Novel transduction enhancers and uses thereof

Abstract

To provide novel transduction enhancers and uses thereof.SOLUTION: The present invention relates to a method for transducing a target cell, the method comprising the step of contacting a target cell with a retroviral vector and a compound capable of enhancing transduction efficiency or a combination of such compounds, wherein the target cell is pre- and / or co-stimulated by pre- and / or co-incubation with a transduction enhancing compound or a combination of transduction enhancing compounds prior to and / or during contacting the target cell with the retroviral vector.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for transducing target cells, comprising the step of contacting the target cells with a retroviral vector and a compound or combination of such compounds that is capable of enhancing transduction efficiency, wherein the target cells are pre-stimulated and / or co-stimulated by pre-incubation and / or co-incubation with a transduction-enhancing compound or combination of transduction-enhancing compounds before and / or during contacting the target cells with the retroviral vector. [Background technology]

[0002] Background of the Invention In gene addition gene therapy, virus-derived vectors are used to introduce a corrective gene (called a transgene) in the form of a cDNA into cells that have a genetic loss-of-function or limiting-function mutation that causes a disease. This introduced cDNA copy of the mutated gene consists of the healthy (unmutated) sequence of the defective gene. In the treated cells, the activity of the introduced transgene compensates for the missing activity of the defective gene.

[0003] For gene therapy of diseases arising from the hematopoietic system (i.e., red blood cell diseases, platelet diseases, and immune system diseases) and for skin diseases, the creation of skin grafts from gene-tagged epidermal / keratinocyte stem cells using retroviral vectors is the state of the art. These vectors are used to engineer hematopoietic stem cells (HSCs) or epidermal stem cells (ESCs). The process by which retroviral vectors stably integrate into the recipient HSC or ESC genome to add corrective cDNA (i.e., transgenes) is called transduction. After intravenous reinfusion of the engineered HSCs into the patient, these HSCs engraft in the bone marrow, where the added corrective cDNA is transmitted to all daughter cells upon HSC proliferation and subsequent differentiation into different blood and immune system cells. Correspondingly, transgenic skin is maintained after transplantation of skin grafts produced from transgenic ESCs.

[0004] Today, primarily self-inactivating lentiviral (HIV-based) vectors pseudotyped with the vesicular stomatitis virus (VSV-G) envelope are used for gene addition into HSCs (Cartier et al., (2009) Science, PMID: 19892975; Cavazzana-Calvo et al., (2010) Nature 467:318-22; Aiuti et al., (2013) Science 341:1233151).

[0005] The clinical success of gene therapy depends primarily on the efficiency of gene addition, i.e., the level of transgene insertion mediated by the retroviral vector, and the dose of corrected cells that can be administered to patients. The efficiency of gene addition per cell depends on the fold excess of viral vector over target cells used during the transduction process, called the multiplicity of infection (MOI). Successful gene addition results can be quantified by determining the average number of integrated vector copies per cell in a cell population, called the vector copy number (VCN). The latter directly depends on the quality of the retroviral transduction process; i.e., the higher the VCN, the better the optimal retroviral transduction conditions can be achieved. For example, a VCN of 0.5 indicates that, on average, one out of two cells in a transduced cell population acquires one copy of the therapeutic gene. A VCN of 2 corresponds to an average of two copies of the therapeutic gene per cell in a transduced cell population.

[0006] The retroviral transduction process involves the following series of events: contact of virus-derived therapeutic particles with cells in cell culture (ex vivo), binding of the viral particles to the cell surface of the target cells, introduction of the therapeutic retroviral RNA into the target cells, reverse transcription of the retroviral RNA into proviral double-stranded DNA containing the therapeutic cDNA sequence, and successful integration into the genome of the target cells. Cell culture conditions during ex vivo retroviral transduction are paramount to the efficiency of transduction. Optimal cell culture conditions during transduction should 1) maintain cell identity (e.g., stemness of HSCs), 2) preserve the ability of cells, e.g., HSCs, to engraft, for example, in the bone marrow, upon reinfusion into the patient, and 3) allow efficient transduction (i.e., result in high levels of gene addition).

[0007] To enhance retroviral transduction, various approaches have been proposed in the past, focusing on (1) improving the contact between the retroviral vector and the target cell and (2) the entry of the retrovirus into the target cell.

[0008] Thus, there is a need for new compounds and approaches to increase the transduction efficiency of human cells with gene therapy vectors, particularly lentiviral vectors encoding therapeutic transgenes of interest.

[0009] In particular, there is a need for compounds or combinations of compounds that result in increased transduction efficiency.

[0010] Furthermore, there is a need in the art for clinically safe compounds that increase the transduction efficiency of gene therapy vectors.

[0011] The present invention provides such novel compounds and approaches. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Cartier et al. (2009) Science, PMID:19892975 [Non-patent document 2] Cavazzana-Calvo et al. (2010) Nature 467:318-22 [Non-patent document 3] Aiuti et al. (2013) Science 341:1233151 Summary of the Invention [Means for solving the problem]

[0013] Summary of the Invention The present invention is characterized in the embodiments and claims provided herein.

[0014] In particular, the present invention relates to, inter alia, the following aspects: 1. A method for transducing target cells, comprising the step of contacting the target cells with a retroviral vector and a compound or combination of such compounds that can enhance transduction efficiency, wherein the target cells are pre-stimulated and / or co-stimulated by pre-incubation and / or co-incubation with the transduction-enhancing compound or combination of transduction-enhancing compounds before and / or during contacting the target cells with the retroviral vector. 2. The method according to aspect 1, wherein the pre-incubation period is from 0.5 hours to 10 hours, particularly from 1 hour to 5 hours, particularly 2 hours. 3. The method of embodiment 1 or embodiment 2, wherein the transduction-enhancing compound is selected from the group consisting of silibinin, midostaurin, amphotericin B, nystatin, and natamycin, or a combination thereof. 4. The method of aspect 1 or aspect 2, wherein said transduction-enhancing compound is selected from the group consisting of resveratrol, everolimus, and prostaglandin E2, or a combination thereof. 5. The method of aspect 3, wherein the final concentration of the transduction-enhancing compound is about 0.05 μM to 500 μM, particularly 0.1 μM to 10 μM, for silibinin, midostaurin, amphotericin B and natamycin, and 50 μM to 150 μM for nystatin. 6. The method of aspect 1 or aspect 2, wherein said transduction enhancing compound is a poloxamer-based polymer, preferably poloxamer synperonic F108, or poloxamer 407 having a molecular weight of between 11 kDa and 15 kDa. 7. The method of aspect 5, wherein the final concentration of the transduction enhancing compound is between about 50 μg / ml and 5,000 μg / ml. 8. The method of embodiment 1 or embodiment 2, wherein the transduction-enhancing compound is a mixture of deoxyribonucleosides comprising 2'-deoxythymidine, 2'-deoxyadenosine, 2'-deoxyguanosine, and 2'-deoxycytidine. 9. The method of embodiment 8, wherein the final concentration of each deoxyribonucleoside is between about 0.1 mM and 10 mM of each deoxynucleoside. 10. The method of aspect 1 or aspect 2, wherein the transduction-enhancing compound is a polymer selected from the group consisting of a PEG-PCL-PEG polymer, a PEG-PLGA-PEG polymer, and a PEG-PLA-PEG polymer. 11. The method of aspect 10, wherein the final concentration of the transduction-enhancing compound is about 20 μg / ml to 5,000 μg / ml. 12. The method of embodiment 10 or embodiment 11, wherein a combination of a transduction-enhancing compound is used, comprising silibinin and a polymer selected from the group consisting of PEG-PCL-PEG polymer, PEG-PLGA-PEG polymer, and PEG-PLA-PEG polymer, particularly at a final concentration of about 0.1 μM to 25 μM silibinin and about 20 μg / ml to 5,000 μg / ml of polymer. 13. The method of embodiment 10 or embodiment 11, wherein a combination of transduction-enhancing compounds is used comprising midostaurin and a polymer selected from the group consisting of PEG-PCL-PEG polymers, PEG-PLGA-PEG polymers, and PEG-PLA-PEG polymers, particularly at a final concentration of about 0.05 μM to 20 μM midostaurin and about 20 μg / ml to 5000 μg / ml of polymer. 14. The method of embodiment 10 or embodiment 11, wherein a combination of transduction-enhancing compounds is used comprising amphotericin B and a polymer selected from the group consisting of PEG-PCL-PEG polymer, PEG-PLGA-PEG polymer, and PEG-PLA-PEG polymer, particularly at a final concentration of about 0.1 μM to 20 μM amphotericin B and about 20 μg / ml to 5,000 μg / ml of polymer. 15. The method of embodiment 10 or embodiment 11, wherein a combination of transduction-enhancing compounds is used comprising nystatin and a polymer selected from the group consisting of PEG-PCL-PEG polymers, PEG-PLGA-PEG polymers, and PEG-PLA-PEG polymers, particularly at a final concentration of about 5 μM to 500 mM nystatin and about 20 μg / ml to 5,000 μg / ml of polymer. 16. The method of embodiment 10 or embodiment 11, wherein a combination of transduction-enhancing compounds is used comprising natamycin and a polymer selected from the group consisting of PEG-PCL-PEG polymers, PEG-PLGA-PEG polymers, and PEG-PLA-PEG polymers, particularly at a final concentration of about 0.1 μM to 20 μM natamycin and about 20 μg / ml to 5,000 μg / ml of polymer. 17. The method of embodiment 9, 10, or 11, wherein a combination of transduction-enhancing compounds is used, comprising a polymer selected from the group consisting of PEG-PCL-PEG polymer, PEG-PLGA-PEG polymer, and PEG-PLA-PEG polymer, and a mixture of deoxyribonucleosides comprising 2'-deoxythymidine, 2'-deoxyadenosine, 2'-deoxyguanosine, and 2'-deoxycytidine, particularly at a final concentration of about 0.1 mM to 10 mM of each deoxyribonucleoside and about 20 μg / ml to 5000 μg / ml of polymer. 18. The method of any one of aspects 10-17, wherein the polymer is a functionalized polymer in which one or both termini of the polymer are covalently attached to a cationic group selected from the group consisting of an amino group, lysine, arginine, and histidine. 19. The method of embodiment 18, wherein the cationic group consisting of lysine, arginine and / or histidine is present as a monomer or as a polymer. 20. The method of any one of aspects 1-19, wherein the target cell is a cell selected from the group consisting of lymphocytes, tumor cells, lymphoid lineage cells, neuronal cells, epithelial cells, endothelial cells, primary cells, T cells, hematopoietic cells, and stem cells. 21. The method of aspect 20, wherein the target cells are hematopoietic cells of human origin. 22. The method of aspect 20 or aspect 21, wherein said target cells are hematopoietic stem cells. 23. A method according to aspect 20 or aspect 21, wherein said target cells are CD34+ cells or a cell population enriched for CD34+ cells. 24. The method of aspect 20, wherein the target cell is a T cell. 25. The method of embodiment 24, wherein said target cells are T cells defined by surface presentation of CD3, CD4 and / or CD8. 26. The method of any one of aspects 1-7, 10-16 and 18 and 19, wherein said target cells are monocytes, macrophages, tissue-resident macrophages or an enriched population of microglial cells, microglia-like cells or dendritic cells. 27. The method of any one of aspects 1-26, wherein said retroviral vector is a lentiviral vector. 28. The method of aspect 27, wherein the lentiviral vector is a self-inactivating lentiviral vector. 29. The method of any one of aspects 1 to 28, wherein the vector comprises a transgene under the control of the miR223 promoter. 30. The method of any one of aspects 1-29, wherein said vector comprises, in whole or in part, a cDNA encoding the p47phox, gp91phox, p22phox, p67phox or p40phox protein. 31. A method for transducing target cells, comprising the step of contacting the target cells with a retroviral vector and a compound or combination of such compounds that can enhance transduction efficiency, wherein prior to contacting the target cells with the retroviral vector, the target cells are pre-stimulated by pre-incubation with the transduction-enhancing compound or combination of transduction-enhancing compounds. 32. A method for transducing target cells, comprising the step of contacting the target cells with a retroviral vector and a compound or combination of such compounds that can enhance transduction efficiency, wherein the target cells are costimulated by co-incubation with the transduction-enhancing compound or combination of transduction-enhancing compounds during and during contacting of the target cells with the retroviral vector. 33. A method for transducing target cells according to aspect 31 and aspect 32, wherein the transduction enhancing compound is selected from the group consisting of silibinin, midostaurin, amphotericin B, nystatin, natamycin, or a combination thereof. 34. A method for transducing target cells according to aspect 31 or aspect 32, wherein said transduction-enhancing compound is selected from the group consisting of resveratrol, everolimus and prostaglandin E2, or a combination thereof. 35. A method for transducing target cells according to aspect 31 or aspect 32, wherein said transduction enhancing compound is a poloxamer-based polymer, preferably poloxamer synperonic F108, or poloxamer 407 having a molecular weight of between 11 kDa and 15 kDa. 36. A method for transducing target cells according to aspect 31 or aspect 32, wherein the transduction enhancing compound is a mixture of deoxyribonucleosides comprising 2'-deoxythymidine, 2'-deoxyadenosine, 2'-deoxyguanosine and 2'-deoxycytidine. 37. A method for transducing target cells according to aspect 31 or aspect 32, wherein the transduction enhancing compound is a polymer selected from the group consisting of a PEG-PCL-PEG polymer, a PEG-PLGA-PEG polymer, and a PEG-PLA-PEG polymer. 38. A polymer selected from the group consisting of PEG-PCL-PEG polymers, PEG-PLGA-PEG polymers and PEG-PLA-PEG polymers; (i) silibinin, particularly at a final concentration of about 0.1 μM to 25 μM; or (ii) midostaurin, particularly at a final concentration of about 0.05 μM to 20 μM; or (iii) amphotericin B, particularly at a final concentration of about 0.1 μM to 20 μM; or (iv) nystatin, particularly at a final concentration of about 5 μM to 5 mM; or (v) natamycin, particularly at a final concentration of about 0.1 μM to 20 μM; or (vi) a mixture of deoxyribonucleosides, particularly 2'-deoxythymidine, 2'-deoxyadenosine, 2'-deoxyguanosine, and 2'-deoxycytidine, at a final concentration of about 0.1 mM to 10 mM of each deoxyribonucleoside; 38. The method of embodiment 37, wherein a combination of transduction-enhancing compounds is used, comprising: 39. A method for transducing target cells according to aspect 31 or aspect 32, wherein said transduction enhancing compound is a mixture of everolimus and amphotericin B. 40. The method of embodiment 39, wherein amphotericin B is present at a final concentration of about 0.1 μM to 20 μM, and everolimus is present at a final concentration of about 0.1 μM to 20 μM. 41. The method according to aspect 31, wherein the pre-incubation period is from 0.5 hours to 10 hours, particularly from 1 hour to 5 hours, particularly 2 hours. 42. The method according to aspect 32, wherein the co-incubation period is from 8 hours to 48 hours, in particular from 10 hours to 24 hours, but in particular 12 hours. 43. A method for treating a disease or disorder comprising ex vivo or in vivo transduction of a retroviral therapeutic vector into a population of hematopoietic stem cells and / or enriched CD34-positive bone marrow cells, wherein said transduction is carried out using a method according to any one of aspects 1 to 41. [Brief explanation of the drawings]

[0015] [Figure 1]Figure 1 summarizes the results of three independent experiments in which human CD34+ HSCs were transduced with a lentiviral self-inactivating gene therapy vector. This vector encodes human p47phox cDNA under the control of the miR223 internal promoter. Cells were transduced in the presence of protamine sulfate alone (resulting VCN set at 100%) or in the presence of protamine sulfate (PS) plus one or more compounds being tested for transduction enhancer activity. Ten to 12 days after transduction, DNA was isolated from transduced cells, and VCN was quantified by qPCR. The increase in VCN relative to that achieved upon transduction in the presence of protamine sulfate alone was expressed as "fold induction relative to PS." Left panel: Transduction at an MOI of 1. Right panel: Transduction at an MOI of 3. PGE2: prostaglandin E2; AmphoB: amphotericin B.

[0016] [Figure 2] Figure 2 summarizes the results of three independent experiments in which human CD34-positive HSCs were transduced with lentiviral self-inactivating gene therapy vectors. Cells were transduced in X-Vivo 10 medium either in the absence of a transduction enhancer or in the presence of a compound to be tested for transduction enhancer activity. Ten to twelve days after transduction, DNA was isolated from transduced cells and VCN was quantified by qPCR. The solid line represents the average VCN obtained using Lentiboost at the recommended concentration of 1 mg / mL. PCL: PEG-PCL-PEG (14.2 kDa); PLA: PEG-PLA-PEG (14.2 kDa).

[0017] [Figure 3]Figure 3 summarizes the results of three independent experiments in which human CD34-positive HSCs were transduced with lentiviral self-inactivating gene therapy vectors. Cells were transduced in BESP1366F medium either in the absence of a transduction enhancer or in the presence of a compound to be tested for transduction enhancer activity. 10–12 days post-transduction, DNA was isolated from transduced cells and VCN was quantified by qPCR. The solid line represents the average VCN obtained using Lentiboost® at the recommended concentration of 1 mg / mL.

[0018] [Figure 4] Figure 4 summarizes the results of three independent experiments in which human CD34-positive HSCs were transduced with lentiviral self-inactivating gene therapy vectors. Cells were transduced in BESP1366F medium in the presence of protamine, amphotericin B, or a combination of these. Ten to twelve days after transduction, DNA was isolated from transduced cells, and VCN was quantified by qPCR. The solid line represents the average VCN obtained under comparable conditions with Lentiboost® at the recommended concentration of 1 mg / mL (see solid line in Figure 3). The dashed line represents the average VCN obtained with protamine alone.

[0019] [Figure 5] Figure 5 summarizes the results of three independent experiments in which human CD34-positive HSCs were transduced with lentiviral self-inactivating gene therapy vectors. Cells were transduced in BESP1366F medium in the presence of Lentiboost®, amphotericin B, silibinin, midostaurin, or combinations of these containing Lentiboost® at a concentration of 1 mg / mL. Ten to 12 days post-transduction, DNA was isolated from transduced cells and VCN was quantified by qPCR. The solid line represents the average VCN obtained with Lentiboost® at the recommended concentration of 1 mg / mL.

[0020] [Figure 6]Figure 6 summarizes the results of three independent experiments in which human CD34-positive HSCs were transduced with lentiviral self-inactivating gene therapy vectors. Cells were transduced in X-Vivo 10 medium in the presence of protamine, PEG-PCL-PEG (14.2 kDa), poloxamer F108, or a combination of these containing protamine. Ten to twelve days after transduction, DNA was isolated from transduced cells, and VCN was quantified by qPCR. The solid line represents the average VCN obtained under comparable conditions using Lentiboost® at the recommended concentration of 1 mg / mL (see Figure 2). The dashed line represents the average VCN obtained with protamine alone.

[0021] [Figure 7] Figure 7 summarizes the results of three independent experiments in which human CD34-positive HSCs were transduced with lentiviral self-inactivating gene therapy vectors. Cells were transduced in X-Vivo 10 medium in the presence of Lentiboost®, amphotericin B, protamine, silibinin, midostaurin, or combinations of these containing Lentiboost. Ten to twelve days post-transduction, DNA was isolated from transduced cells and VCN was quantified by qPCR. The solid line represents the average VCN obtained using Lentiboost® at the recommended concentration of 1 mg / mL (see Figure 2).

[0022] [Figure 8] Figure 8 summarizes the results of three independent experiments in which human CD34+ HSCs were transduced with lentiviral self-inactivating gene therapy vectors. Cells were transduced in X-Vivo 10 medium in the presence of amphotericin B, Lentiboost®, poloxamer F108, PEG-PCL-PEG (14.2 kDa), or combinations of these containing amphotericin B. Ten to twelve days after transduction, DNA was isolated from transduced cells, and VCN was quantified by qPCR. The solid line represents the average VCN obtained under comparable conditions with Lentiboost® at the recommended concentration of 1 mg / mL. The dashed line represents the average VCN obtained with amphotericin B alone.

[0023] [Figure 9] Figure 9 summarizes the results of three independent experiments in which human CD34-positive HSCs were transduced with lentiviral self-inactivating gene therapy vectors. Cells were transduced in X-Vivo 10 medium in the presence of silibinin, Lentiboost®, PEG-PCL-PEG (14.2 kDa), poloxamer F108, or a combination of these containing silibinin. Ten to twelve days after transduction, DNA was isolated from transduced cells, and VCN was quantified by qPCR. The solid line represents the average VCN obtained under comparable conditions with Lentiboost® at the recommended concentration of 1 mg / mL. The dashed line represents the average VCN obtained with silibinin alone.

[0024] [Figure 10] Figure 10 summarizes the results of three independent experiments in which human CD34-positive HSCs were transduced with lentiviral self-inactivating gene therapy vectors. Cells were transduced in X-Vivo 10 medium in the presence of midostaurin, Lentiboost®, poloxamer F108, PEG-PCL-PEG (14.2 kDa), or combinations of these containing midostaurin. Ten to 12 days after transduction, DNA was isolated from transduced cells and VCN was quantified by qPCR. The solid line represents the average VCN obtained under comparable conditions with Lentiboost® at the recommended concentration of 1 mg / mL. The dashed line represents the average VCN obtained with midostaurin alone.

[0025] [Figure 11]Figure 11 summarizes the results of three independent experiments in which human CD34-positive HSCs were transduced with lentiviral self-inactivating gene therapy vectors. Cells were transduced in X-Vivo 10 medium in the presence of PEG-PCL-PEG (14.2 kDa), protamine, amphotericin B, silibinin, midostaurin, or combinations of these containing PEG-PCL-PEG. Ten to twelve days after transduction, DNA was isolated from transduced cells, and VCN was quantified by qPCR. The solid line represents the average VCN obtained under comparable conditions using Lentiboost® at the recommended concentration of 1 mg / mL (see Figure 2). The dashed line represents the average VCN obtained with PEG-PCL-PEG alone.

[0026] [Figure 12] Figure 12 summarizes the results of three independent experiments in which human CD34-positive HSCs were transduced with lentiviral self-inactivating gene therapy vectors. Cells were transduced in X-Vivo 10 medium in the presence of poloxamer F108, silibinin, midostaurin, amphotericin B, protamine, or combinations of these containing poloxamer F108. Ten to twelve days after transduction, DNA was isolated from transduced cells and VCN was quantified by qPCR. The solid line represents the average VCN obtained under comparable conditions using Lentiboost® at the recommended concentration of 1 mg / mL (see Figure 2). The dashed line represents the average VCN obtained with poloxamer F108 alone.

[0027] [Figure 13] Figure 13 summarizes the results of three independent experiments in which human CD34-positive HSCs were transduced with lentiviral self-inactivating gene therapy vectors at an MOI of 20. Cells were transduced in the absence and presence of 1% DMSO. DETAILED DESCRIPTION OF THE INVENTION

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred embodiments of the compositions, methods and materials are described herein. For purposes of the present invention, the following terms are defined below.

[0029] The articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one or one or more) of the grammatical object of the article.

[0030] "Or" should be understood to mean either one, both, or any combination thereof of the alternatives in question.

[0031] "And / or" should be understood to mean either one or both of the alternatives in question.

[0032] Throughout this specification, unless the context requires otherwise, the terms "comprise", "comprises" and "comprising" will be understood to imply the inclusion of the stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0033] The terms "include" and "comprise" are used interchangeably. "Preferably" means one option in a set of options that does not exclude other options. "For example" means one example that is not limited to the examples stated. "Consisting of" means including and limited to whatever follows the word "consisting of."

[0034] Reference throughout this specification to "one embodiment," "one embodiment," "particular embodiment," "related embodiment," "an embodiment," "additional embodiment," "particular embodiment," or "further embodiment," or combinations thereof, means that the particular features, structures, or characteristics described in connection with that embodiment are included in at least one embodiment of the invention. Thus, the appearances of these phrases in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It is also understood that the affirmative recitation of a feature in an embodiment serves as a basis for excluding that feature in a particular embodiment.

[0035] The present invention provides a method for transducing target cells, comprising the step of contacting the target cells with a retroviral vector and a compound or combination of such compounds capable of enhancing transduction efficiency, wherein the target cells are pre-stimulated and / or co-stimulated by pre-incubation and / or co-incubation with the transduction-enhancing compound or combination of transduction-enhancing compounds before and / or during contacting the target cells with the retroviral vector.

[0036] The methods according to the invention can be carried out in vivo or ex vivo. In some embodiments, the methods are carried out ex vivo.

[0037] That is, the present invention is based, at least in part, on the discovery that the efficiency of transduction of target cells by retroviral vectors can be increased by incubating the target cells with a transduction-enhancing compound or a mixture of transduction-enhancing compounds, To this end, the target cells can be pre- and / or costimulated with a transduction enhancer or combination of transduction enhancers disclosed herein.

[0038] The term "transduction enhancer" refers to any compound whose presence during transduction results in an increase in VCN compared to its absence.

[0039] In some embodiments, target cells are costimulated with a transduction enhancer or combination of transduction enhancers during the transduction step. In the present invention, target cells are said to be costimulated with a transduction enhancer or combination of transduction enhancers if the target cells are incubated in the presence of the transduction enhancer or combination of transduction enhancers while being contacted with a retroviral vector.

[0040] During the costimulation step, the target cells, retroviral vector and transduction enhancer or combination of transduction enhancers are preferably contacted in a co-incubation step in liquid medium, more preferably in liquid cell culture medium.

[0041] The target cells can be co-incubated with the viral vector and transduction enhancer or combination of transduction enhancers for any length of time, however, it is preferred that the target cells are co-incubated with the viral vector and transduction enhancer or combination of transduction enhancers for a period of about 8 hours to about 48 hours, particularly about 10 hours to about 24 hours, but particularly about 12 hours.

[0042] Preferably, costimulation of the target cell with the transduction enhancer or combination of transduction enhancers results in an increase in the susceptibility of the target cell to the retroviral vector.

[0043] In some embodiments, target cells may be pre-incubated with a transduction enhancer or a combination of transduction enhancers prior to the transduction step. In the present invention, target cells are said to be pre-stimulated with a transduction enhancer or a combination of transduction enhancers if the target cells are incubated with a transduction enhancer or a combination of transduction enhancers before the target cells are contacted with a retroviral vector.

[0044] During the pre-stimulation step, the target cells and the transduction enhancer or combination of transduction enhancers are preferably contacted during an incubation step in a liquid medium, more preferably in a liquid cell culture medium.

[0045] The target cells can be pre-incubated with the transduction enhancer or combination of transduction enhancers for any length of time, however, it is preferred that the target cells are pre-incubated with the transduction enhancer or combination of transduction enhancers for a period of about 0.5 hours to about 10 hours, particularly about 1 hour to about 5 hours, but particularly about 2 hours.

[0046] Preferably, pre-stimulation of the target cells with the transduction enhancer or combination of transduction enhancers results in increased susceptibility of the target cells to the retroviral vector in a subsequent transduction step.

[0047] In some embodiments, target cells are pre-stimulated and co-stimulated with a transduction enhancer or combination of transduction enhancers, i.e., target cells can be first pre-incubated with a transduction enhancer or combination of transduction enhancers, and then co-incubated with a retroviral vector and a transduction enhancer or combination of transduction enhancers.

[0048] It should be noted that the transduction enhancer or combination of transduction enhancers may or may not be the same between the pre-stimulation and costimulation steps.

[0049] That is, in some embodiments, target cells can be pre-stimulated and costimulated with the same transduction enhancer or the same combination of transduction enhancers. For example, target cells can first be pre-stimulated for a specified time in a liquid medium containing the transduction enhancer or the combination of transduction enhancers. To initiate the costimulation step, a retroviral vector can be added to the liquid medium containing the target cells and the transduction enhancer or the combination of transduction enhancers. Alternatively, the target cells can be isolated from the pre-stimulation medium after a specified time and transferred to a fresh costimulation medium containing the same transduction enhancer or the same combination of transduction enhancers, and optionally a retroviral vector. That is, the costimulation medium can already contain the retroviral vector when the target cells are resuspended therein, or the retroviral vector can be added after the target cells are resuspended in the fresh costimulation medium. The concentration and / or ratio of the transduction enhancer or the combination of transduction enhancers can be different or the same between the pre-stimulation medium and the costimulation medium.

[0050] In some embodiments, target cells can be contacted with a first transduction enhancer or a first combination of transduction enhancers in a pre-stimulation step, and then contacted with a second transduction enhancer or a second combination of transduction enhancers in a costimulation step, preferably by pre-incubating the target cells in a medium containing the first transduction enhancer or the first combination of transduction enhancers, then isolating the target cells from the pre-stimulation medium and transferring them to a costimulation medium containing the second transduction enhancer or the second combination of transduction enhancers and, optionally, a retroviral vector.

[0051] It should be understood that the pre-stimulation step does not necessarily have to be immediately followed by a costimulation step, i.e., the cells can be incubated in medium that does not contain a transduction enhancer between the pre-stimulation and costimulation steps.

[0052] The efficiency of a transduction experiment can be determined as known in the art. Preferably, transduction efficiency can be measured by determining the vector copy number (VCN) in a single cell after the transduction experiment or by measuring the average VCN in a population of cells after the transduction experiment. "Vector copy number" or "VCN" refers to the copy number of a vector or a portion thereof in the genome of a cell. The average VCN can be determined from a population of cells or from individual cell colonies. Exemplary methods for determining VCN include any form of polymerase chain reaction (PCR), such as qPCR or digital droplet PCR, and flow cytometry. For example, VCN can be determined as described in Charrier et al., "Quantification of lentiviral vector copy numbers in individual hematopoietic colony-forming cells shows vector dose-dependent The effects on the frequency and level of transduction, Gene Ther, 2011, 18(5), p. 479-487 or as described in Example 1 or 5.

[0053] Some of the transduction enhancers and transduction enhancer combinations reported herein result in increased transduction efficiency. "Increased transduction efficiency" refers to an increase in VCN during transduction of a cell population with a gene therapy vector in the presence of a transduction enhancer compared to the absence of the transduction enhancer.

[0054] The target cell can be any cell that can be targeted with a retroviral vector, however, it is preferred that the target cell is a mammalian cell, particularly a human cell.

[0055] In a particular embodiment, the present invention relates to a method according to the present invention, wherein the target cell is a cell selected from the group consisting of lymphocytes, tumor cells, lymphoid lineage cells, neuronal cells, epithelial cells, keratinocytes, endothelial cells, primary cells, T cells, hematopoietic cells and stem cells.

[0056] A "target cell" can be a single cell of any of the cell types disclosed herein. However, it should be understood that the method of the present invention can also be applied to a population of cells. That is, the target cell can be a homogeneous population of cells, preferably any one of the cell types disclosed herein. However, the method of the present invention can also be performed using a heterogeneous cell population, such as a cell population obtained in an enrichment step. It is known in the art that enrichment of a particular cell type does not result in a 100% pure culture of said cell type. However, it is preferred that such a heterogeneous population of cells comprises at least one cell type disclosed herein.

[0057] The target cells may preferably be mammalian cells, more particularly cells derived from any germ layer, such as endoderm, ectoderm or mesoderm.

[0058] The term "endodermal cell" refers to a cell that can differentiate into an endodermal organ such as the liver, pancreas, intestine, lung, thyroid, parathyroid, or urinary tract. The term "ectodermal cell" refers to a cell that can differentiate into an ectodermal organ such as the brain, spinal cord, adrenal medulla, epidermis, hair / nail / skin glands, sensory organs, peripheral nerves, skin, or lens. As used herein, the term "mesodermal cell" refers to a multipotent stem cell of mesodermal origin that gives rise to bone, cartilage, tendon, muscle, adipose tissue, and vascular endothelium during development.

[0059] In some embodiments, the target cell may be a fibroblast. As used herein, the term "fibroblast" refers to a cell of mesenchymal origin. Fibroblasts are found in connective tissue. Fibroblasts synthesize actin-myosin filaments, matrix elements (collagen, reticular fibers, and elastic fibers), and glycosaminoglycans and glycoproteins secreted as amorphous intercellular substances. Fibroblasts include connective tissue stem cells, matrix and other protein-synthesizing cells, contractile cells, and phagocytes. Active fibroblasts are characterized by their abundant endoplasmic reticulum (ER), Golgi complex, and ribosomes.

[0060] In some embodiments, the target cell can be a smooth muscle cell or a non-smooth muscle cell.

[0061] In some embodiments, the target cell can be an epithelial cell. As used herein, the term "epithelial cell" refers to the cuboidal nucleated cells that cover the free surface of an organ (skin, mucus or serous) or line the ducts or cavities in an animal body, and is consistent with the art-recognized definition of epithelial cells in epithelium. The layer of epithelial cells generally functions to provide a protective lining and / or surface that can also be involved in transport processes.

[0062] In some embodiments, target cells can be endothelial cells. The term "endothelial cells" used herein encompasses all endothelial cell types, such as the cells that line all blood vessels and form a single cell layer that regulates blood flow and exchange between surrounding tissues. There are many endothelial cell types, and their phenotypes vary between different organs, between different segments of vascular loops within the same organ, and between adjacent endothelial cells of the same organ and blood vessel type. Non-limiting examples of such endothelial cells include hepatic sinusoidal endothelial cells (LSECs), (capillary) endothelial cells from the lung, heart, intestine, skin, and retina, arterial endothelial cells such as endothelial cells from the pulmonary artery, aorta, umbilical artery, and umbilical vein, extrahepatic endothelial cells from certain vascular beds, blood-brain barrier ECs, bone marrow ECs, and high endothelial venule cells (HEVs).

[0063] In some embodiments, the target cell may be a neuronal cell. As used herein, the term "neuronal cell" or "neuron" refers to a nervous system cell that includes a central cell body or soma and two types of extensions or processes: dendrites, through which the majority of neuronal signals are generally conducted to the soma, and axons, through which the majority of neuronal signals are generally conducted from the soma to target neurons or effector cells, such as muscles. Neurons can transmit information from tissues and organs into the central nervous system (afferent or sensory neurons) and from the central nervous system to effector cells (efferent or motor neurons). Other neurons, called interneurons, connect neurons within the central nervous system (brain and spinal column). Specific examples of types of neurons that may be subjected to treatments or methods, either ex vivo or in vivo, or a combination of ex vivo and in vivo, in accordance with the present invention include cerebellar granule neurons, dorsal root ganglion neurons, and cortical neurons, or any other cell type of the central or peripheral nervous system.

[0064] In some embodiments, the target cell may be a tumor cell. As used herein, the term "tumor cell" refers to a cell that is a neoplasm. Tumor cells may be benign, i.e., do not form metastases, invade adjacent normal tissues, or do not destroy adjacent normal tissues, or malignant, i.e., can invade surrounding tissues, generate metastases, recur after attempted removal, and potentially cause host death. Preferably, the tumor cells subjected to the methods of the present invention may be derived from any germ layer (endoderm, ectoderm, mesoderm). In particular, tumor cells may be derived from epithelial, hematopoietic, germ cell, or mesenchymal origin, such as, but not limited to, tumor cells derived from skin cells, lung cells, intestinal epithelial cells, colon epithelial cells, testicular cells, breast cells, prostate cells, brain cells, bone marrow cells, blood lymphocytes, ovarian cells, gonadal and extragonadal-associated cells, or thymocytes.

[0065] In some embodiments, the target cell can be a cell derived from the lymphoid lineage or a cell derived from the myeloid lineage. Cells derived from the lymphoid lineage are cells derived from a common lymphoid progenitor cell, such as a natural killer cell, a T cell, or a B cell. Cells derived from the myeloid lineage are cells derived from a common myeloid progenitor cell, such as a megakaryocyte, a platelet, an erythrocyte, a mast cell, a myeloblast, a basophil, a neutrophil, an eosinophil, a monocyte, or a macrophage.

[0066] The term "primary cells" as used herein is known in the art to refer to cells isolated from tissues and established for in vitro growth. The corresponding cells have undergone few, if any, population doublings and are therefore more representative of the main functional components of the tissue from which they are derived than continuous cell lines, and therefore represent a more representative model of in vivo conditions. Methods for obtaining samples from various tissues and establishing primary cell lines are well known in the art (see, for example, Jones and Wise, Methods Mol Biol. 1997). Primary cells for use in the methods of the present invention can be derived from, for example, bone marrow, blood, skin, lymphoma, and epithelial tumors.

[0067] In some embodiments, the target cell may be a lymphocyte. As used herein, the term "lymphocyte" has its ordinary meaning in the art and refers to any of the mononuclear, non-phagocytic white blood cells found in blood, lymph, and lymphoid tissues, i.e., NK cells, B cells, and T cells.

[0068] In a specific embodiment, the present invention relates to a method according to the present invention, wherein the target cell is a T cell. As used herein, the term "T cell" refers to a type of lymphocyte that plays a central role in cell-mediated immunity. T cells, also called T lymphocytes, can be distinguished from other lymphocytes, such as B cells and natural killer cells, by the presence of a T cell receptor (TCR) on the cell surface. There are several subsets of T cells with different functions, including, but not limited to, T helper cells, cytotoxic T cells, memory T cells, regulatory T cells, and natural killer T cells. In some embodiments, the target cell can be a T cell defined by the surface expression of CD3, CD4, and / or CD8.

[0069] Thus, in some embodiments, the target cell may be a T cell characterized by the expression of CD3. As used herein, the term "CD3" refers to the cluster of differentiation 3 (CD3) T cell coreceptor of all mammalian species, preferably humans. In mammals, CD3 comprises a CD3 zeta chain, a CD3 delta chain, and two CD3 epsilon chains. Thus, the target cell of the present invention may be any T cell that expresses a T cell coreceptor in addition to the T cell receptor.

[0070] In some embodiments, the target cell may be a T cell characterized by the expression of CD4. As used herein, the term "CD4" refers to cluster of differentiation 4, a glycoprotein expressed on the surface of T helper cells, monocytes, macrophages, and dendritic cells. CD4 is a co-receptor that assists T cell receptors (TCRs) in conjunction with antigen-presenting cells. Thus, in some embodiments, the target cell may be a T helper cell.

[0071] In some embodiments, the target cell may be a T cell characterized by expression of CD8. As used herein, the term "CD8" refers to cluster of differentiation 8, a transmembrane glycoprotein that serves as a coreceptor for the T cell receptor (TCR) expressed on cytotoxic T cells (CTLs). Thus, in some embodiments, the target cell may be a cytotoxic T cell.

[0072] In some embodiments, the methods of the present invention can be used in the production of CAR T, CAR M, or CAR NK cells. That is, T cells, particularly cytotoxic CD8+ T cells or CD4+ T helper cells, monocytes, macrophages, or NK cells, can be pre-stimulated and / or costimulated with the transduction enhancers or combinations of transduction enhancers of the present invention disclosed herein. During the costimulation step, the T cells, monocytes, macrophages, or NK cells can then be contacted with a retroviral vector, particularly a lentiviral vector, containing a nucleic acid encoding a chimeric antigen receptor (CAR).

[0073] In a particular embodiment, the present invention relates to a method according to the present invention, wherein the target cells are hematopoietic cells, in particular hematopoietic cells of human origin. As used herein, the term "hematopoietic cells" refers to any type of cell of the hematopoietic system, including, but not limited to, undifferentiated cells such as hematopoietic stem and progenitor cells, and differentiated cells, such as leukocytes (e.g., granulocytes, monocytes, NK cells, and lymphocytes).

[0074] In a particular embodiment, the present invention relates to a method according to the present invention, wherein the target cells are hematopoietic stem cells. The term "hematopoietic stem cells" is used in the broadest sense to refer to stem cells from which blood cells are derived, including pluripotent stem cells, lymphoid and myeloid stem cells.

[0075] Hematopoietic stem cells can develop into any cell lineage present in the blood or tissues. The term, as used herein, refers to both the earliest renewable hematopoietic cell populations involved in generating the cell populations in the blood (e.g., CD34- / CD133+, CD34- / AC133- / lineage-, CD34+ / AC133+ cells, e.g., lineage-CD34+CD38-CD90+CD45RA- (Majeti R., Park CY & Weissman IL (2007) Cell Stem Cell 1:635-645), lineage-CD133+CD38-CD33- (Goetz et al. (2007) Exp Hemat. 35:1408-14)), and very early hematopoietic progenitor cells (HSPCs) (e.g., CD34+ cells, particularly CD34+CD38- cells), which are somewhat more differentiated but are not yet committed and can readily revert to become part of the earliest renewable hematopoietic cell populations. In healthy humans, hematopoietic pluripotent stem cells and lineage-specific progenitor cells Most of these cells are CD34+. Most cells are CD34+CD38+, and a minority of cells (<10%) are CD34+CD38-. The CD34+CD38- stem cell fraction contains the most immature hematopoietic cells capable of self-renewal and multilineage differentiation. This fraction contains more long-term culture-initiating cells (LTC-ICs), which exhibit longer maintenance of stemness and delayed proliferative response to cytokines compared to cells in the CD34+CD38+ cell fraction. Preferably, certain embodiments are applied to cell populations enriched for human CD34+ cells.

[0076] In some embodiments, the target cells are hematopoietic progenitor cells. The term "hematopoietic progenitor cells" refers to any cell population that includes the progeny of pluripotent hematopoietic stem cells that are committed to a specific lineage of differentiation or pluripotent hematopoietic stem cells that are capable of self-renewal and multilineage differentiation. These committed progenitor cells can produce only one or a few blood cell types, e.g., red blood cells. , irreversibly determined as the progenitor of megakaryocytes, monocytes, or granulocytes.

[0077] In some embodiments, the target cells are hematopoietic precursor cells. As used herein, the term "hematopoietic precursor cells" includes any cells that give rise to hematopoietic stem cells, hematopoietic progenitor cells, or cells of hematopoietic lineage (e.g., lymphoid, myeloid). Examples of hematopoietic precursor cells are CFU-GEMM (colony-forming unit-granulocyte-erythrocyte-megakaryocyte-monocyte), CFU-GM (colony-forming unit-granulocyte-monocyte), CFU-E (colony-forming unit-erythrocyte), BFU-E (burst-forming unit-erythrocyte), CFU-G (colony-forming unit-granulocyte), CFU-eo (colony-forming unit-eosinophil), and CFU-Meg (colony-forming unit-megakaryocyte).

[0078] In a specific embodiment, the present invention relates to a method according to the present invention, wherein the target cells are CD34+ cells or cells contained in a CD34+ enriched cell population. As used herein, the term "CD34" refers to a surface antigen cluster present on certain cells in the human body. CD34 is a cell surface glycoprotein and functions as a cell-cell adhesion molecule. CD34 can also mediate stem cell attachment to the bone marrow extracellular matrix or directly to stromal cells. Cells expressing CD34 (CD34+ cells) are typically found in the umbilical cord and bone marrow as hematopoietic cells, endothelial hematopoietic progenitor cells, and vascular endothelial cells, which are subsets of mesenchymal stem cells, but not lymphoid cells. Therefore, the term "CD34+ cells" as used herein preferably refers to hematopoietic stem cells and progenitor cells derived from human bone marrow that are "positive for" the hematopoietic stem cell antigen CD34, i.e., "express" the hematopoietic stem cell antigen CD34. Furthermore, the target cells can be any cells contained in a CD34+ enriched cell population. Those skilled in the art are familiar with methods for enriching CD34+ cells. Additionally, commercially available kits are available for enriching CD34+ cell populations. Certain embodiments may be applied to cell populations enriched for human CD34+ cells.

[0079] In a particular embodiment, the present invention relates to a method according to the invention, wherein the target cells are monocytes, macrophages, tissue-resident macrophages, microglial cells or dendritic cells.

[0080] That is, in certain embodiments, target cells can be monocytes or cells contained within an enriched population of monocytes. As used herein, the term "monocyte" refers to a type of white blood cell that has two main functions in the immune system: (1) recruiting resident macrophages and dendritic cells under normal conditions; and (2) in response to inflammatory signals, monocytes rapidly migrate (approximately 8-12 hours) to sites of infection in tissues and differentiate into macrophages and dendritic cells, which can elicit an immune response. Half of them are stored in the spleen. Monocytes are typically identified in stained smears by their large, bilobed nuclei. In addition to expressing CD14, monocytes also express one or more of the following surface markers: 125I-WVH-1, 63D3, adipophilin, CB12, CD11a, CD11b, CD14, CD16, CD54, CD163, cytidine deaminase, and Flt-1. Methods and commercially available kits for enriching monocytes are known in the art.

[0081] In some embodiments, target cells can be macrophages or cells contained in macrophage-enriched populations.The term "macrophage" used herein refers to CD14+ positive cells derived from monocyte differentiation, characterized as phagocytic cells that act both in non-specific defense (innate immunity) and in supporting the initiation of vertebrate specific defense mechanisms (adaptive immunity).Their role is to phagocytose (engulf and then digest) cellular debris and pathogens as either stationary or migratory cells, and stimulate lymphocytes and other immune cells to respond to pathogens.

[0082] In some embodiments, the macrophages may be tissue-resident macrophages, such as those resident in the brain or kidney. In addition to expressing CD14, macrophages also express one or more of the following surface markers: CD11b, F4 / 80 (mouse) / EMR1 (human), lysozyme M, MAC-1 / MAC-3, 27E10, carboxypeptidase M, cathepsin K, CD163, CD86, CD206, CD209, Mer, and CD68. These markers can be determined by flow cytometry or immunohistochemical staining. Methods and commercially available kits for enriching macrophages are known in the art. It should be noted that certain types of tissue-resident macrophages may not be derived from monocytes but may be derived from other cell types or tissues, such as the yolk sac. However, such non-monocyte-derived tissue-resident macrophages may also be used in the methods of the present invention.

[0083] In some embodiments, the target cells may be dendritic cells, particularly myeloid dendritic cells, or cells contained in an enriched population of dendritic cells, particularly myeloid dendritic cells. As used herein, the term "myeloid dendritic cells" refers to a population of dendritic cells derived from monocytes, including, but not limited to, mDC-1 and mDC-2. In addition to expressing CD14, myeloid dendritic cells also express one or more of the following surface markers: thrombomodulin / CD141 / BDCA-3, CD1c / BDCA-1, neuropilin-1 / BDCA-4, DC-SIGN / CD209, SIRPa / CD172a, ADAM19, BDCA-2, CD1a, CD11c, CD21, CD86, CD208, clusterin, and estrogen receptor-alpha. Methods and commercially available kits for enriching macrophages are known in the art.

[0084] In some embodiments, the target cells may be microglial cells or cells contained in an enriched population of microglial cells. As used herein, the term "microglia" refers to the smallest glial cells, which act as phagocytes and can clear debris located in the CNS. They are considered to be a type of immune cell found in the brain and are characterized by Iba1, CD11b, CD45, CD11c, ferritin, CD68, TMEM2, and / or CD33 expression (Hopperton et al., (2018) Mol. Psych 23:177-198). Microglia are closely related to other phagocytes, including macrophages and dendritic cells. Like macrophages, microglia originate from myeloid progenitor cells derived from the bone marrow.

[0085] In some embodiments, the target cells may be microglia or microglia-like cells, or cells contained in an enriched population of microglia-like cells. The term "microglia-like cells" refers to blood-derived monocytes / macrophages that can cross the blood-brain barrier, especially when infused into patients after pretreatment with busulfan or treosulfan. Microglia-like cells have been reported to enter the brain during neuroinflammatory conditions (Mendiola AS et al., (2020) Nat Immunol 21:513-524, PMID 32284594) and during the progression of brain metastases (Schulz M. et al., (2020) iScience 23:101178. doi:10.1016 / j.isci.2020.101178.), thereby promoting phagocytosis and innate immune functions equivalent to and / or complementary to those of brain tissue-resident microglia.

[0086] Various methods for identifying and / or enriching any of the cell types listed above are known in the art.For example, the cells of specific cell types can be enriched by flow cytometry based on the expression of specific cell surface markers or a combination of cell surface markers.In addition, the cells of specific cell types can be identified by various microscopic methods known in the art or based on their cytokine secretion profile.There are various commercially available kits for identifying and / or enriching specific cell types.

[0087] The methods of the present invention can be used to improve transduction of target cells by retroviral vectors.

[0088] The term "vector" is used herein to refer to a nucleic acid molecule capable of introducing or transporting another nucleic acid molecule. As will be apparent to those skilled in the art, the term "viral vector" is used broadly to refer to either a nucleic acid molecule (e.g., a transfer plasmid) that typically contains viral-derived nucleic acid elements that facilitate the introduction or integration of a nucleic acid molecule into the genome of a cell, or a viral particle that mediates nucleic acid introduction. Viral particles typically contain various viral components, and sometimes also contain host cell components in addition to the nucleic acid(s).

[0089] The term viral vector can refer to either a virus or viral particle that can introduce nucleic acid into cells, or the introduced nucleic acid itself.Viral vectors and transfer plasmids contain structural and / or functional genetic elements that are primarily derived from viruses.The term "retroviral vector" refers to a viral vector or plasmid that is used in the form of a plasmid for transient cell transfection of cells for virus production, or that is used for stable integration into the genome of a cell to generate stable virus-producing cells that contain structural and functional genetic elements or parts thereof that are primarily derived from retroviruses.

[0090] As used herein, the term "retrovirus" refers to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and then covalently integrates the genomic DNA into the host genome. Retroviruses are common tools for gene delivery (Miller, 2000, Nature. 357:455-460). Once a virus is integrated into the host genome, it is called a "provirus." The provirus serves as a template for RNA polymerase II, directing the expression of viral RNA molecules encoded by the host cell. Exemplary retroviruses include, but are not limited to, Moloney murine leukemia virus (MoMLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumaviruses including foamy viruses, Friend murine leukemia virus (FMLV), murine stem cell virus (MSCV) and Rous sarcoma virus (RSV), alpha-retroviruses and lentiviruses. That is, the retroviral vectors used in the methods of the present invention can be derived from any of the retroviruses disclosed herein. Furthermore, the term "retrovirus" refers to any pseudotyped retroviral particle, including, for example, vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped retroviral particles, or envelopes decorated with syncytin-related proteins, preferably syncytin-2 protein (Esnault C. et al. (2008) PNAS 105:17532-17537), and retroviral particles that do not contain pseudotyped viral glycoproteins (Boeker KO et al. (2018) Mol Ther. 26:634-647).

[0091] In a particular embodiment, the present invention relates to a method according to the invention, wherein the retroviral vector is a lentiviral vector.

[0092] The term "lentiviral vector" refers to a retroviral vector or plasmid that contains structural and functional genetic elements, or portions thereof, including primarily LTRs derived from a lentivirus.

[0093] As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Exemplary lentiviruses include, but are not limited to, HIV (human immunodeficiency virus; including HIV types 1 and 2), Visna-Maedi virus (VMV), Caprine Arthritis-Encephalitis Virus (CAEV), Equine Infectious Anemia Virus (EIAV), Feline Immunodeficiency Virus (FIV), Bovine Immunodeficiency Virus (BIV), and Simian Immunodeficiency Virus (SIV).

[0094] The term lentiviral vector also includes hybrid vectors. The term "hybrid" refers to a vector containing both retroviral, e.g., lentiviral, and non-lentiviral viral sequences, LTRs (long terminal repeats), or other nucleic acids. For example, a hybrid vector can refer to a vector or transfer plasmid that contains retroviral, e.g., lentiviral, sequences for reverse transcription, replication, integration, and / or packaging, as well as alphavirus subgenomic promoter sequences, nonstructural proteins, and / or polymerase recognition sites. Another example of a hybrid vector is a pseudotyped lentiviral vector that contains lentiviral elements for reverse transcription and integration, but is covered by an envelope protein of a different origin, such as vesicular stomatitis virus glycoprotein (VSV-G) or a different viral envelope protein.

[0095] In a further aspect, the invention relates to an integration-defective retroviral vector comprising an inactive form of a retroviral integrase enzyme that is used to provide a "template DNA" to a cell for targeted genome editing by sequence-specific insertion of a single-strand break (nicking) and / or a double-strand break in the genome, in combination with a template DNA, a coding sequence flanking the locations of single-strand breaks (nicking) and / or double-strand breaks, and a supply of desired sequences between the "template DNA" that can be transiently provided to a cell by the integration-defective retroviral vector.

[0096] In a particular embodiment, the present invention relates to a method according to the invention, wherein the lentiviral vector is a self-inactivating lentiviral vector.

[0097] A "self-inactivating" (SIN) vector is a replication-deficient vector, e.g., a retroviral or lentiviral vector, in which the right (3') LTR enhancer-promoter region, known as the U3 region, has been modified (e.g., by deletion and / or substitution) to prevent viral transcription beyond the first round of viral replication. As a result, the vector can infect and then integrate into the host genome only once and cannot be further transmitted. This is because the right (3') LTR U3 region, which contains a deletion of the viral promoter / enhancer sequence, is used as a template for the left (5') LTR U3 region during viral reverse transcription; therefore, new viral transcripts from the integrated SIN vector cannot be generated without the U3 enhancer-promoter. Without viral transcripts, they cannot be processed or packaged into virions, thus terminating the viral life cycle. Therefore, because the right (3') LTR U3 region is modified to prevent viral transcription beyond the first round of replication, SIN vectors greatly reduce the risk of generating unwanted replication-competent virus, thus eliminating the ability of the virus to be transmitted.

[0098] In further and / or alternative embodiments of the invention, the 3' LTR may be modified such that the U5 region is replaced with, for example, a heterologous or synthetic poly(A) sequence, one or more insulator elements, and / or an inducible promoter. Note that modifications to the LTR, such as modifications to the 3' LTR, the 5' LTR, or both the 3' and 5' LTRs, are also encompassed by the present invention.

[0099] Further safety enhancement is provided by replacing the U3 region of the 5'LTR with a heterologous promoter to drive transcription of the viral genome during viral particle production. Examples of heterologous promoters that can be used include the viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) thymidine kinase promoter. Typical promoters can drive high-level transcription in a Tat-independent manner. This replacement reduces the possibility of recombination to generate replication-competent viruses due to the absence of a complete U3 sequence in the viral production system.

[0100] The term "long terminal repeat (LTR)" refers to a domain of base pairs located at the end of retroviral DNA, which is a direct repeat in its natural sequence context and contains the U3, R, and U5 regions. LTRs generally provide essential functions for retroviral gene expression (e.g., promotion, initiation, and polyadenylation of gene transcripts) and viral replication. LTRs contain multiple regulatory signals, including transcriptional control elements, polyadenylation signals, and sequences required for viral genome replication and integration. The U3 region contains enhancer and promoter elements. The U5 region is located between the primer binding site and the R region and contains a polyadenylation sequence. The R (repeat) region is flanked by the U3 and U5 regions. At the DNA level, LTRs, composed of the U3, R, and U5 regions, appear at both the 5' and 3' ends of the viral genome. The 5' LTR is flanked by sequences required for reverse transcription of the genome (tRNA primer binding site) and efficient packaging of viral RNA into particles (Psi site).

[0101] Also encompassed by the present invention is a method for transducing target cells, comprising contacting the target cells with a gene therapy vector and a compound or combination of such compounds capable of enhancing transduction efficiency, wherein the target cells are pre-stimulated and / or costimulated by pre-incubation and / or co-incubation with the transduction-enhancing compound or combination of transduction-enhancing compounds before and / or during contacting the target cells with the gene therapy vector. It is understood that the combination of any gene therapy vector disclosed herein with any target cell disclosed herein and / or any transduction enhancer or combination of transduction enhancers disclosed herein is encompassed by the present invention.

[0102] The term "gene therapy vector" includes all vectors used as vehicles for delivering genetic information into target cells within gene therapy approaches, where the genetic information to be delivered is predetermined by the design of the gene therapy vector. The gene therapy vector may be an adenoviral vector, an adeno-associated viral vector, a herpes viral vector, a foamy viral vector, or a retroviral vector, and in particular, the retroviral vector is a lentiviral vector.

[0103] The gene therapy vector or retroviral vector of the present invention can contain a nucleotide sequence of interest intended to be introduced into target cells. The nucleotide sequence of interest is not limited within the present invention and can be any nucleotide sequence that can be introduced into target cells. However, it is preferred that the nucleotide sequence of interest contains a transgene, and more preferably, the regulatory elements required for the expression of the transgene in target cells.

[0104] The term "transgene" as used herein refers to a specific nucleic acid sequence encoding a polypeptide or a portion of a polypeptide to be expressed in a cell into which the nucleic acid sequence is inserted, i.e., a target cell of the present invention. Furthermore, it should be understood that a transgene can encode multiple polypeptides, such as polypeptides constituting a chimeric antigen receptor (CAR). However, typically, to reduce the amount of a specific polypeptide in a cell into which the nucleic acid sequence is inserted, the transgene can also be expressed as RNA. These RNA molecules include, but are not limited to, molecules that exert their function through RNA interference (shRNA, RNAi), microRNA regulatory (miR), catalytic RNA, antisense RNA, RNA aptamer, long non-coding RNA, etc. Of note, expression of a transgene can be limited to a subset of cells into which the nucleic acid sequence is inserted. The term transgene is meant to include (1) a nucleic acid sequence not naturally found in a cell (i.e., a heterologous nucleic acid sequence); (2) a nucleic acid sequence that is a mutant form of a nucleic acid sequence naturally found in a cell into which the nucleic acid sequence has been introduced; (3) a nucleic acid sequence that serves to add additional copies of an identical (i.e., homologous) or similar nucleic acid sequence naturally occurring in a cell into which the nucleic acid sequence has been introduced; or (4) a silent naturally occurring or homologous nucleic acid sequence whose expression is induced in a cell into which the nucleic acid sequence has been introduced; or (5) a sequence that serves as a "template DNA" for targeted homologous recombination during gene editing by targeted insertion of single- and / or double-strand breaks. "Mutant form" refers to a nucleic acid sequence that contains one or more nucleotides that differ from the wild-type or naturally occurring sequence; i.e., a mutant nucleic acid sequence contains one or more nucleotide substitutions, deletions, and / or insertions. In some cases, a transgene may also include a sequence encoding a leader peptide or signal sequence so that the transgene product is secreted from the cell.

[0105] In some embodiments, the transgene can be a nucleic acid encoding a naturally occurring polypeptide that is not expressed or is expressed at a reduced level in target cells due to congenital or acquired genetic defects.In other embodiments, the transgene can encode a chimeric antigen receptor (CAR).When the transgene encodes a CAR, the target cell is preferably a T cell, a monocyte or macrophage, or a NK cell.

[0106] In some embodiments, a transgene can be operably linked to a promoter. The term "promoter" refers to a nucleic acid sequence that directly or indirectly regulates the transcription of the corresponding nucleic acid coding sequence (e.g., transgene) to which the promoter is operably linked. A promoter can function alone to regulate transcription, or can act in concert with one or more other regulatory sequences (e.g., enhancers or silencers). In the context of the present application, a promoter is typically operably linked to a transgene to regulate the transcription of the transgene.

[0107] As used herein, the term "operably linked" refers to the arrangement of various nucleic acid molecular elements relative to each other such that they are functionally connected and can interact with each other. Such elements may include, but are not limited to, promoters, enhancers, polyadenylation sequences, one or more introns, and the coding sequence of the gene of interest to be expressed (i.e., the transgene). When properly oriented or operably linked, nucleic acid sequence elements can act together to modulate each other's activity, ultimately affecting the level of transgene expression. Modulating means increasing, decreasing, or maintaining the level of activity of a particular element. The position of each element relative to other elements may be expressed in terms of the 5' and 3' ends of each element, and the distance between any particular elements may be referenced by the number of nucleotides or base pairs intervening between the elements. As will be understood by those skilled in the art, operably linked refers to functional activity and does not necessarily refer to the linkage of the natural position. Indeed, when used in a vector, regulatory elements are typically located immediately upstream of the promoter (although this is generally the case, it should not be construed as an absolute limitation or exclusion of location within the vector), although this may not necessarily be the case in vivo.

[0108] The promoter contained in the retroviral vector or gene therapy vector of the present invention can be any promoter known in the art, preferably a promoter that can induce transcription of a transgene in the target cell of the present invention.The promoter can be a naturally occurring promoter or a synthetic promoter.The promoter can be a ubiquitous promoter, i.e., a promoter that is active in a wide range of cells, tissues, and cell cycles.Alternatively, the promoter can be a promoter that is active only in a certain cell type or a single cell type, or only in a certain stage of the cell cycle.Furthermore, the promoter can be a constitutive promoter or a promoter for conditional expression.

[0109] As used herein, the term "constitutive promoter" refers to a promoter that allows continuous or sequential transcription of an operably linked sequence. A constitutive promoter can be a "ubiquitous promoter" that allows expression in a wide variety of cell and tissue types, or a "tissue-specific promoter" that allows expression in a limited variety of cell and tissue types. Exemplary ubiquitous promoters include the cytomegalovirus (CMV) immediate early promoter, the viral simian virus 40 (SV40) (e.g., early or late), the Moloney murine leukemia virus (MoMLV) LTR promoter, the Rous sarcoma virus (RSV) LTR, the herpes simplex virus (HSV) thymidine kinase promoter, the H5, P7.5, and P11 promoters from vaccinia virus, the elongation factor 1-alpha (EF1a) promoter, the early These include, but are not limited to, growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70 kDa protein 5 (HSPA5), heat shock protein 90 kDa β, member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), β-kinesin (β-KIN), human ROSA26 locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), ubiquitin C promoter (UBC), phosphoglycerate kinase 1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin (CAG) promoter, β-actin promoter, and U6 and H1 shRNA promoters.

[0110] In certain embodiments, it may be desirable to use tissue-specific promoters to achieve cell type-, lineage-, or tissue-specific expression of a desired polynucleotide sequence (e.g., to express a particular nucleic acid encoding a polypeptide only in a subset of cell types or tissues or during a specific stage of development). Examples of tissue-specific promoters include, but are not limited to, the B29 promoter (B cell expression), the runt transcription factor (CBFa2) promoter (stem cell-specific expression), the CD14 promoter (monocytic cell expression), the CD43 promoter (leukocyte and platelet expression), the CD45 promoter (hematopoietic cell expression), the CD68 promoter (macrophage expression), the CYP450 promoter (hematopoietic cell ... 3A4 promoter (hepatocyte expression), desmin promoter (muscle expression), elastase 1 promoter (pancreatic acinar cell expression), endoglin promoter (endothelial cell expression), fibroblast-specific protein 1 promoter (FSP1) promoter (fibroblast expression), fibronectin promoter (fibroblast expression), fms-related tyrosine kinase 1 (FLT1) promoter (endothelial cell expression), glial fibrillary acidic protein (GFAP) promoter (astrocytic expression), insulin promoter (pancreatic beta cell expression), integrin alpha 2b (ITGA2B) promoter (megakaryocytes), intercellular adhesion molecule 2 (ICAM-2) promoter (endothelial cells), interferon These include the IFN-β promoter (hematopoietic cells), keratinocyte expression, myoglobin (MB) promoter (muscle expression), myogenic differentiation 1 (MYOD1) promoter (muscle expression), nephrin promoter (podocyte expression), bone gamma-carboxyglutamic acid protein 2 (OG-2) promoter (osteoblast expression), 3-oxoacid CoA transferase 2B (Oxct2B) promoter (haploid spermatid expression), surfactant protein B (SP-B) promoter (lung expression), synapsin promoter (neuron expression), and Wiskott-Aldrich syndrome protein (WASP) promoter (hematopoietic cell expression).In one embodiment, the vector of the present invention comprises a tissue-specific promoter and / or enhancer, such as the MND promoter, that expresses a desired polypeptide in microglial cells. In some embodiments, the retroviral or gene therapy vector of the present invention may comprise a transgene under the control of the miR223 promoter.

[0111] In certain embodiments, the promoter contained in the retroviral vector can be any one of the vectors disclosed in EP 2021499 or Santilli et al. (2010) Mol Ther 19:122-32; PMID 20978475) or any vector comprising a chimeric promoter mentioned in PMID 20978475 and consisting of a fused promoter sequence derived from the cFES and cathepsin G promoter sequences.

[0112] As used herein, "conditional expression" can refer to any type of conditional expression, including, but not limited to, inducible expression; repressible expression; expression in cells or tissues with a specific physiological, biological or disease state, etc. This definition is not intended to exclude cell type or tissue-specific expression. Some aspects of the present invention provide conditional expression of a polynucleotide of interest, for example, expression is controlled by subjecting a cell, tissue, organism, etc. to a treatment or condition that causes the polynucleotide to be expressed or the expression of the polynucleotide encoded by the polynucleotide of interest to be increased or decreased.

[0113] Examples of inducible promoters / systems include, but are not limited to, steroid-inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone), metallothionine promoters (inducible by treatment with various heavy metals), MX-1 promoters (inducible by interferon), the "GeneSwitch" mifepristone-regulatable system (Sirin et al., 2003, Gene, 323:67), cumate-inducible gene switches (WO 2002 / 088346), tetracycline-dependent regulatory systems, and the like.

[0114] Conditional expression can also be achieved by using site-specific DNA recombinases. According to certain embodiments of the present invention, a vector contains at least one (typically two) site for recombination mediated by a site-specific recombinase. As used herein, the term "recombinase" or "site-specific recombinase" includes excisive or integrative proteins, enzymes, cofactors, or related proteins involved in recombination reactions involving one or more recombination sites (e.g., 2, 3, 4, 5, 7, 10, 12, 15, 20, 30, 50, etc.), and may be wild-type proteins (see Landy, Current Opinion in Biotechnology 3:699-707 (1993)) or mutants, derivatives (e.g., fusion proteins containing the recombinant protein sequence or fragments thereof), fragments, and variants thereof. Illustrative examples of recombinases suitable for use in particular embodiments of the present invention include, but are not limited to, Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, ΦC31, Cin, Tn3 resolvase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCE1, and ParA.

[0115] A variety of promoters have been described in the art, and those skilled in the art can identify the promoter that is particularly suitable for specific applications.However, it should be noted that neither the selection of transgene nor the selection of promoter are the limiting features of the method claimed herein.Therefore, the nucleotide of interest that is contained in gene therapy vector or retroviral vector can be any nucleotide, as long as the size of nucleotide does not exceed the genetic load of vector.

[0116] The methods of the present invention can be used to increase the transduction efficiency of target cells by retroviral vectors. The retroviral vector can contain any transgene.

[0117] In a particular embodiment, the present invention relates to a method according to the invention, wherein the vector comprises, in whole or in part, a cDNA encoding the p47phox, gp91phox, p22phox, p67phox or p40phox protein.

[0118] That is, the retroviral vector may contain a cDNA encoding any one of the proteins p47phox, gp91phox, p22phox, p67phox, or p40phox. In other embodiments, the retroviral vector may contain a fragment of a cDNA encoding any one of the proteins p47phox, gp91phox, p22phox, p67phox, or p40phox. The cDNA fragment may be the result of alternative splicing, or may be generated by genetic engineering or chemical synthesis, or may be any fusion construct containing a cDNA encoding the protein p47phox, gp91phox, p22phox, p67phox, or p40phox. The fragment may contain 50%, 60%, 70%, 80%, 90%, or 95% of the cDNA encoding the protein p47phox, gp91phox, p22phox, p67phox, or p40phox. Preferably, the variants of p47phox, gp91phox, p22phox, p67phox or p40phox expressed from the cDNA fragments have the same biological function as the respective full-length proteins.

[0119] In a particular embodiment, the present invention relates to a method according to the present invention, wherein the vector comprises a transgene encoding a chimeric antigen receptor (CAR).

[0120] That is, the retroviral vector used in the method of the present invention can contain a nucleic acid encoding a CAR. The CAR is not limiting in this method and can be any CAR known in the art.

[0121] In one embodiment of the invention, the transgene of interest, in particular p47phox, is under the control of an internal promoter, in particular the myeloid-specific miR223 promoter, an internal promoter selected from the group consisting of simian virus 40 (SV40) (e.g. early or late), cytomegalovirus (CMV) (e.g. immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV) and herpes simplex virus (HSV) (thymidine kinase) promoters, in particular the myeloid-specific miR223 promoter.

[0122] In one embodiment of the present invention, a retroviral vector, particularly a lentiviral-SIN vector, comprising a p47phox transgene under the control of a myeloid-specific promoter, particularly the miR223 promoter, is used in a method according to the present invention for the treatment of a disease or disorder associated with p47phox deficiency, particularly for the treatment of p47phox-deficient forms of chronic granulomatous disease. In one embodiment, a lentiviral vector encoding a cDNA encoding gp91phox, p22phox, p67phox, or p40phox under the control of the miR223 promoter is used in a method according to the present invention.

[0123] The term "miR223 promoter" refers to a DNA sequence that is 250 nt or more in length and has greater than 70%, 75%, 80%, 85%, 90%, 95% sequence homology to the following sequences: ACTTGTACAGCTTCACAGGGCTCCATGCTTAGAAGGACCCCACACTTAGTTTAATGTTCTGCTGTCATCATCTTGATATTCTTAATTTTTAAATAAAGGGCCTATCGTTTTCATTTTTTACTGGGCCTTGCAAATTATGTAGCTGGTTCTGTATGCCAGGAGAGAAGTTGGAAGTAAAATGGTATTCCAGGACCAGGAGGCATTCTGGCAGAGTGAAAGAACATGTGATTTGGAGTCCATGGGGATGGGTTTAAATTTCAGCTTTCCACTAATTTGCTTTGTGATACTGAGTATTTCCTTTTATCCCTCAGAGGCTCTGTTTCTCAATTTTGACTACGGGTTTTTTCATTAGATAATGTCTCAGTTCTGGTATTCCAGGTTTCCCTCAATTATTCTGGGAAAACCTCCTTGACCCACAGGCAGAGCCTAGGGCAGCCAGGTGCTTTCTACTCTCTCTCTCTCTGCAGCTTGGAAAGTTAGTGTCTGTTGAAGGTCAGCTGGGAGTTGGTGGAGGCAGGGCAGTGGCCTGCTACTATTGCTGCAGTAGCAGACCCTTTCACAACAGCATTGTTTTGTCATTTTGCATCCAGATTTCCGTTGGCTAACCTCAGTCTTATCTTCCTCATTTCTGTTTCCTGTTGAAGACACCAAGGGCCCTTCAAAACACAGAAGCTTCTTGCTCACGGCAGAAAGCCCAATTCCATCTGGCCCCTGCAGGTTGGCTCAGCACTGGGGAATCAGAGTCCCCTCCATGACCAAGGCACCACTCCACTGACAGGGATCCAAGCTTGCCACC(SEQ ID NO: 1).

[0124] The term "p47phox protein" refers to any protein of 26 amino acids or more in length that contains a sequence having greater than 70%, 75%, 80%, 85%, 90%, 95% homology to any of the isoforms and / or splice variants encoded by the human neutrophil cytosol factor 1 (NCF-1) gene having NCBI GeneID 653361, and / or any protein sequence having greater than 70%, 75%, 80%, 85%, 90%, 95% homology to the following protein sequences: [ka]

[0125] When target cells are pre-stimulated and / or co-stimulated with a transduction enhancer or a combination of transduction enhancers, the target cells are preferably incubated in the presence of the transduction enhancer or the combination of transduction enhancers in a liquid medium, preferably a liquid cell culture medium.

[0126] Those skilled in the art will recognize that the selection of cell culture medium depends on the type of target cell.That is, cell culture medium is preferably the medium that can maintain and / or grow target cell in it.Various cell culture mediums suitable for maintaining and / or growing cells of specific cell types have been described in the art and are commercially available.

[0127] In some embodiments, the target cells are hematopoietic stem cells (HSCs). Various media for culturing HSCs are known in the art. In some embodiments, HSCs can be pre-stimulated and / or costimulated with a transduction enhancer or a combination of transduction enhancers by incubating the HSCs in a liquid medium containing X-Vivo 10 medium (Lonza), X-Vivo 20 medium (Lonza), or BESP1366F medium (modified X-VIVO 20 without antibiotics (gentamicin); Lonza).

[0128] In one embodiment, target cells, particularly HSCs, can be incubated with a transduction enhancer or a combination of transduction enhancers in cell culture medium, particularly X-VIVO 10, X-VIVO 20, or BESP1366F medium, the cell culture medium containing 1% human serum albumin, 300 ng / ml stem cell factor (SCF), 200 ng / ml or 300 ng / ml fms-like tyrosine kinase 3 (FLT-3) ligand (Flt3-lig), and / or 100 ng / ml thrombopoietin (TPO).

[0129] In one embodiment, target cells, particularly HSCs, can be incubated with a transduction enhancer or a combination of transduction enhancers in X-VIVO 10 medium containing 1% human serum albumin, 300 ng / ml stem cell factor (SCF), 300 ng / ml fms-like tyrosine kinase 3 (FLT-3) ligand (Flt3-lig) and / or 100 ng / ml thrombopoietin (TPO).

[0130] In some embodiments, target cells, particularly HSCs, may be incubated with a transduction enhancer or a combination of transduction enhancers in X-VIVO 20 medium containing 1% human serum albumin, 300 ng / ml stem cell factor (SCF), 200 ng / ml fms-like tyrosine kinase 3 (FLT-3) ligand (Flt3-lig) and / or 100 ng / ml thrombopoietin (TPO).

[0131] In one embodiment, target cells, particularly HSCs, can be incubated with a transduction enhancer or a combination of transduction enhancers in BESP1366F medium containing 1% human serum albumin, 300 ng / ml stem cell factor (SCF), 300 ng / ml fms-like tyrosine kinase 3 (FLT-3) ligand (Flt3-lig) and / or 100 ng / ml thrombopoietin (TPO).

[0132] Target cells can be pre-stimulated and / or costimulated with a transduction enhancer or combination of transduction enhancers at any cell density or concentration.

[0133] That is, target cells, particularly HSCs, are cultured at a density of about 1E3 to about 1E10 cells / cm. 2 and a cell density in the range of about 1E4 to about 1E8 cells / cm. 2 and more preferably about 1E5 to about 1E7 cells / cm. 2 and most preferably at a cell density in the range of about 2E6 cells / cm 2 At a cell density of 1000 x g, the cells can be incubated with a transduction enhancer.

[0134] Alternatively, target cells, particularly HSCs, may be incubated with the transduction enhancer at a concentration ranging from about 1E3 to about 1E10 cells / mL, preferably from about 1E4 to about 1E8 cells / mL, more preferably from about 1E5 to about 1E7 cells / mL, and most preferably from 0.1E6 to 4E6 cells / mL.

[0135] Several novel compounds and compound combinations were tested for their potential to increase the transduction efficiency of human cells with gene therapy vectors, with a particular focus on compound combinations with retroviral vectors, particularly lentiviral-SIN vectors, encoding transgenes of interest, such as, but not limited to, p47phox.

[0136] In certain embodiments, the present invention refers to the compound amphotericin B for use as a transduction enhancer. That is, the present invention is based, at least in part, on the surprising finding that amphotericin B can enhance the transduction efficiency of target cells with gene therapy vectors, particularly retroviral vectors. Thus, in certain embodiments, the present invention relates to methods according to the present invention, wherein the transduction enhancer is amphotericin B.

[0137] Amphotericin B is an antifungal drug used to treat serious fungal infections and leishmaniosis. Fungal infections that amphotericin B is used to treat include aspergillosis, blastomycosis, candidiasis, coccidioidomycosis, and cryptococcosis. Amphotericin B is typically given by intravenous injection. It was isolated from Streptomyces nodosus in 1955 and entered medical use in 1958. Amphotericin B is listed on the World Health Organization's list of essential medicines and is among the safest and most effective medicines needed in the healthcare system. Amphotericin B is not indicated for use as a transduction enhancer.

[0138] The present inventors have demonstrated that amphotericin B enhances the transduction efficiency of target cells when contacted with the target cells at a concentration of 0.5 to 1 μg / mL. Therefore, in certain embodiments, amphotericin B can be used as a transduction enhancer at a concentration ranging from about 0.05 to about 10 μg / mL, preferably from about 0.1 to about 5 μg / mL, more preferably from about 0.5 to about 2 μg / mL, and most preferably at a concentration of about 0.75 μg / mL. In certain embodiments, amphotericin B can be used as a transduction enhancer at a concentration ranging from about 0.05 μM to about 500 μM, preferably from about 0.1 μM to about 10 μM, more preferably from about 0.1 to about 3 μM, and most preferably at a concentration of 0.811 μM. Preferably, amphotericin B is contacted with hematopoietic cells, more preferably HSCs, in the pre-stimulation or costimulation step at any of the concentrations and / or densities disclosed above. In certain embodiments, amphotericin B is used at a concentration of 0.5 to 1E6 cells / mL or at a density of 2E6 cells / cm. 2 HSCs at a concentration on the cell culture surface can be pre-stimulated and / or costimulated with amphotericin B at a concentration of 0.5 μg / mL. In some embodiments, HSCs are cultured at a concentration of 0.5-1E6 cells / mL or 2E6 cells / cm. 2HSCs at a concentration on the cell culture surface can be pre-stimulated and / or costimulated with amphotericin B at a concentration of 0.75 μg / mL. In some embodiments, HSCs are cultured at a concentration of 0.5-1E6 cells / mL or 2E6 cells / cm. 2 HSCs at cell culture surface concentration can be pre-stimulated and / or costimulated with amphotericin B at a concentration of 1 μg / mL.

[0139] In one aspect, the present invention refers to the compound silibinin for use as a transduction enhancer. That is, the present invention is based, at least in part, on the surprising finding that silibinin can enhance the transduction efficiency of target cells with gene therapy vectors, particularly retroviral vectors. Thus, in a particular aspect, the present invention relates to a method according to the present invention, wherein the transduction enhancer is silibinin.

[0140] Silybinin, also known as silybin (both derived from the common name of the plant from which it is extracted, Silybum), is the primary active component of silymarin, a standardized extract of milk thistle seeds containing a mixture of flavonolignans consisting of silibinin, isosilibinin, silychristin, and silydianin. Silybinin itself is a roughly equimolar mixture of two diastereomers, silybin A and silybin B. Commercially available silibinin has the chemical name 2,3-dihydro-3-(4-hydroxy-3-methoxyphenyl)-2-(hydroxymethyl)-6-(3,5,7-trihydroxy-4-oxobenzopyran-2-yl)benzodioxin (CAS number: 22888-70-6).

[0141] Silibinin has been reported to inhibit hepatitis B virus entry into HepG2-NTCP-C4 cells (Umetsu et al., (2018) Biochem Biophys Rep. 14:20-25) and hepatitis C virus infection of primary human hepatocytes (Liu et al., (2017) Gut 66:1853-1861). Silybin (SO), a major compound from S. marianum L., has been reported to inhibit influenza A virus (IAV) infection of MDCK cells (Dai et al., (2013) Antimicrob Agents Chemother. 57:4433-443). Exposure of T cells during virus adsorption to Legalon-SIL (SIL), a water-soluble derivative of silibinin (but not silibinin), has been reported to block HIV infection (McClure et al., (2014) Virology 449:96-103).

[0142] There have been no reports of silibinin being used as a transduction enhancer. Moreover, in view of the antiviral activity of silibinin disclosed above, it is quite surprising that silibinin can be used to enhance the transduction of target cells with gene therapy vectors, particularly retroviral vectors.

[0143] The present inventors have shown that silibinin enhances the transduction efficiency of target cells when contacted with the target cells at a concentration of 1 to 5 μM. Thus, in certain embodiments, silibinin can be used as a transduction enhancer at a concentration ranging from about 0.05 to about 500 μM, preferably from about 0.05 to about 25 μM, more preferably from about 0.05 to about 10 μM, even more preferably from about 1 to about 10 μM, and most preferably at a concentration of about 3 μM. Preferably, silibinin is contacted with hematopoietic cells, more preferably HSCs, in the pre-stimulation or costimulation step at any of the concentrations and / or densities disclosed above. In certain embodiments, silibinin is used at a concentration of 0.5 to 1E6 cells / mL or 2E6 / cm. 2HSCs at a density of 0.5-1E6 cells / mL or 2E6 / cm can be pre-stimulated and / or costimulated with silibinin at a concentration of 3 μM. In some embodiments, HSCs at a density of 0.5-1E6 cells / mL or 2E6 / cm can be pre-stimulated and / or costimulated with silibinin at a concentration of 3 μM. 2 HSCs at a density of 100 μM can be pre-stimulated and / or costimulated with silibinin at a concentration of 5 μM.

[0144] In one aspect, the present invention refers to the compound midostaurin for use as a transduction enhancer. That is, the present invention is based, at least in part, on the surprising finding that midostaurin can enhance the transduction efficiency of target cells with gene therapy vectors, particularly retroviral vectors. Thus, in a particular aspect, the present invention relates to a method according to the present invention, wherein the transduction enhancer is midostaurin.

[0145] The protein kinase inhibitor midostaurin, also known as CGP 41251, was first developed as an anticancer drug (Meyer et al., (1989) Int J Cancer 43:851-6). This compound has been reported to reactivate HIV-1 expression from latently infected ACH2 cell lines and primary resting CD4+ T cells (Ao et al. (2016) Virol J 13:177). This drug has not been tested for its efficacy in increasing transduction efficiency.

[0146] The present inventors have demonstrated that midostaurin enhances the transduction efficiency of target cells when contacted with the target cells at a concentration of 100 to 400 nM. Thus, in certain embodiments, midostaurin can be used as a transduction enhancer at a concentration ranging from about 50 to about 500,000 nM, preferably from about 50 to about 25,000 nM, more preferably from about 50 to about 10,000 nM, even more preferably from about 50 to about 5,000 nM, even more preferably from about 50 to about 1,000 nM, even more preferably from about 50 to about 500 nM, and most preferably at a concentration of about 200 nM. Preferably, midostaurin is contacted with hematopoietic cells, more preferably HSCs, in the pre-stimulation or costimulation step at any of the concentrations and / or densities disclosed above. In certain embodiments, midostaurin is contacted with hematopoietic cells, more preferably HSCs, at a concentration of 0.5E6 cells / mL or 2E6 cells / cm. 2 HSCs at a density of 0.5E6 to 1E6 cells / mL or 2E6 / cm can be pre-stimulated and / or costimulated with midostaurin at a concentration of 100 nM. In some embodiments, HSCs at a density of 0.5E6 to 1E6 cells / mL or 2E6 / cm can be pre-stimulated and / or costimulated with midostaurin at a concentration of 100 nM. 2 HSCs at a density of 0.5E6 to 1E6 cells / mL or 2E6 / cm can be pre-stimulated and / or costimulated with midostaurin at a concentration of 200 nM. In some embodiments, HSCs at a density of 0.5E6 to 1E6 cells / mL or 2E6 / cm can be pre-stimulated and / or costimulated with midostaurin at a concentration of 200 nM. 2 HSCs at a density of 1000 nM can be pre-stimulated and / or costimulated with midostaurin at a concentration of 400 nM.

[0147] In one aspect, the present invention refers to the compound nystatin for use as a transduction enhancer. That is, the present invention is based, at least in part, on the surprising finding that nystatin can enhance the transduction efficiency of target cells with gene therapy vectors, particularly retroviral vectors. Thus, in a particular aspect, the present invention relates to a method according to the present invention, wherein the transduction enhancer is nystatin.

[0148] Nystatin is commonly used in cell culture due to its antifungal activity (Fassler et al., (2013) PLoS One 8:e76092). Nystatin is also a cholesterol-binding reagent known to disrupt caveolin-mediated endocytosis. Nystatin was previously tested for its ability to inhibit transduction of 293T cells with wild-type lentiviral vectors. No inhibition of vector entry by nystatin was observed, confirming that wild-type lentiviral vectors use clathrin-mediated endocytosis to enter cells (Lee, Dang, Joo, & Wang (2011) Virus Res. 160:340-50). Treating lentiviral particles with nystatin and then repurifying them from nystatin significantly reduced the infectivity of the repurified lentiviral particles (Guyader et al., (2002) J Virol. 76:10356-64). The transduction-enhancing effect of nystatin on retroviral transduction has not been reported previously.

[0149] The present inventors have shown that nystatin enhances the transduction efficiency of target cells when contacted with the target cells at a concentration of 100 μM. Thus, in certain embodiments, nystatin can be used as a transduction enhancer at a concentration ranging from about 10 to about 1000 μM, preferably from about 25 to about 500 μM, more preferably from about 50 to about 250 μM, and most preferably at a concentration of about 100 μM. Preferably, nystatin is contacted with hematopoietic cells, more preferably HSCs, in the pre-stimulation or costimulation step at any of the concentrations and / or densities disclosed above. In certain embodiments, nystatin is contacted with hematopoietic cells, more preferably HSCs, at a concentration of 0.5 to 1E6 cells / mL or 2E6 / cm. 2 HSCs at a density of 100 μM can be pre-stimulated and / or costimulated with nystatin at a concentration of 100 μM.

[0150] In one aspect, the present invention refers to the compound natamycin for use as a transduction enhancer. That is, the present invention is based, at least in part, on the surprising finding that natamycin can enhance the transduction efficiency of target cells with gene therapy vectors, particularly retroviral vectors. Thus, in a particular aspect, the present invention relates to a method according to the present invention, wherein the transduction enhancer is natamycin.

[0151] Natamycin is an antifungal agent used in the food industry for surface treatment of sausages and cheeses (Juneja, Dwivedi & Yan (2012) Annu Rev Food Sci Technol. 3:381-403). Natamycin has never been reported in the context of viral transduction.

[0152] The present inventors have shown that natamycin enhances the transduction efficiency of target cells when contacted with the target cells at a concentration of 3 μM. Thus, in one embodiment, natamycin can be used as a transduction enhancer at a concentration ranging from about 0.05 to about 500 μM, preferably from about 0.05 to about 10 μM, more preferably from about 1 to about 5 μM, and most preferably at a concentration of about 3 μM. In one embodiment, natamycin can be used as a transduction enhancer at a concentration ranging from about 0.1 to about 20 μM. Preferably, natamycin is contacted with hematopoietic cells, more preferably HSCs, in the pre-stimulation or costimulation step at any of the concentrations and / or densities disclosed above. In one embodiment, natamycin is used at a concentration of 0.5 to 1E6 cells / mL or 2E6 / cm. 2 HSCs at a density of about 100 μM can be pre-stimulated and / or co-stimulated with natamycin at a concentration of about 3 μM.

[0153] In one aspect, the present invention refers to the compound everolimus for use as a transduction enhancer. That is, the present invention is based at least in part on the surprising finding that everolimus can enhance the transduction efficiency of target cells with gene therapy vectors, particularly retroviral vectors. Thus, in a particular aspect, the present invention relates to a method according to the present invention, wherein the transduction enhancer is everolimus.

[0154] Everolimus is a drug used as an immunosuppressant to prevent rejection of organ transplants and in the treatment of renal cell carcinoma and other tumors. Everolimus and other mTOR inhibitors have also been the subject of much research as targeted therapies for use in many cancers.

[0155] The present inventors have shown that everolimus enhances the transduction efficiency of target cells when contacted with the target cells at a concentration of 1 μM. Thus, in certain embodiments, everolimus can be used as a transduction enhancer at a concentration ranging from about 0.1 to about 10 μM, preferably from about 0.2 to about 7.5 μM, more preferably from about 0.5 to about 5 μM, and most preferably at a concentration of about 1 μM. Preferably, everolimus is contacted with hematopoietic cells, more preferably HSCs, in the pre-stimulation or costimulation step at any of the concentrations and / or densities disclosed above. In certain embodiments, everolimus is used at a concentration of 0.5 to 1E6 cells / mL or 2E6 / cm. 2 HSCs at a density of 1 μM can be pre-stimulated and / or costimulated with everolimus at a concentration of 1 μM.

[0156] In certain embodiments, the present invention refers to compound deoxyribonucleosides for use as transduction enhancers. That is, the present invention is based, at least in part, on the surprising finding that deoxyribonucleosides can enhance the transduction efficiency of target cells with gene therapy vectors, particularly retroviral vectors. Thus, in certain embodiments, the present invention relates to methods according to the present invention, wherein the transduction enhancer is a deoxyribonucleoside.

[0157] The term "deoxyribonucleoside" refers to the chemical structure of 2'-deoxythymidine, i.e., having CAS number 50-89-5 and synonyms thymine deoxyriboside, 1-(2-deoxy-β-D-ribofuranosyl)-5-methyluracil, 1-(2-deoxy-β-D-ribofuranosyl)thymine, dT), and 2'-deoxyadenosine, i.e., having CAS number 958-09-8 and synonyms 9-(2-deoxy-β-D-ribofuranosyl)thymine, dT. The deoxyribonucleosides include any composition of 2'-deoxyguanosine (i.e., 9-(2-deoxy-β-D-ribofuranosyl)adenine, adenine deoxyriboside), 2'-deoxyguanosine (i.e., CAS No. 312693-72-4 and synonymous 9-(2-deoxy-β-D-ribofuranosyl)guanine, guanine-2'-deoxyriboside), and 2'-deoxycytidine (i.e., CAS No. 951-77-9 and synonymous cytosine deoxyriboside). Each deoxyribonucleoside may be present at the same concentration or at different concentrations. In a preferred embodiment, all four deoxyribonucleosides listed above are present in equimolar amounts. Deoxyribonucleosides have not been suggested as transduction enhancers for hematopoietic stem cells.

[0158] The present inventors have demonstrated that deoxyribonucleosides, when contacted with target cells at a final concentration of 0.3 to 2.5 mM, have the ability to enhance the transduction efficiency of hematopoietic stem cells as target cells. Thus, in certain embodiments, deoxyribonucleosides can be used as transduction enhancers at concentrations ranging from about 0.1 to about 10 mM, preferably from about 0.25 to about 7.5 mM, more preferably from about 0.5 to about 5 mM, and most preferably at a concentration of about 2.5 mM. Preferably, deoxyribonucleosides are contacted with hematopoietic cells, more preferably HSCs, in the pre-stimulation or costimulation step at any of the concentrations and / or densities disclosed above. In certain embodiments, deoxyribonucleosides are contacted with hematopoietic cells, more preferably HSCs, at a concentration of 0.5 to 1E6 cells / mL or 2E6 cells / cm. 2HSCs at a density of 0.3 mM can be pre-stimulated and / or costimulated with deoxyribonucleosides at a concentration of 0.3 mM.

[0159] In certain embodiments, the present invention refers to the use of BAB-type triblock copolymers as transduction enhancers. That is, the present invention is based, at least in part, on the surprising finding that BAB-type triblock copolymers can enhance the transduction efficiency of target cells with gene therapy vectors, particularly retroviral vectors. Thus, in certain embodiments, the present invention relates to methods according to the present invention, wherein the transduction enhancer is a BAB-type triblock copolymer.

[0160] The term "BAB triblock copolymer" refers to any polymer consisting of a linear arrangement of three blocks, each consisting of a polymeric form of repeating elements, in which a central hydrophobic polymer block "A" is flanked on either side by flanking hydrophilic polymer units designated "B." Polymers referred to herein as "BAB triblock copolymers" are synthesized by the covalent bonding of two "BA" diblock copolymers through a linker designated "L," which is preferably, but not limited to, hexamethylene diisocyanate (HMDI). In "BAB triblock copolymers," the terminal blocks "B" preferably have the formula -(CH2-CH2-O) x The "A" block may be formed by a polymer of ethylene glycol with -, and the "A" block may comprise or consist of poly(D,L-lactic-co-glycolic acid) (PLGA), of poly(lactide) (PLA) or of poly(-caprolactone) (PCL). The corresponding "BAB-type triblock copolymers" are referred to as "PEG-PLGA-PEG," "PEG-PLA-PEG," and "PEG-PCL-PEG" polymers, respectively.

[0161] The term "PEG-PLGA-PEG" polymer refers to a BAB type triblock copolymer, where the "B" block is -(CH2-CH2-O) x-, where the "A" block comprises or consists of poly(D,L-lactic-co-glycolic acid). The resulting polymer is called "methoxypoly(ethylene glycol)-b-poly(D,L-lactic-co-glycolic acid)-b-methoxypoly(ethylene glycol)" ("mPEG-PLGA-mPEG") and has the formula CH3-O-(CH2-CH2-O) x -(CO-CH2-O) y -(CO-CHCH3-O) z -L-(O-CHCH3-CO) z -(O-CH2-CO) y -(O-CH2-CH2) x -O-CH3 (in the case of the HMDI linker, L = CO-NH-CH2-(CH2)4-CH2-NH-CO, where x, y, and z are the numbers of monomers in the polymer). The same molecule can also be called poly(ethylene glycol)-b-poly(D,L-lactic-co-glycolic acid)-b-poly(ethylene glycol) ("PEG-PLGA-PEG") and has the formula CH3-(O-CH2-CH2) x -(O-CO-CH2) y -(O-CO-CHCH3) z -OLO-(CHCH3-CO-O) z -(CH2-CO-O) y -(CH2-CH2-O) x -CH3 (in the case of the HMDI linker, L = CO-NH-CH2-(CH2)4-CH2-NH-CO, where x, y, z are the number of monomers in the polymer).

[0162] In the polymers referred to herein as "PEG-PLGA-PEG" polymers, "L" comprises, or preferably, but not limited to, -CO-NH-CH-(CH)-CH-NH-CO-, in the case of the HMDI linker. Polymers referred to herein as "PEG-PLGA-PEG" may have a molecular weight of 10,000 to 16,000 daltons, preferably about 10,000 daltons, about 11,000 daltons, about 12,000 daltons, about 13,000 daltons, about 14,000 daltons, about 15,000 daltons, or about 16,000 daltons. The "B" block may comprise, preferably, but not limited to, a polymer formed by polymerizing ethylene glycol. The PEG portion of the polymer may contribute more than 50% but less than 95% to the total molecular weight of the polymer. That is, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the total molecular weight of the polymer "PEG-PLGA-PEG" can be attributed to PEG polymers. In some embodiments, PEG-PLGA-PEG can refer to poly(ethylene glycol)-b-poly(D,L-lactic-co-glycolic acid)-b-poly(ethylene glycol) (PEG-PLGA-PEG), which has 5 kDa poly(ethylene glycol) blocks at both ends and a central 4.2 kDa poly(D,L-lactic-co-glycolic acid) block, designated PEG5k-b-PLGA4.2k-b-PEG5k.

[0163] PEG-PLGA-PEG polymers have been described to form micelles in a concentration- and temperature-dependent manner (Jeong, Bae, & Kim (1999) Colloids and Surfaces B: Biointerfaces 16:185-93) and can be used for drug delivery (Tyagi et al. (2004) Pharm Res. 21:832-7). The above-mentioned PEG-PLGA-PEG polymers have not been reported to have viral transduction-enhancing activity.

[0164] PEG-PLGA-PEG can be used as a transduction enhancer at a concentration ranging from about 20 μg / ml to about 5000 μg / ml, preferably from about 100 μg / ml to about 3500 μg / ml, more preferably from about 500 μg / ml to about 2000 μg / ml, and most preferably at a concentration of about 1000 μg / ml. Preferably, PEG-PLGA-PEG is contacted with hematopoietic cells, more preferably HSCs, in a pre-stimulation or costimulation step at any of the concentrations and / or densities disclosed above. In some embodiments, PEG-PLGA-PEG is used at a concentration of 0.5 to 1E6 cells / mL or 2E6 cells / cm. 2 HSCs at a density of 1000 μg / ml can be pre-stimulated and / or co-stimulated with PEG-PLGA-PEG at a concentration of 1000 μg / ml.

[0165] The term "PEG-PLA-PEG" polymer refers to a BAB type triblock copolymer, where the "B" block is -(CH2-CH2-O) n -, and the "A" block comprises or consists of a polymeric form of lactic acid. Polymeric forms of lactic acid include the polymeric forms of the enantiomeric L- and / or D-lactic acid, also known as poly(L-lactide) (PLLA) and poly(D-lactide) (PDLA). The resulting polymer is called methoxypoly(ethylene glycol) / poly(lactide) / methoxypoly(ethylene glycol) (PEG-PLA-PEG or mPEG-PLA-mPEG, collectively referred to herein as PEG-PLA-PEG), and has the formula CH3-O-(CH2-CH2-O) n -(CO-CCH3-O) m -L-(O-CCH3-CO) m -(O-CH2-CH2) n It can be summarized by -O-CH3 (in the case of HMDI linker, L = CO-NH-CH2-(CH2)4-CH2-NH-CO, n, m are the number of monomers in the polymer).

[0166] In the polymers referred to herein as "PEG-PLA-PEG" polymers, "L" can comprise, or preferably, but not limited to, CO-NH-CH-(CH)-CH-NH-CO, in the case of the HMDI linker. The polymers referred to herein as "PEG-PLA-PEG" polymers can have a molecular weight of 10,000 to 16,000 daltons, preferably about 10,000 daltons, about 11,000 daltons, about 12,000 daltons, about 13,000 daltons, about 14,000 daltons, about 15,000 daltons, or about 16,000 daltons. The "B" block can comprise, preferably, but not limited to, a polymer formed by polymerizing ethylene glycol. The PEG portion of the polymer can contribute more than 50% but less than 95% to the total molecular weight of the polymer. That is, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the total molecular weight of the polymer "PEG-PLA-PEG" can be attributed to PEG polymers. In some embodiments, PEG-PLA-PEG can refer to a poly(ethylene glycol) / poly(lactide) / poly(ethylene glycol) (PEG-PLA-PEG) having 5 kDa poly(ethylene glycol) blocks at both ends and a 4.2 kDa poly(lactide) block in the middle, designated PEG5k-b-PLA4.2k-b-PEG5k.

[0167] The present inventors have shown that PEG-PLA-PEG enhances the transduction efficiency of target cells when contacted with the target cells at a concentration of 10 μg / mL. Thus, in certain embodiments, PEG-PLA-PEG can be used as a transduction enhancer at a concentration ranging from about 0.1 μg / mL to about 5000 μg / mL, preferably from about 1 μg / mL to about 2500 μg / mL, more preferably from about 5 μg / mL to about 1000 μg / mL, and most preferably at a concentration of about 50 μg / mL. In certain embodiments, PEG-PLA-PEG can be used as a transduction enhancer at a concentration ranging from about 1 μg / mL to about 100 μg / mL, preferably from about 5 μg / mL to about 50 μg / mL. Preferably, PEG-PLA-PEG is contacted with hematopoietic cells, more preferably HSCs, at any of the concentrations and / or densities disclosed above during the pre-stimulation or costimulation step. In one embodiment, a concentration of 0.5 to 1E6 cells / mL or 2E6 / cm 2 HSCs at a density of 100 μg / ml can be pre-stimulated and / or co-stimulated with PEG-PLA-PEG at a concentration of 50 μg / ml.

[0168] The term "PEG-PCL-PEG" polymer refers to a BAB type triblock copolymer, where the "B" block is -(CH2-CH2-O) n -, and the "A" block comprises or consists of the polymeric form of e-caprolactone. The resulting polymer may be referred to as "methoxypoly(ethylene glycol)-poly(e-caprolactone)-methoxypoly(ethylene glycol)" (PEG-PCL-PEG) and has the formula CH3-O-(CH2-CH2-O) n -(CO-CH2-CH2-CH2-CH2-CH2-O) m -L-(O-CCH3-CO) m -(O-CH2-CH2) n It can be summarized by -O-CH3 (in the case of HMDI linker, L = CO-NH-CH2-(CH2)4-CH2-NH-CO, n, m are the number of monomers in the polymer).

[0169] In the polymers referred to herein as "PEG-PCL-PEG" polymers, "L" can comprise, in the case of the HMDI linker, L=CO-NH-CH2-(CH2)4-CH2-NH-CO, or preferably, but not limited to, L=CO-NH-CH2-(CH2)4-CH2-NH-CO. Polymers referred to herein as "PEG-PCL-PEG" polymers can have a molecular weight of 10,000 to 16,000 daltons, preferably about 10,000 daltons, about 11,000 daltons, about 12,000 daltons, about 13,000 daltons, about 14,000 daltons, about 15,000 daltons, or about 16,000 daltons. The "B" block can comprise, preferably, but not limited to, a polymer formed by polymerizing ethylene glycol. The PEG portion of the polymer can contribute more than 50% but less than 95% to the total molecular weight of the polymer. That is, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the total molecular weight of the polymer "PEG-PCL-PEG" can be attributed to PEG polymers. In some embodiments, PEG-PCL-PEG can refer to poly(ethylene glycol)-poly(e-caprolactone)-poly(ethylene glycol) (PEG-PCL-PEG), which has 5 kDa poly(ethylene glycol) blocks at both ends and a central 4.2 kDa poly(e-caprolactone) block, designated PEG5k-b-PCL4.2k-b-PEG5k. In some embodiments, PEG-PCL-PEG may refer to poly(ethylene glycol)-poly(e-caprolactone)-poly(ethylene glycol) (PEG-PCL-PEG) having 5.3 kDa poly(ethylene glycol) blocks at both ends and a central 2.4 kDa poly(e-caprolactone) block, designated NH2-PEG5.3k-b-PCL2.4k-b-PEG5.3k-NH2.

[0170] The present inventors have shown that PEG-PCL-PEG enhances the transduction efficiency of target cells when contacted with the target cells at a concentration of 10 μg / mL. Thus, in certain embodiments, PEG-PCL-PEG can be used as a transduction enhancer at a concentration ranging from about 0.1 μg / mL to about 5000 μg / mL, preferably from about 1 μg / mL to about 2500 μg / mL, more preferably from about 5 μg / mL to about 1000 μg / mL, and most preferably at a concentration of about 10 μg / mL. In certain embodiments, PEG-PCL-PEG can be used as a transduction enhancer at a concentration ranging from about 1 μg / mL to about 100 μg / mL, preferably from about 5 μg / mL to about 50 μg / mL. Preferably, PEG-PCL-PEG is contacted with hematopoietic cells, more preferably HSCs, at any of the concentrations and / or densities disclosed above in the pre-stimulation or costimulation steps. In one embodiment, a concentration of 0.5 to 1E6 cells / mL or 2E6 / cm 2 HSCs at a density of 10 μg / ml can be pre-stimulated and / or co-stimulated with PEG-PCL-PEG at a concentration of 10 μg / ml.

[0171] The present invention further encompasses the use of functionalized BAB-type triblock polymers for use as transduction enhancers. The term "functionalized" polymer refers to "BAB-type triblock copolymers," including "PEG-PLGA-PEG," "PEG-PLA-PEG," and "PEG-PCL-PEG" polymers, that have been "functionalized" by the covalent attachment of cationic groups to one and / or both ends of the polymer. In these functionalized "BAB-type triblock copolymers," the cationic groups may comprise or consist of molecules containing amino groups, such as, but not limited to, monomeric and / or polymeric forms of lysine, arginine, and / or histidine.

[0172] In one aspect, the present invention refers to the compound resveratrol for use as transduction enhancer.That is, the present invention is based at least in part on the surprising finding that resveratrol can enhance the transduction efficiency of target cells with gene therapy vectors, particularly retroviral vectors.Therefore, in a particular aspect, the present invention relates to the method according to the present invention, wherein the transduction enhancer is resveratrol.

[0173] Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is a naturally occurring phenol and stilbenoid, a phytoalexin produced by some plants in response to wounding or when the plant is under attack by pathogens such as bacteria or fungi. Resveratrol has been studied for its potential therapeutic uses, with little evidence of anti-disease effects or health benefits in humans.

[0174] The present inventors have shown that resveratrol enhances the transduction efficiency of target cells when contacted with the target cells at a concentration of 5 μM. Thus, in certain embodiments, resveratrol can be used as a transduction enhancer at a concentration ranging from about 0.1 to about 10 μM, preferably from about 1 to about 7.5 μM, more preferably from about 2.5 to about 7.5 μM, and most preferably at a concentration of about 5 μM. Preferably, resveratrol is contacted with hematopoietic cells, more preferably HSCs, in the pre-stimulation or costimulation step at any of the concentrations and / or densities disclosed above. In certain embodiments, resveratrol is used at a concentration of 0.5 to 1E6 cells / mL or 2E6 / cm. 2 HSCs at a density of 100 μM can be pre-stimulated and / or co-stimulated with resveratrol at a concentration of 5 μM.

[0175] In some embodiments, the present invention refers to the compound prostaglandin E2 for use as a transduction enhancer.That is, the present invention is based at least in part on the surprising finding that prostaglandin E2 can enhance the transduction efficiency of target cells with gene therapy vectors, particularly retroviral vectors.Therefore, in certain embodiments, the present invention relates to the method according to the present invention, wherein the transduction enhancer is prostaglandin E2.

[0176] Prostaglandin E2 (PGE2), also known as dinoprostone, is a naturally occurring prostaglandin with oxytocin-like properties used as a drug. Dinoprostone is used in labor induction, postpartum hemorrhage, abortion, and to keep the ductus arteriosus open in newborns. In infants, dinoprostone is used in those with congenital heart defects until surgery can be performed. Dinoprostone is also used to manage gestational trophoblastic disease.

[0177] The present inventors have shown that prostaglandin E2 enhances the transduction efficiency of target cells when contacted with the target cells at a concentration of 10 μM. Thus, in certain embodiments, prostaglandin E2 can be used as a transduction enhancer at a concentration ranging from about 1 to about 100 μM, preferably from about 2 to about 50 μM, more preferably from about 5 to about 25 μM, and most preferably at a concentration of about 10 μM. Preferably, prostaglandin E2 is contacted with hematopoietic cells, more preferably HSCs, in the pre-stimulation or costimulation step at any of the concentrations and / or densities disclosed above. In certain embodiments, prostaglandin E2 is contacted with hematopoietic cells, more preferably HSCs, at a concentration of 0.5 to 1E6 cells / mL or 2E6 / cm. 2 HSCs at a density of 10 μM can be pre-stimulated and / or costimulated with prostaglandin E2 at a concentration of 10 μM.

[0178] In some embodiments, the present invention refers to the compound poloxamer synperonic F108 for use as a transduction enhancer. That is, the present invention is based at least in part on the surprising finding that poloxamer synperonic F108 can enhance the transduction efficiency of target cells with gene therapy vectors, particularly retroviral vectors. Thus, in certain embodiments, the present invention relates to a method according to the present invention, wherein the transduction enhancer is poloxamer synperonic F108.

[0179] Poloxamer Synperonic F108 is a non-ionic polymeric surfactant.

[0180] The present inventors have demonstrated that poloxamer synperonic F108 enhances the transduction efficiency of target cells when contacted with the target cells at a concentration of 0.5 to 2 mg / mL. Thus, in certain embodiments, poloxamer synperonic F108 can be used as a transduction enhancer at a concentration ranging from about 0.1 to about 10 mg / mL, preferably from about 0.25 to about 5 mg / mL, more preferably from about 0.5 to about 2 mg / mL, and most preferably at a concentration of about 1 mg / mL. Preferably, poloxamer synperonic F108 is contacted with hematopoietic cells, more preferably HSCs, at any of the concentrations and / or densities disclosed above in the pre-stimulation or costimulation step. In certain embodiments, HSCs at a concentration of 0.5E6 cells / mL can be pre-stimulated and / or costimulated with poloxamer synperonic F108 at a concentration of 0.5 mg / mL. In one embodiment, a concentration of 0.5 to 1E6 cells / mL or 2E6 / cm 2 HSCs at a density of 1000-10 ... 2 HSCs at a density of 1000 μg / mL can be pre-stimulated and / or costimulated with poloxamer synperonic F108 at a concentration of 2 mg / mL.

[0181] In some embodiments, the present invention refers to the compound dimethyl sulfoxide (DMSO) for use as a transduction enhancer. That is, the present invention is based at least in part on the surprising finding that DMSO can enhance the transduction efficiency of target cells with gene therapy vectors, particularly retroviral vectors. Thus, in certain embodiments, the present invention relates to a method according to the present invention, wherein the transduction enhancer is DMSO.

[0182] Dimethyl sulfoxide (DMSO) is an organosulfur compound with the formula (CH3)2SO. This colorless liquid is an important polar aprotic solvent that dissolves both polar and nonpolar compounds and is miscible with a wide range of organic solvents and water.

[0183] The present inventors have shown that DMSO enhances the transduction efficiency of target cells when contacted with the target cells at a concentration of 1% (v / v). Thus, in certain embodiments, DMSO can be used as a transduction enhancer at a concentration ranging from about 0.1 to about 10% (v / v), preferably at a concentration ranging from about 0.25 to about 5% (v / v), more preferably at a concentration ranging from about 0.5 to about 2% (v / v), and most preferably at a concentration of about 1% (v / v). Preferably, DMSO is contacted with hematopoietic cells, more preferably HSCs, in the pre-stimulation or costimulation step at any of the concentrations and / or densities disclosed above. In certain embodiments, HSCs at a concentration of 0.5E6 cells / mL can be pre-stimulated and / or costimulated with DMSO at a concentration of 0.5% (v / v). In certain embodiments, DMSO can be used at a concentration of 0.5 to 1E6 cells / mL or at a concentration of 2E6 / cm. 2 HSCs at a density of 0.5-1E6 cells / mL or 2E6 / cm can be pre-stimulated and / or costimulated with DMSO at a concentration of 1% (v / v). In some embodiments, HSCs at a density of 0.5-1E6 cells / mL or 2E6 / cm can be pre-stimulated and / or costimulated with DMSO at a concentration of 1% (v / v). 2 HSCs at a density of 1000 μg / ml can be pre-stimulated and / or co-stimulated with DMSO at a concentration of 2% (v / v).

[0184] Surprisingly, the inventors have shown that certain combinations of compounds disclosed herein can result in enhanced transduction efficiency and should be considered suitable for future applications of multiple transduction enhancers in all these procedures.

[0185] Furthermore, the commercially available compound Lentiboost® has been described to enhance the transduction of various human target cells by lentiviral vectors. The inventors surprisingly demonstrated that the transduction efficiency of Lentiboost® can be further increased by combining Lentiboost® with an additional transduction enhancer. Lentiboost® consists of a combination of poloxamer F108 and polybrene, but the exact ratio of the two compounds has never been disclosed in the art. An exemplary mixture of the two compounds is disclosed in Example 2.

[0186] Thus, in one embodiment, the present invention relates to a method according to the invention, wherein the transduction enhancer is a combination of Lentiboost® and amphotericin B.

[0187] Lentiboost® is recommended for use at a concentration of 1 mg / mL. Transduction of HSCs with lentiviral vectors at an MOI of 10 in the presence of 1 mg / mL Lentiboost® results in a vector copy number (VCN) of approximately 5. We have shown that the combination of 1 mg / mL Lentiboost® with 0.5–1 μg / mL amphotericin B results in a VCN in the range of 8–10 under comparable conditions. Interestingly, simply increasing the Lentiboost® concentration to 2 mg / mL only resulted in a VCN of approximately 6. Thus, we surprisingly demonstrated that the combination of Lentiboost® and amphotericin B results in increased transduction efficiency.

[0188] Lentiboost® may be used in combination with amphotericin B as a transduction enhancer at any suitable concentration. In some embodiments, the combination of Lentiboost® and amphotericin B may include Lentiboost® at a concentration ranging from about 0.1 mg / mL to about 10 mg / mL, and amphotericin B at a concentration ranging from about 0.1 μg / mL to about 10 μg / mL. In some embodiments, the combination of Lentiboost® and amphotericin B may include Lentiboost® at a concentration ranging from about 0.1 mg / mL to about 10 mg / mL, and amphotericin B at a concentration ranging from about 0.1 μg / mL to about 10 μg / mL. In certain embodiments, Lentiboost® may be added to target cells at a final concentration ranging from about 0.1 mg / mL to about 10 mg / mL in combination with amphotericin B at a final concentration ranging from about 0.1 μg / mL to about 10 μg / mL. Preferably, Lentiboost® may be added to target cells at a final concentration ranging from about 0.1 mg / mL to about 3 mg / mL in combination with amphotericin B at a final concentration ranging from about 0.1 μg / mL to about 3 μg / mL. More preferably, Lentiboost® may be added to target cells at a final concentration ranging from about 0.5 mg / mL to about 2 mg / mL in combination with amphotericin B at a final concentration ranging from about 0.5 μg / mL to about 2 μg / mL. Most preferably, Lentiboost® may be added to target cells at a final concentration of about 1 mg / mL in combination with amphotericin B at a final concentration of about 0.75 μg / mL. Preferably, the combination of Lentiboost® and amphotericin B is contacted with hematopoietic cells, more preferably HSCs, in a pre-stimulation or costimulation step at any of the concentrations and / or densities disclosed above. In certain embodiments, the combination is at a concentration of 0.5 to 1E6 cells / mL or 2E6 / cm. 2 HSCs at a density of 1 mg / mL may be pre-stimulated and / or costimulated with a combination of 1 mg / mL Lentiboost® and 0.5 μg / mL amphotericin B. In some embodiments, HSCs at a concentration of 0.5-1E6 cells / mL or 2E6 / cm 2HSCs at a density of 1 mg / mL may be pre-stimulated and / or costimulated with a combination of 1 mg / mL Lentiboost® and 0.75 μg / mL amphotericin B. In some embodiments, HSCs at a concentration of 0.5-1E6 cells / mL or 2E6 / cm 2 HSCs at a density of 1 mg / mL can be pre-stimulated and / or co-stimulated with a combination of 1 mg / mL Lentiboost® and 1 μg / mL amphotericin B.

[0189] In one embodiment, the invention relates to a method according to the invention, wherein the transduction enhancer is a combination of Lentiboost® and silibinin.

[0190] The present inventors have shown that when Lentiboost® is used in combination with silibinin, it results in higher transduction efficiency compared to Lentiboost® alone. Lentiboost® can be used in combination with silibinin as a transduction enhancer at any appropriate concentration. In some embodiments, the combination of Lentiboost® and silibinin may include Lentiboost® at a concentration ranging from about 0.1 mg / mL to about 10 mg / mL and silibinin at a concentration ranging from about 0.1 to about 25 μM. In some embodiments, Lentiboost® can be added to target cells at a final concentration ranging from about 0.1 μg / mL to about 10 mg / mL in combination with silibinin at a final concentration ranging from about 0.1 to about 25 μM. Preferably, Lentiboost® may be added to target cells at a final concentration ranging from about 0.1 mg / mL to about 3 mg / mL in combination with silibinin at a final concentration ranging from about 0.1 to about 10 μM. More preferably, Lentiboost® may be added to target cells at a final concentration ranging from about 0.5 mg / mL to about 2 mg / mL in combination with silibinin at a final concentration ranging from about 1 to about 10 μM. Most preferably, Lentiboost® may be added to target cells at a final concentration of about 1 mg / mL in combination with silibinin at a final concentration of about 5 μM. Preferably, the combination of Lentiboost® and silibinin is contacted with hematopoietic cells, more preferably HSCs, in the pre-stimulation or costimulation step at any of the concentrations and / or densities disclosed above. In certain embodiments, the concentration ranges from 0.5 to 1E6 cells / mL or 2E6 / cm. 2 HSCs at a density of 1000 μg / cm may be pre-stimulated and / or costimulated with a combination of 1 mg / mL Lentiboost and 1 μM silibinin. In some embodiments, HSCs at a concentration of 0.5-1E6 cells / mL or 2E6 / cm may be pre-stimulated and / or costimulated with a combination of 1 mg / mL Lentiboost and 1 μM silibinin. 2 HSCs at a density of 1 mg / mL can be pre-stimulated and / or co-stimulated with a combination of 1 mg / mL Lentiboost and 5 μM silibinin.

[0191] In one embodiment the invention relates to a method according to the invention, wherein the transduction enhancer is a combination of Lentiboost® and midostaurin.

[0192] The present inventors have shown that when Lentiboost® is used in combination with midostaurin, it results in higher transduction efficiency compared to Lentiboost® alone. Lentiboost® can be used in combination with midostaurin as a transduction enhancer at any appropriate concentration. In some embodiments, the combination of Lentiboost® and midostaurin can include Lentiboost® at a concentration ranging from about 0.1 mg / mL to about 10 mg / mL and midostaurin at a concentration ranging from about 50 to about 20,000 nM. In some embodiments, Lentiboost® can be added to target cells at a final concentration ranging from about 0.1 μg / mL to about 10 mg / mL in combination with midostaurin at a final concentration ranging from about 50 to about 20,000 nM. Preferably, Lentiboost® may be added to target cells at a final concentration ranging from about 0.1 mg / mL to about 3 mg / mL in combination with midostaurin at a final concentration ranging from about 50 to about 5,000 nM. More preferably, Lentiboost® may be added to target cells at a final concentration ranging from about 0.5 mg / mL to about 2 mg / mL in combination with midostaurin at a final concentration ranging from about 50 to about 500 nM. Most preferably, Lentiboost® may be added to target cells at a final concentration of about 1 mg / mL in combination with midostaurin at a final concentration of about 400 nM. Preferably, the combination of Lentiboost® and midostaurin is contacted with hematopoietic cells, more preferably HSCs, in the pre-stimulation or costimulation step at any of the concentrations and / or densities disclosed above. In certain embodiments, the concentration ranges from 0.5 to 1E6 cells / mL or 2E6 / cm. 2HSCs at a density of 1 mg / mL may be pre-stimulated and / or costimulated with a combination of 1 mg / mL Lentiboost® and 100 nM midostaurin. In some embodiments, HSCs at a concentration of 0.5-1E6 cells / mL or 2E6 / cm may be pre-stimulated and / or costimulated with a combination of 1 mg / mL Lentiboost® and 100 nM midostaurin. 2 HSCs at a density of 1 mg / mL may be pre-stimulated and / or costimulated with a combination of 1 mg / mL Lentiboost® and 200 nM midostaurin. In some embodiments, HSCs at a concentration of 0.5-1E6 cells / mL or 2E6 / cm 2 HSCs at a density of 1 mg / mL can be pre-stimulated and / or co-stimulated with a combination of 1 mg / mL Lentiboost® and 400 nM midostaurin.

[0193] In one embodiment, the invention relates to a method according to the invention, wherein the transduction enhancer is a combination of poloxamer F108 and amphotericin B.

[0194] The present inventors have shown that when poloxamer F108 is used in combination with amphotericin B, it results in higher transduction efficiency compared to either of the two compounds alone. Poloxamer F108 can be used in combination with amphotericin B as a transduction enhancer at any suitable concentration. In some embodiments, the combination of poloxamer F108 and amphotericin B can include poloxamer F108 at a concentration ranging from about 0.1 mg / mL to about 10 mg / mL and amphotericin B at a concentration ranging from about 0.1 μg / mL to about 10 μg / mL. In some embodiments, poloxamer F108 can be added to target cells at a final concentration ranging from about 0.1 mg / mL to about 10 mg / mL in combination with amphotericin B at a final concentration ranging from about 0.1 μg / mL to about 10 μg / mL. Preferably, poloxamer F108 can be added to target cells at a final concentration ranging from about 0.1 mg / mL to about 3 mg / mL in combination with amphotericin B at a final concentration ranging from about 0.1 μg / mL to about 3 μg / mL. More preferably, poloxamer F108 can be added to target cells at a final concentration ranging from about 0.5 mg / mL to about 2 mg / mL in combination with amphotericin B at a final concentration ranging from about 0.5 μg / mL to about 2 μg / mL. Most preferably, poloxamer F108 can be added to target cells at a final concentration of about 1 mg / mL in combination with amphotericin B at a final concentration of about 0.75 μg / mL. Preferably, the combination of poloxamer F108 and amphotericin B is contacted with hematopoietic cells, more preferably HSCs, in the pre-stimulation or costimulation step at any of the concentrations and / or densities disclosed above. In certain embodiments, the concentration ranges from 0.5 to 1E6 cells / mL or 2E6 / cm. 2 HSCs at a density of 1 mg / mL may be pre-stimulated and / or costimulated with a combination of 1 mg / mL poloxamer F108 and 0.5 μg / mL amphotericin B. In some embodiments, HSCs at a concentration of 0.5-1E6 cells / mL or 2E6 / cm may be pre-stimulated and / or costimulated with a combination of 1 mg / mL poloxamer F108 and 0.5 μg / mL amphotericin B. 2 HSCs at a density of 1 mg / mL may be pre-stimulated and / or costimulated with a combination of 1 mg / mL poloxamer F108 and 0.75 μg / mL amphotericin B. In some embodiments, HSCs at a concentration of 0.5-1E6 cells / mL or 2E6 / cm2 HSCs at a density of 1 mg / mL can be pre-stimulated and / or costimulated with a combination of 1 mg / mL poloxamer F108 and 1 μg / mL amphotericin B.

[0195] In one embodiment, the present invention relates to a method according to the present invention, wherein the transduction enhancer is a combination of poloxamer F108 and silibinin.

[0196] The present inventors have demonstrated that the use of poloxamer F108 in combination with silibinin results in higher transduction efficiency compared to either compound alone. Poloxamer F108 can be used in combination with silibinin as a transduction enhancer at any appropriate concentration. In some embodiments, the combination of poloxamer F108 and silibinin may contain poloxamer F108 at a concentration ranging from about 0.1 mg / mL to about 10 mg / mL and silibinin at a concentration ranging from about 0.1 to about 25 μM. In some embodiments, poloxamer F108 can be added to target cells at a final concentration ranging from about 0.1 μg / mL to about 10 mg / mL in combination with silibinin at a final concentration ranging from about 0.1 to about 25 μM. Preferably, poloxamer F108 can be added to target cells at a final concentration ranging from about 0.1 mg / mL to about 3 mg / mL in combination with silibinin at a final concentration ranging from about 0.1 to about 10 μM. More preferably, poloxamer F108 can be added to target cells at a final concentration ranging from about 0.5 mg / mL to about 2 mg / mL in combination with silibinin at a final concentration ranging from about 1 to about 10 μM. Most preferably, poloxamer F108 can be added to target cells at a final concentration of about 1 mg / mL in combination with silibinin at a final concentration of about 5 μM. Preferably, the combination of poloxamer F108 and amphotericin B is contacted with hematopoietic cells, more preferably HSCs, in the pre-stimulation or costimulation step at any of the concentrations and / or densities disclosed above. In one embodiment, the concentration ranges from 0.5 to 1E6 cells / mL or 2E6 / cm. 2 HSCs at a density of 1 mg / mL can be pre-stimulated and / or costimulated with a combination of 1 mg / mL poloxamer F108 and 5 μM silibinin.

[0197] In one embodiment, the present invention relates to a method according to the present invention, wherein the transduction enhancer is a combination of poloxamer F108 and midostaurin.

[0198] The present inventors have demonstrated that the use of poloxamer F108 in combination with midostaurin results in higher transduction efficiency compared to either compound alone. Poloxamer F108 can be used in combination with midostaurin as a transduction enhancer at any appropriate concentration. In some embodiments, the combination of poloxamer F108 and midostaurin can include poloxamer F108 at a concentration ranging from about 0.1 mg / mL to about 10 mg / mL and midostaurin at a concentration ranging from about 50 to about 20,000 nM. In some embodiments, poloxamer F108 can be added to target cells at a final concentration ranging from about 0.1 μg / mL to about 10 mg / mL in combination with midostaurin at a final concentration ranging from about 50 to about 20,000 nM. Preferably, poloxamer F108 can be added to target cells at a final concentration ranging from about 0.1 mg / mL to about 3 mg / mL in combination with midostaurin at a final concentration ranging from about 50 to about 5,000 nM. More preferably, poloxamer F108 can be added to target cells at a final concentration ranging from about 0.5 mg / mL to about 2 mg / mL in combination with midostaurin at a final concentration ranging from about 50 to about 500 nM. Most preferably, poloxamer F108 can be added to target cells at a final concentration of about 1 mg / mL in combination with midostaurin at a final concentration of about 400 nM. Preferably, the combination of poloxamer F108 and midostaurin is contacted with hematopoietic cells, more preferably HSCs, in the pre-stimulation or costimulation step at any of the concentrations and / or densities disclosed above. In certain embodiments, the concentration ranges from 0.5 to 1E6 cells / mL or 2E6 / cm. 2 HSCs at a density of 100 μM may be pre-stimulated and / or costimulated with a combination of 1 mg / mL poloxamer F108 and 100 μM midostaurin. In some embodiments, HSCs at a concentration of 0.5 to 1E6 cells / mL or 2E6 / cm 2HSCs at a density of 1 mg / mL may be pre-stimulated and / or costimulated with a combination of 1 mg / mL poloxamer F108 and 200 μM midostaurin. In some embodiments, HSCs at a concentration of 0.5 to 1E6 cells / mL or 2E6 / cm 2 HSCs at a density of 1 mg / mL can be pre-stimulated and / or costimulated with a combination of 1 mg / mL poloxamer F108 and 400 μM midostaurin.

[0199] In one embodiment, the present invention relates to a method according to the present invention, wherein the transduction enhancer is a combination of silibinin and PEG-PCL-PEG.

[0200] The present inventors have demonstrated that the use of silibinin in combination with PEG-PCL-PEG results in higher transduction efficiency compared to either of the two compounds alone. Silibinin can be used in combination with PEG-PCL-PEG as a transduction enhancer at any appropriate concentration. In some embodiments, the combination of silibinin and PEG-PCL-PEG may contain silibinin at a concentration ranging from about 0.1 to about 25 μM and PEG-PCL-PEG at a concentration ranging from about 0.1 μg / ml to about 5,000 μg / ml. In some embodiments, silibinin can be added to target cells at a final concentration ranging from about 0.1 to about 25 μM in combination with PEG-PCL-PEG at a final concentration ranging from about 0.1 μg / ml to about 5,000 μg / ml. Preferably, silibinin is added to target cells at a final concentration ranging from about 0.1 μM to about 10 μM in combination with PEG-PCL-PEG at a final concentration ranging from about 0.1 to about 5,000 μg / ml. More preferably, silibinin is added to target cells at a final concentration ranging from about 1 to about 10 μM in combination with PEG-PCL-PEG at a final concentration ranging from about 5 μg / ml to about 1,000 μg / ml. Most preferably, silibinin is added to target cells at a final concentration of about 5 μM in combination with PEG-PCL-PEG at a final concentration of about 10 μg / ml. Preferably, the combination of silibinin and PEG-PCL-PEG is contacted with hematopoietic cells, more preferably HSCs, in the pre-stimulation or costimulation step at any of the concentrations and / or densities disclosed above. In certain embodiments, the concentration is 0.5 to 1E6 cells / mL or 2E6 / cm. 2 HSCs at a density of 10 μM can be pre-stimulated and / or co-stimulated with a combination of 5 μM silibinin and 10 μg / ml PEG-PCL-PEG.

[0201] In one embodiment, the invention relates to a method according to the invention, wherein the transduction enhancer is a combination of amphotericin B and everolimus.

[0202] The present inventors have shown that the use of amphotericin B in combination with everolimus results in higher transduction efficiency compared to either of the two compounds alone. Amphotericin B can be used in combination with everolimus as a transduction enhancer at any appropriate concentration. In some embodiments, the combination of amphotericin B and everolimus can include amphotericin B at a concentration ranging from about 0.1 μg / mL to about 10 μg / mL and everolimus at a concentration ranging from about 0.1 to about 10 μM. In some embodiments, amphotericin B can be added to target cells at a final concentration ranging from about 0.1 μg / mL to about 10 μg / mL in combination with everolimus at a final concentration ranging from about 0.1 to about 10 μM. Preferably, amphotericin B may be added to target cells at a final concentration ranging from about 0.1 μg / mL to about 3 μg / mL in combination with everolimus at a final concentration ranging from about 0.2 to about 7.5 μM. More preferably, amphotericin B may be added to target cells at a final concentration ranging from about 0.5 μg / mL to about 2 μg / mL in combination with everolimus at a final concentration ranging from about 0.5 to about 5 μM. Most preferably, amphotericin B may be added to target cells at a final concentration of about 1 μg / mL in combination with everolimus at a final concentration of about 1 μM. Preferably, the combination of amphotericin B and everolimus is contacted with hematopoietic cells, more preferably HSCs, in the pre-stimulation or costimulation step at any of the concentrations and / or densities disclosed above. In certain embodiments, the concentration is 0.5 to 1E6 cells / mL or 2E6 / cm. 2 HSCs at a density of 0.5 μg / mL may be pre-stimulated and / or costimulated with a combination of 0.5 μg / mL amphotericin B and 1 μM everolimus. In some embodiments, HSCs at a concentration of 0.5-1E6 cells / mL or 2E6 / cm 2 HSCs at a density of 0.75 μg / mL may be pre-stimulated and / or costimulated with a combination of 0.75 μg / mL amphotericin B and 1 μM everolimus. In some embodiments, HSCs at a concentration of 0.5-1E6 cells / mL or 2E6 / cm may be pre-stimulated and / or costimulated with a combination of 0.75 μg / mL amphotericin B and 1 μM everolimus. 2 HSCs at a density of 1 μg / mL can be pre-stimulated and / or costimulated with a combination of 1 μg / mL amphotericin B and 1 μM everolimus.

[0203] In certain embodiments, the present invention relates to a combination of a protamine salt with one or more of the transduction enhancers disclosed herein for use as a transduction enhancer.

[0204] As used herein, "protamine" refers to a group of strongly basic proteins present in sperm cells in salt-like combinations with nucleic acids. Protamine can be obtained, for example, from salmon (salmine), rainbow trout (iridin), herring (clupein), sturgeon (sutulin), or Spanish mackerel or tuna (chinin), and a wide variety of protamine salts are commercially available. It should be understood that the peptide composition of a particular protamine may vary depending on the family, genus, or species of fish from which the protamine is obtained. Protamine typically contains four major components, i.e., single-chain peptides of approximately 30-32 residues, of which approximately 21-22 are arginine residues. The N-terminus of each of the four major components is proline, and no other amino groups are present in the sequence. Therefore, chemical modification of protamine with a particular salt is expected to be homogeneous in this context.

[0205] Within the present invention, protamine salts used in the methods of the present invention may include, but are not limited to, chloride, sulfate, acetate, bromide, caproate, trifluoroacetate, HCO3, propionate, lactate, formate, nitrate, citrate, monohydrogen phosphate, dihydrogen phosphate, tartrate, or perchlorate salts of protamine, or a mixture of any two protamine salts.

[0206] In one embodiment, the protamine salt used in the method of the present invention is derived from salmon. In another embodiment, the protamine salt used in the method of the present invention is derived from herring. In yet another embodiment, the protamine salt used in the method of the present invention is derived from rainbow trout. In another embodiment, the protamine salt used in the method of the present invention is derived from tuna.

[0207] Protamine can be added to pre-incubation medium and / or co-incubation medium in any salt form, as long as the anionic component of the salt does not inhibit the transduction efficiency of target cells when in solution.The preferred protamine salts that can be used in the method of the present invention are protamine chloride and protamine sulfate.In some embodiments, protamine is added to pre-incubation medium and / or co-incubation medium as GMP-grade protamine chloride.

[0208] The present inventors have shown that protamine salts enhance the transduction efficiency of target cells when contacted with the target cells at a concentration of 4 μg / mL. Thus, in certain embodiments, protamine salts can be used as transduction enhancers at concentrations ranging from about 0.05 μg / mL to about 25 μg / mL, preferably from about 0.1 μg / mL to about 10 μg / mL, more preferably from about 1 μg / mL to about 10 μg / mL, and most preferably at a concentration of about 4 μg / mL. Preferably, protamine salts are contacted with hematopoietic cells, more preferably HSCs, in the pre-stimulation and / or costimulation steps at any of the concentrations and / or densities disclosed above. In certain embodiments, protamine salts are contacted with hematopoietic cells, more preferably HSCs, at any of the concentrations and / or densities disclosed above at concentrations of 0.5 to 1E6 cells / mL or 2E6 / cm. 2 HSCs at a density of 100 μg / mL can be pre-stimulated and / or costimulated with protamine salts at a concentration of 4 μg / mL.

[0209] In one embodiment, the invention relates to a method according to the invention, wherein the transduction enhancer is a combination of a protamine salt and amphotericin B.

[0210] The present inventors have shown that when protamine salts are used in combination with amphotericin B, they result in higher transduction efficiency compared to either of the two compounds alone. Protamine salts can be used in combination with amphotericin B as a transduction enhancer at any appropriate concentration. In certain embodiments, the combination of protamine salts and amphotericin B can include protamine salts at a concentration ranging from about 0.05 μg / mL to about 25 μg / mL and amphotericin B at a concentration ranging from about 0.1 μg / mL to about 10 μg / mL. In certain embodiments, protamine salts can be added to target cells at a final concentration ranging from about 0.05 μg / mL to about 25 μg / mL in combination with amphotericin B at a final concentration ranging from about 0.1 μg / mL to about 10 μg / mL. Preferably, protamine salts may be added to target cells at a final concentration ranging from about 0.1 μg / mL to about 10 μg / mL in combination with amphotericin B at a final concentration ranging from about 0.1 μg / mL to about 3 μg / mL. More preferably, protamine salts may be added to target cells at a final concentration ranging from about 1 μg / mL to about 10 μg / mL in combination with amphotericin B at a final concentration ranging from about 0.5 μg / mL to about 2 μg / mL. Most preferably, protamine salts may be added to target cells at a final concentration of about 4 μg / mL in combination with amphotericin B at a final concentration of about 1 μg / mL. Preferably, the combination of protamine salts and amphotericin B is contacted with hematopoietic cells, more preferably HSCs, in the pre-stimulation and / or costimulation steps at any of the concentrations and / or densities disclosed above. In certain embodiments, the combination is at a concentration of 0.5 to 1E6 cells / mL or 2E6 / cm. 2 HSCs at a density of 0.5-1E6 cells / mL or 2E6 / cm may be pre-stimulated and / or costimulated with a combination of 4 μg / mL protamine salts and 0.5 μg / mL amphotericin B. In some embodiments, HSCs at a concentration of 0.5-1E6 cells / mL or 2E6 / cm may be pre-stimulated and / or costimulated with a combination of 4 μg / mL protamine salts and 0.5 μg / mL amphotericin B. 2 HSCs at a density of 0.5-1E6 cells / mL or 2E6 / cm may be pre-stimulated and / or costimulated with a combination of 4 μg / mL protamine salts and 0.75 μg / mL amphotericin B. In some embodiments, HSCs at a density of 0.5-1E6 cells / mL or 2E6 / cm may be pre-stimulated and / or costimulated with a combination of 4 μg / mL protamine salts and 0.75 μg / mL amphotericin B. 2HSCs at a density of 1 μg / mL can be pre-stimulated and / or co-stimulated with a combination of 4 μg / mL protamine salts and 1 μg / mL amphotericin B.

[0211] In one embodiment, the present invention relates to a method according to the present invention, wherein the transduction enhancer is a combination of protamine salt and PEG-PCL-PEG.

[0212] The present inventors have shown that the use of protamine salt in combination with PEG-PCL-PEG results in higher transduction efficiency compared to either of the two compounds alone. Protamine salt can be used in combination with PEG-PCL-PEG as a transduction enhancer at any appropriate concentration. In some embodiments, the combination of protamine salt and PEG-PCL-PEG can include protamine salt at a concentration ranging from about 0.05 μg / mL to about 25 μg / mL and PEG-PCL-PEG at a concentration ranging from about 0.1 μg / mL to about 5,000 μg / mL. In some embodiments, protamine salt can be added to target cells at a final concentration ranging from about 0.05 μg / mL to about 25 μg / mL in combination with PEG-PCL-PEG at a final concentration ranging from about 0.1 μg / mL to about 5,000 μg / mL. Preferably, protamine salts are added to target cells at a final concentration ranging from about 0.1 μg / mL to about 10 μg / mL in combination with PEG-PCL-PEG at a final concentration ranging from about 1 μg / mL to about 2,500 μg / mL. More preferably, protamine salts are added to target cells at a final concentration ranging from about 1 μg / mL to about 10 μg / mL in combination with PEG-PCL-PEG at a final concentration ranging from about 5 μg / mL to about 1,000 μg / mL. Most preferably, protamine salts are added to target cells at a final concentration of about 4 μg / mL in combination with PEG-PCL-PEG at a final concentration of about 10 μg / mL. Preferably, the combination of protamine salts and PEG-PCL-PEG is contacted with hematopoietic cells, more preferably HSCs, in the pre-stimulation and / or costimulation steps at any of the concentrations and / or densities disclosed above. In certain embodiments, the concentration ranges from 0.5 to 1E6 cells / mL or 2E6 cells / cm. 2HSCs at a density of 10 μg / ml can be pre-stimulated and / or co-stimulated with a combination of 4 μg / ml protamine salts and 10 μg / ml PEG-PCL-PEG.

[0213] That is, in one aspect, the present invention relates to a method according to the present invention, wherein the transduction enhancer is a combination of a protamine salt and silibinin.

[0214] The present inventors have demonstrated that the use of protamine salts in combination with silibinin results in higher transduction efficiency compared to either of the two compounds alone. Protamine salts can be used in combination with silibinin as a transduction enhancer at any appropriate concentration. In some embodiments, the combination of protamine salts and silibinin may contain protamine salts at a concentration ranging from about 0.05 μg / mL to about 25 μg / mL and silibinin at a concentration ranging from about 0.1 to about 25 μM. In some embodiments, protamine salts can be added to target cells at a final concentration ranging from about 0.05 μg / mL to about 25 μg / mL in combination with silibinin at a final concentration ranging from about 0.1 to about 25 μM. Preferably, protamine salts can be added to target cells at a final concentration ranging from about 0.1 μg / mL to about 10 μg / mL in combination with silibinin at a final concentration ranging from about 0.1 to about 10 μM. More preferably, protamine salts are added to target cells at a final concentration ranging from about 1 μg / mL to about 10 μg / mL in combination with silibinin at a final concentration ranging from about 1 μM to about 10 μM. Most preferably, protamine salts are added to target cells at a final concentration of about 4 μg / mL in combination with silibinin at a final concentration of about 5 μM. Preferably, the combination of protamine salts and silibinin is contacted with hematopoietic cells, more preferably HSCs, at any of the concentrations and / or densities disclosed above in the pre-stimulation and / or costimulation steps. In one embodiment, the combination is at a concentration of 0.5 to 1E6 cells / mL or 2E6 / cm. 2 HSCs at a density of 10 μg / mL can be pre-stimulated and / or costimulated with a combination of 4 μg / mL protamine salts and 5 μM silibinin.

[0215] In one embodiment, the invention relates to a method according to the invention, wherein the transduction enhancer is a combination of protamine salt and resveratrol.

[0216] The present inventors have demonstrated that the use of protamine salt in combination with resveratrol results in higher transduction efficiency compared to either of the two compounds alone. Protamine salt can be used in combination with resveratrol as a transduction enhancer at any appropriate concentration. In some embodiments, the combination of protamine salt and resveratrol can include protamine salt at a concentration ranging from about 0.05 μg / mL to about 25 μg / mL and resveratrol at a concentration ranging from about 0.1 to about 10 μM. In some embodiments, protamine salt can be added to target cells at a final concentration ranging from about 0.05 μg / mL to about 25 μg / mL in combination with resveratrol at a final concentration ranging from about 0.1 to about 25 μM. Preferably, protamine salt can be added to target cells at a final concentration ranging from about 0.1 μg / mL to about 10 μg / mL in combination with resveratrol at a final concentration ranging from about 1 to about 7.5 μM. More preferably, protamine salts are added to target cells at a final concentration ranging from about 1 μg / mL to about 10 μg / mL in combination with resveratrol at a final concentration ranging from about 2.5 to about 7.5 μM. Most preferably, protamine salts are added to target cells at a final concentration of about 4 μg / mL in combination with resveratrol at a final concentration of about 5 μM. Preferably, the combination of protamine salts and resveratrol is contacted with hematopoietic cells, more preferably HSCs, in the pre-stimulation and / or costimulation steps at any of the concentrations and / or densities disclosed above. In certain embodiments, the combination is at a concentration of 0.5 to 1E6 cells / mL or 2E6 / cm. 2 HSCs at a density of 4 μg / mL can be pre-stimulated and / or co-stimulated with a combination of 4 μg / mL protamine salts and 5 μM resveratrol.

[0217] In one embodiment, the invention relates to a method according to the invention, wherein the transduction enhancer is a combination of a protamine salt and midostaurin.

[0218] The present inventors have demonstrated that the use of protamine salt in combination with midostaurin results in higher transduction efficiency compared to either of the two compounds alone. Protamine salt can be used in combination with midostaurin as a transduction enhancer at any appropriate concentration. In some embodiments, the combination of protamine salt and midostaurin may include protamine salt at a concentration ranging from about 0.05 μg / mL to about 25 μg / mL and midostaurin at a concentration ranging from about 50 to about 20,000 nM. In some embodiments, protamine salt can be added to target cells at a final concentration ranging from about 0.05 μg / mL to about 25 μg / mL in combination with midostaurin at a final concentration ranging from about 50 to about 20,000 nM. Preferably, protamine salt can be added to target cells at a final concentration ranging from about 0.1 μg / mL to about 10 μg / mL in combination with midostaurin at a final concentration ranging from about 50 to about 5,000 nM. More preferably, protamine salts are added to target cells at a final concentration ranging from about 1 μg / mL to about 10 μg / mL in combination with midostaurin at a final concentration ranging from about 50 to about 500 nM. Most preferably, protamine salts are added to target cells at a final concentration of about 4 μg / mL in combination with midostaurin at a final concentration of about 400 nM. Preferably, the combination of protamine salts and midostaurin is contacted with hematopoietic cells, more preferably HSCs, in the pre-stimulation and / or costimulation steps at any of the concentrations and / or densities disclosed above. In certain embodiments, the concentration ranges from 0.5 to 1E6 cells / mL or 2E6 / cm. 2 HSCs at a density of 0.5-1E6 cells / mL or 2E6 / cm may be pre-stimulated and / or costimulated with a combination of 4 μg / mL protamine salts and 100 nM midostaurin. In some embodiments, HSCs at a density of 0.5-1E6 cells / mL or 2E6 / cm may be pre-stimulated and / or costimulated with a combination of 4 μg / mL protamine salts and 100 nM midostaurin. 2 HSCs at a density of 0.5-1E6 cells / mL or 2E6 / cm may be pre-stimulated and / or costimulated with a combination of 4 μg / mL protamine salts and 200 nM midostaurin. In some embodiments, HSCs at a density of 0.5-1E6 cells / mL or 2E6 / cm may be pre-stimulated and / or costimulated with a combination of 4 μg / mL protamine salts and 200 nM midostaurin. 2 HSCs at a density of 4 μg / mL can be pre-stimulated and / or costimulated with a combination of 4 μg / mL protamine salts and 400 nM midostaurin.

[0219] In one embodiment, the invention relates to a method according to the invention, wherein the transduction enhancer is a combination of a protamine salt and nystatin.

[0220] The present inventors have demonstrated that the use of protamine salts in combination with nystatin results in higher transduction efficiency compared to either of the two compounds alone. Protamine salts can be used in combination with nystatin as a transduction enhancer at any appropriate concentration. In some embodiments, the combination of protamine salts and nystatin can include protamine salts at a concentration ranging from about 0.05 μg / mL to about 25 μg / mL and nystatin at a concentration ranging from about 1 to about 1,000 μM. In some embodiments, protamine salts can be added to target cells at a final concentration ranging from about 0.05 μg / mL to about 25 μg / mL in combination with nystatin at a final concentration ranging from about 1 to about 1,000 μM. Preferably, protamine salts can be added to target cells at a final concentration ranging from about 0.1 μg / mL to about 10 μg / mL in combination with nystatin at a final concentration ranging from about 5 to about 500 μM. More preferably, protamine salts are added to target cells at a final concentration ranging from about 1 μg / mL to about 10 μg / mL in combination with nystatin at a final concentration ranging from about 50 to about 150 μM. Most preferably, protamine salts are added to target cells at a final concentration of about 4 μg / mL in combination with nystatin at a final concentration of about 100 μM. Preferably, the combination of protamine salts and nystatin is contacted with hematopoietic cells, more preferably HSCs, in the pre-stimulation and / or costimulation steps at any of the concentrations and / or densities disclosed above. In one embodiment, the concentration ranges from 0.5 to 1E6 cells / mL or 2E6 / cm. 2 HSCs at a density of 100 μg / mL can be pre-stimulated and / or co-stimulated with a combination of 4 μg / mL protamine salts and 100 μM nystatin.

[0221] In one embodiment, the invention relates to a method according to the invention, wherein the transduction enhancer is a combination of a protamine salt and natamycin.

[0222] The present inventors have demonstrated that the use of protamine salts in combination with natamycin results in higher transduction efficiency compared to either of the two compounds alone. Protamine salts can be used in combination with natamycin as a transduction enhancer at any appropriate concentration. In some embodiments, the combination of protamine salts and natamycin can include protamine at a concentration ranging from about 0.05 μg / mL to about 25 μg / mL and natamycin at a concentration ranging from about 0.05 μg / mL to about 500 μM. In some embodiments, protamine salts can be added to target cells at a final concentration ranging from about 0.05 μg / mL to about 25 μg / mL in combination with natamycin at a final concentration ranging from about 0.05 μg / mL to about 500 μM. Preferably, protamine salts can be added to target cells at a final concentration ranging from about 0.1 μg / mL to about 10 μg / mL in combination with natamycin at a final concentration ranging from about 0.05 μM to about 10 μM. More preferably, protamine salts may be added to target cells at a final concentration ranging from about 1 μg / mL to about 10 μg / mL in combination with natamycin at a final concentration ranging from about 1 μM to about 5 μM. Most preferably, protamine salts may be added to target cells at a final concentration of about 4 μg / mL in combination with natamycin at a final concentration of about 3 μM. Preferably, the combination of protamine salts and natamycin is contacted with hematopoietic cells, more preferably HSCs, in the pre-stimulation and / or costimulation steps at any of the concentrations and / or densities disclosed above. In one embodiment, the combination is at a concentration of 0.5 to 1E6 cells / mL or 2E6 / cm. 2 HSCs at a density of 10 μg / mL can be pre-stimulated and / or co-stimulated with a combination of 4 μg / mL protamine salts and 3 μM natamycin.

[0223] In one embodiment, the invention relates to a method according to the invention, wherein the transduction enhancer is a combination of protamine salts, amphotericin B and everolimus.

[0224] The present inventors have shown that when protamine salts are used in combination with amphotericin B and everolimus, they result in higher transduction efficiency compared to either of these compounds alone. Protamine salts can be used in combination with amphotericin B and everolimus as a transduction enhancer at any suitable concentration. In some embodiments, the combination of protamine salts, amphotericin B, and everolimus can include protamine at a concentration ranging from about 0.05 μg / mL to about 25 μg / mL, amphotericin B at a concentration ranging from about 0.1 μg / mL to about 10 μg / mL, and everolimus at a concentration ranging from about 0.1 to about 10 μM. In some embodiments, protamine salts may be added to target cells at a final concentration ranging from about 0.05 μg / mL to about 25 μg / mL in combination with amphotericin B at a final concentration ranging from about 0.1 μg / mL to about 10 μg / mL and everolimus at a final concentration ranging from about 0.1 μg / mL to about 10 μM. Preferably, protamine salts may be added to target cells at a final concentration ranging from about 0.1 μg / mL to about 3 μg / mL in combination with amphotericin B at a final concentration ranging from about 0.2 μg / mL to about 7.5 μM and everolimus at a final concentration ranging from about 0.1 μg / mL to about 10 μg / mL. More preferably, protamine salts may be added to target cells at a final concentration ranging from about 1 μg / mL to about 10 μg / mL in combination with amphotericin B at a final concentration ranging from about 0.5 μg / mL to about 2 μg / mL and everolimus at a final concentration ranging from about 0.5 μg / mL to about 5 μM. Most preferably, protamine salts may be added to target cells at a final concentration of about 4 μg / mL in combination with amphotericin B at a final concentration of about 1 μg / mL and everolimus at a final concentration of about 1 μM. Preferably, the combination of protamine salts, amphotericin B, and everolimus is contacted with hematopoietic cells, more preferably HSCs, in the pre-stimulation and / or costimulation steps at any of the concentrations disclosed above. In certain embodiments, the combination is at a concentration of 0.5 to 1E6 cells / mL or 2E6 / cm. 2HSCs at a density of 0.5-1E6 cells / mL or 2E6 / cm may be pre-stimulated and / or costimulated with a combination of 4 μg / mL protamine salts, 0.5 μg / mL amphotericin B, and 1 μM everolimus. In some embodiments, HSCs at a concentration of 0.5-1E6 cells / mL or 2E6 / cm may be pre-stimulated and / or costimulated with a combination of 4 μg / mL protamine salts, 0.5 μg / mL amphotericin B, and 1 μM everolimus. 2 HSCs at a density of 0.5-1E6 cells / mL or 2E6 / cm may be pre-stimulated and / or costimulated with a combination of 4 μg / mL protamine salts, 0.75 μg / mL amphotericin B, and 1 μM everolimus. In some embodiments, HSCs at a concentration of 0.5-1E6 cells / mL or 2E6 / cm may be pre-stimulated and / or costimulated with a combination of 4 μg / mL protamine salts, 0.75 μg / mL amphotericin B, and 1 μM everolimus. 2 HSCs at a density of 10 μg / mL can be pre-stimulated and / or co-stimulated with a combination of 4 μg / mL protamine salts, 1 μg / mL amphotericin B, and 1 μM everolimus.

[0225] Several compounds and combinations of compounds have been identified that enhance the transduction of human cells with gene therapy vectors. In particular, novel compounds have been identified that have been shown to mediate increased retroviral transduction efficacy of target cells, particularly human CD34+ HSCs, when contacted with retroviral vectors, particularly lentiviral self-inactivating (SIN) vectors, containing a cDNA encoding a transgene of interest, particularly p47phox, under the control of an internal promoter, such as the myeloid-specific miR223 promoter, simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoter, but particularly the myeloid-specific miR223 promoter.

[0226] In a specific embodiment of the present invention, a lentivirus self-inactivating (SIN) vector, in which the viral promoter / enhancer is deleted in the 3' long terminal repeat (LTR) at the plasmid level, can be used in the method of the present invention. The expression of the transgene of interest can be driven by an internal promoter, such as simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV) and herpes simplex virus (HSV) (thymidine kinase) promoter or myeloid-specific miR223 promoter, but particularly myeloid-specific miR223 promoter.

[0227] In a specific embodiment of the present invention, human CD34-positive HSCs are transduced with a lentiviral self-inactivating gene therapy vector containing a cDNA under the control of the miR223 promoter encoding p47phox.

[0228] In a specific embodiment of the present invention, the pre-incubation medium may be further supplemented with protamine sulfate or protamine chloride, preferably at the concentrations indicated herein. In a specific embodiment of the present invention, the co-incubation medium may be further supplemented with protamine sulfate or protamine chloride, preferably at the concentrations indicated herein. In a specific embodiment of the present invention, the pre-incubation and / or co-incubation medium may be supplemented with 4 μg / mL of protamine sulfate or protamine chloride.

[0229] In another specific embodiment, the pre-incubation medium or co-incubation medium may be further supplemented with polybrene, preferably at a concentration ranging from about 0.1 to about 20 μg / mL, and / or poly-L-lysine, preferably at a concentration ranging from about 0.1 to about 20 μg / mL.

[0230] In various embodiments of the invention, the transduction enhancing compound is silibinin, particularly at a concentration of 5 μM, resveratrol, particularly at a concentration of 5 μM, everolimus, particularly at a concentration of 1 μM, midostaurin, particularly at a concentration of 0.4 μM, amphotericin B, particularly at a concentration of 1 μM, nystatin, particularly at a concentration of 100 μM, natamycin, particularly at a concentration of 3 μM, prostaglandin E2, particularly at a concentration of 10 μM, poloxamer Symperonic F108®, particularly at a concentration of 1,000 μg / ml, Specifically, poly(ethylene glycol)-b-poly(D,L-lactic-co-glycolic acid)-b-poly(ethylene glycol) (PEG-PLGA-PEG) with 5 kDa poly(ethylene glycol) blocks at both ends and a central 4.2 kDa poly(D,L-lactic-co-glycolic acid) block, designated PEG5k-b-PLGA4.2k-b-PEG5k, at a concentration of 1,000 μg / ml; Specifically, methoxypoly(ethylene glycol)-poly(e-caprolactone)-methoxypoly(ethylene glycol) (PEG-PCL-PEG), with 5 kDa poly(ethylene glycol) blocks at both ends and a central 4.2 kDa poly(e-caprolactone) block, designated PEG5k-b-PCL4.2k-b-PEG5k, at a concentration of 10 μg / ml; Specifically, methoxypoly(ethylene glycol)-poly(e-caprolactone)-methoxypoly(ethylene glycol) (PEG-PCL-PEG), having a central 2.4 kDa poly(e-caprolactone) block and terminal 5.3 kDa poly(ethylene glycol) blocks both covalently attached to amino groups, designated NH2-PEG5.3k-b-PCL2.4k-b-PEG5.3k-NH2, at a concentration of 10 μg / ml; In particular, poly(ethylene glycol) / poly(lactide) / poly(ethylene glycol) (PEG-PLA-PEG) having 5 kDa poly(ethylene glycol) blocks at both ends and a 4.2 kDa poly(lactide) block in the middle, designated PEG5k-b-PLA4.2k-b-PEG5k, at a concentration of 50 μg / ml, and deoxyribonucleosides, each having a final concentration of 300 μM, or combinations thereof.

[0231] Lentiboost® is widely recognized as the compound of choice for transducing human cells with retroviral vectors, particularly lentiviral vectors. However, despite being or having been the subject of various clinical trials, Lentiboost® has not received regulatory approval for therapeutic use to date. Furthermore, Lentiboost® contains synthetic polymers, which pose a risk of accumulation of non-degradable compounds in cells treated with Lentiboost®, and thus far have unpredictable results in humans. Therefore, there is a need in the art for safer transduction enhancers.

[0232] The inventors have surprisingly shown that several compounds approved for therapeutic use in humans are well suited as transduction enhancers and may therefore be preferable to Lentiboost® for use in therapeutic applications.

[0233] For example, the inventors have shown that the approved therapeutic compounds silibinin (Legalon), midostaurin (Rydapt), amphotericin B (AmBisome), nystatin (Mycostatin), natamycin (Natacyn), ruxolitinib (Jakavi), and fludarabine (Fludara) are efficient transduction enhancers. Thus, in a specific embodiment, the present invention relates to a method according to the present invention, wherein the transduction enhancer is silibinin, midostaurin, amphotericin B, nystatin, natamycin, ruxolitinib, fludarabine, or any combination thereof. In a preferred embodiment, the present invention relates to a method according to the present invention, wherein the transduction enhancer is silibinin, midostaurin, amphotericin B, nystatin, natamycin, ruxolitinib, fludarabine, or any combination thereof, in particular, wherein the combination is everolimus and amphotericin B. In certain embodiments, silibinin, midostaurin, amphotericin B, nystatin, natamycin, ruxolitinib, fludarabine or any combination thereof may be combined with a protamine salt, particularly protamine sulfate or protamine chloride, at any of the concentrations disclosed herein.

[0234] In a particular embodiment, the present invention relates to a method according to the present invention, wherein the transduction enhancer is silibinin, midostaurin, amphotericin B, nystatin, natamycin, or any combination thereof. In another embodiment, the present invention relates to a method according to the present invention, wherein the transduction enhancer is silibinin, everolimus, midostaurin, amphotericin B, nystatin, natamycin, or any combination thereof, in particular, the combination is everolimus and amphotericin B. In one embodiment, silibinin, midostaurin, amphotericin B, nystatin, natamycin, or any combination thereof may be combined with a protamine salt, in particular protamine sulfate or protamine chloride, at any concentration disclosed herein.

[0235] Furthermore, the present inventors have identified that certain compounds can increase the transduction efficiency of Lentiboost®. In particular, the present inventors have surprisingly found that the combination of Lentiboost® with amphotericin B, silibinin and / or midostaurin results in an increased transduction efficiency compared to Lentiboost® alone. Thus, in a particular embodiment, the present invention relates to a method according to the present invention, wherein the transduction enhancer is Lentiboost® in combination with amphotericin B, silibinin and / or midostaurin. In another embodiment, the present invention relates to a method according to the present invention, wherein the transduction enhancer is Lentiboost® in combination with amphotericin B and / or midostaurin. In another embodiment, the present invention relates to a method according to the present invention, wherein the transduction enhancer is Lentiboost® in combination with amphotericin B, or Lentiboost® in combination with midostaurin, or Lentiboost® in combination with silibinin. Lentiboost® may be combined with amphotericin B, silibinin and / or midostaurin at any of the concentrations disclosed herein.

[0236] Furthermore, the inventors have found that certain combinations of transduction enhancers result in increased transduction efficiency compared to Lentiboost® when used at the recommended concentration of 1 mg / mL.

[0237] We found that transduction of HSCs with lentiviral vectors at an MOI of 10 resulted in a VCN of 3.5 in X-Vivo 10 medium and 5 in BESP1366F medium when pre-incubated and co-incubated with 1 mg / mL Lentiboost® (see Figures 2 and 3).

[0238] To the inventors' surprise, the combination of protamine salt and amphotericin B, under the same conditions as Lentiboost®, resulted in a VCN of 5.4 in BESP1366F medium (see Figure 4). Thus, in a particular embodiment, the invention relates to a method according to the invention, wherein the transduction enhancer is a protamine salt in combination with amphotericin B.

[0239] Furthermore, the inventors found that when treated under the same conditions as Lentiboost®, the combination of protamine salt and PEG-PCL-PEG resulted in a VCN of 4 in X-Vivo 10 medium (see Figure 6). Thus, in a particular embodiment, the present invention relates to a method according to the invention, wherein the transduction enhancer is protamine salt in combination with PEG-PCL-PEG.

[0240] Furthermore, the inventors have found that when treated under the same conditions as Lentiboost®, the combination of amphotericin B and poloxamer F108 results in a VCN of 6.8 in X-Vivo 10 medium (see Figure 8). Thus, in a particular embodiment, the invention relates to a method according to the invention, wherein the transduction enhancer is amphotericin B in combination with poloxamer F108.

[0241] Furthermore, the present inventors found that when treated under the same conditions as Lentiboost®, the combination of silibinin and PEG-PCL-PEG resulted in a VCN of 5 in X-Vivo 10 medium (see Figure 9). Thus, in a particular embodiment, the present invention relates to a method according to the present invention, wherein the transduction enhancer is silibinin in combination with PEG-PCL-PEG.

[0242] Furthermore, the inventors found that when treated under the same conditions as Lentiboost®, the combination of silibinin and poloxamer F108 resulted in a VCN of 7.2 in X-Vivo 10 medium (see Figure 9). Thus, in a particular embodiment, the present invention relates to a method according to the invention, wherein the transduction enhancer is silibinin in combination with poloxamer F108.

[0243] Furthermore, the inventors found that when treated under the same conditions as Lentiboost®, the combination of midostaurin and poloxamer F108 resulted in a VCN of 9.7 in X-Vivo 10 medium (see Figure 10). Thus, in a particular embodiment, the invention relates to a method according to the invention, wherein the transduction enhancer is midostaurin in combination with poloxamer F108.

[0244] Thus, in a particular embodiment, the present invention relates to a method according to the present invention, wherein the transduction enhancer is protamine salt in combination with amphotericin B, protamine salt in combination with PEG-PCL-PEG, amphotericin B in combination with poloxmer F108, silibinin in combination with PEG-PCL-PEG, silibinin in combination with poloxmer F108, or midostaurin in combination with poloxamer F108, preferably at any of the concentrations disclosed herein.

[0245] In a particular embodiment, the present invention relates to a method according to the present invention, wherein the transduction enhancer is protamine salt in combination with amphotericin B, protamine salt in combination with PEG-PCL-PEG, amphotericin B in combination with poloxmer F108, silibinin in combination with PEG-PCL-PEG, silibinin in combination with poloxmer F108, midostaurin in combination with poloxamer F108, Lentiboost® in combination with amphotericin B, Lentiboost® in combination with silibinin or Lentiboost® in combination with midostaurin, preferably at any of the concentrations disclosed herein.

[0246] In certain aspects, the present invention provides a method for producing a transduction-enhancing compound comprising: · Silibinin, especially at concentrations of 0.05 μM to 500 μM; Midostaurin, especially at concentrations between 2 nM and 500,000 nM; · Nystatin, especially at concentrations of 0.1-1000 μM; · Natamycin, especially in concentrations of 0.05-500 μM; · PEG-PCL-PEG polymers, especially at concentrations of 1 μg / ml to 5,000 μg / ml; PEG-PLGA-PEG polymers, especially at concentrations between 1 μg / ml and 5,000 μg / ml; PEG-PLA-PEG polymers, particularly in concentrations between 1 μg / ml and 5,000 μg / ml; and / or Any combination of these The present invention relates to a method in which the composition is selected from the group consisting of:

[0247] Furthermore, the inventors have surprisingly found that amphotericin B can be used as a transduction enhancer, which has not been previously suggested. Thus, in a particular embodiment, the invention relates to a method according to the invention, wherein the transduction-enhancing compound is amphotericin B.

[0248] Amphotericin B can be contacted with target cells to induce transduction efficiency with retroviral vectors during the pre-incubation and / or co-incubation steps at any of the concentrations disclosed herein. Thus, in certain embodiments, the present invention relates to methods according to the invention, wherein target cells are contacted with amphotericin B during the pre-incubation and / or co-incubation steps at a concentration of about 0.05 to about 500 μM, particularly at a concentration of about 0.1 to about 10 μM, or any of the concentrations disclosed herein.

[0249] Additionally, amphotericin B can be combined with any transduction-enhancing compound known in the art or described herein, preferably at any of the concentrations disclosed herein. Thus, in certain embodiments, the invention relates to methods according to the invention, wherein amphotericin B is used in combination with one or more additional transduction-enhancing compounds.

[0250] In certain embodiments, amphotericin B may be used in combination with a protamine salt to improve the efficiency of transduction of target cells by a retroviral vector. Thus, in certain embodiments, the present invention relates to a method according to the present invention, wherein the additional transduction-enhancing compound is a protamine salt.

[0251] The protamine salt can be any protamine salt known in the art, so long as the anionic component of the salt does not interfere with the transduction efficiency of target cells when in solution. Thus, in certain embodiments, the invention relates to methods according to the invention, wherein the protamine salt is protamine chloride or protamine sulfate.

[0252] The protamine salt may be contacted with the target cells at any concentration disclosed herein. That is, in certain embodiments, the present invention relates to methods according to the present invention, wherein the target cells are contacted with a protamine salt at a concentration of about 0.05 μg / mL to about 25 μg / mL, particularly about 0.1 μg / mL to about 10 μg / mL, during the pre-incubation and / or co-incubation steps.

[0253] That is, in a preferred embodiment, the present invention relates to a method according to the present invention, wherein target cells are contacted with a combination of amphotericin B and a protamine salt during the pre-incubation and / or co-incubation steps, wherein amphotericin is contacted with the target cells at a concentration of about 0.05 to about 500 μM, particularly about 0.1 to about 10 μM, and the protamine salt is contacted with the target cells at a concentration of about 0.05 μg / mL to about 25 μg / mL, particularly about 0.1 μg / mL to about 10 μg / mL.

[0254] When two or more transduction-enhancing compounds are added in combination to target cells, it is preferred that all compounds are present simultaneously in the pre-incubation and / or co-incubation medium. However, it should be noted that two or more transduction-enhancing compounds can be added sequentially to the pre-incubation and / or co-incubation medium, as long as the compounds used in combination are present simultaneously in the pre-incubation and / or co-incubation steps at least one time point during the pre-incubation and / or co-incubation steps.

[0255] It has further been shown that the transduction efficiency of amphotericin B can be further increased when used in combination with one or more additional transduction-enhancing compounds. Thus, in certain embodiments, the present invention provides a method for treating amphotericin B comprising administering to a subject a therapeutically effective amount of amphotericin B, wherein the one or more additional transduction-enhancing compounds are: Lentiboost®, especially in concentrations of 0.1 mg / ml to 5,000 mg / ml; · Poloxamer F108, especially in concentrations from 0.1 mg / ml to 5,000 mg / ml; · Silibinin, especially at concentrations of 0.05 μM to 500 μM; Midostaurin, especially at concentrations between 2 nM and 500,000 nM; · PEG-PLA-PEG, especially in concentrations from 1 μg / ml to 5,000 μg / ml; · PEG-PGLA-PEG, especially in concentrations of 1 μg / ml to 5,000 μg / ml; · PEG-PCL-PEG, especially at concentrations of 1 μg / ml to 5,000 μg / ml; · Nystatin, especially at concentrations of 0.1-1000 μM; · Natamycin, especially in concentrations of 0.05-500 μM; Ruxolitinib, especially at concentrations of 0.01 to 10,000 μM; Fludarabine, especially in concentrations of 0.01-10,000 μM; Everolimus, especially at concentrations of 0.1-10 μM; · Resveratrol, especially in concentrations of 0.1-25 μM; · Prostaglandin E, especially at concentrations of 1-100 μM; deoxyribonucleosides, especially at concentrations of 0.1 mM to 10 mM of each nucleoside; and / or Any combination of these The present invention relates to a method in which the composition is selected from the group consisting of:

[0256] Additionally, it should be noted that both amphotericin B and the compounds listed above may be combined at any of the concentrations disclosed herein.

[0257] In a particular embodiment, the invention relates to a method according to the invention, wherein amphotericin B is combined with the transduction enhancer DMSO, particularly at a concentration of 0.1 to 10% (v / v), optionally in combination with one or more of any of the above-listed compounds at the concentrations listed above.

[0258] In a particular embodiment, the present invention relates to a method according to the invention, wherein the at least one additional transduction enhancing compound is selected from the group consisting of Lentiboost®, poloxamer F108 and / or PEG-PCL-PEG polymer.

[0259] Furthermore, the present inventors have surprisingly found that silibinin can be used as a transduction enhancer, which has not been previously suggested. Thus, in a particular embodiment, the present invention relates to a method according to the present invention, wherein the transduction-enhancing compound is silibinin.

[0260] To induce transduction efficiency with a retroviral vector, silibinin can be contacted with target cells during the preincubation and / or co-incubation steps at any of the concentrations disclosed herein. Thus, in certain embodiments, the present invention relates to methods according to the present invention, in which target cells are contacted with silibinin at a concentration of about 0.05 to about 500 μM or at any of the concentrations disclosed herein during the preincubation and / or co-incubation steps. Furthermore, silibinin can be combined with any transduction-enhancing compound known in the art or described herein, preferably at any of the concentrations disclosed herein. Thus, in certain embodiments, the present invention relates to methods according to the present invention, in which silibinin is used in combination with one or more additional transduction-enhancing compounds.

[0261] In certain embodiments, the present invention provides that the one or more additional transduction-enhancing compounds used in combination with silibinin are: Lentiboost®, especially in concentrations of 0.1 mg / ml to 5,000 mg / ml; · Poloxamer F108, especially in concentrations from 0.1 mg / ml to 5,000 mg / ml; · Protamine salts, especially in concentrations of 0.05-25 μg / mL; Amphotericin B, especially in concentrations of 0.05 μM to 500 μM; Midostaurin, especially at concentrations between 2 nM and 500,000 nM; · PEG-PLA-PEG polymers, especially at concentrations of 1 μg / ml to 5,000 μg / ml; · PEG-PGLA-PEG polymers, especially at concentrations of 1 μg / ml to 5,000 μg / ml; · PEG-PCL-PEG polymers, especially at concentrations of 1 μg / ml to 5,000 μg / ml; · Nystatin, especially at concentrations of 0.1-1000 μM; · Natamycin, especially in concentrations of 0.05-500 μM; Ruxolitinib, especially at concentrations of 0.01 to 10,000 μM; Fludarabine, especially in concentrations of 0.01-10,000 μM; Everolimus, especially at concentrations of 0.1-10 μM; · Resveratrol, especially in concentrations of 0.1-25 μM; · Prostaglandin E, especially at concentrations of 1-100 μM; deoxyribonucleosides, especially at concentrations of 0.1 mM to 10 mM of each nucleoside; and / or Any combination of these The present invention relates to a compound selected from the group consisting of:

[0262] It should be noted that both silibinin and the compounds listed above may be combined at any of the concentrations disclosed herein.

[0263] In a particular embodiment, the present invention relates to a method according to the invention, wherein silibinin is combined with the transduction enhancer DMSO, in particular at a concentration of 0.1-10% (v / v), optionally combined with one or more of any of the above-listed compounds at the concentrations listed above.

[0264] Preferably, silibinin may be combined with Lentiboost®, poloxamer F108 or PEG-PCL-PEG polymer at any of the concentrations disclosed herein.

[0265] Furthermore, the inventors have surprisingly found that midostaurin can be used as a transduction enhancer, which has not been previously suggested. Thus, in a particular embodiment, the invention relates to a method according to the invention, wherein the transduction-enhancing compound is midostaurin.

[0266] Midostaurin can be contacted with target cells to induce transduction efficiency with retroviral vectors during the pre-incubation and / or co-incubation steps at any of the concentrations disclosed herein. Thus, in certain embodiments, the present invention relates to methods according to the present invention, in which target cells are contacted with midostaurin at a concentration of 2 nM to 500,000 nM or any of the concentrations disclosed herein during the pre-incubation and / or co-incubation steps. Furthermore, midostaurin can be combined with any transduction-enhancing compound known in the art or any of the transduction-enhancing compounds described herein, preferably at any of the concentrations disclosed herein. Thus, in certain embodiments, the present invention relates to methods according to the present invention, in which midostaurin is used in combination with one or more additional transduction-enhancing compounds.

[0267] In certain embodiments, the present invention provides that the one or more additional transduction enhancing compounds used in combination with midostaurin are: Lentiboost®, especially in concentrations of 0.1 mg / ml to 5,000 mg / ml; · Poloxamer F108, especially in concentrations from 0.1 mg / ml to 5,000 mg / ml; · Protamine salts, especially in concentrations of 0.05-25 μg / mL; Amphotericin B, especially in concentrations of 0.05 μM to 500 μM; · Silibinin, especially at concentrations of 0.05 μM to 500 μM; · PEG-PLA-PEG polymers, especially at concentrations of 1 μg / ml to 5,000 μg / ml; · PEG-PGLA-PEG polymers, especially at concentrations of 1 μg / ml to 5,000 μg / ml; · PEG-PCL-PEG polymers, especially at concentrations of 1 μg / ml to 5,000 μg / ml; · Nystatin, especially at concentrations of 0.1-1000 μM; · Natamycin, especially in concentrations of 0.05-500 μM; Ruxolitinib, especially at concentrations of 0.01 to 10,000 μM; Fludarabine, especially in concentrations of 0.01-10,000 μM; Everolimus, especially at concentrations of 0.1-10 μM; · Resveratrol, especially in concentrations of 0.1-25 μM; · Prostaglandin E, especially at concentrations of 1-100 μM; deoxyribonucleosides, especially at concentrations of 0.1 mM to 10 mM of each nucleoside; and / or Any combination of these The present invention relates to a compound selected from the group consisting of:

[0268] It should be noted that both midostaurin and the compounds listed above may be combined at any of the concentrations disclosed herein.

[0269] In a particular embodiment, the present invention relates to a method according to the invention, wherein midostaurin is combined with the transduction enhancer DMSO, in particular at a concentration of 0.1 to 10% (v / v), optionally in combination with one or more of any of the above-listed compounds at the concentrations listed above.

[0270] Preferably, midostaurin may be combined with Lentiboost® or poloxamer F108 at any of the concentrations disclosed herein.

[0271] Furthermore, the inventors have surprisingly found that nystatin can be used as a transduction enhancer, which has not been previously suggested. Thus, in a particular embodiment, the present invention relates to a method according to the invention, wherein the transduction-enhancing compound is nystatin.

[0272] Nystatin may be contacted with target cells to induce transduction efficiency with retroviral vectors during the preincubation and / or co-incubation steps at any of the concentrations disclosed herein. Thus, in certain embodiments, the present invention relates to methods according to the present invention, in which target cells are contacted with nystatin at a concentration of 0.1-1000 μM or any of the concentrations disclosed herein during the preincubation and / or co-incubation steps. Furthermore, nystatin may be combined with any transduction-enhancing compound known in the art or described herein, preferably at any of the concentrations disclosed herein. Thus, in certain embodiments, the present invention relates to methods according to the present invention, in which nystatin is used in combination with one or more additional transduction-enhancing compounds.

[0273] In certain embodiments, the present invention provides that the one or more additional transduction enhancing compounds used in combination with nystatin are: Lentiboost®, especially in concentrations of 0.1 mg / ml to 5,000 mg / ml; · Poloxamer F108, especially in concentrations from 0.1 mg / ml to 5,000 mg / ml; · Protamine salts, especially in concentrations of 0.05-25 μg / mL; Amphotericin B, especially in concentrations of 0.05 μM to 500 μM; · Silibinin, especially at concentrations of 0.05 μM to 500 μM; · PEG-PLA-PEG polymers, especially at concentrations of 1 μg / ml to 5,000 μg / ml; · PEG-PGLA-PEG polymers, especially at concentrations of 1 μg / ml to 5,000 μg / ml; · PEG-PCL-PEG polymers, especially at concentrations of 1 μg / ml to 5,000 μg / ml; Midostaurin, especially at concentrations between 2 nM and 500,000 nM; · Natamycin, especially in concentrations of 0.05-500 μM; Ruxolitinib, especially at concentrations of 0.01 to 10,000 μM; Fludarabine, especially in concentrations of 0.01-10,000 μM; Everolimus, especially at concentrations of 0.1-10 μM; · Resveratrol, especially in concentrations of 0.1-25 μM; · Prostaglandin E, especially at concentrations of 1-100 μM; deoxyribonucleosides, especially at concentrations of 0.1 mM to 10 mM of each nucleoside; and / or Any combination of these The present invention relates to a compound selected from the group consisting of:

[0274] It should be noted that both nystatin and the compounds listed above may be combined at any of the concentrations disclosed herein.

[0275] In a particular embodiment, the present invention relates to a method according to the invention, wherein nystatin is combined with the transduction enhancer DMSO, in particular at a concentration of 0.1 to 10% (v / v), optionally in combination with one or more of any of the above-listed compounds at the concentrations listed above.

[0276] Furthermore, the inventors have surprisingly found that natamycin can be used as a transduction enhancer, which has not been previously suggested. Thus, in a particular embodiment, the invention relates to a method according to the invention, wherein the transduction enhancing compound is natamycin.

[0277] Natamycin may be contacted with target cells to induce transduction efficiency with retroviral vectors during the pre-incubation and / or co-incubation steps at any of the concentrations disclosed herein. Thus, in certain embodiments, the present invention relates to methods according to the present invention, in which target cells are contacted with natamycin at a concentration of 0.05-500 μM or any of the concentrations disclosed herein during the pre-incubation and / or co-incubation steps. Furthermore, natamycin may be combined with any transduction-enhancing compound known in the art or any of the transduction-enhancing compounds described herein, preferably at any of the concentrations disclosed herein. Thus, in certain embodiments, the present invention relates to methods according to the present invention, in which natamycin is used in combination with one or more additional transduction-enhancing compounds.

[0278] In certain embodiments, the present invention provides a method for treating natamycin comprising administering to a subject therapies comprising administering to said subject the subject therapies comprising administering to said subject the subject the compounds comprising: Lentiboost®, especially in concentrations of 0.1 mg / ml to 5,000 mg / ml; · Poloxamer F108, especially in concentrations from 0.1 mg / ml to 5,000 mg / ml; · Protamine salts, especially in concentrations of 0.05-25 μg / mL; Amphotericin B, especially in concentrations of 0.05 μM to 500 μM; · Silibinin, especially at concentrations of 0.05 μM to 500 μM; · PEG-PLA-PEG polymers, especially at concentrations of 1 μg / ml to 5,000 μg / ml; · PEG-PGLA-PEG polymers, especially at concentrations of 1 μg / ml to 5,000 μg / ml; · PEG-PCL-PEG polymers, especially at concentrations of 1 μg / ml to 5,000 μg / ml; Midostaurin, especially at concentrations between 2 nM and 500,000 nM; · Nystatin, especially at concentrations of 0.1-1000 μM; Ruxolitinib, especially at concentrations of 0.01 to 10,000 μM; Fludarabine, especially in concentrations of 0.01-10,000 μM; Everolimus, especially at concentrations of 0.1-10 μM; · Resveratrol, especially in concentrations of 0.1-25 μM; · Prostaglandin E, especially at concentrations of 1-100 μM; deoxyribonucleosides, especially at concentrations of 0.1 mM to 10 mM of each nucleoside; and / or Any combination of these The present invention relates to a compound selected from the group consisting of:

[0279] It should be noted that both natamycin and the compounds listed above may be combined at any of the concentrations disclosed herein.

[0280] In a particular embodiment, the present invention relates to a method according to the invention, wherein natamycin is combined with the transduction enhancer DMSO, particularly at a concentration of 0.1 to 10% (v / v), optionally in combination with one or more of any of the above-listed compounds at the concentrations listed above.

[0281] Furthermore, the inventors have surprisingly found that fludarabine can be used as a transduction enhancer, which has not been previously suggested. Thus, in a particular embodiment, the invention relates to a method according to the invention, wherein the transduction enhancing compound is fludarabine.

[0282] Fludarabine may be contacted with target cells to induce transduction efficiency with retroviral vectors during the pre-incubation and / or co-incubation steps at any of the concentrations disclosed herein. Thus, in certain embodiments, the present invention relates to methods according to the present invention, in which target cells are contacted with fludarabine at a concentration of 0.01-10,000 μM or any of the concentrations disclosed herein during the pre-incubation and / or co-incubation steps. Furthermore, fludarabine may be combined with any transduction-enhancing compound known in the art or described herein, preferably at any of the concentrations disclosed herein. Thus, in certain embodiments, the present invention relates to methods according to the present invention, in which fludarabine is used in combination with one or more additional transduction-enhancing compounds.

[0283] In certain embodiments, the present invention provides a method for treating fludarabine comprising administering to a patient a therapeutically effective amount of one or more additional transduction enhancing compounds used in combination with fludarabine, comprising administering to a patient a therapeutically effective amount of one or more additional transduction enhancing compounds. Lentiboost®, especially in concentrations of 0.1 mg / ml to 5,000 mg / ml; · Poloxamer F108, especially in concentrations from 0.1 mg / ml to 5,000 mg / ml; · Protamine salts, especially in concentrations of 0.05-25 μg / mL; Amphotericin B, especially in concentrations of 0.05 μM to 500 μM; · Silibinin, especially at concentrations of 0.05 μM to 500 μM; · PEG-PLA-PEG polymers, especially at concentrations of 1 μg / ml to 5,000 μg / ml; · PEG-PGLA-PEG polymers, especially at concentrations of 1 μg / ml to 5,000 μg / ml; · PEG-PCL-PEG polymers, especially at concentrations of 1 μg / ml to 5,000 μg / ml; · Nystatin, especially at concentrations of 0.1-1000 μM; · Natamycin, especially in concentrations of 0.05-500 μM; Ruxolitinib, especially at concentrations of 0.01 to 10,000 μM; Midostaurin, especially at concentrations between 2 nM and 500,000 nM; Everolimus, especially at concentrations of 0.1-10 μM; · Resveratrol, especially in concentrations of 0.1-25 μM; · Prostaglandin E, especially at concentrations of 1-100 μM; deoxyribonucleosides, especially at concentrations of 0.1 mM to 10 mM of each nucleoside; and / or Any combination of these The present invention relates to a compound selected from the group consisting of:

[0284] It should be noted that both fludarabine and the compounds listed above may be combined at any of the concentrations disclosed herein.

[0285] In a particular embodiment, the invention relates to a method according to the invention, wherein fludarabine is combined with the transduction enhancer DMSO, in particular at a concentration of 0.1 to 10% (v / v), optionally in combination with one or more of any of the above-listed compounds at the concentrations listed above.

[0286] Furthermore, the inventors have surprisingly found that ruxolitinib can be used as a transduction enhancer, which has not been previously suggested. Thus, in a particular embodiment, the invention relates to a method according to the invention, wherein the transduction-enhancing compound is ruxolitinib.

[0287] Ruxolitinib can be contacted with target cells to induce transduction efficiency with retroviral vectors during the pre-incubation and / or co-incubation steps at any of the concentrations disclosed herein. Thus, in certain embodiments, the present invention relates to methods according to the present invention, wherein target cells are contacted with ruxolitinib at a concentration of 0.01-10,000 μM or any of the concentrations disclosed herein during the pre-incubation and / or co-incubation steps. Furthermore, ruxolitinib can be combined with any transduction-enhancing compound known in the art or any of the transduction-enhancing compounds described herein, preferably at any of the concentrations disclosed herein. Thus, in certain embodiments, the present invention relates to methods according to the present invention, wherein ruxolitinib is used in combination with one or more additional transduction-enhancing compounds.

[0288] In certain embodiments, the present invention provides that the one or more additional transduction enhancing compounds used in combination with ruxolitinib are: Lentiboost®, especially in concentrations of 0.1 mg / ml to 5,000 mg / ml; · Poloxamer F108, especially in concentrations from 0.1 mg / ml to 5,000 mg / ml; · Protamine salts, especially in concentrations of 0.05-25 μg / mL; Amphotericin B, especially in concentrations of 0.05 μM to 500 μM; · Silibinin, especially at concentrations of 0.05 μM to 500 μM; · PEG-PLA-PEG polymers, especially at concentrations of 1 μg / ml to 5,000 μg / ml; · PEG-PGLA-PEG polymers, especially at concentrations of 1 μg / ml to 5,000 μg / ml; · PEG-PCL-PEG polymers, especially at concentrations of 1 μg / ml to 5,000 μg / ml; Midostaurin, especially at concentrations between 2 nM and 500,000 nM; · Natamycin, especially in concentrations of 0.05-500 μM; · Nystatin, especially at concentrations of 0.1-1000 μM; Fludarabine, especially in concentrations of 0.01-10,000 μM; Everolimus, especially at concentrations of 0.1-10 μM; · Resveratrol, especially in concentrations of 0.1-25 μM; · Prostaglandin E, especially at concentrations of 1-100 μM; deoxyribonucleosides, especially at concentrations of 0.1 mM to 10 mM of each nucleoside; and / or Any combination of these The present invention relates to a compound selected from the group consisting of:

[0289] It should be noted that both ruxolitinib and the compounds listed above can be combined at any of the concentrations disclosed herein.

[0290] In a particular embodiment, the invention relates to a method according to the invention, wherein ruxolitinib is combined with the transduction enhancer DMSO, particularly at a concentration of 0.1 to 10% (v / v), optionally in combination with one or more of any of the above-listed compounds at the concentrations listed above.

[0291] Furthermore, the inventors have surprisingly found that PEG-PCL-PEG polymers can be used as transduction enhancers, which has not been previously suggested. Thus, in certain embodiments, the present invention relates to methods according to the present invention, wherein the transduction-enhancing compound is a PEG-PCL-PEG polymer disclosed herein.

[0292] The PEG-PCL-PEG polymer can be contacted with target cells to induce transduction efficiency with retroviral vectors during the pre-incubation and / or co-incubation steps at any of the concentrations disclosed herein. Thus, in certain embodiments, the present invention relates to methods according to the present invention, in which target cells are contacted with the PEG-PCL-PEG polymer at a concentration of 1 μg / ml to 5,000 μg / ml or any of the concentrations disclosed herein during the pre-incubation and / or co-incubation steps. Furthermore, the PEG-PCL-PEG polymer can be combined with any transduction-enhancing compound known in the art or described herein, preferably at any of the concentrations disclosed herein. Thus, in certain embodiments, the present invention relates to methods according to the present invention, in which the PEG-PCL-PEG polymer is used in combination with one or more additional transduction-enhancing compounds.

[0293] In certain embodiments, the present invention provides that the one or more additional transduction enhancing compounds used in combination with the PEG-PCL-PEG polymer are: Lentiboost®, especially in concentrations of 0.1 mg / ml to 5,000 mg / ml; · Poloxamer F108, especially in concentrations from 0.1 mg / ml to 5,000 mg / ml; · Protamine salts, especially in concentrations of 0.05-25 μg / mL; Amphotericin B, especially in concentrations of 0.05 μM to 500 μM; · Silibinin, especially at concentrations of 0.05 μM to 500 μM; · PEG-PLA-PEG polymers, especially at concentrations of 1 μg / ml to 5,000 μg / ml; PEG-PLGA-PEG polymers, especially at concentrations between 1 μg / ml and 5,000 μg / ml; · Natamycin, especially in concentrations of 0.05-500 μM; Midostaurin, especially at concentrations between 2 nM and 500,000 nM; · Nystatin, especially at concentrations of 0.1-1000 μM; Ruxolitinib, especially at concentrations of 0.01 to 10,000 μM; Fludarabine, especially in concentrations of 0.01-10,000 μM; Everolimus, especially at concentrations of 0.1-10 μM; · Resveratrol, especially in concentrations of 0.1-25 μM; · Prostaglandin E, especially at concentrations of 1-100 μM; deoxyribonucleosides, especially at concentrations of 0.1 mM to 10 mM of each nucleoside; and / or Any combination of these The present invention relates to a compound selected from the group consisting of:

[0294] It should be noted that both the PEG-PCL-PEG polymer and the compounds listed above can be combined at any of the concentrations disclosed herein.

[0295] In a particular embodiment, the present invention relates to a method according to the invention, wherein PEG-PCL-PEG is combined with the transduction enhancer DMSO, particularly at a concentration of 0.1-10% (v / v), and optionally combined with one or more of any of the above-listed compounds at the concentrations listed above.

[0296] Preferably, the PEG-PCL-PEG polymer may be combined with protamine salts or silibinin at any of the concentrations disclosed herein.

[0297] Furthermore, the inventors have surprisingly found that PEG-PLGA-PEG polymers can be used as transduction enhancers, which has not been previously suggested. Thus, in certain embodiments, the present invention relates to methods according to the present invention, wherein the transduction-enhancing compound is a PEG-PLGA-PEG polymer disclosed herein.

[0298] The PEG-PLGA-PEG polymer can be contacted with target cells to induce transduction efficiency with retroviral vectors during the pre-incubation and / or co-incubation steps at any of the concentrations disclosed herein. Thus, in certain embodiments, the present invention relates to methods according to the present invention, in which target cells are contacted with the PEG-PLGA-PEG polymer at a concentration of 1 μg / ml to 5,000 μg / ml or any of the concentrations disclosed herein during the pre-incubation and / or co-incubation steps. Furthermore, the PEG-PLGA-PEG polymer can be combined with any transduction-enhancing compound known in the art or described herein, preferably at any of the concentrations disclosed herein. Thus, in certain embodiments, the present invention relates to methods according to the present invention, in which the PEG-PLGA-PEG polymer is used in combination with one or more additional transduction-enhancing compounds.

[0299] In certain embodiments, the present invention provides that the one or more additional transduction enhancing compounds used in combination with the PEG-PLGA-PEG polymer are: Lentiboost®, especially in concentrations of 0.1 mg / ml to 5,000 mg / ml; · Poloxamer F108, especially in concentrations from 0.1 mg / ml to 5,000 mg / ml; · Protamine salts, especially in concentrations of 0.05-25 μg / mL; Amphotericin B, especially in concentrations of 0.05 μM to 500 μM; · Silibinin, especially at concentrations of 0.05 μM to 500 μM; · PEG-PLA-PEG polymers, especially at concentrations of 1 μg / ml to 5,000 μg / ml; · PEG-PCL-PEG polymers, especially at concentrations of 1 μg / ml to 5,000 μg / ml; · Natamycin, especially in concentrations of 0.05-500 μM; Midostaurin, especially at concentrations between 2 nM and 500,000 nM; · Nystatin, especially at concentrations of 0.1-1000 μM; Ruxolitinib, especially at concentrations of 0.01 to 10,000 μM; Fludarabine, especially in concentrations of 0.01-10,000 μM; Everolimus, especially at concentrations of 0.1-10 μM; · Resveratrol, especially in concentrations of 0.1-25 μM; · Prostaglandin E, especially at concentrations of 1-100 μM; deoxyribonucleosides, especially at concentrations of 0.1 mM to 10 mM of each nucleoside; and / or Any combination of these The present invention relates to a compound selected from the group consisting of:

[0300] It should be noted that both the PEG-PLGA-PEG polymer and the compounds listed above can be combined at any of the concentrations disclosed herein.

[0301] In a particular embodiment, the present invention relates to a method according to the invention, wherein PEG-PLGA-PEG is combined with the transduction enhancer DMSO, particularly at a concentration of 0.1-10% (v / v), optionally combined with one or more of any of the above-listed compounds at the concentrations listed above.

[0302] Furthermore, the inventors have surprisingly found that PEG-PLA-PEG polymers can be used as transduction enhancers, which has not been previously suggested. Thus, in certain embodiments, the present invention relates to methods according to the present invention, wherein the transduction-enhancing compound is a PEG-PLA-PEG polymer as disclosed herein.

[0303] The PEG-PLA-PEG polymer can be contacted with target cells to induce transduction efficiency with retroviral vectors during the pre-incubation and / or co-incubation steps at any of the concentrations disclosed herein. Thus, in certain embodiments, the present invention relates to methods according to the present invention, in which target cells are contacted with the PEG-PLA-PEG polymer at a concentration of 1 μg / ml to 5,000 μg / ml or any of the concentrations disclosed herein during the pre-incubation and / or co-incubation steps. Furthermore, the PEG-PLA-PEG polymer can be combined with any transduction-enhancing compound known in the art or described herein, preferably at any of the concentrations disclosed herein. Thus, in certain embodiments, the present invention relates to methods according to the present invention, in which the PEG-PLA-PEG polymer is used in combination with one or more additional transduction-enhancing compounds.

[0304] In certain embodiments, the present invention provides that the one or more additional transduction enhancing compounds used in combination with the PEG-PLA-PEG polymer are: Lentiboost®, especially in concentrations of 0.1 mg / ml to 5,000 mg / ml; · Poloxamer F108, especially in concentrations from 0.1 mg / ml to 5,000 mg / ml; · Protamine salts, especially in concentrations of 0.05-25 μg / mL; Amphotericin B, especially in concentrations of 0.05 μM to 500 μM; · Silibinin, especially at concentrations of 0.05 μM to 500 μM; PEG-PLGA-PEG polymers, especially at concentrations between 1 μg / ml and 5,000 μg / ml; · PEG-PCL-PEG polymers, especially at concentrations of 1 μg / ml to 5,000 μg / ml; · Natamycin, especially in concentrations of 0.05-500 μM; Midostaurin, especially at concentrations between 2 nM and 500,000 nM; · Nystatin, especially at concentrations of 0.1-1000 μM; Ruxolitinib, especially at concentrations of 0.01 to 10,000 μM; Fludarabine, especially in concentrations of 0.01-10,000 μM; Everolimus, especially at concentrations of 0.1-10 μM; · Resveratrol, especially in concentrations of 0.1-25 μM; · Prostaglandin E, especially at concentrations of 1-100 μM; deoxyribonucleosides, especially at concentrations of 0.1 mM to 10 mM of each nucleoside; and / or Any combination of these The present invention relates to a compound selected from the group consisting of:

[0305] It should be noted that both the PEG-PLA-PEG polymer and the compounds listed above can be combined at any of the concentrations disclosed herein.

[0306] In a particular embodiment, the present invention relates to a method according to the invention, wherein PEG-PLA-PEG is combined with the transduction enhancer DMSO, particularly at a concentration of 0.1-10% (v / v), optionally combined with one or more of any of the above-listed compounds at the concentrations listed above.

[0307] It is understood that all compounds and combinations of compounds listed in the above embodiments are disclosed herein at any of the concentrations or concentration ranges disclosed elsewhere herein. [Example]

[0308] Example 1: Transduction at an MOI of 1, 3 or 5 1% human serum albumin, 300ng / ml stem cell factor (SCF), 300ng / ml 0.5E6 cells / cm in 96-well plates containing X-Vivo 20 medium supplemented with fms-like tyrosine kinase 3 (FLT-3) ligand (Flt3-lig) and 100 ng / ml thrombopoietin (TPO). 2 Thawed cells from healthy donors were cultured at a density of 1E6 cells / ml for 22 hours and, if necessary, subsequently pre-stimulated with the compounds listed below for 2 hours. Cells were then incubated for 12 hours for transduction with lentiviral SIN gene therapy vectors in the presence of the compounds listed below and protamine sulfate. After the 12-hour transduction period, the medium was replaced with the medium described above, and cells were transferred to 12-well plates and cultured in 1 ml of medium for 5–7 days depending on cell density. The medium was then replaced with fresh medium, and cells were cultured in 2 ml of medium for an additional 6 days. On day 12 post-transduction, DNA was isolated, and vector copy number (VCN) was quantified by qPCR. All experiments were performed in triplicate. For negative controls, triplicate non-transduced cells were cultured as described. To determine a baseline for transduction efficacy in the presence of protamine sulfate alone, cells were incubated with lentiviral SIN vectors at an MOI of 1, 3, or 5 in the presence of 4 μg / mL protamine sulfate. For transduction in the presence of potential transduction enhancers, i) 5 μM silibinin, 4 μg / mL protamine sulfate; ii) 5 μM resveratrol, 4 μg / mL protamine sulfate; iii) 1 μM everolimus, 4 μg / mL protamine sulfate; iv) 0, 4 μM midostaurin, 4 μg / mL protamine sulfate; v) 1 μg / mL amphotericin B, 4 μg / mL protamine sulfate; vi) 100 μM nystatin, 4 μg / mL protamine sulfate; vii) 3 μM natamycin, 4 μg / mL protamine sulfate; viii) 10 μM prostaglandin E2, 4 μg / mL protamine sulfate; ix) 1mg / ml Lentiboost®, 4μg / mL protamine sulfate; x) 1,000 μg / ml poloxamer Synperonic F108®, 4 μg / mL protamine sulfate; xi) 1,000 μg / ml poly(ethylene glycol)-b-poly(D,L-lactic-co-glycolic acid)-b-poly(ethylene glycol) (PEG-PLGA-PEG) with 5 kDa poly(ethylene glycol) blocks at both ends and a central 4.2 kDa poly(D,L-lactic-co-glycolic acid) block, designated PEG5k-b-PLA4.2k-b-PEG5k; xii) 10 μg / ml methoxypoly(ethylene glycol)-poly(e-caprolactone)-methoxypoly(ethylene glycol) (PEG-PCL-PEG) with 5 kDa poly(ethylene glycol) blocks at both ends and a central 4.2 kDa poly(e-caprolactone) block, designated PEG5k-b-PCL4.2k-b-PEG5k; xiii) 50 μg / mL poly(ethylene glycol) / poly(lactide) / poly(ethylene glycol) (PEG-PLA-PEG) with 5 kDa poly(ethylene glycol) blocks at both ends and a central 4.2 kDa poly(lactide) block, designated PEG5k-b-PLA4.2k-b-PEG5k; xiv) 50 μg / ml of methoxypoly(ethylene glycol)-poly(e-caprolactone)-methoxypoly(ethylene glycol) (PEG-PCL-PEG), designated NH2-PEG5.3k-b-PCL2.4k-b-PEG5.3k-NH2, with a central 2.4 kDa poly(e-caprolactone) block and terminal 5.3 kDa poly(ethylene glycol) blocks, both covalently linked to amino groups; xv) deoxyribonucleosides, each with a final concentration of 300 μM; or a combination of these; Cells were incubated with lentiviral SIN vectors at an MOI of 1, 3, or 5 in the presence of .

[0309] After 10–12 days, unintegrated proviral DNA was diluted due to cell growth in the plates, and VCN was subsequently quantified by qPCR. All conditions were tested in triplicate, and the mean VCN of the triplicates for each individual condition was calculated. To evaluate the transduction-enhancing activity of the tested compounds, the mean VCN of cells transduced in the presence of protamine sulfate was set as 1, and transduction enhancement was expressed as an X-fold increase over transduction in the presence of protamine sulfate alone.

[0310] The inventors surprisingly observed the following increase in the efficiency of lentiviral transduction (see Figure 1): MOI of 1: Silibinin (4.8 times) Resveratrol (4.5 times) Everolimus (13.5x) Midostaurin (4.2 times) Amphotericin B (3x) Nysatin (2.6 times) Natamycin (3x) Lentiboost (9.8x) MOI of 3: Everolimus (2.5x) Amphotericin B (2.2 times) Everolimus amphotericin B (4.4x) Lentiboost (6.4x)

[0311] Replenishing CD34+ cells with 300 μM of each deoxyribonucleoside or 10 μg / mL of the BAB triblock polymer PCL increased VCN by 1.89-fold or 1.81-fold, respectively, compared with transduction without the transduction enhancer.

[0312] Example 2: Transduction in the presence of poloxamer F108 and polybrene (MOI 5) Human CD34+ HSCs were transduced at a density of 1E6 cells / ml in X-Vivo 20 medium supplemented with 1% human serum albumin, 300 ng / ml SCF, 300 ng / ml Flt3-ligand, and 100 ng / ml TPO with a lentiviral self-inactivating gene therapy vector containing a cDNA encoding human p47phox under the control of the miR223 promoter. The transduction process involved a 2-hour pre-stimulation period in the presence of poloxamer F108 (1,000 μg / ml) and polybrene (8 μg / ml), followed by a 12-hour incubation of the pre-stimulated cells with the gene therapy vector at an MOI of 5 in the presence of poloxamer F108 (1,000 μg / ml) and polybrene (8 μg / ml).

[0313] Example 3: Transduction in the presence of PEG-PCL-PEG polymer and midostaurin (MOI 5) Human CD34+ HSCs were transduced with lentiviral self-inactivating gene therapy vectors at a density of 1E6 cells / ml in X-Vivo 20 medium supplemented with 1% human serum albumin, 300 ng / ml SCF, 300 ng / ml Flt3-ligand, and 100 ng / ml TPO. The transduction process involved a 2-hour pre-stimulation period in the presence of protamine sulfate (4 μg / ml) plus PEG-PCL-PEG polymer (4 μg / ml) and midostaurin (0.4 μM), followed by a 12-hour incubation of the pre-stimulated cells with the gene therapy vector at an MOI of 5 in the presence of protamine sulfate (4 μg / ml) plus PEG-PCL-PEG polymer (4 μg / ml) and midostaurin (0.4 μM).

[0314] Example 4: Transduction in the presence of amphotericin B (MOI 5) Human CD34+ HSCs were transduced at a density of 1E6 cells / ml in X-Vivo 20 medium supplemented with 1% human serum albumin, 300 ng / ml SCF, 200 ng / ml Flt3-ligand, and 100 ng / ml TPO with a lentiviral self-inactivating gene therapy vector containing a cDNA encoding p47phox under the control of the miR223 promoter. The transduction process involved a 2-hour pre-stimulation period in the presence of 1 μg / ml amphotericin B and 4 μg / ml protamine sulfate, followed by a 12-hour incubation of the pre-stimulated cells with the gene therapy vector at an MOI of 5 in the presence of 1 μg / ml amphotericin B and 4 μg / ml protamine sulfate.

[0315] Example 5: Transduction at an MOI of 10 Commercially available, anonymized human CD34+ hematopoietic stem cells (HSCs) were used to test the enhancement of retroviral transduction efficiency by transduction enhancers or combinations of transduction enhancers. All experimental conditions were tested in triplicate, and the final results were expressed as the average of the individual results for each triplicate.

[0316] For transduction, a lentiviral self-inactivating (SIN) vector containing the miR223 promoter as an internal promoter and a cDNA encoding p47phox was used. HSCs were retrovirally transduced at a density of 0.5E6 cells / cm² and a concentration of 1E6 cells / ml in either X-Vivo 10 medium or BESP1366F medium supplemented with 1% human serum albumin, 300 ng / ml stem cell factor (SCF), 300 ng / ml fms-like tyrosine kinase 3 (FLT3-ligand) and 100 ng / ml thrombopoietin (TPO). Three independent experiments were performed at an MOI of 10.

[0317] Before transduction, HSCs were cultured in 96-well plates with the above medium and cytokines for 22 hours. If necessary, they were then pre-stimulated for 2 hours with the compounds listed below in the above medium without the gene therapy vector. For transduction, the cells were then incubated with the lentiviral SIN gene therapy vector (with or without compounds) at an MOI of 10 for 12 hours without changing the medium. After the 12-hour transduction period, the medium was replaced with the above medium (with supplements), and the cells were transferred to 12-well plates and cultured in 1 ml of the above medium (with supplements) for 5–7 days depending on the cell density. The medium was then replaced with fresh medium (with supplements) of the above composition, and the cells were cultured in 2 ml of medium for an additional 6 days.

[0318] Twelve days after transduction, cell numbers were determined, DNA was isolated, and VCN was quantified by qPCR. The number of cells generated within 12 days from transduced CD34+ HSCs was compared to the number of cells generated from untransduced HSCs. Using compounds to enhance transduction, the number of transduced cells that did not reach 65% of the number of untransduced cells was used as an indicator of procedural toxicity. These samples were excluded from analysis.

[0319] For transduction in the presence of potential transduction enhancers, i) 4, 6, or 8 μg / mL protamine; ii) 0.5, 0.75, 1, 1.5, 2 or 2.5 mg / ml Lentiboost®; iii) 0.5, 0.75, or 1 μg / ml amphotericin B; iv) 1 or 5 μM silibinin; v) 100, 200, or 400 nM midostaurin; vi) 4 or 10 μg / ml PCL; vii) 0.5, 1 or 2 mg / ml poloxamer F108; or viii) 10 μg / ml PLA The cells were incubated in the presence of

[0320] The results are summarized in Figures 2 and 3.

[0321] Additionally, combinations of transduction enhancers were tested. For transduction in the presence of potential transduction enhancers, i) 4 μg / mL protamine and 0.75 μg / mL amphotericin B; or ii) 4 μg / mL protamine and 10 μg / ml PCL; or iii) 4 μg / mL protamine and 1 mg / mL poloxamer F108; or iv) 1 mg / ml Lentiboost® and 1 μg / ml amphotericin B; or v) 1 mg / ml Lentiboost® and 0.75 μg / ml amphotericin B; or vi) 1 mg / ml Lentiboost® and 0.5 μg / ml amphotericin B; or vii) 1 mg / ml Lentiboost® and 1 μM silibinin; or viii) 1 mg / ml Lentiboost® and 100 nM midostaurin; or ix) 1 mg / ml Lentiboost® and 200 nM midostaurin; or x) 1 mg / ml Lentiboost® and 400 nM midostaurin; or xi) 1 mg / ml Lentiboost® and 4 μg / mL protamine; or xii) 1 mg / ml Lentiboost® and 5 μM silibinin; or xiii) 1 μg / mL amphotericin B and 1 mg / ml poloxamer F108; or xiv) 1 μg / mL amphotericin B and 10 μg / ml PCL; or xv) 5 μM silibinin and 10 μg / ml PCL; or xvi) 5 μM silibinin and 1 mg / ml poloxamer F108; or xvii) 400 nM midostaurin and 1 mg / ml poloxamer F108; or xviii) 400 nM midostaurin and 10 μg / ml PCL The cells were incubated in the presence of

[0322] The results for the transduction enhancer combinations are summarized in Figures 4-12.

[0323] Example 5: Transduction at an MOI of 20 Human CD34+ HSCs were transduced with lentiviral self-inactivating gene therapy vectors in X-Vivo 20 medium supplemented with 1% human serum albumin, 300 ng / ml SCF, 300 ng / ml Flt3-lig, and 100 ng / ml TPO at a density of 1E6 cells / ml. The transduction process involved a 2-hour pre-stimulation period in the presence of 1% DMSO, followed by a 16-hour incubation of the pre-stimulated cells with the gene therapy vector at an MOI of 20 in the presence of 1% DMSO.

[0324] The vector copy number (VCN) in the absence of DMSO was 0.915 and in the presence of DMSO was 1.16. Therefore, DMSO was shown to have a transduction-enhancing effect. The results are summarized in Figure 13. In certain embodiments, for example, the following are provided: (Item 1) A method for transducing target cells, comprising the step of contacting the target cells with a retroviral vector and a compound or combination of such compounds that can enhance transduction efficiency, wherein the target cells are pre-stimulated and / or co-stimulated by pre-incubation and / or co-incubation with the transduction-enhancing compound or combination of transduction-enhancing compounds before and / or during contacting the target cells with the retroviral vector. (Item 2) 2. The method according to item 1, wherein the transduction-enhancing compound is amphotericin B, and in particular, the target cells are contacted with amphotericin B at a concentration of about 0.05 to about 500 μM, in particular at a concentration of about 0.1 to about 10 μM, during the pre-incubation and / or co-incubation steps. (Item 3) 3. The method of item 2, wherein amphotericin B is used in combination with one or more additional transduction-enhancing compounds. (Item 4) 4. The method of claim 3, wherein the additional transduction enhancing compound is a protamine salt, in particular, the protamine salt is protamine chloride or protamine sulfate. (Item 5) 5. The method according to item 4, wherein the target cells are contacted with a protamine salt at a concentration of about 0.05 μg / mL to about 25 μg / mL, in particular at a concentration of about 0.1 μg / mL to about 10 μg / mL, during the pre-incubation and / or co-incubation steps. (Item 6) the one or more additional transduction enhancing compounds Lentiboost®, especially in concentrations of 0.1 mg / ml to 5,000 mg / ml; · Poloxamer F108, especially in concentrations from 0.1 mg / ml to 5,000 mg / ml; · Silibinin, especially at concentrations of 0.05 μM to 500 μM; Midostaurin, especially at concentrations between 2 nM and 500,000 nM; · PEG-PLA-PEG, especially in concentrations from 1 μg / ml to 5,000 μg / ml; · PEG-PLGA-PEG, especially at concentrations of 1 μg / ml to 5,000 μg / ml; · PEG-PCL-PEG, especially at concentrations of 1 μg / ml to 5,000 μg / ml; · Nystatin, especially at concentrations of 0.1-1000 μM; · Natamycin, especially in concentrations of 0.05-500 μM; Ruxolitinib, especially at concentrations of 0.01 to 10,000 μM; Fludarabine, especially in concentrations of 0.01-10,000 μM; Everolimus, especially at concentrations of 0.1-10 μM; · Resveratrol, especially in concentrations of 0.1-25 μM; · Prostaglandin E, especially at concentrations of 1-100 μM; Deoxyribonucleosides, especially at concentrations of 0.1 mM to 10 mM of each nucleoside; DMSO, especially at a concentration of 0.1-10% (v / v); and / or Any combination of these is selected from the group consisting of 6. The method of any one of items 3 to 5, in particular, wherein the one or more additional transduction enhancing compounds are selected from the group consisting of Lentiboost®, poloxamer F108 and / or PEG-PCL-PEG polymer. (Item 7) the transduction-enhancing compound is · Silibinin, especially at concentrations of 0.05 μM to 500 μM; Midostaurin, especially at concentrations between 2 nM and 500,000 nM; · Nystatin, especially at concentrations of 0.1-1000 μM; · Natamycin, especially in concentrations of 0.05-500 μM; · PEG-PCL-PEG polymers, especially at concentrations of 1 μg / ml to 5,000 μg / ml; PEG-PLGA-PEG polymers, especially at concentrations between 1 μg / ml and 5,000 μg / ml; · PEG-PLA-PEG polymers, especially at concentrations of 1 μg / ml to 5,000 μg / ml; DMSO, especially at a concentration of 0.1-10% (v / v); and / or Any combination of these The method according to item 1, wherein the compound is selected from the group consisting of: (Item 8) The transduction enhancing compound is used in combination with one or more additional transduction enhancing compounds, particularly when the one or more additional transduction enhancing compounds are Lentiboost®, especially in concentrations of 0.1 mg / ml to 5,000 mg / ml; · Poloxamer F108, especially in concentrations from 0.1 mg / ml to 5,000 mg / ml; Everolimus, especially at concentrations of 0.1-10 μM; · Resveratrol, especially in concentrations of 0.1-25 μM; · Prostaglandin E, especially at concentrations of 1-100 μM; Protamine salts, especially in concentrations of 0.05 μg / mL to 25 μg / mL; deoxyribonucleosides, particularly at a concentration of 0.1 mM to 10 mM for each nucleoside; and / or · any combination of these; 8. The method according to item 7, selected from the group consisting of: (Item 9) 9. The method of any one of items 1 to 8, wherein the target cells are co-incubated with the transduction-enhancing compound or combination of transduction-enhancing compounds while the target cells are in contact with the retroviral vector for a period of about 8 hours to about 48 hours, particularly about 10 hours to about 24 hours, but particularly about 12 hours. (Item 10) 10. The method of any one of items 1 to 9, wherein the target cells are pre-incubated with the transduction-enhancing compound or combination of transduction-enhancing compounds before contacting the target cells with the retroviral vector for a period of about 0.5 hours to about 10 hours, particularly about 1 hour to about 5 hours, but particularly about 2 hours. (Item 11) 11. The method according to any one of items 1 to 10, wherein the target cells are mammalian cells, particularly human cells. How to post. (Item 12) 12. The method according to any one of items 1 to 11, wherein the target cells are cells selected from the group consisting of lymphocytes, tumor cells, lymphoid lineage cells, neuronal cells, epithelial cells, keratinocytes, endothelial cells, primary cells, T cells, hematopoietic cells and stem cells. (Item 13) 13. The method of item 12, wherein the hematopoietic cells are hematopoietic stem cells, hematopoietic progenitor cells, CD34+ cells, monocytes, macrophages, tissue-resident macrophages, microglial cells, keratinocytes, or dendritic cells. (Item 14) 13. The method of claim 12, wherein the T cells are characterized by surface presentation of CD3, CD4 and / or CD8. (Item 15) 15. The method according to any one of items 1 to 14, wherein the retroviral vector is a lentiviral vector, particularly a self-inactivating lentiviral vector. (Item 16) 16. The method of any one of items 1 to 15, wherein the vector comprises a transgene under the control of a promoter, in particular wherein the transgene encodes a therapeutic protein or a chimeric antigen receptor (CAR).

Claims

[Claim 1] The invention described in the specification.

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