WO2020078274A1 - 纹蛋白互作蛋白抑制剂及其在制备抗肿瘤药物中的用途 - Google Patents

纹蛋白互作蛋白抑制剂及其在制备抗肿瘤药物中的用途 Download PDF

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WO2020078274A1
WO2020078274A1 PCT/CN2019/110713 CN2019110713W WO2020078274A1 WO 2020078274 A1 WO2020078274 A1 WO 2020078274A1 CN 2019110713 W CN2019110713 W CN 2019110713W WO 2020078274 A1 WO2020078274 A1 WO 2020078274A1
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striatin
inhibitor
protein
virus
interaction protein
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李恭楚
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杭州功楚生物科技有限公司
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Priority to US17/286,486 priority Critical patent/US20220275370A1/en
Priority to EP19874712.3A priority patent/EP3888692A4/en
Publication of WO2020078274A1 publication Critical patent/WO2020078274A1/zh

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Definitions

  • the invention relates to the technical field of biomedicine, in particular to the application of striatin interacting protein (STRIP) as a target in the preparation of anti-tumor drugs, carriers and drugs containing the same.
  • STRIP striatin interacting protein
  • Oncolytic virus refers to a virus that can selectively infect and damage tumor tissues.
  • Oncolytic viruses have a long history. So far, oncolytic viruses belonging to 10 virus families have entered clinical trials, including Adenovirus, Coxackie virus, and Herpes simplex virus (HSV) ), Measles virus, Newcastle disease, Parvovirus, Poliovirus, Reovirus, Vaccinia virus and vesicular mouth Inflammatory virus (Vesicular stomatitis virus). At present, the herpes virus T-VEC has been approved by the FDA to treat melanoma. Overall, oncolytic viruses have shown considerable safety and effectiveness in clinical trials.
  • HSV Herpes simplex virus
  • Vaccinia virus is a large double-stranded DNA virus in the poxvirus family. Compared with other oncolytic viruses, it has the following advantages: (1) It is unique among DNA viruses because it only replicates in the cytoplasm to minimize integration Risks within the host genome; (2) Vaccinia virus was first made into a vaccine against smallpox virus, and its safety is guaranteed; (3) Vaccinia virus has a large cloning ability, which can allow gene fragments of larger length Insert; (4) Strong virus replication ability.
  • Oncolytic vaccinia virus has shown anti-tumor efficacy.
  • most common oncolytic vaccinia viruses have been attenuated, including thymidine kinase TK gene deletion or TK / vaccinia virus growth factor gene double deletion virus strains.
  • the deletion of the TK gene makes vaccinia virus replication more dependent on intracellular TK levels, and cancer cells tend to have higher levels of TK than normal cells; in addition, vaccinia virus replication also depends on epidermal growth factor receptor EGFR /
  • the drive of the Ras pathway makes it highly selective for cancer cells.
  • Oncolytic vaccinia virus shows the ability to target cancer cells to aggregate in experimental animals and humans, so it has become an ideal viral vector for cancer treatment.
  • Striatin interacting protein is a component of the complex of striatin interacting phosphatase and kinase (Striatin interacting and kinases, STRIPAK) (Goudreault, M., LMD'Ambrosio, et.al. 2009 .A PP2A phosphatase high interaction density interaction network identification striatin-interacting phosphatase and kinase complex linked to the cerebral cavernous malformation 3 (CCM3) protein.Mol Cell Proteomics 8 (1): 157-171.Gen contains: 157-171.Gen NM_033088.3) and STRIP2 (GenBank No.
  • NM_020704.2 also known as FAM40A and FAM40B.
  • Drosophila STRIP participates in the negative regulation of CLOCK protein dephosphorylation, thereby participating in the regulation of day and night rhythm (Andreazza, S., S. Boulleau, et al. 2015. Daytime CLOCK Dephosphorylation Is Controlled by STRIPAK Complexes in Drosophila. Cell Rep 11 (8): 1266-1279.).
  • STRIP1 is an important factor regulating the normal movement of mouse embryonic mesoderm (Bazzi, H., E. Soroka, et al.
  • STRIP2 performs key functions during the differentiation of murine embryonic stem cells (Sabour, D., SPSrinivasan, et al. 2017.STRIP2 IsIndispensable for the Onset of Embryonic Stem Cell Differentiation. .
  • studies on breast cancer mouse models have shown that inhibiting the level of STRIP1 can increase the transfer of breast cancer cells in mice, while increasing the level of STRIP1 can inhibit the transfer of breast cancer cells.
  • inhibiting the level of STRIP2 inhibited the metastasis of breast cancer cells in mice (Madsen, CD, S. Hooper, et al. 2015. ): 68-80.).
  • STRIP plays an important role in cell differentiation of lower and higher organisms, and it has a certain relationship with the metastasis of breast cancer cells, but whether there is a regulatory relationship between STRIP and the growth and inhibition of other tumors has not been reported.
  • oncolytic vaccinia virus as a carrier and STRIP, whether there is only a vector expression relationship between the two, or whether STRIP can also regulate the replication of oncolytic vaccinia virus in tumor cells, has not yet been reported.
  • This study regulates the replication level of oncolytic vaccinia virus in tumor cells by inhibiting STRIP, and significantly improves the oncolytic effect of oncolytic vaccinia virus, and provides a new technical approach for the development of oncolytic virus antitumor drugs.
  • the first objective of the present invention is to study the relationship between STRIP and tumor cells, and to provide new applications of inhibitors of STRIP (STRIP); another object of the present invention is to The study of the regulatory relationship between STRIP and oncolytic vaccinia virus invading tumor cells provides a new technical approach for the development of anti-tumor drugs for oncolytic viruses.
  • the siRNA interference of STRIP1 and STRIP2 can significantly reduce the antiviral factor Interferon-induced protein with tetratricopeptide repeats 1 (IFIT1), DExH-Box Helicase 58 (DHX58) and Interferon-stimulated in the malignant glioblastoma U87MG cell Gene 15 (ISG15) transcription level (as shown in Figure 1).
  • IFIT1 Interferon-induced protein with tetratricopeptide repeats 1
  • DHX58 DExH-Box Helicase 58
  • ISG15 Interferon-stimulated in the malignant glioblastoma U87MG cell Gene 15 transcription level (as shown in Figure 1).
  • STRIP1 shRNA interference can cause the expression level of antiviral factor IFI16 in liver cancer cell SMMC-7721 to be significantly reduced (as shown in Figure 2).
  • the interference of STRIP1 increases the expression of caspase 3 and caspase 8, indicating that the interference of STRIP1 helps to increase the sensitivity of tumor cells to apoptosis induction (see Figure 4).
  • a striatin interaction protein inhibitor in the preparation of anti-tumor drugs.
  • the striatin interaction protein inhibitor is any substance that can reduce the activity of STRIP, reduce the stability of STRIP, inhibit the expression of STRIP, reduce the effective time of STRIP, or inhibit the transcriptional processing of STRIP, including but not limited to: Small interfering molecules that specifically interfere with STRIP gene expression and processing, such as shRNA molecules, siRNA molecules, antisense nucleotides, etc .; STRIP antagonists, down-regulators, blockers, blockers, etc. Small interfering RNA molecules, short hairpin RNAs or antisense nucleotides that specifically interfere with the expression of the STRIP gene are preferred, and short hairpin RNAs with simple structures are more preferred.
  • the coding DNA sequence of the short hairpin RNA is shown in the table below:
  • a recombinant carrier for inhibitors of striated protein interacting proteins is provided and its use in the preparation of anti-tumor drugs.
  • the recombinant protein inhibitor vector for striatum interaction protein includes an expression vector and a coding DNA sequence inserted into the STRIP siRNA, STRIP shRNA or STRIP antisense nucleotide provided on the expression vector.
  • the vectors include viral vectors and non-viral vectors.
  • the "viral vector” includes adenovirus, adeno-associated virus, lentivirus, coxsackie virus, herpes simplex virus, measles virus, Newcastle disease virus, parvovirus, poliovirus, reovirus, vaccinia virus With vesicular stomatitis virus and so on. Suitable viral vectors are well known to those of ordinary skill in the art.
  • non-viral vector includes liposomes or lipid complexes, cationic polymers, chitosan polymers, nanoparticle carriers, etc. Suitable non-viral vectors are well known to those of ordinary skill in the art.
  • the inventors discovered through research that STRIP1 shRNA interference significantly inhibited the activation of ISRE transcriptional activity by vaccinia virus, indicating that STRIP1 inhibition can reduce the level of tumor cell response to vaccinia virus (as shown in Figure 3); oncolytic vaccinia virus The replication level of oncoVV-STRIP1 shRNA in tumor cells is significantly higher than the control virus (as shown in Figure 5).
  • the third aspect of the present invention particularly provides an oncolytic vaccinia virus STRIP shRNA recombinant vector and its use in the preparation of anti-tumor drugs.
  • the vaccinia virus is vaccinia virus Western Reserve strain, vaccinia virus Tiantan strain, vaccinia virus Wyeth strain, vaccinia virus Copenhagen strain, vaccinia virus Lister strain or vaccinia virus NYCBH strain.
  • an anti-tumor pharmaceutical composition comprising an active component and a medically acceptable excipient, carrier or diluent.
  • the active component includes a striatin interaction protein inhibitor or a striatin interaction protein inhibitor recombinant carrier.
  • the striatin interaction protein inhibitor, the striatin interaction protein inhibitor recombinant carrier, oncoVV-STRIP1 shRNA of the invention can inhibit liver cancer, glioma, breast cancer, lung cancer, gastrointestinal carcinoid, prostate cancer, etc. Most tumor cells grow.
  • the recombinant virus of the present invention and pharmaceutically acceptable excipients together form an anti-tumor pharmaceutical composition, so as to exert a more stable therapeutic effect.
  • These preparations can ensure the conformational integrity of the amino acid core sequence of the bispecific antibody disclosed in the present invention, while It is necessary to protect the multifunctional groups of proteins and prevent their degradation (including but not limited to coagulation, deamination or oxidation).
  • liquid preparations can be kept stable at 2 ° C-8 ° C for at least one year, and lyophilized preparations are stable at 30 ° C for at least six months.
  • the preparation can be suspension, water injection, lyophilization and other preparations commonly used in the pharmaceutical field.
  • the dosage when the recombinant virus and its composition are administered to animals including humans, the dosage varies according to the age and weight of the patient, the characteristics and severity of the disease, and the route of administration. Refer to the results of animal experiments In various situations, the total dose cannot exceed a certain range.
  • the present invention provides the use of STRIP inhibitors in the preparation of anti-tumor drugs. It has been experimentally confirmed that STRIP inhibitors can reduce the antiviral ability of tumor cells, which is conducive to a large amount of virus replication in tumor cells. , STRIP shRNA interference significantly inhibited the activation of vaccinia virus on ISRE transcription activity, indicating that the inhibition of STRIP1 can reduce the response level of tumor cells to vaccinia virus, while the oncoVV-STRIP1 oncolytic vaccinia virus shRNA replication level in tumor cells is significantly higher For control virus.
  • the suppression of STRIP of the present invention can be used to prepare anti-tumor virus drugs, especially recombining it with vaccinia virus into a recombinant vector, which can combine the advantages of STRIP inhibitor and vaccinia virus, and provides a new target for the viral treatment of tumors. Has broad clinical application prospects.
  • Figure 1 shows that the siRNA interference of STRIP1 and STRIP2 on the antiviral factor Interferon-induced proteins with tetratricopeptide repeats 1 (IFIT1), DExH-Box Helicase 58 (DHX58) and Interferon-stimulated gene 15 (ISG15) in malignant glioblastoma U87MG cells ) The effect of the level of transcription.
  • IFIT1 Interferon-induced proteins with tetratricopeptide repeats 1
  • DHX58 DExH-Box Helicase 58
  • ISG15 Interferon-stimulated gene 15
  • Figure 2 shows the effect of STRIP1 shRNA interference on the expression level of antiviral factor IFI16 in hepatoma cells SMMC-7721.
  • Figure 3 shows the effect of STRIP1 after shRNA interference on the activation of interferon-stimulated response element ISRE after U87MG cells were infected with vaccinia virus.
  • Figure 4 shows the effect of shRNA interference on STRIP1 in U87MG cells on the expression levels of caspase 3 and caspase 8.
  • Figure 5 shows that the growth of transplanted tumors in mice was significantly inhibited by U87MG cells after STRIP1 shRNA interference.
  • Figure 6 is a map of pCB-STRIP1 shRNA plasmid.
  • Fig. 7 is a comparison result of the replication levels of oncoVV-STRIP1 shRNA # 2, oncoVV-STRIP1 shRNA # 3 and control virus oncoVV in tumor cells SHG44 (A), BEL-7404 (B) and U87MG (C).
  • Figure 8 shows the detection of oncoVV-STRIP1 oncoVV-STRIP1, shRNA # 2 and oncoVV-STRIP1 shRNA # 3 by MTT method on tumor cells MDA-MB-231 (A), BEL-7404 (B), U87MG (C), A549 in vitro (D), HCT116 (E), HT-29 (F) and DU145 (G) growth inhibition.
  • Figure 9 shows the inhibitory effect of oncoVV-STRIP1 shRNA # 3 on tumor transplantation of U87MG mice in vivo, with PBS and oncoVV-GM-CSF as controls.
  • Figure 10 shows the effect of oncoVV-STRIP1 shRNA # 1 and oncoVV-STRIP1 shRNA # 3 on survival time of U87MG tumor-bearing mice. PBS and oncoVV-GM-CSF are controls.
  • Example 1 siRNA interference of STRIP1 and STRIP2 results in a significant reduction of antiviral factor levels in malignant glioblastoma U87MG cells
  • STRIP1 or STRIP2 siRNA purchased from Santa Cruz Biotechnology was transfected into glioma cells U87MG, non-specific RNA (Santa Cruz Biotechnology) was used as a control.
  • the cell transfection process is as follows:
  • Reverse transcription PCR method was used to detect the intracellular antiviral factors Interferon-induced proteins with tetratricopeptides repeats 1 (IFIT1), DExH-Box Helicase 58 (DHX58) and Interferon-stimulated genes 15 (ISG15), GAPDH as internal reference.
  • IFIT1 Interferon-induced proteins with tetratricopeptides repeats 1
  • DHX58 DExH-Box Helicase 58
  • ISG15 Interferon-stimulated genes 15
  • RNA interference plasmid of STRIP1 was transfected into SMMC-7721 hepatocellular carcinoma cells, stable expression cell lines were established. Non-specific RNA plasmids were used as controls to establish control cell lines.
  • the method of plasmid transfection is the same as that of Example 1.
  • the transfected cells are screened with puromycin to establish a stable transfected cell line.
  • the cell screening method is as follows:
  • the Western blot method is as follows:
  • Blocking allows antibodies to bind to specific proteins alone.
  • the common blocking solution in the laboratory is 5% BSA or skimmed milk powder, which is dissolved with TBST. We use 5% skimmed milk powder. After the membrane transfer is completed, the membrane is taken out and soaked in the prepared blocking solution, incubated on a shaker for 2 hours, or overnight in a refrigerator at 4 ° C.
  • the secondary antibody can be recovered after incubation.
  • the membrane is washed three times with TBST for 10 minutes each time, so that the non-specific binding of the secondary antibody can be washed away. Insufficient washing at this step will also make the background of the membrane scanning result high.
  • STRIP1 shRNA interference can inhibit the activation of interferon-stimulated response element ISRE in tumor cells caused by vaccinia virus infection
  • the well-grown U87MG cells were spread on a 24-well plate at 80,000 cells per well and 0.5 mL of fresh culture medium. After the cells adhere to the wall, co-transfect the cells with the ISRE firefly luciferase reporter gene plasmid and the Renilla luciferase plasmid at a concentration of 500: 1 (follow the above cell transfection steps), in which the firefly luciferase report
  • the gene plasmid is 400ng per well. 6-8h after transfection, aspirate the culture solution and add 0.5mL of fresh culture solution to reduce the toxicity of the transfection reagent to the cells. Then, the cells were lysed in the culture plate.
  • STRIP1 shRNA interference can increase the expression of caspase 3 and caspase 8.
  • the U87MG cell line stably expressing STRIP1 shRNA was lysed, and the cells expressing non-specific RNA were used as controls.
  • the total protein was collected and the caspase 3 and caspase 8 of the cells were detected by Western blot method.
  • the Western blot method is as described above.
  • the pCB-STRIP1 shRNA recombinant plasmid was constructed according to the plasmid map shown in FIG. 6. Among them, the coding DNA sequence of shRNA specifically inhibiting the expression of STRIP1 is shown in Table 1 below. Of course, the RNA sequence can also be used for the construction of recombinant plasmids.
  • telomere sequences 1, 2 and 3 of the STRIP1 shRNA sequence shown in the table above into the pCB plasmid through the Bgl II and Xba I sites respectively to obtain pCB-STRIP1 shRNA # 1, pCB-STRIP1 shRNA # 2 and pCB-STRIP1 shRNA # 3 Plasmid.
  • the vTK-L and vTK-R regions in the pCB plasmid homologously recombine with the thymidine kinase TK region of the wild-type virus, inserting the foreign gene sequence into the TK region and causing TK deletion.
  • Vaccinia virus replication depends on TK, and the TK level of tumor cells is much higher than that of normal cells. Therefore, TK-deleted vaccinia virus has the characteristics of specific replication in tumor cells.
  • the plasmid also carries the xanthine-guanine phoshporibosyl transferase (gpt) gene as a selection gene.
  • the gpt gene comes from Escherichia coli.
  • MPA mycophenolic acid
  • the virus or cell can block the synthesis of guanine due to MPA, so that the nucleic acid synthesis of the virus or cell cannot proceed normally and die.
  • cells or viruses can use hypoxanthine and xanthine to synthesize guanine through alternative pathways, making nucleic acid synthesis unrestricted.
  • Purified recombinant virus is obtained by adding mycophenolic acid, hypoxanthine and xanthine to the culture medium to clear the wild-type virus.
  • Inoculate 293A cells in good growth condition in a petri dish and the cell density can reach about 80% -90% the next day.
  • the virus solution collected each time is repeatedly screened 3 to 4 times according to the above method.
  • Example 7 OncoVV-STRIP1 oncolytic vaccinia virus shRNA replication levels in tumor cells are significantly higher than the control virus
  • Hepatoma cells BEL-7404, glioma cells U87MG and SHG44 were inoculated into 96-well plates at a density of 5 ⁇ 10 3 / well, and 90 ⁇ L of cell culture fluid was added to each well for overnight cultivation, and 5MOI oncoVV-STRIP1 shRNA # 2, OncoVV-STRIP1 shRNA # 3 or control virus oncoVV, set more than 3 replicate wells per time gradient.
  • the cells and culture medium were collected together, and the replication efficiency of the virus in tumor cells was detected by the TCID50 method (half the amount of tissue culture infection). Methods as below:
  • HEK-293A was plated in a 96-well plate, 4000 cells per well.
  • Infected virus samples Infect the diluted virus samples in sequence in 96-well plates, repeat 8 replicate wells for each concentration gradient, and add 100 ⁇ L of virus solution to each well. Place in a 5% CO 2 incubator at 37 ° C for 5-8 days.
  • s sum of positive ratios (from the first 10-fold dilution);
  • Example 8 MTT method was used to detect the in vitro inhibitory effect of oncoVV-STRIP1 shRNA # 3 and oncoVV-STRIP1 shRNA # 2 on tumor cells in vitro
  • breast cancer cells MDA-MB-231, liver cancer cells BEL-7404, glioma cells U87MG, lung cancer cells A549, colorectal cancer cells HCT116 and HT-29, and prostate cancer cells DU145 were selected according to 5 ⁇ 10 3 / The density of the wells was inoculated into 96-well plates, and 90 ⁇ L of cell culture fluid was added to each well for overnight cultivation. 2MOI, 5MOI or 10MOI viruses were added respectively, and 6 replicate wells were set. Cell culture medium.
  • Cell survival rate (OD value of treatment group-OD value of zero-adjusted group) / (OD value of control group-OD value of zero-adjusted group) ⁇ 100%.
  • the glioma cells U87MG were used to establish subcutaneously transplanted tumors in nude mice.
  • OncoVV-GM-CSF, oncoVV-STRIP1, shRNA # 3 or equal volume of PBS were injected intraperitoneally, and the tumor volume was measured regularly.
  • oncoVV-GM-CSF is similar to the oncolytic vaccinia virus drug Pexa-Vec (formerly known as JX-594) that has entered clinical phase III, and is built on the basis of literature reports (Parato, KA, CJ Breitbach, et al. 2012. The oncolytic poxvirus JX-594 selectively replicates and destroys cancer cells driven by genetic pathways commonly activated cancers. Mol Ther 20 (4): 749-758.). Animal experiment methods are as follows:
  • the glioma cells U87MG were used to establish subcutaneous xenograft tumors in nude mice. Intraperitoneal injection of 1 ⁇ 10 7 pfu oncoVV-GM-CSF, oncoVV-STRIP1 shRNA # 1, oncoVV-STRIP1 shRNA # 3 or equal volume of PBS was used to record the time of mouse death.
  • the animal experiment method is the same as described in Example 9.

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Abstract

提供了纹蛋白互作蛋白(STRIP)抑制剂在制备抗肿瘤药物中的用途、纹蛋白互作蛋白抑制剂重组载体及其在制备抗肿瘤药物中的用途、以及以纹蛋白互作蛋白抑制剂或纹蛋白互作蛋白抑制剂重组载体作为活性组分的抗肿瘤药物组合物。

Description

纹蛋白互作蛋白抑制剂及其在制备抗肿瘤药物中的用途 技术领域
本发明涉及生物医药技术领域，具体涉及纹蛋白互作蛋白(Striatin interacting protein,STRIP)作为靶点在制备抗肿瘤药物中的应用、包含其的载体和药物。
背景技术
溶瘤病毒是指能够选择性感染并损伤肿瘤组织的有治疗价值的病毒。溶瘤病毒有着悠久的历史，至今已有分属10个病毒家族的溶瘤病毒进入临床试验，包括腺病毒(Adenovirus)、柯萨奇病毒(Coxackie virus)、单纯疱疹病毒(Herpes simplex virus,HSV)、麻疹病毒(Measles virus)、新城疫病毒(Newcastle disease virus)、细小病毒(Parvovirus)、脊髓灰质炎病毒(Poliovirus)、呼肠孤病毒(Reovirus)、痘苗病毒(Vaccinia virus)与水疱性口炎病毒(Vesicular stomatitis virus)。目前已有疱疹病毒T-VEC被FDA批准治疗黑色素瘤。总体上，溶瘤病毒在临床试验中表现出了可观的安全性和有效性。
痘苗病毒是痘病毒家族中的大双链DNA病毒，相比其他溶瘤病毒有以下几个优点：(1)其在DNA病毒中是独一无二的，因为它只在细胞质中复制，以尽量减少整合在宿主基因组内的风险；(2)痘苗病毒最早被制成疫苗来对抗天花病毒，其安全性有较大的保障；(3)痘苗病毒的克隆能力大，可以允许较大长度的基因片段的插入；(4)病毒复制能力强。
溶瘤痘苗病毒已经表现出抗肿瘤效力。目前常见的溶瘤痘苗病毒大都经过减毒改造，包括胸腺嘧啶激酶TK基因缺失或TK/痘苗病毒生长因子基因双缺失的病毒株等。TK基因的缺失使痘苗病毒的复制更依赖于细胞内的TK水平，而癌细胞相比于正常细胞往往具有更高水平的TK；另外，痘苗病毒的复制还依赖于表皮生长因子受体EGFR/Ras通路的驱动，从而使其对癌细胞具有很强的选择性。溶瘤痘苗病毒在实验动物和人体上均表现出向癌组织靶向性聚集的能力，因此已成为癌症治疗的理想病毒载体。
纹蛋白互作蛋白(Striatin interacting protein,STRIP)是纹蛋白互作磷酸酶与激酶(Striatin interacting phosphatases and kinases,STRIPAK)复合体中的成分(Goudreault,M.,L.M.D'Ambrosio,et al.2009.A PP2A phosphatase high density interaction network identifies a novel striatin-interacting phosphatase and kinase complex linked to the cerebral cavernous  malformation 3(CCM3)protein.Mol Cell Proteomics 8(1):157-171.)，包含STRIP1(GenBank No.NM_033088.3)和STRIP2(GenBank No.NM_020704.2)，又名FAM40A和FAM40B。已有研究表明，果蝇STRIP参与负调控CLOCK蛋白去磷酸化，从而参与调节日夜节律(Andreazza,S.,S.Bouleau,et al.2015.Daytime CLOCK Dephosphorylation Is Controlled by STRIPAK Complexes in Drosophila.Cell Rep 11(8):1266-1279.)。STRIP1是调节小鼠胚胎中胚层正常移动的重要因子(Bazzi,H.,E.Soroka,et al.2017.STRIP1,a core component of STRIPAK complexes,is essential for normal mesoderm migration in the mouse embryo.Proc Natl Acad Sci U S A 114(51):E10928-E10936.)。STRIP2在鼠胚胎干细胞的分化过程中执行关键功能(Sabour,D.,S.P.Srinivasan,et al.2017.STRIP2 Is Indispensable for the Onset of Embryonic Stem Cell Differentiation.Mol Ther Methods Clin Dev 5:116-129.)。此外，在乳腺癌小鼠模型上的研究显示，抑制STRIP1的水平可提高乳腺癌细胞在小鼠体内的转移，而提高STRIP1的水平则可抑制乳腺癌细胞的转移。相反的，抑制STRIP2的水平却抑制了乳腺癌细胞在小鼠体内的转移(Madsen,C.D.,S.Hooper,et al.2015.STRIPAK components determine mode of cancer cell migration and metastasis.Nat Cell Biol 17(1):68-80.)。
由此可知，STRIP在低等和高等生物的细胞分化中均起到重要作用，与乳腺癌细胞的转移也有一定关系，但STRIP与其他肿瘤的生长与抑制之间是否存在调控关系尚未见相关报道。此外，将溶瘤痘苗病毒作为载体和STRIP相配合，二者之间是否只是单纯存在载体表达关系，还是STRIP也能够对溶瘤痘苗病毒在肿瘤细胞中的复制进行调控，也尚未见相关报道。
本研究通过抑制STRIP来调控溶瘤痘苗病毒在肿瘤细胞内的复制水平，并显著提高溶瘤痘苗病毒的溶肿瘤效果，为溶瘤病毒抗肿瘤药物的研发提供了一条新的技术途径。
发明内容
本发明的第一目的在于，对纹蛋白互作蛋白(STRIP)与肿瘤细胞之间关系进行研究，提供纹蛋白互作蛋白(STRIP)抑制剂的新用途；本发明的另一目的在于，对STRIP与侵入肿瘤细胞中的溶瘤痘苗病毒的调控关系进行研究，为溶瘤病毒抗肿瘤药物的研发提供了一条新的技术途径。
本发明通过研究，STRIP1和STRIP2的siRNA干扰能够显著降低恶性胶质母细胞瘤U87MG细胞内抗病毒因子Interferon-induced protein with tetratricopeptide repeats 1 (IFIT1)、DExH-Box Helicase 58(DHX58)以及Interferon-stimulated gene 15(ISG15) 的转录水平(如图1所示)。
STRIP1的shRNA干扰能够导致肝癌细胞SMMC-7721内抗病毒因子IFI16的表达水平显著降低(如图2所示)。
STRIP1的干扰提高caspase 3和caspase 8的表达，表明STRIP1的干扰有助于提高肿瘤细胞对凋亡诱导的敏感度(见图4所示)。
由此可知，通过抑制STRIP可显著降低肿瘤细胞的抗病毒能力，可用于制备抗肿瘤的病毒药物。
因此，本发明的第一方面，提供了一种纹蛋白互作蛋白抑制剂在制备抗肿瘤药物中的用途。
所述的纹蛋白互作蛋白抑制剂为任何能够降低STRIP的活性、降低STRIP的稳定性、抑制STRIP的表达、减少STRIP的有效作用时间或抑制STRIP的转录活加工的物质，包括但不限于：特异性干扰STRIP基因表达、加工的小干扰分子，如shRNA分子、siRNA分子、反义核苷酸等；STRIP的拮抗剂、下调剂、阻滞剂、阻断剂等。优选特异性干扰STRIP基因表达的小干扰RNA分子、短发夹RNA或反义核苷酸，更优选结构简单的短发夹RNA。
该短发夹RNA的序列如下表所示：
Figure PCTCN2019110713-appb-000001
该短发夹RNA的编码DNA序列如下表所示：
Figure PCTCN2019110713-appb-000002
本发明的第二方面，提供了纹蛋白互作蛋白抑制剂重组载体及其在制备抗肿瘤药物中的用途。
该纹蛋白互作蛋白抑制剂重组载体包括表达载体以及插入设置在该表达载体上的STRIP siRNA、STRIP shRNA或STRIP反义核苷酸的编码DNA序列。
所述的载体包括病毒载体和非病毒载体。
所述的“病毒载体”，包括腺病毒、腺相关病毒、慢病毒、柯萨奇病毒、单纯疱疹病毒、麻疹病毒、新城疫病毒、细小病毒、脊髓灰质炎病毒、呼肠孤病毒、痘苗病毒与水疱性口炎病毒等。合适的病毒载体是本领域普通技术人员所熟知的。
所述的“非病毒载体”，包括脂质体或脂类复合物、阳离子多聚物、壳聚糖聚合物及纳米粒子载体等。合适的非病毒载体是本领域普通技术人员所熟知的。
此外，发明人通过研究发现，STRIP1的shRNA干扰显著抑制了痘苗病毒对ISRE转录活性的激活，表明STRIP1的抑制可降低肿瘤细胞对痘苗病毒的应答水平(如图3所示)；溶瘤痘苗病毒oncoVV-STRIP1 shRNA在肿瘤细胞内的复制水平显著高于对照病毒(如图5所示)。
因此，本发明的第三方面，特别提供了溶瘤痘苗病毒STRIP shRNA重组载体及其在制备抗肿瘤药物中的用途。
优选的，痘苗病毒为痘苗病毒Western Reserve株、痘苗病毒天坛株、痘苗病毒Wyeth株、痘苗病毒Copenhagen株、痘苗病毒Lister株或痘苗病毒NYCBH株。
本发明的第四方面，提供了抗肿瘤药物组合物，该抗肿瘤药物组合物包括活性组分以及医学上可接受的赋形剂、载体或稀释剂。该活性组分包括纹蛋白互作蛋白抑制剂或纹蛋白互作蛋白抑制剂重组载体。
本发明的纹蛋白互作蛋白抑制剂、纹蛋白互作蛋白抑制剂重组载体、溶瘤痘苗病毒oncoVV-STRIP1 shRNA等可以抑制肝癌、胶质瘤、乳腺癌、肺癌、肠胃类癌、前列腺癌等大部分肿瘤细胞的生长。
本发明的重组病毒和药学上可以接受的辅料一起组成抗肿瘤药物组合物，从而更稳定地发挥疗效，这些制剂可以保证本发明公开的双特异性抗体氨基酸核心序列的构像完整性，同时还要保护蛋白质的多官能团，防止其降解(包括但不限于凝聚、脱氨或氧化)。
通常情况下，液体制剂可以在2℃-8℃条件下保存至少稳定一年，冻干制剂在30℃至少六个月保持稳定。制剂可为制药领域常用的混悬、水针、冻干等制剂。
本发明中重组病毒及其组合物在对包括人在内的动物给药时，给药剂量因病人的年 龄和体重，疾病特性和严重性，以及给药途径而异，可以参考动物实验的结果和种种情况，总给药量不能超过一定范围。
本发明的有益保障及效果：
本发明提供了纹蛋白互作蛋白(STRIP)抑制剂在制备抗肿瘤药物中的用途，通过实验证实，STRIP抑制剂能够导致肿瘤细胞抗病毒能力降低，有利于病毒在肿瘤细胞内大量复制，此外，STRIP的shRNA干扰显著抑制了痘苗病毒对ISRE转录活性的激活，表明STRIP1的抑制可降低肿瘤细胞对痘苗病毒的应答水平，同时溶瘤痘苗病毒oncoVV-STRIP1 shRNA在肿瘤细胞内的复制水平显著高于对照病毒。因此本发明针对STRIP的抑制可用于制备抗肿瘤的病毒药物，尤其将其和痘苗病毒重组成重组载体，能够结合STRIP抑制剂和痘苗病毒的优点，为肿瘤的病毒治疗提供了新的靶点，具备广阔的临床应用前景。
附图说明
图1为STRIP1和STRIP2的siRNA干扰对恶性胶质母细胞瘤U87MG细胞内抗病毒因子Interferon-induced protein with tetratricopeptide repeats 1(IFIT1)、DExH-Box Helicase 58(DHX58)以及Interferon-stimulated gene 15(ISG15)的转录水平的影响。
图2为STRIP1的shRNA干扰对肝癌细胞SMMC-7721内抗病毒因子IFI16的表达水平的影响。
图3为STRIP1进行shRNA干扰后对U87MG细胞感染痘苗病毒后干扰素刺激应答元件ISRE的激活作用的影响。
图4为对U87MG细胞内STRIP1进行shRNA干扰后对caspase 3和caspase 8的表达水平的影响。
图5为U87MG细胞进行STRIP1 shRNA干扰后在小鼠体内的移植瘤生长受到明显抑制。
图6为pCB-STRIP1 shRNA质粒图谱。
图7为溶瘤痘苗病毒oncoVV-STRIP1 shRNA#2、oncoVV-STRIP1 shRNA#3及对照病毒oncoVV在肿瘤细胞SHG44(A)、BEL-7404(B)和U87MG(C)内的复制水平比较结果。
图8为MTT法检测溶瘤痘苗病毒oncoVV-STRIP1 shRNA#2和oncoVV-STRIP1 shRNA#3在体外对肿瘤细胞MDA-MB-231(A)、BEL-7404(B)、U87MG(C)、A549(D)、 HCT116(E)、HT-29(F)和DU145(G)的生长抑制作用。
图9为溶瘤痘苗病毒oncoVV-STRIP1 shRNA#3在体内对U87MG小鼠移植瘤的抑制作用，PBS和oncoVV-GM-CSF为对照。
图10为溶瘤痘苗病毒oncoVV-STRIP1 shRNA#1和oncoVV-STRIP1 shRNA#3对U87MG荷瘤小鼠存活时间的影响，PBS和oncoVV-GM-CSF为对照。
具体实施方式
以下实施例、实验例对本发明进行进一步的说明，不应理解为对本发明的限制。实施例不包括对传统方法的详细描述，如PCR方法，那些用于构建载体和质粒的方法，将编码蛋白的基因插入到这样的载体和质粒的方法或将质粒引入宿主细胞的方法。这样的方法对于本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，包括Sambrook,J.,Fritsch,E.F.and Maniais，T.(1989)Molecular Cloning：A Laboratory Manual，2 ndedition，Cold spring Harbor Laboratory Press。
除非另外说明，否则百分比和份数按重量计算。除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明，具体实施方式文中所述的较佳实施方法与材料仅作示范之用。
实施例1、STRIP1和STRIP2的siRNA干扰导致恶性胶质母细胞瘤U87MG细胞内抗病毒因子水平显著降低
将STRIP1或STRIP2的siRNA(购自Santa Cruz Biotechnology)转染入胶质瘤细胞U87MG，非特异RNA(Santa Cruz Biotechnology)作为对照。细胞转染过程如下：
按照QIAGEN公司的Attractene Transfection Reagent Handbook说明书进行操作：
(1)转染之前，向24孔板里每孔接种3×10 4个细胞，每孔加入含1％双抗和10％胎牛血清的DMEM培养基500μL。37℃，5％CO 2培养箱内培养。
(2)待细胞贴壁后(一般需要2～3h，也可视不同细胞不同情况而定)转染，根据siRNA说明书将适量RNA加入到0.5mL离心管中。
(3)向上面的离心管中加入1.5μL的转染试剂，再加入无血清无双抗的培液至终体积为60μL，涡旋振荡器上振荡10s混匀。室温静置孵育10～15min。
(4)用移液枪轻轻吸弃每孔中原本的细胞培养液，加入新鲜的含10％胎牛血清及1％双抗的细胞培养液500μL。
(5)将孵育好的转染溶液加入细胞中，37℃，5％CO 2培养箱内培养。6～18h后换液。
用逆转录PCR方法检测上述细胞内抗病毒因子Interferon-induced protein with tetratricopeptide repeats 1(IFIT1)、DExH-Box Helicase 58(DHX58)以及Interferon-stimulated gene 15(ISG15)的转录水平，GAPDH作为内参。
实验结果如图1所示：STRIP1的siRNA干扰导致IFIH1、DHX58和ISG15转录水平显著下降，STRIP2的siRNA干扰导致DHX58和ISG15转录水平显著下降，表明STRIP1和STRIP2的抑制均可降低肿瘤细胞的抗病毒能力。
实施例2、STRIP1的shRNA干扰下调肝癌细胞SMMC-7721内抗病毒因子IFI16的表达水平
将STRIP1的shRNA干扰质粒转染入SMMC-7721肝癌细胞后建立稳定表达细胞株。非特异RNA质粒作为对照，建立对照细胞株。质粒转染方法同实施例1方法。转染后的细胞用嘌呤霉素进行筛选，建立稳定转染的细胞株，细胞筛选方法如下：
(1)筛选前先用不同的浓度梯度的嘌呤霉素加入待筛选的细胞中。
(2)一般48h后观察哪个浓度梯度嘌呤霉素下的细胞存活率为零，然后用该浓度的嘌呤霉素筛选稳转株细胞。
(3)转染待筛选的细胞，24～48h后观察细胞的转染率(本实验所用的目的质粒带有绿色荧光，可根据绿色荧光的量判断转染率)，当转染率达到50％左右，向细胞中加入上述浓度的嘌呤霉素进行筛选细胞。
(4)观察到没有转染进目的质粒的细胞全部死亡，即存活的细胞全部为带绿色荧光的细胞，并一直用筛选出的嘌呤霉素浓度来维持细胞，直至存活的全部细胞均为转染进目的质粒的细胞。
利用Western blot方法检测两种细胞内STRIP1和IFI16的表达水平，actin作为内参。
Western blot方法如下：
(1)蛋白样品的制备
将3×10 5个细胞的肝癌细胞BEL-7404铺于6孔板中，培养过夜后，每孔分别加入20MOI、50MOI的Ad-EGFP、Ad-CTTNBP2NL感染细胞，用加等体积的PBS加入细胞做对照，48h后，收集上清及还贴壁的细胞，900rpm，3min离心，弃上清加入100μL的已加入PMSF(终浓度为100μg/mL)的SDS裂解液，置于冰上15min，12000rpm，离心10min，收集上清后进行蛋白浓度测定，于-20℃保存。
(2)蛋白定量(参照BCA蛋白定量试剂盒比色法操作)
在96孔板中加入20μL的蛋白标准品或蛋白样品，每个孔中加入100μL的BCA工作试剂，轻微震荡混匀30s，用不干胶纸覆盖96孔板；37℃温育30min。取出96孔板，放冷至室温。在酶标仪检测上OD为562nm的读数。制作标准曲线，根据标准曲线进行蛋白样品浓度的计算。
(3)蛋白样品预处理
电泳前需要把上样量以及上样体积计算好，在蛋白样品中加入1×SDS Loading Buffer。并在金属浴上95℃，煮沸5min。
(4)SDS聚丙烯酸胺凝胶的配制
根据要检测的分子量大小不同配制相应浓度的分离胶，将两块玻璃板排齐，装入密封用硅胶架，用ddH 2O检测是否会漏胶，按照下面配方配制12％的分离胶以及5％的浓缩胶，灌胶前再加TEMED。将检漏的ddH 2O倒掉后加入配好的分离胶，并在胶上面缓慢的加无水乙醇，使胶凝集的更好。约40min分离胶凝集，弃去无水乙醇后加入配好的5％的浓缩胶，插入干净的10孔1.5mm的梳子。
(5)SDS聚丙烯酸胺凝胶加样及电泳
等到胶凝好以后，把玻璃板夹入到电泳仪内，加入电泳缓冲液，垂直向上轻轻的拔出梳子，将制备好的蛋白样品依次加入各孔，上样完成后连接好正负极，盖紧盖子，80V电压跑浓缩胶约30min，直至样品到浓缩胶与分离胶的交接处，换120V电压，约1h。
(6)转膜
等蓝色的Loading到达胶的底部，电泳完成，关闭电源，取出玻璃板后用刮刀在两块板中轻撬使玻璃板和胶分离，采用半干转的方法使胶中的蛋白质转移到膜上。将滤纸和海绵浸泡到预冷的电转液中，合适大小的膜在甲醇中浸泡1min激活后浸泡在电转液里，按照夹子白色的一面在下，依次铺上海绵，两张滤纸，甲醇激活的膜，蛋白胶，两张滤纸，海绵，注意放置的时候膜在正极一侧且不要出现气泡，如果膜与胶之间有气泡可以滴加少许电转液将气泡赶走，动作要轻柔，切勿拖拽膜和蛋白胶，确定完全铺好且没有气泡，就可以把夹子夹好，放入电转槽中，加满预冷的电转液后盖好盖子，电转槽置于冰中以利于降温，接通电源，100V，转膜2h。
(7)封闭
封闭可以使抗体单独和特异的蛋白结合，实验室里常见封闭液是5％的BSA或脱脂奶粉，都用TBST溶解。我们使用的是5％的脱脂奶粉，转膜完成后，将膜取出浸泡在已经配 好的封闭液中，置于摇床上孵育2h，也可以4℃冰箱中过夜。
(8)一抗的孵育
按照合适的稀释比例将所要孵育的一抗配好，一般用5％的BSA(TBST溶解)按1:4000的比例稀释10mL的一抗孵育液，置于干净的抗体孵育盒中备用，取出封闭好的膜，在TBST中把多余的封闭液漂洗干净后，使膜浸润在一抗稀释液中，注意不要有气泡，冰箱4℃孵育过夜。
(9)洗涤
用TBST洗涤3次，每次10min，可以将一抗与抗原的非特异性结合洗去，若洗不干净，则会使扫膜结果背景高。
(10)二抗的孵育
根据一抗选择相应的二抗，用TBST按照1:5000的比例稀释，摇床上室温轻摇孵育2h。
(11)洗涤
二抗孵育结束后可回收，膜用TBST洗涤3次，每次10min，这样可以将二抗的非特异性结合洗去，此步洗涤不充分也会使扫膜结果背景高。
(12)膜的扫描
将A、B显影液按照1:1的比例混匀，用多少配制多少，避免浪费，避光存放，在膜上滴加显影液，使膜完全浸润后，放入超灵敏化学发光仪中曝光显影。再在白光下拍摄marker，将两张图合并后对照marker条带大小进行分析。
实验结果如图2所示：STRIP1的shRNA干扰导致SMMC-7721细胞内抗病毒因子IFI16的表达水平显著降低，结果进一步表明STRIP1的抑制可降低肿瘤细胞的抗病毒能力。
实施例3、STRIP1的shRNA干扰可抑制痘苗病毒感染导致的肿瘤细胞内干扰素刺激应答元件ISRE的激活
构建STRIP1 shRNA干扰的质粒，转染入U87MG细胞后建立稳定表达细胞株。表达非特异RNA的质粒作为对照，转染细胞后建立对照细胞株。两种细胞感染或不感染痘苗病毒，并转染入ISRE-Luc报告质粒，收集细胞蛋白进行报告基因检测，方法如下：
将生长状态良好的U87MG细胞按每孔8万个细胞，0.5mL新鲜培养液铺24孔板。待细胞贴壁后，用ISRE萤火虫荧光素酶报告基因质粒和海肾荧光素酶质粒按500:1的浓度进行共转染细胞(按照上述细胞转染步骤进行操作)，其中萤火虫荧光素酶报告基因的质粒每孔为400ng。转染6-8h后，吸弃培养液，加入0.5mL新鲜的培养液，减少转染试剂对细胞的毒性。然后，在培养板中裂解细胞。首先清洗细胞，小心吸弃培养液，沿板壁缓慢加入足 量的PBS，上下轻轻晃动培养板几次，然后将PBS尽量吸取干净。接着按每孔100μL加入1×裂解缓冲液，将培养板放置在一个轻轻摆动的摇床上以确保裂解缓冲液能够完全覆盖单层细胞，室温摇动15min后将样品分别转移到干净的1.5mL离心管中。将样品进行适当倍数的稀释后利用化学发光酶标仪进行测定，测定过程中保证萤火虫荧光素和海肾荧光素的底物是充足过量的(底物现配现用)。
实验结果如图3显示：STRIP1的shRNA干扰显著抑制了痘苗病毒对ISRE转录活性的激活，表明STRIP1的抑制可降低肿瘤细胞对痘苗病毒的应答水平。
实施例4、STRIP1的shRNA干扰可提高caspase 3和caspase 8的表达
裂解稳定表达STRIP1 shRNA的U87MG细胞株，表达非特异RNA的细胞作为对照，收集总蛋白，用Western blot方法检测细胞的caspase 3和caspase 8。Western blot方法如前所述。
结果如图4显示：STRIP1的干扰提高caspase 3和caspase 8的表达，表明STRIP1的干扰有助于提高肿瘤细胞对凋亡诱导的敏感度。
实施例5、STRIP1的shRNA干扰显著延缓U87MG细胞株在小鼠体内移植瘤的生长
本研究检测实施例3所述构建的U87MG/STRIP1 shRNA细胞与对照细胞在裸鼠体内移植瘤的生长情况。动物实验操作严格参照NIH实验动物指导进行，选取4周龄雌性BALB/c裸鼠，在其前肢腋端皮下注射上述肿瘤细胞，每只小鼠注射1x10 7个/100μL的肿瘤细胞。注射细胞后用千分尺定期测量肿瘤体积，肿瘤的体积(mm 3)＝(长x宽 2)/2。
结果如图5显示：STRIP1的shRNA干扰显著延缓了U87MG细胞裸鼠皮下移植瘤的生长，但经过一段时间潜伏后最终会达到对照细胞的水平。
实施例6、表达STRIP1 shRNA的痘苗病毒oncoVV-STRIP1 shRNA的构建和鉴定
1、构建pCB-STRIP1 shRNA重组质粒
根据如图6所示的质粒图谱构建pCB-STRIP1 shRNA重组质粒。其中，特异性抑制STRIP1表达的shRNA的编码DNA序列如下表1所示，当然也可以将RNA序列用于重组质粒构建。
表1特异性抑制STRIP1表达的shRNA的编码DNA序列汇总
Figure PCTCN2019110713-appb-000003
Figure PCTCN2019110713-appb-000004
将上表所示的STRIP1 shRNA序列的编码DNA序列1、2和3分别通过Bgl II和Xba I位点插入pCB质粒，获得pCB-STRIP1 shRNA#1、pCB-STRIP1 shRNA#2和pCB-STRIP1 shRNA#3质粒。pCB质粒中的vTK-L与vTK-R区通过与野生型病毒的胸腺嘧啶激酶TK区同源重组，将外源基因序列插入TK区，同时造成TK缺失。痘苗病毒的复制依赖TK，而肿瘤细胞的TK水平远高于正常细胞，因此TK缺失的痘苗病毒具有在肿瘤细胞内特异复制的特点。
此外，该质粒还带有黄嘌呤鸟嘌呤磷酸转移酶(xanthine-guanine phoshporibosyl transferase，gpt)基因作为筛选基因。gpt基因来自大肠埃希菌，病毒或细胞在霉酚酸(MPA)存在的情况下，由于MPA可以阻断鸟嘌呤合成，使病毒或细胞核酸合成不能正常进行而死亡。而在gpt基因存在下，细胞或病毒可以利用次黄嘌呤(hypoxanthine)和黄嘌呤(xanthine)通过替代途径合成鸟嘌呤，使核酸合成不受限制。通过在培液中添加霉酚酸、次黄嘌呤和黄嘌呤进行野生型病毒的清除，获得纯化的重组病毒。
2、Western Reserve(WR)株痘苗病毒和pCB-STRIP1 shRNA#1、pCB-STRIP1 shRNA#2或pCB-STRIP1 shRNA#3质粒的重组
(1)在面积6cm 2的培养皿内接种适当数量的293A细胞，使之能于次日长至80-90％成片；
(2)弃去培养液，沿侧壁轻轻加入1mL Western Reserve(WR)株痘苗病毒(0.05～0.1MOI，用含2％血清的培养基稀释病毒液)，置于37℃，5％CO 2培养箱中培养2～4小时，其间大约每15min摇匀一次，以防细胞局部干死。
(3)细胞转染按照试剂盒(Effectene)说明书进行操作，步骤如下：
将1μg的pCB-STRIP1 shRNA补加buffer EC至150μL，再分别加8μL Enhance buffer，振荡1s，室温静置5min；在上述三份混合物中分别加入25μL的Effectene buffer，颠倒混匀5次，振荡10s，室温静置5～10min，然后向上述混合物中分别加入1mL的新鲜培养液(可含血清，抗生素)，上下颠倒两次；与此同时，将步骤2中的培液弃去，加入4mL10％FBS的新鲜培养液，并将混合好的转染液分别加入其中；将培养皿置于37℃，5％CO 2培养箱中培养6～18个小时后吸弃培养液，PBS洗一次，加入5mL的新鲜培养液继续培养。
(4)细胞完全病变后，在生物安全柜中收集病毒液，分装到离心管中，标记好，将 离心管置-80℃和37℃反复冻融三次，彻底裂解细胞释放病毒，2000rmp离心5min收集上清，放置于-80℃超低温冰箱中保存备用。
3、重组病毒的筛选
(1)将生长状态良好的293A细胞接种于培养皿中，次日细胞密度可达约80％-90％。
(2)准备三种筛选药物：黄嘌呤、次黄嘌呤、霉酚酸。
(3)向(1)中每个培养皿沿侧壁小心加入500μL之前包装出来的病毒液，置于37℃，5％CO 2培养箱中培养2-4h。约2～4h后，吸弃悬浮的病毒液，加入3mL新鲜培养液，其中含7.5μL的(1×)霉酚酸、75μL的(1×)黄嘌呤与7.5μL的(1×)次黄嘌呤。
(4)每天观察细胞病变情况，大约两三天后在生物安全柜中收集全部病变的细胞液，反复冻融三次，放置于-80℃超低温冰箱中保存备用。
(5)每次收集的病毒液按照以上方法重复筛选3～4次。
4、病毒挑空斑及鉴定
(1)5％低溶点胶的配制：称取0.25g的低熔点胶溶于5mL的PBS中，将其121℃高压灭菌20min，然后放于4℃冰箱保存备用。
(2)将状态良好的293A细胞接种于六孔板中，次日，待细胞密度达到90％左右时，将病毒液按照10 -4～10 -6梯度进行系列稀释，之后弃去六孔板中旧的培养液，向每孔加入1mL稀释好的病毒液使病毒吸附，置于培养箱中培养2～4h后将已煮沸的低熔点胶放置40℃水浴锅中保温，然后放入超净台，加入三倍体积的DMEM培养液使其最终浓度为1.25％，然后用吸管快速混匀并迅速用移液枪将板中的悬浮病毒液吸弃，用吸管沿侧壁小心加入2mL含1.25％低熔点胶的培液，注意不要将细胞吹起，然后将其置于37，5％CO 2的细胞培养箱培养。
(3)每天在倒置显微镜下观察细胞病变情况，如果有孤立的病毒空斑出现，将其挑取置于提前铺好293A细胞的12孔板中，标记好，将其置于37℃，5％CO 2的细胞培养箱培养，待其充分病变后，在安全柜收集病毒液于1.51mL离心管，置于-80℃超低温冰箱保存，以备下一步鉴定。
(4)利用野生型病毒具有完整的TK区而重组病毒不具有的特征进行PCR鉴定，获得纯化的痘苗病毒oncoVV-STRIP1 shRNA#1、oncoVV-STRIP1 shRNA#2和oncoVV-STRIP1 shRNA#3。
实施例7、溶瘤痘苗病毒oncoVV-STRIP1 shRNA在肿瘤细胞内的复制水平显著高于对照病毒
将肝癌细胞BEL-7404、胶质瘤细胞U87MG和SHG44按5×10 3/孔的密度接种到96孔板中，每孔加入90μL细胞培养液培养过夜，分别加入5MOI oncoVV-STRIP1 shRNA#2、oncoVV-STRIP1 shRNA#3或对照病毒oncoVV，每个时间梯度设置3个以上重复孔。将细胞和培液一起收集，利用TCID50法(半数组织培养感染量)对病毒在肿瘤细胞内的复制效率进行了检测。方法如下：
(1)细胞铺板，在96孔板中铺入HEK-293A，每孔4000个细胞。
(2)制备病毒稀释液：于超净台内无菌操作进行病毒样品稀释：将纯化后的病毒用无血清的DMEM分别稀释成10 -3、10 -4、10 -5、10 -6、10 -7、10 -8、10 -9和10 -10等几个不同的梯度。
(3)感染病毒样品：将稀释好的病毒样品依次感染96孔板，，每个浓度梯度重复8个复孔，每孔内加100μL病毒液。置于37℃，5％CO 2培养箱中正常培养5-8天。
(4)结果计算
病毒滴度计算公式：对于100μL样品，滴度T＝10 1+d(s-0.5)；
d＝log10稀释度＝1(对于10倍的稀释度而言)；
s＝阳性比率之和(从第一个10倍稀释度算起)；
按照以下公式，将TCID50/mL转换为PFU/mL：
T＝a×10 bTCID50/mL＝a×10 b-0.7PFU/mL；
检测结果如图7所示：oncoVV-STRIP1 shRNA#3在上述三种肿瘤细胞内的复制水平都显著优于oncoVV-STRIP1 shRNA#2和对照病毒oncoVV。
实施例8、MTT法检测oncoVV-STRIP1 shRNA#3和oncoVV-STRIP1 shRNA#2对肿瘤细胞的体外抑制效果
本实验选用乳腺癌细胞MDA-MB-231、肝癌细胞BEL-7404、胶质瘤细胞U87MG、肺癌细胞A549、结直肠癌细胞HCT116和HT-29以及前列腺癌细胞DU145，分别按5×10 3/孔的密度接种到96孔板中，每孔加入90μL细胞培养液培养过夜，分别加入2MOI、5MOI或10MOI病毒，设置6个重复孔，实验对照组为不加病毒的细胞，空白组为不含细胞的培液。
37℃，5％CO 2培养，设置48h和72h两个个时间梯度，到相应的时间点时避光每孔加入20μL MTT溶液(5mg/mL)。培养箱静置4h，吸去每组的培液后，每孔加入150μL二甲基亚砜，放在摇床上振荡10min，使结晶物充分溶解。置酶联检测仪上测定OD值，检测波长 490nm。
根据所测OD值计算细胞存活率，公式为：
细胞存活率＝(处理组OD值-调零组OD值)/(对照组OD值-调零组OD值)×100％。
分析结果如图8所示，oncoVV-STRIP1 shRNA#3和oncoVV-STRIP1 shRNA#2在体外均显著抑制肿瘤细胞的增殖，且呈剂量和时间依赖。
实施例9、oncoVV-STRIP1 shRNA显著消除U87MG小鼠移植瘤
利用胶质瘤细胞U87MG建立裸鼠皮下移植瘤，腹腔注射oncoVV-GM-CSF、oncoVV-STRIP1 shRNA#3或等体积PBS，定期测量肿瘤体积。oncoVV-GM-CSF类似于已进入临床III期的溶瘤痘苗病毒药物Pexa-Vec(先前名为JX-594)，依据文献报导自行构建(Parato,K.A.,C.J.Breitbach,et al.2012.The oncolytic poxvirus JX-594 selectively replicates in and destroys cancer cells driven by genetic pathways commonly activated in cancers.Mol Ther 20(4):749-758.)。动物实验方法如下：
本研究所有的动物实验操作严格参照NIH实验动物指导进行，选取4周龄雌性BALB/c裸鼠，按照每只小鼠注射1x10 7个/100μL细胞，在其前肢腋端皮下注射U87MG细胞。在注射细胞之后每天观察肿瘤体积的长势，并用千分尺进行测量，计算肿瘤体积((mm 3)＝(长×宽 2)/2)。待肿瘤长至稳定状态时，将其分为3组：PBS组，oncoVV-GM-CSF组及oncoVV-STRIP1 shRNA#3组，每组各6-8只裸鼠。分完组之后，每只裸鼠分别进行1次腹腔注射1x10 7pfu病毒或等体积PBS，然后定期测量肿瘤体积。
结果如图9所示：相比于PBS与oncoVV-GM-CSF对照，oncoVV-STRIP1 shRNA#3显著消除U87MG小鼠移植瘤。
实施例10、oncoVV-STRIP1 shRNA显著延长小鼠存活时间
利用胶质瘤细胞U87MG建立裸鼠皮下移植瘤，腹腔注射1×10 7pfu oncoVV-GM-CSF、oncoVV-STRIP1 shRNA#1、oncoVV-STRIP1 shRNA#3或等体积PBS，记录小鼠死亡时间。动物实验方法同实施例9所述。
结果如图10所示：相比于PBS，oncoVV-GM-CSF、oncoVV-STRIP1 shRNA#1和oncoVV-STRIP1 shRNA#3都显著延长荷瘤小鼠存活时间，但oncoVV-STRIP1 shRNA#3显著优于oncoVV-STRIP1 shRNA#1和oncoVV-GM-CSF。
以上已对本发明创造的较佳实施例进行了具体说明，但本发明创造并不限于所述实施 例，熟悉本领域的技术人员在不违背本发明创造精神的前提下还可做出种种的等同的变型或替换，这些等同的变型或替换均包含在本申请权利要求所限定的范围内。
Figure PCTCN2019110713-appb-000005
Figure PCTCN2019110713-appb-000006

Claims (11)

  1. 纹蛋白互作蛋白抑制剂在制备抗肿瘤药物中的用途。
  2. 根据权利要求1所述的纹蛋白互作蛋白抑制剂在制备抗肿瘤药物中的用途，其特征在于：所述用途为纹蛋白互作蛋白抑制剂在降低肿瘤细胞内抗病毒因子表达水平和抗病毒应答水平中的用途。
  3. 根据权利要求1或2所述的纹蛋白互作蛋白抑制剂在制备抗肿瘤药物中的用途，其特征在于：
    其中，所述纹蛋白互作蛋白抑制剂为任何能够降低纹蛋白互作蛋白的活性或稳定性、抑制纹蛋白互作蛋白基因表达或加工、减少纹蛋白互作蛋白有效作用时间或抑制纹蛋白互作蛋白基因转录的物质。
  4. 根据权利要求3所述的纹蛋白互作蛋白抑制剂在制备抗肿瘤药物中的用途，其特征在于：
    其中，所述纹蛋白互作蛋白抑制剂为特异性干扰纹蛋白互作蛋白基因表达的小干扰RNA分子、短发夹RNA或反义核苷酸。
  5. 根据权利要求4所述的纹蛋白互作蛋白抑制剂在制备抗肿瘤药物中的用途，其特征在于：
    其中，所述纹蛋白互作蛋白抑制剂为短发夹RNA，该短发夹RNA的序列如SEQ ID NO.1、SEQ ID NO.2或SEQ ID NO.3所示。
  6. 一种纹蛋白互作蛋白抑制剂重组载体，其特征在于，所述重组载体包括表达载体以及插入设置在该表达载体上的STRIP siRNA、STRIP shRNA或STRIP反义核苷酸。
  7. 根据权利要求6所述的纹蛋白互作蛋白抑制剂重组载体，其特征在于：
    其中，所述表达载体为质粒载体、粘粒载体、噬菌体载体或病毒载体，所述病毒载体选自腺病毒、腺相关病毒、慢病毒、柯萨奇病毒、单纯疱疹病毒、麻疹病毒、新城疫病毒、细小病毒、脊髓灰质炎病毒、呼肠孤病毒、痘苗病毒与水疱性口炎病毒。
  8. 根据权利要求6所述的纹蛋白互作蛋白抑制剂重组载体，其特征在于：
    其中，所述表达载体为痘苗病毒，所述痘苗病毒为痘苗病毒Western Reserve株、痘苗病毒天坛株、痘苗病毒Wyeth株、痘苗病毒Copenhagen株、痘苗病毒Lister株或痘苗病毒NYCBH株。
  9. 权利要求6所述的纹蛋白互作蛋白抑制剂重组载体在制备抗肿瘤药物中的用途。
  10. 根据权利要求9所述的纹蛋白互作蛋白抑制剂重组载体在制备抗肿瘤药物中的用途，其特征在于：
    其中，所述纹蛋白互作蛋白抑制剂重组载体为STRIP shRNA痘苗病毒。
  11. 一种抗肿瘤药物组合物，其特征在于，该抗肿瘤药物组合物包括活性组分以及医学上可接受的赋形剂、载体或稀释剂，
    其中，所述活性组分为权利要求1～5任一项所述的纹蛋白互作蛋白抑制剂或权利要求6～10任一项所述的纹蛋白互作蛋白抑制剂重组载体。
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