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.
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.
Liu
D
D
Enzo Biochem, Inc., Farmingdale, New York 11735, USA. dl@enzobio.com
Donegan
J
J
Nuovo
G
G
Mitra
D
D
Laurence
J
J
D000163:Acquired Immunodeficiency Syndrome / Q000628:therapy*; D001483:Base Sequence; D015704:CD4 Antigens / Q000032:analysis; D015496:CD4-Positive T-Lymphocytes / Q000821:virology*; D004279:DNA, Viral / Q000032:analysis; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D016372:RNA, Antisense / Q000502:physiology*; D012342:RNA, Small Nuclear / Q000502:physiology*; D014162:Transfection
D015704:CD4 Antigens; D004279:DNA, Viral; D016372:RNA, Antisense; D012342:RNA, Small Nuclear