Efficient transduction of primary human B lymphocytes and nondividing myeloma B cells with HIV-1–derived lentiviral vectors

Abstract

IntroductionEfficient delivery of genes into primary human B lymphocytes would allow the investigation of gene functions in these cells for the purposes of research and the development of gene therapies. One could then test in mature B cells the promoters/genes potentially suitable for stem cell-based therapies for immunodeficiencies. 1 Vectors achieving the efficient transfection of primary B cells would most likely also be suitable for the delivery of genes into freshly collected B tumor cells-for example, for the development of immune-based anti-B-tumor therapies. 2 Various viral vectors are currently being studied for their ability to transduce hematopoietic cells. [3][4][5] Retroviral vectors derived from murine leukemia virus (MLV) 6,7 can transfer genes into immortal human B-cell lines, such as lymphoblastoid cells, 8 and primary B precursors, 9 but they are inefficient for mature human B cells. 10,11 These simple retroviruses can transduce genes only into actively dividing cells, 12 but a potent T-independent mitogen for human B cells in vitro, such as lipopolysaccharide (LPS) for murine B cells, has not been found. 13 In addition, MLV vectors might not be well adapted for human B cells because of the host species difference. HIV-1 and HIV-derived pseudotyped lentiviral vectors efficiently integrate into human cells, irrespective of cell division. 14-22 High transgene expression from such vectors in human T cells or total lymphocytes has been reported. [23][24][25] Generally, productive HIV infection or lentivectormediated transduction of truly quiescent lymphocytes has not been observed; activation, at least from G 0 to G 1 , seems to be required. 23,[25][26][27][28] Efficient transduction of primary acute lymphoblastic leukemia cells with a bicistronic HIV vector, leading to the expression of a cytokine (granulocyte macrophage-colonystimulating factor [GM-CSF]) and an immunostimulatory molecule (CD80), has also been achieved, 29 indicating a potential use of such vectors in novel anti-B-tumor therapies.In this study we investigated the transduction of peripheral blood B cells with multiply attenuated HIV vectors pseudotyped with vesicular stomatitis virus (VSV) G glycoprotein. 21 Efficient transduction of such B cells occurred after their activation in a culture system using murine EL-4 B5 thymoma cells as helper T cells in conjunction with human cytokines. [30][31][32][33] This system leads to proliferation and subsequent plasmocytic differentiation of all naive and memory human B subsets. 33 Nondividing, freshly isolated multiple myeloma cells were also efficiently transduced by HIV vector. By contrast, an MLV vector pseudotyped with VSV G protein was inefficient even in dividing B cells. F.B. and P.S. contributed equally to this work. D.T. has declared a financial interest as consultant to Cell Genesis, a company whose potential product is related to the HIV-1 vectors used in this study.Reprints: R. Zubler, Division of Hematology, University Hospital, 1211 Geneva-14, Switzerland; e-mail: rudolf.zubl...

Triadin and CLIMP-63 form a link between triads and microtubules in muscle cells

“…These viruses were engineered and produced either by the Gene Vector Production Network at Genethon III (Evry, France) or by the Atlantic Gene Therapies (Nantes, France). Lentiviral vectors carrying either the rat Trisk 95 cDNA, the human CLIMP-63 cDNA or the cDNAs of the mutants CLIMP-MT(+) or CLIMP-MT(−) were produced in the laboratory by a triple transfection of pWPXLd, psPAX2 and pCMV-VSV-G (Addgene, Cambridge, MA) into HEK293T cells, as described previously (Bovia et al, 2003;Zufferey et al, 1997). Titers of viruses in cell culture supernatants were determined as previously described (Nguyen et al, 2005).…”

Cisplatin activates Akt in small cell lung cancer cells and attenuates apoptosis by survivin upregulation

“…28 Recombinant viruses were produced by calcium transfection into 293T cells. Supernatants were collected after 24 and 48 hr, cleared by low-speed centrifugation (2,000 rpm for 10 min) and immediately added to cells.…”

Efficient and stable transduction of resting B lymphocytes and primary chronic lymphocyte leukemia cells using measles virus gp displaying lentiviral vectors

“…However, up to now, long-term gene transfer into primary human B cells has been notoriously difficult, [1][2][3] and lentiviral vector (LV) transduction of truly quiescent B cells has not yet been reported. It is now generally accepted that efficient lentiviral transduction of hematopoietic stem cells and T cells requires a minimal stimulation with cytokines or other factors leading to entry of the cells into the G 1 b phase of the cell cycle.…”

“…[4][5][6][7][8] Yet primary B cells remain very poorly transducible with VSVG-pseudotyped LVs even when stimulated into proliferation by crosslinking of CD40 in the presence of various cytokines. [1][2][3] Such vectors allow only efficient transduction of primary B cells on coculture with murine EL-4 B5 thymoma cells as helper T cells. However, this system led to strong proliferation and plasmocytic differentiation of all human B-cell subsets.…”

“…Of utmost importance, these H/F-LVs allowed also high-level gene transfer into B-cell chronic lymphocytic leukemia (B-CLL) cells, a target that up to now was not permissive to lentiviral transduction. 1 dilutions of vector preparations were added to 293T cells. The infectious titers are expressed as 293T transducing units per milliliter (TU/mL).…”