WO2000027858A1 - Oligonucleotides antisens inhibant l'activite de la telomerase et leurs applications - Google Patents

Oligonucleotides antisens inhibant l'activite de la telomerase et leurs applications Download PDF

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WO2000027858A1
WO2000027858A1 PCT/CN1999/000173 CN9900173W WO0027858A1 WO 2000027858 A1 WO2000027858 A1 WO 2000027858A1 CN 9900173 W CN9900173 W CN 9900173W WO 0027858 A1 WO0027858 A1 WO 0027858A1
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seq
hest212
oligonucleotide
telomerase
oligonucleotide according
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PCT/CN1999/000173
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Chinese (zh)
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Shengqi Wang
Xiaofei Zheng
Baozhen Zhu
Ruiyun Xing
Wei Guan
Zhixian Sun
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Institute Of Radiation Medicine, Academy Of Military Medecine Science
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    • 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
    • C12N15/1137—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 enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Antisense oligonucleotide for inhibiting telomerase activity and application thereof
  • the invention relates to antisense oligonucleotides, in particular to antisense oligonucleotides that inhibit human telomerase activity, a pharmaceutical composition containing the antisense oligonucleotides, and the antisense oligonucleotides. Application in inhibiting the growth of malignant tumors.
  • telomeres are the ends of chromosomes in eukaryotic cells
  • telomere DNA A DNA sequence whose function is to maintain the stability of a chromosome.
  • the length and stability of telomere DNA determine the life span of cells, and are closely related to cell aging and canceration.
  • Telomeres are synthesized by telomerase. Studies have found that telomerase activity is found in immortal cells and 85% of human malignant tumor cells, while telomerase is not expressed in most normal human cells, so telomerase is expected to become a marker for cancer diagnosis Targets for cancer treatment and cancer (Shay JW et al. Curr Opin oncol, 1996, 8 (l): 66-71). Telomerase is an enzyme composed of a nucleoprotein complex composed of RNA and proteins.
  • telomere synthesis its own RNA component provides a template for telomere synthesis at the ends of chromosomes.
  • WEST2 protein subunit with reverse transcriptase activity is the main rate-limiting step for cells to regulate telomerase activity (Meyerson S et al. Cell, 1997, 90: 785-788).
  • Antisense Oligonucleotide refers to a nucleotide oligomer that specifically complements and hybridizes with the meaningful strand of a specific gene or the RNA transcribed from it, and is used to treat tumors and viral infectious diseases (Stein et al. Science 1993, 262 (60 (: 1004-1012))
  • ASON Antisense Oligonucleotide
  • the inventors believe that the antisense oligonucleotide targeting human telomerase hEST2 protein subunit gene structure can inhibit the activity of telomerase, thereby achieving the purpose of inhibiting the growth of tumor cells. Based on this concept, the present inventors conducted a lot of in-depth research and finally completed the present invention.
  • an object of the present invention is to provide an antisense oligonucleotide designed and prepared based on a gene sequence encoding a human telomerase hEST2 protein subunit.
  • a further object of the present invention is to provide a pharmaceutical composition comprising one or more antisense oligonucleotides of the present invention, which can be used as a cancer treatment drug for inhibiting the growth of malignant tumor cells expressing telomerase activity .
  • Another object of the present invention is to provide a test kit comprising the antisense oligonucleotide of the present invention, which can be used to detect the telomerase hEST2 RNA component or the separation of DNA and nucleic acid encoding the telomerase hEST2.
  • an antisense oligonucleotide which is specifically related to a gene encoding human telomerase having a reverse transcriptase active protein subunit (hEST2) gene or an mRNA transcribed therefrom
  • hEST2 reverse transcriptase active protein subunit
  • a part of the HEST2 gene is complementary and hybridized with it, and a part of the hEST2 gene is preferably 3 'and 5' untranslated regions, and the length of the antisense oligonucleotide is 10-30 nucleotides, preferably 15- 25 nucleotides, most preferably 18-22 nucleotides.
  • a pharmaceutical composition comprising a therapeutically effective amount of one or more antisense oligonucleotides of the present invention and a pharmaceutically acceptable carrier, which can be used to inhibit expression Telomerase Activity Cancer Therapy for Malignant Tumor Cell Growth
  • the cancers are, for example, liver cancer, lung cancer, stomach cancer, breast cancer and glioma.
  • a detection kit comprising the antisense oligonucleotide of the present invention, which can be used to detect telomerase hEST2 RNA components or to isolate DNA and nucleic acid encoding telomerase hEST2.
  • the antisense oligonucleotide is labeled with a portion as a signal to facilitate detection.
  • the antisense oligonucleotide of the present invention is a human telomerase encoding reverse transcriptase active protein subunit (hEST2) determined according to, for example, Morin et al. (Morin GB, et al. Science 1997,277 (5328): 955-959).
  • the gene structure is designed to have a length of 10-30 nucleotides, preferably 15-25 nucleotides, and most preferably 18-22 nucleotides.
  • the antisense oligonucleotide of the invention is complementary to the 3, and 5 'untranslated regions of the hEST2 gene.
  • the antisense oligonucleotide of the present invention specifically hybridizes to a part of the hEST2 gene or specifically to a part of the transcribed mRNA, thereby inhibiting its expression.
  • Some illustrative but non-limiting examples of antisense oligonucleotides of the invention are shown in Table 1 below.
  • the oligonucleotides of the invention have the sequences shown in SEQ ID NOS: 6, 16, 17, and 18.
  • the antisense oligonucleotides of the invention are modified.
  • Antisense oligonucleotides have been extensively developed as a new class of drugs (Saghir Akhtar and Sudhir Agrawal, TiPs, 1997; 18: 12-18). Experiments have shown that natural antisense oligonucleotides (0-ASON) are easily degraded by nucleases in vivo and in vitro, which greatly affects their biological activity (Sands H, Gorey-Feret LJ, Cocuzza AJ et al, Mol. Pharmacol 1994; 45: 922-43).
  • Y may be oxygen, or sulfur, or methoxy, etc., preferably Y is sulfur, therefore, the antisense oligonucleotide of the present invention is preferably all thio oligonucleotides.
  • the modification may involve all phosphodiester bonds, or it may involve partial phosphodiester bonds.
  • the antisense oligonucleotide of the present invention may also be modified by 3, such as Y acid, 3 'cholesterol modification or 3' fatty chain modification, preferably 3 'phosphorylation modification.
  • the hybridization of the antisense oligonucleotide of the present invention with a partial sequence of the hEST2 gene or its transcribed mRNA will prevent the synthesis of the hEST2 protein subunit, thereby inhibiting the growth of malignant tumor cells.
  • hEST212 (SEQ ID NO: 17) is used as an example to determine the antisense oligonucleotide antiproliferation effect of different tumor cells of the present invention, and the results prove that the antisense oligonucleotide of the present invention Glycosides and their modifications can inhibit the growth of tumor cells such as liver cancer, lung cancer, glioma, gastric cancer and breast cancer cells and the growth of tumors in nude mice inoculated with tumor cells. Therefore, it was found that the antisense oligonucleotide of the present invention has a good effect in inhibiting the growth of malignant tumor cells, and has a malignant tumor for inhibiting the expression of telomerase activity.
  • another aspect of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a pharmaceutically effective amount of the antisense oligonucleotide of the present invention or a modification thereof, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the present invention can be used for inhibition Growth of Malignant Tumor Cells Expressing Telomerase Activity.
  • the cancers are, for example, liver cancer, lung cancer, stomach cancer, breast cancer and glioma.
  • the antisense oligonucleotide of the present invention when the antisense oligonucleotide of the present invention is immobilized on a solid support according to a known technique, it can be used for the isolation of nucleic acids and its own telomerase hEST2 RNA component or encoding telomerase hEST2 Detection of DNA. Therefore, the present invention also relates to a detection kit comprising the antisense oligonucleotide of the present invention, wherein the oligonucleotide used for detection of the present invention can be labeled with a portion serving as a signal according to known technical methods, Such as radioisotopes, alcohols, fluorescent compounds and so on.
  • Figure 1 shows the inhibition of HepG2 cell growth by the thiotelomerase hEST2 antisense oligonucleotide (hEST21- hEST220).
  • the numbers listed on the horizontal axis in the figure are 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 represent the thio antisense oligonucleotides hEST21, hEST23, hEST25, hEST27, hEST29, hEST211, hEST213, hEST215, hEST219 of the present invention, respectively, and the other antisense oligonucleotides are arranged in numerical order. .
  • Figure 2 shows the inhibition of tumor growth of thiotelomerase hEST2 antisense oligonucleotides on nude mice inoculated with HepG2 liver cancer cells.
  • Figure 3 shows the inhibition rate of thiotelomerase hEST2 antisense oligonucleotides on tumors in nude mice inoculated with HepG2 liver cancer cells.
  • Figure 4 shows the effect of thiotelomerase hEST2 antisense oligonucleotides on tumorigenicity in nude mice inoculated with HepG2 liver cancer cells.
  • FIG. 5 shows the weight gain of nude mice in the thiotelomerase hEST2 antisense oligonucleotide administration group and the control group.
  • FIG. 6 is a photograph of tumors in nude mice administered with a thiotelomerase hEST2 antisense oligonucleotide and a control group.
  • Figure 7 shows the tumor growth inhibition curves of telomerase hEST212 thioantisense oligonucleotides in different doses on nude mice inoculated with HepG2 liver cancer cells.
  • Fig. 8 shows the tumor suppressive rate of telomerase hEST212 thioantisense oligonucleotides in different dose administration groups inoculated with HepG2 liver cancer cells in nude mice.
  • Fig. 9 shows the effects of telomerase hEST212 thioantisense oligonucleotides administered in different doses and control groups on tumorigenicity in nude mice inoculated with HepG2 liver cancer cells.
  • Figure 10 shows the weight gain of telomerase hEST212 thioantisense oligonucleotides administered to different dose groups and control groups to nude mice inoculated with HepG2 liver cancer cells.
  • Figure 11 is a photograph of tumors in nude mice administered with telomerase hEST212 thioantisense oligonucleotide at different doses.
  • Figure 12 is a thin layer chromatogram of the degradation products of modified antisense oligonucleotides.
  • Figure 13 shows the effects of various chemical modifications on the cell proliferation inhibitory activity of hEST212, see Example 4.
  • Figure 14 shows the inhibitory activity of thio antisense oligonucleotides at different sites on HepG2 cell proliferation, see Example 5.
  • Figure 15 shows the sequence specificity of hEST212, see Example 6.
  • Figure 16 shows the antitumor spectrum of hEST212, see Example 7.
  • Figure 17 shows the persistence of hEST212 action, see Example 8.
  • Figure 18 shows the effect of dosing times on hEST212 inhibitory activity, see Example 9.
  • All antisense oligonucleotides used in the examples are based on the cDNA sequence of the human telomerase hEST2 protein subunit ⁇ U (Morin GB, et al. Science 1997,277 (5328) : 955-959) were synthesized using an Applied Biosystems 391 DNA Synthesizer.
  • modified oligonucleotides various modifications are made during synthesis, synthetic and chemical modifications
  • the raw materials are the products of American Glen Company. After synthesis, the solution was deprotected with concentrated ammonia water at 55 ° C for 15 hours, and then purified by chromatography using a Micro Pure II reversed-phase purification column (purchased from Solid Phase Sciences).
  • the cell culture, liposome transfection, and cell proliferation inhibitory activity assays used in the examples are as follows:
  • the various cells used in the examples were obtained from the Institute of Microbiology and Epidemiology of the Academy of Military Medical Sciences.
  • the cells were cultured in a DMEM medium containing 10% calf serum and 100 U / ml penicillin and 100 ⁇ g / ml streptomycin at 37 ° C in a 5% CO 2 incubator.
  • a 96-well cell culture plate each well was seeded with 5 ⁇ 10 3 cells / 0.2 ml. After 50-60% of the cells were confluent, antisense oligonucleotides were transfected with Lipofectm (GIBCO, 1 mg / ml) reagent in a serum-free state according to the instructions.
  • GIBCO Lipofectm
  • antisense oligonucleotides and liposomes were diluted in serum-free DMEM medium in a sterile 1.5 ml centrifuge tube, and left at room temperature for 30 minutes. The solution was mixed and left for 30 minutes at room temperature to form a liposome-antisense oligonucleotide complex, and then added serum-free DMEM medium. The cultured cells were washed twice with serum-free DMEM medium, and a total volume of 100 ⁇ l of liposome-antisense oligonucleotide complex solution was added to each well for 5 hours. The cells were replaced with DMEM medium containing 10% calf serum Incubate for 19 hours.
  • Antisense oligonucleotides were used in each well at a final concentration of 0.5 w mol / L, liposome lg, and the total volume was 100 ⁇ . Three antisense oligonucleotides were set up for each cell, and cells and liposomes were set up. Negative control. Light absorption was measured at 490 nm. The effect of antisense oligonucleotides on cell growth was evaluated by the number of cell growth doublings.
  • the antisense oligonucleotides used are hEST21, hEST22, hEST23, hEST24, hEST25, hEST26, hEST27, hEST28, hEST29 hEST210 hEST211, hEST212, hEST213 hEST214, hEST215, hEST216, hEST217, hEST218, hEST219, hEST220, all oligonucleotides are all thioantisense oligonucleotides.
  • thioantisense oligonucleotides hEST21-hEST220 that inhibit the growth of HepG2 cells.
  • Twenty thioantisense oligonucleotides have different programs for inhibiting HepG2 cells. Among them, hEST21, hEST22T, and hEST218 inhibit the growth of HepG2 cells by 62.3%, 55.4%, and 53.2%, respectively; hEST25, hEST26, hEST28, hEST29, hEST210, hEST214, hEST217, and hEST219 all inhibited the growth of HepG2 cells by more than 30%.
  • telomerase hEST2 antisense oligonucleotide can effectively inhibit the growth of HepG2 liver cancer cells.
  • Example 2 Inhibition of tumor growth by antisense oligonucleotides in nude mice inoculated with HepG2 liver cancer cells
  • HepG2 cells were cultured in DMEM medium containing 10% calf serum. The cells were collected, washed twice with PBS, and resuspended in physiological saline. 35 6-week-old female nude mice (purchased from Beijing Medical University Animal Center), weighed mice, each mouse injected hind armpit 5 X 10 5 HepG2 cells /0.2ml. Ten days later, they were randomly divided into 7 groups, 5 in each group, and started to be administered by intraperitoneal injection. One group was the control group and was injected intraperitoneally with physiological saline 0.1ml. The second group was hEST21 group and was administered 500 wg / 0.1ml daily.
  • Three groups are hEST24 group, daily administration of 500 ⁇ g / 0.1ml; four groups are hEST29 group, daily administration of 500 ⁇ g / 0.1ml; five groups are hEST212 group, daily administration of 500 yg / 0.1ml; Six groups were hEST217 group, daily administration of 500 ⁇ g / 0.1ml; seven groups were hEST218 group, daily administration of 500 ⁇ g / 0.1ml. Tumor size was measured regularly for a total of 18 days. Nude mice were sacrificed, tumor size was measured, and rat weight and tumor weight were weighed. All oligonucleotides used were all thioantisense oligonucleotides.
  • FIG. 4 shows the effect of each antisense oligonucleotide on tumor formation in nude mice inoculated with HepG2 cells.
  • the tumor rate of the control composition was 100%, and the tumor formation rates of the hEST21, hEST24, hEST29, hEST212, hEST217, and hEST218 groups were 60%, 80%> 60 60%, 100%, and 60%.
  • Figure 5 shows the growth of nude mice in each group without significant differences between groups.
  • Figure 6 shows the tumor pictures of each group.
  • telomerase hEST2 thio antisense oligonucleotide can inhibit tumor growth in nude mice inoculated with HepG2 liver cancer cells, and can be applied to inhibit tumor cell growth.
  • Example 3 Dependence of hEST212 thioantisense oligonucleotide on tumor growth inhibition in nude mice inoculated with HepG2 liver cancer cells
  • HepG2 cells were cultured with DMEM medium containing 10% calf serum. The cells were collected, washed twice with PBS, and resuspended in physiological saline. Forty six 6-week-old female nude mice were weighed, and each mouse was injected with IX 10 6 HepG2 cells / 0.2 ml under the axils of the hind legs. Five days later, they were randomly divided into four groups. Ten mice in each group were started by intraperitoneal injection. The control group was injected with 0.1 ml of normal saline daily. The experimental group was injected with thioantisense oligonucleotide hEST212. After 35 days of administration, 500 ⁇ g / 0.1ml, 100 ⁇ g / 0.1ml and 20 ⁇ g / 0.1ml were administered to nude mice, the tumor size was measured, and the tumor weight and tumor weight were weighed.
  • the tumor growth rate has a significant dependence on the dose of antisense oligonucleotides.
  • the inhibition rates of thioantisense oligonucleotide hEST212 on tumors of nude mice inoculated with HepG2 cells were 78.7%, 52.5% and 5.9%, respectively.
  • the tumor formation rate was 100% in the control group, and the tumor formation rates were 90%, 90%, and 80% as the dose was increased.
  • telomerase thioantisense oligonucleotides have a significant inhibitory effect on tumor growth in nude mice inoculated with HepG2 liver cancer cells, and may be used to inhibit the growth of tumor cells. Therefore, telomerase thioantisense oligonucleotide may be a potentially specific and broad-spectrum drug for inhibiting tumor cell growth, which is confirmed in the following examples.
  • Example 4 Effects of chemical modification on the stability and biological activity of antisense oligonucleotides 1. Chemical modification of antisense oligonucleotides
  • the hEST212 sequence was selected for the synthesis of natural and thioantisense oligonucleotides, and was subjected to 3 'phosphorylation and cholesterol modification, respectively.
  • the thiohEST212 sequence was also modified for the 3' end fatty chain.
  • a partially thio-modified hEST212, a hybrid backbone antisense oligonucleotide with 4 nucleotides at the 3 'and 5' ends and a natural oligonucleotide structure in the middle region was synthesized. 2.
  • HepG2 hepatoma cells were inoculated into 96-well plates, and liposome-mediated administration was used.
  • the final concentration of each of the modified antisense oligonucleotides was 200 mol / L, 400 mol / L, 800 ⁇ 1 / ⁇ , and the total volume was 100 ⁇ 1.
  • Cell and liposome controls were set.
  • the inhibitory activity of different modified antisense oligonucleotides was evaluated by cell growth inhibition.
  • hEST212-S in 4 kinds of liquids reacted for 24 hours without any obvious degradation.
  • the results of the P reaction were consistent with hEST212.
  • hEST212-0 was degraded in undiluted calf serum for 4 hours, and degradation gradually became apparent as the reaction time became longer. It degraded significantly in the supernatant of HepG2 cell lysis, and began to degrade in 0.5 hours. It is basically completely degraded, and is basically stable in DMEM and water containing 10% fetal bovine serum.
  • hEST212-Cap degraded significantly in the lysed supernatant of HepG2 cells, but was basically stable in undiluted calf serum, DMEM containing 10% fetal calf serum, and water, as shown in Figure 12.
  • hEST212 had the highest inhibitory activity under the same experimental conditions. The inhibition rate reached 71.6%. HEST212-3T, hEST212-0, hEST212-0-3, and hEST212-Cap showed mild inhibitory activity, while fatty chain and cholesterol-modified antisense oligonucleotides had essentially no inhibitory activity. The experimental results show that thio-modified antisense oligonucleotides have the best inhibitory activity, and other modifications have mild effects on antisense oligonucleotides. Example 5 Optimization of Antisense Oligonucleotide Sequences
  • telomerase hEST2 protein subunit mRNA sequence twelve antisense oligonucleotides (see Table 3) were designed, and the oligonucleotides were all thiooligonucleotides.
  • the activity measurement HepG2 hepatocellular carcinoma cells were seeded in a 96-well plate, and liposome-mediated administration was used.
  • the final concentrations of thioantisense oligonucleotides used in each well were 0.2 mol / L, 0.4 mol / L, and 0.8 mol / L.
  • the total volume is 100 ⁇ l, 3 wells per antisense oligonucleotide, and cell and liposome controls.
  • Cell growth inhibition was evaluated by the number of cell growth doublings of thioantisense oligonucleotides at different targets.
  • hEST212d 3569-3584 ACTCAGGCCTCAGACT Note: hEST2 : human telomerase has reverse transcriptase activity.
  • the sequence of the protein subunits in the table is all thio products. As can be seen from FIG. (See Table 3) 11 of them have different degrees of inhibition on the proliferation of HepG2 hepatocellular carcinoma cells, and they have a good dose dependence.
  • thioantisense oligonucleotides targeting the non-coding region of the 3 ′ end of the hEST2 gene have a strong inhibitory effect and have a significant relationship with the length.
  • the hEST212a lacking 2 bases at the 3 ′ end has little difference in the inhibitory effect at high concentrations.
  • hEST212b 3 'missing 4 bases
  • hEST212 c 5' missing 2 bases
  • hEST212d 5 'missing 4 bases
  • Targeting other targets including the 5, cap and translation initiation regions of thio antisense oligonucleotides of different lengths have no significant inhibitory effect.
  • the above results indicate that antisense oligonucleotides targeting specific sites of the telomerase hEST2 gene can effectively inhibit the growth of HepG2 liver cancer cells.
  • Example 6 Specific analysis of the effect of antisense oligonucleotides
  • Sensed, random and mismatched thio-oligonucleotides of hEST212 were synthesized (see Table 4). Cell culture, liposome transfection, and tumor cell growth inhibitory activity assays were performed using the methods described above.
  • the anti-tumor spectrum is another evaluation index of anti-tumor drugs.
  • this example uses thiohEST212 as an example to determine its inhibitory effect on the proliferation of different tumor cells.
  • human liver cancer (HepG2), lung cancer (GLC), glioma (BT325), gastric cancer (BGC823), and breast cancer (MCF-7) cells were respectively inoculated into 96-well plates, and cultured and cultured according to the methods described above.
  • the sense oligonucleotide liposome was transfected and administered twice. Logistic software was used to calculate IC50 ( ⁇ mol / L).
  • hEST212 inhibited liver cancer (HepG2), lung cancer (GLC), glioma (BT325), gastric cancer (BGC823), and breast cancer (MCF-7) cells to varying degrees.
  • HepG2 liver cancer
  • LLC lung cancer
  • BGC823 gastric cancer
  • MCF-7 breast cancer
  • IC50 0.56 ⁇ mol / L
  • 0.58 ⁇ mol / L 0.51 ⁇ mol
  • hEST212 has a poor inhibitory effect on gliomas with IC50 of 0.77 mol / L, as shown in Figure 16.
  • this example uses thiohEST212 as an example to determine the duration of the antisense oligonucleotide action.
  • HepG2 cells were seeded in a 96-well plate. After 50-60% of the cells were confluent, thiohEST212 was transfected one time, with a final concentration of 0.4 w mol / L. Respectively On the 1st to 15th days after transfection, 3 wells of each cell were taken to determine the inhibition of cell proliferation, and the duration of action was observed.
  • this example uses thiohEST212 as an example to determine the effect of the number of administrations on the antisense oligonucleotide inhibitory activity.
  • HepG2 cells were seeded in 96-well plates. After 50-60% of the cells were confluent, they were transfected with thiohEST212 1-2 times, once / day, with a final concentration of 0.4 mol / L. Twenty-four hours after transfection, three wells of each cell were taken to determine the inhibition of cell proliferation, and the effect of the number of doses on cell proliferation was observed.
  • the number of administrations had little effect on the tumor cell proliferation inhibitory activity of hEST212. As shown in FIG. 18, only at low and medium concentrations, the tumor cell proliferation inhibitory activity tended to increase slightly with the number of administrations. At high concentrations of 0.8 u mol / L, there were no significant differences between 3 and 2 consecutive doses.

Abstract

L'invention concerne des oligonucléotides antisens inhibant l'activité de la télomérase humaine, une composition pharmaceutique contenant ces oligonucléotides antisens et leur application dans l'inhibition de la croissance de tumeurs malignes.
PCT/CN1999/000173 1998-11-09 1999-10-29 Oligonucleotides antisens inhibant l'activite de la telomerase et leurs applications WO2000027858A1 (fr)

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CN100411689C (zh) * 2006-02-14 2008-08-20 南京吉脉生物技术有限公司 抑制端粒酶逆转录酶hTERT表达的ShRNA在制备抗肿瘤药物中的应用
CN102218028B (zh) * 2011-03-24 2013-08-14 杭州天龙药业有限公司 一种治疗肝癌的反义寡核苷酸注射剂及其制备方法
AU2016377398B2 (en) 2015-12-23 2022-10-13 Repluca Pty Ltd Nucleic acid oligomers and uses therefor
CN109182558B (zh) * 2018-11-12 2020-08-25 中山大学孙逸仙纪念医院 能预示和鉴定绵羊羊毛自然长度的分子标记引物对及应用
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WO2000063429A2 (fr) * 1999-04-15 2000-10-26 Bayer Aktiengesellschaft Test rapide automatisable pour la detection de cancers a base d'arnm de telomerase(htc), ainsi qu'amorces et sondes specifiques
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