Enhanced transgene expression in cord blood CD34(+)-derived hematopoietic cells, including developing T cells and NOD/SCID mouse repopulating cells, following transduction with modified TRIP lentiviral vectors

The recent development of lentivirus-derived vectors is an important breakt hrough in gene transfer technology because these vectors allow transduction of nondividing cells such as hematopoietic stem cells (HSC), due to an act ive nuclear import of reverse-transcribed vector DNA. We recently demonstra ted that addition of the central DNA flap of HIV-1 to an HIV-derived lentiv iral vector strikingly increases transduction of CD34(+) cells. We now desc ribe improvements of the transduction protocol designed to preserve HSC pro perties and two modifications of the previously described TRIP-CMV vector. First, deletion of the enhancer/promotor of the 3 ' LTR in the TRIP-CMV vec tor resulted in a safer vector (TRIP Delta U3-CMV) with conserved transduct ion efficiency and increased EGFP transgene expression. Second, the origina l internal CMV promoter was replaced with the promoter for the ubiquitously expressed elongation factor 1 alpha (EF1 alpha). This promoter substitutio n resulted in a significantly more homogeneous expression of the EGFP trans gene in all hematopoietic cell types, including CD34(+)-derived T lymphocyt es, in which the CMV promoter was inactive, and NOD/SCID mouse repopulating cells. We thus present here an HIV-derived lentiviral vector, TRIP Delta U 3-EF1 alpha, which can very efficiently transduce human cord blood HSC and results in high long-term transgene expression in CD34(+)-derived T, B, NK, and myeloid hematopoietic cells.