Therapeutic Effect Of RANTES-KDEL And SDF-KDEL On Inhibition Of HIV-1 In CD34~+ Human Hematopoietic Stem Cells (hHSC)

Acquired immunodeficiency syndrome (AIDS) and human immunodeficiency virus (HIV) infection continue to be major global health concerns, with 1,000,000 Chinese citizens infected by the end of 2002. Although highly active antiretroviral therapy (HAART) has led to profound and prolonged reductions in circulating virus levels in many individuals, high cost, side effects and increasingly the emergence of multidrug-resistant virus strains limit the use of HAART. Gene therapy for HIV infection has got rapid progress in recent years. Several strategies have been approved and are being conducted in clinical trial.Human immunodeficiency virus type 1 (HIV-1) entry into target cells is a multi-step process involving the interaction of viral envelope proteins with cell surface receptors, such as the CCR5 and CXCR4 chemokine receptors. CCR5 and CXCR4 are G protein-coupled receptors for CC- and CXC-chemokines, such as MIP-1α, RANTES and SDF-1, and are also the coreceptors of human immunodeficiency virus (HIV). In concert with CD4, CCR5 and CXCR4 mediate the binding of the viral envelope protein gp120 to the cell surface and allow HIV subsequent entry into target cells.The study by Chen SY et al mimicked the natural resistance of the individuals with the genetic CCR5 defect by inactivating CCR5 using a novel intracellular chemokine (intrakine) strategy. The CC-chemokine,RANTES,a ligand for CCR-5, has been targeted to the lumen of endocytoplasmic reticulum (ER) using a KDEL fusion termed RANTES-KDEL and this construct was found to effectively prevent the transport of newly synthesized CCR-5 to the cell surface. The lymphocytes expressing the CC-intrakine (RANTES-K) were resistant to M-tropic HIV-1 infection, while retaining normal cell functions. In an accompanied study, SDF-1a, a ligand for CXCR4, was also targeted to the ER to inactivate CXCR4, and the viable lymphocytes expressing CXC-intrakine (SDF-K) were found to resist T-tropic HIV-1 infection. These results indicate the therapeutic application of this genetic intrakine strategy to control HIV-1 infection.In the previous study by Bai XF and Zhang Y, a novel"intrakine"strategy was utilized to co-inactivate genetically both CCR5 and CXCR4 in HIV-1 target cells. The principle of co-inactivation of CCR5 and CXCR4 was illustrated by targeting the CC-intrakine and CXC-intrakine to the lumen of the endoplasmic reticulum for intracellular blockade of the transport of newly synthesized chemokine coreceptors to the cell surface. They constructed bicistronic eukaryotic vector and retroviral vector, which harbor the genes of HIV-1 coreceptors ligands RANTES and SDF-1. In order to examine co-expression of RANTES and SDF-1 by the bicistronic vector, several cell strains were transfected with various expression vector DNA. Finally, Env-mediated syncytium formation, envelope complementation assay and p24 detection were carried out to detect anti-HIV-1 activity of the co-inactivation of CCR5 and CXCR4. In summary, they utilized an intrakine strategy to co-inactivate both the principal M-tropic and T-tropic HIV-1 coreceptors in target cells. The lymphocytes with the phenotypic knockout of CCR5 and CXCR4 broadly resisted the infection of various HIV-1 viruses. Thus, intrakine provides a promising therapeutic strategy toward the goal of long-term control of HIV-1 infection.Recent years, some new vectors arosed with the prominent merits and of them, lentiviral vectors have emerged as potent and versatile tools of gene transfer for basic and applied research and are able to transduce nondividing cells and maintain sustained long-term expression of transgenes. For this reason, we constructed the HIV-based lentiviral vector expressing RANTES-KDEL and SDF-KDEL, pLenti6/V5-R-K and pLenti6/V5-S-K, and cotransfected them with the ViraPowerTM Packaging Mix (pLP1, pLP2, and pLP/VSVG) into 293FT cells to produce the replication-incompetent lentivirus stocks. Furthermore, we titrated the lentiviral stocks using HeLa cells, and detected the expression of the gene of interest, RANTES and SDF-1, by indirect immumofluorescence. The results are as following:1,The construction of lentiviral expression vectors, pLenti6/V5-R-K andpLenti6/V5-S-K, was confirmed by enzymatic digestion and sequencing. 2,We constructed lentivirus stocks expressing pLenti6/V5-R-K and pLenti6/V5-S-K with the ViraPower? Packaging Mix in a 293 FT cell line and titrated them in HeLa cell line. We measured the titre of the lentivirus stocks, which was 8.67×105 transduced units (TU)/ml and 8.56×105 transduced units (TU)/ml.3,We detected the RANTES and SDF-1 protein in HeLa cells and CD34+hHSC respectively by a Midi-MACS CD34 Isolation Kit(the purity was 96.8% as evaluated by flow cytometry) transfected with pLenti6/V5-R-K- and pLenti6/V5-S-K-expressing lentivirus using goat-anti-human RANTES and SDF-1 antibody with rabbit-anti-goat IgG-FITC secondary antibody. The majority of RANTES and SDF-1 were expressed in the cytoplasm, especially in the perinuclear region.4,The HIV-1 DP1 / 27 strain was amplified in MT4 cells and the virus titer was 10-4.2/ ml (TCID50)/ 10-4.1/ ml (TCID50). After 6d infection, syncytia were observed in the untransfected cells and in the CD34+hHSC transduced with empty plasmid. In contrast, CD34+hHSC transfected with lentivirus expressing pLenti6/V5-R-K and pLenti6/V5-S-K had no syncytia. We also detected p24 antigen levels of cell culture supernatants on the 4th, 7th, and 10th day, with 103 TCID50 HIV-1 DP-infected CD34+hHSC. The cells transfected with pLenti6/V5-R-K and pLenti6/V5-S-K had a significant reduction of HIV-1 DP transmission with a decrease of 51%,50% on the 4th day, 58%,57% on the 7th day and 60%,58% on the 10th day (P<0.05) compared to the untransfected control cells. The cells transfected with empty plasmid had no discernable decrease of p24 antigen (P>0.05) compared to the untransfected control cells.In summary, we utilized an intrakine strategy to inactivate the principal M-tropic and T-tropic HIV-1 coreceptors in target cells. The CD34+hHSC with the phenotypic knockout of CCR5 and CXCR4 broadly resisted the infection of various HIV-1 viruses. Thus, intrakine provides a promising therapeutic strategy toward the goal of long-term control of HIV-1 infection. These findings demonstrated the ability of lentiviral vectors to transduce multiple genes into human hematopoietic progenitor cells, and the potential therapeutic strategies for the treatment of human diseases.