Improved accumulation and activity of ribozymes expressed from a tRNA-based RNA polymerase III promoter

Thompson, James D.; Ayers, David F.; Malmstrom, Terra A.; McKenzie, Timothy; Ganousis, Louis; Chowrira, Bharat M.; Couture, Larry A.; Stinchcomb, Dan T.

doi:10.1093/nar/23.12.2259

Cited by 79 publications

(55 citation statements)

References 34 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Recently it was shown that modifications of the 3′ end of ribozymes dramatically affected the intracellular stability and cleavage efficiency. 25 The RNAs transcribed by RNA polymerase II have long poly (A) tails which may affect the structure of the ribozyme and subsequently disturb the effective association with target RNA and the cleavage reaction. In addition, retroviral transcripts are often generated from long terminal repeat (LTR) to LTR, 27,28 providing very bulky flanking RNA sequences that may interfere with catalytic and/or binding activation.…”

Section: Discussionmentioning

confidence: 99%

A novel SV40-based vector successfully transduces and expresses an alpha 1-antitrypsin ribozyme in a human hepatoma-derived cell line

et al. 1999

View full text Add to dashboard Cite

Alpha 1-antitrypsin (␣1AT) deficiency disease is one of the ribozyme cleavage. Ribozymes were effective in inhibiting more common hereditary disorders that affects the liver ␣1AT expression in a human hepatoma cell line using a and lung. The liver disease of ␣1AT deficiency is generally newly developed simian virus (SV40) vector system. In thought to be caused by the accumulation of an abnormal addition, the hepatoma cell line was stably transduced with ␣1AT protein in hepatocytes, whereas the lung disease is a modified ␣1AT cDNA that was capable of producing wildthought to be due to a relative lack of the normal protein type ␣1AT protein, but was not cleaved by the ribozyme in the circulation. Therefore, one possible approach to prethat decreased endogenous ␣1AT expression. These vent and treat ␣1AT disease is to both inhibit the results suggest that ribozymes can be employed for the expression of the mutated ␣1AT gene, and to provide a specific inhibition for an abnormal ␣1AT gene product, the means of synthesizing the normal protein. To do this, we first step in designing a gene therapy for the disease. The designed specific hammerhead ribozymes that were capfindings also suggest that the novel SV40-derived vector able of cleaving the ␣1AT mRNA at specific sites, and conmay represent a fundamental improvement in the gene structed a modified ␣1AT cDNA not susceptible to therapeutic armarmentarium.

show abstract

Section: Discussionmentioning

confidence: 99%

A novel SV40-based vector successfully transduces and expresses an alpha 1-antitrypsin ribozyme in a human hepatoma-derived cell line

et al. 1999

View full text Add to dashboard Cite

show abstract

“…A experimental organisms and as potential expression casspecific and promising example of this has been provided settes for small RNAs in gene therapy. 10,11,[19][20][21] An by embedding a ribozyme in a pseudoviral transcript that additional advantage of the U6 gene is that individual might be copackaged with the bona fide retroviral trangenes are heavily expressed in human cells. Transcription scripts, thus providing directed access to the target of only a few copies of the U6 gene (out of 200 or more sequence.…”

mentioning

confidence: 99%

Expression of small, therapeutic RNAs in human cell nuclei

et al. 1997

View full text Add to dashboard Cite

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

show abstract

“…The transcriptional context of the ribozyme had a dramatic impact on its in vitro cleavage efficiency, as has been observed by others (Thompson et al, 1995). Included in the set is the 252 multimer linked 3Ј to the ORF of the bar selectable marker gene (RPA85).…”

Section: Discussionmentioning

confidence: 66%

Ribozymes Targeted to Stearoyl–ACP Δ9 Desaturase mRNA Produce Heritable Increases of Stearic Acid in Transgenic Maize Leaves

Merlo¹,

Cowen²,

Delate³

et al. 1998

Plant Cell

View full text Add to dashboard Cite

Ribozymes are RNAs that can be designed to catalyze the specific cleavage or ligation of target RNAs. We have explored the possibility of using ribozymes in maize to downregulate the expression of the stearoyl-acyl carrier protein ( ⌬ 9) desaturase gene. Based on site accessibility and catalytic activity, several ribozyme constructs were designed and transformed into regenerable maize lines. One of these constructs, a multimer hammerhead ribozyme linked to a selectable marker gene, was shown to increase leaf stearate in two of 13 maize lines. There were concomitant decreases in ⌬ 9 desaturase mRNA and protein. The plants with the altered stearate phenotype were shown to express ribozyme RNA. The ribozyme-mediated trait was heritable, as evidenced by stearate increases in the leaves of the R 1 plants derived from a high-stearate line. The increase in stearate correlated with the presence of the ribozyme gene. A catalytically inactive version of this ribozyme did not produce any significant effect in transgenic maize. This is evidence that ribozymes can be used to modulate the expression of endogenous genes in maize.

show abstract

Improved accumulation and activity of ribozymes expressed from a tRNA-based RNA polymerase III promoter

Cited by 79 publications

References 34 publications

A novel SV40-based vector successfully transduces and expresses an alpha 1-antitrypsin ribozyme in a human hepatoma-derived cell line

A novel SV40-based vector successfully transduces and expresses an alpha 1-antitrypsin ribozyme in a human hepatoma-derived cell line

Expression of small, therapeutic RNAs in human cell nuclei

Ribozymes Targeted to Stearoyl–ACP Δ9 Desaturase mRNA Produce Heritable Increases of Stearic Acid in Transgenic Maize Leaves

Contact Info

Product

Resources

About