CN115227674A - Encapsulated oncolytic viral genetic material and uses thereof - Google Patents
Encapsulated oncolytic viral genetic material and uses thereof Download PDFInfo
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Abstract
The present application discloses encapsulated oncolytic viral genetic material and uses thereof. The present application constructs a lipid nanoparticle with genetic material of an oncolytic virus encapsulated in a liposome. The lipid nanoparticles are used for resisting tumors, so that the problem of virus resistance caused by the fact that viruses activate the innate immunity of patients and gain immunity in the process of directly using an oncolytic virus system for resisting tumors can be solved, and the lipid nanoparticles have better anti-tumor effect. In addition, the application discloses a preparation method of the anti-tumor medicament, which not only reduces the batch difference of the production batches of the anti-tumor medicament and the risk of exogenous factor pollution; the storage and transportation stability of the antitumor drug is enhanced; is beneficial to the further expansion of the injection mode of the antitumor drug.
Description
Technical Field
The present application relates to the field of anti-tumor technology using oncolytic viruses, in particular to encapsulated oncolytic viral genetic material and applications thereof.
Background
The principle of the oncolytic virus is that the oncolytic virus is adopted to resist tumors and is more and more approved, the specific oncolytic virus is prepared by carrying out gene modification on some viruses with weak pathogenicity in the nature, and the inactivated or defective antiviral genes or cancer suppressor genes in target cells are utilized to selectively infect tumor cells, and a large amount of the oncolytic virus is replicated in the tumor cells and finally destroys the tumor cells. At the same time, it can stimulate immune response, attract more immune cells to continuously kill the residual cancer cells. In recent decades, oncolytic virus therapy has attracted much attention and related research has made tremendous progress.
Herpes simplex viruses, including herpes simplex virus type I and herpes simplex virus type II, are often engineered into oncolytic viruses due to their tumor-avid nature, which recognize and selectively infect tumor cells, eventually leading to swelling of the cells and destruction of the tumor cells, but cannot replicate in normal body cells without killing, theoretically having higher anti-tumor effects and lower side effects.
Among the oncolytic viruses, recombinant oncolytic herpes simplex virus is a promising oncolytic virus for cancer immunotherapy, such as human GM-CSF oncolytic type II herpes simplex virus (hereinafter "OH2 virus"), which can selectively infect tumor cells, replicate in the tumor cells, finally lyse, kill the tumor cells, and release progeny virus particles to further infect the surrounding tumor cells, and this process also facilitates the release of tumor-associated antigens. The anti-tumor effect of the OH2 virus is not only shown in the direct toxic effect of killing tumor cells or virus proteins directly by the replication of the virus; recent evidence shows that OH2 virus can regulate the micro-environment of immunosuppressive tumor, and is beneficial to breaking immune tolerance and exciting anti-tumor immune response. Lysis of tumor cells results in the release of Tumor Associated Antigens (TAAs), which induce systemic anti-tumor immune responses in vivo.
At present, the preparation method of the oncolytic virus mainly comprises the steps of culturing cells, inoculating the cells after the cells are cultured to a certain stage, and then harvesting and purifying the oncolytic virus, but the existing preparation method of the oncolytic virus has the problems of complicated process, higher cost, larger batch difference, higher risk of microbial contamination and the like.
The oncolytic virus with the replication capacity is extremely challenging in clinical systemic drug administration, when the oncolytic virus is used as an anti-tumor drug and is injected into a patient intravenously, the oncolytic virus can cause innate and acquired immune strong antiviral effects, so that the patient generates nonspecific inflammatory reaction, and the generated neutralizing oncolytic virus antibody can prevent the oncolytic virus from infecting and dissolving tumor cells, so that the anti-tumor effect is reduced.
There is still an unmet need in the art for replication competent, effective antitumor oncolytic viruses or related vaccines, pharmaceuticals, and there is therefore a need to provide novel substances and/or compositions for antitumor use, and methods of producing the same.
Disclosure of Invention
The present inventors provide a novel method for producing an anti-tumor substance, which is the genetic material of an encapsulated oncolytic virus, but is not a live virus, and a novel method for producing the same. Preferred oncolytic viruses for this application are recombinant oncolytic I or type II herpes simplex viruses. In certain embodiments, a more effective recombinant human GM-CSF oncolytic type II herpes simplex virus, i.e., an OH2 virus, is selected. After the genetic material (including DNA or RNA) of the OH2 virus is encapsulated by LNP or other materials, the formed encapsulation body can enter cells, and then the live OH2 virus is formed in vivo, thereby generating the anti-tumor effect.
In a first aspect, embodiments of the present application disclose a Lipid Nanoparticle (LNP) comprising the following components:
genetic material encoding an oncolytic virus; the oncolytic virus comprises a recombinant oncolytic I or II herpes simplex virus; and liposomes; the liposomes comprise a cationic lipid compound in combination with an accessory molecule; the liposome encapsulates the genetic material to form the lipid nanoparticle.
Preferably, the genetic material of the oncolytic virus is one of DNA or RNA.
Furthermore, the oncolytic virus is recombinant oncolytic I-type or II-type herpes simplex virus, and the genetic material of the oncolytic virus is DNA.
Preferably, the oncolytic virus is a recombinant oncolytic type II herpes simplex virus, the genetic material of which is DNA.
More preferably, the genetic material is DNA encoding a recombinant human GM-CSF oncolytic herpes simplex II virus (OH 2 virus); wherein the OH2 virus strain is designated H2d3d4-hGF, proposed taxonomic nomenclature: the Type II Herpes Simplex Virus (Herpes Simplex Virus Type 2) is preserved in the general microbiological center of the China Committee for culture Collection of microorganisms, is located in the microbiological research institute of China academy of sciences No.3 of Xilu No. 1 North Chen of the Chao Yangye, beijing, has the preservation number of CGMCC No.3600 and has the preservation date of 2010, 2 months and 3 days.
Further, the cationic lipid compound comprises one or a combination of heptadecan-9-yl 8- ((2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) caprylate (SM 102), (2, 3-dioleoyl-propyl) -trimethylammonium-chloride salt (DOTAP).
Further, the accessory molecules include distearoyl phosphatidylcholine (DSPC), cholesterol, and 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000).
Further, the molar ratio of the cationic lipid compound and the accessory molecule is: cationic lipid compound: DSPC: cholesterol: DMG-PEG2000 is (40-55): (5-15): (30-45): (1-5); the preferred molar ratios are: cationic lipid compound: DSPC: cholesterol: DMG-PEG2000 is 50:10:38:2.
in a second aspect, the present application provides a method for preparing Lipid Nanoparticles (LNPs) according to the first aspect, including:
primary extraction of oncolytic virus genetic material; the oncolytic virus is recombinant oncolytic I type herpes simplex virus or recombinant oncolytic II type herpes simplex virus;
purifying and recovering oncolytic virus genetic materials;
preparing a liposome solution; and encapsulating genetic material of the oncolytic virus by using the prepared liposome solution to obtain the Lipid Nanoparticle (LNP).
Furthermore, there are various ways to prepare the oncolytic virus genetic material, and the oncolytic virus genetic material can be prepared by infecting Vero cells or using genetic engineering (gene synthesis, plasmid preparation, gene editing, polymerase Chain Reaction (PCR) amplification, in vitro transcription IVT, etc.).
Further, the oncolytic virus is a recombinant oncolytic type I or II herpes simplex virus.
Preferably, the oncolytic virus is recombinant human GM-CSF oncolytic type II herpes simplex virus (OH 2 virus) and the genetic material is DNA.
Further, the liposome solution comprises the cationic lipid compound and the auxiliary molecule in a molar ratio of: cationic lipid compound: DSPC: cholesterol: DMG-PEG2000 is (40-55): (5-15): (30-45): (1-5).
Preferably, the liposome solution comprises the cationic lipid compound and the auxiliary molecule in a molar ratio of: cationic lipid compound: DSPC: cholesterol: DMG-PEG2000 is 50:10:38:2.
further, the volume ratio of the liposome solution to the encapsulated oncolytic viral DNA is 1: 4.
In a third aspect, the present application discloses the application of the Lipid Nanoparticle (LNP) of the first aspect in tumor resistance.
In a fourth aspect, the present application discloses a medicament or vaccine, wherein the medicament or vaccine comprises the Lipid Nanoparticle (LNP) of the first aspect and a pharmaceutically acceptable excipient.
In a fifth aspect, the present application discloses the use of the drug or vaccine of the fourth aspect in resisting tumor.
Compared with the prior art, the application has at least one of the following beneficial effects:
the present application relates to encapsulated oncolytic viral genetic material and uses thereof. In the present application, genetic material of oncolytic virus is extracted and encapsulated by lipid to form Lipid Nanoparticles (LNP) encapsulating the oncolytic virus genetic material, such as recombinant human GM-CSF oncolytic type II herpes simplex virus (OH 2 virus) self-developed by the inventors of the present application, by producing genetic material DNA of OH2 virus and encapsulating with liposomes to obtain Lipid Nanoparticles (LNP) encapsulating OH2 virus DNA. The Lipid Nanoparticles (LNP) are used for resisting tumors, so that the problem of innate induced by oncolytic virus system administration and strong antiviral effect caused by acquired antiviral immunity can be solved or partially solved, and the oncolytic virus has a better antitumor effect. In addition, the present application discloses a preparation method of the Lipid Nanoparticle (LNP), which can be used for efficiently preparing an anti-tumor drug, such as an LNP drug encapsulating OH2 virus genetic material DNA. The method not only reduces the batch difference of the production batches of the anti-tumor drugs and the risk of exogenous virus and microbial pollution, but also enhances the storage and transportation stability of the anti-tumor drugs; is beneficial to the further expansion of the injection mode of the antitumor drug.
Drawings
FIG. 1 is a line graph showing the change in tumor volume in mice receiving different treatment regimens as provided in the examples of the present application.
Figure 2 is a scatter plot of the mean tumor volume in vivo for mice on day 28 receiving different treatment regimens as provided in the examples herein.
Figure 3 is a graph of the mean tumor volume in vivo results for mice receiving different treatment regimens for 28 days, provided in the examples of the present application.
FIG. 4 shows the removal of a tumor with an initial tumor volume of 150cm or more according to the embodiments of the present application 3 The in vivo tumor volume change line of the mouse.
FIG. 5 shows the removal of a tumor with an initial tumor volume of 150cm or more according to the present embodiment 3 Mean volume scatter plot of tumors in vivo at 28 days in mice.
FIG. 6 shows the removal of a tumor with an initial tumor volume of 150cm or more according to the present embodiment 3 In vivo mean tumor outcome at day 28 in mice.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more clearly understood, the present application is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application. Reagents not individually described in detail in this application are conventional and commercially available; methods not specifically described in detail are all routine experimental methods and are known from the prior art.
It should be noted that the terms "first", "second", and the like in the description and claims of the present invention and in the drawings are used for distinguishing similar objects, and do not necessarily have to be used for describing a specific order or sequence or have a substantial limitation on technical features thereafter. It is to be understood that the data so used is interchangeable under appropriate circumstances such that the embodiments of the invention described herein are capable of operation in other sequences than those illustrated or described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed, but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
Description of Lipid Nanoparticles (LNP)Ming dynasty
In the present examples, the Lipid Nanoparticles (LNPs) are prepared by liposome encapsulation of genetic material encoding an oncolytic virus.
In the examples of the present application, the oncolytic virus is a recombinant herpes simplex virus, and in the previous research and description, herpes Simplex Virus (HSV) has a natural advantage as an oncolytic virus, and is specifically represented as follows:
(1) The host is wide, and can infect various histiocytes and induce the immune response of the organism;
(2) Has the characteristics of replicating, reproducing and cracking host cells in the host cells;
(3) The replication time is short, and the oncolytic effect is ensured;
(4) Can carry exogenous gene, further promote the oncolytic effect;
(5) Can remove a plurality of non-essential genes, reduce the neurotoxicity of the oncolytic virus and increase the selectivity of tumor cell lysis;
(6) The safety is high, and the infection of the oncolytic virus is easy to control;
(7) Is not genetically fused to the host cell and replicates in the host cell in episomal form.
Since herpes simplex viruses are classified into herpes simplex virus type I and herpes simplex virus type II, in the examples of the present application, the oncolytic viruses may be further classified into recombinant oncolytic herpes simplex virus type I and recombinant oncolytic herpes simplex virus type II.
In the embodiment of the application, the method for constructing the oncolytic virus by the herpes simplex virus is to knock out at least one non-essential gene of the original herpes simplex virus, such as the ICP34.5 gene of the neurotoxicity gene, the propagation of the gene in tumor cells is non-essential, and after the gene is knocked out, the oncolytic virus loses neurotoxicity and can selectively replicate in the tumor cells.
In certain embodiments, in addition to knock out non-essential genes of herpes simplex virus, novel expression sequences are introduced in a construction strategy to construct recombinant oncolytic herpes simplex virus as a novel oncolytic virus.
In certain embodiments, the liposome comprises DNA encoding an oncolytic virus that is a recombinant human GM-CSF oncolytic type II herpes simplex virus (OH 2 virus). Wherein the OH2 virus is derived from the common microorganism center of China Committee for culture Collection of microorganisms, the address of the virus strain is located in the microorganism research institute of China academy of sciences No.3 of Xilu No. 1 of Beijing, chaozhou, the rising area of the Chaoyang, the name of the virus strain is H2d3d4-hGF, and the suggested classification names are as follows: the Type II Herpes Simplex Virus (Herpes Simplex Virus Type 2) has the preservation number of CGMCC No.3600 and the preservation date of 2010, 2 months and 3 days.
In some embodiments, the OH2 virus is constructed by knocking out ICP34.5 gene and ICP47 gene based on a standard strain of II herpes simplex virus, and inserting a human granulocyte-macrophage colony stimulating factor (GM-CSF factor) expression cassette; wherein the content of the first and second substances,
the ICP34.5 gene is knocked out, so that the oncolytic virus loses neurotoxicity and can selectively replicate in tumor cells;
the ICP47 gene is knocked out, so that an organism can be induced to specifically resist tumor immune response, and the expression of a downstream US11 gene (which has a promoting effect on the replication of oncolytic virus in tumor cells) can be promoted to be up-regulated;
the GM-CSF factor has the function of promoting the recruitment and activation of dendritic cells (DC cells), and can also increase the amount of major histocompatibility antigen peptides on the surfaces of the DC cells, thereby further enhancing the antigen presenting capacity of the DC cells; GM-CSF can stimulate proliferation and differentiation of myeloid precursor cells, and enhance specific immune response of organisms; in addition, GM-CSF can enhance the cytotoxic effect of antibody-dependent immune cells, peripheral blood lymph and mononuclear cells, and effectively enhance the anti-tumor immune response of the body.
After the genetic material of the OH2 virus is encapsulated by LNP or other materials, the formed encapsulated body can enter cells, and then the live OH2 virus is formed in vivo, thereby generating the anti-tumor effect. Compared with the direct application of the oncolytic virus as an anti-tumor drug, the application of the genetic material of the oncolytic virus for resisting tumor can solve or partially solve the problem of innate induction of systemic administration of the oncolytic virus and strong anti-virus effect caused by acquired anti-virus immunity, so that the oncolytic virus has better anti-tumor effect.
In the present embodiment, the liposome is composed of a cationic lipid compound and an accessory molecule.
DSPC and cholesterol are mainly helper lipids, contributing to the formation of bilayers; the DMG-PEG2000 lipids formulated provide enhanced stability of the LNP during manufacture and storage, and these DMG-PEG2000 lipids generally contain short acyl chains, which facilitate rapid separation of the DMG-PEG2000 lipid from the LNP after injection, avoid aggregation, and facilitate the interaction of the LNP with cells.
In the examples of the present application, the cationic lipid compound is one or a combination of SM102, (2, 3-dioleoyl-propyl) -trimethylammonium-chloride (DOTAP).
In the present embodiment, the accessory molecule is at least one or a combination of Distearoylphosphatidylcholine (DSPC), cholesterol, and 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000).
In the present embodiment, the preferred molar ratio of the cationic lipid compound and the accessory molecule contained in the lipid nanoparticle is: cationic lipid compound: DSPC: cholesterol: DMG-PEG2000 is (40-55): (5-15): (30-45): (1-5).
In certain embodiments, the more preferred molar ratio of the cationic lipid compound and the accessory molecule comprised in the lipid nanoparticle is: cationic lipid compound: DSPC: cholesterol: DMG-PEG2000 is 50:10:38:2.
preparation of Lipid Nanoparticles (LNPs)
The main reagent sources are as follows:
DSPC (Avicent pharmaceutical science and technology Co., ltd., AVT for short, product number B81229); cholesterol (AVT, product No. B80859); DMG-PEG2000 (AVT, product number: E00S-DMG-20043-08-25); SM102 (Xiamen Sainuang Nippon lattice, product code: M2104000880005)
1. Oncolytic virus infection of African green monkey kidney cells (Vero cells)
And infecting the Vero cells by using OH2 virus.
Vero cells were cultured in a T300 flask under conditions of 5% FBS complete medium, 37 ℃ and 5% CO 2 When the cell confluence reaches 60-80%, 1 × 10 cells are inoculated into the cell confluence 6 CCID 50 The OH2 virus and the OH2 virus are self-developed by the inventor of the application, the development batch is BVT-OH2-201912A-SXT-NS, and the titer is 1 multiplied by 10 7 CCID 50 The cells were cultured overnight for 16 hours.
2. Primary extraction of oncolytic virus DNA.
And (3) extracting the OH2 virus DNA by adopting a DNAzol kit.
(1) Preparing 75% alcohol: 21mL of 100% ethanol was aspirated and mixed with 7mL of nuclease-free water (DEPC).
(2) And (3) cracking and homogenizing: pouring out the culture medium in the expanded cell culture bottle (T300), adding 10mL of DNAzol reagent, and blowing and beating uniformly for multiple times until the cracked cells on the bottle wall are blown and beaten completely.
(3) Centrifuging and taking a supernatant: transferring the obtained solution into a 50mL centrifuge tube, leveling, centrifuging for 10min at 5000rmp and 4 ℃ by using a high-speed refrigerated centrifuge, and transferring the obtained supernatant into a new centrifuge tube.
(4) And (3) precipitation: adding 12mL of 75% alcohol into a centrifuge tube, turning the centrifuge tube upside down for 10 times, fully mixing uniformly, and standing for 3 minutes; then, the mixture was centrifuged at 4000g and 4 ℃ for 2min.
(5) Rinsing: the supernatant was discarded, 2mL of 75% ethanol was added thereto, and the mixture was whipped several times and then centrifuged at 4000g at 4 ℃ for 1min.
(6) Repeat 5 steps 2 times.
(7) Dissolving: removing the alcohol as much as possible, standing for 2min, adding 2mL of DEPC water for redissolving after the alcohol is completely volatilized, and storing in a refrigerator at the temperature of-20 ℃ after the DNA is redissolved.
3. DNA electrophoresis
Since OH2 virus DNA obtained by DNAzol method is not pure, it will contain Vero cell genome. This step was to purify the OH2 viral DNA (there was no photography of the nucleic acid gel, since UV was a concern for affecting the DNA).
(1) Preparing glue: (1 g agarose +100mL 1 XTAE solution) was dissolved and then + 10. Mu.L GelRED was added to prepare a gel for large wells.
(2) Sample application: 2mL samples were taken from 5. Mu.L of 15000bp DNA Marker (where each 600. Mu.L sample was mixed with 20. Mu.L of 6 × loading buffer).
(3) Electrophoresis: voltage 120V, electrophoresis for 30min.
4. OH2 viral DNA was extracted using a Gel recovery Kit (Fast Pure Gel DNA Extraction Mini Kit).
(1) Cutting the glue: after the electrophoresis was finished, the gel was cut with a blue shield visible light transilluminator (target band more than 15000 bp), and the cut gel was collected with a 5mL EP tube.
(2) And (3) glue recovery: according to the operation of the rapid purification DNA kit specification, OH2 virus DNA is recovered, and finally DEPC water is adopted to redissolve the OH2 virus DNA to obtain a solution.
(4) The solution concentration of OH2 viral DNA was measured using a Qubit.
5. Preparation of Liposome solutions
In the examples of the present application, the cationic lipid compound SM102 and the auxiliary molecule were mixed in ethanol solvent in proportion, wherein the auxiliary molecule was Distearoylphosphatidylcholine (DSPC), cholesterol and 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG 2000), and the ratio of SM102 to other auxiliary molecules is shown in table 1.
TABLE 1
The total concentration of the prepared organic phase (LNP phase) was 12.5mmol/L.
6. Encapsulating DNA of the oncolytic virus by using the prepared liposome solution to obtain the Lipid Nanoparticle (LNP).
In the present embodiment, the encapsulation method may be selected according to the amount of the Lipid Nanoparticles (LNPs). When the dosage is small, the OH2 virus DNA-LNP compound is prepared by adopting a dropping method, namely; in larger amounts, the aforementioned Lipid Nanoparticles (LNPs) are mass-produced using microfluidic devices.
In the embodiment of the present application, the dropping method comprises the following steps: OH2 viral DNA was dissolved in a sodium acetate buffer (pH 5.0, 20 mmol/L) to adjust the OH2 viral DNA concentration to 20. Mu.g/mL, and then the mixture was concentrated in a volume ratio of 4:1 (sodium acetate buffer phase: LNP phase), slowly dripping the LNP phase into a sodium acetate buffer solution dissolved with OH2 DNA, slowly shaking in the dripping process, blowing and uniformly mixing by using a gun head after finishing dripping, incubating for 5-10 min, and adding a PBS buffer solution (0.01M, pH7.4) to dilute to the required concentration.
Application of Lipid Nanoparticles (LNP) in tumor resistance
The embodiment of the application discloses application of Lipid Nanoparticles (LNP) in resisting mouse colon cancer cells, wherein the Lipid Nanoparticles (LNP) comprise genome DNA of recombinant human GM-CSF oncolytic II type herpes simplex virus.
1. And (4) establishing a tumor model.
Selecting female Balb/C mice of 6-8 weeks old, implanting CT26-iRFP (100 mu L/mouse, 1X 10) 7 one/mL) cells, to construct a CT26 tumor model.
2. Treatment protocol grouping for CT26 tumor mice
The CT26 tumor mice are cultured on day 9 (day 1 when the CT26-iRFP cells are implanted) and used as the 1 st day of the anti-tumor treatment, the anti-tumor treatment is divided into three times, and the anti-tumor agent is injected on the 1 st day, the 4 th day and the 7 th day of the treatment respectively. In the present example, the CT26 tumor mice were divided into 9 groups and given different treatment regimens, respectively, with the grouping and treatment regimens shown in table 2.
TABLE 2
In the above groups, 1 to 4 groups injected antitumor agent is "SM102-OH2 DNA", i.e. genomic DNA Lipid Nanoparticle (LNP) containing recombinant human GM-CSF oncolytic type II herpes simplex virus, the Lipid Nanoparticle (LNP) is prepared by the aforementioned method, the injection DNA dosage of 1 to 3 groups is 500ng, 50ng and 5ng respectively, the injection mode is "intratumoral administration route (i.t.)", the injection DNA dosage of 4 groups is 500ng, and the injection mode is "intravenous administration (i.v.)"; the 5 groups of antitumor agents for injection are SM102 liposome, the dosage of DNA is 0, and the injection mode is 'i.t.'; 6 groups are blank control groups, the injected antitumor agent is PBS buffer solution, the DNA dosage is 0, and the injection mode is "i.t."; the anti-tumor agent injected in the 7 groups is 'OH 2 DNA', namely DNA of unencapsulated OH2 virus, the using amount of the DNA is 500ng, and the injection mode is 'i.t.'; the anti-tumor agent injected in group 8 is "Virus (CCID) 50 ) ", i.e., OH2 virus, was injected at 1X 10 6 CCID 50 The injection mode is "i.t."; the anti-tumor agent injected in group 9 is "DOTAP-OH2 DNA", another genomic DNA lipid nanoparticle encapsulating recombinant human GM-CSF oncolytic type II herpes simplex virus, wherein in the preparation process of our LNP-2, except for replacing SM102 lipid with DOTAP, other preparation links are consistent with the Lipid Nanoparticle (LNP) mentioned above, the amount of the injected DNA is 500ng, and the injection mode is "i.t.".
3. The size of the tumor volume in the mice was observed on days 1, 4, 7, 10, 14, 21, and 28 of the antitumor treatment, respectively.
4. Results and analysis of antitumor therapy.
FIGS. 1 to 3 show the results of the tumor volume changes of mice receiving different treatment regimens, i.e., the experimental results of groups 1 to 9. Fig. 1 shows the tumor volume (mean) change of the mice at different times, fig. 2 shows the tumor volume of the mice in the different groups at 28 days of treatment, and fig. 2 shows that the group 8 (OH 2 Virus (i.t.) group) has 4 tumor-free mice, the group 1 (SM 102-OH2 DNA 500ng (i.t.) group) has 3 tumor-free mice, the group 2 (SM 102-OH2 DNA 50ng (i.t.) group) has one tumor-free mouse, the group 4 SM102-OH2 DNA 500ng (i.v.) group has one tumor-free mouse, which indicates that the OH2 DNA encapsulated by SM102, i.e. the aforementioned Lipid Nanoparticles (LNP), can achieve a certain therapeutic effect.
As shown in FIG. 3, the mean tumor volumes of the groups other than group 8 (OH 2 Virus (i.t.) group) were not significantly different, except that the mean tumor volumes were smaller, i.e., the conclusion of FIG. 2 was not drawn based on the total mean tumor volume at 28 days, which is mainly due to the uneven tumor size during the tumor implantation process, which is generally required when the mean tumor volume is greater than 100mm 3 The injection of the drug is started (this volume is the recommended volume for guiding the efficacy in the clinical precursor, and some documents refer to the refractory tumor, the volume is 50mm 3 Treatment started immediately), and at the time of the ninth day of tumor implantation, some mice had tumor volumes exceeding 200mm 3 Even larger, the optimal treatment time may have been missed and not have been effective.
Based on the reasons, the inventor of the application processes the original experimental data and eliminates the tumor volume of more than or equal to 150mm at the ninth day after the tumor implantation (the first day of treatment) 3 Then another set of experimental data was obtained, as shown in fig. 4-6. It can be seen that the effect of group 1 (SM 102-OH2 DNA 500ng (i.t.) group) was close to that of group 8 (OH 2 Virus (i.t.) group) both in terms of the number of tumor-free mice and in terms of the mean tumor volume size. The above results also suggest that in anti-tumor therapy, the initial tumor volume needs to be strictly controlled to ensure that the initial tumor volume is not too large and as uniform as possible.
In view of the foregoing, the present application discloses encapsulated oncolytic viral genetic material and uses thereof. In this application, the genetic material of the oncolytic virus is extracted and encapsulated by lipid to form Lipid Nanoparticles (LNP) encapsulating the genetic material of the oncolytic virus, such as recombinant human GM-CSF oncolytic type II herpes simplex virus (OH 2 virus) self-developed by the inventors of the present application, by producing genetic material DNA of the OH2 virus and encapsulating with liposomes to obtain Lipid Nanoparticles (LNP) encapsulating the OH2 virus DNA. The Lipid Nanoparticle (LNP) is used for resisting tumor, so that the problem that the patient generates nonspecific inflammatory reaction due to activation of the innate immune pathway of the patient by oncolytic virus in the process of directly resisting tumor by the oncolytic virus can be solved, and the Lipid Nanoparticle (LNP) has better anti-tumor effect. In addition, the application discloses a preparation method of the Lipid Nanoparticle (LNP), which can be used for efficiently preparing antitumor drugs or vaccines, such as LNP drugs encapsulating OH2 virus genetic material DNA. The method not only reduces the batch-to-batch difference of the production batches of the anti-tumor drugs and the risk of exogenous factor pollution, but also enhances the storage and transportation stability of the anti-tumor drugs; is beneficial to the further expansion of the injection mode of the antitumor drug.
The above description is only for the preferred embodiment of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present application should be covered within the scope of the present application.
Claims (12)
1. A lipid nanoparticle comprising the following components:
genetic material encoding an oncolytic virus; the oncolytic virus comprises a recombinant oncolytic type I or II herpes simplex virus; and
a liposome; the liposomes comprise a cationic lipid compound in combination with an accessory molecule;
the liposome encapsulates the genetic material to form the lipid nanoparticle.
2. The lipid nanoparticle of claim 1, wherein the genetic material is one of DNA or RNA.
3. The lipid nanoparticle of claim 1, wherein the oncolytic virus is a recombinant oncolytic type I or type II herpes simplex virus, the genetic material of which is DNA.
4. The lipid nanoparticle of claim 1, wherein the oncolytic virus is a recombinant oncolytic type II herpes simplex virus, the genetic material of which is DNA.
5. The lipid nanoparticle of claim 4, wherein the recombinant oncolytic herpes simplex virus comprises a recombinant human GM-CSF oncolytic type II herpes simplex virus, the genetic material of which is DNA.
6. The lipid nanoparticle of claim 1, wherein the cationic lipid compound comprises one or a combination of heptadecan-9-yl 8- ((2-hydroxyethyl) (6-oxo-6- (undecyloxy) hexyl) amino) caprylate, (2, 3-dioleoyl-propyl) -trimethylammonium-chloride salt.
7. The lipid nanoparticle of claim 1, wherein the accessory molecule comprises distearoylphosphatidylcholine, cholesterol, and 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000.
8. The lipid nanoparticle of claim 7, wherein the molar ratio of the cationic lipid compound and the accessory molecule is: cationic lipid compound: distearoyl phosphatidylcholine: cholesterol: 1, 2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (40-55): (5-15): (30-45): (1-5).
9. A method for preparing the lipid nanoparticle according to any one of claims 1 to 8, comprising: primary extraction of oncolytic virus genetic material; the oncolytic virus is recombinant oncolytic I or II type herpes simplex virus; purifying and recovering oncolytic virus genetic materials; preparing a liposome solution; wherein the liposome solution comprises a cationic lipid compound and an accessory molecule in a molar ratio consistent with the ratio of claim 8; and encapsulating the genetic material of the oncolytic virus by using the prepared liposome solution to obtain the lipid nanoparticle.
10. Use of the lipid nanoparticle of any one of claims 1 to 8 for anti-tumor purposes.
11. A medicament or vaccine comprising the lipid nanoparticle of any one of claims 1 to 8 and a pharmaceutically acceptable excipient.
12. Use of the medicament or vaccine of claim 11 against tumors.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116554046A (en) * | 2023-04-17 | 2023-08-08 | 武汉滨会生物科技股份有限公司 | Ionizable lipid compound and lipid nanoparticle thereof |
| WO2024027697A1 (en) * | 2022-08-05 | 2024-02-08 | 武汉滨会生物科技股份有限公司 | Encapsulated oncolytic virus genetic material and use thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080102087A1 (en) * | 2006-07-20 | 2008-05-01 | Vical, Incorporated | Compositions and methods for vaccinating against hsv-2 |
| CN102146418A (en) * | 2010-02-09 | 2011-08-10 | 武汉滨会生物科技有限公司 | Recombinant II type herpes simplex virus vector, preparation method of recombinant II type herpes simplex virus vector, recombinant virus, medicinal composition and application |
| US20150284691A1 (en) * | 2012-10-29 | 2015-10-08 | The Regents Of The University Of California | Composition of viral vectors in lecithin liposomes, preparation method and treatment methods |
| WO2022081764A1 (en) * | 2020-10-14 | 2022-04-21 | RNAimmune, Inc. | PAN-RAS mRNA CANCER VACCINES |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019226650A1 (en) * | 2018-05-23 | 2019-11-28 | Modernatx, Inc. | Delivery of dna |
| CN113453699A (en) * | 2019-01-04 | 2021-09-28 | 昂克诺斯公司 | Encapsulated RNA polynucleotides and methods of use |
| MX2021010808A (en) * | 2019-03-08 | 2021-12-15 | Massachusetts Inst Technology | Synthetic oncolytic lnp-replicon rna and uses for cancer immunotherapy. |
| JP2022086385A (en) * | 2020-11-30 | 2022-06-09 | 雄行 濱田 | Composition for virus introduction |
| CN115227674B (en) * | 2022-08-05 | 2023-07-04 | 武汉滨会生物科技股份有限公司 | Encapsulated oncolytic viral genetic material and uses thereof |
-
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-
2023
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080102087A1 (en) * | 2006-07-20 | 2008-05-01 | Vical, Incorporated | Compositions and methods for vaccinating against hsv-2 |
| CN102146418A (en) * | 2010-02-09 | 2011-08-10 | 武汉滨会生物科技有限公司 | Recombinant II type herpes simplex virus vector, preparation method of recombinant II type herpes simplex virus vector, recombinant virus, medicinal composition and application |
| US20150284691A1 (en) * | 2012-10-29 | 2015-10-08 | The Regents Of The University Of California | Composition of viral vectors in lecithin liposomes, preparation method and treatment methods |
| WO2022081764A1 (en) * | 2020-10-14 | 2022-04-21 | RNAimmune, Inc. | PAN-RAS mRNA CANCER VACCINES |
Non-Patent Citations (4)
| Title |
|---|
| FU X, 等: "Delivery of herpes simplex virus vectors through liposome formulation", 《MOLECULAR THERAPY : THE JOURNAL OF THE AMERICAN SOCIETY OF GENE THERAPY》 * |
| FU X, 等: "Delivery of herpes simplex virus vectors through liposome formulation", 《MOLECULAR THERAPY : THE JOURNAL OF THE AMERICAN SOCIETY OF GENE THERAPY》, vol. 4, no. 5, 30 November 2001 (2001-11-30), pages 450 * |
| 吴振, 等: "重组Ⅱ型溶瘤单纯疱疹病毒纯化工艺的建立", 中国生物制品学杂志, vol. 31, no. 05, pages 552 - 554 * |
| 孙美玲, 等: "重组Ⅱ型单纯疱疹病毒(OH2)基因组修饰稳定性研究", 湖北科技学院学报(医学版), vol. 31, no. 02, pages 97 - 99 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2024027697A1 (en) * | 2022-08-05 | 2024-02-08 | 武汉滨会生物科技股份有限公司 | Encapsulated oncolytic virus genetic material and use thereof |
| CN116554046A (en) * | 2023-04-17 | 2023-08-08 | 武汉滨会生物科技股份有限公司 | Ionizable lipid compound and lipid nanoparticle thereof |
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