NOVEL TRANSDUCTION ENHANCERS AND USES THEREOF

Abstract

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 said transduction enhancing compound or a combination of transduction enhancing compounds prior to and/or during contacting the target cell with the retroviral vector.

Claims

1. A method for transducing a target cell, the method comprising a 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 said transduction enhancing compound or a combination of transduction enhancing compounds prior to and/or during contacting the target cell with the retroviral vector.

2. The method of claim 1, wherein the transduction enhancing compound is Amphotericin B, in particular wherein the target cell is contacted with Amphotericin B during the pre-incubation and/or co-incubation step at a concentration of about 0.05 to about 500 μM, in particular at a concentration of about 0.1 to about 10 μM.

3. The method of claim 2, wherein Amphotericin B is used in combination with one or more additional transduction enhancing compounds.

4. The method of claim 3, wherein the additional transduction enhancing compound is a protamine salt, in particular wherein the protamine salt is protamine chloride or protamine sulfate.

5. The method of claim 4, wherein the target cell is contacted with the protamine salt during the pre-incubation and/or co-incubation step at a concentration of about 0.05 μg/mL to about 25 mg/mL, in particular at a concentration of about 0.1 μg/mL to about 10 μg/mL.

6. The method claim 3, wherein the one or more additional transduction enhancing compound is selected from the group consisting of: Lentiboost®, in particular at a concentration between 0.1 mg/ml and 5,000 mg/ml; poloxamer F108, in particular at a concentration between 011 mg/ml and 5,000 mg/ml; Silibinin, in particular at a concentration between 0.05 μM and 500 μM; Midostaurin, in particular at a concentration between 2 nM and 500,000 nM; PEG-PLA-PEG, in particular at a concentration between 1 ng/ml and 5,000 ng/ml; PEG-PLGA-PEG, in particular at a concentration between 1 ng/ml and 5,000 ng/ml; PEG-PCL-PEG, in particular at a concentration between 1 ng/ml and 5,000 ng/ml; Nystatin, in particular at a concentration between 0.1 and 1000 μM; Natamycin, in particular at a concentration between 0.05 and 500 μM; Ruxolitinib, in particular at a concentration between 0.01 and 10,000 μM; Fludarabine, in particular at a concentration between 0.01 and 10,000 μM; Everolimus, in particular at a concentration between 0.1 and 10 μM; Resveratrol, in particular at a concentration between 0.1 and 25 μM; Prostaglandin E, in particular at a concentration between I and 100 μM; Deoxyribonucleosides, in particular at a concentration between 0.1 mM and 1OmM of each nucleoside; DMSO, in particular at a concentration between 0.1 and 10% (v/v); and/or any combination thereof; in particular wherein the one or more additional transduction enhancing compound is selected from the group consisting of: Lentiboost®, poloxamer F108 and/or a PEG-PCL-PEG polymer.

7. The method according to claim 1, wherein the transduction enhancing compound is selected from a group consisting of: Silibinin, in particular at a concentration between 0.05 μM and 500 μM; Midostaurin, in particular at a concentration between 2 nM and 500,000 nM; Nystatin, in particular at a concentration between 0.1 and 1000 μM; Natamycin, in particular at a concentration between 0.05 and 500 μM; a PEG-PCL-PEG polymer, in particular at a concentration between 1 μg/ml and 5,000 μg/ml a PEG-PLGA-PEG polymer, in particular at a concentration between 1 μg/ml and 5,000 μg/ml a PEG-PLA-PEG polymer, in particular at a concentration between 1 μg/ml and 5,000 μg/ml DMSO, in particular at a concentration between 0.1 and 10% (v/v); and/or any combination thereof.

8. The method according to claim 7, wherein the transduction enhancing compound is used in combination with one or more additional transduction enhancing compounds, in particular wherein the one or more additional transduction enhancing compound is selected from a group consisting of: Lentiboost®, in particular at a concentration between 0.1 mg/ml and 5,000 mg/ml; poloxamer F108, in particular at a concentration between O.lmg/ml and 5,000 mg/ml; Everolimus, in particular at a concentration between 0.1 and 10 μM; Resveratrol, in particular at a concentration between 0.1 and 25 μM; Prostaglandin E, in particular at a concentration between 1 and 100 μM; A protamine salt, in particular at a concentration between 0.05 μg/mL and 25 μg/mL; Desoxyribonucleosides, in particular at a concentration between 0.1 mM and 10 mM of each nucleoside; and/or any combination thereof.

9. The method of claim 1, wherein the target cell is co-incubated with the transduction enhancing compound or the combination of transduction enhancing compounds during contacting the target cell with a retroviral vector for a period between about 8 hours and about 48 hours, particularly between about 10 hours and about 24 hours, but particularly about 12 hours.

10. The method of claim 1, wherein the target cell is pre-incubated with the transduction enhancing compound or the combination of transduction enhancing compounds prior to contacting the target cell with a retroviral vector for a period between about 0.5 hours and about 10 hours, particularly between about 1 hour and about 5 hours, but particularly about 2 hours.

11. The method of claim 1, wherein the target cell is a mammalian cell, in particular a human cell.

12. The method of claim 1, wherein the target cell is a cell selected from the group consisting of a lymphocyte, a tumor cell, a lymphoid lineage cell, a neuronal cell, an epithelial cell, a keratinocyte, an endothelial cell, a primary cell, a T cell, a haematopoietic cell, and a stem cell.

13. The method of claim 12, wherein the haematopoietic cell is a haematopoietic stein cell, a haematopoietic progenitor cell, a CD34+ cell, a monocyte, a macrophage, a tissue resident macrophage, a microglial cell, a keratinocyte or a dendritic.

14. The method of claim 12, wherein the T cell is characterized by surface presentation of CD3, CD4 and/or CD8.

15. The method of claim 1, wherein the retroviral vector is a lentiviral vector, in particular a self-inactivating lentiviral vector.

16. The method of claim 1, wherein the vector comprises a transgene under control of a promoter, in particular wherein the transgene encodes a therapeutic protein or a chimeric antigen receptor (CAR).

Description

BRIEF DESCRIPTION OF THE FIGURES

[0514] FIG. 1 summarizes the results of three independent experiments, in which human CD34-positive HSC were transduced with a lentiviral self-inactivating gene therapy vector. This vector encodes human p47phox cDNA under control of the miR223 internal promoter. Cells were transduced in presence of protamine sulfate only (resulting VCN set to 100%), or in presence of protamine sulfate (PS) plus one or more compounds tested for transduction enhancer activity. After 10 to 12 days post transduction, DNA from transduced cells was isolated, and VCNs were quantified by qPCR. The increase in VCN relative to the VCN achieved upon transduction in presence of protamine sulfate only was expressed as “fold induction rel. to PS”. Left panel: Transduction at an MOI of 1. Right panel. Transduction at an MOI of 3. PGE2: prostaglandin E2; AmphoB: Amphotericin B.

[0515] FIG. 2 summarizes the results of three independent experiments, in which human CD34-positive HSC were transduced with a lentiviral self-inactivating gene therapy vector. Cells were transduced in X-Vivo 10 medium either in the absence of a transduction enhancer or in the presence of compounds that are tested for transduction enhancer activity. After 10 to 12 days post transduction, DNA from transduced cells was isolated, and VCNs were quantified by qPCR. The solid line represents the average VCN obtained with Lentiboost at the recommended concentration of 1 mg/mL. PCL: PEG-PCL-PEG (14.2 kDa); PLA: PEG-PLA-PEG (14.2 kDa).

[0516] FIG. 3 summarizes the results of three independent experiments, in which human CD34-positive HSC were transduced with a lentiviral self-inactivating gene therapy vector. Cells were transduced in BESP1366F medium either in the absence of a transduction enhancer or in the presence of compounds that are tested for transduction enhancer activity. After 10 to 12 days post transduction, DNA from transduced cells was isolated, and VCNs were quantified by qPCR. The solid line represents the average VCN obtained with Lentiboost® at the recommended concentration of 1 mg/mL.

[0517] FIG. 4 summarizes the results of three independent experiments, in which human CD34-positive HSC were transduced with a lentiviral self-inactivating gene therapy vector. Cells were transduced in BESP1366F medium in the presence of protamine, Amphotericin B or a combination thereof. After 10 to 12 days post transduction, DNA from transduced cells was isolated, and VCNs were quantified by qPCR. The solid line represents the average VCN obtained with Lentiboost® at the recommended concentration of 1 mg/mL under comparable conditions (see solid line in FIG. 3). The dashed line represents the average VCN obtained with protamine alone.

[0518] FIG. 5 summarizes the results of three independent experiments, in which human CD34-positive HSC were transduced with a lentiviral self-inactivating gene therapy vector. Cells were transduced in BESP1366F medium in the presence of Lentiboost®, Amphotericin B, Silibinin, Midostaurin or combinations thereof comprising Lentiboost® at a concentration of 1 mg/mL. After 10 to 12 days post transduction, DNA from transduced cells was isolated, and VCNs were quantified by qPCR. The solid line represents the average VCN obtained with Lentiboost® at the recommended concentration of 1 mg/mL.

[0519] FIG. 6 summarizes the results of three independent experiments, in which human CD34-positive HSC were transduced with a lentiviral self-inactivating gene therapy vector. Cells were transduced in X-Vivo 10 medium in the presence of protamine, PEG-PCL-PEG (14.2 kDa), poloxamer F108 or combinations thereof comprising protamine. After 10 to 12 days post transduction, DNA from transduced cells was isolated, and VCNs were quantified by qPCR. The solid line represents the average VCN obtained with Lentiboost® at the recommended concentration of 1 mg/mL under comparable conditions (see FIG. 2). The dashed line represents the average VCN obtained with protamine alone.

[0520] FIG. 7 summarizes the results of three independent experiments, in which human CD34-positive HSC were transduced with a lentiviral self-inactivating gene therapy vector. Cells were transduced in X-Vivo 10 medium in the presence of Lentiboost®, Amphotericin B, protamine, Silibinin, Midostaurin or combinations thereof comprising Lentiboost. After 10 to 12 days post transduction, DNA from transduced cells was isolated, and VCNs were quantified by qPCR. The solid line represents the average VCN obtained with Lentiboost® at the recommended concentration of 1 mg/mL (see FIG. 2).

[0521] FIG. 8 summarizes the results of three independent experiments, in which human CD34-positive HSC were transduced with a lentiviral self-inactivating gene therapy vector. 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 thereof comprising Amphotericin B. After 10 to 12 days post transduction, DNA from transduced cells was isolated, and VCNs were quantified by qPCR. The solid line represents the average VCN obtained with Lentiboost® at the recommended concentration of 1 mg/mL under comparable conditions. The dashed line represents the average VCN obtained with Amphotericin B alone.

[0522] FIG. 9 summarizes the results of three independent experiments, in which human CD34-positive HSC were transduced with a lentiviral self-inactivating gene therapy vector. Cells were transduced in X-Vivo 10 medium in the presence of Silibinin, Lentiboost®, PEG-PCL-PEG (14.2 kDa), poloxamer F108 or combinations thereof comprising Silibinin. After 10 to 12 days post transduction, DNA from transduced cells was isolated, and VCNs were quantified by qPCR. The solid line represents the average VCN obtained with Lentiboost® at the recommended concentration of 1 mg/mL under comparable conditions. The dashed line represents the average VCN obtained with Silibinin alone.

[0523] FIG. 10 summarizes the results of three independent experiments, in which human CD34-positive HSC were transduced with a lentiviral self-inactivating gene therapy vector. Cells were transduced in X-Vivo 10 medium in the presence of Midostaurin, Lentiboost®, poloxamer F108, PEG-PCL-PEG (14.2 kDa) or combinations thereof comprising Midostaurin. After 10 to 12 days post transduction, DNA from transduced cells was isolated, and VCNs were quantified by qPCR. The solid line represents the average VCN obtained with Lentiboost® at the recommended concentration of 1 mg/mL under comparable conditions. The dashed line represents the average VCN obtained with Midostaurin alone.

[0524] FIG. 11 summarizes the results of three independent experiments, in which human CD34-positive HSC were transduced with a lentiviral self-inactivating gene therapy vector. 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 thereof comprising PEG-PCL-PEG. After 10 to 12 days post transduction, DNA from transduced cells was isolated, and VCNs were quantified by qPCR. The solid line represents the average VCN obtained with Lentiboost® at the recommended concentration of 1 mg/mL under comparable conditions (see FIG. 2). The dashed line represents the average VCN obtained with PEG-PCL-PEG alone.

[0525] FIG. 12 summarizes the results of three independent experiments, in which human CD34-positive HSC were transduced with a lentiviral self-inactivating gene therapy vector. Cells were transduced in X-Vivo 10 medium in the presence of poloxamer F108, Silibinin, Midostaurin, Amphotericin B, protamine or combinations thereof comprising poloxamer F108. After 10 to 12 days post transduction, DNA from transduced cells was isolated, and

[0526] VCNs were quantified by qPCR. The solid line represents the average VCN obtained with Lentiboost® at the recommended concentration of 1 mg/mL under comparable conditions (see FIG. 2). The dashed line represents the average VCN obtained with poloxamer F108 alone.

[0527] FIG. 13 summarizes the results of three independent experiments, in which human CD34-positive HSC were transduced with a lentiviral self-inactivating gene therapy vector at MOI 20. Cells were transduced in the absence and presence of 1% DMSO.

EXAMPLES

Example 1: Transduction at MOI 1, MOI 3 or MOI 5

[0528] Thawed cells from healthy donors were cultured in 96 well plates comprising X-Vivo 20 medium supplemented with 1% human serum albumin, 300 μg/ml stem cell factor (SCF), 300 μg/ml fins like tyrosine kinase 3 (FLT-3) ligand (Flt3-lig) and 100 μng/ml Thrombopoietin (TPO) at a density of 0.5 E6 cells/cm.sup.2, and at a concentration of 1E6 cells/ml for 22h, optionally followed by 2h of pre-stimulation with below mentioned compounds. Afterwards, cells were incubated for transduction with the lentiviral SIN gene therapy vector for 12 h in presence of the compounds indicated below and protamine sulfate. After the 12 h transduction period, the medium was exchanged by above mentioned medium, and cells were transferred to 12-well plates in which they were cultured in a volume of 1 ml for 5 to 7 days depending on cell density. Thereafter, medium was exchanged by fresh medium, and cells were cultured in 2 ml for another 6 days. At day 12 post transduction, DNA was isolated, and vector copy number (VCN) was quantified by qPCR. All experiments were conducted in triplicates. For negative control, a triplicate of non-transduced cells was cultured as described. To determine the baseline of transduction efficacy in presence of only protamine sulfate, cells were incubated with MOI 1, MOI 3 or MOI 5 of the lentiviral SIN vector in presence of 4 μg/mL protamine sulfate. For transduction in the presence of potential transduction enhancers, cells were incubated with MOI 1, MOI 3 or MOI 5 of the lentiviral SIN vector in presence of [0529] i) 5 μM Silibinin, 4 μg/mL protamine sulfate; [0530] ii) 5 μM Resveratrol, 4 μg/mL protamine sulfate; [0531] iii) 1 μM Everolimus, 4 μg/mL protamine sulfate; [0532] iv) 0,4 μM Midostaurin, 4 μg/mL protamine sulfate; [0533] v) 1 μg/mL Amphotericin B, 4 μg/mL protamine sulfate; [0534] vi) 100 μM Nystatin, 4 μg/mL protamine sulfate; [0535] vii) 3 μM Natamycin, 4 μg/mL protamine sulfate; [0536] viii) 10 μM Prostaglandin E2, 4 μg/mL protamine sulfate; [0537] ix) 1 mg/ml Lentiboost®, 4 μg/mL protamine sulfate; [0538] x) 1,000 μg/ml Poloxamer Symperonic F108®, 4 μg/mL protamine sulfate; [0539] xi) 1,000 μg/ml poly(ethylene glycol)-b-poly(D,L-lactic acid-co-glycolic acid)-b-poly(ethylene glycol) (PEG- PLGA-PEG) with 5 kDa poly(ethylene glycol) block on both ends, and a central 4.2 kDa poly(D,L-lactic acid-co-glycolic acid) block, termed PEG5k-b-PLA4.2k-b-PEG5k; [0540] xii) 10 μg/ml methoxypoly(ethylene glycol)-poly(e-caprolacton)-methoxypoly(ethylene glycol) (PEG- PCL-PEG) with 5 kDa poly(ethylene glycol) block on both ends, and a central 4.2 kDa poly(e- caprolacton) block, termed PEG5k-b-PCL4.2k-b-PEG5k; [0541] xiii) 50 μg/mL poly(ethylene glycol)/poly(lactide)/poly(ethylene glycol) (PEG-PLA-PEG) with 5 kDa poly(ethylene glycol) blocks on both ends, and a central 4.2 kDa poly(lactide) block, termed PEG5k-b-PLA4.2k-b-PEG5k [0542] xiv) 50 μg/ml methoxypoly(ethylene glycol)-poly(e-caprolacton)-methoxypoly(ethylene glycol) (PEG- PCL-PEG) with a central 2.4 kDa poly(e-caprolacton) block, and a 5.3 kDa poly(ethylene glycol) block on both ends, which both were covalently linked to an amino group, termed 2-PEG5.3k-b- PCL2.4k-b-PEG5.3k-NH2; [0543] xv) Deoxyribonucleosides with a final concentration of 300 μM of each, or with combinations thereof.

[0544] After 10 to 12 days, non-integrated pro-viral DNA was diluted out due to the proliferation of cells in the plate and VCNs were quantified thereafter by qPCR. Every condition was tested in triplicates and the average of VCNs of triplicates for each individual condition was calculated. To evaluate the transduction enhancement activity of the tested compounds, average VCNs of cells transduced in the presence of protamine sulfate was set as 1, and enhancement of transduction was expressed as X-fold increase relative to transduction in the presence of protamine sulfate only.

[0545] The inventors surprisingly observed the following increases in the efficiency of lentiviral transduction (see FIG. 1):

[0546] MOI of 1:

TABLE-US-00003 Silibinin (factor 4.8) Resveratrol (factor 4.5) Everolimus (factor 13.5) Midostaurin (factor 4.2) Amphotericin B (factor 3) Nysatin (factor 2.6) Natamycin (factor 3) Lentiboost (factor 9.8)

[0547] MOI of 3:

TABLE-US-00004 Everolimus (factor 2.5) Amphotericin B (factor 2.2) Everolimus + Amphotericin B (factor 4.4) Lentiboost (factor 6.4)

[0548] Supplementation of CD34-positive cells with 300 μM of each deoxyribonucleoside or with 10 μg/mL of BAB-type triblock polymer PCL increased VCN by factor 1.89 or by factor 1.81, respectively, relative to transduction without transduction enhancer.

Example 2: Transduction in the Presence of Poloxamer F108 and Polybrene (MOI 5)

[0549] Human CD34+HSC were transduced by a lentiviral self-inactivating gene therapy vector, comprising a cDNA under control of the miR223 promoter encoding human p47phox, 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 comprised a 2 h pre-stimulation period in presence of poloxamer F108 (1,000 μg/ml) and polybrene (8 μg/ml), followed by 12 h incubation of pre-stimulated cells with the gene therapy vector with a MOI 5 in presence of poloxamer F108 (1,000 μg/ml) and polybrene (8 μg/ml).

Example 3: Transduction in the Presence of PEG-PCL-PEG Polymer and Midostaurin MOI 5)

[0550] Human CD34+HSC were transduced by a lentiviral self-inactivating gene therapy vector, in X-Vivo 20 medium supplemented with 1% human serum albumin, 300 μg/ml SCF, 300 ng/ml Flt3-lig and 100 ng/ml TPO at a density of 1E6 cells/ml. The transduction process comprised a 2 h pre-stimulation period in presence of protamine sulfate (4 μg/mL) plus and PEG-PCL-PEG polymer (4 μg/mL) and midostaurin (0.4 μM) followed by 12 h incubation of pre-stimulated cells with the gene therapy vector with an MOI 5 in presence of protamine sulfate (4 μg/mL) plus and PEG-PCL-PEG polymer (4 μg/mL) and midostaurin (0.4 μM).

Example 4: Transduction in the Presence of Amphotericin B (MOI 5)

[0551] Human CD34+HSC were transduced by a lentiviral self-inactivating gene therapy vector, comprising cDNA, under control of the miR223 promoter, encoding p47phox, in X-Vivo 20 medium supplemented with 1% human serum albumin, 300 ng/ml SCF, 200 ng/ml Flt3-lig and 100 ng/ml TPO at a density of 1E6 cells/ml. The transduction process comprised a 2 h pre-stimulation period in presence of 1 μg/mL Amphotericin B and 4 μg/mL protamine sulfate, followed by 12 h incubation of pre-stimulated cells with the gene therapy vector with an MOI 5 in presence of 1 μg/mL Amphotericin B, and 4 μg/mL protamine sulfate.

Example 5: Transduction at MOI 10

[0552] Commercially available, anonymized human CD34+ haematopoietic stem cells (HSC) were used to test the enhancement of retroviral transduction efficiency by transduction enhancers, or by combinations of transduction enhancers. All experimental conditions were tested in triplicates, and final results were expressed as average of the individual results of each triplicate.

[0553] For transduction, a lentiviral self-inactivating (SIN) vector was used, comprising the miR223 promoter as internal promoter, and p47phox encoding cDNA as transgene. Three tests with MOI 10 were conducted independently, in which HSC were retrovirally transduced in presence of either X-Vivo 10 medium or in presence of BESP1366F medium, supplemented with 1% human serum albumin, 300 ng/ml stem cell factor (SCF), 300 ng/ml fins like tyrosine kinase 3 (FLT-3) ligand (Flt3-lig), and 100 ng/ml Thrombopoietin (TPO) at a density of 0.5 E6 cells/cm2, and at a concentration of 1E6 cells/ml.

[0554] Before transduction, HSC were cultured in 96 well plates for 22 h with above mentioned media and cytokines, optionally followed by 2 h of pre-stimulation with below mentioned compounds in above mentioned medium without gene therapy vector, after which cells were incubated for transduction without change of medium (with/without compounds) with the lentiviral SIN gene therapy vector at an MOI 10 for 12 h. After the 12 h transduction period, the medium was exchanged by above mentioned medium (including supplements), and cells were transferred to 12-well plates in which they were cultured in a volume of 1 ml of above mentioned medium (including supplements) for 5 to 7 days, depending on cell density. Thereafter, medium was exchanged by fresh medium of abovementioned composition (including supplements), and cells were cultured in 2 ml medium for another 6 days.

[0555] At day 12 post transduction, cell numbers were determined, DNA was isolated, and VCN was quantified by qPCR. Cell numbers arising within 12 days from transduced CD34+HSC were compared to cell numbers arising from non-transduced HSC. Transduced cell numbers that did not reach 65% of cell number of non-transduced cells were taken as indication for toxicity of the procedure, using compounds for transduction enhancement. Those samples were excluded from analysis.

[0556] For transduction in the presence of potential transduction enhancers, cells were incubated in the presence of: [0557] i) 4, 6 or 8 μg/mL protamine; [0558] ii) 0.5, 0.75, 1, 1.5, 2, or 2.5 mg/ml Lentiboost®; [0559] iii) 0.5, 0.75, or 1 μg/ml Amphotericin B; [0560] iv) 1 or 5 μM Silibinin; [0561] v) 100, 200 or 400 nM Midostaurin; [0562] vi) 4 or 10 μg/ml PCL; [0563] vii) 0.5, 1, or 2 mg/ml Poloxamer F108; or [0564] viii) 10 μg/ml PLA.

[0565] Results are summarized in FIGS. 2 and 3.

[0566] Further, combinations of transduction enhancers were tested. For that, transduction in the presence of potential transduction enhancers, cells were incubated in the presence of: [0567] i) 4 μg/mL protamine and 0.75 μg/ml Amphotericin B; or [0568] ii) 4 μg/mL protamine and 10 μg/m1PCL; or [0569] iii) 4 μg/mL protamine and 1 mg/ml Poloxamer F108; or [0570] iv) 1 mg/ml Lentiboost® and 1 μg/ml Amphotericin B; or [0571] v) 1 mg/ml Lentiboost® and 0.75 μg/ml Amphotericin B; or [0572] vi) 1 mg/ml Lentiboost® and 0.5 μg/ml Amphotericin B; or [0573] vii) 1 mg/ml Lentiboost® and 1 μM Silibinin; or [0574] viii) 1 mg/ml Lentiboost® and 100 Midostaurin; or [0575] ix) 1 mg/ml Lentiboost® and 200 nM Midostaurin; or [0576] x) 1 mg/ml Lentiboost® and 400 Midostaurin; or [0577] xi) 1 mg/ml Lentiboost® and 4 μg/mL protamine; or [0578] xii) 1 mg/ml Lentiboost® and 5 μM Silibinin; or [0579] xiii) 1 μg/mL Amphotericin B and 1 mg/ml Poloxamer F108; or [0580] xiv) 1 μg/mL Amphotericin B and 10 μg/ml PCL; or [0581] xv) 5 μM Silibinin and 10 μg/ml PCL; or [0582] xvi) 5 μM Silibinin and 1 mg/ml Poloxamer F108; or [0583] xvii) 400 nM Midostaurin and 1 mg/ml Poloxamer F108; or [0584] xviii) 400 Midostaurin and 10 μg/ml PCL.

[0585] Results for the combinations of transduction enhancers are summarized in FIGS. 4-12.

Example 5: Transduction at MOI 20

[0586] Human CD34+HSC were transduced by a lentiviral self-inactivating gene therapy vector 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 comprised a 2 h pre-stimulation period in presence of 1% DMSO, followed by 16 h incubation of pre-stimulated cells with the gene therapy vector with an MOI 20 in presence of 1% DMSO.

[0587] The vector copy number (VCN) in the absence of DMSO was 0.915 and 1.16 in the presence of DMSO. DMSO was thus shown to have a transduction enhancing effect. The results are summarized in FIG. 13.