Expression of small, therapeutic RNAs in human cell nuclei

Abstract

Effective intracellular expression of small RNA therapeutics depends on a number of factors. The RNA, whether anti-sense, ribozyme, or RNA aptamer, must be efficiently transcribed, stabilized against rapid degradation, folded correctly, and directed to the part of the cell where it can be most effective. To overcome a number of these problems e have ben testing expression cassettes based on the human tRNA(met) and U6 snRNA promoters, in which transcripts encoding small RNA inserts are protected against attack from the 3' end. Transient expression in cultured cells result in 103-2 x 107 full length transcripts per cell, depending partially on the promotor construct used but also on the nature of the insert RNA. 5' γ-Phosphate methylation (capping) depended, as expected, on the inclusion of specific U6 snRNA sequence from positions +19 to +27. In situ localization of the transcripts shows that both tRNA and U6 promoter transcripts give primarily punctate nuclear patterns, and that capping of transcripts is not required for nuclear retention. Several different insert RNAs directed against HIV-1 were tested by contransfection with HIV-1 provirus and assay for subsequent viral reverse transcriptase production. These include antisense RNA, hairpin and hammerhead ribozymes, and RNA ligands (aptamers) for Tat and Rev RNA binding proteins. Results show that Rev-binding RNAs efficiently block HIV-1 gene expression, whereas other RNAs have little or no effect when expressed in these cassettes.