High level inhibition of HIV replication with combination RNA decoys expressed from an HIV-Tat inducible vector

Intracellular immunization, an antiviral gene therapy approach based on the introduction of DNA into cells to stably express molecules for the inhibit ion of Viral gene expression and replication, has been suggested for inhibi tion of HIV infection. Since the Tat and Rev proteins play a critical role in HIV regulation, RNA decoys and ribozymes sequences have potential as the rapeutic molecular inhibitors. In the present study, we have generated seve ral anti-HIV molecules; a tat-ribozyme, RRE, RWZ6 and decoys and combinatio ns of decoys, and tested them for inhibition of HIV-1 replication in vitro. We used T cell specific CD2 gene elements and regulatory the HIV inducible promoter to direct high level expression and a 3' UTR sequence for mRNA st abilization. We show that HIV replication was most strongly inhibited with the combination TAR+RRE decoy when compared with the single decoys or the t at-ribozyme. We also show that the Tat-inducible HIV promoter directs a hig her level of steady-state transcription of decoys and inhibitors and that h igher levels of expression directly relate to increased levels of I inhibit ion of HIV infection. Furthermore, a stabilization of the 3' end of TAR+RRE inhibitor transcripts using a p-globin 3' UTR sequence leads to an additio nal 15-fold increase in steady-state RNA levels. This cassette when used to express the best combination decoy inhibitor TAR+RRE, yields high level HI V inhibition for greater than 3 weeks. Taken together, both optimization fo r high level expression of molecular inhibitors and use of combinations of inhibitors suggest better therapeutic application in limiting the spread of HIV.