STABLE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 (HIV-1) RESISTANCE IN TRANSFORMED CD4(-1 ANTISENSE SEQUENCES INCORPORATED INTO U1 SNRNA() MONOCYTIC CELLS TREATED WITH MULTITARGETING HIV)

We have approached the development of a human immunodeficiency virus t ype 1 (HTV-1) therapeutic product by producing immune cells stably res istant to HIV-1. Promonocytic CD4(+) cells (U937) were made resistant to HTV-1 by the introduction of a DNA construct (pNDU1A,B,C) that cont ained three independent antisense sequences directed against two funct ional regions, transactivation response and tat/rev, of the HIV-1 targ et, 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 transcrip ts accumulated in the nucleus, These cells were subjected to two succe ssive challenges with HIV-1 (BAL strain), The surviving cells showed n ormal growth characteristics and have retained their CD4(+) phenotype, In situ hybridization assays showed that essentially all of the survi ving cells produced U1/HIV antisense RNA. No detectable p24 antigen wa s 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 i nfection, As a further demonstration that the antisense RNA directed a gainst HTV-1 was functioning in these transfected immune cells, Tat-ac tivated expression of chloramphenicol acetyltransferase was shown to b e specifically inhibited in cells expressing Tat and transactivation r esponse region antisense sequences.