WO2024027697A1 - 包封的溶瘤病毒遗传物质及其应用 - Google Patents

包封的溶瘤病毒遗传物质及其应用 Download PDF

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WO2024027697A1
WO2024027697A1 PCT/CN2023/110521 CN2023110521W WO2024027697A1 WO 2024027697 A1 WO2024027697 A1 WO 2024027697A1 CN 2023110521 W CN2023110521 W CN 2023110521W WO 2024027697 A1 WO2024027697 A1 WO 2024027697A1
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genetic material
virus
oncolytic
tumor
oncolytic virus
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刘滨磊
倪鹏
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武汉滨会生物科技股份有限公司
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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    • 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
    • A61K35/763—Herpes virus
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    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
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    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
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    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
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    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16621—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16632—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2
    • C12N2710/16634—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present application relates to the field of anti-tumor technology using oncolytic viruses, specifically the encapsulated oncolytic virus genetic material and its application.
  • Oncolytic viruses are a type of tumor-killing virus with the ability to replicate.
  • the use of oncolytic viruses to fight tumors is increasingly recognized.
  • the principle is to genetically modify some viruses with weak pathogenicity that exist in nature to make special viruses.
  • Oncolytic viruses exploit the inactivation or defects of antiviral genes or tumor suppressor genes in target cells to selectively infect tumor cells, replicate in large numbers and ultimately destroy tumor cells. At the same time, it can also stimulate an immune response and attract more immune cells to continue killing remaining cancer cells.
  • oncolytic virus therapy has attracted widespread attention, and related research has made great progress.
  • Herpes simplex viruses including herpes simplex virus type I and herpes simplex virus type II, are often used to transform into oncolytic viruses due to their tumor-tropic properties. They recognize and selectively infect tumor cells, ultimately causing cell swelling and destroying tumors. cells, but cannot replicate in normal body cells without killing them. In theory, it has higher anti-tumor effect and lower side effects.
  • oncolytic herpes simplex virus is an oncolytic virus with great development potential for cancer immunotherapy, such as human GM-CSF oncolytic herpes simplex virus type II (hereinafter referred to as "OH2 virus”). "), it can selectively infect tumor cells, replicate in tumor cells, eventually lyse and kill tumor cells, and release progeny virus particles to further infect surrounding tumor cells. This process also contributes to tumor-associated antigens of release.
  • OH2 virus human GM-CSF oncolytic herpes simplex virus type II
  • OH2 virus can regulate the immunosuppressive tumor microenvironment, which is beneficial to breaking immune tolerance and Stimulate anti-tumor immune response. Lysis of tumor cells leads to the release of tumor-associated antigens (TAA), inducing a systemic anti-tumor immune response in the body.
  • TAA tumor-associated antigens
  • the preparation method of oncolytic viruses is mainly by culturing cells, then injecting the virus after the cells are cultured to a certain stage, and then harvesting and purifying the oncolytic viruses.
  • the current preparation methods of oncolytic viruses have cumbersome processes and relatively high costs. High, batch-to-batch variation, and high risk of microbial contamination.
  • oncolytic viruses will induce strong antiviral effects on innate and acquired immunity when injected intravenously into patients, leading to A non-specific inflammatory response is produced, and the neutralizing oncolytic virus antibodies produced will prevent the oncolytic virus from infecting and dissolving tumor cells, reducing the anti-tumor effect.
  • the inventor of the present application provides a brand-new production of anti-tumor substances and methods thereof.
  • the anti-tumor substances are the genetic material of encapsulated oncolytic viruses, not live viruses.
  • the preferred oncolytic virus in this application is recombinant oncolytic herpes simplex virus type I or II.
  • the more effective recombinant human GM-CSF oncolytic herpes simplex virus type II, OH2 virus is used.
  • the genetic material of OH2 virus including DNA or RNA
  • the encapsulated body can enter cells and then form viable OH2 virus in the body, thereby producing anti-tumor effects.
  • lipid nanoparticle which includes the following components:
  • Genetic material encoding an oncolytic virus including a recombinant oncolytic I or II herpes simplex virus; and a liposome comprising a cationic lipid compound in combination with an auxiliary molecule, the liposome
  • the genetic material is encapsulated to form the lipid nanoparticles.
  • the genetic material of the oncolytic virus is one of DNA or RNA.
  • the oncolytic virus is a recombinant oncolytic type I or type II herpes simplex virus, and its genetic material is DNA.
  • the oncolytic virus is a recombinant oncolytic herpes simplex virus type II, and its genetic material is DNA.
  • the genetic material is DNA encoding recombinant human GM-CSF oncolytic herpes simplex virus type II (OH2 virus); wherein the OH2 virus strain is named H2d3d4-hGF, and the recommended classification name is: II Herpes Simplex Virus Type 2, the depository unit is the General Microbiology Center of the China Microbial Culture Collection Committee, located at No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is CGMCC No .3600, deposited on February 3, 2010.
  • the cationic lipid compound includes heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octyl
  • SM102 acid ester
  • DOTAP (2,3-dioleoyl-propyl)-trimethylammonium chloride
  • the accessory molecules include distearoylphosphatidylcholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG -PEG2000).
  • DSPC distearoylphosphatidylcholine
  • DMG -PEG2000 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000
  • the molar ratio of the cationic lipid compound and the auxiliary molecule is: cationic lipid compound: DSPC: cholesterol: DMG-PEG2000 is (40-55): (5-15): (30-45) (1-5);
  • the preferred molar ratio is: cationic lipid compound: DSPC: cholesterol: DMG-PEG2000 is 50:10:38:2.
  • the cationic lipid compound is heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octyl acid ester
  • embodiments of the present application provide a method for preparing the lipid nanoparticles (LNP) described in the first aspect, including: preparing a liposome solution, wherein the liposome solution contains a cationic lipid compound and an auxiliary molecule; The prepared liposome solution is used to encapsulate the genetic material of the oncolytic virus, thereby obtaining the lipid nanoparticles (LNP).
  • the above preparation method further includes one or more of the following steps: extraction, purification and recovery of oncolytic virus genetic material.
  • the genetic material of the oncolytic virus can be prepared in a variety of ways.
  • the oncolytic virus can be used to infect African green monkey kidney cells (Vero cells) or genetic engineering (gene synthesis, plasmid preparation, gene editing, It is prepared by polymerase chain reaction (PCR amplification, in vitro transcription IVT, etc.).
  • the cationic lipid compound includes heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octyl
  • acid ester 2,3-dioleoyl-propyl)-trimethylammonium-chloride salt.
  • the accessory molecules include distearoylphosphatidylcholine, cholesterol, and 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000.
  • the oncolytic virus is a recombinant oncolytic herpes simplex virus type I or II.
  • the oncolytic virus is recombinant human GM-CSF oncolytic herpes simplex virus type II (OH2 virus), and its genetic material is DNA.
  • the liposome solution contains a cationic lipid compound and an auxiliary molecule in a molar ratio of: cationic lipid compound: DSPC: cholesterol: DMG-PEG2000: (40-55): (5-15) :(30-45) :(1-5).
  • the liposome solution contains a cationic lipid compound and an auxiliary molecule in a molar ratio of: cationic lipid compound:DSPC:cholesterol:DMG-PEG2000 of 50:10:38:2.
  • the cationic lipid compound corresponding to this ratio may be heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl) Amino)octanoate.
  • the preparation method further includes: dissolving the oncolytic virus genetic material in sodium acetate buffer to obtain a solution containing the oncolytic virus genetic material.
  • using the prepared liposome solution to encapsulate the genetic material of the oncolytic virus to obtain the lipid nanoparticles specifically includes: adding the liposome solution dropwise to a certain volume ratio. into the solution containing the genetic material of the oncolytic virus to obtain the lipid nanoparticles.
  • the volume ratio of the liposome solution to the encapsulated oncolytic viral DNA is 1:4.
  • the concentration of the liposome solution is 12.5 mol/L, and the concentration of the solution containing the genetic material of the oncolytic virus is 20 ⁇ g/mL.
  • embodiments of the present application disclose a drug or vaccine, wherein the drug or vaccine includes the lipid nanoparticles (LNP) described in the first aspect and pharmaceutically acceptable excipients.
  • LNP lipid nanoparticles
  • embodiments of the present application disclose the use of lipid nanoparticles as described in the embodiments of the first aspect or drugs or vaccines as described in the fourth aspect in anti-tumor applications.
  • embodiments of the present application disclose the use of lipid nanoparticles as described in the embodiments of the first aspect or drugs or vaccines as described in the fourth aspect in the preparation of anti-tumor drugs.
  • embodiments of the present application disclose a method for treating tumors, which includes administering to a subject in need a therapeutically effective amount of lipid nanoparticles as described in the embodiments of the first aspect or a drug as described in the fourth aspect. or vaccine.
  • this application has at least one of the following beneficial effects:
  • the present application relates to encapsulated oncolytic viral genetic material and uses thereof.
  • the genetic material of the oncolytic virus is extracted and encapsulated by lipids to form lipid nanoparticles (LNP) that encapsulate the genetic material of the oncolytic virus.
  • LNP lipid nanoparticles
  • the recombinant human GM- CSF oncolytes herpes simplex virus type II (OH2 virus) by producing the genetic material DNA of the OH2 virus and encapsulating it with liposomes to obtain lipid nanoparticles (LNP) encapsulating the OH2 virus DNA.
  • lipid nanoparticles for anti-tumor can solve or partially solve the strong antiviral effect caused by innate and acquired antiviral immunity induced by systemic administration of oncolytic viruses, allowing oncolytic viruses to have better anti-tumor effects. tumor effects.
  • the present application discloses the preparation of lipid nanoparticles (LNP). Preparation method, the method can be used to efficiently prepare anti-tumor drugs, such as LNP drugs that encapsulate the DNA, the genetic material of the OH2 virus.
  • the method not only reduces the batch-to-batch variation of anti-tumor drug production batches and the risk of exogenous virus and microbial contamination, but also enhances the storage and transportation stability of anti-tumor drugs; it is conducive to the further expansion of anti-tumor drug injection methods.
  • Figure 1 is a line chart of tumor volume changes in vivo in mice receiving different treatment regimens provided by the embodiments of the present application.
  • Figure 2 is a scatter plot of the average tumor volume in vivo of mice receiving different treatment regimens for 28 days provided by the embodiments of the present application.
  • Figure 3 shows the results of the average tumor volume in vivo of mice receiving different treatment regimens for 28 days provided by the examples of this application.
  • Figure 4 is a line chart of in vivo tumor volume changes in mice whose initial tumor volume is ⁇ 150cm3 provided by the embodiment of the present application.
  • Figure 5 is a scatter plot of the average tumor volume in vivo of mice 28 days after removal of initial tumor volume ⁇ 150 cm 3 provided by the embodiment of the present application.
  • Figure 6 shows the average tumor results in vivo at 28 days after removal of mice with initial tumor volume ⁇ 150 cm 3 provided by the embodiments of the present application.
  • LNP lipid nanoparticles
  • the lipid nanoparticles are prepared by liposomes encapsulating genetic material encoding oncolytic viruses.
  • the oncolytic virus is a recombinant herpes simplex virus.
  • herpes simplex virus HSV
  • herpes simplex virus is divided into type I herpes simplex virus and type II herpes simplex virus, in the embodiments of the present application, the oncolytic virus can be further divided into recombinant oncolytic type I herpes simplex virus and recombinant oncolytic type II simplex virus.
  • Herpes virus since herpes simplex virus is divided into type I herpes simplex virus and type II herpes simplex virus, in the embodiments of the present application, the oncolytic virus can be further divided into recombinant oncolytic type I herpes simplex virus and recombinant oncolytic type II simplex virus.
  • Herpes virus Herpes virus.
  • the method of constructing an oncolytic virus from herpes simplex virus is to knock out at least one non-essential gene of the original herpes simplex virus, such as the neurotoxic gene ICP34.5 gene, which is non-essential for its propagation in tumor cells. Moreover, after knocking out this gene, the oncolytic virus loses its neurotoxicity and can selectively replicate in tumor cells.
  • new expression sequences are also introduced to construct recombinant oncolytic herpes simplex viruses as new oncolytic viruses.
  • the liposomes comprise DNA encoding an oncolytic virus whose genetic material is recombinant human GM-CSF oncolytic herpes simplex virus type II (OH2 virus).
  • OH2 virus is derived from the General Microbiology Center of the Chinese Microbial Culture Collection Committee, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the virus strain of the Institute of Microbiology, Chinese Academy of Sciences, named H2d3d4-hGF, as suggested Classification and naming: Herpes Simplex Virus Type 2, the deposit number is CGMCC No. 3600, and the deposit date is February 3, 2010.
  • type II herpes simplex virus is selected, and the ICP34.5 gene and ICP47 gene are deleted based on its standard strain, and human granulocyte-macrophage colony-stimulating factor is inserted.
  • GM-CSF factor human granulocyte-macrophage colony-stimulating factor
  • Knocking out the ICP47 gene can induce the body's specific anti-tumor immune response, and can also increase the expression of the downstream US11 gene (which promotes the replication of oncolytic viruses in tumor cells);
  • GM-CSF factor can promote the recruitment and activation of dendritic cells (DC cells), and can also increase the amount of major histocompatibility antigen peptides on the surface of DC cells, further enhancing the antigen presentation ability of DC cells; GM-CSF factor It can stimulate the proliferation and differentiation of myeloid-derived precursor cells and enhance the body's specific immune response; in addition, GM-CSF factors can enhance the cytotoxic effect mediated by antibody-dependent immune cells, peripheral blood lymph and monocyte cytotoxic effects. , effectively enhance the body’s anti-tumor immune response.
  • DC cells dendritic cells
  • the encapsulated body can enter cells and then form viable OH2 virus in the body, thereby producing anti-tumor effects.
  • this application uses the genetic material of oncolytic viruses to fight tumors, which can solve or partially solve the strong anti-viral effects caused by innate and acquired anti-viral immunity induced by systemic administration of oncolytic viruses. function, making oncolytic viruses have better anti-tumor effects.
  • the liposome is composed of a cationic lipid compound and an auxiliary molecule.
  • DSPC and cholesterol are mainly auxiliary lipids that help form a bilayer; the DMG-PEG2000 lipids in the prescription can improve the stability of LNP during preparation and storage, and these DMG-PEG2000 lipids generally contain short acyl chains , helps DMG-PEG2000 lipids to quickly separate from LNP after injection, avoid aggregation, and promote the interaction between LNP and cells.
  • the cationic lipid compound is one or a combination of SM102 and (2,3-dioleoyl-propyl)-trimethylammonium-chloride (DOTAP).
  • DOTAP (2,3-dioleoyl-propyl)-trimethylammonium-chloride
  • the auxiliary molecules are distearoylphosphatidylcholine (DSPC), cholesterol and 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 ( DMG-PEG 2000) at least one or a combination.
  • DSPC distearoylphosphatidylcholine
  • DMG-PEG 2000 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000
  • the preferred molar ratio of the cationic lipid compound and the auxiliary molecule contained in the lipid nanoparticles is: cationic lipid compound: DSPC: cholesterol: DMG-PEG2000 is (40-55): ( 5-15): (30-45): (1-5).
  • a more preferred molar ratio of the cationic lipid compound and the auxiliary molecule contained in the lipid nanoparticle is: cationic lipid compound: DSPC: cholesterol: DMG-PEG2000 is 50:10:38:2 .
  • DSPC DSPC (AVT (Shanghai) Pharmaceutical Technology Co., Ltd., hereinafter referred to as AVT, product number: B81229); cholesterol (AVT, product number: B80859); DMG-PEG2000 (AVT, product number: E00S-DMG-20043-08- 25); SM102 (Xiamen Sanobange, product number: M2104000880005)
  • Oncolytic virus infects African green monkey kidney cells (Vero cells)
  • Vero cells were infected with OH2 virus.
  • Vero cells were cultured in T300 culture flasks.
  • the culture conditions were 5% FBS complete medium, 37°C, and 5% CO 2 .
  • 1 ⁇ 10 6 CCID 50 OH2 were added to them.
  • the virus, OH2 virus was independently developed by the inventor of the present application.
  • the development batch was "BVT-OH2-201912A-SXT-NS", the titer was 1 ⁇ 10 7 CCID 50 , and the virus was cultured overnight for 16 hours.
  • OH2 virus DNA was extracted using DNAzol kit.
  • Rinse Discard the supernatant, add 2 mL of 75% alcohol, pipet several times, and then centrifuge at 4000g and 4°C for 1 min.
  • Dissolution Remove as much alcohol as possible and let it sit for 2 minutes. After the alcohol has completely evaporated, add 2 mL of DEPC water to reconstitute it. After the DNA is reconstituted, store it in a -20°C refrigerator.
  • the OH2 virus DNA obtained by the DNAzol method is impure, it will contain the genome of Vero cells. This step is to purify the OH2 virus DNA (we are concerned that UV will affect the DNA, so we did not take photos of the nucleic acid gel).
  • Electrophoresis voltage 120V, electrophoresis 30 minutes.
  • the aforementioned cationic lipid compound SM102 and auxiliary molecules are mixed in ethanol solvent in proportion, and the auxiliary molecules are distearoylphosphatidylcholine (DSPC), cholesterol and 1,2-
  • DSPC distearoylphosphatidylcholine
  • DMG-PEG2000 dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000
  • the total concentration of the prepared organic phase (LNP phase) is 12.5mmol/L.
  • the encapsulation method can be selected according to the dosage of lipid nanoparticles (LNP).
  • the dosage is small, the dropwise method is used to prepare the OH2 virus DNA-LNP complex, that is; when the dosage is large, microfluidic equipment is used to prepare the aforementioned lipid nanoparticles (LNP) in large batches.
  • the steps of the dripping method are as follows: dissolve OH2 virus DNA in sodium acetate buffer (pH 5.0, 20 mmol/L), adjust the OH2 virus DNA concentration to 20 ⁇ g/mL, and then use a volume ratio of 4 : 1 (sodium acetate buffer phase: LNP phase), slowly add the LNP phase dropwise to the sodium acetate dissolved with OH2 DNA In the buffer, shake slowly during the dropwise addition. After the dropwise addition is complete, use a pipette tip to mix evenly. After incubating for 5-10 minutes, add PBS buffer (0.01M, pH7.4) to dilute to the required concentration.
  • sodium acetate buffer pH 5.0, 20 mmol/L
  • LNP phase sodium acetate buffer phase
  • PBS buffer (0.01M, pH7.4
  • LNP lipid nanoparticles
  • the embodiments of the present application disclose the application of lipid nanoparticles (LNP) against mouse colon cancer cells.
  • the lipid nanoparticles (LNP) contain the genomic DNA of recombinant human GM-CSF oncolytic type II herpes simplex virus.
  • CT26-iRFP 100 ⁇ L/mouse, 1 ⁇ 10 7 cells/mL
  • CT26 tumor mouse culture day 1 when CT26-iRFP cells were implanted
  • the 9th day of CT26 tumor mouse culture was regarded as the first day of anti-tumor treatment.
  • the anti-tumor treatment was divided into three times, on the 1st, 4th and 4th days of treatment respectively.
  • the treatment method is injection of anti-tumor agent.
  • CT26 tumor mice were divided into 9 groups and given different treatment plans respectively. The groupings and treatment plans are shown in Table 2.
  • the anti-tumor agent injected was "SM102-OH2 DNA", which is the genomic DNA lipid nanoparticle (LNP) containing recombinant human GM-CSF oncolytic herpes simplex virus type II.
  • the lipid nanoparticles (LNP) were prepared by the aforementioned method.
  • the injection DNA dosages in groups 1-3 were 500ng, 50ng, and 5ng respectively.
  • the injection method was "intratumoral administration route (it)".
  • the injection DNA dosage in group 4 was 500ng, the injection method is "intravenous administration (iv)"; the anti-tumor agent injected in the 5 groups is SM102 liposome, the DNA dosage is 0, the injection method is "it”; group 6 is the blank control group, the anti-tumor reagent injected is PBS buffer, the DNA dosage is 0, and the injection method is "it”; the anti-tumor reagent injected in group 7 is "OH2 DNA”, that is, it has not been The DNA of the encapsulated OH2 virus, the DNA dosage is 500ng, and the injection method is "it”; the anti-tumor reagent injected in the 8 groups is "Virus (CCID 50 )", that is, the OH2 virus, the injection dosage is 1 ⁇ 10 6 CCID 50 , The injection method is "it”; the anti-tumor agent injected in group 9 is "DOTAP-OH2 DNA", another genomic DNA lipid nanoparticle that encapsulates recombinant human GM-
  • LNP-2 lipid nanoparticles
  • Figures 1-3 show the results of tumor volume changes in mice receiving different treatment regimens, that is, the experimental results of groups 1-9.
  • Figure 1 shows the changes in mouse tumor volume (average) at different times.
  • Figure 2 shows the tumor volume of each mouse in different groups when they received treatment for 28 days.
  • the inventor of the present application processed the original experimental data, eliminated mice with tumor volume ⁇ 150 mm 3 on the ninth day after tumor transplantation (the first day of treatment), and then obtained another set of experimental data, as shown in Figure As shown in 4-6. It can be seen that whether in terms of the number of tumor-free mice or the average tumor volume, the difference between group 1 (SM102-OH2 DNA 500ng (it) group) and group 8 (OH2 Virus (it) group) The effects are all close.
  • group 1 SM102-OH2 DNA 500ng (it) group
  • group 8 OH2 Virus (it) group
  • this application discloses encapsulated oncolytic virus genetic material and its application.
  • the genetic material of the oncolytic virus is extracted and encapsulated by lipids to form lipid nanoparticles (LNP) that encapsulate the genetic material of the oncolytic virus.
  • LNP lipid nanoparticles
  • the recombinant human GM- CSF oncolytes herpes simplex virus type II (OH2 virus) by producing the genetic material DNA of the OH2 virus and encapsulating it with liposomes to obtain lipid nanoparticles (LNP) encapsulating the OH2 virus DNA.
  • OH2 virus herpes simplex virus type II
  • lipid nanoparticles for anti-tumor can solve the problem of non-specific inflammatory reactions in patients due to the oncolytic virus activating the patient's innate immune pathway in the process of directly using oncolytic viruses to fight tumors, and has a better anti-tumor effect.
  • this application discloses a preparation method of the lipid nanoparticles (LNP), which can be used to efficiently prepare anti-tumor drugs or vaccines, such as LNP drugs that encapsulate the DNA of the genetic material of the OH2 virus.
  • the method not only reduces the batch-to-batch variation of anti-tumor drug production batches and the risk of exogenous factor contamination, but also enhances the storage and transportation stability of anti-tumor drugs; it is conducive to the further expansion of anti-tumor drug injection methods.

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Abstract

公开了包封的溶瘤病毒遗传物质及其应用，具体包括通过包封溶瘤病毒遗传物质以制备脂质纳米颗粒的方法，包括：制备脂质体溶液，其中所述脂质体溶液包含阳离子脂质化合物和辅助分子；和用制备的脂质体溶液包封溶瘤病毒的遗传物质，以得到所述的脂质纳米颗粒。

Description

包封的溶瘤病毒遗传物质及其应用
相关申请的交叉引用
本申请基于申请号为202210938251.9、申请日为2022年8月5日和申请号为202210961161.1、申请日为2022年8月11日的中国专利申请提出，并要求该中国专利申请的优先权，该中国专利申请的全部内容在此引入本申请作为参考。
技术领域
本申请涉及采用溶瘤病毒抗肿瘤技术领域，具体涉及包封的溶瘤病毒遗传物质及其应用。
背景技术
溶瘤病毒是一类具有复制能力的肿瘤杀伤型病毒，采用溶瘤病毒来抗肿瘤越来越得到认可，其原理是通过对自然界存在的一些致病力较弱的病毒进行基因改造制成特殊的溶瘤病毒，利用靶细胞中抗病毒基因或抑癌基因的失活或缺陷从而选择性地感染肿瘤细胞，在其内大量复制并最终摧毁肿瘤细胞。同时它还能激发免疫反应，吸引更多免疫细胞来继续杀死残余癌细胞。近几十年来，溶瘤病毒治疗引起了广泛关注，相关研究取得了巨大进展。
单纯疱疹病毒，包括I型单纯疱疹病毒和II型单纯疱疹病毒，由于其嗜肿瘤特性而常被用来改造成溶瘤病毒，它识别并选择性地感染肿瘤细胞，最终导致细胞溶胀而摧毁肿瘤细胞，但无法在正常机体细胞内复制而不具有杀伤作用，理论上具有更高的抗肿瘤效应和更低的副作用。
在溶瘤病毒中，其中重组溶瘤单纯疱疹病毒是一种大有发展潜力的用于癌症免疫治疗手段的溶瘤病毒，如人GM-CSF溶瘤II型单纯疱疹病毒(后简称“OH2病毒”)，它能选择性地感染肿瘤细胞，并在肿瘤细胞中复制，最终裂解、杀死肿瘤细胞，并释放出子代病毒颗粒进一步感染周围的肿瘤细胞，此过程还有助于肿瘤相关抗原的释放。OH2病毒的抗肿瘤效应不仅表现在病毒的复制直接杀伤肿瘤细胞或病毒蛋白的直接毒性作用；最近越来越多的证据表明OH2病毒可以调节免疫抑制性肿瘤微环境，有利于打破免疫耐受而激发抗肿瘤免疫反应。肿瘤细胞的裂解导致肿瘤相关抗原(TAA)的释放，诱导体内系统性的抗肿瘤免疫反应。
目前，溶瘤病毒的制备方法主要是通过培养细胞，再在细胞培养到一定阶段后接毒，然后收获并纯化溶瘤病毒，然而现行的溶瘤病毒制备方法存在工艺繁琐、成本较 高、批间差较大、微生物污染风险较高等问题。
具有复制能力的溶瘤病毒在临床系统给药极具挑战，溶瘤病毒作为抗肿瘤药物，在对患者进行静脉注射时，溶瘤病毒会引起先天及获得免疫强烈的抗病毒作用，从而导致患者产生非特异性炎症反应，且产生的中和溶瘤病毒抗体会阻止溶瘤病毒对肿瘤细胞的感染及溶解作用，降低了抗肿瘤的效果。
在本领域中，具有复制能力的、有效抗肿瘤的溶瘤病毒或相关疫苗、药品仍然处于未满足的需求，因此，亟需提供全新的用于抗肿瘤物质和/或组合物，及生产该物质和/或组合物的方法。
发明内容
本申请发明人提供了一种全新的生产抗肿瘤物质及其方法，所述的抗肿瘤物质为包封的溶瘤病毒的遗传物质，并非活的病毒。本申请优选的溶瘤病毒为重组溶瘤I或II型单纯疱疹病毒。在某些实施例中，选用了效果更佳的重组人GM-CSF溶瘤II型单纯疱疹病毒，即OH2病毒。将OH2病毒的遗传物质(包括DNA或RNA)经过LNP或者其他材料包封后，形成的包封体能够进入细胞中，然后在体内形成活的OH2病毒，进而产生抗肿瘤的作用。
第一方面，本申请实施例公开了一种脂质纳米颗粒(LNP)，所述脂质纳米颗粒包括以下组分：
编码溶瘤病毒的遗传物质，所述溶瘤病毒包括重组溶瘤I或II单纯疱疹病毒；和脂质体，所述脂质体包括阳离子脂质化合物与辅助分子的组合，所述脂质体包封所述遗传物质以形成所述脂质纳米颗粒。
在一些实施例中，所述溶瘤病毒的遗传物质为DNA或RNA的一种。
在一些实施例中，所述溶瘤病毒为重组溶瘤I型或II型单纯疱疹病毒，其遗传物质为DNA。
在一些实施例中，所述溶瘤病毒为重组溶瘤II型单纯疱疹病毒，其遗传物质为DNA。
在一些实施例中，所述遗传物质为编码重组人GM-CSF溶瘤II型单纯疱疹病毒(OH2病毒)的DNA；其中所述OH2病毒毒株命名为H2d3d4-hGF,建议的分类命名：II型单纯疱疹病毒(Herpes Simplex Virus Type2)，保藏单位为中国微生物菌种保藏管理委员会普通微生物中心，地址位于北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏编号为CGMCC No.3600，保藏日期为2010年2月3日。
在一些实施例中，所述阳离子脂质化合物包括十七烷-9-基8-((2-羟乙基)(6-氧代-6-(十一烷氧基)己基)氨基)辛酸酯(SM102)、(2,3-二油酰基-丙基)-三甲基铵-氯盐(DOTAP)中的一种或组合。
在一些实施例中，所述辅助分子包括二硬脂酰磷脂酰胆碱(DSPC)、胆固醇及1,2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇2000(DMG-PEG2000)。
在一些实施例中，所述阳离子脂质化合物和辅助分子的摩尔比例是：阳离子脂质化合物：DSPC：胆固醇：DMG-PEG2000为(40-55)：(5-15)：(30-45)：(1-5)；优选的摩尔比例是：阳离子脂质化合物：DSPC：胆固醇：DMG-PEG2000为50：10：38：2。
在一些实施例中，所述阳离子脂质化合物为十七烷-9-基8-((2-羟乙基)(6-氧代-6-(十一烷氧基)己基)氨基)辛酸酯，所述阳离子脂质化合物和辅助分子的摩尔比例是：十七烷-9-基8-((2-羟乙基)(6-氧代-6-(十一烷氧基)己基)氨基)辛酸酯：二硬脂酰磷脂酰胆碱：胆固醇：1,2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇2000＝50:10:38:2。
第二方面，本申请实施例提供了第一方面所述脂质纳米颗粒(LNP)的制备方法，包括：配制脂质体溶液，其中所述脂质体溶液包含阳离子脂质化合物和辅助分子；和用制备的脂质体溶液包封溶瘤病毒的遗传物质，即得到所述的脂质纳米颗粒(LNP)。
在一些实施例中，上述制备方法还包括以下步骤中的一个或多个：溶瘤病毒遗传物质的提取、纯化和回收。本申请实施例中，所述溶瘤病毒遗传物质的制备有多种方式，可采用溶瘤病毒侵染非洲绿猴肾细胞(Vero细胞)或者利用基因工程(基因合成、质粒制备、基因编辑、聚合酶链反应PCR扩增、体外转录IVT等)的方式制得。
在一些实施例中，所述阳离子脂质化合物包括十七烷-9-基8-((2-羟乙基)(6-氧代-6-(十一烷氧基)己基)氨基)辛酸酯、(2,3-二油酰基-丙基)-三甲基铵-氯盐中的一种或组合。
在一些实施例中，所述辅助分子包括二硬脂酰磷脂酰胆碱、胆固醇及1,2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇2000。
在一些实施例中，所述溶瘤病毒为重组溶瘤I或II型单纯疱疹病毒。
在一些实施例中，所述溶瘤病毒为重组人GM-CSF溶瘤II型单纯疱疹病毒(OH2病毒)，其遗传物质为DNA。
在一些实施例中，所述脂质体溶液包含的阳离子脂质化合物和辅助分子的摩尔比例是：阳离子脂质化合物：DSPC：胆固醇：DMG-PEG2000为(40-55)：(5-15)：(30-45)：(1-5)。
在一些实施例中，所述脂质体溶液包含的阳离子脂质化合物和辅助分子的摩尔比例是：阳离子脂质化合物：DSPC：胆固醇：DMG-PEG2000为50：10：38：2。在一些实施例中，该比例对应的阳离子脂质化合物可以为十七烷-9-基8-((2-羟乙基)(6-氧代-6-(十一烷氧基)己基)氨基)辛酸酯。
在一些实施例中，制备方法还包括：将所述溶瘤病毒遗传物质溶解在醋酸钠缓冲液，以获得含所述溶瘤病毒的遗传物质的溶液。
在一些实施例中，用制备的脂质体溶液包封溶瘤病毒的遗传物质，以得到所述的脂质纳米颗粒具体包括：以一定的体积比，将所述脂质体溶液滴加至所述含所述溶瘤病毒的遗传物质的溶液中，以得到所述的脂质纳米颗粒。
在一些实施例中，所述脂质体溶液与包封的溶瘤病毒DNA的体积比为1：4。
在一些实施例中，所述脂质体溶液的浓度为12.5mol/L，所述含所述溶瘤病毒的遗传物质的溶液的浓度为20μg/mL。
第三方面，本申请实施例公开了一种药物或疫苗，其中所述药物或疫苗包含第一方面所述的脂质纳米颗粒(LNP)及药学可接受的辅料。
第四方面，本申请实施例公开了如第一方面实施例所述的脂质纳米颗粒或如第四方面所述药物或疫苗在抗肿瘤中的应用。
第五方面，本申请实施例公开了如第一方面实施例所述的脂质纳米颗粒或如第四方面所述药物或疫苗在制备抗肿瘤药物中的应用。
第六方面，本申请实施例公开了一种肿瘤治疗方法，包括向有需要的受试者施用治疗有效量的如第一方面实施例所述的脂质纳米颗粒或如第四方面所述药物或疫苗。
与相关技术相比，本申请至少具有以下有益效果之一：
本申请中涉及包封的溶瘤病毒遗传物质及其应用。本申请中，提取溶瘤病毒的遗传物质，并通过脂质进行包封，形成包封溶瘤病毒遗传物质的脂质纳米颗粒(LNP)，如采用本申请发明人自研的重组人GM-CSF溶瘤II型单纯疱疹病毒(OH2病毒)，通过生产OH2病毒的遗传物质DNA，并用脂质体进行包封，获得包封OH2病毒DNA的脂质纳米颗粒(LNP)。利用所述脂质纳米颗粒(LNP)进行抗肿瘤，可以解决或部分解决溶瘤病毒系统给药诱导的先天及获得的抗病毒免疫导致的强烈抗病毒作用，使溶瘤病毒拥有更好的抗肿瘤效果。另外，本申请公开了脂质纳米颗粒(LNP)的制 备方法，所述方法可用于高效制备抗肿瘤药物，如包封OH2病毒遗传物质DNA的LNP药物。所述方法不仅降低抗肿瘤药物生产批次的批间差和外源病毒和微生物污染的风险，还增强了抗肿瘤药物储存和运输稳定性；有利于抗肿瘤药物注射方式的进一步拓展。
附图说明
图1为本申请实施例提供的接受不同治疗方案的小鼠的体内肿瘤体积变化折线图。
图2为本申请实施例提供的接受不同治疗方案28天时的小鼠的体内肿瘤平均体积散点图。
图3为本申请实施例提供的接受不同治疗方案28天时的小鼠的体内平均肿瘤体积结果。
图4为本申请实施例提供的去除初始肿瘤体积≥150cm3的小鼠的体内肿瘤体积变化折线图。
图5为本申请实施例提供的去除初始肿瘤体积≥150cm3的小鼠28天时的体内肿瘤平均体积散点图。
图6为本申请实施例提供的去除初始肿瘤体积≥150cm3的小鼠28天时的体内平均肿瘤结果。
具体实施方式
为了使本申请的目的、技术方案及优点更加清楚明白，以下结合实施例对本申请进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本申请，并不用于限定本申请。本申请中未详细单独说明的试剂均为常规试剂，均可从商业途径获得；未详细特别说明的方法均为常规实验方法，可从现有技术中获知。
需要说明的是，本申请的说明书和权利要求书及上述附图中的术语“第一”、“第二”等是用于区别类似的对象，而不必用于描述特定的顺序或先后次序，也不对其后的技术特征起到实质的限定作用。应该理解这样使用的数据在适当情况下可以互换，以便这里描述的本申请的实施例能够以除了在这里图示或描述的那些以外的顺序实施。此外，术语“包括”和“具有”以及他们的任何变形，意图在于覆盖不排他的包含，例如，包含了一系列步骤或单元的过程、方法、系统、产品或设备不必限于清楚地列出的那些步骤或单元，而是可包括没有清楚地列出的或对于这些过程、方法、产品或设备固有的其它步骤或单元。
脂质纳米颗粒(LNP)的说明
在本申请实施例中，所述的脂质纳米颗粒(LNP)是由脂质体包封编码溶瘤病毒的遗传物质制得的。
在本申请实施例中，所述溶瘤病毒为重组单纯疱疹病毒，在以往的研究记载中，单纯疱疹病毒(HSV)作为溶瘤病毒有着天然的优势，具体表现在：
(1)宿主广泛，能够感染多种组织细胞，并诱导机体免疫应答；
(2)具备在宿主细胞复制、繁殖、裂解宿主细胞的特性；
(3)复制时间短，保证溶瘤效果；
(4)可携带外源基因，进一步提升溶瘤效果；
(5)可剔除多个非必需基因，降低溶瘤病毒的神经毒性，增加裂解肿瘤细胞的选择性；
(6)安全性较高，溶瘤病毒的感染易控制；
(7)不与宿主细胞基因融合，以游离的形式在宿主细胞中复制。
由于单纯疱疹病毒分为I型单纯疱疹病毒和II型单纯疱疹病毒，因此，本申请实施例中，所述溶瘤病毒进一步可分为重组溶瘤I型单纯疱疹病毒和重组溶瘤II型单纯疱疹病毒。
本申请实施例中，以单纯疱疹病毒构建溶瘤病毒的方法是敲除至少一个原单纯疱疹病毒的非必需基因，如神经毒性基因ICP34.5基因，其在肿瘤细胞内的繁殖是非必需的，而且敲除该基因后，溶瘤病毒丧失了神经毒性，而且可以选择性在的肿瘤细胞内复制。
在某些实施例中，在构建策略上，除了敲出单纯疱疹病毒的非必需基因，同时还引入了新的表达序列，构建重组溶瘤单纯疱疹病毒，作为新的溶瘤病毒。
在某些实施例中，所述脂质体包含编码溶瘤病毒的遗传物质是重组人GM-CSF溶瘤II型单纯疱疹病毒(OH2病毒)的DNA。其中所述OH2病毒来源于中国微生物菌种保藏管理委员会普通微生物中心，地址位于北京市朝阳区北辰西路1号院3号中国科学院微生物研究所的病毒毒株，名为H2d3d4-hGF，建议的分类命名：II型单纯疱疹病毒(Herpes Simplex Virus Type2)，保藏编号为CGMCC No.3600，保藏日期为2010年2月3日。
在一些实施例中，构建上述OH2病毒时，选用的是II型单纯疱疹病毒，在其标准株的基础上敲除ICP34.5基因和ICP47基因，并插入人粒细胞-巨噬细胞集落刺激因子(GM-CSF因子)表达盒；其中：
敲除ICP34.5基因使溶瘤病毒丧失了神经毒性，而且可以选择性在的肿瘤细胞内复制；
敲除ICP47基因可诱导机体特异性地抗肿瘤免疫反应，还能促使下游US11基因(对溶瘤病毒在肿瘤细胞内的复制有促进作用)表达上调；
GM-CSF因子具有促进树突状细胞(DC细胞)募集活化的作用，也可以增加DC细胞表面主要组织相容性抗原肽的量，进一步增强了DC细胞的抗原递呈能力；GM-CSF因子可以刺激髓源性前体细胞增殖分化，增强机体特异性免疫反应；另外，GM-CSF因子可增强抗体依赖的免疫细胞介导的细胞毒效应、外周血的淋巴以及单核细胞的细胞毒效应，有效增强机体的抗肿瘤免疫反应。
将OH2病毒的遗传物质经过LNP或者其他材料包封后，形成的包封体能够进入细胞中，然后在体内形成活的OH2病毒，进而产生抗肿瘤的作用。相比于溶瘤病毒直接作为抗肿瘤药物，本申请以溶瘤病毒的遗传物质进行抗肿瘤，可以解决或部分解决溶瘤病毒系统给药诱导的先天及获得的抗病毒免疫导致的强烈抗病毒作用，使溶瘤病毒拥有更好的抗肿瘤效果。
在本申请实施例中，所述的脂质体由阳离子脂质化合物与辅助分子组成。
DSPC与胆固醇主要是辅助脂质，有助于形成双分子层；处方中的DMG-PEG2000脂质，可提高LNP在制备和储存中的稳定性，而这些DMG-PEG2000脂质一般含有短酰基链，有助于DMG-PEG2000脂质在注射后迅速从LNP中分离，避免聚集，促进LNP与细胞的相互作用。
在本申请实施例中，所述阳离子脂质化合物为SM102、(2,3-二油酰基-丙基)-三甲基铵-氯盐(DOTAP)中的一种或组合。
在本申请实施例中，所述辅助分子为二硬脂酰磷脂酰胆碱(DSPC)、胆固醇及1，2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇2000(DMG-PEG 2000)的至少一种或组合。
在本申请实施例中，所述脂质纳米颗粒中包含的阳离子脂质化合物和辅助分子优选的的摩尔比例是：阳离子脂质化合物：DSPC：胆固醇：DMG-PEG2000为(40-55)：(5-15)：(30-45)：(1-5)。
在某些实施例中，所述脂质纳米颗粒中包含的阳离子脂质化合物和辅助分子更优选的摩尔比例是：阳离子脂质化合物：DSPC：胆固醇：DMG-PEG2000为50：10：38：2。
脂质纳米颗粒(LNP)的制备
主要试剂来源：
DSPC(艾伟拓(上海)医药科技有限公司，后简称AVT，产品编号：B81229)；胆固醇(AVT，产品编号：B80859)；DMG-PEG2000(AVT，产品编号：E00S-DMG-20043-08-25)；SM102(厦门赛诺邦格，产品编号：M2104000880005)
1、溶瘤病毒侵染非洲绿猴肾细胞(Vero细胞)
采用OH2病毒侵染Vero细胞。
Vero细胞在T300培养瓶中培养，培养条件为5％FBS完全培养基、37℃、5％CO2，待细胞汇合度达到60-80％时，向其中接入1×106CCID50个OH2病毒，OH2病毒为本申请发明人自行研制，研制批次为“BVT-OH2-201912A-SXT-NS”，滴度为1×107CCID50，过夜培养16小时。
2、溶瘤病毒DNA的初提取。
采用DNAzol试剂盒提取OH2病毒DNA。
(1)配置75％的酒精：吸取21mL 100％的酒精与7mL的无核酸酶水(DEPC)混匀。
(2)裂解、匀浆：倒出扩培细胞培养瓶(T300)中的培养基，加入10mL的DNAzol试剂，多次吹打混匀，直至将瓶壁上裂解后的细胞完全吹打下来。
(3)离心取上清：将上述得到的溶液转移至50mL的离心管中，配平后用高速冷冻离心机在5000rmp、4℃条件下离心10min，将得到的上清转移至新离心管中。
(4)沉淀：离心管加入12mL的75％的酒精，上下颠倒10次充分混匀后静置3分钟；然后4000g、4℃条件下离心2min。
(5)漂洗：弃上清，加入2mL 75％的酒精，吹打几次，然后在4000g、4℃条件下离心1min。
(6)重复5步骤2次。
(7)溶解：尽可能的去掉酒精，静置2min，待酒精完全挥发掉后，加入2mL的DEPC水复溶，待DNA复溶后放入-20℃冰箱中保存。
3、DNA电泳
由于用DNAzol法得到的OH2病毒DNA不纯，会含有Vero细胞的基因组。此步为纯化OH2病毒DNA(担心UV会对DNA造成影响，所以没有进行核酸胶拍照)。
(1)制胶：(1g琼脂糖+100mL 1×TAE溶液)溶解后+10μL GelRED，制成大加样孔的胶。
(2)点样：采用5μL 15000bp DNA Marker，上2mL的样品(其中每600μL样品与20μL 6×loading buffer混匀上样)。
(3)电泳：电压120V，电泳30min。
4、采用胶回收试剂盒(Fast Pure Gel DNA Extraction Mini Kit)提取OH2病毒DNA。
(1)切胶：电泳结束后，用蓝盾可见光透射仪切胶(目标条带大于15000bp)，将切好的胶用5mL EP管收集。
(2)胶回收：按照快速纯化DNA试剂盒说明书操作，回收OH2病毒DNA，最后采用DEPC水复溶OH2病毒DNA得到溶液。
(3)用Qubit检测OH2病毒DNA的溶液浓度。
5、制备脂质体溶液
在本申请实施例中，将前述阳离子脂质化合物SM102和辅助分子按比例混合于乙醇溶剂中制得，其中的辅助分子为二硬脂酰磷脂酰胆碱(DSPC)、胆固醇及1,2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇2000(DMG-PEG2000)，SM102与其它辅助分子的比例如表1所示。
表1
制备的有机相(LNP相)总浓度为12.5mmol/L。
6、用制备的脂质体溶液包封溶瘤病毒的DNA，即得到前述的脂质纳米颗粒(LNP)。
在本申请实施例中，可以根据脂质纳米颗粒(LNP)的用量大小，选择包封方法。用量较小时，采用滴加法来制备OH2病毒DNA-LNP复合物，即；用量较大时，使用微流控设备大批量制备前述的脂质纳米颗粒(LNP)。
在本申请实施例中，滴加法操作步骤如下：将OH2病毒DNA溶解在醋酸钠缓冲液(pH5.0、20mmol/L)中，调整OH2病毒DNA浓度为20μg/mL，然后以体积比为4：1(醋酸钠缓冲液相：LNP相)，将LNP相缓慢滴加至溶有OH2 DNA的醋酸钠 缓冲液中，滴加过程中缓慢摇晃，滴加完成后用枪头吹打混匀，孵育5-10min后，加入PBS缓冲液(0.01M，pH7.4)稀释至所需浓度。
脂质纳米颗粒(LNP)在抗肿瘤的应用
本申请实施例公开了脂质纳米颗粒(LNP)在抗小鼠结肠癌细胞的应用，所述的脂质纳米颗粒(LNP)包含重组人GM-CSF溶瘤II型单纯疱疹病毒的基因组DNA。
1、肿瘤模型建立。
选取6-8周龄的雌性Balb/C小鼠，在体内植入CT26-iRFP(100μL/只，1×107个/mL)细胞，以此来构建CT26肿瘤模型。
2、CT26肿瘤小鼠治疗方案分组
CT26肿瘤小鼠培养第9天(以植入CT26-iRFP细胞时计为第1天)作为抗肿瘤治疗的第1天，抗肿瘤治疗分三次，分别在治疗的第1天、第4天、第7天，治疗方式为注射抗肿瘤试剂。在本申请实施例中，将CT26肿瘤小鼠分为9组，分别给予不同的治疗方案，分组及治疗方案见表2所示。
表2
以上组别中，1-4组中，注射的抗肿瘤试剂为“SM102-OH2 DNA”，即包含重组人GM-CSF溶瘤II型单纯疱疹病毒的基因组DNA脂质纳米颗粒(LNP)，所述脂质纳米颗粒(LNP)用前述方法进行制备，1-3组的注射DNA用量分别为500ng、50ng、5ng，注射方式为“瘤内给药途径(i.t.)”，4组注射DNA用量为500ng，注射方式为“静脉给药(i.v.)”；5组注射的抗肿瘤试剂为SM102脂质体，DNA用量为0，注射方式 为“i.t.”；6组为空白对照组，注射的抗肿瘤试剂为PBS缓冲液，DNA用量为0，注射方式为“i.t.”；7组注射的抗肿瘤试剂为“OH2 DNA”，即未被包封的OH2病毒的DNA，DNA用量为500ng，注射方式为“i.t.”；8组注射的抗肿瘤试剂为“Virus(CCID50)”，即OH2病毒，注射用量为1×106CCID50，注射方式为“i.t.”；9组注射的抗肿瘤试剂为“DOTAP-OH2 DNA”，即另一种包裹重组人GM-CSF溶瘤II型单纯疱疹病毒的基因组DNA脂质纳米颗粒，这里我们LNP-2，LNP-2的制备过程中，除了将SM102脂质换为DOTAP，其它制备环节与前述脂质纳米颗粒(LNP)一致，此组注射DNA用量为500ng，注射方式为“i.t.”。
3、分别在抗肿瘤治疗的第1、4、7、10、14、21、28天观察小鼠体内肿瘤体积大小。
4、抗肿瘤治疗结果与分析。
图1-3所示的为接受不同治疗方案的小鼠的肿瘤体积变化结果，即1-9组的实验结果。图1所示的是按不同时间显示的小鼠肿瘤体积(平均值)变化，图2所示的是不同组别内各小鼠在接受治疗28天时的肿瘤体积，从图2可以看出第8组(OH2 Virus(i.t.)组)有4只无瘤小鼠，第1组(SM102-OH2 DNA 500ng(i.t.)组)有3只无瘤小鼠，第2组(SM102-OH2 DNA 50ng(i.t.)组)有一只无瘤小鼠，第4组SM102-OH2 DNA 500ng(i.v.)组有一只无瘤小鼠，这说明用SM102包封的OH2 DNA即前述的脂质纳米颗粒(LNP)能够起到一定的治疗效果。
如图3所示，除了第8组(OH2 Virus(i.t.)组)肿瘤的平均体积较小外，其余组肿瘤平均体积没有明显差别，即根据28天时肿瘤的总平均体积水平来看，并不能得出图2的结论，出现这种情况的主要原因在植瘤的过程中肿瘤大小不均一，一般要求在肿瘤平均体积大于100mm3开始注射药物(此体积是临床前体内药效的指导原则推荐体积，有些文献上对于难治疗的肿瘤，体积在50mm3就开始给药治疗)，而在植瘤第九天的时候，有的小鼠肿瘤体积已经超过200mm3甚至更大，这时可能已经错过了最佳的治疗时间，不能起到很好的治疗效果。
基于上述原因，本申请发明人将原始实验数据进行处理，剔除了在植瘤后第九天(治疗第一天)肿瘤体积≥150mm3的小鼠，然后得到了另一组实验数据，如图4-6所示。可以看出，不管是从无瘤小鼠数量还是从平均肿瘤体积大小两方面来看，第1组(SM102-OH2 DNA 500ng(i.t.)组)与第8组(OH2 Virus(i.t.)组)的效果均接近。上述结果也提示在抗肿瘤治疗时，需要严格控制初始的肿瘤体积大小，尽量保证初始肿瘤体积不要过大且尽量均一。
综上所述，本申请公开了包封的溶瘤病毒遗传物质及其应用。本申请中，提取溶瘤病毒的遗传物质，并通过脂质进行包封，形成包封溶瘤病毒遗传物质的脂质纳米颗粒(LNP)，如采用本申请发明人自研的重组人GM-CSF溶瘤II型单纯疱疹病毒(OH2病毒)，通过生产OH2病毒的遗传物质DNA，并用脂质体进行包封，获得包封OH2病毒DNA的脂质纳米颗粒(LNP)。利用所述脂质纳米颗粒(LNP)进行抗肿瘤，可以解决直接用溶瘤病毒抗肿瘤的过程中由于溶瘤病毒激活患者的先天免疫途径而导致患者产生非特异性炎症反应的问题，拥有更好的抗肿瘤效果。另外，本申请公开了所述所述脂质纳米颗粒(LNP)的制备方法，所述方法可用于高效制备抗肿瘤药物或疫苗，如包封OH2病毒遗传物质DNA的LNP药物。所述方法不仅降低抗肿瘤药物生产批次的批间差和外源因子污染的风险，还增强了抗肿瘤药物储存和运输稳定性；有利于抗肿瘤药物注射方式的进一步拓展。
以上所述，仅为本申请较佳的具体实施方式，但本申请的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本申请揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本申请的保护范围之内。

Claims (21)

  1. 一种脂质纳米颗粒，包括以下组分：
    编码溶瘤病毒的遗传物质，其中所述溶瘤病毒包括重组溶瘤I或II型单纯疱疹病毒；和
    脂质体，所述脂质体包括阳离子脂质化合物与辅助分子的组合，
    所述脂质体包封所述遗传物质以形成所述脂质纳米颗粒。
  2. 根据权利要求1所述的脂质纳米颗粒，其中所述遗传物质为DNA或RNA的一种。
  3. 根据权利要求1或2所述的脂质纳米颗粒，其中所述溶瘤病毒为重组溶瘤I型或II型单纯疱疹病毒，其遗传物质为DNA。
  4. 根据权利要求1至3中任一项所述的脂质纳米颗粒，其中所述溶瘤病毒为重组溶瘤II型单纯疱疹病毒，其遗传物质为DNA。
  5. 根据权利要求4所述的脂质纳米颗粒，其中所述重组溶瘤单纯疱疹病毒包括重组人GM-CSF溶瘤II型单纯疱疹病毒，其遗传物质为DNA。
  6. 根据权利要求1至5中任一项所述的脂质纳米颗粒，其中所述阳离子脂质化合物包括十七烷-9-基8-((2-羟乙基)(6-氧代-6-(十一烷氧基)己基)氨基)辛酸酯、(2,3-二油酰基-丙基)-三甲基铵-氯盐中的一种或组合。
  7. 根据权利要求1至6中任一项所述的脂质纳米颗粒，其中所述辅助分子包括二硬脂酰磷脂酰胆碱、胆固醇及1,2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇2000。
  8. 根据权利要求7所述的脂质纳米颗粒，其中所述阳离子脂质化合物和辅助分子的摩尔比例是：阳离子脂质化合物：二硬脂酰磷脂酰胆碱：胆固醇：1,2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇2000为(40-55)：(5-15)：(30-45)：(1-5)。
  9. 根据权利要求8所述的脂质纳米颗粒，其中所述阳离子脂质化合物为十七烷-9-基8-((2-羟乙基)(6-氧代-6-(十一烷氧基)己基)氨基)辛酸酯，所述阳离子脂质化合物和辅助分子的摩尔比例是：十七烷-9-基8-((2-羟乙基)(6-氧代-6-(十一烷氧基)己基)氨基)辛酸酯：二硬脂酰磷脂酰胆碱：胆固醇：1,2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇2000＝50:10:38:2。
  10. 如权利要求1-9中任一项所述的脂质纳米颗粒的制备方法，包括：
    制备脂质体溶液，其中所述脂质体溶液包含阳离子脂质化合物和辅助分子；和
    用制备的脂质体溶液包封溶瘤病毒的遗传物质，以得到所述的脂质纳米颗粒，
    其中，所述溶瘤病毒为重组溶瘤I或II型单纯疱疹病毒。
  11. 根据权利要求10所述的方法，其中所述阳离子脂质化合物包括十七烷-9-基8-((2-羟乙基)(6-氧代-6-(十一烷氧基)己基)氨基)辛酸酯、(2,3-二油酰基-丙基)-三甲基铵-氯盐中的一种或组合。
  12. 根据权利要求10或11所述的方法，其中所述辅助分子包括二硬脂酰磷脂酰胆碱、胆固醇及1,2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇2000。
  13. 根据权利要求12所述的方法，其中所述阳离子脂质化合物和辅助分子的摩尔比例是：阳离子脂质化合物：二硬脂酰磷脂酰胆碱：胆固醇：1,2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇2000为(40-55)：(5-15)：(30-45)：(1-5)。
  14. 根据权利要求13所述的方法，其中所述阳离子脂质化合物为十七烷-9-基8-((2-羟乙基)(6-氧代-6-(十一烷氧基)己基)氨基)辛酸酯，所述阳离子脂质化合物和辅助分子的摩尔比例是：十七烷-9-基8-((2-羟乙基)(6-氧代-6-(十一烷氧基)己基)氨基)辛酸酯：二硬脂酰磷脂酰胆碱：胆固醇：1,2-二肉豆蔻酰-rac-甘油-3-甲氧基聚乙二醇2000＝50:10:38:2。
  15. 根据权利要求10至14中任一项所述的方法，还包括以下步骤中的一个或多个：溶瘤病毒遗传物质的提取、纯化和回收。
  16. 根据权利要求15所述的方法，还包括：将所述溶瘤病毒遗传物质溶解在醋酸钠缓冲液，以获得含所述溶瘤病毒的遗传物质的溶液。
  17. 根据权利要求16所述的方法，其中用制备的脂质体溶液包封溶瘤病毒的遗传物质，以得到所述的脂质纳米颗粒具体包括：
    将所述脂质体溶液和所述含所述溶瘤病毒的遗传物质的溶液以1：4的体积比，将所述脂质体溶液滴加至所述含所述溶瘤病毒的遗传物质的溶液中，以得到所述的脂质纳米颗粒，
    其中所述脂质体溶液的浓度为12.5mol/L，所述含所述溶瘤病毒的遗传物质的溶液的浓度为20μg/mL。
  18. 一种药物或疫苗，包含权利要求1-9中任一项所述的脂质纳米颗粒及药学可接受的辅料。
  19. 如权利要求1至9中任一项所述的脂质纳米颗粒或如权利要求18所述的药物或疫苗在抗肿瘤中的应用。
  20. 如权利要求1至9中任一项所述的脂质纳米颗粒或如权利要求18所述的药物或疫苗在制备抗肿瘤药物中的应用。
  21. 一种肿瘤治疗方法，包括向有需要的受试者施用治疗有效量的如权利要求1至9中任一项所述的脂质纳米颗粒或如权利要求18所述的药物或疫苗。
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