title: The human immunodeficiency virus type-1 central DNA flap is a crucial determinant for lentiviral vector nuclear import and gene transduction of human hematopoietic stem cells.
abstract: 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).
authors: Institut National de la Santé et de la Recherche Médicale (INSERM) U362, Institut Gustave Roussy, Villejuif, France.
authors: A
authors: A
authors: Sirven
authors: F
authors: F
authors: Pflumio
authors: V
authors: V
authors: Zennou
authors: M
authors: M
authors: Titeux
authors: W
authors: W
authors: Vainchenker
authors: L
authors: L
authors: Coulombel
authors: A
authors: A
authors: Dubart-Kupperschmitt
authors: P
authors: P
authors: Charneau
mesh_terms: D000328:Adult; D000818:Animals; D001692:Biological Transport; D001854:Bone Marrow Cells / Q000378:metabolism / Q000821:virology; D002455:Cell Division; D002467:Cell Nucleus / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism*; D004279:DNA, Viral / Q000737:chemistry / Q000378:metabolism*; D005312:Fetal Blood / Q000166:cytology; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D006085:Graft Survival; D015497:HIV-1 / Q000235:genetics*; D006410:Hematopoiesis; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000378:metabolism* / Q000821:virology; D006801:Humans; D007231:Infant, Newborn; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D016133:Polymerase Chain Reaction; D013045:Species Specificity; D013329:Structure-Activity Relationship; D014162:Transfection / Q000379:methods*; D014183:Transplantation, Heterologous
chemical_list: D018076:DNA, Complementary; D004279:DNA, Viral