Expression of small, therapeutic RNAs in human cell nuclei

Abstract

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

Tissue distribution of liposome-mediated epidermal growth factor receptor antisense gene therapy

“…Several studies have examined the expression of small, potentially therapeutic RNA using the U6 small nuclear RNA promoter. 25,26 This is the first report investigating the tissue distribution of a gene expressed under the control of the U6 promoter. There- fore, the widespread distribution and persistent detection of the EGFR transgene in tissues distant from the injection site may be due, in part, to this strong promoter.…”

Discriminatory aptamer reveals serum response element transcription regulated by cytohesin-2

“…An alternative strategy uses transfection of expression plasmids that are translocated into the nucleus so that the expression of the aptamers takes place in the nucleus. This method has been used to investigate effects of inhibitory aptamers on nuclear proteins (30); to address proteins that reside in the cytoplasm, it would be necessary to equip the expressed aptamer with an RNA sequence that serves as a nuclear export signal (31). Despite the fact that such signal sequences are scarce, it will have to be ensured that each aptamer remains active within the context of additional RNA sequences.…”

Inhibition of viral gene expression by human ribonuclease P

“…The DNA sequences coding for TK104, TK109, and TK112 were subcloned and placed under the control of the promoter for small nuclear U6 RNA+ This promoter is transcribed by RNA polymerase III and its transcripts are highly expressed and primarily present in the nucleus, where RNase P is localized (Das et al+, 1988;Yuan et al+, 1992;Liu & Altman, 1995;Bertrand et al+, 1997;Good et al+, 1997)+ To construct cell lines that express EGSs, human 143tk Ϫ cells were cotransfected with each of these three EGS DNA constructs and a plasmid containing a neomycin resistance gene+ These cells were then selected in culture medium that contained neomycin and cells that exhibited neomycin resistance were cloned+ The presence of EGS genes in these constructed cell lines was confirmed by detecting the EGS RNA transcript (Fig+ 3)+ An additional cell line was also constructed which expressed an EGS, CAT101, that targeted the CAT mRNA (Yuan et al+, 1992) 3. Detection of the expression of EGS TK104 from human cultured cells by RNase protection analyses+ RNase protection analyses were carried out using RNA isolated from the nuclei of parental human 143tk Ϫ cells (lane 1) and three cloned cell lines that expressed TK104 (lanes 2-4) and from the cytoplasm of one cloned cell line (lane 5)+ 2 mg of either nuclei or cytoplasmic RNA was used in each lane+ 59 end-[ 32 P]-labeled fragments obtained from digestion of pUC19 DNA with MspI were also separated on the same gel and the positions of the three fragments of 141, 110, and 67 nt were shown at the right as size markers+ The RNA probe contained a sequence complementary to TK104+ Products of the RNase protection assay were separated in 8% polyacrylamide gels that contained 8M urea and the gels were then subjected to autoradiography+ not cytoplasmic RNA fractions (Fig+ 3, lanes 4 and 5)+ Similar results were observed with cell clones which expressed TK109 and TK112 (data not shown)+ The different level of TK104 RNA expression in each individual clone (e+g+ clones 41, 42, and 43) (Fig+ 3, lanes 2-4) is presumably due to random incorporation of the EGS gene into the host chromosome, and its expression is influenced by the flanking sequence in the insertion site (Sambrook et al+, 1989)+ The nature of the doubled bands of TK104 RNA and of TK109 and TK112 (data not shown) is unknown+ Primer extension experiments with reverse transcriptase to map the 59 terminus of TK104 showed that the extension terminated prematurely before it reached the 59 end (data not shown)+ This observation suggested that some unknown modifications (e+g+ modified nucleotides) at this region of the EGS blocks the reverse transcription+ Alternatively, the nature of the doubled bands may possibly be attributed to the heterogeneity of the 39 termini of these transcripts or yet other unknown modification of EGS RNAs in the cultured cells+ To determine whether the EGSs expressed in cultured cells were still active to direct RNase P to cleave TK mRNA, total RNAs were isolated from the EGSexpressing cells and incubated with substrate tk46 and human RNase P in vitro+ Cleavage of tk46 was observed in RNAs from cells that expressed TK112 and TK104 (Fig+ 4, lanes 5 and 6) but not from those that did not express an EGS or expressed CAT101 or TK109 (Fig+ 4, lanes 2-4)+ Cleavage products generated in the presence of these cellular RNA fractions comigrated with those generated in the presence of TK104 synthesized in vitro (Fig+ 4, lane 1), su...…”