RETROVIRAL TRANSDUCTION USING POLOXAMERS
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.
1 . A method for transducing a target cell, the method comprising the step of contacting a target cell in vitro or ex vivo with a retroviral vector and a poloxamer, said poloxamer having an average molecular weight of 12.8 kDa to about 15 kDa, wherein “about” refers to −10% to +20% of said average molecular weight, and said poloxamer having the formula HO(C 2 H 4 O) x (C 3 H 6 O) z (C 2 H 4 O) y H, wherein the average value of z is at least 43, and the average value of x+y is at least 230, and wherein said poloxamer is in a fluid state during the contacting step.
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, and a stem cell.
3 . The method of claim 2 , wherein the 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 claim 1 , wherein the retroviral vector is a lentiviral vector.
5 . The method of claim 4 , wherein the lentiviral vector is pseudotyped with at least one vesicular stomatitis virus glycoprotein (VSV-G) and/or with an antibody fragment fused to VSV-G.
6 . The method of claim 1 , 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.
7 . The method of claim 6 , 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.
8 . The method of claim 7 , wherein the polycationic substances are 1,5-dimethyl-1,5-diaza-undeca-methyl-polymethobromide and/or protamine sulphate.
9 . The method of claim 1 , wherein said target cell is not brought into contact with one or more polycationic substances selected from the group of polycationic polymers or polycationic peptides.
10 . The method of claim 1 , wherein said poloxamer is provided at a concentration of about 50 to 5000 μg/ml.
11 . The method of claim 1 , wherein said poloxamer is provided at a concentration of about 500 to 1000 μg/ml.
12 . The method of claim 1 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 . The method of claim 1 , wherein z is in the range of 44 to 50 and x+y is in the range of 235 to 266.
14 . The method of claim 1 , wherein said retroviral vector and said poloxamer are added simultaneously or sequentially to said target cell.
15 . The method of claim 1 , wherein said target cells contacted with said retroviral vector and said poloxamer exhibit a higher transduction rate without noticeable toxicity compared with target cells contacted with said retroviral vector without said poloxamer under the same conditions.