RETROVIRAL TRANSDUCTION USING POLOXAMERS

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 poloxamer having a molecular weight of 12.8 kDa to about 15 kDa. Further, the invention relates to the use of a poloxamer as defined herein, optionally in combination with a polycationic substance as defined herein, for transducing a target cell with a retroviral vector and a kit comprising a retroviral vector, a poloxamer as defined herein and, optionally, instructions for use.

Claims

1. A method for transducing a target cell, the method comprising the step of contacting a target cell with a retroviral vector and a poloxamer having a molecular weight of 12.8 kDa to about 15 kDa.

2. 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, an endothelial cell, a primary cell, a stem cell.

3. The method of claim 2, wherein lymphocyte is a primary lymphocyte and/or wherein the tumor cell is a hematopoietic tumor cell, a neuronal tumor cell or an epithelial tumor cell.

4. The method of any one of claims 1 to 3, wherein the retroviral vector is a lentiviral vector.

5. The method of claim 4, wherein the lentiviral vector is pseudotyped with VSV-G and/or with an antibody fragment fused to VSV-G.

6. The method of any one of claims 1 to 5, wherein the poloxamer has the formula HO[CH2CH2O]x-[CH2C2H4O]z-[CH2CH2O]y, wherein x+y=265.45 and z=50.34 on average; or wherein the poloxamer has the formula HO[CH2CH2O]x-[CH2C2H4O]z-[CH2CH2O]y, wherein x+y=236.36 and z=44.83 on average.

7. The method of any one of claims 1 to 6, wherein said target cell is further brought into contact with one or more polycationic substances selected from the group of polycationic polymers or polycationic peptides.

8. The method of claim 7, wherein said polycationic polymers are selected from the group consisting of poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLL) and 1,5-dimethyl-1,5-diaza-undeca-methyl-polymethobromide (Polybrene); and/or said polycationic peptides are selected from the group consisting of protamine sulphate and poly-l-lysin (PLL) having a mean molecular weight from 1 to 300 kDa.

9. The method of claim 8, wherein the polycationic substances are 1,5-dimethyl-1,5-diaza-undeca-methyl-polymethobromide and/or protamine sulphate.

10. The method of any one of claims 1 to 9, wherein said poloxamer is provided at a concentration of about 50 to 5000 g/ml.

11. The method of claim 10, wherein said poloxamer is provided at a concentration of about 500 to 1000 g/ml.

12. The method of any one of claims 1 to 11 comprising the further step of spinoculating said retroviral vector with said target cell prior to, concomitant with or after contacting said target cell with said poloxamer.

13. Use of a poloxamer as defined in any one of claims 1 to 12, optionally in combination with a polycationic substance as defined in any one of claims 7 to 9, for transducing a target cell with a retroviral vector.

14. A kit comprising a poloxamer as defined in any one of claims 1 to 12, a polycationic polymer and/or a polycationic peptide as defined in any one of claims 7 to 9, and, optionally, instructions for use.

15. The kit of claim 14 further comprising a retroviral vector as defined in any one of claims 1 to 12.

Description

[0059] The figures show:

[0060] FIG. 1: Cell proliferation assay (WST-1) of HEK293T cells incubated with substances from the class of polycations (a: chloroquine, PEA, dextran, chloresteryl-carbamate and L-ornithine) or poloxamers and related substances (b: synperonic L122, pluronic F68 and F127, synperonic P85, F108 and NP30, arkopal) and normalized to untreated control (black line=100%, 3 different experimentsstandard error (s.e.m.)).

[0061] FIG. 2: Substance-assisted cellular toxicity and infectivity. HEK293T cells were incubated with increasing concentrations of selected transduction substances from the class of polycations (PEA) or poloxamers (pluronic F68, F127 and synperonic F108); phase microscopy (scale bar=50 m) is used for cell viability check up; right row: Green fluorescent light emission of LV-transduced (GP) HEK293T cells at MOI 2.5 in the presence substances (polybrene at 10 g/ml, others at 1000 g/ml).

[0062] FIG. 3: Substance-assisted lentiviral infectivity in HEK293T cells. (a) Representative Dot Blots of cytofluorometric analysis incubated with decreasing MOI of GFP-coding lentivirus (GP) with and without transduction adjuvants from the class of polycations (PEA (at 1000 g/ml)) or poloxamers (pluronic F68 (at 1000 g/ml), F127 (at 1000 g/ml) and synperonic F108 (at 1000 g/ml)) in well-tolerable concentrations, polybrene was used at 10 g/ml; (b) Statistical quantification of transduction experiments described under a, in the presence of polybrene (10 g/ml), synperonic F108 (1000 g/ml) and both substances (3 different experimentsstandard error (s.e.m.)); (c) Synperonic F108 demonstrated better infection than polybrene-assisted LV infection. Synperonic F108 alone or in combination with Polybrene (10 g/ml) increased the mean amount of infected cells (two independent experiments) when compared to polybrene-induced transduction.

[0063] FIG. 4: Adjuvant assisted lentiviral gene transfer in anaplastic large cell lymphoma (ALCL) cells. (a) Four ALCL cell lines were infected with equal MOI and 10 g/ml polybrene; (b) Statistical quantification of transduction efficiency in KARPAS-299 and SR-786 cells in the presence of polybrene (10 g/ml), synperonic F108 (1000 g/ml) or both substances (3 different experiments t standard error (s.e.m.)); (c) Analysis of cellular PI-permeability and apoptosis rate in SR-786 cells; (d) Statistical quantification of PI permeability and apoptosis induction in KARPAS-299 and SR-786 cells (2 different experimentsstandard error (s.e.m.)); (e) Synperonic F108 up to a maximum applied concentration (5000 g/ml) for facilitating lentiviral infection of KARPAS-299 and SR-786 lymphoma cells. Synperonic F108 demonstrated better infection used alone or in combination with polybrene, compared to polybrene-assisted LV infection alone; (f) The effect of the spinoculation step during LV infection was tested in combination with the use of adjuvants for facilitating lentiviral infection of KARPAS-299 and SR-786 lymphoma cells. The spinoculation protocol resulted in better infection of cells as compared to cells infected without the spinoculation step.

[0064] FIG. 5: Synperonic F108 enhanced transduction of primary lymphoid cells of T-cell origin. Representative Dot Blots of cytofluorometric analysis from primary lymphocytes of two donors (a: donor #1, b: donor #2) incubated with decreasing MOI of GFP-coding lentivirus (GP) with adjuvants polybrene (at 10 g/ml), synperonic F108 (at 1000 g/ml) and a combination of both in indicated concentrations; in graphs, statistical quantification of transduction experiments s depicted as mean value of 2 different experiments t standard error (s.e.m.).

[0065] FIG. 6: Adjuvant-assisted cellular toxicity and infectivity in pancreatic carcinoma cells. Combined phase and fluorescence microscopy (scale bar=50 m) of LV-transduced (SIH1) pancreatic carcinoma AsPC-1 and PANC-1 cells at MOI 2.5 incubated with no substance, polybrene (10 g/ml), synperonic F108 (1000 g/ml) or both substances; in indicated cases, cells were additionally centrifuged for 60 min at 1,000 g.

[0066] FIG. 7: Adjuvant assisted lentiviral gene transfer in further ALCL cells. (a) Statistical quantification of LV-transduction efficiency in SUDHL-1 and SUP-M2 cells in the presence of polybrene (10 g/ml), synperonic F108 (1000 g/ml) or a combination of both substances (3 different experimentsstandard error (s.e.m.)); (b) Statistical quantification of PI permeability and apoptosis induction in SUDHL-1 and SUP-M2 cells (2 different experimentsstandard error (s.e.m.)).

[0067] The examples illustrate the invention:

EXAMPLE 1: METHODS

Cell Lines and Chemicals

[0068] The human embryonic kidney cells HEK293T were grown in DMEM supplemented with 10% (vol/vol) fetal calf serum (FCS) and 2 mM glutamine. The anaplastic large cell lymphoma cell lines KARPAS-299, SUDHL-1, SR-786 and SUP-M2 were cultured in RPMI 1640 supplemented with 10% FCS and 2 mM glutamine, pancreatic carcinoma cell line AsPC-1 in RPMI 1640 with 20% FCS, 2 mM glutamine and 1 mM sodium pyruvate and PANC-1 in DMEM complemented with 10% FCS and 4 mM glutamine. All chemical adjuvant candidates were purchased from Sigma-Aldrich and dissolved in water to obtain 100 mg/ml stock solutions.

Cell Proliferation Assay (WST-1 Assay)

[0069] In 6-well plates, triplicates of 210.sup.5 cells per well were seeded and treated with defined adjuvant concentrations ranging from 1 g/ml to 1000 g/ml. After 24 hours incubation (at 37 C. with 5% CO.sub.2) the medium was exchanged with fresh medium and cells were incubated for additional 48 hours. Cell proliferation was determined with the WST-1 colorimetric cell proliferation assay (Roche), according to manufacturer's instructions. Shortly, cells were trypsinized following 100 l cell incubation with 10 l WST-1 substrate (at 37 C. with 5% CO.sub.2) for 2 hours. The assay was performed in 96-well microtiter plates and absorbance was determined at 490 nm using a TECAN-Infinite microplate reader (TECAN).

Lentivirus Production

[0070] The lentiviral transduction vectors pGreenPuro (pGP) and pSIH1-H1-copGFP (pSIH1) (System Biosciences) allow expression of copGFP driven by an internal CMV promoter. Replication-defective lentiviral particles (GP) were produced by transient co-transfection of HEK293T cells in a 10 cm petri dish with 16 g, 8 g and 4 g of packaging plasmids pMDLg/pRRE, pRSV.Rev and pMD2.G (a kind gift from D. Trono, cole polytechnique fdrale de Lausanne) and 8 g of pGP vector using Lipofectamine 2000 (Life Technologies) as transfection reagent according to the manufacturer's instructions. Secondly, lentiviral virus particles (SIH1) were produced in HEK293T cells using a pPackH1-plasmid packaging mix according to the company's instructions (System Biosciences).

[0071] The virus particles were harvested 48 hours after transfection, cleared of cell debris by low-speed centrifugation, and filtered via 0.45 m Stericup filters. The lentivirus was concentrated by ultrafiltration using Amicon-20 columns (both from Millipore) in accordance to the manufacturer's guidelines. Concentrated virus aliquots were stored at 80 C. Virus titers were determined by cytofluorimetric analysis of copGFP expression in HEK293T cells infected with serially diluted virus.

Viral Infection of Cell Lines

[0072] HEK293T cells (210.sup.5 cells per well) were covered with 1 ml medium containing lentivirus (GP) with or without adjuvants. Pancreatic carcinoma AsPC-1 and PANC-1 cells (10.sup.4 cells per well) were covered with 250 l supernatant containing lentivirus (SIH1) with or without adjuvants. In given case, plates were centrifuged at 1,000 g for 60 min. After 24 hours incubation (at 37 C. with 5% CO.sub.2) the supernatant was exchanged with fresh medium and incubated for additional 48 hours. Combined microscopic analysis and documentation was done after 48 hours post infection (HBO 50/AC and AxioCam MRC, Carl Zeiss AG).

[0073] KARPAS 299, SUDHL-1, SR-786 and SUP-M2 suspension cells (10.sup.6 cells per well) were resuspended in 1 ml medium containing lentivirus (GP) with or without adjuvants. Plates were centrifuged at 800 g for 90 min. SUDHL-1 cells were washed and resuspended in fresh medium directly after centrifugation and incubated for 48 hours. KARPAS-299, SR-786 and SUP-M2 cells were incubated in 1 ml medium containing lentivirus over night after centrifugation, then washed and resuspended in fresh culture medium for additional 48 hours incubation.

Viral Infection of Primary Lymphoid Cells of T-Cell Origin

[0074] PBMCs of two healthy donors were collected according to approvals and the requirements of the local ethical board and the principles expressed in the Helsinki Declaration. PBMCs were isolated via ficoll gradient centrifugation, cultivated over three days in RPMI medium supplemented with human serum, 50 U/ml IL-2 (Chiron Vaccines) and 50 ng/ml OKT3 (LGC Standards). After activation, 510.sup.5 cells per well were resuspended in 500 l of supplemented medium containing lentivirus (GP) with or without adjuvants. Plates were centrifuged at 800 g for 90 min, incubated over night, then washed and resuspended in fresh supplemented medium for additional 24 hours incubation.

Cytofluorimetric Analysis

[0075] After lentiviral transduction, cells were washed in PBS and resuspended in PBS with 1 g/ml propidium iodide (Invitrogen). After 10 min incubation on ice, 30,000 events were analyzed for forward/sideward scatter characteristics, green fluorescence light emission at 530 nm and red fluorescence light emission at 610 nm in FACSDiva (BD Biosciences). For apoptosis measurements, cells were washed in PBS and resuspended in 70% (vol/vol) ethanol followed by 45 min incubation on ice. After fixation, cells were centrifuged and pellets resuspended in PBS with 40 g/ml propidium iodide and 100 g/ml RNAse (Qiagen). Cells were analyzed in FACSDiva only for red fluorescence light emission, 30,000 events were analyzed to determine the Sub-G1 proportion of apoptotic cells (<2n).

Statistical Analysis

[0076] All experiments were performed at least in doublets. In corresponding graphs mean valuesstandard error (s.e.m.) are depicted if not stated otherwise.

EXAMPLE 2: CELLULAR TOXICITY PROFILES OF POLYCATION AND POLOXAMER ADJUVANTS

[0077] From potential adjuvant substances for lentiviral gene delivery we selected five representatives from the class of polycation-like and seven from the class of poloxamer-like chemical substances (Tab. 1). HEK293T cells were co-incubated with defined substance concentration for 24 hours, than medium was changed and cells were incubated for additional 48 hours prior measurements of cell proliferation. To test specific HEK293T growth inhibition by different substances we measured incorporation of WST1 substrate and correlated to control cell population without any substance co-incubation (FIG. 1). This set-up was arranged according to common transduction protocols including 24 hours incubation of LV with target cells before LV and adjuvant (polybrene) are washed out.

[0078] For the class of polycations all substances demonstrated non-toxic behavior at 10 g/ml (FIG. 1a). With increasing concentrations, two substances (polybrene and cholesterylcarbamate) induced massive reduction in cell proliferation. Finally only the polycation PEA remained non-toxic at 1000 g/ml. Therefore, only 1 of 5 polybrene-like polycations had an advantageous toxicity profile at very high concentrations. As second substance class, poloxamers were well tolerated at 10 g/ml (FIG. 1b). With increasing amount, three substances (pluronic F68, F127 and synperonic F108) could be administered even at 1000 g/ml without reducing the cellular viability of HEK293T cells. In contrast, poloxamer-related synperonic NP30 and Arkopal showed cell proliferation inhibition already at low concentrations and were not considered for further experiments. All together, only 4 out of 12 adjuvant candidates provided an improved toxicity profile compared to polybrene. Therefore, we have used PEA as well as pluronic F68, F127 and synperonic F108 for subsequent LV transduction of HEK293T cells (FIG. 2). Only synperonic F108 showed comparable transduction efficiency to polybrene, whereas PEA showed low transduction rates. Interestingly, pluronic F68 and F127 exhibit no enhancement in viral transduction (as polybrene and synperonic F108 do). However, they are not inhibiting virus infection itself.

TABLE-US-00001 TABLE 1 Adjuvant candidates for lentiviral transduction from class I, polycations (polybrene like), and class II, poloxamers and related substances, described with names, chemical terms, molecular weight (MW) and configurations. Class Polycations like polybrene Name Polybrene Chemical term 1,5-dimethyl-1,5-diaza-undeca-methylene-polymethobromide MW (kDa) 4-6 Configuration [00001] Class Polycations like polybrene Name Cholesterylcarbamate Chemical term Cholesteryl-3-N-(dimethyl-amino-ethyl)-carbamate-hydrochloride MW (kDa) 0.7 Configuration [00002] Class Polycations like polybrene Name Dextran Chemical term Diethyl-aminoethyl-dextran-hydrochloride MW (kDa) >500 Configuration [00003] Class Polycations like polybrene Name PEA Chemical term L--Phosphatidyl-ethanolamine-dioleoyl MW (kDa) 0.7 Configuration [00004] Class Polycations like polybrene Name Chloroquine Chemical term N-(7-choroquinolin-4-yl)-N,N-diethyl-pentane-1,4-diamine- diphosphate MW (kDa) 0.5 Configuration [00005] Class Polycations like polybrene Name L-Ornithine Chemical term Poly-L-2,5-diamino-pentanoic acetate-hydrobromide MW (kDa) 5-15 Configuration [00006] Class Poloxamers and related substances Name Synperonic L122 Synperonic P85 Pluronic F68 Pluronic F127 Synperonic F108 Chemical term Polyethylene-oxide-polypropylene-oxide MW (kDa) 4.4 4.6 8.4 12.6 14.6 Configuration [00007] Class Poloxamers and related substances Name Synperonic NP30 Arkopal (NP100) Chemical term 4-Nonyl-phenyl-polyethylene-glycol MW (kDa) 0.7 0.8 Configuration [00008]

EXAMPLE 3: ADJUVANT-ASSISTED LENTIVIRAL GENE TRANSFER INTO HEK293T CELLS

[0079] HEK293T cells were covered (for 24 hours) with a mixture of LV with specified adjuvant in a maximum tolerable concentration (1000 g/ml). LV carrying a CopGFP-coding transgene were applied in decreasing MOI from 2.5 to 0.0025. After medium change, cells were cultivated for another 48 hours and measured for green fluorescent light emission by flow cytometry. In FIG. 3a four representative experiments are depicted in dot blots. Adjuvant-free transduction showed low transduction rates over the whole MOI range. Polybrene had the ability to increase transduction rates from 31.5% to 48.4% (MOI 0.25) at the recommended concentration of 10 g/ml.

[0080] Other substances failed to further increase this transduction level (PEA is shown as example in FIG. 3a), expect one adjuvant candidate: Synperonic F108. At well tolerated concentration of 1000 g/ml this poloxamer demonstrated better transduction rates than polybrene-assisted LV infection. Synperonic F108 increased the medial amount of transduced cells to 61.4% (MOI 0.25) and even worked at MOI 0.025 increasing transduction from 15.5 to 19.6% if compared to polybrene-induced transduction. Furthermore, we asked whether polybrene and synperonic F108 could combine their individual transduction capacities and co-administrated both to LV premixes alone or in combination for subsequent HEK293T cell infections (FIG. 3b). Using this combination we could increase synperonic F108 induced total transduction rates up to additionally 5%. As further proof, adherent pancreatic carcinoma cell lines AsPC-1 and PANC-1 indicated higher amounts of GFP-positive cells when co-treated with polybrene and synperonic F108 during infection. Additionally, a spinoculation step increased basal transduction levels and led to maximum transduction rates in combinations of polybrene and synperonic F108 (FIG. 6).

EXAMPLE 4: ADJUVANT-ASSISTED LENTIVIRAL GENE TRANSFER IN LYMPHOMA CELLS

[0081] Many established tumor cell lines in pre-clinical research offer only poor transfection rates with common viral and non-viral tools. Anaplastic large cell lymphoma (ALCL) cell lines that grow in suspension cultures belong to this hard-to-infect subset of tumor cell lines.sup.8. With a modified protocol including a spinoculation step during LV infection, we tested synperonic F108 for facilitating lentiviral infection for KARPAS-299, SR-786, SUDHL-1 and SUP-M2 lymphoma cells (FIG. 4b). Notably, with same median MOI rates (1.5) Karpas-299 cells were 20.8% infected in presence of polybrene and SR-786 showed already 58.8% infection rate (FIG. 4a). KARPAS-299 cells showed Increase in GFP-positive cells from 58.4% in presence of polybrene to 85.2% in presence of synperonic F108 in the premix with LV (FIG. 4b). In lower MOI ranges the rates of polybrene-assisted transduction are enhanced from 21.9% to 33.7% or respectively to 41.0% in combined treatment (polybrene and synperonic F108 addition). The transduction rate of SR-786 cells increased similar to KARPAS-299 cells at MOI 1.5 from 64.9% (polybrene) to 84.1% (synperonic F108). Other lymphoma cell lines (SUDHL-1 and SUP-M2) showed similar effects in enhancement of LV delivery by synperonic F108 (FIG. 7a).

EXAMPLE 5: IMPACT OF SYNPERONIC F108 ON PERMEABILIZATION OF TARGET CELL MEMBRANES

[0082] In the previous experiments the non-ionic poloxamer synperonic F108 demonstrated better efficiency in assisting lentiviral transduction than polybrene known as leading adjuvant in the field. Poloxamers are described to directly interact with cell membranes, so we asked whether the permeabilization status of the target cells is changed by synperonic F108. As a method for monitoring the permeabilization ability of lymphoma cell membranes, we administered propidium iodide (PI) for incorporation in the cells after incubation with polybrene, synperonic F108 or both substances. In general, PI only accumulates in highly permeable or otherwise leaky cells (FIG. 4c). All lymphoma cell lines demonstrated markedly higher permeability for PI in the presence of synperonic F108 when compared to polybrene treatment with the exception of SUDHL-1 cells (FIG. 4d and FIG. 7b). To monitor the background of apoptotic cells (<2n) we measured in parallel fragmented DNA (FIG. 4c,d). These findings indicate a favorable non-toxic membrane effect for synperonic F108 allowing successful transduction of lentiviral particles through cellular membranes.

EXAMPLE 6: SYNPERONIC F-108 ENHANCES TRANSDUCTION IN PRIMARY LYMPHOCYTES

[0083] In cell line experiments, synperonic F108 demonstrated high effectiveness in lentiviral infections especially in combination with the polycation Polybrene. As primary cells differ substantially in their susceptibility towards viral gene transfer, we evaluated vitality and lentiviral transduction rates of primary lymphocytes isolated from the blood of two healthy donors. Both samples showed lower, donor-dependent transduction rates even with high MOI levels (from 2.5 to 25) in comparison to cell lines treated within the same spinoculation protocol (FIG. 5). Synperonic F108 alone and in polybrene-combination succeeded to increase transduction of primary lymphocytes by factor 2 (from 22.2% to 41.7% for donor #1, FIG. 5a) or even by factor 4 (from 5.2% to 19.2% for donor #2, FIG. 5b). Neither synperonic F108 alone nor combined treatment induced remarkable cell death in those target cells. The results obtained in lymphoma cell lines and primary lymphocytes underline the promising role of synperonic F108 as transduction adjuvant for lentiviral particles.

EXAMPLE 7: ADJUVANT-ASSISTED LENTIVIRAL GENE TRANSFER INTO HEK293T CELLS

[0084] HEK293T cells were covered (for 24 hours) with a mixture of LV as used in example 3 with specified adjuvant up to a maximum applied concentration of 5000 g/ml Synperonic F108. Lentivirus particles (GP) as used in example 3, carrying a CopGFP-coding transgene were applied at MOI 0.25 and 0.025. After medium change, cells were cultivated for another 48 hours and measured for green fluorescent light emission by flow cytometry. Synperonic F108 was well tolerated at all concentrations tested without noticeable toxicity (1000 g/ml, 2500 g/ml, and 5000 g/ml). Synperonic F108 demonstrated better infection than polybrene-assisted LV infection (FIG. 3c). Synperonic F108 alone or in combination with Polybrene (10 g/ml) increased the mean amount of infected cells (two independent experiments) when compared to polybrene-induced transduction. Notably, at an MOI of 0.25 the amount of infected cells increasing from 49.5% (polybrene 10 g/ml) alone to 66.2% when Synperonic F108 was used at a concentration of (1000 g/ml), to 68.2% at a concentration of 2500 g/ml, and to 64.3% at a concentration of 5000 g/ml. When Synperonic F108 was used in combination with polybrene (10 g/ml) the mean infection increased further to 69.7% (1000 g/ml Synperonic F108), 71.2% (2500 g/ml Synperonic F108), and 64.8% (5000 g/ml Synperonic F108). Without adjuvant a mean transduction of 23% of cells was observed when using a MOI of 0.25. Statistical analysis with the paired two-sided student's t-test (Table 2, below) from 3 independent biologic replicates demonstrated that polybrene (10 g/ml) and Synperonic F108 (1000 g/ml), both mediated a highly significant (p<0.0) increase in infection of HEK293 cells (MOI 0.25), and Synperonic F108 (1000 g/ml) significantly (p<0.05) increased the infection of HEK293 cells compared to polybrene (10 g/ml).

TABLE-US-00002 TABLE 2 Statistical analysis of the effect of adjuvants on infection of HEK293 cells Polybrene Synperonic F108 Synperonic versus no versus no F108 versus adjuvant adjuvant polybrene MOI 0.25 0.007 0.0007 0.0227 Students t-test p value

EXAMPLE 8: ADJUVANT-ASSISTED LENTIVIRAL GENE TRANSFER IN LYMPHOMA CELLS

[0085] With a modified protocol including a spinoculation step during LV infection, synperonic F108 was tested up to a maximum applied concentration (5000 g/ml) for facilitating lentiviral infection of KARPAS-299 and SR-786 lymphoma cells (FIG. 4e). Lentivirus particles (GP) as used in example 3 carrying a CopGFP-coding transgene were applied at MOI 2.5 and 0.25. Synperonic F108 was well tolerated at all concentrations tested without noticeable toxicity (1000 g/ml, 2500 g/ml, and 5000 g/ml) and demonstrated better infection used alone or in combination with polybrene, compared to polybrene-assisted LV infection alone. Notably, at the MOI 2.5, 59.5% of Karpas-299 cells were infected in presence of polybrene (10 g/ml) alone, whereas 83.3% were infected in the presence of Synperonic F108 at a concentration of 1000 g/ml, 81.7% at a concentration of 2500 g/ml, and 77.5% at a concentration of 5000 g/ml. When KARPAS-299 cells were infected with the same MOI (2.5) using a combination of polybrene (10 g/ml) with Synperonic F108, the amount of infected cells increased further to 86% (1000 g/ml Synperonic F108, 10 g/ml polybrene), 87.2% at a concentration of (2500 g/ml Synperonic F108, 10 g/ml polybrene), and 78.9% (5000 g/ml Synperonic F108, 10 g/ml polybrene), (FIG. 4e).

[0086] At the same MOI 2.5, 62.2% SR-786 cells were infected in presence of polybrene (10 g/ml) alone, whereas 82.3% were infected in the presence of Synperonic F108 at a concentration of 1000 g/ml, 83.6% at a concentration of 2500 g/ml, and 79.9% at a concentration of 5000 g/ml, respectively. At the same MOI 2.5, 88% were infected when a combination of polybrene (10 g/ml) with Synperonic F108 (1000 g/ml) was used, 87.2% at a concentration of Synperonic F108 (2500 g/ml), and 81% at a concentration of Synperonic F108 (5000 g/ml), (FIG. 4e). Without adjuvant a mean of 32.2% SR-786, and 37.2% KARPAS-299 cells, respectively, were infected at MOI 2.5.

EXAMPLE 9: IMPACT OF SPINOCULATION ON LENTIVIRAL GENE TRANSFER IN LYMPHOMA CELLS

[0087] The effect of the spinoculation step during LV infection was tested in combination with the use of adjuvants for facilitating lentiviral infection of KARPAS-299 and SR-786 lymphoma cells (FIG. 4f). Lentivirus particles (GP) as used in example 3 carrying a CopGFP-coding transgene were applied at two different MOIs (2.5 and 0.25) in the presence of adjuvants with and without application of a spinoculation (centrifugation at 800 g for 90 min at room temperature) protocol. At all MOIs tested the spinoculation protocol resulted in better infection of cells. Notably, with the spinoculation protocol at the MOI 2.5, 59.5% of Karpas-299 cells were infected in presence of polybrene (10 g/ml) alone, whereas 83.2% were infected in the presence of Synperonic F108 at a concentration of 1000 g/ml, and 86% when polybrene and Synperonic F108 were used both. At the MOI 2.5, 62.2% of SR-786 cells were transduced in the presence of polybrene (10 g/ml) alone, 82.3% with Synperonic F108 (1000 g/ml), and 88% when both adjuvants were used.

[0088] When infecting Karpas-299 cells with a MOI of 2.5 without spinoculation, 17.8% of cells were infected in presence of polybrene (10 g/ml), whereas 50% were infected in the presence of Synperonic F108 at a concentration of 1000 g/ml, and infection further increased when polybrene and Synperonic F108 were used both (FIG. 4f). When infecting Karpas-299 cells with a MOI of 0.25 without spinoculation, 5.7% of cells were infected in presence of polybrene (10 g/ml), whereas 10.5% were infected in the presence of Synperonic F108 at a concentration of 1000 g/ml, and infection further increased to 13.4% when polybrene and Synperonic F108 were used both (FIG. 4f). When SR-786 cells were infected at a MOI 2.5 without spinoculation, 36.3% of SR-786 cells were transduced in the presence of polybrene (10 g/ml) alone, 58.2% with Synperonic F108 (1000 g/ml), and 60.9% when both adjuvants were used together. When SR-786 cells were infected at a MOI 0.25 without spinoculation, 8.8% of SR-786 cells were transduced in the presence of polybrene (10 g/ml) alone, 14.1% with Synperonic F108 (1000 g/ml), and 17.6% when both adjuvants were used together (FIG. 4f).

[0089] Without adjuvant but with spinoculation, 32.6% of SR-786 cells were infected at an MOI of 2.5, whereas without adjuvant and without spinoculation only 13% of cells were infected. Without adjuvant but spinoculation 37.2% of Karpas-299 cells were infected using a MOI of 2.5, whereas only 3% of cells were infected without adjuvant and without spinoculation.

REFERENCES

[0090] (1) Burns, J. C. et al. Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc. Natl. Acad. Sci. USA 90, 8033-8037 (1993). [0091] (2) Funke, S. et al. Targeted cell entry of lentiviral vectors. Mol. Ther. 16, 1427-1436 (2008). [0092] (3) Bukrinsky, M. I. et al. A nuclear localization signal within HIV-1 matrix protein that governs infection of non-dividing cells. Nature 365, 666-669 (1993). [0093] (4) Dull, T. et al. A third-generation lentivirus vector with a conditional packaging system. J. Virol. 72, 8463-8471 (1998). [0094] (5) Gruber, A., Kan-Mitchell, J., Kuhen, K. L., Mukai, T. & Wong-Staal, F. Dendritic cells transduced by multiply deleted HIV-1 vectors exhibit normal phenotypes and functions and elicit an HIV-specific cytotoxic T-lymphocyte response in vitro. Blood 96, 1327-1333 (2000). [0095] (6) Rouas, R. et al. Lentiviral-mediated gene delivery in human monocyte-derived dendritic cells: optimized design and procedures for highly efficient transduction compatible with clinical constraints. Cancer Gene Ther. 9, 715-724 (2002). [0096] (7) Millington, M., Arndt, A., Boyd, M., Applegate, T. & Shen, S. Towards a clinically relevant lentiviral transduction protocol for primary human CD34 hematopoietic stem/progenitor cells. PLoS One 4, e6461 (2009). [0097] (8) Anastasov, N. et al. Efficient shRNA delivery into B and T lymphoma cells using lentiviral vector-mediated transfer. J. Hematop. 2, 9-19 (2009). [0098] (9) Anastasov, N. et al. C/EBPbeta expression in ALK-positive anaplastic large cell lymphomas is required for cell proliferation and is induced by the STATS signaling pathway. Haematologica 95, 760-767 (2010). [0099] (10) Wurm, M. et al. The influence of semen-derived enhancer of virus infection on the efficiency of retroviral gene transfer. J. Gene Med. 12, 137-146 (2010). [0100] (11) Burns, J. C., Friedmann, T., Driever, W., Burrascano, M. & Yee, J. K. Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc. Natl. Acad. Sci. USA 90, 8033-8037 (1993). [0101] (12) Hesse, J., Ebbesen, P. & Kristensen, G. Correlation between polyion effect on cell susceptibility to in vitro infection with murine C-type viruses and polyion effect on some membrane-related functions. Intervirology 9.173-183 (1984). [0102] (13) Castro, B. A., Weiss, C. D., Wiviott, L. D. & Levy, J. A. Optimal conditions for recovery of the human immunodeficiency virus from peripheral blood mononuclear cells. J. Clin. Microbiol. 26, 2371-2376 (1988). [0103] (14) Aubin, R. J., Weinfeld, M. & Paterson, M. C. Factors influencing efficiency and reproducibility of polybrene-assisted gene transfer. Somat. Cell Mol. Genet. 14, 155-167 (1988). [0104] (15) Lee, R. C., River, L. P., Pan, F. S., Ji, L. & Wollmann, R. L. Surfactant-induced sealing of electro-permeabilized skeletal muscle membranes in vivo. Proc. Natl. Acad. Sci. USA 89, 4524-4528 (1992). [0105] (16) Lu, G. W., Jun, H. W., Dzimianski, M. T., Qiu, H. C. & McCall, J. W. Pharmacokinetic studies of methotrexate in plasma and synovial fluid following i.v. bolus and topical routes of administration in dogs. Pharm. Res. 12, 1474-1477 (1995). [0106] (17) Hannig, J. et al. Surfactant sealing of membranes permeabilized by ionizing radiation. Radiat. Res. 154, 171-177 (2000). [0107] (18) Gebhart, C. L. et al. Design and formulation of polyplexes based on pluronic-polyethyleneimine conjugates for gene transfer. Bioconjug. Chem. 13, 937-944 (2002). [0108] (19) Kabanov, A., Zhu, J. & Alakhov, V. Pluronic Block Copolymers for Gene Delivery. Adv. Genet. 53, 231-261 (2005). [0109] (20) Dishart, K. L. et al. Third-generation lentivirus vectors efficiently transduce and phenotypically modify vascular cells: implications for gene therapy. J. Mol. Cell Cardiol. 35, 739-748 (2003). [0110] (21) Strappe, P. M., Hampton, D. W., Cachon-Gonzalez, B., Fawcett, J. W. & Lever, A. Delivery of a lentiviral vector in a Pluronic F127 gel to cells of the central nervous system. Eur. J. Pharm. Biopharm. 61, 126-133 (2005). [0111] (22) Jones, S. et al. Lentiviral Vector Design for Optimal T Cell Receptor Gene Expression in the Transduction of Peripheral Blood Lymphocytes and Tumor-Infiltrating Lymphocytes. Hum. Gene Ther. 20, 630-640 (2009). [0112] (23) Hudecek, M., Anderson, L. D., Nishida, T. & Riddell, S. R. Adoptive T-cell therapy for B-cell malignancies. Expert. Rev. Hematol. 2, 517-532 (2009). [0113] (24) Rosenberg, S. A., Restifo, N. P., Yang, J. C., Morgan, R. A. & Dudley, M. E. Adoptive cell transfer: a clinical path to effective cancer immunotherapy. Nat. Rev. Cancer 8, 299-308 (2008). [0114] (25) Batrakova, E. V. et al. Mechanism of pluronic effect on P-glycoprotein efflux system in blood-brain barrier: contributions of energy depletion and membrane fluidization. J. Pharmacol. Exp. Ther. 299, 483-493 (2001). [0115] (26) Krylova, O. O. et al. Pluronic L61 accelerates flip-flop and transbilayer doxorubicin permeation. Chemistry 9, 3930-3936 (2003). [0116] (27) Pec, E. A., Wout, Z. G. & Johnston, T. P. Biological activity of urease formulated in poloxamer 407 after intraperitoneal injection in the rat. J. Pharm. Sci. 81, 626-630 (1992). [0117] (28) Carter, K. C., Gallagher, G., Baillie, A. J. & Alexander, J. The induction of protective immunity to Leishmania major in the BALB/c mouse by interleukin 4 treatment. Eur. J. Immunol. 19, 779-782 (1989). [0118] (29) Stefaneanu, L. & Kovacs, K. Effects of drugs on pituitary fine structure in laboratory animals. J. Electron. Microsc. Tech. 19, 80-89 (1991). [0119] (30) Millington M, Arndt A, Boyd M, Applegate T, Shen S, 2009 Towards a Clinically Relevant Lentiviral Transduction Protocol for Primary Human CD34+ Hematopoietic Stem/Progenitor Cells. PLoS ONE 4(7): e6461. doi:10.1371/journal.pone.0006461