Expression of chimeric tRNA-driven antisense transcripts renders NIH 3T3 cells highly resistant to Moloney murine leukemia virus replication.

Sullenger, Bruce A.; Lee, Thomas C.; Smith, Clayton A.; Ungers, Grace; Gilboa, Eli

doi:10.1128/mcb.10.12.6512

Cited by 81 publications

(41 citation statements)

References 49 publications

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“…The transition of such an intracellular inhibitor to in vivo models requires one of two approaches. Gene therapeutic methods utilizing viral constructs may be used to "express" small, therapeutic RNAs following gene transfer into cells (27). Alternatively, an aptamer may be selected de novo with nuclease-resistant nucleotides and delivered locally or systemically.…”

Section: Intimal Hyperplasiamentioning

confidence: 99%

Developing aptamers into therapeutics

White

Sullenger²,

Rusconi³

2000

J. Clin. Invest.

267

195

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Section: Intimal Hyperplasiamentioning

confidence: 99%

Developing aptamers into therapeutics

White

Sullenger²,

Rusconi³

2000

J. Clin. Invest.

267

195

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“…To express ribozymes, RNA polymerase II (pol II) and III (pol III) promoters have been used. RNA pol III-based expression cassettes, containing the tRNA, U6, and adenoviral VA1 promoters, have been extensively used to express small RNAs such as antisense RNAs (Jennings and Molloy 1987;Sullenger et al 1990a), trans-cleaving ribozymes (Ojwang et al 1992), RNA decoys (Sullenger et al 1990b), and siRNAs (Lee et al 2002;Miyagishi and Taira 2002;Paul et al 2002;Sui et al 2002). RNA pol III transcripts accumulate to high levels in transduced mammalian cells.…”

Section: Introductionmentioning

confidence: 99%

Efficient and specific repair of sickle β-globin RNA by trans-splicing ribozymes

Byun¹,

Lan²,

Long³

et al. 2003

RNA

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Previously we demonstrated that a group I ribozyme can perform trans-splicing to repair sickle ␤-globin transcripts upon transfection of in vitro transcribed ribozyme into mammalian cells. Here, we sought to develop expression cassettes that would yield high levels of active ribozyme after gene transfer. Our initial expression constructs were designed to generate trans-slicing ribozymes identical to those used in our previous RNA transfection studies with ribozymes containing 6-nucleotide long internal guide sequences. The ribozymes expressed from these cassettes, however, were found to be unable to repair sickle ␤-globin RNAs. Further experiments revealed that two additional structural elements are important for ribozyme-mediate RNA repair: the P10 interaction formed between the 5 end of the ribozyme and the beginning of the 3 exon and an additional base-pairing interaction formed between an extended guide sequence and the substrate RNA. These optimized expression cassettes yield ribozymes that are able to amend 10%-50% of the sickle ␤-globin RNAs in transfected mammalian cells. Finally, a ribozyme with a 5-bp extended guide sequence preferentially reacts with sickle ␤-globin RNAs over wild-type ␤-globin RNAs, although the wild-type ␤-globin transcript forms only a single mismatch with the ribozyme. These results demonstrate that trans-splicing ribozyme expression cassettes can be generated to yield ribozymes that can repair a clinically relevant fraction of sickle ␤-globin RNAs in mammalian cells with greatly improved specificity.

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“…44 In fact, studies conducted in the last years have shown that insertion of independent transcriptional units within the two viral LTRs in several instances results in silencing of ribozyme expression, possibly due to competition between the 5 0 LTR Figure 9 Suppression of B3A2 bcr -abl transcripts in primary CML bone marrow cells. Bone marrow mononuclear cells from three patients with CML with the B3A2 bcr -abl translocation were mock transduced ( mock ) or transduced with the retroviral vector expressing the VA1 -RibB3A2 ribozyme ( VA1 -Rib ), and analyzed for bcr -abl gene expression at 2 and 5 days after transduction.…”

Section: Discussionmentioning

confidence: 99%

Purging of chronic myelogenous leukemia cells by retrovirally expressed anti–bcr-abl ribozymes with specific cellular compartmentalization

et al. 2002

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In patients with chronic myelogenous leukemia ( CML ), abnormal expansion of myeloid cells is maintained by expression of the p210 bcr -abl fusion protein. Thus, this protein and its mRNA represent primary targets to inhibit proliferation of these cells. Here we describe the properties of a ribozyme against the bcr -abl mRNA, expressed as a fusion transcript with the human U1 small nuclear RNA or the adenovirus VA1 RNA and delivered to the cells through retroviral vectors. These fusion ribozymes are specifically localized in the nucleus or in the cytoplasm, respectively. Transduction of 32D -LG7 myeloid cells, whose growth is IL -3 independent thanks to deregulated bcr -abl expression, imposed strong negative selective pressure on cell growth and induced restoration of an IL -3 -dependent phenotype. Although expressed at a level similar to that of the U1 -fusion ribozyme, the cytoplasmic VA1 ribozyme was a more powerful inhibitor of p210 bcr -abl gene expression. In cells transduced with the vector expressing this ribozyme, the levels of the bcr -abl transcript were reduced up to 10 4 -fold, the p210 bcr -abl protein became undetectable, and the cells underwent massive apoptosis when cultured in the absence of IL -3. Transduction of primary hematopoietic cells obtained from bone marrow of patients with CML resulted in remarkable reduction of bcr -abl mRNA levels, starting a few days after transduction. These results show the feasibility and efficacy of vector -expressed anti -bcr -abl ribozymes for purging of CML cells.

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Expression of chimeric tRNA-driven antisense transcripts renders NIH 3T3 cells highly resistant to Moloney murine leukemia virus replication.

Cited by 81 publications

References 49 publications

Developing aptamers into therapeutics

Developing aptamers into therapeutics

Efficient and specific repair of sickle β-globin RNA by trans-splicing ribozymes

Purging of chronic myelogenous leukemia cells by retrovirally expressed anti–bcr-abl ribozymes with specific cellular compartmentalization

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