Efficient gene transfer into primary lymphocytes

Details Efficient gene transfer into primary lymphocytes Efficient gene transfer into primary lymphocytes

C - Chemistry, Metallurgy

12

N

C12N 15/86 (2006.01) C12N 5/10 (2006.01) C12N 15/85 (2006.01) C12N 15/867 (2006.01) C12N 15/87 (2006.01)

Patent

CA 2168202

The present invention pertains to a method for efficiently introducing exogenous genes into primary lymphoid cells without drug selection which comprises the steps (a) deriving a retroviral vector and a helper cell combination that will yield a level of virus production in the range from 5 x 106 to 5 x 107 units/ml by transfecting a vector into a helper cell followed by selection, isolation of cell clones, and determination of viral titers to identify which virus-producing cell lines produce a virus titer in the range from 5 x 106 to 5 x 107 units/ml; (b) isolating a lymphoid cell subpopulation which can repopulate a specific lymphoid lineage or is a long-lived population by treating a suspension of lymphoid cells with a monoclonal antibody which removes undesired lymphoid cells to obtain an enriched lymphoid subpopulation; (c) culturing the enriched lymphoid subpopulation from step (b) with growth factors specific to the lymphoid subpopulation; (d) co-cultivating the lymphoid subpopulation from step (c) with a lawn of irradiated virus-producing cell line from step (a) to produce an infected lymphoid subpopulation; and (e) harvesting the infected lymphoid subpopulation. The invention further relates to a population of transfected lymphocytes, in whihch greater than about 90 % of the lymphocytes contain a provirus.

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