Effective intracellular expression of small RNA therapeutics inclusion of specific U6 snRNA sequences from positions depends on a number of factors. The RNA, whether anti-+19 to +27. In situ localization of the transcripts shows that sense, ribozyme, or RNA aptamer, must be efficiently tranboth tRNA and U6 promoter transcripts give primarily puncscribed, stabilized against rapid degradation, folded cortate nuclear patterns, and that capping of transcripts is not rectly, and directed to the part of the cell where it can be required for nuclear retention. Several different insert most effective. To overcome a number of these problems RNAs directed against HIV-1 were tested by cotransfection we have been testing expression cassettes based on the with HIV-1 provirus and assay for subsequent viral reverse human tRNA met and U6 snRNA promoters, in which trantranscriptase production. These include antisense RNA, scripts encoding small RNA inserts are protected against hairpin and hammerhead ribozymes, and RNA ligands attack from the 3′ end. Transient expression in cultured (aptamers) for Tat and Rev RNA binding proteins. Results cells results in 10 3 -2 × 10 7 full-length transcripts per cell,show that Rev-binding RNAs efficiently block HIV-1 gene depending partially on the promoter construct used but expression, whereas other RNAs have little or no effect also on the nature of the insert RNA. 5′ ␥-Phosphate when expressed in these cassettes. methylation (capping) depended, as expected, on the
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