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Lentiviral transduction of human hematopoietic cells by HIV-1- and SIV-based vectors containing a bicistronic cassette driven by various internal promoters.
J Gene Med. 2005 Sep; 7(9):1158-71.JG

Abstract

BACKGROUND

Lentiviral gene transfer into hematopoietic cells has been mostly optimized with vectors carrying a single reporter gene. For many clinical applications, lentiviral vectors should contain more than one gene because transduced cells should be enriched by a selectable marker or killed for safety reasons after use. Thus, we compared various vectors containing a bicistronic cassette driven by different ubiquitous promoters for their ability to transduce human T-lymphocytes, CD34+-cells, and dendritic cells (DCs) derived from CD34+-cells or monocytes.

METHODS

We designed HIV or SIV constructs containing a bicistronic cassette composed of two reporter genes (thy1/GFP) linked by an internal ribosome entry site sequence and driven by the cytomegalovirus (CMV) or elongation factor 1alpha (EF1alpha) promoters. The woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) was or not inserted within the constructs, the Vpx accessory protein was or not used for SIV vectors. Target cells were infected at the same multiplicity of infection, transduction efficiency was analyzed both by flow cytometry and vector integration.

RESULTS

For T-cells, HIV-based vectors/WPRE+ in which the thy1/GFP cassette was driven by the EF1alpha promoter were more efficient than SIV-based vectors. For CD34+-cells and CD34+-derived DCs, better thy1/GFP expression was achieved when the CMV promoter drove the cassette inserted into HIV-based vectors/WPRE+. Conversely, for monocyte-derived DCs, the cassette yielded better thy1/GFP expression when inserted into SIV-based vectors/WPRE+ and driven by the CMV or EF1alpha promoters, the use of Vpx significantly improving the expression levels.

CONCLUSIONS

Our results provide guidelines for improving the transduction of T-cells, CD34+-cells or DCs with lentiviral bicistronic vectors designed for clinical applications.

Authors+Show Affiliations

UPMC/CNRS UMR 7087, Bat CERVI, Hôpital Pitié-Salpêtrière, 83 bd de l'hôpital, 75651 Paris cedex 13, France.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

15880619

Citation

Dupuy, Franck P., et al. "Lentiviral Transduction of Human Hematopoietic Cells By HIV-1- and SIV-based Vectors Containing a Bicistronic Cassette Driven By Various Internal Promoters." The Journal of Gene Medicine, vol. 7, no. 9, 2005, pp. 1158-71.
Dupuy FP, Mouly E, Mesel-Lemoine M, et al. Lentiviral transduction of human hematopoietic cells by HIV-1- and SIV-based vectors containing a bicistronic cassette driven by various internal promoters. J Gene Med. 2005;7(9):1158-71.
Dupuy, F. P., Mouly, E., Mesel-Lemoine, M., Morel, C., Abriol, J., Cherai, M., Baillou, C., Nègre, D., Cosset, F. L., Klatzmann, D., & Lemoine, F. M. (2005). Lentiviral transduction of human hematopoietic cells by HIV-1- and SIV-based vectors containing a bicistronic cassette driven by various internal promoters. The Journal of Gene Medicine, 7(9), 1158-71.
Dupuy FP, et al. Lentiviral Transduction of Human Hematopoietic Cells By HIV-1- and SIV-based Vectors Containing a Bicistronic Cassette Driven By Various Internal Promoters. J Gene Med. 2005;7(9):1158-71. PubMed PMID: 15880619.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Lentiviral transduction of human hematopoietic cells by HIV-1- and SIV-based vectors containing a bicistronic cassette driven by various internal promoters. AU - Dupuy,Franck P, AU - Mouly,Enguerran, AU - Mesel-Lemoine,Mariana, AU - Morel,Céline, AU - Abriol,Julien, AU - Cherai,Mustapha, AU - Baillou,Claude, AU - Nègre,Didier, AU - Cosset,François-Loïc, AU - Klatzmann,David, AU - Lemoine,François M, PY - 2005/5/10/pubmed PY - 2005/11/8/medline PY - 2005/5/10/entrez SP - 1158 EP - 71 JF - The journal of gene medicine JO - J Gene Med VL - 7 IS - 9 N2 - BACKGROUND: Lentiviral gene transfer into hematopoietic cells has been mostly optimized with vectors carrying a single reporter gene. For many clinical applications, lentiviral vectors should contain more than one gene because transduced cells should be enriched by a selectable marker or killed for safety reasons after use. Thus, we compared various vectors containing a bicistronic cassette driven by different ubiquitous promoters for their ability to transduce human T-lymphocytes, CD34+-cells, and dendritic cells (DCs) derived from CD34+-cells or monocytes. METHODS: We designed HIV or SIV constructs containing a bicistronic cassette composed of two reporter genes (thy1/GFP) linked by an internal ribosome entry site sequence and driven by the cytomegalovirus (CMV) or elongation factor 1alpha (EF1alpha) promoters. The woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) was or not inserted within the constructs, the Vpx accessory protein was or not used for SIV vectors. Target cells were infected at the same multiplicity of infection, transduction efficiency was analyzed both by flow cytometry and vector integration. RESULTS: For T-cells, HIV-based vectors/WPRE+ in which the thy1/GFP cassette was driven by the EF1alpha promoter were more efficient than SIV-based vectors. For CD34+-cells and CD34+-derived DCs, better thy1/GFP expression was achieved when the CMV promoter drove the cassette inserted into HIV-based vectors/WPRE+. Conversely, for monocyte-derived DCs, the cassette yielded better thy1/GFP expression when inserted into SIV-based vectors/WPRE+ and driven by the CMV or EF1alpha promoters, the use of Vpx significantly improving the expression levels. CONCLUSIONS: Our results provide guidelines for improving the transduction of T-cells, CD34+-cells or DCs with lentiviral bicistronic vectors designed for clinical applications. SN - 1099-498X UR - https://www.unboundmedicine.com/medline/citation/15880619/Lentiviral_transduction_of_human_hematopoietic_cells_by_HIV_1__and_SIV_based_vectors_containing_a_bicistronic_cassette_driven_by_various_internal_promoters_ L2 - https://doi.org/10.1002/jgm.769 DB - PRIME DP - Unbound Medicine ER -