WO2016187908A1 - 重组溶瘤腺病毒及其应用 - Google Patents

重组溶瘤腺病毒及其应用 Download PDF

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WO2016187908A1
WO2016187908A1 PCT/CN2015/081560 CN2015081560W WO2016187908A1 WO 2016187908 A1 WO2016187908 A1 WO 2016187908A1 CN 2015081560 W CN2015081560 W CN 2015081560W WO 2016187908 A1 WO2016187908 A1 WO 2016187908A1
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tumor
gene
oncolytic adenovirus
recombinant oncolytic
cells
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金宁一
李霄
鲁会军
李昌
田明尧
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金宁一
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/861—Adenoviral vectors

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  • the present invention relates to the field of biotechnology and gene therapy, and in particular to a novel recombinant oncolytic adenovirus and application.
  • malignant tumors are dynamic processes involving multiple factors.
  • the experience of tumor gene therapy research over the years has shown that how to achieve victory in the race against malignant tumors is the key to effective treatment. Therefore, the multi-factors and specificities in the occurrence and development of malignant tumors should be taken as the entry point to achieve the dynamic balance of the tumor-inhibiting process, and finally achieve accurate and rapid tumor suppression and effectively treat malignant tumors.
  • Surgical treatment, chemotherapy and radiotherapy are the preferred methods for current cancer treatment, but they will cause irreversible trauma and physiological secondary effects on the body, and the radical cure is incomplete. Therefore, at the beginning of the research on new anti-tumor methods, the traditional scheme of decoupling Dongxi should be abandoned, and the safety, specificity and effectiveness of drug candidates should be achieved by focusing on specificity.
  • chemotherapeutic drugs and radiation play a role in the p53 gene, and 50 to 60% of various tumors have p53 gene mutations, which seriously affects the therapeutic effect.
  • the bcl-2 gene can inhibit apoptosis and thus become the material basis for some tumor chemotherapy resistance. Therefore, exploring new anti-tumor biological effect substances that are not dependent on p53 and not affected by bcl-2 gene, fundamentally solve the problem of drug resistance in some patients with malignant tumors, has become an urgent problem to be solved.
  • malignant tumors can occur in almost any part of the body. 70% of all cases of malignant tumor deaths occur in low- and middle-income countries, and treatment in developed countries at a cost-free manner can only increase the 5-year average survival rate of individual malignant tumor types to 50-60%. Malignant tumors such as cervical cancer, breast cancer and colon cancer can be effectively treated if they are found in time and supplemented with appropriate therapies. Due to the current public health level, medical quality and treatment costs and other restrictions, it will be difficult to effectively treat malignant tumors that are easy to control, and ultimately make China a veritable “cancer power”. Therefore, in the development of anti-tumor Production costs and patient tolerance should be fully considered in the drug candidate process.
  • Gene therapy vectors mainly include two types of non-viral vectors and viral vectors.
  • Non-viral vectors have small side effects, but low transduction efficiency and transient expression characteristics make their development limited. Therefore, more than 75% of gene therapy studies use viral vectors.
  • the success of tumor gene therapy lies not only in its effectiveness, but also in its specificity as an important indicator. Increased copy number of some genes was found in many immortalized cell lines, indicating the presence of tumor-specific promoters. Tumor-specific promoters can drive high-level expression of genes in tumor cells, suggesting that the high efficiency and specificity of such promoters are suitable for targeted expression in tumor cells, providing a new strategy for the clinical application of tumor gene therapy.
  • Apoptin is a small molecule protein derived from chicken anemia virus. Apoptosis can specifically induce apoptosis of various tumor cells without affecting normal cells. In addition, most chemotherapeutic drugs and radiation induce apoptosis through wild-type p53, and 50 to 60% of tumor types have p53 gene mutations, thus seriously affecting the therapeutic effect. The study found that apoptin can effectively induce tumor cell apoptosis regardless of whether there is a mutation in the p53 gene of tumor cells. In addition, overexpression of bcl-2 gene can inhibit apoptosis, so overexpression of bcl-2 has become an important material basis for some tumor cells to develop resistance to chemotherapeutic drugs.
  • Another object of the present invention is to provide a use of a recombinant oncolytic adenovirus for the preparation and treatment of a tumor drug.
  • the recombinant oncolytic adenovirus provided by the present invention comprises an oncolytic adenovirus vector and an expression cassette inserted therein, the expression cassette comprising an expression cassette consisting of a human telomerase reverse transcriptase promoter (hTERTp) and an E1a gene, and An expression cassette consisting of a eukaryotic promoter and a chicken anemia virus VP3 gene.
  • hTERTp human telomerase reverse transcriptase promoter
  • E1a gene an expression cassette consisting of a eukaryotic promoter and a chicken anemia virus VP3 gene.
  • the 209th nucleotide is mutated from cytosine to thymine
  • the 347th nucleotide is mutated from avian to adenine
  • the 352th nucleotide is mutated from thymine to Cytosine, the nucleotide sequence of which is shown in Seq ID No. 1.
  • the human telomerase reverse transcriptase promoter wherein the nucleotide of position 220 is cytosine, the nucleotide of position 266 is guanine, the nucleotide of the 268th nucleotide is guanine, and the nucleotide sequence thereof is Seq. ID No. 2 is shown.
  • the VP3 gene of the invention adopts the mutated VP3 gene, thereby effectively improving the anti-tumor ability, and the mutant human telomerase reverse transcriptase promoter is used to effectively improve the stability thereof.
  • the eukaryotic promoter used in the present invention is preferably a human cytomegalovirus promoter derived from human type 5 adenovirus.
  • the recombinant oncolytic adenovirus of the present invention comprises an oncolytic adenovirus vector, a human telomerase reverse transcriptase promoter, an E1a gene, a human cytomegalovirus promoter, a chicken anemia virus VP3 gene and polyadenylation Acid sequence composition.
  • the recombinant oncolytic adenovirus of the present invention has a sequence as shown in Seq ID No. 3.
  • the invention also provides the use of the recombinant oncolytic virus for preparing anti-tumor drugs and preventing post-operative tumor recurrence.
  • the present invention further provides an antitumor drug prepared from the recombinant oncolytic virus and a drug for preventing postoperative tumor recurrence.
  • the medicament can be prepared into a dosage form such as an injection, a spray or an smear.
  • the novel recombinant oncolytic virus provided by the invention has the dual specificity of tumor-specific replication and tumor-specific killing, can specifically replicate in tumor cells, and simultaneously express an apoptin gene with specific killing ability to tumor cells, thereby Improves its safety by enhancing tumor specificity.
  • the replication of recombinant oncolytic virus and the apoptin gene itself Its ability to kill, improve its tumor killing ability.
  • Figure 1 is a graph showing the results of different concentrations of inhibition in a 72-hour MTT assay in Example 3 of the present invention.
  • Example 2 is a graph showing the results of different time suppression of the MTT test at 100 MOI in Example 3 of the present invention.
  • Fig. 3 is a graph showing experimental results of tumor growth tendency of model animals in Example 4 of the present invention.
  • Figure 4 is a graph showing the average survival test results of model animals in Example 4 of the present invention.
  • E1a upstream primer 5'-GCCTGCAGACCACCATGGGACATATTATCTGCCAC-3'
  • E1a downstream primer 5'-GCGGATCCTTATGGCCTGGGGCGTTTACAGC-3'
  • the reaction conditions of each step and the optimal concentration of the reagents involved were optimized, and DNA amplification was performed on a PCR machine.
  • the total volume of the reaction was 50 ⁇ L: 10 ⁇ L of PCR buffer, 5 ⁇ L/L.
  • Each of the downstream primers was 1 ⁇ L, 5 ⁇ L of template DNA, 5 ⁇ L of dNTPs (2.5 mmol/L each), 4 ⁇ L of 25 mmol/L MgCl 2 , 1 ⁇ L of 1 U/ ⁇ L Ex-Taq DNA polymerase, and 27 ⁇ L of ddH 2 O.
  • the PCR working procedure was screened and determined: 94 ° C for 4 min; then 94 ° C for 30 s, 57 ° C for 45 s, 72 ° C for 1 min, 10 cycles; finally 72 ° C for 10 min, 4 ° C for incubation.
  • the amplified products were ligated to the pMD18-T vector, respectively, and the constructed plasmid pMD18-E1a was subjected to nucleotide sequence determination.
  • VP3 gene sequence (NC_001427) published in GenBank, the following primers were designed to mutate the 209th nucleotide from cytosine to thymine, and the 347th nucleotide to guanine to adenine, the 352th nucleoside
  • the acid is mutated from thymine to cytosine and used to amplify the VP3 gene:
  • the PCR working procedure was screened and determined: 94 ° C for 4 min; then 94 ° C for 30 s, 57 ° C for 45 s, 72 ° C for 1 min, 10 cycles; finally 72 ° C for 10 min, 4 ° C for incubation.
  • the amplified products were ligated to the pMD18-T vector, respectively, and the constructed plasmid pMD18-VP3 was subjected to nucleotide sequence determination.
  • the hTERT promoter was synthesized according to the nucleotide sequence of human telomerase reverse transcriptase promoter hTERT (EU650197) disclosed in GenBank.
  • the 220th nucleotide is cytosine and the 266th nucleotide is guanine.
  • the 268 nucleotide was guanine, and it was ligated to the pKS vector (purchased from Stratagen) to construct a plasmid pKS-hTERT containing hTERTp.
  • the plasmid pMD18-E1a was digested with Pst I/BamH I to obtain an E1a gene fragment, and ligated with the same double-digested plasmid pKS-hTERTp to construct plasmid pKS-hTERTp-E1. Then, Xba I/Xho I double-digested the plasmid pIRES-neo (purchased from Invitrogen) to obtain a Poly A nucleotide fragment, which was filled in and ligated with pKS-hTERTp-E1 digested with Hind III to construct a plasmid. pKS-PolyA-hTERTp-E1.
  • Hind III was digested with the plasmid pacAd5CMV K-N pA, digested with EcoR I, and ligated with the VP3 gene fragment obtained by double digestion of pMD18-VP3 with EcoR I/EcoR V to construct plasmid pAd-VP3.
  • BamH I was digested with pAd-VP3, digested with Spe I, and linearized pAd-VP3 was recovered;
  • Xho I was digested with pKS-PolyA-hTERTp-E1, and after digestion, it was digested with Spe I to obtain -PolyA
  • a fragment of the -hTERTp-E1a nucleotide was ligated to the linearized pAd-VP3 to construct the shuttle vector pAd-Apoptin-PolyA-hTERTp-E1, designated pAd-ATV.
  • the plasmid pAd-ATV was linearized with Nhe I by the following method: Mix appropriate amount of plasmid DNA with appropriate amount of water, and add 4 U restriction endonuclease Nhe I and 10 ⁇ l of corresponding 10 ⁇ restriction endonuclease reaction buffer. The total volume was 100 ⁇ l, the wall of the flick tube was mixed and centrifuged, and placed in a 37 ° C water bath overnight.
  • the transfected HEK-293 cells were further cultured for 7 to 14 days, and the culture medium was changed every 48 to 72 hours (depending on the state of the cells). During the period of 7 to 14 days of the continued culture, if the cells showed lesions, the cells were resuspended, collected in a 1.5 ml centrifuge tube, and repeatedly frozen and thawed at -80 ° C / 37 ° C for 3 times, and stored frozen for use.
  • the cells do not show lesions during this period, resuspend the cells in 10 ml of complete medium and continue to culture for 7 to 14 days in a 10 cm cell culture dish. If lesions appear during this period, follow the above procedure. If no lesions appear, repeat the transfer. Dyeing operation.
  • Monolayers of HEK-293 cells were prepared as above, and the cells were discarded when the cells were grown to 80% confluence and washed twice with Hank's solution.
  • 500 ⁇ l of the above recombinant virus stock solution was inoculated into HEK-293 cells in a 6-well plate, placed in a 37 ° C, 5% CO 2 cell incubator for 4 h, supplemented with DMEM complete medium to 3 ml, and continued to culture until lesions appeared.
  • the cells in the independent lesions were scraped and placed in 500 ⁇ l of serum-free, antibiotic-free DMEM medium, and frozen and thawed for 3 times.
  • Monolayers of HEK-293 cells were prepared in 24-well cell culture plates, and the cells were discarded when the cells were grown to 80% confluence and washed twice with Hank's solution.
  • 300 ⁇ l of monoclonal recombinant adenovirus was inoculated into HEK-293 cells in 24-well plates, placed in a 37 ° C, 5% CO 2 cell incubator for 4 h, supplemented with DMEM complete medium to 3 ml, and continued to culture until lesions appeared. Resuspend the diseased cells and freeze-thaw the cells three times.
  • HEK-293 cells Inoculate HEK-293 cells in 25ml cell culture flasks, place them in a 37°C, 5% CO 2 cell incubator for 4 hours, add DMEM complete medium to 3ml, and continue to culture. A lesion has appeared. The diseased cells were resuspended and frozen and thawed three times, and stored at -80 °C for storage, and the recombinant oncolytic adenovirus ATV was obtained.
  • Monolayers of HEK-293 cells were prepared in 96-well cell culture plates, and the cells were discarded when the cells were grown to 80% confluence and washed twice with Hank's solution.
  • the prepared monoclonal recombinant virus was diluted 10 3 to 10 14 with Hank's solution, and 30 ⁇ l of HEK-293 cells seeded in 96-well plates were placed in a 37 ° C, 5% CO 2 cell incubator for 4 h, supplemented with DMEM completely.
  • the culture solution was adjusted to 200 ⁇ l and culture was continued for 72 to 96 hours.
  • the culture solution was aspirated, and DMEM containing 1% methylcellulose and 2% calf serum (FCS) was added as a maintenance solution, and culture was continued for 24 to 48 hours in a 37 ° C, 5% CO 2 incubator.
  • Aspirate the culture solution wash twice with PBS, fix with 1% formaldehyde for 15 min at room temperature, rinse with distilled water, and stain with 0.1% crystal violet for 5 min. Rinse with distilled water and count the number of virus plaques under an inverted microscope. Calculate per ml according to the following formula. Plaque forming units (PFU) contained in the virus solution:
  • PFU (number of virus plaques ⁇ dilution factor) / inoculation volume
  • the anti-tumor bispecific recombinant oncolytic adenovirus ATV was successfully constructed, and the above recombinant adenovirus was identified by RT-PCR, Western blot and IFA. It was proved that the foreign gene carried by the recombinant adenovirus can be efficiently transcribed and expressed. .
  • the stability of the above recombinant adenovirus was identified by serial passage. The results showed that the recombinant oncolytic adenovirus constructed by the present invention has good stability, and the virus toxicity can be maintained at a level of 10 7 to 10 8 .
  • Digestion of human lung cancer cell A549 in logarithmic growth phase Count and adjust the cell concentration to 5 ⁇ 10 4 /ml with complete cell culture medium, and inoculate 100 ⁇ l/well in 96-well cell culture plate (ie, 5 ⁇ 10 3 /well), after the cells are attached (about 24 h).
  • the culture solution was aspirated and washed twice with Hank's solution.
  • the ATV titers were adjusted to 1 x 10 7 PFU/ml, 1 x 10 6 PFU/ml, and 1 x 10 5 PFU/ml with serum-free and antibiotic-free RPMI-1640 medium.
  • Example 2 50 ⁇ l (ie, 100 moi, 10 moi, and 1 moi) of the virus dilution prepared in Example 2 was added to the corresponding wells of tumor cells cultured in a 96-well cell culture plate washed with Hank's solution at 37 ° C, 5% CO 2 cell incubator. After 4 h of internal action, add complete RPMI-1640 medium to 200 ⁇ l/well. Cells that were not treated were used as controls.
  • Killing rate (%) (control hole A value - experimental hole A value) / control hole A value
  • the recombinant adenovirus Ad-VT without unmutated VP3 and hTERTp and the adenovirus Ad-mock not inserted into the expression cassette were used as controls, and the method was the same as ATV.
  • FIG. 1 and FIG. 2 The results are shown in FIG. 1 and FIG. 2, and the experiments show that the recombinant oncolytic adenovirus ATV obtained by the present invention inhibits human lung cancer cells within 24 hours, and the effect thereof is enhanced with the increase of the concentration of action and the prolongation of action time. Complete inhibition can be achieved after 72 hours, and it can be used as a gene therapy for tumor gene therapy. It has broad application prospects, and its inhibitory effect on tumor cells is significantly better than that of unmutated recombinant adenovirus Ad-VT.
  • Example 4 In vivo antitumor effect of recombinant oncolytic adenovirus
  • LLC lung cancer cells were washed twice with serum-free Hanks solution, adjusted to a cell concentration of 1 ⁇ 10 7 cells/ml, and the right hind limb was subcutaneously inoculated with 0.1 ml of LLC lung cancer cells, ie, 1 ⁇ 10 6 tumor cells. . After 10 days, the nodule of rice size grew, indicating that the tumor was successful.
  • mice When the tumor-bearing mice grew to a diameter of about 5 mm, they were randomly divided into 5 groups, 10 in each group.
  • the groupings were as follows: Group 1 was the saline control group; Group 2 was the Ad-mock control group; Group 3 was the ATV treatment group; Group 4 was the Ad-VT treatment group.
  • the recombinant adenovirus is diluted with physiological saline to 1 ⁇ 10 10 PFU/ml, and the treatment group is intratumorally injected with 100 ⁇ l virus/only/time (ie, 1 ⁇ 10 9 PFU/time/time); the saline control group is injected intratumorally. 100 ⁇ l saline/only/time, the mice were sacrificed 16 days after the last treatment, and the indicators were tested.
  • mice The mental state, feeding status and survival of the mice were observed daily. After tumor-bearing, both tumor volume was measured, once every 2 days, as follows: The vernier caliper measures the long diameter and short diameter of the tumor (including skin thickness). Calculate the tumor volume using the following formula:
  • Tumor volume (V) A 2 ⁇ B ⁇ 0.52
  • the tumor inhibition rate was calculated based on the tumor volume of the tumor-bearing mice at the last time.
  • the calculation formula is as follows:
  • Tumor inhibition rate (%) [(control tumor volume - experimental group tumor volume) / control tumor volume] ⁇ 100%
  • the recombinant oncolytic adenovirus provided by the invention has the dual specificity of tumor-specific replication and tumor-specific killing, can specifically replicate in tumor cells, and simultaneously express an apoptin gene having specific killing ability to tumor cells, thereby Improves its safety by enhancing tumor specificity.
  • the tumor killing ability is improved.

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Abstract

本发明提供一种新型的重组溶瘤腺病毒及应用。所述重组溶瘤腺病毒具有肿瘤特异性复制和肿瘤特异性杀伤的双重特异性，可以在肿瘤细胞内特异性复制，同时表达对肿瘤细胞具有特异性杀伤能力的凋亡素基因，从而通过增强肿瘤特异性提高其安全性。同时，通过重组溶瘤腺病毒的复制和凋亡素基因本身的杀伤能力，提高其肿瘤杀伤能力。

Description

重组溶瘤腺病毒及其应用 技术领域
本发明涉及生物技术和基因治疗领域，具体地说，涉及一种新型的重组溶瘤腺病毒及应用。
背景技术
恶性肿瘤的发展是多因素参与的动态过程，多年来对肿瘤基因治疗研究的经验表明，怎样在与恶性肿瘤的竞速中取得完胜是实现有效治疗的关键。因此，应以恶性肿瘤发生、发展过程中的多因素、特殊性为切入点，使抑瘤过程达到动态平衡，最终实现准确快速抑瘤，有效治疗恶性肿瘤。
手术治疗、化疗和放疗是目前肿瘤治疗的首选方式，但会对机体造成不可逆创伤和生理的次生影响，且根治不完全。因此，在抗肿瘤新方法研究伊始，即应摒弃拆东补西的传统方案，通过将特异性作为研究重点达到候选药物的安全性、特异性和有效性。
多数化疗药物和射线通过p53基因发挥作用，而各类肿瘤中50～60％存在p53基因突变，从而严重影响治疗效果。另外，bcl-2基因可抑制细胞凋亡，因此成为一些肿瘤化疗耐药的物质基础。因此，探索不依赖p53且不受bcl-2基因影响的新型抗肿瘤生物效应物质，从根本上解决某些恶性肿瘤患者的耐药问题，成为亟待解决的问题。
恶性肿瘤超过100种，几乎可以发生在机体的任何部位。全世界70％的恶性肿瘤死亡病例发生在中低收入国家，发达国家以不计成本的方式治疗只能将个别恶性肿瘤类型的5年平均生存率提高至50～60％。宫颈癌、乳腺癌和结肠癌等恶性肿瘤，若发现及时，辅以适当疗法，可以得到有效治疗。由于目前的公共卫生水平、医疗质量和诊治费用等诸多限制，将使极易控制的恶性肿瘤也难以得到有效治疗，最终使中国成为名副其实的“癌症大国”。因此，在研发抗肿瘤 候选药物过程中，应充分考虑生产成本和患者承受能力。
近年来，以基因治疗、体细胞治疗、抗体治疗和生物化疗为研究重点的肿瘤生物治疗发展迅速，其中又以基因治疗研究最为广泛。基因治疗载体主要包括非病毒载体和病毒载体两类。非病毒载体副作用小，但低下的转导效率和瞬时表达的特点使其发展受限。因此，75％以上的基因治疗研究应用病毒载体。肿瘤基因治疗的成功不仅在于其有效性，特异性也是重要的评测指标之一。在很多永生化细胞系中发现一些基因拷贝数增加，说明存在肿瘤特异性启动子。肿瘤特异性启动子可在肿瘤细胞内驱动基因高水平表达，提示该类启动子的高效性和特异性适用于在肿瘤细胞中靶向表达，为肿瘤基因治疗的临床应用提供了新策略。
凋亡素是来源于鸡贫血病病毒的一种小分子蛋白。凋亡素能够在不影响正常细胞的前提下，特异性地诱导多种肿瘤细胞凋亡。另外，多数化疗药物和射线是通过野生型p53诱导细胞凋亡的，而50～60％的肿瘤类型存在p53基因突变，因此严重影响治疗效果。研究发现，无论肿瘤细胞的p53基因是否存在突变，凋亡素能够有效地诱导肿瘤细胞凋亡。另外，bcl-2基因的过表达可抑制细胞凋亡，因此bcl-2的过表达也成为一些肿瘤细胞对化疗药物产生耐药性的重要物质基础。而研究发现，bcl-2基因的过表达不仅对凋亡素的凋亡诱导作用无影响，反而能增强其凋亡诱导功能。这种促进作用不是二者直接作用的结果，因为即使正常细胞过表达bcl-2，凋亡素也不会对其产生影响。因此，凋亡素可以有效地避免耐药性的产生。
发明内容
本发明的目的是提供一种新型的重组溶瘤腺病毒，用于肿瘤的治疗。
本发明的另一目的在于提供重组溶瘤腺病毒在制备和治疗肿瘤药物方面的应用。
本发明提供的重组溶瘤腺病毒，包括溶瘤腺病毒载体和插入其中的表达盒，所述表达盒包括由人端粒酶逆转录酶启动子(hTERTp)与E1a基因组成的表达盒以及由真核启动子与鸡贫血病病毒VP3基因组成的表达盒。
其中，所述鸡贫血病病毒VP3基因，第209位核苷酸由胞嘧啶突变为胸腺嘧啶，第347位核苷酸由鸟嘌呤突变为腺嘌呤，第352位核苷酸由胸腺嘧啶突变为胞嘧啶，其核苷酸序列如Seq ID No.1所示。
所述人端粒酶逆转录酶启动子，其第220位核苷酸为胞嘧啶，第266位核苷酸为鸟嘌呤，第268位核苷酸为鸟嘌呤，其核苷酸序列如Seq ID No.2所示。
本发明采用突变的VP3基因，有效提高了其抗肿瘤能力，采用突变的人端粒酶逆转录酶启动子，有效提高了其稳定性。
本发明中使用的真核启动子优选为人类巨细胞病毒启动子，所述溶瘤腺病毒载体源自人5型腺病毒。
优选地，本发明的重组溶瘤腺病毒，由溶瘤腺病毒载体、人端粒酶逆转录酶启动子、E1a基因、人类巨细胞病毒启动子、鸡贫血病病毒VP3基因和多聚腺苷酸序列组成。
更优选地，本发明的重组溶瘤腺病毒，其序列如Seq ID No.3所示。
本发明还提供所述重组溶瘤病毒在制备抗肿瘤药物和预防术后肿瘤复发药物中的应用。
本发明进一步提供由所述重组溶瘤病毒制备的抗肿瘤药物和预防术后肿瘤复发药物。所述药物可制备成注射剂、喷雾剂或涂抹剂等剂型。
本发明提供的新型重组溶瘤病毒具有肿瘤特异性复制和肿瘤特异性杀伤的双重特异性，可以在肿瘤细胞内特异性复制，同时表达对肿瘤细胞具有特异性杀伤能力的凋亡素基因，从而通过增强肿瘤特异性提高其安全性。同时，通过重组溶瘤病毒的复制和凋亡素基因本身 的杀伤能力，提高其肿瘤杀伤能力。
附图说明
图1为本发明实施例3中72小时MTT实验检测不同浓度抑制结果。
图2为本发明实施例3中100MOI时MTT实验检测不同时间抑制结果。
图3为本发明实施例4中模型动物肿瘤生长趋势实验结果。
图4为本发明实施例4中模型动物平均存活期实验结果。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。实施例中大肠杆菌感受态细胞的制备与转化、质粒的提取及限制性内切酶消化、DNA片段的回收、线性DNA片段的连接、重组质粒的筛选与鉴定、PCR扩增反应等参照金冬雁、黎孟枫等译《分子克隆实验指南》第二版相关章节进行。
实施例1 重组溶瘤腺病毒穿梭载体质粒的制备
1、E1a基因的克隆
根据GenBank中公开的人5型腺病毒E1a基因序列(NC_001406)，设计如下引物，用于扩增E1a基因：
E1a上游引物：5’-GCCTGCAGACCACCATGGGACATATTATCTGCCAC-3’
E1a下游引物：5’-GCGGATCCTTATGGCCTGGGGCGTTTACAGC-3’
优化各步反应条件及参与反应试剂的最佳浓度，在PCR仪上进行DNA扩增。反应总体积50μL：10×PCR缓冲液5μL、20μmol/L上。下游引物各1μL，模板DNA 5μL、dNTP(各2.5mmol/L)5μL，25mmol/L MgCl24μL，1U/μL Ex-Taq DNA聚合酶1μL、ddH2O 27μL。筛选并确定PCR工作程序：94℃ 4min；然后94℃ 30s，57℃ 45s，72℃ 1min，10个循环；最后72℃延伸10min，4℃保温。扩增产物分别连接至pMD18-T载体上，并对所构建的质粒pMD18-E1a进行核 苷酸序列测定。
2、VP3基因的克隆
根据GenBank中公开的VP3基因序列(NC_001427)，设计如下引物，将第209位核苷酸由胞嘧啶突变为胸腺嘧啶，第347位核苷酸由鸟嘌呤突变为腺嘌呤，第352位核苷酸由胸腺嘧啶突变为胞嘧啶，用于扩增VP3基因：
Apoptin上游引物：5’-GCGATATCACCACCATGGACGCTCTCCAA-3’
Apoptin下游引物：5’-GCGAATTCTTACAGTCTTATACGCCTTTTTGCGGTTCGGGGTCGGCTGGGAGTAGTGGTAATCAAGCTTTCTTTTAGCTCGCTTACCCTGTACTCGGAGGGGTCGCAGGATCGCTTCTTCGAGGGAGGCTTGGGTTGATCGGTCCTCAAGTCCGGCACATTCTTGAAACCA-3’
优化各步反应条件及参与反应试剂的最佳浓度，在PCR仪上进行DNA扩增。反应总体积50μL：10×PCR缓冲液5μL、20μmol/L上、下游引物各1μL，模板DNA 5μL、dNTP(各2.5mmol/L)5μL，25mmol/L MgCl24μL，1U/μL Ex-Taq DNA聚合酶1μL、ddH2O 27μL。筛选并确定PCR工作程序：94℃ 4min；然后94℃ 30s，57℃ 45s，72℃ 1min，10个循环；最后72℃延伸10min，4℃保温。扩增产物分别连接至pMD18-T载体上，并对所构建质粒pMD18-VP3进行核苷酸序列测定。
3、肿瘤特异性启动子的合成
分别根据GenBank中公开的人端粒酶逆转录酶启动子hTERT(EU650197)核苷酸序列人工合成hTERT启动子，第220位核苷酸为胞嘧啶，第266位核苷酸为鸟嘌呤，第268位核苷酸为鸟嘌呤，并将其连接于pKS载体(购自Stratagen公司)，构建含有hTERTp的质粒pKS-hTERT。
4、穿梭质粒的构建
Pst I/BamH I双酶切质粒pMD18-E1a，获得E1a基因片段，并与经同样双酶切的质粒pKS-hTERTp连接，构建质粒pKS-hTERTp-E1。 然后Xba I/Xho I双酶切质粒pIRES-neo(购自Invitrogen公司)，获得Poly A核苷酸片段，补平，与经Hind III酶切并补平的pKS-hTERTp-E1连接，构建质粒pKS-PolyA-hTERTp-E1。
Hind III酶切质粒pacAd5CMV K-N pA，补平后用EcoR I酶切，与经EcoR I/EcoR V双酶切pMD18-VP3后获得的VP3基因片段连接，构建质粒pAd-VP3。
BamH I酶切pAd-VP3，补平后用Spe I酶切，回收线性化的pAd-VP3；Xho I酶切pKS-PolyA-hTERTp-E1，补平后用Spe I酶切，获得含-PolyA-hTERTp-E1a核苷酸的片段，将其与线性化的pAd-VP3连接，构建穿梭载体pAd-Apoptin-PolyA-hTERTp-E1，命名为pAd-ATV。
实施例2 重组溶瘤腺病毒的制备
1、共转染
质粒pAd-ATV用Nhe I进行酶切线性化，方法如下：将适量质粒DNA与适量水混匀，同时加入4U限制性内切酶Nhe I及10μl相应的10×限制性内切酶反应缓冲液，使其总体积为100μl，轻弹管壁混匀并离心，置37℃水浴过夜。
将100μl饱和酚加入线性化的质粒中，适度振荡，于4℃、12000rpm条件下离心10min；取上清，加入100μl饱和酚/氯仿/异戊醇(25:24:1)，适度振荡，于4℃、12000rpm条件下离心10min；取上清，加入100μl氯仿/异戊醇(24:1)，适度振荡，于4℃、12000rpm条件下离心10min；取上清，加入200μl无水乙醇和20μl醋酸钠，-20℃放置30min，于4℃、12000rpm条件下离心10min；弃上清，加入200μl70％乙醇洗涤沉淀一次；室温干燥后加入50μl无菌TE(10mMTris，0.1mM EDTA，pH8.0)溶解沉淀。将人5型腺病毒基因组与线性化的pAd-ATV共转染HEK-293细胞。
2、细胞内同源重组
转染后的HEK-293细胞继续培养7～14d，每48～72h更换培养液(视细胞状态确定)。在该继续培养的7～14d期间，若细胞出现病变既重悬细胞，收集于1.5ml离心管内，并于-80℃/37℃反复冻融3次，冻存备用。
若细胞在此期间未出现病变，用10ml完全培养液重悬细胞，并置10cm细胞培养皿内继续培养7～14d，此期间若出现病变则按上法操作，若未出现病变则需重复转染操作。
3、重组溶瘤腺病毒的筛选
按上法制备单层HEK-293细胞，至细胞长至80％融合时弃去培养液，用Hank's液洗两次。取上述重组病毒原液500μl接种于6孔板的HEK-293细胞，置37℃、5％CO2细胞培养箱内作用4h，补加DMEM完全培养液至3ml，继续培养至出现病变。刮取独立病变处细胞，并置于500μl无血清、无抗生素的DMEM培养液中，按上法冻融3次备用。
4、重组腺病毒的扩增及制备
于24孔细胞培养板制备单层HEK-293细胞，至细胞长至80％融合时弃去培养液，用Hank's液洗两次。取单克隆重组腺病毒300μl接种于24孔板的HEK-293细胞，置37℃、5％CO2细胞培养箱内作用4h，补加DMEM完全培养液至3ml，继续培养至出现病变。重悬病变细胞并冻融3次，接种于25ml细胞培养瓶内的HEK-293细胞，置37℃、5％CO2细胞培养箱内作用4h，补加DMEM完全培养液至3ml，继续培养至出现病变。重悬病变细胞并冻融3次，置-80℃冻存备用，即得重组溶瘤腺病毒ATV。
5、重组腺病毒的毒价测定
于96孔细胞培养板制备单层HEK-293细胞，至细胞长至80％融合时弃去培养液，用Hank's液洗两次。取制备的单克隆重组病毒以Hank's液进行103～1014稀释，取30μl接种于96孔板的HEK-293细胞， 置37℃、5％CO2细胞培养箱内作用4h，补加DMEM完全培养液至200μl，继续培养72～96h。吸弃培养液，补加含1％甲基纤维素和2％小牛血清(FCS)的DMEM作为维持液，37℃、5％CO2培养箱中继续培养24～48h。吸弃培养液，用PBS洗涤2次，室温下1％甲醛固定15min，蒸馏水冲洗后用0.1％结晶紫染色5min，蒸馏水冲洗后在倒置显微镜下统计病毒空斑数，按如下公式计算出每毫升病毒液中所含的空斑形成单位(Plaque forming units,PFU)：
PFU＝(病毒空斑数×稀释倍数)/接种体积
成功构建了抗肿瘤双特异重组溶瘤腺病毒ATV，应用RT-PCR、Western blot和IFA等方法，分别对以上重组腺病毒进行了鉴定，证明重组腺病毒所携带外源基因能够有效转录并表达。通过连续传代的方法，对上述重组腺病毒的稳定性进行鉴定。结果表明，本发明所构建的重组溶瘤腺病毒具有良好稳定性，且病毒毒价可维持在107～108的水平。
实施例3 重组溶瘤腺病毒对肿瘤细胞的杀伤作用(MTT实验)
消化处于对数生长期的人肺癌细胞A549。计数并用完全细胞培养液调整细胞浓度至5×104个/ml，按100μl/孔接种于96孔细胞培养板(即5×103个/孔)，待细胞贴壁后(24h左右)，吸弃培养液，用Hank’s液洗涤2次。用无血清无抗生素的RPMI-1640培养液调整ATV滴度至1×107PFU/ml、1×106PFU/ml和1×105PFU/ml。取实施例2制备的病毒稀释液50μl(即100moi、10moi和1moi)，加入经Hank’s液洗涤的培养于96孔细胞培养板的肿瘤细胞相应孔内，于37℃、5％CO2细胞培养箱内作用4h，补加完全RPMI-1640培养液至200μl/孔。以未做任何处理的细胞作为对照。
分别于感染24h、48h、72h和96h，于每孔加入MTT溶液(5mg/ml，溶于PBS中，0.22μm滤器过滤除菌)20μl，于37℃、5％CO2细胞培养箱内作用4h，小心吸弃孔内原培养上清液。每孔加入150μl DMSO， 于37℃条件下放置10min(使结晶物充分溶解)，取溶解有紫色结晶有DMSO 135μl，置96孔酶标板中，于490nm处酶联免疫检测仪上测各孔A值。按以下公式计算杀伤率：
杀伤率(％)＝(对照孔A值-实验孔A值)/对照孔A值
每种处理均设3个重复孔(n＝3)，所得数据进行统计学分析。
以未突变VP3和hTERTp的重组腺病毒Ad-VT和未插入表达盒的腺病毒Ad-mock做为对照，操作方法同ATV。
结果如图1和图2所示，实验表明，本发明获得的重组溶瘤腺病毒ATV在24小时内对人肺癌细胞产生抑制作用，且其作用随作用浓度增加和作用时间延长而加强，至72小时后可实现完全抑制，可作为肿瘤基因治疗的药物，应用前景广阔，其对肿瘤细胞的抑制作用明显优于未突变的重组腺病毒Ad-VT。
实施例4 重组溶瘤腺病毒的体内抑瘤作用研究
取对数生长期的LLC肺癌细胞，用无血清Hanks液洗涤2遍后，调整细胞浓度为1×107个/ml，右后肢皮下接种LLC肺癌细胞0.1ml，即1×106个肿瘤细胞。10d后长出米粒大小的结节，表明荷瘤成功。
待荷瘤小鼠肿瘤生长至直径为5mm左右时，随机分为5组，每组10只。分组情况如下：第1组为生理盐水对照组；第2组为Ad-mock对照组；第3组为ATV治疗组；第4组为Ad-VT治疗组。
分组后既进行治疗，每10天一次，共接种三次。方法如下：用生理盐水稀释重组腺病毒至1×1010PFU/ml，治疗组瘤内注射100μl病毒/只/次(即1×109PFU/只/次)；生理盐水对照组瘤内注射100μl生理盐水/只/次，末次治疗后16d处死小鼠，进行指标检测。
每日观察小鼠的精神状态、采食情况和存活情况。荷瘤后既开始测量肿瘤体积，每2天一次，方法如下：游标卡尺测量肿瘤长径及短径长度(包括皮肤厚度在内)。应用下列公式计算肿瘤体积：
肿瘤体积(V)＝A2×B×0.52
式中，A为肿瘤短径长度；B为肿瘤长径长度。
根据末次测量荷瘤小鼠的肿瘤体积计算抑瘤率，计算公式如下：
抑瘤率(％)＝[(对照组肿瘤体积-实验组肿瘤体积)/对照组肿瘤体积]×100％
结果如图3和图4所示，结果表明，本发明的重组溶瘤腺病毒ATV可显著延缓动物模型实体肿瘤的生长，提高荷瘤动物模型生存率。另外，ATV对实体肿瘤的抑瘤作用，显著高于Ad-VT和其它对照组。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
工业实用性
本发明提供的重组溶瘤腺病毒具有肿瘤特异性复制和肿瘤特异性杀伤的双重特异性，可以在肿瘤细胞内特异性复制，同时表达对肿瘤细胞具有特异性杀伤能力的凋亡素基因，从而通过增强肿瘤特异性提高其安全性。同时，通过重组溶瘤腺病毒的复制和凋亡素基因本身的杀伤能力，提高其肿瘤杀伤能力。
Figure PCTCN2015081560-appb-000001
Figure PCTCN2015081560-appb-000002
Figure PCTCN2015081560-appb-000003
Figure PCTCN2015081560-appb-000004
Figure PCTCN2015081560-appb-000005
Figure PCTCN2015081560-appb-000006
Figure PCTCN2015081560-appb-000007
Figure PCTCN2015081560-appb-000008
Figure PCTCN2015081560-appb-000009
Figure PCTCN2015081560-appb-000010
Figure PCTCN2015081560-appb-000011
Figure PCTCN2015081560-appb-000012

Claims (8)

  1. 重组溶瘤腺病毒，其特征在于，包括溶瘤腺病毒载体和插入其中的表达盒，所述表达盒包括由人端粒酶逆转录酶启动子与E1a基因组成的表达盒以及由真核启动子与鸡贫血病病毒VP3基因组成的表达盒；
    其中，所述鸡贫血病病毒VP3基因的核苷酸序列如Seq ID No.1所示，所述人端粒酶逆转录酶启动子的核苷酸序列如Seq ID No.2所示。
  2. 根据权利要求1所述的重组溶瘤腺病毒，其特征在于，所述真核启动子为人类巨细胞病毒启动子。
  3. 根据权利要求1所述的重组溶瘤腺病毒，其特征在于，所述溶瘤腺病毒载体源自人5型腺病毒。
  4. 根据权利要求3所述的重组溶瘤腺病毒，其特征在于，其由溶瘤腺病毒载体、人端粒酶逆转录酶启动子、E1a基因、人类巨细胞病毒启动子、鸡贫血病病毒VP3基因和多聚腺苷酸序列组成。
  5. 根据权利要求4所述的重组溶瘤腺病毒，其特征在于，其序列如Seq ID No.3所示。
  6. 权利要求1-5任一项所述重组溶瘤腺病毒在制备抗肿瘤药物和预防术后肿瘤复发药物中的应用。
  7. 由权利要求1-5任一项所述重组溶瘤腺病毒制备的抗肿瘤药物和预防术后肿瘤复发药物。
  8. 根据权利要求7所述的药物，其为注射剂、喷雾剂或涂抹剂。
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