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

Abstract

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.

The p53 isoform, Δ133p53α, stimulates angiogenesis and tumour progression

“…27 The EF1a promoter was replaced by the tetracyclin-inducible pTRE promoter coming from the vector pTRE-tight (Clontech, Issy-Les-Moulineaux, France) and a multiple cloning site was inserted in place of the green fluorescent protein (GFP) gene (Supplementary Figure S4). The resulting lentivector plasmids were pTRIP-TRE-D133p53a, -D133P53b, and -D133P53g.…”

Macrophages and HIV-1: dangerous liaisons

“…In HIV-1, the central DNA flap is a 99-nucleotide overlap. This structure enhances the nuclear import of the PIC, increasing the transduction efficiency of lentiviral vectors (Sirven et al, 2000;Zennou et al, 2000). The increased efficiency of cPPT-containing vectors has been attributed at least in part to enhanced nuclear import (Van Maele et al, 2003;Zennou et al, 2000).…”

Lentiviral transduction of human T-lymphocytes with a RANTES intrakine inhibits human immunodeficiency virus type 1 infection

“…These results are in accordance with two previously published papers, demonstrating an enhanced eGFP expression in human T cells transduced with EF1␣-containing lentivectors as compared with corresponding vectors containing an internal CMV-promoter. 18,21 Thus, LOX RANTES-SK was used for further analysis.…”