A SIMPLE ASSAY SYSTEM FOR EXAMINATION OF THE INHIBITORY POTENTIAL IN-VIVO OF DECOY RNAS, RIBOZYMES AND OTHER DRUGS BY MEASURING THE TAT-MEDIATED TRANSCRIPTION OF A FUSION GENE COMPOSED OF THE LONG TERMINAL REPEAT OF HIV-1 AND A GENE FOR LUCIFERASE

Nucleic acid-based drugs, including antisense RNA and DNA, ribozymes a nd decoys appear to have potential for the suppression of the expressi on of specific genes. To allow the examination of the potential of suc h agents in vivo as anti-HIV drugs in standard laboratories, where fac ilities for handling live virions are not available, we constructed a simple assay system (HIV-1 model) that allows measurement of the exten t of inhibition of Tat-mediated transcription of HIV-1 by nucleic acid -based drugs and other agents. In cells that harbor a stable chimeric long terminal repeat (LTR)-Luc construct (a fusion gene consisting of the LTR of HIV-1 and the gene for luciferase), total luciferase activi ty in an aliquot of cell lysate is dose- and promoter-dependent on tra nsfection with a Tat expression plasmid, reflecting the character of t he LTR promoter of HIV. When HeLa cells were co-transfected with the T at expression plasmid and another plasmid that encoded the U6 promoter or the promoter of the gene for tRNA(Val) linked to the trans-activat ing response (TAR) sequence, total luciferase activity was inhibited b y 60 or 40%, respectively. The inhibition was also dependent on the do se of the TAR expression plasmid. These results demonstrate the useful ness of this simple assay system for detection of the efficacy of a de coy RNA or a ribozyme in vivo, without a requirement for HIV-infected cells, by measurement of luciferase activity in vitro. (C) 1998 Elsevi er Science B.V.