WO2021249035A1 - 一种复制型人腺病毒及其应用 - Google Patents

一种复制型人腺病毒及其应用 Download PDF

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WO2021249035A1
WO2021249035A1 PCT/CN2021/088761 CN2021088761W WO2021249035A1 WO 2021249035 A1 WO2021249035 A1 WO 2021249035A1 CN 2021088761 W CN2021088761 W CN 2021088761W WO 2021249035 A1 WO2021249035 A1 WO 2021249035A1
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cancer
nbov1901
srfi
e3gp19k
adenovirus
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陈凌
关素华
刘波
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广州恩宝生物医药科技有限公司
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Definitions

  • the present invention relates to the field of gene therapy, in particular to a recombinant oncolytic adenovirus, a recombinant oncolytic adenovirus vector for preparing a recombinant oncolytic adenovirus, and applications thereof.
  • Cancer is caused by self factors (genetic mutations, natural aging, etc.) or environmental factors (such as smoking, ultraviolet radiation, chemical substances, viral infections, etc.).
  • the WHO report tumors are now the second leading cause of death in the world after cardiovascular diseases, and nearly 1/6 of deaths worldwide are caused by cancer.
  • the global number of cancer deaths reached 9.6 million. It is estimated that by 2025, the number of new cancer cases worldwide will reach 24.49 million each year.
  • lung cancer has the largest number of deaths, followed by colorectal cancer, gastric cancer, liver cancer and breast cancer.
  • the classic methods of treating cancer are mainly surgery, chemotherapy, and radiotherapy.
  • Surgical resection has limited effect and has no effect on metastatic cancer cells.
  • Radiotherapy and chemotherapy have large side effects.
  • target drugs have improved the quality of life of patients to a large extent, tolerability is a problem that chemotherapy has always faced.
  • immunotherapy has become a hot spot in the field of cancer treatment.
  • the principle of immunotherapy is to destroy tumor cells by activating the body's own immune system. It has the advantages of good curative effect, small side effects and prevention of recurrence.
  • Oncolytic virus is a type of virus that has the ability to replicate and can directly lyse tumor cells.
  • Italian doctors discovered that a patient with cervical cancer was infected with rabies virus and the tumor subsided.
  • China approved the world's first oncolytic adenovirus (H101) for marketing, but the clinical efficacy has not yet been recognized internationally.
  • the virus knocked out most of the genes in the E1B-55K and E3 regions.
  • E1B-55K protein can inhibit the expression of anti-oncogene p53 and prevent P53-mediated apoptosis of infected cells.
  • the deletion of the E1B-55K gene allows the virus to replicate specifically in tumor cells lacking functional p53.
  • the E1B-55K protein is a protein necessary for viral mRNA to export from the nucleus. Therefore, the replication ability and oncolytic ability of H101 are significantly weaker than that of the wild strain.
  • T-vec deletes the ⁇ 34.5 gene to make it tumor-targeting, and it also carries GM-CSF (granulocyte-macrophage colony factor) to enhance anti-tumor immunity .
  • GM-CSF granulocyte-macrophage colony factor
  • Oncolytic virus is an important branch of immunotherapy. It can kill tumor cells through a variety of different ways of action, and destroy the microenvironment of tumor immunosuppression to induce long-term tumor-specific immune response. At the same time, it can also make it by carrying therapeutic genes. It is specifically expressed in tumor cells for a long time, thereby enhancing the anti-tumor efficacy.
  • oncolytic viruses and other anti-cancer drugs such as combined immune checkpoint inhibitors, have shown good anti-tumor effects.
  • many viruses including vaccinia virus, reovirus, measles virus, herpes simplex virus, Newcastle disease virus, and Coxsackie virus have undergone clinical trials as oncolytic agents.
  • Adenovirus is a kind of particles with a diameter of 70-90nm without envelope, composed of 252 capsular particles arranged in icosahedrons. The diameter of each shell particle is 7-9nm. Inside the capsid is a linear double-stranded DNA molecule, about 4.7 kb, with inverted terminal repeats about 100 to 600 bp in length at each end.
  • Human adenoviruses (HAdV) belong to the Adenoviridae family (Adenoviridae). According to their immunological, biological, and biochemical characteristics, they are divided into 7 subspecies from A to G. There are 52 serotypes and different serotypes. There are different organ affinity and cause corresponding clinical manifestations.
  • Adenovirus can cause at least three types of infections after invading host cells: chronic and latent infections: often occur in lymphocytes, the amount of virus released during latent infection is very small, and cell necrosis is not obvious, so clinical infection is not obvious. The mechanism of latent infection is unclear. Soluble infection: The virus undergoes a replication process in cells such as human epithelial cells, and causes cell death by lysing the cells. Tumor-like mutation: At this time, virus reproduction only takes the first few steps, and then the adenovirus DNA and cell DNA are integrated and replicated, but no infectious virus is produced, and the antigenicity of adenovirus is relatively stable.
  • oncolytic adenovirus can be specific sexually infect tumor cells and complete the infection-replication cycle in tumor cells, thereby specifically killing and lysing tumor cells but not damaging other normal cells and tissues, so it has become one of the most promising anti-tumor drugs at present.
  • Different viruses have complex biological characteristics, and the virus resources that humans are fully familiar with are still relatively scarce.
  • the purpose of the present invention is to overcome at least one of the deficiencies in the prior art and provide an oncolytic adenovirus vector and its application.
  • the first aspect of the present invention provides:
  • a replicating human adenovirus the full sequence of which is shown in SEQ ID NO.: 21, and the resistance screening gene can be replaced with a well-known screening gene.
  • the full sequence of the replicating human adenovirus is shown in SEQ ID NO.: 21.
  • the E1 and E3 regions of the virus are deleted.
  • the virus can replicate in animal cells, human-derived cells, or humans.
  • the method for constructing the replicating human adenovirus includes:
  • PCR amplification obtains the left and right ends of the NBOV1901 genome, which are connected to the resistance vector plasmid A to obtain pT-NBOV1901(L+R), and recombine with the NBOV1901 genome after linearization to obtain the genome plasmid pNBOV1901;
  • PCR amplification obtains E3gp19K upstream fragments: SrfI to E3gp19K, and E3gp19K downstream fragments: E3gp19K to E3 regions contain E3-14.7K gene fragments, E3gp19K upstream fragments, E3gp19K downstream fragments and digested
  • the three fragments of the constructed vector were recombined and connected between the left and right arms of pNBOV1901 ⁇ E3-SrfI. After linearization, they were recombined with pNBOV1901 ⁇ E3-SrfI to obtain the genomic plasmid pNBOV1901 ⁇ E3gp19K with modified SrfI restriction site;
  • the left and right arms of the E1 region obtained by PCR amplification were connected to the resistance vector plasmid B, and after linearization, they were recombined with pNBOV1901 ⁇ E3gp19K digested with SrfI to obtain the genomic plasmid pNBOV1901 ⁇ E3gp19K ⁇ E1 with the E1 region removed;
  • PCR amplification obtains the E1 region genome with the E1b19K and pRB genes removed, and connects it to the resistance vector plasmid B containing the left and right arms of the E1 region. After linearization, it recombines with the digested pNBOV1901 ⁇ E3gp19K ⁇ E1 to obtain a knockout
  • the genomic plasmid pNBOV1910 of E1b19K and pRB genes is the replicating human adenovirus.
  • the resistance vector plasmid A and the resistance vector plasmid B have different resistances.
  • the resistance vector plasmid A and the resistance vector plasmid B are independently selected from an ampicillin resistance vector plasmid or a kanamycin resistance vector plasmid. These resistance plasmids are more mature, easy to obtain, and the screening cost is also lower.
  • Some examples include:
  • PCR amplification obtains the left and right ends of the NBOV1901 genome, which are connected to the ampicillin resistance vector plasmid to obtain pT-NBOV1901(L+R), and after linearization, they are recombined with the NBOV1901 genome to obtain the genome plasmid pNBOV1901;
  • PCR amplification obtains the left and right arms from SrfI to the entire E3 region, connects to the kanamycin resistance vector plasmid, and recombines with PacI digested NBOV1901 after linearization to obtain the genomic plasmid pNBOV1901 ⁇ E3 from which SrfI is removed to the entire E3 region -SrfI;
  • PCR amplification obtains E3gp19K upstream fragments: SrfI to E3gp19K, and E3gp19K downstream fragments: E3gp19K to E3 regions contain E3-14.7K gene fragments, E3gp19K upstream fragments, E3gp19K downstream fragments and digested
  • the three fragments of the constructed vector were recombined and connected between the left and right arms of pNBOV1901 ⁇ E3-SrfI. After linearization, they were recombined with pNBOV1901 ⁇ E3-SrfI to obtain the genomic plasmid pNBOV1901 ⁇ E3gp19K with modified SrfI restriction site;
  • PCR amplification obtains the E1 region genome with the E1b19K and pRB genes removed, connects to the kanamycin resistance vector plasmid containing the left and right arms of the E1 region, and recombines with the digested pNBOV1901 ⁇ E3gp19K ⁇ E1 after linearization.
  • the genomic plasmid pNBOV1910 with knockout E1b19K and pRB genes was obtained.
  • the second aspect of the present invention provides:
  • the recombinant oncolytic virus vector is loaded with a nucleic acid sequence expressing the following molecules:
  • the molecule having a therapeutic effect on tumors is selected from tumor suppressor, tumor killer, tumor microenvironment regulator, tumor cell surface specific binding factor;
  • the biological reporter tracer molecule is selected from ⁇ -galactosidase, secreted alkaline phosphatase, green fluorescent protein, and luciferase.
  • the recombinant oncolytic virus vector is also loaded with a nucleic acid sequence expressing a tumor-specific recognition factor.
  • the tumor-specific recognition factor is selected from a tumor epitope-specific recognition factor and a tumor cell selective replication factor.
  • the tumor is selected from lung cancer, liver cancer, gastric cancer, colorectal cancer, pancreatic cancer, ovarian cancer.
  • At least one region of the original E1 region and E3 region of the vector is used to integrate foreign genes.
  • the third aspect of the present invention provides:
  • composition containing the replicating human adenovirus according to the first aspect of the present invention is a composition containing the replicating human adenovirus according to the first aspect of the present invention.
  • the composition includes: a) an immunomodulator; b) an immunomodulatory cell; c) a radiotherapy; d) a phototherapy compound; or e) at least one of a chemotherapeutic compound.
  • the composition also contains a pharmaceutically acceptable adjuvant, diluent or excipient.
  • the fourth aspect of the present invention provides:
  • An oncolytic preparation comprising the replicating human adenovirus according to the first aspect of the present invention.
  • the replicating human adenovirus is loaded with nucleic acid sequences expressing the following molecules:
  • the molecule having a therapeutic effect on tumors is selected from tumor suppressor, tumor killer, tumor microenvironment regulator, tumor cell surface specific binding factor;
  • the biological reporter tracer molecule is selected from ⁇ -galactosidase, secreted alkaline phosphatase, green fluorescent protein, and luciferase.
  • the recombinant oncolytic virus vector is also loaded with a nucleic acid sequence expressing a tumor-specific recognition factor.
  • the tumor specific recognition factor is selected from tumor epitope specific recognition factor, tumor cell selective replication factor.
  • the tumor is selected from lung cancer, liver cancer, gastric cancer, colorectal cancer, pancreatic cancer, ovarian cancer.
  • the fifth aspect of the present invention provides:
  • composition is used in the preparation of preparations for preventing or treating tumors or tumor detection, and the composition is as described in the third aspect of the present invention.
  • the tumor is selected from breast cancer, brain cancer, lung cancer, nasopharyngeal cancer, head and neck cancer, kidney cancer, bladder cancer, liver cancer, esophageal cancer, stomach cancer, colorectal cancer, pancreatic cancer, ovarian cancer, skin cancer , Prostate cancer, fallopian tube cancer, urethral cancer, genitourinary system cancer, endometriosis, cervical cancer, bone cancer, or metastatic lesions of cancer.
  • the replicating human adenovirus described in some examples of the present invention can be directly injected into solid tumors after being loaded with relevant nucleic acid sequences, including but not limited to H1299, but also Skov-3, U251 and other tumors. After identification, the adenovirus It can not only replicate in tumor cells and directly kill tumor cells, but also infect surrounding tumor cells after the infected tumor cells are lysed; at the same time, it has no killing effect on normal human cells and has an unexpected oncolytic effect.
  • the replicating human adenovirus described in some examples of the present invention has dual targeting properties to a variety of tumor cells H1299, U251, Skov-3 and the like.
  • Figure 1 is the principle of genomic circularization of NBOV1901 by homologous recombination (A), and the result of restriction digestion identification of pNBOV1901 (B);
  • Figure 2 is a schematic diagram of the genome of knocking out NBOV1901 from SrfI to the entire E3 region (A), and the PCR and restriction enzyme digestion results of pNBOV1901 ⁇ E3-SrfI (B);
  • Figure 3 is a schematic diagram of knocking out the NBOV1901E3gp19K gene (A) and the result of pNBOV1901 ⁇ E3gp19K digestion (B);
  • Figure 4 is the schematic diagram of knocking out the NBOV1901E1 gene (A) and the PCR and restriction enzyme digestion identification results of pNBOV1901 ⁇ E3gp19K (B);
  • Figure 5 is the schematic diagram of knocking out NBOV1901E1B19K, pRB gene (A) and the result of pNBOV1910 digestion (B);
  • Figure 6 is the production and purification results of replicating adenovirus NBOV1901 recombinant adenovirus
  • Figure 7 shows the results of bidirectional screening of replicating adenovirus NBOV1901 recombinant adenovirus in various tumor cells
  • Figure 8 shows the effect of modification of the non-core region of the replicating adenovirus NBOV1901 recombinant adenovirus on the oncolytic effect of H1299 cells;
  • Figure 9 is the experimental results of replicating adenovirus NBOV1901 recombinant adenovirus on the H1299 mouse tumor model
  • Figure 10 shows the oncolytic effect of existing oncolytic adenoviruses.
  • M stands for 15000bp DNA Marker.
  • the novel adenovirus vector pNBOV1901 of the present invention is modified from a strain of adenovirus NBOV1901 isolated from human throat swab samples (genBank with full sequence: MH558113.1). NBOV1901 is different from the currently reported adenovirus and is a new type of human adenovirus.
  • the E1A gene of adenovirus is an essential gene for the virus to regulate the cell cycle.
  • the CR2 binding site on the E1A gene binds to the Rb protein of the cell, so that the cell cycle enters the S phase from the G1 phase.
  • the tumor-targeting adenovirus vector pNBOV1910 of the present invention deletes E1A-CR2, so that the E1A of the virus cannot be combined with the Rb protein of the host cell. Therefore, pNBOV1910 can only selectively replicate in cells in the division phase, such as tumor cells, but not in normal cells, thereby increasing the specificity and safety of oncolysis.
  • the E1B19K of adenovirus is homologous to the cell Bcl-2 gene, and can inhibit cell apoptosis by binding to Beclin-1.
  • the E3gp19K protein can bind to the heavy chain of MHC class I molecules in the endoplasmic reticulum to prevent its transport to the cell surface, and can delay the expression of MHC I. Therefore, deleting these two genes can improve the tumor selectivity of the virus and the ability to spread in the tumor, and activate the host immune response, thereby enhancing the killing ability of the virus on tumor cells.
  • This adenovirus transcription unit encodes a series of polypeptides with related functions.
  • the E1A unit encodes two main proteins, which activate transcription and induce host cells to enter the S phase of the cell cycle; E1B encodes two proteins that block apoptosis; E2 encodes Three proteins directly involved in DNA replication; E3 encodes a product that regulates the host's response to infection; the later family of mRNAs is related to the production and assembly of capsid components.
  • the E4 product mediates transcription, RNA splicing and translation regulation; mRNA nuclear export and regulates DNA replication and apoptosis.
  • the deletion of E1B19K and E3 will not affect the replication of adenovirus and can make room for the insertion of foreign genes
  • a replicating adenovirus vector pNBOV1901 is prepared by the following method: circularizing the NBOV1901 genome and knocking out E3gp19K, E1b19K, and pRB genes.
  • genes encoding RGD peptides are introduced into adenovirus plasmids to form targeted backbone plasmids.
  • a preparation method of replicating human NBOV1901 oncolytic adenovirus vector includes the following steps:
  • PCR amplification obtains the left and right ends of the NBOV1901 genome, which are connected to the ampicillin resistance vector plasmid to obtain pT-NBOV1901(L+R), and after linearization, they are recombined with the NBOV1901 genome to obtain the genome plasmid pNBOV1901;
  • PCR amplification obtains the left and right arms from SrfI to the entire E3 region, connects to the kanamycin resistance vector plasmid, and recombines with PacI digested NBOV1901 after linearization to obtain the genomic plasmid pNBOV1901 ⁇ E3 from which SrfI is removed to the entire E3 region -SrfI;
  • PCR amplification obtains E3gp19K upstream fragments: SrfI to E3gp19K, and E3gp19K downstream fragments: E3gp19K to E3 regions contain E3-14.7K gene fragments, E3gp19K upstream fragments, E3gp19K downstream fragments and digested
  • the three fragments of the constructed vector were recombined and connected between the left and right arms of pNBOV1901 ⁇ E3-SrfI. After linearization, they were recombined with pNBOV1901 ⁇ E3-SrfI to obtain the genomic plasmid pNBOV1901 ⁇ E3gp19K with modified SrfI restriction site;
  • PCR amplification obtains the E1 region genome with the E1b19K and pRB genes removed, connects to the kanamycin resistance vector plasmid containing the left and right arms of the E1 region, and recombines with the digested pNBOV1901 ⁇ E3gp19K ⁇ E1 after linearization.
  • the genomic plasmid pNBOV1910 with knockout E1b19K and pRB genes was obtained.
  • ampicillin resistance vector plasmid and the kanamycin resistance vector plasmid can also be replaced with other well-known screening plasmids, as long as the product can be effectively separated.
  • step S1 the specific method of step S1 is:
  • PCR amplification obtained the left and right ends of the NBOV1901 genome, L-NBOV1901 and R-NBOV1901 as recombination arms connected to the linearized T vector to obtain pT-NBOV1901(L+R), and at the same time in pT-NBOV1901( EcoRI and BamHI were introduced between the left and right arms of L+R) as restriction sites, and pT-NBOV1901(L+R) was linearized with EcoRI+BamHI and recombined with the NBOV1901 genome to obtain pNBOV1901.
  • step S1 is:
  • PCR amplifies the left and right ends of the NBOV1901 genome to obtain L-NBOV1901 and R-NBOV1901.
  • L-NBOV1901 and R-NBOV1901 are recombined with linearized T vector into three fragments to obtain pT-NBOV1901( L+R).
  • EcoRI and BamHI were introduced between the left and right arms of pT-NBOV1901(L+R) as restriction sites.
  • pT-NBOV1901(L+R) was linearized with EcoRI+BamHI double restriction
  • the genome of NBOV1901 was recombined to obtain pNBOV1901.
  • step S2 is:
  • PCR amplification obtained from SrfI to the entire E3 region left and right arm genes L- ⁇ E3-SrfI and R- ⁇ E3-SrfI, connected to the linearized T vector to obtain pT- ⁇ E3-SrfI(L+R ), linearized and recombined with the pNBOV1901 genome digested with PacI to obtain the genome plasmid pNBOV1901 ⁇ E3-SrfI that knocked out ⁇ E3-SrfI and introduced the only restriction site PacI at the position of ⁇ E3-SrfI.
  • step S2 is:
  • PCR amplification was performed to obtain the homologous arms L- ⁇ E3-SrfI and R- ⁇ E3-SrfI at the left and right ends of the E3-SrfI region.
  • the three fragments were ligated using Exnase recombinase to obtain pVax- ⁇ E3-SrfI.
  • homologous recombination with the linearized pNBOV1901 by PacI was obtained.
  • the plasmid pNBOV1901 ⁇ E3-SrfI was obtained that knocked out the E3-SrfI region gene and introduced the only linearization site swaI.
  • step S3 is:
  • E3gp19K upstream fragments SrfI to E3gp19K
  • E3gp19K downstream fragments E3gp19K to E3 region, including E3-14.7K gene fragments
  • Exnase recombinase three fragments were recombined and connected to pVAX- ⁇ E3-SrfI (L+R)
  • the pVAX- ⁇ E3gp19K vector plasmid was obtained.
  • the E3gp19K knockout pNBOV1901 ⁇ was obtained.
  • step S4 is:
  • step S4 is:
  • PCR amplification obtained the homologous recombination arms L- ⁇ E1 and R- ⁇ E1 of the E1 gene and the pVAX vector as a template PCR to obtain the backbone of pVAX.
  • the three fragments were ligated using Exnase recombinase to obtain pVAX- ⁇ E3gp19K ⁇ E1( L+R), pVAX- ⁇ E3gp19K ⁇ E1(L+R) was double digested with Bstz17I+SgrAI, pNBOV1901 ⁇ E3gp19K was digested with SrfI for linearization, and the fragments recovered by digestion of the two were subjected to homologous recombination to obtain knockout E1 At the same time, the gene was introduced into the genome plasmid pNBOV1901 ⁇ E3gp19K ⁇ E1, which is the only restriction site SrfI.
  • step S5 is:
  • NBOV1901 genome Using the NBOV1901 genome as a template, PCR amplified to obtain the E1 region genes lacking E1b19K and pRB genes, which were connected between the left and right arms of pVAX- ⁇ E3gp19K ⁇ E1(L+R), and linearized with pNBOV1901 ⁇ E3gp19K ⁇ digested with SrfI
  • the E1 genome undergoes homologous recombination, and the genomic plasmid pNBOV1910 with the E1b19K and pRB genes knocked out is obtained through the screening of ampicillin resistance genes.
  • step S5 is:
  • pVAX- ⁇ E3gp19K ⁇ E1(L+R) linearized pVAX- ⁇ E3gp19K ⁇ E1(L+R) with deletion of E1b19K and pRB genes by PCR amplification and recombination with Exnase recombinase to obtain pVAX- ⁇ E1B19K_pRB
  • pVAX- ⁇ E1B19K_pRB was digested with Bstz17I+SgrAI double digestion
  • pNBOV1901 ⁇ E3gp19K ⁇ E1 was digested with SrfI for linearization
  • the genome plasmid pNBOV1910 lacking E1b19K and pRB genes was screened by ampicillin resistance gene. .
  • the recombinant adenoviral vector pNBOV1910 is linearized by AsisI digestion, recovered by ethanol precipitation, transfected into 293 cells for virus rescue, amplified and cultured, and purified by CsCl density gradient centrifugation to obtain NBOV1910.
  • the term "foreign sequence" in the present invention refers to any DNA sequence derived from a non-NBOV1901 adenovirus.
  • the exogenous sequence may be an expression cassette of an exogenous gene, or an expression cassette of shRNA or miRNA, or the like.
  • the foreign gene expression cassette may include a eukaryotic promoter, a foreign gene coding sequence, and a transcription terminator.
  • the coding sequence of the exogenous gene can be, but is not limited to, the coding sequence of green fluorescent protein, other viral antigens, shRNA and the like.
  • the left arm (L-NBOV1901) and the right arm (R-NBOV1901) of the NBOV1901 genome were obtained by PCR.
  • L-NBOV1901-F gcgggatccgaattcttaatgcgatcgccatcatcaataatataccttat (SEQ ID NO.: 1)
  • L-NBOV1901-R tatctgcatgagcatgatgatatcctttgacccggaacgcgg (SEQ ID NO.: 2)
  • PCR conditions 95°C, 3min; 95°C, 30s; 60°C 30s; 72°C, 30s; cycles 28; 72°C, 5min; 12°C storage.
  • R-NBOV1901-F ccgcgttccgggtcaaaggatatcatcatgctcatgcagata (SEQ ID NO.: 3)
  • R-NBOV1901-R gaagcgagatcgaattcttagcgatcgccatcatcaataaatacctta (SEQ ID NO.: 4)
  • PCR conditions 95°C, 3min; 95°C, 30s; 60°C 30s; 72°C, 1min; cycles 28; 72°C, 5min; 12°C storage.
  • pT-NBOV1901(L+R) was digested and linearized with EcoRI+BamHI, and then co-transformed with the genome of NBOV1901 into BJ5183 competent cells for recombination, and the ampicillin resistance plate was subjected to resistance screening, and the monoclonal amplification obtained after screening was extracted and extracted
  • the plasmid was transformed into XL competent cells, and the plasmid was extracted to obtain pNBOV1901, which was identified by different enzyme digestion methods ( Figure 1B).
  • pNBOV1901 introduced two AsisI restriction sites on both sides of the genome to facilitate subsequent modification of the modified NBOV1901 The genome is linearized for virus rescue.
  • the left arm (L- ⁇ E3_SrfI) and the right arm (R- ⁇ E3_SrfI) of the genes in the SrfI to E3 region were obtained by PCR.
  • L- ⁇ E3_SrfI-F gatatacgcgtgtatac cttcccaggatggcaccca (SEQ ID NO.: 5)
  • L- ⁇ E3_SrfI-R gtaagtaatttattgtgtgtttatg ttaattaa ctgtgtgaccgctgctgt (SEQ ID NO.: 6)
  • PCR conditions 95°C, 3min; 95°C, 30s; 60°C 30s; 72°C, 30s; cycles 28; 72°C, 5min; 12°C storage.
  • R- ⁇ E3_SrfI-F acagcagcggtcacacag ttaattaa cataaacacacacacaataaattacttac (SEQ ID NO.: 7)
  • R- ⁇ E3_SrfI-R ccgcccagtagaagcgccggtg ccgcccgttttaatttccatgtt (SEQ ID NO.: 8)
  • PCR conditions 95°C, 3min; 95°C, 30s; 60°C 30s; 72°C, 50s; cycles 28; 72°C, 5min; 12°C storage.
  • pVax- ⁇ E3_SrfI(L+R) was double digested with Bstz17I+SgrAI
  • pNBOV1901 was digested with PacI
  • the fragments recovered from both digestion were co-transformed into BJ5183 competent cells for recombination, and the ampicillin resistance plate was used for resistance screening.
  • the plasmid was extracted and transformed into XL competent cells.
  • the plasmid was extracted to obtain pNBOV1901 ⁇ E3_SrfI, which was identified by different enzyme digestion methods ( Figure 2B).
  • pNBOV1901 ⁇ E3_SrfI introduced the unique restriction site PacI in the knockout gene region, which is convenient Subsequent cloning operations.
  • PCR amplification obtains gene fragments from SrfI (site) to the entire E3 region except for E3gp19K.
  • E3gp19K-F1 gtcacagggtgcggtcgccaggacagggtataactcacct (SEQ ID NO.: 9)
  • E3gp19K-R1 ggaatacaagcaagcggaaaa tcatcttggatgtcgcccccag (SEQ ID NO.: 10)
  • PCR conditions 95°C, 3min; 95°C, 30s; 60°C 30s; 72°C, 30s; cycles 28; 72°C, 5min; 12°C storage.
  • E3gp19K-F2 ctgggggcgacatccaagatga ttttccgcttgcttgtattcc (SEQ ID NO.: 11)
  • E3gp19K-R2 ccgcccagtagaagcgccggtgccgcccgttttaatttccatgtt (SEQ ID NO.: 8)
  • P1-E3-SrfI ⁇ E3gp19K, P2-E3-SrfI ⁇ E3gp19K and PacI digested pVAX- ⁇ E3-SrfI(L+R) were ligated into three fragments using Exnase enzyme to obtain pVAX- ⁇ E3gp19K.
  • pVAX- ⁇ E3gp19K was digested with Bstz17I+SgrAI double enzyme, pNBOV1901 ⁇ E3_SrfI was digested with PacI, the fragments recovered by both digestion were co-transformed into BJ5183 competent cells for recombination, and the ampicillin resistance plate was used for resistance screening. After clone amplification, the plasmid was extracted to transform XL competent cells, and the plasmid was extracted to obtain pVAX- ⁇ E3gp19K, which was identified by different enzyme digestion methods (Figure 3B).
  • PCR amplification obtains the homologous recombination arms L- ⁇ E1 and R- ⁇ E1 of the E1 gene.
  • L- ⁇ E1-FW cagatatacgcgtgtataccatcatcaataatatacct (SEQ ID NO.: 12)
  • L- ⁇ E1-RW agacagcaagacacttgctatcgatttttagtcccggtgttgga (SEQ ID NO.: 13)
  • PCR conditions 95°C, 3min; 95°C, 30s; 60°C 30s; 72°C, 30s; cycles 28; 72°C, 5min; 12°C storage.
  • R- ⁇ E1-FW tccaacaccgggactaaaaatcgatagcaagtgtcttgctgtct (SEQ ID NO.: 14)
  • PCR conditions 95°C, 3min; 95°C, 30s; 60°C 30s; 72°C, 30s; cycles 28; 72°C, 5min; 12°C storage.
  • M- ⁇ E1, R- ⁇ E1 and Bstz17I+SgrAI double digestion pVax vector obtained the plasmid backbone using Exnase enzyme for three-fragment ligation to obtain pVAX- ⁇ E3gp19K ⁇ E1 (L+R).
  • pVAX- ⁇ E3gp19K ⁇ E1(L+R) was double digested with Bstz17I+SgrAI
  • pNBOV1901 ⁇ E3gp19K was digested with SrfI
  • the fragments recovered from the digestion were co-transformed into BJ5183 competent cells for recombination and carried out with ampicillin resistant plates. Resistance screening, after screening the monoclonal amplification, the plasmid is extracted and transformed into XL competent cells. The plasmid is extracted to obtain pNBOV1901 ⁇ E3gp19K ⁇ E1, which is identified by different enzyme digestion methods ( Figure 4B).
  • pNBOV1901 ⁇ E3gp19K ⁇ E1 is knocking
  • the unique restriction site SrfI is introduced in the gene-removing region to facilitate subsequent cloning operations.
  • Example 5 Construction of the vector pNBOV1901 ⁇ E3gp19K ⁇ E1B19K_pRB, namely pNBOV1910
  • PCR amplification obtains the E1 region gene lacking E1b19K and pRB genes.
  • P1- ⁇ E1B19K_pRB-FW caacaccgggactaaaaatgagacatattatctgcc (SEQ ID NO.: 16)
  • PCR conditions 95°C, 3min; 95°C, 30s; 60°C 30s; 72°C, 30s; cycles 28; 72°C, 5min; 12°C storage.
  • P2- ⁇ E1B19K_pRB-RW gatgggtttcttcgctccat gaggtcaaatgtaaccaaga (SEQ ID NO.: 19)
  • PCR conditions 95°C, 3min; 95°C, 30s; 60°C 30s; 72°C, 30s; cycles 28; 72°C, 5min; 12°C storage.
  • PCR conditions 95°C, 3min; 95°C, 30s; 60°C 30s; 72°C, 30s; cycles 28; 72°C, 5min; 12°C storage.
  • P1- ⁇ E1B19K_pRB, P2- ⁇ E1B19K_pRB, P3- ⁇ E1B19K_pRB and SrfI digested pVAX- ⁇ E1(L+R) were ligated in four fragments using Exnase enzyme to obtain pVAX- ⁇ E1B19K_pRB.
  • pVAX- ⁇ E1B19K_pRB was digested with Bstz17I+SgrAI double digestion
  • pNBOV1901 ⁇ E3gp19K ⁇ E1 was digested with SrfI
  • the fragments recovered from both digestion were co-transformed into BJ5183 competent cells for recombination, and the ampicillin resistance plate was used for resistance screening.
  • the plasmid was extracted to transform XL competent cells, and the plasmid was extracted to obtain pNBOV1910, which was identified by different enzyme digestion methods (Figure 5B).
  • pNBOV1910 is shown in SEQ ID NO.: 21.
  • Example 6 Production and purification results of replicating adenovirus NBOV1901 recombinant adenovirus
  • pNBOV1910 was linearized with AsisI for virus rescue. After the virus was rescued, the culture was gradually expanded. After the virus species was identified and confirmed by PCR and restriction digestion, it was subjected to large expansion culture, cells were collected, and the virus was purified by CsCl density gradient centrifugation. The results are shown in Figure 6. Show, collect the virus and store it at -80 degrees. It can be seen from the figure that pNBOV1910 retains the production performance of replicating adenovirus NBOV1901, and the replication ability is not weakened.
  • Example 7 Oncolytic effect of different doses of NBOV1910 and NBOV1901 on tumor cells such as H1299
  • Each tumor cell in the logarithmic growth phase was plated on 5*10 ⁇ 3cell/well, cultured for 24h, the virus amount per well was 1250, 250 , 50, 10, 2, 0 respectively.
  • the virus was diluted in a gradient of 100 ⁇ l per well. The cells were incubated for 1 hour and cultured for 24-72 hours (72hpi) or 96hpi to determine the tumor cell killing effect.
  • the experimental results are shown in Figure 7.
  • the CCK-8 test results 72 hours after exposure to the virus showed that the killing effect on tumor cells was stronger as the virus concentration increased.
  • the modified NBOV1910 has a good oncolytic effect, and has a better oncolytic effect in certain dose intervals.
  • Example 8 Oncolytic effect of different doses of NBOV1901 ⁇ E3 ⁇ E1B19K_pRb, NBOV1910, NBOV1901 on H1299 tumor cells
  • Each tumor cell in the logarithmic growth phase was plated on 5 ⁇ 10 ⁇ 3cell/well, cultured for 24h, the virus amount per well was 1250, 250 , 50, 10, 2, 0, respectively.
  • the virus was diluted in a gradient of 100 ⁇ l per well. The cells were incubated for 1 hour and cultured for 72 hours (72hpi) or 96hpi to determine the tumor cell killing effect.
  • Figure 8 shows the CCK-8 test results 72 hours after inoculation. The results show that the modification of the variable region of the replicating oncolytic adenovirus NBOV1901 does not affect its oncolytic effect.
  • Balb/c_nude mice 5-6-week-old Balb/c_nude mice were inoculated with 5 ⁇ 10 ⁇ 6 cells of H1299 cells at the junction of the thigh and the dorsal abdomen or the loose skin under the ribs of the forelegs.
  • 1 ⁇ 10 11 VP virus was injected into the tumor with PBS, NBOV1910 and NBOV1901 respectively, and then the tumor size and tumor clearance rate were observed.
  • Figure 10 shows the oncolytic effect of existing oncolytic adenoviruses (see Taki M, Kagawa S, Nishizaki M, et al. Enhanced oncolysis by a tropism-modified telomerase-specific replication-selective adenoviral agent OBP-405('Telomelysin-RGD) ')[J].Oncogene,2005,24(19):3130-3140.), it can be seen that NBOV1910 and NBOV1901 have better effects on non-small cell lung cancer cells.

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Abstract

提供了一种复制型人腺病毒及其应用。腺病毒全序列如SEQ ID NO.：21所示。该复制型人腺病毒，在负载相关核酸序列后，可以直接注射于实体肿瘤，包括但不限于H1299，还包括Skov-3、U251等肿瘤后，可以在肿瘤细胞中复制并直接杀死肿瘤细胞，在所感染肿瘤细胞裂解后感染其周围肿瘤细胞；同时对正常人体细胞没有杀伤作用，具有溶瘤效果。

Description

一种复制型人腺病毒及其应用 技术领域
本发明涉及基因治疗领域，特别涉及重组溶瘤腺病毒、用于制备重组溶瘤腺病毒的重组溶瘤腺病毒载体及其应用。
背景技术
癌症由自身因素(遗传基因突变，自然衰老等)或环境因素(如吸烟，紫外辐射，化学物质，病毒感染等)引起。根据WHO报道，肿瘤目前已经是继心血管疾病的世界第二大死因，全球有近1/6的死亡是由癌症造成的。2018年全球癌症死亡人数达960万。预计到2025年，全球每年新增癌症病例数将高达2449万例。其中，肺癌的死亡人数最多，随后是结直肠癌，胃癌，肝癌和乳腺癌。
治疗癌症的经典方法主要是手术，化学疗法，和放射性疗法。手术切除效果有限，对转移癌细胞没有作用；放疗和化疗副作用大，尽管靶点药物在很大程度上改善了患者生活质量，但耐受性是化疗一直面临的一个难题。近年来，免疫疗法已成为癌症治疗领域的热点。免疫疗法的原理是通过激活机体自身的免疫系统来消灭肿瘤细胞，具有疗效好，副作用小和防止复发等优点。
近几年溶瘤病毒在癌症治疗领域又重新兴起。溶瘤病毒(oncolytic virus)，是一类具有复制能力，能够直接裂解肿瘤细胞的病毒。早在1912年，意大利医生就发现一名子宫颈癌患者在感染狂犬病病毒后肿瘤随之消退。2005年中国批准了世界上第一个溶瘤腺病毒(H101)上市，但临床疗效目前还未得到国际认可。该病毒敲除了E1B-55K和E3区大部分基因。E1B-55K蛋白能够抑制抗癌基因p53的表达，阻止P53介导的感染细胞的凋亡。由于正常的细胞中的P53蛋白功能完好，而大部分肿瘤丧失P53功能，因此，E1B-55K基因的删除使病毒能特异性在缺乏有功能的p53的肿瘤细胞内复制。然而，E1B-55K蛋白是病毒mRNA从细胞核输出必需的蛋白，因此，H101复制能力和溶瘤能力比野生毒株明显减弱。2015年，FDA批准了第一个溶瘤病毒疗法T-vec(单纯疱疹病毒-1)用于治疗手术切除后复发的黑色素瘤。作为第三代溶瘤病毒，T-vec删除了γ34.5基因，使其具有肿瘤细胞靶向性，同时它还携带了GM-CSF(粒细胞-巨噬细胞集落因子)增强抗肿瘤免疫能力。
溶瘤病毒是免疫疗法的一个重要分支，其可以通过多种不同的作用途径杀伤肿瘤细胞，并破坏肿瘤免疫抑制的微环境诱导长期的肿瘤特异性免疫反应，同时还可以通过携带治疗基因使其在肿瘤细胞中特异地，长时间地表达，从而增强抗肿瘤疗效。另外，溶瘤病毒与其他抗癌药物的联合用药的实验结果如联合免疫检查点抑制剂等方面都显现出良好的抗肿瘤效果。近年来，许多病毒包括痘苗病毒、呼肠孤病毒、麻疹病毒、单纯疱疹病毒、新城疫病毒以及柯萨奇病毒已经作为溶瘤剂进行了临床试验。
腺病毒(adenovirus)是一种没有包膜的直径为70～90nm的颗粒，由252个壳粒呈廿面体排列构成。每个壳粒的直径为7～9nm。衣壳里是线状双链DNA分子，约含4.7kb，两端各有长约100～600bp的反向末端重复序列。人腺病毒(Human adenoviruses，HAdV)属于腺病毒科(Adenoviridae)，根据免疫学、生物学、生物化学特性不同，将其分为A～G7个亚种，共有52个血清型，不同的血清型有不同的器官亲和性并引起相应的临床表现。腺病毒侵入宿主细胞后至少能引起3种感染：慢性、潜伏性感染：常在淋巴细胞内发生，潜伏感染时外放的病毒量极少，细胞坏死也不明显，故临床上感染不明显，潜伏感染的机制尚不清楚。溶解性感染：病毒在细胞内如人类上皮细胞中经历复制过程，通过溶解细胞作用使细胞死亡。肿瘤样变异：此时病毒繁殖只进行最初几步，然后腺病毒DNA与细胞DNA整合并复制，但不产生感染性病毒，腺病毒抗原性较稳定。
周立,何婉婉,朱桢楠,et al.溶瘤腺病毒靶向癌症治疗的临床研究进展[J].中国生物工程杂志,2013,33(12):105-113.指出，溶瘤腺病毒能够特异性地感染肿瘤细胞并在肿瘤细胞中完成感染-复制周期，从而特异性地杀伤和裂解肿瘤细胞但不损伤其他正常细胞和组织，因此成为目前最具研究前景的抗肿瘤药物之一。不同的病毒有着复杂的生物学特性，而目前人类完全熟悉的病毒资源仍旧比较匮乏。尤其大多临床前实验资料均是来自免疫缺陷动物体内人肿瘤移植模型，与人在体内环境、组织细胞的生物学特性等方面都存在很大差异。已有的溶瘤腺病毒之间并无明显的共性，难以基于腺病毒的序列确定其是否具有溶瘤效果，但大多数腺病毒对肿瘤细胞不敏感，不具有溶瘤的特性。
发明内容
本发明的目的在于克服现有技术中的至少一种不足，提供一种溶瘤腺病毒载体及其应用。
本发明所采取的技术方案是：
本发明的第一个方面，提供：
一种复制型人腺病毒，其全序列如SEQ ID NO.：21所示，其中的抗性筛选基因可使用公知的筛选基因替换。
在一些实例中，所述复制型人腺病毒的全序列如SEQ ID NO.：21所示。
在一些实例中，所述病毒的E1区和E3区被删除。
在一些实例中，所述病毒可在动物细胞、人源细胞或人体内复制。
在一些实例中，所述复制型人腺病毒的构建方法包括：
S1)PCR扩增获得NBOV1901基因组的左右两端，连接在抗性载体质粒A得到pT-NBOV1901(L+R)，线性化后与NBOV1901基因组重组得到基因组质粒pNBOV1901；
S2)PCR扩增获得从SrfI至整个E3区左右臂连接至抗性载体质粒B上，线性化后与PacI酶切的NBOV1901进行重组，得到去除SrfI至整个E3区的基因组质粒pNBOV1901△E3-SrfI；
S3)以NBOV1901基因组为模板，PCR扩增获得E3gp19K上游片段：SrfI至E3gp19K，和E3gp19K下游片段：E3gp19K至E3区包含E3-14.7K基因片段，将E3gp19K上游片段、E3gp19K下游片段和酶切后的构建载体三个片段重组连接至pNBOV1901△E3-SrfI左右臂之间，线性化后与pNBOV1901△E3-SrfI进行重组，得到修改了SrfI酶切位点的基因组质粒pNBOV1901△E3gp19K；
S4)PCR扩增获得E1区左右臂连接至抗性载体质粒B上，线性化后与SrfI酶切的pNBOV1901△E3gp19K重组，得到去除E1区的基因组质粒pNBOV1901△E3gp19K△E1；
S5)PCR扩增获得去除E1b19K、pRB基因的E1区基因组，连接至含有E1区左右臂的抗性载体质粒B上，线性化后与酶切后的pNBOV1901△E3gp19K△E1进行重组，得到敲除E1b19K、pRB基因的基因组质粒pNBOV1910，即所述复制型人腺病毒。
在一些实例中，抗性载体质粒A和抗性载体质粒B具有不同的抗性。
在一些实例中，抗性载体质粒A和抗性载体质粒B独立选自氨苄抗性载体质粒或卡那霉素抗性载体质粒。这些抗性质粒更为成熟，易于获得，同时筛选成本也更低。
在一些实例中，包括：
S1)PCR扩增获得NBOV1901基因组的左右两端，连接在氨苄抗性载体质粒得到pT-NBOV1901(L+R)，线性化后与NBOV1901基因组重组得到基因组质粒pNBOV1901；
S2)PCR扩增获得从SrfI至整个E3区左右臂，连接至卡那霉素抗性载体质粒上，线性化后与PacI酶切的NBOV1901进行重组，得到去除SrfI至整个E3区的基因组质粒pNBOV1901ΔE3-SrfI；
S3)以NBOV1901基因组为模板，PCR扩增获得E3gp19K上游片段：SrfI至E3gp19K，和E3gp19K下游片段：E3gp19K至E3区包含E3-14.7K基因片段，将E3gp19K上游片段、E3gp19K下游片段和酶切后的构建载体三个片段重组连接至pNBOV1901△E3-SrfI左右臂之间，线性化后与pNBOV1901ΔE3-SrfI进行重组，得到修改了SrfI酶切位点的基因组质粒pNBOV1901ΔE3gp19K；
S4)PCR扩增获得E1区左右臂，连接至卡那霉素抗性载体质粒上，线性化后与SrfI酶切的pNBOV1901△E3gp19K重组，得到去除E1区的基因组质粒pNBOV1901△E3gp19K△E1；
S5)PCR扩增获得去除E1b19K、pRB基因的E1区基因组，连接至含有E1区左右臂的卡那霉素抗性载体质粒上，线性化后与酶切后的pNBOV1901△E3gp19K△E1进行重组，得到敲除E1b19K、pRB基因的基因组质粒pNBOV1910。
本发明的第二个方面，提供：
本发明第一个方面所述复制型人腺病毒作为重组溶瘤病毒载体的应用。
在一些实例中，所述重组溶瘤病毒载体负载有表达以下分子的核酸序列：
对肿瘤具有治疗效果的分子；
生物学报告示踪分子。
在一些实例中，所述对肿瘤具有治疗效果的分子选自肿瘤抑制因子、肿瘤杀伤因子、肿瘤微环境调节因子、肿瘤细胞表面特异性结合因子；
所述生物学报告示踪分子选自β-半乳糖苷酶、分泌型碱性磷酸酶、绿色荧光蛋白、荧光素酶。
在一些实例中，所述重组溶瘤病毒载体还负载有表达肿瘤特异性识别因子的核酸序列。
在一些实例中，所述肿瘤特异性识别因子选自肿瘤表位特异性识别因子、肿瘤细胞选择性复制因子。
这些分子或序列可是本领域技术人员已知的。
在一些实例中，所述肿瘤选自肺癌、肝癌，胃癌、结直肠癌、胰腺癌、卵巢癌。
在一些实例中，所述载体的原E1区和E3区的至少一个区域用于整合外源基因。
本发明的第三个方面，提供：
一种组合物，该组合物中含有本发明第一个方面所述的复制型人腺病毒。
在一些实例中，所述组合物中包括：a)免疫调控剂；b)免疫调控细胞；c)放射性治疗物；d)光疗法化合物；或e)化学治疗化合物中的至少一种。
在一些实例中，述组合物中还含有药学上可接受的佐剂、稀释剂或赋形剂。
本发明的第四个方面，提供：
一种溶瘤制剂，包括本发明第一个方面所述的复制型人腺病毒。
在一些实例中，所述复制型人腺病毒负载有表达以下分子的核酸序列：
对肿瘤具有治疗效果的分子；
生物学报告示踪分子。
在一些实例中，所述对肿瘤具有治疗效果的分子选自肿瘤抑制因子、肿瘤杀伤因子、肿瘤微环境调节因子、肿瘤细胞表面特异性结合因子；
所述生物学报告示踪分子选自β-半乳糖苷酶、分泌型碱性磷酸酶、绿色荧光蛋白、荧光素酶。
在一些实例中，所述重组溶瘤病毒载体还负载有表达肿瘤特异性识别因子的核酸序列。
在一些实例中，所述肿瘤特异性识别因子选自肿瘤表位特异性识别因子、肿瘤细胞选择性复制因 子。
在一些实例中，所述肿瘤选自肺癌、肝癌，胃癌、结直肠癌、胰腺癌、卵巢癌。
本发明的第五个方面，提供：
组合物在制备预防或治疗肿瘤或肿瘤检测制剂中的应用，所述组合物如本发明第三个方面所述。
在一些实例中，所述肿瘤选自乳腺癌、脑癌、肺癌、鼻咽癌、头颈癌、肾癌、膀胱癌、肝癌、食道癌、胃癌、结直肠癌、胰腺癌、卵巢癌、皮肤癌、前列腺癌、输卵管癌、尿道癌、泌尿生殖系统癌、子宫内膜异位症、宫颈癌、骨癌或癌症的转移性病变。
本发明的有益效果是：
本发明一些实例所述的复制型人腺病毒，在负载相关核酸序列后，可以直接注射于实体肿瘤，包括但不限于H1299，还包括Skov-3、U251等肿瘤后，经鉴定，该腺病毒不仅可以在肿瘤细胞中复制并直接杀死肿瘤细胞，在所感染肿瘤细胞裂解后感染其周围肿瘤细胞；同时对正常人体细胞没有杀伤作用，具有意料之外的溶瘤效果。
本发明一些实例所述的复制型人腺病毒对于多种肿瘤细胞H1299、U251、Skov-3等具有双重靶向性。
附图说明
图1是利用同源重组对NBOV1901的基因组环化的原理(A)，及pNBOV1901的酶切鉴定结果(B)；
图2是敲除NBOV1901从SrfI至整个E3区基因组原理图(A)，及pNBOV1901△E3-SrfI的PCR和酶切鉴定结果(B)；
图3是敲除NBOV1901E3gp19K基因原理图(A)及pNBOV1901△E3gp19K酶切结果(B)；
图4是敲除NBOV1901E1基因原理图(A)及pNBOV1901△E3gp19K的PCR及酶切鉴定结果(B)；
图5是敲除NBOV1901E1B19K、pRB基因原理图(A)及pNBOV1910酶切结果(B)；
图6是复制型腺病毒NBOV1901重组腺病毒的生产及纯化结果；
图7是复制型腺病毒NBOV1901重组腺病毒在各种肿瘤细胞双向筛选结果；
图8是复制型腺病毒NBOV1901重组腺病毒非核心区域改造对H1299细胞溶瘤效果影响；
图9是复制型腺病毒NBOV1901重组腺病毒在H1299小鼠肿瘤模型上的实验结果；
图10是现有溶瘤腺病毒的溶瘤作用。
图1～5中，M代表15000bp DNA Marker。
具体实施方式
本发明的新型腺病毒载体pNBOV1901改造自从人的咽拭子样品中分离到的一株腺病毒NBOV1901(全序列的GenBank:MH558113.1)。NBOV1901不同于目前已报道的腺病毒，为一种新型人腺病毒。腺病毒的E1A基因是病毒调节细胞周期的必需基因，通过E1A基因上CR2结合位点与细胞的Rb蛋白结合，使细胞周期由G1期进入S期。本发明的肿瘤靶向性腺病毒载体pNBOV1910，删除了E1A-CR2，使得该病毒的E1A不能与宿主细胞的Rb蛋白结合。因此，pNBOV1910只能选择性地在处于分裂期的细胞如肿瘤细胞中复制而不在正常细胞中复制，从而增加溶瘤的特异性和安全性。
腺病毒的E1B19K与细胞Bcl-2基因同源，可以通过与Beclin-1结合，抑制细胞凋亡。而E3gp19K蛋白可以在内质网上与MHC I类分子的重链结合阻止其转运到细胞表面，并且可以延缓MHC I的表达。因此，删除这两个基因能够提高病毒的肿瘤选择性及在肿瘤中的扩散能力，并激活宿主免疫反应， 从而增强疫毒对肿瘤细胞的杀伤能力。
本腺病毒转录单位编码一系列具有相关功能的多肽，E1A单元编码两种主要蛋白质，它们激活转录并诱导宿主细胞进入细胞周期的S期；E1B编码两种阻断细胞凋亡的蛋白质；E2编码三种直接参与DNA复制的蛋白质；E3编码调节宿主对感染的反应的产物；较晚的mRNAs家族与衣壳成分的生产和组装有关。E4产物介导转录、RNA剪接和翻译调节；mRNA核输出并调节DNA复制和凋亡。E1B19K和E3的删除不会影响腺病毒的复制并且可以为外源基因的插入腾出空间
一种复制型腺病毒载体pNBOV1901，由如下方法制备得到：将NBOV1901基因组环化，敲除E3gp19K、E1b19K、pRB基因得到。
在一些实例中，在腺病毒质粒中引入编码RGD肽的基因，构成靶向性骨架质粒。
一种复制型人NBOV1901型溶瘤腺病毒载体制备方法，包含如下步骤：
S1)PCR扩增获得NBOV1901基因组的左右两端，连接在氨苄抗性载体质粒得到pT-NBOV1901(L+R)，线性化后与NBOV1901基因组重组得到基因组质粒pNBOV1901；
S2)PCR扩增获得从SrfI至整个E3区左右臂，连接至卡那霉素抗性载体质粒上，线性化后与PacI酶切的NBOV1901进行重组，得到去除SrfI至整个E3区的基因组质粒pNBOV1901ΔE3-SrfI；
S3)以NBOV1901基因组为模板，PCR扩增获得E3gp19K上游片段：SrfI至E3gp19K，和E3gp19K下游片段：E3gp19K至E3区包含E3-14.7K基因片段，将E3gp19K上游片段、E3gp19K下游片段和酶切后的构建载体三个片段重组连接至pNBOV1901△E3-SrfI左右臂之间，线性化后与pNBOV1901ΔE3-SrfI进行重组，得到修改了SrfI酶切位点的基因组质粒pNBOV1901ΔE3gp19K；
S4)PCR扩增获得E1区左右臂，连接至卡那霉素抗性载体质粒上，线性化后与SrfI酶切的pNBOV1901△E3gp19K重组，得到去除E1区的基因组质粒pNBOV1901△E3gp19K△E1；
S5)PCR扩增获得去除E1b19K、pRB基因的E1区基因组，连接至含有E1区左右臂的卡那霉素抗性载体质粒上，线性化后与酶切后的pNBOV1901△E3gp19K△E1进行重组，得到敲除E1b19K、pRB基因的基因组质粒pNBOV1910。
氨苄抗性载体质粒、卡那霉素抗性载体质粒也可以使用其他周知的筛选质粒替换，只要可以实现产物的有效分离即可。
在一些实例中，步骤S1的具体方法为：
以NBOV1901基因组为模板，PCR扩增得到NBOV1901基因组左右两端L-NBOV1901和R-NBOV1901作为重组臂连接到线性化的T载体上，得到pT-NBOV1901(L+R)，同时在pT-NBOV1901(L+R)的左右臂之间引入了EcoRI、BamHI作为酶切位点，EcoRI+BamHI双酶切pT-NBOV1901(L+R)线性化后与NBOV1901基因组重组，得到pNBOV1901。
进一步的，在一些实例中，步骤S1的具体方法为：
以NBOV1901基因组为模板，PCR扩增得到NBOV1901基因组左右两端L-NBOV1901和R-NBOV1901，使用Exnase重组酶将L-NBOV1901和R-NBOV1901和线性化的T载体进行三片段重组得到pT-NBOV1901(L+R)，同时在pT-NBOV1901(L+R)的左右臂之间引入了EcoRI、BamHI作为酶切位点，pT-NBOV1901(L+R)用EcoRI+BamHI双酶切线性化后与NBOV1901基因组重组，得到pNBOV1901。
在一些实例中，步骤S2的具体方法为：
以NBOV1901基因组为模板，PCR扩增获得从SrfI至整个E3区左右臂基因L-ΔE3-SrfI及R- ΔE3-SrfI，连接到线性化的T载体上，得到pT-ΔE3-SrfI(L+R)，线性化后与经过PacI酶切的pNBOV1901基因组进行重组，得到敲除ΔE3-SrfI同时在ΔE3-SrfI位置引入唯一的酶切位点PacI的基因组质粒pNBOV1901△E3-SrfI。
进一步的，在一些实例中，步骤S2的具体方法为：
以NBOV1901基因组为模板，PCR扩增获得E3-SrfI区左右两端同源臂L-△E3-SrfI和R-△E3-SrfI，使用Exnase重组酶进行三片段连接得到pVax-△E3-SrfI。线性化后与PacI酶切线性化的pNBOV1901同源重组，经氨苄抗性筛选后得到敲除E3-SrfI区基因同时引入唯一的线性化酶切位点swaI的质粒pNBOV1901△E3-SrfI。
在一些实例中，步骤S3的具体方法为：
以NBOV1901基因组为模板，PCR扩增获得E3gp19K上游片段(SrfI至E3gp19K)和E3gp19K下游片段(E3gp19K至E3区，包含E3-14.7K基因片段)，Exnase重组酶三片段重组连接到pVAX-ΔE3-SrfI(L+R)左右臂之间，得到pVAX-ΔE3gp19K载体质粒，线性化后与PacI酶切的pNBOV1901△E3-SrfI基因组质粒进行同源重组，经氨苄抗性基因筛选得到敲除E3gp19K的pNBOV1901△E3gp19K基因组质粒。
在一些实例中，步骤S4的具体方法为：
以NBOV1901基因组为模板，PCR扩增得到E1基因同源重组臂L-ΔE1及R-ΔE1，连接到线性化的T载体上，得到pVAX-ΔE1(L+R)，线性化后与SrfI酶切的pNBOV1901ΔE3gp19K同源重组，经氨苄抗性筛选得到敲除E1基因并在E1基因区引入唯一的线性化酶切位点SrfI的质粒pNBOV1901△E3gp19K△E1。
进一步的，在一些实例中，步骤S4的具体方法为：
以NBOV1901基因组为模板，PCR扩增得到E1基因同源重组臂L-ΔE1及R-ΔE1和以pVAX载体为模板PCR获得pVAX的骨架使用Exnase重组酶进行三片段连接得到pVAX-△E3gp19K△E1(L+R)，pVAX-△E3gp19K△E1(L+R)使用Bstz17I+SgrAI双酶切，pNBOV1901ΔE3gp19K使用SrfI进行酶切进行线性化，将两者酶切回收的片段进行同源重组得到敲除E1基因同时引入唯一酶切位点SrfI的基因组质粒pNBOV1901△E3gp19K△E1。
在一些实例中，步骤S5的具体方法为：
以NBOV1901基因组为模板，PCR扩增获得缺失E1b19K、pRB基因的E1区基因，连接到pVAX-△E3gp19K△E1(L+R)左右臂之间，线性化后与SrfI酶切的pNBOV1901△E3gp19K△E1基因组进行同源重组，经氨苄抗性基因筛选得到敲除E1b19K、pRB基因的基因组质粒pNBOV1910。
进一步的，在一些实例中，步骤S5的具体方法为：
以NBOV1901基因组为模板，PCR扩增获得缺失E1b19K、pRB基因的E1区基因并与SrfI酶切线性化的pVAX-△E3gp19K△E1(L+R)使用Exnase重组酶进行重组得到pVAX-△E1B19K_pRB，pVAX-△E1B19K_pRB使用Bstz17I+SgrAI双酶切，pNBOV1901ΔE3gp19KΔE1使用SrfI进行酶切进行线性化，将两者酶切回收的片段进行同源重组经氨苄抗性基因筛选得到缺失E1b19K、pRB基因的基因组质粒pNBOV1910。
在一些实例中，所述重组腺病毒载体pNBOV1910使用AsisI酶切进行线性化，乙醇沉淀回收后转染293细胞进行病毒拯救、扩增培养，以CsCl密度梯度离心法纯化得到NBOV1910。
本发明术语“外源序列”是指非NBOV1901型腺病毒来源的任何DNA序列。本领域普通技术人员应当理解，外源序列可以是外源基因表达框，也可以是shRNA或miRNA的表达框等。
在以下实施例中，根据本领域技术人员的理解，外源基因表达框可包含真核启动子、外源基因编码序列、转录终止子。所述外源基因编码序列可以是但不仅限于绿色荧光蛋白、其他病毒抗原、shRNA等的编码序列。
为了能够更清楚地理解本发明的技术内容，特举以下实施例结合附图详细说明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(NewYork:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。实施例中所用到的各种常用化学试剂，均为市售产品。
除非另有定义，本发明所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不用于限制本发明。
实施例1：NBOV1901基因组的环化
1.构建环化NBOV1901基因组的穿梭质粒pT-NBOV1901(L+R)。
参照图1A，以NBOV1901的基因组为模板，PCR获得NBOV1901基因组的左臂(L-NBOV1901)和右臂(R-NBOV1901)。
L-NBOV1901引物：
L-NBOV1901-F：gcgggatccgaattcttaatgcgatcgccatcatcaataatataccttat(SEQ ID NO.：1)
L-NBOV1901-R：tatctgcatgagcatgatgatatcctttgacccggaacgcgg(SEQ ID NO.：2)
PCR条件：95℃，3min；95℃，30s；60℃30s；72℃，30s；cycles 28；72℃，5min；12℃保存。
R-NBOV1901引物：
R-NBOV1901-F：ccgcgttccgggtcaaaggatatcatcatgctcatgcagata(SEQ ID NO.：3)
R-NBOV1901-R：gaagcgagatcgaattcttagcgatcgccatcatcaataaatacctta(SEQ ID NO.：4)
PCR条件：95℃，3min；95℃，30s；60℃30s；72℃，1min；cycles 28；72℃，5min；12℃保存。
2.构建pNBOV1901。
pT-NBOV1901(L+R)使用EcoRI+BamHI进行酶切线性化，然后和NBOV1901的基因组共转化BJ5183感受态细胞进行重组，氨苄抗性平板进行抗性筛选，将筛选得到单克隆扩增后提取其质粒转化XL感受态细胞，提取质粒得到pNBOV1901，使用不同的酶切方式进行鉴定(图1B)，pNBOV1901在基因组的两侧引入了两个AsisI酶切位点，方便后续对改造后的NBOV1901的基因组进行线性化进行病毒拯救。
实施例2：SrfI至E3区基因的敲除，构建pNBOV1901ΔE3_SrfI质粒的构建
1.构建SrfI至E3区基因敲除的穿梭质粒pVax-ΔE3_SrfI(L+R)。
参照图2A，以NBOV1901的基因组为模板，PCR获得SrfI至E3区基因的左臂(L-ΔE3_SrfI)和右臂(R-ΔE3_SrfI)。
L-ΔE3_SrfI引物：
L-ΔE3_SrfI-F：gatatacgcgtgtatac cttcccaggatggcaccca(SEQ ID NO.：5)
L-ΔE3_SrfI-R：gtaagtaatttattgtgtgtttatg ttaattaa ctgtgtgaccgctgctgt(SEQ ID NO.：6)
PCR条件：95℃，3min；95℃，30s；60℃30s；72℃，30s；cycles 28；72℃，5min；12℃保存。
R-ΔE3引物：
R-ΔE3_SrfI-F：acagcagcggtcacacag ttaattaa cataaacacacaataaattacttac(SEQ ID NO.：7)
R-ΔE3_SrfI-R：ccgcccagtagaagcgccggtg ccgcccgttttaatttccatgtt(SEQ ID NO.：8)
PCR条件：95℃，3min；95℃，30s；60℃30s；72℃，50s；cycles 28；72℃，5min；12℃保存。
L-ΔE3_SrfI、R-ΔE3_SrfI和Bstz17I+SgrAI双酶切pVax载体获得的质粒骨架使用Exnase酶进行三片段连接得到pVax-ΔE3_SrfI(L+R)。
2.构建pNBOV1901ΔE3_SrfI
pVax-ΔE3_SrfI(L+R)使用Bstz17I+SgrAI双酶切，pNBOV1901使用PacI进行酶切，将两者酶切回收的片段共转化BJ5183感受态细胞进行重组，用氨苄抗性平板进行抗性筛选，将筛选得到单克隆扩增后提取其质粒转化XL感受态细胞，提取质粒得到pNBOV1901ΔE3_SrfI，使用不同的酶切方式进行鉴定(图2B)，pNBOV1901ΔE3_SrfI在敲除基因区引入唯一酶切位点PacI，方便后续克隆操作。
实施例3：构建pNBOV1901ΔE3gp19K质粒
1.构建敲除E3gp19K的穿梭载体pVAX-△E3gp19K。
参照图3A，以NBOV1901基因组为模板，PCR扩增获得除E3gp19K外从SrfI(位点)至整个E3区基因片段。
E3gp19K上游片段P1-E3-SrfI△E3gp19K：
E3gp19K-F1：gtcacagggtgcggtcgccaggacagggtataactcacct(SEQ ID NO.：9)
E3gp19K-R1：ggaatacaagcaagcggaaaa tcatcttggatgtcgcccccag(SEQ ID NO.：10)
PCR条件：95℃，3min；95℃，30s；60℃30s；72℃，30s；cycles 28；72℃，5min；12℃保存。
E3gp19K下游片段P2-E3-SrfI△E3gp19K；
E3gp19K-F2：ctgggggcgacatccaagatga ttttccgcttgcttgtattcc(SEQ ID NO.：11)
E3gp19K-R2：ccgcccagtagaagcgccggtg ccgcccgttttaatttccatgtt(SEQ ID NO.：8)
P1-E3-SrfI△E3gp19K、P2-E3-SrfI△E3gp19K和PacI酶切后的pVAX-△E3-SrfI(L+R)使用Exnase酶进行三片段连接得到pVAX-△E3gp19K。
2.构建pNBOV1901ΔE3gp19K质粒
pVAX-△E3gp19K使用Bstz17I+SgrAI双酶切，pNBOV1901ΔE3_SrfI使用PacI进行酶切，将两者酶切回收的片段共转化BJ5183感受态细胞进行重组，用氨苄抗性平板进行抗性筛选，将筛选得到单克隆扩增后提取其质粒转化XL感受态细胞，提取质粒得到pVAX-△E3gp19K，使用不同的酶切方式进行鉴定(图3B)。
实施例4：构建pNBOV1901△E3gp19K△E1质粒
1.构建敲除E1区基因的穿梭载体pVAX-△E3gp19K△E1(L+R)。
参照图4A，以NBOV1901基因组为模板，PCR扩增得到E1基因同源重组臂L-ΔE1及R-ΔE1。
L-ΔE1引物：
L-ΔE1-FW:cagatatacgcgtgtataccatcatcaataatatacct(SEQ ID NO.：12)
L-ΔE1-RW:agacagcaagacacttgctatcgatttttagtcccggtgttgga(SEQ ID NO.：13)
PCR条件：95℃，3min；95℃，30s；60℃30s；72℃，30s；cycles 28；72℃，5min；12℃保存。
R-ΔE1引物：
R-ΔE1-FW:tccaacaccgggactaaaaatcgatagcaagtgtcttgctgtct(SEQ ID NO.：14)
R-ΔE1-RW:gcccagtagaagcgccggtggaattcatctgaactcaaagcgtg(SEQ ID NO.：15)
PCR条件：95℃，3min；95℃，30s；60℃30s；72℃，30s；cycles 28；72℃，5min；12℃保存。
M-ΔE1、R-ΔE1和Bstz17I+SgrAI双酶切pVax载体获得的质粒骨架使用Exnase酶进行三片段连接得到pVAX-△E3gp19K△E1(L+R)。
2.构建pNBOV1901△E3gp19K△E1。
pVAX-△E3gp19K△E1(L+R)使用Bstz17I+SgrAI双酶切，p NBOV1901ΔE3gp19K使用SrfI进行酶切，将两者酶切回收的片段共转化BJ5183感受态细胞进行重组，用氨苄抗性平板进行抗性筛选，将筛选得到单克隆扩增后提取其质粒转化XL感受态细胞，提取质粒得到pNBOV1901△E3gp19K△E1，使用不同的酶切方式进行鉴定(图4B)，pNBOV1901△E3gp19K△E1在敲除基因区引入唯一酶切位点SrfI，方便后续克隆操作。
实施例5：构建载体pNBOV1901△E3gp19K△E1B19K_pRB，即pNBOV1910
1.构建敲除E1B19K、pRB基因的穿梭载体穿梭载体pVAX-△E1B19K_pRB。
参照图5A，以NBOV1901基因组为模板，PCR扩增获得缺失E1b19K、pRB基因的E1区基因。
P1-△E1B19K_pRB引物：
P1-△E1B19K_pRB-FW：caacaccgggactaaaaatgagacatattatctgcc(SEQ ID NO.：16)
P1-△E1B19K_pRB-RW:atcctcgtcgtcactgggtgg atcgaccacctccggtacaa(SEQ ID NO.：17)
PCR条件：95℃，3min；95℃，30s；60℃30s；72℃，30s；cycles 28；72℃，5min；12℃保存。
P2-△E1B19K_pRB引物：
P2-△E1B19K_pRB-FW:ttgtaccggaggtggtcgat ccacccagtgacgacgaggat(SEQ ID NO.：18)
P2-△E1B19K_pRB-RW:gatgggtttcttcgctccat gaggtcaaatgtaaccaaga(SEQ ID NO.：19)
PCR条件：95℃，3min；95℃，30s；60℃30s；72℃，30s；cycles 28；72℃，5min；12℃保存。
P3-△E1B19K_pRB引物：
P3-△E1B19K_pRB-FW:tcttggttacatttgacctc atggagcgaagaaacccatc(SEQ ID NO.：20)
P3-△E1B19K_pRB-RW:tcttggttacatttgacctc atggagcgaagaaacccatc(SEQ ID NO.：20)
PCR条件：95℃，3min；95℃，30s；60℃30s；72℃，30s；cycles 28；72℃，5min；12℃保存。
P1-△E1B19K_pRB、P2-△E1B19K_pRB、P3-△E1B19K_pRB和SrfI酶切后的pVAX-△E1(L+R)使用Exnase酶进行四片段连接得到pVAX-△E1B19K_pRB。
2.构建pNBOV1901△E3gp19K△E1B19K_pRB
pVAX-△E1B19K_pRB使用Bstz17I+SgrAI双酶切，pNBOV1901ΔE3gp19KΔE1使用SrfI进行酶切，将两者酶切回收的片段共转化BJ5183感受态细胞进行重组，用氨苄抗性平板进行抗性筛选，将筛选得到单克隆扩增后提取其质粒转化XL感受态细胞，提取质粒得到pNBOV1910，使用不同的酶切方式进行鉴定(图5B)。
经测序，pNBOV1910如SEQ ID NO.：21所示。
实施例6：复制型腺病毒NBOV1901重组腺病毒的生产及纯化结果
pNBOV1910用AsisI线性化后进行病毒拯救，拯救出毒以后逐步扩大培养，在对毒种进行PCR和酶切鉴定确认后进行大扩培养，收集细胞，CsCl密度梯度离心纯化病毒，结果如图6所示，收集得到的病毒，-80度保存。从图中可以看出，pNBOV1910保留了复制型腺病毒NBOV1901生产性能， 复制能力未有减弱。
实施例7：不同剂量NBOV1910、NBOV1901在H1299等肿瘤细胞溶瘤效果
对数生长期的各肿瘤细胞铺板5*10^3cell/well，培养24h，每孔病毒量 TCID50分别为1250、250、 50、10、2、0梯度进行病毒稀释，稀释后每孔添加100μl感染细胞孵育1h，培养24-72小时(72hpi)或96hpi测定肿瘤细胞杀伤效果。
实验结果如图7所示。接毒72h后CCK-8检测结果，结果显示随着病毒浓度的增加对肿瘤细胞杀伤作用更强。改造得到的NBOV1910具有良好的溶瘤效果，在某些剂量区间具有更好的溶瘤效果。
实施例8：不同剂量NBOV1901ΔE3ΔE1B19K_pRb、NBOV1910、NBOV1901在H1299肿瘤细胞溶瘤效果
对数生长期的各肿瘤细胞铺板5×10^3cell/well，培养24h，每孔病毒量 TCID50分别为1250、250、 50、10、2、0梯度进行病毒稀释，稀释后每孔添加100μl感染细胞孵育1h，培养72小时(72hpi)或96hpi测定肿瘤细胞杀伤效果。
图8为接毒72h后CCK-8检测结果，结果显示复制型溶瘤腺病毒NBOV1901可变区的改造不影响其溶瘤效果。
实施例9：NBOV1910、NBOV1901小鼠体内抗肿瘤效果
用Balb/c_nude小鼠模型，在5-6周龄的Balb/c_nude小鼠大腿与背腹部交界或前腿后肋下皮肤松弛处接种5×10^6cell的H1299细胞。当肿瘤生长到200mm 3时，用1×10 11VP病毒在瘤内注射分别注射PBS、NBOV1910和NBOV1901，然后观察肿瘤大小和肿瘤清除率。
实验结果如图9所示，说明治疗组NBOV1910、NBOV01能够显著抑制肿瘤生长，具有治疗肿瘤的潜力。
图10是现有溶瘤腺病毒的溶瘤作用(参见Taki M,Kagawa S,Nishizaki M,et al.Enhanced oncolysis by a tropism-modified telomerase-specific replication-selective adenoviral agent OBP-405(‘Telomelysin-RGD’)[J].Oncogene,2005,24(19):3130-3140.)，可见NBOV1910、NBOV1901在非小细胞肺癌细胞具有更好的效果。

Claims (8)

  1. 一种复制型人腺病毒，其核苷酸序列如SEQ ID NO.：21所示。
  2. 如权利要求1所述复制型人腺病毒，其特征在于：所述病毒的E1区和E3区被删除。
  3. 如权利要求1所述复制型人腺病毒，其特征在于：所述病毒可在动物细胞、人源细胞或人体内复制。
  4. 一种组合物，其特征在于：其含有权利要求1所述的复制型人腺病毒。
  5. 根据权利要求4所述的组合物，其特征在于，所述组合物中包括：a)免疫调控剂；b)免疫调控细胞；c)放射性治疗物；d)光疗法化合物；或e)化学治疗化合物中的至少一种。
  6. 根据权利要求4所述的组合物，其特征在于，所述组合物中还含有药学上可接受的佐剂、稀释剂或赋形剂。
  7. 组合物在制备预防或治疗肿瘤或肿瘤检测制剂中的应用，其特征在于：所述组合物如权利要求4～6任一项所述。
  8. 根据权利要求7所述的应用，其特征在于：所述肿瘤选自乳腺癌、脑癌、肺癌、鼻咽癌、头颈癌、肾癌、膀胱癌、肝癌、食道癌、胃癌、结直肠癌、胰腺癌、卵巢癌、皮肤癌、前列腺癌、输卵管癌、尿道癌、泌尿生殖系统癌、子宫内膜异位症、宫颈癌、骨癌或癌症的转移性病变。
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