Use of minimally modified antisense oligonucleotides for specific inhibition of gene expression

Uhlmann, Eugen; Peyman, Anusch; Ryte, A. S.; Schmidt, Annette; Buddecke, Eckhart

doi:10.1016/s0076-6879(00)13017-4

Cited by 42 publications

(25 citation statements)

References 41 publications

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“…Antisense and sense oligonucleotides were minimally phosphorothioated as previously described (Uhlmann et al, 2000). Residues that were phosphorothioate-modified are shown in bold: KGFR sense (1372-1390), 3′-caggccaaccagtctgcct-5′; KGFR antisense (1390-1372), 3′-aggcagactggttggcctg-5′ (Post et al, 1996); Bek antisense (first two residues modified for mouse), 3′-tggcagaactgtcaac-5′; Bek sense, 3′-gttgacagttctgcca-5′; antisense to both KGFR/Bek, 5′-gctgaccatggtcac-3′; sense to both KGFR/Bek, 5′gtgaccatggtcagc-3′ (Post et al, 1996).…”

Section: Antisense Experimentsmentioning

confidence: 99%

FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis

et al. 2005

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Branching morphogenesis of mouse submandibular glands is regulated by multiple growth factors. Here, we report that ex vivo branching of intact submandibular glands decreases when either FGFR2 expression is downregulated or soluble recombinant FGFR2b competes out the endogenous growth factors. However, a combination of neutralizing antibodies to FGF1, FGF7 and FGF10 is required to inhibit branching in the intact gland, suggesting that multiple FGF isoforms are required for branching. Exogenous FGFs added to submandibular epithelial rudiments cultured without mesenchyme induce distinct morphologies. FGF7 induces epithelial budding, whereas FGF10 induces duct elongation, and both are inhibited by FGFR or ERK1/2 signaling inhibitors. However, a PI3-kinase inhibitor also decreases FGF7-mediated epithelial budding, suggesting that multiple signaling pathways exist. We immunolocalized FGF receptors and analyzed changes in FGFR, FGF and MMP gene expression to identify the mechanisms of FGF-mediated morphogenesis. FGFR1b and FGFR2b are present throughout the epithelium,although FGFR1b is more highly expressed around the periphery of the buds and the duct tips. FGF7 signaling increases FGFR1b and FGF1expression, and MMP2 activity, when compared with FGF10, resulting in increased cell proliferation and expansion of the epithelial bud, whereas FGF10 stimulates localized proliferation at the tip of the duct. FGF7- and FGF10-mediated morphogenesis is inhibited by an MMP inhibitor and a neutralizing antibody to FGF1, suggesting that both FGF1 and MMPs are essential downstream mediators of epithelial morphogenesis. Taken together,our data suggests that FGFR2b signaling involves a regulatory network of FGFR1b/FGF1/MMP2 expression that mediates budding and duct elongation during branching morphogenesis.

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Section: Antisense Experimentsmentioning

confidence: 99%

FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis

et al. 2005

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“…Other cleavage sites were on the 39-side of C 8 , G 11 , and G 18 in the case of ODN-R, and G 17 in the case of ODN-H. Although endonuclease activity has been described as occurring preferentially at pyrimidine sites, especially with two or more adjacent pyrimidines Uhlmann et al, 2000), it is clear that this was not the case for endonuclease activity of K562 lysate. Besides cleavage at T 3 , C 4 , C 5 , and C 8 , three of the cleavage sites were purines: A 6 , G 11 , and G 18 (or G 17 ).…”

Section: Minimally Modified Oligonucleotides 131mentioning

confidence: 96%

“…Additional PS linkages are placed at internal pyrimidine nucleotides to ensure protection against cellular endonucleases Rait et al, 2000;Uhlmann et al, 2000). We showed in this work that this last modification is not always the best solution.…”

Section: Fret Analysismentioning

confidence: 99%

“…These are phosphodiester ODN protected (1) by two to five PS residues at their 39-end, as the serum enzymes are mainly 39-exonucleases (Shaw et al, 1991), (2) by two PS residues at their 59-end against potential degradation by 59-exonucleases, and (3) by PS linkages at internal pyrimidine nucleotides against degradation by endonucleases. Single-strand specific endonucleases in serum and inside the cells have been shown to cleave oligonucleotides preferentially at pyrimidine sites Rait et al, 2000;Uhlmann et al, 2000), this cleavage becoming dominant in the case of several adjacent pyrimidines . Minimally modified oligonucleotides have a higher cellular uptake than all-PS oligonucleotides (Peyman et al, 1997) and have biologic antisense effects with (Rait et al, 2000) and without (Tanaka et al, 1996) delivery systems.…”

Section: Introduction Mmentioning

confidence: 99%

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Best Minimally Modified Antisense Oligonucleotides According to Cell Nuclease Activity

Samani

Jollès²,

Laigle³

2001

Antisense and Nucleic Acid Drug Development

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Minimally modified oligonucleotides belong to the second-generation antisense class. They are phosphodiester oligonucleotides with a minimum of phosphorothioate linkages in order to be protected against serum and cellular exonucleases and endonucleases. They activate RNase H, have weak interactions with proteins, and have thus a better antisense efficiency. Two of them have been designed from an all-phosphorothioate antisense oligonucleotide directed against mdr1-expressing cells. They are protected against serum and cellular enzymatic degradation by the self-forming hairpin d(GCGAAGC) at their 39 -end and by judiciously located phosphorothioate residues, depending on the cellular composition in exonucleases or endonucleases. Besides their already demonstrated ability to cleave pyrimidine sites, endonucleases show some specificity for CpG sites. Their activity is hindered if specific sites are involved in secondary structure as hairpin.

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“…All ASODNs were synthesized at ABI8909 nucleic acid synthesis system (Applied Biosystems, Foster City, CA, USA) and purified by OPC (Perkin-Elmer, Foster City, CA, USA). Considering that phosphorothiate ASODNs are very resistant to nucleases, 43,44 all these ASODNs had been chemically modified to phosphorithioate ODNs by substituting the oxygen molecules of the phosphate backbone with sulfur. .…”

Section: Asodns Design and Synthesismentioning

confidence: 99%

Whole genome expression profiling of hepatitis B virus-transfected cell line reveals the potential targets of anti-HBV drugs

et al. 2007

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Hepatitis B virus (HBV) infection is a major health concern world wide, and few effective treatments have been developed. It has recently been reported that inhibiting host-cell proteins can prevent viral infection. The human genome may contain more genes required for HBV infection and replication than the viral genome. A systematic approach to find these potential antiviral targets is by host gene expression analysis using DNA microarrays. The aim of this study was to identify and validate novel cellular anti-HBV targets. The Human Whole Genome Bioarray was used to analyze differentially expressed genes in HepG2.2.15 cells and HepG2 cells. Altered gene expression in HepG2.2.15 cells was studied following treatment with the anti-HBV drug, lamivudine. Genes that were differentially expressed during HBV infection and reversed with anti-HBV drugs were validated by semiquantitative reverse transcription-PCR. Bioinformatics analysis revealed ABHD2, EREG, ACVR2B, CDC34, KHDRBS3 and RORA as potential cellular anti-HBV targets. Antisense oligodeoxynucleotides were used to test the antiviral activity of these potential targets. Results strongly suggested that inhibition of ABHD2 or EREG significantly blocked HBV propagation in HepG2.2.15 cells. This study demonstrates that ABHD2 and EREG are essential for HBV propagation and provides strong evidence that these proteins could be used as potential targets for anti-HBV drugs.

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Use of minimally modified antisense oligonucleotides for specific inhibition of gene expression

Cited by 42 publications

References 41 publications

FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis

FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis

Best Minimally Modified Antisense Oligonucleotides According to Cell Nuclease Activity

Whole genome expression profiling of hepatitis B virus-transfected cell line reveals the potential targets of anti-HBV drugs

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