DE10200410A1 - New antisense oligonucleotide, useful for treatment of tumors, inhibits translation of mRNA from the pituitary-tumor transforming gene-1 - Google Patents
New antisense oligonucleotide, useful for treatment of tumors, inhibits translation of mRNA from the pituitary-tumor transforming gene-1Info
- Publication number
- DE10200410A1 DE10200410A1 DE2002100410 DE10200410A DE10200410A1 DE 10200410 A1 DE10200410 A1 DE 10200410A1 DE 2002100410 DE2002100410 DE 2002100410 DE 10200410 A DE10200410 A DE 10200410A DE 10200410 A1 DE10200410 A1 DE 10200410A1
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- Prior art keywords
- cells
- mrna
- antisense oligodeoxynucleotide
- antisense
- pttg
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- Ceased
Links
- 108020004999 messenger RNA Proteins 0.000 title claims abstract description 7
- 238000013519 translation Methods 0.000 title claims abstract description 6
- 206010028980 Neoplasm Diseases 0.000 title claims description 6
- 108091034117 Oligonucleotide Proteins 0.000 title abstract description 4
- 208000007913 Pituitary Neoplasms Diseases 0.000 title abstract description 3
- 239000000074 antisense oligonucleotide Substances 0.000 title abstract 3
- 238000012230 antisense oligonucleotides Methods 0.000 title abstract 3
- 208000010916 pituitary tumor Diseases 0.000 title abstract 2
- 230000001131 transforming effect Effects 0.000 title abstract 2
- 210000005260 human cell Anatomy 0.000 claims abstract 3
- 229940046166 oligodeoxynucleotide Drugs 0.000 claims description 20
- 230000000692 anti-sense effect Effects 0.000 claims description 14
- 210000004027 cell Anatomy 0.000 claims description 14
- 238000004113 cell culture Methods 0.000 claims description 4
- 201000011510 cancer Diseases 0.000 claims description 3
- 230000014509 gene expression Effects 0.000 claims description 3
- RYYWUUFWQRZTIU-UHFFFAOYSA-N Thiophosphoric acid Chemical group OP(O)(S)=O RYYWUUFWQRZTIU-UHFFFAOYSA-N 0.000 claims description 2
- 230000015556 catabolic process Effects 0.000 claims description 2
- 238000006731 degradation reaction Methods 0.000 claims description 2
- 238000010348 incorporation Methods 0.000 claims description 2
- 150000007523 nucleic acids Chemical group 0.000 claims description 2
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical compound [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 claims description 2
- 238000001890 transfection Methods 0.000 claims description 2
- 230000002401 inhibitory effect Effects 0.000 claims 2
- 102000004190 Enzymes Human genes 0.000 claims 1
- 108090000790 Enzymes Proteins 0.000 claims 1
- 108091093037 Peptide nucleic acid Proteins 0.000 claims 1
- YACKEPLHDIMKIO-UHFFFAOYSA-L methylphosphonate(2-) Chemical compound CP([O-])([O-])=O YACKEPLHDIMKIO-UHFFFAOYSA-L 0.000 claims 1
- 102000012152 Securin Human genes 0.000 description 11
- 108010061477 Securin Proteins 0.000 description 11
- 108090000623 proteins and genes Proteins 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 230000002018 overexpression Effects 0.000 description 3
- 210000004881 tumor cell Anatomy 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 102000005446 Anaphase-Promoting Complex-Cyclosome Human genes 0.000 description 2
- 108010031677 Anaphase-Promoting Complex-Cyclosome Proteins 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 230000007515 enzymatic degradation Effects 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 125000005642 phosphothioate group Chemical group 0.000 description 2
- 238000003757 reverse transcription PCR Methods 0.000 description 2
- 125000001424 substituent group Chemical group 0.000 description 2
- 239000012096 transfection reagent Substances 0.000 description 2
- 108010085238 Actins Proteins 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108091060290 Chromatid Proteins 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000012083 RIPA buffer Substances 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 102000005734 Separase Human genes 0.000 description 1
- 108010031091 Separase Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000031016 anaphase Effects 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000001028 anti-proliverative effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 230000010307 cell transformation Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 210000004756 chromatid Anatomy 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 229960003964 deoxycholic acid Drugs 0.000 description 1
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- WSFSSNUMVMOOMR-UHFFFAOYSA-N formaldehyde Substances O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- YACKEPLHDIMKIO-UHFFFAOYSA-N methylphosphonic acid Chemical class CP(O)(O)=O YACKEPLHDIMKIO-UHFFFAOYSA-N 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 108090000765 processed proteins & peptides Chemical class 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000003153 stable transfection Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/31—Chemical structure of the backbone
- C12N2310/315—Phosphorothioates
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- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Oncology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
Die vorliegende Erfindung betrifft drei Oligodesoxynukleotide (ODNs), mit welchen sich wirksam das Wachstum von mit Pituitary tumor transforming gene - 1 (PTTG) - Überexpression assoziierten Tumorzellen hemmen lässt. Insbesondere betrifft die Erfindung drei hochwirksame Antisense-Oligodesoxynukleotide, mit welchen die Translation von PTTG-mRNA in Tumorzellen inhibiert werden kann. The present invention relates to three oligodeoxynucleotides (ODNs) with which effective the growth of with pituitary tumor transforming gene - 1 (PTTG) - Inhibits overexpression associated tumor cells. In particular, the Invention three highly effective antisense oligodeoxynucleotides, with which the Translation of PTTG mRNA in tumor cells can be inhibited.
PTTG-1 Überexpression ist für eine Reihe von Tumoren und Tumorzellinien beschrieben. PTTG wurde erstmals in Ratten-Hypophysentumoren als überexpremiert beschrieben. Eine Überexpression mittels stabiler Transfektion in NIH-3T3 Mausfibroblasten führt zur Zelltransformation in vitro und zur Formation von soliden Tumoren in vivo. PTTG-1 overexpression is for a number of tumors and tumor cell lines described. PTTG was first overexpressed in rat pituitary tumors described. Overexpression using stable transfection in NIH-3T3 Mouse fibroblasts lead to cell transformation in vitro and the formation of solid ones Tumors in vivo.
PTTG hat unter anderem eine essentielle Funktion im Rahmen des Zellzyklus als Securin. Securin verhindert eine frühzeitige Trennung der Schwesterchromatiden durch die Inhibierung der Separase bis zur eigenen Degradierung durch den Anaphasepromoting complex (APC) zu beginn der Anaphase. Among other things, PTTG has an essential function in the context of the cell cycle as Securin. Securin prevents early separation of the sister chromatids the inhibition of the separase up to its own degradation by the Anaphasepromoting complex (APC) at the beginning of anaphase.
Bereits seit längerem ist es bekannt, die Translation von mRNA durch komplementär zur Sequenz einer mRNA ausgerichteter antisense-ODNs zu inhibieren. Neueren Datums ist der Einsatz einzelsträngiger Nukleinsäuren für die selektive Blockade der Genexpression von Transkriptionsfaktoren. Damit derartige ODNs aber selektiver eingesetzt werden können, müssen sie zuvor modifiziert werden, um gegenüber enzymatischem Abbau stabiler zu sein. Hier sind eine Reihe von Modifikationen bekannt, wie Phosphotriester-, Phosphoroamid-, Methylphosphonat-, Phosphorothioat- und Peptidnukleotidsäurebindungen bekannt. Wegen seiner erhöhten Stabilität, guter Löslichkeit und der Aktivierung von RNAse H Aktivität sind Phosphorothioate bevorzugte Kandidaten für eine spezifische Inhibierung der Genexpression. It has been known for a long time to complement the translation of mRNA by Inhibit sequence of an mRNA-targeted antisense ODNs. More recent date is the use of single stranded nucleic acids for the selective blocking of Gene expression of transcription factors. This makes such ODNs more selective can be used, they must be modified beforehand in order enzymatic degradation to be more stable. Here are a number of modifications known such as phosphotriester, phosphoroamide, methylphosphonate, phosphorothioate and peptide nucleotide acid linkages are known. Because of its increased stability, good Solubility and activation of RNAse H activity are preferred for phosphorothioates Candidates for specific inhibition of gene expression.
Der vorliegenden Erfindung liegt daher die Aufgabe zugrunde ODN-Sequenzen zu
finden, die hochwirksam die Translation von PTTG mRNA inhibieren. Erfindungsgemäß
wird die Aufgabe durch die in den folgenden Formeln wiedergegebenen modifizierten
Oligodesoxynukleotide aus 20 Nukleotiden (20-mer) gelöst:
5'-TAG CAA CAT ACC AAA CTT TC-3' (Hannah-02)
5'-TAT CAA CAT AGA TCA GAG TA-3' (Hannah-03)
5'-TCA ACA TCC AGG GTC GAC AG-3' (Hannah-05)
The object of the present invention is therefore to find ODN sequences which highly effectively inhibit the translation of PTTG mRNA. According to the invention, the object is achieved by the modified oligodeoxynucleotides of 20 nucleotides (20-mer) shown in the following formulas:
5'-DAY CAA CAT ACC AAA CTT TC-3 '(Hannah-02)
5'-TAT CAA CAT AGA TCA GAG TA-3 '(Hannah-03)
5'-TCA ACA TCC AGG GTC GAC AG-3 '(Hannah-05)
Als Modifikationen gegen den enzymatischen Abbau kommen prinzipiell die im Stand der Technik bekannten Substituenten, entweder einzeln oder in Kombination, in Frage. Bevorzugt kommen die oben genannten Substituenten zum Einsatz. Die bevorzugte Modifikation ist der Einbau von Phosphothioat. In den folgenden Untersuchungen wurden die Antisense-ODNs in Form ihrer Phosphothioate eingesetzt. As modifications against the enzymatic degradation there are in principle those in the stand substituents known in the art, either individually or in combination. The above-mentioned substituents are preferably used. The preferred Modification is the incorporation of phosphothioate. In the following investigations the antisense ODNs were used in the form of their phosphothioates.
Die Herstellung der Phosphorothioat-modifizierten Hannah-02, Hannah-03 und Hannah- 05 erfolgte mit einem automatischen DANN-Synthesizer (Perseptive 8909; BioSpring GmbH; Frankfurt) nach dem Stand der Technik bekannten Verfahren. Die Deprotektion der Oligonukleotide wurde mit konzentriertem Ammoniak über einen Zeitraum von 16 Stunden bei Raumtemperatur durchgeführt. The production of the phosphorothioate-modified Hannah-02, Hannah-03 and Hannah- 05 was done with an automatic DANN synthesizer (Perseptive 8909; BioSpring GmbH; Frankfurt) according to methods known in the art. Deprotection the oligonucleotide was concentrated with ammonia over a period of 16 Hours at room temperature.
Die in Zellkultur verwendete menschliche Krebszellinie HeLa-S3 wurden von der American Type Cell Culture Collection (ATCC) erworben. 50.000 Zellen wurden in DMEM (Dulbecco's modifiziertem Eagle Medium) suspendiert, mit 10% hitzeinaktiviertem Kälberserum, Antibiotika und 2 mM L-Glutamin versetzte Zellen wurden in 6-Loch Platten ausgesät und bei 37°C in 5% CO2/95% Luftatmosphäre in einem befeuchteten Brutschrank wachsen gelassen. Am darauf folgenden Tag wurden die Zellen mit den jeweiligen, in sterilem deionisiertem Wasser für Zellkulturen gelösten ODNs in Gegenwart von Transfektionsreagenz (Oligofectamin) gemäß den Angaben des Herstellers inkubiert. Die Lebensfähigkeit der Zellen wurde mit der Trypanblau- Ausschlußmethode zu den angegebenen Zeitpunkten überprüft. The human cancer cell line HeLa-S3 used in cell culture was developed by the American Type Cell Culture Collection (ATCC) acquired. 50,000 cells were in DMEM (Dulbecco's modified Eagle Medium) suspended, with 10% heat-inactivated calf serum, antibiotics and 2 mM L-glutamine spiked cells were sown in 6-hole plates and at 37 ° C in 5% CO2 / 95% air atmosphere grown in a humidified incubator. The following day the cells with the respective, dissolved in sterile deionized water for cell cultures ODNs in the presence of transfection reagent (oligofectamine) according to the information incubated by the manufacturer. The viability of the cells was assessed with the trypan blue Exclusion method checked at the specified times.
Die RNA der transfizierten Zellen wurde zu den angegebenen Zeitpunkten mittels RNAeasy-Mini Kit der Firma Qiagen isoliert. Die Qualität der RNA wurde vor Einsatz in der RT-PCR mittels Agarose-Formaldehyd-Gelelektrophorese und anschließender SybrGreen II Färbung überprüft. Nach anschließender Reverser Transkription der RNA wurde die erhaltene cDNA in einer PCR-Reaktion mit spezifischen Primern für PTTG-1 bzw. Glyceraledehyd-3-Phosphat-Dehydrogenase (GAPDH) durchgeführt (5'-ACC TTT GCT TCT CCC ACC TT-3' (PTTG sense), 5'-CAA ATA CAC ACA AAC TCT GAA GCA- 3' (PTTG antisense), 5'-GAT GAC ATC AAG AAG GTG GTG-3' (GAPDH sense), 5'- GCT GTA GCC AAA TTC GTT GTC-3' (GAPDH antisense). Die resultierenden PCR Produkte wurde mittels Agarosegelelektrophorese getrennt und anschließend mit SybrGreen I gefärbt. The RNA of the transfected cells was analyzed at the specified times RNAeasy mini kit from Qiagen isolated. The quality of the RNA was checked before use in the RT-PCR using agarose-formaldehyde gel electrophoresis and subsequent SybrGreen II staining checked. After subsequent reverse transcription of the RNA was obtained the cDNA in a PCR reaction with specific primers for PTTG-1 or glyceraledehyde-3-phosphate dehydrogenase (GAPDH) (5'-ACC TTT GCT TCT CCC ACC TT-3 '(PTTG sense), 5'-CAA ATA CAC ACA AAC TCT GAA GCA- 3 '(PTTG antisense), 5'-GAT GAC ATC AAG AAG GTG GTG-3' (GAPDH sense), 5'- GCT GTA GCC AAA TTC GTT GTC-3 '(GAPDH antisense). The resulting PCR Products were separated by means of agarose gel electrophoresis and then with SybrGreen I colored.
Die Zellen wurden wie oben angegeben behandelt. Nach erfolgter inkubation wurde das zelluläre Protein mittels RIPA-Puffer (1 × PBS, 1% NP-40, 0,5% Natriumdesoxycholat, 0,1% SDS) isoliert. 20 µg zelluläres Protein wurde in mittels SDS-PAGE aufgetrennt. Die Proteine wurden geblottet und anschließend mit PTTG bzw. β-Actin spezifischen Antikörpern inkubiert und mittels Meerettich-Peroxidase konjugierten Sekundärantikörpern und Chemilumineszenz Reagenz visualisiert. The cells were treated as indicated above. After incubation, the cellular protein using RIPA buffer (1 × PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) isolated. 20 µg of cellular protein was separated using SDS-PAGE. The Proteins were blotted and then specific with PTTG or β-actin Antibodies incubated and conjugated using horseradish peroxidase Secondary antibodies and chemiluminescent reagent visualized.
Zusätzlich zu den eingesetzten Oligodesoxynukleotiden wurde sowohl eine Sense als auch eine Transfektionsreagenzkontrolle eingesetzt. Im Verlauf der Experiment konnte kein signifikanter Unterschied zwischen den Kontrollen beobachtet werden. In addition to the oligodeoxynucleotides used, both a sense and a transfection reagent control is also used. In the course of the experiment no significant difference between controls was observed.
Das Dosisabhängige Verhalten der getesteten Oligodesoxynukleotidezeigt sich bereits nach einmaliger Anwendung. Das Ergebnis einer Konzentrationsreihe ist in Fig. 1 gezeigt. The dose-dependent behavior of the tested oligodeoxynucleotides is already evident after a single application. The result of a series of concentrations is shown in FIG. 1.
Wie in Fig. 2 dargestellt findet sich der gleiche Dosisabhängige Effekt ebenfalls auf Proteinlevel. As shown in Fig. 2, the same dose-dependent effect is also found on protein levels.
Zur Ermittlung des Anti-Proliferativen Effektes wurde die Zellen transfiziert und nach 24, 48 und 72 Stunden wurden die lebenden Zellen bestimmt. Hier zeigt sich ein hochsignifikanter Effekt. To determine the anti-proliferative effect, the cells were transfected and after 24, The living cells were determined for 48 and 72 hours. Here shows up highly significant effect.
Transfektionen in Mausfibroblasten zeigten keine vorbeschriebenen Effekte (Daten nicht gezeigt). Alle Daten sind exemplarisch für das Antisense-ODN Hannah-05 wiedergegeben. Transfections in mouse fibroblasts did not show the effects described above (data did not shown). All data are exemplary for the antisense ODN Hannah-05 played.
Claims (7)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE2002100410 DE10200410A1 (en) | 2002-01-08 | 2002-01-08 | New antisense oligonucleotide, useful for treatment of tumors, inhibits translation of mRNA from the pituitary-tumor transforming gene-1 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE2002100410 DE10200410A1 (en) | 2002-01-08 | 2002-01-08 | New antisense oligonucleotide, useful for treatment of tumors, inhibits translation of mRNA from the pituitary-tumor transforming gene-1 |
Publications (1)
| Publication Number | Publication Date |
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| DE2002100410 Ceased DE10200410A1 (en) | 2002-01-08 | 2002-01-08 | New antisense oligonucleotide, useful for treatment of tumors, inhibits translation of mRNA from the pituitary-tumor transforming gene-1 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006088490A3 (en) * | 2004-06-30 | 2007-03-29 | Alnylam Pharmaceuticals Inc | Oligonucleotides comprising a non-phosphate backbone linkage |
| US9227956B2 (en) | 2013-04-17 | 2016-01-05 | Pfizer Inc. | Substituted amide compounds |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001087934A2 (en) * | 2000-05-12 | 2001-11-22 | Cedars-Sinai Medical Center | Treatment of neoplasia/transformation using a pituitary tumor transforming gene carboxy terminal peptides |
-
2002
- 2002-01-08 DE DE2002100410 patent/DE10200410A1/en not_active Ceased
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001087934A2 (en) * | 2000-05-12 | 2001-11-22 | Cedars-Sinai Medical Center | Treatment of neoplasia/transformation using a pituitary tumor transforming gene carboxy terminal peptides |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006088490A3 (en) * | 2004-06-30 | 2007-03-29 | Alnylam Pharmaceuticals Inc | Oligonucleotides comprising a non-phosphate backbone linkage |
| US7615618B2 (en) | 2004-06-30 | 2009-11-10 | Alnylam Pharmaceuticals, Inc. | Oligonucleotides comprising a non-phosphate backbone linkage |
| US7723512B2 (en) | 2004-06-30 | 2010-05-25 | Alnylam Pharmaceuticals | Oligonucleotides comprising a non-phosphate backbone linkage |
| AU2005327517B2 (en) * | 2004-06-30 | 2011-05-26 | Alnylam Pharmaceuticals, Inc. | Oligonucleotides comprising a non-phosphate backbone linkage |
| US8013136B2 (en) | 2004-06-30 | 2011-09-06 | Alnylam Pharmaceuticals, Inc. | Oligonucleotides comprising a non-phosphate backbone linkage |
| US9227956B2 (en) | 2013-04-17 | 2016-01-05 | Pfizer Inc. | Substituted amide compounds |
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