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).

In vitro generated Rhnull red cells recapitulate the in vivo deficiency: A model for rare blood group phenotypes and erythroid membrane disorders

“…All constructs were sequenced on both strands. Vector particles were produced by transient cotransfection of 293T cells (ATCC) with each of the lentiviral constructs described earlier, an encapsidation plasmid lacking all accessory HIV‐1 proteins, and a G protein of the vesicular somatitis virus (VSV‐G) envelope expression‐plasmid . After 72 hr, the culture medium (RPMI, 10% FCS) was purified and concentrated by ultra‐centrifugation (22,000 K, 4°C, 1 hr 30 min).…”

Reprogrammed quiescent B cells provide an effective cellular therapy against chronic experimental autoimmune encephalomyelitis

“…These concerns led us to consider resting B cells rather than activated B cells as a platform for immunosuppressive therapy. Using lentiviral vectors to deliver genes into such non‐dividing hematopoietic cells 30, 31, we report here for the first time that quiescent B cells can be reprogrammed into resting suppressive B cells for adoptive cell therapy against undesirable immune responses. This approach highlights several interesting features for possible clinical application.…”

A Hard Way to the Nucleus

“…During the plus-strand synthesis of the HIV (and other lentiviruses) cDNA, a 99 nucleotide long "central DNA flap" is produced because of additional initiation of the plus DNA synthesis at another polypurine tract in the middle of the HIV genome. This short trimeric structure was proposed as a critical determinant of the PIC passage through the nuclear pore (21) and was reported to dramatically increase the efficiency of lentivirus-mediated gene transfer (22,23). However, later reports demonstrated that the central flap, at least in some cells, is not essential for PIC nuclear import and its effect is dependent on the HIV-1 strain used (24,25).…”