WO2024082262A1 - 一种响应缺氧环境调控病毒复制的可剪接系统及其用途 - Google Patents

一种响应缺氧环境调控病毒复制的可剪接系统及其用途 Download PDF

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WO2024082262A1
WO2024082262A1 PCT/CN2022/126655 CN2022126655W WO2024082262A1 WO 2024082262 A1 WO2024082262 A1 WO 2024082262A1 CN 2022126655 W CN2022126655 W CN 2022126655W WO 2024082262 A1 WO2024082262 A1 WO 2024082262A1
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virus
cancer
gene encoding
protein
viral replication
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French (fr)
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徐建青
张晓燕
丁相卿
廖启彬
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上海鑫湾生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/768—Oncolytic viruses not provided for in groups A61K35/761 - A61K35/766
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/91—Cell lines ; Processes using cell lines

Definitions

  • the present invention belongs to the field of biopharmaceutical technology, and specifically relates to a splicing system that regulates viral replication in response to a hypoxic environment and its use in preparing drugs for treating hypoxic diseases such as solid tumors, especially in preparing oncolytic virus drugs.
  • Tumors are a very complex malignant cell tissue. Due to the mutation of the signaling pathways that control cell growth and immune surveillance, tumor cells proliferate malignantly, thus forming tumor tissue. Most tumors develop into an immunosuppressive environment, which causes the downregulation of immune co-stimulatory molecules and the general high expression of immune checkpoint molecules, which ultimately causes the tumor to "cool down” and escape the body's anti-tumor immune response.
  • Tumor immunotherapy involves immunotherapy methods such as immune checkpoint inhibitors (ICI), T cell receptor modified T cells (TCR-T cells), chimeric antigen receptor T cells (CAR-T cells) and oncolytic viruses (OVs), all of which have shown remarkable effects in anti-tumor clinical studies.
  • ICI immune checkpoint inhibitors
  • TCR-T cells T cell receptor modified T cells
  • CAR-T cells chimeric antigen receptor T cells
  • OVs oncolytic viruses
  • Oncolytic virus is an immunotherapy method that makes full use of the tumor tissue tropism of the virus itself, allowing it to selectively proliferate and lyse tumor cells in tumor tissue, but without harming the normal tissues of the body.
  • the immunosuppressive environment of the tumor is also conducive to the virus being cleared by the body, which also creates the possibility for the application of oncolytic virus anti-tumor immunotherapy.
  • treatment with oncolytic virus alone is often difficult to achieve the desired effect. This is because the mechanism of action of existing oncolytic virus treatment products is relatively simple, and there is a large immunogenicity, and it is impossible to specifically distinguish between normal tissue and tumor tissue.
  • the virus replicates in normal tissue before it reaches the lesion, or the antiviral immune response has already eliminated the virus. Even if the drug is administered multiple times, due to the function of immune memory, a stronger immune response will be produced, and the oncolytic virus will be quickly cleared, but the purpose of treatment will still not be achieved.
  • Oncolytic viruses need to be immunogenic to induce anti-tumor immunity, but also need to avoid immune surveillance so that the virus can replicate in tumor tissue without infecting normal tissue. How to solve the problem of immunogenicity of oncolytic viruses and continuous viral replication has become a major challenge in the field of oncolytic virus therapy.
  • the object of the present invention is to provide a splicing system that regulates viral replication in response to a hypoxic environment and its application in the preparation of drugs for treating hypoxic diseases such as solid tumors, especially in the preparation of oncolytic virus drugs for treating solid tumors.
  • the present invention has developed a modular design of key factors for controlling virus replication, wherein the key factors related to virus replication are controlled by two units, one is a hypoxia sensitive unit, which is used to sense the oxygen environment; the other is a virus replication control unit, which is used to control the replication and proliferation of the virus.
  • the activation of key factors related to modular virus replication is based on two conditions.
  • hypoxia sensing module contains elements that can cut off degradation factors, and the hypoxia sensing module senses the oxygen environment in the tissue environment, and the hypoxia sensitive module is continuously enriched, thereby releasing the key factors related to virus replication, allowing the virus to complete replication and proliferation, and ultimately achieving the purpose of hypoxia-sensitive replication of the virus.
  • splicing means that the protein molecule can be rejoined after being cleaved.
  • oxygen-sensitive protein refers to a protein molecule that is able to sense oxygen.
  • viral replication critical factor refers to a replicase or transcription factor that is critical in the viral proliferation cycle.
  • N-terminal splicing domain refers to the N-terminal domain of an intein that can be spliced with a C-terminal splicing domain.
  • degradation refers to a specific amino acid sequence that can be recognized by intracellular proteases to mediate the degradation and clearance of target proteins.
  • C-terminal splicing domain refers to the C-terminal domain of an intein that can be spliced with the N-terminal splicing domain.
  • hypoxia disease refers to diseases in which tissue oxygen levels fall below 2%, particularly solid tumors.
  • the present invention provides a spliceable system for regulating viral replication in response to a hypoxic environment, which comprises a gene encoding a hypoxia sensing unit and a gene encoding a viral replication control unit, which are separated from or fused to each other, wherein the gene encoding the hypoxia sensing unit comprises an N-terminal splicing domain element and an oxygen-sensitive protein element connected to each other, and the gene encoding the viral replication control unit comprises a C-terminal splicing domain element and a viral replication key factor element connected via a degradation subelement.
  • the hypoxic environment refers to cells or tissues with an oxygen content of less than 2%.
  • Tumor tissues and cells have a great demand for energy substances such as oxygen and glucose.
  • hypoxia of the tumor cells and tissues occurs, and the oxygen content of the cells and tissues is often less than 2%.
  • the splicing system for regulating virus replication in response to hypoxia can respond to a specific condition signal - hypoxia, so as to achieve splicing of key factors of virus replication in response to oxygen environment.
  • the hypoxia sensing unit can respond to a specific condition signal - hypoxia, showing the characteristics of low expression of key factors of virus replication in normal tissue environment and enrichment in tumor microenvironment, that is, key factors of virus replication can be spontaneously/induced to degrade in normal tissues, which promotes the obstruction of virus replication and reduces retention.
  • a gene encoding a linker is included between the N-terminal splicing domain element and the oxygen-sensitive protein element;
  • a gene encoding a linker is included between the degradation sub-element and the C-terminal splicing domain element and between the degradation sub-element and the viral replication key factor element;
  • the linker is (GGS) n , wherein n is 1-3.
  • the N-terminal splicing domain is a protein intron and/or a SpyTag/SpyCatcher self-assembler.
  • the N-terminal splicing domain is a protein intron Int N .
  • nucleotide sequence of the N-terminal splicing domain element is shown in SEQ ID NO: 1.
  • the oxygen-sensitive protein is an oxygen-dependent degradation domain (ODD).
  • ODD oxygen-dependent degradation domain
  • the oxygen-dependent degradation domain is selected from one or more of HIF-1 ⁇ 557-574 (ODD18), HIF-1 ⁇ 530-652 (ODD123) or 4*ODD18 (4 consecutive repeats of ODD18).
  • nucleotide sequence of the oxygen-dependent degradation domain is shown in SEQ ID NO: 2.
  • nucleotide sequence encoding the hypoxia sensing unit is shown as SEQ ID NO: 3.
  • the splicing system for regulating viral replication in response to a hypoxic environment provides a degradable key factor for viral replication, which can be degraded under normal tissue conditions. Since the virus lacks the participation of key factors for its life cycle, viral replication is significantly inhibited and the virus cannot replicate and proliferate.
  • the degradon is selected from one or more of dihydrofolate reductase (DHFR), estrogen receptor (ER), Salmonella type III secretion system effector protein (SopE), plant hormone-induced protein degradon, unstable domain (AD) or light-sensitive protein degradon.
  • DHFR dihydrofolate reductase
  • ER estrogen receptor
  • SopE Salmonella type III secretion system effector protein
  • AD unstable domain
  • the degradon is selected from one or more of dihydrofolate reductase, estrogen receptor or Salmonella type III secretion system effector protein. More preferably, the degradon is dihydrofolate reductase.
  • the nucleotide sequence of the degradation sub-element is shown in one or more of SEQ ID NO: 4, 5 or 6.
  • the C-terminal splicing domain is a protein intron and/or a SpyTag/SpyCatcher self-assembler.
  • the C-terminal splicing domain is a protein intron Int C.
  • nucleotide sequence of the C-terminal splicing domain element is shown in SEQ ID NO: 7.
  • the key factor of viral replication is selected from one or more of polymerase, transcriptase, protein translation enzyme, RNase, protein degrading enzyme, structural protein, assembly element, transcription factor or exocytosis protein.
  • the key factor of viral replication is selected from one or more of polymerase, transcriptase, transcription factor and protein translation enzyme in the early stage of viral replication.
  • nucleotide sequence of the key factor element of viral replication is shown as SEQ ID NO: 8.
  • nucleotide sequence of the gene encoding the viral replication control unit is shown as SEQ ID NO: 9.
  • the splicing system for regulating viral replication in response to a hypoxic environment comprises:
  • a first vector comprising the gene encoding the hypoxia sensing unit
  • a second vector comprising the gene encoding the viral replication control unit.
  • the spliceable system for regulating viral replication in response to a hypoxic environment comprises a vector comprising a fusion of the gene encoding the hypoxia sensing unit and the gene encoding the viral replication control unit.
  • the vector is selected from one or more of vaccinia virus, adenovirus, herpes simplex virus type I (HSV-1), herpes simplex virus type II (HSV-2), vesicular stomatitis virus, echovirus, reovirus, alphavirus, yellow fever virus, coxsackievirus, Newcastle disease virus, measles virus, poliovirus, Zika virus, lymphocytic choriomeningitis virus, M1 virus, Marburg virus, lentivirus or retrovirus.
  • the vector is vaccinia virus.
  • the present invention provides a recombinant virus, the genome of which comprises the splicing system for regulating viral replication in response to a hypoxic environment according to the present invention.
  • the virus is selected from one or more of vaccinia virus, adenovirus, herpes simplex virus type I (HSV-1), herpes simplex virus type II (HSV-2), vesicular stomatitis virus, echovirus, reovirus, alphavirus, yellow fever virus, coxsackievirus, Newcastle disease virus, measles virus, poliovirus, Zika virus, lymphocytic choriomeningitis virus, M1 virus, Marburg virus, lentivirus or retrovirus.
  • HSV-1 herpes simplex virus type I
  • HSV-2 herpes simplex virus type II
  • vesicular stomatitis virus echovirus
  • reovirus alphavirus
  • yellow fever virus coxsackievirus
  • Newcastle disease virus measles virus
  • poliovirus Zika virus
  • lymphocytic choriomeningitis virus M1 virus
  • Marburg virus Marburg virus
  • lentivirus or retrovirus lentivirus
  • the genome of the recombinant virus further comprises coding genes of one or more selected from costimulatory molecules, cytokines, negative regulatory molecules, signal pathway blocking antibodies, chemokines or killer molecules, preferably killer molecules, and more preferably BiTE coding genes.
  • the present invention provides a pharmaceutical composition for treating hypoxic diseases, comprising a splicing system for regulating viral replication in response to a hypoxic environment according to the present invention and a virus for treating a hypoxic disease, or a recombinant virus according to the present invention, and optional pharmaceutically acceptable excipients.
  • the virus for treating hypoxic diseases is selected from one or more of vaccinia virus, adenovirus, herpes simplex virus type I (HSV-1), herpes simplex virus type II (HSV-2), vesicular stomatitis virus, echovirus, reovirus, alphavirus, yellow fever virus, coxsackievirus, Newcastle disease virus, measles virus, poliovirus, Zika virus, lymphocytic choriomeningitis virus, M1 virus, Marburg virus, lentivirus or retrovirus.
  • HSV-1 herpes simplex virus type I
  • HSV-2 herpes simplex virus type II
  • vesicular stomatitis virus echovirus
  • reovirus alphavirus
  • yellow fever virus coxsackievirus
  • Newcastle disease virus measles virus
  • poliovirus Zika virus
  • lymphocytic choriomeningitis virus M1 virus, Marburg virus, lentivirus or retrovirus.
  • the pharmaceutically acceptable excipient is selected from one or more of sodium phosphate, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, sorbitol, inositol, a 0.001% by mass aqueous solution of poloxamer 188, a 0.005% by mass aqueous solution of poloxamer 188, tris(hydroxymethyl)aminomethane (Tris), magnesium chloride or water for injection.
  • the present invention provides use of the splicing system for regulating viral replication in response to a hypoxic environment according to the present invention, the recombinant virus according to the present invention, or the pharmaceutical composition according to the present invention in the preparation of a medicament for treating hypoxic diseases.
  • the hypoxic disease is cancer.
  • the cancer is a solid tumor, such as one or more of neuroblastoma, lung cancer, breast cancer, esophageal cancer, gastric cancer, liver cancer, cervical cancer, ovarian cancer, kidney cancer, pancreatic cancer, nasopharyngeal cancer, small intestine cancer, large intestine cancer, colorectal cancer, bladder cancer, bone cancer, prostate cancer, thyroid cancer or brain cancer.
  • the drug is an oncolytic virus drug.
  • the present invention provides a method for treating hypoxic diseases, comprising administering to a subject in need thereof a therapeutically effective amount of a splicing system for regulating viral replication in response to a hypoxic environment according to the present invention, a recombinant virus according to the present invention, or a pharmaceutical composition according to the present invention.
  • the hypoxic disease is cancer.
  • the cancer is a solid tumor, such as one or more of neuroblastoma, lung cancer, breast cancer, esophageal cancer, gastric cancer, liver cancer, cervical cancer, ovarian cancer, kidney cancer, pancreatic cancer, nasopharyngeal cancer, small intestine cancer, large intestine cancer, colorectal cancer, bladder cancer, bone cancer, prostate cancer, thyroid cancer or brain cancer.
  • Tumor hypoxic microenvironment signals are expected to be used in the development of new technologies for the treatment of hypoxic diseases such as solid tumors.
  • hypoxic microenvironment signals By driving the proliferation of modular oncolytic viruses through hypoxic microenvironment signals, the specificity of tumor treatment can be improved and damage to normal tissues can be avoided or reduced.
  • the splicing control system regulated by hypoxia includes two functional elements: one is an element for connecting and fusion-expressing viral replication-related genes with splicing peptides through degradons, wherein the degradons in the element can effectively degrade viral replication-related factors fused with the degradons, thereby inhibiting viral replication; the splicing peptide can ensure that when another complementary splicing peptide fragment is effectively expressed, the degradons can be cleaved off, thereby saving viral replication-related factors; the second is an element for fusion-expressing a gene fragment that can be degraded in normoxia but not in hypoxia with a splicing peptide sequence, wherein the element ensures effective expression under hypoxic conditions, and cleaves the degradons fused with viral replication-related factors through the action of the splicing peptide, thereby saving viral replication-related factors and enabling the virus to acquire replication ability.
  • hypoxia-regulated spliceable control system responds to a specific condition signal - hypoxia and realizes the splicing of the hypoxia sensing unit and the virus replication unit, showing the following three advantages:
  • the viral replication unit can be spontaneously/induced to degrade, which reduces the retention in normal tissues and reduces the damage to normal tissues;
  • Hypoxia sensing units can respond to specific conditional signals - hypoxia, with the characteristics that key factors for viral replication are low in expression in normal tissue environments, but enriched in tumor microenvironments;
  • Conditionally controlled splicing of key factors for modular viral replication enables specific replication and proliferation in the tumor microenvironment, effectively reducing the on-target off-tumor toxic side effects caused by oncolytic viruses targeting non-tumor tissues.
  • Figures 1a-c show the hypoxia regulation effect achieved by loading oxygen-dependent degradation domain ODD at different vaccinia virus gene sites.
  • Figure 1a is a map of a plasmid (C16-mCherry-ODD) loaded with red fluorescent mCherry protein fused with oxygen-dependent degradation domain ODD (mCherry-ODD) at the C16 gene site of vaccinia virus;
  • Figure 1b is a map of a plasmid (F11-mCherry-ODD) loaded with red fluorescent mCherry protein fused with oxygen-dependent degradation domain ODD (mCherry-ODD) at the F11 gene site of vaccinia virus;
  • Figure 1c shows that mCherry-ODD was inserted into the vaccinia virus genome using recombinant technology, and after infecting cells under normoxic (21% O 2 ) and hypoxic conditions (1% O 2 ) for 24 hours, it can be observed that the red fluorescent protein mCherry can be expressed
  • Figure 2a-e shows the splicing activity of DHFR, SopE and ER.
  • Figure 2a is a map of a plasmid (F11-eGFP-BFP) loaded with green fluorescent protein GFP (eGFP) at the F11 gene site of vaccinia virus;
  • Figure 2b is a map of a plasmid (F11-DHFR-eGFP-BFP) loaded with green fluorescent protein GFP fused with degrader DHFR (DHFR-eGFP) at the F11 gene site of vaccinia virus;
  • Figure 2c is a map of a plasmid (F11-SopE-eGFP-BFP) loaded with green fluorescent protein GFP fused with degrader SopE (SopE-eGFP) at the F11 gene site of vaccinia virus;
  • Figure 2d is a map of a plasmid (F11-ER-eGFP-BFP) loaded with green fluorescent protein GFP fused with degrader
  • Figures 3a-c show that the plaques of TK143 - cell lines infected with vaccinia virus (H5R-KO) after knocking out the H5R gene by gene recombination are significantly smaller than those of vaccinia virus without knocking out the H5R gene (H5R+) ( Figure 3a); and when the vaccinia virus with knocked out the H5R gene (H5R-KO) is used to infect the TK143 - cell line overexpressing H5R, the size of the virus plaques returns to normal ( Figures 3b and c). It can be seen that the H5R gene of vaccinia virus is a key gene for replication, and knocking out the gene can greatly limit the replication of vaccinia virus; and overexpressing H5R can restore the replication ability of the virus.
  • Figure 4a-c shows that the combination of the hypoxia sensing unit (Int N -ODD) and the virus replication control unit (DHFR-Int C -H5R) can mediate the specific replication of the virus under hypoxic conditions.
  • Figure 4a is a map of the plasmid (H5R-DHFR-Int C -H5R) loaded with DHFR-Int C -H5R at the H5R site of the key gene for vaccinia virus replication
  • Figure 4b is a map of the plasmid (C16-Int N -ODD) loaded at the C16 site of vaccinia virus
  • Figure 4c shows that using recombinant technology, Int N -ODD and DHFR-Int C -H5R are inserted into the vaccinia virus genome. After infecting cells under normoxic and hypoxic conditions for 24 hours, it can be observed that the virus can be replicated in large quantities under hypoxic conditions, and the size of the virus plaque
  • the experimental methods in the following examples, unless otherwise specified, are all conventional experimental methods in the art.
  • the experimental materials used in the following examples, unless otherwise specified, are all conventional biochemical reagents purchased from sales companies, including: DMEM culture medium purchased from Corning; fetal bovine serum purchased from BI; LIPOFECTAMINE 3000 transfection kit purchased from Thermo Fisher Scientific; gene synthesis completed by Shanghai Jierui Bioengineering Co., Ltd.; Stabl3 chemical competent cells purchased from Shanghai Weidi Biotechnology Co., Ltd.; endotoxin-free plasmid miniprep kit purchased from OMEGA; TK143 - cells purchased from ATCC, USA; fluorescence microscope purchased from Nikon, Japan.
  • the amino acid sequence of the hypoxia sensing unit is shown in SEQ ID NO: 10, which is composed of the sequences of the oxygen-dependent degradation domain ODD and the N-terminal splicing domain.
  • the specific amino acid sequence is shown in Table 1.
  • the amino acid sequence of the viral replication control unit is shown in SEQ ID NO: 13, which is composed of the sequences of the degradation subunit, the C-terminal splicing domain and the viral replication transcription factor (viral early transcription factor VLTF-4).
  • the specific amino acid sequence is shown in Table 2.
  • the nucleotide sequence of the hypoxia sensing unit is shown in SEQ ID NO: 3, which is formed by connecting the sequence of the hypoxia sensing unit (oxygen-dependent degradation domain ODD) and the N-terminal splicing domain.
  • the specific nucleotide sequence is shown in Table 3.
  • the nucleotide sequence of the viral replication unit is shown in SEQ ID NO: 9, which is composed of the sequences of the degradation subunit, the C-terminal splicing domain and the viral replication transcription factor (viral early transcription factor VLTF-4).
  • the specific nucleotide sequence is shown in Table 4.
  • the nucleotide sequence shown in SEQ ID NO: 19 (the nucleotide sequence of mCherry-ODD) was synthesized by Shanghai Jierui Bioengineering Co., Ltd. and cloned into the corresponding C16 gene recombinant plasmid and F11 gene recombinant plasmid, respectively, to obtain the pSC65-C16-mCherry-ODD-eGFP recombinant plasmid and pSC65-F11-mCherry-ODD-BFP recombinant plasmid carrying the nucleotide sequence SEQ ID NO: 19.
  • the pSC65-C16-mCherry-ODD-eGFP recombinant plasmid used a green fluorescence signal as a recombination screening signal
  • the pSC65-F11-mCherry-ODD-BFP recombinant plasmid used a blue fluorescence signal as a recombination screening signal.
  • nucleotide sequences shown in SEQ ID NOs: 20-23 were synthesized by Shanghai Jierui Bioengineering Co., Ltd.
  • the nucleotide sequences shown in SEQ ID NOs: 24 and 25 were synthesized by Shanghai Jierui Bioengineering Co., Ltd. and cloned into the H5R gene recombinant plasmid and the C16 gene recombinant plasmid to obtain the pSC65-H5R-DHFR-Int C -H5R-mCherry recombinant plasmid carrying the nucleotide sequence SEQ ID NO: 24 and the pSC65-C16-Int N -ODD-eGFP recombinant plasmid carrying the nucleotide sequence SEQ ID NO: 25, wherein the pSC65-H5R-DHFR-Int C -H5R-mCherry recombinant plasmid uses a red fluorescence signal as a recombination screening signal, and the pSC65-C16-Int N -ODD-eGFP recombinant plasmid
  • the nucleotide sequence shown in SEQ ID NO: 24 encodes the degradation subdomain, C-terminal splicing domain and viral replication transcription factor (viral early transcription factor VLTF-4) (see Table 4 for the specific sequence of each component);
  • the nucleotide sequence shown in SEQ ID NO: 25 encodes the N-terminal splicing domain and oxygen condition signal response domain (oxygen-dependent degradation structure ODD) (see Table 3 for the specific sequence of each component).
  • Example 1 The vaccinia gene recombinant plasmid constructed in Example 1 (as shown in Figures 1a-b, 2a-d and 4a-b) was used to recombinant vaccinia virus.
  • TK143 - cells were plated in a 6-well plate, with about 1 ⁇ 106 cells per well. After culturing for about 24 hours, when the cells adhered to the wall and covered the entire bottom surface, the next step was performed.
  • Vaccinia virus incubation Infect cells with the wild-type vaccinia virus Tiantan strain at 0.0125/3 PFU (PFU: plaque forming unit, virus titer)/cell, incubate in a 37°C incubator for 1 hour, remove the cells, aspirate the supernatant, rinse with 1 mL of PBS, and then add 1 mL of DMEM complete medium (DMEM medium + 10% fetal bovine serum (FBS) + 1% penicillin and streptomycin antibiotics (PS)).
  • PFU plaque forming unit, virus titer
  • Plasmid transfection Transfect TK143- cells with vaccinia gene recombinant plasmid and culture in a 37°C incubator for about 48 hours, the specific time depends on the cell pathological condition.
  • a small sample of the recombinant vaccinia virus was amplified by plating TK143 - cells in a six-well plate, with 1 ⁇ 10 6 cells per well. When used, the cells should occupy about 100% of the bottom area of the well plate.
  • DMEM maintenance medium DMEM medium + 2% FBS + 1% PS.
  • VERO cell plating Take a 10 cm dish, with about 5 ⁇ 10 6 cells in each dish, and ensure that the cell density reaches 100% when inoculating vaccinia virus the next day;
  • Collect vaccinia virus discard 8 mL of culture medium in the dish, take 2 mL of maintenance medium to blow off the remaining cells, and collect them in a 15 mL centrifuge tube;
  • TK143 cells were plated in a 24-well plate, with approximately 2 ⁇ 10 5 cells per well. The cell density should reach 100% of the bottom area of the 24-well plate when used;
  • Virus plaque counting First, observe whether the number of virus plaques decreases tenfold, and then count the number of plaques in the two duplicate wells with only single-digit plaques. The sum of the plaque values in the two wells multiplied by the reciprocal value of the dilution corresponding to the well is the titer of the virus in 1 mL.
  • TK143 - cells were plated in 12-well plates at 2 ⁇ 10 5 cells/well, and infected after the cells adhered to the wall the next day.
  • the virus infection multiplicity (MOI) was 0.02. After the virus infected the cells, the cells were placed in hypoxic conditions (1% O 2 concentration) and normoxic conditions (21% O 2 concentration) for 24 hours, and the cytopathic effects were observed. The cytopathic effects were observed under a fluorescence microscope 24 hours later.
  • the mCherry-ODD protein was not expressed or degraded under normoxic conditions (21% O 2 concentration), and could not be observed under a microscope; while under hypoxic conditions (1% O 2 concentration), the red fluorescent protein was not degraded, was enriched, and could be observed under a microscope. It can be seen that after the ODD degradation unit is recombined into the vaccinia virus genome, it has an oxygen-dependent degradation effect.
  • this example carried out an infection experiment in TK143- cells.
  • the pSC65-F11-SopE-eGFP-BFP recombinant plasmid carrying the nucleotide sequence SEQ ID NO: 22
  • the pSC65-F11-ER-eGFP-BFP recombinant plasmid carrying the nucleotide sequence SEQ ID NO: 23 constructed in Example 1 and the method for preparing the recombinant vaccinia virus in Example 2 were used for verification.
  • TK143 - cells were plated in 12-well plates at 2 ⁇ 10 5 cells/well, and infected after the cells adhered to the wall the next day.
  • the virus infection multiplicity (MOI) was 0.02, and the cells were infected with TTV-F11-eGFP-BFP recombinant vaccinia virus, TTV-F11-DHFR-eGFP-BFP recombinant vaccinia virus, TTV-F11-SopE-eGFP-BFP recombinant vaccinia virus and TTV-F11-ER-eGFP-BFP recombinant vaccinia virus, respectively.
  • MOI virus infection multiplicity
  • the cells were infected with the virus, they were incubated in an incubator at 37°C and 5% CO 2 , and the cell lesions were observed under a fluorescence microscope 24 hours after the cell lesions.
  • the expression level of the degradation factor DHFR, SopE or ER fusion eGFP was only 5%, 10% or 12% of the positive control eGFP, and the spontaneous degradation rate was as high as 95%, 90% or 88%. It can be seen from this that after the degradation subunit is recombined into the vaccinia virus genome, it has a significant degradation effect.
  • the pSC65-H5R-DHFR-Int C -H5R-mCherry recombinant plasmid carrying the nucleotide sequence SEQ ID NO: 24 constructed in Example 1 and the method for preparing the recombinant vaccinia virus in Example 2 were used for verification.
  • TK143 - cells were plated into 12-well plates at 2 ⁇ 105 cells/well, and infected after the cells adhered to the wall the next day.
  • the virus infection multiplicity (MOI) was 0.02, and the recombinant vaccinia virus with DHFR-Int C -H5R gene integrated at the H5R site (i.e., H5R knockout vaccinia virus) was infected.
  • the cells were placed in a cell culture incubator and cultured for 24 hours. After the culture was completed, the virus plaques were observed using a fluorescence microscope. As shown in Figure 3a, the plaques of the vaccinia virus with the H5R gene knocked out (H5R-KO) were significantly smaller than those of the vaccinia virus without the H5R gene knocked out (H5R+).
  • the H5R gene of vaccinia virus was artificially overexpressed in the TK143 - cell line ( Figure 3b).
  • the TK143 - cells overexpressing the H5R gene (H5R- TK143- ) were plated into a 12-well plate at 2 ⁇ 10 5 cells/well, and infected after the cells adhered to the wall the next day.
  • the virus infection multiplicity (MOI) was 0.02, and the recombinant vaccinia virus with the DHFR-Int C -H5R gene integrated at the H5R site was infected. After infection, the cells were placed in a cell culture incubator and cultured for 24 hours. After the culture ended, the virus plaques were observed under a fluorescence microscope.
  • the plaques of the H5R-knocked-out vaccinia virus in the wild-type TK143 - cell line were significantly smaller than those in the H5R-overexpressing cell line, that is, the H5R-knocked-out vaccinia virus restored its replication ability (Figure 3c). It can be seen from this that the H5R gene of vaccinia virus is a key gene for replication. Knocking it out can greatly limit the replication of vaccinia virus; and overexpressing H5R can restore the virus's replication ability.
  • a vaccinia virus infection experiment under hypoxic conditions was conducted in TK143- cells to test the effect of hypoxia-regulated spliceable chimeric molecules on viral replication.
  • TK143 - cells were plated in 12-well plates at 2 ⁇ 10 5 cells/well, and infected after the cells adhered to the wall the next day.
  • the virus infection multiplicity (MOI) was 0.02, and the recombinant vaccinia virus with DHFR-Int C -H5R gene integrated at the H5R site and Int N -ODD gene integrated at the C16 site was infected.
  • the cells were cultured under hypoxic conditions (1% O 2 concentration) and normoxic conditions (21% O 2 concentration) for 24 hours, and the virus plaques were observed under a fluorescence microscope after the culture ended. The results are shown in Figure 4c.
  • a clone, 17-3-2-2 strain was screened, which replicated normally under hypoxic conditions (1% O 2 concentration) with obvious double fluorescent plaques, while virus replication was inhibited under normoxic conditions (21% O 2 concentration), and the size of double fluorescent positive plaques was significantly lower than that of vaccinia virus plaques under hypoxic conditions. It can be seen that based on the spliceable system, the recombinant vaccinia virus that simultaneously integrates the hypoxia sensing unit and the virus replication control unit can achieve normal replication under hypoxic conditions, but low-level replication under normoxic conditions.

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Abstract

提供一种响应缺氧环境调控病毒复制的可剪接系统及其用途。该系统包含彼此分开的或融合的编码缺氧感应单元的基因和编码病毒复制控制单元的基因，其中编码缺氧感应单元的基因包括彼此连接的N端剪接域元件和氧敏感蛋白元件，编码病毒复制控制单元的基因包含经由降解子元件连接的C端剪接域元件和病毒复制关键因子元件。该可剪接系统能够响应缺氧这一条件信号，从而实现病毒复制关键因子响应缺氧环境的剪接，从而表现出病毒复制关键因子在正常组织环境下低表达，而在肿瘤微环境下富集的特点。

Description

一种响应缺氧环境调控病毒复制的可剪接系统及其用途 技术领域
本发明属于生物制药技术领域，具体涉及一种响应缺氧环境调控病毒复制的可剪接系统及其在制备治疗实体瘤等乏氧性疾病的药物，特别是在制备溶瘤病毒药物中的用途。
背景技术
肿瘤是一种非常复杂的恶性细胞组织，由于机体控制细胞生长和免疫监视的信号通路发生异变，导致了肿瘤细胞的恶性增殖，从而形成肿瘤组织。大多数肿瘤会发展成免疫抑制的环境，这种环境会致使免疫共刺激分子下调，并且普遍高表达免疫检查点分子，最终致使肿瘤“冷却”，逃逸机体的抗肿瘤免疫应答。
为克服肿瘤的免疫抑制环境，肿瘤的免疫治疗应运而生。肿瘤免疫治疗涉及免疫检查点抑制剂(Immune checkpoint inhibitors，ICI)、T细胞受体修饰T细胞(T cell receptor T cells，TCR-T cells)、嵌合抗原受体T细胞(Chimeric antigen receptor T cells，CAR-T cells)以及溶瘤病毒(oncolytic viruses，OVs)等免疫治疗方法，这些方法在抗肿瘤临床研究中均显示出令人瞩目的效果。
溶瘤病毒是一种充分利用病毒自身肿瘤组织趋向性特征，使其在肿瘤组织内选择增殖并裂解肿瘤细胞，但对机体正常组织没有伤害的免疫治疗方法。并且，肿瘤的免疫抑制环境也有利于病毒免于被机体清除，这也为溶瘤病毒抗肿瘤免疫治疗的应用创造了可能。然而，在临床试验中发现，单独的溶瘤病毒进行治疗往往难以达到预期的效果，这是因为现有的溶瘤病毒治疗产品作用机制比较单一，且存在较大的免疫原性，无法比较特异性地区分正常组织与肿瘤组织，在病毒未到达病灶前在正常组织复制或者抗病毒免疫应答便已将病毒清除殆尽。即使多次给药，由于免疫记忆的功能，也会产生更加强大的免疫应答反应，很快清除溶瘤病毒，依然达不到治疗的目的。
溶瘤病毒既需要有免疫原性来诱导抗肿瘤免疫，又需要避免免疫监视，以便于病毒在肿瘤组织内复制的同时而不感染正常组织。如何解决溶瘤病毒的免疫原性以及病毒持续复制的问题，成为溶瘤病毒治疗领域的一大挑战。
发明内容
鉴于现有技术中存在的上述问题，本发明的目的在于提供一种响应缺氧环境调控病毒复制的可剪接系统及其在制备治疗实体瘤等乏氧性疾病的药物，特别是在制备治疗实体瘤的溶瘤病毒药物中的应用。
实体瘤内的异常血管结构所引起的供血不足以及肿瘤细胞的过度增殖营造了一个相对缺氧的肿瘤微环境，在这种环境下肿瘤组织内的氧水平常常低于2％，所以缺氧是多种实体瘤的一个共有特征信号。因此，如何通过利用肿瘤微环境与正常组织微环境的差异，以此为影响因素严格控制溶瘤病毒的复制，实现其在肿瘤治疗中的临床应用，可解决现有技术无法协调溶瘤病毒免疫原性与病毒持续存在的问题。
本发明根据肿瘤组织微环境缺氧的特征，开发了一种模块化的控制病毒复制关键因子的设计，其中病毒复制相关的关键因子由两个单元进行控制，一个是缺氧敏感单元，其用于感应氧环境；另外一个是病毒复制控制单元，用于控制病毒的复制以及增殖。模块化病毒复制相关的关键因子的激活基于两个条件，一是工程化病毒复制相关的关键因子偶联降解子，复制因子被降解，病毒失去复制增殖的能力；二是缺氧感应模块含有能够剪切掉降解子的元件，通过缺氧感应模块感应组织环境中的氧环境，且缺氧敏感模块不断得以富集，进而将病毒复制相关的关键因子释放出来，病毒得以完成复制与增殖，最终实现病毒缺氧敏感复制的目的。
在本发明中，对部分术语的说明或定义如下，而未定义或说明的术语具有所属技术领域中公知的含义。
术语“可剪接的”是指蛋白质分子可以在被剪切后重新接合。
术语“氧敏感蛋白”是指能够感应氧的蛋白质分子。
术语“病毒复制关键因子”是指在病毒增殖周期中关键的复制酶或者转录因子。
术语“N端剪接域”是指内含肽的N端结构域，可与C端剪接域进行剪接。
术语“降解子”是指一段特定的氨基酸序列，其可被细胞内的蛋白酶识别以介导目标蛋白的降解清除。
术语“C端剪接域”是指内含肽的C端结构域，可与N端剪接域进行剪接。
术语“乏氧性疾病”是指组织氧含量低于2％的疾病，特别是实体肿瘤。
本发明的目的是通过以下技术方案实现的：
一方面，本发明提供一种响应缺氧环境调控病毒复制的可剪接系统，其包含彼此分开的或融合的编码缺氧感应单元的基因和编码病毒复制控制单元的基因，其中所述编码缺氧感应单元的基因包含彼此连接的N端剪接域元件和氧敏感蛋白元件，所述编码病毒复制控制单元的基因包含经由降解子元件连接的C端剪接域元件和病毒复制关键因子元件。
在本发明中，所述缺氧环境是指氧含量低于2％的细胞或组织。肿瘤组织和细胞对氧和葡萄糖等能量物质的需求量很大，随着肿瘤组织供血不足随之出现肿瘤细胞和组织的缺氧，其细胞和组织氧含量往往低于2％。
本发明提供的响应缺氧环境调控病毒复制的可剪接系统能够响应特定条件信号—缺氧，从而实现病毒复制关键因子响应氧环境进行剪接。缺氧感应单元可以响应特定条件信号—缺氧，表现出病毒复制关键因子在正常组织环境下低表达，而在肿瘤微环境下富集的特点，即病毒复制关键因子在正常组织中可自发/诱发降解，促使病毒复制受阻，减少滞留。
根据本发明的一些实施方式，所述N端剪接域元件与氧敏感蛋白元件之间包含编码接头的基因；
根据本发明的一些实施方式，所述降解子元件与C端剪接域元件之间以及所述降解子元件与病毒复制关键因子元件之间包含编码接头的基因；
根据本发明的一些优选实施方式，所述接头为(GGS) n，其中n为1-3。
根据本发明的一些实施方式，所述N端剪接域为蛋白质内含子和/或SpyTag/SpyCatcher自组装子。优选地，所述N端剪接域为蛋白质内含子Int N。
根据本发明的一些优选实施方式，所述N端剪接域元件的核苷酸序列如SEQ ID NO：1所示。
根据本发明的一些实施方式，所述氧敏感蛋白为氧依赖降解结构域(ODD)。优选地，所述氧依赖降解结构域选自HIF-1α 557-574(ODD18)、HIF-1α 530-652(ODD123)或4*ODD18(4个连续重复的ODD18)中的一种或多种。
根据本发明的一些优选实施方式，所述氧依赖降解结构域的核苷酸序列如SEQ ID NO：2所示。
根据本发明的一些更优选实施方式，所述编码缺氧感应单元的核苷酸序列如SEQ ID NO：3所示。
根据本发明的响应缺氧环境调控病毒复制的可剪接系统提供了可降解的病毒复制关键因子，其在正常组织情况下可被降解，病毒由于没有对其生活周期而言关键的因子参与，病毒复制受到显著的抑制，病毒无法复制增殖。
根据本发明的一些实施方式，所述降解子选自二氢叶酸还原酶(DHFR)、雌激素受体(ER)、沙门氏菌III型分泌系统效应蛋白(SopE)、植物激素诱导蛋白降解子、不稳定域(AD)或光敏蛋白降解子中的一种或多种。优选地，所述降解子选自二氢叶酸还原酶、雌激素受体或沙门氏菌III型分泌系统效应蛋白中的一种或多种。更优选地，所述降解子为二氢叶酸还原酶。
根据本发明的一些优选实施方式，所述降解子元件的核苷酸序列如SEQ ID NO：4、5或6中的一种或多种所示。
根据本发明的一些实施方式，所述C端剪接域为蛋白质内含子和/或SpyTag/SpyCatcher自组装子。优选地，所述C端剪接域为蛋白质内含子Int C。
根据本发明的一些优选实施方式，所述C端剪接域元件的核苷酸序列如SEQ ID NO：7所示。
根据本发明的一些实施方式，所述病毒复制关键因子选自聚合酶、转录酶、蛋白翻译酶、RNA酶、蛋白降解酶、结构蛋白、组装元件、转录因子或出胞蛋白中的一种或多种。优选地，所述病毒复制关键因子选自病毒复制早期的聚合酶、转录酶、转录因子和蛋白翻译酶中的一种或多种。
根据本发明的一些优选实施方式，所述病毒复制关键因子元件的核苷酸序列如SEQ ID NO：8所示。
根据本发明的一些更优选实施方案，所述编码病毒复制控制单元的基因的核苷酸序列如SEQ ID NO：9所示。
根据本发明的一些实施方式，所述响应缺氧环境调控病毒复制的可剪接系统包含：
包含所述编码缺氧感应单元的基因的第一载体；和
包含所述编码病毒复制控制单元的基因的第二载体。
根据本发明的一些实施方式，所述响应缺氧环境调控病毒复制的可剪接系统包含一种载体，所述载体包含融合的所述编码缺氧感应单元的基因和所述编码病毒复制控制单元的基因。
根据本发明的一些实施方式，所述载体选自痘苗病毒、腺病毒、I型单纯疱疹病毒(HSV-1)、II型单纯疱疹病毒(HSV-2)、水疱性口炎病毒、埃可病毒、呼肠孤病毒、甲病毒、黄热病毒、柯萨奇病毒、新城疫病毒、麻疹病毒、脊髓灰质炎病毒、寨卡病毒、淋巴细胞脉络丛脑膜炎病毒、M1病毒、马尔堡病毒、慢病毒或逆转录病毒中的一种或多种。优先地，所述载体为痘苗病毒。
再一方面，本发明提供一种重组病毒，其基因组包含根据本发明的响应缺氧环境调控病毒复制的可剪接系统。
根据本发明的一些实施方式，所述病毒选自痘苗病毒、腺病毒、I型单纯疱疹病毒(HSV-1)、II型单纯疱疹病毒(HSV-2)、水疱性口炎病毒、埃可病毒、呼肠孤病毒、甲病毒、黄热病毒、柯萨奇病毒、新城疫病毒、麻疹病毒、脊髓灰质炎病毒、寨卡病毒、淋巴细胞脉络丛脑膜炎病毒、M1病毒、马尔堡病毒、慢病毒或逆转录病毒中的一种或多种。
根据本发明的一些实施方式，所述重组病毒的基因组还包含选自共刺激分子、细胞因子、负调分子、信号通路的阻断抗体、趋化因子或杀伤分子，优选为杀伤分子中的一种或多种的编码基因，更优选为BiTE的编码基因。
又一方面，本发明提供一种用于治疗乏氧性疾病的药物组合物，其包含根据本发明的响应缺氧环境调控病毒复制的可剪接系统和用于治疗乏氧性疾病的病毒、或者根据本发明的重组病毒，以及任选的药学上可接受的辅料。
根据本发明的一些实施方式，所述治疗乏氧性疾病的病毒选自痘苗病毒、腺病毒、I型单纯疱疹病毒(HSV-1)、II型单纯疱疹病毒(HSV-2)、水疱性口炎病毒、埃可病毒、呼肠孤病毒、甲病毒、黄热病毒、柯萨奇病毒、新城疫病毒、麻疹病毒、脊髓灰质炎病毒、寨卡病毒、淋巴细胞脉络丛脑膜炎病毒、M1病毒、马尔堡病毒、慢病毒或逆转录病毒中的一种或多种。
根据本发明的一些实施方式，所述药学上可接受的辅料选自磷酸钠、磷酸氢二钠二水合物、磷酸二氢钠二水合物、氯化钠、山梨糖醇、肌醇、 质量比为0.001％的泊洛沙姆188水溶液、质量比为0.005％的泊洛沙姆188水溶液、三羟甲基氨基甲烷(Tris)、氯化镁或注射用水中的一种或多种。
另一方面，本发明提供根据本发明的响应缺氧环境调控病毒复制的可剪接系统、根据本发明的重组病毒或根据本发明的药物组合物在制备治疗乏氧性疾病的药物中的用途。
根据本发明的一些实施方式，所述乏氧性疾病为癌症。优选地，所述癌症为实体瘤，例如神经母细胞瘤、肺癌、乳腺癌、食管癌、胃癌、肝癌、子宫颈癌、卵巢癌、肾癌、胰腺癌、鼻咽癌、小肠癌、大肠癌、结直肠癌、膀胱癌、骨癌、前列腺癌、甲状腺癌或脑癌中的一种或多种。
根据本发明的一些实施方式，所述药物为溶瘤病毒药物。
相应地，本发明提供一种治疗乏氧性疾病的方法，其包括向有需要的对象给予治疗有效量的根据本发明的响应缺氧环境调控病毒复制的可剪接系统、根据本发明的重组病毒或根据本发明的药物组合物。
根据本发明的一些实施方式，所述乏氧性疾病为癌症。优选地，所述癌症为实体瘤，例如神经母细胞瘤、肺癌、乳腺癌、食管癌、胃癌、肝癌、子宫颈癌、卵巢癌、肾癌、胰腺癌、鼻咽癌、小肠癌、大肠癌、结直肠癌、膀胱癌、骨癌、前列腺癌、甲状腺癌或脑癌中的一种或多种。
本发明人经研究发现，利用肿瘤微环境的内在特征信号驱动模块化溶瘤病毒的增殖更具临床应用价值。肿瘤缺氧微环境信号有望用于实体瘤等乏氧性疾病治疗的新技术的开发，通过缺氧微环境信号驱动模块化溶瘤病毒的增殖，可以提升肿瘤治疗的特异性，避免或减少对正常组织的损伤。
本发明提供的缺氧调控的可剪接控制系统包括两个功能元件：一是将病毒复制相关基因通过降解子与剪接肽进行连接并融合表达的元件，该元件中的降解子能够有效降解与其融合表达的病毒复制相关因子，从而抑制病毒复制；剪接肽能够保证在另一个互补的剪接肽片段有效表达时，降解子能够被剪切掉，从而拯救病毒复制相关因子；二是将常氧可被降解而乏氧不被降解的基因片段与剪接肽序列融合表达的元件，该元件保证在乏氧条件能够有效表达，并通过剪接肽的作用将与病毒复制相关因子融合表达的降解子剪切掉，从而拯救病毒复制相关因子，病毒获得复制的能力。
本发明提供的缺氧调控的可剪接控制系统响应特定条件信号—缺氧并 实现缺氧感应单元和病毒复制单元的剪接，体现出以下三大优势：
1)病毒复制单元可自发/诱发降解，减少了在正常组织中的滞留，降低了对正常组织的损伤；
2)缺氧感应单元可以响应特定条件信号—缺氧，具有病毒复制关键因子在正常组织环境下低表达，而在肿瘤微环境下富集的特点；
3)条件控制的模块化病毒复制关键因子的剪接实现了肿瘤微环境下的特异性复制增殖，有效降低了溶瘤病毒靶向非肿瘤组织所引起的on-target off-tumor毒副作用。
应理解，在本发明范围内，本发明的上述各项技术特征和在下文(如实施例)中具体描述的各项技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅限制，在此不再一一赘述。
附图的简要说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1a-c显示在不同痘苗病毒基因位点装载氧依赖降解结构域ODD实现的缺氧调控作用。其中，图1a为在痘苗病毒C16基因位点装载红色荧光mCherry蛋白融合氧依赖降解结构域ODD(mCherry-ODD)的质粒(C16-mCherry-ODD)图谱；图1b为在痘苗病毒F11基因位点装载红色荧光mCherry蛋白融合氧依赖降解结构域ODD(mCherry-ODD)的质粒(F11-mCherry-ODD)图谱；图1c显示利用重组技术，将mCherry-ODD插入痘苗病毒基因组中，在常氧(21％O 2)和缺氧条件(1％O 2)下感染细胞24h后，可观察到缺氧条件下红色荧光蛋白mCherry可表达富集，而在常氧条件下mCherry蛋白不能富集而被降解。
图2a-e显示DHFR、SopE和ER的剪接活性。图2a为在痘苗病毒F11基因位点装载绿色荧光蛋白GFP(eGFP)的质粒(F11-eGFP-BFP)图谱；图2b为在痘苗病毒F11基因位点装载绿色荧光蛋白GFP融合降解子DHFR(DHFR-eGFP)的质粒(F11-DHFR-eGFP-BFP)图谱；图2c为在痘苗病毒F11基因位点装载绿色荧光蛋白GFP融合降解子SopE(SopE-eGFP)的质粒(F11-SopE-eGFP-BFP)图谱；图2d为在痘苗病毒F11基因位点装载绿色荧光蛋白GFP融合降解子ER(ER-eGFP)的质粒(F11-ER-eGFP-BFP)图谱。图2e显示利用重组技术，将上述基因插入痘苗病毒基因组中，可观察到降解子SopE和ER均有明显的降解蛋白作用，而DHFR的降解效率最高。
图3a-c显示利用基因重组的方法敲除H5R基因后的痘苗病毒(H5R-KO)感染TK143 -细胞系的噬斑要显著小于没有敲除H5R基因(H5R+)的痘苗病毒(图3a)；而用敲除H5R基因后的痘苗病毒(H5R-KO)去感染过表达H5R的TK143 -细胞系，病毒噬斑大小恢复正常(图3b和c)。由此可知，痘苗病毒的H5R基因是复制的关键基因，敲除之后可极大限制痘苗病毒的复制；而过表达H5R后，可以恢复病毒的复制能力。
图4a-c显示缺氧感应单元(Int N-ODD)和病毒复制控制单元(DHFR-Int C-H5R)联合可介导病毒在缺氧条件下的特异性复制。其中，图4a为在痘苗病毒复制关键基因H5R位点装载DHFR-Int C-H5R的质粒(H5R-DHFR-Int C-H5R)图谱；图4b为在痘苗病毒C16位点装载Int N-ODD的质粒(C16-Int N-ODD)图谱；图4c显示利用重组技术，将Int N-ODD和DHFR-Int C-H5R插入痘苗病毒基因组中，在常氧和缺氧条件下感染细胞24h后，可观察到在缺氧条件下病毒得以大量复制，病毒噬斑大小远大于在常氧条件下病毒噬斑大小。
实施发明的最佳方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述更为清楚。以下实施例仅用于说明本发明，但不用来限制本发明的范围。
以下实施例中的实验方法，如无特殊说明，均为本领域的常规实验方法。以下实施例中所使用的实验材料，若无特殊说明，均为常规生化试剂，由销售公司购买所得，其中：DMEM培养基购自Corning公司；胎牛血清购自BI公司；LIPOFECTAMINE 3000转染试剂盒购自Thermo Fisher Scientific公司；基因合成由上海捷瑞生物工程有限公司完成；Stabl3化学感受态细胞购自上海唯地生物技术有限公司；无内毒素质粒小提试剂盒购自OMEGA公司；TK143 -细胞购自美国ATCC；荧光显微镜购自日本尼康。
所述缺氧感应单元的氨基酸序列如SEQ ID NO：10所示，其由氧依赖降解结构域ODD和N端剪接域的序列连接而成，具体的氨基酸序列如表1所示。
表1缺氧感应单元的氨基酸单元序列
Figure PCTCN2022126655-appb-000001
Figure PCTCN2022126655-appb-000002
所述病毒复制控制单元的氨基酸序列如SEQ ID NO：13所示，其由降解子、C端剪接域和病毒复制转录因子(病毒早期转录因子VLTF-4)的序列连接而成，具体的氨基酸序列如表2所示。
表2病毒复制单元的氨基酸序列
Figure PCTCN2022126655-appb-000003
Figure PCTCN2022126655-appb-000004
所述缺氧感应单元的核苷酸序列如SEQ ID NO：3所示，其由缺氧感应单元(氧依赖降解结构域ODD)和N端剪接域的序列连接而成，具体的核苷酸序列如表3所示。
表3缺氧感应单元的核苷酸序列
Figure PCTCN2022126655-appb-000005
Figure PCTCN2022126655-appb-000006
Figure PCTCN2022126655-appb-000007
所述病毒复制单元的核苷酸序列如SEQ ID NO：9所示，其由降解子、C端剪接域和病毒复制转录因子(病毒早期转录因子VLTF-4)的序列连接而成，具体的核苷酸序列如表4所示。
表4病毒复制单元的核苷酸序列
Figure PCTCN2022126655-appb-000008
Figure PCTCN2022126655-appb-000009
Figure PCTCN2022126655-appb-000010
Figure PCTCN2022126655-appb-000011
Figure PCTCN2022126655-appb-000012
实施例1痘苗病毒重组质粒的构建
由上海捷瑞生物工程有限公司合成SEQ ID NO：19所示的核苷酸序列(mCherry-ODD的核苷酸序列)，并分别克隆至对应的C16基因重组质粒和F11基因重组质粒，获得携带核苷酸序列SEQ ID NO：19的pSC65-C16-mCherry-ODD-eGFP重组质粒以及pSC65-F11-mCherry-ODD-BFP重组质粒，其中pSC65-C16-mCherry-ODD-eGFP重组质粒用绿色荧光信号作为重组筛选信号，pSC65-F11-mCherry-ODD-BFP重组质粒用蓝色荧光信号作为重组筛选信号。
由上海捷瑞生物工程有限公司合成SEQ ID NO：20-23所示的核苷酸序列(分别为eGFP、DHFR-eGFP、SopE-eGFP和ER-eGFP的核苷酸序列)，并克隆至对F11基因重组质粒，获得携带核苷酸序列SEQ ID NO：20的pSC65-F11-eGFP-BFP重组质粒、携带核苷酸序列SEQ ID NO：21的pSC65-F11-DHFR-eGFP-BFP重组质粒、携带核苷酸序列SEQ ID NO：22的pSC65-F11-SopE-eGFP-BFP重组质粒和携带核苷酸序列SEQ ID NO：23的pSC65-F11-ER-eGFP-BFP重组质粒，且这四种重组质粒用蓝色荧光信号作为重组筛选信号。
由上海捷瑞生物工程有限公司合成SEQ ID NO：24和25所示的核苷酸序列，并克隆至H5R基因重组质粒和C16基因重组质粒，获得携带核苷酸序列SEQ ID NO：24的pSC65-H5R-DHFR-Int C-H5R-mCherry重组质粒以及携带核苷酸序列SEQ ID NO：25的pSC65-C16-Int N-ODD-eGFP重组质粒，其中pSC65-H5R-DHFR-Int C-H5R-mCherry重组质粒用红色荧光信号作为重组筛选信号，pSC65-C16-Int N-ODD-eGFP重组质粒用绿色荧光信号作为重组筛选信号。其中，SEQ ID NO：24所示的核苷酸序列编码降解子结构域、C端剪接域和病毒复制转录因子(病毒早期转录因子VLTF-4)(各组成部分的具体序列参见表4)；SEQ ID NO：25所示的核苷酸编码N端剪接域和氧条件信号响应结构域(氧依赖降解结构ODD)(各组成部分的具体序列参见表3)。
各个重组质粒图谱如图1a-b、图2a-d和图4a-b所示，合成的核苷酸序列如表5所示。
表5合成的核苷酸序列
Figure PCTCN2022126655-appb-000013
Figure PCTCN2022126655-appb-000014
Figure PCTCN2022126655-appb-000015
Figure PCTCN2022126655-appb-000016
Figure PCTCN2022126655-appb-000017
Figure PCTCN2022126655-appb-000018
Figure PCTCN2022126655-appb-000019
Figure PCTCN2022126655-appb-000020
实施例2痘苗病毒的重组、纯化和滴度测定
1.采用实施例1构建的痘苗基因重组质粒(如图1a-b、图2a-d和图4a-b所示)进行痘苗病毒的重组。
1.1细胞准备：将TK143 -细胞铺在6孔板中，每孔约1×10 6个。培养24小时左右，当细胞贴壁并且铺满整个底面时，进行下一步操作。
1.2痘苗病毒孵育：以0.0125/3 PFU(PFU：空斑形成单位，病毒液滴度)/细胞用野生型痘苗病毒天坛株感染细胞，在37℃孵箱中孵育1小时后取出，吸掉上清，并用1mL PBS冲洗一遍，再加入1mL DMEM完全培养基(DMEM培养基+10％胎牛血清(FBS)+1％青霉素和链霉素抗生素(PS))。
1.3质粒转染：用痘苗基因重组质粒转染TK143 -细胞。在37℃孵箱中培养48小时左右，具体时间根据细胞病变情况而定。
1.4准备病毒铺斑用的2×DMEM维持培养基(含1％PS和2％FBS)，加入2％预热的低熔点琼脂糖。
1.5吸掉6孔板中的上清，将4mL铺斑用的混合物加入6孔板中，每 孔300μL。然后小心放入4℃冰箱促进凝固，待低熔点琼脂糖凝固后再转入37℃孵箱中培养。
1.6在荧光显微镜下挑取重组的病毒噬斑，加入500μL的DMEM完全培养基。在-80℃下反复冻融三次以上，使病毒尽量多的释放。该纯化过程至少需要进行5次。
1.7然后进行重组痘苗病毒的小样扩增，铺TK143 -细胞于六孔板，每孔1×10 6个细胞，使用时细胞约为孔板底面积的100％。
1.8在接种病毒前将孔中的培养基换成2mL DMEM维持培养基(DMEM培养基+2％FBS+1％PS)。将纯化得到的含有荧光的病毒液反复吹打至散开。每孔加入100μL左右病毒液。在37℃孵箱中孵育48小时左右，根据病毒斑形成情况收样。
1.9收样：将孔里的培养基上清小心吸出1mL。用剩下的1mL培养基将细胞充分吹下，收于EP管中，可以用于后续基因组的提取以及作为毒种进行扩增。
2.采用痘苗基因重组质粒重组的痘苗病毒的扩增与纯化
2.1VERO细胞铺板：取10cm皿，每个皿约5×10 6个细胞，保证第二天接种痘苗病毒时细胞密度达100％为宜；
2.2病毒接种前，需将DMEM完全培养基换成8mL维持培养基(DMEM培养基+2％FBS+1％PS)，将病毒接种到维持培养基的细胞中，接种量约为0.02MOI(MOI＝病毒PFU/细胞数)。继续在37℃、5％CO 2的孵箱中培养48小时左右，根据病毒噬斑形成情况收样；
2.3收痘苗病毒：弃掉皿内8mL培养基，取用2mL维持培养基将剩余的细胞吹下，收于15mL离心管中；
2.4冻存24小时后，将收得的病毒液再反复冻融2次，用36％的蔗糖溶液进行密度梯度离心，在16000g、4℃下离心90min，小心倒掉上清，用PBS缓冲液溶解离心管内的病毒沉淀，分装保存于-80℃下，待测定病毒滴度。
3.重组痘苗病毒的滴度测定
3.1 TK143 -细胞的准备：将TK143 -细胞铺在24孔板中，每孔约为2×10 5个细胞，使用时细胞密度达到24孔板底面积的100％；
3.2稀释病毒，用维持培养基稀释痘苗病毒液，从1：100开始，做10 倍比稀释，终体积为1100μL；
3.3弃掉24孔板中的完全培养基，取稀释好的病毒液500μL加入孔中，做两个复孔。在37℃、5％CO 2的孵箱中孵育48小时左右，根据病毒噬斑形成情况决定铺斑时间；
3.4病毒噬斑计数：首先观察病毒噬斑的数目是否呈十倍比的趋势递减，随后统计种毒的两个复孔中仅有个位数噬斑的复孔中的噬斑的数量，得到的两个孔中噬斑数值之和，乘以该孔所对应稀释度的倒数值即为1mL中病毒的滴度。
实施例3缺氧感应单元缺氧响应验证
采用实施例1构建的携带核酸序列SEQ ID NO：19的pSC65-C16-mCherry-ODD-eGFP重组质粒以及pSC65-F11-mCherry-ODD-BFP重组质粒和实施例2中制备重组痘苗病毒的方法进行验证。
将TK143 -细胞按2×10 5个细胞/孔铺到12孔板内，待第二天细胞贴壁后进行感染。病毒感染复数(MOI)为0.02，病毒感染细胞后，细胞分别放置于缺氧条件(1％O 2浓度)和常氧条件(21％O 2浓度)下培养24h，观察细胞病变情况，在细胞病变24小时后在荧光显微镜下观察。由图1c中可知，在不同的痘苗病毒重组位点C16和F11，mCherry-ODD蛋白在常氧条件(21％O 2浓度)下，红色荧光不表达或被降解，显微镜下不能被观测到；而在低氧条件(1％O 2浓度)下，红色荧光蛋白不被降解，得以富集，可在显微镜下被观测到。由此得知，将ODD降解单元重组到痘苗病毒基因组后，其具有氧依赖性降解作用。
实施例4不同降解子自主降解蛋白的表达水平检测
为了验证本发明提供的降解子的自发降解能力，本实施例在TK143 -细胞中进行了感染实验。采用实施例1构建的携带核苷酸序列SEQ ID NO：20的pSC65-F11-eGFP-BFP重组质粒、携带核苷酸序列SEQ ID NO：21的pSC65-F11-DHFR-eGFP-BFP重组质粒、携带核苷酸序列SEQ ID NO：22的pSC65-F11-SopE-eGFP-BFP重组质粒和携带核苷酸序列SEQ ID NO：23的pSC65-F11-ER-eGFP-BFP重组质粒和实施例2中制备重组痘苗病毒的方法进 行验证。
将TK143 -细胞按2×10 5个细胞/孔铺到12孔板内，待第二天细胞贴壁后进行感染，病毒感染复数(MOI)为0.02，分别感染TTV-F11-eGFP-BFP重组痘苗病毒、TTV-F11-DHFR-eGFP-BFP重组痘苗病毒、TTV-F11-SopE-eGFP-BFP重组痘苗病毒和TTV-F11-ER-eGFP-BFP重组痘苗病毒，病毒感染细胞后，在37℃、5％CO 2的孵箱中孵育，观察细胞病变情况，在细胞病变24小时后在荧光显微镜下观察。由图2e中可知，与阳性对照痘苗病毒相比，降解子DHFR、SopE或ER融合eGFP的表达水平仅为阳性对照eGFP的5％、10％或12％，自发降解率高达95％、90％或88％。由此得知，将降解子单元重组到痘苗病毒基因组后，其具有显著的降解作用。
实施例5敲除病毒复制关键基因H5R后可显著影响病毒复制
采用实施例1构建的携带核苷酸序列SEQ ID NO：24的pSC65-H5R-DHFR-Int C-H5R-mCherry重组质粒和实施例2中制备重组痘苗病毒的方法进行验证。
将TK143 -细胞按2×10 5个细胞/孔铺到12孔板内，待第二天细胞贴壁后进行感染。病毒感染复数(MOI)为0.02，感染在H5R位点整合DHFR-Int C-H5R基因的重组痘苗病毒(即H5R敲除痘苗病毒)。感染后，细胞放置于细胞培养箱中培养24h，培养结束后用荧光显微镜观察病毒噬斑的情况。由图3a可知，敲除H5R基因后的痘苗病毒(H5R-KO)噬斑要显著小于没有敲除H5R基因(H5R+)的痘苗病毒。
在TK143 -细胞系中人工过表达痘苗病毒的H5R基因(图3b)，将过表达H5R基因的TK143 -细胞(H5R-TK143 -)按2×10 5个细胞/孔铺到12孔板内，待第二天细胞贴壁后进行感染。病毒感染复数(MOI)为0.02，感染在H5R位点整合DHFR-Int C-H5R基因的重组痘苗病毒。感染后，将细胞放置于细胞培养箱中培养24h，培养结束后用荧光显微镜观察病毒噬斑的情况。敲除H5R的痘苗病毒在野生型TK143 -细胞系中的噬斑要显著小于在H5R过表达的细胞系中的噬斑大小，即敲除H5R的痘苗病毒恢复复制能力(图3c)。由此可知，痘苗病毒的H5R基因是复制的关键基因，敲除之后可极大限制痘苗病毒的复制；而过表达H5R后，可以恢复病毒的复制能力。
实施例6缺氧调控的可剪接嵌合分子导致痘苗病毒差异化复制的检测
本实施例在TK143 -细胞中进行了缺氧条件下的痘苗病毒感染实验，测试缺氧调控的可剪接嵌合分子对病毒复制的影响。
采用实施例1构建的携带核苷酸序列SEQ ID NO：24的pSC65-H5R-DHFR-Int C-H5R-mCherry重组质粒和核苷酸序列SEQ ID NO：25的pSC65-C16-Int N-ODD-eGFP重组质粒和实施例2中制备重组痘苗病毒的方法进行验证。
将TK143 -细胞按2×10 5个细胞/孔铺到12孔板内，待第二天细胞贴壁后进行感染。病毒感染复数(MOI)为0.02，感染在H5R位点整合DHFR-Int C-H5R基因和在C16位点整合Int N-ODD基因的重组痘苗病毒。感染后，细胞分别放置于缺氧条件(1％O 2浓度)和常氧条件(21％O 2浓度)下培养24h，培养结束后用荧光显微镜观察病毒噬斑的情况，结果如图4c所示。
筛选到一株克隆，17-3-2-2毒株，其在缺氧条件(1％O 2浓度)下复制正常，有明显的双荧光噬斑，而在常氧条件(21％O 2浓度)下病毒复制被抑制，双荧光阳性噬斑大小显著低于缺氧条件下痘苗病毒的噬斑。由此可知，基于可剪接的系统，同时整合有缺氧感应单元和病毒复制控制单元的重组痘苗病毒可以实现在缺氧条件下正常复制，但是在常氧条件下低水平复制。
以上所述仅是本发明的优选实施例而已，并非对本发明做任何形式上的限制，虽然本发明已经以优选实施例揭示如上，然而其并非用以限定本发明。任何熟悉本专业的技术人员，在不脱离本发明技术方案的范围内，可利用上述揭示的技术内容作出些许更动或修饰以获得等同变化的等效实施例，但凡是未脱离本发明技术方案的内容，依据本发明的技术实质对以上实施例所作的任何修改、等同变化与修饰均仍落入本发明技术方案。

Claims (10)

  1. 一种响应缺氧环境调控病毒复制的可剪接系统，其包含彼此分开的或融合的编码缺氧感应单元的基因和编码病毒复制控制单元的基因，其中所述编码缺氧感应单元的基因包含彼此连接的N端剪接域元件和氧敏感蛋白元件，所述编码病毒复制控制单元的基因包含经由降解子元件连接的C端剪接域元件和病毒复制关键因子元件。
  2. 根据权利要求1所述的可剪接系统，其中，所述N端剪接域元件与氧敏感蛋白元件之间包含编码接头的基因；
    优选地，所述降解子元件与C端剪接域元件之间以及所述降解子元件与病毒复制关键因子元件之间包含编码接头的基因；
    优选地，所述接头为(GGS) n，其中n为1-3。
  3. 根据权利要求1或2所述的可剪接系统，其中，所述N端剪接域为蛋白质内含子和/或SpyTag/SpyCatcher自组装子；优选地，所述N端剪接域为蛋白质内含子Int N；更优选地，所述N端剪接域元件的核苷酸序列如SEQ ID NO：1所示；
    优选地，所述氧敏感蛋白为氧依赖降解结构域；进一步优选地，所述氧依赖降解结构域选自HIF-1α 557-574、HIF-1α 530-652或4*ODD18中的一种或多种；更优选地，所述氧依赖降解结构域的核苷酸序列如SEQ ID NO：2所示；
    优选地，所述编码缺氧感应单元的基因的核苷酸序列如SEQ ID NO：3所示。
  4. 根据权利要求1至3中任一项所述的可剪接系统，其中，所述降解子选自二氢叶酸还原酶、雌激素受体、沙门氏菌III型分泌系统效应蛋白、植物激素诱导蛋白降解子、不稳定域或光敏蛋白降解子中的一种或多种；优选地，所述降解子选自二氢叶酸还原酶、雌激素受体或沙门氏菌III型分泌系统效应蛋白中的一种或多种；更优选地，所述降解子为二氢叶酸还原酶；进一步优选地，所述降解子元件的核苷酸序列如SEQ ID NO：4、5或6中的一种或多种所示；
    优选地，所述C端剪接域为蛋白质内含子和/或SpyTag/SpyCatcher自组装子；进一步优选地，所述C端剪接域为蛋白质内含子Int C；更优选地，所 述C端剪接域元件的核苷酸序列如SEQ ID NO：7所示；
    优选地，所述病毒复制关键因子选自聚合酶、转录酶、蛋白翻译酶、RNA酶、蛋白降解酶、结构蛋白、组装元件、转录因子或出胞蛋白中的一种或多种；进一步优选地，所述病毒复制关键因子选自病毒复制早期的聚合酶、转录酶、转录因子和蛋白翻译酶中的一种或多种；更优选地，所述病毒复制关键因子元件的核苷酸序列如SEQ ID NO：8所示；
    优选地，所述编码病毒复制控制单元的基因的核苷酸序列如SEQ ID NO：9所示。
  5. 根据权利要求1至4中任一项所述的可剪接系统，其包含：
    包含所述编码缺氧感应单元的基因的第一载体；和
    包含所述编码病毒复制控制单元的基因的第二载体。
  6. 根据权利要求1至4中任一项所述的可剪接系统，其包含一种载体，所述载体包含融合的所述编码缺氧感应单元的基因和所述编码病毒复制控制单元的基因。
  7. 根据权利要求5或6所述的可剪接系统，其中所述载体选自痘苗病毒、腺病毒、I型单纯疱疹病毒、II型单纯疱疹病毒、水疱性口炎病毒、埃可病毒、呼肠孤病毒、甲病毒、黄热病毒、柯萨奇病毒、新城疫病毒、麻疹病毒、脊髓灰质炎病毒、寨卡病毒、淋巴细胞脉络丛脑膜炎病毒、M1病毒、马尔堡病毒、慢病毒或逆转录病毒中的一种或多种；优选地，所述载体为痘苗病毒。
  8. 一种重组病毒，其基因组包含根据权利要求1至4中任一项所述的可剪接系统；
    优选地，所述重组病毒的基因组还包含选自共刺激分子、细胞因子、负调分子、信号通路的阻断抗体、趋化因子或杀伤分子中的一种或多种，优选为杀伤分子的编码基因，更优选为BiTE的编码基因；
    优选地，所述病毒选自痘苗病毒、腺病毒、I型单纯疱疹病毒、II型单纯疱疹病毒、水疱性口炎病毒、埃可病毒、呼肠孤病毒、甲病毒、黄热病毒、柯萨奇病毒、新城疫病毒、麻疹病毒、脊髓灰质炎病毒、寨卡病毒、淋巴细胞脉络丛脑膜炎病毒、M1病毒、马尔堡病毒、慢病毒或逆转录病毒中的一种或多种。
  9. 一种用于治疗乏氧性疾病的药物组合物，其包含根据权利要求1至 7中任一项所述的可剪接系统和用于治疗乏氧性疾病的病毒、或根据权利要求8所述的重组病毒，以及任选的药学上可接受的辅料；
    优选地，所述治疗乏氧性疾病的病毒选自痘苗病毒、腺病毒、I型单纯疱疹病毒、II型单纯疱疹病毒、水疱性口炎病毒、埃可病毒、呼肠孤病毒、甲病毒、黄热病毒、柯萨奇病毒、新城疫病毒、麻疹病毒、脊髓灰质炎病毒、寨卡病毒、淋巴细胞脉络丛脑膜炎病毒、M1病毒、马尔堡病毒、慢病毒或逆转录病毒中的一种或多种。
    优选地，所述药学上可接受的辅料选自磷酸钠、磷酸氢二钠二水合物、磷酸二氢钠二水合物、氯化钠、山梨糖醇、肌醇、质量比为0.001％的泊洛沙姆188水溶液、质量比为0.005％的泊洛沙姆188水溶液、三羟甲基氨基甲烷、氯化镁或注射用水中的一种或多种。
  10. 根据权利要求5至7中任一项所述的可剪接系统、根据权利要求8所述的重组病毒或根据权利要求9所述的药物组合物在制备治疗乏氧性疾病的药物中的用途；
    优选地，所述乏氧性疾病为癌症；进一步优选地，所述癌症为实体瘤，例如神经母细胞瘤、肺癌、乳腺癌、食管癌、胃癌、肝癌、子宫颈癌、卵巢癌、肾癌、胰腺癌、鼻咽癌、小肠癌、大肠癌、结直肠癌、膀胱癌、骨癌、前列腺癌、甲状腺癌或脑癌中的一种或多种。
    优选地，所述药物为溶瘤病毒药物。
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