Methods for gene modification of hematopoietic cells
The present invention relates generally to methods for genetic modification of hematopoietic cells. In particular, the invention relates to use of Prostaglandin E2 (PGE2), poloxamer, and protamine sulfate to enhance transduction by a recombinant retroviral vector. The compositions and methods of the present disclosure are particularly suitable for gene therapy applications, including the treatment of monogenic genetic diseases and disorders.
1 . A method of ex vivo genetic modification of hematopoietic cells comprising pre-stimulating the hematopoietic cells with a recombinant fibronectin fragment, and contacting the hematopoietic cells with a recombinant retroviral vector comprising a sequence encoding a therapeutic protein operably linked to a promoter sequence, Prostaglandin E2 (PGE2) or a derivative thereof, and a poloxamer.
2 . The method of claim 1 , wherein the recombinant retroviral vector is a recombinant lentiviral vector.
3 . The method of claim 1 , wherein the hematopoietic cells are CD34-enriched cells.
4 . The method of claim 1 , wherein the poloxamer is selected from the group consisting of poloxamer 288, poloxamer 335, poloxamer 338, and poloxamer 407.
5 . The method of claim 4 , wherein the poloxamer is poloxamer 338.
6 . The method of claim 1 , wherein the PGE2 or derivative thereof is a modified PGE2.
7 . The method of claim 6 , wherein the PGE2 or derivative thereof is 16,16-dimethyl PGE2 (dmPGE2).
8 . The method of claim 1 , wherein the PGE2 or derivative thereof is an unmodified PGE2.
9 . The method of claim 1 , wherein the cells were transduced by contacting the cells with the Prostaglandin E2 (PGE2) or derivative thereof, the poloxamer, the recombinant fibronectin fragment, and protamine sulfate.
10 . The method of claim 9 , wherein the concentration of the protamine sulfate is 4-10 μg/mL.
11 . The method of claim 10 , wherein the concentration of the protamine sulfate is about 4 μg/mL.
12 . The method of claim 1 , wherein the cells were contacted with the transduction enhancers during the same or an overlapping time period.
13 . The method of claim 1 , wherein the concentration of the PGE2 or derivative thereof is 5-30 μg/mL.
14 . The method of claim 13 , wherein the concentration of the PGE2 or derivative thereof is about 10 μg/mL.
15 . The method of claim 1 , wherein the concentration of the poloxamer is 200-1200 μg/mL.
16 . The method of claim 15 , wherein the concentration of the poloxamer is about 1000 μg/mL.
17 . The method of claim 1 , wherein the concentration of the recombinant fibronectin fragment is 5-50 ug/mL.
18 . The method of claim 17 , wherein the concentration of the recombinant fibronectin fragment is 20 ug/mL.
19 . The method of claim 1 , wherein the hematopoietic cells were obtained from a subject before being contacted ex vivo with the retroviral vector.
20 . The method of claim 19 , wherein the polynucleotide encodes a therapeutic protein that is mutated or lacking in the subject due to a genetic disease or disorder.
21 . The method of claim 20 , wherein the therapeutic protein is selected from the group consisting of Red-cell type Pyruvate Kinase (RPK), Integrin beta 2 (ITGB2), Fanconi Anemia complementation group A protein (FANCA), Fanconi Anemia complementation group C protein (FANCC), Fanconi Anemia complementation group G protein (FANCG), and T cell immune regulator 1, ATPase H+ transporting V0 subunit a3 (TCIRG1), chloride voltage-gated channel 7 (CLCN7), tumor necrosis factor (ligand) superfamily, member 11 (TNFSF11), Pleckstrin homology and RUN domain containing M1 (PLEKHM1), tumor necrosis factor (ligand) superfamily, member 11a (TNFRSF11A) and osteopetrosis associated transmembrane protein 1 (OSTM1).
22 . The method of claim 21 , wherein the disease or disorder is selected from the group consisting of Fanconi Anemia, Leukocyte Adhesion Deficiency Type I, Pyruvate Kinase Deficiency, and Infantile Malignant Osteopetrosis.
23 . The method of claim 22 , wherein the disease or disorder is Fanconi Anemia, and the therapeutic protein is FANCA, FANCC, or FANCG.
24 . The method of claim 22 , wherein the disease or disorder is Leukocyte Adhesion Deficiency Type I, and the therapeutic protein is ITGB2.
25 . The method of claim 22 , wherein the disease or disorder is Pyruvate Kinase Deficiency, and the therapeutic protein is RPK.
26 . The method of claim 22 , wherein the disease or disorder is Infantile Malignant Osteopetrosis, and the therapeutic protein is TCIRG1.
27 . The method of claim 1 , wherein the hematopoietic cells are bone-marrow (BM)-derived cells, cord blood (CB)-derived cells, or mobilized peripheral blood (mPB) cells.