The recent development of lentivirus-derived vectors is an important breakthrough in gene transfer technology because these vectors allow transduction of nondividing cells such as hematopoietic stem cells (HSC), due to an active nuclear import of reverse-transcribed vector DNA. We recently demonstrated that addition of the central DNA flap of HIV-1 to an HIV-derived lentiviral vector strikingly increases transduction of CD34(+) cells. We now describe improvements of the transduction protocol designed to preserve HSC properties and two modifications of the previously described TRIP-CMV vector. First, deletion of the enhancer/promoter of the 3' LTR in the TRIP-CMV vector resulted in a safer vector (TRIPDeltaU3-CMV) with conserved transduction efficiency and increased EGFP transgene expression. Second, the original internal CMV promoter was replaced with the promoter for the ubiquitously expressed elongation factor 1alpha (EF1alpha). This promoter substitution resulted in a significantly more homogeneous expression of the EGFP transgene in all hematopoietic cell types, including CD34(+)-derived T lymphocytes, in which the CMV promoter was inactive, and NOD/SCID mouse repopulating cells. We thus present here an HIV-derived lentiviral vector, TRIPDeltaU3-EF1alpha, 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.

Gene transfer in human hematopoietic stem cells (HSCs) has great potential for both gene therapy and the understanding of hematopoiesis. As HSCs have extensive proliferative capacities, stable gene transfer should include genomic integration of the transgene. Lentiviral vectors are now preferred to oncoretroviral vectors especially because they integrate in nondividing cells such as HSCs, thereby avoiding the use of prolonged cytokine stimulation. Human immunodeficiency virus type-1 (HIV-1) has evolved a complex reverse transcription strategy including a central strand displacement event controlled in cis by the central polypurine tract (cPPT) and the central termination sequence (CTS). This creates, at the center of HIV-1 linear DNA molecules, a 99-nucleotide-long plus-strand overlap, the DNA flap, which acts as a cis-determinant of HIV-1 genome nuclear import. The reinsertion of the DNA flap sequence in an HIV-derived lentiviral vector promotes a striking increase of gene transduction efficiency in human CD34+ hematopoietic cells, and the complementation of the nuclear import defect present in the parental vector accounts for this result. In a short ex vivo protocol, the flap-containing vector allows efficient transduction of the whole hierarchy of human HSCs including both slow-dividing or nondividing HSCs that have multiple lymphoid and myeloid potentials and primitive cells with long-term engraftment ability in nonobese diabetic/severe combined immunodeficiency mice (NOD/SCID).

Gene transfer in human hematopoietic stem cells (HSCs) has great potential for both gene therapy and the understanding of hematopoiesis. As HSCs have extensive proliferative capacities, stable gene transfer should include genomic integration of the transgene. Lentiviral vectors are now preferred to oncoretroviral vectors especially because they integrate in nondividing cells such as HSCs, thereby avoiding the use of prolonged cytokine stimulation. Human immunodeficiency virus type-1 (HIV-1) has evolved a complex reverse transcription strategy including a central strand displacement event controlled in cis by the central polypurine tract (cPPT) and the central termination sequence (CTS). This creates, at the center of HIV-1 linear DNA molecules, a 99-nucleotide-long plus-strand overlap, the DNA flap, which acts as a cis-determinant of HIV-1 genome nuclear import. The reinsertion of the DNA flap sequence in an HIV-derived lentiviral vector promotes a striking increase of gene transduction efficiency in human CD34+ hematopoietic cells, and the complementation of the nuclear import defect present in the parental vector accounts for this result. In a short ex vivo protocol, the flap-containing vector allows efficient transduction of the whole hierarchy of human HSCs including both slow-dividing or nondividing HSCs that have multiple lymphoid and myeloid potentials and primitive cells with long-term engraftment ability in nonobese diabetic/severe combined immunodeficiency mice (NOD/SCID).