WO2012155328A1 - Composition comprenant de l'endostatine et des molécules d'arni et utilisation associée - Google Patents

Composition comprenant de l'endostatine et des molécules d'arni et utilisation associée Download PDF

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WO2012155328A1
WO2012155328A1 PCT/CN2011/074100 CN2011074100W WO2012155328A1 WO 2012155328 A1 WO2012155328 A1 WO 2012155328A1 CN 2011074100 W CN2011074100 W CN 2011074100W WO 2012155328 A1 WO2012155328 A1 WO 2012155328A1
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endostatin
sirna
liposome
peg
pharmaceutical composition
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PCT/CN2011/074100
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Chinese (zh)
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徐根兴
殷妍
郭佳佳
张昌栋
吴稚伟
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南京大学
江苏省基因药物工程技术研究中心
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Priority to PCT/CN2011/074100 priority Critical patent/WO2012155328A1/fr
Publication of WO2012155328A1 publication Critical patent/WO2012155328A1/fr

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    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
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    • 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
    • C12N15/1136—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 growth factors, growth regulators, cytokines, lymphokines or hormones
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    • 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
    • C12N15/1138—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 receptors or cell surface proteins
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    • 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
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • composition comprising endostatin and RNA interference molecules and application thereof
  • the invention belongs to the field of biomedicine and relates to a composition and application of endostatin as a delivery system and a chemically synthesized RNA interference molecule.
  • Endostatin is produced by primary tumor cells and inhibits tumor blood vessel growth (0' Reily et al 1997, Cel l 88 : 277 ). It is a fragment of collagen XVI II composed of 183 amino acids. With a high positive charge, it is a rare protein with a high positive charge in recombinant proteins. At present, there are 183 amino acids, 184 amino acids, 193 amino acids with 6 histidines at the N-terminus, and PEG-modified endostatin have been clinically studied or already on the market. However, there is no corresponding report on the delivery of chemically synthesized RNA interference molecules to the corresponding target tissues by endostatin as a delivery system.
  • Endostatin is currently known to have the following biological functions: (1) Inhibition of angiogenesis by inhibiting proliferation and growth of vascular endothelial cells. Endostatin can specifically inhibit the proliferation of vascular endothelial cells induced by fibroblast growth factor (bFGF), inhibit the migration of endothelial cells, induce apoptosis of endothelial cells, but for non-endothelial cells, such as smooth muscle cells, 3T3 fibrils There is no inhibition of cells and the like. (2) It inhibits the growth of blood vessels and does not contribute to resting blood vessels. (3) inhibit tumor growth and metastasis. A number of experiments have shown that recombinant endostatin or endostatin genes can be used for tumor therapy. It has been reported that endostatin inhibits the growth and metastasis of more than 60 different solid tumors. (4) Endostatin has a certain role in inhibiting tumor cells in vitro.
  • bFGF fibroblast growth factor
  • endostatin itself has a certain therapeutic effect on tumors, clinical use of 7.5 mg/m 2 endostatin alone has no obvious effect. Need to be combined with chemotherapy drugs.
  • RNA interference has attracted much attention since its discovery in 1998.
  • Fire et al (Nature, 1998, 391: 806-811) first discovered double strand RNA (dsRNA) in the nematode to silence gene expression.
  • siRNA synthetic small interfering RNA
  • siRNA can mediate RNA interference in cells, recognize specific mRNA complementary to its sequence, and silence the expression of related genes.
  • the action is specific and efficient, and is a powerful method for specifically inhibiting gene expression.
  • siRNA has become a potential therapeutic tool for many disease-related genes and has become a hot topic in clinical application research. Twelve of the siRNA drugs have entered the I-111 clinical study internationally.
  • aiRNA Asymmetric double-stranded interfering RNA
  • aiRNA Asymmetric double-stranded interfering RNA
  • siRNA has many advantages and may replace siRNA for innovative drug research in the future.
  • siRNA-mediated gene silencing is more efficient, durable, structurally stable, and can reduce the off-target effects of the sense strand.
  • AsiRNA drugs are currently not reported in clinical studies internationally.
  • MicroRNA is a non-coding small RNA of about 22-28 nt in length, and mi RNA passes through the target mRNA 3 ' UTR (Untranslated Regions, the untranslated region, which is a non-coding fragment at both ends of the mRNA molecule).
  • UTR Untranslated Regions, the untranslated region, which is a non-coding fragment at both ends of the mRNA molecule.
  • Complete or incomplete complementary binding results in degradation of target mRNA or translational inhibition, thereby regulating expression of target genes, affecting cell proliferation, differentiation and apoptosis.
  • Their regulation of post-transcriptional gene expression is regulated by miRNA-mediated specific gene silencing leading to degradation of target mRNA and inhibition of protein synthesis. miRNAs have important regulatory roles in cell proliferation, differentiation and apoptosis. Gene expression regulation studies are often performed using microRNA mimics or inhibitors.
  • miRNA mimics are miRNAs that mimic in vivo organisms and are synthesized by chemical synthesis to enhance the function of endogenous miRNAs. miRNA mimics can further enhance the silencing of endogenous miRNAs and reduce the amount of targeted protein expression in cells.
  • MicroRNA inhibitors directly inhibit the function of miRNAs. For example, the Danish pharmaceutical company Santafis Pharma announced that its new treatment for hepatitis C, SPC3649 (locked nucleic acid LNA-antimiRTM-122), has entered the first phase of human clinical trials, the world's first miRNA drug for human clinical trials. Direct inhibition of the function of hepatitis C microRNA-122.
  • RNA interference molecules whether chemically modified or unmodified, two strand-symmetric small nucleic acid interference (siRNA), chemically modified or unmodified two strands paired with asymmetric small nucleic acid interference (asiRNA, aiRNA) ), or a mimetic or inhibitor of microRNA (miRNA).
  • siRNA two strand-symmetric small nucleic acid interference
  • aiRNA asymmetric small nucleic acid interference
  • miRNA mimetic or inhibitor of microRNA
  • the object of the present invention is to provide a high dose, a poor stability, a large toxic side effect or a high manufacturing cost when the siRNA, a siRNA, and miRNA gene drugs characterized by high negative charge in the prior art are administered intravenously.
  • a pharmaceutical composition comprising an endostatin recombinant protein carrying a charge of +5 to +15 and a chemically synthesized RNA interference molecule carrying a charge of -15 to -55, which are combined by positive and negative charges to form a charge value at -20
  • Compositions between +20 preferably form a composition having a charge value between -10 and +10.
  • the pharmaceutical composition further comprises a cationic liposome, a neutral liposome or a PEG-modified long-acting liposome for encapsulating the endostatin recombinant protein and the RNA interference molecule to modulate the amount of positive and negative charges.
  • the composition having a positive or negative electric charge value can also be directly used without being dissolved in the physiological saline solution for injection without liposome encapsulation.
  • the endostatin recombinant protein is a recombinant protein purified and expressed by Escherichia coli, yeast, mammalian or human cells, and a viral vector, and may be all endostatin products currently commercially available, or may be in accordance with the art.
  • the endostatin recombinant protein preferably does not carry a 6 histidine tag of a human-structured endostatin recombinant protein consisting of 183-184 amino acids, N-terminal or / and C-terminal
  • a human-structured endostatin fusion protein consisting of 190-200 amino acids carrying a 6-histidine tag, or a long-acting endothelin formed by PEG modification or albumin modification of the above two types of endostatin recombinant proteins Or a synthetic truncated endostatin (including the major amino acid composition of the 183 amino acid sequence).
  • All types of endostatin recombinant proteins are commonly characterized by carrying a high positive charge from +5 to +15 and have a specific affinity for vascular endothelial cells of active lesions.
  • RNA interference molecule is selected from a chemically modified or unmodified two strand symmetric small nucleic acid interference molecule (siRNA), and the chemically modified or unmodified two strand pairing asymmetric small nucleic acid interference molecule (asiRNA, aiRNA), Or chemically modified or unmodified miRNA.
  • siRNA chemically modified or unmodified two strand symmetric small nucleic acid interference molecule
  • asiRNA aiRNA
  • miRNA chemically modified or unmodified miRNA
  • the two strand-symmetric small nucleic acid interference molecules are double-stranded paired RNAs of the length of the sense strand and the antisense strands, which are 19-25 nt in length, and the two strands of the two strands contain 2_4
  • the UU base or the deoxyribonucleic acid dTdT is single-stranded;
  • the two strands pair asymmetric amino acid interference molecules are chemically synthesized, starting from the target gene symmetric small nucleic acid interference molecule siRNA
  • siRNA By dividing the 5' or 3' end of the siRNA sense strand or the antisense strand by l_5nt bases, the lengths of the two strands are inconsistent, and there are 2-4 UU bases at each 3' end of the strand or dTdT of deoxyribonucleic acid Single-stranded suspension, 14-21 nt double-stranded asymmetric small nucleic acid interference molecule asiRNA
  • the miRNAs include miRNA mimics and miRNA inhibitors.
  • miRNA mimics are miRNAs that mimic in vivo sources and are synthesized by chemical synthesis to enhance the function of endogenous miRNAs.
  • miRNA inhibitors are chemically modified inhibitors specific to target miRNAs in cells that specifically silence a single gene or simultaneously silence multiple related but not identical genes.
  • the chemical modification refers to a thio modification, a methylation modification, a phosphorylation modification, a cholesterol modification, or a fluoro modification.
  • siRNA, or asiRNA, or miRNA involved in the present invention are chemically synthesized RNA interference molecules whether or not chemically modified, and the common feature is that the high negative charge is from -15 to -55, and the expression of the target gene can be regulated.
  • the components of the pharmaceutical composition are separately packaged for use as they are, or directly mixed and packaged together.
  • composition for the preparation of a medicament for treating malignant tumor, hepatitis or AIDS.
  • the components of the pharmaceutical composition are separately packaged for use as they are, or directly mixed and packaged together.
  • the endostatin recombinant protein used in the invention and the RNA interference molecule inhibiting gene expression are combined by positive and negative charges, and can be directly mixed according to the ratio of positive and negative charges, and can be directly mixed according to the positive or negative charge ratio.
  • the charge ratio is mixed and used.
  • the ratio of positive and negative charges can be calculated by electrophoresis and charge measurement, and the proper ratio means that the final composition has a charge value between -20 and +20 after mixing.
  • the composition may also be encapsulated with cationic liposomes, neutral liposomes, or PEGylated long-acting liposomes to adjust the ratio of positive and negative charges to a more suitable range of charge values (ie, charge values between -10 and +10).
  • composition is not precipitated in the physiological saline for injection, does not precipitate, does not affect the activity, is more stable, and is more likely to enter the cells, and can deliver chemically synthesized RNA by subcutaneous injection, local injection, intravenous injection, intravenous infusion.
  • Interfering molecules The target organ regulates the expression of the gene of interest.
  • the recombinant human endostatin provided by the present invention has a high positive charge property, and is combined with a chemically synthesized RNA interference molecule carrying a high negative charge by positive and negative charges to form a composition with or without liposome encapsulation. It does not precipitate in the physiological saline for injection, does not precipitate, does not affect activity, is more stable, and is more likely to enter cells.
  • the pharmaceutical composition greatly reduces the dosage and charge value of endostatin and chemically synthesized RNA interference molecules, and since the delivery of endostatin has the specific property of targeting focal vascular endothelial cells, the composition is in vein
  • the target tissue can be targeted and stably distributed within 1 hour or 24 hours after injection, so that the therapeutic effect is better, the activity is more stable, the effective action time is longer, and the side effects are smaller.
  • the pharmaceutical composition of the present invention has both a targeting effect and a gene regulation function, and plays an active role in the treatment of malignant tumor, hepatitis or AIDS, and can be used for preparing a medicament for treating malignant tumor, hepatitis or AIDS.
  • FIG. 1 Schematic diagram of Bcl2-siRNA, endostatin, PEG liposome (complex lipid) forming a Bcl2_siRNA interference composition that can be administered intravenously.
  • a separate bottle of Bcl2-siRNA, endostatin, and PEG liposomes is mixed together at the time of use.
  • Figure 2. Correspondence diagram between the charge of endostatin and the pH of the solvent. The abscissa is the solvent pH and the ordinate is the endostatin charge.
  • FIG. 4 Zeta potential map of a combination of endostatin and VEGF-a siRNA and PEG liposome-chitosan. Zeta potential is +13.7 mV
  • Figure 5 Electropherogram of the formation of a composition of endostatin and Bcl2-siRNA in the presence of different liposomes.
  • FIG. 1 Electropherogram of endostatin with different gene siRNA, asiRNA, miRNA forming a composition with or without liposome encapsulation.
  • Electropherogram of the composition of VEGF-a siRNA and endostatin in the absence of liposome encapsulation, from left to right, VEGF-a siRNA and endostatin are 1:0, 1:5, 1: 10, 1:15, 1:20, 1:30, 1:40, 1:50, 1:60 mass ratio electrophoretic pattern of the composition formed;
  • Electropherogram of a composition of anti-Has-122 miRNA inhibitor and PEG endostatin in the absence of liposome encapsulation, from left to right, respectively, anti-Has-122 miRNA inhibitor and PEG endothelin An electropherogram of a composition formed by mass ratios of 3:0, 3:5, 3:10, 3:15, 3:20, 3:25, 3:30, 3:40, 3:50.
  • Electropherogram of the composition of Rabllb-siRNA and Endostatin in the absence of liposome encapsulation The lanes from left to right are Rabllb-siRNA and endostatin at 1:0, 1:5, respectively. Electropherogram of the composition formed by 1:10, 1:15, 1:20, 1:25, 1:30, 1:40, 1:50 mass ratio.
  • Figure 7 Particle size of the composition formed by endostatin and chemically synthesized RNA interference molecules.
  • the control group did not receive any transfection, and only different concentrations of cisplatin were added.
  • the B C 12-a siRNA-endostatin group was transfected with B C 12-a siRNA using the endostatin-PEG liposome delivery system followed by the addition of different concentrations of cisplatin.
  • Cytotoxicity of different liposome, endostatin and B C 12-a siRNA forming compositions wherein 1-4 represents a composition formed by Lipo 2000 liposome and B C 12-a siRNA, endostatin-PEG lipid
  • Guang 6 represents: 1 : Lipo 2000 transfected B C 12-a siRNA group; 2 : PEG liposome-endostatin complex transfected Bcl2-a siRNA group; 3: Lipo 2000 transfected Bcl2_a siRNA + low dose cisplatin (1 Micrograms group; 4: PEG liposome-endostatin complex transfected with B C 12-a siRNA + low dose cisplatin group; 5: low dose cisplatin (1 ⁇ g) alone group; 6: doubling dose of cisplatin ( 2 ⁇ g) Individual group.
  • Figure 11 Inhibitory curves of endostatin and chemically modified B C 12-a siRNA forming compositions against tumor volume of H22 liver cancer-bearing mice.
  • the saline group was a blank control for intravenous injection of normal saline; the CTX (15 mg/kg) group and the CTX (30 mg/kg) group were positive controls for intraperitoneal injection of the chemotherapy drug cyclophosphamide; PEG liposome-endostatin-no
  • the relevant siRNA group was a negative control for the formation of a complex of PEG liposome-endostatin with unrelated siRNA in the tail vein; PEG liposome-endostatin-Bcl2-a siRNA group was injected intravenously with PEG liposome-endostatin Complex formed with B C 12-a siRNA; PEG liposome-endostatin_Bcl2-a siRNA+CTX group was a combination group using CTX (15 mg/kg) on the previous group.
  • Figure 12 Inhibition curve of endothelin and chemically synthesized RNA interference molecule forming composition against tumor volume of H22 liver cancer mice.
  • the saline group was a blank control for intravenous saline injection; the CTX (30 mg/kg) group was a positive control for intraperitoneal injection of the chemotherapy drug cyclophosphamide; the PEG liposome group was injected with PEG liposome, PEG liposome.
  • - Endostatin-VEGF-a siRNA group was injected intravenously with PEG liposome-endostatin and VEGF-a siRNA complex.
  • FIG 14. Survival curves of H22 tumor-bearing mice injected with endostatin and chemically synthesized VEGF RNA interference molecules. The samples and injections of each group are the same as in Figure 12.
  • Figure 16 Distribution of endostatin and chemically modified and fluorescently labeled B C 12-a siRNA in various organs of human hepatocarcinoma nude mice and targeting tumor effects. From left to right, the heart, liver, spleen, Lung, kidney, tumor.
  • FIG. 17 Distribution of Endostatin and chemically modified and fluorescently labeled CCR5_a siRNA in various organs of normal nude mice. a: 1 hour and 24 hours of sample injection for quantitative analysis of CCR5_as iRNA residual amount in each organ; b: 1 hour after the injection of the sample, the animal was removed and the viscera was taken for biopsy, from left to right, heart, liver, spleen, Lung, kidney; c: Samples were sacrificed 24 hours after the animals were sacrificed and the viscera was taken for biopsy. From left to right, the heart, liver, spleen, lungs and kidneys were followed. Figure 18. Effect of endostatin and chemically synthesized RNA interference molecule forming composition on human lung cancer A549 xenografts in nude mice. Concrete
  • Fig. 1 The process of forming endothelin and siRNA by positive and negative charge in the presence of liposomes to form a complex is represented by a pattern diagram (Fig. 1), which is divided into three bottles of siRNA, endostatin and liposome.
  • Fig. 1 The advantage is that the quality control of each component is relatively easy, the storage is convenient, but it is troublesome to use. It is also possible to premix the three bottles of the ingredients in a certain proportion of the charge ratio described in the following examples, and combine them in one bottle, which is convenient to use, but the quality control of the mixed components is difficult.
  • Figure 1 shows the siRNA of the tumor anti-apoptosis gene (B-cell lymphoma/le kemia-2, Bcl2, formerly known as B-cell lymphoma/leukemia 2 gene, Bcl2), which can be replaced with asiRNA of this gene or other genes.
  • the liposome exemplified in Fig. 1 may be a cationic liposome, a neutral liposome, or a PEG-modified long-acting liposome, or a liposome, and the endostatin is injected with physiological saline for injection.
  • the mixture is mixed with the RNA interference molecule composition and intravenously instilled.
  • the endostatin recombinant protein used in this embodiment is produced by Jiangsu Zhongzhong Pharmaceutical Group Co., Ltd. Zhongkai Biopharmaceutical Factory, hereinafter referred to as endostatin, consisting of 184 amino acids, the amino acid sequence is: SEQ ID N0. 1, isoelectric The point is 9.3.
  • the mixture is mixed at a mass ratio of 1:10:5 and diluted 15 times.
  • the Zeta is measured by Zeta Sizer 3000 dynamic laser scatterometer. Potential.
  • the zeta potential of PEG liposome-endostatin-B C 12_siRNA is shown in Figure 3. 5 mV ⁇ The average zeta potential of the PEG liposome-endostatin-Bcl2-siRNA was 2. 6mV.
  • VEGF vascular endothelial growth factor
  • VEGF-a siRNA, endostatin, PEG liposome, chitosan, ⁇ -cyclodextrin were mixed at a mass ratio of 1:10:5:15:5, diluted 20-fold to 5 ml with DEPC water, and used 0 The 22 ⁇ m filter was filtered and sealed. A mixture in which PEG liposome, chitosan, and ⁇ -cyclodextrin are formed at 5:15:5 is called chitosan liposome, the same below.
  • the zeta potential of the endostatin-VEGF-a siRNA-chitosan liposome composition was measured, and the average zeta potential was 13.7 mV. The results are shown in Fig. 4.
  • the method determines the charge profile of a composition consisting of an endostatin recombinant protein and an interfering molecule in the presence or absence of liposome encapsulation.
  • RNA loading buffer 100 V, electrophoresis for 30 min.
  • the gel is placed under an ultraviolet lamp to be photographed, and the result is shown in Fig. 5. It can be seen from Fig. 5 that the negatively charged various concentrations of Bcl2-siRNA are mixed with endostatin, and after being encapsulated by liposome and encapsulated by liposome, the electrophoresis strip has hysteresis and appears from negative to negative A certain gradient of positive charge indicates that Bcl2-siRNA forms a positively charged combination with endostatin and liposomes.
  • VEGF-aiRNA powder (sequence and charge value were the same as in Example 2) was dissolved in DEPC water to prepare a 20 nM stock solution.
  • the agarose gel configuration method is the same as above.
  • VEGF-a siRNA and endostatin (sequence and charge value are the same as in Example 2) with a mass ratio of 1:0, 1:5, 1:10, 1:20, 1:30, 1:40, 1: 50, 1:60 were mixed in an RNase-free EP tube, allowed to stand for 15 min, and added to the agarose gel well. Electrophoresis was carried out for 30 min at a constant pressure of 100 V. Take a picture and observe the band. The result is shown in Figure 6a.
  • VEGF-a siRNA sequence, charge value are the same as in Example 2
  • endostatin sequence, charge value are the same as in Example 2
  • PEG liposome mixed at a mass ratio of 1:10:5, respectively, with chitosan :1: 0, 1: 5, 1: 10, 1: 15, 1: 20, 1: 30 mass ratio mixing, and then ⁇ _cyclodextrin with a mass ratio of 1:2.5, 1: 5, in The RNase-free fistula was mixed and allowed to stand for 15 min. Add the agarose gel pores in proportion to the mass ratio from small to large. At 70V constant pressure, electrophoresis for 30min. Photographs were taken to observe the amount of charge carried by the strips and the analysis composition at different ratios. The results are shown in Figure 6b.
  • the electrophoresis of the same method can be used to observe the charge of a composition composed of an endostatin recombinant protein and an RNA interference molecule without liposome encapsulation.
  • Hepatitis-associated Has-122 miRNA inhibitor anti-Has-122 (sequence 5 'ACAAACACC AUUGUC ACACUCCA-3 (SEQ ID N0.2) ) 4.5 ⁇ l, PEG-modified long-acting endostatin (1 mg/ml, Jiangsu Wuzhong Pharmaceutical Group Co., Ltd.
  • Has-122 miRNA inhibitor anti-Has-122 and PEG endostatin are respectively in mass ratio of 3:0, 3:5, 3:10, 3:15, 3: 20, 3: 25, 3: 30, 3: 40, 3: 50 mixed, room temperature for 20min.
  • the above sample was added to RNA loading buffer, and subjected to agarose gel electrophoresis, 100 V, and the gel was placed under ultraviolet light for imaging at 10 min, 15 min, 20 min, and 25 min, respectively, and photographed.
  • the electropherogram is shown in Figure 6c.
  • Different ratios of anti-Has-122 and PEG The composition formed by endostatin exhibits different charge values in electrophoresis, and a composition with a weak positive charge is selected for zeta potential measurement, and the measured potential value is usually within 10 mV, so that it is convenient to find a suitable combination by electrophoresis.
  • the ratio of anti-Has-122 and PEG endostatin corresponding to the charge value of the substance.
  • siRNA receptor-associated gene CCR5 was obtained as asiRNA (sequence: sense strand 5'GUCAAGUCCAAUCUAUGdTdT 3% antisense strand 3 'dTdTCAC AGUUC AGGUUAGAUAC 5'), tumor ion channel-associated Rabllb siRNA (sequence: sense strand 5 'UGUCAGACAGACGCGAAAAdTdT3', antisense strand 3'dTdTACAGUCUGUCUGCGCUUUU 5'), siRNA for the drug resistance gene MDR1 related to tumor chemotherapy drug resistance (sequence: sense chain 5'AAAAUGUUGUCUGGACAAGCAdTdT3', antisense strand 3 'dTdTUUUUACAACAGACCUGUUCGU 5') 1.5 ⁇ 1 and N-terminal endostatin with 6 histidine (lmg/ml, trade name Endu, produced by Shandong Xiansheng Maidjin Bio-Pharmac
  • Bcl2-a siRNA with a charge value of -38 (sequence: sense strand: 5' GAGGCUGGGAUGCCUUUdTdT 3', antisense strand: 3' dTdTGCCUCCGACCCUACGGAAA 5', concentration 20nM) and endostatin (lmg/ml, sequence, charge value
  • PEG liposome (1 mg/ml) was gently mixed at a mass ratio of 1:10:5, and left at room temperature for 15 minutes.
  • the above mixture was diluted 15 times with 0.05 M acetic acid-sodium acetate buffer.
  • the particle size was measured using a Mastersizer FOO particle size analyzer. 4nm ⁇
  • the particle size distribution of the PEG liposome-endostatin-B C 12-a siRNA is relatively uniform, the average particle size is 142.4 nm.
  • the Zeta potential was measured to be +3 mV.
  • VEGF-a siRNA (sequence, charge value are the same as in Example 2), endostatin (1 mg/ml, sequence, charge value are the same as in Example 2), PEG lipid, chitosan (CS), ⁇ -cyclodextrin Fine ( ⁇ -CD) mixed with a mass ratio of 1: 10 : 5 : 15 : 5, that is, 30 ⁇ 1 VEGF-aiRNA was gently mixed with 80 ⁇ 1 endostatin, allowed to stand at room temperature for 5 min, and added with PEG lipid.
  • the present invention used the PEG liposome-endostatin system to transfect the B C 12-a siRNA fragment with the charge value and sequence as in Example 4.
  • the B C 12-a siRNA powder was dissolved in DEPC water to prepare a 20 nM stock solution.
  • human cervical cancer cell HeLaB2 cells purchased from the Institute of Oncology, Chinese Academy of Medical Sciences, Chinese Academy of Medical Sciences
  • DMEM cell culture medium containing calf serum, penicillin and streptomycin.
  • the density of the cells reaches 50%, the original medium is discarded and cultured in DMEM without calf serum and penicillin or streptomycin.
  • the IC50 of cisplatin is significantly reduced, that is, cells transfected with PEG liposome-endostatin-B C 12-a siRNA can inhibit cancer cell proliferation only by lower concentration of cisplatin .
  • Human cervical cancer cell line HeLaB2 with high expression of Bcl2 (purchased from the Chinese Academy of Medical Sciences, Peking Union Medical College, Cancer Research) The cells were inoculated into a 96-well plate and cultured at 37 ° C in an incubator containing 5% CO 2 . When the density of the cells reached 50%, transfection was carried out. The experiment was divided into 4 groups with at least three parallel control wells in each group. Group 1 per well was added 0. 25 ⁇ ILipof ectAMINETM2000 (Lipo 2000) liposome (Life Techonolobies, product number: 11668-019, trade name Inivitrogen, the same below) +0.
  • 25 ⁇ 1 Bcl2_a siRNA (concentration, charge value The sequence is the same as in Example 4, the same below); Group 2 is added 0. 25 ⁇ 1 Bcl2-a siRNA + 0. 5 ⁇ 1 Endostatin (concentration, charge value, sequence are the same as in Example 4, the same below) + 0. 25 ⁇ 1 PEG liposome (concentration as in Example 4, the same below), the zeta potential of the PEG liposome-endostatin-B C 12-a siRNA was determined to be +3 mV; group 3 was added 0 per well. 25 ⁇ 1 Bcl2_a siRNA +0. 5 ⁇ 1 endostatin + 0. 25 ⁇ 1 chitosan liposome; Group 4 is a blank control without any treatment.
  • the culture plate was placed in a 37 V, 5% (0 2 incubator) for 24 h, and the medium was aspirated.
  • the CCK-8 kit instructions 100 ⁇ l of DMEM medium and 10 ⁇ l of CCK were added per well.
  • -8 reagent placed in a 37 ° C incubator for further 1 hour, and 0D450 detection with a microplate reader.
  • the culture plate was placed in a 37 V, 5% C0 2 incubator for 24 h, and then the medium was aspirated.
  • the CCK-8 kit instructions 100 ⁇ l of DMEM medium and 10 ⁇ l of CCK-8 were added per well.
  • the reagent was placed in a 37 ° C incubator for further 1 hour, and the 0D450 was detected by a microplate reader, and the cell viability was calculated as in Example 5.
  • the relationship between the concentration of PEG liposome-endostatin-B C 12-a siRNA complex and cytotoxicity is shown in Figure 6%.
  • the liposomes as PEG - endostatin -B C increased 12-asiRNA complex concentration, cell viability decreased, i.e., increased toxicity to the cells, but not the whole toxicity to cells, when When the concentration of PEG liposome-endostatin-Bcl2-a siRNA complex reached 32 times the normal transfection concentration, the cell viability was about 77%, which was not toxic to cells.
  • Bcl2_a siRNA group was transfected with Lipof ectAMINETM2000 (Lipo 2000) liposome (charge value, sequence is the same as in Example 4); 2: PEG liposome-endostatin complex transfection B C 12-a siRNA group; 3: Lipo 2000 Transfection of B C 12-a siRNA + low dose cisplatin (1 ⁇ g) group; 4: PEG liposome-endothelial complex transfection of B C 12-a siRNA + low dose cisplatin (1 ⁇ g) group; 5 : low dose cis Platinum (1 ⁇ g) alone; 6: doubling dose of cisplatin (2 ⁇ g); 7, blank control group.
  • HelaB2 cells in logarithmic growth phase were inoculated into 96-well plates one day before transfection. When the cell density grew to 50% coverage, the original medium was discarded, and the serum-free and penicillin-free, streptomycin were used. The DMEM medium was washed once, and 100 ⁇ l of DMEM medium containing no calf serum and penicillin or streptomycin was added. Group 1 and Group 3 were transfected as follows: 0.25 ⁇ l of Bcl2-a siRNA (20 nM) was diluted with 25 ⁇ l of serum-free Opti-MEM and gently incubated for 5 minutes at room temperature.
  • Groups 2 and 4 replaced the 0.25 ⁇ l Lipo 2000 liposome in the above transfection method with 0.25 ⁇ l PEG liposome + 0.5 ⁇ l endostatin complex. Other transfection methods are the same as above.
  • the zeta potential of Group 2 PEG liposome-endostatin-Bcl2-a siRNA was determined to be +5 mV.
  • the blank control group was not transfected. After 24 h of transfection, groups 3, 4, and 5 were added with 1 ⁇ g of cisplatin, and group 6 was added with 2 ⁇ g of cisplatin. After 72 h of transfection, CCK-8 assay was performed as described in Example 5.
  • the 0D value of the blank control group was set to 100%, and the relative values of the 0D values of the other groups and the 0D values of the blank control group could reflect the relative levels of the number of cells in each group.
  • the in vitro inhibitory effect of PEG liposome-endostatin-B C 12-a siRNA complex in combination with cisplatin on HelaB2 cells is shown in Figure 10. It can be seen from Figure 10 that the PEG liposome-endostatin complex can also transfect B C 12-a siRNA into cells compared to the commercial transfected liposome Lipo 2000, thereby inhibiting cell growth. Moreover, PEG liposome-endostatin complex transfection with B C 12-a siRNA can inhibit cell proliferation better when combined with low cisplatin, and its inhibition rate on cell growth is higher than that of cisplatin alone. Group ( Figure 10).
  • the interference molecule used in this example was a cholesterol-modified B C 12-a siRNA with a charge value of -35 (sense strand: 5 ' chol-GAGGCUGGGAUGCCUUUdTdT3, antisense strand: 3 'dTdTGCCUCCGACCCUACGGAAA5 ', chol indicates cholesterol modification).
  • the irrelevant siRNA sequence is (sense strand: 5 ' UUCUCCGAACGUGUCACGUdTdT 3 ', antisense strand: 3 'dTdTAAGAGGCUUGCACAGUGCA 5 ').
  • Mouse liver cancer H22 liver cancer ascites cells (Laboratory Animal Center of Shanghai Institute of Materia Medica, Chinese Academy of Sciences) After resuscitation at 37 °C, each mouse was intraperitoneally injected with 0.3 ml, and the second generation was inoculated after 7 days. The second generation of ascites was adjusted, and the cell concentration was adjusted to 5 ⁇ 10 6 /ml with physiological saline. Each mouse was injected subcutaneously into the right side of the forelimb with 0.2 mL, about 1 ⁇ 10 6 tumor cells. The mice were fed ad libitum and fed normally.
  • mice were randomly divided into six groups: saline group, chemotherapy drug cyclophosphamide CTX (15 mg/kg) group, chemotherapy drug cyclophosphamide CTX (30 mg/kg) group, PEG liposome-endostatin-no siRNA group, PEG liposome-endostatin_Bcl2-a siRNA group (Zeta potential measurement +3mV), PEG liposome-endostatin _Bcl2-a siRNA Combined chemotherapy drug cyclophosphamide CTX (15mg/kg) group.
  • the administration was started the next day after the inoculation, and the dose of B C 12-a siRNA was 1 mg/kg, and the mass ratio of B C 12-a siRNA to endostatin and PEG liposome was 1:10:5.
  • the amide CTX (15 mg/kg) group was administered with continuous tail vein for 7 days (0.4 ml/mouse), CTX (15 mg/kg) group and CTX (30 mg/kg) group at the 2nd, 4th, 6th, and 8th after inoculation.
  • Tianjing is administered by intraperitoneal injection.
  • the length and width of the tumor were measured with a vernier caliper on the 5th, 10th, 15th and 20th day of administration, and the volume of the tumor was calculated by the following formula: a*b 2 *0.5 (a is long and b is wide) .
  • the tumor volume curve of each group is shown in Fig. 11. It can be seen from Figure 11 that PEG liposome-endostatin-B C 12-a siRNA can inhibit tumor growth well compared with saline group and unrelated siRNA group, and combined with chemotherapy drug cyclophosphamide CTX, antitumor effect Further increase.
  • the sense chain 5' cholesterol-modified VEGF symmetric and asymmetric RNA interference molecule used in this example the VEGF-a siRNA with a charge value of -38 (19+2/21+2, sense strand 5' cho 1 - GUGAAUGCAGACCAAAGAAdTdT 3', antisense strand 3' dTdTUACACUUACGUCUGGUUUCUU 5' ) and VEGF-siRNA with a charge value of -40 (21+2/21+2, sequence: sense strand 5' AUGUGAAUGCAGACCAAAGAAdTdT3 ', antisense strand 3' dTdTUACACUUACGUCUGGUUUCUU 5 ' ).
  • mice were randomly divided into 5 groups after vaccination: saline group (NS), chemotherapy drug cyclophosphamide CTX group, PEG liposome group, PEG liposome-endostatin _VEGF-a siRNA21/23 (Zeta potential measurement was +13mV). PEG liposome-endostatin-VEGF-siRNA23/23 group (Zeta potential measurement was +15 mV.).
  • the administration was started the next day after inoculation.
  • the doses of siRNA and as i RNA were 1 mg/kg, NS (0.4 ml/mouse), PEG liposome group (0.3 ml/mouse), and continuous tail vein administration for 14 days.
  • CTX (30 mg/kg) was administered intraperitoneally on days 2, 4, 6, 8, 10, and 12 after inoculation.
  • the length and width of the tumor were measured with a vernier caliper, and the volume of the tumor was calculated by the following formula: a*b3 ⁇ 40.5 (a is long and b is wide).
  • the tumor volume of the liposome-VEGF-a siRNA group was significantly smaller than that of the other groups, and it had a good tumor suppressing effect.
  • the tumor volume curve of each group is shown in Fig. 12.
  • mice Male Kunming mice were inoculated, grouped, specifically administered and dosed in the same manner as the Bcl2 fraction in Example 8, using PEG liposome, B C 12-a siRNA, endostatin and PEG liposome-endostatin- The charge values of B C 12-a siRNA were also the same as in Example 8. During the whole treatment, the mice were free to eat and drink water, and the living conditions and survival of the mice were recorded. The number of days was counted from the next day when the tumor cells were inoculated. By the 60th day, the survival time of 60 days or more was also calculated according to 60 days.
  • the median survival time of each group was as follows: saline group: 29 days; chemotherapy drug cyclophosphamide CTX (15 mg/kg) group: 39 days; chemotherapy drug cyclophosphamide CTX (30 mg/kg) group: 17 days; lipid - endostatin-unrelated siRNA group: 34 days; liposome-endostatin-Bcl2-a siRNA group: 41 days; liposome-endostatin-B C 12-a siRNA combined with chemotherapy drug cyclophosphamide CTX (15 mg/kg) Group: 58 days.
  • the survival curves of the mice in each group are shown in Fig. 13.
  • liposome-endostatin-B C 12-a siRNA can significantly prolong the survival of tumor-bearing mice, and when combined with low-dose chemotherapy drugs, when compared with chemotherapy drugs alone The survival period has been extended.
  • Male Kunming mice were inoculated, grouped, specifically administered and dosed in the VEGF fraction of Example 8, PEG liposomes, RNA interference molecules, endostatin and PEG liposome-endostatin-B C
  • the charge values of 12-VEGF-a siRNA21/23, PEG liposome-endostatin_Bcl2-VEGF-siRNA23/23 were also the same as in Example 8.
  • mice were free to eat and drink water, and the living conditions and survival of the mice were recorded. The number of days was counted from the next day when the tumor cells were inoculated. By the 60th day, the survival time of 60 days or more was also calculated according to 60 days.
  • the median survival time of each group was as follows: saline group: 35 days; CTX (30 mg/kg) group: 18 days; PEG liposome group: 20 days; PEG liposome-endostatin-VEGF-a siRNA21/23 Group: 38 days; PEG liposome-endostatin_VEGF_siRNA23/23 group: 32 days.
  • the survival curves of the mice in each group are shown in Fig. 14. From the median survival time and survival curve of each group, PEG liposome-VEGF-a siRNA21/23 could significantly prolong the survival of tumor-bearing mice.
  • the sample was prepared before administration, and PEG liposome-endostatin complex 2ml was taken. (The mass ratio of endostatin to PEG liposome was 2:1) and the fluorescently labeled BCL2-asiRNA-Cy5 20nmol was shaken and incubated for 20 min at room temperature (the PEG liposome-endostatin_Bcl2-a siRNA- The zeta potential of Cy5 was determined to be +7 mV).
  • BALB/C nude mice SPF grade, Shanghai Slack Laboratory Animals Co., Ltd.
  • male weighing 18_20g
  • transplanted tumor is S ⁇ C-7721 liver cancer.
  • Human hepatoma cells S ⁇ C-7721 purchased from Shanghai Chinese Academy of Sciences cell bank) at a concentration of 5 ⁇ 10 6 (0.2 mL/only) were injected subcutaneously into the neck and back of BLBA/c nude mice. Animal experiments were started when the tumor grew to 40-50 mm 3 . 2 ⁇ PEG ⁇ The prepared test drug, the tail vein injection (0.
  • CCR5_a siRNA a chemically modified and Cy5 fluorescently labeled CCR5_a siRNA was used (sequence: sense strand: 5' Choi - (mG) (mU) (mC) AAGUCCAAUCU (FA) (FU) (FG) dT-s-dT- Cy5 3, , Choi is cholesterol modification, m is methylation modification, F is fluoro modification, s is thio modification, antisense strand: 3' dTdTCACAGUUCAGGUUAGAUAC 5' ), by PEG liposome-endo endostatin
  • the delivery system was injected into the tail vein of BALB/c nude mice and observed for distribution in nude mice.
  • PEG liposome-endo endostatin complex 2ml (where Endo endostatin)
  • the mass ratio of PEG liposome is 2:1, the preparation method is the same as PEG liposome-endostatin complex), and the fluorescently labeled chemically modified CCR5- asiRNA - Cy5 20nmol mixed shaker, incubation at room temperature for 20 min, Zeta potential determination It is +10mV.
  • BALB/C nude mice SPF grade, Shanghai Slack Laboratory Animals Co., Ltd.
  • male weighing 18_20g, taking the prepared test drug, normal nude mice tail vein injection (0.
  • CCR5_a siRNA-Cy5 was distributed in all major organs of BLBA/c nude mice at different time points.
  • CCR5-a siRNA-Cy5 was abundantly accumulated by 24 h fluorescence quantification. In the kidney tissue, this product is mainly for renal excretion.
  • RNA interference molecule used in this example is a chemically modified B C 12-a siRNA with a charge value of -35, and the sequence is: sense strand: 5' chol- (mG) (mA) (mG) GCUGGGAUGCC (mU) (mU (mU) dT-s-dT 3', Choi is cholesterol modified, m is methylated, s is thio modified, antisense strand: 3' dTdT GCCUCCGACCCUACGGAAA 5' , endostatin-PEG lipid used The same as Example 2. Forty-two ICR mice were selected according to the weight of the animals according to the randomized grouping method of the group, 20 in each group, half male and half female.
  • This acute toxicity study selected B C 12-a siRNA at a dose of 100 mg/kg as the dose for the acute toxicity study (equivalent to 100 times the effective dose of 10 mg/kg in mice), endostatin-PEG lipid
  • the combined dose was 50 mg/kg (equivalent to 10 times the pharmacodynamically effective dose of 5 mg/kg of endostatin mice); and the negative control group was given 0.1% sterile DEPC water. 0. 4ml / 20g body weight.
  • the test results were as follows: Compared with the negative control group, no significant abnormalities were observed in the clinical symptoms of the animals in the test group, and no significant abnormalities were observed in the weight gain.
  • the minimum lethal dose (LD50) of the test sample B C 12-a siRNA administered to the ICR mice was greater than 100 mg/kg.
  • B C 12-a siRNA, endostatin used in this example, and Example 11, BALB/C nude mice (SPF grade), male, 18-20 g were purchased from Shanghai Slack Laboratory Animals Co., Ltd. Take well-grown A549 human lung cancer solid tumor (provided by Shanghai Pharmaceutical Industry Research Institute), cut into small pieces of about 3mm size under sterile conditions, inoculate a piece of mitral each mouse with right sac, and randomly divide The five groups were: saline group (blank control), chemotherapy drug cyclophosphamide CTX (30 mg/kg) group (positive control), PEG liposome-endostatin-B C 12-a siRNA high dose group, PEG liposome-endostatin-Bcl2-a siRNA in the low dose group of PEG liposome-endostatin-Bcl2_a siRNA.
  • the ratios of the groups were the same, except that the amount injected was 0.4, 0.2, 0. lml, so the zeta potential of the three groups was +3 mV.
  • the tumors were regrouped according to the tumor size, and the animals with too large and too small tumors were eliminated.
  • the average volume of each group of tumors was basically the same, and the administration started.
  • the high-dose group was administered with Bcl2- as iRNA 2 mg/kg, endostatin 15 mg/kg, and PEG liposome 5 mg/kg.
  • the medium dose group was administered 1/2 of the high dose group and the low dose group was 1/4 of the high dose group.
  • the saline group and PEG liposome-endostatin-B C 12-a siRNA were injected into the tail vein for 14 days in the high, medium and low dose groups.
  • the CTX (30 mg/kg) group was given intraperitoneal injection for 7 days.
  • the animals were sacrificed 29 days after inoculation, and the tumor pieces were dissected and photographed.
  • PEG liposome-endostatin-B C 12-a siRNA had a certain anti-tumor effect, and the anti-tumor effect increased with the dose, and the anti-tumor effect of the high-dose group.
  • the tumor segments after dissection in each group are shown in Figure 18.
  • the charge values of all liposome-endostatin recombinant protein-RNA interference molecules involved in Example 12 of the present invention were measured by Zeta potential method using a Zeta Sizer 3000 dynamic laser scatterometer.

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Abstract

La présente invention concerne une composition et son utilisation, la composition comprenant une endostatine qui sert de système d'administration et des molécules d'ARNi chimiquement synthétisées. La composition comprend la protéine recombinante endostatine qui porte de +5 à +10 charges et des molécules d'ARNi chimiquement synthétisées qui ont -15 à -55 charges, et la combinaison des charges positives et négatives des deux permet que la composition acquière -20 à +20 charges.
PCT/CN2011/074100 2011-05-16 2011-05-16 Composition comprenant de l'endostatine et des molécules d'arni et utilisation associée WO2012155328A1 (fr)

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CN101111154A (zh) * 2004-12-09 2008-01-23 表达遗传学公司 联合免疫基因疗法和化学疗法用于治疗癌症和过度增生性疾病

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CN101111154A (zh) * 2004-12-09 2008-01-23 表达遗传学公司 联合免疫基因疗法和化学疗法用于治疗癌症和过度增生性疾病

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MA, CHUNHONG ET AL.: "Inhibition effect of recombinant endostatin combined with anti-sense A targeting PreS2 on hepatoma cells in vivo", CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY, vol. 24, no. 2, February 2004 (2004-02-01), pages 145 *
WANG DONG ET AL.: "Synergetic role of pSilence Ape1 and endostatin in antiangiogenesis of WANG, Dong et al. Synergetic role of pSilence Apel and endostatin in antiangiogenesis of osteosarcoma in animal model", CHINESE JOURNAL OF EXPERIMENTAL SURGERY, vol. 23, no. 8, August 2006 (2006-08-01), pages 990 - 992 *

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