, 13700, 13800, 13900, 14000, 14100, 14200, 14300, 14400, 14500, 14600, 14700, 14800, 14900 or 15000. In some embodiments, the tri-block copolymer (e.g. poloxamer) has an average molecular weight (in g / mol) less than about 15000, 14900, 14800, 14700, 14600, 14500, 14400, 14300, 14200, 14100, 14000, 13900, 13800, 13700, 13600, 13500, 13400, 13300, 13200, 13100, 13000, 12900, 12800, 12700, 12600, 12500, 12400, 12300, 12200, 12100, 12000, 11900, 11800, 11700, 11600, 11500, 11400, 11300, 11200, 11100, 11000, 10900, 10800, 10700, 10600, 10500, 10400, 10300, 10200, 10100, 10000, 9900, or 9800. In some embodiments, the tri-block copolymer (e.g. poloxamer) has an average molecular weight (in g / mol) in a range provided by any two of the previously described upper and / or lower amounts, for example, in some embodiments, the tri-block copolymer (e.g. poloxamer) has an average molecular weight (in g / mol) of between about 10000 and about 15000, between about 12000 and about 15000, between about 12800 and about 15000, between about 14000 and about 15000, or between about 12000 and about 13000. The term ‘average’ in relation to molecular weight is a consequence of the technical difficulty in producing a homogeneous mixture of a given tri-block copolymer (e.g. poloxamer), wherein the copolymers therein are identical and therefore possess identical molecular weight. Copolymers produced according to state of the art methods and / or are obtained from a commercial source will be present as a mixture of copolymers that possess some variability as to the range of molecular weights of the individual copolymer molecules therein, but such mixtures will possess an average molecule weight as described herein. Method for determining molecular weight are well known in the art and are described in standard chemistry textbooks, and for example, include methods such as high performance liquid chromatography (HPLC), or mass spectrometry.

[0271] In some embodiments, the water-soluble non-ionic triblock copolymer (e.g. a poloxamer) has a viscosity (in Pa-s) greater than about 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4 or 3.5. In some embodiments, the poloxamer has a viscosity (in Pa- s) less than about 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1 or 2.0. In some embodiments, the poloxamer has a viscosity (in Pa- s) in range provided by any two of the previously described upper and / or lower amounts, for example, in some embodiments, the poloxamer has a viscosity (in Pa- s) of between about 2.0 and about 3.5, between about 2.5 and about 3.3, or between about 2.7 and about 3.2.

[0272] In one example, the water-soluble non-ionic triblock copolymer is a poloxamer. Poloxamers are nonionic triblock copolymers composed of a central hydrophobic chain of polyoxypropylene (viz. polypropylene oxide)) flanked by two hydrophilic chains of polyoxyethylene viz. poly(ethylene oxide)). Poloxamers are also known by the trade names Synperonics, Pluronic, and Kolliphor. Specific poloxamer copolymers are optionally prefixed with the letter P (for poloxamer) followed by three digits: the first two digits multiplied by 100 gives the approximate molecular mass (g / mol) of the polyoxypropylene core (also referred to as the average polyoxypropylene content), and the last digit multiplied by 10 gives the approximate percentage (%) of polyoxyethylene content. By way of example, poloxamer 407 (or P407) refers to a poloxamer having a molecular mass of approximately 4000 g / mol, and a polyoxyethylene content of approximately 70%. Poloxamers may exist in a variety of physical states at room temperature, for example, a paste, flake or liquid, the predominant form of which for a given poloxamer will depend upon the physical properties and characteristics of that poloxamer. Some trade name poloxamers, for example, Pluronic are prefixed with an L (liquid), P (paste) or F (flake) to indicate the relevant physical state. Herein, unless otherwise specified, poloxamers are referred to by their non-proprietary name. It will be understood that a reference to a non-proprietary poloxamer should be taken to also include a reference to any and all trade names that correspond to or describe the said non- proprietary poloxamer (e.g. a reference to poloxamer 407 should be understood to include a reference to poloxamer Fl 27, as it is known under Pluronic notation).

[0273] In some embodiments, the tri-block copolymer (e.g. poloxamer) has a melting point (in °C) of about or greater than about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60. In some embodiments, the tri-block copolymer (e.g. poloxamer) has a melting point (in °C) less than about 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5 or 1. In some embodiments, the tri-block copolymer (e.g. poloxamer) has a melting point (in °C) in a range provided by any two of the previously described upper and / or lower amounts, for example, in some embodiments, the tri-block copolymer (e.g. poloxamer) has a melting point (in °C) between about 1 and about 60, between about 40 and about 60, or between about 50 and about 60.

[0274] In some embodiments, the poloxamer has an average polyoxypropylene content (in g / mol) of about or greater than about 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000,

[0275] 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400,

[0276] 4500, 4600, 4700, 4800, 4900 or 5000. In some embodiments, the poloxamer has an average polyoxypropylene content (in g / mol) less than about 5000, 4900, 4800, 4700, 4600, 4500, 4400, 4300, 4200, 4100, 4000, 3900, 3800, 3700, 3600, 3500, 3400, 3300,

[0277] 3200, 3100, 3000, 2900, 2800, 2700, 2600, 2500, 2400, 2300, 2200, 2100, 2000, 1900,

[0278] 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100 or 1000. In some embodiments, the poloxamer has an average polyoxypropylene content (in g / mol) in a range provided by any two of the previously described upper and / or lower amounts, for example, in some embodiments, the poloxamer has an average polyoxypropylene content (in g / mol) between about 1000 and about 5000, between about 3800 and about 4200, between about 3000 and about 3600, between about 3000 and about 4200, or between about 3000 and about 4000. In some embodiments, the poloxamer has an average polyoxypropylene content (in g / mol) of about 4000. In some embodiments, the poloxamer has an average polyoxypropylene content (in g / mol) of about 3300. In some embodiments, the poloxamer has an average polyoxypropylene content (in g / mol) of about 3000.

[0279] The term ‘average’ in relation to poly oxypropylene content is a consequence of the technical difficulty in producing a homogeneous mixture of a given poloxamer, wherein the poloxamer molecules therein are identical and therefore possess identical polyoxypropylene content. Poloxamers produced according to state of the art methods and / or are obtained from a commercial source will be present as a mixture of poloxamers that possess some variability as to the range of polyoxypropylene content of individual poloxamer molecules therein, but such mixtures will possess an average polyoxypropylene content as described herein.

[0280] In some embodiments, the poloxamer has an average polyoxyethylene content (in % w / w) of about or greater than about 30, 40, 50, 60, 70, 80 or 90. In some embodiments, the poloxamer has an average polyoxyethylene content (in % w / w) less than about 90, 80, 70, 60, 50, 40 or 30. In some embodiments, the poloxamer has an average polyoxyethylene content (in % w / w) in a range provided by any two of the previously described upper and / or lower amounts, for example, in some embodiments, the poloxamer has an average polyoxyethylene content (in % w / w) between about 30 and about 90, between about 60 and 90, between about 60 and 80, or between about 70 and 90. In some embodiments, the poloxamer has an average polyoxyethylene content (in % w / w) of about 70 or 80. In some embodiments, the poloxamer has an average polyoxyethylene content (in % w / w) of about 70. In some embodiments, the poloxamer has an average polyoxyethylene content (in % w / w) of about 80. It will be understood that average polyoxyethylene content may be given as a % w / w relative to the average total molecular weight of the poloxamer. The term ‘average’ in relation to polyoxyethylene content is a consequence of the technical difficulty in producing a homogeneous mixture of a given poloxamer, wherein the poloxamer molecules therein are identical and therefore possess identical polyoxyethylene content. Poloxamers produced according to state of the art methods and / or are obtained from a commercial source will be present as a mixture of poloxamers that possess some variability as to the range of polyoxyethylene content of individual poloxamer molecules therein, but such mixtures will possess an average polyoxyethylene content as described herein.

[0281] In some embodiments, the poloxamer has an average molecular weight greater than about 10000 g / mol and an average polyoxyethylene content of about or greater than about 50% w / w. In some embodiments, the poloxamer has an average molecular weight greater than about 12000 g / mol and an average polyoxyethylene content of about or greater than about 60% w / w. In some embodiments, the poloxamer has an average molecular weight greater than about 10000 g / mol and an average polyoxypropylene content of about or greater than about 2500 g / mol. In some embodiments, the poloxamer has an average molecular weight greater than about 12000 g / mol and an average polyoxypropylene content of about or greater than about 3000 g / mol.

[0282] In some embodiments, the poloxamer has an average polyoxyethylene content of about or greater than about 50% w / w and an average polyoxypropylene content of about or greater than about 2500 g / mol. In some embodiments, the poloxamer has an average polyoxyethylene content of about or greater than about 60% w / w and an average polyoxypropylene content of about or greater than about 3000 g / mol.

[0283] In some embodiments, the poloxamer is a compound of Formula (II):

[0284] HO— [CH2CH2O — [CH2CH(CH3)O]y— [CH2CH2O]Z— H wherein y is an integer such that the poloxamer has a polyoxypropylene content according to any one the above-described embodiments, and x and z are independently selected integers such that the poloxamer has an average polyoxyethylene content according to any one of the above-described embodiments.

[0285] For example, in one example y is an integer such that the poloxamer has a polyoxypropylene content of about 3000 g / mol viz., in one example, y is about 35. In another example, x and z are independently selected such that the poloxamer has an average polyoxyethylene content (in % w / w) of about 70.

[0286] In some embodiments, y is about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70. In some embodiments, y is greater than 70. In some embodiments, y is between about 50 and about 65. In some embodiments, y is between about 60 and about 70. In some embodiments, y is between about 45 and about 55.

[0287] In some embodiments, x and z are integers independently selected from the integers between 10 and 200, such that x + z is at least about 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390 or 400. In some embodiments, x + z is a range between 180 and 400, between 190 and about 300, or between about 200 and about 280.

[0288] In some embodiments, the poloxamer is selected from the group consisting of poloxamer 105, poloxamer 123, poloxamer 124, poloxamer 182, poloxamer 184, poloxamer 188, poloxamer 234, poloxamer 235, poloxamer 237, poloxamer 238, poloxamer 288, poloxamer 333, poloxamer 334, poloxamer 335, poloxamer 338, poloxamer 403, poloxamer 407, and combinations thereof.

[0289] In some embodiments, the poloxamer is selected from the group consisting of poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 288, poloxamer 335, poloxamer 338, poloxamer 407, and combinations thereof.

[0290] In some embodiments, the poloxamer is selected from the group consisting of poloxamer 288, poloxamer 335, poloxamer 338, poloxamer 407, and combinations thereof.

[0291] In some embodiments, the poloxamer is selected from the group consisting of poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 288, poloxamer 388, poloxamer 407, and combinations thereof.

[0292] In some embodiments, the poloxamer is selected from the group consisting of poloxamer 388, poloxamer 407, and combinations thereof.

[0293] In one example, the cell culture medium is supplemented with poloxamer 237 (also known as F87). In one example, the cell culture medium is supplemented with poloxamer 188 (also known as F68). In one example, the cell culture medium is supplemented with poloxamer 124 (also known as L44). In one example, the cell culture medium is supplemented with poloxamer 388 (also known as F108). In one example, the cell culture medium is supplemented with poloxamer 288 (also known as F98). In one example, the cell culture medium is supplemented with poloxamer 407 (also known as F127).

[0294] In one example, the poloxamer is in the cell culture medium at a concentration of at least 0.01 mg / mL. For example, the poloxamer is in the cell culture medium at a concentration of between 0.01 mg / mL and 10 mg / mL. For example, the poloxamer is in the cell culture medium at a concentration of between 0.1 mg / mL and 5 mg / mL of cell culture medium. In one example, the poloxamer is in the cell culture medium at a concentration of between 0.1 mg / mL and 4 mg / mL of cell culture medium. In one example, the poloxamer is in the cell culture medium at a concentration of between 0.1 mg / mL and 3 mg / mL of cell culture medium. In one example, the poloxamer is in the cell culture medium at a concentration of between 0.1 mg / mL and 2 mg / mL of cell culture medium. For example, the poloxamer is in the cell culture medium at a concentration of between 0.1 mg / mL and 1 mg / mL of cell culture medium. In one example, the poloxamer is in the cell culture medium at a concentration of about 0.1 mg / mL, or about 0.2 mg / mL, or about 0.3 mg / mL, or about 0.4 mg / mL, or about 0.5 mg / mL, or about 0.6 mg / mL, or about 0.7 mg / mL, or about 0.8 mg / mL, or about 0.9 mg / mL, or about 1 mg / mL of cell culture medium. In one example, the poloxamer is in the cell culture medium at a concentration of at least 0.5 mg / mL. For example, the poloxamer is in the cell culture medium at a concentration of about 0.5 mg / mL of cell culture medium. In another example, the poloxamer is in the cell culture medium at a concentration of about 1 mg / mL of cell culture medium.

[0295] According to some examples, the culture medium used has a neutral pH (e.g. comprised between 7 and 7.4, notably 7, 7.1, 7.2, 7.3 or 7.4) conventionally used in the state of the art for cultivating cells and transducing cells with viruses. In one example, the cell culture is at a pH of between 6.0 and 8.0. For example, the pH of the culture medium is 7.1 ± 0.15. In other examples, the method comprises transducing cells in a moderately acid medium. The expression “moderately acid condition” designates the pH of an aqueous solution comprised between 5 and 6.8, for example between 5.5 and 6.5, such as between 5.8 and 6.2. The selected pH will also depend on the buffering power of the culture medium used, which one skilled in the art may easily determine taking into account his / her general knowledge. One skilled in the art is able to modify the pH of a solution.

[0296] Cell harvest and washing

[0297] In one example, following pre- stimulation and transduction, the transduced CD34+cells are harvested and washed. For example, a harvested transduced cell population is produced.

[0298] In one example, the cells are washed to remove additives used in previous steps. For example, the harvested transduced cells are washed.

[0299] Methods of harvesting and washing the cells will be apparent to the skilled person and / or described herein. For example, the cell suspension volume is adjusted in the Prodigy to minimum of 70 mL to achieve the final concentration of 2E6 cells / mL. For example, the volume of the cell suspension (i.e., the suspension comprising the transduced CD34+cells) was reduced to a minimum of 70 mL.

[0300] In one example, the harvested cells are washed with formulation medium (0.9% saline with human serum albumin). In one example, the harvested cells are washed one or more times with formulation medium. For example, the formulation medium comprises 0.9% saline. In another example, the formulation medium comprises human serum albumin.

[0301] In one example, the transduced CD34+-enriched fraction is collected. Final formulation, filling and cryopreservation

[0302] In one example, following harvesting and washing, the transduced CD34+- enriched cells are formulated with a pharmaceutically acceptable carrier. For example, the transduced CD34+-enriched cells are formulated with a cryopreservation medium.

[0303] In one example, the transduced CD34+-enriched cells are centrifuged and resuspended in cryopreservation media (e.g., a mixture of Cryostor® CSB, which contains no DMSO, and Cryostor® CS5, which contains 5% DMSO). For example, the cryopreservation bag is sealed and frozen in a controlled rate freezer down to -90°C for long-term storage in vapor phase liquid nitrogen at < -120 °C.

[0304] In one example, the transduced CD34+-enriched cells are formulated for administration to the subject.

[0305] Production of Enveloped Viruses

[0306] Methods for the production of enveloped viruses will be apparent to the skilled artisan and / or described, for example, in Ansorge et al., (2010) Biochem. Eng. J. 48: 362- 377; Schweizer and Merten (2010) Curr. Gene Ther. 10: 474-486; and Rodrigues et al., (2011) Viral Gene Therapy. Xu, InTech. Chapter 2: 15-40.

[0307] In one example, the virus is a retrovirus, for example, a lentivirus. Exemplary retroviruses are from alpha retroviruses (such avian leukosis virus (ALV)), from beta retroviruses (such as mouse mammary tumor virus (MMTV)), from gamma retroviruses (such as murine leukemia virus (MLV)), from delta retroviruses (such as human T- lymphotropic virus (HTLV)), from epsilon retroviruses (such as Walleye dermal sarcoma virus (WDSV)), from spumavirus (such as human foamy virus (HFV) or simian foamy virus (SFV)), from primate lentiviruses such as the different types of human immunodeficiency viruses (HIV), the different types of simian immunodeficiency viruses (SIV), or from non-primate mammal lentiviruses such as the equine infectious anemia virus (EIAV), from the feline immunodeficiency virus (FIV), the caprine arthritis-encephalitis virus (CAEV), or the ovine visna-maedi virus (VMV).

[0308] In some examples, the enveloped virus, e.g., the retrovirus, is pseudotyped, i.e., it comprises an envelope glycoprotein derived from a virus different from the virus from which it is derived, a modified envelope glycoprotein or a chimeric envelope glycoprotein.

[0309] In one example, the viral vector is derived from the genome of human immunodeficiency virus (HIV). For example, the viral vector is derived from the genome of HIV- 1. It will be apparent to the skilled person that to increase safety, the viral vector only contains HIV genes which are necessary for infection and gene delivery, whilst the genes necessary for replication and virulence factors have been removed. For example, the envelope protein of HIV-1 is exchanged with that of another virus (e.g., VSV-G protein from Vesicular stomatitis Indiana virus (VSV)) to allow infection of a wide range of target cells.

[0310] In some examples, the enveloped virus comprises a transgene introduced into its genome. The transgene will depend on the specific use for which the enveloped viral vector is intended. Exemplary transgenes include a transgene coding for a therapeutic RNA (e.g. encoding an antisense complementary RNA of a target RNA or DNA sequence), a transgene encoding for a protein that is deficient or absent in a subject affected with a pathology, or a transgene used for vaccination with DNA, i.e. a transgene coding for a protein, the expression of which will induce vaccination of the recipient body against said protein. In some examples, the transgene encodes a protein or nucleic acid useful for treating a hemoglobinopathy, e.g., sickle cell disease or a thalassemia. In some examples, the transgene encodes a protein or nucleic acid useful for treating a primary immunodeficiency. In some examples, the transgene encodes a protein or nucleic acid useful for treating sickle cell disease. In some examples, the transgene encodes a protein or nucleic acid useful for treating Wiskott-Aldrich Syndrome. In some examples, the transgene encodes a protein or nucleic acid useful for treating X linked agammaglobulinemia. In some examples, the transgene encodes a protein or nucleic acid useful for treating ADA2 deficiency. In some examples, the transgene encodes a protein or nucleic acid useful for treating sickle cell disease or beta-thalassemia. In some examples, the transgene encodes a protein or nucleic acid useful for treating deficiency of the IL-1 receptor antagonist (DIRA).

[0311] In some examples, an enveloped virus is produced by introducing the four following elements into a host cell: an expression cassette comprising a lentiviral gene gagpol, an expression cassette comprising a lentiviral gene rev, a transgene, all positioned between a lentiviral LTR-5’ and a lentiviral LTR-3’, and an expression cassette encoding envelope glycoprotein(s).

[0312] In some examples, the enveloped virus is produced from a stable line expressing one or several elements required for producing an enveloped virus (Miller (2001) Curr. Protoc. Hum. Genet. Chapter 12: Unit 12.5.; Rodrigues et al. 2011, supra). In one example, the enveloped virus is produced from a mammal host cell transfected transiently with one or several plasmids coding for the elements required for producing the virus. According to an alternative example, the elements are introduced into the cell by means of multiple plasmids: one plasmid bearing an expression cassette comprising a lentiviral gagpol gene, one plasmid bearing an expression cassette comprising a lentiviral rev gene, one plasmid bearing an expression cassette encoding the envelope glycoprotein(s), one plasmid bearing an expression cassette comprising a tetracycline transactivator (iTA) gene, and / or one plasmid bearing an expression cassette comprising a lentiviral tat gene. A transfer plasmid comprising an expression cassette with the transgene, comprised between a lentiviral LTR-5’ and LTR-3’, can be introduced as a concatemer along with a helper plasmid with an antibiotic resistance cassette to confer resistance to the producer cells.

[0313] The host cell may be selected from any cell allowing production of an enveloped virus. According to one example, the host cell for production of the enveloped virus is selected from a human cell (HEK293, HEK293T, HEK293FT, HEK293OX, Te671, HT1080, CEM), a musteli cell (NIH-3T3), a mustelidae cell (Mpf), a canid cell (D17). According to one example, the host cell for production of the enveloped virus is selected from CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY I, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211 A cells.

[0314] According to one example, the host cell for production of the enveloped virus is selected from the GPR, GPRG, GPRT, GPRGT, and GPRTG cell lines. In another example, the host cell for production of the enveloped virus is selected from a cell line derived from any of the above cell lines.

[0315] In one example, the enveloped virus is produced from stable producer cells. Stable producer cells can be derived from packaging cell lines, including as any of the cell lines disclosed herein. In some embodiments the packaging cell lines are GPRG or GPRTG cell lines (Throm et al. (2009) Blood 113(21):5104-5110; and Bonner et al. (2015) Molecular Therapy, Vol. 23, Suppl. 1, S35). In one example, stable producer cell line cells are generated by synthesizing a vector by cloning one or more genes into a recombinant plasmid; forming a concatemeric array from an expression cassette excised from the synthesized vector, and an expression cassette obtained from an antibiotic resistance cassette plasmid; transfecting packaging cell line cells with the formed concatemeric array; and isolating the stable producer cell line cells. Virus is produced by inducing the inducible promoters of the stable producer cell line cells.

[0316] The host cells for production of the enveloped virus are cultivated in a medium suitable for cultivation of mammal cells and for producing an enveloped virus. The host cells for production of the enveloped virus can be cultivated in an adherent environment, e.g., while attached to a surface, or in a suspension environment, e.g., suspended in the medium. The medium may moreover be supplemented with additives known in the field such as antibiotics, serum (notably fetal calf serum, etc.) added in suitable concentrations. The medium may be supplemented with GlutaMax™, Pluronic™ F-68 (ThermoFisher), LONG® R3 IGF-I (Sigma-Aldrich), Cell Boost™ 5, and / or an antidumping agent. The medium used may notably comprise serum or be serum-free. Culture media for mammal cells are known and include, for example, DMEM (Dulbecco’s Modified Eagle’s medium) medium, RPMI1640 or a mixture of various culture media, including for example DMEM / F12, or a serum-free medium like optiMEM®, optiPRO®, optiPRO- SFM®, CD293® (ThermoFisher), TransFx™ (Cytiva), BalanCD® (Irvine), Freestyle F17® (Life Technologies), or Ex-Cell® 293 (Sigma-Aldrich).

[0317] In a process using transiently transfected cells, any agent allowing transfection of plasmids may be used. Exemplary agents include calcium phosphate or polyethyleneimine. The conditions (e.g., amount of plasmid(s), ratio between the plasmids, ratio between the plasmid(s) and the transfection agent, the type of medium, etc.) and the transfection time may be adapted by one skilled in the art according to the characteristics of the produced virus and / or of the transgene introduced into the transfer plasmid.

[0318] According to some examples, the culture medium used has a neutral pH (e.g. comprised between 7 and 7.4, notably 7, 7.1, 7.2, 7.3 or 7.4) conventionally used in the state of the art for cultivating cells and producing viruses. In other examples, the production process used comprises the cultivation of producing cells in a moderately acid medium. The expression “moderately acid condition” designates the pH of an aqueous solution comprised between 5 and 6.8, for example between 5.5 and 6.5, such as between 5.8 and 6.2. The selected pH will also depend on the buffering power of the culture medium used, which one skilled in the art may easily determine taking into account his / her general knowledge. One skilled in the art is able to modify the pH of a solution.

[0319] In one example, the production of the enveloped virus comprises: transient transfection of HEK293T cells or derivatives thereof by means of one or several plasmids coding for the elements required for production of said enveloped vector, or by the use of stable producing cells, e.g., GPRG or GPRTG, producing the vectors constitutively or after induction; culturing the cells in a suitable medium, for which the pH is of about 6 or of about 7; harvesting cell culture medium containing the enveloped virus. Purifying Enveloped Viruses

[0320] In one example, the enveloped virus for use in the present disclosure is purified from the cell culture comprising one or more steps selected from the group consisting of clarification filtration, anion exchange chromatography, concentration and diafiltration.

[0321] The downstream process for purifying and concentrating viral vector from a cell culture includes a harvest filtration step (also known as “clarification filtration” or “harvest clarification filtration” or “bioburden reduction”) to remove cellular debris and components from the harvest, a purification step, e.g., anion exchange chromatography, to reduce overall volume and to separate viral vector from host cell DNA, proteins, and media components, and an ultrafiltration / diafiltration step to concentrate the viral vector into a final formulation buffer. In some examples, the downstream step further includes a sterile filtration step for removal of microorganisms from the final product.

[0322] Pharmaceutical Compositions

[0323] The present disclosure provides a composition comprising a population of transduced (or genetically modified) cells of the disclosure for use as a medicament. In one example, the present disclosure provides a composition for use in gene therapy.

[0324] Accordingly, the present disclosure provides a composition comprising a population of transduced (or genetically modified) cells of the disclosure and a pharmaceutically acceptable carrier.

[0325] Methods for preparing a compound into a suitable form for administration (e.g. a pharmaceutical composition) are known in the art and include, for example, methods as described in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, Pa., 1990).

[0326] An appropriate pharmaceutical composition comprising a population of transduced (or genetically modified) CD34+cells to be administered can be prepared in a physiologically acceptable carrier. For solutions or emulsions, suitable carriers include, for example, aqueous or alcoholic / aqueous solutions, emulsions or suspensions, including saline, cryopreservation solution and buffered media. Parenteral vehicles can include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils or dimethyl sulfoxide (DMSO). A variety of appropriate aqueous carriers are known to the skilled artisan, including water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution and glycine. Intravenous vehicles can include various additives, preservatives (e.g., DMSO), or fluid, nutrient or electrolyte replenishers (See, generally, Remington's Pharmaceutical Science, 16th Edition, Mack, Ed. 1980). The compositions can optionally contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents and toxicity adjusting agents, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride and sodium lactate. The vehicles may contain minor amounts of additives that enhance isotonicity and chemical stability, e.g., buffers and preservatives.

[0327] In one example, the pharmaceutical composition comprises a population of transduced (or genetically modified) CD34+cells and a cryopreservation media. For example, the cryopreservation media is an animal protein-free, serum free cryopreservation media. In one example, the cryopreservation media comprises DMSO. In one example the cryopreservation media comprises 5% DMSO. For example, the cryopreservation media comprises 10% DMSO. Exemplary cryopreservation media will be apparent to the skilled person and includes for example, CryoStor®.

[0328] The optimum concentration of the cells in the chosen medium can be determined empirically, according to procedures known to the skilled artisan, and will depend on the ultimate pharmaceutical formulation desired.

[0329] Upon formulation, compositions of the present disclosure will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The dosage ranges for the administration of the composition of the disclosure are those large enough to produce the desired effect. For example, the composition comprises an effective amount of the population of genetically modified (or transduced) cells. In one example, the composition comprises a therapeutically effective amount of the transduced (or genetically modified) cells.

[0330] The dosage should not be so large as to cause adverse side effects. Generally, the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any complication.

[0331] In one example, the composition is formulated for injection or infusion, e.g., via intravenous infusion.

[0332] The present disclosure is further defined in the following numbered paragraphs. Unless this would result in a contradiction, the embodiments of the following paragraphs can be combined with any of the above examples and provide further details on certain aspects of the disclosure. 1. A method of transducing a cell with an enveloped virus in a cell culture, the method comprising culturing a plurality of cells in a cell culture medium and contacting the cells with the enveloped virus and a transduction enhancer, wherein the transduction enhancer comprises a compound of Formula (I) or a stereoisomer, a racemate, a pharmaceutically acceptable salt or a solvate thereof:

[0333] Formula (I) wherein

[0334] X1and X2are independently selected from O, N, S or CH2;

[0335] L1and L2are independently absent or selected from -Ci-ealkyl-, -Ci ealkcnyl-^ or -Ci ealkynyl-, each of which may be optionally substituted with one or more R4or R5,

[0336] R1is selected from H, -Ci-ealkyl-, -C2-6alkenyl-;>or -C2-6alkynyl-;

[0337] R2and R3are independently selected from H, halogen, or OH; and

[0338] R4and R5are independently selected from halogen, OH, -Ci-4alkyl-, and -Ci- 4alkoxy-

[0339] 2. A method of increasing an enveloped virus transduction efficiency of a cell in a cell culture, the method comprising culturing a plurality of cells in a cell culture medium and contacting the cells with the enveloped virus and a transduction enhancer, wherein the transduction enhancer comprises a compound of Formula (I) or a stereoisomer, a racemate, a pharmaceutically acceptable salt or a solvate thereof:

[0340] Formula (I) wherein

[0341] X1and X2are independently selected from O, N, S or CH2;

[0342] L1and L2are independently absent or selected from -Ci-ealkyl-, -C2-6alkenyl-;>or -C2-6alkynyl-, each of which may be optionally substituted with one or more R4or R5, R1is selected from H, -Ci-6alkyl-, -Ci ealkcnyl-^ or -Ci ealkynyl-;

[0343] R2and R3are independently selected from H, halogen, or OH; and

[0344] R4and R5are independently selected from halogen, OH, -Ci-4alkyl-, and -Ci- 4alkoxy-

[0345] 3. A method of increasing potency of an enveloped virus during transduction of a cell in a cell culture, the method comprising culturing a plurality of cells in a cell culture medium and contacting the cells with the enveloped virus and a transduction enhancer, wherein the transduction enhancer comprises a compound of Formula (I) or a stereoisomer, a racemate, a pharmaceutically acceptable salt or a solvate thereof:

[0346] Formula (I) wherein

[0347] X1and X2are independently selected from O, N, S or CH2;

[0348] L1and L2are independently absent or selected from -Ci-ealkyl-, -Ci ealkcnyl-^ or -Ci ealkynyl-, each of which may be optionally substituted with one or more R4or R5,

[0349] R1is selected from H, -Ci-ealkyl-, -C2-6alkenyl-;>or -C2-6alkynyl-;

[0350] R2and R3are independently selected from H, halogen, or OH; and

[0351] R4and R5are independently selected from halogen, OH, -Ci-4alkyl-, and -Ci- 4alkoxy-

[0352] 4. A method of increasing transduction efficiency of a cell that differentiates into a myeloid cell in a cell culture, the method comprising culturing a plurality of cells in a cell culture medium and contacting the cells with the enveloped virus and a transduction enhancer, wherein the transduction enhancer comprises a compound of Formula (I) or a stereoisomer, a racemate, a pharmaceutically acceptable salt or a solvate thereof:

[0353] Formula (I) wherein

[0354] X1and X2are independently selected from O, N, S or CH2;

[0355] L1and L2are independently absent or selected from -Ci-ealkyl-, -C2-6alkenyl-;>or -C2-6alkynyl-, each of which may be optionally substituted with one or more R4or R5,

[0356] R1is selected from H, -Ci-ealkyl-, -C2-6alkenyl-;>or -C2-6alkynyl-;

[0357] R2and R3are independently selected from H, halogen, or OH; and

[0358] R4and R5are independently selected from halogen, OH, -Ci-4alkyl-, and -Ci- 4alkoxy-.

[0359] 5. A method of increasing vector copy number in a cell in a cell culture, the method comprising culturing a plurality of cells in a cell culture medium and contacting the cells with the enveloped virus and a transduction enhancer, wherein the transduction enhancer comprises a compound of Formula (I) or a stereoisomer, a racemate, a pharmaceutically acceptable salt or a solvate thereof:

[0360] Formula (I) wherein

[0361] X1and X2are independently selected from O, N, S or CH2;

[0362] L1and L2are independently absent or selected from -Ci-ealkyl-, -Ci ealkcnyl-^ or -Ci ealkynyl-, each of which may be optionally substituted with one or more R4or R5,

[0363] R1is selected from H, -Ci-ealkyl-, -C2-6alkenyl-;>or -C2-6alkynyl-;

[0364] R2and R3are independently selected from H, halogen, or OH; and

[0365] R4and R5are independently selected from halogen, OH, -Ci-4alkyl-, and -Ci- 4alkoxy-

[0366] 6. The method of paragraph 5, wherein the cell is a cell that differentiates into an erythroid cell.

[0367] 7. The method of paragraph 5 or paragraph 6, wherein transduction of cells that differentiate into erythroid cells is not reduced. 8. The method of any one of paragraphs 1 to 7, wherein:

[0368] L1has a structure *-L3CH(R4)-, and L2has a structure *-L4CH(R5)-, wherein

[0369] L3and L4are independently absent or selected from -Ci-4alkyl-, -Ci ^alkenyl-, or -Ci-4alkynyl-; and

[0370] * indicates a single bond to the central N.

[0371] 9. The method of paragraph 8, wherein L3and L4are both -Cialkyl-

[0372] 10. The method of any one of paragraphs 1 to 9, wherein X1and X2are both O.

[0373] 11. The method of any one of paragraphs 1 to 10, wherein R4and R5are both OH.

[0374] 12. The method of any one of paragraphs 1 to 11, wherein R1is H.

[0375] 13. The method of any one of paragraphs 1 to 12, wherein R2and R3are both F.

[0376] 14. The method of any one of paragraphs 1 to 9, wherein the compound of Formula

[0377] (I) is nebivolol and / or has the following structure:

[0378] 15. The method of any one of paragraphs 1 to 14, wherein the compound of Formula (I) is L-nebivolol and / or has the following structure:

[0379] 16. The method of any one of paragraphs 1 to 14, wherein the compound of Formula (I) is D-nebivolol and / or has the following structure:

[0380] 17. The method of any one of paragraphs 1 to 16, wherein the transduction enhancer comprises and / or substantially consists of: or pharmaceutically acceptable salts or solvates thereof.

[0381] 18. The method of paragraph 17, wherein the transduction enhancer comprises and / or substantially consists of a racemate of: or pharmaceutically acceptable salts of solvates thereof, in a ratio of between about 4:6 to about 6:4, or between about 4.5:5.5 to about 5.5:4.5, or about 1: 1.

[0382] 19. The method of any one of paragraphs 1 to 18, wherein the transduction enhancer comprises and / or substantially consists of L-nebivolol or:

[0383] 20. The method of any one of paragraphs 1 to 19, wherein the compound is in the cell culture medium at a concentration of at least 1 pM.

[0384] 21. The method of any one of paragraphs 1 to 20, wherein the compound is in the cell culture medium at a concentration of at least 3 pM.

[0385] 22. The method of any one of paragraphs 1 to 21, wherein the compound is in the cell culture medium at a concentration of at least 5 pM.

[0386] 23. The method of any one of paragraphs 1 to 22, wherein the compound is in the cell culture medium at a concentration of at least 10 pM.

[0387] 24. The method of any one of paragraphs 1 to 23, wherein the compound is in the cell culture medium at a concentration of between 1 pM and 20 pM.

[0388] 25. The method of any one of paragraphs 1 to 24, wherein the cell culture further comprises a water-soluble non-ionic triblock copolymer.

[0389] 26. The method of paragraph 25, wherein the water-soluble non-ionic triblock copolymer has an average molecular weight (in g / mol) greater than about 9800, 9900, 10000, 10100, 10200, 10300, 10400, 10500, 10600, 10700, 10800, 10900, 11000, 11100, 11200, 11300, 11400, 11500, 11600, 11700, 11800, 11900, 12000, 12100, 12200, 12300, 12400, 12500, 12600, 12700, 12800, 12900, 13000, 13100, 13200, 13300, 13400, 13500, 13600, 13700, 13800, 13900, 14000, 14100, 14200, 14300, 14400, 14500, 14600, 14700, 14800, 14900 or 15000.

[0390] 27. The method of paragraph 26, wherein the water-soluble non-ionic triblock copolymer has an average molecular weight greater than about 12000 g / mol.

[0391] 28. The method of paragraph 27, wherein the water-soluble non-ionic triblock copolymer has an average molecular weight of about 12600 g / mol. 29. The method of paragraph 28, wherein the water-soluble non-ionic triblock copolymer has a viscosity (in Pa- s) of between about 2.0 and about 3.5.

[0392] 30. The method of any one of paragraphs 25 to 29, wherein the water-soluble nonionic triblock copolymer is a poloxamer.

[0393] 31. The method of paragraph 30, wherein the poloxamer has an average polyoxypropylene content (in g / mol) of about or greater than about 1000, 1100, 1200,

[0394] 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600,

[0395] 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000,

[0396] 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900 or 5000.

[0397] 32. The method of paragraph 30 or 31, wherein the poloxamer has an average polyoxypropylene content of about or greater than about 3000 g / mol.

[0398] 33. The method of any one of paragraphs 30 to 32, wherein the poloxamer has an average polyoxyethylene content (in % w / w) of about or greater than about 30, 40, 50, 60, 70, 80 or 90.

[0399] 34. The method of any one of paragraphs 30 to 33, wherein the poloxamer has an average polyoxyethylene content of about or greater than about 70% w / w.

[0400] 35. The method of any one of paragraphs 30 to 34, wherein the poloxamer is selected from the group consisting of poloxamer 105, poloxamer 123, poloxamer 124, poloxamer 182, poloxamer 184, poloxamer 188, poloxamer 234, poloxamer 235, poloxamer 237, poloxamer 238, poloxamer 288, poloxamer 333, poloxamer 334, poloxamer 335, poloxamer 338, poloxamer 403, poloxamer 407, and combinations thereof.

[0401] 36. The method of any one of paragraphs 30 to 35, wherein the poloxamer is selected from the group consisting of poloxamer 108, poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 288, poloxamer 388, poloxamer 407, and combinations thereof.

[0402] 37. The method of any one of paragraphs 30 to 36, wherein the poloxamer is poloxamer 338. 38. The method of any one of paragraphs 30 to 37, wherein the poloxamer is poloxamer 407.

[0403] 39. The method of any one of paragraphs 30 to 38, wherein the poloxamer is in the cell culture medium at a concentration of at least 0.01 mg / mL.

[0404] 40. The method of any one of paragraphs 30 to 39, wherein the poloxamer is in the cell culture medium at a concentration of between about 0.01 mg / mL and 10 mg / mL.

[0405] 41. The method of paragraph 40, wherein the poloxamer is in the cell culture medium at a concentration of at least 0.1 mg / mL.

[0406] 42. The method of paragraph 41 , wherein the poloxamer is in the cell culture medium at a concentration of at least 0.5 mg / mL.

[0407] 43. The method of paragraph 42, wherein the poloxamer is in the cell culture medium at a concentration of about 1 mg / mL.

[0408] 44. The method of any one of paragraphs 1 to 43, wherein the method comprises contacting the cells with the transduction enhancer for a period of time prior to contacting the cells with the enveloped virus.

[0409] 45. The method of paragraph 44, wherein the method comprises contacting the cells with the transduction enhancer for a period of at least 30 minutes prior to contacting the cells with the enveloped virus.

[0410] 46. The method of paragraph 45, wherein the method comprises contacting the cells with the transduction enhancer for about 1 hour prior to contacting the cells with the enveloped virus.

[0411] 47. The method of any one of paragraphs 1 to 43, wherein the method comprises contacting the cells with the enveloped virus and a transduction enhancer simultaneously.

[0412] 48. The method of any one of paragraphs 1 to 47, wherein the method comprises contacting the cells with the enveloped virus for a period of between 15 and 30 hours. 49. The method of any one of paragraphs 1 to 48, wherein the method comprises contacting the cells with the enveloped virus for a period of between 16 and 24 hours.

[0413] 50. The method of any one of paragraphs 1 to 49, wherein the cell culture medium further comprises protamine sulphate.

[0414] 51. The method of any one of paragraphs 1 to 50, wherein the cell culture medium further comprises one or more cytokines selected from the group consisting of stem cell factor (SCF), thrombopoietin (TPO), flt3 / flk2 ligand (Flt3L) and combinations thereof.

[0415] 52. The method of paragraph 51, wherein the one or more cytokines is in the cell culture medium at a concentration of between about 10 ng / mL and 1 mg / ml.

[0416] 53. The method of paragraph 51 or 52, wherein the one or more cytokines is in the cell culture medium at a concentration of between about 1 ng / mL and 1000 ng / mL.

[0417] 54. The method of paragraph 53, wherein the one or more cytokines is in the cell culture medium at a concentration of between about 10 ng / mL and 300 ng / mL.

[0418] 55. The method of any one of paragraphs 51 to 54, wherein the one or more cytokines is in the cell culture medium at a concentration of about 100 ng / mL.

[0419] 56. The method of any one of paragraphs 1 to 55, wherein the cell culture medium further comprises human serum albumin (HSA).

[0420] 57. The method of paragraph 56, wherein the HSA is in the cell culture medium at a concentration of between 0.1% and 10%.

[0421] 58. The method of paragraph 56 or 57, wherein the HSA is in the cell culture medium at a concentration of about 2%.

[0422] 59. The method of any one of paragraphs 1 to 58, wherein the cells are pre-stimulated for a period of between 16 and 24 hours in cell culture medium before transduction with the enveloped virus. 60. The method of any one of paragraphs 1 to 59, wherein the plurality of cells are in the cell culture medium at a density of between about 1 x 105cells / mL and 1 x IO10cells / mL.

[0423] 61. The method of any one of paragraphs 1 to 60, wherein the plurality of cells are in the cell culture medium at a density of between about 1 x 105cells / mL and 1 x 107cells / mL.

[0424] 62. The method of any one of paragraphs 1 to 61, wherein the plurality of cells are in the cell culture medium at a density of between about 1.0 x 106cells / mL and 4.0 x 106cells / mL.

[0425] 63. The method of any one of paragraphs 1 to 62, wherein the plurality of cells are in the cell culture medium at a density of about 2 x 106cells / mL.

[0426] 64. The method of any one of paragraphs 1 to 63, wherein the virus is added to the cell culture at a multiplicity of infection (MOI) of at least 0.01.

[0427] 65. The method of any one of paragraphs 1 to 64, wherein the virus is added to the cell culture at a MOI of at least 0.1.

[0428] 66. The method of any one of paragraphs 1 to 65, wherein the virus is added to the cell culture at a MOI of at least 1.

[0429] 67. The method of any one of paragraphs 1 to 66, wherein the cell culture is operated for a period of at least 24 hours.

[0430] 68. The method of any one of paragraphs 1 to 67, wherein the method further comprises harvesting one or more cells transduced with the enveloped virus.

[0431] 69. The method of paragraph 68, wherein the method further comprises washing the harvested transduced cells with a formulation medium.

[0432] 70. The method of paragraph 69, wherein the formulation medium comprises 0.9% saline and / or human serum albumin. 71. The method of any one of paragraphs 1 to 70, wherein the method results in a transduction efficiency of at least 20%.

[0433] 72. The method of any one of paragraphs 1 to 71, wherein the method results in a transduction efficiency of at least 30%.

[0434] The method of any one of paragraphs 1 to 72, wherein the method results in a transduction efficiency of at least 40%.

[0435] 74. The method of any one of paragraphs 1 to 73, wherein the method results in a transduction efficiency of at least 50%.

[0436] The method of any one of paragraphs 1 to 74, wherein the method results in a transduction efficiency of at least 60%.

[0437] 76. The method of any one of paragraphs 1 to 75, wherein the method results in a transduction efficiency of at least 70%.

[0438] 77. The method of any one of paragraphs 1 to 76, wherein the method results in a transduction efficiency of at least 80%.

[0439] 78. The method of any one of paragraphs 1 to 77, wherein the method results in a transduction efficiency of at least 90%.

[0440] 79. The method of any one of paragraphs 1 to 78, wherein the method results in at least a 1.5-fold increase in potency of the enveloped virus.

[0441] 80. The method of any one of paragraphs 1 to 79, wherein the method results in at least a 2-fold increase in potency of the enveloped virus.

[0442] 81. The method of any one of paragraphs 1 to 80, wherein the method results in at least a 2.5-fold increase in potency of the enveloped virus.

[0443] 82. The method of any one of paragraphs 1 to 81, wherein the method increases the vector copy number (VCN) in the plurality of cells. 83. The method of paragraph 82, wherein the method further comprises seeding a population of between 100 and 500 transduced cells in a differentiation medium for a period of at least 14 days to produce one or more colony forming units (CFUs) of differentiated transduced cells.

[0444] 84. The method of paragraph 83, wherein the cells differentiate into myeloid or erythroid lineage.

[0445] 85. The method of paragraph 83 or 84, wherein the method further comprises isolating one or more CFUs.

[0446] 86. The method of paragraph 85, wherein the method increases the vector copy number (VCN) in an isolated CFU of myeloid or erythroid transduced cells as determined by ddPCR, compared to a cell transduced in the absence of the transduction enhancer.

[0447] 87. The method of paragraph 86, wherein the method increases the VCN by at least 10%, compared to a cell transduced in the absence of the transduction enhancer.

[0448] 88. The method of paragraph 86 or 87, wherein the method increases the VCN by at least 20%, compared to a cell transduced in the absence of the transduction enhancer.

[0449] 89. The method of any one of paragraphs 1 to 88, wherein the method increases the transduction efficiency of a cell that differentiates into a myeloid cell, compared to a cell transduced in the absence of the transduction enhancer.

[0450] 90. The method of paragraph 89, wherein the transduction of cells that differentiate into an erythroid cell is not reduced.

[0451] 91. The method of any one of paragraphs 1 to 90, wherein the enveloped virus comprises a transgene introduced into its genome.

[0452] 92. The method of paragraph 91, wherein the method results in an increase in expression of the transgene by the cell by at least about 10% or more than a method in the absence of the transduction enhancer. 93. The method of any one of paragraphs 69 to 92, wherein the transduced cell has a viability of at least 75% for at least 24 hours, or for at least 48 hours, or for at least 72 hours after transduction with the enveloped virus.

[0453] 94. The method of any one of paragraphs 1 to 93, wherein the cell culture has a volume of between about 100 uL and 500 mL.

[0454] 95. The method of any one of paragraphs 1 to 94, wherein the cell culture has a volume of about 125 uL, or about 250 uL, or about 500 uL, or about 1 mL, or about 2 mL, or about 5 mL, or about 10 mL, or about 20 mL, or about 30 mL, or about 50 mL, or about 80 mL, or about 100 mL, or about 200 mL, about 250 mL, or about 300 mL.

[0455] 96. The method of any one of paragraphs 1 to 95, wherein the cell culture is at a pH of between 6.0 and 8.0 and / or at a temperature of between 35-39°C and / or < 5% CO2.

[0456] 97. The method of any one of paragraphs 69 to 82 or 91 to 96, wherein the method further comprises formulating the population of harvested transduced cells with a cryopreservation medium.

[0457] 98. The method of paragraph 97, wherein the cryopreservation medium is an animal protein-free, serum free cryopreservation medium.

[0458] 99. The method of paragraph 98, wherein the cryopreservation medium comprises DMSO.

[0459] 100. The method of any one of paragraphs 1 to 99, wherein the cell is a hematopoietic stem cell (HSC) or a hematopoietic progenitor cell (HPC).

[0460] 101. The method of paragraph 100, wherein the HSC or HPC is a CD34 positive (CD34+) cell.

[0461] 102. The method of paragraph 100 or 101, wherein the HSC or HPC are of an erythroid lineage (burst- forming unit-erythroid; BFU-E) and / or a myeloid lineage (colony forming unit that generates granulocyte, erythrocyte, monocyte, megakaryocyte cells; CGU- GEMM). 103. The method of any one of paragraphs 1 to 102, wherein the enveloped virus is a retrovirus.

[0462] 104. The method of paragraph 103, wherein the retrovirus is a lentivirus.

[0463] 105. A genetically modified cell expressing a transgene, wherein the genetically modified cell is transduced with an enveloped virus comprising the transgene by a method of any one of paragraphs 1 to 82 or 91 to 104.

[0464] 106. A method of producing a genetically modified cell for gene therapy, the method comprising transducing a cell with an enveloped virus comprising a polynucleotide expressing a transgene by a method of any one of paragraphs 1 to 82 or 91 to 104, thereby producing a genetically modified cell.

[0465] 107. A composition comprising a population of genetically modified cells of paragraph 105 for use as a medicament.

[0466] The present disclosure is described further in the following non-limiting examples.

[0467] EXAMPLES

[0468] Example 1: Transduction enhancers for lenti viral transduction

[0469] Some widely used transduction enhancers (TE) include protamine sulfate, polybrene, prostaglandin E2, dmPGE2, cyclosporin H, cyclosporin A, caraphenol A, rapamycin, and staurosporine. Poloxamer 388 (F108) has also recently been shown to be effective (Sirion, Bluebird).

[0470] The effect of poloxamer 407 (F127) was investigated on lentiviral transduction and was found to be comparable to F108 (Figure 1).

[0471] Example 2: Identification of nebivolol as a lentiviral transduction enhancer alone or in combination with F127

[0472] CD34+cells were pre- stimulated overnight (16-24h) at 2E6 cells / mL in prestimulation media (HSC Brew + IX HSC Brew Supplement) plus 2% human serum albumin (HSA) or stem cell growth medium (SCGM) plus 2% HSA, containing 100 ng / pL each of thrombopoietin (TPO), flt3 / flk2 ligand (FLT3L), and stem cell factor (SCF) at 37°C under 5% CO2.

[0473] Cells were washed and seeded for transduction at 2E6 cells / mL in pre-stimulation media (as above). Cells were transduced overnight (16-24h) with GFP lentiviral vector (LVV) at multiplicity of infection (MOI) of 1 alone (with 1% DMSO) or in combination with transduction enhancers (F127 at 0.5 mg / mL) to establish benchmarks (Figures 2A- C). Post-transduction cells were washed and seeded at 0.5E6 cells / mL into myeloid expansion media (StemSpan SFEM II with IX Supplement) for 48 hours prior to assessment of GFP expression by flow cytometry.

[0474] Using Fl 27 as the current benchmark, the combination of nebivolol and Fl 27 outperforms F127 alone at nebivolol concentrations greater than 3uM (Figure 2A).

[0475] The additive effect was independent of the transduction media used (Figure 2B and C).

[0476] Example 3: Lentiviral transduction in the presence of nebivolol, and L- and D- enantiomers

[0477] CD34+cells were pre- stimulated overnight (16-24h) at 2E6 cells / mL in prestimulation media (HSC Brew + IX HSC Brew Supplement + 2% HSA or SCGM + 2% HSA containing 100 ng / pL each of TPO, FLT3L, and SCF) at 37°C under 5% CO2. Cells were washed and seeded for transduction at 2E6 cells / mL in pre-stimulation media (as above). Cells were transduced overnight (16-24h) with GFP LVV at MOI 1 alone (with 1% DMSO) or in combination with transduction enhancers at indicated concentrations. Post-transduction cells were assessed for viability and fold-growth prior to being washed and seeded at 0.5E6 cells / mL into myeloid expansion media (StemSpan SFEM II with IX Supplement). After an additional 48 hours, cells were assessed for viability and fold-growth by AOPI staining

[0478] Maximum tolerable concentration (before viability and growth impacts) is lOuM. Cell death observed at concentrations > 15uM. L-nebivolol is somewhat more tolerable than D-nebivolol or racemic mixtures when assessing both viability and fold growth (Figure 3A to 3D).

[0479] Example 4: Synergistic effect of nebivolol and F127

[0480] CD34+cells were pre- stimulated overnight (16-24h) at 2E6 cells / mL in prestimulation media (HSC Brew + IX HSC Brew Supplement + 2% HSA or SCGM + 2% HSA containing 100 ng / pL each of TPO, FLT3L, and SCF) at 37°C under 5% CO2. Cells were washed and seeded for transduction at 2E6 cells / mL in pre-stimulation media (as above). Cells were transduced overnight (16-24h) with GFP LVV at MOI 1 alone (with 1% DMSO) or in combination with transduction enhancers (F127 at 0.5 mg / mL and / or lOpM Nebivolol). Post-transduction cells were washed and seeded at 0.5E6 cells / mL into myeloid expansion media (StemSpan SFEM II with IX Supplement) for 48 hours prior to assessment of GFP expression by flow cytometry.

[0481] The effect of nebivolol at lOuM in combination with 0.5mg / ml F127, compared with 0.5mg / ml F127 alone was investigated.

[0482] As shown in Figure 4A, the combination effect significantly increases transduction, and is independent of media type (Figures 4B and 4C).

[0483] Example 5: Multiplicity of infection (MOI) curves

[0484] CD34+cells were pre- stimulated overnight (16-24h) at 2E6 cells / mL in prestimulation media (HSC Brew + IX HSC Brew Supplement + 2% HSA or SCGM + 2% HSA containing 100 ng / pL each of TPO, FLT3L, and SCF) at 37°C under 5% CO2. Cells were washed and seeded for transduction at 2E6 cells / mL in pre-stimulation media (as above). Cells were transduced overnight (16-24h) with GFP LVV at range of MOIs indicated, either alone or in combination with transduction enhancers at indicated concentrations. Post-transduction cells were washed and seeded at 0.5E6 cells / mL into myeloid expansion media (StemSpan SFEM II with IX Supplement) for 48 hours prior to assessment of GFP expression by flow cytometry.

[0485] As shown in Figure 5, the combination of lOuM nebivolol and 0.5 mg / ml F127 resulted in an increase in lentivirus potency (left shift) by 2-3 fold, and increase in lentivirus transduction capacity (upward shift).

[0486] Example 6: Synergistic effect of nebivolol isomers and F127

[0487] CD34+cells were pre- stimulated overnight (16-24h) at 2E6 cells / mL in prestimulation media (HSC Brew + IX HSC Brew Supplement + 2% HSA or SCGM + 2% HSA containing 100 ng / pL each of TPO, FLT3L, and SCF) at 37°C under 5% CO2. Cells were washed and seeded for transduction at 2E6 cells / mL in pre-stimulation media (as above). Cells were transduced overnight (16-24h) with GFP LVV at MOI 1 alone (with 1% DMSO) or in combination with transduction enhancers at indicated concentrations (alone or with F127 at 0.5 mg / mL) Post-transduction cells were washed and seeded at 0.5E6 cells / mL into myeloid expansion media (StemSpan SFEM II with IX Supplement) for 48 hours prior to assessment of GFP expression by flow cytometry. As shown in Figure 6, transduction with 10 pM nebivolol was comparable to transduction with 10 pM L-nebivolol, D-nebivolol, and racemic-nebivolol. The additive effect of combination nebivolol and Fl 27 was comparable to the additive effect of combination L-nebivolol, D-nebivolol, and racemic -nebivolol with F127.

[0488] Example 7: Timing of addition of transduction enhancer

[0489] CD34+cells were pre- stimulated overnight (16-24h) at 2E6 cells / mL in prestimulation media (SCGM + 2% HSA containing 100 ng / pL each of TPO, FLT3L, and SCF) at 37°C under 5% CO2. Cells were washed and seeded for transduction at 2E6 cells / mL in pre- stimulation media (as above). Cells were then either treated with 10 pM Nebivolol at Ihr prior to LVV addition or left in culture without transduction enhancers for Ihr. Immediately prior to addition of LVV, TE-treated cells were either washed and re- seeded at 2E6 cells / mL or left in culture media containing TEs. Cells were transduced overnight (16-24h) with GFP LVV at MOI 1 alone or in combination with transduction enhancers (10 pM Nebivolol and / or 0.5 mg / mL F127). Post-transduction cells were washed and seeded at 0.5E6 cells / mL into myeloid expansion media (StemSpan SFEM II with IX Supplement) for 48 hours prior to assessment of GFP expression by flow cytometry.

[0490] As shown in Figure 7, pre- stimulating cells for 1 hour was comparable to adding the transduction enhancers simultaneously with LVV, both with F127 and without F127.

[0491] Example 8: Nebivolol is synergistic with both F127 and F108

[0492] CD34+cells were pre- stimulated overnight (16-24h) at 2E6 cells / mL in prestimulation media (HSC Brew + IX HSC Brew Supplement + 2% HSA or SCGM + 2% HSA containing 100 ng / pL each of TPO, FLT3L, and SCF) at 37°C under 5% CO2. Cells were washed and seeded for transduction at 2E6 cells / mL in pre-stimulation media (as above). Cells were transduced overnight (16-24h) with GFP LVV at MOI 1, either with Img / mL F108 or F127 in concentrations of 0.5 mg / mL, 0.1 mg / mL, or 0.05 mg / mL, alone or in combination with lOuM nebivolol.

[0493] As shown in Figure 8, there was comparable activity between F127 and F108 with and without lOuM nebivolol at MOI 1. There was increased activity for both Fl 27 and F108 with the addition of nebivolol. The effect of nebivolol was readily observable at all tested concentrations of F127 between 0.05 mg / mL and 0.5 mg / mL.

[0494] Example 9: Transduction efficiency in erythroid and myeloid colony forming units

[0495] CD34+cells were pre- stimulated overnight (16-24h) at 2E6 cells / mL in prestimulation media (SCGM + 2% HSA containing 100 ng / pL each of TPO, FLT3L, and SCF) at 37°C under 5% CO2. Cells were washed and seeded for transduction at 2E6 cells / mL in pre- stimulation media (as above). Cells were transduced overnight (16-24h) with GFP LVV at MOI 1 alone or in combination with transduction enhancers (0.5 mg / mL F127 + 8 pg / mL Protamine Sulphate ± 10 pM Nebivolol or L-Nebivolol).

[0496] Post-transduction cells were washed and seeded into CFU assay: 250 or 125 cells were seeded into MethoCult (semi-permeable culture media) for 14-day differentiation protocol. After 14 days, approximately 30-35 CFU were picked and transferred into individual wells for DNA extraction and ddPCR for viral transgene and reference gene expression. Data is representative of multiple CFU defined by Erythroid (BFU-E type) or Myeloid (CFU-G / GM / GEMM type) with Average data representing combination of both CFU types.

[0497] As shown in Figure 9A, significantly higher vector copies were obtained from Erythroid CFU following transduction with combination of Fl 27 and nebivolol or L- nebivolol.

[0498] As shown in Figure 9B, significantly higher percentages of transduction were observed in erythroid CFU, myeloid CFU, and on average, following transduction with combination of Fl 27 and nebivolol or L-nebivolol.

[0499] Example 10: Transduction Enhancers at large scale with a head-to-head comparison of standard process

[0500] Transduction enhancers F127 (0.5 mg / mL) and Protamine Sulfate (8 ug / mL) with and without the addition of Nebivolol (10 uM) were tested at large scale utilizing SCGM media supplemented with 2% HSA and a GFP vector at MOI 3. As shown in Figure 10, nebivolol increases the VCN of erythroid CFU above that obtained with Fl 27 alone during transduction at large scale, corroborating what is observed at small scale.

Claims

CLAIMS:

1. A method of transducing a cell with an enveloped virus in a cell culture, the method comprising culturing a plurality of cells in a cell culture medium and contacting the cells with the enveloped virus and a transduction enhancer, wherein the transduction enhancer comprises a compound of Formula (I) or a stereoisomer, a racemate, a pharmaceutically acceptable salt or a solvate thereof:Formula (I) whereinX1and X2are independently selected from O, N, S or CFh;L1and L2are independently absent or selected from -Ci-ealkyl-, -Ci ealkcnyl-^ or -Ci ealkynyl-, each of which may be optionally substituted with one or more R4or R5,R1is selected from H, -Ci-ealkyl-, -Ci ealkcnyl-^ or -Ci ealkynyl-;R2and R3are independently selected from H, halogen, or OH; andR4and R5are independently selected from halogen, OH, -Ci-4alkyl-, and -Ci- 4alkoxy-2. A method of increasing an enveloped virus transduction efficiency of a cell in a cell culture, the method comprising culturing a plurality of cells in a cell culture medium and contacting the cells with the enveloped virus and a transduction enhancer, wherein the transduction enhancer comprises a compound of Formula (I) or a stereoisomer, a racemate, a pharmaceutically acceptable salt or a solvate thereof:Formula (I) whereinX1and X2are independently selected from O, N, S or CH2;L1and L2are independently absent or selected from -Ci-ealkyl-, -Ci ealkcnyl-^ or -Ci ealkynyl-, each of which may be optionally substituted with one or more R4or R5,R1is selected from H, -Ci-ealkyl-, -Ci ealkcnyl-^ or -Ci ealkynyl-;R2and R3are independently selected from H, halogen, or OH; andR4and R5are independently selected from halogen, OH, -Ci-4alkyl-, and -Ci- 4alkoxy-3. A method of increasing potency of an enveloped virus during transduction of a cell in a cell culture, the method comprising culturing a plurality of cells in a cell culture medium and contacting the cells with the enveloped virus and a transduction enhancer, wherein the transduction enhancer comprises a compound of Formula (I) or a stereoisomer, a racemate, a pharmaceutically acceptable salt or a solvate thereof:Formula (I) whereinX1and X2are independently selected from O, N, S or CH2;L1and L2are independently absent or selected from -Ci-ealkyl-, -C2-6alkenyl-;>or -C2-6alkynyl-, each of which may be optionally substituted with one or more R4or R5,R1is selected from H, -Ci-ealkyl-, -C2-6alkenyl-;>or -C2-6alkynyl-;R2and R3are independently selected from H, halogen, or OH; andR4and R5are independently selected from halogen, OH, -Ci-4alkyl-, and -Ci- 4alkoxy-4. A method of increasing transduction efficiency of a cell that differentiates into a myeloid cell in a cell culture, the method comprising culturing a plurality of cells in a cell culture medium and contacting the cells with the enveloped virus and a transduction enhancer, wherein the transduction enhancer comprises a compound of Formula (I) or a stereoisomer, a racemate, a pharmaceutically acceptable salt or a solvate thereof:Formula (I) whereinX1and X2are independently selected from O, N, S or CH2;L1and L2are independently absent or selected from -Ci-ealkyl-, -Ci ealkcnyl-^ or -Ci ealkynyl-, each of which may be optionally substituted with one or more R4or R5,R1is selected from H, -Ci-ealkyl-, -C2-6alkenyl-;>or -C2-6alkynyl-;R2and R3are independently selected from H, halogen, or OH; andR4and R5are independently selected from halogen, OH, -Ci-4alkyl-, and -Ci- 4alkoxy-5. A method of increasing vector copy number in a cell in a cell culture, the method comprising culturing a plurality of cells in a cell culture medium and contacting the cells with the enveloped virus and a transduction enhancer, wherein the transduction enhancer comprises a compound of Formula (I) or a stereoisomer, a racemate, a pharmaceutically acceptable salt or a solvate thereof:Formula (I) whereinX1and X2are independently selected from O, N, S or CH2;L1and L2are independently absent or selected from -Ci-ealkyl-, -C2-6alkenyl-;>or -C2-6alkynyl-, each of which may be optionally substituted with one or more R4or R5,R1is selected from H, -Ci-ealkyl-, -C2-6alkenyl-;>or -C2-6alkynyl-;R2and R3are independently selected from H, halogen, or OH; andR4and R5are independently selected from halogen, OH, -Ci-4alkyl-, and -Ci- 4alkoxy-6. The method of claim 5, wherein:(i) the cell is a cell that differentiates into an erythroid cell; and / or(ii) transduction of cells that differentiate into erythroid cells is not reduced.

7. The method of claim 1, wherein:L1has a structure *-L3CH(R4)-, and L2has a structure *-L4CH(R5)-, whereinL3and L4are independently absent or selected from -Ci-4alkyl-, -Ci ^alkenyl-, or -Ci-4alkynyl-; and* indicates a single bond to the central N.

8. The method of claim 7, wherein L3and L4are both -Ci alkyl-.

9. The method of claim 1, wherein:(i) X1and X2are both O; and / or(ii)R4and R5are both OH; and / or(iii)R1is H; and / or(iv)R2and R3are both F.

10. The method of claim 1, wherein the compound of Formula (I) is:(i) nebivolol and / or has the following structure:and / or(ii) L-nebivolol and / or has the following structure:and / or(iii)D-nebivolol and / or has the following structure:

11. The method of claim 1, wherein the transduction enhancer comprises and / or substantially consists of:or pharmaceutically acceptable salts or solvates thereof.

12. The method of claim 11, wherein the transduction enhancer comprises and / or substantially consists of a racemate of:or pharmaceutically acceptable salts of solvates thereof, in a ratio of between about 4:6 to about 6:4, or between about 4.5:5.5 to about 5.5:4.5, or about 1: 1.

13. The method of claim 1, wherein the transduction enhancer comprises and / or substantially consists of L-nebivolol or:

14. The method of claim 1, wherein the compound is in the cell culture medium at a concentration of at least 1 pM, or at least 3 pM, or at least 5 pM, or at least 10 pM, or between 1 pM and 20 pM.

15. The method of claim 1, wherein the cell culture further comprises a water-soluble non-ionic triblock copolymer.

16. The method of claim 15, wherein the water-soluble non-ionic triblock copolymer has:(A) an average molecular weight (in g / mol):(i) greater than about 9800, 9900, 10000, 10100, 10200, 10300, 10400, 10500, 10600, 10700, 10800, 10900, 11000, 11100, 11200, 11300, 11400, 11500, 11600, 11700, 11800, 11900, 12000, 12100, 12200, 12300, 12400, 12500, 12600, 12700, 12800, 12900, 13000, 13100, 13200, 13300, 13400, 13500, 13600, 13700, 13800, 13900, 14000, 14100, 14200, 14300, 14400, 14500, 14600, 14700, 14800, 14900 or 15000;(ii) greater than about 12000 g / mol; and / or(iii) of about 12600 g / mol; and / or(B) a viscosity (in Pa-s) of between about 2.0 and about 3.5.

17. The method of claim 15, wherein the water-soluble non-ionic triblock copolymer is a poloxamer.

18. The method of claim 17, wherein the poloxamer has:(A) an average polyoxypropylene content (in g / mol) of:(i) about or greater than about 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900 or 5000; and / or(ii) about or greater than about 3000 g / mol; and / or(B) an average polyoxyethylene content (in % w / w) of:(i) about or greater than about 30, 40, 50, 60, 70, 80 or 90; and / or(ii) about or greater than about 70% w / w.

19. The method of claim 17, wherein the poloxamer is:(i) selected from the group consisting of poloxamer 105, poloxamer 123, poloxamer 124, poloxamer 182, poloxamer 184, poloxamer 188, poloxamer 234, poloxamer 235, poloxamer 237, poloxamer 238, poloxamer 288, poloxamer 333, poloxamer 334, poloxamer 335, poloxamer 338, poloxamer 403, poloxamer 407, and combinations thereof; or(ii) selected from the group consisting of poloxamer 108, poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 288, poloxamer 388, poloxamer 407, and combinations thereof; or(iii)poloxamer 338; or(iv) poloxamer 407.

20. The method of claims 17, wherein the poloxamer is in the cell culture medium at a concentration of at least 0.01 mg / mL, or between about 0.01 mg / mL and 10 mg / mL, or at least 0.1 mg / mL, or at least 0.5 mg / mL, or about 1 mg / mL.

21. The method of claim 1, wherein the method comprises:(i) contacting the cells with the transduction enhancer for a period of time prior to contacting the cells with the enveloped virus; and / or(ii) contacting the cells with the transduction enhancer for a period of at least 30 minutes prior to contacting the cells with the enveloped virus; and / or(iii)contacting the cells with the transduction enhancer for about 1 hour prior to contacting the cells with the enveloped virus.

22. The method of claim 1, wherein the method comprises:(i) contacting the cells with the enveloped virus and a transduction enhancer simultaneously; and / or(ii) contacting the cells with the enveloped virus for a period of between 15 and 30 hours; and / or(iii)contacting the cells with the enveloped virus for a period of between 16 and 24 hours.

23. The method of claim 1, wherein the cell culture medium further comprises:(i) protamine sulphate; and / or(ii)one or more cytokines selected from the group consisting of stem cell factor (SCF), thrombopoietin (TPO), flt3 / flk2 ligand (Flt3L) and combinations thereof.

24. The method of claim 23, wherein the one or more cytokines is in the cell culture medium at a concentration of:(i) between about 10 ng / mL and 1 mg / ml; and / or(ii) between about 1 ng / mL and 1000 ng / mL; and / or(iii)between about 10 ng / mL and 300 ng / mL; and / or(iv) about 100 ng / mL.

25. The method of claim 1 , wherein the cell culture medium further comprises human serum albumin (HSA).

26. The method of claim 25, wherein the HSA is in the cell culture medium at a concentration of between 0.1% and 10%, or about 2%.

27. The method of claim 1, wherein the cells are pre-stimulated for a period of between 16 and 24 hours in cell culture medium before transduction with the enveloped virus.

28. The method of claim 1, wherein the plurality of cells are in the cell culture medium at a density of:(i) between about 1 x 105cells / mL and 1 x 1010cells / mL; and / or(ii) between about 1 x 105cells / mL and 1 x 107cells / mL; and / or(iii)between about 1.0 x 106cells / mL and 4.0 x 106cells / mL; and / or(iv)about 2 x 106cells / mL.

29. The method of claim 1, wherein the virus is added to the cell culture at a multiplicity of infection (MOI) of at least 0.01, or at least 0.1, or at least 1.

30. The method of claim 1, wherein the cell culture is operated for a period of at least 24 hours.

31. The method of claim 1, wherein the method further comprises:(i) harvesting one or more cells transduced with the enveloped virus; and / or(ii) washing the harvested transduced cells with a formulation medium.

32. The method of claim 31, wherein the formulation medium comprises 0.9% saline and / or human serum albumin.

33. The method of claim 1, wherein the method results in a transduction efficiency of at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%.

34. The method of claim 1, wherein the method results in at least a 1.5-fold increase in potency of the enveloped virus, or at least a 2-fold increase in potency of the enveloped virus, or at least a 2.5-fold increase in potency of the enveloped virus.

35. The method of claim 1, wherein the method increases the vector copy number (VCN) in the plurality of cells.

36. The method of claim 35, wherein the method further comprises seeding a population of between 100 and 500 transduced cells in a differentiation medium for a period of at least 14 days to produce one or more colony forming units (CFUs) of differentiated transduced cells.

37. The method of claim 36, wherein the cells differentiate into myeloid or erythroid lineage.

38. The method of claim 37, wherein the method further comprises isolating one or more CFUs.

39. The method of claim 38, wherein the method:(i) increases the vector copy number (VCN) in an isolated CFU of myeloid or erythroid transduced cells as determined by ddPCR, compared to a cell transduced in the absence of the transduction enhancer; and / or(ii) increases the VCN by at least 10%, compared to a cell transduced in the absence of the transduction enhancer; and / or(iii)increases the VCN by at least 20%, compared to a cell transduced in the absence of the transduction enhancer.

40. The method of claim 1, wherein the method increases the transduction efficiency of a cell that differentiates into a myeloid cell, compared to a cell transduced in the absence of the transduction enhancer.

41. The method of claim 40, wherein the transduction of cells that differentiate into an erythroid cell is not reduced.

42. The method of claim 1, wherein the enveloped virus comprises a transgene introduced into its genome.

43. The method of claim 42, wherein the method results in an increase in expression of the transgene by the cell by at least about 10% or more than a method in the absence of the transduction enhancer.

44. The method of claim 31, wherein the transduced cell has a viability of at least 75% for at least 24 hours, or for at least 48 hours, or for at least 72 hours after transduction with the enveloped virus.

45. The method of claim 1, wherein the cell culture has a volume of:(i) between about 100 uL and 500 mL; and / or(ii) about 125 uL, or about 250 uL, or about 500 uL, or about 1 mL, or about 2 mL, or about 5 mL, or about 10 mL, or about 20 mL, or about 30 mL, or about 50 mL, or about 80 mL, or about 100 mL, or about 200 mL, about 250 mL, or about 300 mL.

46. The method of claim 1, wherein the cell culture is at a pH of between 6.0 and 8.0 and / or at a temperature of between 35-39°C and / or < 5% CO2.

47. The method of claims 31, wherein the method further comprises formulating the population of harvested transduced cells with a cryopreservation medium.

48. The method of claim 47, wherein the cryopreservation medium:(i) is an animal protein-free, serum free cryopreservation medium; and / or(ii) comprises DMSO.

49. The method of claim 1, wherein the cell is a hematopoietic stem cell (HSC) or a hematopoietic progenitor cell (HPC).

50. The method of claim 49, wherein the HSC or HPC:(i) is a CD34 positive (CD34+) cell; and / or(ii)are of an erythroid lineage (burst-forming unit-erythroid; BFU-E) and / or a myeloid lineage (colony forming unit that generates granulocyte, erythrocyte, monocyte, megakaryocyte cells; CGU-GEMM).

51. The method of claim 1, wherein the enveloped virus is a retrovirus.

52. The method of claim 51, wherein the retrovirus is a lentivirus.

53. A genetically modified cell expressing a transgene, wherein the genetically modified cell is transduced with an enveloped virus comprising the transgene by a method of claim 1.

54. A method of producing a genetically modified cell for gene therapy, the method comprising transducing a cell with an enveloped virus comprising a polynucleotide expressing a transgene by a method of claim 1, thereby producing a genetically modified cell.

55. A composition comprising a population of genetically modified cells of claim 53 for use as a medicament.

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