Intracellular expression of RNA transcripts complementary to the human immunodeficiency virus type 1 gag gene inhibits viral replication in human CD4+ lymphocytes

Abstract

Intracellular expression of antisense transcripts was evaluated for its potential to interfere with human immunodeficiency virus type 1 (HIV-1) replication. Retroviral vectors encoding HIV-1 ⌿-gag complementary sequences downstream of a selectable gene (neo, puromycin gene, or Lyt2 gene) were stable and yielded high titers. Human CEMSS T cells were transduced with amphotropic retroviral vectors to express RNA complementary to the ⌿-gag sequence of HIV-1. Replication of laboratory-adapted HIV-1 strains was inhibited by more than 1 order of magnitude (log 10 ) in these transduced cells even at high inoculation doses (4 ؋ 10 4 50% tissue culture infective doses). Antisense-mediated anti-HIV efficacy was further demonstrated by survival of CD4 ؉ cells in these cultures relative to controls. The level of anti-HIV-1 activity of the ⌿-gag antisense sequence correlated with the length of the antisense transcript. Maximal anti-HIV efficacy was observed with complementary sequence more than 1,000 nucleotides long, whereas transcripts less than 400 nucleotides long failed to inhibit HIV-1 replication. Expression of ⌿-gag antisense RNA also reduced HIV-1 JR-CSF replication 10-fold in primary CD4 ؉ lymphocytes. These results obtained with a T-cell line and primary peripheral blood lymphocytes indicate the potential of long antisense RNAs as an efficient anti-HIV-1 therapeutic agent for gene therapy.

Comparative Analyses of Intracellularly Expressed Antisense RNAs as Inhibitors of Human Immunodeficiency Virus Type 1 Replication

“…Peripheral blood mononuclear cells were isolated from healthy donor buffy coats by density gradient centrifugation in Isoprep (Robbins Scientific Corporation, Sunnyvale, Calif.). CD4 ϩ cells were enriched as described previously (36) by depletion with biotinylated anti-CD8 and anti-CD19 antibodies followed by streptavidin-conjugated magnetic beads (Dynabeads M-280; Dynal A.S., Oslo, Norway) and were cultured in Iscove's modified Dulbecco minimal essential medium. Stimulated, CD4-enriched PBLs (2 ϫ 10 6 cells) were transduced by centrifugation-enhanced retroviral gene transfer in the presence of Polybrene (8 g/ml) (36).…”

“…CD4 ϩ cells were enriched as described previously (36) by depletion with biotinylated anti-CD8 and anti-CD19 antibodies followed by streptavidin-conjugated magnetic beads (Dynabeads M-280; Dynal A.S., Oslo, Norway) and were cultured in Iscove's modified Dulbecco minimal essential medium. Stimulated, CD4-enriched PBLs (2 ϫ 10 6 cells) were transduced by centrifugation-enhanced retroviral gene transfer in the presence of Polybrene (8 g/ml) (36). Lyt2 expression was analyzed 48 h after transduction by flow cytometry with anti-CD8 phycoerythrin-conjugated monoclonal antibodies.…”

“…Lyt2 expression was analyzed 48 h after transduction by flow cytometry with anti-CD8 phycoerythrin-conjugated monoclonal antibodies. PBLs were further expanded and reactivated (36), and the CD4 ϩ Lyt2 ϩ cells were isolated by FACS (Vantage cell sorter; Becton Dickinson). After cell sorting, greater than 90% of the cell population was CD4 and Lyt2 positive.…”

“…HIV-1 infections. A total of 10 6 transduced CEM-SS cells were inoculated in a 1-ml volume with various doses (4 ϫ 10 2 to 4 ϫ 10 5 50% tissue culture infective doses [TCID 50 ]/ml) of HIV-1 (HXB3 or SF2) as described previously (36). The TCID 50 of the HIV-1 isolates was defined by endpoint titration on CEM-SS cells.…”

“…In a previous study (36) we demonstrated that retrovirally expressed RNA complementary to the gag gene sequence (6) of HIV-1 is a very potent inhibitor of viral replication, even at high inoculation doses. In an extension of that initial study, the antiviral activities of sequences complementary to the pol, vif, and env genes as well as the 3Ј long terminal repeat (LTR) were compared in HIV-1 infection experiments using a human CD4 ϩ T-cell line (CEM-SS) and primary CD4 ϩ T lymphocytes (PBLs).…”