Functional synergetic bionic gene editing carrier system as well as preparation method and application thereof

Abstract

The invention relates to the field of bioengineering, in particular to a function-synergetic bionic gene editing carrier system as well as a preparation method and application thereof. The invention provides a gene editing expression box. The gene editing expression box comprises a cancer cell specific promoter, a CXCL9 gene, an sgBAG3 gene and an sgHSP70 gene. A bionic lipid carrier with protein adsorption resistance, cancer cell targeting and membrane fusion delivery is constructed, and an integrated gene editing CRISPR system capable of highly expressing CXCL9 protein factors in cancer cells and double editing tumor cell BAG3 and HSP70 genes is entrapped, so that a multifunctional synergetic bionic gene editing carrier system is constructed; according to the invention, in-vivo delivery stability of the CRISPR system, cancer cell targeting accuracy and intracellular release high efficiency are realized, liver cancer cell gene editing efficiency is improved, a T cell killing enhancement effect tumor immune microenvironment is constructed, and effective cancer resistance of the CRISPR system in a tumor environment is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering, in particular to a functional synergistic biomimetic gene editing carrier system and a preparation method and application thereof. BACKGROUND

[0002] Traditional therapies for liver cancer, such as chemotherapy, radiotherapy, surgical resection, etc., have certain limitations, such as drug resistance of cancer cells to chemotherapeutic drugs, which is one of the main reasons for treatment failure, and the possibility of adverse side effects on internal organs after drug delivery. So far, it is still a challenge to develop a liver cancer treatment drug with low toxicity and specificity. Exploring a new precise treatment for liver cancer, improving treatment effect, reducing patient pain and reducing medical resource consumption is an urgent task for liver cancer treatment.

[0003] Clustered regularly interspaced short palindromic repeats / associated protein 9 (CRISPR / Cas9) is the third generation of gene editing tools, which has been widely used in the research of various gene disease treatments. Studies have shown that CRISPR / Cas9 technology has the potential to precisely edit liver cancer-related oncogenes, inhibit their function, reduce cancer cell proliferation, and achieve liver cancer gene editing therapy. However, how to construct a delivery system for the CRISPR system to achieve efficient delivery and precise gene editing of cancer cells in vivo is one of the key problems of liver cancer gene editing therapy. Nanocarriers have programmable and multifunctional advantages and are widely used in the research of in vitro and in vivo delivery of CRISPR systems, but there are problems such as unstable delivery, low targeting and insufficient intracellular release efficiency, which limit its practical application in gene editing technology. SUMMARY

[0004] Therefore, the present application provides a functional synergistic biomimetic gene editing carrier system and a preparation method and application thereof. The present application constructs a biomimetic lipid carrier with anti-protein adsorption, cancer cell targeting and membrane fusion delivery, and encapsulates an integrated gene editing CRISPR system that can highly express CXCL9 protein factor and double edit tumor cell BAG3 and HSP70 genes, thereby constructing a multifunctional synergistic biomimetic gene editing carrier system, achieving stability of in vivo delivery of the CRISPR system, precision of cancer cell targeting, and high efficiency of intracellular release, improving the efficiency of liver cancer cell gene editing, constructing a T cell killing enhanced effect tumor immune microenvironment, and realizing effective anticancer of the CRISPR system in the tumor environment.

[0005] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0006] The application further provides a liposome outer layer, comprising: distearoylphosphatidylethanolamine-polyethylene glycol-GE11, dioleoylphosphatidylcholine and bromide trimethyl-2, 3-dioleoyloxypropylammonium.

[0007] In some embodiments of the application, in the above-mentioned liposome outer layer, the molar ratio of the distearoylphosphatidylethanolamine-polyethylene glycol-GE11, the dioleoylphosphatidylcholine and the bromide trimethyl-2, 3-dioleoyloxypropylammonium is (5~10): (80~90): (5~10).

[0008] In some embodiments of the application, in the above-mentioned liposome outer layer, the molar ratio of the distearoylphosphatidylethanolamine-polyethylene glycol-GE11, the dioleoylphosphatidylcholine and the bromide trimethyl-2, 3-dioleoyloxypropylammonium is 5:85:10.

[0009] The application further provides a preparation method of the above-mentioned liposome outer layer, mixing the distearoylphosphatidylethanolamine-polyethylene glycol-GE11, the dioleoylphosphatidylcholine and the bromide trimethyl-2, 3-dioleoyloxypropylammonium, and obtaining the liposome outer layer by the thin film hydration method.

[0010] In some embodiments of the application, in the above-mentioned preparation method, the temperature of the mixing is 35℃, and the rotation speed is 50 revolutions / minute.

[0011] The application provides an expression cassette, comprising: a cancer cell specific promoter, a CXCL9 gene, a sgBAG3 gene and a sgHSP70 gene.

[0012] In some embodiments of the application, in the above-mentioned expression cassette, the cancer cell specific promoter comprises: a Sur promoter and / or a Tyr promoter.

[0013] In some embodiments of the application, in the above-mentioned expression cassette, the cancer cell specific promoter is a Tyr promoter.

[0014] In some embodiments of the application, in the above-mentioned expression cassette, the sequence of the CXCL9 gene is as shown in SEQ ID NO:1; the gRNA sequence of the sgBAG3 gene is as shown in SEQ ID NO:2; and the gRNA sequence of the sgHSP70 gene is as shown in SEQ ID NO:3.

[0015] The application further provides an expression vector, comprising: the above-mentioned expression cassette and a backbone plasmid.

[0016] In some embodiments of the application, in the above-mentioned expression vector, the expression cassette and the backbone plasmid comprise: pTC333 / sgBH and / or pXG333.

[0017] The application also provides a liposome comprising the expression vector described above; and

[0018] The outer layer of the liposome described above and / or the outer layer of the liposome obtained by the preparation method described above.

[0019] The application also provides a preparation method of the liposome described above, comprising the following steps:

[0020] S1: calcium precipitation and hybridization of the expression vector to obtain a calcium precipitation hybrid expression vector inner core suspension;

[0021] S2: dispersing the calcium precipitation hybrid expression vector inner core suspension in the outer layer of the liposome, and purifying through a filter membrane with gradient pore size to obtain the functional synergistic biomimetic gene editing carrier system.

[0022] In some embodiments of the application, in the preparation method described above, the calcium precipitation uses 0.5M CaCl2.

[0023] In some embodiments of the application, in the preparation method described above, the hybridization uses 0.01M Na2CO3.

[0024] In some embodiments of the application, in the preparation method described above, the purification uses a filter membrane with a pore size of 200-800nm.

[0025] The application also provides the use of the expression cassette described above, the expression vector described above, the outer layer of the liposome described above, the outer layer of the liposome obtained by the preparation method described above, the functional synergistic biomimetic gene editing carrier system described above and / or the functional synergistic biomimetic gene editing carrier system obtained by the preparation method described above in the preparation of an anti-tumor product.

[0026] In some embodiments of the application, in the use described above, the anti-tumor includes killing cancer cells.

[0027] In some embodiments of the application, in the use described above, the killing of cancer cells includes down-regulating the expression of anti-apoptotic proteins and / or up-regulating the expression of pro-apoptotic proteins.

[0028] The application also provides a product comprising the expression cassette described above, the expression vector described above, the outer layer of the liposome described above, the outer layer of the liposome obtained by the preparation method described above, the functional synergistic biomimetic gene editing carrier system described above and / or the functional synergistic biomimetic gene editing carrier system obtained by the preparation method described above.

[0029] The beneficial effects of the application include:

[0030] (1) The multi-level targeting design of the CRISPR system carrier shell inner core can improve the accuracy of gene editing of cancer tissues.

[0031] (2) Anti-protein adsorption membrane fusion liposome construction can overcome non-specific protein adsorption of the carrier, bypass the "endocytosis-lysosome" pathway, and improve the intracellular release efficiency of the CRISPR system.

[0032] (3) The construction of the integrated enhanced T cell immunotherapy gene editing CRISPR system creates a microenvironment with strong anti-tumor effect, and realizes the effective anti-cancer of the CRISPR system in the tumor environment. BRIEF DESCRIPTION OF DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below.

[0034] Figure 1 Figure 1 shows the construction of the CRISPR system pTC333 / sgBH of the integrated enhanced T cell immunotherapy;

[0035] Figure 2 Figure 2 shows the schematic diagram of intracellular efficient and precise delivery of the cancer-targeting anti-protein adsorption membrane fusion liposome-mediated gene editing system;

[0036] Figure 3 Figure 3 shows the invention mode diagram; in vivo delivery process: cancer-targeting anti-protein adsorption membrane fusion liposome GAPMFL pTC333 / sgBH Overcoming serum protein adsorption, realizing membrane fusion intracellular delivery, and multi-stage cancer cell targeting precise guidance mediated by cancer-targeting ligand and cancer cell-specific CRISPR plasmid, opening BAG3 and HSP70 gene editing and CXCL9 high expression in cancer cells, regulating the tumor microenvironment, improving T cell tumor immunotherapy, and avoiding unnecessary gene editing in non-cancer cells;

[0037] Figure 4 Figure 4 shows the determination of the nanoparticle size and potential of three kinds of liposomes obtained by different formulations: a: particle size of three kinds of liposomes GAPMFL1, GAPMFL2 and GAPMFL3; b: zeta potential of GAPMFL1, GAPMFL2 and GAPMFL3 (n = 3 biological independent samples); * p < 0.05, ns: not statistically significant;

[0038] Figure 5 Figure 5 shows the TEM images of three kinds of GAPML systems with core-shell structure;

[0039] Figure 6Membrane fusion efficiency of three kinds of liposome GAPMFL1, GAPMFL2, GAPMFL3 screened out by confocal display; wherein: a shows the fluorescence microscope graph of the cell after the red fluorescence labeled three kinds of liposome transfecting the green fluorescence labeled cell membrane; b shows the analysis of the colocalization coefficient of three kinds of liposome to cell membrane based on the fluorescence microscope graph, using ImageJ software; c shows the fluorescence intensity of three kinds of liposome fusion on cell membrane based on the fluorescence microscope graph, using ImageJ software;

[0040] Figure 7 Delivery of functional verification by membrane fusion bypassing the traditional "endosome-lysosome" pathway;

[0041] Figure 8 Anti-protein adsorption capacity of three kinds of liposome under different serum concentrations;

[0042] Figure 9 Specific transfection experiment of three kinds of liposome GAPMFL1, GAPMFL2, GAPMFL3 in cancer cells and non-cancer cells;

[0043] Figure 10 Construction schematic diagram of two kinds of cancer cell specific edited CRISPR plasmids pSG333 and pTG333;

[0044] Figure 11 Screening of CRISPR plasmid specifically expressed in cancer cells; wherein: a shows the fluorescence protein expression intensity of three different cancer cell specific gene editing plasmids pTG333, pSG333, pXG333 in 6 kinds of cancer cells; b shows the fluorescence protein expression intensity of three different cancer cell specific gene editing plasmids pTG333, pSG333, pXG333 in 6 kinds of non-cancer cells; c shows the flow cytometry graph of positive cells of fluorescence protein expression of three different cancer cell specific gene editing plasmids pTG333, pSG333, pXG333 in 6 kinds of cancer cells; d shows the flow cytometry graph of positive cells of fluorescence protein expression of three different cancer cell specific gene editing plasmids pTG333, pSG333, pXG333 in 6 kinds of non-cancer cells; e shows the flow cytometry quantitative columnar graph of positive cells of fluorescence protein expression of three different cancer cell specific gene editing plasmids pTG333, pSG333, pXG333 in 6 kinds of cancer cells; f shows the flow cytometry quantitative columnar graph of positive cells of fluorescence protein expression of three different cancer cell specific gene editing plasmids pTG333, pSG333, pXG333 in 6 kinds of non-cancer cells;

[0045] Figure 12 Construction schematic diagram of gene editing system pTC333 capable of simultaneously expressing CXCL9 protein;

[0046] Figure 13 CRISPR system pTC333 / sgBH for integrated enhanced T cell immunotherapy

[0047] Figure 14 Schematic diagram of CRISPR system pTC333 / sgBH for integrated enhanced T cell immunotherapy remodeling tumor immune microenvironment; wherein: a: schematic diagram of CRISPR system pTC333 / sgBH for integrated enhanced T cell immunotherapy transfected cells; b: CXCL9 protein is specifically highly expressed in tumor cells, improves the recruitment effect of T cells to the tumor environment; BAG3 and HSP70 double gene editing destruction, reduces the resistance effect of tumor cells on T cells, thereby shaping a strong anti-tumor effect microenvironment, realizing the effective anticancer effect of CRISPR system in the tumor environment;

[0048] Figure 15 Verification of the synergistic effect of pTC333 / sgBH system on T cell recruitment; wherein: a: is a schematic diagram of the recruitment experiment; b: is a bar chart of the percentage of T cells recruited by pTC333 / sgBH, pTC333, pTG333 / sgBH and control groups.

[0049] Figure 16 Functional synergistic biomimetic liposome GAPMFL pTC333 / sgBH Verification of the killing effect on cancer cells; wherein a: is a schematic diagram of the killing experiment; b: is a graph of the killing effect of different treatments on cancer cells verified by live and dead staining; c: is a Western Blot graph of Cas9 protein specifically expressed in cancer cells in different treatment groups; d: is a quantitative bar graph corresponding to the graph c; e: is a Western Blot graph of two target genes HSP70 and BAG3 protein, inhibition of apoptosis proteins BCL2, BCL2L1, XIAP and pro-apoptotic proteins BAX, CleavedCASP3 in different treatments; f-I: corresponding to the Western Blot graph of two target proteins HSP70 and BAG3, and inhibition of apoptosis proteins BCL2, BCL2L1, XIAP and pro-apoptotic proteins BAX, CleavedCASP3 after different treatments; and the quantitative expression bar graph. DETAILED DESCRIPTION

[0050] The application discloses a functional synergistic biomimetic gene editing vector system, a preparation method and application thereof.

[0051] It should be understood that the expression "one or more of" includes individually each of the objects recited after the expression and various combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and / or" in connection with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0052] The use of the terms "including," "comprising," or "having" and variations thereof, as used in this document, are intended to be open-ended and non-limiting, for example, by not excluding other, unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0053] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the application remains operable. Moreover, two or more steps or actions can be conducted simultaneously.

[0054] The use of any and all examples, or exemplary language herein, for example, "such as" or "including", is intended merely to better illustrate the application and does not indicate a limitation on the scope of the application unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the application.

[0055] Further, the numerical ranges and parameters setting forth the broadest scope of the application are approximations, and are understood to be inherently imprecise. Thus, unless otherwise specifically stated, any numerical values are approximations that are understood to be inherently imprecise. It is intended that each numerical range and parameter be understood as having the same minimum and maximum values as those for the various numerical ranges. It is intended, for example, that any numerical values be understood to be approximations that are understood to be inherently imprecise. It is specifically intended that any numerical values disclosed herein be understood to encompass about the specific value enunciated, for example, to the tenth of the unit of the specific value. Unless otherwise indicated, all ranges, amounts, numbers, and percentages are approximations.

[0056] Traditional liver cancer treatment methods have many limitations, and the side effects are obvious. CRISPR technology provides a new solution for gene editing treatment of liver cancer: traditional treatment methods for liver cancer include chemotherapy, radiotherapy, and surgical resection. However, these methods have certain limitations, such as drug resistance in patients with chemotherapy, limited dose in radiotherapy due to low tolerance of the liver to radiation, and challenges in early diagnosis and complete resection of locally advanced tumors. These limitations force us to seek new treatment strategies, such as targeted therapy and gene editing technology, to improve the treatment effect and survival rate of liver cancer patients. However, how to regulate the CRISPR gene editing system in vivo, improve its stability, targeting accuracy, intracellular release efficiency, enhance gene editing efficiency, and reduce off-target effects, is still a key obstacle to liver cancer gene editing treatment.

[0057] Anti-protein adsorption function of the carrier is one of the important prerequisites for improving the stability of in vivo delivery of the CRISPR system: In biomedical science, nanocarriers, especially cationic lipid nanocarriers, are often used for drug and nucleic acid delivery. However, in biological fluids, the surface of nanocarriers is easily covered by biological components, which seriously affects the direct action of the delivery system on the life system. For example, in a serum environment rich in proteins, nanocarriers inevitably interact with serum proteins, resulting in the formation of a protein corona on the surface of the carrier. Excessive protein adsorption, especially non-purpose protein adsorption, can limit the direct contact of the surface components of the nanocarrier with the cell membrane, change its endocytosis pathway, and even affect the cell uptake efficiency. Ensuring the stability and effectiveness of in vivo delivery of the CRISPR system is an important prerequisite for improving the efficiency of intracellular uptake of the CRISPR system. Therefore, it is particularly urgent to construct a nanocarrier with anti-protein adsorption, inhibition of surface protein corona formation, maintenance of the integrity of the functional components on the surface of the carrier, and prevention of serum protein contamination, to ensure the stability and effectiveness of in vivo delivery of the CRISPR gene editing system.

[0058] Multi-stage targeting function of the carrier is an important guarantee for achieving precision in vivo delivery of the CRISPR system and reducing off-target effects: In biological systems, the activity of gene editing systems is still difficult to accurately control in time and space dimensions. When using the CRISPR / Cas9 system for editing, non-specific time or non-targeted location gene editing should be avoided to cause unnecessary gene toxicity. One of the important reasons for off-target effects or gene toxicity of CRISPR / Cas9 gene editing is the inability to accurately regulate the spatial activity of CRISPR / Cas9. To minimize gene toxicity and off-target effects and maximize therapeutic effects, the accuracy of CRISPR / Cas9 targeting in complex biological systems must be improved. To solve the above problems, scientists actively explore various means such as physics, chemistry, and gene regulation to design targeted delivery systems for CRISPR and regulate the spatial activity of gene editing, such as biologically responsive carriers, light-controlled, heat-activated control, ultrasound control, small molecule inhibitors, and small molecule activators. However, these methods have their own advantages and disadvantages. For example, small molecule activators regulate the spatial and temporal activity of CRISPR, and their toxicity cannot distinguish between target cells and non-target cells, which may cause unnecessary damage. The light signal of light-targeted control needs to penetrate biological tissues to reach the target position, which faces the problems of light scattering and light signal weakening, resulting in a decrease in the actual effect on the target position. Therefore, based on previous work, it is urgent to develop new multi-stage targeted synergistic delivery carriers to improve the precise editing of CRISPR system on target tissues and reduce unnecessary physiological results caused by off-target effects.

[0059] The bypassing of the "endocytosis-lysosome" pathway function of the carrier is a "powerful booster" for realizing the intracellular efficient release of the CRISPR system: at present, viral vectors and non-viral vectors are often used for the delivery of the CRISPR system. Among them, the non-viral vector can be programmed and modified, and is widely used for the targeted delivery of the gene editing system. The currently disclosed non-viral vector mainly mediates the intracellular delivery of the carrier through the "endocytosis-lysosome" pathway, but the low lysosome escape rate makes the delivered gene editing system easy to be degraded, resulting in the reduction of the actual intracellular release efficiency and the overall gene editing level. Lysosome escape has become a key step for limiting the delivery efficiency of nanomedicine. Exploring a new pathway that can bypass the "endocytosis-lysosome" to avoid the risk of endocytosis of the gene drug being degraded by the lysosome and improve the efficient intracellular release efficiency of the macromolecular CRISPR editing system has become a problem that needs to be solved.

[0060] The construction of a T cell killing enhanced tumor immune microenvironment realizes the effective guarantee of the CRISPR system for exerting the anticancer effect in the tumor environment: CXCL9 is a cell tropism factor, and the concentration difference of the factor in the environment can change the targeting of T cells, and the editing of the BAG3 and HSP70 double genes can reduce the resistance effect of tumor cells on T cells. Therefore, how to construct an integrated gene editing CRISPR system that can highly express CXCL9 in the tumor microenvironment and double edit BAG3 and HSP70 of tumor cells to form a strong anti-tumor microenvironment and realize the effective anticancer of the CRISPR system in the tumor environment.

[0061] Based on the above background and research basis, the applicant puts forward a scientific hypothesis: construct a biomimetic lipid carrier with anti-protein adsorption, cancer cell targeting and membrane fusion delivery, and load an integrated gene editing CRISPR system that can highly express CXCL9 protein factor in cancer cells and double edit BAG3 and HSP70 genes of tumor cells, so as to construct a multifunctional synergistic biomimetic gene editing carrier system, realize the stability of the in vivo delivery of the CRISPR system, the precision of the cancer cell targeting, the high efficiency of the intracellular release, improve the gene editing efficiency of hepatocellular carcinoma cells, construct a T cell killing enhanced tumor immune microenvironment, and realize the effective anticancer of the CRISPR system in the tumor environment.

[0062] The experimental steps of the present application include:

[0063] (1) Construction of cancer targeting anti-protein adsorption membrane fusion biomimetic liposome

[0064] Membrane fusogenic liposome (MFL) is a new drug delivery carrier with low toxicity and high efficiency. MFL has the potential to bypass the "endocytosis-lysosome" pathway, directly introduce the encapsulated contents into the cytoplasm through membrane fusion, avoid the risk of lysosomal degradation during endocytosis, and improve the efficiency of intracellular release of the cargo. Like other lipid nanocarriers, in a complex biological environment, MFL is also prone to form a protein corona on the surface, thereby limiting its direct contact with the cell membrane and inhibiting the membrane fusion process. It has been reported that zwitterionic materials have excellent performance in resisting non-specific adsorption of biological components. Due to electrostatic-induced hydration, a strong hydration layer can be formed on the surface of zwitterionic materials, preventing protein adsorption and thus obtaining antifouling performance. Based on this, we hypothesized that the introduction of zwitterionic groups on the surface of MFL would help to improve its anti-protein adsorption capacity and maintain its membrane fusion performance, thereby preparing a membrane fusogenic liposome with anti-protein adsorption (APMFL).

[0065] Tumor cell epidermal growth factor (EGF) is a cytokine closely related to tumor development and progression. GE11 is a synthetic polypeptide sequence that can have high affinity with tumor cell epidermal growth factor receptor EGFR, achieving targeting of tumor cells. Using this property, a cancer-targeting distearoylphosphatidylethanolamine-polyethylene glycol-GE11 (DSPE-PEG-GE11) can be designed as one of the functional lipid components. Subsequently, DSPE-PEG-GE11, bromo-trimethyl-2,3-dioleoyloxypropylammonium (DOTAP), and zwitterionic group-terminated dioleoylphosphatidylcholine (DOPC) are dissolved in chloroform. The mixture is mixed in a round-bottom flask according to the preset molar ratio, and the solvent is removed using a rotary evaporator to form a lipid film on the bottom of the flask. Subsequently, the above-mentioned membrane-shaped lipid mixture is dispersed with a core suspension under ultrasonic vibration, and is purified by sequentially passing through filters with pore sizes of 800 nm, 600 nm, 400 nm, and 200 nm. Finally, a cancer-targeting anti-protein adsorption membrane fusogenic liposome GAPMFL is obtained.

[0066] (2) Construction of a gene editing CRISPR system for integrated enhanced T cell immunotherapy

[0067] Construction of cancer cell-specific CRISPR plasmid: The gene sequence of cancer cell-specific promoter Tyr is searched from Genebank, and the sequence is synthesized. The original promoter "Chicken β-actin" on the CRISPR plasmid pXG333 is replaced by molecular cloning technology to obtain a recombinant plasmid pX333-Tyr-EGFP of the cancer cell-specific promoter, which is abbreviated as pTG333. Figure 10). Then, the EGFP protein on the pTG333 plasmid was replaced by CXCL9 through cloning technology, so as to construct the CRISPR expression plasmid pX333-Tyr-CXCL9 (abbreviated as pTC333) which can specifically express CXCL9 in cancer cells. Figure 12 Finally, the gRNAs targeting HSP70 and BAG3 double genes were designed through the CRISPRdesign tool website, and the gRNAs of different targets were constructed into the CRISPR / Cas9 plasmid pTC333 which can specifically express in cancer cells, to obtain the recombinant plasmid pTC333 / sgBAG3-sgHSP70 (abbreviated as pSC333 / sgBH) targeting HSP70 and BAG3 double genes. Figure 13 The pTC333 / sgBH plasmid has a CRISPR system that can specifically highly express CXCL9 in the tumor microenvironment and double edit BAG3 and HSP70 of tumor cells. The high expression of CXCL9 in the tumor microenvironment causes a concentration difference of the factor, which can promote the recruitment of T cells to the tumor environment. In addition, the editing of BAG3 and HSP70 double genes can improve the tumor killing effect of T cells, thereby shaping a strong anti-tumor microenvironment, and realizing the effective anti-cancer of CRISPR system in the tumor environment.

[0068] (3) Construction of the core in the vector

[0069] Preparation of calcium precipitation hybrid editing plasmid pTC333 / sgBH core: CaCl2 (0.5 M) solution, CRISPR plasmid pTC333 / sgBH and ddH2O were mixed to form A liquid, and B liquid was formed by mixing and diluting Na2CO3 (0.01 M) solution with ddH2O. According to a certain molar ratio, B liquid was added dropwise into A liquid, and the obtained C mixed liquid was placed at room temperature for 15 min, which was the calcium precipitation hybrid CRISPR plasmid core suspension.

[0070] (4) Construction of cancer targeting anti-protein adsorption membrane fusion lipid carrier GAPMFL pTC333 / sgBH

[0071] ​DSPE-PEG-GE11, bromide trimethyl-2,3-dioleoyloxypropyl ammonium (DOTAP) and zwitterionic group-capped dioleoyl lecithin (DOPC) are dissolved in chloroform respectively. The mixture is mixed in a round-bottom flask according to the preset molar ratio, the solvent is removed by a rotary evaporator, and then vacuum drying is performed to form a cancer-targeting anti-protein adsorption membrane fusion lipid film (GAPMFL) on the bottom of the flask. The lipid film is rehydrated with a cancer cell-specific plasmid pTC333 / sgBH hybrid solution under ultrasonic vibration, and the obtained mixed solution is repeatedly extruded under a polycarbonate nanoporous membrane with gradually reduced pore size to obtain a cancer-targeting anti-protein adsorption membrane fusion lipid carrier (GAPMFL) pTC333 / sgBH The design is constructed with a biomimetic lipid carrier with anti-protein adsorption, cancer cell targeting and membrane fusion delivery, which can realize the stability of in vivo delivery of the CRISPR system, the precision of cancer cell targeting, the high efficiency of intracellular release, and improve the target gene editing efficiency of liver cancer Figure 2 .

[0072] In summary, the application uses a cancer-targeting anti-protein adsorption membrane fusion biomimetic liposome GAPMFL to load a cancer-specific promoter-controlled integrated enhanced T cell immunotherapy gene editing CRISPR system pTC333 / sgBH to prepare a functionally synergistic biomimetic gene editing carrier system GAPMFL pTC333 / sgBH . Finally, the antiserum protein adsorption function of the cancer-targeting anti-protein adsorption membrane fusion biomimetic liposome GAPMFL realizes the stable delivery of the CRISPR system pTC333 / sgBH in vivo; the cancer-targeting biomimetic liposome is a primary targeting, and the cancer cell-selective gene editing controlled by the cancer-specific promoter is a secondary targeting, so as to realize the precise aggregation of the CRISPR system pTC333 / sgBH in liver cancer cells; and the CRISPR system pTC333 / sgBH shapes a microenvironment with strong anti-tumor effect, enhances the T cell tumor immunotherapy Figure 3 . The application aims to develop a multifunctional synergistic biomimetic gene carrier, realize accurate control of the spatial activity of the CRISPR / Cas9 system, and perform gene editing treatment on tumor cells, so as to regulate the tumor microenvironment and provide a new basis for T cell tumor immunotherapy.

[0073] In the embodiments 1 to 4, the raw materials and reagents used can be purchased from the market.

[0074] The application is further described below in combination with embodiments:

[0075] Example 1: Functionally synergistic biomimetic liposome screening process

[0076] Step 1: Preparation of the outer layer of liposome: Tumor cell epidermal growth factor (EGF) is a cytokine closely related to tumor development and progression. GE11 is a synthetic 12-amino acid polypeptide sequence (YHWYGYTPQNVI) that can have a high affinity with tumor cell epidermal growth factor receptor EGFR, thereby achieving targeting of tumor cells. Using this property, a cancer-targeting distearoylphosphatidyl ethanolamine-polyethylene glycol-GE11 (DSPE-PEG-GE11) can be designed as one of the functional lipid components. Subsequently, a zwitterionic group-capped dioleoyl lecithin (DOPC) is selected as the phospholipid structural lipid, bromide trimethyl-2,3-dioleoyloxypropyl ammonium (DOTAP) is selected as the cationic functional lipid, and DSPE-PEG-GE11 is selected as the cancer-targeting functional lipid molecule. The three molar ratios of DOPC: DOTAP: DSPE-PEG2000-GE11 are mixed in a round-bottom flask according to the preset ratios as shown in Table 1 (85:10:5; 90:5:5; 80:10:10). The solvent is removed by a rotary evaporator at 35°C at a speed of 50 revolutions per minute to form a lipid film on the bottom of the flask.

[0077] Step 2: Preparation of the inner core suspension: In a mixture of a constant-speed oscillation or stirring soluble CaCl2 (0.5 M) solution and a gene-editing CRISPR plasmid, a mixture of soluble carbonate Na2CO3 (0.01 M) solution B is added dropwise to obtain a calcium precipitate hybrid gene-editing plasmid solution C. The obtained C mixture is placed at room temperature for 15 min to obtain a calcium precipitate hybrid CRISPR plasmid inner core suspension.

[0078] Step 3: Subsequently, the inner core suspension obtained in step 2 is dispersed in the film-shaped lipid mixture of step 1 under ultrasonic vibration, and is sequentially purified through filter membranes with pore sizes of 800 nm, 600 nm, 400 nm, and 200 nm. Finally, cancer-targeting anti-protein adsorption membrane fusion liposomes GAPMFL1, GAPMFL2, and GAPML3 are obtained. The three screened lipid molecules are measured by a nanoparticle size analyzer for particle size (Size: 140-160 nm), particle size uniformity (PDI: 13-16), and electric potential (5.6-10.4) (Table 1; Figure 4 ).

[0079] Table 1 Three GAFML systems prepared using different molar ratios of lipid components

[0080]

[0081] In addition, the selected lipid nanoparticles are photographed by transmission electron microscopy (TEM) ( Figure 5), which proved the integrity of its core-shell structure.

[0082] Example 2 Functional verification of functional synergistic biomimetic liposome

[0083] 1. Cell membrane fusion function verification experiment: 2 x 10 5 liver cells were inoculated in a 6-well plate and incubated at 37°C for 24 hours. Then the culture medium was replaced with three kinds of liposomes GAPMFL1, GAPMFL2, and GAPMFL3 labeled with DSPE-PEG-Cy5 (red fluorescence) and co-incubated for 2 hours, and then the cells were washed with PBS for 3 times. Then the cell nucleus was stained with DAPI for 10 min (blue), and the cell membrane was stained with Cytomembrane (green). After washing with PBS for 3 times, the cells were imaged by confocal laser scanning microscope (Leica TSC SP8, Leica) Figure 6 ). As shown in the figure, the screened GAPMFL1 showed the highest co-localization of cell membrane (green) and liposome (red) after co-incubation (a) of Figure 6 ; the co-localization coefficients of the three kinds of liposomes on the cell membrane were analyzed by ImageJ software, and the co-localization coefficients of GAPMFL1, GAPMFL2, and GAPMFL3 were 0.85, 0.67, and 0.53, respectively, further proving that GAPMFL1 has higher membrane fusion efficiency (b) of Figure 6 Figure 6 c also showed red fluorescence signal of liposomes aggregated on the cell membrane.

[0084] 2. Membrane fusion delivery function verification experiment: To verify whether the constructed liposome can directly deliver the gene editing plasmid to the cytoplasm through membrane fusion, bypassing the traditional "endocytosis-lysosome" pathway. We labeled the plasmid carried in the core with dye DiYO-1 (green), and still used three kinds of liposomes GAPMFL1, GAPMFL2, and GAPMFL3 labeled with DSPE-PEG-Cy5 (red fluorescence) to co-incubate with cells for 4 hours, and then washed the cells with PBS for 3 times. Then the cell nucleus was stained with DAPI for 10 min (blue). After washing with PBS for 3 times, the cells were imaged by confocal laser scanning microscope (Leica TSC SP8, Leica) Figure 7 ). Figure 7 ​The confocal results show that the GAPMFL1 and GAPMFL2 liposomes (red) after co-transfection show a semicircle of cell membrane outline, meaning good fusion with the cell membrane, while the DiYO-1 green-labeled delivery vector plasmid is scattered in the cytoplasm in a diffuse manner, with the strongest diffuse green fluorescence of GAPMFL1, and the red and green areas are not co-localized. GAPMFL3 does not show a semicircle of cell membrane outline during delivery, while the DiYO-1 green-labeled delivery vector plasmid is present in a highly aggregated form, and co-localization analysis shows that the red fluorescence and green fluorescence are highly coincident. This means that GAPMFL1 and GAPMFL2 liposomes are delivered through the traditional "endocytosis-lysosome" pathway.

[0085] 3. Anti-protein adsorption experiment verification: To verify the anti-protein adsorption efficiency of the three kinds of liposomes GAPMFL1, GAPMFL2, and GAPMFL3, the three kinds of liposomes were placed in culture medium containing 1%, 5%, 8%, 10%, 20%, 40%, 60%, 80%, 90%, and 100% serum protein concentrations. Subsequently, the mixture was incubated at 37°C with a rotation speed of 100xg for 1 hour. To remove unbound serum proteins, the protein-adsorbed liposomes were precipitated by centrifugation (21,000xg, 4°C, 50 minutes). Then, the precipitate was carefully collected and washed with sterile deionized water, and the protein concentration on the precipitated liposomes was then calculated by the BCA method. If Figure 8 The results show that even in 100% serum, the protein adsorption content of GAPMFL1 is only 30μg / mg, which is significantly lower than that of GAPMFL2 (50μg / mg) and GAPMFL3 (60μg / mg), indicating that GAPMFL1 is the best solution for anti-protein adsorption.

[0086] 4. Cancer targeting ability verification: To verify the cancer targeting ability of the three kinds of liposomes, we transfected the three kinds of GAPMFL1, GAPMFL2, and GAPMFL3 liposomes loaded with the plasmid pEGP expressing green fluorescent protein into liver cancer cells HepG2, non-cancer cells including embryonic fibroblast cell line NIH3T3, Schwann cell RSC96, and mesenchymal stem cell BMSC. After 48 hours of transfection, the cells were collected, and the number of cells successfully expressing EGFP fluorescent protein was detected by flow cytometry to evaluate the difference in targeting ability and transfection ability of cancer cells and non-cancer cells. Figure 9Results showed that GAPMFL3 exhibited higher transfection efficiency of 30%-40% in both cancer cells HepG2 and non-cancer cells (NIH3T3, RSC96 and BMSC); GAPMFL2 exhibited a certain cancer targeting specificity with transfection efficiency of 29% in cancer cells HepG2 and transfection efficiency of 15%-22% in non-cancer cells (NIH3T3, RSC96 and BMSC); GAPMFL1 exhibited the strongest cancer targeting ability with transfection efficiency of 31% in cancer cells HepG2 and transfection efficiency of 5%-9% in non-cancer cells (NIH3T3, RSC96 and BMSC).

[0087] In summary, we determined that GAPMFL1 is the best formula of plasmid delivery vector with cancer targeting, anti-protein adsorption and membrane fusion multifunctional synergistic.

[0088] Example 3 Construction of a one-piece enhanced T cell immunotherapy gene editing CRISPR system

[0089] Based on the successful construction of the above multifunctional synergistic biomimetic liposome, we further designed the one-piece enhanced T cell immunotherapy gene editing CRISPR plasmid loaded in the core.

[0090] 1. Construction of cancer cell-specific gene editing CRISPR plasmid: In order to further strengthen the function of the synergistic biomimetic gene editing vector, we constructed a cancer cell-specific gene editing plasmid in the core. In order to verify the specificity of the constructed CRISPR gene editing system, we first amplified the green fluorescent protein sequence EGFP from the pX458 plasmid (Addgene #48138) and connected it to the CRISPR / Cas9 plasmid pX333 (Addgene #64073) through genetic engineering technology to obtain a CRISPR plasmid pX333-EGFP (abbreviated as pXG333) that can express green fluorescent protein. Subsequently, the pXG333 plasmid was used as the basis for modification. Two cancer cell-specific promoter sequences were found in Genebank, Sur promoter (ID: 37310) and Tyr promoter (ID: 11323206), which replaced the original promoter "Chicken β-actin" in the CRISPR plasmid pXG333 through molecular cloning technology. Two cancer cell-specific promoter-controlled gene editing plasmids pX333-Sur-EGFP, abbreviated as pSG333, and pX333-Tyr-EGFP, abbreviated as pTG333, were obtained for the first time (as shown in Figure 10

[0091] ​2. Validation of Cancer Cell-Specific Expression of Constructed Gene-Editing Plasmids: CRISPR / Cas9 plasmids pXG333 (without cancer cell-specific promoters) and pSG333 and pTG333 (with cancer cell-specific promoters), respectively, were transfected into six cancer cell types (HepG2, Hepa1-6, HeLa, 4T1, B16, PC3) and six non-cancer cell types (NIH3T3, RSC-96, BMSC, HEK293T, PC12, PNT1A) using Lipofectamine™ 3000 transfection reagent. Samples were collected 48 hours post-transfection, and the EGFP fluorescence expression intensity of different plasmids in different cells was observed using an inverted fluorescence microscope. The positive rate of EGFP expression after transfection of different plasmids into different cells was quantified using flow cytometry to evaluate the specificity of which cancer cell-specific plasmid expressed the protein in hepatocytes.

[0092] Figure 11 a, c, and e represent the inverted fluorescence spectra of EGFP expression obtained by transfecting six cancer cells (HepG2, Hepa1-6, HeLa, 4T1, B16, and PC3) with CRISPR systems controlled by the three different promoter plasmids mentioned above. Figure 11 (a), and its corresponding flow cytogram ( Figure 11 c) and the quantitative map of EGFP after flow cytometry results ( Figure 11 (e). The results showed that pTG333 expression was strongest in six types of cancer cells, according to both fluorescence images and flow cytometry data.

[0093] Figure 11 b, d, and f are inverted fluorescence images of EGFP expression measured by transfecting six non-cancer cells (NIH3T3, RSC-96, BMSC, HEK293T, PC12, and PNT1A) with CRISPR systems controlled by the above three different promoter plasmids. Figure 11 b), and its corresponding flow cytogram ( Figure 11 (d) and the quantitative map of EGFP after flow cytometry results ( Figure 11 (f). The results showed that pTG333 expression was lowest in six non-cancer cells, according to both fluorescence images and flow cytometry data.

[0094] In summary, the constructed CRISPR plasmid pTG333 exhibits the strongest specificity for cancer cell expression.

[0095] 3. Construction of CRISPR system of integrated enhanced T cell immunotherapy based on pTG333: The sequence of CXCL9 is searched from genebank (the specific sequence is shown in Table 2), and the sequence of EGFP protein expression element on the pTG333 plasmid is replaced by the sequence of CXCL9 through cloning technology, so that the gene editing system plasmid capable of simultaneously expressing CXCL9 protein is obtained and named as pTC333 Figure 12 )

[0096] Table 2 CXCL9 gene sequence

[0097]

[0098] Then, the gRNAs targeting the BAG3 and HSP70 double genes are designed through the CRISPR design tool website, respectively, which are sgBAG3: 5-CACCGACAACAGCCGCACCACTACG-3 (as shown in SEQ ID NO: 2) and sgHSP70: 5-CACCGAACCGGCATGGCCAAAGCCG-3 (as shown in SEQ ID NO: 3). Through molecular cloning technology, they are constructed one by one on the pTC333 capable of simultaneously expressing CXCL9 protein which has been constructed, so that the recombinant plasmid pTC333 / sgBAG3-sgHSP70 (referred to as pTC333 / sgBH) targeting the BAG3 and HSP70 double genes is obtained Figure 13 ). The pTC333 / sgBH plasmid has the CRISPR system capable of specifically highly expressing CXCL9 in the tumor microenvironment and double editing BAG3 and HSP70 of tumor cells.

[0099] The expression of CXCL9 and the double gene editing of BAG3 and HSP70 are innovatively constructed into a CRISPR system of cancer cell specific gene editing, so as to establish the CRISPR system pTC333 / sgBH of integrated enhanced T cell immunotherapy Figure 14 ). It is hoped to achieve the specific high expression of CXCL9 protein in tumor cells, promote the recruitment of T cells to the tumor environment, and achieve the synergistic effect of destroying BAG3 and HSP70 double gene editing, improve the killing effect of T cells on tumor cells, shape a strong anti-tumor effect microenvironment, and realize the effective anticancer of CRISPR system in the tumor environment Figure 15 ).

[0100] Example 4 Verification of potential synergistic effect of integrated enhanced T cell immunotherapy gene editing pTC333 / sgBH system

[0101] The gene editing pTC333 / sgBH of the integrated enhanced T cell immunotherapy screened in Example 3 is taken as the experimental group. In addition, the pTC333 system of only overexpressing CXCL9 but not editing the BAG3 and HSP70 double genes and the pTG333 / sgBH system of only preparing the edited BAG3 and HSP70 genes but not overexpressing CXCL9 are taken as the control groups, and PBS is added as the Control group to evaluate whether the overexpression of CXCL9 and the editing of the BAG3 and HSP70 double genes have the synergistic ability of promoting T recruitment. The specific experiment is as follows: we establish a Tanswell model by co-culturing HepG2 with T cells (a) of Example 5. Figure 16 First, HepG2 is inoculated in a 24-well plate (5x10 4 cells per well), and cultured for 24 hours. The cells are treated with PBS, pTC333 / sgBH, pTC333 and pTG333 / sgBH, respectively, and the transfection amount of the three plasmids is 1 μg / well. After transfection for 24 hours, the Transwell is placed in the corresponding 24-well plate, so that the cancer cells are inoculated in the lower chamber of the Transwell, and 1.0x10 6 tumor infiltrating T cells are added in the upper chamber of the Transwell. After co-culturing for two days, it is observed which treatment can drive the T cells in the upper chamber to the lower chamber.

[0102] The results show that the distribution of T cells recruited from the upper chamber to the lower chamber in different treatments is as follows: the Control group only recruits 5% of T cells, the pTG333 / sgBH group of only preparing the edited BAG3 and HSP70 double genes but not overexpressing CXCL9 can recruit 23% of T cells, the pTC333 system of only overexpressing CXCL9 but not editing the BAG3 and HSP70 double genes can recruit 30% of T cells, but the pTC333 / sgBH system of overexpressing CXCL9 and editing the BAG3 and HSP70 double genes can recruit 40% of T cells, which is significantly higher than that of other groups. It can be seen that the overexpression of CXCL9 and the editing of the BAG3 and HSP70 double genes have the potential synergistic ability of promoting T recruitment when they are simultaneously constructed on one editing system, thereby changing the tumor immune microenvironment.

[0103] Example 5 Verification of the functional synergistic biomimetic liposome GAPMFL pTC333 / sgBH killing cancer cell effect verification

[0104] GAPMFL1, the blank CRISPR system in the inner core of GAPMFL1 was replaced by the CRISPR system pTC333 / sgBH for integrated enhanced T cell immunotherapy screened in Example 4, and named as GAPMFL pTC333 / sgBH . In addition, we delivered the gene editing system pXC333 / sgBH and pSC333 / sgBH expressed in non-cancer cells in Example 3, and named as GAPMFL pXC333 / sgBH and GHAPMFL pSC333 / sgBH As a control, PBS was added as the Control group to evaluate its driving ability on T cells and killing ability on cancer cells. The specific experiment is as follows: we established a Tanswell model by co-culturing HepG2 with T cells Figure 16 a). First, HepG2 was inoculated in a 24-well plate (5x10 4 cells per well), and cultured for 24 hours. Then, the cells were treated with PBS, GAPMFL pXC333 / sgBH , GHAPMFL pSC333 / sgBH , and GAPMFL pTC333 / sgBH , respectively, and transfected for 24 hours. Subsequently, the Transwell was placed in the corresponding 24-well plate, so that the cancer cells were seeded in the lower chamber of the Transwell, and 1.0x10 5 tumor infiltrating T cells were added in the upper chamber of the Transwell. After co-culturing for two days, it was observed which treatment could drive the T cells in the upper chamber to kill the cancer cells in the lower layer.

[0105] After being treated with live and dead staining, the GAPMFL pTC333 / sgBH group showed the strongest cell death signal (red) compared with the control group, indicating that the CRISPR system pTC333 / sgBH for integrated enhanced T cell immunotherapy delivered by the cancer-targeting anti-protein adsorption membrane fusion liposome GAPMFL1 played a significant killing effect Figure 16 b). Subsequently, we performed WB detection on the specific expression of the key gene editing protein Cas9 in tumor cells in each group Figure 16 c, d), and the results showed that the GAPMFL pTC333 / sgBH group presented the highest expression of Cas9 protein in cancer cells, indicating that the cancer cell-specific editing function we constructed played a good role and reduced the potential off-target effect.

[0106] We also performed WB detection and quantitative analysis on the expression of the target proteins corresponding to the two target genes HSP70 and BAG3 Figure 16 e, f, g). The results showed that the GAPMFLpTC333 / sgBH After group treatment, two target genes HSP70 and BAG3 indeed caused damage and interference, leading to a significant decrease in the expression of their corresponding proteins Figure 16 f,g).

[0107] We further analyzed the anti-apoptotic proteins BCL2, BCL2L1 and XIAP and pro-apoptotic proteins BAX, Cleaved CASP3 in cells after treatment in each group Figure 16 e), the quantitative analysis results show that, compared with other groups, GAPMFL pTC333 / sgBH After group treatment, the anti-apoptotic proteins BCL2, BCL2L1 and XIAP all showed the lowest expression Figure 16 g,h,i), while the pro-apoptotic proteins BAX, Cleaved CASP3 all showed the highest expression ​ j,k,l).

[0108] In summary, the functional synergistic biomimetic liposome GAPMFL pTC333 / sgBH has the advantages of specifically editing target genes in cancer cells, driving T cells to kill cancer cells, and causing the death of tumor cells by down-regulating the expression of anti-apoptotic proteins and up-regulating the expression of pro-apoptotic proteins.

[0109] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A lipid exfoliating layer, characterized in that, include: Distearate phosphatidylethanolamine-polyethylene glycol-GE11, dioleoyl lecithin and trimethyl-2,3-dioleoyloxypropylammonium bromide.

2. The lipid exolayer as described in claim 1, characterized in that, The molar ratio of the distearate phosphatidylethanolamine-polyethylene glycol-GE11, the dioleoyl lecithin, and the trimethyl-2,3-dioleoyloxypropylammonium bromide is (5~10):(80~90):(5~10).

3. The method for preparing the lipid exogenous layer as described in claim 1 or 2, characterized in that, The lipid ex vivo layer was obtained by mixing the distearate phosphatidylethanolamine-polyethylene glycol-GE11, the dioleoyl lecithin, and the trimethyl-2,3-dioleoyloxypropylammonium bromide and then performing a thin-film hydration method.

4. An expression box, characterized in that, include: Cancer cell-specific promoters, CXCL9 gene, sgBAG3 gene and sgHSP70 gene.

5. The expression box as described in claim 4, characterized in that, The cancer cell-specific promoters include: the Sur promoter and / or the Tyr promoter.

6. An expression carrier, characterized in that, include: The expression cassette and backbone plasmid as described in claim 4 or 5.

7. The expression vector as described in claim 6, characterized in that, The backbone plasmid includes pXG333.

8. A functionally synergistic biomimetic gene editing vector system, characterized in that, include: The lipid exolayer as described in claim 1 or 2 and / or the lipid exolayer obtained by the preparation method as described in claim 3; and The expression vector as described in claim 6 or 7.

9. The method for preparing the functionally synergistic biomimetic gene editing vector system as described in claim 8, characterized in that, Includes the following steps: S1: The expression vector is subjected to calcium precipitation and hybridization to obtain a calcium-precipitated hybrid expression vector core suspension; S2: Disperse the calcium precipitation hybrid expression vector core suspension in the lipid explant layer, purify it, and obtain the functional synergistic biomimetic gene editing vector system.

10. The application of the lipid exolayer as described in claim 1 or 2, the lipid exolayer obtained by the preparation method as described in claim 3, the expression cassette as described in claim 4 or 5, the expression vector as described in claim 6 or 7, the functionally synergistic biomimetic gene editing vector system as described in claim 8, and / or the functionally synergistic biomimetic gene editing vector system obtained by the preparation method as described in claim 9 in the preparation of antitumor products.

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