WO2000027858A1 - Antisense oligonucleotides which inhibit telomerase's activity and their uses - Google Patents

Antisense oligonucleotides which inhibit telomerase's activity and their uses 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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French (fr)
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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Priority to AU64591/99A priority Critical patent/AU6459199A/en
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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.

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Abstract

The invention discloses antisense oligonucleotides which inhibit the activity of human telomerase, pharmaceutical composition containing said antisense oligonucleotides and the use of antisense oligonucleotides in inhibiting the growth of malignant tumor.

Description

抑制端粒酶活性的反义寡核苷酸及其应用 发明领域  Antisense oligonucleotide for inhibiting telomerase activity and application thereof
本发明涉及反义寡核苷酸， 具体地说涉及抑制人端粒酶活性的反 义寡核苷酸， 含有所述反义寡核苷酸的药物组合物及所述反义寡核苷 酸在抑制恶性肿瘤生长中的应用。  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.
背景技术  Background technique
癌症是威胁人类生命的重大疾病， 对癌症的治疗一直是科学界关 注的问题。 由于目前广泛使用的放疗和化疗等手段不仅缺少对癌细胞 良好的特异性杀伤力， 而且有较大的毒副作用， 在很大程度上使治疗 的范围和效果都受到了限制。 因此， 寻找一种特异性高、 毒副作用小 的新型癌症治疗药物对癌症的治疗具有重要的现实意义。 近来研究表 明端粒酶与癌症有着极为密切的关系。 端粒是真核细胞染色体末端的 Cancer is a major disease that threatens human life. The treatment of cancer has always been a concern of the scientific community. As the currently widely used methods of radiotherapy and chemotherapy not only lack good specific killing power for cancer cells, but also have large toxic and side effects, the scope and effect of treatment have been largely restricted. Therefore, finding a new cancer treatment drug with high specificity and low toxic and side effects has important practical significance for cancer treatment. Recent studies have shown that telomerase has a very close relationship with cancer. Telomeres are the ends of chromosomes in eukaryotic cells
DNA序列， 其功能是保持染色体的稳定性。 端粒 DNA的长短和稳定 性决定了细胞的寿命， 并与细胞的衰老和癌变密切相关。 端粒是由端 粒酶合成的。 研究发现在永生细胞和 85 %的人恶性肿瘤细胞中均有端 粒酶的活性， 而在绝大多数正常人体细胞中却没有端粒酶的表达， 因 而端粒酶就有望成为癌症诊断的标志物和癌症治疗的靶点（ Shay J W et al.Curr Opin oncol,1996,8(l):66-71 ) 。 端粒酶是一种由 RNA和蛋白质 组成的核蛋白复合体构成的酶， 在端粒合成时由自身的 RNA组分提 供染色体末端端粒合成的模板。 其中具有逆转录酶活性的 WEST2蛋白 亚基的表达是细胞调节端粒酶活性的主要限速步骤 （Meyerson S et al. Cell, 1997,90:785-788 ) 。 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. During telomere synthesis, its own RNA component provides a template for telomere synthesis at the ends of chromosomes. Among them, the expression of 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).
反义寡核苷酸 （Antisense Oligonucleotide, ASON) 是指能够与特 定基因的有意义链或由它转录的 RNA特异性互补并能杂交的核苷酸 寡聚物，是治疗肿瘤和病毒性传染病的潜在新型药物（Stein et al.Science 1993,262(60(: 1004-1012)o 国内外已尝试采用反义寡核苷酸治疗恶性肿 瘤。 本发明人认为可以通过针对编码人端粒酶 hEST2蛋白质亚基基因 结构的反义寡苷酸抑制端粒酶的活性， 从而达到抑制肿瘤细胞生长的 目的。 基于这种理念， 本发明人进行了大量深入的研究， 终于完成了 本发明。 Antisense Oligonucleotide (ASON) 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)) At home and abroad, attempts have been made to use antisense oligonucleotides to treat malignant tumors Tumor. 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.
因此， 本发明的一个目的是提供根据编码人端粒酶 hEST2蛋白质 亚基基因序列设计并制备的反义寡核苷酸。  Therefore, 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.
本发明的再一目的在于提供一种药物组合物， 其包括本发明的一 种或多种反义寡核苷酸， 其可做为抑制表达端粒酶活性的恶性肿瘤细 胞生长的癌症治疗药物。  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 .
本发明的又一目的在于提供一种包括本发明的反义寡核苷酸的检 测试剂盒，其可用于检测端粒酶 hEST2 RNA组分或编码端粒酶 hEST2 的 DNA及核酸的分离。  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.
发明概述  Summary of invention
根据本发明的一个方面， 提供了反义寡核苷酸， 所述的反义寡核 苷酸特异地与编码人端粒酶具有逆转录酶活性蛋白质亚基 （hEST2 ) 基因或其转录的 mRNA的一部分互补并与之杂交， 所述的 hEST2基 因的一部分优选地为 3 ' 及 5 ' 非翻译区， 所述的反义寡核苷酸长度 为 10— 30个核苷酸， 优选为 15— 25个核苷酸， 最优选为 18— 22个 核苷酸。  According to an aspect of the present invention, there is provided 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 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.
根据本发明的另一方面， 提供了一种药物组合物， 其包括治疗有 效量的本发明的一种或多种反义寡核苷酸以及药物学可接受的载体， 其可做为抑制表达端粒酶活性的恶性肿瘤细胞生长的癌症治疗药物。 所述的癌症例如肝癌、 肺癌、 胃癌、 乳腺癌和脑胶质瘤等。  According to another aspect of the present invention, there is provided 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.
根据本发明的又一方面， 提供了一种包括本发明的反义寡核苷酸 的检测试剂盒， 其可用于检测端粒酶 hEST2 RNA 组分或编码端粒酶 hEST2 的 DNA及核酸的分离， 其中所述的反义寡核苷酸与作为信号 的部分标记以便于检测。 发明详述 According to yet another aspect of the present invention, there is provided 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. Detailed description of the invention
本发明的反义寡核苷酸是根据例如 Morin 等 （Morin GB， et al.Science 1997,277(5328):955-959) 测定的编码人端粒酶具有逆转录酶 活性蛋白质亚基 （hEST2 ) 基因结构设计的， 其长度为 10— 30个核苷 酸， 优选为 15— 25个核苷酸， 最优选为 18— 22个核苷酸。 优选地， 本发明的反义寡核苷酸与 hEST2基因的 3， 和 5 ' 非翻译区互补。 在 正常生理条件下， 本发明的反义寡核苷酸与 hEST2基因的一部分特异 杂交或与转录的 mRNA 的一部分特异杂交， 由此抑制其表达。 本发 明的反义寡核苷酸的一些举例性但非限制性的例子见下表 1。 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. Preferably, the antisense oligonucleotide of the invention is complementary to the 3, and 5 'untranslated regions of the hEST2 gene. Under normal physiological conditions, 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.
表 1 反义寡核苷酸序列及性质 Table 1 Antisense oligonucleotide sequences and properties
SEQ ID 名称 hEST2靶位 长度 特性 寡核苷酸序列  SEQ ID Name hEST2 Target Length Characteristics Oligonucleotide Sequence
NO . (nt) (nt)  NO. (Nt) (nt)
1 hEST21 1-20 20 A 5 ' GCAGCAGGACGCAGCGCTGC 1 hEST21 1-20 20 A 5 'GCAGCAGGACGCAGCGCTGC
2 hEST21a 1-15 15 A 5 ' GCAGCAGGACGCAGC2 hEST21a 1-15 15 A 5 'GCAGCAGGACGCAGC
3 hEST21b 6-20 15 A 5 ' AGGACGCAGCGCTGC3 hEST21b 6-20 15 A 5 'AGGACGCAGCGCTGC
4 hEST22 11-30 20 A 5 ' TCCCACGTGCGCAGCAGGAC4 hEST22 11-30 20 A 5 'TCCCACGTGCGCAGCAGGAC
5 hEST23 30-49 20 A 5 ' GGTGGCCGGGGCCAGGGCTT5 hEST23 30-49 20 A 5 'GGTGGCCGGGGCCAGGGCTT
6 hEST24 46-65 20 A 5 ' CGCGCGGCATCGCGGGGGTG6 hEST24 46-65 20 A 5 'CGCGCGGCATCGCGGGGGTG
7 hEST24a 49-65 17 A 5 ' GCGGCATCGCGGGGGTG7 hEST24a 49-65 17 A 5 'GCGGCATCGCGGGGGTG
8 hEST24b 46-62 17 A 5 ' CGCGCGGCATCGCGGGG8 hEST24b 46-62 17 A 5 'CGCGCGGCATCGCGGGG
9 hEST24c 46-60 15 A 5 ' CGCGCGGCATCGCGG9 hEST24c 46-60 15 A 5 'CGCGCGGCATCGCGG
10 hEST25 71-90 20 A 5 ' GAGCGCACGGCTCGGCAGCG10 hEST25 71-90 20 A 5 'GAGCGCACGGCTCGGCAGCG
11 hEST26 94-113 20 A 5 ' CGCGGTAGTGGCTGCGCAGC11 hEST26 94-113 20 A 5 'CGCGGTAGTGGCTGCGCAGC
12 hEST27 3421-3440 20 A 5 ' TCTTGAAGTCTGAGGGCAGT12 hEST27 3421-3440 20 A 5 'TCTTGAAGTCTGAGGGCAGT
13 hEST28 3432-3451 20 A 5 ， GTCCAGGATGGTCTTGAAGT13 hEST28 3432-3451 20 A 5, GTCCAGGATGGTCTTGAAGT
14 hEST29 3441-3460 20 A 5 ， TGGCCATCAGTCCAGGATGG14 hEST29 3441-3460 20 A 5, TGGCCATCAGTCCAGGATGG
15 hEST210 3464-3483 20 A 5 ' CTCTCGGCCTGGCTGTGGGC15 hEST210 3464-3483 20 A 5 'CTCTCGGCCTGGCTGTGGGC
16 hEST211 3480-3499 20 A 5 ， GGGCTGCTGGTGTCTGCTCT16 hEST211 3480-3499 20 A 5, GGGCTGCTGGTGTCTGCTCT
17 hEST212 3565-3584 20 A 5 ' ACTCACTCAGGCCTCAGACT17 hEST212 3565-3584 20 A 5 'ACTCACTCAGGCCTCAGACT
18 hEST212a 3565-3582 18 A 5 ' ACTCACTCAGGCCTCAGA18 hEST212a 3565-3582 18 A 5 'ACTCACTCAGGCCTCAGA
19 hEST212b 3565-3580 16 A 5 ' ACTCACTCAGGCCTCA19 hEST212b 3565-3580 16 A 5 'ACTCACTCAGGCCTCA
20 hEST212c 3567-3584 18 A 5 ' TCACTCAGGCCTCAGACT20 hEST212c 3567-3584 18 A 5 'TCACTCAGGCCTCAGACT
21 hEST212d 3569-3584 16 A 5 ， ACTCAGGCCTCAGACT21 hEST212d 3569-3584 16 A 5, ACTCAGGCCTCAGACT
22 hEST213 3610-3629 20 A 5 ' GCCGGACACTCAGCCTTCAG22 hEST213 3610-3629 20 A 5 'GCCGGACACTCAGCCTTCAG
23 hEST214 3679-3698 20 A 5 ' CTGTGGGGAAGTGAAGACGG23 hEST214 3679-3698 20 A 5 'CTGTGGGGAAGTGAAGACGG
24 hEST215 3721-3740 20 A 5 ， TGGTGAGGAAAAGCTGGCCC24 hEST215 3721-3740 20 A 5 ， TGGTGAGGAAAAGCTGGCCC
25 hEST216 3756-3775 20 A 5 ， ATGTGGGGAGTGGAA.GCCGG25 hEST216 3756-3775 20 A 5 ， ATGTGGGGAGTGGAA.GCCGG
26 hEST217 3764-3783 20 A 5 ' CTATTCCTATGTGGGGAGTG26 hEST217 3764-3783 20 A 5 'CTATTCCTATGTGGGGAGTG
27 hEST219 3789-3808 20 A 5 ， GAACAATGGCGAATCTGGGG27 hEST219 3789-3808 20 A 5 ， GAACAATGGCGAATCTGGGG
28 hEST218 3841-3860 20 A 5 ， GTCTCCACCTGGATGGTGGG28 hEST218 3841-3860 20 A 5, GTCTCCACCTGGATGGTGGG
29 hEST220 3975-3996 20 A 5 ' TCAGTATTTTACTCCCACAG29 hEST220 3975-3996 20 A 5 'TCAGTATTTTACTCCCACAG
A: 反义 hEST2: 人端粒酶具有逆转录酶活性蛋白亚基 在一些优选的实施方案中， 本发明的寡核苷酸具有 SEQ ID NOS: 6、 16、 17和 18所示的序列。 A: antisense hEST2: human telomerase has a reverse transcriptase active protein subunit In some preferred embodiments, the oligonucleotides of the invention have the sequences shown in SEQ ID NOS: 6, 16, 17, and 18.
在另一些优选的实施方案中， 本发明的反义寡核苷酸被修饰。 反 义寡核苷酸已被作为一类新型药物进行广泛地开发研究（Saghir Akhtar and Sudhir Agrawal, TiPs, 1997; 18: 12-18 ) 。 实验证明天然反义寡核苷 酸 （0— ASON ) 在体内、 体外实验中易被核酸酶降解， 从而极大地 影响其生物活性 （Sands H, Gorey-Feret L J, Cocuzza A J et al, Mol. Pharmacol. 1994; 45:922-43 ) 。 通过特定的化学修饰可改变反义寡核 苷酸的物理化学特性， 从而增加其抗病毒、 抗肿瘤及对其他特定基因 的表达抑制活性 （王升启， 生物化学与生物物理进展， 1995 ; 22(6):506-11 ) 。 因此， 在一些优选的实施方案中， 本发明的抑制端粒 酶活性的反义寡核苷酸可以通过修饰来增加其稳定性。 已知寡核苷酸 由分子式为一 0— P ( =0 ) ( Y) 一 0—的磷酸二酯键连接， 在本发 明中 Y可以是氧、 或硫、 或甲氧基等， 优选地 Y是硫， 因此， 本发 明的反义寡核苷酸优选地是全部硫代寡核苷酸。 所述的修饰可以涉及 全部磷酸二酯键， 也可以涉及部分磷酸二酯键。 另外， 本发明的反义 寡核苷酸也可以经 3， 修饰， 如 y 磯酸化、 3 ' 胆固醇修饰或 3 ' 脂 肪链修饰， 优选为 3 ' 磷酸化修饰。 本领域技术人员根据本文的教导 可以对反义寡核苷酸作各种修饰， 所有这些修饰都属于本发明范畴。  In other preferred embodiments, 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). The specific chemical modification can change the physicochemical properties of antisense oligonucleotides, thereby increasing their antiviral, antitumor, and inhibitory activities on the expression of other specific genes (Wang Shengqi, Advances in Biochemistry and Biophysics, 1995; 22 (6 ): 506-11). Therefore, in some preferred embodiments, the antisense oligonucleotide of the present invention that inhibits telomerase activity can be modified to increase its stability. It is known that oligonucleotides are linked by a phosphodiester bond having a formula of 0—P (= 0) (Y) —0. In the present invention, 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. In addition, 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. Those skilled in the art can make various modifications to the antisense oligonucleotide based on the teachings herein, all of which are within the scope of the present invention.
本发明的反义寡核苷酸与 hEST2基因或其转录的 mRNA的部分 序列杂交后会阻止 hEST2蛋白质亚基合成， 从而抑制恶性肿瘤细胞的 生长。 在本发明的一个实施例中， 以 hEST212 ( SEQ ID NO: 17) 为 例测定了本发明的反义寡核苷酸对不同肿瘤细胞的增殖抑制作用， 其 结果证明本发明的反义寡核苷酸及其修饰物可以抑制肿瘤细胞如肝 癌、 肺癌、 脑胶质瘤、 胃癌及乳腺癌细胞的生长和接种肿瘤细胞裸鼠 恶性肿瘤的生长。 故此发现本发明的反义寡核苷酸在抑制恶性肿瘤细 胞生长中有良好的作用， 具有成为用于抑制表达端粒酶活性的恶性肿 瘤细胞生长的新型生物工程药物的潜力。 因此， 本发明的另一方面涉 及包括药物学有效量的本发明的反义寡核苷酸或其修饰物以及药物学 可接受的载体的药物组合物， 本发明的药物组合物能用于抑制表达端 粒酶活性的恶性肿瘤细胞生长。 所述的癌症例如肝癌、 肺癌、 胃癌、 乳腺癌和脑胶质瘤等。 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. In one embodiment of the present invention, 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. Potential of Novel Bioengineering Drugs for Tumor Cell Growth Therefore, another aspect of the present invention relates to 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.
另一方面， 本发明的反义寡核苷酸当依照己知的技术方法固定在 固体支持物上时， 可用于核酸的分离及其自身对端粒酶 hEST2 RNA 组分或编码端粒酶 hEST2 DNA 的检测。 因此本发明还涉及一种检测 试剂盒， 其包含本发明的反义寡核苷酸， 其中本发明用于检测的寡核 苷酸可根据已知的技术方法用一个做为信号的部分标记， 如放射性同 位素、 醇、 荧光化合物等。  On the other hand, 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.
本发明参照附图及下列实施例进行解释， 这些实施例的目的是只 是为了解释而不是以任何方式限制本发明。  The invention is explained with reference to the drawings and the following examples, which are intended for explanation only and are not intended to limit the invention in any way.
附图简述  Brief description of the drawings
图 1示出硫代的端粒酶 hEST2反义寡核苷酸 (hEST21— hEST220) 对 HepG2细胞生长的抑制， 图中水平轴上列出的数字 1、 3、 5、 7、 9、 11、 13、 15、 17、 19分别代表本发明的硫代的反义寡核苷酸 hEST21、 hEST23、 hEST25 hEST27、 hEST29、 hEST211、 hEST213、 hEST215、 hEST219, 其它反义寡核苷酸按数字大小依次排列。  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. .
图 2为硫代的端粒酶 hEST2反义寡核苷酸对接种 HepG2肝癌细 胞裸鼠体内肿瘤生长抑制曲线。  Figure 2 shows the inhibition of tumor growth of thiotelomerase hEST2 antisense oligonucleotides on nude mice inoculated with HepG2 liver cancer cells.
图 3示出硫代的端粒酶 hEST2反义寡核苷酸对接种 HepG2肝癌 细胞裸鼠体内肿瘤的抑制率。  Figure 3 shows the inhibition rate of thiotelomerase hEST2 antisense oligonucleotides on tumors in nude mice inoculated with HepG2 liver cancer cells.
图 4示出硫代的端粒酶 hEST2反义寡核苷酸对接种 HepG2肝癌 细胞裸鼠体内成瘤率的作用。  Figure 4 shows the effect of thiotelomerase hEST2 antisense oligonucleotides on tumorigenicity in nude mice inoculated with HepG2 liver cancer cells.
图 5示出硫代的端粒酶 hEST2反义寡核苷酸给药组及对照组裸鼠 体重增量。 图 6为硫代的端粒酶 hEST2反义寡核苷酸给药组及对照组裸鼠肿 瘤照片。 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.
图 7为端粒酶 hEST212硫代反义寡核苷酸不同剂量给药组对接种 HepG2肝癌细胞裸鼠体内肿瘤生长抑制曲线。  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.
图 8示出端粒酶 hEST212硫代反义寡核苷酸不同剂量给药组对接 种 HepG2肝癌细胞裸鼠体内肿瘤抑制率。  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.
图 9示出端粒酶 hEST212硫代反义寡核苷酸不同剂量给药组及对 照组对接种 HepG2肝癌细胞裸鼠体内成瘤率的作用。  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.
图 10示出端粒酶 hEST212硫代反义寡核苷酸不同剂量给药组及 对照组对接种 HepG2肝癌细胞裸鼠体重增量。  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.
图 11 为端粒酶 hEST212硫代反义寡核苷酸不同剂量给药组裸鼠 肿瘤照片。  Figure 11 is a photograph of tumors in nude mice administered with telomerase hEST212 thioantisense oligonucleotide at different doses.
图 12为修饰的反义寡核苷酸降解产物的薄层色谱图。  Figure 12 is a thin layer chromatogram of the degradation products of modified antisense oligonucleotides.
图 13示出各种化学修饰对 hEST212的细胞增殖抑制活性的影响， 见实施例 4。  Figure 13 shows the effects of various chemical modifications on the cell proliferation inhibitory activity of hEST212, see Example 4.
图 14示出不同作用位点的硫代反义寡核苷酸对 HepG2细胞增殖 抑制活性， 见实施例 5。  Figure 14 shows the inhibitory activity of thio antisense oligonucleotides at different sites on HepG2 cell proliferation, see Example 5.
图 15 示出 hEST212作用的序列特异性， 见实施例 6。  Figure 15 shows the sequence specificity of hEST212, see Example 6.
图 16示出 hEST212的抗瘤谱， 见实施例 7。  Figure 16 shows the antitumor spectrum of hEST212, see Example 7.
图 17示出 hEST212作用的持续性， 见实施例 8。  Figure 17 shows the persistence of hEST212 action, see Example 8.
图 18示出给药次数对 hEST212抑制活性的影响， 见实施例 9。 实施例  Figure 18 shows the effect of dosing times on hEST212 inhibitory activity, see Example 9. Examples
实施例中所用的所有反义寡核苷酸 （包括硫代寡核苷酸） 均根据 人端粒酶 hEST2 蛋白亚基的 cDNA 序歹 U ( Morin GB， et al.Science 1997,277(5328):955-959) 用 Applied Biosystems 391 DNA合成仪合成。 在修饰的寡核苷酸情况下， 在合成时进行各种修饰， 合成及化学修饰 原料均为美国 Glen公司产品。合成后先用浓氨水 55 °C脱保护 15小时， 然后用 Micro Pure II反相纯化柱 （购自 Solid Phase Sciences公司） 层 析纯化。 All antisense oligonucleotides (including thiooligonucleotides) used in the examples are based on the cDNA sequence of the human telomerase hEST2 protein subunit 歹 U (Morin GB, et al. Science 1997,277 (5328) : 955-959) were synthesized using an Applied Biosystems 391 DNA Synthesizer. In the case of 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).
实施例中所用的细胞培养、 脂质体转染及细胞增殖抑制活性测定 方法如下：  The cell culture, liposome transfection, and cell proliferation inhibitory activity assays used in the examples are as follows:
实施例所用各种细胞得自军事医学科学院微生物流行病研究所。 细胞在含 10%小牛血清及 100U/ml青霉素， 100 μ g/ml链霉素的 DMEM 培养基中， 置 37°C， 5 %C02孵箱培养。 在 96孔细胞培养板中， 每孔 接种 5 X 103细胞 /0.2ml。 待 50— 60 %细胞融合后， 在无血清状态下， 采用 Lipofectm (GIBCO,lmg/ml) 试剂并参照说明书操作进行反义寡 核苷酸转染。 转染时， 在消毒的 1.5ml 离心管中， 将反义寡核苷酸与 脂质体分别用无血清 DMEM培养基稀释， 室温放置 30分钟后， 将脂 质体与反义寡核苷酸溶液混匀， 室温下再放置 30 分钟， 以便形成脂 质体一反义寡核苷酸复合物， 然后加入无血清 DMEM 培养基。 培养 细胞用无血清 DMEM培养基洗两次， 每孔加入总体积 ΙΟΟ μ Ι的脂质 体一反义寡核苷酸复合物溶液， 作用 5 小时， 换含 10 %小牛血清的 DMEM培养基培养 19小时。 然后同上重新换液和加入脂质体与反义 寡核苷酸溶液两次，于培养 72小时后，每孔加 20 μ 1 MTS (Promega)， 并继续培养 90分钟， 于 490nm测定光吸收， 通过细胞生长倍增数量 评价反义寡核苷酸对细胞生长的影响， 计算对细胞增殖的抑制率。 实施例 1 端粒酶 hEST2反义寡核苷酸对 HepG2细胞生长的抑制 如上所述将 HepG2肝癌细胞接种于 96孔板中， 采用脂质体介导 给药。 每孔细胞用反义寡核苷酸终浓度为 0.5 w mol/L， 脂质体 l g， 总体积为 100 μ ΐ, 每条反义寡核苷酸设置 3孔， 并设置细胞和脂质体 阴性对照。 于 490nm测定光吸收。 通过细胞生长倍增数量评价反义寡 核苷酸对细胞生长的影响。 所用反义寡核苷酸为 hEST21、 hEST22、 hEST23、 hEST24、 hEST25、 hEST26、 hEST27、 hEST28、 hEST29 hEST210 hEST211、 hEST212、 hEST213 hEST214、 hEST215、 hEST216、 hEST217、 hEST218、 hEST219、 hEST220， 所有寡核苷酸 均为全部硫代反义寡核苷酸。 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 μg / ml streptomycin at 37 ° C in a 5% CO 2 incubator. In 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. During transfection, 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 μl 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. Then change the solution again and add the liposome and antisense oligonucleotide solution twice. After 72 hours of culture, add 20 μ 1 MTS (Promega) to each well, and continue the culture for 90 minutes. Determine the light absorption at 490nm. The effect of antisense oligonucleotides on cell growth was evaluated by the number of cell growth doublings, and the inhibition rate on cell proliferation was calculated. Example 1 Inhibition of HepG2 cell growth by telomerase hEST2 antisense oligonucleotide HepG2 liver cancer cells were seeded in a 96-well plate as described above, and liposome-mediated administration was used. 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 μΐ. 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.
从图 1 中可见 hEST21— hEST220共二十条硫代反义寡核苷酸对 HepG2 细胞生长的抑制率。 二十条硫代反义寡核苷酸对 HepG2 细胞 均有不同程序的抑制作用，其中 hEST21、hEST22T、hEST218对 HepG2 细胞生长的抑制率分别达到 62.3 % , 55.4 %和 53.2 %； hEST25、 hEST26、 hEST28、 hEST29、 hEST210、 hEST214、 hEST217和 hEST219 对 HepG2细胞生长的抑制率均超过 30%。 结果表明端粒酶 hEST2反 义寡核苷酸可以有效地抑制 HepG2肝癌细胞的生长。 实施例 2 反义寡核苷酸对接种 HepG2肝癌细胞裸鼠体内肿瘤生 长的抑制  From Figure 1, we can see that there are 20 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%. The results show that 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细胞用含 10%小牛血清的 DMEM培养基进行细胞培养。 收集细胞， 用 PBS洗两次， 重悬于生理盐水中。 35只 6周龄雌性裸 鼠 （购自北京医科大学动物中心） ， 称量鼠重， 每只鼠后腿腋下注射 5 X 105HepG2细胞 /0.2ml。 十天后随机分为 7组， 每组 5只， 开始腹 腔注射给药， 一组为对照组， 每日腹腔注射生理盐水 0.1ml; 二组为 hEST21组， 每日给药 500 w g/0.1ml; 三组为 hEST24组， 每日给药 500 μ g/0.1ml;四组为 hEST29组，每日给药 500 μ g/0.1ml;五组为 hEST212 组， 每日给药 500 y g/0.1ml; 六组为 hEST217 组， 每日给药 500 μ g/O.lml; 七组为 hEST218组， 每日给药 500 μ g/0.1ml。 定期测量肿瘤 大小， 共给药 18天。 处死裸鼠， 测量肿瘤大小， 称量鼠重、 瘤体重。 所用寡核苷酸均为全部硫代反义寡核苷酸。 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 μg / 0.1ml; four groups are hEST29 group, daily administration of 500 μg / 0.1ml; five groups are hEST212 group, daily administration of 500 yg / 0.1ml; Six groups were hEST217 group, daily administration of 500 μg / 0.1ml; seven groups were hEST218 group, daily administration of 500 μg / 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.
从图 2可以看出， hEST21、 hEST24、 hEST29、 hEST212、 hEST217 和 hEST218各组裸鼠肿瘤生长均小于对照组，尤其是 hEST21、hEST24 和 hEST212反义寡核苷酸给药组肿瘤在裸鼠体内的生长速度明显低于 对照组。 但 hEST217组接近于对照组。 图 3显示了各条反义寡核苷酸 对裸鼠肿瘤重量的抑制状况。 hEST21、 hEST24、 hEST29、 hEST212、 hEST217和 hEST218的抑制率分别为 87.6%、 76.4%、 49.4%、 4.5 % 和 34.8%。 图 4显示了各条反义寡核苷酸对接种 HepG2细胞裸鼠肿 瘤形成的作用， 对照组成瘤率为 100%， hEST21、 hEST24、 hEST29、 hEST212、 hEST217和 hEST218各组的成瘤率分别为 60%、 80% > 60 60%、 100%和 60%。 图 5 显示了各组裸鼠增长状况， 组间无显 著差异。 图 6给出了各组肿瘤图片。 以上结果说明端粒酶 hEST2硫代 反义寡核苷酸对接种 HepG2 肝癌细胞的裸鼠体内肿瘤生长具有抑制 作用， 可应用于抑制肿瘤细胞的生长。 实施例 3 hEST212硫代反义寡核苷酸对接种 HepG2肝癌细胞裸 鼠体内肿瘤生长的抑制剂量依赖关系 It can be seen from Figure 2 that the tumor growth of nude mice in the hEST21, hEST24, hEST29, hEST212, hEST217, and hEST218 groups is smaller than that in the control group, especially hEST21, hEST24. And hEST212 antisense oligonucleotide administration group tumor growth rate in nude mice was significantly lower than the control group. But hEST217 group was close to the control group. Figure 3 shows the inhibition of tumor weight in nude mice by each antisense oligonucleotide. The inhibition rates of hEST21, hEST24, hEST29, hEST212, hEST217, and hEST218 were 87.6%, 76.4%, 49.4%, 4.5%, and 34.8%, respectively. Figure 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. The above results indicate that the 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细胞用含 10%小牛血清的 DMEM培养液进行细胞培养。 收集细胞， 用 PBS洗两次， 重悬于生理盐水中。 40只 6周龄雌性裸 鼠， 称量鼠重， 每只鼠后腿腋下注射 I X 106 HepG2细胞 /0.2ml。 五天 后随机分为四组， 每组十只， 开始腹腔注射给药， 对照组每日注射生 理盐水 0.1ml; 实验组分别注射硫代反义寡核苷酸 hEST212， 每日腹 腔注射给药剂量分别为 500 μ g/0.1ml、 100 μ g/0.1ml和 20 μ g/0.1ml， 经过 35天给药， 处死裸鼠， 测量肿瘤大小 ， 称量鼠重、 瘤体重。 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 μg / 0.1ml, 100 μg / 0.1ml and 20 μg / 0.1ml were administered to nude mice, the tumor size was measured, and the tumor weight and tumor weight were weighed.
从图 7— 11可以看出， 腹腔注射接种 HepG2细胞的裸鼠， 经过 35 天给药， 肿瘤的生长速度同反义寡核苷酸的剂量有显著的依赖关系。 在以 500 w g/d、 100 g/d 和 20 g/d 剂量给药， 硫代反义寡核苷酸 hEST212对接种 HepG2细胞的裸鼠肿瘤的抑制率分别为 78.7%、 52.5 %和5.9%， 成瘤率对照组为 100%， 随给药剂量增加成瘤率分别为 90 %、 90%和 80%。 实验结果表明端粒酶硫代反义寡核苷酸对接种 HepG2 肝癌细胞 的裸鼠体内肿瘤生长具有显著的抑制作用， 有可能用于抑制肿瘤细胞 的生长。 因此端粒酶硫代反义寡核苷酸可能是一种潜在的特异、 广谱 的抑制肿瘤细胞生长的药物， 这一点在以下的实施例中得以证实。 实施例 4 化学修饰对反义寡核苷酸稳定性和生物活性的影响 1、 反义寡核苷酸的化学修饰 It can be seen from Figures 7-11 that after 35 days of administration of nude mice inoculated with HepG2 cells by intraperitoneal injection, the tumor growth rate has a significant dependence on the dose of antisense oligonucleotides. In the doses of 500 wg / d, 100 g / d and 20 g / d, 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. The experimental results show that 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
选择 hEST212序列进行天然及硫代反义寡核苷酸的合成， 并分别 对其进行 3' 端磷酸化及胆固醇修饰，还对硫代 hEST212序列进行 3 ' 端脂肪链的修饰。 此外， 合成了一条部分硫代修饰的 hEST212即 3 ' 与 5' 端分别有 4个核苷酸硫代修饰而中间区域保持天然寡核苷酸结 构的混合骨架反义寡核苷酸， 见表 2。  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. In addition, 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.
hEST212的修饰方法  hEST212 modification method
名称 修饰方法  Name Modification method
hEST212-S 硫代修饰  hEST212-S thio modification
hEST 212-S-3' P 硫代 +3' 磷酸化  hEST 212-S-3 'P thio +3' phosphorylation
hEST 212-S-3' Chi 硫代 +3' 胆固醇修饰  hEST 212-S-3 'Chi Thio + 3' Cholesterol Modification
hEST 212-S-3' R 硫代 +3' 脂肪链修饰  hEST 212-S-3 'R thio +3' fatty chain modification
hEST212 -0 天然  hEST212 -0 Natural
hEST 212-0-3' P 天然 +3' 磷酸化  hEST 212-0-3 'P Natural +3' Phosphorylated
hEST212-0-3' Chi 天然 +3' 胆固醇修饰  hEST212-0-3 'Chi Natural +3' Cholesterol Modification
hEST212-Cap 两端部分硫代修饰  hEST212-Cap partial thio modification
2、 反义寡核苷酸的降解反应  2.Antisense oligonucleotide degradation reaction
首先制备 HepG2细胞裂解上清， 取 HepG2细胞 2 X 106， 加细胞 裂解缓冲液（主要含 0.5%CHAPS， 进口分装） lml， 冰浴裂解半小时， 10，000g离心 5分钟，弃沉渣。上述 8条反义寡核苷酸各自取 4个 10 g， 真空（Oligo Prep OP120， SAVANT)干燥后， 分别加未稀释小牛血清、 HepG2细胞裂解上清、 含 10%胎牛血清的 NMEM及水 10 μ 1， 混匀， 37°C孵育， 分别于反应后 0.5、 4、 8及 24小时各取 2 μ 1样品， 一70 C贮存备用。 First, prepare the lysate supernatant of HepG2 cells, take 2 × 10 6 HepG2 cells, add 1ml of cell lysis buffer (mainly containing 0.5% CHAPS, imported separately), lyse for half an hour in an ice bath, centrifuge at 10,000g for 5 minutes, and discard the sediment. Take four 10 g of the above eight antisense oligonucleotides, dry them under vacuum (Oligo Prep OP120, SAVANT), add undiluted calf serum, HepG2 cell lysed supernatant, 10% fetal bovine serum-containing NMEM and water 10 μ 1, mix well, incubate at 37 ° C, take 2 μ 1 samples at 0.5, 4, 8 and 24 hours after the reaction, a 70 C Store for future use.
3、 降解产物的高效薄层色谱分析  3. High-performance thin layer chromatography analysis of degradation products
取上述储存样品 ΐ μ ΐ ( l g / μ ΐ) 用加样器点于高效薄层层析板 (Silica gel-60 GF254 10 X 20cm, EMER 公司） 并用吹风机吹干， 然 后放入层析缸 （瑞士 CAMAG公司产品） 中展开 10cm左右， 展开剂 为异丙醇一水一浓氨水 （65: 25： 10) 。 展开完毕， 取出吹干后用凝 胶成像仪 （Bio-Rad Gel DOC 1000) 照相并打印照片。  Take the above storage sample ΐ μ ΐ (lg / μ ΐ) point the high-performance thin-layer chromatography plate (Silica gel-60 GF254 10 X 20cm, EMER company) with a sampler, blow dry with a hair dryer, and put it into the chromatography cylinder ( Product of Swiss CAMAG Co., Ltd.) was developed about 10cm, and the developing agent was isopropyl alcohol-water-concentrated ammonia water (65:25:10). After unfolding, remove and blow dry, take a photo with a gel imager (Bio-Rad Gel DOC 1000), and print the photo.
4、 修饰的反义寡核苷酸的活性评价  4. Evaluation of the activity of modified antisense oligonucleotides
取 HepG2肝癌细胞接种于 96孔板， 采用脂质体介导给药。 上述 每条修饰反义寡核苷酸终浓度分别为 200 mol/L、 400 mol/L、 800μπιο1/ί, 总体积为 100μ1。 并设置细胞和脂质体对照。 通过细胞生 长抑制评价不同修饰反义寡核苷酸的抑制活性。  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 μπιο1 / ί, and the total volume was 100 μ1. Cell and liposome controls were set. The inhibitory activity of different modified antisense oligonucleotides was evaluated by cell growth inhibition.
结果  The result
1、 化学修饰对反义寡核苷酸的稳定性的影响  1. The effect of chemical modification on the stability of antisense oligonucleotides
hEST212-S在 4种液体中， 反应 24小时， 均未见明显的降解。 hEST212-S-3' R、 hEST212-S-3 ' Chi及 hEST212-S-3， P反应结果与 hEST212—致。 hEST212— 0在未稀释小牛血清中， 反应 4小时， 即 可见有降解， 随反应时间长， 降解逐渐明显， 其在 HepG2 细胞裂解 上清中降解明显， 反应 0.5小时开始有降解， 24小时时基本上完全降 解， 而在含 10%胎牛血清的 DMEM及水中基本上稳定。 hEST212— 0 - 3' P 及 hEST212— 0— 3 ' Chi 反应结果与 hEST212— 0 —致。 hEST212-Cap在 HepG2细胞裂解上清中降解明显， 而在未稀释小牛 血清中、 含 10%胎牛血清的 DMEM及水中基本上稳定， 见图 12。  hEST212-S in 4 kinds of liquids reacted for 24 hours without any obvious degradation. hEST212-S-3 'R, hEST212-S-3' Chi, and hEST212-S-3. 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. The results of hEST212— 0-3 'P and hEST212— 0— 3' Chi were consistent with hEST212— 0 —. 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.
2、 化学修饰对反义寡核苷酸生物活性的影响  2. Effect of chemical modification on the biological activity of antisense oligonucleotides
如图 13 所示， 在相同实验条件下， hEST212 的抑制活性最高， 抑制率达 71.6%， hEST212-3T、 hEST212-0、 hEST212-0-3 及 hEST212-Cap呈现轻度抑制活性， 而脂肪链及胆固醇修饰的反义寡核 苷酸基本上无抑制活性。 实验结果显示硫代修饰的反义寡核苷酸抑制 活性最好， 其它修饰对反义寡核苷酸呈现轻度的影响。 实施例 5反义寡核苷酸序列的优化 As shown in Figure 13, 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
根据人端粒酶 hEST2蛋白亚基 mRNA序列， 设计 12条反义寡核 苷酸 （见表 3 ) ， 所述寡核苷酸均为硫代寡核苷酸。 活性测定时， 取 HepG2肝癌细胞接种于 96 孔板中， 采用脂质体介导给药。 每孔细胞 用硫代反义寡核苷酸终浓度分别为 0.2 mol/L、 0.4 mol/L、 0.8 mol/L。 总体积为 100 μ 1， 每条反义寡核苷酸设置 3 孔， 并设置细胞 和脂质体对照。 通过细胞生长倍增数量评价不同作用靶位的硫代反义 寡核苷酸对细胞生长的抑制作用。  According to the human telomerase hEST2 protein subunit mRNA sequence, twelve antisense oligonucleotides (see Table 3) were designed, and the oligonucleotides were all thiooligonucleotides. In 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 μl, 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.
表 3 反义寡核苷酸序列及性质  Table 3 Antisense oligonucleotide sequences and properties
名称 靶位 序列 （5， 一3， )  Name Target sequence (5, a 3,)
hEST21 1-20 GCAGCAGGACGCAGCGCTGC  hEST21 1-20 GCAGCAGGACGCAGCGCTGC
hEST21a 1-15 GCAGCAGGACGCAGC  hEST21a 1-15 GCAGCAGGACGCAGC
hEST21b 6-20 AGGACGCAGCGCTGC  hEST21b 6-20 AGGACGCAGCGCTGC
hEST24 46-65 CGCGCGGCATCGCGGGGGTG  hEST24 46-65 CGCGCGGCATCGCGGGGGTG
hEST24a 49-65 GCGGCATCGCGGGGGTG  hEST24a 49-65 GCGGCATCGCGGGGGTG
hEST24b 46-62 CGCGCGGCATCGCGGGG  hEST24b 46-62 CGCGCGGCATCGCGGGG
hEST24c 46-60 CGCGCGGCATCGCGG  hEST24c 46-60 CGCGCGGCATCGCGG
hEST212 3565-3584 ACTCACTCAGGCCTCAGACT  hEST212 3565-3584 ACTCACTCAGGCCTCAGACT
hEST212a 3565-3582 ACTCACTCAGGCCTCAGA  hEST212a 3565-3582 ACTCACTCAGGCCTCAGA
hEST212b 3565-3580 ACTCACTCAGGCCTCA  hEST212b 3565-3580 ACTCACTCAGGCCTCA
hEST212c 3567-3584 TCACTCAGGCCTCAGACT  hEST212c 3567-3584 TCACTCAGGCCTCAGACT
hEST212d 3569-3584 ACTCAGGCCTCAGACT 注： hEST2: 人端粒酶具有逆转录酶活性蛋白质亚基 表中序列为全部硫代产品 从图 14可见， 以端粒酶 hEST2基因为靶设计的 12条硫代反义寡 核苷酸 （见表 3 ) 中有 11 条对 HepG2肝癌细胞增殖有不同程度的抑 制作用， 并呈较好的剂量依赖性。 其中针对 hEST2基因 3 ' 端非编码 区的硫代反义寡核苷酸抑制作用较强并与长度有明显的关系， 3 ' 端 缺少 2个碱基的 hEST212a抑制效果高浓度时差异不大， 在中等和低 浓度时略优于 hEST212;其它序列包括 hEST212b (3 '缺少 4个碱基）、 hEST212 c (5 ' 缺少 2个碱基） 和 hEST212d (5 ' 缺少 4个碱基） ， 不如 hEST212。 靶向其它靶位包括 5， 帽区和翻译起始区的各种不同 长度的硫代反义寡核苷酸抑制效果均不明显。 上述结果表明以端粒酶 hEST2 基因特定位点为靶的反义寡核苷酸可以有效地抑制 HepG2 肝 癌细胞的生长。 实施例 6 反义寡核苷酸作用的特异性分析 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. Among them, 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. Slightly better than hEST212 at medium and low concentrations; other sequences include hEST212b (3 'missing 4 bases), hEST212 c (5' missing 2 bases), and hEST212d (5 'missing 4 bases) are not as good as hEST212 . 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
分别合成硫代的 hEST212的正义、 随机及错配硫代寡核苷酸 （见 表 4) 采用上述方法进行细胞培养、 脂质体转染及肿瘤细胞生长抑制 活性测定。  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.
表 4 特异性分析中所用的反义寡核苷酸序列及性质  Table 4 Antisense oligonucleotide sequences and properties used in specificity analysis
名称 性质 GC:AT 序列 （5 ' -3 ' ) *  Name Property GC: AT sequence (5 '-3') *
hEST212 反义 10： 8 ACTCACTCAGGCCTCAGACT  hEST212 Antisense 10: 8 ACTCACTCAGGCCTCAGACT
hEST212S 反义 10： 8 AGTCTGAGGCCTGAGTGAGT  hEST212S Antisense 10: 8 AGTCTGAGGCCTGAGTGAGT
hEST212R 随机 10： 8 AACTCGACTCTAGGCCACTC  hEST212R Random 10: 8 AACTCGACTCTAGGCCACTC
hEST212Ml 错配 10： 8 ACTCtCTCAcGCCTgAGACT  hEST212Ml Mismatch 10: 8 ACTCtCTCAcGCCTgAGACT
hEST212M2 错配 10： 8 ACTCACTCAccCCTCAGACT  hEST212M2 Mismatch 10: 8 ACTCACTCAccCCTCAGACT
表中序列为全部硫代产品 为证明 hEST212作用的特异性， 分别合成了其正义、 随机及错配 的序列并进行同样的修饰 （见表 4) 。 设计时使其 GC 含量相同， 其 中随机与反义序列各碱基组成也一致。 图 15 实验结果显示正义、 随 机及错配的反义寡核苷酸均无明显的肿瘤细胞增殖抑制活性， 说明了 hEST212作用的序列特异性。 实施例 7反义寡核苷酸的抗瘤谱分析 The sequences in the table are all thio products In order to prove the specificity of hEST212's action, the positive, random and mismatched sequences were synthesized and modified the same (see Table 4). The GC content is designed to be the same, and the random and antisense sequences have the same base composition. Figure 15 The experimental results show that no positive, random, and mismatched antisense oligonucleotides have significant tumor cell proliferation inhibitory activity, demonstrating the sequence specificity of hEST212 action. Example 7 Antitumor Profile Analysis of Antisense Oligonucleotides
抗瘤谱是抗肿瘤药物的另一个评价指标， 考虑到端粒酶在大多数 肿瘤细胞均高表达， 因此本实施例以硫代的 hEST212为例测定了其对 不同肿瘤细胞的增殖抑制作用。 具体地， 分别接种人肝癌 （HepG2 ) 、 肺癌（GLC )、脑胶质瘤（BT325 )、胃癌（BGC823 )及乳腺癌（MCF-7 ) 细胞于 96 孔板内， 按上述方法进行培养及反义寡核苷酸脂质体转染 给药， 共给药 2次。 采用 Logistic软件计算 IC50 ( μ mol/L) 。  The anti-tumor spectrum is another evaluation index of anti-tumor drugs. Considering that telomerase is highly expressed in most tumor cells, this example uses thiohEST212 as an example to determine its inhibitory effect on the proliferation of different tumor cells. Specifically, 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 (μmol / L).
测定结果显示 hEST212对肝癌 （HepG2 ) 、 肺癌 （GLC ) 、 脑胶 质瘤 （BT325 ) 、 胃癌 （BGC823 ) 及乳腺癌 （MCF— 7 ) 细胞均有不 同程度的抑制作用。 其中对胃癌抑制作用最强， IC50 仅为 0.25 μ mol/L, 对肺癌、 肝癌和乳腺癌的具有中等强度的抑制作用， IC50 分 别为 0.56 μ mol/L, 0.58 μ mol/L和 0.51 μ mol/L。相对其它肿瘤 hEST212 对脑胶质瘤抑制作用较差， IC50为 0.77 mol/L， 见图 16。 上述实验 数据表明本发明的以端粒酶 hEST2基因为靶的反义寡核苷酸具有较广 的抗瘤谱。 实施例 8 反义寡核苷酸作用持续时间的测定  The results showed that hEST212 inhibited liver cancer (HepG2), lung cancer (GLC), glioma (BT325), gastric cancer (BGC823), and breast cancer (MCF-7) cells to varying degrees. Among them, it has the strongest inhibitory effect on gastric cancer, with IC50 of only 0.25 μ mol / L, and moderate-intensity inhibitory effect on lung cancer, liver cancer, and breast cancer, with IC50 of 0.56 μ mol / L, 0.58 μ mol / L, and 0.51 μ mol, respectively. / L. Compared with other tumors, hEST212 has a poor inhibitory effect on gliomas with IC50 of 0.77 mol / L, as shown in Figure 16. The above experimental data show that the antisense oligonucleotide of the present invention targeting the telomerase hEST2 gene has a broad antitumor spectrum. Example 8 Determination of duration of action of antisense oligonucleotides
与上述实施例 Ί一样， 本实施例以硫代的 hEST212为例测定反义 寡核苷酸作用的持续时间。 HepG2细胞接种于 96孔板， 待 50— 60 % 细胞融合后， 转染硫代的 hEST212—次， 终浓度为 0.4 w mol/L。 分别 于转染后第 1一 5 天， 各取 3 孔细胞测定细胞增殖抑制情况， 观察其 作用持续时间。 As in Example VII above, 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.
作用的持续时间不仅是评价反义核酸药物的一个重要指标， 同时 对于给药方案具有重要的参考价值。 图 17可以看出肺癌细胞 （GLC) 中， 终浓度 0.4 mol/L的 hEST212脂质体介导转染给药 1次后， 1一 4天抑制活性逐渐增加， 第 4天达到 62.8 %的峰值， 然后缓慢下降， 但直到第 6天仍保持 53.8 %的抑制率。 肝癌细胞 （HepG2) 中， 给药 后第 2天抑制率即达到 58.3 %的峰值， 然后快速下降， 第 5天时仅保 留 15.6%的抑制活性。 实施例 9 给药次数对反义寡核苷酸抑制活性的影响分析  The duration of the effect is not only an important indicator for evaluating antisense nucleic acid drugs, but also has important reference value for the administration schedule. Figure 17 shows that in lung cancer cells (GLC), hEST212 liposome-mediated transfection at a final concentration of 0.4 mol / L once, the inhibitory activity gradually increased from 1 to 4 days, reaching a peak of 62.8% on the 4th day , And then slowly decreased, but still maintained a 53.8% inhibition rate until the 6th day. In hepatocellular carcinoma (HepG2), the inhibition rate reached a peak of 58.3% on the second day after administration, and then decreased rapidly, and only 15.6% of the inhibitory activity was retained on the fifth day. Example 9 Analysis of the effect of the number of administrations on the inhibitory activity of antisense oligonucleotides
与上述实施例 7—样， 本实施例以硫代的 hEST212为例测定给药 次数对反义寡核苷酸抑制活性的影响。 HepG2 细胞接种于 96孔板， 待 50— 60%细胞融合后， 分别转染硫代的 hEST212 1— 2次， 一次 /天， 终浓度为 0.4 mol/L。 分别于转染后 24h后， 各取 3孔细胞测定细胞 增殖抑制情况， 观察给药次数对细胞增殖的影响。  As in Example 7 above, 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.
给药次数对 hEST212的肿瘤细胞增殖抑制活性的影响不大。 如图 18所示， 仅在低浓度和中浓度时， 其肿瘤细胞增殖抑制活性随给药次 数的增加略有增加的趋势。 高浓度 0.8 u mol/L时连续给药 3次与 2次 也无显著性差异。  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.

Claims

权利要求 Rights request
1、 一种反义寡核苷酸， 其长度为 10— 30个核苷酸， 其特征是- 其与编码人端粒酶具有逆转酶活性蛋白质亚基 hEST2的基因或其转录 的 mRNA的一部分互补。  1. An antisense oligonucleotide, which is 10-30 nucleotides in length, and is characterized in that-it has a gene encoding human telomerase that reverses the activity of the protein subunit hEST2 or part of its transcribed mRNA Complementary.
2、 根据权利要求 1 的寡核苷酸， 其包括 SEQ ID NO: 1—29的 序列。  2. The oligonucleotide according to claim 1, comprising the sequence of SEQ ID NOs: 1-29.
3、根据权利要求 2的寡核苷酸，其为 hEST211 ( SEQ ID NO: 16)、 hEST24 (SEQ ID NO: 6) 、 hEST212 (SEQ ID NO: 17) 、 hEST212a The oligonucleotide according to claim 2, which is hEST211 (SEQ ID NO: 16), hEST24 (SEQ ID NO: 6), hEST212 (SEQ ID NO: 17), hEST212a
(SEQ ID NO: 18) 。 (SEQ ID NO: 18).
4、 根据权利要求 1一 3 中任一项所述的寡核苷酸， 其为修饰的寡 核苷酸。  4. The oligonucleotide according to any one of claims 1 to 3, which is a modified oligonucleotide.
5、 根据权利要求 4所述的寡核苷酸， 其中所述的修饰为核苷酸 间磷酸二酯键部分的单键氧位置是硫或甲基。  5. The oligonucleotide according to claim 4, wherein the modification is that the single bond oxygen position of the internucleotide phosphodiester bond moiety is sulfur or methyl.
6、 根据权利要求 5所述的寡核苷酸， 其为硫代寡核苷酸。  6. The oligonucleotide according to claim 5, which is a thiooligonucleotide.
7、 根据权利要求 6所述的寡核苷酸， 其为硫代的 hEST211 (SEQ ID NO: 16) 、 hEST24 (SEQ ID NO: 6) 、 hEST212 (SEQ ID NO: 17) 、 hEST212a (SEQ ID NO: 18) 。  7. The oligonucleotide according to claim 6, which is thio-hEST211 (SEQ ID NO: 16), hEST24 (SEQ ID NO: 6), hEST212 (SEQ ID NO: 17), hEST212a (SEQ ID NO: 18).
8、 根据权利要求 5 所述的寡核苷酸， 其中该硫代寡核苷酸进一 步被化学修饰， 所述的修饰为 3 ' 憐酸化。  8. The oligonucleotide according to claim 5, wherein the thiooligonucleotide is further chemically modified, and the modification is 3 'phosphorylation.
9、 一种药物组合物， 其特征在于包括药物学有效量的权利要求 1 一 8任一项所述的寡核苷酸， 及药物学上可接受的载体。  9. A pharmaceutical composition, comprising a pharmaceutically effective amount of the oligonucleotide according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.
10、 一种用于检测端粒酶 hEST2 RNA组分或编码端粒酶 hEST2 的 DNA的试剂盒， 其包括权利要求 1一 8任一项所述的寡核苷酸。  10. A kit for detecting a telomerase hEST2 RNA component or a DNA encoding the telomerase hEST2, comprising the oligonucleotide according to any one of claims 1 to 8.
11、 根据权利要求 10 的试剂盒， 其中所述的寡核苷酸被放射性 同位素、 酶或荧光化合物标记。  11. The kit according to claim 10, wherein said oligonucleotide is labeled with a radioisotope, an enzyme or a fluorescent compound.
12、 根据权利要求 1一 8 中任一项所述的寡核苷酸在制备治疗肿 瘤的药物中的应用。 13、 权利要求 12所述的应用， 其中所述的肿瘤选自肝癌、 肺癌、 胃癌、 乳腺癌和脑胶质瘤。 12. Use of the oligonucleotide according to any one of claims 1 to 8 in the preparation of a medicament for treating tumors. 13. The use of claim 12, wherein said tumor is selected from the group consisting of liver cancer, lung cancer, gastric cancer, breast cancer and glioma.
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