Lentivirus-mediated gene transfer in primary T cells is enhanced by a central DNA flap

Retroviral vectors have become the primary tool for gene delivery into hema topoietic cells, including T lymphocytes. Lentiviral vectors offer an advan tage over moloney murine leukemia virus (MuLV) vectors because of their abi lity to translocate across an intact nuclear membrane and integrate into th e genome of nonproliferating cells. We have recently demonstrated that a ce ntral strand displacement event, controlled by the central polypurine tract (cPPT) and the central termination sequence (CTS), results in the formatio n of a central DNA flap which acts as a cis-determinant of HIV-1 genome nuc lear import Here, we show that insertion of this DNA determinant in a class ical lentiviral vector resulted in a significantly higher level of transduc tion in activated T cells (51 +/- 12.7% versus 15 +/- 1.4%). CD4(+) and CD8 (+) T cells were transduced at equivalent levels. Importantly, freshly isol ated T cells stimulated only during the 12-h transduction period could be e fficiently transduced with this new flap-containing lentiviral vector, brit not with the parental lentiviral vector nor an MuLV vector. Transgene expr ession in the flap-containing lenfiviral vector under the control of either an internal cytomegalovirus or the elongation factor-1 alpha (EF1 alpha) p romoter was significant and expression remained elevated in resting T cells . Thus, this system allows stable expression of transgenes in T lymphocytes following a short ex vivo transduction protocol.