The human immunodeficiency virus type-1 central DNA flap is a crucial determinant for lentiviral vector nuclear import and gene transduction of humanhematopoietic stem 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 gen omic integration of the transgene, Lentiviral vectors are now preferred to oncoretroviral vectors especially because they integrate in nondividing cel ls 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 contr olled in cis by the central polypurine tract (cPPT) and the central termina tion sequence (CTS). This creates, at the center of HIV-1 linear DNA molecu les, 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 DN A flap sequence in an HIV-derived lentiviral vector promotes a striking inc rease of gene transduction efficiency in human CD34(+) hematopoietic cells, and the complementation of the nuclear import defect present in the parent al vector accounts for this result. In a short ex vivo protocol, the flap-c ontaining vector allows efficient transduction of the whole hierarchy of hu man HSCs including both stow-dividing or nondividing HSCs that have multipl e lymphoid and myeloid potentials and primitive cells with long-term engraf tment ability in nonobese diabetic/severe combined immunodeficiency mice (N OD/SCID). (C) 2000 by The American Society of Hematology.