WO2016011840A9 - VGSC β3蛋白质用于癌症预防、治疗及诊断检测的靶点 - Google Patents

VGSC β3蛋白质用于癌症预防、治疗及诊断检测的靶点 Download PDF

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WO2016011840A9
WO2016011840A9 PCT/CN2015/079356 CN2015079356W WO2016011840A9 WO 2016011840 A9 WO2016011840 A9 WO 2016011840A9 CN 2015079356 W CN2015079356 W CN 2015079356W WO 2016011840 A9 WO2016011840 A9 WO 2016011840A9
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vgsc
tumor
protein
expression
inhibitor
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WO2016011840A1 (zh
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刘岩峰
崔勇
张景海
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沈阳药科大学
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Priority to US15/329,128 priority patent/US11155820B2/en
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Definitions

  • the present application relates to the association between VGSC ⁇ 3 protein and tumors, in particular, an inhibitor of VGSC ⁇ 3 protein and its use for the prevention and treatment of tumors, in particular cancer, with which inhibitors are used for the prevention and treatment of tumors, in particular cancers.
  • Voltage-gated ion channels are one of the most complex superfamilies involved in signal transduction processes known to date, and currently More than 140 members (CATTERALL W A, CHANDY K G, CLAPHAM D E, et al. International Union of Pharmacology: Approaches to the Nomenclature of Voltage-Gated Ion Channels [J]. Pharmacol Rev, 2003, 55(4): 573 -574), and new members are constantly being discovered. Purification, cloning and assays indicate that the ion channel protein is composed of multiple subunits. Voltage-gated ion channels have subunits such as ⁇ , ⁇ , ⁇ , and ⁇ , but the composition of different ion channels is slightly different.
  • the voltage-gated sodium channel consists of the alpha subunit and the beta subunit (see Figure 1) (CATTERALL W A. From ionic currents to molecular mechanisms: the structure and run ion of voltage-gated sodium channels [J]. Neuron, 2000, 26(11): 13-25, CATTERALL WA, GOLDIN AL, WAXMAN S G. International Union of Pharmacology. XXXIX. Compendium of Voltage-Gated Ion Channels: Sodium Channels [J]. Pharmacol Rev, 2003, 55 (4 ): 575-578).
  • the alpha subunit has a molecular weight of approximately 260 kDa and consists of four homologous transmembrane domains (I-IV), each of which contains six transmembrane helices (S1 to S6), S4 is a voltage receptor, and between S5 and S6.
  • the short peptides participate in the formation of the pores and act as gates to participate in mediating the depolarization process.
  • VGSC contains, in addition to the alpha subunit, one or two small polypeptides called beta subunits.
  • the sodium channel consists of an alpha subunit (Na v 1.1 to Na v 1.9) and one or more accessory beta subunits ( ⁇ 1 , ⁇ 2 , ⁇ 3 and ⁇ 4 (33 ⁇ 36kDa))
  • alpha subunit Na v 1.1 to Na v 1.9
  • accessory beta subunits ⁇ 1 , ⁇ 2 , ⁇ 3 and ⁇ 4 (33 ⁇ 36kDa)
  • TTX sensitive Na v 1.1, Na v 1.2, Na v 1.3
  • Na v 1.4 Na v 1.6, Na v 1.7
  • TTX insensitive Na v 1.5, Na v 1.8, Na v 1.9
  • the ⁇ subunit can regulate the transport and localization of the ⁇ subunit on the cell membrane and regulate the late current of the Na + channel of the myocardium.
  • diseases caused by mutations in the beta subunit are gradually being incorporated into the study of gene-phenotype interactions.
  • the ⁇ 1 and ⁇ 3 subunits have high homology, bind to the ⁇ subunit in a non-covalent bond, and have high homology to the ⁇ 2 and ⁇ 4 subunits, in a disulfide bond manner with the ⁇ subunit. Combine.
  • the functions of the ⁇ 1 and ⁇ 2 subunits have been identified as cell adhesion molecules (CAM), and the functions of the ⁇ 3 and ⁇ 4 subunits are not yet clear.
  • CAM cell adhesion molecules
  • the ⁇ 3 subunit is the same as the ⁇ 1 subunit, the effect is different between the form of the non-covalent bond and the ⁇ subunit, and the ⁇ 3 subunit is regulated by antagonizing the endoplasmic reticulum-release/recovery signal.
  • the voltage-gated Na + channel Nav1.8 contains an ER-retention/retrieval signal antagonized by the ⁇ 3 subunit [J]. J Cell Sci , 2008, 121: 3243-3252). Moreover, the ⁇ 3 subunit does not have a cell adhesion effect, and the ⁇ 3 subunit does not participate in trans-homologous cell-cell adhesion, nor does it bind to the anchoring protein ankyrinG (MCEWEN DP, CHEN C, MEADOWS LS, etc. The voltage-gated Na + channel ⁇ 3 subunit does not mediate trans homophilic cell adhesion or associate with the cell adhesion molecule contactin. Neurosci Lett, 2009, 462: 272-275).
  • the present invention finds for the first time that VGSC ⁇ 3 has a certain correlation with tumors, and can act as a target for diagnosis and treatment of tumors.
  • a first aspect of the invention relates to an inhibitor of VGSC ⁇ 3 which can be used to prevent or treat a tumor in a subject.
  • a second aspect of the invention relates to a pharmaceutical composition for preventing or treating a tumor in a subject comprising an inhibitor of VGSC ⁇ 3, preferably the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, optionally further comprising Other tumor therapeutics that synergistically act on VGSC beta 3 inhibitors.
  • a third aspect of the invention relates to a method of preventing or treating a tumor in a subject using an inhibitor of VGSC ⁇ 3 comprising administering to the subject an amount of a VGSC ⁇ 3 inhibitor, said amount effective to inhibit expression and/or activity of VGSC ⁇ 3.
  • the present invention also relates to the use of an inhibitor of VGSC ⁇ 3 for preventing or treating a tumor in a subject, and the use of an inhibitor of VGSC ⁇ 3 for the preparation of a pharmaceutical composition for preventing or treating a tumor in a subject.
  • a fourth aspect of the invention relates to a method of modulating one or more activities in a cell expressing VGSC ⁇ 3 comprising contacting the cell with an amount of a VGSC ⁇ 3 inhibitor effective to modulate one or more activities.
  • a fifth aspect of the invention relates to a method for diagnosing and detecting tumors using VGSC ⁇ 3 protein, using a tool for detecting VGSC ⁇ 3 protein (for example, a tool for measuring DNA level or mRNA level or protein expression level of VGSC ⁇ 3 subunit) Methods for diagnosing and detecting tumors, use of a tool for detecting VGSC ⁇ 3 protein in the diagnosis and detection of tumors, and use of a tool for detecting VGSC ⁇ 3 protein in preparing a kit for diagnosis and detection of tumors.
  • a tool for detecting VGSC ⁇ 3 protein for example, a tool for measuring DNA level or mRNA level or protein expression level of VGSC ⁇ 3 subunit
  • a sixth aspect of the invention relates to a method for inhibiting proliferation of tumor cells by using an inhibitor of VGSC ⁇ 3, an inhibitor of VGSC ⁇ 3 for inhibiting proliferation of tumor cells, and an inhibitor of VGSC ⁇ 3 for producing tumor cell proliferation Use in a pharmaceutical composition or kit.
  • a seventh aspect of the invention relates to the design, screening and preparation of an active substance for preventing or treating a tumor in a subject, and a pharmaceutical composition comprising the same, with the VGSC ⁇ 3 protein or its expression control sequence as a target.
  • the invention also relates to a method of identifying a tumor suppressor, wherein the tumor is characterized by differential expression of VGSC ⁇ 3 compared to a control.
  • the method comprises contacting a cell expressing VGSC ⁇ 3 with a candidate compound to determine whether VGSC ⁇ 3-related activity is modulated. Regulation of VGSC ⁇ 3-related activity is an indicator of cancer inhibitors.
  • the invention also relates to a kit for detecting or diagnosing a tumor comprising means for measuring the level of DNA or mRNA or protein expression of a VGSC ⁇ 3 subunit.
  • the tumor is associated with VGSC ⁇ 3, preferably a cancer, more preferably selected from the group consisting of lung cancer, liver cancer or leukemia.
  • the VGSC ⁇ 3 inhibitor is selected from the group consisting of an inhibitor capable of inhibiting DNA encoding a VGSC ⁇ 3 protein, an inhibitor capable of inhibiting expression of the gene by modulating upstream and downstream regulatory sequences of a VGSC ⁇ 3 protein-encoding gene, RNAi agent for VGSC ⁇ 3 protein, antibody binding to VGSC ⁇ 3 protein, small molecule inhibitor of VGSC ⁇ 3 protein, antisense oligonucleotide, (poly)peptide and other nucleic acids or simulations capable of inhibiting VGSC ⁇ 3 protein expression or activity Things.
  • the inhibitor is an RNAi inhibitor, preferably a dsRNA, more Preferably it is siRNA.
  • the siRNA double-stranded region is 9-50 bp, 15-36 bp, 15-30 bp, 15-26 bp, 15-23 bp, 15-22 bp, 15-21 bp, 15-20 bp, 15-19 bp, 15-18 bp.
  • the target gene is a target gene encoding a VGSC ⁇ 3 protein
  • the VGSC ⁇ 3 is from a mammal, preferably from a human
  • the amino acid sequence of the VGSC ⁇ 3 is SEQ ID NO
  • the nucleotide sequence of the target gene encoding the VGSC ⁇ 3 protein is shown in SEQ ID NO: 2.
  • the RNAi agent is an siRNA selected from the group consisting of:
  • the inhibitor is an antibody that binds to a VGSC ⁇ 3 protein, preferably the antibody is a monoclonal antibody, more preferably a humanized monoclonal antibody.
  • the VGSC ⁇ 3 protein bound to the antibody is from a mammal, preferably from a human, most preferably The amino acid sequence is shown in SEQ ID NO: 1.
  • the inhibitor is a peptide, such as an analgesic anti-tumor ⁇ ⁇ ⁇ (AGAP).
  • AGAP an analgesic anti-tumor ⁇ ⁇ ⁇
  • the cell expressing VGSC ⁇ 3 is a tumor cell.
  • the tumor cells are preferably cancer cells, more preferably liver cancer cells or lung cancer cells, most preferably HepG2 cells or A549 cells.
  • the means for detecting the VGSC ⁇ 3 protein is a tool for measuring the level of DNA or mRNA or protein expression of a VGSC ⁇ 3 subunit, including a detectable antibody capable of detecting VGSC ⁇ 3 protein expression and activity, Small molecule, oligonucleotide, bait, mimetic or probe.
  • the invention relates to the following specific embodiments:
  • a method of detecting or diagnosing a tumor associated with a VGSC ⁇ 3 subunit in a patient using a VGSC ⁇ 3 subunit comprising:
  • kits for measuring a DNA level or mRNA level or protein expression level of a VGSC ⁇ 3 subunit in the preparation of a kit for detecting or diagnosing a tumor associated with a VGSC ⁇ 3 subunit in a patient, wherein in the patient's The edge-encoded DNA level or mRNA level or protein expression level of the VGSC ⁇ 3 subunit measured in the sample is higher than that in normal control cells. The level indicates that the patient has a tumor.
  • embodiment 4 The method of embodiment 1 or 3, or the use of embodiment 2 or 3, wherein the tumor is cancer, preferably liver cancer, leukemia or lung cancer, most preferably the liver cancer is HepG2 type liver cancer, or the lung cancer is A549 type lung cancer .
  • any one of embodiments 1, 3, 4, 5, or the use of any one of embodiments 2, 3, 4, 5, wherein the means for measuring the level of DNA or mRNA or protein is included
  • a probe or primer that encodes a DNA level or mRNA level of the VGSC ⁇ 3 subunit, or a detectable antibody, small molecule, oligonucleotide, bait, mimetic or probe capable of detecting VGSC ⁇ 3 protein expression and activity is included.
  • a method of preventing or treating a tumor in a subject using an inhibitor of VGSC ⁇ 3, comprising administering to the subject an amount of a VGSC ⁇ 3 inhibitor, the amount effective to inhibit expression and/or activity of VGSC ⁇ 3.
  • VGSC ⁇ 3 inhibitor is selected from the group consisting of an inhibitor capable of inhibiting DNA encoding a VGSC ⁇ 3 protein, capable of regulating a gene encoding a VGSC ⁇ 3 protein Upstream and downstream regulatory sequences to inhibit expression of the gene, RNAi agents against VGSC ⁇ 3 protein, antibodies to VGSC ⁇ 3 protein, small molecule inhibitors of VGSC ⁇ 3 protein, inhibit VGSC ⁇ 3 protein expression or activity Antisense oligonucleotides, (poly)peptides and other nucleic acids or mimetics.
  • the inhibitor is an RNAi inhibitor, preferably a dsRNA, more preferably an siRNA, and most preferably, the siRNA duplex region is 9-50 bp, 15-36 bp 15-30 bp, 15-26 bp, 15-23 bp, 15-22 bp, 15-21 bp, 15-20 bp, 15-19 bp, 15-18 bp, 15-17 bp, 18-30 bp, 18-26 bp, 18-23 bp, 18 -22bp, 18-21bp, 18-20bp, 19-30bp, 19-26bp, 19-23bp, 19-22bp, 19-21bp, 19-20bp, 19bp, 20-30bp, 20-26bp, 20-25bp, 20 -24 bp, 20-23 bp, 20-22 bp, 20-21 bp, 21-30 bp, 21-26 bp, 21-25 bp, 21-24 bp, 20-23 bp, 20-22 b
  • the gene is a target gene encoding a VGSC ⁇ 3 protein, preferably the VGSC ⁇ 3 is from a mammal, preferably from a human, and preferably the amino acid sequence of the VGSC ⁇ 3 is as shown in SEQ ID NO: 1, most preferably the coding
  • SEQ ID NO: 2 the amino acid sequence of the target gene of the VGSC ⁇ 3 protein is shown in SEQ ID NO: 2.
  • RNAi agent is an siRNA selected from the group consisting of:
  • the inhibitor is an antibody or fragment thereof that binds to a VGSC ⁇ 3 protein
  • the antibody is a monoclonal antibody or fragment thereof, more preferably a humanized monoclonal
  • the antibody or fragment thereof, preferably, the VGSC ⁇ 3 protein that binds to the antibody is from a mammal, preferably from a human, most preferably the amino acid sequence thereof is SEQ ID NO: 1 is shown.
  • the tumor is a tumor associated with a VGSC ⁇ 3 subunit, preferably a cancer, more preferably liver cancer, leukemia or lung cancer, most preferably the liver cancer is HepG2 type liver cancer, or the lung cancer is A549 type lung cancer.
  • a method of modulating one or more activities in a cell expressing VGSC beta3 comprising contacting the cell with an amount of a VGSC beta3 inhibitor effective to modulate one or more activities.
  • VGSC ⁇ 3 inhibitor is selected from the group consisting of an inhibitor capable of inhibiting DNA encoding a VGSC ⁇ 3 protein, capable of regulating upstream of a gene encoded by a VGSC ⁇ 3 protein Downstream regulatory sequences to inhibit expression of the gene, RNAi agents against VGSC ⁇ 3 protein, antibodies that bind to VGSC ⁇ 3 protein, small molecule inhibitors of VGSC ⁇ 3 protein, can inhibit the antisense of VGSC ⁇ 3 protein expression or activity Oligonucleotides, (poly)peptides and other nucleic acids or mimetics.
  • the inhibitor is an RNAi inhibitor, preferably a dsRNA, more preferably an siRNA, and most preferably, the siRNA duplex region is 9-50 bp, 15-36 bp, 15- 30bp, 15-26bp, 15-23bp, 15-22bp, 15-21bp, 15-20bp, 15-19bp, 15-18bp, 15-17bp, 18-30bp, 18-26bp, 18-23bp, 18-22bp, 18-21 bp, 18-20 bp, 19-30 bp, 19-26 bp, 19-23 bp, 19-22 bp, 19-21 bp, 19-20 bp, 19 bp, 20-30 bp, 20-26 bp, 20-25 bp, 20-24 bp, 20-23 bp, 20-22 bp, 20-21 bp, 21-30 bp, 21-26 bp, 21-25 bp, 21-24 bp, 20-22 bp, 20-21 bp
  • RNAi agent is an siRNA selected from the group consisting of:
  • the inhibitor is an antibody or a fragment thereof that binds to a VGSC ⁇ 3 protein
  • the antibody is a monoclonal antibody or a fragment thereof, more preferably a humanized monoclonal antibody or Fragment
  • the VGSC ⁇ 3 protein that binds to the antibody is from a mammal, preferably from a human, most preferably the amino acid sequence thereof is set forth in SEQ ID NO: 1.
  • the tumor is a tumor associated with a VGSC ⁇ 3 subunit, preferably a cancer, more preferably liver cancer, leukemia or lung cancer, most preferably the liver cancer is HepG2 type liver cancer Or the lung cancer is A549 type lung cancer.
  • tumor cell is a liver cancer cell or a lung cancer cell, preferably a HepG2 cell or an A549 cell.
  • VGSC ⁇ 3 or its gene regulatory sequences as targets, designed and prepared for pre-preparation in subjects
  • a method for preventing or treating a tumor active substance which comprises designing an active substance capable of inhibiting expression or activity of a VGSC ⁇ 3 protein based on an amino acid sequence of a VGSC ⁇ 3 protein and a nucleic acid coding sequence.
  • a method of screening for an active substance for preventing or treating a tumor in a subject comprising contacting a cell expressing VGSC ⁇ 3 with a candidate active substance to determine whether inhibition of expression, activity and/or related biology of VGSC ⁇ 3 Whether the activity inhibits one or more cancer cell indicators such as cancer cell growth, wherein if the expression, activity and/or related biological activity of VGSC ⁇ 3 is inhibited or one or more cancer cell indicators are inhibited, This indicates that the candidate active substance is an active substance for preventing or treating a tumor.
  • a method of screening for an anticancer agent or an inhibitor that inhibits proliferation of tumor cells comprising contacting a cell expressing VGSC ⁇ 3 with a candidate compound, determining whether the expression, activity, and/or related biological activity of VGSC ⁇ 3 is modulated or Whether one or more cancer cell indicators such as cancer cell growth are inhibited, wherein if the expression, activity, and/or related biological activity of VGSC ⁇ 3 is inhibited or one or more cancer cell indicators are inhibited, the candidate is indicated
  • the active substance is an anticancer agent or an inhibitor that inhibits proliferation of tumor cells.
  • the active substance is selected from the group consisting of an inhibitor capable of inhibiting DNA encoding a VGSC ⁇ 3 protein, capable of inhibiting the upstream and downstream regulatory sequences of a gene encoding a VGSC ⁇ 3 protein.
  • an inhibitor of gene expression an RNAi agent against VGSC ⁇ 3 protein, an antibody that binds to VGSC ⁇ 3 protein, a small molecule inhibitor of VGSC ⁇ 3 protein, an antisense oligonucleotide capable of inhibiting expression or activity of VGSC ⁇ 3 protein, Many) peptides and other nucleic acids or mimetics.
  • RNAi inhibitor preferably a dsRNA, more preferably an siRNA
  • the siRNA duplex region is 9-50 bp, 15-36 bp, 15-30 bp, 15-26 bp, 15-23 bp, 15-22 bp, 15-21 bp, 15-20 bp, 15-19 bp, 15-18 bp, 15-17 bp, 18-30 bp, 18-26 bp, 18-23 bp, 18-22 bp, 18- 21bp, 18-20 bp, 19-30 bp, 19-26 bp, 19-23 bp, 19-22 bp, 19-21 bp, 19-20 bp, 19 bp, 20-30 bp, 20-26 bp, 20-25 bp, 20-24 bp, 20-23 bp, 20-22 bp, 20-21 bp, 21-30 bp, 21-26 bp, 21-26 bp, 21-30 bp, 21-26 bp,
  • RNAi agent is an siRNA selected from the group consisting of:
  • VGSC ⁇ 3 protein is from a mammal, preferably from a human, most preferably its amino acid sequence is set forth in SEQ ID NO: 1.
  • the tumor is a tumor associated with a VGSC ⁇ 3 subunit, preferably a cancer, more preferably liver cancer, leukemia or lung cancer, most preferably the liver cancer is HepG2 type liver cancer Or the lung cancer is A549 type lung cancer.
  • cancer cell is a cancer cell associated with a VGSC ⁇ 3 subunit, preferably a liver cancer cell or a lung cancer cell, more preferably a HepG2 cell or an A549 cell.
  • VGSC ⁇ 3 as a target in the preparation of a pharmaceutical composition for preventing or treating a tumor, and use in the preparation of a reagent for diagnosing or detecting a tumor.
  • a kit for detecting or diagnosing a tumor associated with a VGSC ⁇ 3 subunit in a patient comprising means for measuring a DNA level or mRNA level or protein expression level of a VGSC ⁇ 3 subunit.
  • kit of embodiment 40 wherein the kit further comprises one or more of the following: a control (positive and/or negative), a control container, a photograph of a representative example of a positive and/or negative result or description.
  • kits of embodiment 40 or 41, wherein the tumor is cancer, preferably liver cancer, leukemia or lung cancer, most preferably the liver cancer is HepG2 type liver cancer, or the lung cancer is A549 type lung cancer.
  • kits of any of embodiments 40-42, wherein the means for measuring the level of DNA or mRNA or protein is comprised of a probe or primer that can be used to measure the level or mRNA level of the DNA of the VGSC ⁇ 3 subunit, Alternatively, a detectable antibody, small molecule, oligonucleotide, bait, mimetic or probe capable of detecting VGSC ⁇ 3 protein expression and activity.
  • FIG. 1 Structure of a voltage-gated sodium ion channel (ROGER S, POTIER M, VANDIER C, et al. Voltage-Gated Sodium Channels: New Targets in Cancer Therapy? [J]. Curr Pharm Des, 2006, 12 (28): 3681-3695);
  • SCN3B a gene encoding the VGSC ⁇ 3 subunit, is expressed in different leukemias
  • FIG. 6 Effect of cell disruption on the VGSC ⁇ 3 subunit: D: blank HepG2 cell flow cytometry results; E: flow cytometry results after 48 h of SCN3B interference in HepG2 cells; F: SCN3B in HepG2 cells Flow cytometry results after 60h interference; G: D, E, F plots of p3 peak and p5 peak area after integration, the number of cells, where p3 is G0/G1 phase, p5 is fragment peak (apoptotic peak) );
  • Figure 8 Protein expression of VGSC ⁇ 3 subunit gene in lung cancer cell line A549 after interference
  • FIG. 10 Expression of VGSC ⁇ 3 subunit protein in Hep3B cells after transfection and expression of VGSC ⁇ 3 subunit protein after AGAP administration: lane 1: transfection with pcDNA3.0-hSCN3B; lane 2: transfection with empty plasmid pcDNA3.0 Dye 3: Effect on VGSC ⁇ 3 expression after administration of AGAP to Hep3B cells transfected with VGSC ⁇ 3; GAPDH was used as an internal control.
  • Figure 11 Detection of cell proliferation levels after transfection of VGAC ⁇ 3 subunit in Hep3B cells and detection of cell proliferation after administration of AGAP;
  • Figure 12 Effect of VGSC ⁇ 3 subunit interference on cell cycle and cell morphology: A: Photo of normal blank HepG2 cells; B: Photograph of SCN3B interference in HepG2 cells 48 h later; C: Photograph of SCN3B interference in HepG2 cells after 60 h; D: Flow cytometry results of normal blank HepG2 cells; E: flow cytometry results after 48 min of SCN3B interference in HepG2 cells; flow cytometry results after 60 min of SCN3B interference in Fep HepG2 cells; G: D, E, F
  • the statistical results of the area integration of the p3 peak and the p5 peak represent the number of cells, wherein p3 is the G0/G1 phase and p5 is the fragment peak (apoptotic peak);
  • FIG. 13 Effect of ANEXIN V on the apoptosis of VGSC ⁇ 3 subunit: A-D is the result of Annexin V-FITC-PI staining.
  • E-H is the result of Hoechst33342-PI staining.
  • Figure 14 A plasmid map comprising a nucleic acid sequence encoding a VGSC ⁇ 3 subunit (i.e., SCN3B, as set forth in SEQ ID NO: 2);
  • Figure 15 is a flow chart showing the construction of the plasmid described in Figure 14.
  • VGSC ⁇ 3 refers to the ⁇ 3 subunit of any voltage-sensitive ion channel known in the art, preferably mammalian VGSC ⁇ 3, more preferably VGSC ⁇ 3 protein is derived from humans.
  • Exemplary VGSC ⁇ 3 that has been characterized in the art includes Gene ID 55800, NM 018400 (http://www.ncbi.nlm.nih.gov/gene/55800).
  • Exemplary VGSC ⁇ 3 mutant proteins are also described in Dan Hu MD, Hector Barajas-Martines, Maria Burashnikov BS, et al. A mutation in the ⁇ 3 subunit of the cardiac sodium channel associated with brugada ECG phenotype. Circ Cardiovasc Genet. 2009 2 (3 ): 270-278.
  • the human VGSC ⁇ 3 protein comprises the following amino acid sequence, or the amino acid sequence thereof is as follows:
  • SEQ ID NO: 1 Amino acid sequence of human VGSC ⁇ 3 protein:
  • SEQ ID NO: 2 Nucleic acid sequence encoding human VGSC ⁇ 3 protein:
  • VGSC ⁇ 3 inhibitor includes any substance capable of inhibiting DNA, mRNA or protein of VGSC ⁇ 3, such as interfering RNA (RNAi active agents), antibodies, small molecule inhibitors, (poly)peptides and nucleic acids, Antisense oligonucleotides, or mimetics.
  • RNAi active agents interfering RNA
  • the VGSC ⁇ 3 protein is capable of inhibiting the expression, activity, and/or related biological activity of the VGSC ⁇ 3 protein by inhibiting its coding DNA sequence, its upstream and downstream regulatory sequences, mRNA, and VGSC ⁇ 3 protein.
  • VGSC ⁇ 3 refers to any statistically significant reduction in the biological activity, biological level, activity, and/or expression of VGSC ⁇ 3, including complete blockade of activity and/or expression.
  • inhibiting can mean that the VGSC ⁇ 3 level, activity, and/or expression is reduced by at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%.
  • the term “inhibiting” refers to a significant decrease in level, activity, and/or expression, while targeting any other biologically active agent or composition.
  • the inhibitor of VGSC ⁇ 3 inhibits VGSC ⁇ 3 biological activity by 25%, 50%, 75%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% compared to the control. . In some embodiments, the inhibitor of VGSC ⁇ 3 inhibits expression of VGSC ⁇ 3 by at least 25%, 50%, 75%, 80%, 90%, 95%, 97%, 98%, 99% or 100 compared to the control. %.
  • an inhibitor of VGSC ⁇ 3 disclosed herein comprises any inhibitor disclosed herein or known in the art capable of inhibiting VGSC ⁇ 3 and administered to a patient in need thereof, eg, suffering from a tumor Patients (especially cancer).
  • VGSC ⁇ 3 associated disease “reduced” indicates a statistically significant decrease in such levels. Such reduction can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more.
  • Treatment with the VGSC ⁇ 3 RNAi agent of the disclosure herein may specifically reduce the level or expression of VGSC ⁇ 3 to the literature, if the level or expression of VGSC ⁇ 3 is elevated for a particular disease or for an individual suffering from a particular disease. It is considered to be a normal range of levels for individuals without such conditions.
  • the level or expression of VGSC ⁇ 3 can be measured by evaluation of mRNA (eg, by Northern blot or PCR) or protein (eg, Western blot).
  • RNAi agents on VGSC ⁇ 3 expression can be determined by measuring the rate of transcription of the VGSC ⁇ 3 gene (eg, by Northern blotting; or reverse transcriptase polymerase chain reaction or real-time polymerase chain reaction). Direct measurement can be performed on the level of (cell surface expressed) VGSC ⁇ 3, for example, by Western blotting a tissue in which VGSC ⁇ 3 is expressed.
  • downstream-regulation refers to any statistically significant reduction in the biological activity and/or expression of VGSC ⁇ 3, including complete blocking activity (ie, complete inhibition) and/or expression.
  • “downregulation” can mean that the VGSC ⁇ 3 level, activity, and/or expression is reduced by at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%.
  • the term “inhibiting” VGSC ⁇ 3 refers to any statistically significant reduction in the biological level, activity, and/or expression of VGSC ⁇ 3, including complete blockade of activity and/or expression.
  • “inhibiting” can mean that the VGSC ⁇ 3 level, activity, and/or expression is reduced by at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100%.
  • the term “inhibiting” refers to a significant decrease in level, activity, and/or expression, while targeting any other biologically active agent or composition.
  • Level means that the VGSC ⁇ 3 RNAi agent or other inhibitor can alter VGSC ⁇ 3 Level, for example, the level of VGSC ⁇ 3 DNA or the level of mRNA or the level of VGSC ⁇ 3 protein, or the level of activity of VGSC ⁇ 3.
  • VGSC ⁇ 3 where the disease is characterized by overexpression and/or hyperactivity of VGSC ⁇ 3, administration of an inhibitor of VGSC ⁇ 3, such as an RNAi agent, reduces the level, expression and/or activity of VGSC ⁇ 3.
  • an inhibitor of VGSC ⁇ 3 such as an RNAi agent
  • Normal or “almost normal” for levels, expression and/or activity means at least about 50%, about 60%, about 70 of the level, expression or activity of VGSC ⁇ 3 in healthy cells, tissues or organs. %, about 80%, about 90%, and/or about 100%; and/or no more than about 100%, about 120%, about 130%, about 140%, or about 150%.
  • VGSC beta 3 inhibitors of the disclosure can be used to inhibit levels of VGSC ⁇ 3 expression, activity, and/or levels, depending on the disease condition and biological environment.
  • the VGSC ⁇ 3 inhibitor is an interfering RNA (RNAi) active agent.
  • RNAi interfering RNA
  • RNA interference techniques are well known in the art, and the design and acquisition of RNAi active agents are also known in the art, see for example the details in WO 2011/073326 or WO 2011/131707.
  • RNA interference is a method of reducing the expression of a gene of interest with small single-stranded or double-stranded RNA molecules.
  • Interfering RNA includes double-stranded or single-stranded small interfering RNA (ds siRNA or ss siRNA), microRNA (miRNA), small hairpin RNA (shRNA), and the like.
  • ds siRNA or ss siRNA double-stranded small interfering RNA
  • miRNA microRNA
  • shRNA small hairpin RNA
  • RNA interference appears to occur in vivo, and double-stranded RNA precursors are cleaved into small RNAs of approximately 20-25 nucleotides in length. Cleavage is accomplished by RNAeIII-RNA helicase dicase.
  • the sense strand of the siRNA i.e., the strand having the sequence identical to the sequence of the mRNA of interest, is removed, leaving an "antisense strand" complementary to the sequence of the mRNA of interest to reduce mRNA expression.
  • the antisense strand of siRNA appears to direct a protein complex called RISC (RNA-induced silencing complex) to the mRNA, and then the Argonaute protein of RISC cleaves the mRNA, thereby reducing protein production of the mRNA.
  • RISC RNA-induced silencing complex
  • Interfering RNA has catalytic activity, sub-chemical dose of mRNA-related stem Interfering with RNA can reduce mRNA expression.
  • a decrease in mRNA expression can also occur through mechanisms of transcription and translation.
  • RNAi agents of the present disclosure target (eg, bind, anneal, etc.) VGSC ⁇ 3 mRNA.
  • VGSC ⁇ 3-specific RNAi agents results in a decrease in VGSC ⁇ 3 activity, levels and/or expression, such as a “knock-down” or “knock-out” target gene or target sequence.
  • the RNAi comprises a single strand (eg, a shRNA as described herein).
  • one or both strands are slit.
  • a single-stranded RNAi agent oligonucleotide or polynucleotide may comprise a sense and/or antisense strand. See, for example, Sioud 2005 J. Mol. Biol. 348: 1079-1090, and references cited therein. Accordingly, the disclosure encompasses RNAi agents having a single strand comprising a sense or antisense strand of an RNAi agent described herein.
  • siRNAs particularly useful in the present disclosure include those that can specifically bind to a VGSC ⁇ 3 mRNA region, and that have one or more of the following properties: binding in the coding region of VGSC ⁇ 3; binding at or near the 5' untranslated region and The junction at the beginning of the coding segment; at or near the translation initiation site of the mRNA; binding to, covering or close to the junction of the exon and intron; mRNA or transcript with little or no binding to other genes (almost no or no "off-target effect"); binding of VGSC ⁇ 3 mRNA in or near one or more regions that are not double-stranded or stem, for example, in a loop or single-stranded portion; with little or no immunogenicity; In the segment of the VGSC ⁇ 3 mRNA sequence that is conserved between various animal species (including human, mouse, rat, cynomolgus, etc.), the presence of conserved sequences facilitates the use of multiple experimental animal tests; Double-stranded region
  • double-stranded RNA refers to an RNAi agent comprising a first strand and a second strand; for example, an RNA molecule or molecule complex comprising a region having a hybrid duplex a composition of a body (ie, a region of nucleotide base pairing of a first strand and a second strand) comprising two antiparallel and substantially complementary nucleic acid strands, referred to as having a relative to the target RNA
  • a body ie, a region of nucleotide base pairing of a first strand and a second strand
  • two antiparallel and substantially complementary nucleic acid strands referred to as having a relative to the target RNA
  • the direction of justice and anti-sense When it comes to mRNA targets, the antisense strand is also called the “guide” chain, and the sense strand is also called the "passenger” chain.
  • the passenger strand can include at least one or more of the following properties: one or more additional nucleotides (eg, bulges or 1 nt loops) compared to other strands, gaps, vacancies, etc., compared to other strands.
  • the RNAi agent comprises a first strand and a second strand.
  • the first strand is the sense strand and the second strand is the antisense strand.
  • the first strand is an antisense strand and the second strand is a sense strand.
  • the duplex region can be any length that allows for specific degradation of the desired target RNA by the RISC pathway, but typically ranges from 9 to 36 base pairs ("bp") in length, for example, 15-30 bp in length.
  • the duplex may be of any length within the range, such as 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 bp, and any subranges therebetween, including but not limited to 15-30 bp, 15 -26bp, 15-23bp, 15-22bp, 15-21bp, 15-20bp, 15-19bp, 15-18bp, 15-17bp, 18-30bp, 18-26bp, 18-23bp, 18-22bp, 18-21bp 18-20 bp, 19-30 bp, 19-26 bp, 19-23 bp, 19-22 bp, 19-21 bp, 19-20 bp, 19 bp, 20
  • the length of dsRNA produced in cells by Dicer and similar enzyme processing is typically in the range of from about 19 to about 22 bp.
  • One strand of the duplex region of the dsRNA comprises a sequence that is substantially complementary to the region of the target RNA.
  • the two strands forming the duplex structure may be derived from a single RNA molecule having at least one self-complementary duplex region, or may be formed from two or more isolated RNA molecules that hybridize to form a duplex.
  • the duplex region is formed by two self-complementary regions of a single molecule, the molecule may have a single-stranded nucleotide (referred to herein as a "hairpin loop", eg, found in the shRNA structure).
  • the single-stranded nucleotide is located between the 3' end of one strand forming the duplex structure and the 5' end of the corresponding other strand.
  • the hairpin loop may comprise at least one unpaired nucleotide; in some embodiments, the hairpin loop may comprise at least 3, at least 4, at least 5, at least 6, at least 7, At least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides.
  • two substantially complementary dsRNA strands are contained in an isolated RNA molecule, these molecules are not necessarily covalently linked.
  • shRNA When the two strands are covalently linked by a hairpin loop, the structure is generally referred to herein as "shRNA".
  • the joint structure is referred to as a "joint.”
  • RNAi agents RNAi agents
  • RNAi agents sense strands, antisense strands and (optional) overhangs
  • the RNAi agent comprises a first strand and a second strand, eg, the sense strand and the antisense strand and, optionally, at one or both ends of the duplex are referred to herein as Unpaired nucleotides at the ends.
  • antisense strand refers to a strand of an RNAi agent that comprises a region that is substantially complementary to a target sequence.
  • complementary region refers to a region on the antisense strand that is substantially complementary to a sequence as defined herein (eg, a target sequence).
  • the mismatch can be located in the interior or terminal region of the molecule. In general, the most tolerated mismatch is located in the internal region, e.g., within 5, 4, 3 or 2 nucleotides of the 5' and/or 3' end.
  • RNAi agent refers to a strand of an RNAi agent that comprises a region that is substantially complementary to the region of the antisense strand of the term as defined herein.
  • the sequence of the gene may be different between individuals, particularly at the swing position within the coding segment, or in the untranslated region; the coding sequences of the individual may also differ from each other, resulting in additional differences in mRNA.
  • the sense and antisense strand sequences of the RNAi agent can be designed to correspond to the sequence of the individual patient, if desired.
  • the sequence of the RNAi agent can also be modified to reduce immunogenicity, binding to undesirable mRNAs (e.g., "off-target effects") or to increase stability in the blood. These sequence variants are not dependent on chemical modification of the base or 5' or 3' or other end cap of the RNAi agent.
  • the RNAi agent may also have an overhang of 0, 1 or 2 overhangs, and in the case of a 0 nt overhang, the active agent is blunt ended.
  • the RNAi agent can have 0, 1 or 2 blunt ends. In In the "blunt-ended RNAi agent", both strands end in base pairs; therefore, the blunt-ended molecule lacks a 3' or 5' single-stranded nucleotide overhang.
  • the term "overhang” or “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the end of at least one of the two strands of the duplex structure of the RNAi agent. For example, when the 3' end of one strand of the dsRNA extends beyond the 5' end of the other strand or vice versa, the unpaired nucleotide forms an overhang.
  • the dsRNA may comprise an overhang of at least one nucleotide; alternatively, the overhang may comprise at least two nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides or more .
  • the overhang may comprise a nucleotide/nucleoside analog or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside.
  • the overhang can be located on the sense strand, the antisense strand, or any combination thereof.
  • the overhanging nucleotide may be located at the 5' end, 3' end or both ends of the antisense strand or the sense strand of the dsRNA.
  • the RNAi agent may also optionally comprise a cap.
  • cap or the like includes a chemical moiety attached to a double-stranded nucleotide duplex, but is used herein to exclude a chemical moiety that is a nucleotide or nucleoside.
  • the "3" cap is attached to the 3' end of the nucleotide or oligonucleotide.
  • the "5" cap is attached to the 5' end of the nucleotide or oligonucleotide.
  • the 3' end caps are as disclosed, for example, in WO 2005/021749 and WO 2007/128477.
  • RNAi agents that comprise an antisense strand (which may be contiguous or linked by a linker or loop) in the RNAi agent.
  • the RNAi agent comprises an antisense strand and a sense strand, and together comprise a double stranded or complementary region.
  • one or two overhangs and/or one or two caps may also optionally be included.
  • RNAi agents are used to induce RNA interference by VGSC ⁇ 3.
  • RNAi agent of the disclosure targets (eg, specifically binds, anneals, etc.) an mRNA encoding the gene VGSC ⁇ 3.
  • the use of VGSC ⁇ 3-specific RNAi agents results in a decrease in VGSC ⁇ 3 activity, levels and/or expression, such as a “knock-down” or “knock-out” target gene or target sequence.
  • a “knock-down” or “knock-out” target gene or target sequence is particularly in one embodiment, where the disease state is characterized by overexpression or hyperactivity of VGSC ⁇ 3
  • administration of an RNAi agent against VGSC ⁇ 3 knocks down the VGSC ⁇ 3 gene to a level sufficient to restore normal VGSC ⁇ 3 activity and/or Or normal Na + reabsorption levels.
  • target sequence or "target gene” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of a gene (eg, a gene encoding a VGSC ⁇ 3 protein), including for a primary transcription product. mRNA for the product of RNA processing.
  • the target portion of the sequence will be at least long enough to serve as a substrate for iRNA directed cleavage in the vicinity of the portion or portion.
  • the target sequence will typically be 9-36 nucleotides (“nt”) long, such as 15-30 nt long, including all sub-regions therebetween.
  • the target sequence can be 15-30 nt, 15-26 nt, 15-23 nt, 15-22 nt, 15-21 nt, 15-20 nt, 15-19 nt, 15-18 nt, 15-17 nt, 18-30 nt, 18-26nt, 18-23nt, 18-22nt, 18-21nt, 18-20nt, 19-30nt, 19-26nt, 19-23nt, 19-22nt, 19-21nt, 19-20nt, 19nt, 20-30nt, 20-26nt, 20-25nt, 20-24nt, 20-23nt, 20-22nt, 20-21nt, 20nt, 21-30nt, 21-26nt, 21-25nt, 21-24nt, 21-23nt or 21-22nt, 21nt, 22nt or 23nt.
  • the sense and antisense strands of RNAi comprise sequences complementary to the target nucleic acid VGSC ⁇ 3.
  • the term "complementary" means an oligonucleotide or polynucleotide comprising a first nucleotide sequence under certain conditions and an oligonucleoside comprising a second nucleotide sequence.
  • Such conditions can be, for example, stringent, for example, 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50 ° C or 70 ° C for 12-16 hours, followed by washing.
  • Other conditions such as physiologically relevant conditions that may be encountered in a living organism, are also applicable. The skilled artisan will be able to determine the set of conditions that are most suitable for testing the complementarity of the two sequences based on the final application of the hybridized nucleotides.
  • “Complementary” sequences when used herein, may also include non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, or entirely by non-Watson-Crick base pairs and / Or base pairs formed from non-natural and modified nucleotides are formed as long as the above requirements regarding their ability to hybridize can be satisfied.
  • Such non-Watson-Crick base pairs include, but are not limited to, G: U Wobble or Hoogstein base pairing.
  • complementary may also be referred to herein as base matching between the sense strand and the antisense strand of the dsRNA or between the antisense strand and the target sequence of the RNAi agent. Used in base matching, as understood from the context of its use.
  • a polynucleotide that is "substantially complementary to at least a portion of a messenger RNA (mRNA)” refers to a polynucleotide that is substantially complementary to a contiguous portion of a target mRNA (eg, an mRNA encoding VGSC ⁇ 3).
  • mRNA messenger RNA
  • a polynucleotide is complementary to at least a portion of a VGSC ⁇ 3 mRNA if the sequence is substantially complementary to a non-interrupted portion of the mRNA encoding VGSC ⁇ 3.
  • RNAi agent eg, within a dsRNA as described herein
  • oligonucleotide or polynucleotide comprising a first nucleotide sequence and an oligonucleotide or polynucleotide comprising a second nucleotide sequence Base pairing over the entire length of one or two nucleotide sequences.
  • Such sequences may be referred to herein as being “completely complementary" to each other.
  • the first sequence is referred to herein as being “substantially complementary” to the second sequence, the two sequences may be fully complementary, or they may form when hybridizing to a duplex of up to 30 base pairs.
  • a duplex comprising one oligonucleotide of 21 nucleotides in length and another oligonucleotide of 23 nucleotides in length (of which, longer Oligonucleotides comprising a 21 nucleotide sequence that is fully complementary to a shorter oligonucleotide can still be considered "fully complementary”.
  • the term overhang describes a nucleotide that is unpaired at the 3' or 5' end of a double-stranded nucleotide duplex, as described above. In one embodiment, the overhang is 0 to 4 nt long and is located at the 3' end.
  • an RNAi agent of the disclosure is complementary or substantially complementary to a target sequence in a target VGSC ⁇ 3 and is double-stranded, comprising a sense strand and an antisense strand (which may be contiguous, linked by a loop, or linked), wherein the double-stranded region is 9 to 36 bp long (especially, for example, 19-22 bp or 19-23 bp long), and may further optionally comprise a 3' or 5' overhang, and the RNAi active agent may further comprise a 3' cap.
  • RNAi agents mediate RNA interference, down-regulate or inhibit the level, expression and/or activity of VGSC ⁇ 3, and/or establish or re-establish almost normal ENaC levels and/or VGSC ⁇ 3 activity, or other biological functions associated with ENaC .
  • RNAi active agents that reduce VGSC ⁇ 3 levels, expression and/or activity
  • RNAi agents for targeting VGSC ⁇ 3 include an active agent that binds to the VGSC ⁇ 3 sequence provided herein and acts by the RNAi mechanism to reduce VGSC ⁇ 3.
  • exemplary siRNAs for VGSC ⁇ 3 are provided, for example, in Table 1.
  • RNAi agent of the present disclosure silences, inhibits the expression of the VGSC ⁇ 3 gene, down-regulates the expression of the VGSC ⁇ 3 gene, and/or suppresses the expression of the VGSC ⁇ 3 gene, such that an almost normal level of VGSC ⁇ 3 activity, expression and/or level is achieved, And / or Na + reabsorption.
  • RNAi agents of the disclosure can be used to establish VGSC ⁇ 3 expression, activity, and/or levels below normal, or above normal levels, depending on the disease condition and biological environment. Level.
  • Changes in VGSC ⁇ 3 activity, levels, and/or expression induced by VGSC ⁇ 3 siRNA can be measured using any method known in the art. Measurements can be performed at various time points before, during or after administration of the siRNA to determine the effect of the siRNA.
  • VGSC ⁇ 3 gene refers to the VGSC ⁇ 3 gene. In this context, it is meant to be at least partially repressed by the expression of the VGSC ⁇ 3 gene, which can be isolated or detected in a first cell or group of cells in which the VGSC ⁇ 3 gene is transcribed and has been treated to inhibit expression of the VGSC ⁇ 3 gene. A decrease in the amount of VGSC ⁇ 3 mRNA is obtained, which is compared to a second cell or group of cells (control cells) that is substantially identical to the first cell or group of cells but not subjected to such treatment. The degree of inhibition is usually expressed as:
  • the degree of inhibition can be given as a decrease in parameters associated with functional expression of the VGSC ⁇ 3 gene, for example, the amount of protein encoded by the VGSC ⁇ 3 gene, changes in lung fluid levels or mucus levels, and the like.
  • VGSC ⁇ 3 gene silencing can be determined by any appropriate assay in any constitutive or genomically engineered cell expressing VGSC ⁇ 3.
  • the assays provided in the examples below can be used as such references when determining whether the given RNAi agent inhibits expression of the VGSC ⁇ 3 gene to a certain extent, whether or not it is encompassed by the present disclosure. .
  • the expression of the VGSC ⁇ 3 gene is inhibited by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40% by administering a characteristic RNAi agent of the present disclosure. 45% and 50%.
  • the VGSC ⁇ 3 gene is inhibited by at least about 60%, 70%, or 80% by administering a RNAi agent that is characteristic of the disclosure.
  • the VGSC ⁇ 3 gene is inhibited by at least about 85%, 90%, or 95% or more by administering an RNAi agent described herein.
  • RNAi agent to inhibit VGSC ⁇ 3 can be tested first in vitro (eg, using test cells such as H441).
  • RNAi agents can also be tested in animal tests. Test and control animals include animals overexpressing or underexpressing VGSC ⁇ 3 as described, for example, in Hummer et al, 2005 J. Am. Soc. Nephrol. 16: 3160-3166; Randrianarison et al, 2007 Am. J. Physiol. Cell. Mol. Physiol. 294: 409-416; Cao et al, 2006 Am. J. Physiol. Renal Physiol., and references cited therein. RNAi agents that inhibit or alter the level, activity and/or expression of VGSC ⁇ 3 can be used in the treatment of a variety of VGSC ⁇ 3-related diseases.
  • RNAi agent Down-regulation of the expression of a particular protein in a cell using an RNAi agent or composition comprising an antisense nucleic acid is generally known in the art.
  • An RNAi agent includes a sequence that is complementary to, and capable of binding to, the coding strand (e.g., mRNA) of another nucleic acid.
  • the RNAi agents of the present disclosure encompass any RNAi agent that targets (eg, is complementary thereto, capable of hydrogen binding, etc.) such as the sequences set forth in Table 1.
  • the antisense sequence complementary to the mRNA may be complementary to the coding region, the 5' or 3' untranslated region of the mRNA, and/or the region encoding the coding region and the untranslated region, and/or portions thereof.
  • RNAi activity The agent or portion thereof may be complementary to a regulatory region of a gene encoding an mRNA, such as a transcriptional or translational initiation sequence or regulatory element.
  • the RNAi agent or portion thereof may be complementary to a region preceding the start codon of the coding strand, or to a region comprising a start codon, or to a region in the 3' untranslated region of the mRNA.
  • RNAi active agent molecules can be designed according to Watson and Crick base pairing rules.
  • the RNAi agent may be complementary to the complete coding region of VGSC ⁇ 3 mRNA, but more particularly an oligonucleotide that is only antisense to a portion of the coding region or non-coding region of VGSC ⁇ 3 mRNA.
  • an antisense oligonucleotide can be complementary to a region located around the translation initiation site of the VGSC ⁇ 3 mRNA.
  • the antisense oligonucleotide can be, for example, about 5, 10, 15, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • the RNAi agent can be modified internally or at one or both ends. Modification of the ends can help stabilize the RNAi agent and protect it from nuclease degradation in the blood.
  • the RNAi agent can optionally be directed to a region of the VGSC ⁇ 3 mRNA that is known or predicted to be close to or located at a gene splice site; for example, an exon-intron junction (eg, as described in Saxena et al., 1998).
  • the RNAi agent can also optionally be designed to anneal to known or predicted exposure and/or single-stranded regions (eg, loops) of the mRNA.
  • RNAi agents can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, naturally occurring nucleotides or a plurality of modified nucleotides can be used (designed to reduce off-target effects, and/or increase the biological stability of the molecule, or to increase the formation of duplexes between antisense and sense nucleic acids) Physical stability), to chemically synthesize RNAi agents, for example, phosphorothioate derivatives, and acridine-substituted nucleotides can be used.
  • G”, “C”, “A”, “T”, and “U” each generally represent a nucleotide comprising guanine, cytosine, adenine, thymidine, and uracil as bases, respectively.
  • ribonucleotide or “nucleotide” may also mean a modified nucleotide or a surrogate replacement moiety. It is well known to those skilled in the art that guanine, cytosine, adenine and uracil can be replaced by other moieties without substantially altering the base pairing properties of oligonucleotides comprising nucleotides having such replacement moieties.
  • a nucleotide comprising inosine as its base can be base paired with a nucleotide containing adenine, cytosine or uracil.
  • a nucleotide containing uracil, guanine or adenine may be substituted with a nucleotide containing, for example, inosine.
  • adenine and cytosine at any position in the oligonucleotide can be replaced by guanine and uracil, respectively, to form a G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for use in the compositions and methods of the present disclosure.
  • RNA molecule or "ribonucleic acid molecule” includes not only RNA molecules that are found or expressed in nature (ie, naturally occurring), but also non-naturally occurring ones that are described herein or that are known in the art. RNA analogs and derivatives of one or more ribonucleotide/ribonucleoside analogs or derivatives are known. Strictly speaking, “ribonucleoside” includes nucleobases and ribose, while “ribonucleotides” are ribonucleosides with one, two or three phosphate moieties. However, the terms “ribonucleoside” and “ribonucleotide” are used herein to be considered equivalent.
  • RNA can be modified in a nucleobase structure or in a ribose-phosphate backbone structure, for example as described herein below.
  • molecules comprising ribonucleoside analogs or derivatives must retain the ability to form duplexes.
  • the RNA molecule can also include at least one modified ribonucleoside, including but not limited to: a 2'-O-methyl modified nucleotide, a core comprising a 5' thiophosphate group a nucleoside, a terminal nucleoside linked to a cholesteryl derivative or a dodecanoic acid diamide group, a locked nucleoside, an abasic nucleoside, a 2'-deoxy-2'-fluoro modified nucleoside, 2'- Amino-modified nucleosides, 2'-alkyl modified nucleosides, morpholino nucleosides, unlocked ribonucleotides (eg, acyclic nucleomonomers, as described in WO 2008/147824), phosphoramidates Or a nucleoside comprising a non-natural base, or any combination thereof.
  • a 2'-O-methyl modified nucleotide e.g, acycl
  • the RNA molecule can comprise at least two modified ribonucleosides, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15 At least 20 or more, up to the full length of the dsRNA molecule.
  • the modifications need not be the same.
  • the modified RNA contemplated for use in the methods and compositions described herein is capable of forming the desired duplex structure and allows or mediates specific degradation of the target RNA via the RISC pathway.
  • PNA Peptide nucleic acid
  • modified nucleotides that can be used to produce an RNAi active agent include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4- Acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, ⁇ -D -galactosyl Q-nucleoside ( ⁇ -D-galactosylqueosine), inosine, N6-isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine , 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil , 5-methoxyaminomethyl-2-thiouracil,
  • the disclosure encompasses any modified variant of the modification of any of the RNAi agents disclosed herein.
  • Modified variants contain the same sequence, but may be modified to include modifications in phosphoric acid, sugars, bases, nucleotides, and the like.
  • a modified variant may contain one or more of the modified nucleotides recited herein, e.g., C substituted with a 2'-modified C.
  • the modified ribonucleoside comprises a deoxyribonucleoside.
  • the RNAi agent may comprise one or more deoxynucleosides including, for example, a deoxynucleoside overhang, or one or more deoxynucleosides within the dsRNA duplex portion.
  • RNAi active agent does not in any case include double-stranded DNA molecules.
  • siRNA duplex having a 3' overhang of two nucleotides with deoxyribonucleotides did not adversely affect RNAi activity.
  • Replacing up to four nucleotides per siRNA at each end with deoxyribonucleotides has been well tolerated, while complete substitution with deoxyribonucleotides results in no RNAi activity.
  • International PCT Publication No. WO 00/44914 and Beach et al., International PCT Publication No. WO 01/68836 initially suggest that siRNA can include modifications to the phosphate-sugar backbone or nucleoside to include at least one nitrogen or thia atom.
  • RNA-dependent protein kinase PKR double-stranded RNA-dependent protein kinase PKR
  • 2'-amino or 2'O- Base nucleotides as well as nucleotides containing a 2'-O or 4'-C methylene bridge. Additional 3'-terminal nucleotide overhangs include dt (deoxythymidine), 2'-O, 4'-C-vinyl thymidine (eT) and 2-hydroxyethyl phosphate (hp).
  • Parrish reported that inosine incorporation into either strand produced a substantial reduction in interfering activity. Parrish also reported that the incorporation of 5-iodouracil and 3-(aminoallyl)uracil into the antisense strand resulted in a substantial decrease in RNAi activity.
  • oligonucleotides are modified to enhance stability and/or enhance biological activity by employing nuclease resistant groups (eg, 2'-amino, 2'-C-allyl, 2'-fluoro, Modification of 2'-O-methyl, 2'-O-allyl, 2'-H), nucleotide Base modification is achieved (for a review, see Usman and Cedergren 1992 TIBS. 17:34; Usman et al, 1994 Nucleic Acids Symp. Ser. 31: 163; Burgin et al, 1996 Biochemistry 35: 14090). Sugar modifications to nucleic acid molecules are widely described in the art.
  • nuclease resistant groups eg, 2'-amino, 2'-C-allyl, 2'-fluoro, Modification of 2'-O-methyl, 2'-O-allyl, 2'-H
  • nucleotide Base modification is achieved (for a review, see Usman and Cedergren 1992 TIBS. 17:34; Usman
  • RNAi agents can also be chemically modified (including conjugation to other molecules) to improve in vivo pharmacokinetic residence time and efficiency.
  • the RNAi agent for VGSC ⁇ 3 comprises: at least one 5'-uridine-adenine-3' (5'-ua-3') dinucleotide, wherein the uridine is 2'- Modified nucleotide; at least one 5'-uridine-guanine-3' (5'-ug-3') dinucleotide, wherein 5'-uridine is a 2'-modified nucleotide; a 5'-cytidine-adenine-3' (5'-ca-3') dinucleotide, wherein 5'-cytidine is a 2'-modified nucleotide; and/or at least one 5'- Uridine-uridine-3' (5'-uu-3') dinucleotide, wherein 5'-uridine is a 2'-modified nucleotide.
  • the RNAi agent comprises a 2'-modification selected from the group consisting of 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2 '-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'- O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) and 2' -ON-Methylacetamido (2'-O-NMA).
  • the RNAi comprises a gap or a lost base.
  • a phosphate-sugar backbone can exist but bases are lost.
  • the RNAi agent has a single stranded nick (eg, a broken or missing bond in the backbone).
  • the single stranded gap can be in the sense strand or the antisense strand or both strands.
  • the nick can, for example, in the sense strand, produce small internal fragmented interfering RNA, or siRNA, which can have less off-target effects than the corresponding RNAi active agent without the nick.
  • the antisense nucleic acid or RNAi agent may also have an alternative backbone, such as a locked nucleic acid (LNA), a morpholino, a peptide nucleic acid (PNA), a threose nucleic acid (TNA), or a glycol nucleic acid (GNA), and / or it can be labeled (eg radiolabeled or tagged).
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • TAA threose nucleic acid
  • GNA glycol nucleic acid
  • One or both chains may contain an alternative backbone.
  • the RNAi agent utilized by the methods of the disclosure may comprise an alpha-anomeric nucleic acid molecule.
  • the ⁇ -anomeric nucleic acid molecule forms a specific double-stranded hybrid with the complementary RNA, wherein, contrary to the usual ⁇ -unit, the strands are arranged in parallel with each other. Gaultier et al., 1987 Nucleic Acids. Res. 15: 6625-6641.
  • Antisense nucleic acid molecules may also comprise 2'-o-methylribonucleotides (Inoue et al., 1987 Nucleic Acids Res. 15: 6131-6148) or chimeric RNA-DNA analogs (Inoue et al., 1987 FEBS Lett) .215:327-330).
  • the RNAi agent is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity that cleave single-stranded nucleic acids, such as mRNA, with complementary regions to them.
  • ribozymes e.g., hammerhead ribozyme (described in Haselhoff et al., 1988, Nature 334:585-591)] can be used to catalytically cleave VGSC ⁇ 3 mRNA transcripts, thereby inhibiting translation of VGSC ⁇ 3 mRNA.
  • gene expression can be inhibited by targeting a nucleotide sequence complementary to a regulatory region of VGSC ⁇ 3 (eg, a promoter and/or enhancer) to form a triple helix structure that prevents transcription of the VGSC ⁇ 3 gene.
  • a nucleotide sequence complementary to a regulatory region of VGSC ⁇ 3 eg, a promoter and/or enhancer
  • a triple helix structure that prevents transcription of the VGSC ⁇ 3 gene.
  • the RNAi agent can be biologically produced using an expression vector in which the nucleic acid is subcloned in an antisense orientation (i.e., the RNA transcribed from the inserted nucleic acid is in an antisense orientation to the target nucleic acid of interest).
  • the RNAi agent can also be biologically produced using an expression vector in which the nucleic acid is subcloned into a shRNA construct (ie, the RNA transcribed from the inserted nucleic acid has a first region in an antisense orientation to the target nucleic acid of interest, including A second region of the loop or hinge and a third region that is in a normal orientation to the target nucleic acid, wherein the first and third regions of the transcript preferably hybridize to themselves to form a stem-loop structure).
  • a shRNA construct ie, the RNA transcribed from the inserted nucleic acid has a first region in an antisense orientation to the target nucleic acid of interest, including A second region of the loop or hinge and a third region that is in a normal orientation to the target nucleic acid, wherein the first and third regions of the transcript preferably hybridize to themselves to form a stem-loop structure.
  • RNAi active agents are generally known in the art and are of ordinary skill in the art. Available to members.
  • Kits for the synthesis of RNAi are commercially available, for example, from New England Biolabs and Ambion.
  • the RNAi agent is a dsRNA, preferably a double strand length of 9-50 (eg 15-36 bp, 15-30 bp, 15-26 bp, 15-23 bp, 15-22 bp, 15-21 bp) 15-20 bp, 15-19 bp, 15-18 bp, 15-17 bp, 18-30 bp, 18-26 bp, 18-23 bp, 18-22 bp, 18-21 bp, 18-20 bp, 19-30 bp, 19-26 bp, 19 -23 bp, 19-22 bp, 19-21 bp, 19-20 bp, 19 bp, 20-30 bp, 20-26 bp, 20-25 bp, 20-24 bp, 20-23 bp, 20-22 bp, 20-21 bp, 21-30 bp, 21 -26bp, 21-25bp, 21-24bp, 21-23bp, 21-22bp, 20-
  • stringent hybridization conditions means that a probe, primer or oligonucleotide will hybridize to its target sequence under such conditions, but hybridize to a minimal amount of other sequences.
  • Stringent conditions are sequence dependent and vary from one environment to another. Longer sequences hybridize at a higher temperature to their appropriate complementary sequences. Generally, stringent conditions are selected to be about 5 ° C below the thermal melting point (Tm) of the particular sequence at a defined ionic strength and pH. Tm is the temperature at which 50% of the probe complementary to the target sequence hybridizes to the target sequence to equilibrium (at defined ionic strength, pH, and nucleic acid concentration).
  • stringent conditions can be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01-1.0 M sodium ion (or other salt), pH 7.0-8.3, and for short probes, primers or oligos. Nucleotides (e.g., 10-50 nucleotides), the temperature is at least about 30 ° C, and for longer probes, primers, or oligonucleotides, the temperature is at least about 60 °C. Stringent conditions can also be achieved by the addition of destabilizing agents such as formamide.
  • Moderately stringent conditions refers to conditions under which the probe, primer or oligonucleotide can hybridize to its target sequence, but hybridize to a limited number of other sequences.
  • Moderately stringent conditions are sequence dependent and vary from one environment to another. Moderate conditions are generally known to those skilled in the art and are described in particular in Maniatis et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory; 2nd Edition (December 1989)) and the like.
  • the invention also relates to nucleic acids, expression cassettes, expression vectors, and host cells comprising the RNAi agent.
  • the VGSC beta 3 inhibitor is a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a human antibody, a humanized antibody, a single chain antibody, or an antibody fragment, such as a Fab fragment.
  • Antibodies against VGSC ⁇ 3 are known in the art and can be obtained, for example, ABcam (Aibo (Shanghai) Trading Co., Ltd.
  • the antibody can be labeled with, for example, an enzyme, a radioisotope or a fluorophore.
  • the binding affinity of the antibody to a polypeptide other than VGSC ⁇ 3 is less than about 1 ⁇ 10 5 Ka.
  • the VGSC ⁇ 3 inhibitor is a monoclonal antibody that binds to VGSC B3 with an affinity of at least 1 x 10 8 Ka.
  • antibody refers to monoclonal and polyclonal antibodies specific for a target protein or fragment thereof, single chain antibodies, chimeric antibodies, bifunctional/bispecific antibodies, humanized antibodies, human antibodies. And complementarity determining regions (CDRs) for grafting antibodies.
  • CDRs complementarity determining regions
  • the term “antibody” also includes in vivo therapeutic antibody gene transfer.
  • the invention also provides antibody fragments, including Fab, Fab', F(ab')2, scFv, and Fv.
  • the term "monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for the possible naturally occurring mutations present in small amounts. Monoclonal antibodies are highly specific and target a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations comprising different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant of the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized without being contaminated by other antibodies.
  • a monoclonal antibody used in accordance with the present invention can be prepared by a hybridoma method.
  • Antibodies which are first described in Kohler et al, Nature 256:495 (1975), or can be produced by recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567).
  • phage antibody libraries for example, described in Clackson Human monoclonal 352: 624-628 (1991) and the technique of Marks et al, J. Mol. Biol. 222: 581-597 (1991) isolate "monoclonal antibodies”.
  • Monoclonal antibodies herein specifically include “chimeric” antibodies as well as fragments of such antibodies, as long as they exhibit desirable biological activities in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequence of the antibody,
  • the antibody is derived from a particular species or belongs to a particular antibody class or subclass, and the remaining strand is identical or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass (U.S. Patent No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
  • the chimeric antibodies of interest herein include "primatized" antibodies comprising variable region domain antigen binding sequences derived from non-human primates (e.g., Old World monkeys, apes, etc.) and humans Constant region sequence.
  • antibody fragment includes a portion of an intact antibody, preferably including its antigen binding or variable region.
  • antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linearized antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); single chain antibodies a molecule; and a multispecific antibody formed from an antibody fragment.
  • the antibody of the invention is a humanized antibody.
  • Humanized antibodies can be achieved by a variety of methods including, for example: (1) transplantation of non-human complementarity determining regions (CDRs) into human framework regions and constant regions (a process known in the art as "humanization"), or (2) The entire non-human variable domain is grafted, but they are "cloaked” by human-like surfaces by replacing surface residues (a process known in the art as “veneering").
  • humanized antibodies can include “humanized” and "mosaic” antibodies.
  • human antibodies can be produced by introducing a human immunoglobulin locus into a transgenic animal, for example, a mouse that is partially or completely inactivated with an endogenous immunoglobulin gene.
  • an antibody of the invention can function as an inhibitor/antagonist of a polypeptide of the invention.
  • the antibodies of the invention may be used alone or in combination with other compositions.
  • the antibody may also be recombinantly fused to the heterologous polypeptide at the N- or C-terminus, or chemically conjugated to the polypeptide or other composition (including Valence and non-covalent conjugation).
  • an antibody of the invention can be used as a fusion or conjugation of a molecule for the detection of a marker and an effector molecule, such as a heterologous polypeptide, drug, radionuclide or toxin. No. 5,314,995;
  • a fully human antibody can be derived from a transgenic mouse having a human immunoglobulin gene (see, for example, U.S. Patent Nos. 6,075,181, 6,091,001 and 6,114,598, incorporated herein by reference), or From phage display libraries of human immunoglobulin genes (see, for example, McCafferty et al, Nature, 348:552-554 (1990). Clackson et al, Nature, 352:624-628 (1991), and Marks et al, J. Mol. Biol., 222: 581-597 (1991)).
  • antibodies can be produced and identified by a scFv-phage display library. Antibody phage display library technology is available from commercial sources, such as Xoma (Berkeley, CA).
  • Monoclonal antibodies can be prepared using the method of Kohler et al., (1975) Nature 256:495-496, or modifications thereof. Typically, mice are immunized with a solution containing the antigen. Immunization can be carried out by mixing or emulsifying the antigen-containing solution in saline, preferably in an adjuvant such as Freund's complete adjuvant, and parenterally injecting the mixture or emulsion.
  • the monoclonal antibodies of the invention can be obtained using any immunological method known in the art. After immunizing the animal, the spleen (optionally, some large lymph nodes) is removed and separated into single cells. Splenocytes are screened by applying the cell suspension to a plate or well coated with the antigen of interest.
  • B cells expressing antigen-specific membrane-bound immunoglobulin bind to the plate and are not washed away. Then, the obtained B cells or all dissociated splenocytes are induced to fuse with myeloma cells to form a hybridoma, and cultured in a selective medium.
  • the cells obtained by plating are plated by serial or limited dilution, and the production of antibodies that specifically bind to the antigen of interest (and does not bind to an unrelated antigen) is determined.
  • the selected monoclonal antibody (mAb) secreting hybridomas are then cultured in vitro (e.g., in tissue culture flasks or hollow fiber reactors) or in vivo (in mouse ascites).
  • antibodies can be produced in a cell line, such as a CHO or a myeloma cell line, as disclosed in U.S. Patent Nos. 5,545,403; 5,545,405 and 5,998,144 each incorporated herein by reference. Briefly, cell lines were transfected with vectors capable of expressing light and heavy chains, respectively. Chimerism can be produced by transfecting two proteins on different vectors antibody. Immunol. 147:8; Banchereau et al, (1991) Clin. Immunol. Spectrum 3:8; and Banchereau et al, (1991) Science 251:70; both incorporated herein by reference.
  • Human antibodies can be produced using techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et al, CJ Mol. Biol., 222: 581 (1991). )). The techniques of Cole et al. and Boerner et al. can also be used to prepare human monoclonal antibodies (Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al, J. Immunol., 147 (1): 8695 (1991)).
  • Humanized antibodies can be achieved by a variety of methods including, for example: (1) transplantation of non-human complementarity determining regions (CDRs) into human framework regions and constant regions (a process known in the art as “humanization"), or (2) The entire non-human variable domain is grafted, but they are "cloaked” by human-like surfaces by replacing surface residues (a process known in the art as “veneering”).
  • humanized antibodies can include "humanized” and "mosaic” antibodies.
  • human antibodies can be produced by introducing a human immunoglobulin locus into a transgenic animal, for example, a mouse that is partially or completely inactivated with an endogenous immunoglobulin gene.
  • Humanized antibodies can also be produced by engineering transgenic animals containing human immunoglobulin loci.
  • WO 98/24893 discloses transgenic animals having a human Ig locus in which the animal does not produce a functional endogenous immunoglobulin due to inactivation of the endogenous heavy and light chain loci.
  • WO 91/10741 also discloses transgenic non-primate mammalian hosts capable of producing an immune response to an immunogen, wherein the antibody has a primate constant region and/or a variable region, and wherein the locus encoding the endogenous immunoglobulin is Is replaced or inactivated.
  • WO 96/30498 discloses the use of the Cre/Lox system to modify mammalian immunoglobulin loci, for example to replace all or part of a constant region or variable region, to form a modified antibody molecule.
  • WO 94/02602 discloses a non-human mammalian host having an inactivated endogenous Ig locus and a functional human Ig locus.
  • U.S. Patent No. 5,939,598 discloses a method of making transgenic mice in which the mouse lacks an endogenous heavy chain and expresses an exogenous immunoglobulin locus comprising one or more heterologous constant regions.
  • the antibodies of the invention can also be produced using the human engineering techniques disclosed in U.S. Patent 5,766,886, incorporated herein by reference.
  • an immune response against a selected antigen molecule can be produced, and antibody producing cells can be removed from the animal and used to produce a hybridoma secreting a human monoclonal antibody.
  • Immunization protocols, adjuvants and the like are known in the art for immunizing transgenic mice such as those described in WO 96/33735. The ability of a monoclonal antibody to inhibit or neutralize the biological activity or physiological effects of a corresponding protein can be tested.
  • the antibodies of the invention can be administered to a subject by in vivo therapeutic antibody gene transfer as discussed by Fang et al, (2005), Nat. Biotechnol. 23, 584-590.
  • a recombinant vector can be produced to deliver a polycistronic expression cassette comprising a peptide that mediates an enzyme-independent, co-translational autocleavage of the polypeptide, the polypeptide being located in the MAb heavy and light chain coding sequences between. Expression of two MAb chains that produce a stoichiometric amount.
  • a preferred example of a peptide that mediates an enzyme-independent, co-translational autocleavage is the foot-and-mouth disease-derived 2A peptide.
  • the antibody fragment retains the desired affinity of the full length antibody.
  • a fragment of an anti-VGSC ⁇ 3 antibody retains the ability to bind VGSC ⁇ 3.
  • Such fragments are characterized by properties similar to those of the corresponding full-length anti-VGSC ⁇ 3 antibodies, ie, the fragments specifically bind to human VGSC ⁇ 3 antigen expressed on the surface of human cells.
  • the antibody binds to one or more epitopes of VGSC ⁇ 3. In some embodiments, the antibody modulates one or more VGSC ⁇ 3-related biological activities. In some embodiments, the antibody inhibits one or more cancer cell growth, tumor formation, and cancer cell proliferation.
  • antisense therapy is a form of treatment for a genetic disorder or infection.
  • a nucleic acid strand DNA, RNA, or chemical analog
  • mRNA messenger RNA
  • This synthetic nucleic acid is referred to as an "antisense” oligonucleotide because its base sequence is complementary to the gene's messenger RNA (mRNA), the latter being referred to as a “sense” sequence (such a sensed mRNA segment "5 '-AAGGUC-3'” can be blocked by the antisense mRNA segment "3'-UUCCAG-5'").
  • mRNA messenger RNA
  • the oligonucleotide is complementary to a region, domain, portion or fragment of the VGSC ⁇ 3 gene or gene product. In some embodiments, the oligonucleotide comprises from about 5 to about 100 nucleotides, from about 10 to about 50 nucleotides, from about 12 to about 35 nucleotides, and from about 18 to about 25 nucleotides . In some embodiments, the oligonucleotide is at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, at least 96% of the region, portion, domain, or fragment of the VGSC ⁇ 3 gene or gene product. At least 97%, at least 98%, at least 99%, or 100% homologous.
  • oligonucleotide hybridizes to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 2 under moderate or stringent hybridization conditions.
  • the VGSC ⁇ 3 inhibitor is a mimetic.
  • mimetic refers to a compound that mimics the activity of a peptide.
  • the mimetic is not a peptide, but may comprise an amino acid joined by a non-peptide bond.
  • U.S. Patent No. 5,637,677, issued toJ.S. Jane In other words, the three-dimensional structure of the peptide is replicated by a molecule other than the peptide, which interacts specifically with the three-dimensional structure of VGSC ⁇ 3.
  • the VGSC ⁇ 3 inhibitor is a small molecule.
  • small molecule refers to an organic or inorganic non-polymeric compound having a molecular weight of less than about 10 kilodaltons. Examples of small molecules include peptides, oligonucleotides, organic compounds, inorganic compounds, and the like. In some embodiments, the small molecule has a molecular weight of less than about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 kiloDalton.
  • the VGSC ⁇ 3 inhibitor is a (poly)peptide.
  • the peptide is an analgesic anti-tumor ⁇ ⁇ ⁇ (AGAP).
  • the gene encoding AGAP is cloned into an E. coli expression vector, transformed into E. coli cells, and a recombinant strain is constructed to realize expression of the active peptide in E. coli, and purified by chromatography to obtain recombinant analgesic anti-tumor Glycopeptide.
  • a recombinant strain is constructed to realize expression of the active peptide in E. coli, and purified by chromatography to obtain recombinant analgesic anti-tumor Glycopeptide.
  • analgesic activity Liu YF1, Ma RL, Wang SL, Duan ZY, Zhang JH, Wu LJ. Wu CF.
  • Tumor refers to any tissue mass resulting from excessive cell growth or proliferation, either benign (non-cancerous) or malignant (cancer), including precancerous lesions.
  • cancer refers to a primary or metastatic cancer, leukemia or lymphoma.
  • cancer cell refers to a transformed cell. These cells can be isolated from patients with cancer, or these cells are transformed into cancerous cells in vitro. Cancer cells can be derived from a variety of types of samples, including any tissue or cell culture line. In some embodiments, the cancer cell is a hyperplasia, a tumor cell, or a neoplasm.
  • the cancer cells are isolated from liver cancer, lung cancer, colon cancer, testicular cancer, thymic cancer, breast cancer, skin cancer, esophageal cancer, pancreatic cancer, prostate cancer, uterine cancer, cervical cancer, bladder cancer, ovarian cancer, Multiple myeloma and melanoma.
  • the cancer cells are obtained from an already existing cell line that is publicly available.
  • the cancer cells are isolated from an existing patient sample or isolated from a library comprising cancer cells.
  • the cancer cells are isolated and then implanted into different hosts, such as xenografts.
  • the cancer cells are transplanted and used in a SCID mouse model.
  • the cancer is liver cancer, lung cancer, or leukemia.
  • VGSC ⁇ 3-related tumors refers to any tumor involved in the level, expression and/or activity of VGSC ⁇ 3, and/or any level, expression and/or regulation of VGSC ⁇ 3 by regulation (particularly inhibition) Or active treatment and / or remission of tumors.
  • VGSC ⁇ 3-related cancer refers to a cancer characterized by a cell difference (particularly increased) expression of VGSC ⁇ 3 relative to a non-cancerous cell.
  • the present invention can also be applied to any tumor cell type in which VGSC ⁇ 3 plays a role in cancer cell growth and the like.
  • the cancer is leukemia, colon cancer, liver cancer, testicular cancer, thymic cancer, breast cancer, skin cancer, esophageal cancer, pancreatic cancer, prostate cancer, uterine cancer, cervical cancer, lung cancer, bladder cancer, ovarian cancer, Multiple myeloma and melanoma.
  • the cancer is liver cancer, leukemia or lung cancer.
  • Such cancers exhibit at least about 25%, at least about 50%, at least about 75%, at least about 100%, at least about 150%, at least about 200%, or at least about 300% differential expression of VGSC ⁇ 3 (especially increased expression).
  • a "pharmaceutical composition” includes a pharmaceutically effective amount of one or more VGSC beta 3 inhibitors, a pharmaceutically acceptable carrier, and, optionally, other tumor therapeutic agents that act synergistically with a VGSC beta 3 inhibitor.
  • pharmaceutically effective amount means that the amount of VGSC beta 3 inhibitor is effective to produce the desired pharmacological, therapeutic or prophylactic result. For example, if there is at least a 10% reduction in measurable parameters associated with the disease or condition, then a given clinical treatment is considered to be effective, and a therapeutically effective amount of the drug used to treat the disease or condition is such that the parameter Produces an amount necessary for at least a 10% reduction.
  • a therapeutically effective amount of a VGSC ⁇ 3 inhibitor that targets VGSC ⁇ 3 can reduce VGSC ⁇ 3 protein levels by at least 10%.
  • a therapeutically effective amount of a medicament for treating the disease or condition is to produce at least 15, 20, 25, 30, 35, 40 of the parameters, respectively. The amount necessary to reduce the 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95%.
  • pharmaceutically acceptable carrier refers to a carrier to which a therapeutically active agent is administered.
  • Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the term specifically excludes cell culture media.
  • pharmaceutically acceptable carriers include, but are not limited to, pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binders, lubricants, sweeteners, flavoring agents, coloring agents, and preservatives. .
  • Suitable inert diluents include sodium and calcium carbonates, sodium and calcium phosphates, and lactose, while corn starch and alginic acid are suitable disintegrants.
  • the binder may include starch and gelatin, while the lubricant, if present, is typically magnesium stearate, stearic acid or talc.
  • the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract.
  • the active agents included in the pharmaceutical preparations are also in this paper. Described in.
  • compositions comprising VGSC beta 3 inhibitors may be in solid form, such as powders, granules, tablets, pills, soft capsules, capsules, liposomes, suppositories, chewable forms or patches.
  • Pharmaceutical compositions comprising VGSC beta 3 inhibitors may also be presented in liquid form, such as solutions, emulsions, suspensions, elixirs or syrups.
  • suitable liquid supports can be, for example, water, organic solvents (for example polyhydric alcohols such as glycerol or ethylene glycol, including propylene glycol and polyethylene glycol) or ethanol, polyoxyethylene castor oil (Cremophor EL) or mixtures thereof (more Proportion, in water).
  • the composition may comprise nano-sized amorphous or crystalline particles coated with aluminum or a surfactant.
  • Suitable supports may include, for example, antibacterial and antifungal agents, buffers, calcium phosphate, cellulose, methyl cellulose, chlorobutanol, cocoa butter, colorants, dextrin, emulsified Agents, enteric coatings, flavoring agents, gelatin, isotonic agents, lecithin, magnesium stearate, fragrances, polyols such as mannitol, injectable organic esters, such as ethyl oleate, Paraben, phenol sorbic acid, polyethylene glycol, polyvinylpyrrolidine, phosphate buffered saline (PBS), preservative, propylene glycol, sodium carboxymethylcellulose , sodium chloride, sorbitol, various sugars (including but not limited to sucrose, fructose, galactose, lactose and trehalose), starch, suppository wax, talc, vegetable oils such as olive oil and corn oil, Vitamins, waxes and/or wetting
  • the biologically inert portion of the pharmaceutical composition can optionally be erodible to allow release of the VGSC beta3 inhibitor over time.
  • the pharmaceutical composition may include other components that contribute to delivery, stability, efficacy, or reduced immunogenicity.
  • a pharmaceutical composition comprising a VGSC beta 3 inhibitor can be by buccal, inhalation (including insufflation or deep inhalation), nasal, oral, parenteral, implantation, injection or infusion (via epidural, intra-arterial, intra-articular, vesicle) Internal, intracardiac, intraventricular, intracranial, intradermal, intramuscular, intraorbital, intraperitoneal, spine Internal, intrasternal, intrathecal, intravenous, subarachnoid, subcapsular, subcutaneous, subepidermal, transendothelial, transtracheal, transvascular, rectal, sublingual, topical, and/or vaginal routes. This can be done by injection, infusion, dermal patch or any other method known in the art.
  • the formulation may be powdered, nebulized, aerosolized, granulated or suitably prepared for delivery. If it is a liquid, administration can be carried out slowly, or by bolus injection, although in some cases known in the art, bolus injections may result in loss of material through the kidneys.
  • compositions comprising VGSC beta 3 inhibitors can be administered using medical devices known in the art.
  • a VGSC beta 3 inhibitor can be administered using a needleless hypodermic injection device, such as the devices disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556.
  • a needleless hypodermic injection device such as the devices disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556.
  • Examples of well-known implants and modules that can be used in the present disclosure include: U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing medicament at a controlled rate; U.S. Patent No.
  • U.S. Patent No. 4,447,233 which discloses a medicament infusion pump for delivering a medicament at a precise infusion rate
  • U.S. Patent No. 4,447,224 which discloses A variable flow rate implantable infusion device for continuous drug delivery
  • U.S. Patent No. 4,439,196 which discloses a osmotic drug delivery system having a multi-chamber compartment
  • U.S. Patent No. 4,475,196 which discloses a osmotic drug Delivery system.
  • Many other such implants, delivery systems and modules are known to those skilled in the art.
  • a pharmaceutical composition comprising a VGSC beta 3 inhibitor can be formulated to ensure proper distribution in vivo.
  • Administration of a VGSC ⁇ 3 inhibitor against VGSC ⁇ 3 may be systemic (systemic) or in particular tissues or organs that target expression (or overexpress or exhibit hyperactivity) VGSC ⁇ 3, such as lung, kidney, colon and gland .
  • Methods of targeting these particular tissues or organs are described herein, and/or are known in the art.
  • they can be formulated in liposomes.
  • Liposomes can comprise one or more moieties that are selectively transported into a particular cell or organ, thereby enhancing targeted drug delivery (see, eg, V.V. Ranade (1989) J. Clin. Pharmacol. 29:685).
  • Exemplary targeting moieties include folic acid or biotin (see, for example, US Patent of Low et al. 5,416,016); Mannoside (Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153: 1038); Antibiotics (PG Bloeman et al. (1995) FEBS Lett. 357: 140; M. Owais et al. (1995) Antimicrob. Agents Chemother. 39: 180); Surfactant Protein A Receptor (Briscoe et al. (1995) Am. J. Physiol.
  • compositions comprising one or more VGSC ⁇ 3 inhibitors against VGSC ⁇ 3, which may optionally comprise various modifications and/or other components for the treatment of VGSC ⁇ 3-relevance disease.
  • a therapeutically effective amount of a VGSC beta 3 inhibitor of the present disclosure is administered to a patient in need thereof.
  • an “effective amount” or “therapeutically effective amount” is an amount that treats a disease or medical condition of an individual, or, more generally, an amount that provides a nutritional, physiological or medical benefit to an individual.
  • the phrases “therapeutically effective amount” and “prophylactically effective amount” mean in the treatment, prevention or management of the apparent symptoms of pathological processes mediated by VGSC ⁇ 3 expression or pathological processes mediated by VGSC ⁇ 3 expression.
  • the amount that provides a therapeutic benefit can be readily determined by one of ordinary practitioners and can vary depending on factors known in the art, such as the type of pathological process mediated by VGSC ⁇ 3 expression, the patient's medical history and age. , the stage of pathological processes mediated by ⁇ -ENaC expression, and the administration of other active agents that inhibit pathological processes mediated by VGSC ⁇ 3 expression.
  • the patient is at least about 1, 3, 6 or 9 months, or 1, 5, 10, 20, 30, 40, 50, 55, 60, 65, 70 or 75. year old. In various embodiments, the patient is no more than about 1, 3, 6 or 9 months, or 1, 5, 10, 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 90 Or 100 years old. In various embodiments, the patient has at least about 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260 , 280, 300, 320, 340, 360, 380 Or 400lbs of weight.
  • the patient has no more than about 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380 or 400 lbs of body weight.
  • the dose [measured only by the active ingredient] can be at least about 1, 5, 10, 25, 50, 100, 200, 250, 300, 250, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 ng, 1, 5, 10, 25, 50, 100, 200, 250, 300, 250, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 micrograms, 1, 5, 10, 25, 50, 100, 200, 250, 300, 250, 400, 450, 500, 550, 600, 650, 700, 750 , 800, 850, 900, 950 or 1000 mg.
  • the dosage may not exceed about 10, 25, 50, 100, 200, 250, 300, 250, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 mg.
  • the dosage can be at least once a day, daily, weekly, weekly, weekly, monthly, and/or every 2, 3, 4, 5, 6, 7, 8, 9, Apply at 10, 11 or 12 months, or a combination thereof.
  • the dosage is related to the individual's body weight or body surface area.
  • the actual dosage level can be varied to achieve an amount of active active agent that is effective for a particular patient, composition, and mode of administration but not toxic to the patient.
  • the selected dose will depend on a variety of pharmacokinetic factors, including the activity of the particular VGSC ⁇ 3 inhibitor used, the route of administration, the rate of VGSC ⁇ 3 inhibitor excretion, the duration of treatment, other drugs used in combination with the VGSC ⁇ 3 inhibitor, Compounds and/or materials, age, sex, weight, condition, general health and prior medical history of the patient, and similar factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine the effective amount of a desired VGSC beta 3 inhibitor.
  • a suitable dose will be that amount, in fact, the lowest dose that is effective for producing a therapeutic effect, or a dose that is low enough to produce a therapeutic effect but does not cause side effects.
  • the present invention provides methods comprising the combination of other active ingredients with a VGSC ⁇ 3 inhibitor of the invention.
  • the method further comprises administering to the patient one or more conventional cancer therapeutic agents.
  • the methods of the invention further comprise treating the patient with one or more chemotherapy, radiation therapy, hormone ablation, or surgery.
  • the invention also provides methods and compositions for treating, inhibiting, and managing cancer or other tumors, such as surgery, chemotherapy, radiation therapy, hormonal therapy, and biological therapy, for existing or standard cancer treatments, Has been partially or completely resisted.
  • the invention provides a method for treating and/or preventing a tumor or a symptom of a cancer or cancer in a subject comprising administering to the subject a therapeutically effective amount of one or more VGSC beta 3 inhibitors of the invention.
  • the cancer is a cancer associated with VGSC ⁇ 3.
  • the cancer is leukemia, colon cancer, liver cancer, testicular cancer, thymic cancer, breast cancer, skin cancer, esophageal cancer, pancreatic cancer, prostate cancer, uterine cancer, cervical cancer, lung cancer, bladder cancer, ovarian cancer, Multiple myeloma or melanoma.
  • the cancer is located within a non-hormone regulated tissue.
  • the subject has been diagnosed with or susceptible to cancer.
  • the cancer is liver cancer, lung cancer, or leukemia.
  • Symptoms of cancer are generally known to those skilled in the art including, but not limited to, weight loss, anemia, abdominal pain, intestinal obstruction, blood in the stool, diarrhea, constipation, other changes in bowel habits, colon metastasis, death, weakness, excessive fatigue, eating Difficulties, loss of appetite, chronic cough, worsening of dyspnea, hemoptysis, hematuria, nausea, vomiting, liver metastasis, lung metastasis, bone metastasis, abdominal distension, bloating, abdominal effusion, vaginal bleeding, bloating, colon perforation, acute Peritonitis (infection, fever, pain), pain, hematemesis, severe sweating, fever, high blood pressure, jaundice, dizziness, cold, tendon, lung metastasis, bladder metastasis, liver metastasis, bone metastasis, renal metastasis, and pancreas metastasis, Difficulty swallowing, etc.
  • the therapeutically effective amount of the modulating compound can be determined empirically according to procedures generally known to pharmacists, and depending on the age of the patient, the severity of the condition, and the desired final pharmaceutical formulation.
  • the therapeutic compositions of the present invention may also be administered as part of a combination therapy with other known anticancer agents or other known anti-bone disease treatment regimens.
  • the invention also provides a method of inhibiting the growth of cancer cells in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of one or more VGSC beta 3 inhibitors.
  • Suitable assays for measuring the growth of VGSC ⁇ 3-related cells are known to those skilled in the art and are presented in the context.
  • the invention also provides methods of inhibiting cancer in a patient in need thereof.
  • the method comprises administering to the patient a therapeutically effective amount of one or more VGSC beta 3 inhibitors.
  • the invention also provides methods of inhibiting cancer in a patient diagnosed or suspected of having cancer.
  • the method comprises administering to the patient a therapeutically effective amount of one or more VGSC beta 3 inhibitors.
  • the invention also provides methods of modulating one or more cancer symptoms in a patient.
  • the method comprises administering to the patient a therapeutically effective amount of a VGSC ⁇ 3 composition described herein.
  • the invention also provides a method for inhibiting cell growth in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a VGSC beta 3 inhibitor.
  • the invention also provides a method for inhibiting migration of cancer cells in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a VGSC beta 3 inhibitor.
  • the invention also provides a method for inhibiting adhesion of cancer cells in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a VGSC beta 3 inhibitor.
  • the present invention also provides a method of prophylactic treatment of a patient who is prone to develop cancer, cancer metastasis or which already has metastasis and thus is prone to recurrence or reproduction.
  • the method is particularly useful for high risk individuals, for example, having a family history of cancer or metastatic tumors, or exhibiting a genetic predisposition to cancer metastasis.
  • the tumor is a VGSC ⁇ 3-related tumor.
  • the method is used to prevent recurrence of VGSC ⁇ 3-related tumors in patients who have had VGSC ⁇ 3-related tumors that have been removed by surgical resection or treated with conventional cancer treatment.
  • the invention also provides a method of inhibiting cancer progression and/or causing regression of cancer comprising administering to the patient a therapeutically effective amount of a VGSC beta 3 inhibitor.
  • a patient in need of anti-cancer therapy is treated with a VGSC beta 3 inhibitor of the invention in combination with chemotherapy and/or radiation therapy.
  • a patient can also be treated with a therapeutically effective amount of anti-cancer radiation.
  • the combination provides a chemotherapeutic treatment and a VGSC beta 3 inhibitor.
  • the VGSC beta 3 inhibitor is administered in combination with chemotherapy and radiation therapy.
  • the method of treatment comprises administering to the patient a single or multiple doses of one or more VGSC beta 3 inhibitors.
  • the VGSC beta 3 inhibitor is administered as an injectable pharmaceutical composition, the composition being sterile, non-pyrogenic, and comprising a combination with a pharmaceutically acceptable carrier or diluent VGSC ⁇ 3 inhibitor.
  • the treatment regimen of the invention is used with conventional treatment regimens for cancer, including but not limited to surgery, radiation therapy, hormone ablation, and/or chemotherapy.
  • Administration of the VGSC ⁇ 3 inhibitor of the present invention can be carried out before, concurrently with, or after conventional cancer treatment.
  • two or more different VGSC ⁇ 3 inhibitors are administered to the patient.
  • the amount of VGSC ⁇ 3 inhibitor administered to the patient is effective to inhibit one or more of neutral cell growth, cancer cell growth, tumor formation, cancer cell proliferation, cancer cell metastasis, and VGSC ⁇ 3 expression. In some embodiments, the amount of VGSC ⁇ 3 inhibitor administered to the patient is effective to increase cancer cell death by apoptosis.
  • an inhibitor of VGSC ⁇ 3 of the disclosure herein is administered to a patient in need thereof with one or more additional pharmaceutically active agents suitable for treating a tumor, particularly a cancer.
  • a patient suffering from cancer can be administered a pharmacologically effective amount of one or more inhibitors of VGSC beta 3 with a pharmacologically effective amount of one or more of any of the cancer treatments listed herein and/or any other known in the art. Cancer treatment.
  • one or more inhibitors of VGSC ⁇ 3 and one or more other tumor therapeutic agents can be administered in any order, simultaneously or sequentially, or in multiple doses over time.
  • Administration of the inhibitor of VGSC ⁇ 3 and other therapeutic agents can be, for example, simultaneous, concurrent, separate or sequential.
  • Simultaneous administration can be carried out, for example, in the form of a fixed combination of two or more active ingredients, or can be carried out by simultaneous administration of two or more active ingredients formulated separately.
  • Sequential use (administration) preferably means that the one (or more) components of the combination are applied at one time point, the other components are applied at different time points, ie, in a long-term staggered manner, preferably such that the combination shows a single Compounds are administered with higher efficiency (especially showing synergy).
  • Separate use preferably means that the combined components are administered independently of one another at different points in time, preferably indicating that components (a) and (b) are administered such that the measurable overlap of blood levels of the two compounds is not The overlap mode (simultaneously) exists.
  • a combination of two or more of sequential, separate and simultaneous administration is possible, preferably such that the combined component-drug exhibits a combined therapeutic effect over that found when the combined component-drug is used independently at time intervals. Synergistic effects are particularly preferred, to the extent that they are too large to find an interaction with their therapeutic efficiency.
  • Combination therapeutic activity or “combination therapeutic effect” means that the compounds may be such that they preferably exhibit (preferably synergistic) interactions (combination therapeutic effects) in the warm-blooded animal (especially human) to be treated.
  • the compartments are administered separately (in a long-term staggered manner, especially in a sequential-specific manner). Whether this is the case can be determined by monitoring blood levels (showing that both compounds are present in the blood of the person to be treated, at least during certain time intervals) and the like.
  • a conventional cancer therapeutic is administered with a composition of the invention.
  • Conventional cancer therapeutics include:
  • Cancer chemotherapeutic agents include, but are not limited to, alkylating agents such as carboplatin and cisplatin; nitrogen nervating agents; nitrosourea alkylating agents such as carmustine (BCNU); antimetabolites such as methotrexate; Acyltetrahydrofolate; anthraquinone analog antimetabolite, purine; pyrimidine analog antimetabolite, such as fluorouracil (5-FU) and gemcitabine Hormone antineoplastic agents such as goserelin, leuprolide and tamoxifen; natural antineoplastic agents such as aldesleukin, interleukin-2, docetaxel, etoposide (VP-16), interference Alpha, paclitaxel And retinoic acid (ATRA); antibiotics for natural antineoplastic agents such as bleomycin, dactinomycin, daunorubicin, doxorubicin, daunorubicin and mitomycin (including mito
  • alkaloids natural antineoplastic agents such as vinblastine, vincristine, vindesine; hydroxyurea; acetaldehyde lactone; adriamycin, ifosfamide, Notitabine, cyclosalicysteine, arubicin, amphetamine, nimustine, procarbazine hydrochloride, carbofuran, carboplatin, carmofur, leucomycin A3, anti-tumor polysaccharide, anti-tumor Platelet factor, cyclophosphamide Szofiram, cytarabine (cytosine arabinoside), dacarbazine, thioinosine, thiotepa, tegafur, dolastatin, dolastatin analogs such as auristatin, CPT-11 Liticon), mitoxantrone, vinorelbine, teniposide, aminopterin, erythromycin, espomycin (see,
  • Neocarcin No. 4,675,187
  • Neocarcin OK-432
  • Bo Levomycin Fluoroiron
  • bromouridine busulfan
  • diethylstilbestrol tetrasodium albinomycin
  • phenylbutyrate Interferon- ⁇ memantine, dibromomannitol, melphalan, laminin peptide, lentinan, Coriolus versicolor extract, tegafur/uracil, estramustine (female) Hormone / nitrogen mustard).
  • active agents that can be used as treatments for cancer patients include EPO, G-CSF, ganciclovir; antibiotics, leuprolide; meperidine; zidovudine (AZT); interleukins 1 to 18, including variants and Analogs; interferons or cytokines such as interferon alpha, beta and gamma; hormones such as luteinizing hormone releasing hormone (LHRH) and analogs, and gonadotropin releasing hormone (GnRH); growth factors such as transforming growth factor - ⁇ (TGF- ⁇ ), fibroblast growth factor (FGF), nerve growth factor (NGF), growth hormone releasing factor (GHRF), epidermal growth factor (EGF), fibroblast growth factor homologous factor (FGFHF) , hepatocyte growth factor (HGF) and insulin growth factor (IGF); tumor necrosis factor- ⁇ & ⁇ (TNF- ⁇ & ⁇ ); invasion inhibitor-2 (IIF-2); bone morphogenetic protein 1-7 (BMP 1-7) ; somatostatin
  • Prodrug means a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic or non-cytotoxic to tumor cells than the parent drug and that can be enzymatically activated or converted to activated or The active parent form. See, for example, Wilman, "Prodrugs in Cancer Chemotherapy” Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Harbor (1986) and Stella et al., “Prodrugs: A Chemical Approach to Targeted Drug Delivery,” Directed Drug Delivery, Borchardt et al. (eds.), pp. 247-267, Humana Press (1985).
  • Prodrugs include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acids Modified prodrug, glycosylated prodrug, prodrug containing beta-lactam, prodrug containing optionally substituted phenoxyacetamide or prodrug containing optionally substituted phenylacetamide a prodrug of 5-fluorocytosine or other 5-fluorouridine (its Can be converted to a more activated cytotoxic free drug).
  • cytotoxic drugs that can be derivatized into prodrug forms for use herein include, but are not limited to, the chemotherapeutic agents described above.
  • the invention also provides methods of diagnosing tumors, such as cancer and/or predicting tumors, such as cancer progression, using the inventive VGSC ⁇ 3 to diagnose a tumor or cancer in a subject.
  • the method includes:
  • the protein or gene content of VGSC ⁇ 3 in the sample or cell to be tested is higher than that of the control, indicating that the subject has a tumor.
  • the present invention also provides a method of diagnosing a VGSC ⁇ 3-related tumor (e.g., cancer) in a subject using the VGSC ⁇ 3 of the present invention, the method comprising
  • the protein or gene content of VGSC ⁇ 3 in the tumor cells to be tested is higher than that of the control normal cells, indicating that the subject has a VGSC ⁇ 3-related tumor (for example, cancer).
  • the means for measuring a DNA level or mRNA level or protein level comprises a probe or primer capable of measuring a DNA level or mRNA level of a VGSC ⁇ 3 subunit, or capable of detecting VGSC ⁇ 3 protein expression and activity
  • the sample is a sample of tissue suspected of having a tumor, preferably a tumor biopsy or a cell extract thereof.
  • the cell extract comprises an extract of circulating cells of a solid tumor.
  • Circulating cells are typically isolated from patient samples using one or more separation methods known in the art including, for example, immunomagnetic separation, CellTrack (TM) systems, microfluidic separation, FACS, density gradient centrifugation, and elimination.
  • TM CellTrack
  • FACS density gradient centrifugation
  • the patient sample includes whole blood, serum, plasma, sputum, bronchial irrigation Lotions, urine, nipple aspirate, lymph, saliva and/or fine needle aspirate samples.
  • a whole blood sample is separated into plasma or serum components and cellular components (ie, cell pellets).
  • the cellular components typically contain circulating cells of red blood cells, white blood cells, and/or solid tumors, such as CTCs, CECs, CEPCs, diffuse tumor cells of lymph nodes, and/or CSCs, and combinations thereof.
  • Plasma or serum components typically contain nucleic acids (eg, DNA, RNA) and proteins released by circulating cells of solid tumors.
  • the invention provides a tool for imaging and/or detecting a gene or gene product associated with VGSC ⁇ 3 overexpression, or for measuring a DNA level or mRNA level or protein expression level of a VGSC ⁇ 3 subunit Tools, and kits containing the tools.
  • the tools of the invention can include detectable antibodies, small molecules, oligonucleotides, baits, mimetics or probes.
  • the kit of the invention comprises the tool and instructions for carrying out the method of the invention.
  • the kit may also contain one or more of the following: a photograph or description of a representative example of a control (positive and/or negative), a control container, a positive and/or negative result.
  • the present invention also provides a method for designing and preparing an active substance for preventing or treating a tumor in a subject by using VGSC ⁇ 3 as a target, the method comprising, according to the amino acid sequence of the VGSC ⁇ 3 protein and the nucleic acid coding sequence, the design can inhibit An antibody, small molecule inhibitor, (poly)peptide and nucleic acid, antisense oligonucleotide, or mimetic of expression or activity of a VGSC ⁇ 3 protein.
  • the method further comprises the steps of preparing the above-described designed active substance and determining whether it inhibits the expression, activity and/or related biological activity of VGSC ⁇ 3. In some embodiments, inhibition of one or more cancer cell indicators, such as cancer cell growth, is indicative of the active substance described above.
  • the present invention also provides a preparation of the above-mentioned active substance prepared as a pharmaceutical composition.
  • the invention also provides a method of screening for an active substance for preventing or treating a tumor in a subject, the method comprising contacting a cell expressing VGSC ⁇ 3 with a candidate active substance, determining whether the expression, activity and/or inhibition of VGSC ⁇ 3 is inhibited A step related to biological activity. In some embodiments, inhibition of one or more cancer cell markers, such as cancer cell growth, is indicative of the above activity substance.
  • the present invention also provides a preparation of the above-mentioned active substance prepared as a pharmaceutical composition.
  • the invention also provides methods for screening for anticancer agents or inhibitors that inhibit tumor cell proliferation.
  • the method comprises contacting a cell expressing VGSC ⁇ 3 with a candidate compound to determine whether the expression, activity and/or related biological activity of VGSC ⁇ 3 is modulated.
  • inhibition of one or more cancer cell indicators, such as cancer cell growth, is indicative of an anticancer agent.
  • the term "about” refers to a value of +/- 20%, +/- 10%, or +/- 5%.
  • polypeptide or "protein” is used interchangeably and refers to a polymeric form of amino acid of any length, which may include both encoded and non-coding amino acids, chemically or biochemically modified or derivatized amino acids, and modified peptides.
  • the polypeptide of the main chain includes fusion proteins including, but not limited to, fusion proteins having a heterologous amino acid sequence, fusions having heterologous and homologous leader sequences (with or lacking an N-terminal methionine residue); immunolabeled proteins ;Wait.
  • subject refers to any subject, particularly a human, in need of diagnosis, treatment or treatment.
  • Other subjects may include cattle, dogs, cats, guinea pigs, rabbits, rats, mice, horses, and the like.
  • the subject is a human.
  • sample refers to a biological material from a patient.
  • the samples determined by the present invention are not limited to any particular type.
  • Samples include, by way of non-limiting example, single cells, multiple cells, tissues, tumors, biological fluids, biological molecules, or any of the above extracts or supernatants. Examples include removal of tissue for biopsy, tissue removed during resection, blood, urine, lymphoid tissue, lymph, cerebrospinal fluid, mucus, and stool samples.
  • the sample used can be varied depending on the method of assay, the method of detection, and the nature of the tumor, tissue, cell or extract to be assayed. Methods for preparing samples are generally known in the art and can be conveniently adjusted to obtain samples that are compatible with the method used.
  • biological molecule includes, but is not limited to, polypeptides, nucleic acids, and sugars.
  • a “gene product” is a biological polymerization product that is expressed or produced by a gene.
  • the gene product can be, for example, unspliced RNA, mRNA, spliced variant mRNA, polypeptide, post-translationally modified polypeptide, splice variant polypeptide, and the like.
  • the term also encompasses biopolymerization products produced using the RNA gene product (ie, cDNA of RNA) as a template.
  • the gene product can be enzymatic, recombinant, chemically produced, or produced within the natural cells of the gene.
  • the biological product is biologically active if the gene product is protein.
  • the gene product if the gene product is a nucleic acid, it can be translated into a gene product of a protein that exhibits biological activity.
  • biological molecule includes, but is not limited to, polypeptides, nucleic acids, and sugars.
  • the terms “differential expression in cancer cells” and “polynucleotides differentially expressed in cancer cells” are used interchangeably herein to refer to when the same cell type is not cancerous.
  • the representative gene or the polynucleotide corresponding to the gene is differentially expressed in the cancerous cell, for example, the level of mRNA is found to be at least about 25%, at least about 50% to about 75%, at least about 90%, At least about 1.5 times, at least about 2 times, at least about 5 times, at least about 10 times, or at least about 50 times or more different (eg, higher or lower).
  • Comparisons can be made in the tissue, for example using in situ hybridization or another assay to allow for some degree of differentiation of cell types in the tissue. It is also possible or optional to compare between cells removed from their tissue source, or between an in situ cell and a second cell removed from its tissue source. In some embodiments, the gene of the oncogene is up-regulated compared to normal cells.
  • FGF21-associated cancer is "suppressed” if at least one symptom or clinical endpoint of cancer is alleviated, terminated, slowed, or prevented. As used herein, FGF21-associated cancer is also “suppressed” if it reduces, slows, delays, or prevents metastasis or recurrence of cancer.
  • Clinical endpoint refers to a measurable event that is an indicator of cancer.
  • Clinical endpoints include, but are not limited to, time to first metastasis, time to secondary metastasis, size and/or number of metastases, size and/or number of tumors, location of the tumor, aggressiveness of the tumor, quality of life, pain Wait. The ability of a person skilled in the art to determine and measure clinical endpoints is credible.
  • fragment refers to a physically contiguous portion of the primary structure of a biomolecule.
  • the sense moiety refers to at least 3-5 amino acids, at least 8-10 amino acids, at least 11-15 amino acids, at least 17-24 amino acids, at least 25-30 amino acids, and at least 30-45 amino acids.
  • a portion is defined by a contiguous portion of the nucleic acid sequence of the oligonucleotide, meaning at least 9-15 nucleotides, at least 18-30 nucleotides, at least 33-45 nucleosides Acid, at least 48-72 nucleotides, at least 75-90 nucleotides, and at least 90-130 nucleotides.
  • a portion of a biomolecule is biologically active.
  • the FGF21 polypeptide fragment does not comprise the entire FGF21 polypeptide sequence set forth in SEQ ID NO: 2.
  • an epitope refers to an antigenic determinant of a polypeptide.
  • an epitope can include three or more amino acids in an epitope-specific spatial conformation.
  • the epitope is a linear or conformational epitope.
  • an epitope consists of at least 4, at least 6, at least 8, at least 10 and at least 12 such amino acids, more typically 8-10 such amino acids.
  • Methods for determining the spatial conformation of an amino acid are known in the art and include, for example, x-ray crystals and 2-dimensional nuclear magnetic resonance.
  • complementarity determining region refers to an amino acid sequence that collectively determines the binding affinity and specificity of the native Fv region of a native immunoglobulin binding site. See, for example, Chothia et al, J. Mol. Biol. 196:901-917 (1987); Kabat et al, U.S. Dept. of Health and Human Services NIH Publication No. 91-3242 (1991).
  • constant region refers to a portion of an antibody molecule that produces an effector function.
  • oligonucleotide refers to a series of linked nucleotide residues. Oligonucleotides include, but are not limited to, antisense and siRNA oligonucleotides.
  • the oligonucleotide comprises a portion of the DNA sequence and has at least about 10 nucleotides and up to about 500 nucleotides. In some embodiments, the oligonucleotide comprises from about 10 nucleotides to about 50 nucleotides, from about 15 nucleotides to about 30 nucleotides, from about 20 nucleotides to about 25 nucleotides. Oligonucleotides can be chemically synthesized and can also be used as probes.
  • the oligonucleotide is single stranded. In some embodiments, the oligonucleotide comprises at least a portion that is double stranded. In some embodiments, the oligonucleotide is an antisense oligonucleotide (ASO). In some embodiments, the oligonucleotide is an RNA interference oligonucleotide (RNAi oligonucleotide).
  • ASO antisense oligonucleotide
  • RNAi oligonucleotide RNA interference oligonucleotide
  • the term “combination” or “combination” refers to application with other treatment regimens.
  • the FGF21 modulator of the present invention is used.
  • detecting means establishing, discovering, or confirming evidence of activity (eg, gene expression) or biomolecules (eg, polypeptides).
  • homologous nucleotide sequence or “homologous amino acid sequence” or variant thereof, refers to a sequence characterized by at least a certain percentage of homology at the nucleotide or amino acid level, with " Sequence identity is used interchangeably.
  • Homologous nucleotide sequences include those encoding protein isoforms. Such isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, isoforms can be encoded by different genes.
  • a homologous nucleotide sequence includes a nucleotide sequence of a protein encoding a species other than a human, including but not limited to a mammal.
  • Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variants and mutations of the nucleotide sequences set forth herein.
  • a homologous nucleotide sequence encodes a polypeptide having the same or similar binding characteristics and/or activity as a wild-type sequence.
  • Homologous amino acid sequences include those amino acid sequences containing conservative amino acid substitutions having polypeptides having the same or similar binding characteristics and/or activity as the wild type sequence.
  • the nucleotide or amino acid sequence is homologous if it has at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.
  • nucleotide or amino acid sequence It is homologous. In some embodiments, the homologous amino acid sequence has no more than 5 or no more than 3 conservative amino acid substitutions.
  • Percent homology or identity can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Software Package, UNIX Version 8, Genetics Computer Group, University Research Park, Madison WI) and using default settings using Smith And Waterman's algorithm (Adv. Appl. Math., 1981, 2, 482-489).
  • the homology between the probe and the target is between about 75% and about 85%.
  • the nucleic acid has at least about 85%, about 90%, about 92%, about 94%, about 95%, about 97%, about 98%, about 99%, and about SEQ ID NO: 1 or a portion thereof 100% homologous nucleotide.
  • Complementary sequences of such sequences are also provided.
  • the complementary sequence is the complete and fully complementary sequence of the nucleotide sequence.
  • Homology can also be at the polypeptide level.
  • the polypeptide is at least about 85%, about 90%, about 92%, about 94%, about 95%, about 97%, about 98%, about 99%, and about 100 with SEQ ID NO: 2 or a portion thereof % homologous.
  • the polypeptide has up to 5, up to 10, up to 15, up to 20 or up to 30 amino acid insertions, deletions or substitutions.
  • the term "probe” refers to a nucleic acid sequence of variable length.
  • the probe comprises at least about 10 and up to about 6000 nucleotides.
  • the probe comprises at least 12, at least 14, at least 16, at least 18, at least 20, at least 25, at least 50, or at least 75 contiguous nucleotides.
  • Probes are used to detect identical, similar or complementary nucleic acid sequences. Longer probes are typically obtained from natural or recombinant sources, have high specificity to the target sequence, and hybridize much more slowly to the target than oligomers. Probes can be single-stranded or double-stranded and designed to be specific in PCR, membrane-based hybridization, in situ hybridization (ISH), fluorescence in situ hybridization (FISH) or ELISA-like techniques.
  • ISH in situ hybridization
  • FISH fluorescence in situ hybridization
  • binding refers to a physical or chemical interaction between two or more biomolecules or compounds. Binding includes ionic, nonionic, hydrogen bonding, van der Waals forces, hydrophobic interactions, and the like. Binding can be direct or indirect; indirect through or due to the effects of another biomolecule or compound. Direct binding refers to interactions that do not pass or are not affected by another biomolecule or compound, and instead do not require other basic chemical mediators.
  • sample includes any biological sample obtained from a patient.
  • Samples include, but are not limited to, whole blood, plasma, serum, red blood cells, white blood cells (eg, cells of peripheral blood mononuclear cells), saliva, urine, feces (ie, excretion), sputum, bronchial lavage, tears, nipples.
  • Absorbents lymph (eg, diffuse tumor cells of lymph nodes), fine needle aspirate, any other body fluid, tissue samples (eg, tumor tissue), such as tumor biopsy (eg, needle biopsy) and their cell extraction Things.
  • the sample is whole blood or a component thereof, such as plasma, serum or cell pellet.
  • the sample is obtained from circulating cells of whole blood molecular solid tumors or cellular components thereof using any technique known in the art.
  • the sample is a formalin fixed paraffin embedded (FFPE) tumor tissue sample, such as a solid tumor from the lung, colon or rectum.
  • FFPE formalin fixed paraffin embedded
  • Biopsy refers to the process of taking a tissue sample for diagnostic or prognostic evaluation, and also refers to the tissue sample itself. Any biopsy technique known in the art can be applied to the methods and compositions of the present invention. Place The biopsy technique applied will generally depend on the type of tissue to be assessed and the size and type of tumor (ie, solid or suspended (ie, blood or ascites)). Representative biopsy techniques include excisional biopsy, incisional biopsy, needle biopsy (eg, core needle biopsy, fine needle aspiration biopsy, etc.), surgical biopsy, and bone marrow biopsy. Biopsy techniques are described, for example, in Harrison's Princpiles of Internal Medicine, edited by Kasper et al, 16th edition, 2005, Chapter 70, and throughout Section V.
  • I.1 pcDNA3.0-hSCN3B purchased from Origene Corporation (OriGene Technologies, 9620 Medical Center Dr., Suite 200, Rockville, MD 20850.1.888.267.4436)
  • I.2 cell line Hep3B, HepG2, HL7702, purchased from Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences
  • the I.5 ⁇ 3-specific primers were synthesized by Kingsray Technology and the sequences are as follows:
  • the gene of GENBANK gene sequence number AF464898 was cloned into E. coli expression vector, transformed into E. coli cells, recombinant bacteria were constructed, and the active peptide was expressed in E. coli. After purification by chromatography, recombinant analgesic anti-tumor sorghumin was obtained.
  • I.7 GIBCO RPMI1640 medium (Cat. No.: LM-R1645), newborn bovine serum (Cat. No. 16010-159), fetal bovine serum (Cat. No. 10099-141), purchased from lLife Technologies, (Life Sciences Solutions Group, Thermo Fisher Scientific, Carlsbad, California, USA
  • BIOZOL RNA extraction kit was purchased from BioFlux (Cat. No. R1020-01) (BIOFLUX SRL Address: 54 Ceahlau street, kann-square 400488, Bulgaria, European Union, Europe)
  • KGP2100 Protein Extraction Kit
  • Reverse Transcription Kit Category No.: KGEA-01
  • Kaiji Company Najing Kaiji Biotechnology Development Co., Ltd., Zijin Fangshan, No. 18, Zhilan Road, Jiangning District, Nanjing Community Building 6
  • I.10 ⁇ 3-specific antibody was purchased from abcam (article number: ab48552)
  • I.11 Invitrogen Lipofectamine 2000 Transfection Kit was purchased from Life Technologies (Cat. No. 11668019) (Life Sciences Solutions Group, Thermo Fisher Scientific, Carlsbad, CA, USA)
  • I.12 Transwell chamber was purchased from Corning, Inc. (Corning Incorporated One Riverfront Plaza Corning, NY 14831 USA).
  • I.13 Matrigel was purchased from BD (BD Biosciences)
  • Example 1 Expression of ⁇ 3 subunit in tumor cells
  • the total mRNA of different cell lines (HepG2, HL7702) was extracted, 10 ⁇ g of mRNA was reverse transcribed, and 2 ⁇ L of cDNA was used as a template to specifically amplify mRNA transcribed from ⁇ 3 gene (PCR conditions and primers used: Candenas L, Seda M, Noheda P) , Buschmann H, Cintado CG, Martin JD, et al. Molecular diversity of voltage-gated sodium channels alpha and beta subunit mRNAs in human tissues. Eur J Pharmacol. 2006; 542: 9-16.), agarose gel electrophoresis , detecting mRNA levels in individual cell lines.
  • the total protein of different cell lines was extracted, the protein content was determined by Bradford method, 20-70 ⁇ g protein electrophoresis was carried out, and the ⁇ 3 subunit gene was expressed at the protein level by western blotting after electrophoresis. Specifically, the steps of Bradford method are as follows:
  • bovine serum albumin 1.
  • bovine serum albumin 1.0 mg/ml
  • standard protein solutions were added with 0 ⁇ l, 10 ⁇ l, 20 ⁇ l, 30 ⁇ l, 40 ⁇ l, 50 ⁇ l and 100 ⁇ l, respectively, and less than 100 ⁇ l were made up to 100 ⁇ l with physiological saline.
  • Coomassie Brilliant Blue G-250 solution (weigh 50mg of Coomassie Brilliant Blue G-250 dissolved in 25ml of 95% ethanol, add 50ml of 85% phosphoric acid, and finally dilute to 500ml with distilled water, filter paper), with normal saline As a blank control;
  • the plate was shaken on a shaker for 30 sec, shaken again after 2 min, and then determined by colorimetry at 595 nm (the colorimetric should be completed within 1 h). Taking the protein concentration (mg/ml) as the abscissa and the absorbance as the ordinate, plotting the standard curve;
  • the corresponding protein concentration can be found on the standard curve, and the sample dilution factor is the actual concentration of the sample.
  • Sample processing load the loading buffer and the protein to be tested in the same volume, vortex and mix, boil water for 5 min, and centrifuge;
  • the already concentrated sample can be mixed with 2 ⁇ loading buffer, and heated at 100 ° C for 5 min to denature the protein. Samples with a relatively low concentration need to be enriched with trichloroacetic acid, and then the protein is precipitated with acetone;
  • the electrophoresis device is connected to the power source (the positive electrode should be connected to the lower tank), and electrophoresis until the bromophenol blue reaches the bottom of the separation gel, and then the power is turned off;
  • the glass plate is quickly opened with a rubber cutting board, and the glue is kept flat.
  • the desired target strip is cut according to the pre-dyed Marker, and the cutting range contains the target protein, which is slightly wider; Angle, mark; measure its length and width;
  • the film transfer time was set according to the molecular weight of the protein, and the film was transferred for 1 hour and 40 minutes.
  • SCN3B The expression of SCN3B in different types of leukemia cell lines and normal cells in the ONCOMINE database (www.oncomine.com) was detected.
  • the data analysis using the gene chip database (www.oncomine.org) showed that there was also abnormal SCN3B mRNA in VGSC in leukemia. expression.
  • the gene is SCN3B
  • the comparison object is leukemia vs. normal tissue.
  • SCN3B encodes a voltage-gated sodium channel ⁇ 3 subunit.
  • the following figure shows the expression of SCN3B in different types of leukemia. The results show that except for the sixth group of myelodysplastic syndromes (SCN3B expression in Myelodysplastic Syndrome is not significantly different from normal cells).
  • the remaining 7 different types of leukemia (Acute Myeloid Leukemia, B-cell Acute lymphoblastic Leukemia, B-Cell Childhood Acute Lymphoblastic Leukemia), Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Pro-B Acute Lymphoblastic Leukemia, T-Cell Acute Lymphoblastic Leukemia Both showed high expression of SCN3B (P ⁇ 0.05), and the expression levels of different types of leukemia SCN3B were also different (Fig. 3).
  • RNAi was used to interfere with the expression of the ⁇ 3 subunit of Hep G2 cell line, and the expression of ⁇ 3 subunit was down-regulated, and the proliferation of hepatoma cells was also inhibited.
  • RNAi interferes with ⁇ 3 subunit gene expression
  • the cells were inoculated one day before the interference experiment, and the cells should reach 30% to 50% coverage on the day of interference.
  • the interference experiment was performed according to the instructions of Lipofectamine 2000, and the two pairs of ⁇ in the aforementioned I.3 3 specific small interfering RNA was transfected into cells at the same concentration (50 nM) to interfere with VGSC ⁇ 3 expression, using the same amount of the negative sequence in random sequence I.4 as a control.
  • liposome Lipofectamine 2000 reagent and plasmid siRNA 25-50 nM
  • the above mixture was directly added to the medium, gently mixed by shaking, and the cells were cultured under normal conditions for 4-6 hours, and then the culture was replaced by replacing the fresh medium for 2-3 days.
  • Example 2 Using the method described in Example 1, the total cell protein obtained in 2.1.1 was extracted and the protein content was determined, and the loading buffer and the protein to be tested (20-70 ⁇ g protein) were loaded in equal volume, and vortexed. Mix well, boil water for 5 min, and centrifuge. After electrophoresis, the gel was transferred to a PVDF membrane, and western blotting analysis was performed using the VGSC ⁇ 3 specific antibody obtained from Abcam in I.10.
  • the cell proliferation level was determined by MTT method: cells with good growth state and in logarithmic growth phase were blunted into single cell suspension after routine trypsinization, and the concentration was adjusted to 10 5 /ml after counting by blood cell counting plate ( HepG2 cells, inoculated into 96-well culture plates, 100 ⁇ l per well, and set blank zero-adjusted wells (only 100 ⁇ l of culture medium, no cells added); 37 ° C, 5% CO 2 incubator for 24 h, add 0.5 per well MTT of mg/ml, 100 ⁇ l, continue to culture for 4h, discard the supernatant, add 150 ⁇ l of DMSO to each well, and shake it at low speed for 10min on a small shaker. After the crystals are fully dissolved, use the microplate reader to detect each at 490nm. The absorbance value of the well.
  • Inhibition rate (%) (control group - loading group) / (control group - blank zero hole) ⁇ 100
  • the cell concentration was adjusted to 10 6 /ml, centrifuged again (1000 rpm, centrifuged for 5 minutes), fixed with -20 ° C pre-cooled 70% ethanol, fixed at -20 ° C overnight;
  • the cells were resuspended in PI staining solution (10 6 /500 ⁇ L) and stained at room temperature for 15 to 30 minutes.
  • a single cell suspension was prepared through a 200 mesh screen and detected by flow cytometry: the excitation wavelength was 488 nm and the emission wavelength was 615 nm.
  • the total protein of the HepG2 cells in 2.1.2 was extracted and the protein content was determined, and 20-70 ⁇ g of the protein was taken as the protein to be tested, and the loading buffer and the protein to be tested were equal in volume. Add the sample, vortex and mix, boil water for 5 min, and centrifuge. After electrophoresis, the gel was transferred to PVDF membrane and analyzed by western blotting with specific antibody. The results showed that the expression level of cyclin cylin D1 (SANTA CRUZ; sc753) was down-regulated (Fig. 7).
  • RNAi was used to interfere with the expression of the ⁇ 3 subunit of lung cancer cell lines, and the expression of VGSC ⁇ 3 subunit was down-regulated, and lung cancer cell proliferation was also inhibited.
  • RNAi interferes with ⁇ 3 subunit gene expression
  • the cells were inoculated one day before the interference experiment, and the cells should reach 30% to 50% coverage on the day of interference.
  • Interference experiments were performed according to the instructions of Lipofectamine 2000, using two pairs of ⁇ 3-specific small interfering RNAs described in I.3 to interfere with the expression of VGSC ⁇ 3, and the random sequence, the negative sequence in I.4, was used as a control (method is the same as 2.1.1).
  • Example 2 Using the method described in Example 1, extract the total protein of the cells obtained in 2.2.1 and determine the protein content, load the loading buffer and the protein to be tested (take 20-70 ⁇ g) in equal volume, and vortex and mix. , boiling water bath for 5 min, centrifugation. After electrophoresis, the gel was transferred to PVDF membrane, and western blotting analysis was performed using VGSC ⁇ 3 specific antibody in I.10.
  • the cell proliferation level was determined by the MTT method (as described in 2.1.3), and the results were determined according to the test results.
  • the inhibition rate was calculated according to the following formula.
  • Inhibition rate (%) (control group - loading group) / (control group - blank zero hole) ⁇ 100
  • the cell proliferation was measured by MTT assay, and the two pairs of siRNA doses were transfected at a concentration of 50 nM respectively (method is the same as 2.1.1).
  • the cell proliferation inhibition rate is 50%.
  • Example 3 Active peptide AGAP inhibits liver cancer cells by inhibiting protein expression of VGSC ⁇ 3
  • the analgesic anti-tumor ⁇ ⁇ ⁇ (AGAP) was 0, 5, 10, 15, 20, 25, 30, 35, 40 ⁇ M, 8 concentrations with liver cancer cell line HepG 2 (96 well plates, 10 4 cells / Wells, after 24h, the above-mentioned different concentrations of AGAP were administered, and each concentration was repeated for at least 3 wells) for 48 hours, then the expression of VGSC ⁇ 3 subunit was detected by Western blotting using the antibody in I.10, and the cell proliferation level was determined by MTT assay. .
  • the active peptide AGAP inhibits cell proliferation by inhibiting the expression of the VGSC ⁇ 3 subunit.
  • the VGSC ⁇ 3 gene was used as the plasmid shown in Figure 14 (in which the gene SCN3B encoding VGSC ⁇ 3 is shown as SEQ ID NO: 2, the plasmid construction procedure is shown in Figure 15, and the pcDNA3.0 plasmid was purchased. From Beijing Origene (ORIGENE, Adderess: 9620 Medical Center Dr., Suite 200 Rockville, MD 20850), the following cell transfection experiments were used to transfect into Hep3B cell lines that did not express VGSC ⁇ 3 gene. Western blotting revealed ⁇ 3 subunits. Protein level expression was achieved ( Figure 10).
  • Solution A The DNA was diluted with serum-free and antibiotic medium RPMI1640 to a concentration of 4 ug, and the final amount was 500 ul.
  • the plasmid DNA included a plasmid containing the gene of interest, and the empty plasmid pcDNA3.0 plasmid was used as a negative control.
  • Solution B Lipofectamine 2000 8 ul was diluted with double medium without serum and antibiotics, the final amount was 500 ul, and allowed to stand at room temperature for 5 min. The ratio of the amount of plasmid to the Lipo transfection reagent was 1:2.
  • Hep3B is transfected with VGSC ⁇ 3 subunit gene and expresses VGSC ⁇ 3 (transfected protein sheet) Figure 10, lane 1), the effect on cell proliferation was determined by the MTT method as described above. From the results shown in Figure 11, column 2, the proliferation of cells was promoted by transfection of the ⁇ 3 subunit in Hep3B cells (**P ⁇ 0.01). It can be seen that the VGSC ⁇ 3 subunit can promote cancer cell proliferation.
  • siRNA of the invention inhibits tumor growth in nude mice
  • Example 6 Influencing cell morphology and cell cycle by inhibiting expression of VGSC ⁇ 3 subunit in HepG2 cells
  • Example 7 Influencing apoptosis by inhibiting the expression of VGSC ⁇ 3 subunit in HepG2 cells
  • the cells were resuspended in normal medium, mixed, transferred to a centrifuge tube, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, the cells were collected, and the cells were gently resuspended in PBS and counted.
  • Annexin V-FITC was green fluorescence and PI was red fluorescence.

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Abstract

本申请公开了VGSC β3蛋白质的抑制剂以及其用途，利用所述VGSC β3蛋白质或其抑制剂诊断、预防和治疗癌症的方法，以及利用VGSC β3蛋白质或其调控序列筛选抗肿瘤药物的方法。

Description

VGSC β3蛋白质用于癌症预防、治疗及诊断检测的靶点 技术领域
本申请涉及VGSC β3蛋白质与肿瘤之间的关联，特别地，涉及VGSC β3蛋白质的抑制剂以及其用于预防和治疗肿瘤，特别是癌症的用途，利用所述抑制剂预防和治疗肿瘤特别是癌症的方法，利用VGSC β蛋白质或其调控序列筛选抗肿瘤药物的方法，以及利用VGSC β蛋白质对肿瘤进行诊断、检测以方法和用途。
背景技术
电压门控性离子通道，或称电压依赖性和电压敏感性(voltage-dependent and voltage-sensitive)离子通道，是迄今人类所了解的参与信号转导过程的最复杂的超家族之一，目前其成员已超过140个(CATTERALL W A，CHANDY K G，CLAPHAM D E，等人International Union of Pharmacology：Approaches to the Nomenclature of Voltage-Gated Ion Channels[J].Pharmacol Rev，2003，55(4)：573-574)，并且还有新的成员不断被发现。经纯化、克隆和测定表明，离子通道蛋白是由多个亚基构成复合体。电压门控离子通道有α、β、γ、δ等亚基构成，但不同的离子通道的组成略有差异。
电压门控性钠离子通道有α亚基和β亚基组成(见图1)(CATTERALL W A.From ionic currents to molecular mechanisms：the structure and run ion of voltage-gated sodium channels[J].Neuron，2000，26(11)：13-25，CATTERALL W A，GOLDIN A L，WAXMAN S G.International Union of Pharmacology.XXXIX.Compendium of Voltage-Gated Ion Channels：Sodium Channels[J].Pharmacol Rev，2003，55(4)：575-578)。α亚基分子量约260kDa，由四个同源跨膜结构域(I-IV)构成，而每个结构域含有六次跨膜螺旋(S1～S6)，S4为电压感受器，S5和S6之间的短肽参与构成孔道，起到闸 门的作用，参与介导去极化过程。一般来说，VGSC除了α亚单位以外，还含有1～2个被称之为β亚单位的小分子多肽。对于哺乳动物的脑神经细胞来说，钠通道由一个α亚单位(Nav1.1～Nav1.9)与一个或更多的辅助β亚单位(β1、β2、β3及β4(33～36kDa))组成的复合体所构成。
根据钠离子通道对其阻滞剂河豚毒素(tetrodoxin，TTX)的敏感性不同，将钠离子通道的各种亚型分为两类：TTX敏感型(Nav1.1，Nav1.2，Nav1.3，Nav1.4，Nav1.6，Nav1.7)和TTX不敏感型(Nav1.5，Nav1.8，Nav1.9)(ROGER S，POTIER M，VANDIER C，等人Voltage-Gated Sodium Channels：New Targets in Cancer Therapy？[J].Curr Pharm Des，2006，12(28)：3681-3695)。β亚基有多种功能，它不仅能够调节电压门控、离子通道在质膜上的表达，还能够作为细胞黏附分子与细胞骨架蛋白、细胞外基质和其他细胞黏附分子相互作用(KAUFMANN S G，WESTENBROEK R E，ZECHNER C.Functional protein expression of multiple sodium channel alpha-and beta-subunits isoforms in neonatal cardiomyocytes[J].J Mol Cell Cardiol，2009，48(1)：261-269；BRACKENBURY W J，DAVIS T H，CHEN C，等人Voltage-gated Na+ channel betal subunit-mediated neurite outgrowth requires Fyn kinase and contributes to postnatal CNS development in vivo[J].J Neurosci，2008，28(12)：3246-3256)。近年来，人们逐渐认识到β亚基作为辅助亚基对通道功能的重要性，如β亚基可以调节α亚基在细胞膜上的转运和定位、调节心肌Na+通道晚期电流。除此之外，基于β亚基突变所导致的疾病也逐步被纳入到基因-表型相互作用的研究中来。
β1和β3亚基同源性较高，以非共价键的方式与α亚基相结合，β2和β4亚基同源性较高，以二硫键的方式与α亚基相结合。β1和β2亚基功能已经明确为细胞粘附分子(CAM，cell adhesion molecules)，β3和β4亚基的功能尚不明确。虽然β3亚基与β1亚基一样，通过非共价键的形式与α亚基结果，但两者的作用却是不同的，β3亚基通过拮抗内质网-释放/回收信号调节细胞表面的Nav1.8的表达(ZHANG Z N，LI Q，LIU C，等人The voltage-gated Na+ channel Nav1.8 contains an ER-retention/retrieval signal  antagonized by the β3 subunit[J].J Cell Sci，2008，121：3243-3252)。而且β3亚基不具有细胞粘附的作用，β3亚基没有参与反式-嗜同种细胞-细胞粘附，也不与锚定蛋白ankyrinG结合(MCEWEN D P，CHEN C，MEADOWS L S，等人The voltage-gated Na+ channel β3 subunit does not mediate trans homophilic cell adhesion or associate with the cell adhesion molecule contactin.Neurosci Lett，2009，462：272-275)。
综上所述，电压门控钠离子通道β亚基的研究也愈来愈受到人们的重视，功能辅助亚基如β亚基已成为疾病治疗的新靶点以及认识疾病发病过程的新视角。但是，现有技术中没有涉及过β3亚基与肿瘤的关联，也没有涉及过其在肿瘤的诊断以及治疗方面的用途。
发明内容
本发明首次发现了VGSC β3与肿瘤之间具有一定的关联性，能够作用为肿瘤的诊断和治疗的靶点。
本发明的第一个方面涉及VGSC β3的抑制剂，其可以用于在对象中预防或治疗肿瘤。
本发明的第二个方面涉及包含VGSC β3的抑制剂的用于在对象中预防或治疗肿瘤的药物组合物，优选地，所述药物组合物还包含可药用载体，任选地还包含与VGSC β3抑制剂协同作用的其他肿瘤治疗剂。
本发明的第三个方面涉及使用VGSC β3的抑制剂在对象中预防或治疗肿瘤的方法，包括向对象施用一定量的VGSC β3抑制剂，所述量有效抑制VGSC β3的表达和/或活性。
本发明还涉及VGSC β3的抑制剂在在对象中预防或治疗肿瘤中的用途，以及VGSC β3的抑制剂在制备用于在对象中预防或治疗肿瘤的药物组合物中的用途。
本发明的第四个方面涉及调节表达VGSC β3的细胞中的一种或多种活性的方法，包括将细胞与有效调节一种或多种活性的量的VGSC β3抑制剂接触。
本发明的第五个方面涉及利用VGSC β3蛋白质诊断和检测肿瘤的方法，利用检测VGSC β3蛋白质的工具(例如用于测量VGSC β3亚基的编码DNA水平或mRNA水平或蛋白质表达水平的工具)来诊断和检测肿瘤的方法，检测VGSC β3蛋白质的工具在诊断和检测肿瘤中的用途，以及检测VGSC β3蛋白质的工具在制备用于诊断和检测肿瘤的试剂盒中的用途。
本发明的第六个方面涉及利用VGSC β3的抑制剂抑制肿瘤细胞的增殖的方法，VGSC β3的抑制剂在抑制肿瘤细胞增殖中的用途，以及VGSC β3的抑制剂在制备用于抑制肿瘤细胞增殖的药物组合物或试剂盒中的用途。
本发明的第七个方面涉及以VGSC β3蛋白质或其表达调控序列为靶点，设计、筛选以及制备用于在对象中预防或治疗肿瘤的活性物质以及包含所述活性物质的药物组合物。
本发明还涉及鉴定肿瘤抑制剂的方法，其中肿瘤的特征是与对照相比VGSC β3的差异表达。方法包括将表达VGSC β3的细胞与候选化合物接触，确定是否调节了VGSC β3-相关的活性。VGSC β3-相关活性的调节是癌症抑制剂的指标。
本发明还涉及试剂盒，其用于检测或诊断肿瘤，其包括用于测量VGSC β3亚基的编码DNA水平或mRNA水平或蛋白质表达水平的工具。
在具体的实施方案中，肿瘤与VGSC β3相关，优选为癌症，更优选选自肺癌、肝癌或白血病。
在具体的实施方案中，VGSC β3抑制剂选自能够抑制编码VGSC β3蛋白质的DNA的抑制剂、能够通过调节VGSC β3蛋白质编码基因的上游和下游调控序列来抑制所述基因表达的抑制剂、针对VGSC β3蛋白质的RNAi活性剂、与VGSC β3蛋白质结合的抗体、VGSC β3蛋白质的小分子抑制剂，能够抑制VGSC β3蛋白质表达或活性的反义寡核苷酸、(多)肽和其它核酸或者模拟物。
在一个实施方案中，所述抑制剂为RNAi抑制剂，优选为dsRNA，更 优选为siRNA。最优选地，所述siRNA双链区域为9-50bp、15-36bp、15-30bp、15-26bp、15-23bp、15-22bp、15-21bp、15-20bp、15-19bp、15-18bp、15-17bp、18-30bp、18-26bp、18-23bp、18-22bp、18-21bp、18-20bp、19-30bp、19-26bp、19-23bp、19-22bp、19-21bp、19-20bp、19bp、20-30bp、20-26bp、20-25bp、20-24bp、20-23bp、20-22bp、20-21bp、21-30bp、21-26bp、21-25bp、21-24bp、21-23bp、21-22bp、21bp、22bp或23bp个核苷酸长度，且包含反义链和与反义链实质上互补的正义链，其中反义链与靶基因的15、16、17、18、10、20或21个或更多个连续核苷酸至少约80％-90％互补或与之在严格条件下杂交，或者，更优选地、与靶基因的15、16、17、18、10、20或21个或更多个连续核苷酸至少约90-95％互补或与之在严格条件下杂交，并且最优选与靶基因的15、16、17、18、10、20或21个或更多个连续核苷酸至少约95％、96％、97％、98％或99％互补或与之在严格条件下杂交，或与之完全相同，其中所述靶基因为编码VGSC β3蛋白质的靶基因，优选地，所述VGSC β3来自哺乳动物，优选来自人，还优选地所述VGSC β3的氨基酸序列如SEQ ID NO：1所示，最优选地所述编码VGSC β3蛋白质的靶基因的核苷酸序列如SEQ ID NO：2所示。最优选地，所述RNAi活性剂为选自下述的siRNA：
Figure PCTCN2015079356-appb-000001
Figure PCTCN2015079356-appb-000002
或
Figure PCTCN2015079356-appb-000003
在另一个实施方案中，所述抑制剂为与VGSC β3蛋白质结合的抗体，优选地抗体为单克隆抗体，更优选地为人源化的单克隆抗体。优选地，与抗体结合的所述VGSC β3蛋白质来自哺乳动物，优选来自人，最优选其 氨基酸序列如SEQ ID NO：1所示。
在另一个实施方案中，所述抑制剂为肽，例如镇痛抗肿瘤缬精甘肽(AGAP)。在具体的实施方案中，表达VGSC β3的细胞为肿瘤细胞。
在具体的实施方案中，肿瘤细胞优选为癌细胞，更优选为肝癌细胞或肺癌细胞，最优选为HepG2细胞或A549细胞。
在具体的实施方案中，检测VGSC β3蛋白质的工具为用于测量VGSC β3亚基的编码DNA水平或mRNA水平或蛋白质表达水平的工具，包括能够检测VGSC β3蛋白质表达和活性的可检测的抗体、小分子、寡核苷酸、诱饵、模拟物或探针。
本发明的其他方面和实施方案在如下发明详述中阐释，或将会因其变得显而易见。
本发明的实施方案
具体地，本发明涉及以下具体实施方案：
1.利用VGSC β3亚基检测或诊断患者中与VGSC β3亚基相关的肿瘤的方法，其包括：
a)从患者中体内分离样品；
b)测量a)中获得的样品与正常对照细胞中的VGSC β3亚基的编码DNA水平、mRNA水平或蛋白质水平；
c)比较b)中测量的VGSC β3亚基的编码DNA水平、mRNA水平或蛋白质水平；
d)待测样品或细胞中VGSC β3的蛋白质或基因含量比对照正常高表明对象患有VGSC β3相关肿瘤。
2.用于测量VGSC β3亚基的编码DNA水平或mRNA水平或蛋白质表达水平的工具在制备用于检测或诊断患者中与VGSC β3亚基相关的肿瘤的试剂盒中的用途，其中在患者的样品中测量到的VGSC β3亚基的边编码DNA水平或mRNA水平或蛋白质表达水平高于正常对照细胞中的 水平表明患者患有肿瘤。
3.实施方案1的方法或实施方案2的用途，其中所述样品是怀疑患有肿瘤的组织的样品，优选肿瘤活检或其细胞提取物。
4.实施方案1或3的方法或实施方案2或3的用途，其中所述肿瘤为癌症，优选肝癌、白血病或肺癌，最优选所述肝癌为HepG2型肝癌，或所述肺癌为A549型肺癌。
5.实施方案1、3和4中任一项的方法或实施方案2、3和4中任一项的用途，其中利用逆转录PCR方法测量mRNA水平或利用Western印迹测量蛋白质水平。
6.实施方案1、3、4、5中任一项的方法或实施方案2、3、4、5中任一项的用途，其中测量编码DNA水平或mRNA水平或蛋白质水平的工具包括能够用于测量VGSC β3亚基的编码DNA水平或mRNA水平的探针或引物，或者能够检测VGSC β3蛋白质表达和活性的可检测的抗体、小分子、寡核苷酸、诱饵、模拟物或探针。
7.用于在对象中预防或治疗肿瘤的VGSC β3的抑制剂。
8.使用VGSC β3的抑制剂在对象中预防或治疗肿瘤的方法，包括向对象施用一定量的VGSC β3抑制剂，所述量有效抑制VGSC β3的表达和/或活性。
9.VGSC β3的抑制剂在在对象中预防或治疗肿瘤中的用途，或者VGSC β3的抑制剂在制备用于在对象中预防或治疗肿瘤的药物组合物中的用途。
10.实施方案7的抑制剂或实施方案8的方法或实施方案9的用途，其中所述VGSC β3抑制剂选自能够抑制编码VGSC β3蛋白质的DNA的抑制剂、能够通过调节VGSC β3蛋白质编码基因的上游和下游调控序列来抑制所述基因表达的抑制剂、针对VGSC β3蛋白质的RNAi活性剂、与VGSC β3蛋白质结合的抗体、VGSC β3蛋白质的小分子抑制剂，能够抑制VGSC β3蛋白质表达或活性的反义寡核苷酸、(多)肽和其它核酸或者模拟物。
11.实施方案10的抑制剂或方法或用途，其中所述抑制剂为RNAi抑制剂，优选为dsRNA，更优选为siRNA，最优选地，所述siRNA双链区域为9-50bp、15-36bp、15-30bp、15-26bp、15-23bp、15-22bp、15-21bp、15-20bp、15-19bp、15-18bp、15-17bp、18-30bp、18-26bp、18-23bp、18-22bp、18-21bp、18-20bp、19-30bp、19-26bp、19-23bp、19-22bp、19-21bp、19-20bp、19bp、20-30bp、20-26bp、20-25bp、20-24bp、20-23bp、20-22bp、20-21bp、21-30bp、21-26bp、21-25bp、21-24bp、21-23bp、21-22bp、21bp、22bp或23bp个核苷酸长度，且包含反义链和与反义链实质上互补的正义链，其中反义链与靶基因的15、16、17、18、10、20或21个或更多个连续核苷酸至少约80％-90％互补或与之在严格条件下杂交，或者，更优选地、与靶基因的15、16、17、18、10、20或21个或更多个连续核苷酸至少约90-95％互补或与之在严格条件下杂交，并且最优选与靶基因的15、16、17、18、10、20或21个或更多个连续核苷酸至少约95％、96％、97％、98％或99％互补或与之在严格条件下杂交，或与之完全相同，其中所述靶基因为编码VGSC β3蛋白质的靶基因，优选地，所述VGSC β3来自哺乳动物，优选来自人，还优选地所述VGSC β3的氨基酸序列如SEQ ID NO：1所示，最优选地所述编码VGSC β3蛋白质的靶基因的核苷酸序列如SEQ ID NO：2所示。
12.实施方案11的抑制剂或方法或用途，其中所述RNAi活性剂为选自下述的siRNA：
Figure PCTCN2015079356-appb-000004
Figure PCTCN2015079356-appb-000005
或
Figure PCTCN2015079356-appb-000006
13.实施方案10的抑制剂或方法或用途，其中所述抑制剂为与VGSC β3蛋白质结合的抗体或其片段，优选地抗体为单克隆抗体或其片段，更优选地为人源化的单克隆抗体或其片段，优选地，与抗体结合的所述VGSC β3蛋白质来自哺乳动物，优选来自人，最优选其氨基酸序列如SEQ ID  NO：1所示。
14.实施方案10的抑制剂或方法或用途，其中所述抑制剂为多肽，优选地镇痛抗肿瘤缬精甘肽(AGAP)。
15.实施方案7-14中任一项的抑制剂或方法或用途，其中所述肿瘤为与VGSC β3亚基相关的肿瘤，优选癌症，更优选肝癌、白血病或肺癌，最优选所述肝癌为HepG2型肝癌，或所述肺癌为A549型肺癌。
16.调节表达VGSC β3的细胞中的一种或多种活性的方法，包括将细胞与有效调节一种或多种活性的量的VGSC β3抑制剂接触。
17.实施方案16的方法，其中所述细胞为肿瘤细胞。
18.利用VGSC β3的抑制剂抑制肿瘤细胞的增殖的方法。
19.VGSC β3的抑制剂在抑制肿瘤细胞增殖中的用途。
20.VGSC β3的抑制剂在制备用于抑制肿瘤细胞增殖的药物组合物或试剂盒中的用途。
21.实施方案17或18的方法或实施方案19或20的用途，其中所述VGSC β3抑制剂选自能够抑制编码VGSC β3蛋白质的DNA的抑制剂、能够通过调节VGSC β3蛋白质编码基因的上游和下游调控序列来抑制所述基因表达的抑制剂、针对VGSC β3蛋白质的RNAi活性剂、与VGSC β3蛋白质结合的抗体、VGSC β3蛋白质的小分子抑制剂，能够抑制VGSC β3蛋白质表达或活性的反义寡核苷酸、(多)肽和其它核酸或者模拟物。
22.实施方案21的方法或用途，其中所述抑制剂为RNAi抑制剂，优选为dsRNA，更优选为siRNA，最优选地，所述siRNA双链区域为9-50bp、15-36bp、15-30bp、15-26bp、15-23bp、15-22bp、15-21bp、15-20bp、15-19bp、15-18bp、15-17bp、18-30bp、18-26bp、18-23bp、18-22bp、18-21bp、18-20bp、19-30bp、19-26bp、19-23bp、19-22bp、19-21bp、19-20bp、19bp、20-30bp、20-26bp、20-25bp、20-24bp、20-23bp、20-22bp、20-21bp、21-30bp、21-26bp、21-25bp、21-24bp、21-23bp、21-22bp、21bp、22bp或23bp个核苷酸长度，且包含反义链和与反义 链实质上互补的正义链，其中反义链与靶基因的15、16、17、18、10、20或21个或更多个连续核苷酸至少约80％-90％互补或与之在严格条件下杂交，或者，更优选地、与靶基因的15、16、17、18、10、20或21个或更多个连续核苷酸至少约90-95％互补或与之在严格条件下杂交，并且最优选与靶基因的15、16、17、18、10、20或21个或更多个连续核苷酸至少约95％、96％、97％、98％或99％互补或与之在严格条件下杂交，或与之完全相同，其中所述靶基因为编码VGSC β3蛋白质的靶基因，优选地，所述VGSC β3来自哺乳动物，优选来自人，还优选地所述VGSC β3的氨基酸序列如SEQ ID NO：1所示，最优选地所述编码VGSC β3蛋白质的靶基因的核苷酸序列如SEQ ID NO：2所示。
23.实施方案22的方法或用途，其中所述RNAi活性剂为选自下述的siRNA：
Figure PCTCN2015079356-appb-000007
Figure PCTCN2015079356-appb-000008
或
Figure PCTCN2015079356-appb-000009
24.实施方案21的方法或用途，其中所述抑制剂为与VGSC β3蛋白质结合的抗体或其片段，优选地抗体为单克隆抗体或其片段，更优选地为人源化的单克隆抗体或其片段，优选地，与抗体结合的所述VGSC β3蛋白质来自哺乳动物，优选来自人，最优选其氨基酸序列如SEQ ID NO：1所示。
25.实施方案21的方法或用途，其中所述抑制剂为多肽，优选地镇痛抗肿瘤缬精甘肽(AGAP)。
26.实施方案17-25中任一项的方法或用途，其中所述肿瘤为与VGSC β3亚基相关的肿瘤，优选癌症，更优选肝癌、白血病或肺癌，最优选所述肝癌为HepG2型肝癌，或所述肺癌为A549型肺癌。
27.实施方案18-26中任一项的方法或用途，其中所述肿瘤细胞为肝癌细胞或肺癌细胞，优选地为HepG2细胞或A549细胞。
28.利用VGSC β3或其基因调控序列为靶点，设计和制备用于在对象中预 防或治疗肿瘤的活性物质的方法，所述方法包括，根据VGSC β3蛋白质的氨基酸序列以及核酸编码序列，设计能够抑制VGSC β3蛋白质的表达或活性的活性物质。
29.实施方案28的方法，还包括制备上述设计出的活性物质并且确定其是否抑制了VGSC β3的表达、活性和/或相关生物学活性或者是否抑制了对癌细胞生长等一种或多种癌细胞指标的步骤。
30.筛选用于在对象中预防或治疗肿瘤的活性物质的方法，所述方法包括将表达VGSC β3的细胞与候选活性物质接触，确定是否抑制了VGSC β3的表达、活性和/或相关生物学活性或者是否抑制了对癌细胞生长等一种或多种癌细胞指标的步骤，其中如果VGSC β3的表达、活性和/或相关生物学活性被抑制或者一种或多种癌细胞指标被抑制，则表明该候选活性物质为预防或治疗肿瘤的活性物质。
31.筛选抗癌剂或抑制肿瘤细胞增殖的抑制剂的方法，所述方法包括将表达VGSC β3的细胞与候选化合物接触，确定是否调节了VGSC β3的表达、活性和/或相关生物学活性或者是否抑制了对癌细胞生长等一种或多种癌细胞指标，其中如果VGSC β3的表达、活性和/或相关生物学活性被抑制或者一种或多种癌细胞指标被抑制，则表明该候选活性物质为抗癌剂或抑制肿瘤细胞增殖的抑制剂。
32.实施方案28-31中任一项的方法，其中所述活性物质选自能够抑制编码VGSC β3蛋白质的DNA的抑制剂、能够通过调节VGSC β3蛋白质编码基因的上游和下游调控序列来抑制所述基因表达的抑制剂、针对VGSC β3蛋白质的RNAi活性剂、与VGSC β3蛋白质结合的抗体、VGSC β3蛋白质的小分子抑制剂，能够抑制VGSC β3蛋白质表达或活性的反义寡核苷酸、(多)肽和其它核酸或者模拟物。
33.实施方案32的用途，其中所述活性物质为RNAi抑制剂，优选为dsRNA，更优选为siRNA，最优选地，所述siRNA双链区域为9-50bp、15-36bp、15-30bp、15-26bp、15-23bp、15-22bp、15-21bp、15-20bp、15-19bp、15-18bp、15-17bp、18-30bp、18-26bp、18-23bp、18-22bp、18-21bp、 18-20bp、19-30bp、19-26bp、19-23bp、19-22bp、19-21bp、19-20bp、19bp、20-30bp、20-26bp、20-25bp、20-24bp、20-23bp、20-22bp、20-21bp、21-30bp、21-26bp、21-25bp、21-24bp、21-23bp、21-22bp、21bp、22bp或23bp个核苷酸长度，且包含反义链和与反义链实质上互补的正义链，其中反义链与靶基因的15、16、17、18、10、20或21个或更多个连续核苷酸至少约80％-90％互补或与之在严格条件下杂交，或者，更优选地、与靶基因的15、16、17、18、10、20或21个或更多个连续核苷酸至少约90-95％互补或与之在严格条件下杂交，并且最优选与靶基因的15、16、17、18、10、20或21个或更多个连续核苷酸至少约95％、96％、97％、98％或99％互补或与之在严格条件下杂交，或与之完全相同，其中所述靶基因为编码VGSC β3蛋白质的靶基因，优选地，所述VGSC β3来自哺乳动物，优选来自人，还优选地所述VGSC β3的氨基酸序列如SEQ ID NO：1所示，最优选地所述编码VGSC β3蛋白质的靶基因的核苷酸序列如SEQ ID NO：2所示。
34.实施方案33的方法或用途，其中所述RNAi活性剂为选自下述的siRNA：
Figure PCTCN2015079356-appb-000010
Figure PCTCN2015079356-appb-000011
或
Figure PCTCN2015079356-appb-000012
35.实施方案32的用途，其中所述活性物质为与VGSC β3蛋白质结合的抗体，优选地抗体为单克隆抗体，更优选地为人源化的单克隆抗体，优选地，与抗体结合的所述VGSC β3蛋白质来自哺乳动物，优选来自人，最优选其氨基酸序列如SEQ ID NO：1所示。
36.实施方案32的用途，其中所述活性物质为多肽，优选地镇痛抗肿瘤缬精甘肽(AGAP)。
37.实施方案28-36中任一项的方法或用途，其中所述肿瘤为与VGSC β3亚基相关的肿瘤，优选癌症，更优选肝癌、白血病或肺癌，最优选所述肝癌为HepG2型肝癌，或所述肺癌为A549型肺癌。
38.实施方案29-37中任一项的方法或用途，其中所述癌细胞为与VGSC β3亚基相关的癌细胞，优选地肝癌细胞或肺癌细胞，更优选地为HepG2细胞或A549细胞。
39.VGSC β3作为靶点，在制备用于预防或治疗肿瘤的药物组合物中的用途，以及在制备用于诊断或检测肿瘤的试剂中的用途。
40.试剂盒，其用于检测或诊断患者中与VGSC β3亚基相关的肿瘤，包含用于测量VGSC β3亚基的编码DNA水平或mRNA水平或蛋白质表达水平的工具。
41.实施方案40的试剂盒，其中所述试剂盒还包含一种或多种下述：对照(阳性和/或阴性)、对照的容器、阳性和/或阴性结果的代表性实例的照片或描述。
42.实施方案40或41的试剂盒，其中所述肿瘤为癌症，优选肝癌、白血病或肺癌，最优选所述肝癌为HepG2型肝癌，或所述肺癌为A549型肺癌。
43.实施方案40-42中任一项的试剂盒，其中测量编码DNA水平或mRNA水平或蛋白质水平的工具包括能够用于测量VGSC β3亚基的编码DNA水平或mRNA水平的探针或引物，或者能够检测VGSC β3蛋白质表达和活性的可检测的抗体、小分子、寡核苷酸、诱饵、模拟物或探针。
附图说明
图1：电压门控钠离子通道的结构(ROGER S，POTIER M，VANDIER C，等人Voltage-Gated Sodium Channels：New Targets in Cancer Therapy？[J].Curr Pharm Des，2006，12(28)：3681-3695)；
图2：β3在不同肝癌细胞株中表达；
图3，编码VGSC β3亚基的基因——SCN3B在不同白血病中表达；
图4，western blotting检测干扰后蛋白水平β3基因表达；
图5，MTT法测定VGSC β3的RNAi干扰对Hep G2细胞增殖的影响；
图6，干扰VGSC β3亚基的基因后对细胞周期的影响：D：空白HepG2细胞流式细胞术结果；E：HepG2细胞中SCN3B干扰48h后，流式细胞术结果；F：HepG2细胞中SCN3B干扰60h后，流式细胞术结果；G：D，E，F图p3峰和p5峰的面积积分后统计结果，代表细胞数量，其中p3为G0/G1期，p5为碎片峰(凋亡峰)；
图7，VGSC β3亚基的基因被干扰干扰后，HepG2细胞周期蛋白cylin D1表达水平下调；
图8：肺癌细胞株A549中的VGSC β3亚基基因被干扰后蛋白水平表达情况
图9 AGAP对Hep G2细胞增殖及VGSC β3亚基表达的的影响：
A：AGAP对Hep G2细胞增殖的抑制作用；
B：AGAP对Hep G2细胞VGSC β3亚基表达的的抑制作用。
图10：Hep3B细胞中转染后VGSC β3亚基蛋白水平表达以及给予AGAP后VGSC β3亚基蛋白表达情况：道1：用pcDNA3.0-hSCN3B转染；道2：用空质粒pcDNA3.0转染；道3：在转染VGSC β3的Hep3B细胞给予AGAP后对VGSC β3表达的影响；使用GAPDH作为内部对照。
图11：Hep3B细胞中转染VGAC β3亚基后细胞增殖水平检测以及给予AGAP后细胞增殖检测；
图12：VGSC β3亚基干扰后对细胞周期和细胞形态的影响：A：正常空白HepG2细胞照片；B：HepG2细胞中SCN3B干扰48h后照片；C：HepG2细胞中SCN3B干扰60h后照片；D：正常空白HepG2细胞流式细胞术结果；E：HepG2细胞中SCN3B干扰48h后，流式细胞术结果；F：HepG2细胞中SCN3B干扰60h后，流式细胞术结果；G：D，E，F图p3峰和p5峰的面积积分后统计结果，代表细胞数量，其中p3为G0/G1期，p5为碎片峰(凋亡峰)；
图13：ANEXIN V检测VGSC β3亚基对细胞凋亡的影响：A-D为Annexin V-FITC-PI染色结果。A：HepG2细胞未干扰结果；B：HepG2细胞干扰48h结果；C：HepG2细胞干扰60h结果；D：HepG2细胞干扰72 h结果。
E-H为Hoechst33342-PI染色结果。A：HepG2细胞未干扰结果；B：HepG2细胞干扰48h结果；C：HepG2细胞干扰60h结果；D：HepG2细胞干扰72h结果。
I：Q2和Q4的统计结果，其中Q2代表晚期凋亡，Q4代表早期凋亡。
图14：包含有编码VGSC β3亚基的核酸序列(即SCN3B，如SEQ ID NO：2所示)的质粒图谱；
图15：图14所述的质粒的构建流程图。
具体实施方案
本发明可以通过参考以下详细描述的本发明优选实施方案及本文所包含实施例而更容易地理解。除非另外说明，本文中所用术语将根据相关领域普通技术人员的习惯用法加以理解。除了下文提供的术语定义外，分子生物学中常用术语的定义也可以在Rieger等，1991 Glossary of genetics：classical和molecular，第五版，Berlin：Springer-Verlag；和在Current protocols in Molecular Biology，F.M.Ausubel等编著，Current Protocols，Greene Publishing Associates，Inc.与John Wiley&Sons，Inc.的合资企业(1998增刊)中找到。应当理解本文中使用的术语仅仅旨在描述具体实施方案而并非意味对其限制性。
在本申请通篇范围内，参考了多种出版物。全部这些出版物及这些出版物中引用的那些参考文献的公开内容完整地并入本申请作为参考，旨在更充分地描述本发明所涉及领域的现状。用于克隆、DNA分离、扩增和纯化的标准技术、用于涉及DNA连接酶、DNA聚合酶、限制性内切核酸酶等的酶促反应的标准技术，以及多种分离技术是本领域技术人员已知和通常使用的。一些标准技术描述在Sambrook等人，1989 Molecular Cloning，第二版，Cold Spring Harbor Laboratory，Plainview，N.Y.；Maniatis等人，1982 Molecular Cloning，Cold Spring Harbor Laboratory，Plainview，N.Y.；Wu(编著)1993 Meth.Enzymol.218，部分I；Wu(编著)1979 Meth Enzymol.68；Wu 等人，(编著)1983 Meth.Enzymol.100和101；Grossman和Moldave(编著)1980 Meth.Enzymol.65；Miller(编著)1972 Experiments in Molecular Genetics，Cold Spring Harbor Laboratory，Cold Spring Harbor，N.Y.；Old和Primrose，1981 Principles of Gene Manipulation，University of California Press，Berkeley；Schleif和Wensink，1982 Practical Methods in Molecular Biology；Glover(编著)1985 DNA Cloning卷I和II，IRL Press，Oxford，UK；Hames Higgins(编著)1985 Nucleic Acid Hybridization，IRL Press，Oxford，UK；以及Setlow和Hollaender 1979 Genetic Engineering：Principles and Methods，卷1-4，Plenum Press，New York；Remington′s Pharmaceutical Sciences，第18版(Easton，Pennsylvania：Mack Publishing Company，1990)；Methods In Enzymology(S.Colowick和N.Kaplan编著，Academic Press，Inc.)和Handbook of Experimental Immunology，第I-IV卷(D.M.Weir和C.C.Blackwell编著，1986，Blackwell Scientific Publications)。当使用时，缩写和命名被认为是本领域中标准的并且在专业杂志例如本文中引用的那些专业杂志中经常使用的。
I.VGSC β3蛋白质
如本文所述，术语“VGSC β3”是指本领域已知的任何电压敏感性离子通道的β3亚基，优选地哺乳动物的VGSC β3，更优选地VGSC β3蛋白质来自于人。本领域已经表征的示例性的VGSC β3包括Gene ID55800，NM 018400(http：//www.ncbi.nlm.nih.gov/gene/55800)。示例性的VGSC β3突变蛋白质还参见Dan Hu MD，Hector Barajas-Martines，Elena Burashnikov BS，et al.A mutation in the β3 subunit of the cardiac sodium channel associated with brugada ECG phenotype.Circ Cardiovasc Genet.2009 2(3)：270-278。
例如，人VGSC β3蛋白质包含如下氨基酸序列，或其氨基酸序列如下所示：
SEQ ID NO：1：人VGSC β3蛋白质的氨基酸序列：
Figure PCTCN2015079356-appb-000013
SEQ ID NO：2：编码人VGSC β3蛋白质的核酸序列：
Figure PCTCN2015079356-appb-000014
II.VGSC β3的抑制剂
如本文所述，术语“VGSC β3抑制剂”包括任何能够抑制VGSC β3的DNA，mRNA或蛋白质的物质，例如干扰RNA(RNAi活性剂)、抗体、小分子抑制剂、(多)肽和核酸、反义寡核苷酸、或模拟物。具体地，VGSC β3蛋白质能够通过抑制其编码DNA序列、其上下游的调控序列、mRNA以及VGSC β3蛋白质来抑制VGSC β3蛋白质的表达、活性和/或相关生物学活性。
术语“抑制”VGSC β3指VGSC β3的生物学活性、生物学水平、活性和/或表达的任何统计学显著的减少，包括完全阻断活性和/或表达。例如，“抑制”可以指VGSC β3水平、活性和/或表达减少至少约10、20、30、40、50、60、70、80、90或100％。如本文中使用的，术语“抑制”类似的指水平、活性和/或表达的显著的减少，同时针对任何其他的生物学活性剂或组合物。
在一些实施方案中，与对照相比VGSC β3的抑制剂抑制VGSC β3生物学活性25％、50％、75％、80％、90％、95％、97％、98％、99％或100％。 在一些实施方案中，与对照相比，VGSC β3的抑制剂抑制VGSC β3的表达至少25％、50％、75％、80％、90％、95％、97％、98％、99％或100％。
在一种具体的实施方式中，本文公开内容的VGSC β3的抑制剂包含本文公开的或本领域已知的任何能够抑制VGSC β3的抑制剂，并被施用给需要其的患者，例如，遭受肿瘤(特别是癌症)的患者。
在VGSC β3相关性疾病的症状的上下文中，“降低”表示此类水平的统计上显著的减少。这种减少可例如为至少10％、至少20％、至少30％、至少40％或更多。针对特定疾病或针对遭受特定疾病的个体，如果VGSC β3的水平或表达升高，则用本文公开内容的VGSC β3 RNAi活性剂进行的治疗可特别地将VGSC β3的水平或表达降低至处于文献中认为对没有此类病症的个体来说正常的范围内的水平。VGSC β3的水平或表达可通过对mRNA(例如通过Northern印迹或PCR)或蛋白质(例如Western印迹)的评价来测量。RNAi活性剂对VGSC β3表达的影响可通过测量VGSC β3基因转录速率来测定(例如通过Northern印迹；或逆转录酶聚合酶链式反应或实时聚合酶链式反应)。直接测量可对(细胞表面表达的)VGSC β3的水平进行，例如通过对其中表达VGSC β3的组织进行Western印迹来进行。
如本文中使用的，“下调”指VGSC β3的生物学活性和/或表达的任何统计学显著的减少，包括完全阻断活性(即，完全抑制)和/或表达。例如，“下调”可以指VGSC β3水平、活性和/或表达减少至少约10、20、30、40、50、60、70、80、90或100％。
如本文中使用的，术语“抑制”VGSC β3指VGSC β3的生物学水平、活性和/或表达的任何统计学显著的减少，包括完全阻断活性和/或表达。例如，“抑制”可以指VGSC β3水平、活性和/或表达减少至少约10、20、30、40、50、60、70、80、90或100％。如本文中使用的，术语“抑制”类似的指水平、活性和/或表达的显著的减少，同时针对任何其他的生物学活性剂或组合物。
“水平”意指VGSC β3 RNAi活性剂或其它抑制剂可以改变VGSC β3的 水平，例如，VGSC β3 DNA的水平或mRNA的水平或VGSC β3蛋白质的水平，或者VGSC β3的活性水平。
在一个实施方案中，在疾病的特征是VGSC β3过表达和/或活性过高的情况下，施用VGSC β3的抑制剂，例如RNAi活性剂降低了VGSC β3的水平、表达和/或活性。
对于水平、表达和/或活性而言，“正常”或“几乎正常”意指在健康细胞、组织或器官中的VGSC β3的水平、表达或活性的至少约50％、约60％、约70％、约80％、约90％和/或约100％；和/或不超过约100％、约120％、约130％、约140％或约150％。
此外，在多个实施方案中，根据疾病病症和生物学环境，可接受使用本公开内容的VGSC β3抑制剂来抑制VGSC β3表达、活性和/或水平的水平。
II-1 干扰RNA
在一些实施方案中，VGSC β3抑制剂是干扰RNA(RNAi)活性剂。
RNA干扰技术是本领域熟知的技术，RNAi活性剂的设计以及获得也是本领域已知的技术，参见例如WO2011/073326或WO2011/131707中的详细介绍。
RNA干扰被称作“RNAi”，是一种以小的单链或双链RNA分子实现降低目的基因的表达的方法。干扰RNA包括双链或单链的小干扰RNA(ds siRNA或ss siRNA)、微小RNA(miRNA)、小发夹RNA(shRNA)等等。不希望被理论束缚，RNA干扰似乎发生在体内，双链RNA前体被切割成长度约为20-25个核苷酸的小RNA。切割是由RNAeIII-RNA解旋酶切丁酶完成的。siRNA的有义链，即有与目的mRNA序列完全相同序列的链被除去，留下与目的mRNA序列互补的“反义链”来降低mRNA的表达。siRNA的反义链似乎引导被称作RISC(RNA诱导沉默复合体)的蛋白复合体到达mRNA，然后RISC的Argonaute蛋白对mRNA进行切割，从而降低该mRNA的蛋白质产生。干扰RNA具有催化活性，亚化学剂量的mRNA相关的干 扰RNA就能降低mRNA的表达。mRNA表达的降低也可通过转录和翻译的机制发生。
本公开内容的RNAi活性剂靶向(例如，结合、退火等)VGSC β3 mRNA。使用VGSC β3特异性的RNAi活性剂导致VGSC β3活性、水平和/或表达的减少，例如“敲低(knock-down)”或“敲除(knock-out)”靶基因或靶序列。
在一个实施方案中，RNAi包含单链(例如本文所述的shRNA)。
在多种实施方案中，一条或两条链是带切口的。
在一个实施方案中，单链RNAi活性剂寡核苷酸或多核苷酸可以包含正义和/或反义链。参见例如，Sioud 2005 J.Mol.Biol.348：1079-1090，及其中引用的参考文献。因此，本公开内容涵盖了具有这样的单链的RNAi活性剂，所述单链包含本文所述RNAi活性剂的正义或反义链。
特别用于本公开内容的siRNA包括可以特异性结合VGSC β3 mRNA区域的，和具有一个或多个下列性质的那些：结合在VGSC β3的编码区段中；结合在或靠近5’非翻译区和编码区段起始的连接处；结合在或靠近mRNA的翻译起始位点；结合在、覆盖或靠近外显子和内含子的连接处；几乎不或不结合其他基因的mRNA或转录物(几乎没有或没有“脱靶效应”)；在不是双链或茎的一个或多个区域内或附近结合VGSC β3 mRNA，例如，在环或单链部分；几乎不或不引发免疫原性；结合在在多种动物物种(包括人、小鼠、大鼠、食蟹猴等)之间保守的VGSC β3 mRNA序列的区段中，因为保守序列的存在有利于使用多种实验动物测试；结合mRNA的双链区域；结合富含AT的区域(例如，至少约50、51、52、53、54、55、56、57、58、59或60％AT丰富的)；和/或缺少已知或怀疑减少siRNA活性的特定序列，例如，在5’端存在GG序列，其可能减少siRNA双链部分的分离。在一个实施方案中，VGSC β3特异性的RNAi活性剂可以是具有任何一种或多种这些性质的双链RNA。
如本文中使用的，术语“双链RNA”或“dsRNA”指包含第一链和第二链的RNAi活性剂；例如，包含具有杂交双链体区域的RNA分子或分子复合 体的组合物(即，第一链和第二链的核苷酸碱基配对的区域)，所述区域包含两条反向平行和基本互补的核酸链，被称为相对于靶RNA具有“正义”和“反义”方向。涉及mRNA靶时，反义链也被称为“指导”链，正义链也被称为“过客”链。过客链可以包括至少一个或多个下列性质：相比其他链的一个或多个额外核苷酸(例如，凸起或1nt环)、相比其他链的缺口、空位等。在多个实施方案中，RNAi活性剂包含第一链和第二链。在多种实施方案中，第一链是正义链，第二链是反义链。在其他实施方案中，第一链是反正义链，第二链是正义链。
双链体区域可以是允许通过RISC通路特异性降解所需靶RNA的任何长度，但通常范围是长度从9至36对碱基对(“bp”)，例如，长度15-30bp。考虑到在9和36bp之间的双链体，双链体可以是该范围内的任何长度，例如9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35或36bp，及其之间的任何子范围，包括但不限于15-30bp、15-26bp、15-23bp、15-22bp、15-21bp、15-20bp、15-19bp、15-18bp、15-17bp、18-30bp、18-26bp、18-23bp、18-22bp、18-21bp、18-20bp、19-30bp、19-26bp、19-23bp、19-22bp、19-21bp、19-20bp、19bp、20-30bp、20-26bp、20-25bp、20-24bp、20-23bp、20-22bp、20-21bp、21-30bp、21-26bp、21-25bp、21-24bp、21-23bp、21-22bp、21bp、22bp或23bp。通过Dicer和类似酶加工，在细胞内产生的dsRNA的长度通常在约19至约22bp的范围内。dsRNA的双链体区域的一条链包含与靶RNA的区域基本互补的序列。形成双链体结构的两条链可以来自具有至少一个自身互补的双链体区域的单个RNA分子，或者可以自两个或多个杂交形成双链体的分离的RNA分子形成。当双链体区域是由单个分子的2个自身互补区域形成时，分子可以具有被单链核苷酸(在本文中称为“发夹环”，例如，可见于shRNA结构中的)隔开的双链体区域，所述单链核苷酸位于形成双链体结构的一条链的3’端和相应另一条链的5’端之间。发夹环可以包含至少一个未成对的核苷酸；在一些实施方案中，发夹环可以包含至少3个、至少4个、至少5个、至少6个、至少7个、 至少8个、至少9个、至少10个、至少20个、至少23个或更多个未配对的核苷酸。当2条基本互补的dsRNA链包含在分离的RNA分子中时，这些分子不必但可以是共价连接的。当两条链是通过发夹环共价连接之时，所述结构在本文和本领域中一般被称为“shRNA”。当两条链是通过发夹环以外的方式共价连接之时，所述连接结构被称为“接头”。
本文描述的dsRNA分子(RNAi活性剂)可用于VGSC β3的RNA干扰。
(A)RNAi活性剂的特征：正义链、反义链和(任选的)突出端
在多个实施方案中，RNAi活性剂包含第一链和第二链，例如，正义链和反义链以及任选的，在双链体的一个或两个末端含有在本文中被称为突出端的未配对的核苷酸。
术语“反义链”指RNAi活性剂的这样的链，所述链包含基本与靶序列互补的区域。如本文中使用的，术语“互补的区域”指反义链上与本文定义的序列(例如，靶序列)基本互补的区域。当互补的区域与靶序列不完全互补时，错配可以位于分子的内部或末端区域。一般而言，最耐受的错配位于内部区域中，例如，在5’和/或3’末端的5、4、3或2个核苷酸内。
如本文中使用的，术语“正义链”指RNAi活性剂的这样的链，所述链包含与本文定义的术语的反义链的区域基本互补的区域。
基因序列在个体之间可以是不同的，尤其是在编码区段内的摆动位置，或在非翻译区中；个体的编码序列也可以彼此不同，导致mRNA的额外差异。因此需要时，RNAi活性剂的正义链和反义链序列可被设计为与个体患者的序列对应。还可以修饰RNAi活性剂的序列来降低免疫原性、与不理想的mRNA的结合(例如，“脱靶效应”)或增加在血液中的稳定性。这些序列变体不依赖于对RNAi活性剂的碱基或5’或3’或其他末端帽的化学修饰。
RNAi活性剂还可以具有0、1或2个突出端的突出端，在0nt突出端的情况下，活性剂是平端的。RNAi活性剂可以具有0、1或2个平端。在 “平端RNAi活性剂”中，两条链都以碱基对结束；因此，平端分子缺少3’或5’单链核苷酸突出端。
如本文中使用的，术语“突出端”或“核苷酸突出端”指从RNAi活性剂的双链体结构两条链中的至少一条的末端突出来的至少一个未配对的核苷酸。例如，当dsRNA的一条链的3’端延伸超过另一条链的5′端或反之时，未配对的核苷酸形成突出端。dsRNA可以包含至少一个核苷酸的突出端；可选的，突出端可以包含至少两个核苷酸、至少3个核苷酸、至少4个核苷酸、至少5个核苷酸或更多。突出端可以包含核苷酸/核苷类似物，或由核苷酸/核苷类似物组成，包括脱氧核苷酸/核苷。突出端可以位于正义链、反义链或其任意组合上。此外，突出端的核苷酸可以位于dsRNA的反义链或正义链的5′端、3′端或两端。
RNAi活性剂还可以任选的包含帽。术语“帽”等包括与双链核苷酸双链体连接的化学部分，但在本文中用于排除是核苷酸或核苷的化学部分。“3”帽连接在核苷酸或寡核苷酸的3’端。“5”帽连接在核苷酸或寡核苷酸的5’端。在一个实施方案中，3’端帽如例如WO 2005/021749和WO 2007/128477中公开的。
因此，本公开内容考虑了VGSC β3特异性的RNAi活性剂，其在所述RNAi活性剂中包含反义链(可以是通过接头或环连续的或连接的)。在更具体的实施方案中，RNAi活性剂包含反义链和正义链，并且一起包含双链或互补区域。在一个实施方案中，还可以任选的包含一个或两个突出端和/或一个或两个帽。RNAi活性剂用于诱导VGSC β3的RNA干扰。
(B)靶和互补序列
本公开内容的RNAi活性剂靶向(例如，特异性结合、退火等)编码基因VGSC β3的mRNA。使用VGSC β3特异性的RNAi活性剂导致VGSC β3活性、水平和/或表达的减少，例如“敲低(knock-down)”或“敲除(knock-out)”靶基因或靶序列。特别是在一个实施方案中，在疾病状态的特征是VGSC β3过表达或活性过高的情况下，施用针对VGSC β3的RNAi 活性剂敲低VGSC β3基因至足以恢复正常的VGSC β3活性水平和/或正常的Na+重吸收水平。
在本文中使用时，“靶序列”或“靶基因”指在基因(例如编码VGSC β3蛋白质的基因)转录期间形成的mRNA分子的核苷酸序列的连续部分，包括是对一级转录产物进行RNA加工的产物的mRNA。序列的靶部分将至少长到足以用作为在该部分或该部分附近iRNA指导的切割的底物。例如，靶序列将通常为9-36个核苷酸(“nt”)长，例如15-30nt长，这包括其间的所有子区域。作为非限制性的例子，靶序列可15-30nt，15-26nt、15-23nt、15-22nt、15-21nt、15-20nt、15-19nt、15-18nt、15-17nt、18-30nt、18-26nt、18-23nt、18-22nt、18-21nt、18-20nt、19-30nt、19-26nt、19-23nt、19-22nt、19-21nt、19-20nt、19nt、20-30nt、20-26nt、20-25nt、20-24nt、20-23nt、20-22nt、20-21nt、20nt、21-30nt、21-26nt、21-25nt、21-24nt、21-23nt或21-22nt、21nt、22nt或23nt。RNAi的正义链和反义链包含与靶核酸VGSC β3互补的序列。
在本文中使用时，并且如无另外指明，术语“互补”表示包含第一核苷酸序列的寡核苷酸或多核苷酸在某些条件下与包含第二核苷酸序列的寡核苷酸或多核苷酸杂交并形成双链体结构的能力。此类条件可例如是严格的，例如400mM NaCl，40mM PIPES pH 6.4，1mM EDTA，50℃或70℃进行12-16小时，接着进行洗涤。其它条件，例如在生物体内可能遇到的生理相关条件也可应用。技术人员将能根据杂交的核苷酸的最终应用来确定最适用于测试两条序列的互补性的条件组。
“互补”序列在本文中使用时还可包括非Watson-Crick碱基对和/或从非天然和经修饰的核苷酸形成的碱基对，或完全由非Watson-Crick碱基对和/或从非天然和经修饰的核苷酸形成的碱基对形成，只要能满足关于其杂交能力的上述要求即可。此类非Watson-Crick碱基对包括但不限于G：U Wobble或Hoogstein碱基配对。
术语“互补”、“完全互补”和“实质上互补”在本文中还可在提到dsRNA的正义链和反义链之间的碱基匹配或RNAi活性剂的反义链和靶序列之间 的碱基匹配时使用，如从关于其用途的上下文中所理解的那样。
在本文中使用时，与信使RNA(mRNA)“的至少部分实质上互补”的多核苷酸指与目标mRNA(例如编码VGSC β3的mRNA)的连续部分实质上互补的多核苷酸。例如，如果序列与编码VGSC β3的mRNA的非间断部分实质上互补的话，多核苷酸就与VGSC β3mRNA的至少部分互补。
RNAi活性剂内(例如本文所述的dsRNA内)的互补序列包括包含第一核苷酸序列的寡核苷酸或多核苷酸与包含第二核苷酸序列的寡核苷酸或多核苷酸在一条或两条核苷酸序列全长上的碱基配对。在本文中此类序列可被称为互相“完全互补”。但是，当第一序列在本文中被称为与第二序列“实质上互补”时，两条序列可完全互补，或者它们可在针对多至30个碱基对的双链体进行杂交时形成一个或多个但通常不超过5、4、3或2个错配的碱基对，但保持在与它们的最终应用(例如，经由RISC途径抑制基因表达)最相关的条件下杂交的能力。但是，当两条寡核苷酸被设计来在杂交期间形成一个或多个单链突出端时，在测定互补性时此类突出端将被不认为是错配。例如，就本文所述的目的而言，包含长度为21个核苷酸的一条寡核苷酸和长度为23个核苷酸的另一寡核苷酸的双链体(其中，较长的寡核苷酸包含与较短的寡核苷酸完全互补的21个核苷酸的序列)仍可被认为是“完全互补”的。术语突出端描述了在双链核苷酸双链体的3′或5′端未配对的核苷酸，如上所述。在一个实施方案中，突出端是0至4nt长，并位于3’端。
因此，本公开内容的RNAi活性剂与靶VGSC β3中的靶序列互补或基本互补，且是双链的，包含正义链和反义链(可以是连续的、通过环连接、或连接的)，其中双链区是9至36bp长(特别是例如，19-22bp或19-23bp长)，并可以进一步任选的包含3′或5′突出端，并且RNAi活性剂可以进一步包含3’帽。RNAi活性剂介导RNA干扰，下调或抑制VGSC β3的水平、表达和/或活性，和/或建立或重新建立几乎正常的ENaC水平和/或VGSC β3活性，或其他与ENaC相关的生物学功能。
(C).降低VGSC β3水平、表达和/或活性的RNAi活性剂
用于靶向VGSC β3的RNAi活性剂包括结合本文提供的VGSC β3序列并通过RNAi机制作用为降低VGSC β3的活性剂。VGSC β3的示例性siRNA提供在例如表1中。
本公开内容的RNAi活性剂沉默、抑制VGSC β3基因的表达，下调VGSC β3基因的表达，和/或阻抑VGSC β3基因的表达，使得实现几乎正常水平的VGSC β3活性、表达和/或水平，和/或Na+重吸收。
此外，在多个实施方案中，根据疾病病症和生物学环境，可接受使用本公开内容的RNAi活性剂来建立低于正常水平、或高于正常水平的VGSC β3表达、活性和/或水平的水平。
可使用本领域已知的任何方法测量由VGSC β3 siRNA诱导的VGSC β3活性、水平和/或表达的改变。测量可以在施用siRNA之前、之间或之后的多个时间点实施，以确定siRNA的效应。
术语“沉默”、“抑制......的表达”、“下调......的表达”、“阻抑......的表达”等等在提到VGSC β3基因的时候在本文中是表示对VGSC β3基因表达的至少部分阻抑，表现为：在VGSC β3基因在其中转录并且已经受到处理以抑制VGSC β3基因表达的第一细胞或细胞组中，可分离或检测到的VGSC β3 mRNA量的降低，这是较之与第一细胞或细胞组基本上相同但是未受此类处理的第二细胞或细胞组(对照细胞)而言的。抑制的程度通常表示为：
Figure PCTCN2015079356-appb-000015
   等式1
可选的，抑制程度可以作为与VGSC β3基因表达功能性相关的参数的降低来给出，例如，由VGSC β3基因编码的蛋白质的量，肺部液体水平或粘液水平的改变，等。原则上，可以在任何组成型或通过基因组工程表达VGSC β3的细胞中，通过任何恰当的测定，来确定VGSC β3基因沉默。然 而，当为了确定所给出的RNAi活性剂是否抑制VGSC β3基因表达至某程度，从而是否涵盖在本公开内容中，而需要参照或对照时，下文实施例中提供的测定可作为此类参考。
例如，在某些例子中，通过施用本公开内容中特征性的RNAi活性剂，抑制VGSC β3基因的表达至少约10％、15％、20％、25％、30％、35％、40％、45％和50％。在一些实施方案中，通过施用本公开内容中特征性的RNAi活性剂，抑制VGSC β3基因至少约60％、70％或80％。在一些实施方案中，通过施用本文所述的RNAi活性剂，抑制VGSC β3基因至少约85％、90％或95％或更多。
可以首先在体外测试RNAi活性剂抑制VGSC β3的能力(例如，使用测试细胞，如H441)。
然后，使用例如PBMC(外周血单核细胞)测定，可以测试能够在体外抑制针对VGSC β3的RNAi活性剂的免疫刺激作用。还可以在动物测试中测试RNAi活性剂。测试和对照动物包括过表达或低表达VGSC β3的动物，描述在例如，Hummer等人，2005 J.Am.Soc.Nephrol.16：3160-3166；Randrianarison等人，2007 Am.J.Physiol.Lung Cell.Mol.Physiol.294：409-416；Cao等人，2006 Am.J.Physiol.Renal Physiol.，及其中引用的参考文献。抑制或改变VGSC β3的水平、活性和/或表达的RNAi活性剂可用于治疗多种VGSC β3-相关性疾病的药物中。
(D)RNAi活性剂的类型及其修饰
使用包含反义核酸的RNAi活性剂或组合物下调特定蛋白质在细胞内的表达是本领域普遍已知的。RNAi活性剂包括与另一种核酸的编码链(例如，mRNA)互补并且能够与之氢结合的序列。因此，在多个实施方案中，本公开内容的RNAi活性剂涵盖了任何靶向(例如，与之互补，能够氢结合等)例如表1所展示的序列的RNAi活性剂。
与mRNA互补的反义序列可以与编码区、mRNA的5′或3′非翻译区，和/或桥接编码区和非翻译区的区域，和/或其部分互补。此外，RNAi活性 剂或其部分可以与编码mRNA的基因的调控区互补，所述调控区例如转录或翻译起始序列或调控元件。特别的是，RNAi活性剂或其部分可以与位于编码链的起始密码子之前的区域，或与包含起始密码子的区域，或与mRNA的3′非翻译区中的区域互补。
可以根据Watson和Crick碱基配对规则，设计RNAi活性剂分子。RNAi活性剂可以与VGSC β3 mRNA的完全编码区互补，但更特别的是仅与VGSC β3 mRNA的编码区或非编码区的一部分反义的寡核苷酸。例如，反义寡核苷酸可以与位于VGSC β3 mRNA翻译起始位点周围的区域互补。反义寡核苷酸可以是例如长度约5、10、15、19、20、21、22、23、24、25、30、35、40、45或50个核苷酸。
RNAi活性剂可以在内部或在一端或两端具有修饰。末端的修饰可以帮助稳定RNAi活性剂，保护其免受血液中的核酸酶降解。RNAi活性剂可任选的针对VGSC β3 mRNA中已知或预测靠近或位于基因剪接位点的区域；例如，外显子-内含子连接(例如，Saxena等人，1998中所述)。
RNAi活性剂还可以任选的被设计为与mRNA的已知或预测的暴露和/或单链区域(例如，环)退火。
可使用本领域已知的流程，使用化学合成和酶促连接反应，来构建RNAi活性剂。例如，可使用天然存在的核苷酸或经多种修饰的核苷酸(设计来减少脱靶效果，和/或增加分子的生物稳定性，或增加反义和正义核酸之间形成的双链体的物理稳定性)，来化学合成RNAi活性剂，例如可使用硫代磷酸衍生物，和经吖啶取代的核苷酸。
“G”、“C”、“A”、“T”和“U”每个通常代表分别含有鸟嘌呤、胞嘧啶、腺嘌呤、胸腺嘧啶核苷和尿嘧啶作为碱基的核苷酸。但是术语“核糖核苷酸”或“核苷酸”还可表示经修饰的核苷酸或替代置换部分(surrogate replacement moiety)。本领域技术人员熟知鸟嘌呤、胞嘧啶、腺嘌呤和尿嘧啶可被其它部分替换，而不会实质上改变包含具有此类置换部分的核苷酸的寡核苷酸的碱基配对性质。例如但不限于，包含肌苷作为其碱基的核苷酸可与含有腺嘌呤、胞嘧啶或尿嘧啶的核苷酸碱基配对。因此，在本文 公开内容所涉及的dsRNA的核苷酸序列中，含有尿嘧啶、鸟嘌呤或腺嘌呤的核苷酸可被含有例如肌苷的核苷酸置换。在另一实例中，在寡核苷酸中任何位置的腺嘌呤和胞嘧啶可被鸟嘌呤和尿嘧啶分别替换，以与靶mRNA形成G-U Wobble碱基配对。含有此类置换部分的序列适用于本文公开内容所涉及的组合物和方法。
技术人员将认识到，术语“RNA分子”或“核糖核酸分子”不仅包括在自然界中发现或表达的RNA分子(即天然存在的)，其还包括非天然存在的包含有本文所述或本领域已知的一个或多个核糖核苷酸/核糖核苷类似物或衍生物的RNA类似物和衍生物。严格地说，“核糖核苷”包括核苷碱基和核糖，而“核糖核苷酸”是核糖核苷与一个、两个或三个磷酸部分。但是，术语“核糖核苷”和“核糖核苷酸”在本文中使用时可被认为是等同的。RNA可在核苷碱基结构或在核糖-磷酸主链结构中被修饰，例如如本文下文所述的。但是，包含核糖核苷类似物或衍生物的分子必须保持形成双链体的能力。作为非限制性的例子，RNA分子还可包括至少一个经修饰的核糖核苷，这包括但不限于：2′-O-甲基修饰的核苷酸、包含5’硫代磷酸基团的核苷、与胆固醇基衍生物或十二烷酸二癸酰胺基团连接的末端核苷、锁定核苷、无碱基核苷、2′-脱氧-2′-氟修饰的核苷、2′-氨基-修饰的核苷、2′-烷基修饰的核苷、吗啉代核苷、解锁核糖核苷酸(例如无环核苷酸单体，如WO 2008/147824所述)、氨基磷酸酯或包含非天然碱基的核苷，或它们的任何组合。或者，RNA分子可包含至少两个经修饰的核糖核苷，至少3个、至少4个、至少5个、至少6个、至少7个、至少8个、至少9个、至少10个、至少15个、至少20个或更多个，多至dsRNA分子的全长。对于RNA分子中多个经修饰的核糖核苷中的每个而言，修饰无须是相同的。在一种实施方式中，考虑用于本文所述的方法和组合物中的经修饰的RNA是具有形成所需双链体结构的能力并且允许或介导经由RISC途径对靶RNA的特异性降解的肽核酸(PNA)。
可用于产生RNAi活性剂的经修饰的核苷酸的例子包括5-氟尿嘧啶、5-溴尿嘧啶、5-氯尿嘧啶、5-碘尿嘧啶、次黄嘌呤、黄嘌呤(xantine)、4- 乙酰基胞嘧啶、5-(羧基羟基甲基)尿嘧啶、5-羧基甲基氨基甲基-2-硫尿苷、5-羧基甲基氨基甲基尿嘧啶、二氢尿嘧啶、β-D-半乳糖基Q核苷(β-D-galactosylqueosine)、肌苷、N6-异戊烯基腺嘌呤、1-甲基鸟嘌呤、1-甲基肌苷、2，2-二甲基鸟嘌呤、2-甲基腺嘌呤、2-甲基鸟嘌呤、3-甲基胞嘧啶、5-甲基胞嘧啶、N6-腺嘌呤、7-甲基鸟嘌呤、5-甲基氨基甲基尿嘧啶、5-甲氧基氨基甲基-2-硫代尿嘧啶、β-D-甘露糖基Q核苷、5′-甲氧基羧基甲基尿嘧啶、5-甲氧基尿嘧啶、2-甲基硫代-N6-异戊烯基腺嘌呤、尿嘧啶-5-羟乙酸(v)、wybutoxosine、假尿嘧啶、Q核苷(queosine)、2-硫代胞嘧啶、5-甲基-2-硫代尿嘧啶、2-硫代尿嘧啶、4-硫代尿嘧啶、5-甲基尿嘧啶、尿嘧啶-5-羟乙酸甲基酯、尿嘧啶-5-羟乙酸(v)、5-甲基-2-硫代尿嘧啶、3-(3-氨基-3-N-2-羧基丙基)尿嘧啶、(acp3)w和2，6-二氨基嘌呤。
在一个实施方案中，本公开内容涵盖了本文公开的任何RNAi活性剂的修饰的任何修饰的变体。修饰的变体含有相同的序列，但可以经过修饰含有在磷酸、糖、碱基、核苷酸等中的修饰。例如，修饰的变体可以含有一个或多个本文列举的修饰的核苷酸，例如用2’-修饰的C取代C。
在一个方面，经修饰的核糖核苷包括脱氧核糖核苷。在这种情况下，RNAi活性剂可包含一个或多个脱氧核苷，包括例如，脱氧核苷突出端，或dsRNA双链部分内的一个或多个脱氧核苷。但是，不言而喻地，术语“RNAi活性剂”在任何情况下都不包括双链DNA分子。
用脱氧核糖核苷酸替换具有两个核苷酸的3’突出端的21体siRNA双链体的3’末端核苷酸突出端区段，对RNAi活性没有不利影响。用脱氧核糖核苷酸替换siRNA每个末端的多至四个核苷酸已被良好容许，而用脱氧核糖核苷酸进行完全取代会导致没有RNAi活性。国际PCT公开No.WO 00/44914和Beach等人，国际PCT公开No.WO 01/68836初步暗示，siRNA可将修饰包括至磷酸-糖主链或核苷上，以包括至少一个氮或硫杂原子。Kreutzer等人，加拿大专利申请No.2,359,180也描述了用于dsRNA构建体的某些化学修饰，以抵消双链RNA依赖性的蛋白质激酶PKR的活化，特别是2’-氨基或2’O-甲基核苷酸，以及含有2’-O或4’-C亚甲桥的核苷酸。 另外的3’-末端核苷酸突出端包括dt(脱氧胸苷)、2’-O，4’-C-乙烯胸苷(eT)和2-羟乙基磷酸酯(hp)。
Parrish等人2000 Molecular Cell 6：1077-1087使用长(＞25nt)siRNA转录本，在秀丽隐杆线虫(C.elegans)中测试了靶向unc-22基因的某些化学修饰。作者描述了通过用T7和T3 RNA聚合酶并入硫代磷酸核苷酸类似物，将硫代磷酸残基引入这些siRNA转录本，并且观察到，具有两个经硫代磷酸酯修饰的碱基的RNA与RNAi一样在有效性上也具有相当的减少。此外，Parrish等人报道，对超过两个残基进行硫代磷酸修饰在体外使得RNA被大大去稳定，从而干扰活性不可测定。Id.at 1081。作者还测试了长siRNA转录本中核苷酸糖2’-位的某些修饰，并发现用脱氧核苷酸取代核糖核苷酸，使得干扰活性大为减少，尤其是在尿苷到胸苷和/或胞苷到脱氧胞苷取代的情况下。此外，作者测试了某些碱基修饰，包括在siRNA的正义链和反义链中用4-硫代尿嘧啶、5-溴尿嘧啶、5-碘尿嘧啶和3-(氨基烯丙基)尿嘧啶取代尿嘧啶以及用肌苷取代鸟苷。虽然4-硫代尿嘧啶和5-溴尿嘧啶取代显示出是容许的，Parrish报道称，肌苷并入任一链时产生了干扰活性的实质性减少。Parrish还报道称，5-碘尿嘧啶和3-(氨基烯丙基)尿嘧啶并入反义链导致RNAi活性也实质减少。
本领域技术人员将认识到，可使用本领域已知的任何常规方法(见Henschel等人，2004 DEQOR：a web-based tool for the design and quality control of siRNA.Nucleic Acids Research 32(Web Server Issue)：W113-W120)，按需求合成和修饰siRNA。此外，本领域技术人员还显而易见，有多种调控序列(例如组成型或可诱导启动子、组织特异性启动子或其功能片段等等)可用于反义寡核苷酸、siRNA或shRNA表达构建体/载体。
本领域中有多个例子描述了可被引入核酸分子并且显著增强了其核酸酶稳定性和效力的糖、碱基、磷酸和主链修饰。例如，修饰寡核苷酸以增强稳定性和/或增强生物活性，这通过采用核酸酶抗性基团(例如，2’-氨基、2’-C-烯丙基、2’-氟代、2’-O-甲基、2’-O-烯丙基、2’-H)的修饰，核苷酸 碱基修饰来实现(综述见Usman和Cedergren 1992 TIBS.17：34；Usman等人，1994 Nucleic Acids Symp.Ser.31：163；Burgin等人，1996 Biochemistry 35：14090)。对核酸分子的糖修饰在本领域中被广泛描述。
对RNAi活性剂的另外的修饰和缀合已被描述过。Soutschek等人，2004 Nature 432：173-178展示了通过吡咯烷接头将胆固醇缀合到siRNA分子的正义链的3’端，由此产生共价和不可逆缀合物。还可对RNAi活性剂进行化学修饰(包括与其它分子缀合)，以改进体内药物动力学驻留时间和效率。
在多种实施方式中，针对VGSC β3的RNAi活性剂包含：至少一个5’-尿苷-腺嘌呤-3’(5’-ua-3’)二核苷酸，其中尿苷是2’-修饰的核苷酸；至少一个5’-尿苷-鸟嘌呤-3’(5’-ug-3’)二核苷酸，其中5’-尿苷是2’-修饰的核苷酸；至少一个5’-胞苷-腺嘌呤-3’(5’-ca-3’)二核苷酸，其中5’-胞苷是2’-修饰的核苷酸；和/或至少一个5’-尿苷-尿苷-3’(5’-uu-3’)二核苷酸，其中5’-尿苷是2’-修饰的核苷酸。
在多种实施方式中，RNAi活性剂包含选自下组的2’-修饰，所述组由2′-脱氧、2′-脱氧-2’-氟代、2’-O-甲基、2′-O-甲氧基乙基(2′-O-MOE)、2′-O-氨基丙基(2′-O-AP)、2′-O-二甲基氨基乙基(2′-O-DMAOE)、2′-O-二甲基氨基丙基(2′-O-DMAP)、2′-O-二甲基氨基乙氧基乙基(2′-O-DMAEOE)和2′-O-N-甲基乙酰氨基(2′-O-NMA)构成。
在另一实施方式中，RNAi包含缺口或丢失的碱基。例如，磷酸-糖主链可存在，但碱基丢失。
在另一实施方式中，RNAi活性剂具有单链切口(例如主链中的断裂或丢失的键)。在多个实施方案中，单链缺口可以在正义链或反义链或两条链中。
该切口可例如在正义链中，产生小的内部片断化的干扰RNA，或siRNA，其可比没有切口的相应RNAi活性剂具有更少的脱靶效果。
反义核酸或RNAi活性剂还可具有替代性的主链，例如锁核酸(LNA)、吗啉代、肽核酸(PNA)、苏糖核酸(TNA)或乙二醇核酸(GNA)，和 /或其可被标记(例如经放射性标记或加标签的)。
一条链或两条链可包含替代性的主链。
在另一实施方式中，本文公开内容的方法所利用的RNAi活性剂可包括α-异头核酸分子。α-异头核酸分子与互补RNA形成特异性双链杂交体，其中，与通常的β-单元相反，链互相平行排列。Gaultier等人，1987 Nucleic Acids.Res.15：6625-6641。
反义核酸分子还可包含2′-o-甲基核糖核苷酸(Inoue等人，1987 Nucleic Acids Res.15：6131-6148)或嵌合RNA-DNA类似物(Inoue等人，1987 FEBS Lett.215：327-330)。
在还一实施方式中，RNAi活性剂是核酶。核酶是具有核糖核酸酶活性的催化性RNA分子，其能切割与它们具有互补区域的单链核酸，例如mRNA。因此，核酶[例如锤头核酶(被描述于Haselhoff等人，1988，Nature 334：585-591中)]可用于催化性切割VGSC β3 mRNA转录本，由此抑制VGSC β3 mRNA的翻译。
或者，可通过靶向与VGSC β3的调控区域(例如启动子和/或增强子)互补的核苷酸序列，以形成防止VGSC β3基因转录的三螺旋结构，来抑制基因表达。一般而言，见Helene 1991 Anticancer Drug Des.6(6)：569-84；Helene等人，1992 Ann.N.Y.Acad.Sci.660：27-36；和Maher 1992，Bioassays 14(12)：807-15。
(E)生产RNAi活性剂
可以使用表达载体生物学生产RNAi活性剂，所述表达载体中以反义方向亚克隆了核酸(即，从插入核酸转录的RNA与目标靶核酸为反义方向)。还可以使用表达载体生物学生产RNAi活性剂，所述表达载体中被亚克隆了核酸为shRNA构建体(即，从插入的核酸转录的RNA具有与目标靶核酸反义方向的第一区，包括环或铰链的第二区和与目标靶核酸正义方向的第三区，其中转录物的第一和第三区优选与其自身杂交，从而形成茎环结构)。
生产RNAi活性剂的方法是本领域普遍已知的，是本领域普通技术人 员可获得的。
用于合成RNAi的试剂盒是可以从例如New England Biolabs和Ambion商业获得的。
(F)本发明的RNAi活性剂
在本发明的一个实施方案中，RNAi活性剂为dsRNA，优选地为双链长度为9-50(例如15-36bp、15-30bp、15-26bp、15-23bp、15-22bp、15-21bp、15-20bp、15-19bp、15-18bp、15-17bp、18-30bp、18-26bp、18-23bp、18-22bp、18-21bp、18-20bp、19-30bp、19-26bp、19-23bp、19-22bp、19-21bp、19-20bp、19bp、20-30bp、20-26bp、20-25bp、20-24bp、20-23bp、20-22bp、20-21bp、21-30bp、21-26bp、21-25bp、21-24bp、21-23bp、21-22bp、21bp、22bp或23bp个核苷酸长度，或9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50)个核苷酸长度的siRNA，其包含反义链和与反义链实质上互补的正义链，其中反义链与编码VGSC β3的核苷酸序列的15、16、17、18、10、20或21个或更多个连续核苷酸至少约80％-90％互补或与之在严格条件下杂交，或者，更优选地、与靶基因的15、16、17、18、10、20或21个或更多个连续核苷酸至少约90-95％互补或与之在严格条件下杂交，并且最优选与靶基因的15、16、17、18、10、20或21个或更多个连续核苷酸至少约95％、96％、97％、98％或99％互补或与之在严格条件下杂交，或与之完全相同。
如本文中使用的，词组“严格杂交条件”或“严格条件”指在所述条件下探针、引物或寡核苷酸将与其靶序列杂交，但与最小量的其它序列杂交。严格条件是序列依赖性的，随不同环境而不同。更长的序列在更高温度下特异性的与其适当的互补序列杂交。通常，严格条件选为在定义的离子强度和pH下，低于特定序列的热解链点(Tm)约5℃。Tm是50％的与靶序列互补的探针与靶序列杂交达到平衡的温度(在定义的离子强度、pH和核酸浓度下)。由于靶序列通常过量存在，在Tm时，50％的探针与其 互补序列杂交达到平衡。通常，严格条件可以是这样的条件，其中盐浓度少于约1.0M钠离子，通常约0.01-1.0M钠离子(或其他盐)，pH 7.0-8.3，并且对于短的探针、引物或寡核苷酸(例如，10-50个核苷酸)，温度是至少约30℃，对于更长的探针、引物或寡核苷酸，温度是至少约60℃。严格条件还可以通过添加去稳定剂例如甲酰胺来实现。
如本文中使用的，术语“中度严格条件”指在所述条件下探针、引物或寡核苷酸可以与其靶序列杂交，但与有限量的其它序列杂交的条件。中度严格条件是序列依赖性的，随不同环境而不同。中度条件是本领域技术人员普遍已知的，特别描述在Maniatis等人(Molecular Cloning：A Laboratory Manual，Cold Spring Harbor Laboratory；第2版(1989年12月))等中。
本发明还涉及包含所述RNAi活性剂的核酸、表达盒、表达载体以及宿主细胞。
II-2 抗体
一些实施方案中，VGSC β3抑制剂是单克隆抗体、多克隆抗体、嵌合抗体、人抗体、人源化抗体、单链抗体或抗体片段，例如Fab片段。本领域已知并且可以获得针对VGSC β3的抗体，例如ABcam公司(艾博抗(上海)贸易有限公司
Room 5401，Floor 4，Building 5，No.338 Galileo Road，Pudong New Area，Shanghai.Postal Code：201203)可商购的抗VGSC β3的抗体，如
Ho C et al.Differential expression of sodium channelβsubunits in dorsal root ganglion sesory neurons.J Biol Chem.2012 287(18)：15044-15053和Hu D等人.A mutation in the β3 subunit of the cardiac sodium channel associated with brugada ECG phenotype.Circ Cardiovasc Genet.2009 2(3)：270-278所提及的抗体等。
抗体可以用例如酶、放射性同位素或荧光团标记。在一些实施方案中，抗体和VGSC β3以外的多肽的结合亲和力小于约1x 105Ka。在一些实施 方案中，VGSC β3抑制剂是单克隆抗体，其与VGSC B3结合的亲和力为至少1x 108Ka。
如本文中使用的，术语“抗体”指特异性针对靶蛋白或其片段的单克隆和多克隆抗体，单链抗体、嵌合抗体、双功能/双特异性抗体、人源化抗体、人抗体，和互补决定区(CDR)移植抗体。术语“抗体”还包括体内治疗性抗体基因转移。本发明还提供抗体片段，包括Fab、Fab’、F(ab’)2、scFv和Fv。
如本文中使用的，术语“单克隆抗体”指从基本同质的抗体群体中获得的抗体，即，除了小量存在的可能的天然发生突变外，包含群体的个体抗体是相同的。单克隆抗体是高度特异性的，针对单个抗原性位点。此外，与包括针对不同决定子(表位)的不同抗体的多克隆抗体制品相反，每种单克隆抗体针对抗原的单个决定子。除了它们的特异性之外，单克隆抗体的优势在于可以被合成而不被其它抗体污染。修饰语单克隆”表示抗体的特征，所述特征是从基本同质的抗体群体获得，而不认为需要通过任何特定方法来生产抗体。例如，可以通过杂交瘤方法制备根据本发明使用的单克隆抗体，所述方法首先描述在Kohler等人，Nature 256：495(1975)中，或者可以通过重组DNA方法生产(参见例如，美国专利号4,816,567)。还可以从噬菌体抗体文库中利用例如描述在Clackson等人，Nature 352：624-628(1991)和Marks等人，J.Mol.Biol.222：581-597(1991)中的技术分离“单克隆抗体”。
本文中的单克隆抗体特别包括“嵌合”抗体以及此类抗体的片段，只要其表现出理想的生物学活性，其中重链和/或轻链的一部分与抗体相应序列相同或同源，所述抗体源自特定物种或属于特定的抗体类或亚类，而剩余的链与下述抗体相应序列相同或同源，所述抗体源自另一种物种或属于另一种抗体类或亚类(美国专利号4,816,567；和Morrison等人，Proc.Natl.Acad.Sci.USA，81：6851-6855(1984))。本文中的目标嵌合抗体包括“灵长源化的(primatized)”抗体，其包含源自非人灵长类的可变区结构域抗原结合序列(例如，旧大陆猴、类人猿等)和人的恒定区序列。
“抗体片段”包括完整抗体的部分，优选的包括它的抗原结合区或可变区。抗体片段的实例包括Fab、Fab’、F(ab’)2和Fv片段；双抗体；线性化抗体(Zapata等人，Protein Eng.8(10)：1057-1062[1995])；单链抗体分子；和由抗体片段形成的多特异性抗体。
在一些实施方案中，本发明中的抗体是人源化抗体。人源化抗体可以通过多种方法实现，包括例如：(1)将非人互补决定区(CDR)移植到人构架区和恒定区(本领域称为“人源化”的过程)，或可选的(2)移植整个非人可变结构域，但通过替换表面残基用人-类似表面“隐匿(cloaking)”它们(本领域称为“镶嵌(veneering)”的过程)。在本发明中，人源化抗体可以包括“人源化的”和“镶嵌的”抗体。类似的，可以通过将人免疫球蛋白基因座引入转基因动物来产生人抗体，例如，内源性免疫球蛋白基因部分或完全失活的小鼠。基于刺激，观察了人抗体生产，其在所有方面都严格模拟在人中观察的，包括基因重排、聚集和抗体谱。该方法描述在例如美国专利号5,545,807、5,545,806、5,569,825、5,625,126、5,633,425、5,661,016和下列科学出版物中：Marks等人，Bio/Technology 10，779-783(1992)；Lonberg等人，Nature 368 856-859(1994)；Morrison，Nature 368，812-13(1994)；Fishwild等人，Nature Biotechnology 14，845-51(1996)；Neuberger，Nature Biotechnology 14，826(1996)；Lonberg和Huszar，Intern.Rev.Immunol.13 65-93(1995)；Jones等人，Nature 321：522-525(1986)；Morrison等人，Proc.Natl.Acad.Sci，U.S.A.，81：6851-6855(1984)；Morrison和Oi，Adv.Immunol.，44：65-92(1988)；Verhoeyer等人，Science 239：1534-1536(1988)；Padlan，Molec.Immun.28：489-498(1991)；Padlan，Molec.Immunol.31(3)：169-217(1994)；和Kettleborough，C.A.等人，Protein Eng.4(7)：773-83(1991)，其每个通过引用整合到本文中。
在一些实施方案中，本发明的抗体可以作为本发明的多肽的抑制剂/拮抗剂发挥作用。
本发明的抗体可以单独使用或与其它组合物组合使用。抗体还可以在N-或C-末端重组融合异源多肽，或与多肽或其它组合物化学缀合(包括共 价的和非共价的缀合)。例如，本发明的抗体可以重组的融合或缀合用作检测测定中的标记的分子和效应分子，例如异源多肽、药物、放射性核素或毒素。参见例如，PCT公开WO 92/08495；WO 91/14438；WO 89/12624；美国专利号5,314,995；和EP 396,387。
除了嵌合抗体和人源化抗体外，完全人抗体可以来自具有人免疫球蛋白基因的转基因小鼠中(参见例如，美国专利号6,075,181、6,091,001和6,114,598，其通过引用整合到本文中)，或从人免疫球蛋白基因的噬菌体展示文库中(参见例如，McCafferty等人，Nature，348：552_554(1990)。Clackson等人，Nature，352：624_628(1991)，和Marks等人，J.Mol.Biol.，222：581-597(1991))。在一些实施方案中，可以通过scFv-噬菌体展示文库生产和鉴定抗体。抗体噬菌体展示文库技术从商业来源是可获得的，例如Xoma(Berkeley，CA)。
可以利用Kohler等人，(1975)Nature 256：495-496的方法，或其修饰制备单克隆抗体。通常，用含有抗原的溶液免疫小鼠。可以通过在盐水中混合或乳化含抗原溶液来实施免疫，优选的在佐剂中例如弗氏完全佐剂中，并将混合物或乳剂肠胃外注射。可以使用本领域已知的任何免疫方法获得发明的单克隆抗体。在免疫动物后，移除脾脏(任选的，一些大淋巴结)，分离成单细胞。通过将细胞悬液施于用目标抗原包被的平板或孔上，筛选脾细胞。表达针对抗原特异的膜结合免疫球蛋白的B细胞与平板结合，并不会被冲走。然后，将获得的B细胞或所有解离的脾细胞诱导与骨髓瘤细胞融合，形成杂交瘤，并在选择性培养基中培养。通过系列或有限稀释，铺板获得的细胞，测定与目标抗原特异性结合的抗体(且不结合不相关抗原)的生产。然后，在体外(例如，在组织培养瓶或中空纤维反应器中)或体内(在小鼠腹水中)培养选定的分泌单克隆抗体(mAb)的杂交瘤。
作为利用杂交瘤表达的替代方案，可以在细胞系例如CHO或骨髓瘤细胞系中生产抗体，如美国专利号5,545,403；5,545,405和5,998,144中公开的；均通过引用整合到本文中。简而言之，用能够分别表达轻链和重链的载体转染细胞系。通过转染位于不同载体上的两种蛋白质，可以生产嵌合 抗体。Immunol.147：8；Banchereau等人，(1991)Clin.Immunol.Spectrum 3：8；和Banchereau等人，(1991)Science 251：70；均通过引用整合到本文中。
可以利用本领域已知的技术生产人抗体，包括噬菌体展示文库(Hoogenboom和Winter，J.Mol.Biol.，227：381(1991)；Marks等人，C.J.Mol.Biol.，222：581(1991))。Cole等人和Boerner等人的技术也可用于制备人单克隆抗体(Cole等人，Monoclonal Antibodies and Cancer Therapy，Alan R.Liss，第77页(1985)和Boerner等人，J.Immunol.，147(1)：8695(1991))。人源化抗体可以通过多种方法实现，包括例如：(1)将非人互补决定区(CDR)移植到人构架区和恒定区(本领域称为“人源化”的过程)，或可选的(2)移植整个非人可变结构域，但通过替换表面残基用人-类似表面“隐匿(cloaking)”它们(本领域称为“镶嵌(veneering)”的过程)。在本发明中，人源化抗体可以包括“人源化的”和“镶嵌的”抗体。类似的，可以通过将人免疫球蛋白基因座引入转基因动物来产生人抗体，例如，内源性免疫球蛋白基因部分或完全失活的小鼠。基于刺激，观察了人抗体生产，其在所有方面都严格模拟在人中观察的，包括基因重排、聚集和抗体谱。该方法描述在例如美国专利号5,545,807、5,545,806、5,569,825、5,625,126、5,633,425、5,661,016和下列科学出版物中：Marks等人，Bio/Technology 10，779-783(1992)；Lonberg等人，Nature 368 856-859(1994)；Morrison，Nature 368，812-13(1994)；Fishwild等人，Nature Biotechnology 14，845-51(1996)；Neuberger，Nature Biotechnology 14，826(1996)；Lonberg和Huszar，Intern.Rev.Immunol.13 65-93(1995)；Jones等人，Nature 321：522-525(1986)；Morrison等人，Proc.Natl.Acad.Sci，U.S.A.，81：6851-6855(1984)；Morrison和Oi，Adv.Immunol.，44：65-92(1988)；Verhoeyer等人，Science 239：1534-1536(1988)；Padlan，Molec.Immun.28：489-498(1991)；Padlan，Molec.Immunol.31(3)：169-217(1994)；和Kettleborough，C.A.等人，Protein Eng.4(7)：773-83(1991)，其通过引用整合到本文中。利用可商购来源例如Morphosys(Martinsried/Planegg，Germany)，可以在筛选测定中鉴定完全人源化的抗体。
还可以利用改造含有人免疫球蛋白基因座的转基因动物生产人源化抗体。例如，WO98/24893公开了具有人Ig基因座的转基因动物，其中，由于内源重链和轻链基因座的失活，动物不产生功能性的内源免疫球蛋白。WO91/10741还公开了能够对免疫原产生免疫应答的转基因非灵长类哺乳动物宿主，其中抗体具有灵长类恒定区和/或可变区，并且其中编码内源性免疫球蛋白的基因座是取代了或失活的。WO96/30498公开了利用Cre/Lox系统修饰哺乳动物的免疫球蛋白基因座，例如替换全部或部分的恒定区或可变区，形成修饰的抗体分子。WO94/02602公开了具有失活的内源性Ig基因座和功能性人Ig基因座的非人哺乳动物宿主。美国专利号5,939,598公开了制造转基因小鼠的方法，其中小鼠缺失了内源性重链，表达包含一个或多个异种恒定区的外源免疫球蛋白基因座。还可以利用美国专利5,766,886中公开的人改造技术生产本发明的抗体，其通过引用整合到本文中。
利用上述转基因动物，可以产生针对选定抗原分子的免疫应答，并且可以从动物移除抗体生产细胞，并将其用于生产分泌人单克隆抗体的杂交瘤。免疫规程，佐剂等是本领域已知的，用于免疫例如WO96/33735中描述的转基因小鼠。可以测试单克隆抗体抑制或中和相应蛋白质的生物学活性或生理学效应的能力。
通过Fang等人，(2005)，Nat.Biotechnol.23，584-590讨论的体内治疗性抗体基因转移，可以向受试者施用本发明的抗体。例如，可以产生重组载体来递送多顺反子表达盒，所述表达盒包含介导对多肽进行不依赖酶的、共翻译自体切割的肽，所述多肽位于MAb重链和轻链编码序列之间。表达产生化学计量的量的两种MAb链。介导不依赖酶的、共翻译自体切割的肽的优选实例是口蹄疫衍生的2A肽。
在一些实施方案中，抗体片段保留了全长抗体的理想亲和力。因此，在一些实施方案中，抗-VGSC β3抗体的片段保留了结合VGSC β3的能力。此类片段的特征是与相应全长抗VGSC β3抗体相似的性质，即所述片段特异性结合在人细胞表面表达的人VGSC β3抗原。
在一些实施方案中，抗体结合VGSC β3的一个或多个表位。在一些实施方案中，抗体调节一种或多种VGSC β3-相关的生物学活性。在一些实施方案中，抗体抑制一种或多种癌细胞生长、肿瘤形成和癌细胞增殖。
II-3 反义寡核苷酸
根据本发明的一个或多个实施方案，反义疗法是用于遗传紊乱或感染的一种治疗形式。当已知特定基因的遗传序列为特定疾病的病因时，可以合成与该基因产生的信使RNA(mRNA)结合并使之失活的核酸链(DNA、RNA或化学类似物)，从而“关闭”该基因。这是因为mRNA为进行翻译必需是单链。这种合成的核酸称为“反义”寡核苷酸，因为其碱基序列互补于基因的信使RNA(mRNA)，后者称为“有义”序列(这样，有义mRNA区段“5’-AAGGUC-3’”可以被反义mRNA区段“3’-UUCCAG-5’”封闭)。
在一些实施方案中，寡核苷酸是与VGSC β3基因或基因产物的区域、结构域、部分或片段互补的。在一些实施方案中，寡核苷酸包括约5至约100个核苷酸，约10至约50个核苷酸，约12至约35个核苷酸，和约18至约25个核苷酸。在一些实施方案中，寡核苷酸与VGSC β3基因或基因产物的区域、部分、结构域、或片段至少50％、至少75％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％，或100％同源。在一些实施方案中，在VGSC B3基因或基因产物的至少15、20、25、30、35、40、50或100个连续核苷酸上具有基本或完全的序列同源性。在一些实施方案中，在完整长度的VGSC β3基因或基因产物上具有基本或完全的序列同源性。在一些实施方案中，寡核苷酸在中度或严格杂交条件下与具有SEQ ID NO：2的核苷酸序列的核酸分子杂交。
II-4 模拟物
在一些实施方案中，VGSC β3抑制剂是模拟物。如本文中使用的，术语“模拟物”指模拟肽的活性的化合物。模拟物不是肽，但可以包含通过非肽键连接的氨基酸。1997年6月10日申请的美国专利号5,637,677，及其母申请，均通过引用整合到本文中，包括对模拟物生产的详细指导。简而 言之，通过不是肽的分子复制肽的三维结构，所述结构与VGSC β3的三维结构特异性相互作用。
II-5 小分子
在一些实施方案中，VGSC β3抑制剂是小分子。如本文中使用的，术语“小分子”指分子量小于约10千道尔顿的有机或无机非聚合化合物。小分子的实例包括肽、寡核苷酸、有机化合物、无机化合物等。在一些实施方案中，小分子具有小于约9、约8、约7、约6、约5、约4、约3、约2或约1千道尔顿的分子量。
在一些实施方案中，VGSC β3抑制剂是(多)肽。在优选的实施方案中，肽为镇痛抗肿瘤缬精甘肽(AGAP)。在本文中，镇痛抗肿瘤缬精甘肽是指从东亚钳蝎蝎毒中分离纯化得到的活性肽，其基因可以从东亚钳蝎中克隆得到，在GENBANK中的基因序列号为AF464898。
在一个实施方案中，将编码AGAP的基因克隆到大肠杆菌表达载体中，转化大肠杆菌细胞，构建重组菌，实现活性肽在大肠杆菌中表达，经层析纯化后获得重组镇痛抗肿瘤缬精甘肽。体内实验显示活性肽既具有抗肿瘤活性，又具有镇痛活性(Liu YF1，Ma RL，Wang SL，Duan ZY，Zhang JH，Wu LJ.Wu CF.Expression of an antitumor-analgesic peptide from the venom of Chinese scorpion Buthus martensii karsch in Escherichia coli.Protein Expr Purif.2003 Feb；27(2)：253-8；Qinghong Mao，1 Jiaping Ruan，1，* Xueting Cai，1，2 Wuguang Lu，1，2 Juan Ye，1，2 Jie Yang，1，2 Yang Yang，1，2 Xiaoyan Sun，1，2 Junli Cao，3 and Peng Cao1，2，*Published online Nov 14，2013.doi：10.1371/journal.pone.0078239PMCID：PMC3828337Antinociceptive Effects of Analgesic-Antitumor Peptide(AGAP)，a Neurotoxin from the Scorpion Buthus martensii Karsch，on Formalin-Induced Inflammatory Pain through a Mitogen-Activated Protein Kinases-Dependent Mechanism in Mice PLoS One.2013；8(11)：e78239)。体外实验显示活性肽对人恶性胶质瘤细胞SHG-44的增殖和迁移具有抑制作用(Zhao Y，Cai X，Ye T，Huo J，Liu C，等人(2011)Analgesic-antitumor peptide inhibits proliferation and migration of  SHG-44 human malignant glioma cells.J Cell Biochem 112：2424-2434)。
III.肿瘤
如本文所用的“肿瘤”是指由于过度细胞生长或增殖而产生的任何组织块，或良性(非癌变)或恶性的(癌变)，包括癌前病变。
如本文中使用的，“癌症”指原发性或转移性癌症、白血病或淋巴瘤。术语“癌细胞”指转化的细胞。可以从患有癌症的患者分离这些细胞，或者这些细胞是在体外转化成为癌性的细胞。癌细胞可以源自多种类型的样品，包括任何组织或细胞培养系。在一些实施方案中，癌细胞是超常增生、肿瘤细胞或赘生物。在一些实施方案中，癌细胞分离自肝癌、肺癌、结肠癌、睾丸癌、胸腺癌、乳腺癌、皮肤癌、食管癌、胰腺癌、前列腺癌、子宫癌、宫颈癌、膀胱癌、卵巢癌、多发性骨髓瘤和黑色素瘤。在一些实施方案中，癌细胞取自公众可获得的已经存在的细胞系。在一些实施方案中，癌细胞分离自已存在的患者样品，或分离自包含癌细胞的文库。在一些实施方案中，癌细胞是分离的，然后植入不同的宿主内，例如异种移植。在一些实施方案中，癌细胞是移植的，并用于SCID小鼠模型。在一些实施方案中，癌症是肝癌、肺癌或白血病。
通过本发明的方法和组合物可以治疗的肿瘤，特别是癌症的特定实例包括但不限于，VGSC β3-相关肿瘤，特别是VGSC β3-相关癌症。如本文中使用的，“VGSC β3-相关肿瘤”是指任何涉及VGSC β3的水平、表达和/或活性的肿瘤，和/或任何可以通过调节(特别是抑制)VGSC β3的水平、表达和/或活性治疗和/或缓解的肿瘤。具体地，“VGSC β3-相关癌症”指癌症的特征是相对于非癌性细胞，细胞差异(特别是增加)的表达VGSC β3。本发明还可用于任何肿瘤细胞类型，其中VGSC β3在癌细胞生长等中发挥作用。在一些实施方案中，癌症是白血病、结肠癌、肝癌、睾丸癌、胸腺癌、乳腺癌、皮肤癌、食管癌、胰腺癌、前列腺癌、子宫癌、宫颈癌、肺癌、膀胱癌、卵巢癌、多发性骨髓瘤和黑色素瘤。在一些实施方案中，癌症是肝癌、白血病或肺癌。在一些实施方案中，相对于对照， 此类癌症表现出至少约25％、至少约50％、至少约75％、至少约100％、至少约150％、至少约200％或至少约300％的VGSC β3的差异表达(特别是增加的表达)。
VI.药物组合物
如本文中使用的，“药物组合物”包括药学上有效量的一种或多种VGSC β3抑制剂、可药用载体和，任选的与VGSC β3抑制剂协同作用的其他肿瘤治疗剂。如本文中使用的，“药学上有效量”、“治疗有效量”或简单的“有效量”指VGSC β3抑制剂的量有效的产生预期的药理学、治疗或预防结果。例如，如果与疾病或病症相关的可测量参数存在至少10％的降低，则给定的临床治疗被认为是有效的，用于治疗所述疾病或病症的药物的治疗有效量是使所述参数产生至少10％的降低所必需的量。在该实施方案中，治疗有效量的靶向VGSC β3的VGSC β3抑制剂可以降低VGSC β3蛋白质水平至少10％。在其他实施方案中，当与疾病或病症相关的可测量参数存在至少15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90或95％的降低，则给定的临床治疗被认为是有效的，用于治疗所述疾病或病症的药物的治疗有效量是分别使所述参数产生至少15、20、25、30、35、40、45、50、55、60、65、70、75、80、85、90或95％的降低所必需的量。
术语“可药用载体”指的是施用治疗活性剂的载体。此类载体包括但不限于盐水、缓冲盐水、葡萄糖、水、丙三醇、乙醇、和它们的组合。术语明确排除细胞培养基。对于经口施用的药物，可药用载体包括但不限于可药用赋形剂，例如惰性稀释剂、崩解剂、粘合剂、润滑剂、甜味剂、调味剂、着色剂和防腐剂。适当的惰性稀释剂包括钠和钙的碳酸盐、钠和钙的磷酸盐、以及乳糖，而玉米淀粉和褐藻酸则是适当的崩解剂。粘合剂可以包括淀粉和明胶，而润滑剂(如果存在的话)通常为硬脂酸镁、硬脂酸或滑石粉。如果需要的话，片剂可以用例如单硬脂酸甘油酯或二硬脂酸甘油酯的材料包衣来延迟在胃肠道的吸收。药物制剂中包括的活性剂也在本文 中被描述。
包含VGSC β3抑制剂的药物组合物可以是固体形式的，例如粉末、颗粒、片剂、丸剂、软胶囊、胶囊、脂质体、栓剂、咀嚼形式或贴剂。包含VGSC β3抑制剂的药物组合物还可呈现为液体形式，例如溶液、乳液、悬浮液、酏剂或糖浆。合适的液体支持物可以是例如水、有机溶剂(例如多元醇，例如甘油或乙二醇，包括丙二醇和聚乙二醇)或乙醇、聚氧乙烯蓖麻油(Cremophor EL)或其混合物(以多种比例，在水中)。组合物可包含纳米尺寸的无定型或晶体颗粒，其被铝或表面活性剂涂覆。
合适的支持物可包括例如，抗细菌和抗真菌剂，缓冲剂，磷酸钙，纤维素，甲基纤维素，氯丁醇(chlorobutanol)，可可脂(cocoa butter)，着色剂，糊精，乳化剂，肠溶衣(enteric coatings)，调味剂，明胶，等张剂，卵磷脂，硬脂酸镁，芳香剂，多元醇例如甘露醇，可注射的有机酯类，例如油酸乙酯，对羟基苯甲酸酯(paraben)，山梨酸苯酚(phenol sorbic acid)，聚乙二醇，聚乙烯吡咯烷(polyvinylpyrrolidine)，磷酸缓冲盐溶液(PBS)，防腐剂，丙二醇，羧甲基纤维素钠，氯化钠，山梨糖醇，多种糖(包括但不限于蔗糖、果糖、半乳糖、乳糖和海藻糖)，淀粉，栓剂蜡(suppository wax)，滑石，植物油，例如橄榄油和玉米油，维生素，蜡和/或润湿剂。对于VGSC β3抑制剂，优选的支持物包含右旋糖酐(dextran)和水，例如，水中5％右旋糖(D5W)。
药物组合物的生物惰性部分可任选地是易蚀的，以允许VGSC β3抑制剂随时间释放。
药物组合物可以包括其他组分，所述组分有助于递送、稳定性、功效或降低免疫原性。
VI-1.VGSC β3抑制剂的施用
包含VGSC β3抑制剂的药物组合物可通过颊、吸入(包括吹入或深度吸入)、鼻、口服、肠胃外、植入、注射或输注(经由硬膜外、动脉内、关节内、囊内、心内、脑室内、颅内、皮内、肌内、眶内、腹膜内、脊柱 内、胸骨内、鞘内、静脉内、蛛网膜下、被膜下、皮下、表皮下、经内皮、经气管、经血管、直肠、舌下、局部和/或阴道途径)施用。这可通过注射、输注、皮肤贴片或本领域已知的任何其它方法来进行。制剂可以是粉末化的、雾化的、气溶胶化的、颗粒化的或者经合适制备用于递送的。如果是液体的话，施用可以缓慢进行，或者通过推注进行，虽然在本领域已知的一些情况下，推注注射可能导致材料经肾脏损失。
可用本领域已知的医学设备来施用包含VGSC β3抑制剂的药物组合物。例如，在特定的实施方式中，可用无针头皮下注射设备，例如美国专利Nos.5,399,163、5,383,851、5,312,335、5,064,413、4,941,880、4,790,824或4,596,556中公开的设备，来施用VGSC β3抑制剂。可用于本文公开内容中的公知植入体和模块的例子包括：美国专利No.4,487,603，其公开了可植入的微输注泵，用于以受控速率来分配药剂；美国专利No.4.,486,194，其公开了用于经由皮肤施用药剂的治疗设备；美国专利No.4,447,233，其公开了用于以精确输注速率递送药剂的药剂输注泵；美国专利No.4,447,224，其公开了用于连续药物递送的可变流速可植入输注装置；美国专利No.4,439,196，其公开了具有多腔区室的渗透性药物递送系统；以及美国专利No.4,475,196，其公开了渗透性药物递送系统。很多其它此类植入体、递送系统和模块是本领域技术人员已知的。
在某些实施方案中，可以配制包含VGSC β3抑制剂的药物组合物以保证在体内的正确分布。施用针对VGSC β3的VGSC β3抑制剂可以是全身的(全身)或特别是靶向表达(或过表达或展示出活性过高的)VGSC β3的组织或器官，例如肺、肾、结肠和腺体。靶向这些特定组织或器官的方法描述在本文中，和/或是本领域已知的。例如，可以将它们配制在脂质体中。就制造脂质体的方法，参见例如美国专利4,522,811；5,374,548和5,399,331。脂质体可包含被选择性运输进特别的细胞或器官的一种或多种部分，由此增强靶向药物递送(见例如V.V.Ranade(1989)J.Clin.Pharmacol.29：685)。
示例性的靶向部分包括叶酸或生物素(见例如Low等人的美国专利 5,416,016)；甘露糖苷(Umezawa等人.，(1988)Biochem.Biophys.Res.Commun.153：1038)；抗生素(P.G.Bloeman等人(1995)FEBS Lett.357：140；M.Owais等人(1995)Antimicrob.Agents Chemother.39：180)；表面活性剂蛋白A受体(Briscoe等人(1995)Am.J.Physiol.1233：134)，其不同种类可包含本文公开内容的制剂以及所发明的分子的组分；p120(Schreier等人(1994)J.Biol.Chem.269：9090)；还见K.Keinanen；M.L.Laukkanen(1994)FEBS Lett.346：123；J.J.Killion；I.J.Fidler(1994)Immunomethods 4：273。
因此，本公开内容涵盖了包含一种或多种针对VGSC β3的VGSC β3抑制剂的药物组合物，其可以任选的包含多种修饰和/或其他组分，用于治疗VGSC β3-相关性疾病。
VI-2 VGSC β3抑制剂的剂量和有效量
向需要的患者施用治疗有效量的剂量的本公开内容的VGSC β3抑制剂。
“有效量”或“治疗有效量”是治疗个体的疾病或医学状况的量，或者更一般性地，向个体提供营养、生理或医学益处的量。在本文中使用时，短语“治疗有效量”和“预防有效量”表示在对VGSC β3表达所介导的病理过程或VGSC β3表达所介导的病理过程的明显症状的治疗、预防或管理中提供治疗性益处的量。具体的治疗有效的量可容易地被普通执业医师确定，并且可取决于本领域已知的因素而变动，所述因素例如VGSC β3表达所介导的病理性过程的类型、患者的病史和年龄、β-ENaC表达所介导的病理性过程的阶段以及抑制VGSC β3表达介导的病理性过程的其它活性剂的施用。
在本文公开内容的多种实施方式中，患者年龄为至少大约1、3、6或9个月，或1、5、10、20、30、40、50、55、60、65、70或75岁。在多种实施方式中，患者年龄不超过大约1、3、6或9个月，或1、5、10、20、30、40、50、55、60、65、70、75、80、90或100岁。在多种实施方式中，患者具有至少大约5、10、15、20、30、40、50、60、70、80、90、100、120、140、160、180、200、220、240、260、280、300、320、340、360、380 或400lbs的体重。在多种实施方式中，患者具有不超过大约5、10、15、20、30、40、50、60、70、80、90、100、120、140、160、180、200、220、240、260、280、300、320、340、360、380或400lbs的体重。
在本文公开内容的多种实施方式中，剂量[仅测量活性成分]可以是至少大约1、5、10、25、50、100、200、250、300、250、400、450、500、550、600、650、700、750、800、850、900、950或1000ng，1、5、10、25、50、100、200、250、300、250、400、450、500、550、600、650、700、750、800、850、900、950或1000微克，1、5、10、25、50、100、200、250、300、250、400、450、500、550、600、650、700、750、800、850、900、950或1000mg。在多种实施方式中，剂量可以不超过大约10、25、50、100、200、250、300、250、400、450、500、550、600、650、700、750、800、850、900、950或1000mg。在多种实施方式中，剂量可以至少每天不止一次，每日、每周不止一次、每周、隔周、每月和/或每2、3、4、5、6、7、8、9、10、11或12个月施用，或其组合施用。
在多种实施方式中，剂量与个体的体重或体表面积相关。实际剂量水平可有所变动，以获得对于特定患者、组合物和施用模式有效但对患者无毒性的有活性的活性剂的量。所选择的剂量将取决于多种药物动力学因素，包括所用的特定VGSC β3抑制剂的活性，施用途径，VGSC β3抑制剂排出速率，治疗持续期，与VGSC β3抑制剂组合使用的其它药物、化合物和/或材料，患者的年龄、性别、体重、状况、一般健康和之前医疗史，以及医学领域公知的类似因素。具有本领域普通技术的医师或兽医可容易地确定需要的VGSC β3抑制剂的有效量。合适的剂量将是这样的量，其实对于产生治疗性效果来说有效的最低剂量，或者足够低到能产生治疗性效果但是不导致副作用的剂量。
IV 预防/治疗肿瘤(特别是癌症)
对于预防和/或治疗与VGSC β3相关的肿瘤，例如癌症，本发明提供了方法，包括其他活性成分与本发明的VGSC β3抑制剂组合。在一些实 施方案中，方法还包括向患者施用一种或多种常规癌症治疗剂。在一些实施方案中，本发明的方法还包括用一种或多种化疗、放射治疗、激素消融或手术来治疗患者。
本发明还提供用于治疗、抑制和管理癌症或其它肿瘤的方法和组合物，所述癌症或肿瘤对于现有或标准的癌症治疗，例如手术、化疗、放射治疗、激素疗法和生物学疗法，已经部分或完全抗拒。
本发明提供了用于治疗和/或预防受试者的肿瘤或癌症或癌症的症状的方法，包括向受试者施用治疗有效量的一种或多种本发明的VGSC β3抑制剂。在一些实施方案中，癌症是与VGSC β3相关的癌症。在一些实施方案中，癌症是白血病、结肠癌、肝癌、睾丸癌、胸腺癌、乳腺癌、皮肤癌、食管癌、胰腺癌、前列腺癌、子宫癌、宫颈癌、肺癌、膀胱癌、卵巢癌、多发性骨髓瘤或黑色素瘤。在一些实施方案中，癌症是位于非激素调节的组织内。在一些实施方案中，已经诊断受试者患有癌症或对癌症易感。在一些实施方案中，癌症是肝癌、肺癌或白血病。
癌症的症状是本领域技术人员普遍已知的，包括但不限于体重减轻、贫血、腹痛、肠梗阻、便血、腹泻、便秘、肠习惯的其它改变、结肠转移、死亡、虚弱、过度疲劳、进食困难、丧失食欲、慢性咳嗽、呼吸困难恶化、咳血、尿血、恶心、呕吐、肝转移、肺转移、骨转移、肚胀、胃气胀、腹腔积液、阴道出血、腹胀、结肠穿孔、急性腹膜炎(感染、发热、疼痛)、疼痛、呕血、重度出汗、发热、高血压、黄疸、晕眩、寒冷、肌痉挛、肺转移、膀胱转移、肝转移、骨转移、肾转移和胰腺转移，吞咽困难等。
根据药物化学师普遍已知的程序，并且根据患者的年龄、病况的严重程度和理想的最终药物制剂等，可以经验地确定调节化合物的治疗有效量。如上文讨论的，本发明的治疗组合物还可以作为组合疗法的一部分，与其它已知的抗癌剂或其他已知的抗骨疾病治疗方案一起施用。
本发明还提供了在需要其的患者中抑制癌细胞生长的方法，包括向患者施用治疗有效量的一种或多种VGSC β3抑制剂。用于测量VGSC β3-相关细胞生长的合适测定是本领域技术人员已知的，并在上下文中提出。
本发明还提供了在需要其的患者中抑制癌症的方法。方法包括向患者施用治疗有效量的一种或多种VGSC β3抑制剂。
本发明还提供了在诊断或怀疑患有癌症的患者中抑制癌症的方法。方法包括向患者施用治疗有效量的一种或多种VGSC β3抑制剂。
本发明还提供了调节患者的一种或多种癌症症状的方法。方法包括向所述患者施用治疗有效量的本文描述的VGSC β3组合物。
本发明还提供了用于在需要其的患者内抑制细胞生长的方法，包括向所述患者施用治疗有效量的VGSC β3抑制剂。
本发明还提供了用于在需要其的患者内抑制癌细胞迁移的方法，包括向患者施用治疗有效量的VGSC β3抑制剂。
本发明还提供了用于在需要其的患者内抑制癌细胞附着的方法，包括向患者施用治疗有效量的VGSC β3抑制剂。
本发明还提供了预防性治疗患者的方法，所述患者易于发展癌症、癌症转移或者其已经具有转移从而因此易于复发或再现。方法特别用于高风险个体，其例如具有癌症或转移性肿瘤的家族史，或表现出对癌症转移的遗传素质。在一些实施方案中，肿瘤是VGSC β3-相关肿瘤。此外，方法用于预防患者的VGSC β3-相关肿瘤复发，所述患者曾具有通过手术切除术移除的或用常规癌症治疗治疗过的VGSC β3-相关肿瘤。
本发明还提供了抑制癌症进展和/或引起癌症消退的方法，包括向患者施用治疗有效量的VGSC β3抑制剂。
在一些实施方案中，用本发明的VGSC β3抑制剂联合化疗和/或放射疗法治疗需要抗癌治疗的患者。例如，在施用VGSC β3抑制剂后，还可以用治疗有效量的抗癌放射治疗患者。在一些实施方案中，组合提供化疗治疗和VGSC β3抑制剂。在一些实施方案中，组合施用VGSC β3抑制剂和化疗以及放射疗法。
治疗方法包括向患者施用单次或多次剂量的一种或多种VGSC β3抑制剂。在一些实施方案中，VGSC β3抑制剂作为可注射的药物组合物施用，所述组合物是无菌的、无致热源，并包含与可药用载体或稀释剂组合的 VGSC β3抑制剂。
在一些实施方案中，本发明的治疗方案与癌症的常规治疗方案一起使用，包括但不限于手术、放射性疗法、激素消融和/或化疗。可以在常规癌症治疗之前、同时或之后进行本发明的VGSC β3抑制剂的施用。在一些实施方案中，向患者施用两种或多种不同的VGSC β3抑制剂。
在一些实施方案中，向患者施用的VGSC β3抑制剂的量有效的抑制中立细胞生长、癌细胞生长、肿瘤形成、癌细胞增殖、癌细胞转移以及VGSC β3表达中的一种或多种。在一些实施方案中，向患者施用的VGSC β3抑制剂的量通过凋亡，有效的增加癌细胞死亡。
V.组合疗法
在一种实施方式中，本文公开内容的VGSC β3的抑制剂与一种或多种适于治疗肿瘤，特别是癌症的额外药物活性剂一起被施用给需要其的患者。例如，遭受癌症的患者可被施用药理有效量的一种或多种VGSC β3的抑制剂与药理有效量的一种或多种本文列出的任何癌症治疗和/或本领域已知的任何其它癌症治疗。
在治疗肿瘤，特别是癌症的情况下，一种或多种VGSC β3的抑制剂和一种或多种其它肿瘤治疗剂可以以任何顺序、同时或依序、或以多个剂量随时间被施用。VGSC β3的抑制剂和其它治疗剂的施用可例如是同时、并行、分开或依序的。
同时施用可例如以两种或多种活性成分的一种固定组合的形式进行，或者可通过同时施用独立配制的两种或多种活性成分来进行。依序使用(施用)优选表示在一个时间点施用组合中的一种(或多种)组分，在不同的时间点施用其它组分，即，以长期交错方式，优选使得组合显示出比单种化合物独立施用更高的效率(尤其是显示出协同作用)。分开使用(施用)优选表示在不同时间点相互独立地施用组合的组分，优选表示：组分(a)和(b)被这样施用，使得两者化合物可测量到的血液水平的重叠没有以重叠方式(同时)存在。
此外，依序、分开和同时施用中的两种或多种的组合是可能的，优选使得组合组分-药物显示出超过组合组分-药物以时间间隔独立使用时发现的效果的联合治疗效果，超过程度大到不能发现对它们治疗效率的相互影响，协同效果是尤其优选的。
“联合治疗活性”或“联合治疗效果”表示化合物可以被以使得它们优选在待被治疗的温血动物(尤其是人)中仍显示出(优选协同的)相互作用(联合治疗效果)的时间间隔分开给予(以长期交错的方式，尤其是顺序特异性方式)。可通过监视血液水平(显示两者化合物至少在某时间间隔期间均存在于待被治疗的人的血液中)等等，来确定是否是这种情况。
在一些实施方案中，常规的癌症治疗剂与本发明的组合物施用。常规的癌症治疗剂包括：
a)癌症化疗剂；
b)其它活性剂；
c)前体药物。
癌症化疗剂包括但不限于，烷化剂例如卡铂和顺铂；氮芥烷化剂；亚硝基脲烷化剂例如卡莫司汀(BCNU)；抗代谢物例如甲氨喋呤；甲酰四氢叶酸；嘌呤类似物抗代谢物，巯嘌呤；嘧啶类似物抗代谢物，例如氟尿嘧啶(5-FU)和吉西他滨
Figure PCTCN2015079356-appb-000016
激素抗肿瘤药，例如戈舍瑞林、亮丙瑞林和他莫昔芬；天然抗肿瘤药，例如阿地白介素、白介素-2、多西他赛、依托泊苷(VP-16)、干扰素α、紫杉醇
Figure PCTCN2015079356-appb-000017
和维甲酸(ATRA)；抗生素天然抗肿瘤药，例如博来霉素、更生霉素、柔红霉素(Daunorubicin)、多柔比星、道诺霉素和丝裂霉素(包括丝裂霉素C)；以及长春花生物碱天然抗肿瘤药，例如长春碱、长春新碱、长春地辛；羟基脲；醋葡醛内酯；亚德里亚霉素(adriamycin)、异环磷酰胺、依诺他滨、环硫雄醇、阿柔比星、安西他滨、尼莫司汀、盐酸丙卡巴肼、卡波醌、卡铂、卡莫氟、色霉素A3、抗肿瘤多糖、抗肿瘤血小板因子、环磷酰胺
Figure PCTCN2015079356-appb-000018
施佐菲兰、阿糖胞苷(胞嘧啶阿拉伯糖苷)、达卡巴嗪、硫代肌苷、塞替派、替加氟、多拉司他汀、多拉司他汀类似物例如auristatin、CPT-11(依立替康)、 米托蒽醌、长春瑞滨、替尼泊苷、氨基蝶呤、洋红霉素、埃斯波霉素(参见例如，美国专利号4,675,187)、新制癌菌素、OK-432、博来霉素、氟铁龙、溴尿苷、白消安、二磷酸己烯雌酚四钠、培洛霉素、苯丁抑制素
Figure PCTCN2015079356-appb-000019
干扰素-β、美雄烷、二溴甘露醇、美法仑、层粘连蛋白肽、香菇多糖、采绒革盖菌(Coriolus versicolor)提取物、替加氟/尿嘧啶、雌莫司汀(雌激素/氮芥)。
可以用作癌症患者治疗的其它活性剂包括EPO、G-CSF、更昔洛韦；抗生素、亮丙瑞林；哌替啶；齐多夫定(AZT)；白介素1到18，包括变体和类似物；干扰素或细胞因子，例如干扰素α、β和γ；激素，例如黄体生成素释放激素(LHRH)和类似物，以及促性腺素释放素(GnRH)；生长因子，例如转化生长因子-β(TGF-β)、成纤维细胞生长因子(FGF)、神经生长因子(NGF)、生长激素释放因子(GHRF)、表皮生长因子(EGF)、成纤维细胞生长因子同源因子(FGFHF)、肝细胞生长因子(HGF)和胰岛素生长因子(IGF)；肿瘤坏死因子-α&β(TNF-α&β)；侵入抑制因子-2(IIF-2)；骨形态发生蛋白1-7(BMP 1-7)；生长抑素；胸腺素-α-1；γ-球蛋白；超氧化物歧化酶(SOD)；补体因子；抗血管发生因子；抗原物质；和前体药物。
“前体药物”指药学活性物质的前体或衍生形式，其与母体药物相比，对肿瘤细胞具有更低的细胞毒性或非细胞毒性，并能够被酶促活化或转变为活化的或更活化的母体形式。参见例如，Wilman，″Prodrugs in Cancer Chemotherapy″Biochemical Society Transactions，14，第375-382页，615th Meeting Belfast(1986)和Stella等人，″Prodrugs：A Chemical Approach to Targeted Drug Delivery，″Directed Drug Delivery，Borchardt等人(编著)，第247-267页，Humana Press(1985)。前体药物包括但不限于，含磷酸盐/酯的前体药物、含硫代磷酸盐/酯的前体药物、含有硫酸盐/酯的前体药物、含有肽的前体药物、D-氨基酸修饰的前体药物、糖基化的前体药物、含有β-内酰胺的前体药物、含有任选取代的苯氧乙酰胺的前体药物或含有任选取代的苯乙酰胺的前体药物、5-氟胞嘧啶或其他5-氟尿苷的前体药物(其 可以转变为更活化的细胞毒性游离药物)。本文中使用的可以衍生成前体药物形式的细胞毒性药物的实例包括但不限于上述化疗剂。
VII.诊断/检测肿瘤
本发明还提供了利用发明的VGSC β3诊断对象中肿瘤或癌症的方法，来诊断肿瘤，例如癌症和/或预测肿瘤，例如癌症进展。所述方法包括：
从对象中获得样品或待测细胞；
利用检测VGSC β3 DNA、mRNA或蛋白质的工具检测样品或待测细胞和对照正常细胞中的VGSC β3的蛋白质或基因含量；
比较测量的结果，待测样品或细胞中VGSC β3的蛋白质或基因含量比对照正常高表明对象患有肿瘤。
本发明还提供了利用本发明的VGSC β3诊断对象中的VGSC β3相关肿瘤(例如癌症)的方法，所述方法包括
从对象中获得待测的样品或肿瘤细胞；
利用检测VGSC β3 DNA、mRNA或蛋白质的工具检测待测的肿瘤细胞和对照正常细胞中的VGSC β3的蛋白质或基因含量；
比较测量的结果，待测肿瘤细胞中VGSC β3的蛋白质或基因含量比对照正常细胞高表明对象患有VGSC β3相关肿瘤(例如癌症)。
在一个实施方案中，其中测量编码DNA水平或mRNA水平或蛋白质水平的工具包括能够用于测量VGSC β3亚基的编码DNA水平或mRNA水平的探针或引物，或者能够检测VGSC β3蛋白质表达和活性的可检测的抗体、小分子、寡核苷酸、诱饵、模拟物或探针。
在一个实施方案中，所述样品是怀疑患有肿瘤的组织的样品，优选肿瘤活检或其细胞提取物。
在一些实施方式中，细胞提取物包含实体瘤的循环细胞的提取物。通常采用本领域已知的一种或多种分离方法从患者样品分离循环细胞，所述方法包括，例如免疫磁性分离、CellTrackTM系统、微流体分离、FACS、密度梯度离心和消除法。
在其它实施方式中，患者样品包括全血、血清、血浆、痰、支气管灌 洗液、尿液、乳头抽吸物、淋巴、唾液和/或细针抽吸物样品。在某些情况中，将全血样品分成血浆或血清组分和细胞组分(即，细胞沉淀)。细胞组分通常含有红细胞、白细胞和/或实体瘤的循环细胞，例如CTC、CEC、CEPC、淋巴结的扩散性肿瘤细胞和/或CSC和它们的组合。血浆或血清组分通常含有实体瘤的循环细胞所释放的核酸(例如，DNA、RNA)和蛋白质。
VIII.试剂盒
在一些实施方案中，本发明提供了用于成像和/或检测与VGSC β3过表达相关的基因或基因产物，或者用于测量VGSC β3亚基的编码DNA水平或mRNA水平或蛋白质表达水平的工具的工具，以及包含所述工具的试剂盒。
本发明的工具可包括可检测的抗体、小分子、寡核苷酸、诱饵、模拟物或探针。本发明的试剂盒包含所述工具以及用于实施发明的方法的说明书。任选的，试剂盒还可以包含一种或多种下述：对照(阳性和/或阴性)、对照的容器、阳性和/或阴性结果的代表性实例的照片或描述。
VIII 筛选的方法
本发明还提供了利用VGSC β3为靶点，设计和制备用于在对象中预防或治疗肿瘤的活性物质的方法，所述方法包括，根据VGSC β3蛋白质的氨基酸序列以及核酸编码序列，设计能够抑制VGSC β3蛋白质的表达或活性的抗体、小分子抑制剂、(多)肽和核酸、反义寡核苷酸、或模拟物。所述方法还包括制备上述设计出的活性物质并且确定其是否抑制了VGSC β3的表达、活性和/或相关生物学活性的步骤。在一些实施方案中，对癌细胞生长等一种或多种癌细胞指标的抑制指示了上述活性物质。本发明还提供了将制备出的上述活性物质配制称为药物组合物。
本发明还提供了筛选用于在对象中预防或治疗肿瘤的活性物质的方法，所述方法包括将表达VGSC β3的细胞与候选活性物质接触，确定是否抑制了VGSC β3的表达、活性和/或相关生物学活性的步骤。在一些实施方案中，对癌细胞生长等一种或多种癌细胞指标的抑制指示了上述活性 物质。本发明还提供了将制备出的上述活性物质配制称为药物组合物。
本发明还提供了用于筛选抗癌剂或抑制肿瘤细胞增殖的抑制剂的方法。方法包括将表达VGSC β3的细胞与候选化合物接触，确定是否调节了VGSC β3的表达、活性和/或相关生物学活性。在一些实施方案中，对癌细胞生长等一种或多种癌细胞指标的抑制指示了抗癌剂。
IX.定义
除上文所涉及的定义外，本公开内容还涉及如下定义：
如本文中使用的，术语“约”指+/-20％，+/-10％，或+/-5％的值。
术语“多肽”或“蛋白质”是可互换的使用的，指任何长度的氨基酸聚合体形式，可以包括编码的和非编码的氨基酸，化学或生物化学修饰或衍生的氨基酸，以及具有修饰的肽主链的多肽。术语包括融合蛋白，包括但不限于，具有异源性氨基酸序列的融合蛋白，具有异源和同源前导序列的融合体(具有或缺少N-末端甲硫氨酸残基)；免疫标记的蛋白质；等。
术语“个体”、“对象”、“宿主”和“患者”是可互换的使用的，指需要诊断、处理或治疗的任何对象，特别是人。其它对象可以包括牛、狗、猫、豚鼠、兔、大鼠、小鼠、马等。在一些优选的实施方案中，对象是人。
如本文中使用的，术语“样品”指来自患者的生物学材料。本发明测定的样品不限于任何特定的类型。样品包括(作为非限制性实例)单细胞、多细胞、组织、肿瘤、生物学液体、生物学分子或任何上述的提取物或上清液。实例包括移除用于活组织检查的组织、在切除过程中移除的组织、血液、尿、淋巴组织、淋巴液、脑脊液、黏液和粪便样品。根据测定方式、检测方法和待测定的肿瘤、组织、细胞或提取物的性质，可以改变使用的样品。用于制备样品的方法是本领域普遍已知的，为了获得与所使用的方法相容的样品可以方便的调整。
如本文中使用的，术语“生物学分子”包括但不限于多肽、核酸和糖类。
“基因产物”是通过基因表达或产生的生物聚合产物。基因产物可以是例如未剪接的RNA、mRNA、剪接的变体mRNA、多肽、翻译后修饰的多肽、剪接变体多肽等。该术语还涵盖了利用RNA基因产物(即，RNA的cDNA)作为模板产生的生物聚合产物。基因产物可以是酶促的、重组的、化学的产生的，或在基因的天然细胞内产生的。在一些实施方案中，如果基因产物是蛋白质的，则表现出生物学活性。在一些实施方案中，如果基因产物是核酸，则可以翻译成表现生物学活性的蛋白质的基因产物。
如本文中使用的，术语“生物学分子”包括但不限于多肽、核酸和糖类。
如本文中使用的，术语“癌细胞中的差异表达”和“在癌细胞中差异表达的多核苷酸”在本文中是可互换的使用的，指当与不是癌性的同一细胞类型的细胞相比时，代表基因或与基因相应的多核苷酸在癌性细胞中是差异表达的，例如，发现mRNA的水平至少约25％、至少约50％至约75％、至少约90％、至少约1.5倍、至少约2倍、至少约5倍、至少约10倍，或至少约50倍或更多的不同(例如，更高或更低)。可以在组织中进行比较，例如利用原位杂交或另一种测定方法允许一定程度的区分组织中的细胞类型。还可以或可选的在从其组织来源移除的细胞之间进行比较，或者在一种原位的细胞和从其组织来源移除的第二种细胞之间进行比较。在一些实施方案中，与正常细胞相比，癌基因的基因是上调的。
如果缓解、结束、减慢或预防了癌症的至少一种症状或临床终点，则“抑制”了FGF21相关癌症。如本文中使用的，如果降低、减慢、延迟或预防了癌症的转移或复发，则也“抑制”了FGF21相关癌症。
如本文中使用的，术语“临床终点”指作为癌症指标的可测量的事件。临床终点包括但不限于，到首次转移的时间、到继发性转移的时间、转移的大小和/或数量、肿瘤的大小和/或数量、肿瘤的位置、肿瘤的侵占性、生活质量、疼痛等。本领域技术人员确定和测量临床终点能力是可信的。
如本文中使用的，术语“片段”指生物分子的初级结构的物理学连续的部分。在蛋白质的情况下，通过该蛋白质的氨基酸序列的连续部分来定 义部分，指至少3-5个氨基酸，至少8-10个氨基酸，至少11-15个氨基酸，至少17-24个氨基酸，至少25-30个氨基酸，和至少30-45个氨基酸。在寡核苷酸的情况下，通过寡核苷酸的核酸序列的连续部分来定义部分，指至少9-15个核苷酸，至少18-30个核苷酸，至少33-45个核苷酸，至少48-72个核苷酸，至少75-90个核苷酸，和至少90-130个核苷酸。在一些实施方案中，生物分子的部分具有生物学活性。在本发明的上下文中，FGF21多肽片段不包括SEQ ID NO：2提出的完整的FGF21多肽序列。
如本文中使用的，术语“表位”指多肽的抗原决定簇。在一些实施方案中，表位可以包括3个或更多个氨基酸，所述氨基酸在表位特异的空间构象中。在一些实施方案中，表位是线性的或构象的表位。通常，表位由至少4个、至少6个、至少8个、至少10个和至少12个此类氨基酸组成，更常见的由8-10个此类氨基酸组成。确定氨基酸空间构象的方法是本领域已知的，包括例如x-射线晶体和2-维核磁共振。
词组“互补决定区”指这样的氨基酸序列，所述序列共同确定天然免疫球蛋白结合位点的天然Fv区域的结合亲和力和特异性。参见例如，Chothia等人，J.Mol.Biol.196：901-917(1987)；Kabat等人，U.S.Dept.of Health and Human Services NIH Publication No.91-3242(1991)。词组“恒定区”指产生效应子功能的抗体分子的部分。
如本文中使用的。术语“寡核苷酸”指一系列相连的核苷酸残基。寡核苷酸包括但不限于反义和siRNA寡核苷酸。寡核苷酸包含部分DNA序列，并具有至少约10个核苷酸和多至约500个核苷酸。在一些实施方案中，寡核苷酸包含从约10个核苷酸到约50个核苷酸，从约15个核苷酸至约30个核苷酸，从约20个核苷酸至约25个核苷酸。寡核苷酸可以是化学合成的，还可用作探针。在一些实施方案中，寡核苷酸是单链。在一些实施方案中，寡核苷酸包含至少一部分是双链。在一些实施方案中，寡核苷酸是反义寡核苷酸(ASO)。在一些实施方案中，寡核苷酸是RNA干扰寡核苷酸(RNAi寡核苷酸)。
如本文中使用的，术语“组合”或“联合”指与其它治疗方案一起施 用本发明的FGF21调节剂。
如本文中使用的，术语“检测”意指建立、发现或确认活性(例如，基因表达)或生物分子(例如，多肽)的证据。
如本文中使用的，词组“同源核苷酸序列”或“同源氨基酸序列”或其变体，指在核苷酸水平或氨基酸水平以至少特定百分比的同源性表征的序列，与“序列同一性”可互换的使用。同源核苷酸序列包括编码蛋白质同工型的那些序列。此类同工型可以在同一生物体的不同组织中表达，作为例如RNA选择性剪接的结果。可选的，可以由不同的基因编码同工型。同源核苷酸序列包括编码人以外的物种的蛋白质的核苷酸序列，所述物种包括但不限于哺乳动物。同源核苷酸序列还包括但不限于，天然存在的等位变体和本文提出的核苷酸序列的突变。在一些实施方案中，同源核苷酸序列编码与野生型序列具有相同或相似结合特征和/或活性的多肽。同源氨基酸序列包括含有保守性氨基酸取代的那些氨基酸序列，其多肽具有与野生型序列具有相同或相似结合特征和/或活性。在一些实施方案中，如果具有至少70％、80％、85％、90％、95％、96％、97％、98％或99％同一性，则核苷酸或氨基酸序列是同源的。在一些实施方案中，如果具有1-10、10-20、20-30、30-40、40-50或50-60个核苷酸/氨基酸取代、添加或缺失，则核苷酸或氨基酸序列是同源的。在一些实施方案中，同源的氨基酸序列具有不超过5个或不超过3个保守性氨基酸取代。
可以通过例如Gap程序(Wisconsin序列分析软件包，UNIX第8版，Genetics Computer Group，University Research Park，Madison WI)和使用缺省设定，来确定百分比同源性或同一性，所述程序使用Smith和Waterman的算法(Adv.Appl.Math.，1981，2，482-489)。在一些实施方案中，探针和靶之间的同源性在约75％至约85％之间。在一些实施方案中，核酸具有与SEQ ID NO：1或其部分至少约85％、约90％、约92％、约94％、约95％、约97％、约98％、约99％和约100％同源性的核苷酸。还提供了此类序列的互补序列。在一些实施方案中，互补序列是核苷酸序列的完整和完全互补序列。
同源性还可以在多肽水平。在一些实施方案中，多肽与SEQ ID NO：2或其部分至少约85％、约90％、约92％、约94％、约95％、约97％、约98％、约99％和约100％同源。在一些实施方案中，多肽具有多达5个、多达10个、多达15个、多达20个或多达30个氨基酸插入、缺失或取代。
如本文中使用的，术语“探针”指可变长度的核酸序列。在一些实施方案中，探针包括至少约10个和至多约6000个核苷酸。在一些实施方案中，探针包括至少12、至少14、至少16、至少18、至少20、至少25、至少50或至少75个连续的核苷酸。探针用于检测相同的、相似的或互补的核酸序列。较长的探针通常获得自天然的或重组的来源，与靶序列具有高特异性，与寡聚物相比慢得多的与靶杂交。探针可以是单链或双链，设计为在PCR、基于膜的杂交、原位杂交(ISH)、荧光原位杂交(FISH)或ELISA-样技术中具有特异性。
如本文中使用的，术语“结合”意指两个或多个生物分子或化合物之间的物理或化学相互作用。结合包括离子的、非离子的、氢键、范德华力、疏水性相互作用等。结合可以是直接的或间接的；间接的是通过或由于另一种生物分子或化合物的影响。直接结合指相互作用不通过或不由于另一种生物分子或化合物的影响而发生，相反不需要其它基本的化学媒介。
本文所用的术语“样品”包括获自患者的任何生物学样本。样品包括但不限于：全血、血浆、血清、红细胞、白细胞(例如，外周血单核的细胞)、唾液、尿液、粪便(即，排泄物)、痰、支气管灌洗液、眼泪、乳头抽吸物、淋巴(例如，淋巴结的弥散性肿瘤细胞)、细针抽吸物、任何其它体液、组织样品(例如，肿瘤组织)，如肿瘤活检(例如，针吸活检)和它们的细胞提取物。在一些实施方式中，样品是全血或其组分，例如血浆、血清或细胞沉淀。在优选的实施方式中，采用本领域已知的任何技术从全血分子实体瘤的循环细胞或其细胞组分而获得样品。在其它实施方式中，样品是福尔马林固定石蜡包埋(FFPE)的肿瘤组织样品，例如来自肺、结肠或直肠的实体瘤。
“活检”指取出组织样品以供诊断或预后评估的过程，也指组织样本自身。可将本领域已知的任何活检技术应用于本发明的方法和组合物。所 应用的活检技术通常取决于待评估的组织类型和肿瘤大小及类型(即，实体或悬浮的(即，血液或腹水))等因素。代表性活检技术包括切除活检、切开式活检、针吸活检(例如，芯针活检、细针抽吸活检等)、手术活检和骨髓活检。活检技术参见，例如Harrison′s Princpiles of Internal Medicine(内科学哈里森原理)，Kasper等编，第16版，2005，第70章，以及整个第V部分。
本文描述的每种专利、专利申请、登录号和出版物都通过引用全文整合到本文中。
除本文描述的以外，根据上述描述，发明的不同修饰对本领域技术人员是显而易见的。此类修饰还意在落入所附实施方案的范围内。出于示例的目的，本发明还展示了下列实施例，并非意在限制本发明的范围。
实施例
材料、试剂(厂家)及仪器型号和生产厂家
以下材料、试剂将在实施例中使用，它们均为商业上可获得的，并且具体公开如下：
I.1 pcDNA3.0-hSCN3B，购自Origene公司(OriGene Technologies，9620 Medical Center Dr.，Suite 200，Rockville，MD 20850·1.888.267.4436)
I.2 细胞株：Hep3B，HepG2，HL7702，购自中国科学院上海生命科学研究院生物化学与细胞生物学研究所
I.3 干扰RNA序列，由上海吉玛制药技术有限公司设计并提供
Figure PCTCN2015079356-appb-000020
I.4 阴性序列，上海吉玛制药技术有限公司设计并提供
Figure PCTCN2015079356-appb-000021
Figure PCTCN2015079356-appb-000022
I.5 β3特异性引物由金斯瑞科技公司合成，序列如下：
Figure PCTCN2015079356-appb-000023
I.6 镇痛抗肿瘤缬精甘肽AGAP：
将GENBANK基因序列号AF464898的基因克隆到大肠杆菌表达载体中，转化大肠杆菌细胞，构建重组菌，实现活性肽在大肠杆菌中表达，经层析纯化后获得重组镇痛抗肿瘤缬精甘肽。
I.7 GIBCO RPMI1640培养基(货号：LM-R1645)，新生牛血清(货号：16010-159)，胎牛血清(货号：10099-141)，购自lLife Technologies，(Life Sciences Solutions Group，Thermo Fisher Scientific公司，美国加州Carlsbad)
I.8 BIOZOL RNA提取试剂盒购自BioFlux(货号：R1020-01)(BIOFLUX SRL Address：54 Ceahlau street，Cluj-Napoca 400488，Romania，European Union，Europe)
I.9 蛋白质提取试剂盒(KGP2100)，逆转录试剂盒(货号：KGEA-01)，购自凯基公司(南京凯基生物科技发展有限公司，南京市江宁区芝兰路18号紫金方山创业社区6号楼)
I.10 β3特异性抗体购自abcam公司(货号：ab48552)
I.11 Invitrogen Lipofectamine 2000转染试剂盒购自Life Technologies公司(货号：11668019)(Life Sciences Solutions Group，Thermo Fisher Scientific公司，美国加州Carlsbad)
I.12 Transwell小室购自Corning公司(CORNING，Corning Incorporated One Riverfront Plaza Corning，NY 14831 USA)，
I.13 Matrigel购自BD公司(BD Biosciences)
I.14 仪器：Thermo二氧化碳培养箱(311型)，北京六一仪器厂DYY-8C电泳仪，北京六一仪器厂DYCZ-40D转膜槽
实施例1：β3亚基在肿瘤细胞中的表达
1.1 β3亚基在肝癌细胞株中的表达
提取不同细胞株(HepG2，HL7702)总mRNA，取10μg mRNA逆转录，然后取2μLcDNA为模板特异性扩增β3基因转录的mRNA(PCR条件及所使用的引物参见：Candenas L，Seda M，Noheda P，Buschmann H，Cintado CG，Martin JD，et al.Molecular diversity of voltage-gated sodium channels alpha and beta subunit mRNAs in human tissues.Eur J Pharmacol.2006；542：9-16.)，琼脂糖凝胶电泳检测，检测各个细胞株中的mRNA水平。
提取不同细胞株总蛋白，使用Bradford法测定蛋白含量，取20-70μg蛋白电泳，电泳后进行western blotting检测β3亚基基因在蛋白水平表达，具体地，Bradford法的步骤如下：
1、精密称取牛血清白蛋白约10mg，置10ml容量瓶中，用蒸馏水溶解并稀释至刻度，摇匀，即得工作液。在标记好的试管中分别加入标准蛋白质溶液(牛血清白蛋白，1.0mg/ml)0μl、10μl、20μl、30μl、40μl、50μl和100μl，不足100μl者用生理盐水补足至100μl。加入5ml考马斯亮蓝G-250溶液(称取50mg的考马斯亮蓝G-250溶于25ml 95％的乙醇中，加入50ml 85％的磷酸，最后加蒸馏水稀释到500ml，滤纸过滤)，以生理盐水为空白对照；
2、把酶标板放在振荡器上振荡30sec，放置2min后再次振荡，然后在595nm下比色测定(比色应在1h内完成)。以蛋白质浓度(mg/ml)为横坐标，吸光值为纵坐标，绘出标准曲线；
3、稀释待测样品至合适浓度，使样品稀释液总体积为100μl，加入考马 斯亮蓝G-250溶液100μl，充分混匀，放置2min后，以标准曲线0号管做参比，在595nm波长下比色，记录吸光值；
4、根据所测样品的吸光值，在标准曲线上即可查得相应的蛋白质浓度，乘以样品稀释倍数即为样品实际浓度。
Western Blot步骤如下：
1、样品处理：将上样缓冲液Loading buffer与待测蛋白等体积加样，涡旋混匀，沸水浴5min，离心；
2、电泳
(1)根据厂家说明书安装玻璃板；
(2)按表1所给数值配制所需浓度的分离胶溶液；
(3)迅速在两玻璃板的间隙中灌注丙烯酰胺溶液，留出灌注浓缩胶所需空间(梳子的齿长再加1cm)。胶聚合完全后，倾出覆盖液体，用去离子水洗涤凝胶顶部数次以除去未聚合的丙烯酰胺。尽可能排去凝胶上的液体，再用纸巾的边缘吸净残留液体；
(4)按表1所给数值配制浓缩胶溶液；
(5)在已聚合的分离胶上直接灌注浓缩胶，然后立即在浓缩胶溶液中插入干净的Teflon梳子，小心避免混入气泡，将凝胶垂直放置于室温下聚合；
(6)当浓缩胶发生聚合时，可把已经浓缩好的样品与2×loading buffer混合，100℃加热5min使蛋白质变性。浓度较稀的样品需用三氯乙酸富集样品，然后用丙酮沉淀蛋白；
(7)浓缩胶聚合完全后，小心移出Teflon梳子，立即用电泳上槽缓冲液洗涤加样槽以除去未聚合的丙烯酰胺。把凝胶固定于电泳装置上，加入电泳缓冲液。排出凝胶底部两玻璃板间的气泡；
(8)按预定顺序将样品加至样品孔底部，每加完一个样品，应将加样注射 器洗涤干净。最后在所有不用的样品孔中加上等体积的凝胶加样缓冲液；
(9)电泳装置与电源连接(正极应接下槽)，电泳直至溴酚蓝到达分离胶底部，然后关闭电源；
(10)从电泳装置上卸下玻璃板，放在纸巾上，用刮勺撬开玻璃板。
表1：分离胶和凝缩胶的组成
Figure PCTCN2015079356-appb-000024
3、切胶、转膜
(1)电泳结束后，用切胶板将玻璃板迅速撬开，注意保持胶的平整，按预染的Marker切取所需目的条带，切割范围包含目的蛋白，比之略宽；对胶切角，进行标记；测量其长宽；
(2)将胶浸入事先准备好的转膜缓冲液中(分子量小的蛋白需缩短在转膜缓冲液中的浸泡时间，分子量大的蛋白需延长在转膜液中的浸泡时间有利于转膜)；
(3)剪取PVDF膜，并在角上做标记，用甲醇浸润5min，放入ddH2O平衡5min，再浸入转膜缓冲液中；
(4)安装转移装置“三明治结构”从下到上依次为；海绵-滤纸→膜→胶→ 滤纸-海绵(每层均需确保无气泡，滤纸用干净的滴管或试管赶，膜和胶层在放置时注意避免气泡的产生)；
(5)以胶面积大小的2倍作为转膜时电流的大小，根据蛋白的分子量设定转膜时间，横流转膜1h40min。
4、免疫反应
(1)转膜后，取出膜，根据条带所在位置剪取膜大小，并标记蛋白所在面；
(2)用5％的脱脂奶粉作为封闭液，于摇床上室温封闭3h；
(3)配制一抗，用牛奶封闭液稀释一抗，将目的蛋白所在面朝下，置于一抗稀释液上，4℃过夜孵育；
(4)弃一抗，TBST洗膜，置于摇床上洗膜三次，每次15min；
(5)二抗孵育，方法同一抗，室温孵育2h左右；
(6)弃二抗，TBST洗膜，置于摇床上洗膜三次，每次15min；
5、显影
(1)将ECL发光液A∶B＝1∶1混合，按说明书所推荐的比例使用；
(2)将发光液均匀的滴加在膜上；
(3)在暗室中取出胶片，置于暗盒中，曝光20min，取出胶片；
(4)将胶片在显影液中浸泡2min，水洗2min，定影液中浸泡2min，水洗2min，观察结果。
实验结果表明，肝癌细胞株HepG2中检测到有VGSC β3亚基(SCN3B)的RNA(图2A)及蛋白的表达(图2B)，而正常的HL7702细胞中则没有VGSC β3亚基的mRNA以及蛋白质。表明在肝癌细胞HepG中，VGSC β3亚基表达高。
1.2 β3亚基在不同白血病细胞修改中表达不同
对ONCOMINE数据库(www.oncomine.com)中不同类型白血病细胞株与正常细胞中SCN3B表达进行检测，应用基因芯片数据库(www.oncomine.org)数据分析显示在白血病中，也存在VGSC的SCN3B mRNA异常表达。数据库筛选选项中，基因为SCN3B，比较对象为白血病vs.正常组织。SCN3B编码电压门控钠离子通道β3亚基，下图为SCN3B在不同类型白血病中表达情况，结果可见除了第六组骨髓增生异常综合征(Myelodysplastic Syndrome中SCN3B表达与正常细胞没有显著性差异之外，其余7种不同类型白血病(急性髓性白血病(Acute Myeloid Leukemia)、B细胞急性淋巴细胞白血病(B-cell Acute lymphoblastic Leukemia)、B细胞儿童急性淋巴细胞白血病(B-Cell Childhood Acute Lymphoblastic Leukemia)、慢性淋巴细胞白血病(Chronic Lymphocytic Leukemia)、慢性髓性白血病(Chronic Myelogenous Leukemia)、前B-急性淋巴细胞白血病(Pro-B Acute Lymphoblastic Leukemia)、T细胞急性淋巴细胞白血病(T-Cell Acute Lymphoblastic Leukemia))均表现出SCN3B的高表达(P＜0.05)，并且不同类型白血病SCN3B表达水平也不同(图3)。
实施例2.下调β3亚基表达影响肿瘤细胞增殖
2.1 下调β3亚基表达影响肝癌细胞增殖
利用I.3中的两对β3特异性小干扰RNA，采用RNAi的方法，干扰肝癌Hep G2细胞株β3亚基的表达，使β3亚基表达下调，肝癌细胞增殖也受抑制。
2.1.1 RNAi干扰β3亚基基因表达
干扰实验前一天接种细胞，进行干扰当天细胞应达到30％～50％覆盖率。干扰实验按Lipofectamine 2000说明书进行操作，将前述I.3中的两对β 3特异性小干扰RNA以同样的浓度(50nM)转染到细胞中，以干扰VGSC β3表达，利用同样量的随机序列I.4中的阴性序列做对照。转染当天，将脂质体Lipofectamine 2000试剂和质粒siRNA(25-50nM)分别用无血清培养基稀释，混匀，室温孵育5min。将上述混合物直接加入培养基中，轻轻晃动混匀，常规条件培养细胞4-6h后，更换新鲜培养基继续培养2-3d后进行实验。
2.1.2 β3亚基被干扰后，Hep G2细胞株蛋白水平表达下调
利用实施例1所述的方法，提取2.1.1的中获得的细胞总蛋白并测定蛋白含量，将上样缓冲液Loading buffer与待测蛋白(取蛋白20-70μg)等体积加样，涡旋混匀，沸水浴5min，离心。电泳后切胶转PVDF膜，以I.10中的获得自abcam公司的VGSC β3特异性抗体进行western blotting分析。
Western blotting结果显示，I.3中的两对siRNA抑制了HepG2细胞中的VGSC β3的表达，也就是说，I.3中的siRNA有效抑制了β3亚基蛋白水平(图4)。
2.1.3 抑制β3基因能够降低HepG2细胞增殖水平
采用MTT法测定细胞增殖水平：取生长状态良好，处于对数生长期的细胞，常规胰酶消化后，吹打成单细胞悬液，血球计数板计数后将浓度调整至105个/ml(HepG2细胞)，接种于96孔培养板，每孔100μl，同时设置空白调零孔(只加100μl培养液，不加细胞)；37℃，5％CO2培养箱内培养24h后，每孔加入0.5mg/ml的MTT，100μl，继续培养4h后，弃去上清，每孔加入150μl的DMSO，置微量振荡器上低速振荡10min，待结晶物充分溶解后，利用酶标仪在490nm处检测各孔的吸光度值。
依据检测结果进行显著性分析，抑制率计算按照下面公式进行。
抑制率(％)＝(对照组-加样组)/(对照组-空白调零孔)×100
在如2.1.2所示利用siRNA干扰HepG2细胞株VGSC β亚基表达后，采用MTT法测定细胞增殖情况变化。由结果可知，HepG2细胞中β3亚基干扰后细胞的增殖水平显著降低(P＜0.05)(图5)。
由上述结果可知，能够通过抑制HepG2细胞中的VGSC β3的蛋白质水平来降低HepG2细胞的增殖水平。
2.1.4 通过干扰VGSC β3基因阻滞HepG2细胞周期
对2.1.2中的干扰后HepG2细胞进行PI染色：细胞处理完毕后，消化后离心收集细胞(0.25％的胰蛋白酶消化液，2mL，消化1～2分钟)，弃上清，PBS(KCl 0.2g/L，KH2PO40.2g/L，NaCl 8g/L，Na2HPO4.12H2O 2.9g/L)重悬后，离心洗涤1次(1000rpm，离心5分钟)；
PBS重悬后，调整细胞浓度为106个/ml，再次离心(1000rpm，离心5分钟)后用-20℃预冷的70％乙醇固定，-20℃固定过夜；
固定结束后，离心除去乙醇，PBS离心洗涤1次。
以PI染色液重悬离心细胞(106个/500μL)常温避光染色15～30min。过200目筛网制成单细胞悬液，使用流式细胞仪检测：激发波长为488nm，发射波长为615nm。
流式细胞仪计数，实验结果显示干扰后48小时细胞周期阻滞在G0/G1期(**P＜0.01)(图6)。可见，能够通过干扰VGSC β3基因阻滞HepG2细胞周期。
2.1.5 通过干扰VGSC β3基因下调HepG2细胞周期蛋白cylin D1表达水平
利用实施1所述的方法，对2.1.2中的干扰后HepG2细胞提取细胞总蛋白并测定蛋白含量，取蛋白20-70μg作为待测蛋白，将上样缓冲液Loading buffer与待测蛋白等体积加样，涡旋混匀，沸水浴5min，离心。电泳后切胶转PVDF膜，以特异性抗体进行western blotting分析，实验结果显示细胞周期蛋白cylin D1(SANTA CRUZ；sc753)表达水平下调(图7)。
2.2 通过下调VGSC β3亚基表达影响肺癌细胞增殖
利用I.3中的两对β3特异性小干扰RNA，采用RNAi的方法，干扰肺癌癌细胞株β3亚基的表达，使VGSC β3亚基表达下调，肺癌细胞增殖也受抑制。
2.2.1 RNAi干扰β3亚基基因表达
干扰实验前一天接种细胞，进行干扰当天细胞应达到30％～50％覆盖率。干扰实验按Lipofectamine 2000说明书进行操作，利用I.3中所述的两对β3特异性小干扰RNA干扰VGSC β3表达，随机序列——I.4中的阴性序列做对照(方法同2.1.1)
2.2.2 VGSC β3亚基干扰后，肺癌细胞株蛋白水平表达下调
利用实施例1所述方法，提取2.2.1中获得的细胞的总蛋白并测定蛋白含量，将上样缓冲液Loading buffer与待测蛋白(取20-70μg)等体积加样，涡旋混匀，沸水浴5min，离心。电泳后切胶转PVDF膜，以I.10中的VGSC β3特异性抗体进行western blotting分析。
Western blotting结果显示，I.3中的两对siRNA有效抑制了肺癌细胞A549中的VGSC β3亚基蛋白水平的表达(图8)。
2.2.3 β3基因干扰后，肺癌细胞增殖水平降低
采用MTT法(如2.1.3所述)测定细胞增殖水平，依据检测结果进行显 著性分析，抑制率计算按照下面公式进行。
抑制率(％)＝(对照组-加样组)/(对照组-空白调零孔)×100
在如2.2.2所示利用siRNA干扰B5492细胞株VGSC β亚基表达后，采用MTT法测定细胞增殖情况变化，在两对siRNA剂量分别以50nM的浓度转染时(方法同2.1.1)，细胞增殖抑制率达50％。
实施例3活性肽AGAP通过抑制VGSC β3的蛋白质表达抑制肝癌细胞
3.1 活性肽下调VGSC β3亚基表达，并且抑制肝癌细胞增殖
将镇痛抗肿瘤缬精甘肽(AGAP)以0、5、10、15、20、25、30、35、40μM，8个浓度与肝癌细胞株HepG2(96孔板，104个细胞/孔，24h后给予上述不同浓度AGAP，每个浓度重复至少3个孔)孵育48h，然后通过western印迹，利用I.10中的抗体检测VGSC β3亚基表达量，并且利用MTT法测定细胞增殖水平。
实验结果显示HepG2细胞给予AGAP后VGSC β3的表达随给药剂量升高而降低，呈剂量依赖性(图9B)，同时，AGAP相应地呈剂量依赖性抑制了肿瘤细胞的增殖，IC50值为10μM(图9A)。相反，作为对照的牛血清白蛋白和细胞色素C(40μM，孵育48h，其它条件与给药AGAP相同)对VGSC β3亚基表达没有影响，也不影响细胞增殖。
可见，活性肽AGAP通过抑制VGSC β3亚基的表达来抑制细胞增殖。
3.2 活性肽AGAP逆转由于VGSC β3亚基表达上调所促进的Hep3B细胞的增殖
将VGSC β3基因利用图14所示的质粒(其中编码VGSC β3的基因SCN3B如SEQ IDNO：2所示，质粒构建流程如图15所示，pcDNA3.0质粒购 自北京Origene公司(ORIGENE，Adderess：9620 Medical Center Dr.，Suite 200 Rockville，MD 20850)利用下述细胞转染实验转染进入不表达VGSC β3基因的Hep3B细胞株中，western blotting结果显示β3亚基实现蛋白水平表达(图10)。
细胞转染实验：
1、采用Lipofectamine 2000转染实验方法，转染前一天，胰酶消化细胞并计数，细胞铺板，以合适的细胞密度6×105个/孔接种到6孔培养板上，使得细胞在转染时要达到80％-90％的汇合度。
2、转染液制备：在EP管中制备以下两液(为转染1个孔细胞所用的量)。
A液：用不含血清和抗生素培养基RPMI1640稀释DNA使浓度为4ug，终量500ul，质粒DNA包括含有目的基因的质粒，其中空质粒pcDNA3.0质粒作为阴性对照。
B液：用不含血清和抗生素的双无培养基稀释Lipofectamine 2000 8ul，终量500ul，室温放置5min。质粒的量与Lipo转染试剂的比例为1∶2。
3、轻轻混合A、B液，室温中置20min，稍后会出现微浊现象，但并不影响转染。
4、在这期间，将6孔板中的细胞用无血清培养基冲洗细胞两遍后，加入1ml无血清无抗生素RPMI1640培养基。
5、将溶液A与溶液B的混合液逐滴加入孔中，摇动培养板，轻轻混匀。在37℃，5％的CO2培养箱中培养6小时。
6、6小时后，更换含有血清和抗生素的完全培养基，在37℃，5％的CO2培养箱中继续培养，48-72h后检测蛋白水平。
Hep3B转染在VGSC β3亚基基因后表达VGSC β3(转染后蛋白表 达为图10，泳道1)，如前文所述采用MTT法测定对细胞增殖的影响。由结果图11，柱2可知，Hep3B细胞中转染β3亚基后促进细胞的增殖(**P＜0.01)。可见，VGSC β3亚基能够促进癌细胞增殖。
Hep3B转染β3亚基基因后，加入活性肽AGAP(10μM)，western blotting显示VGSC β3亚基表达下调(图10，泳道3)。如上所述采用MTT法测定对细胞增殖的影响，结果显示AGAP对转染后细胞增殖有显著抑制作用(图11柱3)。
实施例5本发明的siRNA抑制裸鼠体内肿瘤生长
利用将I.3中所述的两对β3 siRNA和I.4中所述对照siRNA转染HepG2，24h后收集细胞(方法同2.1.1)。选取12只裸鼠，随机分为HIF-1a siRNA实验组和对照组，背部皮下注射相应的HepG2细胞(5×106/只)。每天测量肿瘤大小，计算肿瘤体积，直至21天处死动物。肿瘤体积＝(肿瘤长径×(肿瘤短径)2)/2。
siRNA转染HepG2肝癌细胞在裸鼠皮下生长的肿瘤生长缓慢，从第6-7天开始，实验组和对照组裸鼠在接种肿瘤细胞的皮下部位均出现肿瘤结节，但两组间无差别(t＝690，P＝0.064＞0.05)。第12天开始，HIF-1 α siRNA实验组的肿瘤体积比对照组的肿瘤结节小，差异有统计学意义(t＝15.70，P＝0.021＜0.05)。之后直至21天处死裸鼠，实验组肿瘤体积均小于对照组，差异有统计学意义(17d：t＝17.98，P＝0.014＜0.05；21d：t＝17.43，P＝0.015＜0.05)(表2)。
表2实验组及对照组裸鼠接种后第7、12、17和21天肿瘤体积变化情况
Figure PCTCN2015079356-appb-000025
注意：*P＜0.05 siRNA组相比对照组
实施例6.通过抑制HepG2细胞中VGSC β3亚基表达影响细胞形态以及细胞周期
对如实施例2所述获得的干扰后HepG2细胞进行PI染色，参见PI单染操作步骤(方法同2.1.4)，流式细胞仪(BD FACS AriaTM III)计数，实验结果干扰48小时后细胞体积变小，60小时后出现凋亡小体和细胞碎片。干扰后48小时细胞周期阻滞在G0/G1期(**P＜0.01)，干扰60h后细胞周期阻滞降低(*P＜0.05)，同时出现凋亡峰(*P＜0.01)(图12，p5)。。
实施例7.通过抑制HepG2细胞中VGSC β3亚基表达影响细胞凋亡
如通过实施例2所述利用I.3中的两对siRNA抑制HepG2细胞中VGSC β3亚基的表达并且获得干扰后的细胞。对细胞进行ANEXIN V染色后流式细胞仪分析，包括以下步骤：
1.把细胞培养液吸出至合适离心管内，PBS洗涤铁壁细胞一次，加入适量胰酶细胞消化液(可含EDTA)消化细胞。
2.将细胞用正常培养基重悬后，混匀，转移到离心管内，1000rpm离心5min，弃上清，收集细胞，用PBS轻轻重悬细胞并计数。
3.取100万重悬细胞，1000rpm离心5min，弃上清，加入195μl Annexin V-FITC结合液轻轻重悬细胞。加入5μl Annexin V-FITC，轻轻混匀。
4.室温(20～25℃)避光孵育10min。可以使用铝箔进行避光。
5.1000rpm离心5min，弃上清，加入190μl Annexin V-FITC结合液轻轻重悬细胞。加入10μl碘化丙啶染色液，轻轻混匀，冰浴避光放置。
6.随即进行流式细胞仪检测，Annexin V-FITC为绿色荧光，PI为红色荧光。
由结果图可知(图13)，HepG2细胞在60h后细胞发生早期凋亡，72h后晚期凋亡居多(*P＜0.05，**P＜0.01)。可见，能够通过抑制细胞中VGSC β3亚基的表达来促进细胞凋亡。

Claims (14)

  1. 利用VGSC β3亚基检测或诊断患者中与VGSC β3亚基相关的肿瘤的方法，其包括：
    e)从患者中体内分离样品；
    f)测量a)中获得的样品与正常对照细胞中的VGSC β3亚基的编码DNA水平、mRNA水平或蛋白质水平；
    g)比较b)中测量的VGSC β3亚基的编码DNA水平、mRNA水平或蛋白质水平；
    h)待测样品或细胞中VGSC β3的蛋白质或基因含量比对照正常高表明对象患有VGSC β3相关肿瘤。
  2. 用于测量VGSC β3亚基的编码DNA水平或mRNA水平或蛋白质表达水平的工具在制备用于检测或诊断患者中与VGSC β3亚基相关的肿瘤的试剂盒中的用途，其中在患者的样品中测量到的VGSC β3亚基的边编码DNA水平或mRNA水平或蛋白质表达水平高于正常对照细胞中的水平表明患者患有肿瘤。
  3. 用于在对象中预防或治疗肿瘤的VGSC β3的抑制剂。
  4. 使用VGSC β3的抑制剂在对象中预防或治疗肿瘤的方法，包括向对象施用一定量的VGSC β3抑制剂，所述量有效抑制VGSC β3的表达和/或活性。
  5. VGSC β3的抑制剂在在对象中预防或治疗肿瘤中的用途，或者VGSC β3的抑制剂在制备用于在对象中预防或治疗肿瘤的药物组合物中的用途。
  6. 调节表达VGSC β3的细胞中的一种或多种活性的方法，包括将细胞与有效调节一种或多种活性的量的VGSC β3抑制剂接触。
  7. 利用VGSC β3的抑制剂抑制肿瘤细胞的增殖的方法。
  8. VGSC β3的抑制剂在抑制肿瘤细胞增殖中的用途。
  9. VGSC β3的抑制剂在制备用于抑制肿瘤细胞增殖的药物组合物或试剂 盒中的用途。
  10. 利用VGSC β3或其基因调控序列为靶点，设计和制备用于在对象中预防或治疗肿瘤的活性物质的方法，所述方法包括，根据VGSC β3蛋白质的氨基酸序列以及核酸编码序列，设计能够抑制VGSC β3蛋白质的表达或活性的活性物质。
  11. 筛选用于在对象中预防或治疗肿瘤的活性物质的方法，所述方法包括将表达VGSC β3的细胞与候选活性物质接触，确定是否抑制了VGSC β3的表达、活性和/或相关生物学活性或者是否抑制了对癌细胞生长等一种或多种癌细胞指标的步骤，其中如果VGSC β3的表达、活性和/或相关生物学活性被抑制或者一种或多种癌细胞指标被抑制，则表明该候选活性物质为预防或治疗肿瘤的活性物质。
  12. 筛选抗癌剂或抑制肿瘤细胞增殖的抑制剂的方法，所述方法包括将表达VGSC β3的细胞与候选化合物接触，确定是否调节了VGSC β3的表达、活性和/或相关生物学活性或者是否抑制了对癌细胞生长等一种或多种癌细胞指标，其中如果VGSC β3的表达、活性和/或相关生物学活性被抑制或者一种或多种癌细胞指标被抑制，则表明该候选活性物质为抗癌剂或抑制肿瘤细胞增殖的抑制剂。
  13. VGSC β3作为靶点，在制备用于预防或治疗肿瘤的药物组合物中的用途，以及在制备用于诊断或检测肿瘤的试剂中的用途。
  14. 试剂盒，其用于检测或诊断患者中与VGSC β3亚基相关的肿瘤，包含用于测量VGSC β3亚基的编码DNA水平或mRNA水平或蛋白质表达水平的工具。
PCT/CN2015/079356 2014-07-25 2015-05-20 VGSC β3蛋白质用于癌症预防、治疗及诊断检测的靶点 WO2016011840A1 (zh)

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