High-Level Transduction and Gene Expression in Hematopoietic Repopulating Cells Using a Human Imunodeficiency Virus Type 1-Based Lentiviral Vector Containing an Internal Spleen Focus Forming Virus Promoter

Abstract

Prolonged exposure of human hematopoietic stem cells (HSC) to growth factors for efficient transduction by murine oncoretroviral vectors has major detrimental effects on repopulating activity. In this study, we have used a vesicular stomatitis virus G envelope protein (VSV-G)-pseudotyped human immunodeficiency virus type 1 (HIV-1) lentiviral-based vector system to transduce cord blood (CB) CD34+ cells over a limited time period (< or =24 hours). Under these conditions, significant gene marking was observed in engrafted human lymphoid, myeloid, and progenitor cells in all transplanted Severe Combined Immunodeficient (SCID) mice. To enhance the level of gene expression in hematopoietic cells, we also generated a series of lentiviral vectors incorporating the spleen focus forming virus (SFFV) long terminal repeat (LTR) sequences, and the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). By including the central polypurine tract (cPPT) sequence of HIV-1 we were then able to achieve high levels of transduction (over 80%) and gene expression in vivo after a single exposure to viral supernatant. These results demonstrate that lentiviral vectors are highly effective for gene transfer to human HSC, and that SFFV regulatory sequences can be successfully incorporated to enhance the long-term expression of a transgene in primary human hematopoietic cells in vivo.

Highly efficient EIAV-mediated in utero gene transfer and expression in the major muscle groups affected by Duchenne muscular dystrophy

“…19,20 Cells were grown in DMEM supplemented with 10% fetal calf serum and transfected with plasmid vectors to transiently produce infectious virus particles as previously described. 21 Virus titration was performed on D17 canine cells and human HT1080 cells by seeding cells onto 12-well culture dishes at 8 Â 10 4 cells/well followed by the addition of serially diluted virus.…”

Chemoprotection of human hematopoietic stem cells by simultaneous lentiviral overexpression of multidrug resistance 1 and O6-methylguanine-DNA methyltransferaseP140K

“…BCNU and doxorubicin are administered as treatment of multiple myeloma, 38 ACNU and paclitaxel for non-small cell lung cancer, 39 TMZ and paclitaxel for treatment of malignant melanoma 40 and malignant brain tumors like glioma and medulloblastoma. 41 A severe problem is the development of resistance against alkylating agents by high expression of wild-type 20 In contrast with other studies examining combination gene therapy with MDR1 and MGMT expressed by a gammaretroviral vector, [27][28][29][30] we chose a third generation lentiviral self-inactivating (SIN) vector 32 . Besides the advantageous lentiviral ability of transducing nondividing cells, 32 the risk of vector mobilization and recombination is minimized by the establishment of lentiviral SIN-vectors.…”

“…For efficient gene transfer into non-dividing cells like hematopoietic stem cells, the use of lentiviral vectors would be advantageous. 31,32 Therefore, we have chosen a lentiviral SIN vector of the third generation 32,33 for co-expression of both the transgenes MDR1 and MGMT P140K , linked by an EMCV-IRES element (internal ribosome entry site from encephalomyocarditis virus). HL60 (myeloid leukemia cell line) and human hematopoietic stem cells (CD34 + ) were transduced with this combination vector (HR'SIN-MDR1-IRES-MGMT P140K ) or as controls with singlegene vectors containing either MDR1 or MGMT P140K as transgene.…”

In vivo evaluation of a novel albumin-binding prodrug of doxorubicin in an orthotopic mouse model of prostate cancer (LNCaP)

“…The luciferase-neomycin fusion gene cassette was inserted into a lentiviral backbone vector, 17,18 using BsRGi/SnaBI replacing the original expression cassette. The 293T cells were transfected with this lentiviral backbone and plasmids pCMV VSV-G (encoding the vesicular stomatitis virus surface gene) and pCMV (GAG/REV) using Ca-phosphate precipitates.…”