A murine leukemia virus (MuLV) long terminal repeat derived from rhesus macaques in the context of a lentivirus vector and MuLV gag sequence results in high-level gene expression in human T lymphocytes.
We constructed human immunodeficiency virus type 1 (HIV-1) vectors that will allow higher levels of gene expression in T cells. Gene expression under the control of an internal cytomegalovirus (CMV) immediate-early promoter in a self-inactivating lentiviral vector (CSCG) is 4- to 15-fold lower in T-cell lines (SUPT1 and CEMX174) than in non-lymphoid-cell lines (HeLa and 293T). This is in contrast to a Moloney murine leukemia virus (MoMLV)-based retrovirus vector (SRalphaLEGFP). We therefore replaced the internal CMV promoter of CSCG with three different murine oncoretroviral long terminal repeat (LTR) promoters-murine sarcoma virus (MSV), MoMLV (MLV), and the LTR (termed Rh-MLV) that is derived from the ampho-mink cell focus-forming (AMP/MCF) retrovirus in the serum of one rhesus macaque monkey that developed T-cell lymphoma following autologous transplantation of enriched bone marrow stem cells transduced with a retrovirus vector preparation containing replication-competent viruses (E. F. Vanin, M. Kaloss, C. Broscius, and A. W. Nienhuis, J. Virol. 68:4241-4250, 1994). We found that the combination of Rh-MLV LTR and a partial gag sequence of MoMLV (Deltagag(871-1612)) in CS-Rh-MLV-E gave the highest level of enhanced green fluorescent protein (EGFP) gene expression compared with MLV, MSV LTR, phosphoglycerate kinase, and CMV promoters in T-cell lines, as well as activated primary T cells. Interestingly, there was a further two- to threefold increase in EGFP expression (thus, 10-fold-higher expression than with CMV) when the Rh-MLV promoter and Deltagag(871-1612) were used in a self-inactivating-vector setting that has a further deletion in the U3 region of the HIV-1 LTR. These hybrid vectors should prove useful in gene therapy applications for T cells.
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Department of Microbiology, UCLA School of Medicine, Los Angeles, California 90095-1678, USA.
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D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D005434:Flow Cytometry; D015870:Gene Expression; D015683:Gene Products, gag / Q000378:metabolism; D017930:Genes, Reporter; D015750:Genes, gag; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D009052:Leukemia Virus, Murine / Q000235:genetics*; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008213:Lymphocyte Activation; D008253:Macaca mulatta; D008979:Moloney murine leukemia virus / Q000235:genetics; D011401:Promoter Regions, Genetic; D013601:T-Lymphocytes / Q000378:metabolism* / Q000821:virology; D020079:Terminal Repeat Sequences / Q000235:genetics*
D015683:Gene Products, gag; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins