Stable human immunodeficiency virus type 1 (HIV-1) resistance in transformed CD4+ monocytic cells treated with multitargeting HIV-1 antisense sequences incorporated into U1 snRNA

Abstract

We have approached the development of a human immunodeficiency virus type 1 (HIV-1) therapeutic product by producing immune cells stably resistant to HIV-1. Promonocytic CD4 ؉ cells (U937) were made resistant to HIV-1 by the introduction of a DNA construct (pNDU1A,B,C) that contained three independent antisense sequences directed against two functional regions, transactivation response and tat/rev, of the HIV-1 target. Each sequence was incorporated into the transcribed region of a U1 snRNA gene to generate U1/HIV antisense RNA. Stably transfected cells expressed all three U1/HIV antisense transcripts, and these transcripts accumulated in the nucleus. These cells were subjected to two successive challenges with HIV-1 (BAL strain). The surviving cells showed normal growth characteristics and have retained their CD4 ؉ phenotype. In situ hybridization assays showed that essentially all of the surviving cells produced U1/HIV antisense RNA. No detectable p24 antigen was observed, no syncytium formation was observed, and PCR-amplified HIV gag sequences were not detected. Rechallenge with HIV-1 (IIIB strain) similarly yielded no infection at a relatively high multiplicity of infection. As a further demonstration that the antisense RNA directed against HIV-1 was functioning in these transfected immune cells, Tat-activated expression of chloramphenicol acetyltransferase was shown to be specifically inhibited in cells expressing Tat and transactivation response region antisense sequences.

HSP70 binding protein 1 (HspBP1) suppresses HIV-1 replication by inhibiting NF-κB mediated activation of viral gene expression

“…Jurkat and CEM-GFP cells were infected with HIV-1 NL4-3 virus at various multiplicities of infection (MOI) in the presence of polybrene (1 μg/ml) as described previously ( 53 ). Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of normal seronegative donors obtained from local blood bank using Ficoll-Hypaque (Amersham Biosciences, USA) gradient centrifugation.…”

Progress toward curing HIV infection with hematopoietic cell transplantation

“…For gene therapy applications, CD34 + HSPCs are transduced with an integrative vector that encodes one or more anti-HIV genes. From the late 1990s, several clinical trials were conducted to test the safety, feasibility, and effectiveness of CD34 + HSPCs transduced with gammaretroviral vectors that encoded anti-HIV ribozymes, 27 – 29 antisense transcripts, 30 and RNA decoys. 31 Due to the tendency of gammaretroviral vectors to integrate near and activate oncogenes, 32 more recent approaches have utilized lentiviral vectors, 33 – 37 foamy virus vectors, 38 – 40 or transposons 41 for transgene expression.…”

Human immunodeficiency virus‐1 Nef protein interacts with Tat and enhances HIV‐1 gene expression

“…The culture supernatants collected at the same time were used to determine virus production by p24 gag antigen capture enzyme‐linked immunosorbent assay (ELISA; NEN, USA). CEM cells were infected with HIV‐1 NL4.3 virus using 100 ng of p24 units per 2×10 6 cells as described previously [29].…”