Methods of preconditioning vascular cells for transduction, methods of transduction and methods of preserving transduced cells
Methods and compositions for pre-conditioning, transduction and/or hypothermic preservation of vascular cells transduced with nucleic acid constructs for expressing pro-angiogenic factors are provided. Also provided are uses of such cells in therapy.
1 . A method for transducing human smooth muscle (SM) cells comprising:
(a) preconditioning said human smooth muscle cells with between 0.125 mg/ml and 0.5 mg/ml DEAE-Dextran for 1-4 minutes, so as to obtain preconditioned human smooth muscle cells,
(b) washing said preconditioned SM cells so as to remove said DEAE-Dextran, and
(c) contacting the preconditioned human smooth muscle cells with a viral vector comprising a retroviral nucleic acid construct or a lentivirus nucleic acid construct in a DEAE-Dextran-free transduction medium comprising the viral vector comprising the retroviral or lentiviral nucleic acid construct, wherein the construct comprises a polynucleotide sequence encoding a VEGF 165 polypeptide,
wherein said transduction medium comprises M199 medium,
and wherein following step (c) said SM cells are characterized by at least one of increased transduction rate and increased proliferation following transduction, as compared to similar SM cells preconditioned with 1.0 mg/ml DEAE-Dextran or greater.
2 . The method of claim 1 , wherein said preconditioning is performed with 0.25 mg/ml DEAE-Dextran.
3 . The method of claim 1 , wherein said preconditioning is performed with 0.125 mg/ml DEAE-Dextran and for 1 minute.
4 . The method of claim 1 , wherein said transduction medium further comprises 2 mM Glutamine.
5 . The method of claim 1 , wherein said transduction medium is a serum-free medium.
6 . The method of claim 1 , wherein said human smooth muscle cells are selected from the group consisting of freshly isolated smooth muscle cells from venous tissue, cryopreserved and thawed smooth muscle cells from venous tissue and cultured human smooth muscle cells.
7 . The method of claim 1 , wherein said human smooth muscle cells are cultured in medium comprising Smooth muscle basal medium (SmBM), Fetal Bovine Serum, Insulin, Human Epidermal Growth Factor (hEGF), Human Fibroblast Growth Factor-B (hFGF-B) and Gentamicin plus Amphotericin B prior to and following said transduction.
8 . The method of claim 7 , wherein said culture medium further comprises 5-25% fetal bovine serum (FBS).
9 . The method of claim 7 , wherein said culture medium further comprises 10-20% fetal bovine serum (FBS).
10 . The method of claim 7 , wherein said culture medium further comprises 15% fetal bovine serum (FBS).
11 . The method of claim 1 , wherein said human smooth muscle cells are smooth muscle cells isolated from venous tissue in isolation medium comprising Smooth muscle basal medium (SmBM), Fetal Bovine Serum, Insulin, Human Epidermal Growth Factor (hEGF), Human Fibroblast Growth Factor-B (hFGF-B) and Gentamicin plus Amphotericin B.
12 . The method of claim 11 , wherein said isolation medium comprises 20% fetal bovine serum.
13 . A method for transducing human smooth muscle (SM) cells comprising:
(a) preconditioning said human smooth muscle cells with between 0.125 mg/ml and 0.5 mg/ml DEAE-Dextran for 1-4 minutes, so as to obtain preconditioned human smooth muscle cells,
(b) washing said preconditioned SM cells so as to remove said DEAE-Dextran, and
(c) contacting the preconditioned human smooth muscle cells with a viral vector comprising a retroviral nucleic acid construct or a lentivirus nucleic acid construct in a DEAE-Dextran-free transduction medium comprising the viral vector comprising the retroviral or lentiviral nucleic acid construct,
wherein said transduction medium comprises M199 medium, and
wherein the construct comprises a polynucleotide sequence encoding a VEGF 165 polypeptide, wherein said polynucleotide sequence is as set forth in SEQ ID NO: 1,
and wherein following step (c) said SM cells are characterized by at least one of increased transduction rate and increased proliferation following transduction, as compared to similar SM cells preconditioned with 1.0 mg/ml DEAE-Dextran or greater.