Efficient transduction of myeloid cells by an HIV-1-derived lentiviral vector that packages the Vpx accessory protein

Abstract

Lentiviral vectors are widely used for the stable expression of genes and small hairpin RNA (shRNA)-mediated knockdown and are currently under development for clinical use in gene therapy. Pseudotyping of the vectors with VSV-G allows them to infect a wide range of cell types. However, myeloid cells, such as dendritic cells and macrophages, are relatively refractory to lentiviral vector transduction as a result of the myeloid-specific restriction factor, SAMHD1. SIVmac/HIV-2 and related viruses relieve the SAMHD1-mediated restriction by encoding Vpx, a virion-packaged accessory protein that induces the degradation of SAMHD1 upon infection. HIV-1 does not encode Vpx and cannot package the protein. We report the development of an HIV-1-based lentiviral vector in which the Vpx packaging motif has been placed in the p6 region of the Gag/Pol expression vector that is used to generate the lentiviral vector virions. The virions package Vpx in high copy number and infect myeloid cells with a two-log increase in titer. Transduction of dendritic cells with an shRNA against transportin-3 resulted in >90% knockdown of the encoding mRNA. The system can be applied to any HIV-based lentiviral vector and is useful for laboratory and clinical applications where the efficient transduction of myeloid cells is required.

Fig. 1. Modified HIV-1 Gag/Pol expression vector generates virions that package SIV Vpx

(A) To generate Vpx-cotaining lentiviral vector stock, 293T cells are transfected with a lentiviral vector plasmid, pMDL-X chimeric p6 HIV-1 Gag/pol expression vector and expression vectors for Vpx, VSV-G and HIV-1 Rev. The virus is normalized by infectious titer on 293T cells and used to infect DC differentiated with GM-CSF and IL-4. Approximate location of the inserted Vpx packaging motif is indicated in the Gag/Pol vector as cross-hatched (B) 293T cells were cotransfected with pMDL or pMDL-X containing wild-type (WT) or chimeric p6_SIV(17–26), respectively and pcVpx.myc (+) or pcDNA (−). Two days later, cell lysates and virions were analyzed by immunoblotting. The immunoblot was probed with an antibody to myc-tagged Vpx, HIV-1 p24 CA, or tubulin. Transfection with pcVpx-myc.His alone was included in order to rule out nonspecific release of Vpx. (C) Cotransfection of 293T cells with pMDL or pMDL-X and decreasing amounts of pcVpx.myc. The amount of packaging plasmid harboring p6 remained constant, whereas the amount of pcVpx.myc decreased.

Fig. 2. Effect of Vpx packaging on infection of DC by pLenti-CMV-GFP

(A) DC were infected at an MOI of 2 with the GFP reporter virus produced with pMDL or pMDL-X and decreasing amounts of pcVpx.myc. Whereas the amount of the packaging plasmid containing p6 remained constant, the amount of pcVpx.myc was decreased stepwise 3-fold (1/3, 1/9, 1/27). Three days post-infection, cells were collected and analyzed for GFP expression by FACS. (B) The graph represents the average result of two independent infections of DC derived from PBMCs of two different donors. (C) DC were infected at MOIs of 0.5, 1, 2 or 3 with a GFP reporter virus produced with pMDL-X and either pcVpx.myc or pcDNA. Three days post-infection, cells were collected and analyzed for GFP expression by FACS. The data shown are representative of four independent experiments with different donor DC. The panel on the right depicts 293T cells infected an MOI of 3 to assess the general infectivity of the viruses.

Fig. 3. Lentiviral vector that packages Vpx bypasses the need for VLP pre-treatment

DC were infected at an MOI of 2 with the GFP reporter virus produced with pMDL-X and pcVpx.myc or pcDNA at a plasmid mass ratio of pMDL:pcVpx.myc 1:0.33 (1/3) and 1:01 (1/9), respectively. Where indicated, DC were treated with VLP for 2 h prior to infection with the virus at 5X or 10X p27 to p24, respectively. Three days post-infection, cells were collected and analyzed for GFP expression by FACS. The experiment was done in triplicate and the graphs depict the results of two experiments conducted with DC obtained from two different donors.

Fig. 4. Use of chimeric p6 lentiviral vector system for shRNA mediated knock-down of transportin 3 in DC

DC were infected at an MOI of 5 with pLKO.1-pGK-GFP, harboring an shRNA specific for TNPO3 or a nonspecific, scrambled shRNA control, as well as pMDL-X and pcVpx.myc. Three days post-infection, cells were collected and either analyzed for GFP expression by FACS (A) or for mRNA expression levels of TNPO3 (B). For the latter, RNA was prepared and the reverse transcripts were quantified by qRT-PCR using primers specific for TNPO3. The results were normalized to G6PDH and compared to DC infected with the control shRNA.