Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5

Abstract

Double-stranded RNAs approximately 21 nucleotides long [small interfering RNA (siRNA)] are recognized as powerful reagents to reduce the expression of specific genes. To use them as reagents to protect cells against viral infection, effective methods for introducing siRNAs into primary cells are required. Here, we describe success in constructing a lentivirus-based vector to introduce siRNAs against the HIV-1 coreceptor, CCR5, into human peripheral blood T lymphocytes. With high-titer vector stocks, >40% of the peripheral blood T lymphocytes could be transduced, and the expression of a potent CCR5-siRNA resulted in up to 10-fold inhibition of CCR5 expression on the cell surface over a period of 2 weeks in the absence of selection. In contrast, the expression of another major HIV-1 coreceptor, CXCR4, was not affected. Importantly, blocking CCR5 expression by siRNAs provided a substantial protection for the lymphocyte populations from CCR5-tropic HIV-1 virus infection, dropping infected cells by 3- to 7-fold; only a minimal effect on infection by a CXCR4-tropic virus was observed. Thus, our studies demonstrate the feasibility and potential of lentiviral vector-mediated delivery of siRNAs as a general means of intracellular immunization for the treatment of HIV-1 and other viral diseases.

Figure 1

Construction of a lentivirus-based vector for delivering anti-human CCR5 siRNAs. (A) Schematic diagram of the siRNA-expressing lentiviral vector. The short hairpin form of siRNA is expressed under the control of a human U6-RNA Pol III promoter (Pol III). The vector also contains a human UbiC promoter driving the GFP marker gene for tracking transduced cells. 5′ LTR, HIV-1 5′LTR; Δ3′ LTR, HIV-1 self-inactivating 3′LTR; Flap, HIV-1 DNA flap element; WRE, woodchuck hepatitis B virus RNA regulatory element. (B) Selection of siRNA constructs that can effectively inhibit CCR5 expression in Magi-CCR5 cells. Magi-CCR5 cells were transduced with various lentiviral vectors. The cells were harvested 4 days after virus transduction and analyzed by FACS with anti-human CCR5 or isotype control antibody staining. The productively transduced (GFP+) and nontransduced (GFP−) cells were gated based on their GFP signals. The CCR5 staining is represented by the open curve and the isotype control by the shaded curve. Note that some lower GFP-expressing cells were included in the GFP− gate and, thus, there appeared to be a slight decrease of CCR5 staining in this population of the anti-CCR5 vector-transduced samples. CCR5-siRNA(186), the vector expressing a potent anti-CCR5 siRNA; CCR5-siRNA(809), the vector expressing a weaker anti-CCR5 siRNA; lacZ-siRNA, a nonspecific siRNA vector; FG12, empty vector; Mock, mock transduction control.

Figure 2

Reduction of CCR5 surface expression on human PBLs transduced by anti-CCR5 lentiviral vector. PHA-stimulated and CD8+-depleted PBLs were transduced with various lentiviral vectors as described in Fig. 1. The transduced cells were further cultured in IL-2-containing medium for 8 days before FACS analysis for CCR5 or CXCR4 expression on the cell surface. A representative experiment with Donor B is shown. (A) Cells were stained with allophycocyanin (APC)-labeled anti-human CCR5 mAb (2D7, PharMingen). The results are exhibited as CCR5 vs. GFP dotplots with cell populations in the live lymphocyte gate (typically >75%). The quadrant lines were defined by mock-transduction and isotype-control staining, and the percentage numbers are indicated. (B) Cells were stained with APC-labeled anti-human CXCR4 mAb (CXCR4, PharMingen), and the results are plotted in the same manner as in A.

Figure 3

Inhibition of CCR5-tropic HIV-1 infection in human PBLs transduced by lentiviral vector expressing anti-CCR5 siRNA. PBLs were transduced by various lentiviral vectors as described in Fig. 2. The transduced PBLs were cultured in IL-2-containing medium for 4 days before being challenged with either the CCR5-tropic reporter virus (A) or the CXCR4-tropic reporter virus (B). The cells were harvested 4 days after the virus challenge, and the virus-infected cells were quantitated by FACS analysis for the expression of the HSA marker. The FACS results are presented as HSA vs. GFP dotplots with cell populations in the live lymphocyte gate. The quadrant lines were defined by mock-infection and isotype-control staining, and the percentage numbers are indicated. One representative experiment with Donor B is shown.

Figure 4

Decrease of p24 production in HIV-1-infected cultures of anti-CCR5 lentiviral vector-transduced human PBLs. Culture supernatants were collected 6 days after the HIV-1 virus challenge, as described in Fig. 3, and p24 levels were measured by ELISA in triplicates. Again, the representative result from Donor B is shown.