Efficacy of Morpholino-modified Antisense Oligomers Directed against Tumor Necrosis Factor-α mRNA

Taylor, Margaret; Paulauskis, Joseph; Weller, Dwight D.; Kobzik, Lester

doi:10.1074/jbc.271.29.17445

Cited by 63 publications

(39 citation statements)

References 23 publications

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“…14,39 Most RNase H-independent antisense types can block translation only when targeted against sequences in the region from the 5Ј cap to about 25 bases past the AUG translational start site of an mRNA. 39,42 In contrast, RNase H-competent oligonucleotides, i.e., PS-modified ODNs, can also be effective against sequences elsewhere in the RNA transcript by virtue of their effecting degradation of the paired RNA target sequence by RNase H. 39 Our data showed that swapping the nucleotide backbone (morpholino or PS) does not necessarily result in the same inhibitory effect (Fig. 2c).…”

Section: Discussionmentioning

confidence: 73%

Morpholino antisense oligomer targeting human midkine: Its application for cancer therapy

Takei

Kadomatsu

Yuasa

et al. 2004

Intl Journal of Cancer

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Overexpression of a heparin-binding growth factor, midkine (MK), has been observed in many malignancies, making it an attractive therapeutic target. We used morpholino antisense oligomers to downregulate human MK expression in human prostate (PC-3) and colon carcinoma (SW620) cells, and determined the practical advantages of this anticancer therapeutic. Morpholino antisense oligomers directed against MK caused a dramatic and sequence-specific decrease of the target protein level, resulting in the inhibition of growth and anchorage-independent growth of the transfected cells. Furthermore, MK morpholino antisense oligomers exhibited a significant anticancer effect in the PC-3-and SW620-xenograft models. In comparison with phosphorothioatemodified oligodeoxynucleotide, morpholino oligomers showed 2 major advantages, stability and non-toxicity. MALDI-TOF mass spectrometric analysis showed that morpholino antisense oligomers were completely stable in the presence of serum nuclease(s). Serological examinations demonstrated no toxicity of MK morpholino antisense oligomers. Our study indicates that inhibition of MK expression by morpholino antisense oligomer is a promising novel and safe therapeutic strategy for cancers.

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Section: Discussionmentioning

confidence: 73%

Morpholino antisense oligomer targeting human midkine: Its application for cancer therapy

Takei

Kadomatsu

Yuasa

et al. 2004

Intl Journal of Cancer

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“…Inhibition of translation of TSG and chordin mRNAs was performed as previously described (Taylor et al, 1996). Briefly, 1 µg of each capped mRNA was translated using the Promega Rabbit Reticulocyte System and [ 35 S]methionine in the absence or presence of 2.5 µM of the specified morpholino antisense oligonucleotide.…”

Section: Morpholinos and In Vitro Translationsmentioning

confidence: 99%

Twisted gastrulation loss-of-function analyses support its role as a BMP inhibitor during earlyXenopusembryogenesis

2003

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BMP signals play important roles in the regulation of diverse events in development and in the adult. In amniotes, like the amphibian Xenopus laevis, BMPs promote ventral specification, while chordin and other BMP inhibitors expressed dorsally in the Spemann's organizer play roles in establishment and/or maintenance of this region as dorsal endomesoderm. The activities of chordin are in turn regulated by the secreted proteolytic enzymes BMP1 and Xolloid. Recently, we and others have identified the protein twisted gastrulation (TSG) as a soluble BMP modulator that functions by modifying chordin activity. Overexpression and genetic analyses in Drosophila, Xenopus and zebrafish together with in vitro biochemical studies suggest that TSG might act as a BMP antagonist; but there is also evidence that TSG may promote BMP signaling. Here we report examination of the in vivo function of TSG in early Xenopusdevelopment using a loss-of-function approach. We show that reducing TSG expression using antisense TSG morpholino oligonucleotides (MOs) results in moderate head defects. These defects can be rescued both by a TSG that cannot be inhibited by the MO, and by the BMP antagonists chordin and noggin. Furthermore, while neither the onset of gastrulation nor the expression of marker genes are affected in early gastrulae, dorsal marker gene expression is reduced at the expense of expanded ventral marker gene expression beginning at mid to late gastrula stage. TSG-MO and Chd-MOs also cooperate to strongly repress head formation. Finally, we note that the loss of TSG function results in a shift in tissue responsiveness to the BMP inhibitory function of chordin in both animal caps and the ventral marginal zone, a result that implies that the activity of TSG may be required for chordin to efficiently inhibit BMPs in these developmental contexts. These data, taken together with the biochemistry and overexpression studies, argue that TSG plays an important role in regulating the potency of chordin's BMP inhibitory activity and TSG and chordin act together to regulate the extent of dorsoanterior development of early frog embryos.

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“…This difference could be due to a slow rate of uptake of morpholino oligonucleotides and/or the fact that nuclear and not cytoplasmic target were used in this study. Note, however, that morpholino oligonucleotides taken up by the RAW 264.7 macrophage-like cells were found effective in down-regulating tumor necrosis factor-␣ mRNA (53). Overall, the splicing modification assay provides a more sensitive and stringent test of true antisense activity of the oligonucleotides.…”

Section: Discussionmentioning

confidence: 99%

Antisense Oligonucleotides with Different Backbones

Schmajuk

Sierakowska²,

Kole

1999

Journal of Biological Chemistry

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A novel, positive read-out assay that quantifies only sequence-specific nuclear activity of antisense oligonucleotides was used to evaluate morpholino and 2-Omethyl sugar-phosphate oligonucleotides. The assay is based on modification of the splicing pathway of human ␤-globin pre-mRNA. In addition, scrape-loading of cells with oligonucleotides allows the separate assessment of intracellular antisense activity of the oligonucleotides and their ability to penetrate the cell membrane barrier. The results show that, with scrape-loading, the morpholino oligonucleotides were approximately 3-fold more effective in their intrinsic antisense activity than alternating phosphodiester/phosphorothioate 2-O-methyl-oligoribonucleotides and 6-9-and almost 200-fold more effective than the exclusively phosphorothioate and phosphodiester derivatives, respectively. The morpholino oligonucleotides were over 20-fold more effective than the phosphorothioate 2-O-methyl-oligoribonucleotides in free uptake from the culture media. The antisense activity of the morpholino oligonucleotides was detectable not only in monolayer HeLa cells but also in suspension K562 cells. Time course experiments suggest that both the free uptake and efflux of morpholino oligonucleotides are slow.Antisense oligonucleotides and RNAs show great promise as sequence-specific agents able to down-regulate the expression of targeted genes. In this capacity, extensively reviewed in a recent volume and review articles (1-4), they have advanced not only to clinical trials (5-8) but also to clinical practice (9); they have also proven to be useful as research tools (1). However, the application of antisense technology both in research and in clinical studies presents a number of outstanding issues.In principle, the most important feature of antisense oligonucleotides is their ability to block mRNA function by sequence-specific hybridization to the RNA. Surprisingly, the oligonucleotides may also exert their effects by binding directly to a number of proteins in a sequence-dependent but not sequence-specific manner, resulting in unpredictable and nonspecific effects (10 -14). This non-antisense mechanism of binding was shown to be particularly pronounced in commonly used oligodeoxynucleoside phosphorothioates (15); other mechanisms also contribute to sequence-independent oligonucleotide effects (16).Our ability to select appropriate target sequences within the RNA is still limited (17). Cell-free selection of susceptible sites is not always helpful (18, 19) because in vivo cellular RNAs are always complexed with proteins (20), which may block the sites and/or change the secondary and tertiary structure of the target RNA. Therefore, oligonucleotide targeting is frequently carried out by trial and error, requiring the synthesis of large numbers of compounds (21,22).Equally unsatisfactory is our understanding of the intracellular site of action of oligonucleotides. Most of the antisense oligonucleotides are designed against sequences within mRNA which presumably represents a...

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Efficacy of Morpholino-modified Antisense Oligomers Directed against Tumor Necrosis Factor-α mRNA

Cited by 63 publications

References 23 publications

Morpholino antisense oligomer targeting human midkine: Its application for cancer therapy

Morpholino antisense oligomer targeting human midkine: Its application for cancer therapy

Twisted gastrulation loss-of-function analyses support its role as a BMP inhibitor during earlyXenopusembryogenesis

Antisense Oligonucleotides with Different Backbones

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