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

Abstract

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.

FIG. 1

Comparison of the control of EGFP gene expression in CSCG and SRαLEGFP vectors. HeLa and SUPT1 cells were infected by unconcentrated virus supernatant of CSCG (virus titer, 0.5 × 10⁵ IU/ml; MOI, 0.125) and SRαLEGFP (virus titer, 0.64 × 10⁴ IU/ml; MOI, 0.016). The percentage of EGFP⁺ cells and the MFI of EGFP expression of the infected cells were analyzed by flow cytometry 3 days after infection. FSC, forward scatter; FL, fluorescence.

FIG. 2

Maps of various lentivirus-retrovirus hybrid vectors developed from a SIN HIV-1-based CSCG vector. The internal CMV immediate-early promoter was removed from CSCG and replaced with an oncoretrovirus LTR (MLV, Rh-MLV, or MSV) with or without a partial gag sequence of MoMLV.

FIG. 3

EGFP expression of CS-MLV-E in lymphoid- and non-lymphoid-cell lines. Lymphoid cells (SUPT1 and CEMX174) and non-lymphoid cells (HeLa and 293T) were infected by unconcentrated virus supernatant of CSCG (virus titer, 0.8 × 10⁵ IU/ml; MOI, 0.2), CS-MLV-E (virus titer, 0.96 × 10⁵ IU/ml, MOI, 0.24), and SRαLEGFP (virus titer, 2 × 10⁴ IU/ml; MOI, 0.05). The percentage of EGFP⁺ cells and the MFI of EGFP expression of the infected cells were analyzed by flow cytometry 3 days after infection. FSC, forward scatter; FL, fluorescence.

FIG. 4

EGFP expression of CS-MLV-E in SUPT1 cells is higher than that of CSCG at low MOIs. SUPT1 cells (0.1 × 10⁶) were infected by virus supernatant of CSCG (virus titer, 3 × 10⁵ IU/ml), CS-MLV-E (virus titer, 1.2 × 10⁶ IU/ml), and SRαLEGFP (virus titer, 0.7 × 10⁵ IU/ml) at the indicated dilutions (undiluted [1×, MOI = 0.8] and 1/10× and 1/20× dilutions for CSCG; 1× (MOI = 3.0), 1/20×, 1/50×, and 1/100× dilutions for CS-MLV-E; 1× (MOI = 0.18), 1/10×, and 1/20× dilutions for SRαLEGFP). The percentage of EGFP⁺ cells and the MFI of EGFP expression of the infected cells were analyzed by flow cytometry 3 days after infection. FSC, forward scatter; FL, fluorescence.

FIG. 5

Partial gag sequence of MoMLV in CS-MLV-E and CS-Rh-MLV-E vectors is involved in enhanced EGFP expression in CEMX174 and SUPT1. CEMX174 and SUPT1 cells were infected by virus supernatant of CSCG (virus titer, 0.3 × 10⁶ IU/ml; MOI, 0.075), CS-MLV-E (virus titer, 1.1 × 10⁶ IU/ml; MOI, 0.055), CS-MLVΔ-E (virus titer, 1.3 × 10⁶ IU/ml; MOI, 0.065), CS-Rh-MLV-E (virus titer, 1.9 × 10⁶ IU/ml; MOI, 0.095), and CS-Rh-MLVΔ-E (virus titer, 1.2 × 10⁶ IU/ml; MOI, 0.06). The percentage of EGFP⁺ cells and the MFI of EGFP expression of the infected cells were analyzed by flow cytometry 3 days after infection. FSC, forward scatter; FL, fluorescence.

FIG. 6

CS-Rh-MLV-E vector allows a high level of EGFP expression in primary activated T cells. (A) Flow cytometry analysis of CSCG-, CS-MLV-E-, and CS-Rh-MLV-E-infected T cells on day 3 or day 8 postinfection. (B) EGFP expressions in the infected CD4⁺ and CD8⁺ T-cell subsets were similar. Human PBMC were activated by plate-bound anti-CD3 and anti-CD28 MAbs for 60 h. Activated T-cell blasts were infected by virus supernatant of CSCG (virus titer, 0.3 × 10⁶ IU/ml; MOI, 0.75), CS-MLV-E (virus titer, 1.1 × 10⁶ IU/ml; MOI, 0.55), CS-MLVΔ-E (virus titer, 1.3 × 10⁶ IU/ml; MOI, 0.65), CS-Rh-MLV-E (virus titer, 1.9 × 10⁶ IU/ml; MOI, 0.48), and CS-Rh-MLVΔ-E (virus titer, 1.2 × 10⁶ IU/ml; MOI, 0.3). The percentage of EGFP⁺ cells and the MFI of EGFP expression of the infected cells were analyzed by flow cytometry on days 3 and 8 postinfection. On day 8, aliquots of the infected cells were also stained for surface expression of CD4 and CD8 molecules and analyzed by fluorescence-activated cell sorter. (C) Flow cytometry analysis of the CSCG-, CS-RhMLV-E-, and SRαLEGFP-infected SUPT1 and activated primary T cells. SUPT1 cells were infected by virus supernatant of CSCG (virus titer, 1 × 10⁶ IU/ml; MOI, 2.5), CS-RhMLV-E (virus titer, 1.4 × 10⁶ IU/ml; MOI, 0.07), and SRαLEGFP (virus titer, 7 × 10⁴ IU/ml; MOI, 0.18) at the following dilutions: undiluted (1×) for CSCG, 1/50× dilution for CS-Rh-MLV-E, and 1× for SRαLEGFP. Activated T-cell blasts were infected by undiluted virus supernatant of CSCG (virus titer, 1 × 10⁶ IU/ml; MOI, 2.5) and CS-RhMLV-E (virus titer, 1.4 × 10⁶ IU/ml; MOI, 3.5), except for SRαLEGFP, where 140×-concentrated virus supernatant (virus titer, 9.8 × 10⁶ IU/ml; MOI, 25) was used in the infection. The percentage of EGFP⁺ cells and the MFI of EGFP expression of the infected cells were analyzed by flow cytometry on day 3 postinfection. FSC, forward scatter; FL, fluorescence.

FIG. 6

CS-Rh-MLV-E vector allows a high level of EGFP expression in primary activated T cells. (A) Flow cytometry analysis of CSCG-, CS-MLV-E-, and CS-Rh-MLV-E-infected T cells on day 3 or day 8 postinfection. (B) EGFP expressions in the infected CD4⁺ and CD8⁺ T-cell subsets were similar. Human PBMC were activated by plate-bound anti-CD3 and anti-CD28 MAbs for 60 h. Activated T-cell blasts were infected by virus supernatant of CSCG (virus titer, 0.3 × 10⁶ IU/ml; MOI, 0.75), CS-MLV-E (virus titer, 1.1 × 10⁶ IU/ml; MOI, 0.55), CS-MLVΔ-E (virus titer, 1.3 × 10⁶ IU/ml; MOI, 0.65), CS-Rh-MLV-E (virus titer, 1.9 × 10⁶ IU/ml; MOI, 0.48), and CS-Rh-MLVΔ-E (virus titer, 1.2 × 10⁶ IU/ml; MOI, 0.3). The percentage of EGFP⁺ cells and the MFI of EGFP expression of the infected cells were analyzed by flow cytometry on days 3 and 8 postinfection. On day 8, aliquots of the infected cells were also stained for surface expression of CD4 and CD8 molecules and analyzed by fluorescence-activated cell sorter. (C) Flow cytometry analysis of the CSCG-, CS-RhMLV-E-, and SRαLEGFP-infected SUPT1 and activated primary T cells. SUPT1 cells were infected by virus supernatant of CSCG (virus titer, 1 × 10⁶ IU/ml; MOI, 2.5), CS-RhMLV-E (virus titer, 1.4 × 10⁶ IU/ml; MOI, 0.07), and SRαLEGFP (virus titer, 7 × 10⁴ IU/ml; MOI, 0.18) at the following dilutions: undiluted (1×) for CSCG, 1/50× dilution for CS-Rh-MLV-E, and 1× for SRαLEGFP. Activated T-cell blasts were infected by undiluted virus supernatant of CSCG (virus titer, 1 × 10⁶ IU/ml; MOI, 2.5) and CS-RhMLV-E (virus titer, 1.4 × 10⁶ IU/ml; MOI, 3.5), except for SRαLEGFP, where 140×-concentrated virus supernatant (virus titer, 9.8 × 10⁶ IU/ml; MOI, 25) was used in the infection. The percentage of EGFP⁺ cells and the MFI of EGFP expression of the infected cells were analyzed by flow cytometry on day 3 postinfection. FSC, forward scatter; FL, fluorescence.

FIG. 6

CS-Rh-MLV-E vector allows a high level of EGFP expression in primary activated T cells. (A) Flow cytometry analysis of CSCG-, CS-MLV-E-, and CS-Rh-MLV-E-infected T cells on day 3 or day 8 postinfection. (B) EGFP expressions in the infected CD4⁺ and CD8⁺ T-cell subsets were similar. Human PBMC were activated by plate-bound anti-CD3 and anti-CD28 MAbs for 60 h. Activated T-cell blasts were infected by virus supernatant of CSCG (virus titer, 0.3 × 10⁶ IU/ml; MOI, 0.75), CS-MLV-E (virus titer, 1.1 × 10⁶ IU/ml; MOI, 0.55), CS-MLVΔ-E (virus titer, 1.3 × 10⁶ IU/ml; MOI, 0.65), CS-Rh-MLV-E (virus titer, 1.9 × 10⁶ IU/ml; MOI, 0.48), and CS-Rh-MLVΔ-E (virus titer, 1.2 × 10⁶ IU/ml; MOI, 0.3). The percentage of EGFP⁺ cells and the MFI of EGFP expression of the infected cells were analyzed by flow cytometry on days 3 and 8 postinfection. On day 8, aliquots of the infected cells were also stained for surface expression of CD4 and CD8 molecules and analyzed by fluorescence-activated cell sorter. (C) Flow cytometry analysis of the CSCG-, CS-RhMLV-E-, and SRαLEGFP-infected SUPT1 and activated primary T cells. SUPT1 cells were infected by virus supernatant of CSCG (virus titer, 1 × 10⁶ IU/ml; MOI, 2.5), CS-RhMLV-E (virus titer, 1.4 × 10⁶ IU/ml; MOI, 0.07), and SRαLEGFP (virus titer, 7 × 10⁴ IU/ml; MOI, 0.18) at the following dilutions: undiluted (1×) for CSCG, 1/50× dilution for CS-Rh-MLV-E, and 1× for SRαLEGFP. Activated T-cell blasts were infected by undiluted virus supernatant of CSCG (virus titer, 1 × 10⁶ IU/ml; MOI, 2.5) and CS-RhMLV-E (virus titer, 1.4 × 10⁶ IU/ml; MOI, 3.5), except for SRαLEGFP, where 140×-concentrated virus supernatant (virus titer, 9.8 × 10⁶ IU/ml; MOI, 25) was used in the infection. The percentage of EGFP⁺ cells and the MFI of EGFP expression of the infected cells were analyzed by flow cytometry on day 3 postinfection. FSC, forward scatter; FL, fluorescence.

FIG. 7

Further enhancement of EGFP expression by the Rh-MLV LTR and the partial gag sequence in a SIN vector that has further deletions in the U3 region. SUPT1 cells were infected by virus supernatant of CSCG (virus titer, 0.5 × 10⁶ IU/ml; MOI, 0.025), CS-RhMLV-E (virus titer, 0.5 × 10⁶ IU/ml; MOI, 0.025), RRL-PGK-EGFP-SIN18 (virus titer, 0.3 × 10⁶ IU/ml; MOI, 0.075), and SIN18-Rh-MLV-E (virus titer, 0.6 × 10⁶ IU/ml; MOI, 0.03) at the following dilutions: 1/50× for CSCG, CS-RhMLV-E, and SIN18-Rh-MLV-E and 1/10× for RRL-PGK-EGFP-SIN18. For T-cell blast infection experiments, 1× undiluted virus supernatants of CSCG (MOI, 1.25), CS-Rh-MLV-E (MOI, 1.25), RRL-PGK-EGFP-SIN18 (MOI, 0.75), and SIN-18-Rh-MLV-E (MOI, 1.5) were used. The percentage of EGFP⁺ cells and the MFI of EGFP expression of the infected cells were analyzed by flow cytometry 3 days after infection. FSC, forward scatter; FL, fluorescence.