Enhanced expression and HIV- 1 inhition of chimeric tRNA^Lys3-Ribozymes under dual U6 snRNA and tRNA promoters

We previously demonstrated that chimeric tRNA^Lys3-ribozymes targeting the primer binding site of HIV produced virions with reduced infectivity. To further enhance the anti-HIV efficiency of these ribozymes by increasing their level of transcription, we designed several tRNA^Lys3 promoter variants and compared their expression levels from the internal tRNA^Lys3 promoters and also from an exogenous human U6 snRNA promoter. The dual U6/tRNA promoter constructs gave rise to much higher levels of expression than constructs that used only an internal tRNA promoter. The most abundant expression is produced when a U6 promoter drives a chimeric tRNA^Lys3-ribozyme containing a mutation in the tRNA B box. As detected by fluorescent in situ hybridization, transcripts from a construct with the tRNA promoter alone localized strictly to the cytoplasm, whereas transcripts from dual U6/tRNA promoter were present in both the cytoplasm and the nucleus. Inhibition of HIV-1 correlates well with expression levels of the chimeric constructs. The results presented demonstrate that U6 and tRNA promoters can be placed in tandem for high-level expression of small RNA therapeutic transcripts.